The Role of Adaptive Evolution of Phenotypic Plasticity and Historical Population Genetic Processes in Purple Loosestrife

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

2007 The oler of adaptive evolution of phenotypic plasticity and historical population genetic processes in purple loosestrife (Lythrum salicaria L.) invasion in North America Young Jin Chun Iowa State University

Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Ecology and Evolutionary Biology Commons, and the Genetics and Genomics Commons

Recommended Citation Chun, Young Jin, "The or le of adaptive evolution of phenotypic plasticity and historical population genetic processes in purple loosestrife (Lythrum salicaria L.) invasion in North America" (2007). Retrospective Theses and Dissertations. 15922. https://lib.dr.iastate.edu/rtd/15922

This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected].

The role of adaptive evolution of phenotypic plasticity and historical population genetic processes in purple loosestrife (Lythrum salicaria L.) invasion in North America

Young Jin Chun

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Ecology and Evolutionary Biology

Program of Study Committee: John D. Nason, Co-major Professor Kirk A. Moloney, Co-major Professor Fredric J. Janzen Brian J. Wilsey Dianne H. Cook

Iowa State University

Ames, Iowa

2007

UMI Microform 3274864 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii

Dedication

This dissertation is dedicated to my parents, Jin-Woo Chun and Hyung-Sook Park for their love and support throughout my life and educational endeavors. A special dedication must be made with tears to my dear sister Ji-Yeon Chun, for her unfulfilled life and dreams.

iii

Table of Contents

List of Tables v

List of Figures vi

Chapter 1. General Introduction 1 Introduction 1 Two main streams of invasion biology 1 Research Rationale: What can we learn from invasive species biology? 3 Study System 4 Thesis Organization 5 Literature Cited 7

Chapter 2. Are invasive species “made”? – a summary of mechanistic 10 approaches to the study of invasiveness Abstract 10 Introduction 10 The Evolution of Increased Competitive Ability (EICA) 12 The Evolution of Phenotypic Plasticity 22 Local Adaptation 31 Allelopathy 34 Hybridization / Polyploidization 38 Synthesis of Mechanistic View on Invasiveness 40 Literature Cited 43

Chapter 3. Phenotypic plasticity of native vs. invasive purple loosestrife: 61 a two-state multivariate approach Abstract 61 Introduction 62 Univariate Analysis of Reaction Norms 65 Application of Two-State Multivariate Analysis 67 Methods 69 Results 73 Discussion 77 Acknowledgements 84 Literature Cited 84 Chapter 4. Phenotypic plasticity of native vs. invasive purple loosestrife: 91 iv

an application of path analysis Abstract 91 Introduction 92 Methods 94 Results 100 Discussion 104 Acknowledgements 109 Literature Cited 110

Chapter 5. A method for extracting genomic DNA from purple loosestrife 113 containing high levels of secondary metabolites Abstract 113 Introduction 113 Materials and Methods 116 Results 118 Discussion 120 Acknowledgements 122 Literature Cited 122

Chapter 6. Quantitative and molecular genetic variation of native vs. invasive 127 purple loosestrife populations Abstract 127 Introduction 127 Methods 130 Results 137 Discussion 143 Acknowledgements 145 Literature Cited 146

Chapter 7. General Conclusion 154 Summary 154 Future Research 155

Appendix I. Raw data from common garden experiment in Chapter 3 157 Appendix II. Raw data from common garden experiment in Chapter 4 161 Appendix III. Raw data from AFLP experiment in Chapter 6 167 Vita 222

List of Tables

Chapter 2 1. Comparative studies on the effect of natural enemies on invasive plants. 18 2. Comparative studies on phenotypic plasticity between invasive and non- 24 invasive species. 3. Studies on local adaptation of invasive species. 33 4. Invasive lineages evolved after interspecific hybridization. 39

Chapter 3 1. PCA result (loadings) on log-transformed full data set. 74 2. MANOVA results for full and reduced data sets. 76 3. Summary table analyzing the difference in vector magnitude and direction 76 on full and reduced datasets.

Chapter 4 1. Geographical position of sampling sites. 95 2. Summary of ANOVA with P-values of the corresponding F-tests for each 100 trait. 3. PCA result (loadings) on log-transformed full data set. 104 4. Summary statistics of model fit for each provenance and treatment 108 combination.

Chapter 5 1. Major contaminants (polysaccharides, polyphenols, proteins, and RNA), 115 chemicals used to remove them, advantages and/or drawbacks of using these chemicals, and references describing their use in modifications of Doyle and Doyle’s (1990) CTAB method.

2. Mean (±SE) of DNA yield and A260/A280 and A260/A230 ratios, from purple 119 loosestrife, cactus, and cotton species (n: number of individual samples).

Chapter 6 1. Geographical position of sampling sites with genetic diversity in 131 parenthesis for each hierarchical geographical level. 2. Hierarchical nested analysis of molecular variance (AMOVA; Excoffier et 141 al. 1992). 3. Summary of two-tailed sign test for significant deviance from the 50:50 143

odds if QPT = ΦPT on average, with the number of cases for QPT>ΦPT or

QPT<ΦPT. Significant P values are indicated in bold.

List of Figures

Chapter 2 1. Hypothetical expectations of EICA for invasive and native genotypes 13 (species). Solid and dashed lines represent invasive and native genotypes of an invasive species (c, d, e) or invasive and native species in the introduced range (f). 2. Hypothetical expectations on the fitness of invasive and non-invasive 26 genotypes across two environments, representing adaptive plasticity (a, b, c, f) or non-adaptive plasticity (d, e). Solid and dashed lines denote reaction norms for invasive and non-invasive genotypes, respectively. 3. A conceptual model of the potential mechanisms of invading plant species. 41

Chapter 3 1. Least square means (±SE) of six trait values for plants from native (open 66 circles) and invasive (solid circles) provenance. Environmental treatments:

WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL,

High water, Low nutrient; WHNH, High water, High nutrient. 2. Hypothetical reaction norms for two genotypes or populations (indexed 1 68 and 2) and two environments (open and closed circles) in bivariate (a, b, c, d) and multivariate (e) trait space. Solid and dashed lines denote reaction norm vectors for populations 1 and 2, respectively. In panels a-d, the contrasts between columns (a, c vs. b, d) and between rows (a, b vs. c, d)

demonstrate significant G × E interactions due to the amount (|D1 – D2|)

and angular direction (θ1,2) of phenotypic change, respectively. Panels b-e

exhibit significant G × E due to |D1 – D2| > 0, θ1,2 > 0, or both. 3. Principal component analysis (PCA) plots of (a, c) PC1 vs. PC2 (including 74 size effects) and (b, d) PC2 vs. PC3 (excluding size effects). Plots (a) and (b) include polygons which represent convex hulls for each provenance and treatment combination; solid and dashed lines represent invasive and native populations, respectively. Plots (c) and (d) include arrows which indicate the direction and length of trait vectors in the same PC space.

Environmental treatments are: WLNL, Low water, Low nutrient; WLNH,

Low water, High nutrient; WHNL, High water, Low nutrient; WHNH, High water, High Nutrient. vii

4. PCA plots showing reaction norms significantly different for D and/or θ 79 between native plants and invasive plants. Solid and dashed lines represent invasive and native populations, respectively. Left panels are for PC1 vs. PC2 (including size effects) and right panels are for PC2 vs. PC3

(excluding size effects). Environmental treatments are: WLNL, Low water,

Low nutrient; WLNH, Low water, High nutrient; WHNL, High water, Low

nutrient; WHNH, High water, High Nutrient.

Chapter 4 1. Path analytical models. (a) just-identified (default) model; (b) over- 99 identified (tested) model. Numbers represent standardized estimate of regression weights (path coefficients). The eight paths dropped from the just-identified model are marked as dotted line and coefficients in bold. Traits are: HeightB, seedling height at the time of transplant; NDFF, number of days from sowing to the first flowering; StemsR, number of stems originating from the rootstock; Branch, number of secondary branches; MassT, aboveground biomass, HeightF, final height at harvest, and FlowerN, total number of flowers per plant. 2. Least square means (±SE) of eight trait values for plants from native (open 102 cicles) and invasive (solid circle) provenance. Environmental treatments

are: WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient;

WHNL, High water, Low nutrient; WHNH, High water, High nutrient. 3. Least square means (±SE) of eight trait values across all treatments for 103 native (open circles) and invasive (solid circle) plants from each region. Letters indicate that any two regions with no common characters differ significantly (P<0.05, Tukey’s pairwise test). Regions are: TU, Tübingen; SW, Switzerland; PD, Potsdam; NJ, New Jersey; MI, Michigan; IA, Iowa. 4. Total effects of path coefficients between the effects of six traits to other 105 traits of native (open circles) and invasive (solid circle) plants. Traits are: HeightB, seedling height at the time of transplant; NDFF, number of days from sowing to the first flowering; StemsR, number of stems originating from the rootstock; Branch, number of secondary branches; MassT, aboveground biomass, HeightF, final height at harvest, and FlowerN, total

number of flowers per plant. Environmental treatments are: WLNL, Low

water, Low nutrient; WLNH, Low water, High nutrient; WHNL, High water,

Low nutrient; WHNH, High water, High Nutrient. viii

Chapter 5 1. Genomic DNA from purple loosestrife, cactus, and cotton species using 119 electrophoresis in a TAE-buffered, 1% agarose gel with 1kb DNA ladder. Lanes 1 and 10: 1kb DNA ladder, Lanes 2-5: purple loosestrife, Lanes 6-8: cactus (Cylindropuntia imbricata, Opuntia chlorotica, and Opuntia ficus- indica), Lane 9: cotton (Gossypium barbadense). 2. AFLP fingerprint profile of purple loosestrife, cactus and cotton species. 119 Lanes 1-4: purple loosestrife, Lanes 5-7: cactus (Cylindropuntia imbricata, Opuntia chlorotica, and Opuntia ficus-indica), Lane 8: cotton (Gossypium barbadense).

Chapter 6 1. Unrooted phylogram drawn from Nei’s unbiased genetic distance using 139 neighbor-joining method, with bootstrap values (percentages) estimated for each branch. 2. Principal coordinate plots displaying population structure (a) between 139 dimension 1 and 2, (b) between dimension 2 and 3. 3. Estimated population structure from model-based clustering analysis. 140 (Left) Each sample is represented by a thin vertical line, which is partitioned into K colored segments that represent the sample’s estimated membership fractions in K clusters. Black lines separate different populations. Populations are labeled below the figures. (Right) Clustering pattern diagram based on Q matrix of membership coefficients of populations to each cluster.

4. Pairwise ΦPT between pairs of populations plotted against the geographic 141 distance (km) in natural log scale. (a) invasive provenance, (b) native provenance.

5. Comparison of pairwise QPT and ΦPT estimates for (a) height, (b) number 142 of secondary branches, (c) number of stems originating from the rootstock, (d) leaf area, (e) number of days from sowing to the first flowering, (f) aboveground biomass, (g) ratio of reproductive (flowering) part to the aboveground biomass, and (h) total number of flowers per plant. 1

Chapter 1. General Introduction

Introduction

The introduction and spread of non-native species has become a global ecological and environmental problem. Biological invasion has been raised as an important component of global environmental change, and is considered as serious as other factors including greenhouse gas driven climatic change, nitrogen deposition, and change of land use (Vitousek et al. 1996, 1997). The effects of invasive species are considerable to both human life and the ecological systems, including 1) the spread of infectious diseases such as West Nile, monkeypox, or SARS; 2) the economic loss caused by affecting cropfield, rangeland, and commercial forests; 3) the change in ecosystem processes including primary productivity, decomposition, hydrology, nutrient cycling, or natural disturbance regimes; 4) native species extinctions and reduction of biodiversity from introduced predators and pathogens (Vitousek et al. 1996). The invasion process and success is stimulated by other global environmental problems such as land use change, nitrogen deposition, and climate change (Vitousek et al. 1996, Dukes and Mooney 1999). A recent nationwide estimate is that the economic costs of approximately 50,000 nonindigenous species established in the USA exceed $120 billion per year (Pimentel et al. 2005). Lodge et al. (2006) suggested some actions that need to be taken by the government to reduce current and future damage by invasive species, including the screening and prevention of introduction, early detection and eradication, and the establishment of a national center for invasive species management.

Two main streams of invasion biology

The history of invasion biology may be represented as two paths that are independent but not mutually exclusive – the Eltonian path and the Asilomar path (Davis 2005). The Eltonian path has its root in Elton’s (1958) classic book, The Ecology of Invasions by Animals and Plants, describing the world as the battlefield between invasive and native species and calling for conservation efforts. With a strong environmental 2 movement and conservation biology in the 1970s, many studies were oriented to developing management and control programs for specific invasions, using general ecological theory and principles. This trend is evident from the three main questions raised by SCOPE (Scientific Committee on Problems of the Environment, established by the International Council of Scientific Unions) in 1982: 1) what factors determine whether a species will be an invader or not? 2) what are the characteristics of the environment that make it either vulnerable to or resistant to invaders? 3) how can the knowledge gained from answering the first two questions be used to develop effective management strategies? These questions have been the major objectives for invasion biologists for a long time. Studies on invasion ecology have dramatically increased, especially from the conservation perspective. This was coincidently with the emergence of restoration ecology and environmental concerns in the 1980s. Furthermore, practical benefits to society have been emphasized by funding agencies for ecologists in addition to the scientific value of the research (for example, the mandatory section “broader impacts” for all proposals submitted to NSF). Thus, the major research interests of invasion biologists in the late 20th century have been strongly biased to the conservation perspectives of the Eltonian path. In contrast to the Eltonian path, there have been efforts to use the information of invasion in order to develop a better general ecological theory and principles. This view originated from the Biological Sciences Symposium of International Union of Biological Sciences in 1964 in Asilomar, California, in which geneticists, ecologists, and taxonomists gathered to present ideas about evolutionary changes taking place after a species is introduced to a new range. This Asilomar path was inherited by Simberloff (1981, 1986), who used biological invasions as a way to test ecological theory, e.g., island biogeography theory, and Brown and Marshall (1981), who focused on the evolutionary changes of introduced species. While scientists in the Eltonian path implied some anthropogenic concepts of invasion using terms such as “alien”, “exotic”, “invaders” evoking aggressive and intrusive connotations, other scientists used more unbiased words like “colonizers”, “non-native”, “introduced”, evoking basic ecological ideas. The concern with invasive species targets all introduced species and was established on the notion that introduced species are generally ‘bad’, while native species are ‘good’ 3

(Simberloff 2003a). Some scientists disapproved the anthropocentric and hasty insights that vilify all non-native species as malignancies or ecological abnormalities and often neglect the benefits of certain introduced species (Lodge 1993, Simberloff 2003a). Native species may be considered as “invasive” when they spread into “undesirable” habitats (Randall 1997), or when they increase in abundance in their natural habitats by human- caused changes (Alpert et al. 2000). The Asilomar path is a relatively underrepresented and unexplored perspective compared to the large mass of studies accumulated along Eltonian path. However, it appears that the Eltonian path has already been taken to the limit, as it is still unclear whether a general background knowledge of biological invasion will be helpful in establishing a practical management strategy (Allendorf and Lundquist 2003). Mack et al. (2000) addressed the limitations and ineffectiveness of management strategies, including the adverse and indirect effects of biocontrol. Some researchers argued that there are as many reasons for predominance of invasive species as the number of them (Gray 1879, Alpert et al. 2000). Therefore, it is desirable to see the recently increasing efforts to inform more general ecological understandings and theory, utilizing the massive information collected from biological invasions. Clearly, biological invasions can be viewed as experiments of species colonization and invasive species provide an exceptional opportunity for basic research in population ecology and the short-term evolution of species (Allendorf and Lundquist 2003, Simberloff 2003b).

Research Rationale: What can we learn from invasive species biology?

The first two questions raised by SCOPE in 1982, i.e., “what traits make species invasive?” and “what determines the susceptibility of habitats to invasive species?” have been intensively addressed in a large volume of studies and summarized in some good review papers (Crawley 1987, Rejmánek 1995, 1996, Lonsdale 1999, Alpert et al. 2000, Mack et al. 2000, Sakai et al. 2001). However, from the understanding of pre-adapted traits of invasive species or community characteristics that make them susceptible and vulnerable to invaders, the management and control of invasive species is still a controversial and complex problem. One of the most likely reasons is that invasiveness 4 relies more on the interaction between the characteristics of invasive species and their potential new habitats (Alpert et al. 2000). More specifically, invaders often undergo rapid evolutionary events in response to the novel selective pressures they encounter in the new ranges (Müller-Schärer and Steinger 2004). Thus, we may be able to develop a deeper understanding on the background of invasiveness from the studies of evolutionary changes that invasive species undergo from their introduction to the establishment and aggressive spread. Although this knowledge may not be very helpful for effective management and control of invasive species, it may be useful in identifying potential invaders and predicting future invasions (Reichard and Hamilton 1997, Kolar and Lodge 2001, Allendorf and Lundquist 2003). The ultimate goal of this dissertation is to contribute a deeper knowledge on the ability of invasive species to respond to the novel environment and prevail via evolutionary changes, using purple loosestrife (Lythrum salicaria L. Lythraceae) as a model system.

Study System

Purple loosestrife (Lythrum salicaria L.) is an herbaceous perennial plant first introduced to North America from Europe in the early 1800s (Thompson et al. 1987). It has become a well-known, aggressive species changing the basic structure of most of the wetlands it has invaded (Thompson et al. 1987). Specifically, it is considered a significant environmental problem in North America because of its negative ecosystem impacts, including reducing high quality bird habitat, limiting plant biodiversity, and altering wetland function (Blossey et al. 2001). Competitive stands of purple loosestrife have reduced the biomass of 44 native plants and endangered wildlife that depend on these native plants (Gaudet and Keddy 1988). Purple loosestrife now occupies more than 120,000 hectares of North American wetlands and costs $45 million per year in control costs and forage losses (Blossey et al. 2001, Pimentel et al. 2000). The first mention of purple loosestrife in North America was in 1814, in the first edition of the flora by Torrey and Gray (Stuckey 1980). It has been estimated that purple loosestrife first arrived in North America in the last years of the 18th century, the seeds being transported from Europe via ship ballast and livestock (Thompson et al. 1987). 5

During the second half of the 19th century, purple loosestrife may have been brought in with the European immigrants and cultivated for horticultural and pharmacological use (Thompson et al. 1987). It spread first along rivers, canals, and ditches in the northeastern portion of the United States, moving westward along the newly constructed Erie Canal (Thompson et al. 1987). Many public construction projects greatly increased the area being disturbed, providing large areas of available habitat and travel corridors for invasion. The rapid spread of purple loosestrife began in the 1930s, approximately 130 years after being first introduced into North America. Since beekeepers began to recognize the potential of using purple loosestrife as a honey source in the 1940s, they deliberately imported seeds and developed a variety of cultivars. It may have been repeatedly introduced into North America, with different European genotypes being introduced from different areas of Europe (Edwards et al. 1995, Thompson et al. 1987). Purple loosestrife is established in a wide range of habitats, including freshwater marshes and wetlands, open stream margins, and alluvial floodplains (Thompson et al. 1987). Compared to native wetland species in North America, it possesses massive seed production, faster germination, higher germination rates, and greater seedling growth rate (Blossey et al. 2001). The breeding system of purple loosestrife has been of particular interest, since it is a tristylous species, exhibiting three distinct flower types that require outcrossing for successful fertilization (Ågren and Ericson 1996). In addition, studies on purple loosestrife have been greatly increased after it was recognized as a serious invader in North America, including its biology, physiology, impacts on wetland communities and ecosystems, and the evaluation of control and management programs including biological control with herbivores (Blossey et al. 2001).

Thesis Organization

The next chapter of this dissertation is a review paper summarizing recent studies on the leading hypotheses on the mechanisms for invasiveness: (1) EICA (Evolution of Increased Competitive Ability) (2) the evolution of phenotypic plasticity, (3) local adaptation, (4) allelopathy, (5) hybridization / polyploidization. Since invasive species often confront new biotic and abiotic environments in the introduced range, they are 6 exposed to novel selection pressure created by these environments including different climatic and disturbance regimes and different associations of predators, pathogens, and symbionts (including pollinators). The third chapter tests the evolution of phenotypic plasticity hypothesis with an empirical study on the phenotypic plasticity in native vs. invasive populations subject to experimentally manipulated water and nutrient environments. A novel multivariate approach describing a population’s phenotypic plasticity as a vector of mean trait differences (Collyer and Adams 2007) was adopted in this study. The magnitude and direction of this vector between native and invasive populations was compared to show that invasive and native populations differ in adaptive plasticity and trait covariation. This study has been published in Ecology and expanded into a larger international collaborative project that aims to detect the genetic difference in demography and phenotypic plasticity between native and invasive populations. Essentially this is a reciprocal transplant study with multiple common gardens involving collaborators from New Jersey and Germany (led by co-advisor Kirk Moloney). The results from the the Iowa common garden have been presented in Chapter 4, employing structural equation modeling approach (path analysis) to describe causal relationships between fitness and traits that contribute to fitness (fitness-related traits) considering the ontogeny of purple loosestrife. The fifth chapter is a technical note on the development of a genomic DNA extraction protocol for plants that have a large amount of secondary metabolites including polysaccharides and polyphenols. This protocol proved to be fast and effective after multiple tests with purple loosestrife and some cotton and cactus species. The sixth chapter is a population genetic study on the same populations of purple loosestrife used in Chapter 3, with two objectives. One is to compare the genetic diversity and population genetic structure between native and invasive populations. The other is to examine the effect of major evolutionary forces on invasive populations - whether the formation of invasive populations is purely due to stochastic events such as genetic drift and migration, or instead by disruptive and/or stabilizing selection leading to locally adapted populations in the new provenance (North America). This was explored by FST-

QST analysis contrasting population divergence in quantitative traits with neutral genetic variation among populations. The last chapter is the general conclusion summarizing the 7 findings from the above studies, discussing the evolutionary potential of invasive species, and suggesting future research perspectives.

Literature Cited

Ågren, J., and L. Ericson. 1996. Population structure and morph-specific fitness differences in tristylous Lythrum salicaria. Evolution 50:126-139. Allendorf, F. W., and L. L. Lundquist. 2003. Introduction: population biology, evolution, and control of invasive species. Conservation Biology 17(1):24-30. Alpert, P., E. Bone, and C. Holzapfel. 2000. Invasiveness, invisibility and the role of environmental stress in the spread of non-native plants. Perspectives in Plant Ecology, Evolution and Systematics 3(1):52-66. Blossey, B., L. C. Skinner, and J. Taylor. 2001. Impact and management of purple loosestrife (Lythrum salicaria) in North America. Biodiversity and Conservation 10:1787-1807. Brown, A. H. D. and D. R. Marshall. 1981. Evolutionary changes accompanying colonization in plants. Pages 351-363 in G. G. E. Scudder and J. L. Reveal, editors. Evolution Today. Carnegie-Mellon University Press, Pittsburgh, PA. Crawley, M. J. 1987. What makes a community invasible? Pages 429-454 in M. J. Crawley, P. J. Edwards and A. J. Gray (eds.) Colonisation, succession and stability, Blackwell Scientific Publication, Oxford, UK. Davis, M. A. 2005. Invasion biology 1958-2005: the pursuit of science and conservation. Pages 35-64 in M.W. Cadotte, S.M. McMahon, and T. Fukami, editors. Conceptual ecology and invasion biology: reciprocal approaches to nature. Springer, Dordrecht, The Netherlands. Dukes, J. S., and H. A. Mooney. 1999. Does global change increase the success of biological invaders? Trends in Ecology and Evolution 14(4):135-139. Edwards, K. R., M. S. Adams, and J. Květ. 1995. Invasion history and ecology of Lythrum salicaria in North America. Pages 161-180 in P. Pyšek, K. Prach, M. Rejmánek, and M. Wade (eds.) Plant Invasions - General Aspects and Special Problems. SPB Academic Publishing, Amsterdam, The Netherlands. 8

Elton, C. S. 1958. The Ecology of Invasions by Animals and Plants. Methuen, London. Gaudet, C. L., and P. A. Keddy. 1988. Predicting competitive ability from plant traits: a comparative approach. Nature 334:242-243. Gray, A. 1879. The pertinacity and predominance of weeds. The American Journal of Science and Arts, Vol XVIII. Kolar, C. S., and D. M. Lodge. 2001. Progress in invasion biology: predicting invaders. Trends in Ecology and Evolution 16(4):199-204. Lodge, D. M., S. Williams, H. J. Macisaac, K. R. Hayes, B. Leung, S. Reichard, R. N. Mack, P. B. Moyle, M. Smith, D. A. Andow, J. T. Carlton, and A. McMichael. 2006. Biological invasions: recommendations for U. S. policy and management. Ecological Applications 16(6):2035-2054. Lonsdale, W. M. 1999. Global patterns of plant invasions and the concept of invisibility. Ecology 80(5):1522-1536. Mack, R. N., D. Simberloff, W. M. Lonsdale, H. Evans, M. Clout, and F. A. Bazzaz. 2000. Biotic invasions: causes, epidemiology, global consequences, and control. Ecological Applications 10:689-710. Pimentel, D., L. Lach, R. Zuniga, and D. Morrison. 2000. Environmental and economic costs of nonindigenous species in the Unites States. BioScience 50:53-65. Pimentel, D., R. Zuniga, and D. Morrison. 2005. Update on the environmental and economic costs associated with alien invasive species in the United States. Ecological Economics 52:273-288. Randall, J. M. 1997. Defining weeds of natural areas. Pages 18-25 in J. O. Luken and J. W. Thieret, editors. Assessment and Management of Plant Invasions. Springer- Verlag, New York, NY. Reichard, S. H., and C. W. Hamilton. 1997. Predicting invasions of woody plants introduced in North America. Conservation Biology 11(1):193-203. Rejmánek, M. 1995. What makes a species invasive? Pages 3-13 in P. Pyšek, K. Prach, M. Rejmánek, and P. M. Wade (eds.) Plant Invasions – General Aspects and Special Problems. SPB Academic Publishing, Amsterdam, The Netherlands. Rejmánek, M. 1996. A theory of seed plant invasiveness: the first sketch. Biological Conservation 78:171-181. 9

Sakai, A. K., F. Allendorf, J. Holt, D. Lodge, J. Molofsky, K. With, S. Baughman, R. Cabin, J. Cohen, N. Ellstrand, D. McCauley, P. O’Neil, I. Parker, J. Thompson, and S. Weller. 2001. The population biology of invasive species. Annual Review of Ecology and Systematics 32:305-312. Simberloff, D. 1981. Community effects of introduced species. Pages 53-81 in H. Nitecki, editor. Biotic Crises in Ecological and Evolutionary Time. Academic Press, New York, NY. Simberloff, D. 1986. Introduced insects: a biogeographic and systematic perspective. Pages 3-26 in H. A. Mooney and J. A. Drake, editors. Ecology of Biological Invasions of North America and Hawaii. Springer-Verlag, New York, NY. Simberloff, D. 2003a. Confronting introduced species: a form of xenophobia? Biological Invasions 5:179-192. Simberloff, D. 2003b. How much information on population biology is needed to manage introduced species? Conservation Biology 17(1):83-92. Stuckey, R. L. 1980. Distributional history of Lythrum salicaria (purple loosestrife) in North America. Bartonia 47:3-20. Thompson, D. Q., R. L. Stuckey, and E. B. Thompson. 1987. Spread, Impact, and Control of Purple Loosestrife (Lythrum salicaria) in North American Wetlands. US Fish and Wildlife Service. 55 pages. Jamestown, ND: Northern Prairie Wildlife Research Center Home Page. http://www.npwrc.usgs.gov/resource/1999/loosstrf/loosstrf.htm (Version 04JUN99). Vitousek, P. M., C. M. D’Antonio, L. L. Loope, and R. Westbrooks. 1996. Biological invasions as global environmental change. American Scientist 84(5):468-478. Vitousek, P. M. H. A. Mooney, J. Lubchenco, and J. M. Melillo. 1997. Human domination of Earth’s ecosystems. Science 277:494-499.

Chapter 2. Are invasive species “made”? – a summary of mechanistic approaches to the study of invasiveness

Abstract

Numerous studies have investigated the biological characteristics of invasive species and the susceptibility of natural communities and ecosystems to invasion, typically with the perspective that invaders are pre-adapted to the recipient community. Despite many intensive empirical studies and surveys across a broad range of invasive taxa, there is still no consensus on the nature of successful invasion. Recent studies have begun focusing on the “mechanisms” underlying successful invasion of new environments from the standpoint that invasiveness arose as the consequence of rapid evolutionary changes experienced during or following introduction. Here we summarize five leading hypotheses based on a mechanistic approach to explain the background of invasiveness. Though often treated independently, these hypotheses are not mutually exclusive and are mechanistically related via the biotic processes involved in the adaptation and proliferation of colonizing species in natural ecosystems. To examine each hypothesis we review available empirical studies, especially those contrasting invasive and non-invasive populations. Also, we point out aspects of each hypothesis that have been overlooked and should be tested, potential caveats that we should beware of in designing experimental studies to test each hypothesis, and desirable aspects of future studies.

Keywords: biological invasions, mechanistic approaches, EICA, phenotypic plasticity, local adaptation, allelopathy, hybridization, polyploidization, evolution of invasiveness.

Introduction

For a long time, humans have introduced and transported many species to areas outside their natural ranges (Elton 1958). While most cannot survive outside of 11 cultivation and some establish only locally self-sustaining populations (Williamson and Fitter 1996), a smaller subset of species may proliferate and become invasive, dominating communityies and even causing the extinction of natives through rampant growth and/or indirect effects (Mack et al. 2000). To predict the invasive potential of exotic species, efforts have been made to identify key traits of invaders or the vulnerability of recipient communities to invasion, with the goal of identifying and controlling future invaders (Sakai et al. 2001, Mack et al. 2000). This perspective presupposes that invasive species inherently possess traits pre-adapting them to their invaded ranges, or that some characteristics in the recipient communities make them especially susceptible to invaders. It is still generally unclear, however, why a species may be a successful invader in some grographical areas but not others, and why some apparently vulnerable communities are invaded by some aggressive species but not others. Indeed, there are no communities vulnerable to all invasive species, nor are there communities entirely resistant to invasion; as Crawley (1987) noted, “all communities are invasible”. Many researchers argue that disturbance is important too, predisposing a community to invasion, however, examples exist showing that disturbance is not always required and that the role of disturbance depends on the invasive taxon and community type (Lodge 1993). One of the most likely reasons why the search for pre-adapted traits for invasive species has failed is that invasiveness relies more on the interaction between the characteristics of invasive species and their potential new habitats (Alpert et al. 2000). Studies investigating the background of invasion success have thus been switching their focus from the pre-invasion causes to post-invasion events, as there is accumulating evidence that invaders often undergo rapid evolutionary change in response to novel selective pressures they encounter in their new ranges (Müller-Schärer and Steinger 2004). These mechanistic approaches may provide a key to understanding the major processes enabling an invader to be successful in the new environments. They may be able to explain why some invasive species are not successful in some ranges, and why some communities are not generally vulnerable. They have been independently derived, but involve some processes not mutually exclusive for successful invasion. In this review, we summarize and evaluate leading hypotheses related to the interaction between invasive spcies and their new habitats or the changes experienced by 12 invasive species after arriving in the new range. They are the mechanistic approaches to the study of invasiveness (likelihood of species establishment and spread, Rejmánek 2000) – (1) EICA (Evolution of Increased Competitive Ability) (2) the evolution of phenotypic plasticity, (3) local adaptation, (4) allelopathy, (5) hybridization / polyploidization. To investigate available experimental evidence addressing these five mechanisms, we performed meta-analysis of geographical comparative studies obtained via an extensive search of online databases (Biosis, BioOne, EBSCO, JSTOR, Web of Sciences, AGRICOLA) and published articles. The criteria for article selection are detailed below in sections reviewing each hypothesis. Finally, we synthesize the findings of this review, identifying the primary processes common to these hypotheses to develop a better understanding of the mechanistic underpinnings of successful invasion.

The Evolution of Increased Competitive Ability (EICA)

Natural enemies such as insect herbivores and pathogens can limit plant abundance and habitat distribution through depression of plant growth, survival, and reproduction. Therefore, plant species may increase dramatically in abundance and expand the range of habitats occupied in the absence of natural enemies, as may occur when plants are introduced to areas outside of their native range. The enemy release hypothesis (ERH, Keane and Crawley 2002) suggests that the increased abundance and distribution of invasive plants results from lower loads of natural enemies. Alternatively, invasive plants may be more vigorous and abundant, with greater reproduction in the introduced than in the original range, due to genetic differences (see Willis and Blossey 1999 for review). Connecting these two ideas, Blossey and Nötzold (1995) proposed "the evolution of increased competitive ability" (EICA) hypothesis, which states that increased competitive ability of invasive plants results from the post-invasion evolution of more vigorous, but less well defended, genotypes. When plants are resource-limited, they will exhibit trade-offs in resource allocation between growth and defense (optimal defense hypothesis; Coley et al. 1985, Bazzaz et al. 1987). If herbivores are absent in the new non-native habitats, selection will favor genotypes with improved competitive abilities and reduced allocation of resources to herbivore defenses (Blossey and Nötzold 1995). 13

Consistent with these expectations, evidence exists for increased biomass (Blossey and Nötzold 1995, Radford and Cousens 2000, Siemann and Rogers 2001, Buckley et al. 2003, Leger and Rice 2003, Jakobs et al. 2004), release from herbivory (Bossard and Rejmánek 1994, Paynter et al. 1998, Fenner and Lee 2001, Mitchell and Power 2003), and lower level of defensive chemicals (Willis et al. 1999) in invasive genotypes as compared to native conspecifics.

Expectations and supporting evidence for EICA Since the EICA hypothesis posits that invasive genotypes have evolved greater fitness at the expense of herbivore defense, it makes key predictions that can be tested empirically (Fig. 1):

Figure 1. Hypothetical expectations of EICA for invasive and native genotypes (species). Solid and dashed lines represent invasive and native genotypes of an invasive species (c, d, e) or invasive and native species in the introduced range (f). 14

(a) Invasive genotypes should exhibit greater performance (biomass, seed production, etc.) than non-invasive genotypes when grown in a common garden without natural enemies. (b) Invasive genotypes should experience greater damage than non-invasive genotypes when grown with specialist enemies. (c) Non-invasive genotypes should benefit from excluding natural enemies disproportionately compared to invasive genotypes when grown in their respective ranges. This is because both generalist and specialist enemies negatively impact native genotypes in the native range, whereas only generalists exist in the introduced range. Therefore, the harmful effect of all natural enemies (generalists + specialists) should be greater on plants grown in the native range than introduced range. This can be examined by a parallel experiment – where invasive and native plants are grown in their respective areas and natural enemies are excluded from both areas. (d) In a common garden, invasive genotypes should exhibit greater performance than non-invasive genotypes in the absence of specialists, however, invasive genotypes will not perform as well as native genotypes in the presence of specialists. When protected from specialists, invasive genotypes should outperform native genotypes because they have evolved increased competitive ability at the cost of less defense. In contrast, when specialists are present, invasive genotypes should not perform better than non-invasive genotypes because they evolved increased resource allocation to growth and reproduction at the cost of decreased allocation to defense. Reciprocal transplantation of plants between native and introduced range can also test this hypothesis, if specialists are absent in introduced ranges.

These four core predictions of the EICA hypothesis are not mutually exclusive as they represent different outcomes resulting from the assumption of trade-offs between investment in growth and reproduction (competitive ability) versus defense against specialist enemies. When introduced into a new range, however, though having escaped from specialists, plants will confront novel pressures exerted by the community of native generalists. Understanding the effects of these new generalists is thus critical for 15 evaluating invasion success. With this in mind, in addition to the four key predictions of EICA indicated above, invasion biologists are addressing two additional predictions relating invasion success and responses to generalist enemies.

(e) Invasive genotypes should exhibit greater performance (or less damage) in response to the impact of generalists than their ancestral native genotypes, whereas native genotypes are subject to greater damage by generalists than invasive genotypes. (f) Generalist herbivores in the introduced range should feed more lightly on the invasive species and more heavily on native congeneric species (Schierenbeck et al. 1994, Blaney and Kotanen 2001, Rogers and Siemann 2002, Siemann and Rogers 2003a, Lankau et al. 2004, Parker and Hay 2005). This is different with (e) in that the comparision occurs between invasive genotypes and their ancestral conspecific native genotypes in (e), while this hypothesis compares invasive species and the native species that are resident to the introduced ranges and share the natural enemies. In natural settings, generalists in the introduced range are likely to stick with their native hosts and not readily switch to introduced exotic species (behavioral avoidance, Cornell and Hawkins 1993). In controlled experiment, however, generalists may prefer feeding on invasive species relative to native species when they are forced to choose between them (Siemann and Rogers 2003c, Lankau et al. 2004).

Expectations (e) and (f) are a fine-tuned extension of EICA. Recent studies indicate that invasive species evolve to allocate less defense to specialists, but allocate more defense to generalists. While the defense to specialists (digestibility-reducing compounds: tannins and lignins) is quantitative and energetically expensive, defense to generalists (alkaloids, cyanogenic glycosides, glucosinolates) is qualitative and cheap (Joshi and Vrieling 2005). Consequently, in the ancestral native range, invasive species should allocate more to defense against specialists, whereas, in the newly introduced range, if released from specialists, invasive species should decrease allocation to costly defense against specialists, and instead invest more in cheaper qualitative defense against generalists (Müller-Schärer et al. 2004, Joshi and Vrieling 2005). 16

Efforts are being made to disentangle two different responses of plants to attack from natural enemies – resistance and tolerance (Bossdorf et al. 2004, Müller-Schärer et al. 2004, Stastny et al. 2005). While resistance is related to the reduction in damage due to plant traits affecting the preference or performance of natural enemies (Müller-Schärer et al. 2004), tolerance is the ability to compensate for the effect of damage by repairing or re-growing after damage (Stastny et al. 2005). Some empirical studies have focused on resistance simply by measuring the preference of herbivores or the amount of damage. Others emphasize the ability of invasive genotypes that have evolved to tolerate generalists’ attack in the introduced range, at the cost of lower defense to specialists. Although invaders may have evolved greater resistance or tolerance to resident generalists, some researchers emphasize the role of generalist enemies in limiting establishment of invading plants (Biotic Resistance Hypothesis, BRH, Elton 1958). Indeed, generalists are likely to prey on invasive species and resist their invasion (Maron and Vilá 2001, Agrawal and Kotanen 2003, Siemann and Rogers 2003c, Parker and Hay 2005, Parker et al. 2006). Generalists may act differently depending on phylogenetic relatedness of invaders to resident species. Invasion success may increase when an introduced species is less closely related to the resident species, while it may not be successful if it is more closely related (Strauss et al. 2006). If an invader is closely related to a resident species, it will acquire additional natural enemies (particularly generalists) that previously attacked resident relatives (Mitchell et al. 2006, Parker and Gilbert 2007). Therefore, the probability of invasion success will increase if the invader is phylogenetically distant from the resident native species (Darwin’s naturalization hypothesis, Darwin 1859). In contrast, invaders more closely related to natives might be more likely to succeed in novel environments, because they share some traits with related residents that allow them to thrive in the novel environment (Duncan and Williams 2002). Ricciardi and Ward (2006) reported a reduced effect of generalists on invasive species that are closely related to resident species, suggesting that invaders may share some traits with related resident species that confer similar resistance to generalists. Since introduced species share no close evolutionary history with generalist herbivores in the new range, they will not have experienced selection from these enemies and may lack effective defenses (new 17 associations or increased susceptibility hypothesis, Hokkanen and Pimentel 1989, Colautti et al. 2004). Therefore, an invasive species distantly related to the resident species (and their generalists) should be more suppressed than closely related species, in contrast to Darwin’s naturalization hypothesis. The role of phylogenetic relatedness in the effect of generalists is expanded to the role of coevolution in invasion success, which remains a controversial topic (Parker and Hay 2005, Mitchell et al. 2006). To assess whether empirical studies support the six expectations described above, we summarize 33 published comparative studies (Table 1). We included all studies comparing the performance of invasive and native genotypes (or species) under presence and absence of natural enemies (Table 1). We excluded some studies working on only invasive genotypes. Since EICA addresses genotype-based phenotypic change in invasive genotypes relative to native genotypes (or species), the full test of EICA is achieved by comparative studies. From each study, we selected one or two traits that best represent the fitness of invasive vs. native genotypes and ran a meta-analysis using the effect size d (Gurevitch and Hedges 1993).

2 2 X E − X C 3 (n −1)s + (n −1)s d = J J = 1− s = E E C C (1) s 4(n + n − 2) −1 pooled n − n − 2 C E E C where X C is the mean of the control group and X E is the mean of the experimental group. Control vs. experimental groups depends on the hypothesis examined, and can be the absence vs. presence of specialist enemies, the native vs. invasive regions, and the absence vs. presence of generalist enemies. The correction term for sample sizes (J) and the pooled standard deviation (spooled) were calculated to estimate the effect size d. To combine the effect sizes across studies and correctly estimate the weighted average of effect size estimates (d+), we computed the weights as the reciprocal of effect size variance, and ran a weighted ANOVA to look at the difference between invasive vs. native genotypes using R (R development core team 2006). Generally, invasive genotypes indeed evolve greater fitness than non-invasive genotypes [hypothesis (a): dinvasive,+ = 1.87, dnon-invasive,+ = 1.21, P < 0.001], and there is significant evidence that invasive genotypes really escaped from the attack of specialist enemies [hypothesis (c): dinvasive,+ = 0.42, dnon-invasive,+ = -0.34, P < 0.001]. However, it does not necessarily accompany the reduction of defense to specialists [hypothesis (b): 18

dinvasive,+ = 1.01, dnon-invasive,+ = 1.06, P = 0.543]. When reared in a common garden, invasive genotypes are not significantly different in their performance with non-invasive genotypes either in the presence and absence of specialist enemies [hypothesis (d): dinvasive,+ = 0.35, dnon-invasive,+ = 0.44, P = 0.507]. Invasive genotypes do not evolve more resistance to generalist enemies in the invading range [hypothesis (e): dinvasive,+ = 2.06, dnon-invasive,+ = 2.02, P = 0.723], however, invasive species turned out to be less affected by the generalists than the resident species [hypothesis (f): dinvasive,+ = 0.86, dnon-invasive,+ = 1.04, P = 0.045]. Our results indicate that there is an evidence of escaping natural enemies, however, it does not linked to the evolution of competitive ability by trading off with less defense. Thus, we suggest that the traditional biocontrol method of introducing specialist enemies to control invasive species may not be effective. There is still no consensus on the role of specialists that have been co-introduced with their hosts into the new ranges. Some cases suggest that specialists prefer invasive plants over resident congeners (see review by Maron and Vilá 2001) and thus the invaders may restore the resistant ability in response to the specialists (Zangerl and Berenbaum 2005). However, others argue that introduced herbivores facilitate the abundance and species richness of invasive plants by negatively affecting native plants in the introduced range (Parker et al. 2006). In sum, further evaluation of EICA calls for more carefully designed studies.

Suggestions for testing EICA Though EICA is an appealing hypothesis in many aspects, later studies designed to test it have been inconclusive. This may be due to some points that need to be considered for an unambiguous test of EICA. First, EICA states that invasive genotypes evolved greater “competitive ability” at the cost of reduced herbivore defense. In most studies, this competitive ability has been tested as the size or growth of individuals and damage by natural enemies was measured as the loss of vegetative parts (specifically leaf). Some studies measured the amount of secondary chemicals to assess the defense of plants to attack (Table 1). However, the key effect of escaping from natural enemies may be increased population growth and reproduction, instead of individual size or vegetative growth. Native seed predators generally have significantly greater impacts than do folivores 20

(Maron and Vilá 2001), and introduced populations often exhibit more density than native populations (Maron et al. 2004a, Vilá et al. 2005). Therefore, a test of EICA would require emphasizing on reproductive success and population growth of invasive species. In fact, some studies failed to support that escape from natural enemies does not lead to increased size (Willis and Blossey 1999, Willis et al. 2000, Thébaud and Simberloff 2001, Beckstead and Parker 2003, van Kleunen and Schmid 2003, Maron et al. 2004a, b, Vilá et al. 2005, Lewis et al. 2006), increased growth rate (DeWalt et al. 2004a, b, Genton et al. 2005), or increased ability to win interspecific competition (Bossdorf et al. 2004a) of invasive populations. Other studies suggest that invasive plants are not consistently bigger than their ancestral native counterparts, due to a high variance in size between populations (Willis and Blossey 1999, Vilá et al. 2003). Thus, escaping enemies may not always be interpreted in terms of increased size or vegetative growth. Instead, introduced species may change demography or increase reproduction leading to greater population density and growth (Vilá et al. 2005). That is, escaping enemies and population density may trade off. Only a few studies have addressed this (van Kleunen and Schmid 2003, Bossdorf et al. 2004b, Meyer et al. 2005) with mixed results, which calls for additional studies. Second, many comparative tests of the EICA hypothesis have worked on perennial or woody invasive plants, collecting biological data from plants in a single season of growth in the field, or culturing plants only for a year or two in a common garden. However, a more thorough evaluation of EICA may involve the long-term demographic monitoring of invasive populations. Plant life-history characteristics may affect the ability of specialist herbivores to limit plant abundance (Louda and Potvin 1995). Relatively short-lived plants that rely on current seed production for regeneration are most vulnerable to herbivory that reduces seed production, whereas long-lived perennials that depend on vegetative spread or longer lasting seedbanks are buffered from population-level effects of herbivores (Maron and Vilá 2001). Therefore, common garden experiments or reciprocal transplant experiments may need to be conducted for a long term to test the ultimate effects of natural enemies on invasiveness. Comparative studies in the field may need to consider the demography of each population, since the allocation strategy of a perennial plant may switch between growth and reproduction, depending on 21 the demographic stage. These demographic data may be incorporated into population transition matrices for elasticity analysis and estimation of future population growth (For example, see Jongejans et al. 2006). Often the poor success of biocontrol is due to attacking weeds at life-history stages that are not demographically important (McEvoy and Coombs 1999). Therefore, comprehensive demographic studies may identify critical life-history stages during which control is most successful (Allendorf and Lundquist 2003). Third, future studies should disentangle the impact of specialists and generalists on invasive species. In addition to release from specialists, the effect of generalists in the introduced range may be especially important for successful invasion of introduced species. Are invaders suppressed or not affected by resident generalists? Even though invaders are attacked by generalists, they may compensate for damage by rapid re-growth (tolerance), which calls for the assessment of tolerance as well as resistance (the amount of loss). An important factor to consider here is whether there are resident natives (and their natural enemies) closely related to the invaders. As described above, phylogenetic relatedness may affect the foraging behavior of generalists and the invader’s coevolved response to attack. Most importantly, one first needs to assess if the invasive populations are mostly affected by generalists or specialists, which may be a challenge. Fourth, a more careful experimental design is needed for common garden studies. To test the effect of natural enemies on invasive and non-invasive genotypes, one may expect that invasive genotypes are more negatively affected than native genotypes, since the invasive genotypes have lost the defense ability. However, others may expect that invasive genotypes are less affected because the natural enemies favor to feed on native genotypes. In order to disentangle these two factors (the susceptibility of plants to natural enemies and the behavioral preference of natural enemies), one may need multiple experiments examining both of them (e.g. Siemann and Rogers 2003c). In addition, some common garden studies involve the use of pesticides (insecticide, fungicide) to exclude natural enemies, which may also remove soil mutualists (mycorrhizal fungi, beneficial nematodes), thus confounding the effects of escape from enemies (Parker and Gilbert 2007). 22

In sum, the simple idea of “escape from specialists” of EICA has been expanded to a more general hypothesis dealing with the effect of natural enemies on introduced species and the evolution of invasive genotypes responding to the new selective pressure from generalists and co-introduced specialists in the introduced range. Study designs for testing this more general hypothesis may require more complicated and sophisticated design, considering the background of the specific study system (Maron and Vilá 2001) incorporating a broad range of ecological topics including the assemblage of natural enemies, phylogeny, chemical ecology, and demography.

The Evolution of Phenotypic Plasticity

Plant species introduced outside their native ranges may face different environmental conditions than occur in their native habitats. Soil properties, hydrology, or growing season can all differ, as can differences in herbivore or competitor assemblages. Successful establishment and subsequent spread of an invader may depend on its ability to tolerate and adapt to the new environments. In addition to the considerable adaptive challenges, introduced species often start with small numbers of propagules and in turn exhibit little genetic variation in their new range due to genetic bottlenecks and drift experienced by small founding populations (Barrett and Richardson 1986). Founder effects and genetic drift can generate inter-population differentiation, but intra-populational genetic variation, at least initially, is low (Brown and Marshall 1981). Despite often low levels of intra-population genetic variation, introduced plants frequently occupy extensive new ranges that encompass diverse environments (Mack and Pyke 1983). Phenotypic plasticity for physiological and life-history traits may allow adaptation to spatially or temporally broad range of environments (Marshall and Jain 1968, Baker 1974). Phenotypic plasticity is often favored in disturbed habitats of highly variable, unpredictable, heterogeneous, and rapidly changing environment, where invaders frequently establish (Bradshaw 1965, Bazzaz 1996). Consequently, phenotypic plasticity has historically been seen as a potentially important mechanism for colonization success.

Appropriate hypothesis to investigate the role of phenotypic plasticity To examine the role of phenotypic plasticity in successful invasion, a number of studies have compared phenotypic plasticity between invasive exotic species to a control group under two or more manipulated environments (Table 2). The choice of control group depends on three major hypotheses – (a) invasive exotic species are more plastic than native species, (b) invasive exotic species are more plastic than non-invasive exotic species, (c) invasive species have evolved greater plasticity in the introduced range than in the ancestral native range. The first hypothesis is focused on the general advantage of invasive species exhibiting greater plasticity over the native species in the introduced range. In most cases, native species were chosen from taxa phylogenetically related or ecologically similar to the invasive species, because they are likely to have more traits in common and more overlapping resource requirements than distantly related species. However, since a species native to one area may be invasive in another one, the conclusions drawn from such studies are specific to the particular geographic range considered (Richards et al. 2006). The second hypothesis compares invasive exotic species to the non-invasive exotic congeners. It is a more improved approach by addressing the question of what makes an introduced species invasive in contrast to the non-invasive introduced congeners (Muth and Pigliucci 2006). Since closely-related species are likely to have more traits in common and more overlapping resource requirements than more distantly related species (Goldberg 1987), this is a conservative test of the role of phenotypic plasticity. Identifying such species may be difficult because invasive species are likely to obscure their morphological or physiological characteristics through active hybridization with other introduced congeners or their counterpart native in the introduced range. In addition, this approach may not represent true evidence of the evolution of phenotypic plasticity in invasive species since any differences in phenotypic plasticity can be attributable to phylogenetic or ecological difference between the invasive and non-invasive species. The last hypothesis clearly addresses whether invasive species have evolved phenotypic plasticity for invasiveness by contrasting invasive populations in the introduced range and ancestral populations in the native range. Also, this population-level 24

25 approach can be extended to address how invasive populations have evolved phenotypic plasticity during the invasive process in the new geographical range by comparing populations at different stages of invasion in the introduced range. Only a few recent studies have focused on this hypothesis, but this is an appropriate direction for addressing questions about the role of the evolution of phenotypic plasticity in determining invasion success.

Expectations and supporting evidences of the evolution of phenotypic plasticity To explain the role of phenotypic plasticity in successful invasion, one needs to address whether greater phenotypic plasticity actually leads to greater fitness. If there is genetic variation for plasticity in the invasive populations, and the genotypes with more plasticity confer a fitness advantage in the novel environment, then increased plasticity in the invasive populations will evolve. The mechanistic link between the evolution of phenotypic plasticity and increased fitness is the concept of adaptive plasticity. Adaptive plasticity is a phenotypic response to environmental variation, including highly-specific developmental, physiological and reproductive adjustments, ultimately leading to higher fitness (Sultan 1995, Alpert and Simms 2002, Sultan 2003). If the phenotypic plasticity of underlying traits has evolved to confer greater fitness to invasive genotypes, this evolved plasticity is considered adaptive. Therefore, it will be imperative to test whether invasive populations exhibit greater fitness than non-invasive populations. However, many studies have suggested greater variation or increased performance of physiological or ecological traits in invasive species than native ones, using some traits related to fitness (underlying traits) as surrogates for fitness, which is not readily quantifiable. This approach makes two basic misconceptions: (1) by overemphasizing the role of one or a few underlying traits (not fitness) in the successful invasion, and (2) by erroneously regarding greater plasticity of the underlying trait as the key to successful invasion. A more desirable approach has two aspects: (1) directly comparing fitness between invasive and non-invasive populations (with a reasonable assessment of fitness), (2) investigating the role of phenotypic plasticity of underlying traits leading to greater fitness in the invasive populations. They may be the most important questions that need to be addressed in the study of phenotypic 26 plasticity as a potential mechanism of invasion. We will address the first aspect in this section – a direct comparison of fitness between invasive and non-invasive populations and its link to the successful invasion. Fitness may be the final product of complex interactions between plasticity in underlying traits (Alpert and Simms 2002, Schlichting and Smith 2002). Baker (1974)’s ‘general purpose genotypes’ concept posits that a few invasive genotypes are plastic and thus have greater tolerance across a broad environmental gradient. Rejmánek (2000) and other researchers related this concept to the maintenance of constant fitness as a result of complex interaction of plasticities in underlying traits (fitness homeostasis). Invasive genotypes may exhibit greater fitness than non-invasive genotypes averaged across all environments (Relyea 2002), regardless of whether its plasticity (the slope of reaction norm) is greater or not (Fig. 2a, b). The evolution of phenotypic plasticity facilitating

Figure 2. Hypothetical expectations on the fitness of invasive and non-invasive genotypes across two environments, representing adaptive plasticity (a, b, c, f) or non-adaptive plasticity (d, e). Solid and dashed lines denote reaction norms for invasive and non-invasive genotypes, respectively. 27 increased fitness can enhance invasiveness over a range of environments (Fig. 2a). Invasive genotypes may maintain higher levels of fitness than non-invasive genotypes (fitness homeostasis, Fig. 2b). This outcome can be achieved even without significant G×E effect (Fig. 2c), which is an extreme case where only genetic effects enable invasive genotypes to exhibit higher fitness than non-invasive genotypes. Invasive genotypes may not be successful if their fitness is consistently lower (Fig. 1d, Alpert and Simms 2002) or specifically lower in one environment but higher in another than non-invasive genotypes (Fig. 2e). The latter case may be explained as the ability of invasive genotypes to rapidly take advantage of available resources in favorable environments. Superior ability to capitalize on resources when they are abundant could facilitate the invasion of productive habitats, but would not necessarily promote the invasion of habitats where resources are scarce. However, Fig. 2e represents an extreme case where the mean genotype fitness across environments is the same between invasive and non-invasive populations. If we consider more than three environments and there are more cases where invasive genotypes have greater fitness than non-invasive genotypes, then we can say that invasive genotypes are generally successful since the mean fitness across environments is greater for invasive genotypes than non-invasive genotypes (Fig. 2f). To the contrary, if invasive genotypes win over non-invasive genotypes only in one environment and loses in other environments, the former will be considered as non- adaptive since such limited success more likely reflects specialization for invading a single habitat type, rather than facilitation of the invasion of multiple habitats. In sum, the key point for successful invasion is that invasive genotypes exhibit greater mean fitness than non-invasive genotypes across all environments (Fig. 2a, b, c, f). They are related to the three concepts (Jack-of-all-trades, Master-of-Some, and Jack-and- master) by Richards et al. (2006), but our concepts are more general. Jack-of-all-trades states that invasives are more robust in fitness, which corresponds to Fig. 2b and c. Here we accounted for a possibility that invasives can possess higher fitness than natives without a G×E effect (Fig. 2c). Master-of-some posits that invasives increase fitness, which includes Fig. 2a and f. Here we considered some cases where invasives possess lower fitness in some environments, but have higher overall fitness (Fig. 2f). Jack-and- master is the mixture of the two prior concepts (Fig. 2a, b, c, f). 28

We analyzed all published studies contrasting the performance of ‘invasive’ and ‘non-invasive’ genotypes (or species) under two or more manipulated environments. We excluded some studies working on only invasive genotypes, since phenotypic plasticity and fitness are concepts for some genotypes relative to others. We conducted a meta- analysis on the 24 published studies we reviewed (Table 2), to address two questions: (1) if invasive genotypes possess greater phenotypic plasticity than native counterparts, introduced but non-invasive species, and resident species, (2) if invasive genotypes exhibit greater fitness regardless of the amount of plasticity. We applied the same method described in the former section, but used the control and experimental group as the two extreme treatments in case that there are multiple levels of treatments. In addition, although it will be desirable to test each hypothesis (a, b, c in Table 2) separately and for each type of treatment, we pooled all data since there are only a few studies testing each class of hypothesis and treatment type. We obtained significant P-values for both questions [question (2): dinvasive,+ = 2.02, dnon-invasive,+ = 1.30, P < 0.001; question (2): dinvasive,+ = 2.12, dnon-invasive,+ = 1.87, P = 0.028], which indicates that invasive genotypes are not only more plastic, but also possess greater fitness than their native counterparts, introduced but non-invasive species, and resident species. To explain why invasive species are established and spread across a broader range of habitats in the field, it will be important to investigate whether invasive genotypes win over non-invasive genotypes across a broader range of environmental conditions. In addition, the greater fitness of invasive genotypes is important for invasion success, but the plasticity of fitness (the slope of reaction norm) does not necessarily lead to a conclusion that invasive genotypes are adaptive.

The concept of adaptive plasticity leading to invasion success To investigate the role of phenotypic plasticity in successful invasion, a direct comparison of fitness between invasive and non-invasive populations should be done first, and then an evaluation of the role of phenotypic plasticity conferring greater fitness. In fact, many published studies describe a greater plasticity in underlying traits, assuming that it leads to greater fitness or invasiveness, which is not always true. Some underlying traits may be closely related to fitness, while others may not. For example, total biomass, 29 reproductive effort, or survival are considered to be most closely related to fitness. However, relative growth rate (RGR), nutrient content, biomass allocation, photosynthesis, or minor morphological characteristics are distantly related to fitness and may not be reasonable surrogates of fitness. In addition, underlying traits may not linearly relate to fitness, i.e. high plasticity in underlying traits does not necessarily lead to higher plasticity in fitness, which may be due to the costs or limits of plasticity from possessing the genetic or physiological mechanism for plastic responses, or the consequence of genetic correlations with other traits affecting fitness (Relyea 2002, van Kleunen and Fischer 2005). Therefore, the ability of genotypes to be plastic in the underlying traits related to fitness may not be readily assumed to be adaptive. To test if invasive genotypes possess greater fitness, further studies are needed to investigate how different plasticities of diverse underlying traits interact and finally may result in increased fitness.

Suggestions for testing the role of adaptive plasticity of invasive species To investigate the evolution of phenotypic plasticity for successful invasion, a logical hypothesis is that invasive populations exhibit greater fitness than their ancestral, non-invasive counterparts, and that phenotypic plasticity is evolved to confer greater fitness for invasive populations. In this section we discuss how to empirically test this hypothesis with a reasonable study design. Traditional studies on plasticity involve common garden experiments in the greenhouse or field using samples collected from multiple populations both from introduced and native populations. As successful invasion happens across a broad range of habitats, it raises the need for comprehensive biogeographical studies comparing multiple populations from diverse environmental ranges both in native and invasive provenances (Daehler 2003, Bossdorf et al. 2005, Hierro et al. 2005). Populations should be chosen across a broad range of habitats representing the environmental variability where populations naturally reside. In the introduced range, an intensive sampling of populations at different stages of invasion is needed to further our insights in discovering post-invasion evolution of phenotypic plasticity. Fewer than 10 populations will lack the statistical power to detect the difference between introduced and native populations 30

(Richards et al. 2006). In addition, multiple common gardens need to be set up both in native and invasive provenances to test unambiguously if invasive populations have evolved phenotypic plasticity (Maron et al. 2004b), since only one or two common gardens may not represent the broad range of habitats and are likely to miss the true potential of invasive populations. Samplings from populations should be done carefully to sufficiently represent the entire gene pool of populations. At most times we need to compare plastic responses among sampled populations or higher levels of geographical scale, although phenotypic plasticity is a genotypic property. For a given trait and set of environments, the magnitude and direction of phenotypic plasticity may vary for each genotype (Counts 1993). To describe phenotypic plasticity at the population level, it is common to sample a statistically reasonable number of full-sibs or half-sibs from each population (Richards et al. 2006), and reaction norms at the population level are often calculated as the Euclidean distance between phenotypic ‘means’ from one environment to another. Thus among- genotype source of variation within population are often neglected, although a great deal of genetic variation for plasticity (G×E interactions) may exist among genotypes. For this reason, some researchers simply avoid describing reaction norms at the population level and employ a multivariate approach to detect differences between populations. Determining phenotypic plasticity is also often problematic because the pattern of phenotypic response may vary depending on the set of environmental conditionings (Bradshaw and Hardwick 1989) and the trait sets measured (Schlichting and Levin 1986). An appropriate selection of environmental conditions and trait sets will be crucially important in the comparative study of phenotypic plasticity of an invasive species (Sultan 1995). The environmental factors need to be chosen from the primary factors determining the environmental variability and inducing plastic responses significantly affecting fitness (e.g. light, nutrient, water etc.). The range of environmental treatments needs to consider the environmental variability naturally existing both in the invasive and non-invasive range (Sultan 1995). If the range and variability of environmental conditions are similar between two ranges, conditions may be chosen to represent the lowest and highest (or stressful and favorable, respectively) points along the environmental gradient. On the other hand, if the range and variability of environmental conditions are distinct between 31 two ranges, conditions may be chosen to represent the mean condition from both invasive and non-invasive regions. The former design will address the question how invasive and non-invasive populations differ in the plastic response to the broad range of conditions, while the latter will be informative with respect to whether invasive populations have evolved phenotypic plasticity in response to the novel environment of the introduced range. Trait selection needs to give highest priority to traits closely related to fitness (growth and reproduction) as surrogates of fitness. Fitness needs to be measured at the genotype level for controlled experiments discussed in this context, although fitness has been traditionally measured at population level. A more sophisticated way for the estimation of individual fitness was developed as LRS (Lifetime Reproductive Success,

Clutton-Brock 1988) or λind (individual finite rate of increase, McGraw and Caswell 1996). To understand the role of underlying traits’ plasticity contributing to greater fitness, a set of underlying traits will have to be chosen for measuring. An ideal set of underlying traits may include those which are functionally integrated to contribute to fitness and may have evolved jointly in response to environmental changes (Pigliucci 2004). Finally, to test if invasive populations have evolved phenotypic plasticity, underlying traits may be examined for the environmental effect (phenotypic plasticity to environment, E), population effect (genetic variation among populations, G), and population × environment interaction (genetic variation for plasticity, G×E) using statistical analysis such as ANOVA, MANOVA, or the two-state multivariate analysis developed by Collyer and Adams (2007). The relationship between plasticity of underlying traits and fitness has been descriptive in many studies, though it needs appropriate quantitative methods. A path analysis (Kingsolver and Schemske 1991, Pigliucci et al. 1995, Pigliucci and Kolodynska 2006) or elasticity analysis on a population projection matrix for individuals (McGraw and Caswell 1996) is suggested to investigate how phenotypic plasticity of underlying traits contributes to greater fitness.

Local Adaptation

When the environment varies at a small spatial scale, phenotypic plasticity may be advantageous if the genotype’s response to such environmental heterogeneity keeps its 32 relative fitness more or less constant (van Tienderen 1991). If the environment varies at a scale that is larger than the typical dispersal distance of a species, however, the likely outcome is genetic adaptation to local conditions (Bell 1997). Environmental conditions on a geographical scale may exhibit different selection pressures, for example, climate, altitude, latitude, or land management regime. On this scale, local adaptation often is considered to contribute to the success of introduced plants in their new range (Barrett 1982, Rice and Mack 1991). Local adaptation has intensively been reported for widespread, colonizing plants in heterogeneous environments, though little information is available for invasive plants (Kollmann and Bañuelos 2004). It is partly because local adaptation often requires a wide geographical distribution with high levels of genetic variation, although species introductions often involve population bottlenecks and drift. However, local adaptation has been expected to be an important mechanism for rapid spread into multiple environments (Sakai et al. 2001, Sexton et al. 2002), since successful invasive species exhibit surprisingly high levels of genetic variation (Barrett and Richardson 1986, see review by Bossdorf et al. 2005). Accumulating evidence from empirical studies of population genetics suggest that such variation may actually increase when multiple introductions from a genetically diverse source pool (Warwick et al. 1987, Novak and Mack 1993, Neuffer and Hurka 1999, Pappert et al. 2000, Meekins et al. 2001, Bartlett et al. 2002, Durka et al. 2005). After gaining a foothold, successful invaders expand their range into a broader array of sites, further enhancing genetic diversity from high outcrossing rates and the creation of novel genotypes through gene flow among independently introduced genotypes (Parker et al. 2003). With ample genetic variation and strong selection pressure, populations are subject to natural selection by local environmental conditions, and are genetically differentiated and adapted in respective environments if different genetically-controlled characters are favored in different environments (Antonovics 1976, Galloway and Fenster 2000).

Expectations and supporting evidences for the role of local adaptation Common garden experiments test genetically-based phenotypic differences between plants from different local conditions. However, to test if local adaptation has 33 played a role in driving invasion, reciprocal transplant experiments are required to evaluate whether genetic differentiation between local populations actually let them fit in their respective sites (Parker et al. 2003, Kollmann and Bañuelos 2004, Maron et al. 2004b). If invasive species from different environments are compared in reciprocal transplant experiments, significant home-advantages and away-disadvantages may reflect local specialization by genetic adaptation (Clausen et al. 1940). We identified empirical evidence of local adaptation in which comparisons were made for an invasive species across a broad geographical or climatic gradient (latitude, altitude, etc.; Table 3). The most intriguing study was by Maron et al. (2004b) who found a significant latitudinal cline in plant size and fecundity of invasive St. John’s wort (Hypericum perforatum) through a reciprocal transplant experiment between the U. S. and Europe. In northern US common gardens, plants from northern Europe grew larger and produced more seeds than plants from southern Europe, whereas, in southern U. S. common gardens, plants originating from southern Europe were larger and outperformed in seed production than plants from northern Europe. Using AFLP genotyping, they also found that some introduced genotypes have genetically closest individuals from different latitudes in Europe, eliminating the possibility that all invasive populations established at latitudes similar to their original range. These results indicate that populations of St. John’s wort have been introduced to the U. S. multiple times, some of them may have

Table 3. Studies on local adaptation of invasive species. Invasive species Experiment Varying Sample size Variables References (introduction year) type environment Verbascum thapsus Plant size, biomass CG 16 populations Latitude Reinartz 1984 (1880s) allocation Daucus carota Age of reproduction, RT 9 populations Latitude Lacey 1988 (1600s) age-specific survival Solidago altissima (1738) Plant size, Weber and Schmid RT 24 populations Latitude Solidago gigantean phenology 1988 (1758) Temperature Survival, phenology, Bromus tectorum Six habitat Rice and Mack CG and soil biomass, fecundity, (late 1800s) types 1991 moisture and seed weight Verbascum thapsus Leaf size and CG 10 populations Altitude Parker et al. 2003 (1880s) reflectance Impatiens Kollmann and CG 26 populations Latitude Biomass and height grandulifera (1850s) Bañuelos 2004 Hypericum RT 50 populations Latitude Plant size, fecundity Maron et al. 2004b perforatum (1793) Notes: Experiment types are: CG, common garden experiment; RT, reciprocal transplantation experiment. 34 pre-adapted in similar climatic conditions to those in the original range, whereas others were not pre-adapted and thus may have evolved in response to the new local climatic conditions.

Suggestions for testing the role of local adaptation Although local adaptation has been a potential mechanism enabling exotics to colonize a geographically large area, the following needs to be considered for further studies. First, assessing this hypothesis requires extensive work covering a broad range of environments to sample populations or to set up multiple common gardens, calling for large scale international cooperative work. Second, this hypothesis may be appropriate only for widespread invasive species with a long history of invasion. Local adaptation needs high levels of genetic variation, and probably requires enough time for an invasive species to genetically differentiate and adapt in their local conditions, as all invaders in Table 3 have more than 100 years of invasion history. Third, the results of reciprocal transplant studies suggest that selective regimes of locally adapted populations may not always be apparent throughout habitat and time (Rice and Mack 1991), since spatial or temporal variation in environments is known to affect the direction and intensity of natural selection within a habitat (Kelly 1992). In sum, local adaptation is an appealing mechanism for a widespread invasive species adapted to a broad range of environments for a long time.

Allelopathy

Releases of chemical compounds (allelopathy) by the invader will exhibit harmful effects on members of the recipient plant community. Allelopathy has been suspected as a mechanism by which invasive species eliminate natives, and empirical studies have linked allelopathy to invasion (see review by Hierro and Callaway 2003). But it has been only a few years since active and intensive studies were conducted, such as comparative tests between native and invasive regions, development of physiological mechanisms of allelopathy, and techniques to ameliorate chemical effects. While EICA or adaptive plasticity are “defensive” mechanisms in response to the new selective regime in the introduced range, allelopathy opens up new insight because it 35 is an “offensive” mechanism with which invasive species attack native species. The novel weapons hypothesis (Callaway and Ridenour 2004) proposes that allelopathy is a potential way for an invasive plant to affect natural communities and become successful. This hypothesis specifically suggests that allelopathy may be more important in recipient than in origin communities because the former are more likely to be naïve to the chemicals possessed by newly invading species, while the latter are well-adapted over time and thus not affected.

Supporting evidences for Novel Weapons Hypothesis Callaway and Aschehoug (2000) reported that an invasive species Centaurea diffusa in North America may exude allelochemicals that are relatively ineffective against long-time neighbors in its natural communities, but to which plants in invaded communities lack co-evolved tolerance. Vivanco et al. (2004) identified an allelochemical 8-hydroxyquinoline from C. diffusa, and found that it was three times more concentrated in C. diffusa-invaded North American soils than native Eurasian soils. They also indicated that experimental communities built from North American grass species were far more susceptible to invasion by C. diffusa than communities built from Eurasian species. In addition to the case of C. diffusa, there is more supporting evidence about the difference of susceptibility between plants in native and introduced communities. Bais et al. (2003) reported that germination and growth of European grasses were more resistant to (-)-catechin from the root of C. maculosa than were germination and growth of naïve North American plants of the same genus. Prati and Bossdorf (2004a) found that invasive North American populations of Alliaria petiolata greatly reduced germination of naïve North American Geum laciniatum seeds, but had no effect on European G. urbanum seeds. Even within the introduced range, North American grasses that have survived C. maculosa invasion were affected less by allelochemicals than individuals that did not experience invasion (Callaway et al. 2005). This result indicates that, after native plant communities experienced invasion, they are able to evolve tolerance of allelochemicals and resist invasion. 36

Evidence is also accumulating that invasive species exude more allelochemicals when attacked by herbivores, pathogens, and soil microbes. When C. maculosa had been exposed to herbivory, they had greater negative effects on North American grass Festuca idahoensis than C. maculosa that were kept free from herbivory (Callaway et al. 1999). Exudates from the root of C. maculosa that had been exposed to fungal cell wall had stronger allelochemic activity than exudates from uninfected root (Bais et al. 2002). C. maculosa exuded far higher amounts of (±)-catechin (allelochemical) when attacked by larvae of two different root boring biocontrol herbivores and a parasitic fungus (Thelen et al. 2005). Interestingly, the biomass and reproduction of C. maculosa was not reduced by herbivore or fungal pathogen (Callaway et al. 1999, Thelen et al. 2005). Siemens et al. (2002) indicated that the cost for allelopathy was not apparent, since the induction of herbivore defenses may provide added allelopathic effects. It was also suggested that soil microbes have strong effects on the interaction between C. maculosa and neighboring plants, and exudation induced by herbivory may alter these interactions in ways that provide an advantage to C. maculosa (Thelen et al. 2005). Vivanco et al. (2004) reported that sterilized North American soils suppressed C. diffusa more than sterilized European soils, indicating that the microbes in the North American soil may be a factor promoting C. diffusa invasion. In sum, these results indicate that moderate herbivory stimulates compensatory growth and induces the production of defense chemicals that also have allelopathic effects, suggesting that some biocontrol may not be effective for invasive species, even causing indirect negative effects on native species. Efforts to investigate the physiological background of allelopathy have been made. An allelochemical (±)-catechin was identified from C. maculosa root exudates, and (-)- catechin inhibits seed germination and root growth of some North American species by triggering a wave of reaction oxygen species initiated at the root meristem, which leads to a Ca2+ signaling cascade triggering genome-wide changes in gene expression and ultimately death of the root system (Bais et al. 2002, 2003). Bais et al. (2002) found that the concentration of (±)-catechin in soil extracts in fields containing C. maculosa were far higher than the minimum treatment dose for inhibition, linking the effect of chemicals found in exudates in the laboratory and in the field.

Suggestions for testing Novel Weapons Hypothesis Although the Novel Weapons Hypothesis is interesting, there are more questions to address. First, it is still not clear if allelopathy evolved after introduction or if it is a plastic response induced by stimulation, such as herbivory. While the possibility of selection on biochemical mechanisms was suggested (Hierro and Callaway 2003, Callaway and Ridenour 2004), other studies indicate that allelopathy is stimulated by herbivore or pathogen attack. Second, the hypothesis assumes that naïve communities are more susceptible to allelochemicals than “experienced” communities. It means that it is only when these chemicals are introduced to naïve communities that their toxicity is manifest. Therefore, it is expected that invasive species can be highly successful from the beginning of invasion, not undergoing a lag phase. However, it appears that plants in the introduced range have evolved tolerance to chemicals rapidly, compared to the relatively long history of invasion (Williamson 1990). It can be expected that there will be no suppression effect of allelochemicals in the introduced range, given enough time to evolve tolerance by local native plants. Third, since the hypothesis assumes that allelopathy is expressed only by the invaders, it doesn’t consider the counter-effect of allelopathy expressed by surrounding native species in the recipient communities. Mallik and Pellissier (2000) found that the dominant understory species Vaccinium myrtillis had stronger biochemical effects on the exotic black spruce Picea mariana than on the native Norway spruce Picea abies. The invader should be naïve and highly susceptible to allelopathy it encounters in the newly invaded habitats, giving the invader an apparent disadvantage. Fourth, allelopathy may exhibit detrimental effects to the invaders themselves (autoinhibition). Perry et al. (2005) reported that field concentration of (±)-catechin delayed germination of C. maculosa seedlings. Allelochemicals may inhibit nitrifying bacteria or mycorrhizal fungi (Nilsson et al. 1993, Kraus et al. 2004), subsequently reducing nitrogen availability or disrupting the mutualistic relationships with invaders. Future studies are needed to incorporate the indirect effect of allelochemicals which may be harmful to invasive species. 38

Finally, recent studies of the role of allelopathy in invasiveness have been limited to a few species with strong allelopathic activity. This hypothesis is intriguing in many aspects, broadening our insights into the background of invasiveness, but awaits more empirical studies to be accepted for general theory in invasive species research.

Hybridization / Polyploidization

Recent studies suggest that the genetic architecture of the invading species and its ability to respond to natural selection play a major role in the ultimate establishment of certain invasive species (Lee 2002). Introduced populations may become successful invaders because they evolve increased population fitness (survival and reproduction) relative to populations in the native range. The rate of adaptive evolutionary change should be proportional to the amount of genetic variation for fitness (Fisher 1930), therefore, increased relative fitness in the invasive range is likely to be favored when multiple, independent introductions lead to the formation of diverse, genetically- intermingled populations. Selection could act on this variation to produce locally- specialized ecotypes with genetic recombination among diverse genotypes following introduction and/or interspecific hybridization with native congeners contributing genetic variation leading to the adaptive evolution of increased invasiveness (Thompson 1991, Abbot 1992, Ellstrand and Schierenbeck 2000) and greater resistance to herbivory (Boecklen and Spellenberg 1990, Graham et al. 1995, Vilá and D’Antonio 1998). Hybridization has been regarded as problematic for management and control of invasive species because it has the potential to contaminate the native genotypes with foreign genes and thus confound biocontrol results (Rhymer and Simberloff 1996). Polyploidy may be a stimulus for the evolution of invasiveness. With high levels of genetic diversity (Soltis and Soltis 2000), polyploid plants often possess novel physiological and life-history characteristics not present in their diploid ancestors (Levin 1983, Lumaret 1988). Especially, polyploidy from a hybrid origin may confer new adaptive attributes (Barrett and Richardson 1986, Petit and Thompson 1999, Bleeker and Matthies 2005). Another intriguing result is that polyploids are more resistant to attack by herbivores or pathogens than diploids, possibly creating a barrier to colonization and 39 thereby providing a means for the plant to escape from enemies (evolutionary barrier hypothesis, Reinert et al. 1986, Schoen et al. 1992, Busey et al. 1993). It is explicable by the differences in defensive chemistry (Thompson et al. 2004). However, polyploidy could also provide wider host ranges for some enemies, who continue to use plants of both ploidies as hosts, offering a route to increased diversification in host use (Thompson et al. 1997). Moreover, some enemies could attack polyploids more than diploids (Nuismer and Thompson 2001). Although it is unclear if polyploidy confers more resistance to specialized enemies, if polyploidy affects plant/animal interactions, then repeated evolution of polyploid lineages could create highly dynamic geographical patterns in the ecology and evolution of interspecific interactions (Thompson et al. 2004). However, there are only a few studies that have undertaken comparative evaluations of attack on diploids and polyploids by herbivores (Thompson et al. 2004). As in the case of local adaptation, this hypothesis complies with the argument about the need for “lag time” to accumulate adequate levels of genetic variation and to create novel, invasive genotypes by hybridization and polyploidization. Since Ellstrand and Schierenbeck (2000) summarized a list of 28 examples of hybrid invasive taxa; recent reports from 2000 to present are listed in Table 4.

Table 4. Invasive lineages evolved after interspecific hybridization. Parent taxa Family Evidence other than Reference morphology Myriophyllum heterophyllum × Haloragaceae Organelle DNA Moody and Les 2002 M. pinnatum Tamarix ramosissima × Tamaricaceae Nuclear DNA Gaskin and Schaal 2002 T. chinensis Rorippa austriaca × R. sylvestris Brassicaceae Nuclear and organelle DNA Bleeker 2003 Quercus petraea × Q. robur Fagaceae Organelle DNA Petit et al. 2003 Sarcocornia perennis × S. fruticosa Chenopodiaceae Nuclear DNA Figueroa et al. 2003 Notes: See review by Ellstrand and Schierenbeck (2000) for the cases reported before 2000.

Although hybridization and polyploidization of invasive species has been traditionally studied by evolutionary biologists, there are increasing studies testing the fitness advantage of hybrid invasive plants using common garden studies. Some studies indicate that hybrids are as fit or more fit than their invasive parents (Vilá and D’Antonio 1998, Weber and D’Antonio 1999, Burgess and Husband 2006, Tiébré et al. 2007), while others find that hybrid fitness may increase or decrease, depending on the abundance of 40 parental taxa (Hauser et al. 2003) or ploidy level of the hybrids (Bleeker and Matthies 2005). This may be an important subject that needs well-established cooperative work involving both evolutionists and ecologists. To address how hybridization stimulates the evolution of invasiveness, Ellstrand and Schierenbeck (2000) suggested four mechanisms: evolutionary novelty, increased genetic variation, fixed heterosis, and dumping genetic load. These mechanisms may be tested through quantitative population genetic studies, and the actual increase of fitness can be confirmed by ecological studies employing common garden or reciprocal transplantation. Therefore, comprehensive research integrating comparative ecological, quantitative, and population genetic analyses of populations may allow a deeper understanding of the contribution of evolutionary process to invasiveness.

Synthesis of Mechanistic View on Invasiveness

Ellstrand and Schierenbeck (2000) asked a key question on invasiveness: are invaders “born” (that is, pre-adapted invasive genotypes were introduced) or are they “made” (that is, invasiveness is evolved after introduction)? During the early part of history in the study of invasion, much effort was devoted to figure out the biological characteristics of invasive species and the susceptibility of communities and ecosystems to invasion (see review by Mack et al. 2000, Sakai et al. 2001), from the standpoint that invaders are pre-adapted to the recipient community. However, evidence is accumulating that invaders actively adapt to the new environment in the introduced range, rather than being passively selected and allowed to establish. To figure out the background of invasiveness correctly, we need to trace evolutionary changes at different stages after introduction. Towards this goal, contemporary research is more focused on ‘mechanistic’ insight, testing whether invasiveness has arisen as the consequence of rapid evolutionary change (Thompson 1998, Mooney and Cleland 2001, Lee 2002). A common pitfall for many hypotheses is the difficulty separating cause and effect. For example, if invasive genotypes possess greater tolerance to generalists than native genotypes, this result could be interpreted as a cause of invasion as opposed to a consequence of invasion. Therefore, a comparative study involving native and invasive 41 genotypes at different invasion stage is desirable. If performance of invasive genotypes is better at later stages than earlier stages, and there is no significant difference between earlier-stage invasive genotypes and native genotypes, then invasive genotypes have evolved greater fitness during the course of invasion. The core concept of a “mechanistic” view is that invasive genotypes evolved after an introduction event in the new range and we have outlined five major hypothesis based on this view. To integrate five hypotheses in an extensive framework of invasion biology, we constructed a conceptual diagram showing the potential mechanisms of invading species with interactions between them (Fig. 3). At the beginning of invasion, introduced species should experience release from specialist enemies. Multiple introductions confer a diverse genetic pool for invasive species. Invasive species may be armed with allelochemicals that suppress native species in the introduced range. Invasive species often stay at a fairly low population size for years (lag time) and then spread explosively (Sakai et al. 2001). Lag time is a latent period for invasive species to build up ‘invasive potential’, the unexpressed power for increased invasiveness such as amplified genetic

Figure 3. A conceptual model of the potential mechanisms of invading plant species. 42 variation from multiple introductions, or hybridization / polyploidization. They may also benefit from mutualists (pollinators, seed dispersers, and symbionts such as mycorrhizal fungi or nitrogen-fixing bacteria) that are widespread generalists in the introduced range (Richardson et al. 2000). In addition, invasive plants may compete with native resident species for pollinators or seed dispersers, disrupting mutualistic interactions of resident species (Traveset and Richardson 2006). Likewise, invaders may actively modify habitats to facilitate invasion success for subsequently introduced species, including increased nitrogen availability, soil salinity, and fire frequency (Invasional Meltdown Hypothesis; Simberloff and Von Holle 1999, Reinhart and Callaway 2006). Later at the establishment phase, invasive potential is expressed as a variety of advantages, ultimately leading to greater fitness and invasiveness. Though our conceptual diagram may not be a complete picture of invasion, it serves as a framework for directing future research and for incorporating more factors and interactions. Our conceptual model suggests that the five hypotheses are not independent or mutually exclusive, instead, they are closely interconnected and affect each other with biotic interactions (Mitchell et al. 2006). Introduced species usually encounter a set of benign abiotic and biotic environments including new associations of potential enemies, competitors, and mutualists (Maron et al. 2004b, Mitchell et al. 2006). In addition to this novel selective pressure, the gene pools of introduced species may recombine with genes from native congeners, which is aided by polyploidy. All of these have been considered primary factors driving the evolution of invasive genotypes. In turn they are good reasons why the same process is not likely to occur in the ancestral native ranges. We have reviewed five major hypotheses based on this mechanistic view. However, no one hypothesis is fully convincing on the nature of successful invasion. This may be partly because of the limited amount of comprehensive studies encompassing a broad scale of biogeographical range. Another reason may be that most studies have been limited to a few invasive taxa, rather than encompassing a diversity of invasive species. However, most importantly, empirical studies for each hypothesis have examined a specific aspect of the whole picture. Future studies may need to broaden their emphasis to consider the background of invasiveness in this whole context, rather than testing each 43 specific part. In addition, hypotheses share some key features, such as increased genetic variation, which may be important as the root of building up invasive potential for future studies on invasive species management. Quarantine authorities must assess this potential for introduced species to become aggressive invaders, in order to decide whether to establish policies on eradicating them before they become problematic (Panetta 1993). Invasive species provide an exceptional opportunity for basic research in the population ecology and short-term evolution of species (Allendorf and Lundquist 2003), calling for close cooperative work at the interface of ecology and evolution.

Literature Cited

Abbot, R. J. 1992. Plant invasions, interspecific hybridization and the evolution of new plant taxa. Trends in Ecology and Evolution 7:401-405. Agrawal, A. A., and P. M. Kotanen. 2003. Herbivores and the success of exotic plants: a phylogenetically controlled experiment. Ecology Letters 6:712-715. Allendorf, F. W., and L. L. Lundquist. 2003. Introduction: population biology, evolution, and control of invasive species. Conservation Biology 17(1):24-30. Alpert, P., and E. L. Simms. 2002. Relative advantages of plasticity and fixity in different environments: when is it good for a plant to adjust? Evolutionary Ecology 16:285- 297. Alpert, P., E. Bone, and C. Holzapfel. 2000. Invasiveness, invisibility and the role of environmental stress in the spread of non-native plants. Perspectives in Plant Ecology, Evolution and Systematics 3(1):52-66. Antonovics, J. 1976. The nature of limits to natural selection. Annals of the Missouri Botanical Garden 63:224-247. Bais, H. P., R. Vepachedu, S. Gilroy, R. M. Callaway, and J. M. Vivanco. 2003. Allelopathy and exotic plant invasion: from molecules and genes to species interactions. Science 301:1377-1380. Bais, H. P., T. S. Walker, F. R. Stermitz, R. A. Hufbauer, and J. M. Vivanco. 2002. Enantiomeric-dependent phytotoxic and antimicrobial activity of (±)-catechin. A 44

rhizosecreted racemic mixture from spotted knapweed. Plant Physiology 128:1173- 1179. Baker, H. G. 1974. The evolution of weeds. Annual Review of Ecology and Systematics 5:1-24. Barrett, S. C. H. 1982. Genetic variation in weeds. Pages 73-98 in R. Charudattan and H. Walker, editors. Biological control of weeds with plant pathogens. John Wiley, New York, New York, USA. Barrett, S. C. H., and B. J. Richardson. 1986. Genetic attributes of invading species. Pages 21-33 in R. H. Groves, and J. J. Burdon, editors. Ecology of biological invasions. Cambridge University Press, Cambridge, UK. Bartlett, E., S. J. Novak, and R. N. Mack. 2002. Genetic variation in Bromus tectorum (Poaceae): differentiation in the eastern United States. American Journal of Botany 89:602-612. Bazzaz, F. A. 1996. Plants in Changing Environments. Cambridge University Press, New York. Bazzaz, F. A., N. R. Chiarello, P. D. Coley, and L. F. Pitelka. 1987. Allocating resources to reproduction and defense. Bioscience 37:58-67. Beckstead, J., and I. M. Parker. 2003. Invasiveness of Ammophila arenaria: release from soil-borne pathogens? Ecology 84(11):2824-2831. Bell, G. 1997. Selection: the mechanism of evolution. Chapman & Hall, New York. Blair, A. C., and L. M Wolfe. 2004. The evolution of an invasive plant: an experimental study with Silene latifolia. Ecology 85(11): 3035-3042. Blaney, C. S., and P. M. Kotanen. 2001. Effects of fungal pathogens on seeds of native and exotic plants: a test using congeneric pairs. Journal of Applied Ecology 38:1104-1113. Bleeker, W. 2003. Hybridization and Rorippa austriaca (Brassicaceae) invasion in Germany. Molecular Ecology 12:1831-1841. Bleeker, W., and A. Matthies. 2005. Hybrid zones between invasive Rorippa austriaca and native R. sylvestris (Brassicaceae) in Germany: ploidy levels and patterns of fitness in the field. Heredity 94:664-670. 45

Blossey, B., and R. Nötzold. 1995. Evolution of increased competitive ability in invasive nonindigenous plants: a hypothesis. Journal of Ecology 83:887-889. Boecklen, W. J., and R. Spellenberg. 1990. Structure of herbivore communities in two oak (Quercus spp.) hybrid zones. Oecologia 85:92-100. Bossard, C. C., and M. Rejmánek. 1994. Herbivory, growth, sees production, and resprouting of an exotic invasive shrub. Biological Conservation 67:193-200. Bossdorf, O., D. Prati, H. Auge, and B. Schmid. 2004a. Reduced competitive ability in an invasive plant. Ecology Letters 7:346-353. Bossdorf, O., S. Schröder, D. Prati, and H. Auge. 2004b. Palatability and tolerance to simulated herbivory in native and introduced populations of Alliaria petiolata (Brassicaceae). American Journal of Botany 91(6):856-862. Bossdorf, O., H. Auge, L. Lafuma, W. E. Rogers, E. Siemann, and D. Prati. 2005. Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia 144(1):1-11. Bradshaw A. D. 1965. Evolutionary significance of phenotypic plasticity in plants. Advances in Genetics 13:115-155. Bradshaw, A. D., and K. Hardwick. 1989. Evolution and stress—genotypic and phenotypic components. Biological Journal of the Linnean Society 37:137-155. Brock, M. T., C. Weinig, and C. Galen. 2005. A comparison of phenotypic plasticity in the native dandelion Taraxacum ceratophorum and its invasive congener T. officinale. New phytologist 166:173-183. Brown, A. H. D., and D. R. Marshall. 1981. Evolutionary changes accompanying colonization in plants. Pages 351-363 in G. E. C. Scudder and J. L. Reveal, editors. Evolution Today, Proceedings of the Second International Congress of Systematic and Evolutionary Biology. Hunt Institute for Botanical Documentation, Carnegie- Mellon University, Pittsburgh, Pennsylvania, USA. Buckley, Y. M., P. Downey, S. V. Fowler, R. Hill, J. Memmot, H. Norambuena, M. Pitcairn, R. Shaw, A. W. Sheppard, C. Winks, R. Wittenberg, and M. Rees. 2003. Are invasives bigger? A global study of seed size variation in two invasive shrubs. Ecology 84:1434-1440. 46

Burgess, K. S., and B. C. Husband. 2006. Habitat differentiation and the ecological costs of hybridization: the effects of introduced mulberry (Morus alba) on a native congener (M. rubra). Journal of Ecology 94:1061-1069. Burns, J. H. 2004. A comparison of invasive and non-invasive dayflowers (Commelinaceae) across experimental nutrient and water gradients. Diversity and Distributions 10:387-397. Burns, J. H. and A. A. Winn. 2006. A comparison of plastic responses to competition by invasive and non-invasive congeners in the Commelinaceae. Biological Invasions 8(4):797-807. Buschmann, H., P. J. Edwards, and H. Dietz. 2005. Variation in growth pattern and response to slug damage among native and invasive provenances of four perennial Brassicaceae species. Journal of Ecology 93:322-334. Busey, P., R. M. Giblin-Davis, and B. J. Center. 1993. Resistance in Stenotaphrum to the sting nematode. Crop Science 33:1066-1070. Callaway, R. M., and E. T. Aschehoug. 2000. Invasive plants versus their new and old neighbors: a mechanism for exotic invasion. Science 290:521-523. Callaway, R. M., and W. M. Ridenour. 2004. Novel weapons: invasive success and the evolution of increased competitive ability. Frontiers in Ecology and the Environment 2(8):436-443. Callaway, R. M., T. H. DeLuca, and W. M. Belliveau. 1999. Biological-control herbivores may increase competitive ability of the noxious weed Centaurea maculosa. Ecology 80(4):1196-1201. Callaway, R. M., W. M. Ridenour, T. Laboski, T. Weir, and J. M. Vivanco. 2005. Natural selection for resistance to the allelopathic effects of invasive plants. Journal of Ecology 93:576-583. Carpenter, D., and N. Cappuccino. 2005. Herbivory, time since introduction and the invasiveness of exotic plants. Journal of Ecology 93:315-321. Chun, Y. J., M. L. Collyer, K. A. Moloney, and J. D. Nason. 2007. Phenotypic plasticity of native vs. invasive purple loosestrife: a two state multivariate approach. Ecology 88(6):1499-1512. 47

Clausen, J., D. D. Keck and W. M. Hiesey. 1940. Experimental studies on the nature of species. I. Effects of varied environments on western North America plants. Carnegie Institution of Washington Publication 520:1-452. Clutton-Brock, T. H. (ed.) 1988. Reproductive success. University of Chicago Press, Chicago. Colautti, R. I., A. Ricciardi, I. A. Grigorovich, and H. J. MacIsaac. 2004. Is invasion success explained by the enemy release hypothesis? Ecology Letters 7:721-733. Coley, P. D., J. P. Bryant, and F. S. III. Chapin. 1985. Resource availability and plant antiherbivore defense. Science 230:895-899. Collyer, M. L., and D. C. Adams. 2007. Analysis of two-state multivariate phenotypic change. Ecology 88(3):683-692. Cornell, H. V., and B. A. Hawkins. 1993. Accumulation of native parasitoid species on introduced herbivores: a comparison of hosts as natives and hosts as invaders. American Naturalist 141:847-865. Counts, R. L. 1993. Phenotypic plasticity and genetic variability in annual Zizania spp. along a latitudinal gradient. Canadian Journal of Botany 71(1):145-154. Crawley, M. J. 1987. What makes a community invasible? Pages 429-454 in M. J. Crawley, P. J. Edwards and A. J. Gray (eds.) Colonisation, succession and stability, Blackwell Scientific Publication, Oxford, UK. Daehler, C. C. 2003. Performance comparisons of co-occurring native and alien invasive plants: implications for conservation and restoration. Annual Review of Ecology and Systematics 34:183-211. Daehler, C. C., and D. R. Strong. 1997. Reduced herbivore resistance in introduced smooth cordgrass (Spartina alterniflora) after a century of herbivore-free growth. Oecologia 110:99-108. Darwin, C. 1859. On the Origin of Species by Means of Natural Selection. John Murray, London. DeWalt, S. J., J. S. Denslow, and J. L. Hamrick. 2004a. Biomass allocation, growth, and photosynthesis of genotypes from native and introduced ranges of the tropical shrub Clidemia hirta. Oecologia 138:521-531. 48

DeWalt, S. J., J. S. Denslow, and K. Ickes. 2004b. Natural-enemy release facilitates habitat expansion of the invasive tropical shrub Clidemia hirta. Ecology 85(2):471- 483. Duncan, R. P., and P. A. Williams. 2002. Darwin’s naturalization hypothesis challenged. Nature 417:608-609. Durka, W., O. Bossdorf, D. Prati, and H. Auge. 2005. Molecular evidence for multiple introductions of garlic mustard (Alliaria petiolata, Brassicaceae) to North America. Molecular Ecology 14:1697-1706. Ellstrand, N. C., and K. Schierenbeck. 2000. Hybridization as a stimulus for the evolution of invasiveness in plants? Proceedings of the National Academy of Sciences 97:7043-7050. Elton, C. S., 1958. The ecology of invasions by animals and plants. University of Chicago Press, Chicago, IL. Fan, J., and W. Harris. 1996. Effects of soil fertility level and cutting frequency on interference among Hieracium pilosella, H. praealtum, Rumex acetosella, and Festuca novae-zelandiae. New Zealand Journal of Agricultural Research 39:1-32. Feng, Y.-L., J.-F. Wang, and W.-G. Sang. 2007. Irradiance acclimation, capture ability, and efficiency in invasive and non-invasive alien plant species. Photosynthetica 45(2):245-253. Fenner, M., and W. G. Lee. 2001. Lack of pre-dispersal seed predators in introduced Asteraceae in New Zealand. New Zealand Journal of Ecology 25:95-99. Figueroa, M. E., J. M. Castillo, S. Redondo, T. Luque, E. M. Castellanos, F. J. Nieva, C. J. Luque, A. E. Rubio-Casal, and A. J. Davy. 2003. Facilitated invasion by hybridization of Sarcocornia species in a salt-marsh succession. Journal of Ecology 91:616-626. Fisher, R. A. 1930. The Genetical Theory of Natural Selection, Clarendon Press, Oxford. UK. Galloway, L. F., and C. B. Fenster. 2000. Population differentiation in an annual legume: local adaptation. Evolution 54(4):1173-1181. 49

Gaskin, J. F., and B. A. Schaal. 2002. Hybrid Tamarix widespread in U.S. invasion and undetected in native Asian range. Proceedings of the National Academy of Sciences 99(17):11256-11259. Genton, B. J., P. M. Kotanen, P.-O. Cheptou, C. Adolphe, and J. A. Shykoff. 2005. Enemy release but no evolutionary loss of defence in a plant invasion: an inter-continental reciprocal transplant experiment. Oecologia 146: 404-414. Gerlach, J. D. Jr., and K. J. Rice. 2003. Testing life history correlates of invasiveness using congeneric plant species. Ecological Applications 13(1):167-179. Gleason, S. M., and A. Ares. 2004. Photosynthesis, carbohydrate storage and survival of a native and an introduced tree species in relation to light and defoliation. Tree Physiology 24:1087-1097. Goldberg, D. 1987. Neighborhood competition in an old-field plant community. Ecology 68:1211-1223. Graham, J. H., D. C. Freeman, and E. D. McArthur. 1995. Narrow hybrid zone between two subspecies of big sagebrush (Artemisia tridentate: Asteraceae). II. Selection gradients and hybrid fitness. American Journal of Botany 82:709-716. Hauser, T. P., C. Damgaard, and R. B. Jørgensen. 2003. Frequency-dependent fitness of hybrids between oilseed rape (Brassica napus) and weedy B. rapa (Brassicaceae). American Journal of Botany 90(4): 571-578. Hastwell, G. T., and F. D. Panetta. 2005. Can differential responses to nutrients explain the success of environmental weeds? Journal of Vegetation Science 16:77-84. Hierro, J. L., and R. M. Callaway. 2003. Allelopathy and exotic plant invasion. Plant and Soil 256:29-39. Hierro, J. L., J. L. Maron, and R. M. Callaway. 2005. A biogeographical approach to plant invasions: the importance of studying exotics in their introduced and native range. Journal of Ecology 93:5-15. Hokkanen, H. M. T., and D. Pimentel. 1989. New associations in biological control – theory and practice. Canadian Entomologist 121:829-840. Jakobs, G., W. Weber, and P. J. Edwards. 2004. Introduced plants of the invasive Solidago gigantea (Asteraceae) are larger and grow denser than conspecifics in the native range. Diversity and Distribution 10:11-19. 50

Jongejans, E., A. W. Sheppard, and K. Shea. 2006. What controls the population dynamics of the invasive thistle Carduus nutans in its native range? Journal of Applied Ecology 43:877-886. Joshi, J., and K. Vrieling. 2005. The enemy release and EICA hypothesis revisited: incorporating the fundamental difference between specialist and generalist herbivores. Ecology Letters 8:704-714. Kaufman, S. R., and P. E. Smouse. 2001. Comparing indigenous and introduced populations of Melaleuca quinquenervia (Cav.) Blake; response of seedlings to water and pH levels. Oecologia 127:487-494. Keane, R. M., and M. J. Crawley. 2002. Exotic plant invasions and the enemy release hypothesis. Trends in Ecology and Evolution 17(4):164-170. Kelly, C. A. 1992. Spatial and temporal variation in selection on correlated life-history traits and plant size in Chamaecrista fasciculata. Evolution 46:1658-1673. Kercher, S. M., and J. B. Zedler. 2004. Flood tolerance in wetland angiosperms: a comparison of invasive and noninvasive species. Aquatic Botany 80:89-102. Kingsolver, J. G., and D. W. Schemske. 1991. Path analysis of selection. Trends in Ecology and Evolution 6: 276-280. Kolb, A., and P. Alpert. 2003. Effects of nitrogen and salinity on growth and competition between a native grass and an invasive congener. Biological Invasions 5:229-238. Kollmann, J., and M. J. Bañuelos. 2004. Latitudinal trends in growth and phenology of the invasive alien plant Impatiens glandulifera (Balsaminaceae). Diversity and Distributions 10:377-385. Kraus, T. E. C., R. J. Zasoski, R. A. Dahlgren, W. R. Horwath, C. M. Preston. 2004. Carbon and nitrogen dynamics in a forest soil amended with purified tannins from different plant species. Soil Biology and Biochemistry 36:309-321. Lacey, E. P. 1988. Latitudinal variation in reproductive timing of a short-lived monocarp, Daucus carota (Apiaceae). Ecology 69(1):220-232. Lankau, R. A., W. E. Rogers, and E. Siemann. 2004. Constraints on the utilization of the invasive Chinese tallow tree Sapium sebiferum by generalist native herbivores in coastal prairies. Ecological Entomology 29:66-75. 51

Lee, C. E., 2002. Evolutionary genetics of invasive species. Trends in Ecology and Evolution 17(8):386-391. Leger, E. A., and K. J. Rice. 2003. Invasive California poppies (Eschscholzia californica Cham.) grow larger than native individuals under reduced competition. Ecology Letters 6:257-264. Leger, E. A., and M. L. Forister. 2005. Increased resistance to generalist herbivores in invasive populations of the California poppy (Eschscholzia californica). Diversity and Distributions 11:311-317. Leicht, S. A., and J. A. Silander Jr. 2006. Differential responses of invasive Celastrus orbiculatus (Celastraceae) and native C. scandens to changes in light quality. American Journal of Botany 93(7):972-977. Leishman, M. R., and V. P. Thomson. 2005. Experimental evidence for the effects of additional water, nutrients and physical disturbance on invasive plants in low fertility Hawkesbury Sandstone soils, Sydney, Australia. Journal of Ecology 93:38- 49. Levin, D. A. 1983. Polyploidy and novelty in flowering plants. American Naturalist 122:1-25. Lewis, K. C., F. A. Bazzaz, Q. Liao, and C. M. Orians. 2006. Geographic patterns of herbivory and resource allocation to defense, growth, and reproduction in an invasive biennial, Alliaria petiolata. Oecologia 148: 384-395. Lodge, D. M. 1993. Biological invasions: lessons for ecology. Trends in Ecology and Evolution 8(4):133-137. Louda, S. M., and M. A. Potvin. 1995. Effect of inflorescence-feeding insects on the demography and lifetime fitness of a native plant. Ecology 76:229-245. Luken, J. O., L. M. Kuddes, T. C. Tholemeier, and D. M. Haller. 1997. Comparative responses of Lonicera maackii (Amur Honeysuckle) and Lindera benzoin (Spicebrush) to increased light. American Midland Naturalist 138:331-343. Lumaret, R. 1988. Adaptive strategies and ploidy Levels. Acta Oecologica/Oecologia Plantarum 9:83-93. Mack, R. N., and D. A. Pyke. 1983. The demography of Bromus tectorum: variation in time and space. Journal of Ecology 71:69-93. 52

Mack, R. N., D. Simberloff, W. M. Lonsdale, H. Evans, M. Clout, and F. A. Bazzaz. 2000. Biotic invasions: causes, epidemiology, global consequences, and control. Ecological Applications 10:689-710. Mallik, A. U., and F. Pellissier. 2000. Effects of Vaccinium myrtillus on spruce regeneration: testing the notion of coevolutionary significance of allelopathy. Journal of Chemical Ecology 26(9):2197-2209. Maron, J. L., and M. Vilá. 2001. When do herbivores affect plant invasion? Evidence for the natural enemies and biotic resistance hypothesis. Oikos 95:361-373. Maron, J. L., M. Vilá, and J. Arnason. 2004a. Loss of enemy resistance among introduced populations of St. John’s Wort (Hypericum perforatum). Ecology 85(12):3243- 3253. Maron, J. L., M. Vilá, R. Bommarco, S. Elmendorf, and P. Beardsley. 2004b. Rapid evolution of an invasive plant. Ecological Monographs 74(2):261-280. Marshall, D. R., and S. K. Jain. 1968. Phenotypic plasticity of Avena fatua and A. barbata. American Naturalist 102:457-467. McEvoy, P. B., and E. M. Coombs. 1999. Biological control of plant invaders: regional patterns, field experiments and structured population models. Ecological Applications 9:387-401. McGraw, J. B., and H. Caswell. 1996. Estimation of individual fitness from life-history data. American Naturalist 147(1): 47-64. Meekins, J. F., H. E., Ballard Jr., and B. C. McCarthy. 2001. Genetic variation and molecular biogeography of a North American invasive plant species (Alliaria petiolata, Brassicaceae). International Journal of Plant Sciences 162:161-169. Meyer, G., R. Clare, and E. Weber. 2005. An experimental test of the evolution of increased competitive ability hypothesis in goldenrod, Solidago gigantea. Oecologia 144:299-307. Milberg, P., B. B. Lamont, and M. A. Pérez-Fernández. 1999. Survival and growth of native and exotic composites in response to a nutrient gradient. Plant Ecology 145:125-132. Mitchell, C. E., and A. G. Power. 2003. Release of invasive plants from fungal and viral pathogens. Nature 421:625-627. 53

Mitchell, C. E., A. A. Agrawal, J. D. Bever, G. S. Gilbert, R. A. Hufbauer, J. N. Klironomos, J. L. Maron, W. F. Morris, I. M. Parker, A. G. Power, E. W. Seabloom, M. E. Torchin, and D. P. Vázquez. 2006. Biotic interactions and plant invasions. Ecology Letters 9:726-740. Moody, M. L., and D. H. Les. 2002. Evidence of hybridity in invasive watermilfoil (Myriophyllum) populations. Preceedings of the National Academy of Sciences 99(23):14867-14871. Mooney, H. A., and E. E. Cleland. 2001. The evolutionary impact of invasive species. Preceedings of National Academy of Sciences 98(10):5446-5451. Müller-Schärer, H., and T. Steinger. 2004. Predicting evolutionary change in invasive, exotic plants and its consequences for plant-herbivore interactions. Pages 137-162 in L. E. Ehler, R. Sforza, and T. Mateille, editors. Genetics, Evolution and Biological Control. CABI Publishing, Wallingford, UK. Müller-Schärer, H., U. Schaffner, and T. Steinger. 2004. Evolution in invasive plants: implications for biological control. Trends in Ecology and Evolution 19(8):417-422. Muth, N. Z., and M. Pigliucci. 2006. Traits of invasives reconsidered: phenotypic comparisons of introduced invasive and introduced noninvasive plant species within two closely related clades. American Journal of Botany 93(2):188-196. Neuffer, B., and H. Hurka. 1999. Colonization history and introduction dynamics of Capsella bursa-pastoris (Brassicaceae) in North America: isozymes and quantitative traits. Molecular Ecology 8:1667-1681. Nilsson, M. C., P. Hogberg, O. Zackerisson, and W. Fengyou. 1993. Allelopathic effects by Empetrum hermaphroditum Hagerup on development and nitrogen uptake by roots and mycorrhizas of Pinus sylvestris L. Canadian Journal of Botany 71:620- 628. Novak, S. J., and R. N. Mack. 1993. Genetic variation in Bromus tectorum (Poaceae): comparison between native and introduced populations. Heredity 71:167-176. Nuismer, S. L., and J. N. Thompson. 2001. Plant polyploidy and non-uniform effects on insect herbivores. Proceedings of the Royal Society of London. Series B: Biological sciences 268:1937-1940. 54

Panetta, F. D. 1993. A system of assessing proposed plant introductions for weed potential. Plant Protection Quarterly 8(1):10-14. Pappert, R. A., J. L. Hamrick, and L. A. Donovan. 2000. Genetic variation in Pueraria lobata (Fabaceae), an introduced, clonal, invasive plant of the southeastern United States. American Journal of Botany 87(9):1240-1245. Parker, I. M., and G. S. Gilbert. 2007. When there is no escape: the effects of natural enemies on native, invasive, and noninvasive plants. Ecology 88(5): 1210-1224. Parker, I. M., J. Rodriguez, and M. E. Loik. 2003. An evolutionary approach to understanding the biology of invasions: local adaptation and general-purpose genotypes in the weed Verbascum thapsus. Conservation Biology 17(1):59-71. Parker, J .D., and M. E. Hay. 2005. Biotic resistance to plant invasions? Native herbivores prefer non-native plants. Ecology Letters 8:959-967. Parker, J. D., D. E. Burkepile, and M. E. Hay. 2006. Opposing effects of native and exotic herbivores on plant invasions. Science 311:1459-1461. Pattison, R. R., G. Goldstein, and A. Ares. 1998. Growth, biomass allocation and photosynthesis of invasive and native Hawaiian rainforest species. Oecologia 117:449-459. Paynter, Q., S. V. Fowler, J. Memmott, and A. W. Sheppard. 1998. Factors affecting the establishment of Cytisus scoparius in southern France: implications for managing both native and exotic populations. Journal of Applied Ecology 35:582-595. Perry, L. G., G. C. Thelen, W. M. Ridenour, T. L. Weir, R. M. Callaway, M. W. Paschke, and J. M. Vivanco. 2005. Dual role for an allelochemical: (±)-catechin from Centaurea maculosa root exudates regulates conspecific seedling establishment. Journal of Ecology 93:1126-1135. Petit, C., and J. D. Thompson. 1999. Species diversity and ecological range in relation to ploidy level in the flora of the Pyrenees. Evolutionary Ecology 13:45-66. Petit, R. J., C. Bodénès, A. Ducousso, G. Roussel, and A. Kremer. 2003. Hybridization as a mechanism of invasion in oaks. New Phytologist 161:151-164. Pigliucci, M. 2004. Studying the plasticity of phenotypic integration in a model organism. Pages 155-175 in M. Pigliucci and K. Preston, editors. Phenotypic integration: 55

studying the ecology and evolution of complex phenotypes. Oxford University Press, New York. Pigliucci, M., and A. Kolodynska. 2006. Phenotypic integration and response to stress in Arabidopsis thaliana: a path analytical approach. Evolutionary Ecology Research 8:415-433. Pigliucci, M., J. Whitton, and C. D. Schlichting. 1995. Reaction norms of Aradidopsis. I. Plasticity of characters and correlations across water, nutrient and light gradients. Journal of Evolutionary Biology 8:421-438. Prati, D., and O. Bossdorf. 2004. Allelopathic inhibition of germination by Alliaria petiolata (Brassicaceae). American Journal of Botany 91(2):285-288. R Development Core Team. 2006. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3- 900051-07-0, URL http://www.R-project.org. Radford, I. J., and R. D. Cousens. 2000. Invasiveness and comparative life-history traits of exotic and indigenous Senecio species in Australia. Oecologia 125:531-542. Reinartz, J. A. 1984. Life history variation of common mullein (Verbascum thapsus): II. Plant size, biomass partitioning and morphology. Journal of Ecology 72(3):913-925. Reinert, J. A., P. Busey, and F. G. Bilz. 1986. Old world St. Augustine grasses resistant to the southern chinch bug (Heteroptera: Lygaeidae). Journal of Economic Entomology 79:1073-1075. Reinhart, K. O., and R. M. Callaway. 2006. Soil biota and invasive plants. New Phytologist 170:445-457. Rejmánek, M. 2000. Invasive plants: approaches and predictions. Austral Ecology 25(5):497-506. Relyea, R. A. 2002. Costs of phenotypic plasticity. American Naturalist 159(3):272-282. Rhymer, J. M., and D. Simberloff. 1996. Extinction by hybridization and introgression. Annual Review of Ecology and Systematics 27:83-109. Ricciardi, A., and J. M. Ward. 2006. Comment on “Opposing effects of native and exotic herbivores on plant invasions”. Science 313:298a. Rice, K. J., and R. N. Mack. 1991. Ecological genetics of Bromus tectorum. III. The demography of reciprocally sown populations. Oecologia 88:91-101. 56

Richards, C. L., O. Bossdorf, N. Z. Muth, J. Gurevitch, and M. Pigliucci. 2006. Jack of all trades, master of some? On the role of phenotypic plasticity in plant invasions. Ecology Letters 9:981-993. Richardson, D. M., N. Allsopp, C. M. D'Antonio, S. J. Milton, and M. Rejmánek. 2000. Plant invasions – the role of mutualisms. Biological Reviews 75:65-93. Rogers, W. E., and E. Siemann. 2002. Effects of simulated herbivory and resource availability on native and invasive exotic tree seedlings. Basic and Applied Ecology 3:297-307. Rogers, W. E., and E. Siemann. 2004. Invasive ecotypes tolerate herbivory more effectively than native ecotypes of the Chinese tallow tree Sapium sebiferum. Journal of Applied Ecology 41:561-570. Rogers, W. E., and E. Siemann. 2005. Herbivory tolerance and compensatory differences in native and invasive ecotypes of Chinese tallow tree (Sapium sebiferum). Plant Ecology 181:57-68. Sakai, A. K., F. Allendorf, J. Holt, D. Lodge, J. Molofsky, K. With, S. Baughman, R. Cabin, J. Cohen, N. Ellstrand, D. McCauley, P. O’Neil, I. Parker, J. Thompson, and S. Weller. 2001. The population biology of invasive species. Annual Review of Ecology and Systematics 32:305-312. Schlichting, C. D., and D. A. Levin. 1986. Phenotypic plasticity: an evolving plant character. Biological Journal of the Linnean Society 29:37-47. Schlichting, C. D., and H. Smith. 2002. Phenotypic plasticity: linking molecular mechanisms with evolutionary outcomes. Evolutionary Ecology 16:189-211. Schierenbeck, K. A., R. N. Mack, and R. R. Sharitz. 1994. Effects of herbivory on growth and biomass allocation in native and introduced species of Lonicera. Ecology 75(6):1661-1672. Schoen, D. J., J. J. Burdon, and A. H. D. Brown. 1992. Resistance of Glycine tomentella to soybean leaf rust Phakopsora pachyrhizi in relation to ploidy level and geographic distribution. Theoretical and Applied Genetics 83:827-832. Schweitzer, J. A. and K. C. Larson. 1999. Greater morphological plasticity of exotic honeysuckle species may make them better invaders than native species. Journal of the Torrey Botanical Society 126(1):15-23. 57

Sexton, J. P., J. K. McKay, and A. Sala. 2002. Plasticity and genetic diversity may allow saltcedar to invade cold climates in North America. Ecological Applications 12(6):1652-1660. Siemann, E., and W. E. Rogers. 2001. Genetic differences in growth of an invasive tree species. Ecology Letters 4:514-518. Siemann, E., and W. E. Rogers. 2003a. Herbivory, disease, recruitment limitation, and success of alien and native tree species. Ecology 84(6):1489-1505. Siemann, E., and W. E. Rogers. 2003b. Increased competitive ability of an invasive tree may be limited by an invasive beetle. Ecological Applications 13(6):1503-1507. Siemann, E., and W. E. Rogers. 2003c. Reduced resistance of invasive varieties of the alien tree Sapium sebiferum to a generalist herbivore. Oecologia 135: 451-457. Siemens, D. H., S. H. Garner, T. Mitchell-Olds, and R. M. Callaway. 2002. Cost of defense in the context of plant competition: Brassica rapa may grow and defend. Ecology 83(2):505-517. Simberloff, D., and B. Von Holle. 1999. Positive interactions of nonindigenous species: invasional meltdown? Biological Invasions 1:21-32. Soltis, P. S., and D. E. Soltis. 2000. The role of genetic and genomic attributes in the success of polyploids. Proceedings of the National Academy of Sciences 97(13):7051-7057. Stastny, M., U. Schaffner, and E. Elle. 2005. Do vigour of introduced populations and escape from specialist herbivores contribute to invasiveness? Journal of Ecology 93:27-37. Strauss, S. Y., C. O. Webb, and N. Salamin. 2006. Exotic taxa less related to native species are more invasive. Proceedings of the National Academy of Sciences 103(15):5841-5845. Sultan, S. E. 1995. Phenotypic plasticity and plant adaptation. Acta Botanica Neerlandica 44(4):363-383. Sultan, S. E. 2003. Phenotypic plasticity in plants: A case study in ecological development. Evolution & Development 5(1):25-33. Thébaud, C., and D. Simberloff. 2001. Are plants really larger in their introduced ranges? American Naturalist 157(2):231-236. 58

Thelen, G. C., J. M. Vivanco, B. Newingham, W. Good, H.P. Bais, P. Landres, A. Caesar, and R.M. Callaway. 2005. Insect herbivory stimulates allelopathic exudation by an invasive plant and the suppression of natives. Ecology Letters 8:209-217. Thompson, J. D. 1991. The biology of an invasive plant. Bioscience 41:393-401. Thompson, J. N. 1998. Rapid evolution as an ecological process. Trends in Ecology and Evolution 13(8):329-332. Thompson, J. N., S. L. Nuismer, and K. Merg. 2004. Plant polyploidy and the evolutionary ecology of plant/animal interactions. Biological Journal of the Linnean Society 82:511-519. Thompson, J. N., B. C. Cunningham, K. A. Segraves, D. M. Althoff, and D. Wagner. 1997. Plant polyploidy and insect/plant interactions. American Naturalist 150:730- 743. Tiébré, M.-S., S. Vanderhoeven, L. Saad, and G. Mahy. 2007. Hybridization and sexual reproduction in the invasive alien Fallopia (Polygonaceae) Complex in Belgium. Annals in Botany 99:193-203. Traveset, A., and D. M. Richardson. 2006. Biological invasions as disruptors of plant reproductive mutualisms. Trends in Ecology and Evolution 21(4):208-216. Vilá, M., A. Gómez, and J. L. Maron. 2003. Are alien plants more competitive than native conspecifics? A test using Hypericum perforatum L. Oecologia 137:211-215. Vilá, M., J. L. Maron, and L. Marco. 2005. Evidence for the enemy release hypothesis in Hypericum perforatum. Oecologia 142:474-479. Vilá, M., and C. M. D’Antonio. 1998. Hybrid vigor for clonal growth in Carpobrotus (Aizoaceae) in coastal California. Ecological Applications 8(4):1196-1205. Vivanco, J. M., H. P. Bais, F. R. Stermitz, G. C. Thelen, and R. M. Callaway. 2004. Biogeographical variation in community response to root allelochemistry: novel weapons and exotic invasion. Ecology Letters 7:285-292. van Kleunen, and B. Schmid. 2003. No evidence for an evolutionary increased competitive ability in an invasive plant. Ecology 84(11):2816-2823. van Kluenen, and M. Fischer. 2005. Constraints on the evolution of adaptive phenotypic plasticity in plants. New Phytologist 166:49-60. 59 van Tienderen, P. H. 1991. Evolution of generalists and specialists in spatially heterogeneous environments. Evolution 45:1317-1331. Warwick, S. I., B. K. Thompson, and L. D. Black. 1987. Genetic variation in Canadian and European populations of the colonizing weed species Apera spica-venti. New Phytologist 106(2):301-317. Weber, E., and C. M. D’Antonio. 1999. Phenotypic plasticity in hybridizing Carpobrotus spp. (Aizoaceae) from coastal California and its role in plant invasion. Canadian Journal of Botany 77:1411-1418. Weber, E., and B. Schmid. 1998. Latitudinal population differentiation in two species of Solidago (Asteraceae) introduced into Europe. American Journal of Botany 85(8):1110-1121. Williams, D. G., and R. A. Black. 1994. Drought response of a native and introduced Hawaiian grass. Oecologia 97:512-519. Williamson, G. B. 1990. Allelopathy, Koch’s postulates and the neck riddle. Pages 143- 162 in J. B. Grace and D. Tilman, editors. Perspectives on Plant Competition. Academic Press, Inc. San Diego, CA. Williamson, M., and A. Fitter. 1996. The varying success of invaders. Ecology 77(6):1661-1666. Willis, A. J., and B. Blossey. 1999. Benign environments do not explain the increased vigour of non-indigenous plants: a cross-continental transplant experiment. Biocontrol Science and Technology 9:567-577. Willis, A. J., J. Memmott, and R. I. Forrester. 2000. Is there evidence for the post- invasion evolution of increased size among invasive plant species? Ecology Letters 3:275-283. Willis, A. J., M. B. Thomas, and J. H. Lawton. 1999. Is the increased vigor of invasive weeds explained by a trade-off between growth and herbivore resistance? Oecologia 120:632-640. Wilson, S. B., P. C. Wilson, and J. A. Albano. 2004. Growth and development of the native Ruellia caroliniensis and invasive Ruellia tweediana. HortScience 39(5):1015-1019. 60

Wolfe, L. M. 2002. Why alien invaders succeed: support for the escape-from-enemy hypothesis. American Naturalist 160(6):705-711. Wolfe, L. M., J. A. Elzinga, and A. Biere. 2004. Increased susceptibility to enemies following introduction in the invasive plant Silene latifolia. Ecology Letters 7:813- 820. Yamashita, N., A. Ishida, H. Kushima, and N. Tanaka. 2000. Acclimation to sudden increase in light favoring an invasive over native trees in subtropical islands, Japan. Oecologia 125:412-419. Zangerl, A. R., and M. R. Berenbaum. 2005. Increase in toxicity of an invasive week after reassociation with its coevolved herbivore. Proceedings of the National Academy of Sciences 102(43):15529-15532. 61

Chapter 3. Phenotypic plasticity of native vs. invasive purple loosestrife: a two-state multivariate approach

A paper published in Ecology1

Young Jin Chun2,4, Michael L. Collyer2,3, Kirk A. Moloney2,5, and John D. Nason2

Abstract

The differences in phenotypic plasticity between invasive (North American) and native (German) provenances of the invasive plant Lythrum salicaria (purple loosestrife) were examined using a multivariate reaction norm approach testing two important attributes of reaction norms described by multivariate vectors of phenotypic change: the magnitude and direction of mean trait differences between environments. Data were collected for six life history traits from native and invasive plants using a split-plot design with experimentally manipulated water and nutrient levels. We found significant differences between native and invasive plants in multivariate phenotypic plasticity for comparisons between low and high water treatments within low nutrient levels, between low and high nutrient levels within high water treatments, and for comparisons that included both a water and nutrient level change. The significant G × E effects support the argument that invasiveness of purple loosestrife is closely associated with the interaction of high levels of soil nutrient and flooding water regime. Our results indicate that native and invasive plants take different strategies for growth and reproduction; native plants flowered earlier and allocate more to flower production, while invasive plants exhibited an extended period of vegetative growth before flowering to increase height and allocation to clonal reproduction, which may contribute to increased fitness and invasiveness in subsequent years.

1 Reprinted with permission of Ecology, 2006, 88(6):1499-1512. 2 Department of Ecology, Evolution, and Organismal Biology, 3 Department of Statistics, Iowa State University, Ames, IA 50011 4 Primary researcher and author. 5 Author for correspondence. 62

Keywords: common garden, genetic variation × environment (G × E) interactions, invasive species, Lythrum salicaria, multivariate analysis, native vs. invasive, phenotypic plasticity, purple loosestrife, reaction norm.

Introduction

Invasive species are a source of global environmental change, harming native species and communities, altering ecosystem processes in invaded areas, and causing enormous economic damage (Vitousek et al. 1996, Simberloff 2000). Although considerable research has focused on invasive species (Mooney and Hobbs 2000), little consensus exists about the underlying strategies of species that allow them to become successful invaders (cf., Williamson 1999, Kolar and Lodge 2001, Sakai et al. 2001, Mack et al. 2002). Nonetheless, phenotypic plasticity has historically been seen as a potentially important mechanism for colonization success in environmentally diverse areas and may play a role in the process of invasion (Marshall and Jain 1968, Rice and Mack 1991, Williams et al. 1995, Kaufman and Smouse 2001, Maron et al. 2004). Phenotypic plasticity is simply a phenotypic response to environmental variation, however, adaptive plasticity is a response that ultimately enhances function and maximizes fitness through highly specific developmental, physiological, and reproductive adjustments to local environmental conditions (Sultan 2003). Therefore, adaptive plasticity is a more appropriate concept to explore in examining successful invasion than is phenotypic plasticity. Adaptive plasticity may contribute to invasive ability by allowing the acclimation of invasive genotypes to diverse environments, as well as buffering existing genetic variation from selection, thereby reducing the necessity for local adaptation (Schlichting 1986, Sultan 2003). Therefore, adaptive plasticity may lead to phenotypic homeostasis (tolerance) in fitness, which can be important in the process of successful invasion (Rejmánek 2000, Alpert and Simms 2002). Adaptive plasticity has been detected repeatedly among invasive plant species. As an example, in one study Sultan and Bazzaz (1993a) found that Polygonum persicaria grown under flooding produced dense mats of finely branched superficial and 63 adventitious roots at the soil surface, such that growth was maintained by avoiding the deleterious effects of oxygen deficits. In a second study (Sultan and Bazzaz 1993b), they found that at different nutrient levels, P. p e r si c a r ia maintained the same photosynthetic surface area relative to total biomass and the same leaf nitrogen concentration. Since adaptive plasticity is often expressed through interactions among multiple traits rather than through a single trait, such characteristics in a phenotypic response argue for the analysis of multiple traits contributing to fitness in studying the relationship between adaptive plasticity and invasiveness. In this paper, we use purple loosestrife (Lythrum salicaria L.) as a model system to investigate the relationship between adaptive plasticity and invasiveness. This was done in an experimental study designed to contrast the multivariate plastic response of plants derived from native and invasive source populations to varying environmental conditions. Purple loosestrife is an herbaceous perennial plant and a well-known, aggressive invader of wetlands in North America (Thompson et al. 1987). Introduced to North America in the early 1800s (Stuckey 1980), it has spread rapidly throughout the continent and changed the basic structure of most wetlands it has invaded (Blossey et al. 2001). New recruits into a population are primarily from seed (Stevens et al. 1997b), but as plants increase in size, they can produce multiple shoots originating from a single root crown. The breeding system of purple loosestrife has been of particular interest, since it is a tristylous species, exhibiting three distinct flower types that require outcrossing for successful fertilization (Ågren and Ericson 1996). Seed production is massive, with an individual plant producing approximately 900 seed capsules with 120 seeds per capsule (Shamsi and Whitehead 1974a). Seeds are light (0.5-0.6 mg), but wind dispersal is often limited to 10 m (Thomposon et al. 1987). The most likely mode of spread is by wetland wildlife through ingestion or external adherence of seed with subsequent deposition (Thompson et al. 1987). There is some evidence that purple loosestrife is more vigorous in North America than in Europe. Invasive populations in North America appear to be characterized generally by higher densities (Edwards et al. 1998, Bastlová-Hanzélyová 2001), taller and larger plants (Blossey and Nötzold 1995, Bastlová-Hanzélyová 2001, Bastlová and Květ 2002), and more fertile shoots (Edwards et al. 1998) than populations in native 64

European habitat. Although there has been some work examining purple loosestrife in the context of its response to varying environmental conditions (Shamsi and Whitehead 1974a, 1974b, 1977a, 1977b, Mal et al. 1997a, Edwards et al. 1998, Stevens et al. 1997a, Bastlová et al. 2004), there has not been a study adopting a multivariate reaction norm approach to relate its invasiveness to the evolution of phenotypic plasticity. While classical studies in evolutionary ecology have tended to focus on variation in single characters, there is increasing interest in covariation among characters and the impact this has on phenotypic evolution (Waldmann and Anderson 2000). The idea that an organism’s phenotype cannot be easily characterized by a single trait is illustrated in the concepts of genetic correlation (Falconer and MacKay 1996) and phenotypic integration (Wagner and Altenberg 1996, Pigliucci 2003). An organism’s phenotype may be treated as a set of multivariate variables, whereby the covariation of traits that are important in phenotypic evolution is considered in the analysis of phenotypic differentiation. It is particularly interesting to study the relationship between phenotypic integration and environmental variation, i.e., how the environment can alter the patterns of phenotypic correlation among traits, and how this response may differ between native and non-native populations. The contrast between the adaptive advantages of integrated responses (Schlichting 1986, Schlichting and Pigliucci 1998) versus their nonadaptive roles as indicators of constraints (Gould 1984) is fundamental to our understanding of phenotypic evolution (Pigliucci and Schlichting 1995, 1998). Even though methods have been developed to quantify natural selection acting on multivariate phenotypes (Lande and Arnold 1983), there has been a dearth of applications of these methods to measurements taken in different environments. To address this issue, we adopt a recently developed two-state multivariate vector approach (Collyer and Adams 2007) for comparing phenotypic responses among different source populations. Phenotypic change within a population is depicted through vectors of mean trait differences between contrasting environments (e.g., multivariate reaction norm analysis). The two-state approach contrasts the multivariate response between populations in two ways. First, a comparison of the magnitude of vectors can be used to determine if invasive populations exhibit greater multivariate plasticity than native ones. A second analysis quantifies directional differences between vectors in multivariate trait 65 space to determine if native and invasive populations exhibit different patterns of trait covariation. Such directional differences may provide further insight into the evolution of life-history strategies associated with invasiveness. Utilizing this multivariate approach, we address two basic questions concerning phenotypic plasticity and invasiveness in purple loosestrife: (1) When plants are exposed to a change in water and nutrient levels, do native and invasive populations exhibit different patterns in the magnitude and direction of their multivariate reaction norms? (2) Are the differences in the magnitude and direction of multivariate reaction norms between native and invasive populations consistent with a change in adaptive plasticity of invasive populations, which may produce greater fitness? To address these questions, we conducted a common garden study in which three populations each from the native (Europe) and invasive provenance (North America) were subject to experimentally manipulated water and nutrient levels. Differences between provenances in the amount and direction of multivariate phenotypic change were then determined for a set of six vegetative and reproductive characters.

Univariate Analysis of Reaction Norms

The reaction norm is a function of phenotypic values across environments that can be described for individuals or for populations (Via et al. 1995, Pigliucci 2001). The traditional analysis of reaction norms involves a univariate approach, examining reaction norms for each trait separately. (The same univariate approach has also been applied to synthetic metrics, such as PCA scores, that are often used to reduce the dimensionality of multivariate data sets when there is a high degree of collinearity among traits.) To assess the effect of G (genetic variation), E (environment), and G × E interactions on phenotypic responses, ANOVA is conducted for each trait. Significant G × E effects indicate significant genetic variation for phenotypic plasticity. To infer a biological explanation for genetic variation in phenotypic plasticity, one can examine G × E means plotted trait by trait. For example, four G × E means for six different traits are plotted for purple loosestrife in Fig. 1 (see Methods for a more complete description). Although such plots easily indicate why a significant G × E would be observed for a single trait in an ANOVA (e.g., a change in the rank of phenotypic 66 values between two environments, as in Figs. 1b, d, e, f), it is practically impossible to gain an understanding of whether there is genetic variation in phenotypic plasticity in terms of trait covariation (i.e., plasticity of phenotypic integration). This is especially problematic for traits that are not independent. For univariate data, the reaction norm simply describes the magnitude of difference in trait values between environments. For multivariate data, reaction norms are more complex because of potential negative or positive covariance among traits. Therefore, an analytical approach is needed that accounts for the covariation of multiple traits.

Figure 1. Least square means (±SE) of six trait values for plants from native (open circles) and invasive (solid circles) provenance. Environmental treatments: WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL,

High water, Low nutrient; WHNH, High water, High nutrient.

Application of Two-State Multivariate Analysis

G, E, and G × E effects can be statistically analyzed with multivariate analysis of variance (MANOVA) to determine if there are significant genetic differences in phenotypic plasticity, accounting for trait covariation (e.g., Langerhans and DeWitt 2002). To visualize such differences, one can project multivariate G × E means onto the first two or three principal component axes of the multivariate set of trait values (Fig. 2), providing a rough interpretation of the significance of G, E, and G × E effects. However, this approach does not test specific hypotheses regarding the attributes of phenotypic change for multivariate reaction norms. Specifically, a significant G × E effect in MANOVA does not indicate whether genetic differences in phenotypic plasticity occur because of differences in the amount of phenotypic change (Fig. 2b), differences in the direction of trait covariation (Fig. 2c), or both (Fig. 2d) (as pointed out by Schlichting [1986]). Subsequent analyses are needed to test for differences in multivariate reaction norm magnitude and direction (two-state analysis; e.g., Collyer and Adams 2007). Here we resolved this issue by describing reaction norms as multivariate vectors possessing magnitude and direction in multivariate trait space. Reaction norms for multivariate data can be calculated as vectors (between two phenotypic states) that describe the difference in multivariate G × E means in more than two dimensions (Collyer and Adams 2007). Such vectors have two properties: magnitude (the amount of phenotypic change between two environments) and direction (the way traits covary between two environments) (Fig. 2). Reaction norm vectors for two populations can be compared for these properties by (1) a contrast in their vector length (|D1 – D2|) and (2) the angle between vectors (θ1,2). For a given population, the multivariate reaction norm is defined as a column vector of differences between phenotypic means in environments i and j, ΔX= Xi – X j , where Xi and X j are vectors of trait means in environment i and j, respectively. The

1/ 2 Euclidean distance of a population’s reaction norm is calculated as D = ()ΔXTΔX (where ΔX T is the transpose matrix of ΔX ; Fig. 2). Given two populations (denoted 1 and 2), the similarity in orientation of their reaction norm vectors is described by their 68

Figure 2. Hypothetical reaction norms for two genotypes or populations (indexed 1 and 2) and two environments (open and closed circles) in bivariate (a, b, c, d) and multivariate (e) trait space. Solid and dashed lines denote reaction norm vectors for populations 1 and 2, respectively. In panels a-d, the contrasts between columns (a, c vs. b, d) and between rows (a, b vs. c, d) demonstrate significant G × E interactions due to the amount (|D1 – D2|) and angular direction (θ1,2) of phenotypic change, respectively. Panels b-e exhibit significant G × E due to |D1 – D2| > 0, θ1,2 > 0, or both. 69 vector correlation, which is the inner product of the two vectors scaled to unit length:

T ⎡ ΔX1 ⎤ ⎡ ΔX 2 ⎤ VC1,2 = ⎢ ⎥ ⎢ ⎥ (Schluter 1996). The angle between vectors, θ1,2, is the ⎣ D1 ⎦ ⎣ D2 ⎦ -1 arccosine of the vector correlation, θ1,2 = cos (VC1,2). Small angles imply similar orientation. We used this approach of decomposing multivariate reaction norms into attributes of magnitude and direction for comparison of loosestrife phenotypic plasticity in life history traits between native and invasive provenances. As described below, plants from both provenances were raised in four experimental environments that varied in water and nutrient levels.

Methods

Seed collection We included three European populations (native provenance) and three North American populations (invasive provenance) to contrast native vs. invasive populations. In fall 2002, collections of seeds of L. salicaria were obtained from three populations in North America (Little South Storm Lake, Iowa [IA, 42° 38Ń, 95° 13´W]; Shell Rock, Minnesota [MN, 43° 32Ń, 93° 15´W]; and Fayettville, New York [NY, 42° 49Ń, 76° 49´W]) and three populations from Germany (Schollener See [G1, 52° 39Ń, 12° 11É]; Strodehne [G2, 52° 45Ń, 12° 11É] in the state of Brandenburg; and Meiβendorfer Teiche [G3, 52° 43Ń, 9° 50É] in the state of Saxony). Three populations from each provenance were chosen to represent a range of nutrient and moisture conditions found naturally in the field. We randomly chose seeds from a bulk sample of a large number of plants within each population. As a consequence, our measurements of phenotypic plasticity reflect variability at the population level with three populations as replicates within each provenance.

Common Garden Experiment Comparative studies of differences in plasticity between plants and populations are typically conducted in a single common garden. Although reciprocal transplant studies 70 involving multiple field sites also have advantages in the study of invasiveness (Maron et al. 2004), we have conducted a common garden experiment because it enables us to better control and quantify environmental variation and to interpret the causal basis of phenotypic change. Our experiment was conducted in the Forestry greenhouse at Iowa State University, IA, USA. Seeds from the 6 source populations were planted on May 20, 2004. After 20 days of growth, we selected 40 seedlings (2–4 cm in height) from each population, restricting initial size to reduce variation in traits related to germination and pre-transplant growth (i.e., variation due to maternal effects). Seedlings were individually transplanted into plastic pots (30 cm diameter × 25 cm depth) on June 9, 2004, and placed into plastic wading pools (1.4 m diameter × 30 cm depth). The pots were filled with Sunshine LC1 potting soil (Sun Gro Horticulture Canada, Seba Beach, Alberta, Canada), which has an initial nutrient charge that would correspond to high nutrient levels in natural wetland soils, approximately 3 times the average content of nitrogen as reported in Bridgham et al. (1996) and Bedford et al. (1999). However, the effect of this over the course of the experiment would be minimal, as the initial nutrient charge is highly water soluble, provides the equivalent of approximately one application of a liquid fertilizer, and needs to be supplemented by regular fertilizer treatments soon after potting (Sun Gro Horticulture has provided the authors with proprietary information regarding average levels of the nutrient charge in LC1, through a personal communication.) We applied a split-plot design consisting of five complete blocks, with four wading pools within each block. Each wading pool in a block contained one of four environmental treatment combinations: (1) low water/low nutrient (WLNL), (2) low water/high nutrient (WLNH), (3) high water/low nutrient (WHNL), and (4) high water/high nutrient (WHNH). Two plants from each population were put in each wading pool in each block (12 plants per pool). The total number of experimental units for this experiment was 5 blocks × 4 treatment combinations = 20 units, with 6 populations × 2 plants = 12 observations per experimental unit. Pots in the high water treatments were kept at saturation, similar to standing water conditions in lakes and ponds. Low water treatments are comparable to drier, upland conditions, where plants were watered every other day, letting the soil soak for a few hours, after which the soil was allowed to dry. In the low nutrient treatment, no fertilizer was applied, whereas in the high nutrient 71 treatment 100 g of slow-release 14:14:14 N:P:K Osmocote (The Scotts Company, Marysville, Ohio, USA) was applied once to a pot at the beginning of the experiment (N, 2481 mg/kg soil; P, 563 mg/kg soil; K, 1,320 mg/kg soil). The Osmocote applied in the high nutrient treatment followed the manufacturer’s recommendation for amount and rate (one application for a 3–4 month growing period) to be used in producing high fertilization levels (The Scotts Company). As a consequence, the low and high nutrient treatments were designed to be a reasonable approximation of low and high soil nutrient levels, respectively, which would be encountered under natural field conditions (cf. Bridgham et al. 1996, Thormann and Bayley 1997, Bedford et al. 1999). On 16 August 2004, after 92% of the plants had initiated flowering, we harvested them. As a perennial herb, purple loosestrife usually takes four to five years to be competitively dominant under field conditions (Weiher et al. 1996, Mal et al. 1997b). However, as they begin to increase ramet production from the second year of growth (Mal et al. 1997b), it may be important to test whether native and invasive genotypes exhibit genetically based differences during the first year of growth in response to varying environmental conditions. We randomly chose wading pools across blocks for harvesting to minimize experimental variation due to growth during the harvesting period. The following six traits were measured for each experimental individual: (1) height, (2) number of secondary branches, (3) number of stems originating from the rootstock, (4) total number of flowers per plant, (5) aboveground biomass, and (6) belowground biomass. To determine (4), we haphazardly sampled five flower stalks from each population and treatment combination (one flower stalk from each block) to count the number of flowers per unit length of flower stalk. We measured total length of flower stalks per plant for each individual. Total number of flowers per plant was then calculated by multiplying the number of flowers per unit length by total length of flower stalk per plant, for each population and treatment combination. To determine aboveground and belowground biomass, plants were divided into respective parts and dried in an oven for 24 hours at 60ºC to constant weight.

Application of Multivariate Analyses We analyzed the phenotypic plasticity of purple loosestrife populations from the 72 native and invasive provenance for the four environmental treatment combinations. To avoid pseudoreplication, the data for the two replicate plants in each treatment were averaged prior to analysis (creating 120 total observations). Two of these observations were removed from the study because they were outliers due to individuals showing retarded growth. Data were log-transformed to create variables that were more normally distributed to satisfy assumptions of our analytical methods. We analyzed our data in three ways – PCA, mixed-model MANOVA, and the two- state multivariate analysis of phenotypic plasticity. First, we conducted PCA on the correlation matrix calculated from log-transformed variables to reduce the dimensionality of data because of trait variation due to allometry, resulting in six PC axes. The PCA indicated that four phenotypic traits (number of secondary branches, number of stems originating from rootstock, aboveground biomass, and belowground biomass) were all strongly, positively associated with PC1, thus indicating a collinear relationship of PC1 with plant “size.” Size effects may obscure the actual effects of provenance and environment we are interested in. To explain the phenotypic responses holding the size effect constant, we excluded PC1 and made a new dataset composed of five principal components (PC2 through PC6), hereafter be referred to as the “reduced” dataset, in contrast to the “full” dataset (raw data). Second, we performed MANOVA on the full and reduced datasets to test for differences in phenotype due to the effects of native and invasive provenance (G effect), nutrient and water conditions (E effects), and their interactions (G × E effect). Differences among blocks (main plot) were analyzed using the block by environment (sub-plot) interactions as error terms. A significant G × E interaction indicates that plants from the different provenances differ in their multivariate phenotypic plasticities. Finally, using the two-state methods of Collyer and Adams (2007), we calculated multivariate reaction norms for all six possible pairwise comparisons of G × E means (estimated as least-squares means from the MANOVA model) within provenance. The significance of each reaction norm magnitude was determined by a Hotelling (1931) multivariate T2 test by converting reaction norm Euclidian distances to squared Mahalanobis (1936) distances, using the model error variance/covariance matrix from the MANOVA (see Legendre and Legendre [1998] for test details). Subsequently, reaction 73 norms were compared in magnitude and direction using a permutation procedure that preserves overall genetic (G) and environmental (E) effects, but assumes a null model of Var(G × E) = 0 (See Collyer and Adams 2007 for statistical details). The permutation procedure uses a two stage operation. In the first stage, a linear model that lacks the G×E effect is used to generate predicted values and residuals. These residuals are the permuted units, and with every iteration of the permutation procedure, new values for the response variables are created from the predicted values and random residuals. In the second stage of the procedure, a linear model that contains the G × E effect is used to estimate least squares treatment means. Thus, random reaction norms are calculated at each iteration, holding the overall G and E effects constant. We calculated pairwise contrasts and angles between native and invasive provenances for 4,999 permutations of random vectors under the null hypothesis of Var(G × E) = 0. Along with observed values, this created empirical reference distributions of 5000 contrasts and angles from random vectors. We calculated the probability of finding, by chance, more extreme contrasts or angles than observed values, from the empirical reference distributions created from random pairs of vectors. PCA was performed with R (R Development Core Team 2005) and sums of squares cross-products (SSCP) matrices were calculated with JMP (Sall et al. 2003) to perform the mixed-model MANOVA. The determinants of SSCP matrices were used to calculate Wilks´ Λ. Permutation tests were run for both full and reduced data sets, using the Monte-Carlo analysis of PopTools (Hood 2005). To preserve the error rate of α = 0.05 for the entire set of comparisons, we used sequential Bonferroni corrections for pairwise comparisons (Rice 1989).

Results

PC1 (the principal axis of variation) and PC2 together explained 79% of the total variation in the full data (Table 1). PC1 was positively associated with number of secondary branches, number of stems originating from the rootstock, aboveground biomass, and belowground biomass (plant “size” indicators); whereas PC2 was positively associated with plant height and negatively associated with number of flowers, 74

Table 1. PCA result (loadings) on log-transformed full data set. Variables PC1 PC2 PC3 PC4 PC5 PC6 Height 0.103 0.713 –0.441 0.423 –0.264 –0.196 Branches 0.486 –0.288 0.285 0.106 –0.416 –0.645 Stems 0.455 0.277 0.397 0.266 0.698 –0.012 Flowers 0.269 –0.540 –0.662 0.342 0.284 0.023 Aboveground Biomass 0.535 –0.005 0.100 0.061 –0.414 0.727 Belowground Biomass 0.433 0.203 –0.344 –0.786 0.130 –0.130 Eigenvalue 3.248 1.494 0.613 0.407 0.179 0.059 Cumulative percentage of variance PC1–PC6 54.13 79.03 89.25 96.03 99.02 100.0 PC2–PC6† 54.28 76.57 91.35 97.86 100.0 † Calculated assuming that the total variance (PC2–PC6) is 100.

Figure 3. Principal component analysis (PCA) plots of (a, c) PC1 vs. PC2 (including size effects) and (b, d) PC2 vs. PC3 (excluding size effects). Plots (a) and (b) include polygons which represent convex hulls for each provenance and treatment combination; solid and dashed lines represent invasive and native populations, respectively. Plots (c) and (d) include arrows which indicate the direction and length of trait vectors in the same PC space. Environmental treatments are: WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL, High water, Low nutrient; WHNH, High water, High Nutrient. 75 representing a tradeoff between growth and reproduction. PC3 was negatively associated with height and number of flowers. After removing the variance associated with the size effect of PC1, 76.6% of the total remaining variation was explained by PC2 and PC3 (Table 1). Whether variation due to size allometry was included (Fig. 3a) or excluded (Fig. 3b), differences in phenotype among environmental treatments were obvious. The MANOVA results indicated that overall G, E, and G × E effects were significant for both the full and reduced data sets (Table 2). The only relative difference found between MANOVAs of the two datasets was related to G × E interactions involving water level (W effect), which were significant for the reduced dataset but not for the full. These contradictory results suggest that even though the differences in growth were similar for plants from native and invasive provenances across water treatments, each provenance had different phenotypic plasticities across the same water treatments when the effect of size allometry was removed. All reaction norm magnitudes were significantly greater than zero (P < 0.003), but there were several differences in reaction norm magnitude between provenances (Table 3).

The differences of reaction norm magnitudes between provenances for WLNL – WHNL and WLNH – WHNL were significant for both the full and reduced datasets (Table 3). In both cases, reaction norm magnitudes were greater for native plants than for invasive plants. This indicates that plants from the native provenance exhibited a greater amount of phenotypic change when water conditions changed under low nutrient conditions

(WLNL – WHNL) or when water level and nutrient level changed in the opposite direction

(WLNH – WHNL). Plants of the invasive provenance, in contrast, exhibited greater phenotypic change for the reaction norm for WLNL – WHNH, but only for the full dataset (Table 3), indicating that invasive plants exhibited a greater growth size response between environments that differed in both water and nutrient level in the same way. The directional difference was significant for the WLNH – WHNL and WHNL – WHNH reaction norms for both the full and reduced datasets (Table 3), which indicates that plants from native and invasive provenances differed in the way phenotypic traits covaried under changing nutrient conditions (Table 2). Taken together, our results indicate that native plants demonstrate greater phenotypic plasticity to changing water level, though when water level and nutrient level 76 changed in the same direction, invasive plants demonstrate greater phenotypic plasticity in growth. The two provenances also differ in the way traits covary between environments that differ in nutrient levels (WLNH – WHNL and WHNL – WHNH).

Table 2. MANOVA results for full and reduced data sets. Full data set Reduced data set Source Wilks′ Λ Λobs df1 df2 F P Λobs df1 df2 F P | BE | B 0.0067 24 36.10 4.816 < 0.0001 0.0160 20 37.43 4.643 < 0.0001 | BE + B | | ε | G 0.3656 6 89.00 25.734 < 0.0001 0.3811 5 90.00 29.229 < 0.0001 | ε + G | | ε | E 0.0028 18 252.22 97.765 < 0.0001 0.0344 15 248.85 39.623 < 0.0001 | ε + E | | ε | W 0.0972 6 89.00 137.757 < 0.0001 0.1377 5 90.00 112.696 < 0.0001 | ε + W | | ε | N 0.0599 6 89.00 232.963 < 0.0001 0.1577 5 90.00 96.165 < 0.0001 | ε + N | | ε | W × N 0.0713 6 89.00 193.278 < 0.0001 0.3032 5 90.00 41.375 < 0.0001 | ε + WN | | ε | G × E 0.6194 18 252.22 2.586 0.0006 0.6530 15 248.85 2.769 0.0006 | ε + GE | | ε | G × W 0.8745 6 89.00 2.129 0.0576 0.8755 5 90.00 2.560 0.0326 | ε + GW | | ε | G × N 0.7985 6 89.00 3.744 0.0023 0.8377 5 90.00 3.489 0.0063 | ε + GN | | ε | G × W × N 0.8822 6 89.00 1.980 0.0768 0.8856 5 90.00 2.325 0.0492 | ε + GWN | | ε | B × E 0.2755 72 490.02 1.820 < 0.00010.3408 60 425.21 1.832 0.0004 | ε + BE | Notes: Sums of Squares and Cross Product matrices (SSCP) are denoted as B: block, G: provenance (native vs. invasive), E: environment, W: water, N: nutrient, and ε: error. Values of P < 0.05 are significant.

Table 3. Summary table analyzing the difference in vector magnitude and direction on full and reduced datasets. Full data set Reduced data set Euclidean Euclidean Reaction norm distance P θ P distance P θ P vectors D N, I θ D N, I θ N I N I WLNL–WLNH 0.21 0.19 0.7560 75.88 0.1434 0.09 0.07 0.6702 73.97 0.1368 WLNL–WHNL 1.27 0.96 0.0116 10.91 0.0998 0.40 0.27 0.0062 10.76 0.2098 WLNL–WHNH 0.80 0.99 0.0022 9.59 0.4662 0.15 0.18 0.6746 20.48 0.1274 WLNH–WHNL 1.42 1.07 0.0062 19.33 0.0014 0.48 0.31 0.0012 20.50 0.0056 WLNH–WHNH 0.90 0.93 0.6604 12.62 0.2650 0.22 0.17 0.4054 13.21 0.3046 WHNL–WHNH 1.67 1.68 0.8820 14.24 0.0066 0.29 0.23 0.0368 34.80 0.0120 Notes: Symbols represent: N, native provenance; I, invasive provenance; WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL, High water, Low nutrient; WHNH, High water, High nutrient. PD represents the empirical probability that the absolute difference in the magnitude of random reaction norms is larger than the observed difference. θN, I indicates the observed angle between reaction norms of native (N) and invasive (I) provenances. Pθ represents the probability that the angle between pairs of random reaction norms is larger than that of the observed angle. Bold values indicate where significant differences or angles occurred, after sequential Bonferroni correction to maintain α = 0.05. 77

Discussion

There are few comparative studies of phenotypic plasticity in native vs. invasive populations and little consensus as to whether a distinct plastic response by invasive populations is related to invasiveness (Richards et al. 2006). About half of the studies examining this issue conclude that there is no difference in response, whereas the other half indicates that phenotypic plasticity predisposes invasive populations to be more aggressive than native populations (Bossdorf et al. 2005). Recent studies, in fact, have shown that within a species the interpretation of the importance of plasticity to invasiveness may vary depending upon the trait examined (e.g., Barney et al. 2005, Brock et al. 2005, Burns and Winn 2006). Further empirical studies contrasting phenotypic plasticity between native and invasive populations may increase our understanding of the process of invasion, especially when conducted under conditions that the populations normally experience in the field. Also, by extending the analysis of plasticity to incorporate a multivariate approach, some of the problems recently identified in analyzing multiple traits in a univariate context may be mitigated (cf., Barney et al. 2005, Brock et al. 2005, Burns and Winn 2006). Although there is no previous study comparing the magnitude and direction of multivariate reaction norms of purple loosestrife between native and invasive populations, previous studies of purple loosestrife have found genetic variation for plasticity to water and nutrient availability. In a field study by Edwards et al. (1998), fecundity of purple loosestrife was similar between USA and European populations in nutrient-poor habitats, but was significantly greater in USA populations in nutrient-rich habitats (cf., Burns and Winn 2006). In a study conducted on 11 purple loosestrife populations from different latitudes in Europe, Bastlová et al. (2004) reported significant latitude × nutrient and latitude × water interactions on some growth characteristics, where size-related traits were negatively correlated with latitude. These findings support the idea that purple loosestrife from different geographic provenances exhibit different amounts of plasticity in response to changes in water or nutrient conditions. These studies utilized univariate statistical approaches to analyze phenotypic plasticity, providing useful insights into the invasion biology of purple loosestrife. However, their interpretation is limited to an 78 exploration of the presence or absence of G (genotypic), E (environmental), or G × E effects on single traits. By adding a multivariate component to the analysis, we hoped to gain a deeper understanding of the integrated response among traits to changing environmental conditions. The two-state multivariate analysis applied in our study is advantageous for testing hypotheses concerning population differences in both the amount and direction of phenotypic plasticity in a multivariate trait space. It offers the additional benefit, compared to MANOVA, of interpreting the meaningful aspects of phenotypic change that produce a significant G × E interaction. Description and comparison of magnitude and direction in phenotypic change vectors provides greater insight into the nature of differences in phenotypic plasticity between native and invasive species. In our study comparing native vs. invasive populations of purple loosestrife, we found significant differences in multivariate phenotypic plasticity for comparisons between low and high water treatments within low nutrient levels, between low and high nutrient levels within high water treatments, and for comparisons that included both a water and nutrient level change (Table 3). The significant reaction norm differences occurred because of differences in the magnitude (D) of phenotypic plasticity in response to water level change and different directional responses (θ) to nutrient level change, which were detected via the two-state multivariate methods applied to this study. Based on the statistical results for reaction norm comparisons (Table 3), the covariation of original variables and PCs (Fig. 3), and plots of reaction norms projected onto PCs (Fig. 4), we are able to draw several conclusions concerning differences in the nature of plasticity in native vs. invasive populations. This served to extend the understanding gained through the more traditional univariate approaches (e.g., Fig. 1 and related analyses; Edwards et al. 1998, Bastlová et al. 2004). First, given low nutrient availability, conditions for growth were better under low water conditions (WLNL vs. WHNL), where all of the measured traits were greater in amount, except for height (Fig. 1). For both the full and reduced data sets, reaction norms associated with changing hydrology under low nutrient conditions differed significantly in magnitude, but not direction (WLNL – WHNL; Table 3, Fig. 4a, b). This indicates that the general growth response to changes in hydrology was the same for plants from both 79

Figure 4. PCA plots showing reaction norms significantly different for D and/or θ between native plants and invasive plants. Solid and dashed lines represent invasive and native populations, respectively. Left panels are for PC1 vs. PC2 (including size effects) and right panels are for PC2 vs. PC3 (excluding size effects). Environmental treatments are:

WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL, High water, Low nutrient; WHNH, High water, High Nutrient. 80 provenances, but the amount of change was significantly greater for plants of native provenance (Table 3). This is most obvious in the projection of the reaction norms on PC2, an axis most associated with variation in height and flower production (cf., Table 1, Figs. 1, 3, 4a, b). There is a tradeoff between height and flower production along PC2, where invasive populations tend to increase height while native populations are likely to increase flower production in response to changing water levels with low nutrient conditions (Figs. 1a, d, 3). Second, under constant flooding, conditions for growth were better with high nutrient availability (WHNL vs. WHNH), where, with the exception of height, all of the measured traits were greater in amount (Fig. 1). For both the full and reduced data sets, reaction norms associated with these two treatment combinations differed significantly in direction, but not magnitude (WHNL – WHNH; Table 3, Fig. 4c, d). This directional difference between native and invasive plants represents a difference in the covariation of traits in response to changes in environmental conditions. From WHNL to WHNH, the reaction norm of native plants is oriented approximately in the direction of increased number of branches (Figures 4d, 3d, and 1b), while the reaction norm of invasive plants is directed toward increased aboveground biomass (Figures 4d, 3d, and 1e). In interpreting this result it must be kept in mind that the PC scores were obtained though an analysis of the correlation matrix, which places equal emphasis on all six traits. This may downplay the impact of traits exhibiting the greatest response to the changing treatment conditions, but takes into account significant trends in the plastic response of all 6 traits. Third, reaction norms in which water and nutrient levels changed in opposite directions differed in both magnitude and direction for both data sets (WHNL – WLNH in Table 3, Fig. 4c, d). The change in magnitude was greatest for the native provenance (Table 3), primarily in the direction of PC2 (Figs. 4e, f). An inspection of trait loadings on PC2 suggests that native plants should exhibit a greater change in flower production and height in response to the shift in environmental conditions between WHNL and WLNH (Table 2). A direct examination of mean trait responses indicates a tradeoff between height and flower production along PC2, where native plants suffer reduction in height coupled with a disproportionate gain in flower production as compared to invasive plants, in response to low water, high nutrient conditions (Fig. 1a, d). Native plants also show a 81 decrease in allocation to belowground biomass, which could represent a decrease in allocation to growth for the next growing season. In contrast, the invasive plants increased allocation to belowground biomass under the same conditions. These differential responses result in a significant difference in direction between reaction norms and represent a genetic difference in the response by the invasive versus native provenance to the underlying environmental conditions. It is interesting to note that the

WHNL treatment could be interpreted as representing the conditions most likely experienced by plants in native habitat, whereas the WLNH treatment most closely approximates the situation of plants expanding into more mesic conditions associated with high nutrient inputs, as is the case for some North American populations (K. A. Moloney, personal observation). Greater allocation of resources to fecundity and less to belowground storage under low water conditions by the native plants could represent a stress response that is not being expressed by the invasive plants (cf., Pigliucci and Schlichting 1998). It is interesting to contrast the previous result with the reaction norms between the treatments where water and nutrient levels change in the same direction (WLNL – WHNH). For this reaction norm, the only significant effect was a difference in magnitude for the full data set, representing a generalized response in the same direction for both provenances. In this case, the invasive plants had a greater response than did the native ones. All of the traits, except height, responded by increasing in amount with a concomitant increase in water and nutrient availability. The response to an increase in water levels and a corresponding decrease in nutrients (or vice versa) was more complex representing tradeoffs that differed among traits and provenances. In addition to the environmental conditions resulting in significant differences between native and invasive populations, it is also interesting to consider the two sets of conditions under which differences in phenotypic plasticity were not observed. Under conditions of low water availability but shifting nutrient status (WLNL – WLNH), phenotypic plasticity was minimal for both provenances and data sets (full and reduced; Table 3). This indicates that when water availability is low, the potential for plastic response is limited in both native and invasive populations, regardless of variation in nutrient availability. In contrast, under conditions of high nutrient availability there was a 82

large plastic response to change in water availability (WLNH – WHNH), though this response did not differ in magnitude or direction for either provenance or data set (Table 3). This implies that when nutrients are not limiting, both native and invasive populations can be expected to exhibit similar patterns of phenotypic change in response to environmental variation in water availability. In general, our results indicate that native and invasive purple loosestrife plants exhibit different patterns of plastic responses, depending on specific environmental changes in water and nutrient levels. However, questions can be raised as to whether this difference is accompanied with an increase in plant function and fitness. Greater magnitude of plastic responses does not necessarily mean greater fitness and adaptation (Richards et al. 2006). Native plants produced more flowers in response to low water availability (Fig. 1d), but invasive plants were taller with greater aboveground biomass over all treatment combinations (Fig. 1a, e, and as indicated in Fig. 3, particularly with respect to PC2). Our results agree with Bastlová and Květ (2002) who found that non- native purple loosestrife plants allocate more biomass to aboveground shoots while native plants allocate more to reproductive parts. In addition, Mal et al. (1997a) reported that populations invading Canada significantly increased clonal growth in response to increased water or nutrient levels, but flower production didn’t increase. This may reflect a difference in reproductive strategy between native and non-native populations in the first season of growth; native populations are primarily producing flowers whereas invasive populations rely on clonal reproduction and increased allocation to storage in the roots. Considering that the onset of flowering in invasive plants is approximately ten days later than in native plants (Bastlová and Květ 2002), it appears that invasive plants have developed a strategy of a more extended period of vegetative growth before flowering, which might potentially contribute to increased fitness in successive years. In the first year of growth by a perennial plant, it might be advantageous to allocate more energy to vegetative growth and storage rather than sexual reproduction. Preliminary studies suggest that invasive purple loosestrife plants have a longer lifespan than native plants (H. Dietz and K. A. Moloney, unpublished data). A long-term study of the type presented 83 here may be required to determine if perennial invasive plants exhibit more adaptive plasticity in the long run than native plants. In any case, our results support the argument that invasiveness of purple loosestrife is closely associated with the interaction of high levels of soil nutrient and a flooding water regime. Invasive populations exhibit significantly greater shoot biomass than native populations in nutrient-rich field conditions with a standing water regime (Edwards et al. 1998). Weiher et al. (1996) found that purple loosestrife plants in northeast North America establish and subsequently dominate in local communities when soil fertility is high, in either standing water or a seasonally flooded water regime. However, establishment was lowest when soil fertility was low in seasonally flooded water regimes. In addition, purple loosestrife generally grows taller under high moisture conditions (Lempe et al. 2001, Shadel and Molofsky 2002). Our results agree with these studies, in that height of invasive plants is greatly increased in high water conditions, and most of the other traits are greatly increased in high nutrient, high water conditions. Although purple loosestrife is an aggressive invader in North America, they are mostly found in permanently or periodically flooded habitats, while they have actually a wider ecological range in Europe (Bastlová-Hanzélyová 2001). However, it has been repeatedly reported that invasive populations tend to be tall and vigorous in growth, often forming high-density monocultural stands, while native populations are composed of more widely scattered plants of generally smaller stature (Blossey and Nötzold 1995, Edwards et al. 1998, Bastlová-Hanzélyová 2001, Bastlová and Květ 2002). Our results indicate that invasive plants exhibit greater plasticity than native plants when water and nutrient levels increase simultaneously (WLNL – WHNH, Table 3), especially with respect to an increase in height and aboveground biomass (Fig. 1). In contrast to native plants, invasive purple loosestrife plants may have evolved a greater ability to take advantage of high nutrient levels, produced from disturbances and human activities, especially under flooded conditions. This may help explain the accumulation of invasive potential in the first growing season and the development of aggressiveness in subsequent years in the invaded range of North America.

Acknowledgements

We thank H. Asbjornsen, R. Hall, and S. Mahoney for use of greenhouse facilities. M. Y. Chung, D. G. Kim, D. Tessin, R. Schmitt, and K. Day provided helpful comments and assistance in the greenhouse. We would also like to thank L. Gough, C. Schlichting and an anonymous reviewer for providing constructive comments that greatly improved this paper. This work was financially supported by the EEOB department at Iowa State University and an NSF VIGRE postdoctoral fellowship (from grant DMS 0091953) to MLC.

Literature Cited

Ågren, J., and L. Ericson. 1996. Population structure and morph-specific fitness differences in tristylous Lythrum salicaria. Evolution 50:126-139. Alpert, P., and E. L. Simms. 2002. Relative advantages of plasticity and fixity in different environments: when is it good for a plant to adjust? Evolutionary Ecology 16:285- 297. Barney, J. N., A. Di Tommaso and L. A. Weston. 2005. Differences in invasibility of two contrasting habitats and invasiveness of two mugwort Artemesia vulgaris populations. Journal of Applied Ecology 42:567-576. Bastlová-Hanzélyová, D. 2001. Comparative study of native and invasive plants of Lythrum salicaria L.: population characteristics, site and community relationships. Pages 33-41 in G. Brundu, J. Brock, I. Camarda, L. Child, and M. Wade, editors. Plant invasions: Species ecology and ecosystem management. Backhuys Publishers, Leiden. Bastlová, D., H. Čížková, M. Bastl, and J. Květ. 2004. Growth of Lythrum salicaria and Phragmites australis plants originating from a wide geographical area: Response to nutrient and water supply. Global Ecology and Biogeography 13:259-271. Bastlová, D., and J. Květ. 2002. Differences in dry weight partitioning and flowering phenology between native and non-native plants of purple loosestrife (Lythrum salicaria L.). Flora 197:332-340. 85

Bedford, B. L, M. R. Walbridge, and A. Aldous. 1999. Patterns in nutrient availability and plant diversity of temperate North American wetlands. Ecology 80(7):2151–2169. Blossey, B., and R. Nötzold. 1995. Evolution of increased competitive ability in invasive nonindigenous plants: A hypothesis. Journal of Ecology 83:887-889. Blossey, B., L. C. Skinner, and J. Taylor. 2001. Impact and management of purple loosestrife (Lythrum salicaria) in North America. Biodiversity and Conservation 10:1787-1807. Bossdorf, O., H. Auge, L. Lafuna, W. E. Rogers, E. Siemann, and D. Prati. 2005. Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia 144(1):1-11. Bridgham, S. D., J. Pastor, J. A. Janssens, C. Chapin, and T. J. Malterer. 1996. Multiple limiting gradients in peatlands: a call for a new paradigm. Wetlands 16(1):45-65. Brock, M. T., C. Weinig and C. Galen. 2005. A comparison of phenotypic plasticity in the native dandelion Taraxacum ceratophorum and its invasive congener T. officinale. New Phytologist 166:173-183. Burns, J. H. and A. A. Winn. 2006. A comparison of plastic responses to competition by invasive and non-invasive congeners in the Commelinaceae. Biological Invasions 8:797-807. Collyer, M. L., and D. C. Adams. 2007. Analysis of two-state multivariate phenotypic change. Ecology 88(3):683-692. Edwards, K. R., M. S. Adams, and J. Květ. 1998. Differences between European native and American invasive populations of Lythrum salicaria. Journal of Vegetation Science 9:267-280. Falconer, D. S., and T. F. C. MacKay. 1996. Introduction to quantitative genetics, 4th edition, Longman Group Ltd, Harlow, Essex. Gould. S. J. 1984. Covariance sets and ordered geographic variation in Cerion from Aruba, Bonaire and Curacao: A way of studying nonadaptation. Systematic Zoology 33:217-237. Hood, G. M. 2005. PopTools version 2.6.6. Available on the internet. URL http://www.cse.csiro.au/poptools. 86

Hotelling, H. 1931. The generalization of student's ratio. Annals of Mathematical Statistics 2:360–378. Kaufman, S. R., and P. E. Smouse. 2001. Comparing indigenous and introduced populations of Melaleuca quinquenervia (Cav.) Blake; response of seedlings to water and pH levels. Oecologia 127:487-494. Kolar, C. S., and D. M. Lodge. 2001. Progress in invasion biology: Predicting invaders. Trends in Ecology and Evolution 16:199-204. Lande, R., and S. J. Arnold. 1983. The measurement of selection on correlated characters. Evolution 37:1210-1226. Langerhans, R. B., and T. J. DeWitt. 2002. Plasticity constrained: Overgeneralized induction cues cause maladaptive phenotypes. Evolutionary Ecology Research. 4:857-870. Legendre, P., and L. Legendre. 1998. Numerical ecology, 2nd edition, Elsevier, Amsterdam, The Netherlands. Lempe, J., K. J. Stevens, and R. L. Peterson. 2001. Shoot responses of six Lythraceae species to flooding. Plant Biology 3:186–193. Mack, R. N., S. C. H. Barret, P. L. deFur, W. L. MacDonald, L. V. Madden, D. S. Marshall, D. G. McCullough, P. B. McEvoy, J. P. Nyrop, S. E. H. Reichard, K. J. Rice, and S. A. Tolin. 2002. Predicting invasions of nonindigenous plants and plant pests. National Academy Press, Washington, D. C. Mahalanobis, P. C. 1936. On the generalized distance in statistics. Proceedings of Natural Institute of Sciences 2:49-55. Mal, T. K., J. Lovett-Doust, and L. Lovett-Doust. 1997a. Effect of soil moisture and fertilizer application in clonal growth and reproduction in a tristylous weed, Lythrum salicaria. Canadian Journal of Botany 75:46-60. Mal, T. K., J. Lovett-Doust, and L. Lovett-Doust. 1997b. Time-dependent competitive displacement of Typha angustifolia by Lythrum salicaria. Oikos 79:26-33. Maron, J. L., M. Vila, R. Bommarco, S. Elmendorf, and P. Beardsley. 2004. Rapid evolution of an invasive plant. Ecological Monographs 74(2):261-280. Marshall, D. R., and S. K. Jain. 1968. Phenotypic plasticity of Avena fatua and A. barbata. American Naturalist 102:457-467. 87

Mooney, H. A., and R. J. Hobbs. 2000. Invasive species in a changing world - A Project of SCOPE, the Scientific Committee on Problems of the Environment. Island Press, Washington, D. C. Pigliucci, M. 2001. Phenotypic plasticity: beyond nature and nurture. John Hopkins Univ. Press, Baltimore, Maryland, USA. Pigliucci, M. 2003. Phenotypic integration: studying the ecology and evolution of complex phenotypes. Ecology Letters 6:265-272. Pigliucci, M., and C. D. Schlichting. 1995. Reaction norms of Arabidopsis (Brassicaceae) III. Response to nutrients in 26 populations from a worldwide collection. American Journal of Botany 82(9):1117-1125. Pigliucci, M. and C. D. Schlichting. 1998. Reaction norms of Arabidopsis. V. Flowering time controls phenotypic architecture in response to nutrient stress. Journal of Evolutionary Biology 11(3): 285-301. R Development Core Team. 2005. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3- 900051-07-0, URL http://www.R-project.org. Rejmánek, M. 2000. Invasive plants: approaches and predictions. Austral Ecology 25(5):497-506. Rice, K. J., and R. N. Mack. 1991. Ecological genetics of Bromus tectorum. III. The demography of reciprocally sown populations. Oecologia 88:91-101. Rice, W. R., 1989. Analyzing tables of statistical tests. Evolution 43:223-225. Richards, C. L., O. Bossdorf, N. Z. Muth, J. Gurevitch and M. Pigliucci. 2006. Jack of all trades, master of some? On the role of phenotypic plasticity in plant invasions. Ecology Letters 9: 981-993. Sakai, A. K., F. Allendorf, J. Holt, D. Lodge, J. Molofsky, K. With, S. Baughman, R. Cabin, J. Cohen, N. Ellstrand, D. McCauley, P. O’Neil, I. Parker, J. Thompson, and S. Weller. 2001. The population biology of invasive species. Annual Review of Ecology and Systematics 32:305-312. Sall, J, A. Lehman, and L. Creighton. 2003. JMP Start Statistics version 5.1. Pacific Grove, CA: Duxbury Press. 88

Schlichting, C. D. 1986. The evolution of phenotypic plasticity in plants. Annual Review of Ecology and Systematics 17:667-693. Schlichting, C. D. and M. Pigliucci. 1998. Phenotypic evolution: a reaction norm perspective. Sinauer Associates, Sunderland, MA. Schluter, D. 1996. Adaptive radiation along genetic lines of least resistance. Evolution 50:1766-1774. Shadel, W. P., and J. Molofsky. 2002. Habitat and population effects on the germination and early survival of the invasive weed, Lythrum salicaria L. (purple loosestrife). Biological Invasions 4:413-423. Shamsi, S. R. A., and F. H. Whitehead. 1974a. Comparative eco-physiology of Epilobium hirsutum L. and Lythrum salicaria L. 1. General biology, distribution and germination. Journal of Ecology 62:279-290. Shamsi, S. R. A., and F. H. Whitehead. 1974b. Comparative eco-physiology of Epilobium hirsutum L. and Lythrum salicaria L. 2. Growth and development in relation to light. Journal of Ecology 62:631-645. Shamsi, S. R. A., and F. H. Whitehead. 1977a. Comparative eco-physiology of Epilobium hirsutum L. and Lythrum salicaria L. 3. Mineral nutrition. Journal of Ecology 65:55-70. Shamsi, S. R. A., and F. H. Whitehead. 1977b. Comparative eco-physiology of Epilobium hirsutum L. and Lythrum salicaria L. 4. Effects of temperature and inter-specific competition and concluding discussion. Journal of Ecology 65:71-84. Simberloff, D. 2000. Nonindigenous species: A global threat to biodiversity and stability. Pages 325-334 in P. Raven, and T. Williams, editors. Nature and human society: the quest for a sustainable world. National Academy Press, Washington, D. C. Stevens, K. J., R. L. Peterson, and G. R. Stephenson. 1997a. Morphological and anatomical responses of Lythrum salicaria L. (purple loosestrife) to an imposed water gradient. International Journal of Plant Sciences 158(2):172-183. Stevens, K. J., R. L. Peterson, and G. R. Stephenson. 1997b. Vegetative propagation and the tissues involved in lateral spread of Lythrum salicaria. Aquatic Botany 56:11- 24. 89

Stuckey, R. L. 1980. Distributional history of Lythrum salicaria (purple loosestrife) in North America. Bartonia 47:3-20. Sultan, S. E. 2003. Phenotypic plasticity in plants: A case study in ecological development. Evolution & Development 5(1):25-33. Sultan, S. E., and F. A. Bazzaz. 1993a. Phenotypic plasticity in Polygonum persicaria. II. Norms of reaction to soil moisture and the maintenance of genetic diversity. Evolution 47(4):1032-1049. Sultan, S. E., and F. A. Bazzaz. 1993b. Phenotypic plasticity in Polygonum persicaria. II. The evolution of ecological breadth for nutrient environment. Evolution 47(4):1050-1071. Thompson, D. Q., R. L. Stuckey, and E. B. Thompson. 1987. Spread, Impact, and Control of Purple Loosestrife (Lythrum salicaria) in North American Wetlands. US Fish and Wildlife Service. 55 pages. Jamestown, ND: Northern Prairie Wildlife Research Center Home Page. http://www.npwrc.usgs.gov/resource/1999/loosstrf/loosstrf.htm (Version 04JUN99). Thormann, M. N., and S. E. Bayley. 1997. Response of aboveground net primary plant production to nitrogen and phosphorous fertilization in peatlands in southern boreal Alberta, Canada. Wetlands 17(4):502-512. Via, S., R. Gomulkiewicz, G. De Jong, S. M. Scheiner, C. D. Schlichting, and P. H. Van Tienderen. 1995. Adaptive phenotypic plasticity: Consensus and controversy. Trends in Ecology and Evolution 10:212-217. Vitousek, P. M., C. M. D’Antonio, L. L. Loope, and R. Westbrooks. 1996. Biological invasions as global environmental change. American Scientist 84:468-478. Waldmann, P., and S. Anderson. 2000. Comparison of genetic (co)variance matrices within and between Scabiosa canescens and S. columbaria. Journal of Evolutionary Biology 13:826-835. Wagner, G. P., and L. Altenberg. 1996. Complex adaptations and the evolution of evolvability. Evolution 50:967-976. Weiher, E., I. C. Wisheu, P. A. Keddy, and D. R. J. Moore. 1996. Establishment, persistence, and management implications of experimental wetland plant communities. Wetlands 16(2):208-218. 90

Williams, D. G., R. N. Mack, and R. Black. 1995. Ecophysiology of introduced Pennisteum srtaceum on Hawaii: the role of phenotypic plasticity. Ecology 76:1569-1580. Williamson, M. 1999. Invasions. Ecography 22:5-12. 91

Chapter 4. Phenotypic plasticity of native vs. invasive purple loosestrife: an application of path analysis

Abstract

We examined differences in the response to environmental change (phenotypic plasticity) between invasive (North American) and native (German) plants of Lythrum salicaria (purple loosestrife). We used an in-field common garden study with a split-plot design with experimentally manipulated water and nutrient level conditions. Data were collected for a total of eight traits related to life history (number of dates to first flowering), plant size / architecture (height, number of secondary branches, number of stems from root, and leaf area), and fitness (number of flowers, reproductive: aboveground biomass ratio, and aboveground biomass). Plants from invasive provenances were found to be significantly different from plants from native provenances in phenotypic plasticity and plant performance. Generally, invasive plants exhibited greater height and leaf area than native plants across all treatments. Native plants, in contrast, flowered earlier than invasive plants, thus allocating more resource to flower production. However, given increased nutrients, invasive plants outperformed native plants in total biomass and number of flowers. Structural equation modeling revealed that native plants failed to produce branches and biomass due to the negative effect of early flowering, while invasive plants exhibited greater performance in vegetative traits on behalf of the greater positive effects between them. Our results indicate that invasive plants may have evolved the ability to utilize more resources and accumulate potential for aggressive invasion after introduction to North America.

Keywords: phenotypic plasticity, invasive species, Lythrum salicaria, purple loosestrife, path analysis.

Introduction

Many studies have attempted to identify characteristics that enable an invasive species to be aggressive in new areas. Although appealing hypotheses have been suggested, phenotypic plasticity in particular is a potential mechanism enabling the survival and reproduction of a widespread colonizing species in a broad range of environments (Marshall and Jain 1968, Rice and Mack 1991, Sultan and Bazzaz 1993, Williams et al. 1995), and is one character for predicting invasiveness (Rejmánek and Richardson 1996). However, it is still inconclusive how phenotypic plasticity results in higher fitness (adaptive plasticity) and thus contributes to the invasiveness of an exotic species. This ambiguity may arise from misconceptions about the role of phenotypic plasticity in invasiveness. One view is that plasticity is an absolute barometer of invasiveness. But plasticity itself is the variable phenotypic response to different environments, therefore it does not guarantee that a genotype (or genotypes) with greater plasticity will perform better than others with less plasticity. Additional confusion derives from interpreting the greater performance of invasive species using traits that are not closely related to fitness (or a lack of effort to relate the effect of the trait on increased fitness and invasiveness). A sound understanding of the concept of phenotypic plasticity may aid in constructing a testable hypothesis and experimental design for the role of adaptive plasticity for invasiveness. First, one should be able to evaluate “fitness” in order to determine whether it is maintained at the highest level across a range of environments, although empirical studies of phenotypic plasticity can only measure survival, growth, and reproduction as surrogates of genotype fitness. Also, in reality fitness is the final outcome of these traits, there has not been an attempt to integrate them to assess fitness in a relevant way. It is imperative to establish appropriate criteria for measuring fitness, however, it may depend on the organism’s life-history characteristics. Second, there are many traits related to fitness closely or distantly and contribute to fitness directly or indirectly (hereby called ‘underlying traits’). Underlying traits should not be used as direct measures of fitness, since they may not be linearly related to fitness. In many cases 93 high plasticity of underlying traits does not lead to higher fitness. It may be due to the costs or limits of plasticity from possessing the genetic or physiological mechanism for plastic responses, or a consequence of genetic correlations with other traits affecting fitness (van Kleunen and Fischer 2005). Therefore, it is important to investigate how underlying morphological or physiological traits evolved higher fitness and adaptive plasticity, although there are only few studies addressing this issue in invasion biology. This can be accomplished by adapting a multivariate approach, from the keynote work by Lande and Arnold (1983) proposing a multivariate analysis of the covariance between traits and fitness. This multiple regression assumes a simple causal relationship where all traits affect fitness directly. However, in nature, all morphological or physiological traits may not be directly related to fitness; some of them may be closely related, while others may not. For example, total biomass, reproductive effort, and survival are considered to be closely related to fitness and may be used as surrogates of fitness. However, relative growth rate (RGR), photosynthesis, nutrient contents, biomass allocation, or minor morphological characteristics are indirectly related to fitness and make little sense as reasonable surrogates of fitness. Therefore, a more reasonable model should be able to evaluate both the direct effects of closely related traits and the indirect effects of distantly related traits. In addition, the causal relationships between traits should consider the ontogeny of the study organism, beginning with the effects of early traits such as seedling size and finishing with the effects of late traits such as total biomass and number of flowers. The ideal approach that incorporates these elements is structural equation modeling (path analysis). It is a statistical technique used to examine causal relationships between two or more variables and is frequently applied to ecological studies mainly to understand comparative strengths of direct and indirect relationships among a set of ecological traits (Crespi and Bookstein 1989, Kingsolver and Schemske 1991, Scheiner et al. 2000), as well as among a set of environmental treatments (Scheiner and Callahan 1999, Pigliucci and Kolodynska 2006). Using purple loosestrife (Lythrum salicaria L.) as a model system, we applied a path analytic model to investigate whether invasive and native populations differ in causal relationships leading to fitness. We used a common garden study designed to contrast the plastic response of plants derived from native and invasive source 94 populations to varying environmental conditions. The trickiest part of path analysis is finding the right model to describe the data. It is possible that there is more than one good model that fits the data equivalently. The model should be specified a priori by the researcher, based on theoretical and practical knowledge. To build a reasonably good model, we utilized experience from a prior study on purple loosestrife (Chun et al. 2007), as well as preliminary analysis of this work involving univariate analysis and PCA. In this study, we address two basic questions regarding phenotypic plasticity and trait covariance of invasive vs. native purple loosestrife: (1) Do invasive and native populations exhibit different patterns of univariate phenotypic plasticity either by significant genetic (G) or genetic by environmental (G×E) interactions? (2) Do invasive and native populations exhibit different patterns of causal relationships among traits, ultimately leading to the greater fitness and invasion success of invasive populations?

Methods

Seed collection To contrast native vs. invasive populations, we used three European regions (native provenance) and three North American regions (invasive provenance). Three study regions in North America were chosen to reflect different stages in the invasion history of purple loosestrife, with New Jersey (NJ) populations as the oldest (many dating from before 1900), Michigan (MI) populations being of intermediate age (many appearing from the 1900’s through the 1940’s), and Iowa (IA) populations being the youngest, beginning to establish after the 1940’s (Stuckey 1980, Edwards et al. 1995). Three populations from each region were chosen to represent a range of nutrient and moisture conditions found naturally in the field (Table 1). From each population, we randomly chose 20 plants separated by 3 m to ensure they are genetically distinct. From each plant we separately collected seeds which are half-sibs of the mother plant.

Common Garden Experiment We have conducted a common garden experiment in an open field site at the Bruner Farm, Boone, IA, USA (42° 00´N, 93° 43´E). Seeds from the 18 source 95

Table 1. Geographical position of sampling sites. Provenance Region Population ID Latitude Longitude Collection Boone Folks IA BF 42°17Ń 93°56´W Fall 2004 Iowa Little South Storm Lake IA LS 42°38Ń 95°14´W Manly IA MA 43°16Ń 93°07´W Kellogg Biological Station MI KB 42°21Ń 85°21´W Fall 2004 North Michigan Lake Lansing MI LL 42°46Ń 84°23´W America Rifle Range near Pittsford MI RR 41°51Ń 84°30´W Beaver Run NJ BR 41°09Ń 74°36´W Fall 2004 New Jersey Hainville County Store NJ HV 41°15Ń 74°48´W Walkill River NJ WR 41°03Ń 74°37´W Golm PD GO 52°25Ń 12°57É Fall 2005 Potsdam Grube PD GR 52°27Ń 12°57É Potsdam PD PO 52°29Ń 12°57É Altmatt SW AL 47°20Ń 07°52É Fall 2005 Europe Switzerland North Sihl Lake SW NO 47°36Ń 08°13É Steinbode SW ST 47°09Ń 08°43É Hagelloch Tobel TU HA 48°32Ń 09°01É Fall 2005 Tübingen Reusten "Hinterer See" TU RE 48°33Ń 08°55É Unterjesingen "Wiesbrunn" TU UN 48°31Ń 08°58É populations were planted on 2 May, 2006. After 28 days of growth, we selected 5 half-sib families from each population to include in the experiment. Therefore, our measurements of phenotypic plasticity reflect variability at the population level with 5 half-sib families as replicates within each population, as recommended by Richards et al. (2006). We chose 4 seedlings of 1 – 2 cm in height from each half-sib family per population (18 populations × 5 half-sib families × 4 seedlings = 360) to reduce environmental variation in traits related to germination and pre- / post-transplant growth. On 30 May 2006, seedlings were individually transplanted into plastic pots (30 cm diameter × 25 cm depth) and placed into plastic wading pools (1.4 m diameter × 30 cm depth). The pots were filled with Sunshine LC1 potting soil (Sun Gro Horticulture Canada, Seba Beach, Alberta, Canada), which has an initial nutrient charge that would correspond to high nutrient levels in natural wetland soils, approximating 3 times the average content of nitrogen as reported in Bridgham et al. (1996) and Bedford et al. (1999). As the initial nutrient charge is highly water soluble, providing the equivalent of approximately one application of a liquid fertilizer, nutrients were supplemented by regular fertilizer treatments as described below. Plants were arranged in a split-plot design consisting of five complete blocks, with four subplots within each block. Each subplot consisted of three plastic wading pools that contained one of four environmental treatment combinations: (1) low water/low nutrient

(WLNL), (2) low water/high nutrient (WLNH), (3) high water/low nutrient (WHNL), and 96

(4) high water/high nutrient (WHNH). Three plants from each provenance were put in each wading pool (6 plants per pool). Three populations from each region were distributed among three pools at random, with the restriction that only one population from each region can appear in a pool. Five half-sib families from each population were also randomized across five blocks. The total number of experimental units for this experiment was 5 blocks × 4 treatment combinations = 20 units, with 6 regions × 3 populations = 18 observations per experimental unit. Pots in the high water treatments were kept at saturation, similar to standing water conditions in lakes and ponds. Low water treatments are comparable to drier, upland conditions, where plants were watered to the extent of letting the soil soak for a few hours, after which the soil was allowed to dry. Due to drought conditions at the common garden site, plants in the low water treatments were watered every day until June 27, and then watered three times a week. In addition, 52 seedlings were killed from summer heat between 2 and 22 June and were replaced with seedlings from the same half-sib family. After that period, six plants died and were not considered in the data analysis. In the low nutrient treatment, no fertilizer was applied, whereas in the high nutrient treatment 100 g of slow-release 14:14:14 N:P:K Osmocote (The Scotts Company, Marysville, Ohio, USA) was applied once to a pot at the beginning of the experiment (N, 2481 mg/kg soil; P, 563 mg/kg soil; K, 1,320 mg/kg soil). The Osmocote applied in the high nutrient treatment followed the manufacturer’s recommendation for amount and rate (one application for a 3–4 month growing period) to be used in producing high fertilization levels (The Scotts Company). As a consequence, the low and high nutrient treatments were designed to be a reasonable approximation of low and high soil nutrient levels, respectively, that would be encountered under natural field conditions (cf., Bridgham et al. 1996, Thormann and Bayley 1997, Bedford et al. 1999). On 31 August 2006, after 97% of the plants had initiated flowering, we harvested the aboveground part of the plants. Belowground parts were left for a subsequent year’s study. As a perennial herb, purple loosestrife usually takes from 4-5 years to be competitively dominant under field conditions (Weiher et al. 1996, Mal et al. 1997). However, as they begin to increase ramet production in the second year of growth (Mal et al. 1997), it may be important to test whether native and invasive genotypes exhibit 97 genetically based differences during the first year of growth in response to varying environmental conditions. We randomly chose wading pools across blocks for harvesting to minimize experimental variation due to growth during the harvesting period. The following eight traits were measured for each experimental individual: (1) height, (2) number of secondary branches, (3) number of stems originating from the rootstock, (4) leaf area, (5) number of days from sowing to the first flowering, (6) aboveground biomass, (7) ratio of reproductive (flowering) part to the aboveground biomass, and (8) total number of flowers per plant. To determine (4), we chose one largest leaf from each plant and calculated leaf area by multiplying the maximum length and width of the leaf. To determine (8), we haphazardly sampled six flower stalks from each plant (two each from the apical, middle, and basal part of the stalk), weighed, and counted the number of flowers, in order to calculate the number of flowers per unit biomass of flower stalk. We measured total biomass of flower stalks per plant for each individual. Total number of flowers per plant was then calculated by multiplying the number of flowers per unit biomass by total biomass of flower stalk per plant, for each individual. To determine total biomass of flower stalks and vegetative parts, plants were divided into respective parts and dried in an oven for 24 hours at 60ºC to constant weight.

Univariate and Exploratory Multivariate Analysis We analyzed the phenotypic plasticity of purple loosestrife populations from the native and invasive provenance for the four environmental treatment combinations. The reaction norm is a function of phenotypic values across environments that can be described for individuals or for populations (Via et al. 1995, Pigliucci 2001). To infer a biological explanation for genetic variation in phenotypic plasticity, trait means contrasting four environmental treatments are plotted for each trait. To assess the effect of G (genetic variation), E (environment), and G×E interactions on phenotypic responses, we conducted a mixed model ANOVA for each trait. It is a nested structure with main plot containing block and environmental effects and subplot containing the genetic effects (provenances and regions nested within provenance). All G, E, and G×E interactions are fixed effects, while block and pool were considered as random effects. A significant G 98 effect means that populations are genetically differentiated and a significant E effect confirms the existence of phenotypic plasticity for that environmental factor. Significant G×E indicates significant genetic variation for phenotypic plasticity. To examine multivariate relationships among the eight traits and how they differ among populations, we conducted principal component analysis (PCA) on the correlation matrix calculated from log-transformed trait data.

Path Analysis We conducted structural equation modeling using the AMOS 6.0 software (Arbuckle 2006). We began by considering how life-history traits are related to each other and to measures of reproductive fitness in purple loosestrife. The core concept here is that paths should be in a sequence parallel to ontogeny, beginning with the effects of early traits and finishing with the effects of late traits. Therefore, our model follows an ontogenic cascade from the seedling-stage trait through flowering time to the traits measured at harvest. We considered the total reproductive effort (number of flowers) as a surrogate of fitness. With this in mind, the following seven traits were chosen: (1) seedling height at the time of transplant, (2) number of days from sowing to the first flowering, (3) number of stems originating from the rootstock, (4) number of secondary branches, (5) aboveground biomass, (6) height, and (7) total number of flowers per plant. We first defined a just-identified model, which includes all paths among the observed variables (Fig. 1a) and simplified it by dropping eight paths that were not significant from the preliminary analysis, creating an over-identified model (Fig. 1b). This was done to establish the most parsimonious model that can explain the maximum information in the data with the smallest complexity. The over-identified model was evaluated for model fit, using chi-square statistic (CMIN) and other measures. CMIN is the minimum value of the discrepancy between the tested (over-identified) model and the saturated (just- identified) model. CMIN was 8.56 for our over-identified model and the probability of getting as large a discrepancy as 8.56 was 0.381. This non-significant P value indicates that the fit between the over-identified model and the data is not significantly worse than the fit between the just-identified model and the data. Other measures (RMSEA=0.014, NFI=0.991, CFI=0.999) indicated a good fit of our over-identified model. The Root Mean 99

Square Error of Approximation (RMSEA) estimates lack of fit of the tested model compared to the saturated model. Generally, values of 0.05 or less indicate a good fit. The Normed Fit Index (NFI) measures the difference between the tested and saturated models’ chi-squares divided by the chi-square for the independence model. The Comparative Fit Index (CFI) uses a similar approach as NFI with a non-central chi- square. For both NFI and CFI, values of 0.9 or higher indicate good fit. The Akaike Information Criterion (AIC) for the over-identified model (62.56) was smaller than the AIC of the just-identified model (70.00), which means that the over-identified model is a parsimonious model that can explain the data better than the just-identified model. Therefore, we decided to apply the over-identified model to our data. First it was applied to the invasive and native provenances separately, then to the four subsets of each provenance, one for each combination of environmental treatments. In each case, model

Figure 1. Path analytical models. (a) just-identified (default) model; (b) over-identified (tested) model. Numbers represent standardized estimate of regression weights (path coefficients). The eight paths dropped from the just- identified model are marked as dotted lines and associated coefficients in bold. Traits are: HeightB, seedling height at the time of transplant; NDFF, number of days from sowing to the first flowering; StemsR, number of stems originating from the rootstock; Branch, number of secondary branches; MassT, aboveground biomass, HeightF, final height at harvest, and FlowerN, total number of flowers per plant. 100 performance was evaluated using model fit measures described above. We plotted the reaction norms of path coefficients (estimates of regression weight) representing the direct effects described by the model, with their standard error of regression weight. In this way, we examined how the environmental treatments affect the path coefficients and how this relationship differs between invasive and native provenances.

Results

Analysis of variance revealed that six of the eight traits showed significant genetic variation between native and invasive provenances (Table 2). Within each provenance, seven traits (the exception being height) exhibited significant genetic variation among regions. Significant treatment effects were found for height, leaf area, aboveground biomass, and number of flowers, while number of branches and reproductive: aboveground biomass ratio was characterized by significant nutrient and water effects, respectively. Interestingly, there was no treatment effect on the number of days to first flowering, indicating that flowering phenology is not affected by the levels of nutrient and water used in this experiment.

Table 2. Summary of ANOVA with P-values of the corresponding F-tests for each trait. Source DF HeightF Branch StemsR LeafArea NDFF MassT RatioRT FlowerN Main plot (block and treatments) block, B 4 0.4365 0.2528 0.1516 0.9561 0.3962 0.4002 0.3680 0.2103 water, W 1 <.0001 0.7653 0.0005 0.0030 0.4377 <.0001 <.0001 <.0001 nutrient, N 1 <.0001 <.0001 <.0001 <.0001 0.3340 <.0001 0.4125 <.0001 W×N 1 <.0001 <.0001 0.0658 <.0001 0.0693 <.0001 0.5714 <.0001 error (0.9973×L[B×W×N] 52

+0.0027×residual)† Subplot Provenances provenance, P 1 <.0001 0.0043 0.2848 <.0001 <.0001 <.0001 0.0004 0.2241 P×W 1 0.5304 0.8647 0.5657 0.2325 0.0406 0.9501 0.3544 0.3621 P×N 1 0.2010 0.0103 0.3424 0.4305 0.0658 0.1344 0.1328 0.7113 P×W×N 1 0.6287 0.0259 0.1847 0.0427 0.2740 0.2901 0.6994 0.7506 Regions (R) within provenance R(P) 4 0.0567 <.0001 0.0033 <.0001 <.0001 <.0001 <.0001 <.0001 R×W(P) 4 0.8436 0.2622 0.3450 0.1390 0.5011 0.2630 0.8554 0.8058 R×N(P) 4 0.0578 0.1569 0.8875 0.4910 0.4493 0.0781 0.0026 0.1368 R×W×N(P) 4 0.6928 0.6143 0.3933 0.0002 0.9019 0.5976 0.5798 0.1589 error (residual) 274 Total 353

Notes: Significant P values are indicated in bold. DF: degree of freedom; Traits are: HeightF, final height at harvest; Branch, number of secondary branches; StemsR, number of stems originating from the rootstock; LeafArea, leaf area; NDFF, number of days from sowing to the first flowering; MassT, aboveground biomass; RatioRT, ratio of reproductive (flowering) part to the aboveground biomass; FlowerN, total number of flowers per plant. 101

Across all environmental treatments, invasive plants were generally taller, possessing greater leaf area, and flowering later than native plants (Fig. 2a, d, e). Especially, invasive plants exhibited greater aboveground biomass and number of flowers in high nutrient treatments (Fig. 2f, h). The reproductive: aboveground biomass ratio was greater for natives than invasives, however, the difference became smaller as water and nutrient resources were added, and ultimately there was no difference at WHNH treatment (Fig. 2g). When the trait means across all treatments were compared among regions, a consistent difference between native and invasive provenance was found in height and leaf area (Fig. 3a, d), while considerable variation among regions exist in number of days to first flowering (Fig. 3e). Especially, plants from the Switzerland and Michigan region flowered earlier and exhibited greater reproductive: aboveground biomass ratio than plants from other regions within provenance (Fig. 3e, g). Within the invasive provenance, the number of secondary branches, the number of flowers, and aboveground biomass were greater in Iowa and Michigan than in New Jersey, although it was not significant for the number of branches (Fig. 3b, f, h). PC1 (the principal axis of variation) and PC2 together explained 67% of the total variation in the data (Table 3). PC1 was positively associated with all traits except for the number of days to first flowering. PC2 was positively associated with the number of days to first flowering, while negatively associated with reproductive: aboveground biomass ratio, indicating a tradeoff between flowering time and reproductive effort. PC3 was positively associated with leaf area and negatively associated with number of stems from root. In the PCA plot (not shown), invasive plants positioned to express increased traits associated with PC1 and delayed flowering compared to native plants. The structural equation model revealed more information on how invasive and native plants differ in the way that traits affect each other in causal paths leading to fitness. This was examined by plotting the path coefficients in each environmental treatment (Fig. 4). The effect of seedling height on number of days to first flowering was around zero both for native and invasive plants (Fig. 4a), however, the effect on number of flowers was well below zero for native plants. The number of days to first flowering had a consistently positive effect on number of branches for natives, while it was around 102

Figure 2. Least square means (±SE) of eight trait values for plants from native (open cicles) and invasive (solid circle) provenance. Environmental treatments are: WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL,

High water, Low nutrient; WHNH, High water, High nutrient. zero for invasives (Fig. 4b). It means that early-flowering native plants are likely to produce fewer branches, while the production of branches by invasive plants was not affected by flowering phenology. The effect of flowering phenology on number of flowers was more negative for natives than for invasives, which indicates that early- flowering native plants produce more flowers. The effect of number of stems showed a 103

Figure 3. Least square means (±SE) of eight trait values across all treatments for native (open circles) and invasive (solid circle) plants from each region. Letters indicate that any two regions with no common characters differ significantly (P<0.05, Tukey’s pairwise test). Regions are: TU, Tübingen; SW, Switzerland; PD, Potsdam; NJ, New Jersey; MI, Michigan; IA, Iowa. notable pattern in WHNL, where the effect on number of flowers was positive for natives while negative for invasives (Fig. 4c). Also, the effect on number of branches was positively greater for natives than invasives at WHNH. The effect of number of branches on aboveground biomass was around zero or slightly positive for invasive plants (Fig. 4d). Interestingly, it was consistently decreasing for native plants as water and nutrient 104

Table 3. PCA result (loadings) on log-transformed full data set. Variables PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 HeightF 0.400 –0.108 0.433 0.174 0.423 –0.634 0.147 –0.088 Branch 0.415 0.194 –0.309 –0.477 –0.239 –0.117 0.625 –0.073 StemsR 0.283 0.272 –0.561 0.572 0.402 0.193 0.075 –0.015 LeafArea 0.342 0.249 0.440 0.465 –0.588 0.235 0.081 –0.054 NDFF –0.015 0.552 0.428 –0.319 0.471 0.428 0.070 0.028 MassT 0.507 0.056 –0.074 –0.215 –0.053 –0.043 –0.506 0.654 RatioRT 0.121 –0.635 0.141 0.065 0.164 0.456 0.428 0.374 FlowerN 0.447 –0.321 –0.022 –0.215 0.071 0.314 –0.361 –0.645 Eigenvalue 3.38 1.95 1.06 0.52 0.41 0.35 0.25 0.07 Percentage of Variance 42.26 24.39 13.26 6.52 5.16 4.43 3.07 0.92 Cumulative Percentage of Variance 42.26 66.65 79.91 86.43 91.59 96.02 99.08 100.00 Notes: Traits are: HeightF, final height at harvest; Branch, number of secondary branches; StemsR, number of stems originating from the rootstock; LeafArea, leaf area; NDFF, number of days from sowing to the first flowering; MassT, aboveground biomass; RatioRT, ratio of reproductive (flowering) part to the aboveground biomass; FlowerN, total number of flowers per plant.

resources were added. The effect of aboveground biomass showed a contrasting trend across environments – increasing for invasives and decreasing for natives, ultimately to negative at WHNH (Fig. 4e). The effect of height on aboveground biomass was generally positive but greater for invasives than natives at WLNL and WHNL (Fig. 4f). Its effect on number of flowers was greater for natives than invasives at WLNH, while it was greater for invasives than natives at WHNH.

Discussion

One of the difficulties in studying the background of invasiveness is to establish an appropriate hypothesis comparing the overall performance and fitness between invasive and native populations. A number of studies have compared phenotypic plasticity between invasive exotic species to a control group under two or more manipulated environments, yielding mixed results (Bossdorf et al. 2005, Richards et al. 2006). This outcome is partly due to inappropriate choice of control group and hypotheses, including that invasive exotic species are more plastic than native species, or invasive exotic species are more plastic than non-invasive exotic species. By comparing populations from the invasive range with those from their ancestral native range, we clearly tested whether invasive species have evolved greater fitness in the introduced range than in the ancestral native range. Using purple loosestrife as a model organism, we conducted a common garden study involving native and invasive 105 populations subject to experimentally manipulated water and nutrient environments to show that invasive and native populations differ in adaptive plasticity and trait covariation.

Figure 4. Total effects of path coefficients between the effects of six traits to other traits of native (open circles) and invasive (solid circle) plants. Traits are: HeightB, seedling height at the time of transplant; NDFF, number of days from sowing to the first flowering; StemsR, number of stems originating from the rootstock; Branch, number of secondary branches; MassT, aboveground biomass, HeightF, final height at harvest, and FlowerN, total number of flowers per plant. Environmental treatments are: WLNL, Low water, Low nutrient; WLNH, Low water, High nutrient; WHNL, High water, Low nutrient; WHNH, High water, High Nutrient.

106

Path analysis is one of the appropriate statistical techniques for testing how well specific hypotheses predict observed phenotypic covariances among underlying traits that contributes to fitness (Pigliucci and Kolodynska 2006). However, the specified model needs to be based on sufficient knowledge of the biological and ecological background of the study system. To obtain such information, we conducted a set of preliminary statistical analyses including univariate analyses of variance and an exploratory multivariate approach. The univariate analyses of variance indicate that the native and invasive populations of purple loosestrife are significantly different in the genotypic effect for six out of eight traits (Table 2). Also, the environmental effects significantly influence the growth and reproduction of purple loosestrife. However, the G×E interactions (P×W and P×N) were not significant for most of the traits. This result indicates that the reaction norms of invasive and native populations possess similar slopes with different means, where invasive populations consistently "win over" native populations for aboveground biomass (Fig. 2f), while native populations possess higher reproductive: aboveground biomass ratio (Fig. 2h). It has been repeatedly found that invasive populations tend to be tall and vigorous in growth, often forming high-density monocultural stands, while native populations are composed of more widely scattered plants of generally smaller stature (Blossey and Nötzold 1995, Edwards et al. 1998, Bastlová-Hanzélyová 2001, Bastlová and Květ 2002). In addition, the pairwise comparison among regions revealed that lately- established populations (in Iowa and Michigan) possess greater number of secondary branches, number of flowers, and aboveground biomass than early-established populations (in New Jersey), which indicates that purple loosestrife may have evolved greater fitness in the invasion process (Fig. 3b, f, h). The invasive populations tend to exhibit greater number of branches, aboveground biomass, and number of flowers in response to increased nutrient level (Fig. 2b, f, h), although the P×N (provenance by nutrient) interaction effects were not significant except for the number of branches (Table 2). This finding agrees with prior studies on the physiological response of purple loosestrife to various water and nutrient conditions. Weiher et al. (1996) found that purple loosestrife plants in northeast North America establish and subsequently dominate in local communities when soil fertility is high, in 107 either standing water or a seasonally flooded water regime. Edwards et al. (1998) reported that invasive populations exhibit significantly greater shoot biomass than native populations in nutrient-rich field conditions with a standing water regime. Our results support the argument that invasiveness of purple loosestrife is closely associated with the increase in nutrient level. Principal component analysis indicates that there is a tradeoff between the number of days to first flowering and reproductive: aboveground biomass ratio, and it appears that native and invasive populations have diverged on this tradeoff. Native populations generally flowered earlier than invasive populations and possessed greater reproductive: aboveground biomass ratio, while invasive populations flowered later and allocated less biomass to the reproductive part (Fig. 2e, g). This tradeoff is also strong at the regional level: plants from Switzerland and Michigan flowered earlier and thus possess greater reproductive: aboveground biomass ratio than plants from other regions within each provenance (Fig. 3e, g). Bastlová and Květ (2002) found that non-native purple loosestrife plants allocate more biomass to aboveground shoots while native plants allocate more to reproductive parts. This pattern may reflect a difference in reproductive strategy between native and invasive populations in the first season of growth; native populations primarily produce flowers whereas invasive populations rely on clonal reproduction. However, the gap between native and invasive populations in the reproductive: aboveground biomass ratio became smaller as water and nutrients were added (Fig. 2g), indicating that native and invasive plants are not different in their biomass allocation strategy – both allocate more biomass to vegetative growth than sexual reproduction. As invasive plants generally flowered later than native plants, they may take advantage of longer time for vegetative growth and prepare for vegetative growth in subsequent years with accumulated resources. This strategy of invasive plants was consistent across all treatment. In contrast, native plants under high water and nutrient conditions are likely to invest more biomass in vegetative growth, indicating a more plastic response to environmental change than invasive plants. Structural equation modeling was useful in examining how native and invasive plants differ in the context of causal relationships between ontogenetically early traits and later traits. Native plants exhibited a negative relationship between number of days to 108 first flowering and number of flowers (Fig. 4b). Since native seedlings flowered earlier than invasives, this may have acted as a positive effect on the production of flowers for natives. In addition, natives took advantage of a greater number of stems for production of flowers in WHNL (Fig. 4c). However, native plants are inferior to invasives in vegetative growth. In WLNH, early-flowered native plants produce fewer branches (Fig.

4b), leading to shorter height and smaller biomass (Fig. 4d). In WHNL, native plants exhibit a negative relationship between the number of branches and height, limiting vegetative growth. In contrast, invasive plants benefited from the positive effect of number of stems on the number of branches in WLNH (Fig. 4d). Also, invasive plants with greater biomass resulted in greater flower production in WHNH (Fig. 4e). These results confirm that invasive plants outperform natives in vegetative growth, especially under increased nutrient conditions. Our prior study on purple loosestrife (Chun et al. 2007) and the preliminary analysis of our data enabled us to establish a path model to predict the observed trait covariances under various environmental conditions. Our model generally described the observed covariances well in two environments in invasive populations and three environments in native populations (Table 4; the significant P indicates that the tested model cannot explain the observed covariances well compared to the saturated model.

Table 4. Summary statistics of model fit for each provenance and treatment combination. Chi-square Goodness of fit RMSEA Information criteria CMIN P NFI CFI RMSEA PCLOSE AIC BCC Invasive 65.754 0.000 0.805 0.850 0.078 0.045 281.754 331.147 Native 36.045 0.285 0.901 0.984 0.027 0.815 252.045 300.732 WLNL 19.257 0.014 0.785 0.817 0.179 0.026 73.257 85.257 WLNH 18.244 0.019 0.846 0.887 0.175 0.035 72.244 84.950 Invasive WHNL 13.542 0.095 0.757 0.801 0.127 0.141 67.542 79.885 WHNH 13.542 0.065 0.797 0.849 0.127 0.102 68.711 81.054 WLNL 6.281 0.616 0.930 1.000 0.000 0.685 60.281 72.624 WLNH 5.003 0.625 0.963 1.000 0.000 0.809 59.003 71.346 Native WHNL 5.875 0.661 0.824 1.000 0.000 0.727 59.875 71.875 WHNH 18.889 0.015 0.819 0.857 0.176 0.029 72.889 84.889 Notes: CMIN: minimum value of the discrepancy between tested (over-identified) model and saturated (just-identified) model; P: probability of getting as large a discrepancy as occurred with the corresponding data; NFI: normed fit index (the difference between the tested and saturated models’ chi-squares divided by the chi-square for the independence model. Values of .9 or higher indicate good fit.); CFI: comparative fit index (a similar approach as NFI with a non- central chi-square. Values of .9 or higher indicate good fit.); RMSEA: root mean square error of approximation (It estimates lack of fit compared to the saturated model. Generally, values of .05 or less indicates a good fit); PCLOSE: p- value of testing the null hypothesis that RMSEA is no greater than .05; AIC: Akaike information criterion; BCC: Browne-Cudeck criterion. Significant P and PCLOSE are indicated as bold, in which the tested model is not fit. 109

Significant PCLOSE indicates that the RMSEA is significantly greater than 0.05, indicating a bad fit. Therefore, non-significant P or PCLOSE means “good” fit of the tested model.). However, the tested model significantly deviated from the saturated model in other environments, which may be because the environmental change not only may change the regression weights of each path, but also the causal relationships among traits (Pigliucci and Kolodynska 2006). Exploring different models that fit best to each environment may complicate the interpretation of expected covariances. In general, our results support the view that native and invasive plants have different strategies for growth and reproduction; native plants flower earlier and invest more biomass to flower production, while invasive plants exhibit an extended period of vegetative growth before flowering to increase height and leaf area (Chun et al. 2007). Invasive plants especially benefit from increased nutrients to produce more branches, biomass, and flowers. This increased amount of nutrients may be produced from disturbances and human activities such as agricultural runoff. Path analysis results indicate that native plants stimulate the production of flowers by flowering early and increasing number of stems, but failed to produce branches and biomass due to the negative effect of flowering phenology. Invasive plants exhibited greater performance in vegetative traits on behalf of the greater positive effects between them. They may have evolved to take more advantage of nutrient resources to accumulate more biomass while maintaining consistent biomass allocation for reproduction. It may help understanding the greater ability of invasive plants utilizing more resources and accumulating invasive potential for aggressive invasion in subsequent growth seasons in North America.

Acknowledgements

We thank M. W. Fiscus for use of Bruner farm facilities. We are also grateful for collecting seeds and helpful comments to all collaborators in 2006 common garden project team. We appreciate P. Dixon and M.-Y. Yum for statistical consulting. C. Swensen, P. T. Alemu, C. W. August, F. Knaus, and H. Chae provided assistance in the field work. This research was funded by EEOB Department at Iowa State University and by CGRER Seed Grant Program at University of Iowa to Y. J. Chun. 110

Literature Cited

Arbuckle, J. L. 2006. Amos (Version 6.0) [Computer Program]. Chicago: SPSS. Bastlová-Hanzélyová, D. 2001. Comparative study of native and invasive plants of Lythrum salicaria L.: population characteristics, site and community relationships. Pages 33-41 in G. Brundu, J. Brock, I. Camarda, L. Child, and M. Wade, editors. Plant invasions: Species ecology and ecosystem management. Backhuys Publishers, Leiden. Bastlová, D., and J. Květ. 2002. Differences in dry weight partitioning and flowering phenology between native and non-native plants of purple loosestrife (Lythrum salicaria L.). Flora 197:332-340. Bedford, B. L, M. R. Walbridge, and A. Aldous. 1999. Patterns in nutrient availability and plant diversity of temperate North American wetlands. Ecology 80(7):2151–2169. Blossey, B., and R. Nötzold. 1995. Evolution of increased competitive ability in invasive nonindigenous plants: A hypothesis. Journal of Ecology 83:887-889. Bossdorf, O., H. Auge, L. Lafuma, W. E. Rogers, E. Siemann, and D. Prati. 2005. Phenotypic and genetic differentiation between native and introduced plant populations. Oecologia 144(1):1-11. Bridgham, S. D., J. Pastor, J. A. Janssens, C. Chapin, and T. J. Malterer. 1996. Multiple limiting gradients in peatlands: a call for a new paradigm. Wetlands 16(1):45-65. Chun, Y. J., M. L. Collyer, K. A. Moloney, and J. D. Nason. 2007. Phenotypic plasticity of native vs. invasive purple loosestrife: a two state multivariate approach. Ecology 88(6):1499-1512. Crespi, B. J., and F. L. Bookstein. 1989. A path-analytic model for the measurement of selection on morphology. Evolution 43:18-28. Edwards, K. R., M. S. Adams, and J. Květ. 1995. Invasion history and ecology of Lythrum salicaria in North America. Pages 161-180 in P. Pyšek, K. Prach, M. Rejmánek, and M. Wade (eds.) Plant Invasions - General Aspects and Special Problems. SPB Academic Publishing, Amsterdam, The Netherlands. Edwards, K. R., M. S. Adams, and J. Květ. 1998. Differences between European native and American invasive populations of Lythrum salicaria. Journal of Vegetation Science 9:267-280. 111

Kingsolver, J. G., and D. G. Schemske. 1991. Analyzing selection with path analysis. Trends in Ecology and Evolution 5:276-280. Lande, R., and S. J. Arnold. 1983. The measurement of selection on correlated characters. Evolution 37:1210-1226. Mal, T. K., J. Lovett-Doust, and L. Lovett-Doust. 1997. Time-dependent competitive displacement of Typha angustifolia by Lythrum salicaria. Oikos 79:26-33. Marshall, D. R., and S. K. Jain. 1968. Phenotypic plasticity of Avena fatua and A. barbata. American Naturalist 102:457-467. Pigliucci, M. 2001. Phenotypic plasticity: beyond nature and nurture. John Hopkins Univ. Press, Baltimore, Maryland, USA. Pigliucci, M., and A. Kolodynska. 2006. Phenotypic integration and response to stress Arabidopsis thaliana: a path analytical approach. Evolutionary Ecology Research 8:415-433. Rejmánek, M, and D. M. Richardson. 1996. What attributes make some plant species more invasive? Ecology 77:1655-1661. Rice, K. J., and R. N. Mack. 1991. Ecological genetics of Bromus tectorum. III. The demography of reciprocally sown populations. Oecologia 88:91-101. Richards, C. L., O. Bossdorf, N. Z. Muth, J. Gurevitch, and M. Pigliucci. 2006. Jack of all trades, master of some? On the role of phenotypic plasticity in plant invasions. Ecology Letters 9:981–993. Scheiner, S. M., and H. S. Callahan. 1999. Measuring natural selection on phenotypic plasticity. Evolution 53(6):1704-1713. Scheiner, S. M., R. J. Mitchell, and H. S. Callahan. 2000. Using path analysis to measure natural selection. Journal of Evolutionary Biology 13:423-433. Stuckey, R. L. 1980. Distributional history of Lythrum salicaria (purple loosestrife) in North America. Bartonia 47: 3-20. Sultan, S. E. and F. A. Bazzaz. 1993. Phenotypic plasticity in Polygonum persicaria. III. The evolution of ecological breadth for nutrient environment. Evolution 47:1050-1071. Thormann, M. N., and S. E. Bayley. 1997. Response of aboveground net primary plant production to nitrogen and phosphorous fertilization in peatlands in southern boreal Alberta, Canada. Wetlands 17(4):502-512. 112 van Kluenen, and M. Fischer. 2005. Constraints on the evolution of adaptive phenotypic plasticity in plants. New Phytologist 166:49-60. Via, S., R. Gomulkiewicz, G. De Jong, S. M. Scheiner, C. D. Schlichting, and P. H. Van Tienderen. 1995. Adaptive phenotypic plasticity: Consensus and controversy. Trends in Ecology and Evolution 10:212-217. Weiher, E., I. C. Wisheu, P. A. Keddy, and D. R. J. Moore. 1996. Establishment, persistence, and management implications of experimental wetland plant communities. Wetlands 16(2):208-218. Williams, D. G., R. N. Mack, and R. A. Black. 1995. Ecophysiology of introduced Pennisetum setaceum on Hawaii: the role of phenotypic plasticity. Ecology 76:1569-1580. 113

Chapter 5. A method for extracting genomic DNA from purple

loosestrife containing high levels of secondary metabolites

Abstract

Generating high quality DNA is a concern for all molecular biologists, especially for those working with material containing high concentration of secondary metabolites. Here we present a simple, efficient, and reliable method for isolation of total DNA from leaves of the highly mucilaginous invasive plant, Lythrum salicaria, as well as selected cactus and cotton species. This method involves a modified CTAB procedure that yields large amounts of high-quality DNA free from contaminating polysaccharides and polyphenols. We demonstrate that the DNA generated with this procedure is suitable for further processing, including digestion with restriction endonucleases, PCR amplification, and amplified fragment length polymorphism (AFLP) analysis.

Keywords: DNA extraction, secondary metabolite, polysaccharides, polyphenols, Lythrum salicaria.

Introduction

A major constraint to the application of molecular techniques in plants is extracting sufficient quantities of high quality DNA. A preponderance of medicinal or aromatic plant species produce secondary metabolites such as alkaloids, phenols, polysaccharides, terpenes, and quinines (Khanuja et al. 1999). Among them, polysaccharides and polyphenols present major contamination problems in the purification of plant DNA. Polysaccharides are particularly challenging to remove because they can co-precipitate with DNA during purification forming highly viscous solutions (Demeke and Adams 1992, Pandey et al. 1996) that are difficult to pipette and often remain in wells during electrophoresis, inhibiting movement of DNA (Sharma et al. 2002). Further, these polysaccharides may be inhibitory to many enzymatic reactions used in molecular 114 techniques (Fang et al. 1992, Crowley et al. 2003, Angeles et al. 2005). Polyphenols too are problematic because oxidized forms covalently bind to DNA and proteins, giving the DNA a brown color and making it useless for certain research applications (Alijanabi et al. 1999). A rapid and effective DNA extraction method for plants with high polysaccharide and polyphenol content is necessary because high-quality DNA is required for PCR and restriction-based techniques such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP), as well as microsatellite analysis (Alijanabi et al. 1999). Doyle and Doyle’s (1990) traditional CTAB method for DNA extraction from plants includes a number of chemicals utilized to increase DNA quality. This method has subsequently been modified for plants containing high amounts of polysaccharides and polyphenols, usually with additional steps incorporating detergents, salts, and enzymes. We have summarized many of these modifications to the original CTAB method in Table 1, indicating both their potential advantages and drawbacks. In some cases, they require additional chemicals and labor that may increase cost and time of the extraction. Also, additional purification steps, requiring differential precipitation or extraction, increase the likelihood of DNA loss. Thus, protocols that can successfully remove contaminants from genomic DNA in as few steps as possible are most desirable (Friar 2005). The purpose of this study was to develop an optimized method for acquiring high- purity DNA for AFLP analysis of Lythrum salicaria (purple loosestrife), a plant containing high levels of polysaccharides and polyphenols. L. salicaria is a well-known, aggressive invader of wetlands in North America. In addition to incidental introduction, it has been deliberately brought in for medicinal, horticultural, and beekeeping purposes (Thompson et al. 1987). Though numerous studies have examined the reproductive biology and physiological ecology of L. salicaria, as well as its biocontrol, few have adopted a molecular approach, in part because high concentration of polysaccharides and polyphenols have made it difficult to efficiently isolate high quality DNA. Standard DNA extraction techniques (e.g., Doyle and Doyle’s [1990] CTAB method and Qiagen plant DNeasy plant mini kits) are not effective for L. salicaria and to date the only extraction published for this species (Houghton-Thompson 2001, Houghton-Thompson et al. 2005) used a modified CTAB method using ethanol and sodium acetate instead of isopropanol 115

Table 1. Major contaminants of DNA extraction (polysaccharides, polyphenols, proteins, and RNA), chemicals used to remove them, advantages and/or drawbacks of using these chemicals, and references describing their use in modifications of Doyle and Doyle’s (1990) CTAB method. Comtaminant Chemical Advantages and/or Drawbacks References Is a strong detergent. One-hour incubation Al-Shayji et al. 1994 Peterson et al. 1997 is needed before SDS treatment because Jobes et al. 1995 Dixit 1998 SDS (sodium dodecyl adding SDS directly to the extraction media de la Cruz et al. 1997 Angeles et al. 2005 sulfate) can lead to premature lysis of the cell, Kim et al. 1997 Xu et al. 2005 interfering with tissue dispersal and shearing of the DNA (Jobes et al. 1995). Is a strong detergent used along with Murray and Thompson Sharma et al. 2002 Sarcosyl CTAB, but not necessary because CTAB is 1980 Crowley et al. 2003 (N-lauroyl sarcosine) a strong detergent. Aljanabi et al. 1999 Polysaccharides Tel-Zur et al. 1999 The addition of NaCl with CTAB is known Fang et al. 1992 Porebski et al. 1997 to increase the solubility of polysaccharides Lodhi et al. 1994 Khanuja et al. 1999 High concentration of in ethanol to remove them (Fang et al. Wang et al. 1996 Tel-Zur et al. 1999 NaCl 1992, Paterson et al. 1993). Because high (3-5M compared to concentrations of salt may lower the Doyle and Doyle’s A /A ratio (Manchester 1995) and 1.4M) 260 280 inhibit enzyme activity, additional steps are needed to wash salt out of the DNA. CsCl CsCl density-gradient purification of DNA Murray and Thompson Richards et al. 1994 (cesium chloride) is relatively tedious and time-consuming. 1980 Polysaccharides / Is a detergent-like chemical used along with Dixit 1998 Syamkumar et al. 2005 Proteins PEG CTAB, but not necessary because CTAB is Crowley et al. 2003 (polyethylene glycol) a strong detergent. Phenol denatures and removes proteins but Peterson et al. 1997 Cheng et al. 2003 it is hazardous chemical and may be Porebski et al. 1997 Michiels et al. 2003 omitted without affecting the yield and Aljanabi et al. 1999 Syamkumar et al. 2005 quality of DNA (Dixit 1998). If not Tel-Zur et al. 1999 Phenol removed completely after use, residual phenol inhibits restriction enzymes and Proteins reduces the efficiency of PCR and sequencing (Hiesinger et al. 2001). Is expensive and not necessary when Callahan and Mehta Peterson et al. 1997 chloroform:isoamyl alcohol (24:1) 1991 Porebski et al. 1997 Proteinase K denatures proteins and remove them. Al-Shayji et al. 1994 Jobes et al. 1995 High concentration of Wang et al. 1996 Pirttilä et al. 2001 β-mercaptoethanol Tel-Zur et al. 1999 Puchooa and Khoyratty (1-5% compared to 2004 Doyle and Doyle’s 0.2%) All are strong antioxidants of polyphenols Sodium sulfite or Aljanabi et al. 1999 Puchooa and Khoyratty and can be used as alternatives or sodium metabisulfite 2004 supplements of β-mercaptoethanol. High Al-Shayji et al. 1994 Geuna et al. 2004 DTT (dithiothreitol) concentration of β-mercaptoethanol up to Jobes et al. 1995 Polyphenols 5% is more effective (Puchooa and Khoyratty Jobes et al. 1995 Aljanabi et al. 1999 2004) Wang et al. 1996 Khanuja et al. 1999 PVP (polyvinyl Kim et al. 1997 Pirttilä et al. 2001 pyrrolidone) Peterson et al. 1997 Michiels et al. 2003 Porebski et al. 1997 Puchooa and Khoyratty 2004 Is a water-insoluble and yields less DNA Lodhi et al. 1994 Angeles et al. 2005 PVPP (polyvinyl than water-soluble PVP (Porebski et al. Geuna et al. 2004 polypyrrolidone) 1997). Selectively precipitates RNA and has an Jobes et al. 1995 Geuna et al. 2004 advantage over RNase treatment because Pirttilä et al. 2001 incompletely digested RNA can contribute RNA LiCl (lithium chloride) to the misreading in spectrophotometer or serve as primers in subsequent PCR (Storts 1993). 116 and ammonium acetate, and omitting RNase step. This procedure required large amounts of leaf tissue and downstream AFLP analysis yielded relatively few informative bands per primer pair (Houghton-Thompson et al. 2005). We desired instead a reliable mini- prep method that would provide good yields of high quality DNA and that would more support more efficient collection of AFLP polymorphism data. Therefore, to facilitate molecular genetic analysis of L. salicaria, an optimized DNA extraction protocol was developed. Importantly, we demonstrate the utility of this protocol for extracting DNA from other challenging plant taxa including cactus (Cylindropuntia imbricata, Opuntia chlorotica, and Opuntia ficus-indica) and cotton (Gossypium barbadense) species containing high levels of polysaccharides and polyphenols (Paterson et al. 1993, de la Cruz et al. 1997). This protocol is a modification of Doyle and Doyle’s (1990) CTAB method, specifically including an additional centrifugation step after incubation with CTAB buffer, using a high concentration of β-mercaptoethanol and PVP to remove polyphenols, and eliminating the use of phenol and unnecessary washing steps, as described below.

Materials and Methods

Plant materials We used 60 mg of fresh leaf tissue per sample from L. salicaria and cotton. Though we successfully isolated DNA from the mature leaves of L. salicaria, young leaf tissue is preferable since it may contain fewer polyphenolic and terpenoid compounds than older tissue (Rosenthal and Janzen 1979). For the cacti, all leafless stem succulents, we used epidermal tissue to avoid high concentrations of polysaccharides located in the stem cortex.

Reagents and chemicals CTAB extraction buffer (modified from Doyle and Doyle [1990]) 100mM Tris-HCl, pH 8.0 20mM EDTA (ethylenediaminetetraacetic acid), pH 8.0 1.4M NaCl 117

2% (v/v) β-mercaptoethanol* 2% (w/v) PVP (polyvinylpyrrolidone), Mol. Wt. 40,000* 2% (w/v) CTAB (hexadecyltrimethylammonium bromide)* * β-mercaptoethanol, PVP, and CTAB should be added just before use. Chloroform:isoamyl alcohol (24:1) 100% isopropanol 100% ethanol RNase (10 mg/ml)

Equipment Small mortars and pestles, two heating blocks, microcentrifuge, 1.6 ml microcentrifuge tubes, liquid nitrogen, and ice.

Pre-isolation preparation (at least 30 minutes beforehand) 1. Set one heating block to 65°C to preheat CTAB extraction buffer and sterile distilled water. Set the other heating block to 37°C. 2. Place mortars and pestles in -20°C freezer and isopropanol and ethanol in 4°C refrigerator.

DNA isolation procedure 1. Prepare 700 μl of extraction buffer for each extraction. With a mortar and pestle, grind 60 mg of fresh tissue in liquid nitrogen, to fine powder. Immediately add the powdered tissue to the 700 μl of extraction buffer in a 1.6 ml microcentrifuge tube and vortex to mix thoroughly. 2. Incubate the sample at 65°C for 30 minutes with occasional mixing to avoid aggregation of the homogenate. Centrifuge at 13,000 rpm for 5 minutes to precipitate polysaccharides, then transfer the supernatant to a new microcentrifuge tube. 3. Add 700 μl of chloroform:isoamyl alcohol (24:1) and mix by inverting tube. Centrifuge at 13,000 rpm for 5 minutes, transfer the upper phase to a new microcentrifuge tube excluding green and white layers. Discard lower phase. 4. Add 400 μl of 4°C isopropanol and mix gently by inverting the tube at least 50 times. 118

The tube should not be shaken vigorously because the DNA is very vulnerable to fragmentation at this step. Incubate at -20°C for 30 minutes to precipitate nucleic acids then centrifuge at 13,000 rpm for 10 minutes to pellet DNA. Carefully pipette off the supernatant without losing the pellet and let the pellet air-dried (place tubes below hood door for 15 minutes to dry out remaining liquid completely). Dissolve the pellet with 200 μl of 37°C water. 5. Add 400 μl of 4°C ethanol and carefully invert the tube at least 50 times until white DNA fibers appear. Incubate at -20°C for 30 minutes then centrifuge at 13,000 rpm for 10 min. Carefully remove the supernatant without losing the pellet and let the pellet air-dry (as above). Add 100 μl of 37°C water and 1 μl of RNase. Mix by finger-vortexing to dissolve DNA completely and incubate at 37°C for 1 hr to break down RNA.

Examination of DNA quantity and quality

DNA concentration and purity was determined by A260/A280 and A260/A230 ratios (measures of contamination by proteins and polysaccharides, respectively) using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, 2006). DNA samples were electrophoresed on a 1% agarose gel to see if the extracted DNA was intact.

AFLP analysis Approximately 200 ng of genomic DNA per sample was digested with EcoR I and Mse I restriction enzymes, ligated to the corresponding adaptors, and subjected to pre- amplification and selective amplification according to the original protocol of Vos et al. (1995) with Eco-AAC and Mse-CAA primers. Amplification products were detected by labeling the EcoR I primer with a fluorescent dye (HEX) and genotyped using the ABI 3100 Genetic Analyzer at the DNA facility of Iowa State University.

Results

The method developed for DNA extraction from L. salicaria resulted in consistent DNA yields (mean 387 μg of DNA per gram of leaf material; Table 2). We obtained 119 approximately the same yields of DNA from cotton and half that amount from cacti.

A260/A280 and A260/A230 ratios indicated high quality DNA and minimal or no protein or polysaccharide contamination in the samples. A peak at 260 nm was consistently observed for all samples and no contamination-associated peaks were noted at higher or lower wavelengths.

Table 2. Mean (±SE) of DNA yield and A260/A280 and A260/A230 ratios, from purple loosestrife, cactus, and cotton species (n: number of individual samples).

DNA yield (μg / g plant material) A260/A280 A260/A230 Purple loosestrife (n = 173) 386.88 ± 13.14 2.031 ± 0.005 1.804 ± 0.020 Cactus (n = 23) 176.20 ± 13.73 1.932 ± 0.041 1.660 ± 0.118 Cotton (n = 23) 381.15 ± 37.65 2.055 ± 0.013 2.077 ± 0.066

Fig. 1. Genomic DNA from purple loosestrife, cactus, and cotton Fig. 2. AFLP fingerprint profile of purple species using electrophoresis in a TAE-buffered, 1% agarose gel loosestrife, cactus and cotton species. Lanes 1-4: with 1kb DNA ladder. Lanes 1 and 10: 1kb DNA ladder, Lanes 2- Lythrum salicaria, Lanes 5-7: cactus 5: Lythrum salicaria, Lanes 6-8: cactus (Cylindropuntia imbricata, (Cylindropuntia imbricata, Opuntia chlorotica, Opuntia chlorotica, and O. ficus-indica), Lane 9: cotton and O. ficus-indica), Lane 8: cotton (Gossypium (Gossypium barbadense). barbadense). 120

Agarose gel electrophoresis of extracted DNA showed conspicuous bands of high molecular weight DNA (~50 kb) with minimal degradation and no visible RNA contamination (Fig. 1). After selective AFLP amplification, agarose gel examination indicated smears of amplified fragments in the range of 100 - 500 bp. For a single pair of +3 primers, we were able to score numerous AFLP fragments in each species (Fig. 2), suitable for genotyping and inferring genetic distances between individuals.

Discussion

We modified the DNA extraction procedure of Doyle and Doyle (1990) and associated protocols to obtain high-quality DNA in relatively few steps. The detailed characteristics and advantages of our protocol are summarized as follows: The first step in the DNA extraction process involves breaking or digesting away cell walls to release the cellular constituents. This is followed by disruption of cell membranes to release DNA into the extraction buffer, which is achieved by using the detergent cetyltrimethylammonium bromide (CTAB). The released DNA should be protected from endogenous nucleases. For this purpose, EDTA is included in the extraction buffer to chelate magnesium ions, a necessary cofactor for nucleases. Initial DNA extracts often contain large amounts of polysaccharides, polyphenols, proteins, pigments, and RNA, interfering with restriction and PCR of DNA. A high concentration (up to 2.5 M) of NaCl is known to increase the solubility of polysaccharides in ethanol, effectively decreasing co-precipitation of the polysaccharides and DNA (Fang et al. 1992, Paterson et al. 1993). As in Doyle and Doyle’s (1990) protocol, a NaCl concentration of 1.4 M was effective in removing polysaccharides in our protocol. Precipitating DNA in ethanol has been shown to increase the yield and purity of DNA (Rogers and Bendich 1985). Although Doyle and Doyle (1990) used 0.2% β-mercaptoethanol and 1% of PVP to prevent oxidation of polyphenols and other researchers use sodium (meta)bisulfite, DTT, or PVPP for the same purpose (Al-shayji et al. 1994, Aljanabi et al. 1999, Geuna et al. 2004), we found that high-concentrations of β-mercaptoethanol and PVP (both 2%) effectively eliminated polyphenols (Table 1). 121

While the original CTAB protocol (and others) extracts DNA after treating with CTAB buffer and chloroform:isoamyl alcohol (24:1) in one step, our protocol added a centrifugation step after incubation with CTAB buffer and before addition of chloroform:isoamyl alcohol (24:1). This centrifugation step precipitated polysaccharides and polyphenols effectively, leaving a homogeneous supernatant that was easy to pipette. This step proved useful in removing a majority of the polysaccharides and proteins and made the extraction step with chloroform:isoamyl alcohol (24:1) more effective in clearing away residual contaminants and pigments. Our protocol also employed an RNase step to remove RNA in the final step of the DNA extraction procedure. Although Doyle and Doyle (1990) used 30 minutes of incubation with RNase, Porebski et al. (1997) suggested that it takes one hour to completely degrade RNA into small ribonucleosides. Examination of agarose gels (Fig. 1) indicates that RNA was completely degraded after one-hour incubation. Selective precipitation of RNA has also been achieved using high molar LiCl (Table 1), however, in our experience excessive lithium salts were precipitated with DNA, requiring additional washing steps. Our protocol importantly eliminates the use of phenol in removing proteins. Not only is phenol hazardous, its use after isopropanol precipitation shifts the spectrophotometric peak to 270 nm, lowering the A260/A280 ratio. This shift is caused by residual phenol, which is known to have an intense absorbance at 270 nm (Manchester 1995). Moreover, the use of phenol in DNA extraction has potential downstream disadvantages, including the inhibition of restriction enzymes leading to incomplete digestion in AFLP analysis and reduction in efficiency of PCR and sequencing (Hiesinger et al. 2001). As described above, we were able to effectively precipitate polysaccharides and polyphenols from extracts by adding a centrifugation step. At the subsequent chloroform:isoamyl alcohol (24:1) step, we effectively removed proteins, pigments, and other contaminants. Since we obtained a high A260/A230 ratio, our protocol effectively removed proteins without the use of phenol. Overall, compared to original CTAB and related procedures, by removing unnecessary steps we saved time and effort while maintaining good DNA yield. While these other procedures include washing steps with sodium acetate or ammonium acetate, 122 thereby increasing the risk of DNA loss, such additional steps were not needed in our method since the first two extraction steps were effective in removing contaminants. A benefit of our modified CTAB method is that it does not require expensive chemicals, enzymes, and equipment, or increased sample handling time. Costs are 20-22 times less with our method ($0.14 per sample) than using commercial kits (e.g. Qiagen DNeasy plant mini kit), and processing times are similar: our method takes 6-7 hours to complete DNA extraction of 10-20 samples. During subsequent AFLP procedures, preselective and selective amplification produced excellent-quality DNA fingerprints with consistent amplification and a high number of scorable peaks. This cost-effective extraction procedure thus successfully removes polysaccharides and other contaminants from L. salicaria, cactus, and cotton, while producing genomic DNA suitable for restriction enzyme digestion and PCR. Given the difficulties these plant species have posed for other DNA extraction methods, we expect the procedure described here to be effective for obtaining high quality DNA from other particularly recalcitrant plant species, especially those containing high concentrations of polysaccharides and polyphenols.

Acknowledgements

We are grateful to Ryan Percifield for thoughtful comments and suggestions. We also thank Dr. Wendel for access to his laboratory and Nanodrop Spectrophotometer.

Literature Cited

Aljanabi, S. M., L. Forget, and A. Dookun. 1999. An improved and rapid protocol for the isolation of polysaccharide- and polyphenol-free sugarcane DNA. Plant Molecular Biology Reporter 17:1-8. Al-Shayji, Y., M. Saleem, S. Al-Amad, S. Al-Awadhi, and F. Al-Salameen. 1994. Isolation and analysis of the total genomic DNA from the date palm (P. dactylifera L.) and related species. Acta Biotechnologica 14(2):163-168. 123

Angeles, J. G., A. C. Laurena, and E. M. Tecson-Mendoza. 2005. Extraction of genomic DNA from lipid-, polysaccharide-, and polyphenols-rich coconut (Cocos nucifera L.). Plant Molecular Biology Reporter 23:297a-297i. Callahan, F. E., and A. M. Mehta. 1991. Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.). Plant Molecular Biology Reporter 9(3):252-261. Cheng, Y.-J., W.-W. Guo, H.-L. Yi, X.-M. Pang, and X. Deng. 2003. An efficient protocol for genomic DNA extraction from Citrus species. Plant Molecular Biology Reporter 21:177a-177g. Crowley, T. M., M. S. Muralitharan, and T. W. Stevenson. 2003. Isolating Conifer DNA: a superior polysaccharide elimination method. Plant Molecular Biology Reporter 21:97a-97d. de la Cruz, M., F. Ramirez, and H. Hernandez. 1997. DNA isolation and amplification from Cacti. Plant Molecular Biology Reporter 15:319-325. Demeke, T., R. P. Adams. 1992. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12(3):332-334. Dixit, A. 1998. A simple and rapid procedure for isolation of Amaranthus DNA suitable for fingerprint analysis. Plant Molecular Biology Reporter 16:1-8. Doyle, J. J., and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12:13-15. Fang, G., S. Hammar, and R. Rebecca. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques 13:52-56. Friar, E. A. 2005. Isolation of DNA from plants with large amounts of secondary metabolites. Methods in Enzymology 395:1-12. Geuna, F., C. Maitti, S. Digiuni, and R. Banfi. 2004. A method for extracting genomic DNA suitable for medium-throughput applications from plant tissues in contaminants. Plant Molecular Biology Reporter 22:87a-87f. Hiesinger, M., D. Löffert, C. H. Ritt, and U. Oelmüller. 2001. The effects of phenol on nucleic acid preparation and downstream applications. Qiagen News 5:23-26. 124

Houghton-Thompson, J. 2001. Importance of hybridization and ecological differentiation in the success of Lythrum salicaria in North America. Ph. D. dissertation. Michigan State University. MI, USA. Houghton-Thompson, J., H. H. Prince, J. J. Smith, and J. F. Hancock. 2005. Evidence of hybridization between Lythrum salicaria (purple loosestrife) and L. alatum (winged loosestrife) in North America. Annals of Botany 96:877-885. Jobes, D. V., D. L. Hurley, and L. B. Thien. 1995. Plant DNA isolation: a method to efficiently remove polyphenolics, polysaccharides, and RNA. Taxon 44(3):379-386. Khanuja, S. P. S., A. K. Shasany, M. P. Darokar, and S. Kumar. 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Molecular Biology Reporter 17:1-7. Kim, C. S., C. H. Lee, J. S. Shin, Y. S. Chung, and N. I. Hyung. 1997. A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nucleic Acids Research 25(5):1085-1086. Lodhi, M. A., G. N. Ye, N. F. Weeden, and B. I. Reisch. 1994. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter 12:6-13.

Manchester, K. L. 1995. Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 19(2):208-210. Michiels, A., W. Van den Ende, M. Tucker, L. Van Riet, and A. Van Laere. 2003. Extraction of high quality genomic DNA from latex-containing plants. Analytical Biochemistry 315:85-89. Murray, M. G., and W. F. Thompson. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8:4321-4325. NanoDrop Technologies, 2006. ND-1000 Spectrophotometer V3.3 User’s Manual. NanoDrop Technologies, Inc. Wilmington, Delaware, USA. Pandey, R. N., R. P. Adams, L. E. Flournoy. 1996. Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides. Plant Molecular Biology Reporter 14:17-22. 125

Paterson, A. H., C. L. Brubaker, and J. F. Wendel. 1993. A rapid method for extraction of cotton (Gossypium spp) genomic DNA suitable for RFLP or PCR analysis. Plant Molecular Biology Reporter 11:122-127. Peterson, D. G., K. S. Boehm, and S. M. Stack. 1997. Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), a plant containing high levels of polyphenolic compounds. Plant Molecular Biology Reporter 15(2):148- 153. Pirttilä, A. M., M. Hirsikorpi, T. Kämäräinen, L. Jaakola, and A. Hohtola. 2001. DNA isolation methods for medicinal and aromatic plants. Plant Molecular Biology Reporter 19:273a-f. Porebski, S., L. G. Bailey, and B. R. Baum. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenols components. Plant Molecular Biology Reporter 15(1):8-15. Puchooa, D., and S.-U. S. S. Khoyratty. 2004. Genomic DNA extraction from Victoria amazonica. Plant Molecular Biology Reporter 22:195a-195j. Richards, E., M. Reichardt, and S. Rogers. 1994. Preparation of genomic DNA from plant tissue. Current Protocols in Molecular Biology. Wiley Interscience. Rogers, S. O., and A. J. Bendich. 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Molecular Biology Reporter 19:273a-273f. Rosenthal, G. A., and D. H. Janzen (eds.). 1979. Herbivores, their interaction with secondary plant metabolites. New York. Sharma, A. D., P. K. Gill, and P. Singh. 2002. DNA isolation from dry and fresh samples of polysaccharide-rich plants. Plant Molecular Biology Reporter 20:415a-415f. Syamkumar, S., M. Jose, and B. Sasikumar. 2005. Isolation and PCR amplification of genomic DNA from dried capsules of Cardamom (Elettaria cardamomum M.). Plant Molecular Biology Reporter 23:1a-1e. Tel-Zur, N., S. Abbo, S. Myslabodski, and Y. Mizrahi. 1999. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Molecular Biology Reporter 17:249-254. 126

Thompson, D. Q., R. L. Stuckey, and E. B. Thompson. 1987. Spread, Impact, and Control of Purple Loosestrife (Lythrum salicaria) in North American Wetlands. US Fish and Wildlife Service. 55 pages. Jamestown, ND: Northern Prairie Wildlife Research Center Home Page. http://www.npwrc.usgs.gov/resource/1999/loosstrf/loosstrf.htm (Version 04JUN99). Vos, F. R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new concept for DNA fingerprinting. Nucleic Acids Research 23:4407-4414. Wang, X.-D., Z.-P. Wang, and Y.-P. Zou. 1996. An improved procedure for isolation of nuclear DNA from leaves of wild grapevine dried with silica gel. Plant Molecular Biology Reporter 14(4):369-373. Xu, X., S. Kawasaki, T. Fujimura, C. Wang. 2005. A protocol for high-throughput extraction of DNA from rice leaves. Plant Molecular Biology Reporter 23:291-295. 127

Chapter 6. Quantitative and molecular genetic variation of

native vs. invasive purple loosestrife populations

Abstract

Adaptive evolutionary changes in populations of invasive species can be advanced by the joint application of quantitative and population genetic methods. Using purple loosestrife as a model system, we investigated the relative roles of natural selection versus genetic drift and gene flow by examining phenotypic and genetic differentiation between native European populations and invasive North American populations. In addition, we examined if invasive populations have locally adapted through directional selection on fitness-related traits, which was accomplished by contrasting population divergence in quantitative traits with neutral genetic variation among populations. Our results indicate that invasive populations harbor substantial genetic variation not less than native populations, consistent with multiple independent introductions into North Ameica from a diverse European gene pool. Quantitative genetic analysis suggests significant directional selection for number of flowers, which may have provided the basis for local adaptation and high population fitness for invasive purple loosestrife populations in North America.

Keywords: invasive species, Lythrum salicaria, purple loosestrife, AFLP, genetic variation, quantitative trait, ecological genetics.

Introduction

Elucidating mechanisms by which populations differentiate at different geographical and temporal scales is a primary theme in evolutionary biology. Of special interest are processes influencing levels of genetic variation and the role of natural selection in shaping heritable variation into adaptive differences among populations. Although phenotypic differences between populations are often assumed to be adaptive, 128 both molecular and quantitative variation can be influenced by non-adaptive processes involving random genetic drift and gene flow (Wright 1931, Hartl and Clark 1989, Falconer and Mackay 1996). Founder effects, bottlenecks, and admixture, in particular, can have strong impacts on the distribution of phenotypic variation within and among populations (Lande 1980, Lynch et al. 1999). Under these conditions it can be especially challenging and interesting to determine whether heritable phenotypic differences between populations represent the outcomes of adaptive or non-adaptive processes. Invasive species provide excellent systems for investigating the relative roles of natural selection versus genetic drift and gene flow in population differentiation. Invasive populations of non-native species are having major negative impacts on natural and human-managed landscapes (Vitousek et al. 1996). These populations are expected to be under strong selection to adapt to environmental conditions of the invasive range and, though often relatively recently introduced, have frequently been found to possess genetically-based phenotypic differences from populations in the ancestral range (Baker and Stebbins 1965, Baker 1974, Sakai et al. 2001), indicative of rapid evolutionary change (Lee 2002). While changes in size and fecundity may be adaptive, they may also reflect high rates of genetic drift associated with founding events and post-colonization bottlenecks (Brown and Marshall 1981, Le Page et al. 2000, Lee 2002, DeWalt and Hamrick 2004). Multiple independent introductions from genetically diverse sources and subsequent admixture among them can also occur (Warwick et al. 1987, Novak and Mack 1993, Neuffer and Hurka 1999, Pappert et al. 2000, Meekins et al. 2001, Bartlett et al. 2002, Maron et al. 2004, Durka et al. 2005). Such admixture may increase genetic diversity (Novak and Mack 1993, Amsellem et a. 2000, DeWalt and Hamrick 2004) and so enhance the ability of populations to adapt to and potentially to radiate into novel environments (e.g., Sakai et al. 2001), directly contributing to significant phenotypic differentiation between populations from the invasive and native range by spurring the formation of novel recombinant genotypes (e.g., Hedge et al. 2006). A research program combining ecological and genetic perspectives can potentially distinguish the relative contributions of adaptive and non-adaptive processes to population differentiation. Population genetic methods can be effective for identifying influential features of invasion history including bottlenecks, independent introduction 129 events, and subsequent intermingling of populations during invasive spread (Cruzan 1998, Durka et al. 2005). The adaptive nature of phenotypic differentiation among populations, in turn, can be tested relative to a neutral model of quantitative genetic divergence, where neutral genetic variation is used as the null expectation for the amount of differentiation among populations in quantitative variance (Spitze 1993, Whitlock 1999). This can be achieved by contrasting neutral genetic variation among populations (FST) with its analog for quantitative genetic traits (QST)(Spitze 1993, Whitlock 1999, McKay and Latta 2002).

FST reflects population differentiation due to non-adaptive processes including genetic drift, migration, and mutation, while QST may reflect the action of natural selection, as well as these non-adaptive processes (Spitze 1993). If a quantitative trait is selectively neutral and exhibits a purely additive genetic basis and linkage equilibrium among underlying loci, QST has the same expectation as FST since the trait is affected only by random genetic drift, migration, and mutation (Merilä and Crnokrak 2001). Alternatively, if trait variation is not neutral, then QST ≠ FST with the direction of the difference depending on the form of selection (Reed and Frankham 2001). Stabilizing selection for a common optimal phenotype can homogenize variation across populations resulting in

QST < FST, whereas disruptive selection in response to different local optima results in

QST > FST (Crnokrak and Merilä 2002, McKay and Latta 2002).

The analysis of QST-FST relationship thus provides a useful tool for investigating the role of adaptive versus non-adaptive processes in the spread of invasive species. When introduced populations experience new local selection pressures favoring alternative genotypes that are appropriate to the different local environments, they are expected to diverge and produce genetically distinct, locally specialized ecotypes. Thus, with ample additive genetic variation created by multiple introduced genotypes and strong selection, local adaptation is expected to be an important mechanism for rapid spread into novel environments (Sakai et al. 2001, Sexton et al. 2002). Non-adaptive processes too can promote invasion, including benefits of increased heterozygosity (Mitton 1993), genetic reorganization following bottlenecks (Goodnight 1995), and release from native predators, parasites, and competitors (Maron and Vilá 2001, Mitchell and Power 2003, Reinhart and Callaway 2006). The goal of this study is to investigate the relative importance of different 130 population genetic processes and phenotypic adaptation in the invasive spread of purple loosestrife (Lythrum salicaria L., Lythraceae) in North America. Introduced from Europe in the early 1800’s (Thompson et al. 1987), this aggressive invader is having significant negative impacts including reduction in plant biodiversity and quality of wildlife habitat as well as alteration of wetland function (Blossey et al. 2001). Research on this species has documented phenotypic differences between invasive North American vs. native European populations (e.g., Edwards et al. 1998, Bastlová and Květ 2002) with some differences shown to have a significant genetic basis (Chun et al. 2007). Nevertheless, the relative impact of adaptive and non-adaptive processes to this differentiation has not been tested. Towards this end, this paper specifically addresses the following questions: (1) Do purple loosestrife populations from the invasive provenance exhibit spatial patterns and levels of neutral variation that, relative to native populations, are indicative of single or multiple founding events, bottlenecks, or population admixture? (2) For which fitness- related traits do invasive populations exhibit significant genetically-based differentiation from native populations? (3) For which of these traits is the differentiation between invasive and native provenances adaptive or neutral? (4) For adaptive differences, is the overall pattern of phenotypic variation indicative of disruptive or stabilizing selection?

Methods

Seed collection and germination To contrast native vs. invasive populations, we examined three European regions (native provenance) and three North American regions (invasive provenance). The three study regions in North America were chosen to reflect different stages in the invasion history of purple loosestrife, with New Jersey (NJ) populations as the oldest (many dated to before 1900), Michigan (MI) populations being of intermediate age (many appearing from the 1900’s through the 1940’s), and Iowa (IA) populations being the youngest (established after the 1940’s) (Stuckey 1980, Edwards et al. 1995). Three populations from each region were chosen to represent a range of nutrient and moisture conditions found naturally in the field (Table 1). From each population, we collected seeds from 20 plants separated by at least 3 meters to ensure they were distinct genets. Seeds were 131

Table 1. Geographical position of sampling sites with genetic diversity in parenthesis for each hierarchical geographical level. Provenance Region Population ID Latitude Longitude Number of total / private loci Boone Folks (0.140) IA BF 42°17Ń 93°56´W 915/ 3 Iowa Little South Storm Lake (0.150) IA LS 42°38Ń 95°14´W 915 / 5 (0.176) Manly (0.06) IA MA 43°16Ń 93°07´W 542 / 0 Kellogg Biological Station (0.158) MI KB 42°21Ń 85°21´W 1045 / 3 North America Michigan Lake Lansing (0.156) MI LL 42°46Ń 84°23´W 1015 / 9 (0.204) (0.184) Rifle Range near Pittsford (0.122) MI RR 41°51Ń 84°30´W 810 / 1 Beaver Run (0.175) NJ BR 41°09Ń 74°36´W 1131 / 14 New Jersey Hainville County Store (0.168) NJ HV 41°15Ń 74°48´W 1102 / 5 (0.193) Walkill River (0.171) NJ WR 41°03Ń 74°37´W 1110 / 7 Golm (0.146) PD GO 52°25Ń 12°57É 1032 / 6 Potsdam Grube (0.163) PD GR 52°27Ń 12°57É 1113 / 2 (0.184) Potsdam (0.153) PD PO 52°29Ń 12°57É 1096 / 2 Altmatt (0.179) SW AL 47°20Ń 07°52É 1141 / 16 Europe Switzerland North Sihl Lake (0.151) SW NO 47°36Ń 08°13É 1037 / 0 (0.193) (0.188) Steinbode (0.156) SW ST 47°09Ń 08°43É 1068 / 10 Hagelloch Tobel (0.154) TU HA 48°32Ń 09°01É 1065 / 6 Tübingen Reusten "Hinterer See" (0.160) TU RE 48°33Ń 08°55É 1106 / 15 (0.175) Unterjesingen "Wiesbrunn" (0.152) TU UN 48°31Ń 08°58É 1036 / 4 obtained from at least 1,000 capsules per plant and pooled into maternal families at the individual plant level. Given that plants are obligately outcrossing (trystylous; Ågren and Ericson 1996) and populations are large, seeds within maternal families were considered to be half-sibs. Seeds from each half-sib family were planted in the Bessey greenhouse at Iowa State University on May 2, 2006. After 28 days of growth, we selected eight half- sib families from each population for AFLP analysis, five of which were selected for a common garden experiment.

DNA extraction and AFLP analysis AFLP markers are presumably neutral and have found wide application in analysis of genetic variation below the species level, particularly in investigations of population structure and differentiation (Mueller and Wolfenbarger 1999). Leaves were obtained from one young plant sampled from each of the eight half-sib families from the 18 study populations (total sample size of 144). We extracted genomic DNA from loosestrife using the CTAB method (Doyle and Doyle 1990) with modification to minimize polysaccharides and other contaminating materials (Chapter 5). AFLP procedures were performed essentially following the protocol of Vos et al. (1995) with only minor modification. Genomic DNA (~200 ng) was digested with 10 units each of EcoRI and MseI, incubating at 37ºC for three hours. Double-stranded 132 adaptors were prepared from the following complementary single-stranded oligonucleotides: 5’ CTC GTA TAC TGC GTA CC 3’ (forward) and 5’ AAT TGG TAC GCA GTA 3’ (reverse) for the EcoRI adapter pair, and 5’ GAC GAT GAG TCC TGA G 3’ (forward) and 5’ CTA CTC AGG ACT CAT 3’ (reverse) for the MseI adapter pair. Ligation reactions were performed by adding 75 pmoles each of the EcoRI adapter and MseI adapter, and 20 units of T4 DNA ligase with its buffer to the digested product, and incubating overnight at 16ºC. For the pre-selective polymerase chain reaction (PCR), we added 10 μl of the ligation product to 40 μl of a pre-selective PCR mix consisting of: 13

μl dH2O, 5 μl 10X PCR buffer, 1.5 μl MgCl2 (50 mM), 4 μl dNTP (2.5 mM), 8 ml (5 pmol/μl) of each pre-selective primer, and 0.5 μl of Taq DNA polymerase (5 U/μl). Sequences of pre-selective primers are: EcoRI +A: 5’ TAC TGC GTA CCA ATT CA 3’, and MseI +C: 5’ GAC GAT GAG TCC TGA GTA AC 3’. Pre-selective PCR conditions were a preliminary 75ºC extension for 2 min followed by 20 cycles of 94ºC for 30 sec, 56ºC for 30 sec, 75ºC for 2 min, finishing with one cycle of 60ºC for 30 min. Five μl of this PCR product were electrophoresed through 1% TAE-agarose gels and stained with ethidium bromide to verify adequate pre-selective amplification. The remaining 45 μl were diluted with 180 μl dH2O. For the selective PCR, we added 5 μl of diluted pre- selective PCR product to 20 μl of the selective PCR mix consisting of 11.5 μl dH2O, 2.5

μl 10X PCR buffer, 0.75 μl MgCl2 (50 mM), 3 μl dNTP (2.5 mM), 0.75 μl (5 pmol/μl) each of two EcoRI labeled (6-FAM and HEX) selective primers, 0.5 μl (50 pmol/μl) of one MseI unlabeled primer, and 0.25 μl of Taq DNA polymerase (5 U/μl). We performed selective PCR with four primer pairs: EcoRI +AGC (6-FAM), EcoRI +ACG (HEX), EcoRI +ACA (6-FAM), and EcoRI +AAC (HEX), each paired with MseI +CAA. The PCR profile was one cycle of 94ºC for 2 min, one annealing cycle of 94ºC for 30 sec, 65ºC for 30 sec, and 72ºC for 2 min, followed by nine cycles of a 1ºC decrease in annealing temperature per cycle, followed by 35 cycles of 94ºC for 30 sec, 56ºC for 30 sec, and 72ºC for 2 min, and a final extension at 60ºC for 30 min. For all samples we performed duplicates of entire reactions to verify reproducibility. The total sample size for AFLP analysis was 18 populations × 8 half-sibs × 2 replicates × 4 selective primer pairs = 1152. Selective PCR products were electrophoretically separated using automated sequencing gels on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster 133

City, CA) at the DNA Sequencing and Synthesis Facility at Iowa State University. Colored gel images were analyzed with ABI GeneScan Analysis 2.1 (Applied Biosystems) software. To verify reproducibility, samples were run in a manner that permitted duplicates to be visualized side by side on each gel. All fragments between 35 and 500 bp in length were scored and a presence/absence binary matrix was constructed in a Microsoft Excel 2007 spreadsheet. A preliminary neighbor-joining cluster analysis including the above fragment range appropriately clustered replicates and individuals within populations. Therefore, we used all fragments in the above range for our analysis.

Analysis of Phenotypic Traits We conducted a common garden experiment in an open field site at the Bruner Farm, Boone, IA, USA (42° 00´N, 93° 43´W). We chose four seedlings of 1 – 2 cm in height from each of five half-sib families per population (18 populations × 5 half-sib families × 4 seedlings = 360) to reduce environmental variation in traits related to germination and pre- / post-transplant growth. On 30 May 2006, seedlings were individually transplanted into plastic pots (30 cm diameter × 25 cm depth) and placed into plastic wading pools (1.4 m diameter × 30 cm depth). The pots were filled with Sunshine LC1 potting soil (Sun Gro Horticulture Canada, Seba Beach, Alberta, Canada), which has an initial nutrient charge that would correspond to nutrient levels in natural wetland soils, approximating three times the average content of nitrogen as reported in Bridgham et al. (1996) and Bedford et al. (1999). As the initial nutrient charge is highly water soluble, providing the equivalent of approximately one application of a liquid fertilizer, nutrients were supplemented by regular fertilizer treatments as described below. Plants were arranged in a split-plot design consisting of five complete blocks, with four subplots within each block. Each subplot consisted of three plastic wading pools, which contained one of four environmental treatment combinations: (1) low water/low nutrient (WLNL), (2) low water/high nutrient (WLNH), (3) high water/low nutrient

(WHNL), and (4) high water/high nutrient (WHNH). Three plants from each provenance were put in each wading pool (6 plants per pool). Three populations from each region were distributed among three pools at random, with the restriction that only one population from each region can appear in a pool. Five half-sib families from each 134 population were also randomized across five blocks. The total number of experimental units for this experiment was 5 blocks × 4 treatment combinations = 20 units, with 6 regions × 3 populations = 18 observations per experimental unit. Pots in the high water treatments were kept at saturation, similar to standing water conditions in lakes and ponds. Low water treatments are comparable to drier, upland conditions, where plants were watered to the extent of letting the soil soak for a few hours, after which the soil was allowed to dry. Due to drought conditions at the common garden site, plants in the low water treatments were watered every day until 27 June, and then watered three times a week. In addition, 52 seedlings were killed from summer heat between 2 and 22 June and were replaced with seedlings from the same half-sib family. After that period, six plants died and were not considered in the data analysis. In the low nutrient treatment, no fertilizer was applied, whereas in the high nutrient treatment 100 g of slow-release 14:14:14 N:P:K Osmocote (The Scotts Company, Marysville, Ohio, USA) was applied once to a pot at the beginning of the experiment (N, 2481 mg/kg soil; P, 563 mg/kg soil; K, 1,320 mg/kg soil). The Osmocote applied in the high nutrient treatment followed the manufacturer’s recommendation for amount and rate (one application for a 3–4 month growing period) to be used in producing high fertilization levels (The Scotts Company). As a consequence, the low and high nutrient treatments were designed to be a reasonable approximation of low and high soil nutrient levels, respectively, that would be encountered under natural field conditions (cf., Bridgham et al. 1996, Thormann and Bayley 1997, Bedford et al. 1999). On 31 August 2006, after 97% of the plants had initiated flowering, we harvested the aboveground part of the plants. Belowground parts were left for a subsequent study. We randomly chose wading pools across blocks for harvesting to minimize experimental variation due to growth during the harvesting period. The following eight traits were measured for each experimental individual: (1) height, (2) number of secondary branches, (3) number of stems originating from the rootstock, (4) leaf area, (5) number of days from sowing to the first flowering, (6) aboveground biomass, (7) ratio of reproductive (flowering) part to the aboveground biomass, and (8) total number of flowers per plant. To determine (4), we chose one largest leaf from each plant and calculated leaf area by multiplying the maximum length and width of the leaf. To determine (8), we haphazardly 135 sampled six flower stalks from each plant (two each from apical, middle, and basal part of the stalk) to count the number of flowers per unit biomass of flower stalk. We measured total biomass of flower stalks per plant for each individual. Total number of flowers per plant was then calculated by multiplying the number of flowers per unit biomass by total biomass of flower stalk per plant, for each individual. To determine total biomass of flower stalks and vegetative parts, plants were divided into respective parts and dried in an oven for 24 hr at 60ºC to constant weight.

AFLP data analysis From the four selective primer pairs and 288 samples, 1864 fragments ranging from 35 to 500 bp were scored, of which 254 (13.6%) were monomorphic. The number of fragments per individual ranged from 430 to 621, with an average of 538 fragments. The typical AFLP frequency profile is supposed to produce appropriately clustered individuals within a population, however, a subset (62 out of 288) of samples produced numerous fragments around 300 bp, disrupting the presumed population structure. Therefore, further analysis was conducted without them. From this reduced sample, 1864 fragments ranging from 35 to 500 bp were scored, of which 284 (15.2%) were monomorphic. The number of fragments per individual ranged from 439 to 621, with an average of 546 fragments. To consider the noise from AFLP reactions between replicates from the same individuals, we constructed a data matrix of means for each locus between replicates and calculated the squared Euclidean distance matrix among all samples in a manner of calculating genetic distances among multilocus codominant genotypes (Smouse and Peakall 1999) using R 2.3.1 (R Development Core Team 2006). To quantify genetic diversity in each geographical level, the expected mean heterozygosity was calculated while correcting for sample size (Nei 1987). The estimated genetic diversity was compared between provenances and among regions within provenance using ANOVA (Analysis of Variance) weighted with the inverse of squared standard error. In addition, to test whether genetic diversity has decreased or increased through invasion history, a regression analysis was conducted between the genetic diversity of invasive populations and their geographic distances (in km) from the Easternmost population (NJ WR). 136

We have established a priori null model based on the geographic structure (native European vs. invasive North American provenance) and tested for variation at provenance and regional levels using AMOVA (Analysis of Molecular Variances; Excoffier et al. 1992). The squared Euclidean distance data matrix was used to conduct a hierarchically nested AMOVA model examining how total AFLP molecular genetic variation is partitioned among provenances relative to the total populations (ΦRT), populations within provenances (ΦPR), and between populations (ΦPT) using GenAlEx 6 (Peakall and Smouse 2006). We conducted AMOVA for each provenance separately and calculated pairwise ΦPT for all pairs of populations, which in turn was plotted against the natural log-scaled geographic distances (in km) between populations. We explored an alternative population model from the genetic structure of our data, using the phylogram of genetic distance, PCoA (Principal Coordinates Analysis), and clustering analysis. First, estimates of genetic distances (D) among populations were calculated from the binary data matrix using Nei’s unbiased genetic distance after Lynch and Milligan (1994) with 1,000 bootstraps, which were used to draw an unrooted phylogram with the neighbor-joining method (Saitou and Nei 1987) using the ‘neighbor’ and ‘consense’ programs in the PHYLIP 3.6 (Felsenstein 2005) package and the Phylodendron 0.8d (Gilbert 1997) program. Second, principal coordinates (PCOs) were extracted from the squared Euclidean distance matrix among all samples and plotted using GenAlEx 6 (Peakall and Smouse 2006). Third, we conducted a model-based clustering analysis using the Structure 2.2 (Pritchard et al. 2000) program with a 30,000 replicate burn-in and 100,000 simulations, assuming that each individual purely came from one of the populations (no admixture model) and that allele frequencies were correlated among populations. We ran this simulation program, increasing the number of clusters (K) from two to six where we found a hierarchical diverging pattern of regions and populations and obtained the maximum (optimized) estimated posterior probability of K. The inferred clusters were drawn as colored box plots using distruct (Rosenberg 2004) program. Finally, we evaluated the relative fit of two models (null model and alternative model) to decide which model better explained the population genetic structure of the data. To do so, we estimated the AIC (Akaike Information Criterion) for each model using the least square estimate of residual variance. 137

Analysis of quantitative phenotypic data

QST measures quantitative genetic variation in a manner analogous to FST (McKay and Latta 2002). In practice, measuring QST requires one to distinguish genetic variation among populations from environmental variation (Latta 2003). Therefore, our common garden system is an excellent research design to study QST. The lack of statistical power has been problematic in assessing molecular genetic variability, however this problem can be mitigated by adopting molecular markers of highly variable loci (such as AFLP and microsatellites, c.f. Reed and Frankham 2001). Since our experimental design involves multiple half-sib families nested within population, the additive genetic variance within populations is four times the within- population (among family) variance (Lynch and Walsh 1998). Also, we assumed that the inbreeding coefficient was zero since purple loosestrife is an obligate outcrossing species

(Thompson et al. 1987). Therefore, QST here can be written as:

2 σ g (between) VP VP QST = 2 2 = = = QPT (2) σ g (between ) + 2σ g (within) VP + 2(4VS (P) ) VP + 8VS (P) where VP is the among-population phenotypic variance, VS(P) is the phenotypic variance among half-sib families nested within population. This formula allows us to distinguish genetic variances among (and within) populations from environmental effects and estimate the QPT correctly. We calculated QPT for each of eight quantitative traits, as well as pairwise QPT for all pairs of populations using SAS 9.1 (SAS Institute Inc., Cary, NC).

Pairwise QPT were plotted against pairwise ΦPT and tested for significant deviation from the null hypothesis QPT = ΦPT using two-tailed sign tests to investigate whether natural selection or genetic drift is the leading force in the invasion history of purple loosestrife.

Results

For our data set of 18 populations, the four AFLP primer combinations generated 1864 fragments, 284 (15.2%) of which were monomorphic. The estimated genetic diversity was slightly greater in the invasive provenance than in the native provenance (Table 1), although not significantly so (P = 0.179). Genetic diversity decreased from the ancestral region (NJ) through the intermediate region (MI) to the region where purple loosestrife 138 established most recently (IA), but the regression between genetic diversity and geographic distance from the Easternmost population was not significant (R2 = 0.378, P = 0.078). The unrooted phylogram based on the genetic distance matrix resolved largely two clusters, one containing populations from the invasive provenance, and the other with populations from the native provenance, hence supporting our null hypothesis (Fig. 1). Three clusters formed within the invasive provenance (NJ, IA/MIRR, and MIKB/MILL) and five clusters formed within the native provenance (PDGO/SWST, PDGR/PDPO, TU, SWNO, and SWAL). New Jersey and Tübingen populations were clearly nested within regions. PC1 (the principal coordinate axis) and PC2 together (Fig. 2a) explained 57.5% of the total genetic variation, while PC2 and PC3 together explained 38.2% (Fig. 2b). PC1 extracted 34% of the total genetic variation and revealed several groups that are slightly different from those in the nested phylogram (Fig. 1, 2a): two major groups representing the native and invasive provenances with two populations from the native and invasive provenances (PDGO and MILL) respectively in contact with each other in the middle. PC2 largely reflected the population structures within each provenance (Fig. 2b). For example, SWST, PDGO, MIKB, and MILL populations are located on the positive side of PC2, all of which form the peripheral clusters of native provenances. MIRR, IABF, and IALS are also positive along PC2, which are branched in the higher hierarchy of the invasive cluster. Population structures inferred through simulation (Fig. 3) revealed the sequential split of provenances into regions and populations with increased number of assumed clusters (K). The clustering pattern generally corresponded with the nested phylogram (Fig. 1), dividing population structure into provenances and regions, until the regional structure is almost revealed at K = 6 (Fig. 3). However, the PDGO population shared same membership with populations from the invasive provenance at K = 2 and 3, which is congruent with the PCoA plot displaying the PDGO population in contact with an invasive population (MILL; Fig. 2a). Therefore, we established an alternative model where PDGO belongs to invasive provenance and tested which model is more appropriate. The AIC difference between the null and alternative models was approximately 0.05, indicating that the alternative model does not significantly better explain our genetic data. Our null hypothesis that PDGO belongs to native provenance 139

Figure 1. Unrooted phylogram drawn from Nei’s unbiased genetic distance using neighbor-joining method, with bootstrap values (percentages) estimated for each branch.

Figure 2. Principal coordinate plots displaying population structure (a) between dimension 1 and 2, (b) between dimension 2 and 3. 140

Figure 3. Estimated population structure from model-based clustering analysis. (Left) Each sample is represented by a thin vertical line, which is partitioned into K colored segments that represent the sample’s estimated membership fractions in K clusters. Black lines separate different populations. Populations are labeled below the figures. (Right) Clustering pattern diagram based on Q matrix of membership coefficients of populations to each cluster. was further supported by the high bootstrap value (82%) for the cluster PDGO/SWST in the phylogram (Fig. 1). From our geographical null model, the nested AMOVA for both provenances reflected significant genetic structure of provenances and populations, allowing 8% of the variation (P < 0.0001) to be accounted for among provenances and 19% of the variation (P < 0.0001) among populations within provenance (Table 2). Analyzing provenances separately revealed almost no difference between the native and invasive provenance in population divergence: 22% and 23% of the genetic variation was partitioned among populations in the native and introduced provenances, respectively. However, regional differentiation was greater in the introduced provenance (11%) than in the native provenance (5%). Population differentiation was greater in the native provenance (17%) than in the introduced provenance (12%), which is consistent with the finding that genetic distance (ΦPT) among populations was slightly greater in the native provenance

(mean pairwise ΦPT = 0.195, SD = 0.064) than in the invasive provenance (mean pairwise

ΦPT = 0.189, SD = 0.079). Both native and invasive populations are significantly structured, however, with the majority of genetic variation retained within populations.

Pairwise ΦPT between populations indicated significant pattern of “isolation by distance” for invasive provenance, but not for native provenance (Fig. 4). For the entire dataset, QPT were significantly greater than ΦPT for four traits (number of branches, 141

Table 2. Hierarchical nested analysis of molecular variance (AMOVA; Excoffier et al. 1992). Source df SS MS Variance components % Var Stat P Native and invasive provenances

Among provenances 1 1176.202 1176.202 14.897 8% ΦRT = 0.083 <0.0001 Among populations within provenances 15 5319.506 354.634 34.316 19% ΦPR = 0.207 <0.0001 Within populations 95 12462.950 131.189 131.189 73% ΦPT = 0.273 <0.0001 Total 111 18958.658 1662.025 180.402 Native provenance

Among regions 2 983.542 491.771 7.429 5% ΦGT = 0.047 <0.0001 Among populations within regions 6 1946.508 324.418 26.969 17% ΦPG = 0.178 <0.0001 Within populations 58 7235.563 124.751 124.751 78% ΦPT = 0.216 <0.0001 Total 66 10165.612 940.940 159.149 Invasive provenance

Among regions 2 1066.884 533.442 20.922 11% ΦGT = 0.113 <0.0001 Among populations within regions 5 1322.573 264.515 22.164 12% ΦPG = 0.136 <0.0001 Within populations 37 5227.388 141.281 141.281 77% ΦPT = 0.234 <0.0001 Total 44 7616.844 939.237 184.367

Figure 4. Pairwise ΦPT between pairs of populations plotted against the geographic distance (km) in natural log scale. (a) invasive provenance, (b) native provenance. number of days to first flowering, aboveground biomass, and number of flowers), while

QPT < ΦPT for the number of stem (Fig. 5, Table 3). Within the native provenance, QPT >

ΦPT for number of branches and flowers, while QPT < ΦPT for height and number of stems.

Within the invasive provenance, QPT > ΦPT for number of flowers, while QPT < ΦPT for height and leaf area. Between provenances, QPT > ΦPT for six traits, while QPT < ΦPT for the number of stems. The reproductive: aboveground biomass ratio did not significantly deviate from QPT = ΦPT in any case.

142

Figure 5. Comparison of pairwise QPT and ΦPT estimates for (a) height, (b) number of secondary branches, (c) number of stems originating from the rootstock, (d) leaf area, (e) number of days from sowing to the first flowering, (f) aboveground biomass, (g) ratio of reproductive (flowering) part to the aboveground biomass, and (h) total number of flowers per plant. 143

Table 3. Summary of two-tailed sign test for significant deviance from the 50:50 odds if QPT = ΦPT on average, with the number of cases for QPT>ΦPT or QPT<ΦPT. Significant P values are indicated in bold. Height Branches StemsR LeafArea

QPT>ΦPT QPT<ΦPT P QPT>ΦPT QPT<ΦPT P QPT>ΦPT QPT<ΦPT P QPT>ΦPT QPT<ΦPT P Total 57 73 0.052 67 41 0.007 43 68 0.009 67 64 0.134 Within natives 5 27 <0.001 23 5 0.001 9210.027 15 16 0.280 Within invasives 8 18 0.047 6 120.142 13 60.104 6 220.003 Between 44 28 0.032 38 24 0.042 21 41 0.008 46 26 0.012 provenances NDFF MassT RatioRT FlowerN

QPT>ΦPT QPT<ΦPT P QPT>ΦPT QPT<ΦPT P QPT>ΦPT QPT<ΦPT P QPT>ΦPT QPT<ΦPT P Total 103 33 <0.001 72 48 0.013 74 59 0.060 92 38 <0.001 Within natives 21 15 0.162 11 16 0.194 21 14 0.135 22 11 0.045 Within invasives 18 10 0.098 7 14 0.111 17 9 0.093 23 2 <0.001 Between 64 8 <0.001 54 18 <0.001 36 36 0.187 47 25 0.006 provenances

Discussion

Distinct genetic entities of purple loosestrife were revealed between native and invasive provenances (Table 2), as well as genetic subdivision at the region-wide geographical scale. Although the PCoA (Fig. 2a) and cluster analyses (Fig. 3) suggested an alternative model, where PDGO population belongs to the invasive provenance, the comparison through AIC indicated that this alternative model did not significantly improve compared to the geographical null model. Both in the native and invasive provenances, genetic variation within populations accounted for the majority of total genetic variation, although the partitioning of genetic variation among populations was significant (Table 2). Each population thus may be a mixture of different genetic lineages both in the native and invasive provenances, possibly due to frequent gene flow and genetic drift. Genetic variation among regions was greater in the invasive than in the native provenance (Table 2). The pairwise ΦPT between populations was also significant in invasive provenance, but not in native provenance (Fig. 4). It indicates that invasive populations exhibit more genetically differentiated distribution compared to native populations.

An FST-QST analysis is useful to test whether quantitative traits are affected purely by genetic drift, migration, and mutation, or if they are under directional or stabilizing selection. Our results indicate that some quantitative traits experience episodes of selection either in the native or invasive provenance, while other traits do not (Table 3). Selection acted in different pattern between traits. While height is under stabilizing 144 selection in both provenances, number of flowers experienced directional selection (and thus populations are formed by local adaptation). Number of branches and number of stems from root are influenced by directional and stabilizing selection, respectively in the native provenance, but not in the invasive provenance. In contrast, leaf area underwent stabilizing selection only in the invasive provenance. Natural selection was not involved in three traits (number of days to first flowering, aboveground biomass, and reproductive: aboveground biomass ratio). The variability of these QST results may be explained by the different genetic architecture forming each trait, suggesting that the relationship between selection and drift is specific to each trait (McKay and Latta 2002). The general pattern confirms the expectation that fitness-related traits (for example, number of flowers) should experience strong directional selection and traits more distantly linked to fitness (other morphological traits) should undergo stabilizing selection.

Interpreting the FST-QST contrasts safely requires many assumptions. Specifically, quantitative traits associated with fitness generally have a large amount of dominance and epistatic genetic variance (Crnokrak and Roff 1995). This may be especially problematic in understanding the role of natural selection of fitness-related traits, which is one of the major issues for evolutionary biologists. Indeed, how QST is affected by the presence of dominance and epistasis is controversial. Some researchers suggest that QST can be either inflated or deflated by dominance effects (Whitlock 1999, López-Fanjul et al. 2003); more recent study (Goudet and Büchi 2006) suggests that dominance generally deflates

QST. Moreover, Lynch et al. (1999) suggest that epistasis should inflate QST, while others indicate that epistasis should drive QST downward (Whitlock 1999, López-Fanjul et al.

2003). If nonadditive gene actions tend to deflate QST, our result of QPT>ΦPT for number of flowers is a conservative test of directional selection. In sum, our study suggests that multiple independent introductions of purple loosestrife into North America resulted in substantial molecular genetic variation. The impact of genetic bottlenecks and founder effects depends on the breeding system strongly limiting the genetic diversity of plants relying on selfing or asexual reproduction (Baker 1967, Brown and Marshall 1981). Those species may be limited further by reduced recombination among founding genotypes and thereby maintain low genetic diversity (Lambrinos 2001). In contrast, outbreeding species often exhibit high levels of 145 genetic diversity in both native and introduced ranges (Pappert et al. 2000). Our results indicate that introduced purple loosestrife populations possess genetic diversity at least as great as native populations. Population processes such as founder effects and genetic drift affect introduced purple loosestrife populations resulting in biased frequencies of flower morphs (Eckert and Barrett 1992, Mal and Lovett-Doust 1996), while equal frequencies of three flower morphs are typical in native European populations (Eckert et al. 1996). Whether these processes actually lead to decreased genetic variation in the introduced provenance is unknown, our results suggest that genetic variation decreased during the course of invasion in North America. In any case, the substantial overall genetic variation in the introduced provenance suggests multiple introductions to North America from diverse gene pools in Europe. During the second half of the 19th century, purple loosestrife was introduced by European immigrants as a medicinal, horticultural, and beekeeping plant (Thompson et al. 1987). Thus, it may have been repeatedly introduced, involving various genotypes from different areas in Europe (Edwards et al. 1995). In addition, invasive species often create novel genotypes, increasing genetic diversification through polyploidization and hybridization with other species or among diverse genotypes after introduction (Thompson 1991, Abbot 1992, Ellstrand and Schierenbeck 2000). Hybridization between wild purple loosestrife plants and its cultivars is suggested by crossability, fertility, and morphological evidence (Anderson and Ascher 1993, Ottenbreit and Staniforth 1994, Anderson et al. 1995). Hybrids between purple loosestrife and its native congener (L. alatum) have also been supported using allozyme and AFLP markers (Strefeler et al. 1996, Houghton-Thompson et al. 2005). The considerable genetic variation created by multiple independent introduction events and subsequent intermingling of populations during the invasive spread may have provided substrate for local adaptation of invasive purple loosestrife populations in North America.

Acknowledgements

We thank J. F. Wendel for the use of the facilities in his laboratory. We are grateful to R. Percifield, M. L. Collyer, J. Hawkins, L. Flagel, J. Triplett, and L. Gutierrez for 146 thoughtful comments and suggestions. This research was funded by EEOB Department at Iowa State University and by CGRER Seed Grant Program at University of Iowa to Y. J. Chun.

Literature Cited

Abbot, R. J. 1992. Plant invasions, interspecific hybridization and the evolution of new plant taxa. Trends in Ecology and Evolution 7:401-405. Ågren, J., and L. Ericson. 1996. Population structure and morph-specific fitness differences in tristylous Lythrum salicaria. Evolution 50:126-139. Amsellem, L., J. L. Noyer, T. L. Bourgeois, and M. Hossaert-Mckey. 2000. Comparison of genetic diversity of the invasive weed Rubus alceifolius Poir. (Rosaceae) in its native range and in areas of introduction, using amplified fragment length polymorphism (AFLP) markers. Molecular Ecology 9:443-455. Anderson, N. O., and P. D. Ascher. 1993. Male and female fertility of loosestrife (Lythrum) cultivars. Journal of the American Society for Horticultural Science 118:851-858. Anderson, N. O., P. D. Ascher, and B. E. Liedl. 1995. Importance of introgressive hybridization in the development of invasive Lythrum salicaria. HortScience 30:819. Baker, H. G. 1967. Support for Baker's Law as a rule. Evolution 21:853-856. Baker, H. G. 1974. The evolution of weeds. Annual Review of Ecology and Systematics. 5:1-24. Baker, H. G., and G. L. Stebbins (eds.). 1965. The Genetics of Colonizing Species. Academic Press, New York, USA. Bartlett, E., S. J. Novak, and R. N. Mack. 2002. Genetic variation in Bromus tectorum (Poaceae): differentiation in the eastern United States. American Journal of Botany 89:602-612. Bastlová, D., and J. Květ. 2002. Differences in dry weight partitioning and flowering phenology between native and non-native plants of purple loosestrife (Lythrum salicaria L.). Flora 197:332-340. 147

Bedford, B. L, M. R. Walbridge, and A. Aldous. 1999. Patterns in nutrient availability and plant diversity of temperate North American wetlands. Ecology 80(7):2151–2169. Blossey, B., L. C. Skinner, and J. Taylor. 2001. Impact and management of purple loosestrife (Lythrum salicaria) in North America. Biodiversity and Conservation 10:1787-1807. Bridgham, S. D., J. Pastor, J. A. Janssens, C. Chapin, and T. J. Malterer. 1996. Multiple limiting gradients in peatlands: a call for a new paradigm. Wetlands 16(1):45-65. Brown, A. H. D., and D. R. Marshall. 1981. Evolutionary changes accompanying colonization in plants. Pages 351-363 in G. G. E. Scudder, and J. I. Reveal (eds.) Evolution today. Proceedings of the Second International Congress of Systematic and Evolutionary Biology. Hunt Institute for Botanical Documentation, Pittsburgh, Pennsylvania. Chun, Y. J., M. L. Collyer, K. A. Moloney, and J. D. Nason. 2007. Phenotypic plasticity of native vs. invasive purple loosestrife: a two state multivariate approach. Ecology 88(6):1499-1512. Crnokrak, P., and D. A. Roff. 1995. Dominance variance: associations with selection and fitness. Heredity 75:530-540. Crnokrak, P., and J. Merilä. 2002. Genetic population divergence: markers and traits. Trends in Ecology and Evolution 17:501. Cruzan, M. B. 1998. Genetic markers in plant evolutionary biology. Ecology 79(2):400- 412. DeWalt, S. J., and J. L. Hamrick. 2004. Genetic variation of introduced Hawaiian and native Costa Rican populations of an invasive tropical shrub, Clidemia hirta (Melastomataceae). American Journal of Botany 91(8):1155-1162. Doyle, J. J., and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12:13–15. Durka, W., O. Bossdorf, D. Prati, and H. Auge. 2005. Molecular evidence for multiple introduction of garlic mustard (Alliaria petiolata, Brassicaceae) to North America. Molecular Ecology 14:1697-1706. 148

Eckert, C. G., and S. C. H. Barrett. 1992. Stochastic loss of style morphs from populations of tristylous Lythrum salicaria and Decodon verticillatus (Lythraceae). Evolution 46(4):1014-1029. Eckert, C. G., D. Manicacci, and S. C. H. Barrett. 1996. Genetic drift and founder effect in native versus introduced populations of an invading plant, Lythrum salicaria (Lythraceae). Evolution 50(4):1512-1519. Edwards, K. R., M. S. Adams, and J. Květ. 1998. Differences between European native and American invasive populations of Lythrum salicaria. Journal of Vegetation Science 9:267-280. Edwards, K. R., M. S. Adams, and J. Květ. 1995. Invasion history and ecology of Lythrum salicaria in North America. Pages 161-180 in P. Pyšek, K. Prach, M. Rejmánek, and M. Wade (eds.) Plant Invasions - General Aspects and Special Problems. SPB Academic Publishing, Amsterdam, The Netherlands. Ellstrand, N. C., and K. Schierenbeck. 2000. Hybridization as a stimulus for the evolution of invasiveness in plants? Proceedings of the National Academy of Sciences 97:7043-7050. Excoffier, L., P. Smouse, and J. Quattro. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction sites. Genetics 131: 479-491.

Falconer, D. S., and T. F. C. Mackay. 1996. Introduction to quantitative genetics. 4th ed., Longman Group Ltd., Edinburgh, U. K. Felsenstein, J. 2005. PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle. Fisher, R. A. 1930. The Genetical Theory of Natural Selection, Clarendon Press, Oxford. UK. Gilbert, D. G. 1997. Phylodendron (an application for phylogenetic tree drawing) version 0.8d. Distributed by the author. Biology Department, Indiana University, Bloomington. 149

Goodnight, C. J., 1995. Epistasis and the increase in additive genetic variance: implications for Phase 1 of Wright's shifting-balance process. Evolution 49:502- 511. Goudet, J., and L. Büchi. 2006. The effects of dominance, regular inbreeding and sampling design on Qst, an estimator of population differentiation for quantitative traits. Genetics 172:1337-1347. Hartl, D. L., and A. G. Clark. 1989. Principles of Population Genetics. Sinauer Associates, Sunderland, MA, 682 p. Hedge, S. G., J. D. Nason, J. M. Clegg, N. C. Ellstrand. 2006. The evolution of California's wild radish has resulted in the extinction of its progenitor. Evolution 60: 1187-1197. Houghton-Thompson, J., H. H. Prince, J. J. Smith, and J. F. Hancock. 2005. Evidence of hybridization between Lythrum salicaria (purple loosestrife) and L. alatum (winged loosestrife) in North America. Annals of Botany 96:877-885. Lambrinos, J. G. 2001. The expansion history of a sexual and asexual species of Cortaderia in California, U.S.A. Journal of Ecology 89:88-98. Lande, R. 1980. Genetic variation and phenotypic evolution during allopatric speciation. American Naturalist 116(4):463-479. Latta, R. G. 2003. Gene flow, adaptive population divergence and comparative population structure across loci. New Phytologist. 161:51-58. Le Page, S. L., R. A. Livermore, D. W. Cooper, and A. C. Taylor. 2000. Genetic analysis of a documented population bottleneck: introduced Bennett's wallabies (Macropus rufogriseus rufogriseus) in New Zealand. Molecular Ecology 9(6):753-763. Lee, C. E. 2002. Evolutionary genetics of invasive species. Trends in Ecology and Evolution 17:386-391. López-Fanjul, C., A. Fernández, and M. A. Toro. 2003. The effect of neutral nonadditive gene action on the quantitative index of population divergence. Genetics 164:1627- 1633. Lynch, M. and B. G. Milligan. 1994. Analysis of population genetic structure with RAPD markers. Molecular Ecology 3:91-99. 150

Lynch, M., and B. Walsh. 1998. Genetics and Analysis of Quantitative Traits. Sinaur Associates, Sunderland, MA. Lynch, M., M. Pfrender, K. Spitze, N. Lehman, J. Hicks, D. Allen, L. Latta, M. Ottene, F. Bogue, and J. Colbourne. 1999. The quantitative and molecular genetic architecture of a subdivided species. Evolution 53(1):100-110. Mal, T. K., and J. Lovett-Doust. 1997. Morph frequencies and floral variation in a heterostylous colonizing weed, Lythrum salicaria. Canadian Journal of Botany 75:1034-1045. Mal, T. K., J. Lovett-Doust, and L. Lovett-Doust. 1997. Time-dependent competitive displacement of Typha angustifolia by Lythrum salicaria. Oikos 79:26-33. Maron, J. L., and M. Vilá. 2001. When do herbivores affect plant invasion? Evidence for the natural enemies and biotic resistance hypothesis. Oikos 95:361-373. Maron, J. L., M. Vilá, R. Bommarco, S. Elmendorf, and P. Beardsley. 2004. Rapid evolution of an invasive plant. Ecological Monograph 74(2): 261-280. McKay, J. K., and R. G. Latta. 2002. Adaptive population divergence: markers, QTL and traits. Trends in Ecology and Evolution 17(6):285-291. Meekins, J. F., H. E., Ballard Jr., and B. C. McCarthy. 2001. Genetic variation and molecular biogeography of a North American invasive plant species (Alliaria petiolata, Brassicaceae). International Journal of Plant Sciences 162:161-169. Merilä, J., and P. Crnokrak. 2001. Comparison of genetic differentiation at marker loci and quantitative traits. Journal of Evolutionary Biology 14:892-903. Mitchell, C. E., and A. G. Power. 2003. Release of invasive plants from fungal and viral pathogens. Nature 421:625-627. Mitton, J. B. 1993. Theory and data pertinent to the relationship between heterozygosity and fitness. Pages 17-41 in N. W. Thornhill (ed.) The natural history of inbreeding and outbreeding. University of Chicago Press, Chicago. Mueller, U. G., and L. L. Wolfenbarger. 1999. AFLP genotyping and fingerprinting. Trends in Ecology and Evolution 14(10):389-394. Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. 151

Neuffer, B., and H. Hurka. 1999. Colonization history and introduction dynamics of Capsella bursa-pastoris (Brassicaceae) in North America: isozymes and quantitative traits. Molecular Ecology 8:1667-1681. Novak, S. J., and R. N. Mack. 1993. Genetic variation in Bromus tectorum: comparison between native and introduced populations. Heredity 71:167-176. Ottenbreit, K. A., and R. J. Staniforth. 1994. Crossability of naturalized and cultivated Lythrum taxa. Canadian Journal of Botany 72:337-341. Pappert, R. A., J. L. Hamrick, and L. A. Donovan. 2000. Genetic variation in Pueraria lobata (Fabaceae), an introduced, clonal, invasive plant of the southeastern United States. American Journal of Botany 87(9):1240-1245. Peakall, R., and P. E. Smouse. 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6:288-295. Pritchard, J. K., M. Stephens, and P. Donnelly. 2000. Inference of population structure using multilocus genotype data. Genetics 155:945-959. R Development Core Team. 2006. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3- 900051-07-0, URL http://www.R-project.org. Reed, D. H., and R. Frankham. 2001. How closely correlated are molecular and quantitative measures of genetic variation? A meta-analysis. Evolution 55(6):1095- 1103. Reinhart, K. O., and R. M. Callaway. 2006. Soil biota and invasive plants. New Phytologist 170:445-457. Rosenberg, N. A. 2004. Distruct: a program for the graphical display of population structure. Molecular Ecology Notes 4:137-138. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4(4):406-425. Sakai, A. K., F. Allendorf, J. Holt, D. Lodge, J. Molofsky, K. With, S. Baughman, R. Cabin, J. Cohen, N. Ellstrand, D. McCauley, P. O’Neil, I. Parker, J. Thompson, and S. Weller. 2001. The population biology of invasive species. Annual Review of Ecology and Systematics 32:305-312. 152

Sexton, J. P., J. K. McKay, and A. Sala. 2002. Plasticity and genetic diversity may allow saltcedar to invade cold climates in North America. Ecological Applications 12(6):1652-1660. Smouse, P. E., and R. Peakall. 1999. Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure. Heredity 82:561-573. Spitze, K. 1993. Population structure in Daphnia obtusa: quantitative genetics and allozyme variation. Genetics 135:367-374. Strefeler, M. S., E. Darmo, R. L. Becker, and E. J. Katovich. 1996. Isozyme characterization of genetic diversity in Minnesota populations of purple loosestrife, Lythrum salicaria (Lythraceae). American Journal of Botany 83(3):265-273. Stuckey, R. L. 1980. Distributional history of Lythrum salicaria (purple loosestrife) in North America. Bartonia 47: 3-20. Thompson, D. Q., R. L. Stuckey, and E. B. Thompson. 1987. Spread, Impact, and Control of Purple Loosestrife (Lythrum salicaria) in North American Wetlands. US Fish and Wildlife Service. 55 pages. Jamestown, ND: Northern Prairie Wildlife Research Center Home Page. http://www.npwrc.usgs.gov/resource/1999/loosstrf/loosstrf.htm (Version 04JUN99). Thompson, J. D. 1991. The biology of an invasive plant. Bioscience 41:393-401. Thormann, M. N., and S. E. Bayley. 1997. Response of aboveground net primary plant production to nitrogen and phosphorous fertilization in peatlands in southern boreal Alberta, Canada. Wetlands 17(4):502-512. Vitousek, P. M., C. M. D’Antonio, L. L. Loope, and R. Westbrooks. 1996. Biological invasions as global environmental change. American Scientist 84(5):468-478. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. V. D. Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407-4414. Warwick, S. I., B. K. Thompson, and L. D. Black. 1987. Genetic variation in Canadian and European populations of the colonizing weed species Apera spica-venti. New Phytologist 106(2):301-317. 153

Weiher, E., I. C. Wisheu, P. A. Keddy, and D. R. J. Moore. 1996. Establishment, persistence, and management implications of experimental wetland plant communities. Wetlands 16(2):208-218. Whitlock, M. C. 1999. Neutral additive variance in a metapopulation. Genetical Research 74:215-221. Wright, S. 1931. Evolution in Mendelian populations. Genetics 16: 97-159. 154

Chapter 7. General Conclusion

Summary

Using purple loosestrife as a model organism, a series of comparative experimental studies on the phenotypic plasticity, population genetic structure, and evolution of quantitative traits has been conducted. This approach will provide key insights into the process of invasion and will allow us to deepen our understanding of evolutionary changes of invasive species responding to novel environmental conditions in the introduced range. Our results revealed major differences in life history and morphological traits between native and invasive populations of purple loosestrife. Native populations generally flowered earlier and invested more in sexual reproductive efforts than invasive populations, while the latter flowered later but accumulated more biomass for vegetative growth in subsequent growth seasons. In addition, invasive populations took advantage of excessive water and nutrient resources both for increased vegetative growth and sexual reproduction to overcome the disadvantage of late flowering compared to native populations. The high (excessive) nutrient treatment was designed to approximate wetland soils enriched by nutrients running off from agricultural fields, a common occurrence in the invaded wetlands of North America. Thus, human intervention and disturbance in the establishment and spread of invasive species is indicated and is consistent with the general idea that invasive species perform disproportionately better than native species in response to conditions that increase resource availability (fluctuating resources hypothesis). A comparative study of population genetics indicated that invasive populations possess considerable genetic diversity not less than native populations. In addition, genetic variation within populations accounted for the majority of total genetic variation both in native and invasive provenances. Thus, introduced purple loosestrife populations may have experienced founder effects and genetic drift upon arrival in North America, but these effects were outweighted by accumulation of genetic variation created by multiple introductions of independent genotypes from different regions in Europe, and 155 subsequent blending of different genotypes throughout the invasion process in North America. The assessment of evolutionary adaptive variation of invasive populations revealed significant evidence for directional selection on fitness-related traits (number of flowers), indicating that invasive purple loosestrife populations may have established such that different phenotypes are locally adapted across a broad environmental range.

Future Research

Though this dissertation unveiled a substantial amount of information on the background of phenotypic plasticity and evolutionary processes of purple loosestrife invasion, future works could provide more comprehensive and detailed picture of invasion. First, a long-term study is required to clearly evaluate the competitive ability and invasive potential of this non-native species, since purple loosestrife is a perennial plant taking 4-5 years to be competitively dominant and displace native species in U.S. wetlands. Because it begins to increase ramet production from the second year of growth, the invasive potential for vegetative growth would be examined more definitely with monitoring of demography for at least five years. A long-term study thus would better determine if perennial invasive plants exhibit more adaptive plasticity in the long run than native plants. Second, the phenotypic plasticity study could be expanded to incorporate more levels of environmental treatments. Most invasive species, including purple loosestrife, occupy a diverse range of environments. Therefore, incorporating multiple levels of environmental conditions in a study design would allow greater ability to evaluate characteristics of growth and reproduction under each environmental condition, thus greatly increasing our understanding of the phenotypic plasticity under diverse environments. Third, our understanding of invasion processes would be vastly improved if population genetic studies incorporate more populations both from native and invasive provenance. More broadly sampling potential source populations in the native provenance would aid in identifying the source regions that contribute to the gene pool in the invasive provenance. Likewise, comprehensive sampling in the invasive provenance would clarify introduction points and pathways of dispersal. 156

Despite numerous studies of the background and process of biological invasion, our understanding of invasion patterns is still limited, which may be remedied by comprehensive research involving population ecology and genetics. This dissertation contributes an important step in that direction, serving as a model approach for the study of other invasive species.

157

Appendix I. Raw data from common garden experiment in Chapter 3. Height Number of Number of Number of Aboveground Belowground Block Water Nutrient Population Replicate (cm) branches basal stems flowers biomass (g) biomass (g) B1 L L G1 1 106 64 1 392.414 17.365 2.730 B1 L L G1 2 88 82 1 684.355 22.375 2.300 B2 L L G1 1 88 80 1 729.852 19.275 2.850 B2 L L G1 2 90 102 1 690.042 30.005 3.180 B3 L L G1 1 85 56 1 775.349 10.285 4.440 B3 L L G1 2 128 125 1 2428.416 31.375 7.410 B4 L L G1 1 79 50 1 70.142 14.595 6.220 B4 L L G1 2 73 187 1 174.406 38.055 10.180 B5 L L G1 1 78 150 1 130.805 40.015 7.680 B5 L L G1 2 87 31 1 314.689 9.065 4.690 B1 L H G1 1 85 161 1 418.091 16.345 1.770 B1 L H G1 2 88 192 1 907.924 30.365 2.820 B2 L H G1 1 98 54 1 1531.349 27.375 4.050 B2 L H G1 2 78 61 1 383.456 16.215 2.280 B3 L H G1 1 89 110 1 821.338 28.085 5.560 B3 L H G1 2 103 126 1 1533.823 38.725 6.360 B4 L H G1 1 91 96 1 1177.581 27.795 5.590 B4 L H G1 2 75 81 1 1697.101 40.575 9.760 B5 L H G1 1 91 217 2 1420.024 41.485 7.100 B5 L H G1 2 74 95 1 410.669 23.095 2.370 B1 H L G1 1 116 28 1 111.134 15.885 2.580 B1 H L G1 2 129 21 1 171.172 15.935 N/A B2 H L G1 1 127 23 1 75.367 9.725 2.070 B2 H L G1 2 137 26 1 125.186 11.165 2.260 B3 H L G1 1 125 21 1 65.148 8.955 4.100 B3 H L G1 2 109 16 1 186.501 7.195 3.720 B4 H L G1 1 111 18 1 84.309 10.035 7.600 B4 H L G1 2 120 25 1 75.367 8.435 4.720 B5 H L G1 1 132 24 1 127.741 12.935 8.170 B5 H L G1 2 106 25 1 54.928 10.415 8.440 B1 H H G1 1 120 208 4 436.591 73.395 3.770 B1 H H G1 2 155 230 5 1140.701 81.895 4.960 B2 H H G1 1 108 369 6 207.595 120.385 10.430 B2 H H G1 2 105 414 1 761.894 129.315 9.720 B3 H H G1 1 95 329 3 395.928 83.685 8.470 B3 H H G1 2 100 175 3 420.540 48.045 8.490 B4 H H G1 1 127 290 5 1680.019 103.845 25.060 B4 H H G1 2 95 219 1 N/A 95.205 11.980 B5 H H G1 1 118 293 3 247.188 116.655 24.310 B5 H H G1 2 138 209 3 371.316 124.845 25.740 B1 L L G2 1 89 40 1 555.937 9.425 1.230 B1 L L G2 2 88 107 1 699.827 27.455 2.650 B2 L L G2 1 68 114 1 559.207 31.975 3.970 B2 L L G2 2 77 227 1 420.223 27.895 2.740 B3 L L G2 1 64 58 1 425.128 39.135 4.870 B3 L L G2 2 79 103 1 899.310 43.215 5.310 B4 L L G2 1 98 150 1 580.464 39.135 7.080 B4 L L G2 2 94 114 1 1231.238 33.965 8.340 B5 L L G2 1 69 161 1 408.777 20.535 5.120 B5 L L G2 2 75 107 1 688.381 19.585 6.760 B1 L H G2 1 84 136 1 671.255 22.935 2.470 B1 L H G2 2 57 68 1 469.878 17.365 1.200 B2 L H G2 1 94 85 1 942.339 24.075 2.140 B2 L H G2 2 14 123 1 810.669 18.895 2.180 B3 L H G2 1 102 128 1 3570.560 43.365 6.340 B3 L H G2 2 73 108 1 1332.183 25.485 4.020 B4 L H G2 1 76 140 1 1833.042 32.245 4.220 B4 L H G2 2 67 93 1 438.897 26.785 7.080 B5 L H G2 1 66 235 1 663.510 30.535 8.240 B5 L H G2 2 78 92 1 632.529 15.395 6.190 B1 H L G2 1 106 24 1 36.597 11.285 2.210 B1 H L G2 2 122 24 1 54.164 14.095 2.600 B2 H L G2 1 97 29 1 50.504 6.555 2.030 B2 H L G2 2 101 16 1 45.381 11.465 3.390 158

Appendix I. (continued) Height Number of Number of Number of Aboveground Belowground Block Water Nutrient Population Replicate (cm) branches basal stems flowers biomass (g) biomass (g) B3 H L G2 1 92 20 1 38.793 12.735 4.880 B3 H L G2 2 124 18 1 61.483 10.965 4.960 B4 H L G2 1 105 22 1 33.669 14.405 6.490 B4 H L G2 2 98 21 1 32.206 13.535 6.890 B5 H L G2 1 105 24 1 35.133 14.635 10.240 B5 H L G2 2 91 16 1 28.546 11.535 6.700 B1 H H G2 1 109 295 6 186.110 51.495 3.730 B1 H H G2 2 105 283 7 647.174 78.525 5.340 B2 H H G2 1 104 218 6 222.873 68.715 5.960 B2 H H G2 2 107 218 1 392.900 90.735 10.080 B3 H H G2 1 116 238 4 751.334 108.195 13.750 B3 H H G2 2 113 286 7 274.187 125.725 14.690 B4 H H G2 1 106 230 6 703.084 77.835 13.290 B4 H H G2 2 100 300 6 134.030 109.695 16.230 B5 H H G2 1 104 135 4 217.512 69.735 8.890 B5 H H G2 2 103 137 7 101.097 62.995 9.970 B1 L L G3 1 88 100 1 795.529 24.245 3.410 B1 L L G3 2 105 113 1 984.412 23.715 3.170 B2 L L G3 1 87 102 1 451.096 28.285 5.920 B2 L L G3 2 86 153 1 586.647 23.385 6.740 B3 L L G3 1 103 90 1 935.525 34.795 4.170 B3 L L G3 2 101 49 1 1408.843 28.465 4.750 B4 L L G3 1 83 146 1 937.747 42.895 12.270 B4 L L G3 2 85 280 1 1335.512 45.885 15.240 B5 L L G3 1 80 29 1 248.881 23.715 N/A B5 L L G3 2 142 116 1 1999.934 40.005 23.620 B1 L H G3 1 85 122 1 643.204 30.095 2.000 B1 L H G3 2 63 80 1 212.045 14.785 1.650 B2 L H G3 1 88 81 1 796.348 15.595 N/A B2 L H G3 2 86 79 1 614.931 22.665 2.470 B3 L H G3 1 90 100 1 1199.233 26.765 8.320 B3 L H G3 2 72 130 1 1050.802 26.785 6.380 B4 L H G3 1 66 50 1 749.226 28.385 4.820 B4 L H G3 2 82 69 1 1008.393 17.005 3.040 B5 L H G3 1 82 147 1 2153.437 22.745 4.040 B5 L H G3 2 73 140 1 1338.241 26.185 5.330 B1 H L G3 1 119 25 1 83.427 12.955 2.490 B1 H L G3 2 123 26 1 41.713 13.245 3.010 B2 H L G3 1 126 23 1 99.859 9.265 2.760 B2 H L G3 2 112 21 1 56.882 12.565 2.740 B3 H L G3 1 116 21 1 59.410 6.805 9.540 B3 H L G3 2 111 15 1 38.553 6.415 4.980 B4 H L G3 1 115 24 1 59.410 8.845 7.970 B4 H L G3 2 101 12 1 49.298 11.685 5.610 B5 H L G3 1 101 21 1 29.073 13.795 11.020 B5 H L G3 2 88 15 1 29.705 16.425 12.910 B1 H H G3 1 90 374 6 642.924 69.335 7.300 B1 H H G3 2 113 248 6 425.588 64.645 7.130 B2 H H G3 1 104 307 6 377.368 71.415 7.660 B2 H H G3 2 104 188 1 830.211 81.905 15.050 B3 H H G3 1 78 202 4 74.775 60.265 6.470 B3 H H G3 2 102 232 5 635.936 68.755 10.300 B4 H H G3 1 118 238 7 503.857 70.525 14.900 B4 H H G3 2 95 210 6 581.427 65.895 20.240 B5 H H G3 1 121 188 4 232.711 73.315 14.990 B5 H H G3 2 130 118 5 280.930 65.005 14.720 B1 L L Iowa 1 77 38 1 N/A 7.105 2.140 B1 L L Iowa 2 67 76 1 5.104 14.080 2.840 B2 L L Iowa 1 96 42 1 91.875 21.765 3.890 B2 L L Iowa 2 125 84 1 1340.698 31.355 3.720 B3 L L Iowa 1 105 77 1 399.828 39.835 5.130 B3 L L Iowa 2 150 76 1 1223.302 36.935 9.050 B4 L L Iowa 1 127 84 1 603.995 39.015 10.350 B4 L L Iowa 2 103 96 1 153.125 67.495 9.740 B5 L L Iowa 1 106 82 1 949.378 39.015 3.230 159

Appendix I. (continued) Height Number of Number of Number of Aboveground Belowground Block Water Nutrient Population Replicate (cm) branches basal stems flowers biomass (g) biomass (g) B5 L L Iowa 2 104 114 1 862.607 67.495 11.180 B1 L H Iowa 1 89 53 1 N/A 14.735 1.400 B1 L H Iowa 2 82 85 1 136.464 23.855 2.450 B2 L H Iowa 1 84 134 1 291.298 33.165 2.050 B2 L H Iowa 2 82 91 1 215.193 33.005 6.100 B3 L H Iowa 1 71 58 1 278.176 26.125 7.420 B3 L H Iowa 2 79 78 1 207.320 41.215 8.060 B4 L H Iowa 1 92 82 1 572.098 31.285 7.170 B4 L H Iowa 2 106 100 1 1380.384 42.415 5.370 B5 L H Iowa 1 93 87 1 36.740 14.735 4.660 B5 L H Iowa 2 95 131 1 1033.976 58.495 6.610 B1 H L Iowa 1 123 7 1 73.052 10.115 3.210 B1 H L Iowa 2 162 19 1 80.121 18.685 2.450 B2 H L Iowa 1 114 21 1 84.835 15.045 3.800 B2 H L Iowa 2 112 24 1 91.904 13.745 3.170 B3 H L Iowa 1 141 20 1 127.252 10.485 3.390 B3 H L Iowa 2 111 6 1 14.139 8.405 3.600 B4 H L Iowa 1 160 37 1 282.782 16.885 5.030 B4 H L Iowa 2 126 11 1 106.043 15.155 5.780 B5 H L Iowa 1 139 17 1 108.400 15.795 5.090 B5 H L Iowa 2 128 39 1 122.539 17.485 7.380 B1 H H Iowa 1 140 321 6 278.819 181.345 12.980 B1 H H Iowa 2 29 1 1 N/A 2.035 N/A B2 H H Iowa 1 118 393 1 297.775 220.175 11.830 B2 H H Iowa 2 101 220 5 44.232 94.955 5.930 B3 H H Iowa 1 121 381 5 413.884 147.745 15.870 B3 H H Iowa 2 125 211 1 270.920 95.785 10.400 B4 H H Iowa 1 122 197 5 360.174 169.675 30.580 B4 H H Iowa 2 144 279 6 1661.065 166.685 31.390 B5 H H Iowa 1 139 193 5 1344.333 179.695 15.740 B5 H H Iowa 2 136 173 6 692.703 121.545 10.750 B1 L L Minnesota 1 64 30 1 N/A 13.905 3.670 B1 L L Minnesota 2 94 39 1 371.823 22.135 3.060 B2 L L Minnesota 1 99 52 1 340.489 22.465 3.300 B2 L L Minnesota 2 96 30 1 785.423 27.475 3.340 B3 L L Minnesota 1 112 38 1 597.423 51.305 7.490 B3 L L Minnesota 2 125 83 1 1702.447 56.035 6.970 B4 L L Minnesota 1 116 46 1 609.957 37.985 7.770 B4 L L Minnesota 2 93 132 1 701.868 34.745 5.930 B5 L L Minnesota 1 108 78 1 1742.136 37.985 7.190 B5 L L Minnesota 2 82 39 1 282.000 32.585 5.180 B1 L H Minnesota 1 75 148 1 111.459 23.745 2.000 B1 L H Minnesota 2 71 163 1 117.032 25.185 3.230 B2 L H Minnesota 1 103 80 1 969.693 32.305 4.710 B2 L H Minnesota 2 74 135 1 167.188 32.145 3.590 B3 L H Minnesota 1 116 93 1 1515.842 55.675 10.820 B3 L H Minnesota 2 86 91 1 1064.433 37.985 7.390 B4 L H Minnesota 1 87 41 1 707.764 23.165 7.650 B4 L H Minnesota 2 76 211 1 N/A 73.995 6.060 B5 L H Minnesota 1 86 112 1 482.060 29.365 7.680 B5 L H Minnesota 2 92 128 1 844.301 45.635 6.290 B1 H L Minnesota 1 104 24 1 46.017 10.075 2.120 B1 H L Minnesota 2 115 27 1 47.603 14.385 2.390 B2 H L Minnesota 1 141 30 1 119.008 15.245 2.820 B2 H L Minnesota 2 118 17 1 101.554 12.585 2.890 B3 H L Minnesota 1 133 14 1 133.289 10.075 4.810 B3 H L Minnesota 2 129 21 1 82.512 10.755 4.260 B4 H L Minnesota 1 125 25 1 58.711 20.185 6.720 B4 H L Minnesota 2 123 21 1 106.314 21.225 5.370 B5 H L Minnesota 1 126 26 1 122.182 17.725 8.430 B5 H L Minnesota 2 129 22 1 63.471 18.095 8.300 B1 H H Minnesota 1 112 216 6 82.689 121.085 6.140 B1 H H Minnesota 2 136 206 6 1416.055 182.665 17.480 B2 H H Minnesota 1 138 430 7 1504.382 167.185 10.070 B2 H H Minnesota 2 135 352 5 768.635 218.585 18.450 160

Appendix I. (continued) Height Number of Number of Number of Aboveground Belowground Block Water Nutrient Population Replicate (cm) branches basal stems flowers biomass (g) biomass (g) B3 H H Minnesota 1 135 295 1 957.505 170.735 15.520 B3 H H Minnesota 2 136 202 1 1443.304 142.025 19.280 B4 H H Minnesota 1 148 226 7 880.453 167.185 22.190 B4 H H Minnesota 2 149 223 6 1602.106 218.585 27.860 B5 H H Minnesota 1 159 149 7 1305.176 185.185 10.470 B5 H H Minnesota 2 151 188 6 1385.046 169.755 23.880 B1 L L New York 1 103 79 1 96.173 29.405 2.090 B1 L L New York 2 102 86 1 63.113 22.455 1.840 B2 L L New York 1 101 70 1 N/A 35.595 4.440 B2 L L New York 2 85 108 1 207.372 34.195 4.770 B3 L L New York 1 91 0 1 27.049 37.745 5.720 B3 L L New York 2 106 79 1 688.235 49.675 13.860 B4 L L New York 1 104 130 1 1068.417 41.285 7.110 B4 L L New York 2 87 173 1 255.458 58.405 3.700 B5 L L New York 1 108 106 1 165.297 57.935 7.140 B5 L L New York 2 116 96 1 168.302 34.195 13.300 B1 L H New York 1 94 170 1 N/A 26.395 2.680 B1 L H New York 2 81 125 1 N/A 24.185 2.390 B2 L H New York 1 70 92 1 127.654 25.705 2.770 B2 L H New York 2 70 67 1 N/A 35.775 2.510 B3 L H New York 1 87 256 1 869.026 58.995 7.390 B3 L H New York 2 93 218 1 358.412 78.375 7.760 B4 L H New York 1 118 137 1 589.170 77.455 10.380 B4 L H New York 2 75 100 1 N/A 78.375 8.100 B5 L H New York 1 75 55 2 N/A 43.785 5.240 B5 L H New York 2 87 126 1 1428.738 24.415 N/A B1 H L New York 1 83 44 1 N/A 13.485 2.700 B1 H L New York 2 126 43 1 85.374 11.925 1.160 B2 H L New York 1 107 15 1 94.860 16.795 3.230 B2 H L New York 2 96 3 2 N/A 8.775 1.420 B3 H L New York 1 96 28 1 N/A 9.565 1.930 B3 H L New York 2 131 11 1 82.212 12.885 4.640 B4 H L New York 1 127 8 1 287.742 10.185 1.950 B4 H L New York 2 134 22 1 41.106 15.485 3.060 B5 H L New York 1 157 22 1 41.106 15.055 3.410 B5 H L New York 2 123 36 1 145.452 11.505 5.660 B1 H H New York 1 123 151 1 19.936 54.985 2.980 B1 H H New York 2 127 433 5 462.513 159.295 6.490 B2 H H New York 1 99 292 1 43.859 81.595 4.840 B2 H H New York 2 116 778 7 121.609 192.235 8.970 B3 H H New York 1 113 481 6 242.553 184.395 10.600 B3 H H New York 2 107 373 5 7.310 120.625 7.820 B4 H H New York 1 114 238 5 7.974 125.125 8.170 B4 H H New York 2 95 319 5 41.201 94.935 8.020 B5 H H New York 1 119 279 5 13.955 221.325 16.780 B5 H H New York 2 124 183 5 13.291 85.665 5.960 N/A: data not available.

161

Appendix II. Raw data from common garden experiment in Chapter 4.

B, Block; W, Water; N, Nutrient; P, Pool; REG, region; POP, population; SIB, half-sib family; Height (cm), plant height; Branches, number of branches; Stems, number of stems from root; Leaf (cm2), leaf area; NDFF, Number of days to first flowering; Biomass (g), aboveground biomass; R:B Ratio, reproductive:aboveground biomass ratio; Flowers, Number of flowers. Height Leaf Biomass R:B B W N P REG POP SIB Branches Stems NDFF Flowers (cm) (cm2) (g) Ratio B1 H H 1 IA MA 1 109 243 12 16.20 73 338.66 0.22 7258.49 B1 H H 1 NJ HV 17 88 257 10 15.20 89 245.97 0.09 3205.73 B1 H H 1 PD PO 20 71 452 15 13.11 73 171.05 0.07 2732.52 B1 H H 1 TU HA 18 72 136 5 17.16 68 74.66 0.11 N/A B1 H H 1 MI KB 8 125 200 10 17.40 72 258.58 0.26 6108.11 B1 H H 1 SW ST 4 94 74 9 11.22 65 129.63 0.48 7237.85 B1 H H 2 IA LS 1 90 133 17 12.06 86 242.55 0.16 3724.77 B1 H H 2 NJ BR 18 85 224 9 12.35 79 131.81 0.08 926.94 B1 H H 2 PD GR 2 73 77 11 16.79 75 142.45 0.10 1355.06 B1 H H 2 TU UN 19 71 392 14 14.74 79 192.87 0.03 1011.59 B1 H H 2 MI RR 2 102 200 17 12.18 68 181.45 0.39 8486.62 B1 H H 2 SW AL 1 62 16 6 8.48 62 68.58 0.26 3424.74 B1 H H 3 IA BF 20 92 111 14 14.74 68 204.21 0.34 9480.67 B1 H H 3 NJ WR 17 69 264 21 15.00 86 212.46 0.14 3945.49 B1 H H 3 PD GO 8 69 146 14 17.82 70 146.75 0.22 3815.42 B1 H H 3 TU RE 10 43 85 17 10.62 77 89.17 0.13 1712.01 B1 H H 3 MI LL 6 91 332 N/A 17.43 78 243.42 0.06 1461.54 B1 H H 3 SW NO 13 87 50 4 6.11 60 91.87 0.47 6738.21 B1 H L 1 IA MA 5 42 34 5 7.65 78 12.20 0.01 N/A B1 H L 1 NJ WR 20 80 19 3 5.61 84 11.36 0.32 684.46 B1 H L 1 PD GR 8 40 39 5 6.24 69 8.20 0.10 N/A B1 H L 1 TU UN 17 78 18 5 14.07 76 11.71 0.24 418.05 B1 H L 1 MI LL 12 64 14 5 7.44 94 11.74 0.08 111.98 B1 H L 1 SW ST 3 53 24 5 8.16 62 9.69 0.56 1069.70 B1 H L 2 IA LS 13 89 27 4 14.30 77 12.50 0.15 248.50 B1 H L 2 NJ BR 20 63 33 4 9.49 94 12.18 0.09 N/A B1 H L 2 PD GO 13 57 49 5 8.40 76 14.83 0.07 N/A B1 H L 2 TU RE 20 44 36 7 9.45 65 11.59 0.30 632.68 B1 H L 2 MI RR 9 104 24 3 10.83 73 21.94 0.47 1298.96 B1 H L 2 SW NO 16 44 22 2 4.20 62 18.51 0.54 1638.22 B1 H L 3 IA BF 2 97 29 5 10.98 78 27.86 0.12 549.64 B1 H L 3 NJ HV 1 61 38 7 9.44 106 17.18 0.08 N/A B1 H L 3 PD PO 18 39 27 7 7.65 73 8.61 0.08 N/A B1 H L 3 TU HA 12 70 25 5 5.85 73 12.32 0.18 431.86 B1 H L 3 MI KB 6 73 18 5 7.42 76 14.01 0.24 540.68 B1 H L 3 SW AL 4 51 12 6 7.95 60 7.10 0.26 282.90 B1 L H 1 IA MA 19 79 34 3 7.20 80 156.27 0.23 5806.58 B1 L H 1 NJ BR 16 86 53 10 7.42 86 189.31 0.30 10329.36 B1 L H 1 PD GO 17 N/A N/A N/A N/A N/A N/A N/A N/A B1 L H 1 TU HA 14 60 304 7 5.06 76 85.22 0.11 N/A B1 L H 1 MI KB 13 85 33 5 10.79 77 137.81 0.23 4563.56 B1 L H 1 SW ST 15 73 55 8 6.72 62 81.27 0.62 8311.61 B1 L H 2 IA BF 16 79 117 6 19.53 76 178.44 0.45 9920.62 B1 L H 2 NJ WR 15 82 141 8 23.75 76 132.60 0.43 9964.02 B1 L H 2 PD PO 19 43 158 10 8.82 70 94.24 0.30 4538.10 B1 L H 2 TU RE 1 53 160 10 15.48 81 67.74 0.14 N/A B1 L H 2 MI RR 10 75 22 1 13.68 78 46.74 0.44 3235.63 B1 L H 2 SW NO 15 75 98 7 8.82 65 73.38 0.46 6490.77 B1 L H 3 IA LS 6 92 250 9 24.38 78 246.62 0.42 9508.63 B1 L H 3 NJ HV 6 53 95 9 21.63 98 48.02 0.04 N/A B1 L H 3 PD GR 19 63 81 4 7.28 68 104.45 0.20 2375.04 B1 L H 3 TU UN 16 105 226 7 19.92 75 202.02 0.30 7730.18 B1 L H 3 MI LL 9 92 391 12 24.36 82 329.01 0.10 3407.17 B1 L H 3 SW AL 5 70 31 8 13.49 67 97.49 0.38 3909.73 B1 L L 1 IA MA 17 74 12 8 20.90 85 86.38 0.07 831.69 B1 L L 1 NJ WR 16 98 37 5 23.52 75 59.01 0.17 1557.46 B1 L L 1 PD PO 14 46 23 3 11.73 73 18.45 0.03 91.74 B1 L L 1 TU RE 4 53 42 4 13.11 68 23.14 0.33 1329.72 B1 L L 1 MI LL 3 103 26 11 20.60 72 63.58 0.28 2288.92 B1 L L 1 SW AL 3 84 17 5 8.96 66 59.27 0.47 3153.81 B1 L L 2 IA BF 17 73 19 3 22.54 92 41.70 0.17 964.88 B1 L L 2 NJ HV 20 96 41 7 22.80 85 81.73 0.10 916.46 162

Appendix II. (continued) Height Leaf Biomass R:B B W N P REG POP SIB Branches Stems NDFF Flowers (cm) (cm2) (g) Ratio B1 L L 2 PD GR 11 88 29 10 15.96 68 51.15 0.19 1235.98 B1 L L 2 TU UN 3 80 86 7 19.00 70 52.64 0.30 2161.70 B1 L L 2 MI KB 12 94 N/A 9 7.50 76 93.16 0.22 2712.51 B1 L L 2 SW ST 11 N/A N/A N/A N/A N/A N/A N/A N/A B1 L L 3 IA LS 4 75 114 7 16.80 92 41.24 0.19 1706.98 B1 L L 3 NJ BR 3 82 37 4 14.28 95 42.25 0.09 515.11 B1 L L 3 PD GO 20 78 77 6 17.22 71 83.41 0.24 1730.57 B1 L L 3 TU HA 13 69 84 5 15.39 72 58.69 0.35 2857.55 B1 L L 3 MI RR 3 77 25 3 16.79 73 76.69 0.50 3815.74 B1 L L 3 SW NO 8 69 73 6 11.84 61 31.37 0.46 N/A B2 H H 1 IA BF 2 75 281 15 12.81 77 222.51 0.16 3788.31 B2 H H 1 NJ HV 20 75 249 11 14.80 89 145.73 0.10 1456.76 B2 H H 1 PD PO 19 77 261 26 21.84 71 180.79 0.18 3529.57 B2 H H 1 TU RE 8 87 210 24 12.92 71 166.04 0.14 5451.78 B2 H H 1 MI RR 8 123 131 10 15.41 69 278.89 0.47 13636.53 B2 H H 1 SW ST 11 109 88 8 15.64 63 159.99 0.36 7980.67 B2 H H 2 IA MA 17 107 311 9 14.80 85 300.54 0.17 6712.21 B2 H H 2 NJ BR 19 67 50 9 15.75 88 98.59 0.04 460.66 B2 H H 2 PD GO 17 70 344 15 12.60 69 150.88 0.15 3257.00 B2 H H 2 TU UN 4 49 169 15 13.68 72 128.10 0.08 1792.19 B2 H H 2 MI LL 7 113 304 19 14.96 78 231.03 0.24 8344.90 B2 H H 2 SW NO 16 63 180 10 15.12 64 119.96 0.25 5222.97 B2 H H 3 IA LS 4 106 117 6 25.00 75 312.04 0.24 7950.82 B2 H H 3 NJ WR 16 111 174 9 26.73 75 243.81 0.51 14683.26 B2 H H 3 PD GR 8 92 209 13 10.35 70 230.25 0.13 2576.76 B2 H H 3 TU HA 12 52 225 9 8.45 74 95.20 0.12 1335.17 B2 H H 3 MI KB 13 79 431 12 10.01 86 265.43 0.07 4285.09 B2 H H 3 SW AL 3 81 204 9 11.70 68 131.53 0.20 2239.59 B2 H L 1 IA LS 1 46 37 5 9.35 99 13.85 0.17 342.38 B2 H L 1 NJ BR 18 64 58 4 7.84 94 18.26 0.13 438.21 B2 H L 1 PD PO 14 66 42 4 9.75 67 15.58 0.14 197.79 B2 H L 1 TU RE 10 62 18 14 6.24 72 12.08 0.12 357.47 B2 H L 1 MI KB 4 66 35 5 7.95 77 16.46 0.29 717.48 B2 H L 1 SW ST 4 76 20 3 11.20 63 11.36 0.42 459.42 B2 H L 2 IA MA 16 92 25 8 7.84 77 15.34 0.21 455.34 B2 H L 2 NJ WR 17 59 45 6 7.93 94 11.60 0.11 N/A B2 H L 2 PD GR 19 37 26 5 7.98 69 9.44 0.11 131.05 B2 H L 2 TU UN 16 64 13 3 7.28 76 13.01 0.37 N/A B2 H L 2 MI RR 10 71 13 4 9.52 79 14.19 0.36 721.46 B2 H L 2 SW AL 7 85 15 5 9.75 64 10.62 0.32 357.25 B2 H L 3 IA BF 17 86 37 7 11.90 87 22.79 0.18 353.93 B2 H L 3 NJ HV 6 45 8 7 10.08 N/A 13.69 0.00 N/A B2 H L 3 PD GO 8 54 32 6 8.48 78 17.98 0.07 100.78 B2 H L 3 TU HA 20 64 64 6 8.80 69 18.98 0.38 855.18 B2 H L 3 MI LL 3 56 18 6 7.54 82 25.52 0.28 959.41 B2 H L 3 SW NO 8 38 49 7 9.45 59 12.51 0.39 730.25 B2 L H 1 IA MA 1 99 124 8 19.09 71 193.04 0.44 23439.42 B2 L H 1 NJ HV 1 81 66 6 9.66 82 202.10 0.22 4277.54 B2 L H 1 PD GR 17 96 88 11 18.17 71 201.00 0.34 7783.20 B2 L H 1 TU UN 3 84 68 7 10.44 67 130.49 0.39 5671.67 B2 L H 1 MI RR 2 90 N/A 17 10.60 73 243.05 0.18 5436.24 B2 L H 1 SW ST 3 80 67 8 11.05 64 156.06 0.60 17429.91 B2 L H 2 IA LS 13 64 148 11 23.80 101 76.64 0.09 1078.99 B2 L H 2 NJ BR 20 N/A N/A N/A N/A N/A N/A N/A N/A B2 L H 2 PD PO 20 88 361 15 11.90 76 400.17 0.20 7161.17 B2 L H 2 TU RE 4 38 27 1 11.52 78 16.79 0.09 N/A B2 L H 2 MI LL 6 100 110 4 13.68 82 200.39 0.36 8908.70 B2 L H 2 SW NO 13 87 45 5 15.00 66 138.59 0.56 11858.57 B2 L H 3 IA BF 20 71 186 7 18.48 108 77.59 0.04 235.79 B2 L H 3 NJ WR 20 99 161 7 11.70 88 139.41 0.19 4455.85 B2 L H 3 PD GO 13 75 221 9 7.70 78 151.22 0.32 6251.54 B2 L H 3 TU HA 13 73 258 12 9.01 72 161.63 0.21 7767.25 B2 L H 3 MI KB 8 98 95 9 6.84 85 246.42 0.35 9448.47 B2 L H 3 SW AL 1 67 118 10 12.60 67 123.46 0.49 7588.39 B2 L L 1 IA LS 2 83 95 4 23.52 98 65.63 0.09 707.80 163

Appendix II. (continued) Height Leaf Biomass R:B B W N P REG POP SIB Branches Stems NDFF Flowers (cm) (cm2) (g) Ratio B2 L L 1 NJ WR 4 79 26 7 16.80 75 37.08 0.26 1680.07 B2 L L 1 PD PO 12 57 26 5 8.97 71 23.36 0.14 389.80 B2 L L 1 TU UN 17 75 29 4 9.30 69 27.78 0.36 1196.63 B2 L L 1 MI KB 6 91 123 5 11.02 72 55.00 0.31 2203.57 B2 L L 1 SW NO 4 53 30 1 3.80 60 36.44 0.55 3206.60 B2 L L 2 IA BF 14 85 12 4 14.49 88 71.58 0.11 978.98 B2 L L 2 NJ BR 16 70 22 1 11.20 92 25.26 0.11 471.06 B2 L L 2 PD GR 2 49 12 4 7.20 88 15.12 0.02 68.78 B2 L L 2 TU HA 18 55 25 6 8.85 69 39.66 0.24 1108.55 B2 L L 2 MI LL 9 111 66 1 19.20 72 110.13 0.32 3401.27 B2 L L 2 SW ST 14 62 24 1 6.63 64 33.10 0.57 3263.73 B2 L L 3 IA MA 5 74 33 1 13.32 73 66.75 0.21 1936.62 B2 L L 3 NJ HV 18 83 59 4 18.96 82 57.87 0.14 976.68 B2 L L 3 PD GO 15 49 137 10 9.66 83 53.90 0.14 983.34 B2 L L 3 TU RE 20 69 18 5 9.92 66 29.17 0.45 1511.55 B2 L L 3 MI RR 9 79 15 3 8.64 74 63.69 0.56 6045.29 B2 L L 3 SW AL 4 66 17 4 9.60 67 37.12 0.36 1354.41 B3 H H 1 IA LS 13 99 238 17 25.25 85 268.87 0.13 4583.87 B3 H H 1 NJ HV 18 80 204 N/A 31.50 72 268.98 0.03 790.77 B3 H H 1 PD PO 18 77 134 18 16.34 69 184.46 0.05 572.62 B3 H H 1 TU RE 1 47 106 N/A 10.45 75 170.66 0.02 321.03 B3 H H 1 MI LL 3 111 433 16 16.00 87 205.06 0.05 1335.67 B3 H H 1 SW NO 8 56 90 18 10.79 63 127.56 0.18 4201.03 B3 H H 2 IA BF 17 110 129 11 22.32 85 240.59 0.25 3163.17 B3 H H 2 NJ WR 20 N/A N/A N/A N/A N/A N/A N/A N/A B3 H H 2 PD GR 19 108 221 16 13.86 65 182.10 0.15 5309.28 B3 H H 2 TU HA 14 58 11 N/A 9.35 72 179.93 0.19 5092.52 B3 H H 2 MI RR 9 121 198 9 19.20 72 350.24 0.43 16002.97 B3 H H 2 SW AL 4 57 132 14 5.72 64 76.54 0.23 2509.75 B3 H H 3 IA MA 5 65 214 23 18.90 89 414.56 0.53 N/A B3 H H 3 NJ BR 16 80 96 6 6.38 89 101.59 0.09 1028.62 B3 H H 3 PD GO 15 57 14 11 10.92 73 193.11 0.04 1245.09 B3 H H 3 TU UN 3 95 16 8 15.60 65 179.87 0.07 2532.21 B3 H H 3 MI KB 4 88 369 13 16.72 78 201.82 0.11 4581.60 B3 H H 3 SW ST 14 50 55 5 2.00 64 23.75 0.25 N/A B3 H L 1 IA LS 4 74 58 4 11.36 82 20.05 0.23 693.58 B3 H L 1 NJ HV 20 N/A N/A N/A N/A N/A N/A N/A N/A B3 H L 1 PD GR 11 59 31 4 8.12 72 11.57 0.07 N/A B3 H L 1 TU UN 4 54 20 5 5.04 74 5.12 0.06 N/A B3 H L 1 MI LL 9 98 14 5 11.55 73 11.68 0.44 530.54 B3 H L 1 SW AL 5 69 16 3 7.70 70 16.67 0.28 498.38 B3 H L 2 IA BF 20 74 52 3 10.20 77 24.23 0.24 668.67 B3 H L 2 NJ BR 3 75 60 5 15.96 100 17.10 0.10 N/A B3 H L 2 PD PO 20 49 10 11 7.42 73 16.81 0.13 317.53 B3 H L 2 TU RE 4 41 65 3 4.50 67 13.39 0.36 681.49 B3 H L 2 MI RR 3 109 16 4 8.50 65 21.62 0.46 1430.16 B3 H L 2 SW NO 13 76 7 4 6.72 61 14.83 0.52 1286.33 B3 H L 3 IA MA 1 72 57 4 10.08 74 16.78 0.19 425.93 B3 H L 3 NJ WR 15 80 44 7 14.00 89 13.52 0.10 245.37 B3 H L 3 PD GO 20 30 63 6 6.63 79 8.07 0.04 N/A B3 H L 3 TU HA 18 43 34 7 9.30 65 11.16 0.21 N/A B3 H L 3 MI KB 13 74 12 5 12.00 89 12.76 0.17 266.64 B3 H L 3 SW ST 15 50 24 5 7.42 63 8.06 0.51 N/A B3 L H 1 IA MA 16 125 148 2 18.63 76 391.58 0.40 35683.63 B3 L H 1 NJ BR 19 59 78 3 21.60 94 109.51 0.25 4609.71 B3 L H 1 PD GR 8 92 136 1 15.62 80 187.16 0.35 8026.90 B3 L H 1 TU UN 17 101 73 1 22.95 70 192.99 0.50 11390.71 B3 L H 1 MI RR 8 72 159 2 9.15 68 179.32 0.08 1696.11 B3 L H 1 SW AL 7 96 62 1 17.64 75 103.28 0.50 6285.18 B3 L H 2 IA LS 2 86 134 9 27.00 92 183.78 0.24 6047.57 B3 L H 2 NJ HV 17 98 233 9 14.11 91 208.02 0.17 3258.84 B3 L H 2 PD PO 12 69 373 13 4.77 84 151.71 0.18 3932.21 B3 L H 2 TU RE 8 71 179 17 8.40 76 103.37 0.31 3776.67 B3 L H 2 MI KB 12 N/A N/A N/A N/A N/A N/A N/A N/A B3 L H 2 SW ST 4 81 69 5 12.42 69 172.33 0.52 13117.88 B3 L H 3 IA BF 14 112 222 8 16.32 86 184.42 0.19 4289.83 164

Appendix II. (continued) Height Leaf Biomass R:B B W N P REG POP SIB Branches Stems NDFF Flowers (cm) (cm2) (g) Ratio B3 L H 3 NJ WR 4 98 120 2 7.67 73 226.00 0.36 8918.79 B3 L H 3 PD GO 8 77 167 2 7.80 76 121.41 0.43 7247.73 B3 L H 3 TU HA 12 44 136 2 14.25 79 63.53 0.14 1552.36 B3 L H 3 MI LL 12 71 408 12 8.82 76 150.04 0.15 3198.96 B3 L H 3 SW NO 4 54 52 6 8.26 60 43.52 0.32 2848.95 B3 L L 1 IA BF 16 90 115 2 27.25 82 63.52 0.22 1098.77 B3 L L 1 NJ BR 20 106 37 1 17.38 85 71.19 0.21 1722.29 B3 L L 1 PD GR 17 65 76 1 13.50 71 52.47 0.26 1189.22 B3 L L 1 TU UN 19 78 96 1 14.63 71 97.48 0.33 3316.72 B3 L L 1 MI LL 7 92 91 1 18.05 86 89.93 0.27 2340.26 B3 L L 1 SW NO 15 64 58 1 3.78 64 47.45 0.32 1693.11 B3 L L 2 IA MA 19 99 51 1 10.54 90 76.57 0.17 1398.37 B3 L L 2 NJ WR 17 112 84 1 8.16 77 110.38 0.29 3764.28 B3 L L 2 PD GO 17 72 135 2 12.58 77 121.51 0.14 1642.35 B3 L L 2 TU HA 20 73 20 1 14.44 84 29.27 0.23 567.30 B3 L L 2 MI RR 10 98 32 1 18.85 76 84.74 0.35 4907.09 B3 L L 2 SW AL 1 69 37 1 8.16 71 36.98 0.37 1464.00 B3 L L 3 IA LS 6 69 100 10 18.48 94 33.00 0.10 441.06 B3 L L 3 NJ HV 6 71 31 6 20.60 96 29.83 0.11 476.27 B3 L L 3 PD PO 19 37 145 12 12.96 71 73.29 0.28 1724.32 B3 L L 3 TU RE 10 106 87 3 8.25 68 49.61 0.41 2368.83 B3 L L 3 MI KB 8 110 98 9 21.12 75 68.88 0.40 2931.34 B3 L L 3 SW ST 3 56 22 4 6.75 61 40.04 0.63 5104.21 B4 H H 1 IA BF 14 91 244 14 15.40 88 279.83 0.11 3465.97 B4 H H 1 NJ BR 20 83 274 14 22.63 102 162.90 0.06 1716.51 B4 H H 1 PD GR 17 52 305 N/A 15.01 89 172.34 0.02 343.17 B4 H H 1 TU RE 20 75 218 20 11.34 65 159.31 0.09 4680.00 B4 H H 1 MI KB 12 63 204 N/A 10.26 70 223.80 0.03 570.29 B4 H H 1 SW AL 5 58 92 8 13.68 84 78.53 0.21 2007.07 B4 H H 2 IA LS 6 80 194 9 12.41 85 244.90 0.19 5005.97 B4 H H 2 NJ WR 4 98 239 14 14.00 77 268.84 0.15 4450.21 B4 H H 2 PD GO 13 68 247 14 15.77 75 136.04 0.09 2006.45 B4 H H 2 TU UN 17 92 296 9 17.01 72 192.01 0.14 4933.09 B4 H H 2 MI RR 3 85 98 13 18.48 90 224.24 0.21 4185.88 B4 H H 2 SW NO 15 74 122 8 10.40 63 97.12 0.23 4669.72 B4 H H 3 IA MA 19 83 298 14 19.00 73 249.27 0.11 3006.90 B4 H H 3 NJ HV 6 82 293 20 19.00 94 153.82 0.03 540.62 B4 H H 3 PD PO 12 63 152 11 11.85 70 109.05 0.06 1680.73 B4 H H 3 TU HA 13 84 131 15 14.79 77 147.56 0.19 4270.67 B4 H H 3 MI LL 12 85 275 21 20.16 80 310.93 0.23 10644.44 B4 H H 3 SW ST 15 80 119 10 13.00 61 157.45 0.32 6174.68 B4 H L 1 IA LS 2 52 10 1 7.41 112 19.58 0.10 311.26 B4 H L 1 NJ HV 17 92 68 8 7.81 113 50.20 0.21 2162.96 B4 H L 1 PD GR 2 30 102 6 7.82 76 25.30 0.45 N/A B4 H L 1 TU UN 19 72 26 5 11.34 70 14.44 0.17 298.94 B4 H L 1 MI LL 7 84 6 7 7.84 89 21.34 0.35 727.47 B4 H L 1 SW NO 4 42 25 6 4.10 59 12.45 0.38 586.04 B4 H L 2 IA BF 16 50 43 2 10.80 N/A 13.28 0.00 N/A B4 H L 2 NJ WR 16 74 25 1 6.27 82 14.75 0.07 221.57 B4 H L 2 PD GO 15 68 71 5 8.12 74 9.46 0.14 242.79 B4 H L 2 TU RE 8 43 41 2 7.95 66 8.92 0.21 280.54 B4 H L 2 MI RR 8 63 20 1 10.08 82 14.66 0.35 773.43 B4 H L 2 SW AL 3 48 29 1 8.06 66 11.96 0.19 245.14 B4 H L 3 IA MA 17 37 73 3 12.07 87 19.28 0.10 350.36 B4 H L 3 NJ BR 19 49 22 4 9.15 98 11.04 0.12 N/A B4 H L 3 PD PO 19 34 22 3 4.73 76 10.73 0.11 N/A B4 H L 3 TU HA 14 34 37 3 5.39 70 10.21 0.05 76.24 B4 H L 3 MI KB 8 84 32 1 9.00 76 15.72 0.28 372.03 B4 H L 3 SW ST 11 57 24 1 5.76 64 9.34 0.23 386.24 B4 L H 1 IA MA 5 84 139 6 10.72 69 186.83 0.34 6896.80 B4 L H 1 NJ BR 3 64 200 6 15.20 92 70.31 0.07 449.75 B4 L H 1 PD GO 20 57 267 9 8.40 83 139.63 0.04 641.84 B4 L H 1 TU UN 4 48 137 13 16.94 74 65.66 0.06 384.40 B4 L H 1 MI KB 6 101 170 10 11.70 73 204.31 0.37 7493.82 B4 L H 1 SW NO 8 61 68 9 9.15 63 111.07 0.51 6523.56

165

Appendix II. (continued) Height Leaf Biomass R:B B W N P REG POP SIB Branches Stems NDFF Flowers (cm) (cm2) (g) Ratio B4 L H 2 IA LS 4 92 177 14 13.32 88 142.71 0.20 3779.12 B4 L H 2 NJ HV 18 76 34 10 11.02 77 201.60 0.21 4721.56 B4 L H 2 PD GR 11 103 155 14 8.26 73 172.52 0.20 4010.34 B4 L H 2 TU HA 20 86 175 11 12.58 67 143.13 0.34 6215.98 B4 L H 2 MI LL 3 101 196 10 15.84 73 258.92 0.41 15041.83 B4 L H 2 SW ST 14 92 38 6 11.40 64 117.12 0.70 12314.59 B4 L H 3 IA BF 2 127 297 15 16.38 76 295.29 0.46 19221.12 B4 L H 3 NJ WR 17 86 130 12 18.48 75 198.94 0.29 11252.64 B4 L H 3 PD PO 14 62 102 4 9.01 67 80.89 0.06 715.11 B4 L H 3 TU RE 10 63 104 10 11.73 70 84.21 0.06 867.45 B4 L H 3 MI RR 9 96 114 8 19.75 71 229.35 0.49 16299.36 B4 L H 3 SW AL 4 86 113 7 9.28 61 147.80 0.47 11337.42 B4 L L 1 IA BF 20 88 153 7 23.52 73 81.94 0.33 3300.17 B4 L L 1 NJ BR 18 65 78 14 22.50 92 42.92 0.19 1148.61 B4 L L 1 PD PO 18 92 21 5 13.87 71 24.15 0.04 78.57 B4 L L 1 TU RE 1 66 43 5 12.00 66 62.04 0.36 2594.29 B4 L L 1 MI KB 4 84 8 8 19.76 76 57.03 0.29 1375.11 B4 L L 1 SW ST 4 69 19 2 16.80 67 36.69 0.49 2112.67 B4 L L 2 IA LS 1 89 8 3 24.30 90 94.75 0.15 1296.66 B4 L L 2 NJ HV 1 92 68 9 17.48 75 74.67 0.10 N/A B4 L L 2 PD GO 8 69 76 10 21.39 73 46.10 0.27 1261.63 B4 L L 2 TU HA 12 62 32 1 12.16 69 39.75 0.43 2399.05 B4 L L 2 MI LL 6 124 76 6 20.24 76 93.98 0.17 1268.89 B4 L L 2 SW NO 16 61 22 3 7.04 61 44.34 0.66 4362.58 B4 L L 3 IA MA 16 75 55 5 17.85 74 79.48 0.17 1129.92 B4 L L 3 NJ WR 15 113 32 5 19.80 74 119.68 0.17 2931.87 B4 L L 3 PD GR 19 77 34 5 12.80 72 57.63 0.18 1388.48 B4 L L 3 TU UN 16 85 35 4 10.98 77 27.86 0.63 1828.30 B4 L L 3 MI RR 2 88 59 3 14.30 75 40.75 0.44 3225.23 B4 L L 3 SW AL 7 68 22 4 5.60 67 40.06 0.49 2584.66 B5 H H 1 IA LS 2 92 301 22 26.00 88 346.59 0.12 5481.46 B5 H H 1 NJ WR 15 103 209 28 20.88 76 223.33 0.22 4204.38 B5 H H 1 PD GO 20 91 138 6 17.29 70 194.67 0.24 6223.19 B5 H H 1 TU HA 20 85 112 N/A 11.88 65 183.10 0.38 9772.26 B5 H H 1 MI RR 10 94 142 17 27.84 76 217.63 0.32 9198.08 B5 H H 1 SW AL 7 69 109 12 7.41 64 130.05 0.40 7611.21 B5 H H 2 IA BF 16 78 329 N/A 27.16 88 234.54 0.03 1393.44 B5 H H 2 NJ HV 1 56 248 N/A 14.40 N/A 77.51 0.00 N/A B5 H H 2 PD GR 11 102 N/A 19 19.00 70 277.35 0.12 2502.88 B5 H H 2 TU UN 16 81 156 24 13.68 77 180.79 0.10 2209.55 B5 H H 2 MI LL 9 100 166 17 28.34 76 398.45 0.31 10119.38 B5 H H 2 SW NO 4 92 79 11 8.64 63 81.04 0.27 4458.99 B5 H H 3 IA MA 16 93 336 13 12.58 87 304.77 0.12 5213.20 B5 H H 3 NJ BR 3 82 382 27 15.87 89 220.21 0.10 4221.97 B5 H H 3 PD PO 14 61 335 22 11.20 77 182.12 0.03 609.74 B5 H H 3 TU RE 4 67 N/A N/A 15.39 69 230.03 0.18 6736.93 B5 H H 3 MI KB 6 79 65 24 17.38 86 228.36 0.08 2572.09 B5 H H 3 SW ST 3 76 52 8 8.96 62 140.05 0.35 6352.40 B5 H L 1 IA BF 14 54 5 10 10.50 96 15.84 0.08 204.77 B5 H L 1 NJ WR 4 78 24 4 9.36 83 18.75 0.11 294.06 B5 H L 1 PD GR 17 31 9 6 10.24 N/A 16.28 0.00 N/A B5 H L 1 TU RE 1 43 10 21 4.86 70 11.84 0.22 480.51 B5 H L 1 MI KB 12 31 11 13 8.54 N/A 13.24 0.00 N/A B5 H L 1 SW ST 14 51 14 5 6.00 64 14.73 0.33 556.90 B5 H L 2 IA MA 19 84 32 5 13.50 80 15.04 0.33 713.84 B5 H L 2 NJ BR 16 26 9 1 13.50 N/A 7.57 0.00 N/A B5 H L 2 PD PO 12 42 36 2 8.40 74 13.87 0.11 N/A B5 H L 2 TU UN 3 53 69 8 10.35 68 18.77 0.16 302.23 B5 H L 2 MI RR 2 58 18 3 9.66 68 14.63 0.24 527.68 B5 H L 2 SW AL 1 53 32 4 9.30 73 9.32 0.06 N/A B5 H L 3 IA LS 6 65 24 11 11.10 94 11.15 0.03 N/A B5 H L 3 NJ HV 18 70 11 8 8.64 74 14.23 0.11 266.02 B5 H L 3 PD GO 17 40 46 3 7.20 74 9.81 0.06 N/A B5 H L 3 TU HA 13 30 16 6 7.02 69 9.68 0.18 241.10 B5 H L 3 MI LL 6 117 31 6 10.50 77 15.49 0.25 480.17 B5 H L 3 SW NO 15 42 56 8 3.52 66 8.09 0.51 715.52 166

Appendix II. (continued) Height Leaf Biomass R:B B W N P REG POP SIB Branches Stems NDFF Flowers (cm) (cm2) (g) Ratio B5 L H 1 IA MA 17 109 19 5 9.28 77 161.92 0.09 829.55 B5 L H 1 NJ BR 18 71 110 4 15.40 94 84.50 0.09 319.05 B5 L H 1 PD GO 15 50 366 15 4.40 73 164.88 0.06 1264.32 B5 L H 1 TU HA 18 55 40 15 10.24 71 61.41 0.12 707.63 B5 L H 1 MI KB 4 85 230 10 8.12 82 207.90 0.14 2759.65 B5 L H 1 SW ST 11 57 9 12 8.40 63 62.15 0.44 4925.70 B5 L H 2 IA BF 17 77 88 10 11.88 90 156.83 0.07 1312.68 B5 L H 2 NJ HV 20 106 78 7 14.80 85 206.65 0.18 4640.56 B5 L H 2 PD PO 18 75 47 8 10.08 70 100.43 0.20 2628.25 B5 L H 2 TU RE 20 91 116 8 15.20 70 106.90 0.36 8052.20 B5 L H 2 MI RR 3 118 156 9 21.90 78 317.28 0.48 12746.99 B5 L H 2 SW AL 3 82 47 4 15.40 74 127.34 0.55 6900.98 B5 L H 3 IA LS 1 106 296 11 20.25 96 340.29 0.07 2227.31 B5 L H 3 NJ WR 16 124 207 16 22.80 76 274.95 0.07 2133.57 B5 L H 3 PD GR 2 78 208 13 14.63 72 129.60 0.24 4326.61 B5 L H 3 TU UN 19 85 279 11 18.69 73 201.92 0.16 3349.72 B5 L H 3 MI LL 7 87 211 10 11.36 83 88.82 0.24 1988.84 B5 L H 3 SW NO 16 66 39 4 10.45 63 57.88 0.43 2755.21 B5 L L 1 IA BF 2 73 18 9 14.82 86 74.88 0.23 1960.82 B5 L L 1 NJ BR 19 67 34 8 22.00 95 38.59 0.02 165.18 B5 L L 1 PD PO 20 49 52 2 8.40 82 60.58 0.36 2869.50 B5 L L 1 TU UN 4 54 20 5 13.02 72 28.32 0.32 1036.17 B5 L L 1 MI KB 13 51 148 7 12.60 91 36.27 0.07 355.41 B5 L L 1 SW ST 15 77 46 12 8.40 62 54.56 0.60 7079.33 B5 L L 2 IA LS 13 102 51 3 18.86 74 64.07 0.34 2752.54 B5 L L 2 NJ HV 17 81 24 5 11.90 96 30.22 0.17 632.75 B5 L L 2 PD GO 13 48 17 7 11.16 77 44.98 0.10 526.59 B5 L L 2 TU HA 14 58 12 1 5.50 80 10.86 0.15 195.77 B5 L L 2 MI LL 12 63 30 2 11.25 77 50.42 0.27 1643.49 B5 L L 2 SW NO 13 54 22 2 7.80 63 29.62 0.53 1458.80 B5 L L 3 IA MA 1 64 30 1 9.60 83 58.43 0.23 2788.45 B5 L L 3 NJ WR 20 83 32 5 19.20 85 58.90 0.19 2184.08 B5 L L 3 PD GR 8 68 23 4 13.49 65 54.64 0.28 1497.57 B5 L L 3 TU RE 8 99 23 2 7.80 70 38.31 0.35 2087.91 B5 L L 3 MI RR 8 79 26 3 10.83 73 19.88 0.36 881.21 B5 L L 3 SW AL 5 58 26 5 11.73 65 38.30 0.36 1899.12 N/A: data not available.

167

Appendix III. Raw data from AFLP experiment in Chapter 6.

The first line of each dataset indicates the region, population, half-sib family, and replicate. The next line represents the presence (1) /absence (0) binary data for 1580 polymorphic loci.

IA BF 1 1 01000100001000000110011111111101100111101101111001111001111001010100010111000001111010011101000110001011001111 0101100101000011011101100110110000110110011000011000010011000001100111101111011000001101000001100001110001101 1010000001111001010001101011000001000000000011100011000011000000000001001100001001101000000011100011000000110 000000000000000000000000000100000000000000100000011000011100000000000000010000000000100000000000100011100000 00000101100110111110011100100111111100000011001000001111110011000100101100011110011001010011110011100101111001 1001100110100000000111110000000011000000100011000100001100000110000011001001110000000111100010110000000000111 1000000110011000000011000000000001000111110011011000110110110010001100110110000001000011000000000011000000000 000111000000010000000000000100000000100001000000000000000001000101000000000010010000001101011101010100110111 10111101100111111001110111100110110100100000111100110110111010011010110000011111111001100000000111001110011001 0000001100001110110100100110000011011101110000000100001100001110000000011100000000110110001100000001001100000 001100000110000110011000100000000011001000001100000000110010000000001001000000000000000100000000010000001000 000000001001000000000000000011101111111001101101110111110100101111111110101101111110011101111111101101101001110 11100110000001011110110000001001111101000000110110110110110101100110110010111001101100001111111010011000011011 001000001100010000001100001011000000000011100000010000000001010000100000000010000000100000000000011001011000 0000000001100100000100000000000000000000000000000000

IA BF 1 2 00100000001100000110111111111101100111101101111011111001111001101100010111000001111010011011000110001011001111 0101100011001011011101100100110000110000111000011001000011000101100011101111011001001101100001100001011001101 1010000001111001110001100011000001000000000011100011000011001110000000100100001001100101100011100001000000110 000000000000000000000000000000000000000000010000001000011000000000000000010000000001000000000001100111100000 000001111001111101100111000001111111001010110110000111111100110001011111000011100110011101011100111001101110011 0011001101100000001111100000000110000001100000001000011010001100100010110011100010001111000101100110000101100 0000001100110100000110000000000110000111100010110001101101100100011001101100000010000010000010000110000000000 000110000000100000000000001000000001000001000000000000000010001010000000000010100010011010110111101001111011 01111011001111100011101111001000101001110000011001101101110100110001100000111111100011000000001110011100110010 0000010010101101100010101100000110011011100000000000011010011100000000111000000001001100011000000000011000000 011000001100001100000001000100000010000000011000000000000100000000010000000000000000000000000000000000000000 00000001001000000000000000010101111111001101101110111111101101111111110101011110100011101111101100111101001110 11110110000000011110100100001001111101100000100110110110110101100110110110111001101100001111111010011000011101 101000001100011000000000011011000000000011100000000000000000000000100000000010000000100000000000010001011000 0000000001100100000000000000000000000000000000000000

IA BF 16 1 0100000000110000001111110110110110011110010110011110000101101011010010001011000111100100001000111001010010001 1010110010100111001110110001011001010000011100000110001001100000110110010001010100000110100000000000110100010 110100000011110010010011100111100000010100010110001100010000001000000000101011010000001000000001100110000000 000000000000000000000000000000000000000000001100010110000111000000000000100100000001000000000000101001000000 00100000111001101011110110011001111111001000111010011011110000111011001111100101111110011101010110111011011000 0110010010111101100000110000100001100000001000000110011011011101101000010010001000011001101101100110100000011 100000001100011000000110000011010011000110110011100010000110000001001100000110000000100000000000000110000000 110000100000000000000000000000000000000100000000000010000000000000000011000000000000000001100111101011011101 01011111111000111110001110101111100011001111000101101110110111000011010011100110101110001100000011000011000000 000000110110100011011001011011111000000100011000000000000000000011000000000000000000000011000110000000000000 000000110000110000000000000001000000001100000000101000000000001000000000000000000000000000000000000000000000 0000000000001001000000000000110011011111000010111010101111001101011001101110010110101100011111001101000111000 00011101110110000010100110011000001111101101100000100110100110111101100110110011001000101100001111101010011000 001011001100000010001000000100000011000000000110000000000011000000000001000000000000000000000000000000000000 0001000000000000000000000000000000000011000000000000000010

IA BF 16 2 0100000100000000001101110110110111111110000110011110000101111010110010000000000111100000011001011010100001001 1010110001100011010110110001011000011000011001000110001001111100110110110000100110100110110000000000110110010 110100000011110000101011101110100000001100010110000100010000100000000000101011010000001000000001100010000000 00000000000000000000000000000000000000000000010001001000010100000000000010010000000100000000000010100100000 000100011111001101111110110011001111111000000110111000001110000111111001111100111011110011100110100111011011100 0110000010111101101000110100000001100000000000000110011011011101101100011010011000011001101101100110100000011 100000000001011100000110000010110011000110110001000000000110000001001100000110001000100000000000001100000000 110000100000000000000000000000000000000100000000000100000000000000000001000000000010000001100111101011011111 0100111111100011111000111010110010001100101100000110011011011100001101001100010010111000110000001100000100000 000000011011010001100000001001111100000010001100000000000000000001100000000000000000000001100011000000000000 000000011000011000000000000000000000000000000000000000000000000100000000000000000000000000000000000000000000 0000000000000000110000000000010001101111100001011101110111100110011100100111000011010010001101101110100011000 168

0000110011101100000011101101100000110111011011000001001101001101101011001101101100110001011000011111000100110 000110100011000001000100000010000000010000000001100000000000100000000000010000000000000000000000000000000000 000000000000000000000000000000000000000000000000000000000000

IA BF 17 1 01000000000000000000111111111101110110101100011101100000011111001100111110010001100000011011101110011000001110 1111100010000110011101100000110000000011110011001100010001000001101101010011001100101101000000000001101001111 1010000110011000110001101110011100110000001011011110000000001000000000000101011000000100000000110001000000000 110000000000110000000000000000000000000000110011000100010010000000000100000000000001000000000000100100000000 001011111011111111110111011001111111000000110010000001111100011011101011000101110110111100110110110101111100011 0010110111001000110001100000000001100000000000111010011110001100011000110001000000000011000110110000000011100 000000000110000000110000000000010101100110000011000110110000001000100000010001100000000000000000011000000000 000100000000010000000000000000000000100000000000000000000010000000011000100001000000001100111101001001110110 01111011100111110011100111001000010111111000101111011001111010011010110101110101111001100000000001101110000001 1001110001000110110000100110000000011010110000000000001100000110000000110000000110000110011100000000011000000 001100001110000000000000010010000011000000000100000000000011000000001001000000000000000000000000000000000000 00100000000100100000000000001111111110001011101110111100010111100010111010011110011100111101111100011101100011 01111011000111101111110100011010110110011000010011011011011110101011011001011000101110000111010001001100000101 000000000001001000000110110000000000000001000010000000000000000000000000000000001100000000000000000100000100 00000000001000100000000000000000001000000000000000000

IA BF 17 2 00100000000000000000011111101101110110101100001011100000011111001100111110010001100000011011001110011000001110 1111100011000110011010110000110000000111100111001100000011100001101101010011001100101101000000000001001101111 101000011001100011000110111000010011000000101101110100000000000000000000010101100000010000000011000100000000 011000000000001000000000000000000000000000010000100010001001010000000010000000000000010000000000100010000000 000101001101111110110111101100111111100000011001000000111101001101111101110011111011011010011011011010111110001 1001011011011100111000110000000000110000000000011101001111100110101110011000100000000101100001011000000000110 000000000011000000011000000000001010010011000001100011011000000100010000001000110000000000000000001100000000 000100000000001000000000000000000000010000000000000000000001000000000100000000100000000110011110100100111101 0010110111001111100111001110000000101010100000011110110011110000110101100001101011110011000000000010011100000 011001100001000110110000100110000000011010110000000000001100000110000001100000000100000110011000000000011000 000001100001110000000000000000110000001000000000000000000000011000000001000000000000000000000000000000000000 00000000000000110000000000000001111111110001011100100111100010111100011111000011110011100111101110100011000100 01001111011000100011110110100000010110110011000010011011011011111011011011001111000011110000110100001001000001 001010000000011001000000100000000000000000011000010000000000000000000000000000000001100000000000000000000000 11000000000000000000000000000000000000000000000000000000

IA LS 6 1 01001100010000000011111111111001110110100001111011110001000111001100110000011001111010000011010110011001011011 11010000101001110110111001101100000000010101100011000100110110011011110101110011101111010000110000011000110111 0110001111110011100011111101100001100000000000000110000110000000010000000001110000110000111001000110000000000 000011000000000000000000001000000000000000100000011000010000000000000000100000000000000100000001011000000000 000111111111011011111110110011011110000001100100001011111001111111111011001110101100110011101101110110101100110 01011011010001011111110000000100110001111001101001001110011010110111011000111000000111100110101011000000100001 1000000000100000000000010001111000100110001000000000110000000001101111010110100000000000000000110110000110011 100000000000000000000000000000100000000000000100000000101000000001010000010100000000110011010101101111011101 1011100010111100111010100010001100001100000110011011111101001101001110010000111101110011000000000001000110110 0110100110011011000000011000001100101011000011000000000000111000000111100000000001011000100000001100000000000 110001011000000000000010011000000100100000010000000000001000000000100000000010000000000000000000000000000000 00000010010000000000000000110011111100110111011101110011110011011111100011111001110011010111001000110000101000 11101101011010111100100110011110100011000001000001101101111101101101000101110001111000110011100100111000100110 000000001100110000000000000000000000001100000100000000000010000001100000000100000000000000000000000000011000 000000010000100000000000000000001000000000000000010

IA LS 6 2 01000000000000000001011111111001110110100001011101110001111101011100110000011001111010000010010110011001011011 11010000111000101111011000111100000001011011100011101100110110011011110101110011101111010000111000010001110111 0110001111110011000011111101100001100000010000000110000110001000010000000000110000110000111001000110000000000 000011010000000000000000001000000000000001100000011000010000000000000000100000000000000100000001001000000000 10110011101111101111111011001101111000000110010001101111000101110111101101011000110011001110110010011010110011 0001100111100100111101100000001001110011110011010011011100110100101110110001110000001110001101010110000001000 001000000000100000100000010001111000101100001000000000110000000000101111010110100000000000000001110110000110 011100000000110000000000000000000010000000000000100000000101000000011001000000010000001100111101011011101111 01011111000111110001110111001100110000011000001100110111111010011010001100100001111011100110000000000010001101 100110100110011011000000011000001100101011000011000000010000111000000111000000000001011000100000001100000000 000110000111000000000000010011000000000100000010000000000001000000000100000000000000000000000000000000000000 00000000000011100000000000000110111111001101110111011110111000110011111100110111011010011010111011000110000101 0010110110001001011110010010010111010001100000100000110110111110010110000010111000111100010101110010011100010 010000000000110010000000000000000000000000110000100000000000001000000110000000010000000000000000000000000001 169

1100000000000001000000000000000000001000000000000000010

IA LS 13 1 01000011001000000000111111101101110111100011011011110001101111001100110000000101111001100110011001001001011010 1101000010000111000011100000110000110000100010000000010010011001101110111100101101101101100000011100001001001 101000011101100000100010010000110000000000000000001101100000000000000000111000010000110000000000110000000000 000000110000000011000000000000000000000100011000000100000010000010000000000000000010000000000010010010000000 10000100111111110111101110110111111110100001101100110111010000000111001100001110011000101000110101110011111101 1100000001100001001100011000000001000000111100011100110111100011010110101110100000110000011011001100000101110 0000000111000000000000000011000111100011011001101001000011000000000110110111011110011000001100000011000000000 000000000000001000000000000000000010000000000000010000000010010000000100000001001000000011001101110110110101 11011011100010111000111011111011011000111100000110101010001100001100011000111100111000110011001000000110000000 110011000110001101000111001110000000010000000000110001000000011000000000000000000010101100011000000000011000 000011000101100000000000000001100000010000000010100000000000100000000010000000000000000000000000000000000000 00000000001010011000000000110001011011110001011100110111001110101111111111010111110110001001101111100011001001 1001011011100001011111011001101101110010110000010011011011011000110010011011001100110100001100110001001100001 101100110000001000000000100000001100000000011000000000110000000000000110000000011000001100001100000000100000 10000000000000001100000000000000000011000000000000000000

IA LS 13 2 0000011001011010111000110011110010101100000010111100100010010100110011000001010111100110001100011000010101101 0010100011000011100001110000011000011001010001000000101001001100110110010110011100110110110000001110000111100 010100001111110000011001001100011000111000000000000110110000000000000000011100001000001000000000010000000000 000000011000000000100000000000000000000010000100000010000001100001000000001000000001000000000001000000000011 01011100000010110010101100101011110111101000110010011011111000000001110110100111000110011100010100011001101110 1110000000010100000110001100000001100001011110001110011011110101101011010111101000011000001101100010100001101 1000000011100011000001000001100011110001011100010000000001100000000011011011101101001100000110000000100000000 000000000000000100000000000000000001000000000000001000000001010000000010000000000000000010001111111100111011 00110111100010111100011101111101100110001110000011001101000110000110001100001110011100011001100000000011100000 011001100011000110000001100111000000001000000000011000100000000100000000000000000001100110001100000000001100 000001100001110000000000000000110000001000000000100000000000010000000011000000000000000000000000000000000000 0000000000000000000100000001000111110110000111100110111100111000110110101110101111001100011010111011000110000 0110011010110000000111111110010011011100101100000100110110110100001100100110110011011101100011001000010011000 011010000000000010000000001000000011000000000110000000001100000000000001100000000110000011000011000000000000 0110000000000000000000000000000000000000000000000000000000

IA LS 17 1 01000000000000000001011111100001101110101100000101110111001111001110010111000001111000000010000111000101011011 1011000010110000011001100000110000111000000010000000010011010001101101101101001001001101100011000000001001011 101000011001100000100111001011011000000000000010000000110000011110000101110100010000100000000011000100000000 000000110000000011000000001000000000010000001000101100001011000000000010000010000000000000000000010010000000 000001001110111101111011101100110111100110110011000111111110101101101100100011110010011110111111001101100110011 1001100110000010011111110000000000110000100000011001101111011010000011011100100001100001100110011000000001110 0000000001001100100000000111100011001100110001000000000110000000111101100110110111000000000000000010010000110 001100000000000000000000000000000000100000000000100000100101000000000000000000010000001101011011011100111011 10110101000111111001110101100110011000011100001101100011111010011011011000000111111001110110000110001010000110 000110000000011001000110011000000000101000000000000000000000011000000000000000000011011110110000000000000000 000110000011000000000000000011000000100000000000000000000001000000000100000000000000000000000000000000000000 00000000010000000000000001100011111101110111111110101101100101111011111111010101001000101110110011001110000001 1010100110000001111001111000010101111101100000100110110110111001110110000110011001001000001011011010011000011 011101100000110001011000000000011000110000010000110000000001000000001100000000000000000000001000000000000011 0000000000000000000000000000000000001000000000000000000

IA LS 17 2 0010000000000000001101110110000110111010100000010111001000110100110101011100000110100000001100011101010101101 1101100001011000001100110000011000011000000000000110001001011000110110110110100110100110110000100000000100111 110100001100110000010011000101100100000000000001000000011000001111000010111010001000010000000001100010000000 000000011000000000100000001100000000001000000100010110000010100000000001000000000000000000000000011001000000 000000000111111111011101110110001011110011011101100011111110110110110110010001111011001111001111100110010011001 1100110011000000101111111000000000011000010000001100110111101111010001101110010000110000110011001110000000011 000000000010011011000000001111000110011001100010000000001100000001101011001100101101000000000000000100100001 100001000000000000000000000000000000001000000000001000001001001000000010000000000000000010011101011010011101 10011010110001111100011101101001100110000111000011011000111110010110100000000001111110011101100001100010100001 100001100001000110011000000110000000001010000000000000000000000010000000000000000000110111001100000000000000 000001100000110000000000000000110000001000000000000000000000010000000001000000000000000000000000000000000000 00000000000000110000000000011001111110110001101111110111110010111100111111110101010011001111101100110010000000 0110111001100000011110011110100100011111011000001001101101101110011001000000100110011001000010110110100110000 110110011000000100000110000000000110001000000100001100100000010000000011000000000000000000000000000000000000 110000000000100000000000000000000000000001000000000000000

170

IA MA 19 1 0001011000000000000100111010001111010010001001011011101000110101100010011000000100001001111000100010111011010 110100000100001010111011000001100001000111010100011000100110000100010001011010001101011000001000000000110010 001011000110110000011001100100000100100000000000000000000001100100010101000100011000001100001100000001000000 000000000000000000000000100000000000100000110100000010000000100000000000000000000000000000000000000001000000 00001000111101100010111011111000111101100110010010000001101110000011011011001010100010011100111100101100110100 0011001110110001000000101100000000000101011000011000000001111011000000101101001000000000000110010001011101011 100001101100000011100000001000000000100011001000100100001001000000000100010000000000110000000000000000000000 000000010000000001000000000000000000000000000000000001000000000000000000100000000000000000110011110101101101 01110110101100011111000111010111011001110111100000111101100011100001100010100001000111100111000000000001110001 000110011010000001100000000001100000010010001100000000000000000010000000010000000000000001100010000000000000 000000011000001000000000000000001000000000000000000000000000000100000000000000000000000000000000000000000000 00000000000000001001000000000100011011111100011010101001111011101111001111110001101110110001011011101100110001 0011000111011000011001100001100001010110110110000010011001011011100110010011111001000010110000111100001001100 001001100110000010000000000000000000000000000010100000000000000100000000100000000000000000000000000000000100 00011000000000000000000000000000000000001000000000000000000

IA MA 19 2 1000000000000000001010111110000111011010001000111011100000101101100011011000000100000001111001110000101110101 1100100001000001101110110000011000011001011001000110001000000001000100110110100011001110000010000000001100010 0101100011111000001100110010000110011000000000000000000000110011001011100010010110000110001110000000100000000 000000000000000000000010000000000010000110110000001000001110000000000000000000000000000000000000100010000010 00100001111110001011101110110011111110011000100100000111110100000111100100101110000000111011101001110010111100 1100101111000010000011110000000000011101100001100000000111100100000011001100100000000000001001000101101010110 000110110000001010000001000000000110001101100010001100100100000001101011000000000001100000000000000000000000 000001000000000100000000000000000000000000000000000100000000000000000010000000000000000011001111111100110011 1001100110001011110001110101110100011101111000001111011000111000011000100000000001111001100000000000000100010 001000110100000011000000000011000000000100010000000000000000000110000000110000000000000011001110000000000000 000000110000110000000000000000011000000000000000000000000000001000000000000000000000000000000000000000000000 0000000000000000000000000000100011011111100011100011011110110111011001010100001101100110001110111101000110000 0001000111011000011001110001100000110110110110000010011110011011000110010011011000000101110000111100001001100 001010000100000010000000000000000000000000000011000000000000000000000000110000000000000000100000000000001100 00110000000000000000000000000000000000000000000000000000000

MI KB 4 1 01000000000000100000011111111101110111000011011111011100111101001100110100000001111000110011010111011101111011 0011000010011010011010001100110000110110111100011001010010000101101000100000001001011101000100000001001101111 1011000001111001111001110110110000110000000000000011011001111011110011001011100110001001000001101001000000000 011000001010000000001100000100000000000001110000110000001000100000000000100000000000000000000000100100000000 011000111001111011110111011001111111000100111110000011111000100011111110000110110110011100110100110000001100111 1010000111100000110110000011000011001000000010110000011101001101001110111010100000001011101101110100000011000 000001000011100000000000000000100110100110001000000000110000000000101100010000001000000000011000000000000110 001011000000000000000000000000000000010000000000010000000100000000001000000010010100000110011100111101101011 00110111000111110011011111001101110110111000001101111111111010011010011110001101110001100110011000001110001101 0001100001011110100001100011110000011000000000000000001100001010000000011000000110000110011100000000000000000 001100000110000000000001100110000001100000001100000000000110000001011001000000000000000000000000000000010000 00000000100101000011100000101100101111100101110111011111011110111111111001101100111110110101110001101100100110 0011001110110011110000100000010111010011101011001101011101100011001101001100110101000001110111000100110000110 110010000001000110110000010000000000000001100000100000110000000000011000000000000110001000000000000010000111 000100000000000000000000000000000010000000000010000010

MI KB 4 2 01000000001100100110011111111101110111000011011111111100010101001000110100000001111001110110001011011101011011 0011000010011010011010001100110000110110011000011000001100010101100100010000001000101101000000000001000101111 1011000001111001111001110110110000110000000000000011011001101111110001001001100011001001100001101101000000000 011000001010000000000100000000000000000001010000010000001000100000000000100000000000000000000000101100100101 110101111011111011110111011001111111000100111110000011111000100011111110001101010100011000110100110100001100111 1010100111010101110111000011010011000001100010110000011010101101100110110011000000001011101101010111000111000 000001100011000000000000000000000101100110001000000000110010000000101100010000011000000000011000000000000110 001011000000010000000000000000000000100000000000100000000100000000000110101011110100000110011100111101101110 00110111000111110011011111001011101110111000001101110111111010011010011110001101110001100110011000000010001111 0001100001011110100001100111110000001000000100000001010001100110000000011000000110000110011100000000001000000 001100000110000000000000100110000001100000000100000000000100000001011001000000000000000000000000000000000000 00000000100101001110001101101100101111000101110111011111011110111111111001101100111000111111110001101100000110 00110011101100111100011001000111110100110011010011110111111100110111010011001101110100001101110001001100001101 100000000001001101000000100000000000000011000110000001100000000000110000000000001100110000000000000100001100 00100000000000000000000000100000010000000000001000000

MI KB 8 1 010010001001010000001111111111011101110000011110111111001110011101001111110000011110110001100011111110110110110 171

1110101010000100110111011001100011101011000100000011100110101000110111010111010000011100000111000110000110111 0110000010110001100011001101011111000000000000001100110000000110000000000011011000011010000011000110000000000 110000111100000000001000010000000001010011100000010001101000000000000000000000000000000000000011011000100100 101111110111111111101110110111111110101001101100010111110100010111101110011010111101110000110101010110011001110 0010000110101001101110011000010110110101111100000010110101011011001100100110011000000110011001100111100110000 000000000110000000000000000000000001011000010011000001100000111101011001110000010000000000000000000000000000 010111000000000000000000000000000001000000000000000000000000000010010100100101101000001100111011011011111010 01101110001011100011101101111001011001110001011101011011110100111011110011011011100011000000110000011000001010 001100001101110011001000111000011001001100000000000000000001110000000001000000000010000011100000000000000000 001101001100000110000100000110000010010000000000000000000010000000000001000000000000000000000000000000000000 00000000100100000000001000001100111111000101100111011111101110110110111110101110110001110001100110111100110110 01000011100001001111100001101100110000100000010011011111011100110011110011011000110100001101110001001100001110 000100000010001100000000110000000000100011000110000001100000000000110000000000001100000000000000000100000110 00000000000000000000000000100000000000000000000000000

MI KB 8 2 010010000000000000000111111111111101110000011111101111010110111110001011110110011110010001100011110110110110110 1110001010000110110111011001100000101011000100010011000110101110110111001111011000011101000011000110000010111 0110000010110000110011000101011110100000000000001100110000001110000000000011110000011010000001000010000000000 110000111100000000001000010000000001001010100000010001101000000000000000000000000000000000000011011000100100 101111110011111111101110110111111110000101101100000111101110011111101100000110111111111011110101110110011001110 0001001110110001101110011000011110010011111100000010110011011011000101000011011000000110001001101011000110000 000000000110000000010000000000001001011000010011000001100000111101011001110000110000000000000000000000000000 0101110000001000000000000000000000010000000000000000000000000000001110100000111010000110010111011100111110100 11011100010111000111011011010001110001100010111010110111101001110111100110110111000110000001100000110000010100 0111000110111001100110011100001100100110000000000000000000111000000001100000000001000001110000000000000000000 110100111000011000110000011000001001000000000000000000001000000000000100000000000000000000000000000000100000 00000000101100000000100000110111111100011010011101111110111011011011111001111001100011000110011001110011011011 1000110100010011111000011011001100001000000100110111110111001000111100110110001101000010111000010011000011010 001100000100011000000001100000000000000110000100000011000000000000100000000000011000010000000000001000001000 0000000000000100000000000000000000000000000000000000

MI KB 12 1 01000101010101001000111111111101110111111011011010110001101011001100110100010101111000100011011110011011111011 1111010110000111011011100010110000010001001010000000010011000100101001100001011001011100100010000001111111011 1011000001010000110001111111000110110001000000001101011000000000110000000000000110001100000001100011000000000 000000000000000000000000000000000000100110100001011000111000000000000000010000000000000000000000100100010011 100111111011111111111110011001111111001100111011001011111100111011011110101101000101110101111010110011011100011 0001111000101100110001000000001111000000000000000010011101101101000110000011000000000011101101100110000100000 000010110011001000000011000000001000111001110000000100110000000110101100001100001100000000000000000000000000 000001100000000000000000000000000000100000000000100100000000000001000010000000010100000110011110111101111011 00110111000110010011010110011100110110011100001100011011111000011010011110101111110000000011100000011100011101 1001100001100110000001100111110000001000110000001000001000001110000001100011000100001100011100000000000000000 001100000110000000000100000110000001000000000100000000000100000000011001000000000000000000000000000000000000 00000001001000100000000000110100111111010101100111011101101010110011111011101110010001101101110100101100100011 11000011000000111111011000000111111110110001010111010011011000111010010011011010100110001011100001001100001100 001100000110001000000000000000000000000000000000000001100000000000011000000000001100000000000000000000000100 00000000000100000000000000000000000000000000000000000

MI KB 12 2 01000100110101000000110011111101110111100111011011010001100011001100110000010001111000100110011011001011011011 01110001100001111110011001011100000111100110100010000100110111001011011001010110010111001001010001011111110111 011000001011000010001111110010110110001000000001100011000000010110000000000000010001000000001100001000000000 000000000000000000000000000000000000100111010001010000010100000000000000010000000000000000000010100110010010 101111111011111011111110011001111111000000110111001011111000101011011111101111000110010100111000110110111100011 0010010111000100110001000000011011000000000000000000011101101100000110110011000110000011101101100111000011000 000001110011001000000011000000001000111000110000000000110000000111000100001100001100000000000000000000000000 000101100000000000000000000000000000100000000000100100000000000000000010000000110100010110011000111101111111 00111001000110110011010110001100110110011100001111011011111000010110011110101111110000000011100000011100011101 1001100001100110000001100111110000001000110000001000001100000110000001100011000000001100011100000000000000000 001100000110000000000100010110000001001000000100000000000010000000001001000010000000000000000000000000000000 00000000100100000000000000111100111111011101100101011101101010111011111011101110010000101101110101011100110011 01100011000000111110111000001001111110100001010111101011011000110110010001011001111110001011000001001100001101 000100000010001000000000010000000000100000100000000001100000000000011000000000001100000000000000010000100100 00010000000100000000000000000000001000000000001000010

MI KB 18 1 01000000000000000001111111111111101111100011111011011000110110101100111111011001111000011010010111101001100011 0111100010010110011011100011110011000101101011000001100100000111000001100000011000011111000000000000111000011 1010000011110000010001100110100110110000010110101100011000011011000000000000110100101110011100000001000100000 172

000000000000000000001100000100000000000101110000000000010010000000000000010000000000000000000000100100000000 000011111111111111110110011001111111011010110011011001111100100010111101101111110111111000010001110011011100011 1010100111100101110110000001110111001011001100000000011110101100000110110111000000000011000001010100000011100 011000000001100000000000001101100000110100011000000010010000000001101100110100000000000000011000011000000110 000001100000000000000000000000000000100000000000000000000001000001001001000000011101000100011100111100111111 10110111000111110001110101110100110100011000001101000010111000011011001010101101110001110001011000001110011001 100110001101111010000000011011000001100010000000000000000110110000000000000000010000110000110000000000000000 000110000011000000000000000010000001100000000000000000000001100000000000000000000000000000000000000000000000 0000000001001000000101000000011000011110001011101110111000011101100111110001011100100011101011101110100000001 1100110011000000001110011000001100110001100101010011011110011100110010110011011000110100001100100001001100001 101000000000010001001100000110000000000010011000000000001000000000000011000000000001100000000000000000000000 10000000000000000000000000000000000000000000000000000000

MI KB 18 2 00100000000000100000011111111110100111100011111011011000110110101100011111011001111000011011001111101001100001 0111000010000110011011100011110011000001101011000001100001000001000000100000001110011101000001000000101100011 1010000011011000011001100010100110110000000110101100011000011011000000000000111000101101011100000001000100000 000000001000000000000100000100000000000101010000000000010011000000000000010000000000000000000000100000000000 000111111101111011110110011001111111011100110011011001111110101011111101101111001110011000110000110011011100011 1010100111100100110110100001111111001010000100011000011010101100001110110111000000000011000001110100000011100 011000000001100000010000001101100000110110001000000010010000000000101100010100000000000000011000011000000110 000111100000000000000000000000000000100000000000000000000001000000001000000000010000000110011100111100111011 10110111000111110001110110110100011100011000101111000010111000011011101010101101110001110001011000001100011001 100110001101111010000000011011000001100010000000000000000010110000000000000000010000110000110000000000000000 000110000011000000000000000011000001100000000000000000000001000000000000100000000000000000000000000000000000 00000000010010000000010000000110000111100110111011101110000111011001111100010111001000111010111011001100010011 1001100110000000111100110000001001100011001101100111111110111101100100100110110000101000010111000010011000011 000001000000100001011000001100000000000110110000000000010000000000000100000000000011000000000000000000010011 0000000000000000000000000000000000000000000000000000000

MIKB 20 1 00100000000000000000111010111101100110100011011010111011110110111100011111011011111000000110001110011101011010 1111000110010010111011000000110000100001101100100000010011100110001001100001001110101101000010000001011011001 011100011001000001100110011000011011000000000000001101100000001100000000100000000001110000000010000100000000 01000000010000000000000000000000000000010001000000000000100000000000100000100000000000000000000101001000000 010000000110111110111111110110011111110111001101100000111111011010101101101001110111101110001101101101110111000 1100101100110000110110010000000000110000110001100000000110100011011111101100110000000010111001011001110011010 100110110010010000001100000011011000001101100010100000001100100001001011001100000010000000000000000000000000 100010010000000100100000000000000000001000000000000100000001000001000000000000000001010001100110111011011011 0110110101000111010000110111100100011100111000001100011001111000011011000000000001110001100000000000001000001 101000110100000111001100000001111000001100010000000000000000001010000000000000000000000000000100000000000000 000000100000010000000000000000010000000000000000000000000000011000000000000000000000000000000000000000000000 0000000000001000000000000000100001001011110011011101100110100111101100111111101011100110001101101100010110000 0011100111011000101000011001000000101110010100000100010010011010000111010110011001000101000001011100001001100 001001001001000010001000000000110000000000011011000110000001000000000000000000000000000000000000000000000000 00010000000000001000000000000000000000000000000000000010000

MI KB 20 2 00111000000100000000110011111101100110111011011011111001111001110100111110011011111001000110010110011101011010 1111000010010000111011001000110000100101101100001110010011100110000100100000101110011100100010000001111001011 0111000110011000011001100110000110110000000000000001011000000011000000001000000000011100000001100011000000000 100000001000000000000000000000000000001000110000000000010101000000010000010000000000000000000001100100000000 000011111001111011111111011001111111010110110101001011101101101110110110100101011110111000011010110101111100011 00101000110000111110000001000000110000110001100010000111111011011110101100110000000010111001111001111111010100 110011010101000001100000011011000001101100010000000001100100001011011001100000010000000000000000000010001100 010011000000100100000000000000000001000000000000010000000100001010110000000000101110011000111111110011001101 0110101000111010001110111100100011100011000001100111001111000011011011001101001110001110000000000001000101101 100110010100111001100100001101001000100010000000000000000000110000000000000000000000000000110000000000000000 000110000011000000000000000010000000100000000000000000000011000000000000000000000000000000000000000000000000 00000000000010000000100001100110011111001011011011101101001111011001111110011111001100011010111001101100000011 1001110110001010010110010000001011100101100001100110010110110011110100100010110000100000001111000010011100011 010011010000100010000000001100000000000100110000100000011000000000000000000000000000000000000000000100000001 0000000000000000000000000000000000000000000000000010000

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

0010001101101101101111000011101011010000110110001000000101101100011101110011001100010011001011101001000000010 0000011001111000011001100000000001101101000010000000111110110110011011000100000001000000000110000001100000000 011011000000100000000000100000000001000000000001000000000000000011100000000000100011010101111110110110001000 1011011000101110011010111101100110111011000011001011100111010010100011010001101110000000001110000011100001101 0001100001000111100001100110110001001000110000000001100000001110000001100000000100000111001100000000000000000 001100000110000000011000000110000000001000000100000000000010000000000001000000000000000000000000000000000000 0000000000110100000010000000100001111000110110101011111001001110101111000011101011000101111111000110110000011 01100001100000110100011100000110011011111000011001101101101100111001110001101100111011000110011110100110110110 100000000011000100010000011000110000000000000000000000000000000000000000000000000110000000000000000000000110 000100000000000000000000000000000000000000000000000000

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

000100000000011000000000000010000000000000000000010000000000000000111000010001000000001100011000110001101010 0011010110001101100110101110001001101111010000110110110001110101101010110100011011110011000011010000001000000 0110011000010111101100011001101100111010001000000000000000011011000000000010001100000011110110000000000000000 000011000001100010000010000001100000011000000000001000000000100000000000000000000000000000000000000100000000 0011000000001100000000100000111100010110010011101010111101010011100111111000001010110001101101110100110001000 1100100001000000110110001100000110010011111000101001101101111001100001111001101100011010000110111000100110000 111100110000011000010000000111000110000001100000000011000001000000000001100000000000110000000000000000001100 100000100000000000000000000000000000001000000000000000000

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

010001000000000100110000000110000000001000110000000000000011001100000000000000000000001100001100000000000000 000001000000110000000000000000100000000000000000000000000000010000000000000000000000000000000000000000000000 000000000000000000000000000000011000111100010111001100011100100011000010100000110101100001101001100001000000 0001001000101000111111000001100000001110001100001010010011011010001100011100011101000110100001101000001001100 001100000000000010000000000000110000000000000011000000000000010000000000100000000000001000000000000000000010 00000000000000000000000000000000000000000000000000000000000

MI LL 12 2 01000100100100000000010010111101101111100000011011011101110011010100011100011011111000110110010111011011011011 11001101011001111110100001011100000101110110000011011101011001001110010100010011001111110000010000011000010111 0110000011110110110011111100001111100000001100100110000000001110000000000001100000011011000011000010010000000 000000000000000000000000000000000000001001100000100000001000000000000000000000000000000000001001001100100000 001110110111110111111110110011111110000101111100110011111011010110100111010110001101110001110101010110110000110 0101011010010001101110000010000111111100000001100000111011011011011001100100011010000110010011000000001111000 001000001100000010100000000110000100011100010000000001100110001101011001000011000000000000110000000011000000 000001000000100000000000000000000000000000000000000000000010000001100000000000100000110001011111001111010000 1110110001111110011010111101100110110010010011000011100111000011000011110000001110001100000000000000100000101 000100000000011011000000011000000000000011000000000000001100110000000000000000000000110000110000000000000000 000100000011000000000000000011000000000000000000000000000001000000000000000000000000000000000000000000000000 0000000000000000000000000000010001111110101110010000110000000011001111100000110101100001001001100001000000001 1000001101000111010100001100000010110001100001010010011011010000000011010011100000110100001101000001001100001 100000000000010000000000000110000000000000011000000000000000000000000110000000000000000000000000000000000000 00000000000000000000000000000000000000000000000000000000

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

MI LL 13 2 00000000000000000000001001111000011001111000000111101001110101001100111110011001111000110110011100111011011011 0011000110000110011110000110110001000111000010000001100111001110011001010000101110101001000000000010011001101 101100000101100001100111011000001100000000000001001001100000000000000000000011000000111000001110000100010000 000111000000100000000000000000000000010100101000100100000110000000000000010100000000000000000010010000100000 000000111110111000011111111011001110110001101100101100110110000111111111100001100011001100001101011011110110011 1001010001100100011100100001101011000100011000011000101111100000101111001001000110000111100110110011011001110 000010110001110000000000000000000000011100000100000000011001100100110110001000110000000000000000000000000011 000100110000000000000000000000000000001000000000000000001000100000011000000000000000000101011101111011001100 00111011000101111001101011100110011011001100001100011100011100001101101111000110111000000001110000000110000100 100011000000001101100000001001100000110001100000000000000011001000000000000000000000011000011000000000000000 000010000001100000000000000001100000011000000000000000000001100000000000000000000000000000000000000000000000 00000000000011000000000000110010001111100001110011010111100101011011111100111011101100001011001100011011000001 1011000011000001101100011000000100110111100011010011011011011000110011100011011000110000001100110001000100001 100001000000010000010000000110000000000000011000000000000000000000000000000000000001100000000000000000010001 00000000000000000000000000000000000000000000000000000000

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

0000000000011000000000000000010001111000011110011010110110111011001100100011011110100011010011100001011000001 1111000011000001110000000110001100110111101001010011000011010000000010010011001100110000001100101101001100001 100000000000110000000000000000000100000000000000000000000011000000000100000000000001000000000000000000011001 00000000000000000000000000000000000001000000000000000000

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

MI LL 19 1 01000000000000100000011011100001100111100001011010111101101110011000011100011011111001110010001110001011111011 0111000010000111111011000000010000110010111001100001010101001010001011100000001000111101000001000001001111001 101100000110100001000111011000010001000000000010011001100000011100000000010001001001000100000010000100000000 001000000000000000000010000000000000010101010000000100001010000000100000001000000000000000000010010011001000 010011111101110001111111101100111111100110011011001100111011110111101101111001101100001100111101011101100110001 1011011110100000011000100000100011110101101100010000100101010110100011011001000110000001101110110011000011110 001110100001000000110010000001100110100011000100000000011001100100010010001000100000000000001100000000010001 000000010000001000000000000000000000000000000000000000000000100000001000000000000000001100011000111011101100 0101101100010111000110110110010001101110110000110110010001110001100000110100101101100011000011000000011000010 000001000000001100100000000100000000101000100000000000000000001100000000000000000000001000010000000000000000 000001000000100000000000000000100000001100000000000000000000100000000000000000000000000000000000000000000000 0000000000000000000000000001001100011111001011000110001101010101100101010000011011011000110110011000010010000 0011000001100001011100000011000011001000011000010100100110111100101000101000110000001101000011001000010011000 011000000000000100110000000001000000000000000100000000000000000000000000100000000000010000000000000000000000 0100000000000000000000000000000000000000000000000000000000

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

MI RR 9 1 01001100000001010000011111111101101110111001111011110001111110110110110101000011111000000110001110001010011011 01110000101100111110011111101100001101101110100010001100110001100010010111000110000111010000010001110000110111 011000000010000011001100110000100000000000000000000000001100000111100001000011001001001111100000011000000000 000000000000000000000000000001001000001000110000001000011100000000000000100000000000000000000000100100010000 000100011001011111111110011001111111000110110110011011101010000011110110000111000001111100111110110101011110111 0010000011111101011001100011001001100110000011000000011110101000011110110010000011000011001101100110000001100 000000000011100011000000000000000110110100001011000110110000010000100000010000000100100000000000110000000110 0011011000000000000000000000000000001000000000000000000000010000000111010000000111000011001111010110010111111 10111110001111100011101110101000111000110000111101111001110110111001000001001011110111110010110001110000000001 0010000011111100100000000111010111010011000001010001000000010000000011000000000000011000111011000000000000000 011000001100000000000001000100000010000000000000000000001100000000010010000000000000000000000000000000010000 00000001001001000000000000011011111000010111110101111101111011001111110011011110110001100011101100111000001110 1111011001100001110110000011101110001010000010010111111011001101010011011001100110100001100110001101100001001 177

000110000001001000000000000000000000000011000000000000000000000000011000000000000000000000000000011001101100 00000000000100000000000000000000000000000000000010000

MI RR 9 2 01000100001101010000011101111101101111101001111001110010101111001110110101011011111001010110010110001010011011 01110000111100111110011110111100001100011101100010001100111001100010011011000110010111010000010001110000110011 0110000000110000110011001100011000100000000000000001000011000001111000010000110010010011111000000110000000000 000000000000000000000000000010010000010000100000010000101000000000000001000000000000000000000011001000100000 001001110111111111111100110011111110001101101100110111110100010111101100000110100011111001101101110011001001110 0101101111101010110011000110011011001100000010000000111101011001011101100100000110000110011011001100000011000 000000011011000110000000000000001101101000010110001101100000101001000000100000001001000000000000100000000100 0101110000000000000000000000000000010000000000000000000000010000001110100000001100000010011110111000101111111 01011100011111000111011101010001110001100010110111110011101101101011001010010111101111100101100011110000100010 0100000111111001000000001110101101100110000010110000000000010000000110000000000000110000110110000000000000110 110000011000000000010010011000001100100000000000000000011000000000100100000000001000000000000000000000100000 00000001010010000000000000110111011000110111101011101011110111111111100110111101100011000111011001110000011101 11101101110001111011011101110111000011000001001111111101101110101101001100110001011000110011000100110000100110 011000000100110000000000000000000000001010000101000000000000000001100000000000000000000010000001000110110000 100000000100000000000000000000000000000000000010000

MI RR 16 1 0000000001011011101000110101100010101100001010111101101011011100111011000001100111100100001000111101101001001 1101100001000001111100000101011000011000110001000000110001011011000100100010101101000111000100000011001100101 110110001100110100000011000100001101100000000110000000000011000000100000110000000010110000000001000010000000 000000000000000000000001000000000000001000001000000010000100100000000001000000000000000000000000000101101011 00101101010010110010111100101001111011100101111101011011110000001011100110000111100110111100110110110101111101 1110010000111000101100001100111000101100000000001000000011100111000100110110010100011000000111101100110100101 100000000001101100000000000000000101100110100001001000010110000001000100000010000000000100000000000010000000 010000001000000000000000000000000000000000000000000000000000001000000011000000000000000001101111111110010001 11101100110001011110001110101100110011100111000001111010010111000011010111000000101111001110000010000001100001 100000110000100011011000000000000000001100110000000000000001000110000000100000000000000110000100000000000000 000000100000010000000000000011010000000000101100000000000000010000000000000000000000000000000000000000000000 00000000000001001010000000000000110111110000111100110111110001010110011101100001111101110011100111000111100000 1011001001110011000111111100010110111001001110000100110101100110010100100110110111000100000001101100010011000 010000001100000110011000000001000000000000000100000000010000000000100000100000000000010000000000001000000010 0110000000000001000000000000000000000000000000000000000000

MI RR 16 2 0010000000001100000000101101110011101101100100011011101100101011110111001000011001111000001100111001010100101 1011100001000001101100100101011000000000110001101000110001001011000110100010011100100111000011110011001101101 110110001100100100000011000100001000100000000110000000000011000001100000111000000010110000000011000010000000 00000000000000000000000100000100000000100000010000001000010100000000000100000000000000000000000000100001000 000000011110110001011001111111010110111101110011111011001011111000001111011010011100011110100010110110101111101 1110010100011000101100001100011000101100010000001001001011100110001100110110010100001000101111101100110101011 100000000011011100000110000000001001001110110001001000110010001001000100000010000000000100000000000010000000 110000001000000010000000000000000000000000000000000000000000001000001011101000000000000001100111101011011000 11111101011000111110001110111000110011100011000001101011010111010011010111000100101111011110000011000001110001 101100110000100011011000000000000001101100110000010101010001100111000000110000000010000110000110000000000000 000001110000011000000000010011001000000100101100000000000000011000000000000100000000000000000000000000000000 00000000000001011010000000000110110111111000101110011011111001010110011011101010111101100011100111010111110000 1011001000110011000011111100011011111101101110000100110101100110001010100110010011000010000101011100010011000 010010001100000110011000000011100000000000110010000000110000000000110000110000000000011000000000010100000110 0110000000000001000000000000000000000001010000000000010000

NJ BR 3 1 0000000100110101111011011100110110100001101011100001111100001100001000001000110000011000011000110000111101101 1100101011000001001100000001111000011110110101000101101000111010000001110010010100011110100000110000101100111 110100000011110010110011101100000010000000000010010110000011000000000001000100110110011010000000000010110001 001100000000000000000001000001000000001010000100000000010001000000000000001000000000001000000000010000111010 11010110100101111111011000111101100011111011100011111011000100001000101100011111001100100111101110010101111111 0111111000011001111100101100110000001100011000000001000011001101101010110111001100000001100000100100110000101 000100000110110010000110000000000011001100000001000110110110000000000100001010000001000000000000000000000000 010000100000000010000010000000100000000100000000000000000000100000001010100000000000000001100111101101011100 11001101011000110111000101111000110011000011001101111100111111010011001011101100111111011011000000000011100000 0011001100001011111110100100011000011001000100100000001100000001110000001110000000000000110011100010000011000 010011101000110000010110000001000000001001011000100000000000010001010011001000010101000000100010010010000000 10010100000000100110000000000111111111110011010110110111110110011100101111000011101011101110111111111011100011 00011010011110011011111110001001110111101110100010011011001011010010001010111001100010010000111110001001110000 110100000110001001001000001101100000101000011010110000001100000000000011000001001000001100001110110000100000 01001000000000100000100100000100010000010000100001000010 178

NJ BR 3 2 0000010100110110111010101110011011001000101101110100101100110001100001000001000110000100001100011000100111101 10101010011000110011000000011110000111101101010101100010011110111000011100101101100111011000001100001010001111 100000000111100101100010111100000010000000000100101100000100000110000010001001000100110100000001000100100010 011000000000000000000011000000000000010100001000000000100010000000000000010000000000100000000000100010110101 00101101000110111111011000011101101000111110011000000111011000010010000110000111101100101111101100111101111110 1111110010100111011011011001110011011000001001000010000110111011011101101010011000100011000001000000000000110 101100001101100000000100000000000011001000000010001000101100000000001000010100000010000000000000000000000000 100001000000000100000000000001000000001000000000000000000001000000010010000000001000000011001111011010111011 10111010110001101110001011110001001100000110011011110101111110100110000110011001111110110110000000000110000000 0110011000010111111101001000110000110010001000100000011000010011010000011010000000000001100111000100000110000 100011000001100000001100000010000000010010100001000000000001100011000110010001010010000000001100100100000001 010000000000010010000000000111111111111001100111111011111011001111110111100001111101110111011111111101110101000 01111001111001111111111000101111011110111010101001101101101101001001101010101110001011000011111000100111000011 010000010000100100100000110110000000100001101011000000110000000000001100000000100000010000110010000000000010 001000000001000001000100000100010000010000000000001010

NJ BR 4 1 00000101010101111110000111110110110100011011011011001010111111011000111011001001111001011011001110011000011111 0110100110011010011011001011110000111101111011001101100011100101000010101100101000111011000001100001100001011 100000000111100101100111001000011000000000001100001100001010010110000010000011001100110100000000001100000000 010000000100000000000010000110000000010000010000000100001010000000000000000000000000100000000000001000010101 111011011011101111111110001011010110000111010110000100111110001101100111000001111011001010011111010110111110111 1011100001101100101111010000001100110000011001010000001100110111000011011101100001100110100011110000000010100 000100000011001000001100000000101010110000000110011011011000000000110110011000000100000000000000000000000001 000001100000000000000000000000000000010000010000000000000000010000001111100000010000000010011110110100110111 10111101100011111100010011101011001000001110001111101111111101001110011001011011001101011110000011110011001100 1100110000000011111010110011100001101100011000000100000110000111000000110000000000001011011110000000000000001 001110000011000001011000010011000001111001010000000010000001100011000100100000100000000010001001000001000100 00000000000110010000000000111111111111001111011101011110111001110111101100001110101110111010011011001110010011 10010001111001101100011011000111011011110110101001101101101111011101100011100100011011000010101000010010000011 010000010001100110000000110000010000110000111000100000000000000000001110000000000001100000000011000100000001 001000010000000000100100000100000000010000000000000000

NJ BR 4 2 01000100000000000001011111110001110111100000111010110011001110101100110011011011111010110110010110001000011111 01011000100110100110110000111100001111011110100010010000111000110011111011001110001110110000011000011000010111 000000001111000011001100110000110000000000011000011000011000001100001000000010001001110000000000011000000000 10000000000000000000010000010000000010000001000000100001010000000000000000000000000010000000000010010001000 010010011100010111111111111100011111100001111001000100111110000111100010000001101011001100011011011110111110111 1011100001101010100111100000001100110000011001100100001101010111000011011101100001100110000011110000000000100 000000000111001000001101000000001100010000100110101001011000000000010110011000000010000000000000000000000001 0000011000000000000000000000000000000100000100000000000000000100001011111100001111000000100111101101001101110 01111011000111111001000111010110110000011000101011011111111010011100110001110110011010111100000111100011011001 1001100000000111110000110011000011011000110000001000001100001100000001101000000000010110111100000000000000010 011101000110000010110000010110001000010010100000000100000010000100001001000001001000001100010010000001000100 00000000000100100000000000111111111110011010111010111110110011101111011000101101011101110101110110011100100111 00100011110011011100110000001110110111101101010011011011011110111011000111001000110110000011110001000100000110 100000100011001100000001100000000001100000110001010000000000000000011100000000000011000000000110001000000010 00000010000000000100100000100000000001000000000000000

NJ BR 6 1 01000111001100000110111111101101100110011001011111110001101110101100010011000011111001110110000111011111011011 0101100110001010011000000011110000110111011010001000110011001100010011100000011000111011000001100000110001111 011000000001100000100111011011000000000000001100000010000000011100000010000111000100100000000000001101100000 00100000000000000000100000001000000001010001000000000010001000000000000000000000000000000000000001001001000 01100001011011111001111110011001101111000001110010001011111011100110000110000111010100010110100110101101111110 0111011001011110000010111000011011000010000000001100000011010101101000110100001000011011101010100010100000001 110100000000001100000110000010000011001110000001100000010010000011000101100110000000000000000000000000000000 000000011100000010000000010000000000000100000000000000000000100000000011001000000100000001100111101101001100 11011101111000111110101110111001110011000011100101100111011111010011000011001110111111001111100000110011010000 0001001100011101111110000000011000011001000110000001100001100001110000001000000000000010110111100010001000000 001001100000110000010001101100110000001010010100010000100000010001010001011000001001000000100010000000001000 000000000000000110000001000001111011111100111111101101111101101111011011111001011110110011011111100011011111001 11010100111101110111111110010111101111101111000100010110010111011000010110110011000101100011010100010001000001 0110001001001100110000000000001011000000000101011000000110010000000001100000001100001110000110011100110000011 101000000001100000000100000000000011001000000100000100

NJ BR 6 2 179

01001111001100000111011111100111110111101001011111000010011011000100110000001111001111001100001100101111011011 0101100111010010011000000011110000110101101011001100010011000110010011100000011000111011000001100001010001111 011000000001100000100111001011010010000000000100000000000000011100000010000111000100100000000000000100100000 00100000000000000000000000000000000001010000100000000010001000000000000000000000000000000000000001001001000 01100001001111111001111110011101011110000111010001011111000010011100011000011100101000111100010110110101111101 0111011001100001100110111000010011000010010000001100000011010101101000110110011000011011101010100010000000001 011000011011100010000110000011000001001110000001100000010110000011000101100110000001000000000000000000000000 000000101000000001000000000000000000000100000000000000000000100000000011001100000110000001100111101011011101 110111011110001111110011101110111000110000011001011101111011110100110000110011101111110011111000001101100000000 0010011000111011111100000000110000110010001100000010001011000011100000001000000000000101101111001100010000000 010011000001100000100011001100100000010010011000000001000000100000100010010000010010000001001000000000000000 00000000000000101000001000001011011111101111111110101111101101111011011111101010110110011011111101011011001011 1101011011110011011111111000001110111110010101010011011001011001010001011001001100010110000101110001000100000 101100010010011001100000000000100100000000011010110000001101100000000011000000011000011000001100110001100000 01100000000001100000000100000000000011010000000100000010

NJ BR 15 1 0100000100010110000001111110000110011001000001101011000000011000110011001101000111100000001100111001101101111 01111000011101111111011100011110000000101100010001000010011100000011011100000011000111011000000000000011101101 1010000000011000011001101110110110000000000011000000000000000000000000100001101101001011011100000011011000000 100000000000000000000000001100000000001000100000000000001000000000000000000000000000010000000000100000000000 10000001100111000111111101100110111100111011011000000111110001101100111000010100010001100111011011100001110001 1011100000110010000011000000001100110000000000110000001100110111000011100001100000000110001111000000000000101 000000000001110000000000000000001010111000000110011001011000000000010110001010000000011000000000001100000011 000001100000000000000000000010000000010000000000000000000100100000001110000000000000000010011000110101110011 10110101100011111100111111101011001000001100110110101101111101101100011000110000111100110100000011110001001100 1100110000000011011010110001111000000100011000000100000110000101100000111000000111001000001110000100000010001 101110000011000011011000010011000001100001000001000010000001001101001100100001100100000001101000000000000000 00010000000111010000010000010111011111001101110111011110111001110110101110010101101100111110111011001110010001 10011101101001111101111010001110011111011110111001101101001100011100100101100100011001100110111000100010000010 110000000000110110100000000000000000000000101000000000100000000000001100000000000000010000110011010110000001 000000000000100100000001010100000000010000000101000001

NJ BR 15 2 01000001000101100000011111100011110110100010111011110101010111001100110011010001111010000011101110011011011111 01111000110110110110111000111100001100011000100010000100111100000110011100000110001110110000000000010111011011 010000000011000011001101010010110010000000011000000000000000000000000100001101101001111011100000001011000000 110000000000000000000000000100000000001000100000000000011100000000000000000000000000010000000000100100010001 10011011100111000111111101100110111101011011011000000111110001101000111000010100010001110111111011000001110001 1011100001101110011011100000001100110001000000110000001101010111110011110001100000000110001111010010000000101 000000000101111010000000000000001010010100000110011001011000000000110110011010000010011000000000001100000011 000001100000000000000000000010000000010000000000000000000010100000001110000000011000000100011000101101110111 01110101100011111100110111101011001100001100110110101110011101101100011000110000111100110100000011110101001100 1100110000000011011000110011111000001100011000000100000110000101100000110000000111001000000110000100000000001 101110000011000011011000001011000001100001000001000010000011000110000100100001100100000010110000000000000000 00010000001101010000000000010110011111001101110101011111111001110111101101001101101100111110111011001110000001 00011101110001111101111010001110011111011110111001101101001100011100100100110100001011100110111000100010000010 110000000000110110100000000000000000000001101000000000100000000000001100000000000000010000110010010110000001 100000000000100000000000000000000000010000001000000000

NJ BR 16 1 0100000000000110000001110111100110011001100101101101000000011010110001110100000110001101101100011000111011101 1011100011000011011100000001111000000011000100000000011001100000000000110000011100001101100000110000000110111 1101100110001100001100110111000011001000000000000001101100000000110000100001100110100101101110000000100000000 010000000000000000000000000010000000000100010000000000000110000000000000000000000000000000000000010000000000 00010000010011111011111110000011011110101111100110000011110000000110111100001000000100110001101101110000110001 1101110000001010111101100000000110011000001100001100000110101011000110100100011000000011000000100001100010001 000000000000010000001100000100000011001011000010000001100100000000001011001100000000000000000000000000000001 100000110000000000000000000000000000001000010000000000000010000000010110000000000000000001001111011010111001 10011010110001111101011101110101100110000110000011101111111110100111000110011000011110110111000011111000100011 0110011000010001101100011000110000110010011100001110000011000011110000001000000011000001100111000000000110000 010011000001100000000001001101100000000010010000000000000001100001000010100000000010000000001000000000000100 000000000000010010000000000011110011111001111110011111111011001111111111111001110101110001110110010101010111101 1001110111000110111111100000111001111111000000100110110110110001000010010110010000101100001101100000001000001 011001100000010011000000011000001100000000010000110000011000000000000000000000010000010000010000000000000000 0100000000000100000000000000000000000010000000110000000

NJ BR 16 2 01000100100101100000011111111001100110011001011010110000010111011000111101010001100011011110001110001011011011 0111010111000110111000001011110000110101000010000100110011000100001001100000011000011011000001100000001101111 180

1011001100011011011001101110000110010000000000000011011000000001100000100011001111001001011100000001000000000 100000000000000000000000000100000000001000100000000000001100000000000000000000000000000000000000100100010001 111010111001111111111111000001111111100111110010001011111110001011011110000101110110011000110110110101011000111 0111000100010111110111000000011001100000110000110000011111001100011000110001100000001101000100010110001101100 000100000001000000110000011000001100101100001000000110110000000000101100110000001000000000000000000000000010 000011000000000000000000000100000000100001000000000000001001000001011000000000000000001000111101101011101110 01111011000110110101110111010110011000011000001110101111111010011100011001100001111011011100001111100000001101 1001100001000110110001100011000011001001110000111000001100001011000000100000001100000110001100000000011000010 001100000110000000000101100110000000010001000000000000000010000000001010000000000000000000100000000000001000 00000000000100100000000000101100111100011110111111111101110111011111111110011011011000110101100101011110110010 0111101101001100111111000000110011111011000001001101101101100001001100110110100001011001010011000100110000110 100011000000100110000000110000011000000001100001100000110000000000000000000000100000100000110000000000000001 100000000000100000000000000000000000001000000101000000

NJ BR 18 1 0100000000000000000001110110110110011001000011101111000001011010110010000001100111100000001100111000110111101 1110100011001101001101100001111000000000111100000100001001100001100000110010001100011110100000000000001000110 110000011000110000110011000101100000011000000110000110000110000001100001000010110010011000000000110010000000 001000000000000000000000000011000010000010001100000010000101000000000000000000000000000000000010001001000000 00000110001000101101111110011000101111000000110110001001110000001101000011100011000110111000111110100100111100 0110011000110011000110110000001000101100000000001100000011110001100011110110011000011000001010100000110001101 010000100000011100000110000110000010001110000000001000000010000000001100001010000110000110000000000000000000 010000001000000000000000000000100000000100000000000000000001001000001011000000001100000001100110001001011101 01001101011100111110101110111100011011000011000101110111110111011011011011001100111111000011000000001100001000 0011001110001000110110000110111000000001000100000010000000100001110000001100000000000110110011100010000011000 100011100000110000110010101100110000001010010000000000000000011001000001010000000000000000000010000000000001 00100000010010010100000000000111101111110011110100110111101110111101111011100011011011111011111110110011100000 1110011001110000001111011100000000011110011010101001101101101111001000100101100110001011000110111000110010000 011100000011000100100000000001000000000000001101011000000000000000000001100000000000000110000000000000010000 000100100000000000000000100000000000000000010001100000000

NJ BR 18 2 0100000000000000000000111110110110011011000111101011001100011100110010000001100111100000010100111000110111101 1010100001000101001101100001111000011010101100000100001001100001100000101010001100011111100000000000011000110 110000011000110010110011010101000000011000000110000110000011000001100010000110110010010000000000010010000000 001000000000000000000000000011000010000010001000000010000101000000000000000000000000000000000010001001000000 00000110011001101101111110011000101111000000110110001001110000001101000011100011000110111000110110101000111100 1110011000110000100110110100000000101100010000001100000011110001100011010100011000011000000010010000110001101 010000000000011100000110000011000010000110000000011000000110000000001100001010000010000110000000000000000000 010000011000000000000000000000100000000100000000000000000000101000001011000000000100000001100110001001011101 11001101011100110110101110111100011011000011001011110110110111011011011011001100111111000011000000001100001000 001100110000100011011000011011100000000100010000000000000100000111000000110000000000011011000110000100001100 000001110000011000011000000100011000001101001000000000000000001101100000101000000000000000000001100000000000 100100000000000110100000000000111101111110101111101111111111110111111111011111011011011011111101110110011100100 11100110011010000011110110000000100111110110101010011011011011110111011011011001100010110001101110001100100000 110100000110001000000000000000000000000000011010110000000000000000000011000000000000011100000000000000000000 01100100000000000000000100000000000000000010000100000000

NJ BR 19 1 0100000000000110000111111110001110011000000011100101000010111000010011110100000110011100001100011000101011101 1100110001100011001100000001111000000011101101000100011001100010000000010010001100011110100000000001010011000 1101100110111100001100110001011010000000000000100110100000000001100000010000000110000110101110000000100000110 00000000000000000000000000001000000000010001000000000000101000000000000001010000000000000000010001001000000 00001000001101101111111110011001111111000000110010011001110110100011000010000011110000011000110110000101111100 01101110000010111010001100000000110011001101100000000000111101011000111101111011000000011110111111001100000111 000000000001100000000000000000010100011000000010001101101100000000001000011000000000000000000000000000000001 100000011000000000000000000001000000001000001010000000100001001000000010000000000000000011001111011010010001 10011011110001111100011011110101110110000110001011001100111110100111100110011100011110011000000100011001000110 0000011010110111111100001100110110110110100001000100011000110011000000011000000000000001100111000000000000000 100001000001100001100000001001100000010000100000100000000000100010000010010000001000000001001000000000000010 00001000000001001000001000000011001111100111111001111111100101100011111110110101111110011100011101010111000000 01001100111000110111111101110111001111001101010100110010110110000100010100010011001101100001010110011001100001 101000001100010011000000000100000000000000011100010000011000000000000111000000000001010000011000000001000000 1100100000000100100000100000010000001010000000001000000

NJ BR 19 2 01000000000001100001111111100011100110100000011101110001010110001100111101000001100101000011000110000111011011 1001100110000110011000010011110000110001011010001000110011010100000010100100011000111011000010000011000100001 1011001101111000011001110010110100000000000011001011000000000011000000100000001100000101011100000001000001100 00000000000000000000000000010000000000100001000000000001010000000000000000000000000000000000100010010000000 181

01010011010111011111111100110011111110000001100100110011101110000110000100000110101000110001101110101011111001 11011100001001111000011100000001100110011011000000100001111010110101101111000110000000111101111110011000011110 000000000011000000000000000000011000110000000000011011011000000000010000110000000000000000000000000000000011 000010110000000000000000000010000000010000010100000001000100100000000100000000000000000110011110110100100011 00110111100011111010110110111011101100001100010110011001111010101110001100110000111100110000001000110010001100 0000110101101111111000011001101001101101000000000000101001100110000000110100000000000010000110000000000000001 100110000010000011000000001011000001100000000011000000000001001100000100100000000000000000010000000000000100 00010000000000010000000000000110011111001111110111010111001001001111101101101111111100111000111001101110000001 0001100110100110111111101010111001100001101010100110010110110000100010100010011001101100001110110011001000001 011000001100010011000000000100000000000000011100010000011000000000000111000000000001100000011000000000000000 1100100000000100000000100000100000000011000000000000000

NJ BR 20 1 0000000001010110100001101110001100110010111000100000010010001110001110101100101101110000110001110001101101101 1010110010100001001100000000111000100010111101100110101010111010000100011000001000010101100000110000110110000 010100011011110000110011000100001100000000000011000110000010000000100001000001100010011100000000000010110000 000000000000000000000001000001000010000010001000000000000001000000000000001000000000000000000000010101011010 11001100110110111111010110001111011000011110110000011011010101011000011000010110110100111010111110110101101000 1111111000101001101110110000010001001100000000000001000011110001100011110111001100010001111000000010010001111 011000010011100010000000000000000000001100000000100110010010000001000101010110001000000010000000000001100000 010000000100000010000000100000100000000100000001000000000001001000001001000000000000000000100111101101001100 11001101111000111110101110111010110011000011000011100110111111011011110011001100000111001111100000000010000011 0011001100111000110100000100111000000011110110000000000000000001110000001100000000000010000001100000000001000 011001100000100000010000000100010000001000011000110000000000010000000001001000000000000001000011000000001001 000000000000000101100000000000011001111100111111001111111011100111011111110101111111110010110111101010111110001 11001100111000110111111100000111001101101111001100110010110110000100011100011011000101100000111100011001000001 001000001100010001000000000100001100000000001000010000000000000000000011000000000000010100011000010000000001 1000000000000100000000000000100000000010000010001000000

NJ BR 20 2 00000001110101101001011011100011001100011110110010011101100011100011111011001011101011001100011100011011011011 0101100011000110011011000011110000100101111010001000010011010101011000100000010000101011000011100011001100001 101000110111100001100111001000011000000000000110001100000100000001000010000001001100110100000000000100100000 000000000000000000000010000000000100000100010000000000001010000000000000010000000000000000000000100101110101 11011001101110111010110100111110110000110101100000111110001011110000110000001111001000110101111101101011111001 1101100001010101111101101000100110011000000000000010000111101011000111101100011000100011110000001101100111110 000000000011010000000000000000000000011000000011001100111100000110011000011100000000000000000000000001000000 100000001000000000000000000001000000001000000010000000000010010000010010000000000101110001011101111110011101 11001101110001111111011101111101100110000110001011001101111110110111100110011000001110011110000000000100110110 0110011001110001101000001001110000000011101100000000000000110011110000001000000000001100000011000000000010000 110011000011000000100000001001100000110000110000100000000001100000100110010000000000000010001010000000000010 00000000000000101100000000011011001101110011110001111111111010101111110111100111110100001111011101110111000001 11001100110100110111111100000011011101110111001100110110110110001110110110011011000101100001101100011011000011 011000001100010011000000000000001000000000010000010000000000000000000110000000000000000000011000000000000001 0100000000001000000000000000100000001010000010001000000

NJ HV 1 1 0100000000000100000101111111110111011010000111101101100010011101010001001100110111101000011000011000100001101 1010010010100011011100110001111000001110110101100100000101100010000110110100011100111001100000100000111111100 1101000011001101101100111011011000000000000000000000101111000000110000100001011100100110001110000000100000110 01000000000000000000000000000000000001000000100000000000101000100000000001000000000000100000010001001000100 001000101111111110011111110110011111110010111100110110111111001100110100111101010101001101011111101110111111001 1100101101111010010101101000000110011000000000001100000111101011010001001010110001000000111011001100000000111 100001110000110000000110000000000110000110000010110001101100001100001011000100000000000000000000000000000000 000001110000000100000000000001000000001000000000000000000000000000010111010000001100001011111101010110011001 10111010110001111101011001101101110110000001001011011111111110110110110110101001111110011011011010000110000000 0011011000110111101100011001111100000110001100001110001011000001000000011010000001100011000111000000010110000 100111000001000001000100000101100000010100110000100000000000110010100010010000000000000001000010000000010010 000000100000010011000000000011110111111000111110101111111011011110111111100001111101100011110111110001110000110 01011101101101011111100110010101111110011000001001101101101111011000101111100010001011000011111010100010000110 110011000110110110000000001000000000110001100001000000110000000000001100000000100000100100000000100110100011 101100000011100000000100110100000001010000010000000000

NJ HV 1 2 00000000010010000000100111111110110111011001010011110110011011100110110011001001111110100001100011000110011011 0101001110000110111001100011110000111001101011001100101011000100001101100101111001011011000000000001101101001 1010000110011011011001100010110000000000000000000001011110000011100100100011110111000100011100000001000000100 100000000000000000000000000100000000100000100000000000010100010000000000100000000000010000001000100001000011 000011010111101111000111111110110011111011011111101110110111100110011000111111011110010101111110100011101000111 0010101111001101011110100001011001100000000000110001011111101101101010101011000100000011101100110100000101110 182

000011000011001000011000000000011000001000001101000110110000000000101100010000000000000000000000000000000000 000011000000010000000000000100000000100000000000000000000000000001001101010000000000001101110101101001100110 11101011000111110101100110110111011000001100101101101111111101011011011001100111111011101101101000011000000000 1101100011011110110111100111110000011000110010111001001101100100000001101000000110001100011100000001011000011 011100000110000100010000010110000001010001000010000000000010001010001001000000000000000100001000000010001000 00000000000010110000000000111101111110001101100110111110110111101111111000011101011000111101111001011101000000 1101101101101011111100110001101111110011000001001101101100111001000100111100010001101000001101010100010000010 101011010110110110000000011010001000110001100001001000110000000000001000000000000000100000000000100010010001 100100000011100000000100010100000001010000000100000000

NJ HV 6 1 01001000100001000100011111111101110111100010001010111001000110011000110101011111011000000110010110011100011011 11011001110001110110000000111100001101011110100010001110110111000000110100001110010010110000100000011011011111 0000011100110000110011100101100000100010100110011010111100000010001010000001100110011000000001100010000000001 000000000000000000000000000000000000000000100000000000101000100000000000100000000000000001000001001010100011 001110111111110011111110110011111110011011001010110111010100111110101101100110111000111101101101001011111001110 0110000110001010111110000000110011000001100001100000111111011000001100100010000110001010011001101100001010100 000001100001000000110000000000101001101000000110000101100000000001000001111001110000000000000000000000000000 000100011000100000000000001000000000000000000000000000000000000010110010000010100000011001111010110010101100 111101100011111010111111110011001100001100110111101101111101011101101100110011111100110000110111110010000000110 0110001101111011000000011001000001100011010000000000110000111000000110100000000000011000110000000001100010001 110000011000110000000001011000000100101100011000000000011100001001100011000000000000010010100000000000100000 000010001110110000000011101111111110001111111010111110100111111111101000101110011101111101110100011101100111011 1101110001011111011110010110011001010000001001101101101100011001101000110110001011000010101110100011000110110 011000010100100000000000010000000000001101000100000110000000000011000000000000001101000000010000100000001100 100000000100000000100000100000001010010000100000000

NJ HV 6 2 01000000100101000000011111111101110111100001011110111000100110010100110000001111011000000011001110011100011011 1101100011000011111000000011110000100010111000010000010011000000000011100000111000001011000000000001101001111 100000011001100001100111001011001000100001001100110101111000000100000000000011001100100000000011000100000000 01000000000000000000000000000000000000000000100000000000101000100000000001000000000000000001000001001000000 00100010011111111001111111011001111111001001100101011011111010011011001110110011011100010110110110110101101100 1110011000101000101011111000000011001000000110000110000011011101100001010000010000011000101001100110110000001 010000000110000000000011000000000011000110100000011000010110000000000100000111100111000000000000000000000000 000000010000000010000000000000100000000000000000000000000000000000001011001000001010000001100111101101010110 11001101011000111110101111110100110011000011001101111010011111001111011011001100111111001100001101111100000000 0001001100011011110110000000110110000011000110100001000001100001101000001101000000000000110000100000000011000 000011100010110001100000000010110000001001011000010000000000011000010001000100000000000000100011000000010001 00000000000000100100000000011111111111100001101110110111110110111101110101000011111011101111101110100011101100 1110111101110001011111011110010110011001010000001001101101101100001000101001100010001001000010101110100011000 110110011000010110100000000000010000000000001101000000000110000000000011000000000000000101000000001000100000 001000000000000100000000100000100000001010010000100100000

NJ HV 10 1 01000100101001000001111111110110110111100001111010111000000110011000110100011111111001000110010110001100011011 0001101110000010111001100011110000111001100110000100100011010100011011100100110001011011000000000000001001001 1011001110011010000001110011010000000000000011001101011000100000000001000000101100000100011100110011000000000 000000000000000000000100000000000000101000010000000000010100000000000000000000000000000000001001110100010000 110111111011110001111111011001111111001011010110000011111110110111010001111101010100010100110110100101011100111 0111000110000100111111000011000001100110110000110000011001101110000000111010000000001101101100010010001101110 000000001010000000010000000000011000010000011011000000110000000000101100011100000000000000000000001100000000 0011000000000100000000000000000000001000000000000000000001000000000111110000011100000011111111010110011101101 111101100011111100111111111011001100001100110111010100011101001101101100111011111100110110001100001101110110010 0110000001100011000000011100001101100011000001000010110000111100000110000000000001110000110000001100100011001 110000011010000000000010011000000100100000000000000000001100001001100100000000000000010001100000000000000000 00000000110010000010000000110011110000111011101011111011011111111101111110110101100011000110001001110000011011 01101110011101111100110001111011000110000001001101101101111011000101111100110001011000101111100110010000110110 011000000100010001000000010001000100001100001100000110000000110101000000000000000110000110000000000000001000 000000010000000000100010100000000001010100000000000

NJ HV 10 2 01000101000101000001111111110110110111100001111010111010000110011000110100011111111000100011001111001100011011 0001101101001110111001110011110000011001100110001000010011010100011011100100110011011011000000000000001001011 1011001110011010000001110011010000000000000011001101011000100010000000100000101100001100011100110011000000000 000000000000000000001110000000000000101000110000000000011100000000000000100000000000000000001001110100010011 10111111101111000111111101100111111100101101010000000111110011011101001111011101010001010011011010010101100011 1011100011100010011111100001100000110000011000011000001100110111000001111101000000000110110110000001000000111 000000000001100000011000000000000100001000000111100000011000000000010110101110000000010000000000000110000000 0000110000000010000000000000000000000100000000000000000000100000000011001100001110000001111111101011001110110 183

111110110001111110011111111101100110000110011011110011001110100110110110011101111110011011000110000110110011001 0011000000110101100000001110000110010001100000100010011000011110000001000000000000111000011000000001010001100 011000001101000000000001001100000010010000000000000000000100000100010010000000000000001000110000000000000000 00000000010101100000100011011011111100111101101101111101101111111110110111011010110001100011001100111001001100 01110111000110111110101000111101100001000000100110110110110101100010111110011000101100011010111001001100011011 001100000010001000010000001000100010000110000110000011000000011001100000000000000111000011000000000000000110 0000000010000000000100010000000000001010100000000000

NJ HV 13 1 01000100000101000000111111111101110110100000011010110001100110001100110011011001111000000010001110001001111011 1101100110001011011010000011110000011011011110001000110011000001000011100100111001011011000000000000011001001 1010000110011000011001101110110110000000000011001101000110000101100101000100001001011000011100110011000000100 10000000000000000000000000000000000000000010000000000001010000000000000001000000000000000000000000010001000 001011111101111110111111101100111111100010110010100101111111011011100101111010110000001111011011001000011110011 1011010011000000011000110000001100110000000000011000001101100110111011011000100000000110110110000011000000110 000000000000100000011000010100001100011010000000000011011000000000100000010110011000000000000000000000000000 000001100000001000000000000010000000010000000000000000000000000000101100000000101000000110011110100100110011 011101011000111110101111110110110011000011001101111111011111101011000011001100111111011101101100001100001001100 1101100011011110110001100111000000011000110100010000100000001111000000011100000000010100011100000000011001010 001100110110000000000000010110001001000011000100000100000010001010001001000000000000000100001000000000000001 00000100001100100001010011001101111100011111011110111110110111101011111000011101011101110001110110001101000110 11011011010010011111011000000001111100110001010011011011011010100101011111000100001110000101011101100110001101 100110000001001000000000010000010001000011010110000000000000000000011000000001000011010000100110001100000011 00000000010100000000100010100000001010000000100000000

NJ HV 13 2 01000000000001100000011111111101110110100011001011110011100110010100010011011001011000000011001110001101111011 1101100010000011011001000011110000011111111110000000010011000001100001101000111001011011000000000100011001001 1010000110011000011001110110110110000000000011001101000110000101100101001100001101011000011100110001000000100 10000000000000000000000000000000000000000001000000000001010000000000000010000000000000000000000101010001000 0101111111011111011111111011001111111000111010110001011111110110111001111110111010000011110110110110001111100111 0110100110000000110001100001011001100000000000110000011011101101110110100001000000001101011100000010000101100 000000001001000000110000110000011000110000000000000010110000000000100000101100110000000000000000000000000000 000011000000000000000000000100000000100000000000000000000000000001011000000001010000001100111101001001100110 111010110001111101011111101001100110000110001011101111111110110110110110011001111110111011011000011000111011001 101100011011110110001100011000000011000110100000000100000001101000000001100000000010000001100000000011001010 001101010110000000000000100110001001000011000100000100000010001010001001000000000000000100001000000000000000 10000000000100000000100011001101111100001111011110111110110111101011011000011110011100111101110110001110110110 11011011011010101111011000001000111100110000010011001011011011100101011111000100001110000011011101100110001101 000110000001000000000000010000010000000011010011000000000000000000011000000000000011010000100100001100000011 00000000010100000000101010000000001001000001100000000

NJ HV 15 1 01001000000001000000011111101011110111101001011011111000001111011001110000001101100111000110001110001000011011 1101101010001111011011000011110000011001101010001001110011100100011101100100111001011011000010000000001100001 1010000110011011001001100010000110001100000011000111000110110101100101000000101100000001011100110001001000000 100000000000000000001100000010000000100100100000000001100100110000000000100000000000010000000000100100010000 111111111011100001111111011001111111001111100110011001111100111011011011100111010100011110010111011001011100111 0010100100000101110110100110011001100000110000110000011111101100110110110001000000000001011100100000010101100 000000001000000000110000011000001000110110001101000000110000000000101101101100000100000000000000110000000110 000101100000000000000000000100000000100000000000000000000000000000000010000000110000000110011011101100111011 10110101000111111001110111011100011110011000001111101010111101011110011001100111110001101101011000011110000000 0001100001111001100001100011000000001000110000001000001100001100000011011000000110001100111100000000000001000 011100000100000000000000010110000001000001000100001000001110000000011001000000000000000100000000000000010000 00000000101100000000100000001100101111100111111111011101001011111111011100101011011100101101111110111011100110 00110011001010111111011000001110111001100000010011011011011001110011010011011100110110001011011101000100001110 100110001001001100000000010000010001000010110001000010000000000000010000000000000000000000000000000100000011 00000000000100000000000000000000000010000000100000000

NJ HV 15 2 01001100000101100100011111101011110111101000011010111000101101101100110000001101100110000010001110001000011011 1101101110000111011011000110110000011001101110001000010011110100011101101000111001011011000000000000011000001 1010000110011011011001100010000100001100000011001011000110110101100101000000101100000001011100110001001000000 110000000000000000000100000010000000101000110000000000100100010000000000100000000000000000000000100100000000 101111111011111011111111011001111111000111100010011001111000111011011011100111111100011110010110011000101100011 0011000010000101000110100110011001100000110000110000011011101100110010110010000000000001111101000000000101100 000000001000100000010000110000001000110100001101000000110000000000101101101100000100000000000000010000000110 000100100000000000000000000100000000100000000000000000000000000000011001100000010100001100111101101001100110 11101111000111110101110110110110011110011000001101111011111010011110011001100111110001101101101000011010000000 000110000111100110000110001100000000100011010000100000110000110000000100100000011000110001110000000000000100 184

101110000010000000000000001011000000010000100010000010000111000000000100100000100000000010000000000000000100 01000000000110010000000000010110101111001111001011011010101011111111111110011111101110011110111101101110110011 0001100110000101111110110000011011100011000000100110110110110011100010110110111000101100010110111010001000011 011001100001010011000000000100000100011000011100010000000000000000000100000000000000010100000000000001000000 1100000000000100000000000000000000000000010001000000000

NJ HV 17 1 01000000000001100000011111110011110111100001111011110001100110011100010111011000111000000011001110001100011011 01110000100100110110110000111100010100011011100000011100110101110011111000001100010111011000011000011110010011 0110011000110100110011011111100000000010100000001100111100000000000101000001010000001000111000000110000011001 000000000000000000000000000000110001010001000000010000101000000000000001000000000000100000000001001000000001 001111110111100111101110110011111110000111100100010011011011011111100111001110101100111101111101100010111000110 1110000110001011101101001110000010000101100110010010111000011010011001100011000000011111111001101100000011000 000100010111001000000001100000010000110000011110000101100000000101011001011000000000000000000000000000000000 0010100000001000000000000000000000010000000100000001000000000000101100100000111000000111111110101100111011101 11101100011111000111110100010001100001110000110011101111101001101100100110000111100110000000100110001100000111 0110000001111110000110011111000001100000000000100000111100111000000110100000000000011011110000000000000011001 110000011000000001000100011000000101001100000000000000001100001000100100000000000000010000100000000000000000 000000001110110000000011101101111100011111111010111010110011101111111010101111011000111101110101111100100111101 11011110010101111001100001011110000110000010011001011011000110111011101101000001110001010111101100100001101100 110000001001000000000010000000001100011000011000001100000000000011000000001000001000000000001001000000011001 10000000100000000000000000000000001000100000000000

NJ HV 17 2 01000000000101100110011111110011110111100001111011110001101110010100010111010001111000000011001110001100011011 0111000110000011101011000011110010111001101110000000110011000101001111100000110001011101010001100001011101001 1011001100011010011001101110110000000000010000001110011110000000000001000000111000000100011100000011000001101 100000000000000000000000000000001000101000100000001000100100000000000000000000000000010000000000100100010001 100110111011110011110111011001111111000011110010001001101110001111101011100111010110011110110110110000111100011 0111000011001100110110100011000101100000110011000000011100001100101010110001100000001111101100110000000001100 001010000011100000000000010000001000011000001011000010110000000000101100101100000100000000000000000000000000 000101000000010000000000000000000000100000000000000010000000000001011001000000010000001111111101101001110110 01101011000111110001111110100100011000011100001100111111111001011001000101100001111001100000001001100001000001 1101100000011110100001100111110000011000000100001000001111001110000001101000000000000110110100000000000001010 011100000110000000010000100110000001010011000000000000000010000010001001000000000000000100001000000000000001 00000000000110100000000011101101111100011110111110111010100101101111111010111111011000110001110000011100100111 0011001101001000111101101100111011000011000001001101101100100001100100101100010001011000010001000110011000011 010011000000100100000000001000000000110001100000100000110000000000001100000000010000100000000000000000000001 100100000000000000000001000000000000010000100000000000

NJ HV 18 1 01000000000000000000011111101101110111100000111111111001110101110100110111011001111001000011001110001100011010 1101100010011010011001100011110000100101100010001001110011000100010011100000110001011111000001100000011001001 1010001100011000011001110110110000000000000011001101011110000001101001000011100000001100000000110001000001100 100000000000000000000000000100011000100000010000001000010100000000000000100000000000010000000000100100000000 100101111111101011111111011001111111001011110100001001111100111011011011100111010110011100010110110111001100011 0110110010001101100101100010011001100000000000110000011011001101000110110001100000001011111100110110000001110 000000000001101100000000010000001001110000000000000000110000000000100000011000110000000000000000000000000010 001010000000010000000000000000000000000000000000000000000000000001011001000000110000001111111101011011001110 01111011000111110001110101110110011011011000011101101110111001011011011101100111111001111101101110000001000001 100110000100011011000110001111001101100011000000100010000000011100000001100000000000010000110000000000000000 001110000011000000001000000011000000100001100000000000000001000000001100010000000000000010001000000000000000 10000000000011010000010001110110101110000111111101010111011110110111111110011111101100011110111000101101101000 0001100111000100111101110000011001100001000000100110010110110001110110110010011000101000001111111011001000010 011001100010010011000000000000000000000000110100000000011000000000000110000000000000000000000001100001000001 0001100000000100000000000000000000001001010000010000000

NJ HV 18 2 00100000000101000000011111101101110111100000111111111001101001110100110111010001111010000011001111001110011010 1101100110000010111001100011110000100001100011001000010011000000011011100000110000011001000001100000001101101 101000110001100000100110101011000000000000000100110101111000000100000010001110000000110000000011001100000110 010000000000000000000000000110000100010000010000000100001010000000000000000000000000001000000001010010000000 010001111111111101111111101100111111100101111010000111110100011101011101110011101011001110011011011011101010011 1011010011000010110010110001001100110000000000011000001101110110100011010000110000000111101110011011000000110 000000000000010000000000001000000100111000000000000000011000000000110000001100011000000000000000000000000001 000010000000001000000000000000000000000000000000000000000000000000101110100000001000000111111110101101100011 00110101100011111000111110111011001101101100000110110111111010101101101110110001111100110110101111000000100000 110011000000001101100011000111100110010001100000010001000000001110000000010000000000001000001000000000000000 000111000101100000000000000001100000010000110000000000000000100000000010010000000000000001000100000000000000 00100000000001100000000000110011010111000011101111101011101101011011111110101111110110011111011101010110110000 185

0000110011010010011110111000000100110000110000010011001011011000111011011001101100010110000111010101100100001 001100110000001000100000000000000000000000011000000000001100000000000001000000000000000000000000100000000000 01000100000000100000000000000000000001000010000000000000

NJ HV 20 1 00100000000000000001011111111101110110110111011011111000100110101100010011011101111000000010001110101011011011 1101100010000111011001010011110000111001111010001001110011000000001101100100110101011101000000000000001000101 101000011001101101100110001110000000000000000000000100111000100010000100000100000000100000000000000000000010 00000000000000000000001000000000000000001001000000000000010000000000000000100000000000000000010001001000100 001001111110111110111111110110011111110011111100010010111100001100110110111100111001001110011111101111011011001 1100110000100111001101110011100011011001100000001100010110100011001000101100010000000011110011000100000001011 000000001100100100000010000100000010000001000001110001101100000000101011001000000010000000000000000011000000 000000010000000000000000000001000000001000000010000000000000000000010111010000000000001011001111010110011111 10111001110001111101011111010101100110000110000011001111110110110110110111011001011110111011010111100110110000 0110011000110111101100111001110000110010001101001100001011000011100000110000000000000001100111000000000010000 000011000001100001000100000101100000010010110011000000000000100000000010010000000000000001000010000100000100 000000000000010010000110001110110101111001111111101010111011011111111101101011111101110111110111010111110110011 1001100110110110011110010010111011100001101000100110010110110000110110110011001000101100001100100010001000011 011011000000010011000000000011000000000000110000110000000000000000001100000000010000110000010000000011000001 0000100000000000000000000110000000000000000000000000000

NJ HV 20 2 00000011101101111101101111011011010001011011101100010010100110000110011011100111100010101100011100011011011011 01011000110001111110010100111100001110011110100010001100110001000011111001001101010111110000000000000011010011 010000110011011011001101011110000000000000000001011000110000100100001000011000000001000000000000000000001100 100000000000000000000100000000000000001000100000000001001100000000000000010000000000000000001001010111011011 01011010110111111011001101011010110111100110001011101110011001100110111100011100010011100111111011110110110011 1001100011001110011010110111000100110000000000011000001101000110110010011001000000000101100110101000000010110 000000011001110000000100011000000100000110000101100011011000000001010110011000001100000000000000000110000000 000001100000001000000000000010000000010000000100000000000000000000101110100000001000000110011110101100111011 01110001100011111010111111101011001100001100000110010111101101101101101110110011111101110110110111001101000000 1100110001101111111000110011100001100100011010011000010110000111100001100000000000000011001110000000001100000 000110100011000010001000001001000000100101100010000000000001000000000100100000000000000010000100000000000100 010000000000111010001100011101101011110011110110111111110110111011110111011101111011101111101110110011101110111 0011001110101101111100100101110111000010000001001101101101110001101101100100010011011000011101000100010000110 010111000000100110000000000010011000000000101001100000000000000000011000000000010000101000010000000110000010 100100000000100000000000010000000000010010000000000000

NJ WR 4 1 0001010001010110111000000011010101101101001100101010100011111100110011010001000111101101111000011011110100001 10111000011111011011000011011110000110111001010001101011011000101000001010100101100111011000001100000001101111 1010000110011011011001110110110110001100000011101101011011011000000000000000100100001100000000000001000100000 110000000000000000000000000010000000000000010000001000010100010000000000100000000000100000001000010110101100 10011000000010110110110011000111110110000111011100001111100001101101101101011101110001110111011011101101110001 1001100010100111011011110001100110010011011000010001001101010110100011011101100000000000110011001000000000110 000000000011010010011000000000000010110000000000100011011000000000110000110110000010000000000000011000000001 000001100000001000000000000000000000000000000000000000000000000000101100000000111110000110011110100100110011 01111101101011011010110011101011001111001100001110111011111101111111001100110001111100110110110000110110000000 0110110001111100011000110011000010000100000000011100111000010110100000110000000000000110000110000000001100000 100110000011000110000000010100000001100100100011000000000001000001000000100001000000000010001100000000100000 00000001000110010000010000100110101111000111111011111111011011110011101101011111101100011000110100111110011101 10011101101011011111100110100111011111011000101000000111101111110000101111100110011000000010101100010010000111 101011110000100010000000111000001000100001101000100001100000000000001110000000000001100100110010000110000010 000100000000100000000000001000000000010000000001000000

NJ WR 4 2 00000100010101110110000001010101011011110011001110111000111011001100110010011001111000011011000111111110000011 1011010110111011011000010011110000110110100010001101011011000101000001011000101101101011000001100001001101101 1010000110011001011001110110110110001100000011101101011010011000001100000001100100001100000000000001000100000 010000000000000000000000000100000000000000100000001000000100010000000000010000000000100000001001110110101100 10011010001011010111110011000111101110000111011100000111100001101101101100011101110001110111011011011100011001 100110001010111101101011000100010001001101100001000000110101011010101100100110001000000010101110100000000010 100000111101101001001100000000000010001000000010000001101100000000011000111011000010000000000000001100000000 100001010000000100000000000000000000000000000000000000000000000000010110000100011100000011001111011010011001 10011100110001101101011001110101100111100110001101011101111111011111100110011000111110011011011000011011000000 000101100001111000110101100110000100001000000100110000010001101101000001100000000000001100001100000000011000 011001100000110000100000000101000000011001011000110000000000010000010000001000010000000000100011000000000000 00000000010001100110000110001101101011110001111110110111110110111101110011010111111011000111101101001111100111 01100111011010101101111001100001101111111100001010000001111011110110011011111001100110000000101011000100100001 111010111100001000100000001110000010001000011010001000011000000000000010100000000000011001001100110001000000 186

01000100000000100000000000001000000000001010000001000000

NJ WR 11 1 01001100100110100000011111100101100110111000111011110001110011010100110011011001011010011011010110011110011011 1101000110011011011011001011110000110101111110000000001011010111001101101011011001001101000001100000011001101 101100000111100001000111001000001100110000001110001100001100000000000100001001010000110000000000000100010000 000000000000000000000010000010000000010100001000000110001010000000100000001000000000010000000100110010001000 011011111101100110111111111100110111100011011011000100111100010101100111110111101011001100111010011101100010011 1000110110000110111100110001100110001011011000011001001111010110010011001001100000000000000110101010000001100 000000100101110000011000000000000100010101001100000011011000000000110110101000001010011000000000011000000000 000001100000001000000000000000000000000000010000000001000000010000101101110010101100000110011000100101011011 10111101100011111010110011101110011011001100000110111011111101101111001101010000111100111100000000110001100000 1110110001100111011000011001111000001100010010000000010001100111100000001100000000001000001100000000000100000 001110100011000011000000001111000001100101000010000000000001100001000100100000000000100000000100000000001100 10000000000110110000010001100110111111000101011011011101011001110111110111010111101110111110111000110110110011 11011101110001110111111100100011011110111000001000001101101100001000101111100110000111000101101110100010000010 010011110001000101000001011000000000000000101000100000000000000000011010000000000000100000000000000110000001 000000001000100000000100000000001000001000000000000000

NJ WR 11 2 01000110100101100000011111100101010111101001111011110011111111010100110011011001011110010011010110011101011011 11010001110110110110110011111100001100011111100000000010110001110011001110110010010011010000000000000011011111 011000001111000011001101011010011001100000011101011000001000000000001000010010100000100000000000001000100000 000000000000000000000100000100000000100100010000001000010100000001000000010000000000100000001001110100010000 100111111111001101111111111001110111000110110110000001111000101011001111101011000110011101111010111011001110111 0010101100001101111001000010101010010010100000110000011111101100000110110011000000000101001010010100000001000 000000011011100000111000000000001000101000001000000110110000000001101101010000010100110000000000010000000000 000011000000010000000000000000000000000000100000000010000000100000011001001101011100001100110101001010110110 11111011000110110100100110111100110110011000001101110111111011011110011001100001111011111000000001100011000000 1101100011001110110000100011110000011000100100000000100011101111000000011000000000010000011000000000001000000 011101000110000110000100000110000001000010000100000000000001000000001001000000000001000000001000000000001000 00000000000100000000100011111101111110001010100110110110110011101101101010011111011101110101110001011101100111 0011101110001010111111010100011011110011000001000001101101110001000101101100110000111000110101110100010000011 010010010001000100000000011000000000000001101000100000000000000000011001000000000000100000000000000110000001 000100000000100000000100000000000000001000100000000000

NJ WR 12 1 01000000110000000000011111000001110110111001111011010011111110101101010110000001111001101110000110001000000010 1111000010000001011001100011110000010110100110110000100011000011000001101100101100111101100011100000001001011 1010000110011000011001101110110110001100000011101101000000001101100000000010110000001100000000000001000100000 110000000000000000000000000100000000001000010000000001000100000000000000100000000000100000001000100100000000 10001001100101001111111101100010111100001111011000001111100001101101010000010100110001010111011011001101110001 1001110110110100111001100001100001110000011110000001001100100110000111010011100001100000010110000000000010101 000000000001110000011000000000000100011010000110110011011000000100010010010110000000000000000000000000000011 000001110000000000000000000000000000010000000000000000000000000000001100110000101010001110011110110101110011 10111101100011111100110111011100001100001100010110111100011101101111001101101011111100110110001100110010000000 0110110001100111111000110011100011101100011000011000011000000111000000110100000011000110001110001000001100000 100110100011000110000110001100100000100100100001000000000001100001000000100001000100000110000100000000000000 10000001000111101000010000110110011111001111010011011011001011111111101111010111001101101000111000001110100011 1010100110110110111111101000001101101101100010100110011010111110100010010110001000111100011010111011001000001 010000011000011001010000000001000000010100110000010001011000000000001100001000000000000000001000000010001001 0000000000010100000000100000000000011010000100000000000

NJ WR 12 2 0100000001000000000001111100000111011011100111101111000000101100110011011000000111100110111000011000100000001 1011100001000000101100110001111000011011100011001000001001100001100000110110010110010110110000100000000110101 1101000011001100001100110111011011000110000001100110100000000110110000000011011000000110000000000000100010000 011000000000000000000000000000000000000010001000000000100010000000000000010000000000100000000100010010001000 00001110110110100111111100110011011110000111101100000111110000110110111100001110001000101011101101100110111000 1100101011010011011110011000110000111000001111000000000110011011000001101000110000110000011011000000000000111 100000000000011000001100000000000110001101000010000001101100000010011001011011000001000000000000000000000001 100000111000000000000000000000000000001000000000000000000000000000010110010000010100000111001111011010111001 11011110110001111110011001101110000110000011001011011111001110110111100110110101111110011011000110011000000000 0011011000110011111100001001110000110110001100001100001100000011110000011010000001100011000111000100000110000 010011010001100011000011001110100000010010010000100000000000100000100000010000100010010000100010000000000000 00100000100001110000001000011011001111100011101001101101100101111111010111100111100110001100011100000111001001 11001100111011011011111101010011101101101100000100110011010111110100010010110001000111100011010111011001000001 010000011001011001010000000000000000000100110000010000111000000000001101001000000000010000001000000010010001 0000000000010100000000100000000000011001000100000100000

187

NJ WR 13 1 01001100000101100000011111100011110110111001011011110101100111001100010011011001111010000011001110011011011111 0101000110000011011011000011110000110001111110010000010001000101001111101000001101001011000000000001011001111 1110000110011011000001100110010011001100000011010011000000110011000001000001000100001100000000000001001100000 010000000000000000000000000010000000100000010000000000001100000000000000100000000000000000000100100100010000 000011111001110011101110011001111111000011110110011001111110101011001111100111110100011001110111001011011100011 0111001100001000110111000000000011101010110000110001011101001100000110100011000100000000111101100100000101100 010000110110100000110010110000000001110100001010000010010000000000100001011000001000110000000000110000000000 0000110000000000000000000000000000001000000000000000000000000000000110011100011111000011001111011010111011101 110001100011111110111111011010001011001100001110011010111100111111001100111011111100110110110000110001100000111 0010110011011011000110011000001100101110100000100000000000111000000001100000000000110001110100000000000010001 110000011000000011010010111000101100001100001000000000001000001000010010000000000000010000100000000000100100 00000000010101000001000110110111111000111110101011010111011111011111101011111101100011110110010111110000011101 11001100001011111101010000111011010011010001001101101101111101000101111100110001011000110101101100110000110000 001100001000110000000001010011000000001101000100010110000000000011010000000000000000000010000000110000010000 100000000100000000000000100000000001000000001000000

NJ WR 13 2 01000000000001100000011111100001110111100001011011110110110110111100010001011001111010000110010110101110011111 1001000110000011011011000011110000110001011110000001010000000101001111101000001101001011100000000000001101111 111000011001101100000110101001001100110000001101001100000001001100000100001000010000110000000000000100010000 001000000000000000000000000001000000010000001000000000000110100000000000010000000000000000000010000010001000 01001111110011000111011100110011111110000111100100110011100001011101101111000101101000110011101110010110110000 1101110011000001001101110010010000111000101100001100000111111011000001001000011000000000000111011000000001010 100000001101100000001100000100000001000101100010000000101100000000001000010110000010001100000000001100000000 000001010000000000000000000000000000001000000000000000000000000000000110011100000100000011001111011010111001 10111000111001111101011111101101001101100110001011001101011110011111100110101101111110011101011000011000110000 011100101100100110110000100110000001001010111000000000000000001101000000011000000000001100011100100000000000 100011101000110000000100100101010001011000011000000000000000010000000000100100000000000000100001000000000001 0010000000000110011000000000100110111111000111010011011011011001110010110101011011101100011110110010110110000 01110111001100001011111100100000111011110011000001001101101101111011000101101100010011011000101101101100110000 110000001000001000010000000001010011000000001101000101000110000000000011000000000000000000000010000000110000 001000100000000100000000000000100000000010000001010000000

NJ WR 15 1 00001000010101101111010011100111010100011011011001110011111111001100110011010101101000100011001110001011011011 11010100101000110110110000111100001101011110100100011100111010110011111101001110011111011000011000000011000011 0110001100110110110011010100001110111000000110100110110000000110000010000000001100011000000000000000001001000 000000000000000000000000000000000001000001100000011010001000000000000001000000000000000000000010001001010100 01111110011011101101010010110110100100111001110000011111100011011011111000110100110010100011110011001101100011 0010000110100101100101110000000011100000000000000100111110001100100110011011000000000101101101100110000001010 000000000011101000000000000000001001110000001000000010110000000000100110110000001000000000000000110000000000 000001000000010000000000000000000000100000100000000000000001000001011011000001110000001100111101011011000110 01110011000111110000110111100100011110011000001101110111111101011110011001100011111011110000011110000010000000 1101100011100110110001100110110011011000110000000100100011001110000000000000000110001100001100000000011000010 001101000110000110010000000110000011000010100100000000000011000000000001000010000001000000000000000000010010 00000000000100000000100011001100011110001111110110111101110111101111011000111101111100110001110001001101000111 01110011100011101111110011001010110011100000010011001111101100010011010001000100110010001101011111000100000110 100000110001001100000110010000000000000011000000000000000000000000111000000000000000000000100100000000000010 00010000010100000000000001000001000010000000110000000

NJ WR 15 2 00000000110101101111010011100111110000011011011101110010000111001100110011001101101100000011001110001011011011 1101010010100011011011001011110000110111011010010000010011000011001011111000101101111101100001100000001100001 1011000110011011011001101010000111110000000011010011011000000011000001000000000110001100000000000000000001100 000000000000000000000000000000000000100000010000001101000100000000000000100000000000000000000010000101101010 10011011101101110110101001011011010010011100111000000111100000101101111110011010011001010011111011101010110001 1001000011100110010010110000000001110001000000010000001111010110000011011001100000000000101110111001000000110 000000000001110000000000000000000100111000000100000001011000000000010010101000000100000000000000001000000000 000001010000000000000000000000000000010000010000000000000000100000101101100000101000000110011110101101100011 00111001100011111000011111110010001111001100000110111011111101101111001100110001101101111000001111000001000000 0110110001110011011000010011011001100100011000000010010001100111000000000000000011000110000110000000001100001 000110100011000011001000000011000001100001010001000000000001100000000000100000000000100000000000000000000101 00000000000110000000010001110110001110001111011011011110111011110111111101011110111110011000111000000101000011 10111001110001101111111001100010111001110000001001100111101100001001101000100010011001000110101111100010000011 010000011000100110000011001000000000000001100000000000000000000000011100000000000000010000010011000010000010 000000000010100000000000001000001000000000001110000000

NJ WR 16 1 01000010000000000000011111100001110110100111011011110001111110101100010010011001111010011010001110011111100111 188

1100000010000011011110001011110000110001101110000001010011000000001101100111001100111111000001000000010101001 1011000000011011011001101011010110001100000011100011000001101100111000000010011000001100000000000001000100000 00000000000000000000000000000000000010000001000000100100010100000100000001000000000000000000100010010000000 000010111111110001111111101100111111100000011001000001111000000101101111111011101110001100011110101001101110001 1001110010110010111011000001101001100011000110000000000011100110000011011001110000000010110110001000000000100 000000000100010010000000000000001010000111000100100011001000000000010000100110000100011000000000000000000000 000001100000000000000000000000000000010000000000000000010000000000101100111000001000000111111110110101101011 00110101100011111000111111101101111000001100000110111001111101101111001101010000111100110100110100000010011000 0110110110100011011000011011100000001100111000000000000000000110000001101100000000000110000110000000001100000 001110000011000000100000010101000000010101100010000000000001000000000000100001000100000010001000000100000100 01000001000110010000001001100111011011001001110011111011001011110111101100011111101100110000111100110110110011 1101110110100010111101101000011001101101100000100110000110110000101010010110001000011000010110100010001000001 101001000000010010100000000011000000000000110000000000110000000000001100000000000000000000011000010010000001 0000000000000100000000000000100000000001000100010000000

NJ WR 16 2 01000110000000000000011111100001110110100001011011110001001110101100010101010001111010011110000110011110010111 11000000110000110111100010111100001101011011100000011100110000000011111010110011011111110000010000000010110011 0110000000100110110011101101101100011000000111000110000100011001010000000100110000011000000000000010010000000 00000000000000000000000000000000000100000010000001001000101000001000000010000000000000000001000100100000000 00010011101111001111111101100111111100000011001000001111010000001101111101011101010001010011011101001101110001 1001100001100110111011000001100001100011000110000000000011110110100011011000100000000000110110011000000000110 000000000100010000000000000000001100001011000100000001001000000000010000100110000100011000000000000000000000 000001100000000000000000000000000000010000000000000000010000000000001100111000001000000111111110110101101011 00110101100011111000111111011001101100001100000110111001111100101111001101010000111100110100110100000010011000 0110110010100011011000011011100000001101011000000000000000000110000001101100000000000110000110000000001100001 001110000011000000100000010111000000010101100010000000000001000000000000100001000100100010001000000000000100 01000001000110100000000001100110011011001001110011011011011011110011011100011111101100011000111100110110010011 1011110111000010111101110000011001101101100000100110000110110000101010010110001000011000001110100010001000001 101001000000010011000000000111000000000000110000010000110000000000000100000000000000010000011000010011000001 0000000000000100000000000000100000000010000100000000000

NJ WR 17 1 01001000100000000000111111101101100110100001111011000001111110101100110011010001111000011110001110011111011011 1101000011000000011000001001110000000001111010110001010000000011001111100100101100001011000000000000001101001 101000000001100001100010001100011000000000011110110100000001000110000100001001100000110000000000000100000000 000000000000000000000000000000001101110000011000000000001010000000010000010000000000000000000000010000000000 000010011111110111111111001100110111100000011011000001111100111101101011111011100010001100000111001101101110001 1001100001000010011101110000000001110000011000011000000011000110110011011001110001100001101010101100000000110 000000000000010000011000011000000100000101000100000000011000000000010001110110010100000000000000001000000001 000000100000000000000000000000000000010001000000000000000000000000001100000000001100000010011110110101100011 00110101100011111100111111101011001111001100000111101101011100101101101100110011101101110100001100000110000000 0000110000110111011000110011000001101101000110000000110000100111000000110000000011000000000110000000001100000 000110000011000000110000001111000000101001100000010010000001000000000100010001000100000000010000001000100100 00010000000011000000010001100110111111001111110011010111011001110111101111011111101100111000011000101110010011 1001110111000011111101101010010101100101110001100000111110111100100010110010011000010000011010111010001000001 101001000000010010000000011100000100000000010100000000000000000000011100000000000000110000000000100010000001 0000000000000100000000001000000000001010010000001000000

NJ WR 17 2 01000100000000000000011111101101110110110001111011010001001010101100110011000001111000011010001110011110011011 1101000010000000011011001001110000110001111010110001010000000011001111100100101100001011000000000000001101001 101000000001100001100010101100011000000000011110110100000001000110000010001001000000110000000000000100000000 000000000000000000000000000000001101010000001000000000001010000000000000010000000000000000000001000010000000 00001001111111011011111100110001011110000001101100000111110011110110011111101110101100101001011100110110111000 11001010001100110011101100000000001110000011000011000000011100110110011011011110001100001111110011100000000110 000000000000001000011000001100000100000101000100000000001000000000010001100110001100000000000000001000000001 000000100000000000000000000000000000010000000100000000000000000000001100000000001010000010111110110101100011 00110101100011111100111111101010001111001100000111101111011100101101101100110011101101110100001100000110000000 110011000001011101100011001100000110110100001000000001000010001010000011000000001100000000011000000000010000 000011000001100000011000000001100000010100110000001000000001100000000010001000100010000000000100000010000010 00000000000001100000000000111011011111100111111011101011101100111011110111101111110110011101001100010111001001 1100110011100001111110110101001100110011011000010000011011011110010011011001101100001000000111011101000100000 100100100000001001000000001110000010000000001000000000000000000000000010000000000000011000000000010001000000 10000000000000100000000000000000000001010010000001000000

NJ WR 20 1 00000000010101101010000011110111101110101011011011001011011101011000010110001101101110001111001110011011011011 0100010010000011011011101011110000011101101110110001110011000010011011100000001100111001000001100000001001101 011100011001100011000110111010000000110000000100001100000000000100000010001000010010010000000000000100000000 189

000000000000000000000000000000000100010000001000000100001010000000000010000000000000010000000101001010110101 11011000001110111011010100111101101000110101011000010111011100110110111111011101101110111011101101000110011000 11001100101001110111011110011101110110001000100011000001101100100110001011101100110000000110110101010111010111 000000000111010000001100000000000010010100000100000000001000000000010000100010001000000000000000001000000000 0000001000000000000000000000100000000000000000000000000000000000001011100100001111100001100111101001011011111 011000110001111110011111011100000111100110000011011011111110010111100110011000011110011011000110000011111100011 101000011000110110100100110110000011000110000000000000000001100000001101000000000000000001100000000001000010 001100000110000000000100000110000001000011000010000000000001000000001001000000000000000000000000000000001000 000000000011101010000000000011001111100011111110101111011101110111111110001111110110001111011100110111010000110 1101011100111101111011101000101110011100001010000001011011000010101000011001100010000000111111011000100001100 100110000010000100000110110000000000000011010001000001100000000000010000000000000100000000100010000100000010 00000000000100000000000000000000001010000000000000000

NJ WR 20 2 00000010010101111010000011111011110110001011011011001011001111010100010110001001101110011011001110011010011011 0100010010000011011011100011110000111101101110010101110011000010011011100000001100111011000001100000001001101 0111000110011000011001101110110000001100000011000011000000000011100000100010000101101100000000000001000000000 000000000000000000000000000000001000100000010000001000010100000000000010010000000000100000001001101101101011 00110000101101110110110001111011010001101010110000101110111000101100111110111101101101110111011010000110110001 1001101001101110111011110011101110110001000100011000001101110100100001011101100000000000110110101011110010101 000000000111010000001100000000000010010100000100000000011000000000110010100110011000000000000000011000000000 000000100000000000000000000010000000000000000000000000000000000000101110010000111010000110011110110101100111 01110001100011111100111110111000001111001100010110110011111100101111001100110000111100110110001100000111011000 011010000111001101101001001100110010010001100000000000000000001000000001110000000000000000111000000000110000 100011000001100000000001000001100000010000110001000000000001100000000010010000000000010000000010000000000010 000000000000110010000000000000110011111000111111111111110111011111111111101011111101100011110111001101110100001 1111010111001111011110011000011011100101100000100000010110110000101010000011011000100000001111100110001000011 001001100000100001000000101100000000000000010100010000011000000000000100000000000001000000011001000001000001 0000000000001000000000100000000000000010000000001000000

PD GO 5 1 0100100011010010000100011110000111011000000001101100110001000000110011011000100010000110001100111000110111101 1000111001100111011011000000011000000001011010000010010101100110100110001000110110000110110000000000010011001 011011000001011000001000001110000110000000000000000001100011000011110000000010011000011000000001000010000000 000000000000000000000001000000000000001010001000100011001010000000000000000000001100000000000001001010100010 00000010011111110111101111011001111111000000110011001101111100001111011011000011100011011000110000000001101010 1110000010001000000100011101001000111001000000100000000011011101100001110011001100110000001100011001101000000 100010000100001100000000110000000000000100001000000000001101100000000110110100001000000000000000000000000000 110000001000000000000000000000000001000100000000100000000000000000000110110000001010000001110111101001010111 11001101011000110110011010111001100110110011010011011011101111010011110011110100001101000110000000001101100111 1000001100001100110110001100110000011001110110000001100000011001000000000011000000000000000001100000000000001 100001100000110000010001100000110000001001000000100000000000010000000001001000110000000000000000000001000010 00000000100001101000000000011001100011110001011101010111110110111101111111011111011110001110101110110101100110 01111000011000111101111011000001100110110110001010011011011010001101011011011001100010110001101110001000100001 101000111000001001100000000110000110011010001100001000001100000000000110000000000000100000000000000010000001 10000000000000100000000000000100000001010000001010000000

PD GO 5 2 0100000000010000000010110100000111011000000001111000110000100000110001110001000100000110011001011001100101101 1001011001100111011010000000111000000000011000000001101001110110000110001000110110000110110000000000010110001 011011000010010000010000010110101100000000000000000010100011000011110000000010010000011000000011000010000000 000000001000000000000001000000000000001010001000100011001010000000000000000000001100000000000001010001000100 00000011001101111110111111011001111111000000010110011001111011001111110011100000001110011000110000000001001100 1110000000001000011100001100110000111000100000100100001011011101100001100101001100110000001100110001000100000 000000000100001100000000100000000000001100010000000000000111000000000010110100000100000000000000000000100000 010000001000000000000000000000000000100100000000010000000000000000000110010000001010000001100111101001011010 11001101011000111110011010111001100110110011010011011011110111010110110011111000001110000110000000001101100111 100000110000110011011100010010000001100111011000000100000001100100000000000100000000000000000110000000000000 010000010100011000001000010000001000000100100000010000000000001000000000100100011000000000000000000001000000 1000000001000111010000000000110011000111100110111001101110110101011010101110111110101100011101011000100001001 1001111000101000111011011011000011100110101110001010010011011010001101001011011001100010100001101010000000100 000100000111000001001100000000110000110001010000100001000001101000000000010000000000001001000000000000000000 00010000000000000100000000000000100000001100000001010000000

PD GO 8 1 0100011010010010011101010100110111011010000001110000110011000100110011000000000110110110001100111101110111101 1000110001100111001100011001011000000001011001000010100001100110000111011000010110010011100000000000010011011 011011000000011100010001111010000111000000000000000001011000000011000000000110000000001001100110000010000000 000000000000000000000000000000100000000010001000100100010010000000000000100001000100000000100000001110100000 00100000001111011101101111011001111111000000110011001101100110001111011011010011000011011000010000000001101100 190

0010001110011000010100001000000000100000000001100110000011001100000011110000011100110000001100000110001000000 110000000000001000000000000000000000000111100000110000000001110000000110011110001000000000000000000000000100 110000011000000000000000000000000001001100000000000000000000000000000110010000001000000001100110101101000101 11001101011000111110001011111001100110110011000011001001110111010011000001111001101100000110000000001000000110 0001001100000000110111001100111000011101010000000001100000011101110000001011000000000000110011100000000000001 000001100000110010000000100000110000001000000100001100000000010000000001001000011011000000000000000001100001 0000000001000110100000000000100110001111001110110101011110111000110011101000011101011000111000111011111010000 0110100010110001111011110110000011001100001000010100110110110110011000110100110010000101000011011100011011000 001000000001100100011000000001100000000110100010000000000110000000000001100011000000010010000000000000110000 0010000000000001000000000000000100000001010000001001000010

PD GO 8 2 0100110100010000001100010110110111011010000001110100110010000000110011001001100111100110001100111001110111101 1000110001100110001100011001011000000000011001000010100001100110000111101000010110110011100000000000011011011 011011000000011100001001101011000111000000000000000000011000000011000000000010000000001001100110000010000000 000000100000000000000000000000100000000010001000100100010010000000000000101000100100000000100000010011000000 00000011101110100111111111011001111111000000110011001101111100001111010011000000100110011000111000000001100100 1110001110001000000100001100001001110000000001100011000011001100000001110010011000110000001100000110000000000 100000000000011000000000011000000000000110100000010000000000110000000010010110001000000100000000000000000100 010000001000000000000000000000000001000100000000000000000000000000000110010000010010000001100111101110001100 1100110101100111111001101111100110011011000100001100000011011101010101000011100010110000011000000000000000011 0000000110000000010011100110011100001110101000000000110010000110111000000101100000000000011000110000000000000 100000110000011000100000010000001000000100000010000110000000001000000000100100001001000000000000000001000001 0000000000100011000000000000010011000111100110111001101110001100011010111100000111101100011100001101101101000 0011010001101000110111011011000001100110001100000010010011011110000100011110011010000010110001110010001101100 000100000000110010001000000000010100000001001001000000000010000000000000010000100000001001000000000000001000 00010000000000000100000000000000100000001010000001000000000

PD GO 10 1 0100010000010000001100011110111111011000000101101001100001000000110001010000000011100110111100111000110110001 1000111001101001011001000100011000000010011111000010000001100000100100101000001110001110110000000000000011010 0110110000110110000110011100100001100000011000001100000110110000000000000000011100000110000000000000100000000 000000000000000000000010000000000001010000010000000100001100000000000000000000000000000000000010010110000100 00100100011111101011101100110011111110000001101110011011101100011110110110000101101100110001000000000011110000 110000000011001100001000000000000011000000000010000000011001001100001100101001100110000011110000110001000010 100000000000111000000000001100000000000110100000000000000001110000001100101010001000000000000000000000001100 000000011000000000000000000000000001000100000000000000000000000100000010000000001011010101110111101101011100 11001011011000110110011010111001100110110011000001101111011111000011011011011001101111000000000000000010000001 1000001100000001110110000000111000011001000110000110000000011001101100011001010000000000000111100000000000000 110001100000110000110000000000110000001000000000100100000000010000000000001000010000000000000000000010000010 00000000010001101000000100000001100011110011101101010111111010101101111011111111010010001110101110100101100110 1110001001101000111010001100000111011000110000101001101101111000110001101101100110001001000110011010100110000 011100010110011000010000011010100010001100000100000000010110000000000011000000000000010000000000000000000000 010000100000000100000000000000000000001010010001000000000

PD GO 10 2 0100000000010000000100011111111111011000000101101001100001000100110001011000000011100010111100111000110110001 1000111001101001011001000100011000000010011100000010000001100000001100101000001110000110110000000000000011011 0110110000110110000110011010100001100001111000001100000110110000000000000100001100000110000000000000100000000 000000000000000000000010000000000000010000010000000100001100000000000000000000000000000000000010000001000100 10100000011111100011011100110011111110000001101110011010101100011110110110000011100100110000000001000011110000 010000000011001101000000100011000011000000000000000000011010101100001100011001000110000011110000110001000010 100000000000110100000000001100000000000110110000000000000001101000001100010100001000000000000000000000001100 000000011000000000000000000000000001000100000000000000000000001000000110000000001010000001110111101101011100 1100101101100011011001101011110110011011000100001100101110111101001101101100100111101100000000000000011000000 110000011000000011101101000001110000110010101100001100000000110011011000010010100000000000001011000000000000 010100001000001100001100001000000100000010000000001001000000000100000000000010000100000000000000000000100000 10000000000100011010000001000000011000111100110111001101111110100111011110110111110101100011101011100011101001 1011100110011100000110000011000001100110001100001010011011011110001010011011001001100010010001100010100001100 000101000101100110000100000100010000110001000001000000000001100000000000110000000000000100001000000000000000 00010000100000001000000000000000000000000010010001000000000

PD GO 13 1 0100100110010000001100010100111111011000000001101001100001000000110011000001000111100110001100111000100101101 1001100001100000011101011000011000000000011000000010000001100110001100011000000110001110110000000000000011011 0110110000110110000110011100100001100000000000001100000110110000111100000000100000000110011001100000100000000 000000000000000000000110000000000000000100010001000000000100000000000001000010000000000001000000100010000000 01000000011111100011011110110011111110010001100110011011011000011110110110000111000000110001101011000011000001 000000000011000000000001100000000011000000001100000000000001000000001110000001000000000001100000100101001100 110000000000010000100000001000000000000011010000000000000001100000000110010010001000000000000000000000000000 191

110000011000000000000000000000100001000100000000100000000000000000000110010001001011000001100111101110011000 11001011011001110110011011111100000110110011011111001101101111000101011011111001111011001100000000000000000010 000000110000100111111000000011100000010100000000000000000000000111000000101100000000000000000111000000000000 010000110000011000011000010000001000000100000000000010000000001000000000100100001000000000000000000001000000 0000000001000010010000001000000011000111100100111001101101101101011010101100111100101100011101011101001011000 0011110000101100110011011011010001100110001100001010011011011110000100011110011001100010100001101110001001100 000101000000100001000100000000000000000001000000100000000010000000000000110000100000000000000000000000000000 00010000100000000000000000000000100000001000100001010000000

PD GO 13 2 0100010011000000001100010101111111011110000001101101100001000010110001010001000111100110001100111000000101101 1001010001100000011101011000011000000000011000000010000001100110001100101000000111100111110000000000000011001 0110110000110110000010011011010001100000000001001100000110110000111100000000100000000110011000100000100000000 00000000000000000000001000000000000000010001000100000000010000000000000100001000000000000100000000000100000 00010010001111110101101111011001111111000000110011001101100100001101011001000001100000011000110101100001100001 110000000000100000000101110000000001100001000110010110000010110000000111001000100000000000110001011010100110 010000000100001001110000000100000000001001101000000000000000110000000011001001000010000010000000000000000000 001000001100000001000000000000000000100010000000010000000000000000000011001000000101100000111011111101101100 01100110101100111011001101111110000011011000101110110111101111100001101101111100010111100010000000000000000001 0000000110001100111111000000011100000010100000000000000000000001111000001001100000000000000000111000000000000 110000110000011000011000000000001000000100000000000010000000001000000000000100001000000000000000000001000000 0000000000100011010000001000001011000111100000111001101110011101011000111110111101101100001101011101001011000 0011101000011000110001111011010001100110000100001010001011001010000010011010011001000010100001110110001001100 000101000000110001000100000000010100010001000001000000000010000000000110010000000000000000000000000000000000 00010000000000000000000000000000100000001000000000010000000

PD GO 15 1 01011100110101111111001001100001110110000000011000000000010010111000111100000001111000011011001110011011011011 000100001100010101101000100011000000010010000000010000001110001110100111000010110001110110000000000000110010 011011000011011000110000000100000110000000000000000000011000000000000000000010000000010000100011000000000000 00000000000010000000000000000010000000001000100000000100001000000000000000000000100000000000000001000100001 00011111100111110011110111101100111111100000011110100110110110000110101111100000000011001101100110000000110010 001100000000110000101010000000000000110000000000000000000000000011000101000010000001100000110000111010000000 000000000000000101000000000000000000000001000110000000000000001000000001100101100000000000000000000000000000 000000000010000000000000000000000000000001000000001000010000000000000001100100000000100000111001111011010101 10110111010110011111100010111111011001101100010001110010111101110101011010011011011011110001100100000000011000 011000001100011000110111000000110011011001010110000110000000011001000000000001000000000000100001100000000000 000100000100000110000110000000000010000001100000000100100000000010000001000001000010001000000000000000010000 0000001000000000100100000000000000110001111001101110011011101101010110110111011110001011000111011011011000110 0110110010000110001111001000110000011001101111000010100010110011101001010111100110011001101100011001101000011 000001010001001000010001000000001100000000000000110000100000100000000000000100011000000001000000000000000000 0000011000000000000100000000000000100000001010010000000000000

PD GO 15 2 0100010010010000001100000100000111111010000001101000000001001000110001110000001111100001101100110001101101101 1001010000100011011101000100011000000001111011000010001001110000011100111000010110011110110000000000000010011 011011000011011000110001101110000110000000000000000000011000000000000000000010110000010001100011000010000000 000000100000100000000000010000100000101010001000000111000010000000000000000000001000000000000000000001100010 00001011101101100111101111011001111111000000110111001101111110001111011111010011100010011011001100100001100100 0011000100011000010111001100000000111000000000000000000000101101100000001011011000110000011100011011100000000 100000000000111100000000000000000000001100001000000000000001100000000110011110000000000000000000000000000001 000000011000000000000000000000000000000100000000100000010000001000000110010000000010000001110110101101010100 11011101011000111010001011111101100110110001000101101011110111000011101001101101101101000010010000000001100001 100000110000110011011010000011001101100101011000011000000001100110000000000100000000000010000110000000000000 101000110000011000011000000000001000000110000000010000000000001000000000000100001001000000000000000001000000 00010000000000110100000010000100110001111001110110011011111101010110110111100111001101000011011011011000110011 0110000000110001111010000110000011001101111000010100010110011101001000111100110011000101000011001101000011000 001010001001000010001000000000100000000000000110000100000110000000000000100001000000010000000000000000000000 0111000000000001000000000000000100000001010010000001000000

PD GO 16 1 0100000000010000000100011100111111011000001101101110110000100000110011110000000110000001101100110001100111101 1000111001101111110001011000011000000000011100000011000001100110000000101000001110000110110000000110000011110 011101000011010000011001100111100100000000000000110001011000000011000000010000000000011000000011000010000000 001100000000000000000001000000000000000010001000000000000101000000000000000000100000000001000001001001000000 00000011001111111111101111011001111111000000110011001001101100001111111011101011000010011000010000000001101100 000111000001101011001000110000000010000000000001010000001100110110000111010101100000000000110101011010100000 111001100100001100000000000000000000000110011000011000000001100000000011011010000010000000000001100000000000 001000000100000000000000000000000000000010000000001000000000000000000001000000000001000000111011100110101110 01110110101100011111000101011100110011000001100000101101110111101001110001111100000110100011000000000000110000 192

110110011000000001100000011001110000010110001100000100000000111010000000000100000000000011001110000000000000 000100011001001100001100000000101100000010000001001000000000000100000000100010000000000000000000000000000000 10000000000000011011000000000010011000111101111011011101111101100011101110110111010110110001100011101101101001 0001001100011000111101000001100001100110001010001010011011011001001100011010011001100110100001101010001000100 001111000101100010001100000000010001100000000001000000000000000000000000110000000000000000000000000000000000 00110000000000001000000000000000000000001000010001000000000

PD GO 16 2 01000000000000000011000111101111111110000011011011001101100000001100010111000000100000011011001110011001111011 0001110011011101110010110000010000000010111000001101000011001100000001010000011100001101100000000100000011010 110110000110110000010011000111001100000000000000110010110000000110000000010000000000010000000010000100000000 011000000000000000000010000000000000000100010000000000010100000000000000001001000000000010000010010010000100 00000100111101101101011110110011111110000001100110011001110000011110110010010010100110111001010000000011011000 0001010000110001110110011000010001100000011001001100000110101011000011100110110000000000011000111011010000001 100110010000011001000000000000000000011001100001100000000011000000000100101001010000000000000100000000000000 100000110000000000000000000000000000001000000001000000000000000000001100100000010100000001101110011010111001 10011010110001111100110101110011001100000110000010010111011110100110110111101000011010001100000000000011100111 011001100000000110000001100111000001001000110000010000000001101000000000010000000000001100101000000000000000 100000100100110000010000000000010000001000000100100000000000010000000011001000000000000000000000000000000010 0000000000000010100000000000000110001111001010110011011110111000110010101100110111001000011010111011010110010 0010010000110001110010000011000011001100011100010100110110110100011000110110110011000101100011010100010001000 001001001011000100001000000000100011000000000010000000000000000000000000100000000000000000000000000000000100 0010000000000000000000000000000000000001000010001000000000

PD GO 17 1 0100010000000000001100010100111111011000001101111010110010000010110011000000000110100000111101100001101101101 100011000010000100000001100001100000000110110000010010000110011000000001100000011000011110000000001100010001 1011010000011011000001001101110000111000000000001011001100000100001110010011000000000001101100011000010000000 00010000000000000000000100000000000100101000100010010000001000000000000000000000010000000000000000100000000 00000110000111101011110111101100111111100000011011100110111100000101101101100001111000001100001000110000110010 0111100000001100001111000100000000001100001100011111000001101100010000111001101100000000000110000101110000000 001000000000000110000000000000000000000110001000010100000000110000110111001111000000000000000001000000000000 000000001100000000010000000000000000000010000000000000000000000000000011001000000000000001011011110111000110 0110011000110001101100110101110011001100000110001011011110101110100110000011011011011110011000000110000000000 0110110011000011011100000011001100000110110000000001100000000010111000000000110000000000011000111000000000000 000000011000001100001100000000001100000010000001000000000000000100000000010010000100000000000000000000010000 10000000000000001010000000000000011001101100111011001101111000100011001110110111011111100011000011101111101000 0011101000011100110101100011000001100100000100001010011011011010011100011010001000000010010001100010101000100 000101000101100001000100000000110000000000000000000000000001100000000000110000000000001000000000000000000010 00110100000000001000000000000000000000000010011000000000000

PD GO 17 2 0100010000000000001100010110111111011000001101111000110001000110110000100000000110100000111101100000101101101 100101000010000110000001100000110000000110010000011010000110111000000010100001011000011110000000001000100001 100110000001101100000100110101000011100000000000110100110000000000011000000100000000000010110011100001000000 000010000000000000000000100000000000000001000100010010000001100000000000000000000010000000000000000100010001 00000011000111111111110111101100111111100000011011100110111000000001101101100001010000001110001100110000110010 0111100000000100000011000110000000001100001100001110000001100110110100111001001100000000000110000101100000000 001000000000000110000000000000000000000011001000001000000000010000110111001101000000000000000000100000000000 000000000100000000000000000000000000000010000000000000000000000000000001001000000001000001010011110111000110 01100110101100011111001101011100110011000001100110110111101011101001100000110110110111100110000001100000001000 1101100110000100111010000110011000001101100000000001000000000011110000000001100000000000110000110000000000000 000000110000011000011000000000001000000100000100000000000000001000000000100100001000000000000000000001000001 0000000000000001010000000000000011001111100111011001101101000100011001110110111011110100011010011101100111000 0011101100011100110101100011010001100110001100001010011001011010011100011011011000000010010001100010101000100 000101000100100001000100000000110000000000000000000000000001100000000000010000000000001000000000000000000010 00010000000000001000000000000000000000000010001000000000000

PD GO 20 1 0000100100000000001100011100000111111010000001111011110110101000110001110101000100000000011001011001100101101 1000110001101111011100011000011000000000011100000110010001100010100100101000010110000110100000000000001110010 011101000010110000010001001010001011000000000000010000011011000011000000000000010000010000000100000010000010 000000000000000000000001000000100000000010001000000100001110000000000000000000000000000000100000000101000000 000000110111011111011011100110011111110000001101100110011111100011111100110000111001100110110110110111110110000 1100000010110010110001001001010011000000000010110000000110111010010111001010110001100000011000011101100000101 011000000001110001000000110000000000000100100000100000000001000000000100101000010000000000000000000000100000 000000010000000000000000000000000000001000000010000000000000000000001000000000100000000010001111011100111001 1001101011001111110010011011001100110110011001011000011111011000101100001111000001010000000000000000001000001 100000100001100011000000110010000001000100011000001000000000000110000000001000000000000010010110000000000000 110000110000010000001000010000010000000100000000010000000000001000000000000100000001000000000000000001000000 193

00000000000001110111000000001000110001010001101110011011011101010110011111001111011011001011011010011110110011 0110110011010001110101000110000011001001111000110100110111101100011010111000110011001101000001010100000001000 001000000011000100001000000000011001000000000010000010000000000000000001000000000000010000000010000000000000 0100000000000001100000000000000000000000010000000000000000

PD GO 20 2 01000000010000000101000111011111110110011101111111110101011110011110010111011001110110110011110101011001011001 1001100011000110011000111011011000111000111010011001100110110111001001010100011101011101100000001101100110010 110110000110110000010010010100001110010000000000110000010011000111000100110000100000100000011010000100110100 000000001000000000000010000001000001001100010000001000010100000000000000000000000000000010000000010101000000 00000111011101101111011101111011111110000001101110011001010011011110110110000010000110110110101001001011110101 0110010001110000100000111100000001100001100011110000000110010000010111101100111001100000011000001011010000011 000010001100011000000000011000000000000100010000110000000011010000001100111000010000000000000000000000000000 0000000100000000000000000000000000000010000001000000000000000100000011000011101111100000011011110101111100011 00110101100011111001101011100111011001001101010111100111011100001111000110110010111100000010000100000011001110 110011100101001101000011001100000010010001110001100000011000001010000011000000000000001000111000000000000000 100001000001100001100001000000100000010000000001000000000000100000000000010000000010000000000000000000000000 0000000000001101110000001011001100011110011011100110111101000101100101111010001011010001010110110010101100101 0100110101100011100110111101000110011111011000101001101100101100110101101001100110011001000010101000100010000 010000000110001000010000000000000010000000000100000100000000000000000011000000000000010000000100000000000000 010000000000000100000000000000000000000100000000000000000

PD GR 1 1 00000000001101101010111111001101100000111011010000111101110110000011000000000110111011001100001100011011111011 01110001101100001110001000111100010111011011000010011010110111100011001100001011011111110000011001111001011111 011000011011000011101001110010110000000000000110001111001001000010000000000011000001100000101000001000000000 000000000000000000000000000010000000101000010000000000011100000000000000010010000000000010000101101101101010 00111010110110111001100111111010000001100110011011101111001001100101100100110110000011100011111011001101110011 10010001100001100110101100110110101100111000110000000011111101101000110111011000000000011000111100111110011000 000000110011000000000000000000000000001100001100000000110000000110101100010001101000000000000000000000000000 000000100000010000000000000000000000100000000000000000000000000000011000000000110100011100111101101011010110 01101011000111110001111111011010011110000110001111111110111010011000011101110101111001100000000001100000001101 1001100001100110110101101111000011001010110000001000010101111110000000001010000000001100001110000000000000110 001100000111000110000100100011100001000001000000100000000010000000101011000010000000000000000000010000010000 000000001011111110000000101011001111100010111101101111101100011101110110101111111110001111011101010111011001110 0101011110000111100100101001101110000110001010011011011011000111001011110111100110110001101010001000100000100 100111100110001100100001110001100001000001010110000000000000000000010000000000011100011001100000000001100100 01100000000100000000000000000000011010000000100000000

PD GR 1 2 01001010110101000000011111101111100111101000111011110001111111111000011000000001101111100110001010011001111011 01010001101100001110011010111100001111011011000010001010110101100011001100001011011111110000011000111001010111 0110000110110000111010011100001100000000000001100011110001011000100011000000110000011000000110000010000000000 000000000000000000000000000000000001010001000000000000111000000000000000100100000000000100001001001001100001 111111110001000111111110110011111110001001101100110011111101110110001100111110101000111001111101100110111001110 0101011010001001101011010101101011001100001100000000111011011011101101100110000000000110001111001101000110000 000001100110000000000000000001001001011000110000000001100000001101011000100011010000100000000000000000000000 001001000000100001000000000000000001000000000000000000000000000000110001000000101000111001111011010110101100 11010110001111100101111110111000101100110100011110111101110100110000111011101011110011000000000011000100001011 0011000001011101100111011110000110010101101100010110011001111100000001110100000000001000011100000000000001100 011000001110001100001001000001000010000010000001000000000100000001010110000000000000000000000000100000010000 000000010110101100000000110110011111100101111011011111011000111111101101111011111100011110111010111111010011100 11001111000011110011110000111011010011000101001101101101100110101101100111110001010000110101000100110000110010 011110011000110010000111000110000100000111011000000000000000000001000000000000110000100110000000000110010000 000000000100000000000000000000001010000000000000000

PD GR 2 1 01000101000000100000011111101101101111100101011010010011111001011000011100011011111000111110001110011010011011 1001100110110000011001100011110000110010101110001100010011010001000000100000111001111101000000000011001100001 0111000111011000111101110110010110000000000001101000110110111100000000100000011000001000000001100011000000000 000000000000000000000000000101000001100000010000000000001100000000000010000000010000000010000100100100000000 10100011110110000111111101100111111100100110010001100111100100111100101010011101001111110011011100001101110001 1001011011100100001010100011000001100000001100001000001111010110110011011011000000000011100000001010100101100 001100101011000000010000000000000010111011000110000000011000000000010110011010000100000000000000000001100011 000010110000001000011000000000000000010000000000000000000000000000101101000000101000110110011110110101100011 01101101100011101000111011101011001111101101000110011011111110101101101110110111111100110110000000101100000110 0000110000111011001000110011100001101101011000011100000000000110000000001100000000000110110000000000000000101 000110110011010011000010001011000000110100000001000000000001100000000100100001000100000000010000001000000100 000000010101110111000000001001100111110011110101110110110110011111111011011101011011000111111110110011110000111 01011011110000111100000110001100111100110011010011011011011001111011011011011100110110001101110101001100001101 194

101101100010001101011000010001100001000011000001000001100000000000110000001000001101001000000000010000000110 00000000000100100000000000100000001010000000110000000

PD GR 2 2 01000101000100100100011111101101101111100011011010010011101101110100011100011011011000111110011010011110011011 1001100110110000011011100011110000010010101010001101001011010011000000110000110101111101000000000011001100001 0111000111011000111101110110110110000000000001100000110110111100000000000000011000001000000001100001000000000 00000000000000000000000000000100000010000001000000000000010000000000001000000001000000001000010100010001000 010101111110110101111111101100111111100001111010001101111100110111100111010011100001111100011011100011101110001 1001011011100100000010100010000001100000001100000000001111100110111011011011000000000011100000001000000001000 001100011011000000000001000000000010111011001100000000011000000000110110011010000100000000000000000000100011 000010110000001000001000000000000000010000000000000000000000000000101101010000101000110100011110110101100111 01101101100011111000111011101011001101111101000110011011111110101101101110111010111100110110000000101101000010 0000110000110111011000110011100001101100011000011100000000000010000000000100000000000110110000000000000000011 000110101011000011000010001011000000100100000010000000000011000000000100100001000100000000010000001000000100 00000001000101011100000000100110011111001101010101011111011001110011101101110101101100011110111011001110100011 10011101111000011110000011000110011110011001101001101101101100111101101101101110011011000110111010100110000110 110110110001000110001100001000110000100000110000100000110000000000011000000100000110100100000000001000000110 000000000000100100000000000100000001010000000000000000

PD GR 3 1 01000001000000100000011111101101101111100000111010010001111001110100111100010011111001111110001110011001111011 0010100010110001011101100011110000000110101101100001110011001100011000010000101100011111001101100001001101101 1011010011011000010001110011010110000000000000000000011110011000000000000001101001011001100001100001000000000 000000000000000000000000000100000001101000010000001000010100000000000100000000010010000010000101100100000000 100111011011000011111110011001111111000010110110011001111110101111110011000111100110011100110100111011011100011 0010001111011101000001000011001000001000000001100000011101001101001010110011000010100011000001110010000011010 000000110010100000000000000000000101101100010100000000110000000110101101100000001000000000000000000011000110 0000111000000100000000000000000000001000000000000000000000000000010110111000010100000011111111011010011101101 11010110001111100011111110111000101100110000011011111111110100110010111101001011110011000000000011000110011011 0011000011011101100011001110100001111101100000011000100001111100000011000100000000011000011000000000000001100 001001001101001100101000101100000010010000001001000000000100001001110010000100010000000000000000100000010000 00000101001110010100000011011001111111111101001101111110100111001110110011011110110001001011101100111000100111 1100011010001011100000110001000110001100011010011011011011101110001011001011100110110001101110011001100000101 101101100110000001001001100101110001000000000001000001100000000010010000000001001100001000000000000000001100 01110000000010110000000000100000001010000000100000010

PD GR 3 2 01010000000010100000011111101101011111100000111010010001111001001100011100111011111001111110011010111001111011 0010100010010001011011100011110010000110101111100001110011001000011000110000011100011111001101100001001101101 1011010011011010011001110011110110000000000000000000011110011000000001100000111001011001100001100001100000000 000000000000000000000000000000000001101000010000001000010100000000000010000000001010000010000101001100000000 100111111001100011111110011001111111000010110110011001111110001111110010001111000110011001111010111011011100011 00101011110111011001110001110110000010100011111000000110101011010010101101110110111000110000011000101010111100 000001101101000000000100000000000111001000101000000001100000001101011011000000010000000000000000000110001100 0001110000001000000000000000000000010000000000000000000000000000101100000000101000000111111110101100111011011 10101100011111000111111101010001001001101000110111110111100101101101110110010111100110000000000110001100110110 0110000110111011000110011101000011111011000000110001000011111000000111100000000000110000110000000000000011000 110000011010011000010000011000000101100000101010000000001000001011100100001000000000000000000001000001000000 00001010011100100000000110110011111111111010101011111101001110011101100110111101100011110111011001110000001101 0100111000010111000001100000001100011000110100110110110111011000010110110111001101100001011100110011000011011 011001001100000010110011001011100010000000000010000011000000001100100000000000011000000000000000000000011000 1010000000000110000000000100000001010000000100000010

PD GR 8 1 0100100000000000000001101111111101011010100001110101000011100101010011110001000111101001001100011001100111101 1011010011000001101100110001011000001011001110000100011100010111100000011001101110001110100010000000110010110 1101100001101100001110111011000011011011000000000000101100000110000000000001100001001000000000110001100000000 00000000000000000000000000001000000001010000100000010000010100000000000010010001000100000100001001001000100 000001110110011110011111110110011111110010001111000010011111001000110100100001110011011111010111100111110011000 1100101011011000000001011000000110000100000001100000000111110011010010101101100000010101110001101101100000011 1001110011001100000010000000000011001100110000000100000011010000001011110011001101100000000000000000000100011 000000110000001000000000000000000000010000000000000000000000000000101100010000001000000110011110110101100011 10101101101011111100111111100010111011001110000110011110111111001110101100110000111100000110000000110000000110 0000110000010011011000110011100001100101011000011100001000110111000000000100000000000110000011000000000000101 000010000011000000000010001100110000100000000010000000000001000001000100100000000110000000000000001000000100 00000000000011110100000000100110001111001001010101011101011001110111101101110111101000110110111011011111000011 11101001110000011110000011000010011010011000011001101101101110111001101100100110011011000110111010100110000011 010011110011000010000000000110000000100000100000100001110000110000001000000100000000000100010010000000010110 000100000000110000000000000100000001010000000100000100 195

PD GR 8 2 01000010100110000000111011111111110111100000011011110000110010110100111100000001111011011011000110011011111011 0111100010010011011001100000110000010110011100011000010001100111001000110011011100011111000000000001100101101 1011000011011000011101101010000110110110000000000011011000001100000000000011000010011000000001100011000000000 00000000000000000000000000000000000010100001000000100000101000000000000100000010001000001000010010010001000 011001101100111101111111101100111111100000011110000100111110010001101101000011100101111110111111011110111110001 100100011000000001001011000010011000001000001100000000110101011010010101100110000000001110001101101100000110 0001110011001100000000000000000011001100110000000000000011010000001011110011001100110000000000000000000100011 000010110000001000000000000000000000010000000000000000000000000000101100010000001000000110011110110101101011 01101101100011111100111111100011101111011110000110011110111110101110101100111000111100000110000000110000000110 0000110000010011011000110011100001100101011000011100001000101111000000000000000000000110000111000000000000101 000010000011000000000010000001010001100000000010000000000001000001000100100000000110000000000000001000001000 00000001000011110100000000110110011111001001010011011111001001110111101100110111101100111010111011001110000011 11010001111000011110000011000110011010011000101001101101101110111001101101100110011011000110111010100110000010 110011110011000010000100000110000000100000100000100000110000010000001000000000000000000100110010000000010110 000100000000010000000000000100000001010000000100000100

PD GR 11 1 010000001000101000000111111111110101111000000110111100011011101011001111000110111110111111101101100111011110110 0110001100000100110110000101100010101100110000100010100010001100001110100000101001111110000000000011011010011 0110000000110010110011011110001101101000000001100000111110000000000000000110001100110000000011000110000000000 00000000000000000000100000100000000101000010000000000010010000000000100000001010000000010000100100100010000 001111111001111001111111011001111111000011110110011101111100001111111111001111001110010101110110111001001100111 0010101101011000111001100000001011000110000110000000011011001101001010110111000000000111000011010110000001010 000000000110101000000010000000000000100100000000011000110011000111101100110000001000000000000000000111000000 000100100000010000000000000000000000100000000000000000000000000001011000000000010000001100111101101011000110 01101011000111111001111111010110011011011000001110111101111110011011001110100001111000001100011000110010001100 0001100001110110111001100111000000111010110000110000000000111100000000000110000110000000011100000000000000110 000100110110000010000100010110000001001001000010000000000010000000001001000010001000000000000000010000000000 000000100011011100000000001011000111100110111001111111101100111011011110111011111110011111001101100111100001110 1111011000001011100010000001101110000010000110011011011011010010111011001111100110110001101110001001100000100 100111100010001101000000000101110001011001000001000001100000100000010000000000000101000000000010010000001100 00000000000100010000000000000000001010000100001000100

PD GR 11 2 010000010000001000000111111111101101111000000110111100111011101110000111000110111110111111101101100111011110110 0110001100000100110110000111100101101100110100100000100111001100001101000000110011111110000000000011011110011 0110000000110000110011101110001101100000000001100000111110000000000000000110001100110000000011000010000000000 00000000000000000000100000000000000101000010000000000010010000000000100000001010000000010000100100100000000 0001111110011110111111110110011111110000111011100111011111100011111111110101110111100111011110101110110111001110 0101011010110001110011000000011110001100001100000000111110011011011011101110000000001110000110101100000011000 000000001101010000000000000000000001001000000000110001100110001111011001100000010000000000000000001110000000 001001000000100000000000000000000001000000000000000000000000000010110000000000100000011001111011110110001100 11010110001111100011111101101100110111110000011101110011111100110110011011000011110000011000110001100000011000 0011000001001100110011001110000001110101100001100000000011011000000000100000001100000000111000000000000010100 001010101100000000001000001100000010000010001000000000001100000000010010000100000000000000000000000000000000 00000000001001100000000000011000111100110100010111111101101011011011110110011111110011111001101101111010001110 1111011000001011100000000001101110000111101010011011011011000010011011011011101010110001101010001001100000100 100110100010000101000000000101110000011001000000000001100000100000010000000000000100000000000000010000001100 00000000000100000000000000000000001010000100000000010

PD GR 12 1 01010000000000100000011010111101100111100010111011010011001010101100011100010001111000111110001110101110011011 0101100010110110011011100000110000010001001110010000010011000100011000100011101001101011000000000001000001101 1011000011011000011001111011000110000000000000010001111011000000000000000001000010011000000001100000100000000 00000000000000000000010000001000000010100001000000000001010000000000010000000100000100001000000010010000100 000011001101111111111111101100111111100000011001001100111010000111101111100111100111111100011110101001101110001 1011100110100000000010100001011100100000000011000000001101100110110011011011000000100011100011101011000010110 000000001011001000010000000000011010110110000100000000011110000010010110100000000100000000000000000001100011 000000010000001000000000000000000000010000000000000000000000000000001100000000101000000111111110110101110011 00110101100011111000111111011011001101101100100110111100111101001101100111100000111100110000000000110001001110 000011000000011101100010101110000010110100000000000000100010111000000011000000000000011000111000000000000001 100011010001100000100001000001100000010000000001000000000001100000000010010000000000000000000000000100000000 00000000101001110100000000001011001111000101101010111111101111011001110111101011110110011111011101100011000001 11001110110010001111000000001011001100001100001100110110110110011111110110011011001101100011011101010011000001 101001101000100001010000000001001100010000001000010000100000000000000100000000000011000000000000000100001011 0000000000000000000000100000100000000010000000100000000

PD GR 12 2 196

00100001000100100000111011111101100111100010111011111011101001001100111100010001111000111110001111011010011011 0101100011010110011011100000110000010001101010000000010011000100011000010011101001111011000000000001000001101 1010000011011000011001101110000110000000000000000001111011000000000000000001100110011000000001100001000000000 000000000000000000001100000010000001101000100000000000100100000000000100000011000001000010000001100100000000 000010011011111111111111011001111111000000110110011001110100101111111111000101100111111100010101010010111100011 0111001101000100000111000010111001000000000110000000011011001101100100101110000000000111000110110100001011100 000000110110000000000010000000110011101100110100000000111100000101101101000000001000000000000000000010000110 000001100000000000000000000000000000100000000000000000000000000000011000000001010000101111111101101011101110 01011011000111110001111111010100010110011000001101111001111010011011001101100001111001100000000001100000011100 000110000000111011000011011100000101101000000000000001000010110000000010000000000000110001110000000000000011 000111100011000001000110000011000000100000000010010000000011000000000100100000000000000000000000001000000000 00000001000011110000000000010110011110001011100101111110101110110011101111110111101100111111011011001110010011 10011011100100011110000000010110011000010000011001101101101100111111101100100110111011000110111010100110000110 110010110001000010000000000010011000000000100000100000100000000000001000000000000110000000000000001000010010 000000000000000000000100000100000000010000000000000000

PD GR 17 1 01000000010100000000110011111101100111110001111011011011101110110100111100011001111001101111110111011010011011 0111000001110111011011100011110000010001100010100101011011001110000000110000001101111011000001000011000101101 011100001101100101100110101000001000000000000100001111100001100000000000001101000001100000000100000100000000 000000000000000000000010000000100000010100001000000000000010000000000010000100100000000001000010010111000000 000011111101110101111111001100101111100000011011000110111111110111111001100010101011001010001101011001101110011 1001010110101100011001010110000110000011011011010000001110100110110101010111100000000001101101111011001000110 000000001001100000000000000000011000110110000000000000011010000011110110110000000100000000000000000000000011 000010110000000000000000000000000000010000000000000000000000000000101100000000001000110110011110101101010011 10101111100011111000111111101100001011001100000110011110111101001101101111011000111100010000000000000001100111 1100110000100111001100010011100001101100111000011000000000010110000000111001000000000010110110000000000000011 000111100011000000000000000011000000100000000000000000000001000000001100100001000000000000000000000000000000 00100001000010010000000000000110001111001101010101011111101110110111101101110111111100111110011011001110100011 1011110111000010110000101000011001100001100001100110111110110001100110110011010010101100011010101011011000001 001001111001100011000000001100011100000110011000000001011000001000000110000010000001000000000000000000100001 0000000000000000000000000000000000001000000000000000000

PD GR 17 2 00100000000100000000011011111101100111100000111011110011101111001100111100011001111011101011110110011011011011 0111000010110111011010100011110000010001100011100001011011001110000000110000001101111011000000000011000111101 011100001101100001100110101000001000000000000000001111100001100000000000001101000001100000001100000100000000 000000000000000000000010000000100000010100001000000000000010000000000000000000000000000011000011010010000000 00000110110111010111111100110010111110000001101100011011111101011111100110001110101100111000110101100110110001 11001010110101110111010110110000110000011011011010000001101100110110011010111000000000001101101111011001101110 000000001001100000000001000000011000110110000000000000011010000011110110110000000100000000000000000000000001 000010110000000000000000000000000000010000000000000000000000000000001100000000000000110110011110101101110111 10011111100011111000111111011100001101001100000110011110111101001101101110111000111100101000000000000001100111 1100110000000111001100110011100001011101011000011000000000010110000000011000000000000110110110000000000000011 000111100011000000000000000011000000100000000000000000000001000000001100100001000000000000000000000000000000 00100001000011010000000000100110001111011101000101011111011110110111111101010111111100111110011010101110110011 0111110110100001110000001100011001100001100001100110111110110011000110110011010001101100011010101010011000011 000001111001100001000000001100011100000110110000000000111000001000000110000010000011000000000000000000100001 0000000000000000000000000000000000011000000000000000000

PD GR 19 1 01001001100100100000111011101101100111110011011011110101101110101100101100011001111011110011001111011011111011 0101100010110100111011100011110000010001111100010000010011101100000000110001101101101111000010000010000101101 011100011001100101110101001100011000000000000000001100001100100000000000000110001000100000000100000100000000 000000000000000000000010000001100000010010010000000100000010000000000001000001000000000000000011010001001000 001001011110100011111111101100111111101101011011000111111010010101111011100011101011001110111101011010111110010 1001101001100010100010100110100110110000100100101100001101110110110011011110100000000011101111011011000100110 000010100001110100000001000000110000110100010000000000011000001100110110000000001100000000001100000000000011 000000110000000000000000000000000000010000000000000000000000000000001100010000101000001100011110110101110111 01110001100011111000111011011011001101100100000110110011111101001101100110110000111100000000000000110001011010 010011000000011100100011001100000001110100000000011000000000010110000011000000000000001000011000000000000000 100001000001100000100000100000100000000010000000000000000000100000001000010000000010000000000000000100000010 00000000000001001110000000001011101111100110101001111111100111011111111110111011110110001111001101010111000100 1100011011010010111100010110001000110000110001110011001011011000111011011001101000010000001011010001001100000 100000101100010001100000000000100101001000000000000000001100000000000010000000000001100001001100000000000000 10000010000000100000000000000100000001000000000000000000

PD GR 19 2 01010101100100100000110011101101110111100110011011111101111110101100011100011001111011110011001110001011111011 0101100010110110011010100011010000110001011100011000010011101100000000110011101101101111000000000001000111101 197

011100011001100101110100101100011000000000000000101100001101010000000000000011011000100000000100000100000000 000000000000000000000110000000100000010010001000000100100010000000000001100001000000000000000010010001001000 010011111110110011111111101100111111100011011001100111111010000111111011100111111111001010111101011100111110010 1001010001100100101010110110000110110001100000100100101111010110110011111010100000000011101011011011000100110 0000101101011101000100110000001100001101100010000000000110000011000111100000000001100000100011000000000000110 000101100000000000000000000000000000100000000000000000000000000001000010001010010101001100011011111101111011 10110001000111111001110110110100010110011001101101110111111000011011001101100001111000000000000001100010111101 100110000000111001000011011010000011101000000000010000000000111100000110000000000000010000110000000000000011 000110000011000011000000000011000000100000000000000000000001000000010000100000000100000000000000001000000100 000010000100101100010100001100111011111001101110111011111101110111111111011011111111000111100111011011100100011 0001101101001011110001011000010011000011000111001101101101100001101101100110100000100000011101000100110000110 110101010000100110000000000110011000100000000000100001110000000000010000000000000110000000010000000000000010 000010000001000000000000000100000001000000000010000000

PD PO 10 1 01010100001000000000011111100001100111101001110111001110111101000111100011011101111111101100110110101110011011 0111000010100001011011011011110001010101011010001100010011010110010110110010011001101011000001000011100001011 1011011011011000011001111110000110000110110000000001011111000000011000000001100000011100000001100001000000000 000000000000000000001100000010000000101000010000000000010100000000000100001000100010000000000100100100010000 010111011011101101111110011011111110000111111001011111010101111000011100011110001100111000110110111011011101011 0010101111001001000101001001110100000010110000000000011111101010101010111011011000000111100111100111110011000 0000111100110000000100100000000000111011000010000000001111000110011011000110110010000000000000000000000000000 001011000000000000000000000000000001000000000000000000000000000001110001000010100000011001111011100111001101 11010110001111101011101101111010110000111000011101101111111010110110001011001011110001000000011000011100011011 0011000000001101100011001110000001110101100000010000010010011100000011010100000000011000011000000000000001110 011010001100000000001000101100000010000000001000000000000110000000010010001000010000000000000000010000010000 00000101011101000000000001111001111100111101010101111011100011011111110011011111110001101001100010111001100110 0101011100000111001000110001100111101100000110011011011011100010011011010101100110010001101010001000100000101 000110100010000110000000010101100001000011000011000001100000000000110000000000101100000000000000110000000100 00100000001110000100100000100000001010000001100000000

PD PO 10 2 01010100000100000000011111100001100111101001011011010011111001011000111100011011101111111110001111011101011011 1101010001100001011011011011110000010110011000011100010011000100010110100000011001101011000000000011111001011 1011011011011000011001101110000110000110100000000001011111000000011000000000100000001100000001100001000000000 00000000000000000000010000001000000010010010000000000001010000000000000000100010001000000000010010010001000 001011101100110111111111001100111111100110111111000011111111110111100011100011111011001110001011011101101100001 1001010111100100110010110100111010001101011000000000001101010101010101011101001100000011110000110011010001111 100001111011000001000000000000000011010110000100000000011010001100110110001101100010000000000000000000000000 0000011100000000000000000000000000000100000000000000000000000000000111001100001010000001100111101011011101111 01011011000110110101110110111101010000011000001110111001111110011011000101100101111001100000001100001110001101 1001100000100110110001100111000100011010110010001000010001101110000000101000000000001100001100000000000000111 001100100110000000000100010110000001000000001100000000000010000000001001000100001000000000000000010000001000 00000010101110110000000000111100111110011010101010111110110001101111011001101111011000111100110111101101111011 1101101110001011110000011000010011110110001101001101101101110001001101110100010011001000110111000100110000010 0100110100001001111100000111101100001000001100011000001100000000000110000000001001100001000000000011000001100 01100000001100000010100000100000001010000000100000000

PD PO 12 1 01000100000100100000011111100001111110100001111011011001110111011000011100011001111011111110010110011101111011 0011010110000001011001100000110000000101101100001100010001100111011010010000011001101101101001100001111101111 1111000011011000011001110010110110000000000000110011011011001100000000000001000010001100000001100011000000000 000000000000000000000000000100000000100100010000001000010101000000000000000000000010000000000100101100010000 10000111101110000111111001100111111100010011011001100111110001101001111000011100010011110011000011100101110010 0001000110100011100010110101100100110001000100010010001111010110000001011101111100001111110000110010000110100 001100011001100000000000000000000100000110000100100000001010001100110110011000000100000010000000000000000011 000010010000001000000000000000000000000000000000000000000000000000111101010000001000110110011110101101011111 00111001100011111000111111101010001111001100000110111101111100101101101110111000111101010000001100010001100110 0000110000000111011000110011100001100101011000000100000000010111100000011101000000000011011000000000000000111 000110011011000001000010000011000001100100000110000000000001000000000101100001000110000000000000001000000000 00100001000010011100000000100110101111100001011101011110111110111101111100111011101110011110111001101010000011 1011010110100111011101000100010001101101110110100110110110111001100110110010001001101100011011101010001000001 011001111000110011110010000011001100000100011000010000011000000000000110000000000011000010000000000010000001 0000010000001100000000100000100000001000010000100000000

PD PO 12 2 010101000010101000001111111000111111101000001110110110111110011110000111000110011110011111100011100111011110111 0110001100000010110111000101100001101011010000011000110011001000110100100000110011011010010011000011011011111 111000011011000010001110010110110000000000000110001011011001100000000000001000010001100000000100001000000000 00000000000000000000000000000000000010100001000000100001010100000000000000000000001000000000010010010001000 198

000000111101110001111111001100111111100100011111001100111110001101001111000011101000011110001100111100111110110 0001000110100001110010100101100100100101000000000000001111100101000001011101011100001111110000110010010110100 001100011001100000000001000000000010000110000000100000001010001100110110011000000100000100000000000000000001 000000010000000000000000000000000000000000000000000000000000000000001100010000101000110110011110101101101011 00111001100011111000111111011010001101001100000110111101111101001101100110111000111100110000001100011001100110 0100110000000111011000110011100001100101011000000100000000010111000000110101000000000011011000000000000000011 000110101011000001000010000001000001100100000010000000000001000000000100100001000110000000000000001000000000 00100001010011011100000000100110001111100001011011011111011010110001111101111011101100011110011101101111010011 1011010111000011011100000100010001101110110010100110110110111001100010110110001001101100011011101010001000001 011001101000010011000000000001001100000110001000010000011000000000000100000000000011000010000000000010000001 0000010000001100000000100000100000001000100000100000000

PD PO 14 1 01000100100100000000111010100011110111100000111011110011101010111000111100011001111001100110011110011110011011 1101010110100000011001100011110000010001101110010101110001100101011000110011011100011011000000000001000001111 1111000011011000011001101111010100111100000000000001111100011000000000000011100110001100000000000001000000000 000000000000000000000100000010000000001000010000001000010100000000000100011000010010000000000100100100010000 010111011001100001110110011001111111000011110110111011111000001111011111000111010011011100110010111011011100011 0010001101001100000001101011001101100010000000110001111111100001000110110010000000000011000000100000000001100 000000110000011000000011000000000111001100001000000000110101100110101101100000001100000000000000000000000110 000101100000010000000000000000000000100000000000000000000000000000011001100001010000001100111101011011110111 01101011000111110001110111011100010011011010001100111001111010011101000001110110011100100000001100001111110001 100110000010111011000110011100000011101011000000100000000010100000000110000000000000011010110000000000000011 000010001011000011000000000011000000100000000000000000000001000001000100100000000000000000000000000000001000 00000000010111010000010000100110011110001111011101111111011110111111101100111011101100011010011011010110000011 11101001101000101110000011000100011110011000101001101111101110110001101110110110011011000110111010100110000111 010011110001000010000000110110011000101101101000100000001000000000000000000000000110000100000000000000000010 000000000000000000000100000000000001000010000100000000

PD PO 14 2 01000101000100000000010010100001100110100000111111110011111010111000101100011001111001100110011110001011011011 1101010110110000011011100110110000000001101100011101110001100001011000110011011100011011000001000001000001111 1111000011011000011001101111010100111100000110000001111100011000000000000011100010001100000000000001000000000 000000000000000000001100000010000000000100010000001000010100000000000100011000010010000000000100100100000000 000111011001100001110110011001111111000101111110111011111000101111011111001111000011011100110010111011011100011 0010101101001101000001101011001011100010000000110001111010100101001010110010000000100011000001100100011001100 0000001101000110000001110000000001110011000110010000001101011001101011011001100010001000000000000000000001100 001001000000100000000000000010000001000000000000000000000000000000110010000010100000011001111010110111001110 11010110001111100011101110101000100110110100011001110111110100111010000011101100111011000000011000011111100011 0011000011011101100011001110000001110101100000000000000011010000000001100000000000001101011000000000000000100 001000101100001100000000000100000010000000000000000000000100000100010010000000000000000000000000000000010000 10000001011101010000000011011001111100011101101101111101111011001111010011101110110001111001101100111010001110 1110011100001011100000110001000111100110000110011011111011100100011011001001101010110001101110101000100000100 100111100010000100000001101100110001011011010001000000010000000000000000000000000100001000000000000000001100 00000000000100000000100000000000001000100000100000000

PD PO 15 1 01000101000100000000111110101101010111100101011011010011101110110100111100011001011001111011011110011011011011 0111000010000001011001100011110000010001101000001000010110001100011000100000101101101011000100000011001111001 1011000011011000111001101110110110000000000001100000011001001100000000110011000010011000000001100001001000000 000000000000000000000000000000000000001000010000000000000100000000000010000001010000000000000001100101010001 111111011011111011111111011001111111000000110100011001110100001111011111010111000111111100111110111011011100011 0001001101000100011111000000000110100000000110110001011110101001100110110011000000000111000111100110000111000 011000110011001000000010000000000000110100000000011000110100011111101100100000001000000000000000000000000010 000101100000000000000000000001000000100000000000000000000000000001011000000000010001101100111101101001100111 01011011000111110001111110110110011010011000001110111001111101011011011101100001111000000000000000000011011101 1001100000001110110001100111000011001101100000001000000000110110000000001000000000000100001100000000000001110 000101000110010000000100010010000001000000000100000000000010000010001001000010001000000000000000101000000000 01000010101110100000000000001100111111011011101010111110111101100111010010111111111000111101110101011010110110 0110101101000101110000011000100011000110000011001101101101100111101101110110110001011000110101000100110000010 010110010001100010000000011010011000001100000000100000100000000000001100110000000110000110010001000000000110 001100000000000000000000000100000001001000000100000000

PD PO 15 2 01010100001000000100011111101101100110100101011010010011101101010100111100011001111001111011011110011011011011 0111000011000001011001101011110000010101101100001100010011001100011000110000101101101101000000000011001111001 1011000011011000111001101101110110000000000000110001011001000110000000010011000110011000000001100001000100000 000000001000000000010000000000000000001000010000000000000100000000000010000001010000000000000001100111011000 101111011011111011111111011001111111001100110100011001110101101111011111011111010111111000111110111011011100011 0000101101000000011110100100000101000000000110100001011111001101100110110011000100000111000111100110000011000 199

011000110011001000000110000000000000110100001000011000110100011111101100100000001000100000000000000000000010 000101100000000000000000000000000000100000000000000000000000000001111010100000010001101101111101101001100111 01101011000111110001111110110110011011011010001110111011111110011011011101100001111000000000000000000011001101 1001100000001110110001100111000011001001100000001000000000111110000000000000000000000100001100000000000000110 000101000110010000000100000010000001000000000100000000000010000010001001000010001000000000000000110000000000 01000010001101100000000000001100111111011011100110111110110101101111101011011111111000111101110011001100110110 0100101101000101110000001000000011000010000011001101101101100101101101100110110001011000110101000100110000010 010110110001100010000000001000011000001100000000000000100000000000001000010000000010000110010000000000000110 001100000000000000000000000100000000010000000110000000

PD PO 17 1 01001101000000000110010111111101100110100001011010010001001101101100111100011001111011111011001110111001111011 0101100110100000011001100010110001110001111000001101110011100010011000010000111001101111000000000011001101101 1011000011011001010001101110110010000000000000000010111011110000011000000001100001011100000001100011000000000 000000000000000000000000000010001000001000010000000001001000000000000100001000000000000000000000101110010000 111111111001111101111111011001111111001000110110011001111100011011011011001111000110010101110100110111011100011 001011011100010100001010010100100100000000011000000001101010000100101011101000000000011100000110011000001110 000000011001000000000000000000000000111000000000000001011110001000110110011000000100000000001100000000000011 000000110000001000000000010000000000010000000000000000000000000000001100010000101000000110011110110101110011 10110101100011111010111111101011001101001100000110111100111101101110101100110000111100100000000110000000011110 1100110000110111001000110011100001100111011000011100000110000010100000010100000000000011000110000000000000110 000110000011000011000010001011000001100000000010110000000011000000010100100001000000000000000000100000001000 00000000000101100000000000110110011111101001010011011111011011110011101100011011111100111010111010100110000011 0001010111100111110000000100011001100001000110100110110110110001100010111011011001101100011010100010011000011 001000101001100001000010001011000000010000000000000100000000000000000110000000000011010000001000100100000001 0000100000000100000000000000100000001010000001000000100

PD PO 17 2 01000100000000000010011111111101100110100001011010010001111001010100011100011001111011111110001110111001111011 0101100110100000011011100010110010100001111000011100110011100010011000010000101101101111000000000011000101101 1011000011011000111001101110110010000000000000000001111011110000011000000001010011011100000001100011000000000 00000000000000000000000000001000100000010001000000000100100000000000010000100000000000000000000010010001000 010111111100111101111111101100111111100100011011001100111110001101101101100111101011001110011010011101011110001 1001100000110010110001100101100100110000000011000000001101010000100101011101100000010011100000110011000001110 000000011001000000010000000000000000111000000000000000011110001000110110011000000010000000001100000000000011 000001110000001000000000010000000000010000000000000000000000000000001100000000101000000110011110110101110011 01110101100011111010111111011011001101101100000110111100111101101110101101110000111100110000000110000001101110 1100110000100111011000110011100001100101011000011100000100000010100000011100000000000011000110000000000000110 000110000011000001000000001011000001100000000010010000000011000000010100100001000000000000000000000000000100 00000000000111100000000000100110011110101001010111011101101010110011111100011011111100111110111001000110000011 0001010111100111111000000000011001100001000110100110110110110011000010110110011001101100011010100010011000011 001000101101110001000000001011000000010000000000000100000000000000000010000000000011010000001000100000000001 0000010000000100000000000000100000001010000001000000010

PD PO 18 1 01000111010100000110011111001101100110111000011011010001001001101100110100010001111011111011010110011011111011 0101000110100110111001100011110000010001111000010000010011000101000000010000101101111011000000000001101101101 101101001101100001100110111011011000000000000000000001101100000001000000000000001001100000000110001100000000 00000000000000000000000000000100000001001000100000000000100100000000000100000010100000000100000001001000100 01100111011011100101111111011001111111000000110100011001111000000011110011000111000110011101011011110110101101 0001010101100000001000010101011011001000001000110000000011010101001100110110001100000000111000111100100100011 100011000110011000000100000000000000001101100000000000000110100010110101100110000001000000000011000000110000 110000100100000000000000000000000000000100000000000000000000000000001011000000000011000001101111101011011101 01001110011000111110001111110110111011110011000001110111001111010011011001101100001101001101100001100011000111 1011001100000011110011001100111000011001001110000011101000000111011000001001000000000000000001100000000000000 110001101000110100010000100010110000011000000000000000000000010000000001001000000000000010000000000010000010 00000000000010110100000000000001101011110010011101111111011011101101111011011101111011000110101110110001101000 1110010101110000011000001010000110011000011001101001101101100100110000101110100110001001000110101000100010000 010010000110010100010000000011010010000000100010000111001100000000001001000000000000110000100000000000100000 010000000000000100000000000000000000001010000000100000000

PD PO 18 2 01000101000100000000111111001101100110101000011110010011111010110100111000010001111011111011011010111001111011 0101000110101110011001100010110010110001111000001001010011000100000000100011001101111011000000000001001101101 101101001101100101100110110101011000000000001000000001101100000001000000000000000101100000000010001100000000 000000000000000000000000000011000000010100001000000000001010000000000010000001000000000001000000010010001000 01001111110011001111111110110011111110001001101000110011110011001111100110001101001100110001110111100101011001 0010101011010001011000010000110010010010010001100000000111101010011101111100111000000001110001111001101100111 000110001100110010101000000000000000011011000010000000001101000101101011001100000010000000000110000001100001 100001001000000000000000000000000000001000000000000000000000000000000110000000000100001011011111010110111001 200

10011100110001111100011101101101100111101110000011110110111110100110110001111000011110011011000011010110001111 0110011000000111101100011001110000110010011100000110101000011011100000110110000000000000000011000000000000001 100011010001101000100001000100100000110000000000000000000001100000000010010000000000000100000000000010000010 00000000001010101000000000000011000111100100111010111111010101111011110100110111110110001100011101100011010001 1100101011010010110100110010001100110000110010110011011011011011100011011011001100010010001101010001000100000 100100101100111000100000000100000100000001000100001110011000000000001010000000000001100001000000000010000000 10000000000000000000000000000000000000010000000100000000

PD PO 19 1 01000001000000000000011011101101100111101001011010010001101110101100011100000001111001111110000110011110111011 0101100010010110111001100011110000010110000010001001010011110101000000100000101101111101000000000011001101111 1111010001111001011001110110000110000000000000110001000000000000000000000001100010011100001001100011000000000 01000000000000000000010000000000000010000001000000000100001000000000000010001000001000000000010010010001000 00000101110111110011111110110011111110000001101100110011101011100110011110101110001101101010110101101101011001 0110101011000011100001011000000011010000000000110000000111101011011101101101100000000000110001100001100001111 000000001100110000000000000000000000001011000000000000001100000001101011001100000010000000000110000001100000 100000011000000000000000001000000000001000000000000000000000000000000110000000000100000011001111011010110001 11011010110001111100011111110101100111010110000011001110111110100110000111011001011110000000000011001100000011 1001011000011011100000011001110000000110101100001110001100010111100000000110000000000011001011000000000000001 100011101001100000100001000100000000110010000001000000000000100000000010010000000000000000000000000100000000 00000000000001101000000000000011001111100111111011101111100100111001111111101101110110011100011101010111000000 1001111011110001011100000010001100110000110000110011011011011100111011011001101100010110001101010001000100000 100100111100101001100000001100100110001000001100000000001100000000000001000001001000100000001000000001000000 10000000000000100000000000000100000001010000000100000000

PD PO 19 2 00100000000100000000011011101101100111101001011011010001101110101100011100000001111001111011000110111001111011 0101100110000110111001100011110000010110000010001001110011111111000000100000101101101101000000000011001101101 1111000001111000011001110110000110000000000000110001000000000000000000000001100111011100001001100011000000000 010000000000000000000100000000000000100000010000000001000100000000000000100010000010000000000101100100000000 000001011011101011111111011001111111000000110110011001110101110011001111010111000111110101111010110111001100001 1010101100001101000101100000001101100000000011000000011111001101100110110110000000000011000110000110000111100 000000110010000000110000000000000100101100000000000000110000000111101100110000001000000000001000000110000110 000000100000010000000000100000000000100000000000000000000000000000011000000001010000001100111101011011000110 11011011000111110001110111010110011110011000001100111011111010011000011101100001111000000000001100110000001110 0001100001101110000001100111000000011010110000111000110000111011000000011000000000001100101100000000000000110 001100100110000010000100010000000001000000000100000000000010000000001001000000000000000000000000010000000000 00000000000100100000000000001100111110011011100110111111010101100111111110101111011001111001110111011100000110 0111101111000011110000001000110011000011100011001101101101110011101101100110110001011000110101000100010000010 010011110000100110000000110011011000000001100000000000110000000000000100000100100010000000010000001000000010 000000000000100000000000000100000001010000000000000000

PD PO 20 1 01000000000010100000111010100001100110110110011001111000111111001100011100010001111000011110001111011001111111 0111010010000000011000100011110000010001100010110101010011100111000110100000111001001110100010000011100101101 101100001101101001000110110101011000000000000000000101100111100001000000000010001000100000000010000100000000 000000000000000000000010000000000000000100010000000100001010000000000000001001010001000000000000010010111000 110001001111110011111111001100111111100000111110001110111010110111101111001011100011011010101011011101101110000 0001011011100010011010100011100001100101100000000001001111010001100110011101000000000011101000110011000110110 100011100010000100000001100000000001110100000010000000011000000001010110010000000000000000000100000000100001 000010110000000000000000000000000000010000000000000000000000000000101100110000001000000110011110101101101011 10011101100011111000111110111011001101101101010110110011111100101101101100110000111100110000000000001100001010 0100110000111011011001110011100001101111011000011100000000000100100000110000000000000010011000000000000000010 000010100011000000000001001001000000100100000000000000000001000000000000100000000000000000000000000000000000 000000000001010111000000011001100111110011011101111111101110101111111111110111111011000111100110110011100010011 0110001101000011110000011000110011000011101101001101101101101111101101100110110011001000110101010100010000010 010000110001000010000100000000000000100100000000100000110000000010001000000000000010000000110000000000000010 000000000000100000000100000100000001010000001000000000

PD PO 20 2 01000000000110110000010011100001100110101000011001110010101110101100011100011001111000011010001110111001111111 0111000011000000011000100011110000010101100010110101010001100101100110100000011101011110100000000001010111101 1011000011011010111001101101010110000000000000000001011001111000110000000001100110011000000001100011000000000 000000000000000000000100000000000000001000100000001000010100000000000000011001100010000000000000100100000001 100010011111100111111110011001111111000001111100011101110101101111011110010111000110110101110110111001111100000 0010111111001100110101100111000011000100000000000010011110100011000110110010000000000111010000100110001101111 100000001101011100000011000000000011110000001100000000110000000110101100100000000000000000011000000011000110 001101100000000000000000000000000000100000000000000000000000000001011001100000010000001101111101011011010110 00111011000111110101111111110110011011011001001101100111111010011011011010100001111001110000000000011110011101 0001100001110110110000100111000011011010110000110000000001000111000001100000000000001100010000000000000001010 201

000101000110000000000100010110000001001000000000000000000010000000001011000010000000000000001000010000000000 00000000000110111000000011001100011110011011100111111110110010111111111001101111011000111101110110011010000010 0110001101000011110000001000010011000011101011001101101101101110111101100110110011011000010101010100010000010 010000110001000010001100000010000000100100100000100000110000000010001100000000000110000100110000000000000010 010000000000100000000100000100000001010000001000000000

SW AL 3 1 01000001101001001100110111111101110111100100111011110011101011011000111100011011111000000110011011011011011011 0001100011000001111011100000110000010101110010001100010011110100011001110000011000001100110000000001001011001 1111000011011001011001010110000000110000000000110000011110000001111000010001100011011000000000000000001000000 010000000000000000000000000010001000111000010000000000010100000000010000000000000000000010000000101100010000 01011101101110100111111100000111111100000011011000001111100000101101111100110100111011100011101100001101110001 1000000101100001101000110000011011000011000011000000000101100101100001010001000000000001100011110110000110110 000000001101100001100001000000001010110010000100001000011000000000110110000000100100000011000000000000000001 0000100000000010000000000000000000000100000000000000000000000000100011001100101011110101101111101101011100111 01101011000111111001111111010110011010011000001100111011111010011011000110100111111000000100000000000001101100 1001100001100110110101100111000100001001110000001000011000101111000000111000000000000000001100000000000001010 100111000011000110000010110110000001000000000000001000000110000010001010000001000000101000100000001000001000 000000100011101010000000110011000111100111101001101111111110111111110110111101110110011111001100000111000001110 00110111100010111000000110011011101101100010100110110110111111010110111011011100101100011010101010001000011011 000100000010001110000011011001100010110010100010100011000000011000000000000000000010000000000000000001011000 1100000000101100000100000100000001000000001000000000

SW AL 3 2 0101100011011000000000110110101000000000100000110000001101000000000011110000000100110101100000110001101101010 001011001100000010000001001001100100001010000000000001000111101000001001101000100110001101100000000000011011 001111000011011000011000001011000000110000000000011000011011011101101000000000010001001000000000000000000000 000000000000000000000000000000010001000101000010000000000010100100000100000000000000000000100000000110110110 1000010011101011100000000110011000000010110100000000111010011010111010011100011010011000000001000100011101100 11011010000001110010010001011111110111101000000001101111111000110110110111011000100000000000101001111010000011 0011100000011011001111011011100000001100100110100110100000011011100001100111100011000000000010000000000000000 1100111011100000110000000010000000001011000000000000000010000000010010110011001010111101011011111011010111001 11011010110001111110011111110001100100100110000011001110111110100110110001011001111110000001000000000000011011 0010011000011001101101011001110001000010011100000011000011001111110000001110000000000000000011000001000000001 101001010000110000100000100101100000010010000000000010000000100000100010100000000000000110001000000100000010 00000000100011001000000000111011000111100111101001101111010100111011110110111101110110011111001100010111000001 11000110111010010111000001010011011101101100011100110110110111011110110111011011110101100001010101010001000011 001000100000010011010000011011001100010110010100010100011000000001000000000000000000000000000000000000001001 0001000000000100000000100000100000001000000000100000000

SW AL 4 1 01000100000101101100110011100001100111100000011110100001100010011000111100011011111000000110010110011101111011 001101010100000110000110001011000001010110001000000001000110010000000001000000100000110011000000000100100100 110100000110110000110000010100001000000000000110000000110000000010100000000000000010010010000000000010000000 00000000000000000000001000000010010000101000010000000000010100000000000000000000000000000010000000000100000 00000000001100100000111111001100111011100000011110000001111000001111001011000110100111011100011101000001101100 001100000000100000110001011001000011100001000001100000000001111010010000101100110000000000110000011000000010 011000000001100100000110000110000000000000001000010000000000100000000001011001000010000000000000000000000100 000100001000000000000000000000000000000001000000000000000000000000001010110001100010111000011111111011010111 00111011010110001110110011111110011010101111110100011010110011110100110010011011001111110001000000011111000011 0110001011000010001101101001001110100001110011101000010000010000001110000001110000000000000100011000000000000 000100001001000110010100011000101100000010000000000000000000001100000100010110000000010000000001000000100000 01000000000000001110000000000111011000111000011101001100110011100111111111010101101110110011111101100110111000 0011000010111101001011100010000011101100110100011110011011011011000011111011011001101010101001101010011001100 000110100010100010001101000001110100110011011001000001101011000000000100011000000100001100001000000000010000 10010000000000000100000000100000000000001010000000100000000

SW AL 4 2 01000001111011100100010111100001100111100001011101110101101011010101111100011011111000001110010110011110111011 0011010011000001110001100110110000010101100110001001001101000110000001110000011000001100110000000001011111001 101100001101101001100100101000010000000000001101000001100000000111100000000000000101101100000000000100000000 000000000000000000000100000001001100010100001000000000001010000000000000000000001000000001000000100110001001 00100110110110110011111100111011111110000001111001110111110000011100111110001010011101101001110100000101110000 1100000001110000100111011011000101100011000001100000000101101110110001001100110000000000110000011000000011011 000000001101100000110000110000001101000001000110000000001100000000011011001000011000000000000000000000110000 100001001000000000000000000000000000001000000000000000000000000001010110010001110110001001011111011010111001 10111010110001111110011111110001110110111110101011010110011110100110010011011011111110001100000001111001011011 0110011000011001101101011001110100001110011101000011000101000011010000001110000000000000100011000000000000001 100001001000110010100011000101100000010010000000000000000000100000100010110000010010000100001000000010000010 00000000000001101100001000110011000111000111101001100111011100111111111110101111110110011101100100010111000001 202

1000010011110001011100010110011101100110110001110011011011011000011111011101001101010110001101010011000100000 100100010100010001111000001110100110011011001100001010011000000000100011000000100001100000000000000010000100 10001000000000100000000100000100010011010100000100000000

SW AL 5 1 0100000010010000010110001111011111011100010001101110000000110000110011010001000111100000001100110000010111101 1000110001101000101101110000011000000000011001000011000000110010000100011011001100100111001000000000110011011 011011000011011000010000001110001100000000000000000000011000000001101000000000100110001100000001000000000000 000100000000000000000010000000100000001100000100000000000110000000000000000000000000000001000000000001000100 0000000000110011000110011101100111111100000011001100001111100000101100011010001000001101101011000000000111100 001100000001110000110000011000100001101000100000000000000000100011010000000000100000000000011000001101100001 101100000000000100000011000011000000000001100000001000000000110000000110100101100000000000001000000000000000 0011000011100000000000000000000000000100010000000000000000000000000000101100101010101111010111111110110101110 01110110101100011111100111111101010001101111100000110101110111101001101100111010011110100000010001100001010011 0101100110001100011011010110011111000100100111000000000001000011111100000000110000000000011000110000000000000 001110110110011000011000010001011000000100100000000000000000001000000000101000000100100000100000000000000000 10000000001000011010000000000110110001111001111110101000111011001111111111101011111101100111010001001001110001 01110000101111001110110010001100110011010110000111001001101101100010101101101110111011011000101101110100110000 110100001000001000010000000001010100001101100001000100000110000000000001100000010000000100000000000001000000 110001100000000000000000100000100000001010000000100000000

SW AL 5 2 010001110011111100000101101011011101111001101110111100011001111110001111000110111110000001100010110111101110110 0101001101000010110111000001100001101011000100101010100010011000110011000001100010111001100000000011111011110 111000011110000010000001011011100000000000000001000011000000011001000000001100110001100000001100000000000000 011000000000000000001000000010000000111000010000000000010100000000000000100000000000000010000000000101010000 11011101101110000110011101100111111100000011011000011111101000101101011100001100011011100011101010011101110001 1001100001100001100011100001000111100011000001100000100011011101110000011001000000000001100000110101000110110 000000000011000001100001100000000000110001000100000000011000000011010110111000000100010011000000000000000011 0000100000000000000000000000000000000100000000000000000000000000101011001010001011110101111111101101011100111 01101011000111111001111110100110011011011000001101011101111010011011001101100111111000000100011000000100010100 1001100011000110110001100111010011001001110000000000001000110111000000000100000000000110001100000100000000010 101101100110000110000100010110000001001000000000000000000010000000001010000001000000000000100000000000001000 00000010000110101000000001101100011100001110100110011111010011111111111001111111011000110100010001111101000111 0000001100001101110000001100111011000010000011000100101101101011101101101110111011101000010101110100110000010 010001000000100110000000001010010001101100001000100001110000000000001100000010000000100000000000001000000110 001000000000100000000100000000000010010000000100000000

SW AL 7 1 010000010001000101011011011001001101110000000111000000000011100010000101000010010100000100100001000010000010 100110100001000010001000100100010000000000100001000001000001010010000000010000001000000010000000000010100000 100000100000100100000100000010100000010000000000001000000100000000100000000000000000110010000000010000000000 000000000000000000100101110000010111100001000110100000000000010010100000000000000000000000011001001000011001 001110111110111010010110011000100101100100000001010001101101010000101101001000010000000100100010000000000100 0100001000101001100001111101110001000001010011100100110000000001110010110111101000100001001000010010010010001 001011100010010000100001010010010000010100010000000110100100001000000100110010000000011000000000000000010000 0000111110111011000000000000000000000100111101010000000000000000000000000011001110001111100101101111101101011 10011001101011000111111001111111011010011011111100001101111001101010011011001101100111111000000000000110000000 001100000110000100111000000110011111001100101010000000100000000011010000000000010000000000011000110000000000 000001110110110011000001000001001011000000100100000000001000000001000000000100100000000000000000000000000000 00000000000000000011000000000001100110001110001101110101000110111011111111101101011011101100111111011010000110 0010110000000110100011011000100110001001100001000010110000010111010011000110111011011001101000011010101010011 000001010001100000010000000000000001000000000110010000000000001000000000000010000000000000000000010000000010 0010110001000000000100000000100000000000010010001001000000000

SW AL 7 2 0011001000110011101101010111000010001101110100001101001011100001110100000001101100001100110010110001111101101 1000110011110000101100110000011000001010110001000110101101100011000011110000001000000110111000000111101110110 1011100001101100001100110101111011011000000000011000001100000100100000000000110001000110000000110000000000000 001000000000000000000100000001000000011000001000000000001001101000000000001000000000000001000000011010110110 10110101001111011000111101011000000101011100110110111110000101100000110000111000011011110001110100001110011100 1100110001110000100001110010110101111000000000110000000011101011011001101100110000000000110001011010100011011 000000001100110000110000000000000000011000000010000000001100000001101011000000010001000001100000000000000000 100001001000000100000000000000000000001000000000000000000000000001000110011100011111001011001111011010011010 10011010110001111110011111110101100110111111000011010110001110100110110011011001111110000000000001100001000111 000001100001001110000101100111110011001001100000001000000000110100000000000100000000000110001100000000000000 011101101100110000010000100010110000001001000000000000000000010000000001001000000000000000000000000000000000 0000000000100011000000000001100110001110001101010101000110101011111111101101011011101100111101101001100110001 0110000000110100010111000100100011001100001000011110100110111010011000110110111011000101000011010101010011000 001001000100000010000000000000001000000000110101000000000011000000000000110000000000000000000000000000100001 203

0111000010000000100000000100000000000001010001000100000000

SW AL 10 1 01010101001000000000111011110101100111110110011110110101101011011000111100010101111001100110000111010001111011 1011010101100000011001100011110001010101100011001100010011000100011001010000111000001011000000000000100001111 0111000111011000011000001011000110000000000000110011011000110000000000000011101100001100000001100000100000000 000000000000000000000000000000000000101000010000001000010001000000000000000000010000001000000000101100010001 110111011101001101111110010001111111000000111101101101111100000111111111001011000110111000111010000011011000010 0011000111000110100111000000011001001100110000000000011110001101100000101110000000000011001101100100000111100 000000011110000000000000000000000001100100001000000000110000000101101100111000000000000000000000000000000110 0001000000000000000000000000000000001000000000000000000000000000000110011100010110001011011111010110111111010 110001100011111010111010110011101101111100000110111101111100101110101101111110111100000010101100001110011111000 011000000001101100001001110000001110100000000110000010000001110000000110000000000001000111000000000000001100 011011001100001100000100100100001011010000000000000000000100000000010010000010000000000000000000000000010000 00000101001001000000000111011001111100100111010101111011101111111111110101111110110010111101100111001000001101 1101011010010111100001001000101110000110000110011001011011000101011010001100010010110001011110101001100001100 100010100010000100000000110000000000000000000000010011101000000100110000000000000000001000000000000001100101 00100000000100100000100000100000001010000000000000000

SW AL 10 2 01000101011010000100110011111101100111110110011001110101011010111000111100000101111001100110000111011011111011 0011010011100000011001100011110001110101100011001100010001100100011001110000111000001111000000000001100001111 0111000111011000011000011010000110000000000000110011011000110000000000000011100100001100000000100000100000000 000000000000000000000000000000000000101000010000001000010010000000000000100000010000000000000000101100010001 11011101110101100111111001100111111100000011010110110111110000101111111010101100011011100011101000001101100011 0001100011100111011011100101000100100010011001100000001101000110110000011111100000000001100110110101000110110 000010001111000000000000000000000000110000000100000000011000000010110110011100000100000000000000000000000001 000010000000000000000000000000000000010000000000000000000000000000100111000001001010001011001101111110111010 010110001000111111101110101100111011110111000001101111011111010011011011101111101111000000110011000011100111100 000110000010011011000010011100000011100000000000100000100000010100000001100000000000010001010000000000000001 000110110011000011000000011001000010110100000000000000000001000000000100100000000000000000000000000000000100 00000001010011010000000001110011001111100101111010101110110101011111111100010111101100101111011001001110000011 1011110110100110111001000000011011101011000010100110110110110000110110100011000100011100011010101010001000011 001000111000100001000000001100000000000000000000001100111000000001001100000000000000000000000000000000001001 1000100000000100100000100000100000001000000000000000000

SW AL 11 1 01001101011011111110111011101111110111100000110110011000101111000101100110001101100010101000110000001100011011 011000001001011011100100000011000001000100010010100111000010100100000100000000100000110000000000000000100100 010000000000100000100010010100000000000001001001000000100000000000000000000001011010001000000010000100000000 001000000000000000001010000000000000001010001000000000000100010000000000000000001000000000000000001001000110 01111101101011101111111110011011111110011010010001111111111001001101011000001001000000110000110011000101011000 010000000001001110100100100011000100101010010000000100001000110100101100110110100000000101000100001101100000 010001000010001100100000000111000111000010010001100000000001000000000100010000000101000000000011000000010100 0000110111000000000000000000000000000000100000000000000000000000000001011011001110111111010110111110111100011 11100110001100111111001110111000110011110011010001101110111111010011001011000110101101000010001111000001011001 101100110000100011011001000011001100001100011000000000010101000001010000000010000000000010100110000000000000 001000110000011000011000010000011000001000100000000000000000001000000000100100001000000000000000000001000000 000000000011000110000110100000100110011111101101111111011101101010111111111101111111011000111110110110011100000 11101110011110011011100010011011001110100110000110011011011011001110011010001101010011010000111111101000100001 100100110010001000100000000010000000000010010100001000000101000000000010000000000000100001000000000001000000 10100000000001000000000100000100000010000000000000000000

SW AL 11 2 01001101110001010100111111110101100110100000011010110011101101101100111100011001111000000110001110011011011011 1011000101010111011001101010110000010101100100101101010011100101000111100000011000011111000000000010001101001 101100000001100001101100101000010000000000000011000001100000000000000000000010010000110000000110000100010000 00100000010000000000001000000000000001010000100000010000100000000000000000000001000000000000000000001000100 001011110110110011111111110110011111110000001110100111111101100011111011100000110000000110001110100101110110000 1100101000110001100001010001100011011001101110000000000110000011110101101101000000000000110001010101100000011 000000001100110000000000000000000000111001000010000000001100000000011011010000010010000000000110000000100000 0000000010000000000000000000000000000010000000000000000000000000010101110100101001011101011001110111110001111 11011000100011111100111111100011001101001101000110111011111100101100101100010010110100001000111100000001100110 1100110000000111011000000011011000001100111000000000011001100001011000000010000000000110000110000000000000011 000110000011000011000010001011000001000100000000000000000001000000000100100001000000000100000000001000000000 000000010100111100001000001110110001111101101010101011101101010111111111111101111011001111110110110011011100111 01110011110010101100000111011101110100110000110011001011011001110011010001101110110010001011110101000100000110 100110100001000100000000110000010000011001100011001001101000000000110000000000001100000000000000011000100100 00000000000100000000100000100010011000000000000000000

204

SW NO 5 1 01001011101111000110011111101101100111100000011010110001100001010100111100000001111010011110001110001011011011 0011000011100110011011100011110000000001101100000000001011100000011000100001101101001111000000110000000110001 1011000000011000011001101011000110110000000011000011011010001101100000010011000000011000000001100001000000000 000000000010000000001000000000000000100000010000000000000010000000000000000000010000000100000000000111101101 11110001100100110111111001100111111100000011011111100111100000101100001100001100111001100000000000000111100001 1000000001100001011000110000000111100000000011000000001111010110000000000011000001000001100000110000000000110 000000011100000000000000011000000000110000000000000000011000000001110110011000000100000000000000000000000001 0000100000000000000000000000000000000000000000000000000000000000001011011110111110011101111111101101001100110 11010011000111110001111111011101010110011100101100111110111010011011011111001101111000101100000001101010001100 0001100000000110110001100110000000111010110000011100010011001110000000001100000000000110001100000000000001011 101111000110001010000101100110000001100000001100000001000011000010001001000000001000010000100000010000000000 010000000010101000111011011011101111100100111011101111010100111111111111011011110100011111101100010111000000111 0100011000000111100000010001100110110110001010011011011011001100001011011001100111110001101110101101100000110 100101100010000101000000000100100000011000100011000001100000000000000001100100000000001001100000000011000011 00010000000100000001000000101000011010000000100000000

SW NO 5 2 01011101011011100110011011101101010110100000011010110001101011011000111100011001111010111110010110011011110011 00110001111001100111011000111100100100011110000100011110110011000111001100111110010011110000011000010011010011 0110001100110000110011011100001101101100000110000110110110110011000001000110000000110100000011000010000000000 00000000001000000001000000000000000100000010000000001010010000000000100010000010000000010000000000100010000 01001101101100110111111001100111111100000011011111100111000000111000001000001010111001100011000000101101110001 1000000011110011011011100000100101100100000011000000001111010110100011011111100011000001100000110001000000110 000010001101000000000000111000000000110110000100000000011000000011100110011000000100000000000000000000000011 0000010100000010000000000000000000000000000000000000000000000000001011011100001011000001111111101101011100110 11010011000111110001111111010100010110011100101100111010111010011011011111001101111001101100000001101010001100 1001100000000110110101100110000000111010110000001011010011001110000000011100000000000110001100000000000001011 101111000110001010000101100110000001100000000100000000000010000010001001000000001000100000100000101000000000 01000000001010100000000001001100011110010011101110111101110011111111111001110111011001111110110011011100100011 1010001100000011110000001000110011011011000101001101101101100110000101101100110011111000110111010110110000010 100010110001000010100000001010010000001100100001100001110000000000000000110000000000100100100000000001100001 100010000100101000001100000100000011010000000100000000

SW NO 8 1 01011101100110100000111111101101010111100001011011010000001001101100111100011011111011111110010110001101111011 0101100010110001011011100011110000000101111101100001001001100111001100110000001101101110100000110001000111100 1110000011011000001101101011000110000010000000000011011011001100000000000001100010011000000011000001000000000 010000000000000000000000000000000000101000010000000000000100000000000001000000010000000000000001010100010001 10111101101110111111111101100110111100000011010001100111000000111111011100001000011001100011100001100111110000 000110011010011101100011000010011000000100000100010000110100000000111001010110000000000110000000110000000001 000000001100110000000011100000000000110011000001000000001100001100001011001000000000001000000000000000100000 100000100000000000000000000000000000001000000000000000000000000000000110001000010100000011011111010110111001 110101101100011111010111111111011001111001101100111011110111110101101101110110000111100110000001100101101111110 1100110000000111011000110011100000011101011000010000001000110111000000110001000000000000000011000000000000010 000010110011000011000000000001000000100100000010000000000011010001010001100000000110000000010000001000001000 00010000010111110000000001110110011110001111110101011111011001111011101100010111101100011110011011101110110011 10011101111000011110000011000110111000110011101001101101101110101000101111100111011011000110101000100110000111 010110110000100001100000000110110001000000100000111000110000000010011000000000000001100100000000000000000110 001100000000100000000000000100000001010100000110000100

SW NO 8 2 01011101010000100000011111011101100111100000011110011101011001011000111100011001111011111110011010011001011011 1011000110110001011101100011110000000101011001110001101011100100001000110000001001101111000001100001001111001 101000001101000000101100001100011000010000000000001101101101100000000000001000001001100000000100000100000000 00000000000000000000000000000000100001010000100000000000000100000000000010000001000000000000000010001000100 00110111111011001101111111011001111111001100111100011001110000010110010111001101000110011000111000010011011101 000001100110100111000000100000000011000000000011000000001001000000001101010111000000000001100000001100000000 010000000011010100000000111000000000011000110001100000000011000001000100110010000000000010000000000000001100 001000001000000000000000000000000100000010000000000000000000000000000011101110100101010000110111110110101110 011101101011000111110110111110100100011110011011001110111111111010011011011101100001111001100000011001110011011 1011001100000001110110001100111000000111010110000100000010000111110000001100010000000000000000110000000000000 110000101100110000110000000000010000001001000000100000000000010100010100001000000001100000000100000010000001 000001000000001111100000000111011001111000111101001101111101100111101110110111011111110011111001110110011011001 11001110111100001111000001100011011101011001110100110110110110101100010111011011000101100011010100010111000001 011011011000010000110000000011001000010000010000011100011000000001001100000000000000010010000000000000000001 0000100000000100000000000000100000001010000000101000010

SW NO 9 1 0100010100010101010011101110110110011110000001101110000100100110110011110000000111100000001101011000101111101 205

1011100001110000001100010000011000001000110110011000001001100010101100011000111110110111100000000000010111100 1011100001101100001100101101111011000000000000000000110011000110000010000001100000101100110000110001100000000 00000000000000000000001000000000000001010001000000000000000100000000001000000001000000000100001000001000100 00000000011011101011111111011001111111000000110100011001110000000011010111000111000110111000110000010011011000 0110000001100000000010000000000111001100000110110100000011100001101001010001101000000000111000001110000000001 100000000110010000000000000110000000001101000001100000000110000000110100100000000001000000000011000000001000 010000000100000000000110000000000000000000000000000000000000000000001011001100101011100101101111101101011100 11000111011000111110001110111011011011001011101001100111001111011011000111101100111111000000000000000011000011 1011001100000010110110001100111000000011001110000001000010001101100000001100000000000001100001100000000000000 110000100000110100110000100100110000001001001001100000000000010000000001001000000000000000000000000000000001 00000000010001010100000000010001100111110011010110111111101011101100111011101101111011000111000110110001100110 11101010011010001011100000110001101111101100001010011011011011011001011011111001101010110001101111101001100000 100100001100010001100011000110101100001000000100001000001100000000000010000001000000100000000000000000000001 10000000000000000000000100000100000000010000000100000000

SW NO 9 2 01110101101001100100111011111101100111100001011110110001001001011000111100010011011000110110111010011111011011 1011000110100000111001100000110000010001111000110000001011001111011000110011111000101011000000000001011110001 1011000011110000110001110010110110000000000000000011000110001100001000000011000001011001100000100001000100000 00000000000000000000010000000000000010100001000000000001001000000000010001000010001000000100010000010000000 101001001101111110111111101100111111100000011110001100111100000011001111000011000011011100111000011101101110001 1001000110000011011001100000001100110110011011011000011011001100100101010111000000000011100000111011000010110 000000011011000000000000011000000000111000001100000000011000000011010110000000000010000000000100000000100001 000001110000000000001000000000000000000000000000000000000000000000001100110010101100010110011110110101110011 00110101100011111000111011101010001100101101000111011100111101101100101110110011111100000000000000001100001110 110011000000101101100011001110010000010011100000010000100011011000000011000000000000011000011000000000000010 100001000001101001100001000001100000010010010001000000000001100000000010010000000000000000000000000000000010 00000000000001101010000000010011001111100110101110111111110111011001110110011011111110001110011101100011001101 11011100111100001111000001100011011111001000010100110110110110011010110110011011001101100011011101010011000001 010000011000100011000110001101011000010000010000010000011000000000000100000010010001000000000000000000000001 0000000000000000000000100000100000000010000000100000000

SW NO 13 1 01001101000101100110111011100011100111100110111010110001101001110100110000011011111110011110001110011011011011 01111100111100000110001101101100010101010111100010000010111011100011001100000111010011110000000011011111111010 1110000110110000111011011110001101000000000001010110110010000000110000000011000000011000000011000010000000000 00000000000000000000000000000000000101000010000000001010010000000000000000000010000000000000000100100010000 00011001101100000111111001101111101100000011011011100111000000101100011010001100011011100001000001100111100011 100000000110000001100010000001100011000100000001000011000101000000000001111100000000000111000001000000000011 000000000110100000000000001100000000011000000000000000001100000110011001100000110000000001100000000000000001 1000001110000000000000000001000000000010000000000000000000000000000001101110000101100010110111110101101101011 10110001100011111001011111101110001000111110000110111110111101001101101100101010110100000100000000111101110110 010011000010011101100011001110000110110000000000011000100000011100000000001100000000011000000000000000000010 100011110001100110010001000001100000000011000000000000000000101000000010010000000010000000001000000010000000 00000000000001110000000000110011000111000110111001101111010101011111111110101001110110001000001101101001000001 11101110110100001111000001110000001101100100011100110110110110000110010110011011001101100011011101010011000001 010001000000100001000000000111000000110000010001100000000000000000000000000000000001000000001000000100000001 0001010000000100000000100000100000001010100000100000000

SW NO 13 2 01001101101001000010111011100001100111100101111110110011101010111000110100011011011010011110010110011011011011 01111100111000000110011001101100101101101110100100000010110011000110001100001110011110110000010011011111110011 1110000111100001010011100110001100000000000001100110110110000000110000000110000000011000000001000010000000000 00000000000000000000000000000000000101000010000000001010001000000000000000000010000000000000001000100010000 01001001101100000111111001101111101100000011111011100111000001011000011100101100011011100011001101101101110011 1000000001100000110011100000011000100110000000110000111111100000110000111101000000000001110000110100000110110 000010001101000000000000011000000001010000000000000000110000001100100110000001100000000011000000000000000001 0000011100000000000000000000000000000010000000000000000000000000000011110000010010100010110011110111101100011 10110001000111110001110111101100110001011100011011101101111010011011011001001101111000001000000001110001101100 000110000000111011000110011100001101100000000000110001000000111000000000010000000000110000000000000000000101 000111100011010100000110000011000000001100000000000000000001010000000100100000001100000000010000001000000000 00000000010010000100000001100110000111100111011010101110110101111011111111010101111000110000011011110110110011 11011001101000011110000011100000011011011001111001101101101100010101001100100110111011000110111010100110000011 100010000001000110000000001110000001100000100011000000000000000000000000000000000010000000110000001000000010 101000000000100000000000000100000001000000001100000000

SW NO 15 1 01000000001100000000110011111011100111100101011110010011100110111000110000011011101111100110010110000011011011 1011100101000001111101100010110001110110111100011000010000011110100000110000111000001111000011000001001101001 011100011011000001111100111000010000000000000010001101101100000001000000000000000000100000000011000100000000 206

00000000000000000000000000000000000001000000100000000000101000000000000000000000000000000000000000001000100 00010111011111111001111111011011011111000000111110100001110000001101100111001011000110111000110000111011011000 1100000000011000110100110000100000001001100110000000000010010000001100110111110000000000011001101101010000000 100000110011110000000000000000000000001100000000000000000100000000001001100000011000000000110001000000000000 0100000001000000000000000000000000000000100000000000000000000000000010110111001011110000011011111010110011101 110111001100011111100111011101011001111001110000111111111111101001101100111100000111100110110000000110001011000 100011000010011101100011001110000000110100000000000001100000011100000000001000000000011000000000000000000011 000011000001100000000001000001100000010000000000100000000000110000000010010000000010000000000000000100000000 00000000000001101100000000000011000111100111111010101111100100011111111111111011110110001101101100110111001001 11010110111000001110001100000011011101101000110100110110110110001010110110010011001101100011101111011001000001 101001100000010011000000000001000000000100010000010010000000000000000000000000000000000000000000000000000001 1001000000010100000000000000100000011010000000100000000

SW NO 15 2 01001001101011000100011010111001100111100101011010110011100011011000110000011011111010110110010110011011011011 1011110101100001111101100010110001010110111100001001010000001111000000100000111000001111000000000001111101001 011100011011000011011101011100010000000000000010110101101100000000100000000000000000100000000011000100000000 00000000000000000000000000000000000001000000100000000000101000000000000000000000010000000000000010001000100 011111110110110110011111110110110111110000001111111000011100000011110101110000110001101110001100110110110111001 1011000000110001101101110000000110110011001100000000000101100000111001101111100000000000110011011010100000011 000011100111100000000000000000000001011000100010001000001100000000001011000000110010001001100010000000000001 100001011000000000000000000100000000000100000000000000000000000000010110111100010101000011001111010110010101 11011100110001111110011101101101000110100111000011111111111110100110110011110000011110011011000000011000101100 000001100001001110110001100111000000011010000000000000110000001110000000000100000000001100000000000000000000 110001100000110000001000100000110000001000000000010000000000011000000001001000000001000000000000000010000000 00000000000100110111000000000001100010100011111101010111100010001111111111111101111011000110110110101011100000 11110011011010000111000110010001101110110100001111011011011011000101011011001101101110110001101111101100100000 101100110000001000100000000000100000000011001100011001000000000000000000000000000000000000000000000000010000 11001000000010100000000000000100000011010000000100000000

SW ST 3 1 0010000000000000000010111011111110011010110001111110001110010101100001000001001101100011011001011000111011011 0001100011000000101110110001011000001000110100000110001011100010011000010000011100010101100000000001110110101 101100000111100001100011111000001100000000000001100100110000000000000000000000000000001000000001000010000000 00000000000000000000000000000000000000101000100000000000001000000000010100000010000000000010000000001000000 00000000001101111110110111101001110101100000110111011101110000011011100011000011100011001100001000000101100100 0011000000001100001110011100000000001100110011000000000011011101100000010111101000110000001100011000100000000 010000000000011000000000000011000001101100000000100000000011000110001100110010110000000010000000000000001000 000000001010000000000001000000000000000010000000000000000000000000000011000000010101000000110011110110100110 01100110111100011101001101111110110011000001100011011111100111101001101100111101100110100000000001100110000001 1101100010000100011001000110011111000000111011000001100011000110111000000000100000000000011010110000000100000 010010011100011001001000010001001000001000100000000000000100001000000000100100001000100000000000000010000001 00000000000000110100000000000100110001011001111010011011011101001110101111001110111111001111011011011011010000 0110111100110000011110000000000100011101011000000100110110111100011000010111110011001110100001101101000001000 001010000111000100001000001001101001100110000010000010000000000000000000000000000000001000000011000000000100 0011000100000011000100000100000100010001010000001000000010

SW ST 3 2 00000000000000000001101111111111100110101100011110100001100111011000010000011011011000110110000110011110110110 0011000110000001011101100010110000000001111000001000010111000000110000100000111000101011000000000011001101011 011000001111000011000111010000011000000000000011001101100000000000000000000000000000010000000010000100000000 00000000000000000000000000000000000001010001000000000000010000000000101000000100000000000100000000010000000 0000000001101111110110110101001111101100000101111011100110000001011100011000011000011001100001000000101100100 001100100000110001011010110000000010100011001100000000001101100110000001011011000011000000110001100010000000 011000100000000100000000000001100000110101000000010000000000101001100110011001011000000001000000000000001100 0000000010110000000000011000000000000000010000000000000000000000000000011001110001111000010110011110110101110 01000110111100111101001101101110110011000001110001101111100111101011011000111100000001000000000001100110000001 010000011000000011100000011001110100000010101000000000000000011011100000000010000000000001101001000000000000 001000101000001100000000001000100100000100000000000000000000000100000000010010000000010000000000000000000000 0000000000000011111000000010010001100011110011011101010101011010101100110110011110110110001110110110010101101 0001100011011000000011100001010000100010110110000101001101101111000110001111011100110011011000111001110100110 000010000011000001000010000000011000001000100000010000000000000000000000000000000000000010000000100000000000 000011000100000000000000000000000000000001010000000000000100

SW ST 4 1 00000001000000000000101011111111100101100001011110100110000111011000111100010001111000011110010110000110110011 0011000110100000111001100101110000010101101000001001010111000111001000100000111110101101000000110000001111011 111100001111000011000001010000011011000000000011000101100000000010000000010000000000110000000010000100000000 000000000000000000000000000001000100010100010000000100001000000000000000010000110000000001000000000100000000 00100100110010011011111111100111111100000011011000110111100000100100001100011100011000110011000000000110011000 207

1100000001100000100010110000000110110011001100000000001101110000000111011000100011000000110000011100000000001 000000000110100000000000100000000010001110000010000000001000000000010011001101000010000000000000000000100000 100001001000000000000100000000000000000000000000000000000000000000001100111000010110000011001111011010110001 1101101011000111110011010111001100110110011000011000100101111010011100001111011001011000000000011000000000001 100000110000000111001000110011100000001101011000000100001000000101001000000100000000000000010110000000100000 010000010000011000000000010001011000001100000000000000000000001000000000100100000000000000000000000001000010 00000000000000110111000000001100110001111001001100101011111001010111011111101110111111001111011011011010010000 0110111100110000011110000001100011001101101000010100010110110100001010110110110011001100000011010101010001000 001000001101000100010010000000111001100110100000010000110010000000000000110001010000011000010000000000100000 0100000100000001000100000100000100000010010000001000000100

SW ST 4 2 01000101000100000000111011101101100111100001011110110000000101110100111100000001111000011110001110010101011011 10011100111100001110111001111100000101011011000010010100111001010010001100000101101011110000001100000001111110 1110000011110000110000011110001101100000000001100010110000000001100000001100000000001000000011000110000000000 000000000000000000000000000100110011010000100000010000100000000000000000100000000000000100000010001000100001 111011110111111111111110110010111110000001111100110011110000010110101100000110011001110011000000000101011011110 0000000110011100011011010100011011001101100000000000111101000000110101111110001100000110001011101000000011000 001000110100000000000000000000001001011000001000000001100000000011001001111000010000000000000000001100000100 001011000000000001100000000000000000000000000000000000000000000001100110000101010000011001111011010110001110 11010110011011100111011111011001101100110000110001100011110101101000011110110111010000000000110000000000011000 001100000010110010001101011000000101011100000001000010000001010010001001000000000000001001100000000000000100 000100000110000000000100100010000011000000000000000000000010000000001001000000000000000000000000010000100000 0000000000110101000000001100110001011000101000011001111001001111101111001110111011000111011011011000010010011 011110101000001111000000010001100101010100000010000001011110000100001010011001100010000001110010100000100000 100000010100010001001000000011100110001100000001000011010000000000000101000100000000100001000000000000000000 10000010000001000100000000000100000010010000001000000100

SW ST 11 1 0000001010010010001011011110011101111001100001000111010001100110001001110100101010111000011101101110010010001 000100001011010100100110010010011000110100001000100011010001000000000100011000010000010101000010011001000110 100010000010010000010001010110000010110000110001001000110001001000110100110100010010001000000010000010100000 000010000000000000010010001000000000000010000000000000000001000000010000000000000000000100000000001001110000 00000111010110011111011100111101110010011101100100111001001110011010101101111000010110010001000011001111001001 011000110010000110110000010100000111000010010010001110011010110011010100001100011001000000010000100000010010 011101100100000001000000010000000000101010001000100000000001000111000100110000100000001010000000011100100000 100010000000000000000010010001000000000000001100000000000000000000000001100101000011000001011001111010110011 00111011010110001111100110101111011001101100111000110011010001110101010000000110000011100011000000110111000001 1110110011000000011001100011001110100001110101100000011000100000010000000001100000000000001000011000000010000 011000001000001100000010001000101101000010000000000000000110000100000000000010000000000000010000000000100000 10000000000000011011000000000110011000111100001100001101111101101011001110110111110111100011011101101101011000 0001011110011100110110100000000001101011101100001110010011011110101100001010011001100010000001101110101101100 000101000101100010000100000000111100110011101001001001010001000000001100000000100000000001000001100000000000 10011001100000000100000000100000100010001010001001000000000

SW ST 11 2 00011011101000000001101101011111100110100101011100110000000111011001111100010001111101111110010110011110110101 0011000110010110011101100000110011000101100000001000010011000110000001100001011001001111000000000001100011011 0111000001110000110001110011100110110000000011010010011011000000110000000100000000001000000000000001001000000 01000000000000000000000000000000000010100010010000000000010000000000000000001000000000000000000000100000000 00001100111111011010111000000111111100000011011100110010111000111100101110001100001101110001000000100110110000 1100100001100000111000110011000100110000001101101100001100000000000011011011100000000000111001000011000001011 000000000001100000000000000000000110100011000000000000001100000000010011001000000110000000000000000000000000 100000011000000000001100000100000000000000000000000000000000000000001100111000100110000011001111010110011001 10011010110001111100110101111011001101100110000110111010001110100110000000110100011100011000000011111000001111 011001100001001100110001100111010000111010110000001100010000000100000000010000000000000100001100000001000000 110000100000110000001000100010010000001000000000000000010000010000000000001000000000000000000000000010000000 0000000000000110100000000001100110001111000001000011011111101010110101101101111110111001110111011001000010000 0010111100100000101101000000000011101101101000001100100110110100001000010110110011000100000011010101011011000 001010001011000100001000000000101001100010010010010010010010000000001000000000000000000010000001000000000000 0011001100000001000000000100000100000001010001001000000000

SW ST 14 1 00001001101010000000101101111111100110100000011111110001000111011010111100011001111111010110011101101011110101 00110001100000100111111101011100000100110010100001101001110110000110011000001110010010110000011011000011011111 111000000110000011000111011100110000000000000110010011010011001000000000100000000001100000000100001000000001 00000000000000000001000000001000000010100001000000000100010001000000000001000001000000001000000000100000000 00101100110110011111101000100111110110001111011100110011100000111100111100001010001100110001100000000110110000 1100110001100000110000100000000010110000000000011000001101100000011001011111100011000000110001111011000000011 000000000110000000000000100000000010010001000000000000001101000010001011011001010000000000000000000001100000 208

000001001000000100000000000100000000001000000000010000000000000000011100001000101010000011001110011010111001 10011000110011111110110110110011001101110110100110111011011110100111100011110101110110000000000001100010000011 001001100000010110010001100111000001101000100000000000110001101110000000010000000000000100000100000000010000 000100100000110000011000100000010000011101000000100100000000010000010001001000000001000000000100000010000010 0000000000000110101000000000000110001111000101100011011101101010110011111001101010011000111010011011000110000 0111001101100001100110001101100000001000000000010100100110111100011000011101110010001101100011100101010001000 001110001011000100010000000011000001100100100010000000000011000000000000000011000000110000001010000000100000 0011001000000010100100000000000101000001010000001010000010

SW ST 14 2 00010001101000000001101111111111100110100000011111110010110001011010101100011001111111000110011101000011110011 0011000110000011011111100101100000001111001010000000010011011000011001100000111011001011000001101100001101101 1111000000110000001001110011100110000000000000110011011010011001000000000100000000011001000001100001000000111 00000000000000000000000000001000000010100001000000000100010000000000000000000001000000001000000000100000000 0000000011011001010110100010011111011000111101110011001010000011110010110000110000110011000110000000011011000 011000000011010001000001000000000111000000000000110000011010000000000011010001000110000001100011110011000000 110000000001100000000000001100000000000100010000000000000010000001100100110010111100000000000001100000011000 000000000110000000000000000000000000000010000000000100000000000000000011001010000101100000110011100110100110 01100110101100011111101101111100110011011101100001100110110111101001101000111101011101100000000000011000110100 110010011000000001101100011001110000011010001100000000000100011011100000000010000000000011000001000000000100 000001001000001100000110000100000100000111010000001001000000000100000100010010000000010000010001000000100000 0100000000000001101010000000000001100011110001101000110111111010101101010011011000110110001110100110110001100 0001110011001100011000110001011000000011000000000001001001101111000110000111001100100011001000111011010100010 000010100010110000100010000000110010011000001100100000100000110000000000000000010000000100000010110000001000 000011000000000011000100000000000100000001010000001000000010

SW ST 15 1 1000000100100000000100001100110101011100000001101000000110000000110011110000000111100000011000011000111110101 100110101010010000110011000101100010001101010000010100000110101010010000100001111100110010000000011011001010 010110000011011000011001100011000110000000000000110000011000001110000000000010000000000100000000100001000000 000000000000000000000000000000000000000101000101000000001000100000000000000110000010000000000000000000100000 0000011100010111000011001110110110101110000001101000110011110000001100000100000100000110110001100000000110010 000110010000000100010110001100000011001100000110000000000000010000000001100100011000000000011000111101000000 000100000000011010000000110000000000000001100000000000000000110000010001100100100000000000000110000000000000 000010000000000000000000000000000000000001100000000000000000000000000000110011100010101000001100111111011000 11011001101011000111011011010111001100110111111100011011101000111010101110011011000000111000000000000001100000 001100000110000001011000000110011100000000101011000000100001000110011000000010100000000000010010110000000000 001100010110100011000001000010001001000001000100000000000000000001000000000100100000000100010000010000000000 0001000000000000011010000000000100011000101100011011001101110110101011001110100111101101100011101101100100011 0000011000000101010110111000000000001100110101100001010001011011110000100011110011001100110100001101010100000 100000101000011100010000001000001110000000001010001000000000001100000000000000000000000000000000000000000010 00000010001000000001000100000100000100000001010001001000000000

SW ST 15 2 00000001001000001001100110011111100111000001011110000001100111011101111100100011011000010110000110011111110010 0011010110010000011001100100110000010111011100000000010110000111001000100000111110011011000000001111001111001 011000001011000011000111001110011000000000000011000001100001110000000000010000000001000000000110000100000000 00000000000000000000000000000000000001010001010000000010001000000000000000100000100000000000000000010001000 00110011011011101001110111110011111110000001101100011001110000010110000110000011000011011000110000000011011000 011001000000000000110000110000001101100000011000000000000111000000000110100101000000000001100110110100000000 010000000001101000000011000000000000001010000000000000000011000010000100110010000000000000011000000000000000 001000001000000000000000000000000000000010000000000000000000000000000011001110000101100000110011110101100011 01100110101100011101101101011100110011011111110001101110100011101001111001101100000011100000000000000110000000 010000011000000001100000011001110000000010101100000011000100011001100000000110000000000001001011000000000000 110000011001001100000100001000100100000100010000000000000000000100000000010010000000010000000000000000000000 01000000000000011010000000000010011000111100111011001101110110100111001110110111101101100011101101101100011000 0011001000101000110101100000000001100110110110001010001011011010001100011011011001100110010001101010101000100 000100100011100010000111000000110000000001011001000000000001100000000000000000000000000000000000000000010000 00010001000000001000000000100000100000001010011001000000000

SW ST 18 1 0010110000100000001100001111110101011010000001101001100001000101100011011011000101101000011000111001101101101 1001110011011011001100110001111000000010101100000101000010100010101100010000011111001101100000110000000010100 101110000000110000011011011011001100000000000001100000110110001100001000000011000000011000000001000000000000 01000000000000000000000000000100000000101000010000000000001000000000000000000000010000000100000000000100010 00000000000101110100110011001101101111100001011011111100111000000101101011000001110111011101101100000001100110 0010000000001110001010001100000000100100000011000011000001111100100000110011100000000000001101100110000000000 010000011000001000000000000011000000010000010000000000000011000001010010010000010000000000000001100000001000 001000000100000000000000000000000000000100000000000000000000000000000011001010000101100000110011110110100011 01100110101100011111001101011101110011011110110001100101100111101001101101111100001101100110000000000110000000 209

000000011000000011100100011001110100000110001100000010000000011001100000000000000000000001000000000000000000 001000011001101100000000100000100100000111000000000000000000000100000000010010000000010000000000000010000000 10000000000000011010100000000110011000111100111010001101111110101011001100100110111101100001101101101101111000 0001001000011000111011100000010001101110110010100010001011010011100100001010011001100111000001101010101000100 000101000010100110000010000000000000000000000000100000000010000000000000000000100000000100000000000000100000 00111000000000001000000000000000100000001010000001000000000

SW ST 18 2 0000000100100000000010001111111110011010000001110011001100001101100011011001000101110000011000011000001011011 0001110011011111001010110001111000000011011100000000001011100001001100110000011111000101100000110000000110100 1111100000011000011100110011010010000000000000011000001101100001000110000001100000001100000000010000000000000 100000000000000000000000000001000000010100001000000000000110000000000000000000001000000010000000000100000000 00000000110110100010001100100111111100000111011111110011100000000110101100011000011101110011010000000110010000 000000000101100001100000000000011011000000110000110000010111011000001100001000000000000011100001100000000000 100000000000010000000000000110000000000001000000000000001100000101001000100001100000000000000011000000010000 010000000100000000000000000000000000000100000000000000000000000000000110010100001011000101100111101011000110 11001101011000111110011010111011100110111111000011010110001111010011011011110100000011001100000000001110000000 000000110000001011000000110011100000001100011000000100000000101110000000000100000000000010000000000000000000 101000110001011000000000000001010000001110000000000000000000001000000000100100000000100000000000000100000001 0000000000000011010100000000110011000111100111010001101011110101010001110110110010100100011101011101101011000 000100100001100011001110000001000110111011001010001000101101001010010000101001100110001100000110101000100010 000011100001010011000000000000000000000000000000000000000001100000000000000000010000000110000000000000000000 000110000000000001000000000100000100000011100000001000000000

SW ST 19 1 00000011101000001001101111101111100110100001011111000010100111011001111100011001111000011110010110011011110011 1111000110100110111001100100110000000101111000000000010011000101011001100000110000011011000000000000101111011 011100011011001011000111011000010000000000000011011001100000000010000110000000000001000000000110000100000000 00000000000000000000000000000100000001010000100000000000101000000000001000000000100000001000000001010000000 00000010011101000001111111000011111110000001101110010001111000000110110110001110000110111000110110010011011000 0110010000110000001100010000011011000000100000000000000110110000000000111100010000000000011000111101010000001 100000000110010000000110000000000001000111000000000000000100000000011001100111100000100000000001000000000000 000000101100000000000010000010000000000000000000000000000000000000000111011100001011000001100111101011011100 11100010011001111110011011011001100110111111000011001010000111010011011000111011000011001100000001111100000110 000000110000000011011000000011100000001100011000000100001000110110000000000000000000000011000110000000000000 010000110100011000000000010000011000000000100000000000000000001000000000100100000001000000000000000000000000 00000000000000110000000000001100110001111001110110011001101011011111111011001110111011000110111011011000110000 0011001100110000101011000100000011001101101000010100110110111011011000110100110011000100100011010100010001000 001010000000000100001100000001100000000010000010000010000001000000000000000000000000000000000000000000000000 0001100000000000000000000000000000000011010000001000000000

SW ST 19 2 00000000001000000001101111101111100110100000010110000010100111011000111100010001111000011110010110011101110101 1111000110100110111001100100110000000101110000000000010011000100011001100000110000011011000000000000101111011 0111000110110000110001111110000100000000000000110110011000000001100001100000000000010000000001100001000000000 00000000000000000000000000001000000010100010000000000001010000000000000000000000000000010000000000100010000 00000000111110000010111110000111101100000011011100100011110000111101111100011100001101110001101100000110110000 110110000010000101100010000100011000001000000000000000110100000000000100111010000000000011000111100100000001 100000100110010000000110000000000001000010100000000000000110000000011001100111100000100000000011000000000000 000000001100000000000010000000000000000000000000000000000000000000000110010100001011000001100111101011011100 11101100011000111110011010111001100110111111000001101010001111010011011000111010000011001100000001111100000110 000000110000000011011000000011100000001100011000000000001000110110000000000000000000000011000110000000000000 010000110000011000000000001000011000000001100000000000000000001000000000100100000000100000000000000000000000 00000000000000110000000000001100110001111001111010011000101001011111111111101110011101000110110111011010110010 0011001100110001100111000100000011001101101100010100110110110111001000110110110011000101100011010100010001000 001001000000000100001100000001100000000010000010000010000011000000000000000000000000000000000000000000000000 0001000000000000000100000000000000000010000000001000000000

SW ST 20 1 0001000100000000000110001110111110011010000001111100011010101011100011110001100101100000011001011001101100011 0001100011010000001110110011011000000011110001000010010011001100000000110000011100000101100000011000010001100 101110000001100000100001101100001100000000000001100100110000000001100000000000000000010000000001000010000000 00000000000000000000000100000000000000101000100000000000010100010000010000000000010000000000000000001000100 00011011001101100101011111001001111101100000110011001100111000001011001111100111000110111100011000000001101100 0011001100001100011011100001010000001100000000011010000011011100000000010011110000000000001100110101101000000 110000000001101000000000100000000001101000100000100000000011000110000100110000000000100000000000000000010000 001000000110000000000000000000000000000010000000000000100000000000000010001100001010100000100011100101101110 0111011010110011101100110110111011001101110110000110111100001110101101100110110000110100000000000100000000001 001000001100000000110000001101011000000011000100000000000010001101000000000010000000000000100000000000000000 00010000010000001000000000000010001000000101000000000000000000001000000001001000000000000000000000000000000 210

0000000000000000010100100000000110010000111000100111001100101110101011011101110111101001100111001111001100001 0000011000000101100000110100000010001100111101100001010011011011110000100011110011001000010100000101010000000 100000100000010000010000100000001000000000000011001000000000001100000000000000000100000000000001000000000000 00000010000000000001000000000000000000000000000000000000000000

SW ST 20 2 00010010001010001101110011101111100110000000011111000001101110111001011100011011011000000110011110011011000110 1011000110100001010101100110110000000111000011000100010110011000000001100000111000001011000000110001100011001 011100000011000011000001101000011000000000000011001001100000000011000000000000000000100000000010000100000000 00000000000000000000001000000000000001010001000000000000101000100000100000000000100000000000000000010001000 00110010011101001001111100110011111010000001100110011010110000011110011111000110001100111000100000000011011000 011001100001010001000101000000000001000100000011010000011010000010110010101110000000000001100110001111000000 110000000010111000000000011000000001101000010000100000000011000010000100110000000000100000000000000000110000 001000000110000000000000000000000000000010000000000000000000000000000001001110000101100000110011100101101110 01110110101100011011001101011110110011011101100000111111000011101001101101111100000111110000000001100000000001 010100011000000001100000011001110000000110101100000001000100011011000000000110000000000001001000000000000000 001000001000001100000000000000101100000010010000000000000000000100000000010010000000000000000001000000000000 00000000000000001011100000000111011000111100101011001100011110100111011110111111011110100011001101101100010100 0011000100011100000001100010010001100110110110000110011011011011000100011011011001100010100001101010101000100 000100000010000010000101000001100000010000011001000000000001100000000000000000100000000000000000000000000000 00010001000000001000100000000000100000001000000001000000000

TU HA 12 1 01000101000011000000011010101111110110111100011010110111100011111000111100000011001110110110110110011110011011 1110100110000110111001100110110000110111100110010100010101000110011000110000111000001101000100001100001101011 0110000001110010110001110110000110000100000000110110011100001111100000100001100000101100000001100001000000000 000000000001000000000000000100000000100000010010000000000010000000000010011000011000000000000001000100010001 00001001100110110111011001111101111100000011111001100111111100011011011000011011111001100011000001101100100001 1011100110101001100000110001101100000110011001100000000111101101000011111001000000000001100110000011000010010 000000001000100000000001100000001111010011000100000000010000000000100010001010000010000000000000000000000000 000010110001001000000000000000000000010000000000000000000000000000000000000101001100110110101011011100010011 00111001100011111010111011101011101101100000101111011101111101001101001110111000111100000000001100000110011000 0010110000110011000010110011100010001101011000000111000100110110000000001101000000000110000110000000000000001 000110100010000000001110011000000000101000001010110000000000110000000100100001100000000000000000001000000100 01000001010100000000000001100110001111100111110011011111101001110111101101110111100100011110011000001110010011 1001000110000011011110011000011001101101100010100110110110010011001010111110011001101100011011100010011000001 0100011110011000111100000000110011000101001100000111000110000000000000000011000000010101100000000000000010001 000100000000100000001100100100000001001010000100000010

TU HA 12 2 01000001000011000000010011101101110110111001011110010001101001111000111100000011010110110110110110011110011011 0111100110000111011001100110110000110011100010001000001011000010011000110000111000001101000000001100001101011 1110000001110000110001100110000110001100000000110110011100001111100000100001000000110100000001100001000000000 000000000010000000000000000100000001100000010010000000010010000000000100000000100000000000000000000100000000 10000001100110110111011001111011111100000001111001100111110000011011011000011000111001100011000001011010100001 1011000110110001100000110000101100000110011001100000000111010101010011100001000000000001100110001010000010010 000000001000100000000001100000001101100011000100000000010000000000100010001010000010000000000000000000000000 000000110011001000000000000000000000010000000000000000000000000000000000000100000000110110101111011100010011 00111001100011111010111011100011101101000000101111101101111101001101001110111000111100001000001100000110011000 1010110000100011000000110011100010001101011000000111000001110110000000000100000000000110000110000000000000011 000110100011000000001010011000000000101000010010010000000000110000000100100000100000000000000000001000000100 01000001010010000000000001100110001111100111110011011110111001110111101101010111100100011110111000001110010011 1001000110100011011110011000011001101111000011100110110110010001111010110110011001100100011011100010011000001 001001011001100011010000000011001100010110110000111100011100000000000000001100000001010110000000000000001000 1000100000110100000001100000100000001001000000101000100

TU HA 13 1 01001101101001100000110011101110100110010000011101010001100001001100111110000001111000110110010110011011101011 11110001101001101110011011011100010011111100110000000101010001100011001000001110000110110000010011001011110011 1110001011100101100011101101101100000000000001101000111000010110000011000010000011001000000011000110000000000 00000000001000000000000000100000000100000010000000000000010000000000100010000100000000000000000000100010010 00001011101100100111111001101011111100000011111001100111100000001111011100010000111011100011000001001101110001 1101100010000111011100110000001100000100011000000000110101000100010001011001000000000001110110011010000000010 000000011011000000000000000000000011100100000000000000110000011001100110010000000110000000000000000110000000 000000110000000000000000000000000000010000000000000000000000000000000000000000001100000011010111111101100011 01111001100011011000111111101011101101100011010110111100111101001101101110111000011100000000001100000110011000 0100110000110111000000110011100000011100011000001111000000011110000000001100000000000011000110000000000000011 000010101011000001011010001000000000100100010000010000000001110000000101000001100100000000000000001000000100 00000000010011000000000001100110011111101111110011011111101001111001101101101111011100111001111000101110001011 1101010110100010111110011000110001101101100010110110110110111001111010110011011010101100001010101010001000001 211

011001011000010011010000000011001100110000010000100100101000000000000000000000000011000010000000000000001000 1000100000000010000000100000100000001001010000100000010

TU HA 13 2 0100010010010100000001001110110110011010000001110101000000101110110011111000000111100011001101011001101111101 10111110010100110011001100011110000010111011110010000001011100101001100110000011100001111000000001100010111101 011100010111100001100101001101011000000000000011001001101000110110000001000100000010011000000010000100000000 000000000000100000000000000000000000010000001000000000000011000000000001001000010000000000000000001010001000 11000100111110011111101100110011111110000001101100110011111000000111110001001010011101101000100000101110011000 1110110001100011101100011000000101000000001100000000011100100011011001001101110000000000111001101101100000011 000000001101100000000000000000000000011001000000000000001100001100011001000100000010000000000000000001000000 000000011000000000000000000000000000001000000000000000000000000000000000000010000110000001100101111110110011 11011100110001111101011111101101110110110001101011011110011110010110110111011100011110000000000110000011001100 0010011000011011100000011001110000001110001100000011100000011011000000000110100000000001000011000000000000000 100001010101100000101101000100000000010010001000011000000000011000000010100000110110000000000000000100000010 00000000000001000010000000111011010111110111111001101110101101111100110110110111101110001101111100010110100001 1110101011010001011111001100011000110111100001010011011011011101110101011001001101110110001101010101000100000 101100100100010001101000000001100110001000001000010010001100000000000000000000000001100001000000000000000100 01000100000010010000000100000100000001001010000100000100

TU HA 14 1 0100010100100100000001001010111110011110000001111001000100001001100011110001000111100011011001011001101110011 1101100010101011001100110010111000001011000011000110000000100010000110011000001100000101100000000001100110100 010100000011100000100011000101101100000000000001100100100000000010000001000010000001000000000001000010000000 00000000000000000000000000000000000000100000010000000000000010000000000000010000100000000000000000000100010 0010011000110011000010111100110110111110000001111000110011100000000101101100010000000101111000100000010110110 000110010000100000100100000000000011001000000110000000000000110001000000110000010000000000011001100010010000 000110000000010010000000000000000000000001000100000000000000100001000001000100010000000100000000000000000000 000000000000000000000000100000000000000000000000000000000000000000000000001110000110011001101100010110111011 10111101101011000111110001110110110110011011001100011100111000111010011011011101100001111000000000011000011000 1100000001100001100110110101100111000011011000110000001110001001101100000011010010000000001100001110000000000 000110001101010100000000011100110110000000110000100100000000000011100000001001000010001100000000000000010000 01000000000010100100001000000000001100011111001111100110111110101111100111011011011101011000111100110100011100 0001100010101101000010111100011001110010011110000101001101101101100011001101110110100001001000110101100100010 0000101100111100001100101000000001100111001011001000010110101100000000000010000110001001100001100000000001101 10001001110000000000000000100000100010001001000000100000100

TU HA 14 2 01000101000001000000011010101111100111100000011110110001101001011000111100010001111000110110011010011011100111 1101010110010110111001100101110001010111000110010001001111000111001100110000111000001011000000000011001101000 101000000111000001000110001011011000000000000011001001000000001100000010000100000011000000000010000100000000 000000000000000000000000000000000000010000010000000000000001000000000000000100010000000000000000100110001001 10011100110011011101111100110110111110000001111000110011101000011101101100001110000111101001100000110110110000 1100100011100011101010110000000101011010001100000000000001110010000001110110100000000000110011011101100000000 100000001100100000000000000000000010001001000000000000001000010000010001000100000001000000000000000000000000 0000000000000000000001000000000000000000000000000000000000000000000011100101000101100111100011110110101110111 10110101100011111000111011011011001101101101001110011100011101001101101110110000111100000000001100001100011000 0000110000110011011000110011100001101100011000000111000000110110000001101100000000000110000111000000000000011 000110101011000000001110010011000000110100110001000000000001010000000100100001001110000000000000001000001000 00000000010011010000000000010110001111001111010011011111100111111011101111110111101100011110011010001101000011 0001010110100001111110001100011001101101100010100110110110110001100110111011010001100100011010110010001000001 011001011000010001010000000011001110000110010000101101011000000000000100001100010011000011000000000011011001 1000110000000000000000100000100000001001000000100000100

TU HA 15 1 0100010110100100000001001010110110011010000001111011011010111101100011110001100110100110011000111001101101101 100011001100000010111011001011100100001110000110100000100110001010110000100000110000010010000000011000000010 011011000001111000011001100010110110000000000000000110011000000011000000100001000000110000000000100001000000 00000000000000000000000000000000000000010010001000000000001001000000000000001000010000000001000000000010001 00010011110111110010011111100110011111110000001111000110011111000011110010110101010001100110001110000110110111 0101110100000110110111010110000000110011010101101100000000101100000000001101100100000000000111000011001000000 001100000000000100000000000001100000000000001000000000000001000000000010011000001011000000000000000000001000 000000000001000000000000100000000000000000000000000000000000000000000010000000001100110000011100101111110110 00110011010110001111101011111101101100110100110100111011111011110100110110111011100011110011000000000000110110 000000001100000010110110001100111000000111000001000000110001001101100000000001110000000001100000100000100000 001010001101010110000000011100100110000011001100100000100000000010100000001001000010000100010000000000001000 00100000000000100100000000000000101100011111000111100110111110101111110111011011011101011000111101110011110100 0001110110001100000011111100110001100011011010000111101101101101100110100101101100100111011000110101000100010 000011100000010001010110100000000110011000100000100000100010110000000100000000011000000110100110110000000000 010011000010000000100000000100000100000001001000000100000010 212

TU HA 15 2 01000001101001000000110011101111100111100000011101010110101111011000111100011001101001100110011010011110110011 0011000110000001011101100101110001000111001011010001001101001111011000110000011000001001100000001100100001001 1111000001101000011001100011110110000000000000000110011000000011000000100011000001101000000001100001000000000 100000000000000000000000000000000000101000010000000000010010000000000100001000100000000010000001000100010011 011111111111001001111100011001101111001100111100011001111110010110011111100101000110011000011000011011001101011 1011000111011111101011000000011001101100110110000000010010010001100110110011000000000011100101100100000100010 000000000110000000000010110000001000000110000000000001100000000001001100000101100000000100000000000100000000 000001100000001000010000000000000000100000000000000000000000010001001000000010011000001110010111111011000110 01101011000111110101111111110110011011011010011101111101111001011010001101111001111101100100000000011011100000 0001100000001110110001100111000000111000000000000110000000111100000000000110000000001100000100000100000000110 001101010110000000011100000110000011001100000000100000000011100000001001000010000100000000000000001000001000 00000001000100000000000000001100011111010111100110111101101111110011011011011101011000111101110011110100000111 0100001100000011111010110001100010011110000111101101101101100011100101100110100011011000010101000100010000010 110000000001010110000000000110011001000000100000100000110000000110000000011000000110000100110000000000010011 000110000001100000000100000100000001001000001100000010

TU HA 17 1 00111001100111001001101111001101010000000101001110010110010010001100011110001110111100011011000110101110110010 101101001010100011001011010010110000000101010001100100011001000000100010001001000000100100010010011000000100 010100000100000000100011000101000100100000001001001000010010100100000001000100000010100000000010010010000011 000010000000000000000000000001000000001011101000001110101001000000000001000010001000000000010000010111010111 1010101100101100111001010010110111010100100100100011101111110110111100100110000100110001101010010010000100101 000000011001011011011001110010010110100011001001100010001000110010001100110000100000101000010000101001101001 011100100000100100011100110000000000010000010000100100000010000000110010010000110000010010100000000000000000 0000000011000000001000000000000001101000000000000000000000000000000000000101000011001100000011111111111101100 11110110001100011111000111010111011001101101100001110110101111101001101001100111000111100000000001100110000011 000010011000000011100000011001110000010110000000000011000000010111010000010010000000000001100011000000000000 001100111000001100000000101000101100000110010001000000000000000111000000000010000000010000000000000000100000 00000000000001001000000000000111011000111100110111011101111110101011001111110110111110110011111011100000110100 0011100101011010001101100001100011100110110100001010101011011011100110011011001101100010010001101011101000100 000101100001100001000100000000001100110001000001000000110110000000001000011000000000001101001000000000010000 10011001100000000100000000000000100000001001000000101000010

TU HA 17 2 01111101101111101100010110101111100111100000011101110011000001011000111100011001111000110110010110011110011011 0011000010100111011101100101110000000101110010001100001011000110001100110000001100001111000100000011100101101 1111000001110000011001100010110000110000000000110010000011011011000001100011000000101100000000100001000000000 000000000000000000000000000000000000100000010000000000000100000000000100001000100000000000000000111100010001 01111111111110100111011001101101111100000011010001100111100001101101011100001000111011100011000000001100110000 0001110001100111011001100010001101100110000011000000001101010110110101011000100000100001100110011011000011011 001100000001000000000000000000001111000011000000000000110000000000100110000110000000000000000000000000000000 0000000000000000000000000000000000000000000000000000000000000000001001110001110011000000111111111111011100111 01100011000111110001110110110110011011011000011101101101111010011010011001110001111000000000011001100000110000 000110000000111000000110011100000101100000000000110000000101100100000100100000000000011000110000000000000011 001110000011000000001010001011000001100100001000000000000001110000000000100000000100000000000000001000000000 00000000010010000000000001110110001111001011110011011111011001110011101101101111101100111111011000001110000011 1001010110100010111000011000111001101101000010100010110110111001100110110011011001100100011010111010001000001 011001011000010001000000000001001100000000010000001101100000000010000110000000000011010010000000000000001001 1000100000000100000000000000000000001010001000100000000

TU HA 18 1 01111101101010000000100011101101100111100000011110110011001110011000111100010001111010000110010110011111011011 000110011011011001100110010111000001010110001100000001001100011000100011000000100000101000000000000000010100 001100000010100000100001000101001000000000000000100000100000010010000001000010000000010000000001000010000000 000000000000000000000000000000000000000000000100000000000001000000000000000100001000000000000000010101001100 11011110011011101001110110011011011111010000110110011001111100011010010010000101010110011000110000000011011100 011001100010000101110000110000001100100100011011000000010001000100010000011001000000000000100000000011000010 00000100000100010000000001000000000010010001000000000000001000000000010010000100000001000000000000000000000 000000000100000000000000000000000000000000000000000000000000000000000000010100000100110000001101011111110111 11001011100111001111100011111110101100110110110000111011110111110100110111111011000011110000000000110000110000 000000001100000000110001101100111000011001000110000110000000001011000000001000000000000000110001110000000000 000010001100010110000001000100010110000001010000000000000000000011100000001001000000001000000000000000010000 01000000000000100100000000000000001100111110011011101010111111110111111111011101110111011000111100110101011100 0000110010101100000101110101011001100010011011000101001101101101100011101101100100100011011000110111110100010 000110010000010001000010000100000010011000001100100000111011010000000000000000001000000000000000000000000000 010011000000000000100000000000000100000001000010000100000000

TU HA 18 2 213

0100100100101100000001001010110110011110000001111011000100101101100011110001001011100000011011011001111001101 1001100011011011101110110010111000011001111001100000000101100011000100011010000100000111100000000001000111100 1111100000101001001000111001011011000000000000011000001100001101100000010001100000000110000000110000100000000 00000000000000000000000000000000000001000000100000000000001000000000000000100001000000000000000010001000100 00000110011011101001111110011011011111000000111110011001111100011110110011000100010110111000110000000111011100 0111011100011001011110001100000011011101000110110000000000010001100101100110011000000000011101101110110000101 110011000110110000000001100000000011000101100000000000001100000000011001100110000000100000000000000000000000 000000100000000000000000000000000000000100000000000000000000000000001011001100001011000001100111101101011101 10011110011000111110001111111010110011011011000011101101011111010011011111011100001111000000000011000011001000 000000110000000011000110110011100001100100011000011000000000011100000000100000000000000011000111000000000000 011001110001011000000100010001011000000100100000000000000000001110000000100100000000110000000000000001000001 00000000000010011010000000000000110001110001100110011011110111010111111101110110111101100011110011010101101000 0011010000110000010111010001100110001001101100010110110110110110001100110110011011000101100011011101010001000 001001000011001100001000010000001001100000110010000011101011000000000000000001100000000000000000000000000001 0011001100000000100000000000000100000001000010000100000000

TU HA 19 1 01000101101000000000010010111111101111100000011001110011101001011000110100010001111001100110001010111110010111 0011000101100110011101100111110000010111101010011000011011001111011000110000010000001011000000000011100001101 101100001001000011000111011000011000000000000011010011000001100000000010001100000000110000010110000100000000 001000000000000000000000000000000000010100001000000000000101100000000000000000000000010000000000100010101001 10001110111111110011111110110110111110000011111000010011110000110100011101000110100111110001100000100110110000 1100110001110010111000110000000010011010100001100000000110100110011010101110000000000001110011001101100000011 000000000000100000000000000000000000010010000000000000011000001100110011000011000000000000000000000000000000 000001000000000000000000000000000000001000000000000000000000000000010110011001010110011011001111011110110101 10111000111001111100011101101101100110110010000011001110111110100110110111011001011111000000000000000110000100 010001100000000110110101100110000000001000110000110100110001101100000000000000000000000110001100000000000000 010101101000110000000010100010110000000001000000101100000000011100000001001000010000000000000000000010000010 00010000000100110100000000000001100111110010010100110111101110011101111111011101111011000111100110000011010000 0110010001101000110101100110000010011011011100101001101101101100011001101110110110001001000010101000100110000 010100010010000100010000000001000011000001100000000100000100000000000001100010000000110000110010000000000010 010000000000000100000000000000100000001000000100000000000

TU HA 19 2 01000101101001100000110011111101111111100000011001110011101101011000110100011001111001110110011011011110100111 0011000011000111011101110111110000010111101010010000010001001111011000110000001000001011000000000011100001101 111100001101100001000110110000010000000000000011010011000001100000000010000100000000110000010110000100000000 001000000000000000000000000000000000010100001000000000000101000000000000000100000000010000000000100010101001 111011101111110100111111101101101111100000111110000110111000001101000111101101101001111100011001001011101110001 1001100011100101110011100000100000110101000011000000001111000101100011111100000000000011100110011101000010110 000000000001100000000000000000000010110110000000000000011000011000100110000110000000000000000000000000000000 000001000000000000000000000000000000010000000000000000000000000001101100100010101100110110011110111101101011 01110001110011111000111011011011001101101100000110011110111101001101101110110010111110000000000000001100001000 000011000000001101101011001100000000010001100000101001100010111000000000000000000000001100011000000000000000 101011001001100000000101000101100000000000000000001000000000111000000010000000000000000000000000000000000010 00000000001101101000000000000011001111100100101001101111011100001011111110011111110110011111001100100111000000 1100101011010000111011001100000000110110111000111011001011011000110011011101101100110010000101010001001100000 101000100100001000100000000000000110000110000000001000101000000000000001000001000001100001100100000000000100 10000000000000100000000000000100000001000000000000000000

TUHA 20 1 01000101011000000000110011111101100110110000011010110101001010011000111100011011011001100110010110011111011011 0001100110100111011101100101110001011101101010000000010111001111011000100000111000011011000001001100011011001 111100000111000011000110001000011000000000000011010000000000101100000010000100000001100000000010000000000000 000000000001000000000000000000000000010000001000000000000001000000000000001100001000000001000000010010001000 111011101111101110111111101100111111100001010111001100111101000111001011111110101011011100011000001011001110001 1101000011100111110010110100101100110101000011000000111011000110110110011101000001100001100110110011000010111 000000011001000000000000000000000010110111000000000000010000000000110110001010000010000000000000000000000000 0000001100010000000000000000000000000100000000000000000000000000000011000100100011001111100111101110011000111 01110011000111110001110101110110011011001000001101111000111010011011011101110001111000000000000000111010110001 110110000110011011000110011100000001100011000000000000000110110000000000000000000000110000110000000000000001 000100000011000000001010000011000001010100000000000000000001000000000100100000000000000000000000000100000100 00000000010011010000010000100110101111000101010111000110111001111111111100101111101110011110101101101101000011 0011010110000001111110011000011101101101100010100110110110110001010110110110000010101100011010100010011000001 010000011000010001000010000011000110010000010000110000011000000000000010000000000000000011010000000000000000 1000000000000100000000100000100000001000000000100000010

TU HA 20 2 01000001000101000000010011111101100110100000011110110000111010011000111100011001111001100110001110011101111011 0010100110100110011101100101110000001101101110010000010011001111011000110000111000011011000000001100011011001 214

101100000111000011000110001000011000000000000011010000000000101100000010000100000001100000000010000000000000 00000000000100000000000000000000000001000000100000000000000100000000000000010000100000000100000001001000100 010101110111111011011111110110110111110000001111100110011111100011110011111101010001101111001100100101100111000 11101000011101111110100110110001100110100000011000000111111001100110011111100000001100001100110110101000110111 000000011001000000000000000000000011100111000000000000011000000000100110001110000100000000000000000000000000 000000010001000000000000000000000000010000000000000000000000000001101100000010001100110110011110101101101011 01111001100011111000111010111011001101101100000110111100011101001101101110111000111100000001000000001100011000 101011000011001101101011001110010000010001100000000000000001111000000000010000000000011000011000000000000000 100111010001100000000101000101100000111010000000001000000000110000000010010000100000000000000000000010000010 00000000001101101100000000010011000111100011011001110011011100011111111110011011110111001111010110110110100001 1010101011000001011111001110001110110110110001010011011011011000101011011001100010110110001101010001001100001 101100001100001000100001000000110011011000001000000010011100000000000011000000000000001001101100000000000000 01000000000000100000000100000100000001000000000100000010

TU RE 1 1 00100000001000000000101010101111100110100000011110110001100100011000111100010011111001100110011110011110011011 0011000110000011011101100110110000000001100010001100100011000111001100010000111001001011000001101101001101101 101100001111000100000111101000011000000000000011001001101100000000000010000000000000100001000010000100000000 00000000000000000000000000000000000001010000100000000000100100000000000000100000010000000000000000010000000 0000000001101110001111101001001111101100000110011001100111010001100000011000110100011001100011000000001100100 000000000000110000111100110001000000110000000000000000000101110110000011010010100000000000110000011100000000 001000000000001100000000000000000000010001100000000000000000100000100001011001000000000000000000000000000000 000100001001000000000000000000000000000001000000000000000000000000000010110111010010111011011001111111010010 11110111100110001110110110111110001100110100010100111011111011110100110111110011010011110000001000110010111010 0110110011000010001100100011001110010000111101100000011101100110001011011000011100000000001011011000001010000 000100011000001110101100001000001100100010010000000100001000000010000010000010010000010000000000000000100000 010000100001010111011100000001100110011110001111011111011011011001110111111101111011111100011111111001001110000 00010111101100110110110000011101110011011011000111001101101101100110000101111100110101001010110111000100110000 010110010110001000011100000011010110001100000100000111000001000000000011000010110000010101000000000000000000 010000011000000100100000100000100000001001000100110000000

TU RE 1 2 00000000001000010000100011101101100111100000011110110010000111011000111100010011111001100110011110001110011011 0011000110000111011101100110110000010101100010001000100110000111001000110000111001001011000001101101001101101 101100001111000100000111101000011000000000000011000101101100000000000010000000000000100001000010000100000000 00000000000000000000000000000000000001010000100000000000100100000000000001000000010000000000000000010000000 0001011011101110000111101001001111001100010110111001100111010001100000011100101000011001100011000000001100100 000000100011010000101100110001000000110000000000000000000101100110001011010010100000000000110000011101000000 001000000000001010000000000100000000000001000000000000000001100000100001011001000000000000000000000000000000 000100000101000000000000000000000000000001000000000000000000000000000000010100100000001011001100101111110111 01010101100110001110110110111110001000111100011001011011111011110100110111110011110011110000001000111010111010 0110110011000010001111100001001110000000011101100000011101010111001001011000011000000000001011011000000001000 000100011000001110101100001000000100000010010000001000001000000010000010000010010000010000000000000000100000 01000010000101001111000000000110011001111110101011101101111101100111011111110111101111110001011101101010111001 0000111011011001101101100000111011010110110110001110011011011011010110001011101101100110010100101110001000100 000101101101100010000111000000110101100011000001000001001000010000000000110000101100000100011010000000000000 00001000010100010100100000100000100000001001000100100000000

TU RE 4 1 00001010101000000001101011100011100110100000111111100001001100011000111100011001111001110110011100111110110110 0011001110100110011001100101110000100101101010011000100111000100001000110000011000001011000000000011011000101 111000001111000011000101101011011000000000000000000001100001000000000010001000000001010000000010000100000000 001000000000000000000000000010000000000000010000000000001010000000000010010001000100000000000000001100010000 00001110110011010111111110100101110110000011011100110011111000011101101110011100000000110001100000011110011000 1100100000110001001100110001000001110011001101100000000110110011000011001111100000000000110010011010000011001 000000001000100000000000000000000000101100000000000000001000000000010010010001000010001000000110000000001000 100000011000000000000000000000000000000000000000010000000000000000000110110100010110000011001111011010101111 00111100110001111100011111101001100110110110100011011110011110100110110111011100011110011000000110000110111111 0110011000000011100000011011110000001111101101000011101010110001100000000110000000000011001111000000000000001 100011000001100000110101001001100000010011000011001000000000010000100000010000100001000000000001000010000100 00000000110001110000000000110011001111100110101101101111101101011000111110111011111110011111101100010111000110 11101110110000010111011000000111001100001001110100110110110111111010010111110011001110100011011100010001000001 0110011110001000001110000000110111001101100100000111001011000000000001100110000000110100100000000000100000011 000101000000100100000100000100000001001000000100000000

TU RE 4 2 01000101010100010100110011011011100110100000111011110000011101010100111100011001111001111110010110011011011011 0001001010100110111001100011110000000101101010111001001011100111001100110000111100000110100000000011011100101 101100001101100001100010110101011000000000000000000001100000000000000001001100000000100000000110000100000000 00100000000000000000000000000000000000000000100000000000101000000000000100100010010000000000000000001000100 215

00010111011001101101111111011001111111000001101110011001110000000111011011100010000000110100010000000010111100 0110011000011001110110010101100001011000110110110000001011110101101101100101011000000000011000001101100000101 100000000110011000000000000000000000001111000000000000000110000000001101101001100001000000000001000000001100 0100000011000000000000000000000000000000000000000010000000000000000000111100000100111000011001101111110101111 00111100110001111100011111101001100110110110100011011110011110100110110111011100011110011000000110000110111111 0110011000000101100000011011110000001111101101000011101100110001100000000110100000000011001111000000000000001 100001000001110000110001000000100000010011000001001000000000100000100000010000100001000000000000000010000010 00000000101010000000000000111011001111110001111101101111110100111000111110111011111110011011101100010111001010 11011110110000001111011000000111001100001001110100110110110111011000010111110011000101100011011100010001000001 011000011000100000010100000001011100010100001000010100101100000000000110011000000001010000000000000010000001 0000101000000100100000100000100000001001000000100000000

TU RE 8 1 01001101011001000000011111100001110111100010011110110001000001011000111100010001111010010110010111111011101011 0001010110000110111001100001110111010101110010010000011011000110001001100011111000001011000010000001011001101 1111000111110000001001110100000110001100000000110110011000000011000000100010000000010000000001100011001100000 000000000000000000001100000010000000101000010000000000010011000000000000010000010110000010000000000100010000 00011001111110000111111101100111111100000011111001100111100000101001011000010100011111100110000000001101110111 1001100110000101011101100000001101100000000001100000001111101100000101011001000000000001100000011011000000010 000000011011000011000001111000000001101100000000000000110000000000100100000000100010000000000000000000100011 000000100000000000000000000000000000010000000000000100000000000000001100010000101000110110011110110101100011 01111001100011111000111111101011101101100101010110111011111101001100001110111000111100000000011100000010001110 0000110000000011011000110011100010000111011000001011011000110111000000110011000000000011000110000000010000011 010111100011000100100010110011000001101100000010000010000000100000000100100000000000000000000000000000001000 00000001101011010000000001100110101010001111011110011101011001110101111101111011101100011110011000001101100001 1101010110000010111110000000011101101001100011110110110110111001000010100110011011100100011011111010001000001 001001101001010001110000011101011000110010101000110100101100000000000100011001000011010010000000000000000001 1001100000010100110000000000100000001001000000100000000

TU RE 8 2 00010011101000000001101110100111110111100000011110100001000011011000111100010001111100011110111100011010110110 0011000110000110011001100001110011000101110010111000111011000100001001100011111000001111000000000001011001101 101100001111000001100111010000011000110000000011001001100000001100000010001000000000100000000010000100100000 000000000000000000000010000010000000010100010000000000001010000000000000010000001011000001000000000100010000 00011110111110100011111111100111100110000011001100110011110000101100101100011100001101110011000000000110010011 1100110011000001101010110000000110011000000000110000000101110110000011011000100000000000110000001100000000001 000000000100100001100000111100000000100110000000000000001100000000001010000000010010000000000000000000100000 100000010000000000000000000000000000001000000000000100000000000000000000100000000100011001100111111110010001 10111100110001110100010111110110100111100111001011011110111110100110000111011100011110000000001110000001101111 0000011000000001101100001001110001000010101100000010011010001111100000001001100000000001100011000000001000001 101011110001100001010001011001100000110010000011000001000000100000000010110000000000000000000000000000000010 00000000101001101000000000110011010111100101111101001111100100111000111111011101110110001111001100000111100010 1101101011000001011111000000001110110100110001111011011011011000110001011101001101010010001101011101000100000 100100110100101000011000001110100100011001001000001100110100000000000110001100100001101001000000000000000000 11001100000010100000000000000100000001001010000100000000

TU RE 10 1 01001101010100000000111111100101110111100000011011110011101101001100011100011011111011111011001111001011100011 0111100011110110111010100011110000100001100000110101010011000101100000110000011101100110100000110001001101111 0111000011011011001000101111010110110000000000000001111000000001100000000001000000011000000000100001000100000 000000000100000000000000000000000000110000010000000000011000000000000000000000000000000010000001000100010000 001111011111011111110111011001111111000000111110011001111110011110011110100011000010111000010010010111011100011 0011010111001101110001100000001111000110000000100001111010101101101100101110000010000011000110110110011001100 000000000011000000000110000000000001100110000000000000110000000000100110000001100000100000000000000000000010 000010100000010000000000000000000000100000000000000000000000000001011001110001010000001100111101101010110111 01110011000111110101111110100110011011011000001110111011111010011011011101111001111000000000000001100011010101 1011100000000110110000000111000011001110110000000000010000110100100000011000000001101100010100001100000001010 001111000110000111001100010010001001001000000010100000000001000000001001000000001000000000000000001000010000 00000010110110111000000001001100110110001110100111111100111011101111011001101111011100111111110011001101110011 0100110110000110111101100001100011000011000101010001101101100011110101110110110011001000010111000100010000010 010000110011000010100100011110011001110001100001100000110000000000001000000010000110000100000000001100000011 001100000000100010000000000100000001000010000101000000

TU RE 10 2 00001011010000000000101111101111100111100000011110100011100110011000111100011001111001100110011100011101100011 1011000101010111011101100110110011000111000010110000100101000101100000110000111001001011001001100001011001111 1111000011110011010001011110110110110000000000000011011000000011000000000010000000001000000000100001000110000 010000000100000000000000000000000000110000100000000000011000000000000000100000010000000010000000001000100000 00001001111100100111111101001101111100000011111001100111010001110011011100101000011001100011000000001101100001 1001100001100101011001100000001101000110000000100000100111000110110110011101000000000001100110110010001100110 216

000000000011000000000011000000000100010011000000000000001000000000100110000000100000010000000000000000000001 000001010000001000000000000000000000010000000000000000000000000000001100101000000000000110011110110101011011 01111001100011111000111111010011111101100100000111011101111100101101100110111100111100000000000000110000101010 1101110000000011011000000011100001100111011000000100001000110011010000001100000000100110001010000110000000011 000110100011000011100110001001000100100100000010010010000001000000000101100000000100000000000000001000001000 00000001010011011000000000110110011011001111010011111110010001110111101100111111101110011111111001100110111001 1001010110000010111110010100110001100001100010101000110110110001011010110110011001100100011011100010001000001 001000011001100001010010001101000100011001010000110000011000000000000100000001000011000010000000000110000001 0001100000000110010000000000100000001000010000110000000

TU RE 12 1 0100110110010100000011101110110101011000000001101000000001000100110011110001001111100111101000111001101101101 10111100011000111011001100011110000000101001110001100001011100101001100110000101110001111010000001100101111101 0111000000011000011000111110110110000000000000110000011011000000001100000011000000001000000000100001000000000 000000000000000000000000000000000000101000010000000000010000000000000000100000001000000010000000101100010001 01111101101110110111011101100111111100000011110000010111110000111101111010011000011011110001100001001010110001 1000000001100001111000100000000111110000011001000000111101110110100111011011100000110001100000111000000000010 000000000001100000000000001000000000110000000000000000011000000001110011000110000000000000000100000011000000 000000100000000000000000000000000000010000000000000000000000000000000101000001001000000110001011111101110110 01101001100011111000111010101011101101101101000110111110111101001101101110110110111100000110001100111100000110 000011000000001101100011001110000000011101100000010000100000011000000001000000000000000001111000000000000000 100011001001100001100001000101100000010000001001000000000000100000000010010000000011000000000000000100000000 00000000001001101000000000000011000111111101001101100111100100111011110110101011110110011111001101010110100100 1101111011000001011100000000001100110000110001011101011011011001101001011111001100110110001101011101001100000 100100000000010000011010000110000000000010010100000000001100000001000010001101000001100000000100000001000000 11100100000000100000000100000100000001010000000100000000

TU RE 12 2 00010011001000000001100011101111100110100110011110100010000010011000111100011001011001101110010110011011101011 1111000110000101011001100100110011000111011000001000010111000110011000110001011000001011000000001101011001001 111100000011000011000111110011010000000000000011000001101100000000110000001000000000100000000010000100000000 00000000000000000000000000000000000001010000100000000000100000000000000001000000100000000100000000010001000 00000011011011100001111111011011101111000001101100011001111000011110110111100110000110111000110000011011011100 0110011000101000011000011000000011010000000110110000000011111011010001111111110000011000011000001110000000001 100000000000010000000000000010000000011000000000000000001100000000011001100000100000000000000011000000010000 000000001000000000000000000000000000000100000000000000000000000000010011000010001010000001100111101111011101 10011100011000111110001111111010110011010011010001101111101111010011011011101101101111000001100011001111000001 100000110000000011011000110011100000000111011000000100000000000110000000010000000000000000011110000000000000 001000110010011000011000010001011000000100000000010000000000001000000000100100000000110000000000000001000000 00000000000010011011100000000100110001110010111011111010111001001110110101101011111101100011110001010101110000 0011011110110000010111000001000011001100001110010111010110110110011010010111101011000101100011010111010011000 001001000000000100000010100001100000000000110010000000000011000000010000110001000000001000000001000000010000 0011000100000000100000000000000100000001001000000100000000

TU RE 16 1 0000101010111010001111000100010110010010001001111010000110010101100001110001000111101111111001110000100100100 100010000100100000110001000100100000101001000100111001000010001100010001000000100000010010001000000000010001 000010000001010000010000100010000100000000000000100010010000001100010000100000000000010001001100000001000000 000000000001110100000000000001100000000000101100000000000100100000000000100000000000010100010000000001010100 0110111111110110110011110101100110010110000001001000001011111000011100100100010100100100110001000010000010010 010110011000100000110011000100100010001000010010001000000010010001001100100110010000001000001000000100100001 100100000100100001000000110010000111011000001000000000000000100000010001000100101000001000000000000000000000 000000000000100000000000000000000000000000000000000000000000001100000000011000000000010000000100101111111001 10011011100011100111110001111110110110011011011000001101110111111010011010011101100101111000001101001101111100 1111001001100000010110110001100111000000111010110000001100110000110100000000011000000000000100001100000000000 000110001111000110010000000101100110000011001000000000000000000010000000001001000010000000000000000000010000 00100000000000100111000000000011001100111110011011100110111110110011111111011111011011111000111100110101011100 00001101101011001100111100011010001100110110100000111100011011011011010011011001001101110010000101111101101100 010110100000000110000000110001110100000001100001000111000110100000011000010000000000000101001000000000001000 00011000000010001100100000100000100000001001000000100000000

TU RE 16 2 00001011001000000000101011100110100111100000011110010001000011011000111100011001111101111110011010111011100011 101100011011000101110110001011000001010110100001100010001100010000100010000001000000101100000000000101100110 101110000111100001100011000100001101100000000001100110110000000001100001000000000000100000101000000010000000 00000000000000000000000000000000000000001000010000000000100010000000000000000000000010000010000000001000010 00000111101101100000111111001101110101100000010110000000111110000011011001101110100011001100011000000001100100 1011001100110100011000000110110001101100000011000100000001011011100000110101111100001100001100000110011000010 110000000011000100000000001100000000100001010000000000000010000011000100110111010000100000000000000000000000 000000001010000000000000000000000000000000000000000000000000000000000101101100000001000000010011110110100110 217

01101101001100011111000111111011011001101101100000110111011111101001101000110110000111100000110100110111110011 110100011000000011101100011001110000000110101100000011000010010101001000000110000000000001000011000000000000 010100011010001100000100001011001100000110010000000000000000000100000000010010000100000000000000000000000000 01000000000001001101110000000110011001111000111101011101111011100011111110111111110110110001111011100110111000 00011011010110001001111000111010001001101101100010111000110110110110100010100110011011101100011011111011011000 101001000000001100001001100001111000000010100010000100001101000000010000100000000000001000010000000000010000 0001000000010000100100000010000100000001001000000100000000

TU RE 17 1 0000000110100000000010001110111110011110000001101001000110101011100011110001000110100110011001111000111010101 1010111011000000001110110001011000001001101110000000010011000011100000011000001100100110100000000111000110110 101110000011100001100001011100001100000000000001101100110110000000001000000001000000010000100001000010000000 000000000000000000000001000000000010000110001000000000000100000000000000100100000000100000100000000010000100 01001111011011100001111110010011011111000000011100011001110000000110110111101010000010011000110000000011011100 0110011000010001011001011000000011011000100110000000000110110011000101100111110000000000011000100111010001100 100000000000110000000000000000000000000110000000000000000110000000001001100010101001000000000000000000000000 010000100100000010000000000000000000000100000000001000000000000000000011000001001011000000100110101011010111 00011110011000111110001111111110110011011011000101101100111111010011011011101100101111010110000000001100010000 1011001100001000110110000011111000001011110110000000000001000111110000000001000000000000110001100000000000000 011101101000111000000000100000110000001001000000100000000000010000000001001000000000000000000000000000000000 00000000010100110100000000000001100111110011010110110111100010011101111111011101011111000111011110100011101000 0110010101101000101111101101000000011000011100101000101101101100011001101100100000101011000110101110100110000 010010000010001000000011000001010011000101100110000000001110000000010001000000000000010000000000000000000000 010000000000000100000000000000100000001001000000100000000

TU RE 17 2 0000001110000000000010001110111110010110001001101010000100001101100011110001100110100111011001011001111011001 1010111011000000101110110001011000001001001110000100010011001011000000011000001100101101100001000111000110110 101110000011100000100011011100001100000000000001100100110110000000011000000001000000010000100001000010000000 000000000000000000000001000000000010000110000100000000000100000000000000101000000000100001000000000010100100 01000111111011100001111110110011111011000000101100011001110000000110110111101101000010011000010011000011011100 0110011001011011110101011000000011011000100110000000000110110011000101100111101000000000011000100111010001101 100000100000110000000000000000000000001110000000000000000110000000000101100111101001000000000000000000000000 010000100100000010000000000000000000000100000000001000000000000000000011000100000011100000100111101011010111 00011101011000111110001110111110110011011011000001101110111111001011011001101100101111011100000000001100010000 1011001100001000110110000101111000000111010110000000100010001011110000000001000000000000110001100000000000000 011101101000110000000000100000110000001001000000000000000000010000000001001000000000000000000000000000000000 00000000000100110101000000000101100111100011010111110111110010011111111111010111011111001111011110010011101000 0100010101101000011111101100100000011010111000101000101101110100101001101101100000101011000110101010100110000 010010000010001000000001000001110011000101100110000000010110000000010001000000000000000000000100000000000000 010000000000000100000000000000100000001001000000100000000

TU RE 20 1 00001010000000000001100111111111100110100000011110000011101100011000111100011001100001100110011110111110101011 0011010110000000011101100001110011000111000100010000100001000001001000110000011000001101010000000001001111001 111100001101100000110101001010000000010000000011011001100000001100110000001000001000100000000010000100000000 000000000100000000000000000000000100010000010000000000001001000000000100000000110000000000000000000100000000 00001110111111011011111110110110111110000001101000110011111000111101101111011011000000110001100100000110011000 1100110000110010101000101000000000110000100000000000000101100010001011001100110000000000110011011110100011011 000000000001010000000001100000000001101001000000000000001100001100001010001100010000000001000010000000100000 000000001000000000000000000000000000001000000000000000000000000000110110010100000100011001001111111010101101 10111100110001111100011111111101101110000011001011011001111110010110110110011001111110011000000000011011110000 011001100001001110110001100111000000011000110000100100000001100111000000000100000000000100100000000100000000 010001101000110000100000100000010000001001000000000000000000010000000001001000000000000000000100000000000000 00000000000100110100000000011101101011100011110110111011101110011101111111010111011111001111101110111011011100 0100101101100000110111101100010110011000011000011110101101101100101001101100100110101001000110111000100010000 011010100110011000011100000001000110000010100100000001100110000000000000000010000000110000000000000000100000 011000000000000000000000000000100000001001000000000000000

TU RE 20 2 00000010000000001001100111111111100110100000011111000110100111011000111100011001100001100110011100111110100110 0011000110000001011101100001110011000111000010000000010011000111011000100000011000001101000100000001001111001 111100001111000001100101001010000000110000000011011001100000001100110000001000001000100000000010000100000000 000000000000000000010000000000000100010000010000000000010001000000000100000000110000000000000000000100000000 00000100111111110111111101100111111100000011001000110011111000101101101110011100000000110001000100000110110000 1100110000110010101000100000000000110000000000000000001101100110011011001100100000000000110011011111000011011 000000100011010000000001000000000001100000000000000000001100001100010010001100110000000010000010000000100000 000000001000000000000000000000000000001000000000000000000000000000010110010100010100011001001111011010101101 10111000110001111100011111111101100110000110101011011001111110100110110110011001111110000000000000011011110000 010101100001001110110001100111000000011000110001100100000000110101100000100100000000001101100000000100000000 218

010101101000110000100001100010110000001011000000000000000000010000000001001000000000000000000100000000000001 000000000101001101000000000110011010111100111101101110011011100111111111110110110111110011111111101110100111000 1001111011000001001111000000001100110000110000111101011011011001110011011001101101010110001101110011000100000 101101000100110000101000000010001100000011001000001011001100000000000000000100000001100000000000000001000100 11000000000000000100000000000100000001001000100000000000

TU UN 3 1 01000111000000010100011111101111010111100000011101110001100101111000111100010001111000110110010110011011001111 00011101101101110110011001101110101101110110100010011100110011110000001000001110000011110001010000110011110011 111000011110010110001110010000110000000000000000010000011001000100001100011000000001010000001100001000000000 00000000000000000000000000010000000010100001000000000101000000000000000010100001001000001000000010010001000 00000100110010011011111110110011111110000011011100010011111000011100001100001010001000111001101100110110011000 0000110000110110011000110000000110000000000001100000000001101000010001101111100000000000110011011011000011001 000001000001100011000000110000000001101011000000000000001100001100010011001011011000000000000000000000000001 100000111000000000000000000000000000001000000000000000000000000000010110010001000100011001001111011010001011 11010100111001101101011111110001100111100010001011101111011110100110110110011101011110001000000011000011000011 0110011000111001101100011001110000110011101100000111000100010011010000000101100000000011000011100000000000010 100011110001100000000011001001100000010110000000001001000000100000000010010000100011000000000000000010001100 00000000001001101010000000110011001111100011101111101111101100111000111110111100110110010111111101100111010001 11011110110000001111110101100110001101101100011110110110110111011010010111010011000101100011011100010101100001 011100011001010010010000011101001100010000110000011100100000000000000100000000000011010010011000000010000101 1001100000001000110000100000100000001001000001100010000

TU UN 3 2 01001101000000010100011111101110110111100000011101110001100101111000111100011001111000110110010110011011001111 00011101101101110110011001101110001100100100100010011110110011111000001100001110000011110000010000111011110111 111000111110001010001110010000110000000000000001010001011001100100000100011000000001010000001100001000000000 00000000000000000000000000000000000010100001000000000001000000000000000010000000101000001000001010010001000 00001101110010011011111110110011111110000001011100010011111000111100001110001010000000110001101100110110011000 0001110001100110111000110000000010000011000001100000000111110000111001111011100000000000110001011001000011011 000001000000110011000000110000000001011011000000000000001100001100010011001001011000000000000000000000000001 100000111000000000000000000000000000001000000000000000000000000000010110010000000100011111001111011010001010 11010100111001111101011111110101100110100110001011101111011110100110110110011101011110010100000011000011000011 0110011000110011101100011001110000110011101100000011000100010011010000000101000000000011000011100000000000001 100011110001100000000011001001100000010010000000011000000001100000000110010000100011000000000000000010000010 00000000001000001000000010110011001101000111101110101111101100111110111110111011110110010111011101010111010001 11011110110000010111110101100110101101101110111100110110110111001010110111110011001101100011011100010111000001 011000011001010010110000001101011000110000110000011101100000000000001100000000000011000010011000000010001001 1000100000000110100000100000100000001001000000100010000

TU UN 4 1 01000101001000000000010010101111100111100000111110110000101101011000111101010011011000011110011011011011001011 10111001101111101111011000111100001101011110000110001110110001100110011000010100010111110000010000110110110110 1100000001100101100011111100001100000000000001100110000110010111100000000010000000110000000000000010000000000 000000000000000000001000000000000001010000100010000000110010000000000000100001000000000000000010001000100000 00010011011011001111110011010111111000000111110011001110010011010011110000011100110011000110000110011011100011 0011000011011011110111000011001011000110000111100000110110011000001011111010000000000011000001101000001000100 000000110110000000000000000000000111001000011000000000100000011001001100111100000100000000000000001100000110 000011100000010000000000000000000000010000000000001000000000000000000000000010011000001100011111111011000110 10010011100111110001111111010110011110011000001101111011111010011101110001110000011000000000011001100111001100 1001100000000110110001100111000011001010110000001000011000001101000001110110000000001100001100000000000000111 101101000110000110001100100110000001001000000100100100000001000000011001000000001100000000000000000000010000 000000000001100000000000111011000111110001111101101111101100111111111110101101111110011111011100100111011000110 1011011000110101100110110100010110111100001010011011011011100101001011111001100110010001101110001001100000101 100010000110000101001100110100110011000000100000101110000000000000010001100000000010001000000000001000100110 00100000000000000000100000100000001001000000100010010

TU UN 4 2 01000000000000000000010011101111010111100000111011110001101001010100111101000001111000111110001110001011001111 0110110010111110111011100011110000000001101000001001100011000101011100010000011001001111000000000010001101111 011100000001100001100110111000011000000000000011000110001100110110100000000100000000100000000000000100000000 000000000000000000000010000000000000000100001000100000001100000000000000001000010000000000000000010010001000 00000100110110111111111110110011111110000001111100110011100110110100101100000111001100110000100000000011011000 1100110000110110111001011000100000110001100001111000000110101011010001001110110000000000110000001011000010011 000000001000100000000000000000000000011000000110000100001100000110011001001101000010000000000000000011000001 100000111000000100000000000000000000001000000000000010000000000000000110000000000100000011001111010110110011 10100100111001111100011101110101100111100110000011011111011110100110111100011100000110000000000110011001000011 0000011000000011101000011001110000110110101100000010000110000011010000011001100000000011000011000000000000001 111011010001100011000011001001100000010100000000001000000000100000000010010000000010000000000000000000000010 00000000101000001000000000110011011111100110101110101111010100111110110110111101110110011101001100100111011000 219

11011110110101101111001101100000011101111000110110110110110111001110110111011011001100100011011100010011000011 011000110001100001100001001100001100110000001000001011100000100000001100011000000010000010000000000010000101 1000000000000000000000100000100000001010000000100010100

TU UN 14 1 0010011100001010001001001011110110011010000011101011000100110100100011110001001111100000011001011101101011001 1101101011010011101100110000011010011011000110000010011001101011001010010000111100000101100000000000010001101 1011100001111001011000111111000011000000000000000000001101100001110000000001100000010110000000000001100000000 00000000000000000000000000000100010001010000100000000000100100000000001000010000000000000000000100001000100 01000011011011011101111010011001111111011000110100000001110001000011100110000100100110011100110000011000001100 0110011000100011011110011000000000011100000000111100000011111010100001011101110000000000011000100011100000001 100000000110110000000000000000000000000100100011000000001100000100011001100011000000100000000011000001100000 000000011100000000000000000000000000000010000000000000000000000000000000000000000100000001101011111111001110 11100110011100110110101111111010111011010011100111100110111111010011000011101111110011000000000110000001100111 1001001100000010110110001100111000011001110000000111000010000001111000011001010000000000100011100000000000001 010001111000110000000001100010110000000011000000101100000000001000000011001000010000000000000000000100000000 00100000000100110000000000011001100011111010111110110111110010011111111111001110111011000111101110100011100000 0110111101100000101101100001000110011000011000101010101101101110001011101101100110011001000110111010110010000 010110000110011000110000000011010110000100000100000111001010000000001011000000100000110000100000000000100000 011101100000000010100000100000100000001001000000100010000

TU UN 14 2 01001111001110100111110011111101100111100000111101110010101101011000111100010011111000010110011011011011100011 11010001101001110111011001001110001101101111000011010110111001110110011100010110000111110000010000001000110110 1110000110100001100011111100001100000000000000000010110110010111000000000110000001011000000000000010000000000 000000000000000000000000000100010001001000100000000000100100000000001000010000000000000000000000001001110010 11011011011001101111110011001101111011000111100000001111011000111000110000110100110011000110010011000001100011 0011001101011011111011000011000011001100000111100000010110111000000010111110000000000011001100011010000000100 000000110101000000000000000000000000100110011000000001100000100011001100010000001000000000011000001100000000 000011100000000000000000000000000000100000000000000000000000000000000000000000100000001101101111111001111010 101100111001111101011111111101100110110111001111011101111110100110000111011111100110000000001100000011001111001 0011000000011101100011001110000110011100000001100000010000011110000110110000000000001000111000000000000001100 011110001100001100011000001100000000110000001011000000000100000000110010000100000000000000000001000000010000 000000001011000000000001100110001111001011111011011111011001111111111100011111101100111111011001001110001001101 11101100000101101100001000110011000011000101001101101101110001111101100110110011011000110111010110110000110110 0001100110001100000001111101100001000001000001100110100000000010110000001000001100011000000000110000000111011 00000000010100000100000100000011001000000100010000

TU UN 15 1 01001101011001100100111111100011100111100000111110111000101101011000111100011001111001110110010110011011010011 10110101101011100111010100011100000001101001100110000110110111100110101100010110000011110000000000010111110111 1110000010100000100011111100001100000000000000010000110110000111000011000010110000011000000001000010000000000 00000000000000000000100000000000000100100010001000001001010000000000100001000000000000000000000000100010000 00001001101110110111111101100101111100101110111000110111010001011100001100001100000001010110000001101101110000 0001100010101101011001100000000001100010000001100000001111000100000011011111000000000001100110110101000000010 000000000011000110000000000000000001011100000100000000011010001000100110010100000000000000000100000110000001 0000001100000010000000000000000000000100000000000000000000000000000011001000000010000001111111111011010111011 000100111001111100011111111101100111100110001011111110111110100110110110011111001110001000000000000011011111001 0011000000011101100011001110000110111101100000011000100011011010000000001100000000011000011000000000000010100 011000001100001000001000101100000010010000001001000000000100000000010010000010010000000000000000000000000000 00000010000001000000000110011001111100111101111101111101100111100111110101101110111010111011100000111000001110 11110110100000111111100101000001101101100110100000110110111001010010111110011001101100111011100010011000001011 000001001100001000000011101001000010000011000000000100000000000000100001010000011000010000000000000000001110 1000000000100000001000000100000001010000000100010000

TU UN 15 2 01000111001010100100111111100001100111100000111110111000110101011000111100011001111001110110010110011011011011 10110101101011110111101100011100000001110011100100000010110101100110101100010110000011110000000000010111110111 1110000011100000100011111100001100000000000000010000110110000110100001000101110000011000000001000010000000000 00000000000000000000100000000000000101000010001000001001010000000000100001000000000000000000000100100010000 00001001101110110111111101101110111100110110011000000111010001011100001100001110000001010110000001101101110000 1001100010101101011001100000100001100110000001100000001011101100010101011111100000000001100110110101000000110 000000000011000110000000000000000000111100000100000000010010001000100110010100000000000000001100000110000001 000000110000001000000000000000000000010000000000000000000000000000001100100000000000000111111111101100011011 010010011100111110001111111100110011010011000101111111011111101011011011001111100111001100000000000001111111101 1001100000001110110001100111000011011110110000001100010000110101000000000110000000001100001100000000000000110 001101000110000100001100010110000001011000000101000100000010000000011001000001001000000000000000000000010000 00000000100000100000000011001100111110011110110110111110110011110011111010111111011101011101110011011100000111 01111011010000011111110010100010110110110011010000011011011100101001011011101110110110001101110001001100000101 100001100110000100000101110100110001000001100000000010000000000000110000001000001100001000000000000000000111 220

01000000001000000001000000100000001001000001100010000

TU UN 16 1 01001111001010000000110011101011101111100001111011111000001101001100111100000001111001100110001110011011011011 0111010010000111011011001000110100000111000110001000001011001110011101010000001100011011000000000000110111101 0111000001111000011001111011000110000000000000000000011011000101110000010011000000000110000000100001000000000 00000000000000000000000000000000000010010001000000000001010000000000001000000000000000000000000000010001000 00100100111110011011111100110011111110000101111000000011100100011011110111000110001100110000100000110011011000 1100000001010000011101110000000000111000000110011000001001101011011011001111100000000011110001011001100000011 000000110000100000000000000000000000011001100010001000001100001100011001000111000000000000000110000001000001 100000010000000000000100000000000000001000000000000000000000000000000010100000100110000001100111111110011101 11011100111001111100011111101101100110110110000011101101111110100111010111011011100110000000000110000111001111 0010011000110101101100000111110000110110100000000000100100011011000000000000000000000011000011000000000000001 100001000001100000100001000001100000000011000001011000000000110000000010010000000000000000000000001000000010 00000000001001100000000000110011000111100101111101101111011100111111110110101011110110011111001100110111010000 1100111011010001011000110000001001110000111001010011011011011000100001011011001100110010001101010101100100000 100100000100001000100000001110100110001000001000001011010100000000000010000100000000100001000000000001000000 11001100000000010000000000000100000001001000000110010000

TU UN 16 2 01001111001010000000110011100011100111100001111110111001101101011000111100011001111011100110011010111011011011 1011010010000111011110110000111000110111001011111000001011011110011001010001001000011011000000000010101111001 011100000111000110100111101100011000000000000000000001101100101100000010001000000000110000000010000100000000 00000000000000000000000000000000000001001000100000000000100100000000000100010000000000000000000000001000100 01010111011011001101111110011001111111000010111100001001111100011001111111100101000110011000110000011011001100 0110010000011001101110111000000000011100000011001100001110110011001101100101110000000001011000101100110000001 100000011000110000000000000000000000001100100001000000000100000100011001100011100000000000000001000000101000 110000001000000000000010000000000000000100000000000000000000000000011011000000000011000001100111101101001110 10101110011100111110001111111010110011010011000001111011011111010011100001001100110011000000000011000011100111 1001001100011001110110000011111000011011000000000000010010001101100000000011000000000001100001100000000000000 110000100000110000010000100000110000000001100000101100000000011000000001001000000000000000000000000000000001 00000000000100110100000000011001100011100001110110110111110110101101111011001110111011001111100110101101110000 0110011101101000101100011000000000011000011100101001101101101100001000101100110110001001000010111010110010000 110010000000000100010000000111010011000100000110000011001010000000000001000010000000010000100000000000100000 011001000000000000000000000000100000001001000000100010000

TU UN 17 1 01001111001001010100101011111101110111100000111101111000001010011010111000010001111000011110001110011011011011 001100001000011001100110000001000001010001101001000011001100001000100011000000100000100100000000000000110100 000100000010100000010001000100001000000000000001100000000100000000000000000010000000001000000000000010000000 000000000000000000000001000000100010000010001000000000000001000000000000000000001000000000000000010001000110 00110111011011101101110110011001111111011010111100001101110000001011111010000011000000011100101000000011001100 001001100001000001011001100000000001100000000000000000001101000110000011010011000000000001100010110011000000 110000000011001000000000000000000000000100010000100000000010000001000100100001000000100000000000000000010000 000000000010000100000001000000000000000010000000000000000000000000000000101000000001100000111001011111101011 01110111001110011111000111111011011001101001100010110110010011101001101100110110110111100000000000000000000000 1101100110001100111011000110011100000001100011000000000001000110100000000100000000000000011000110000000000000 011000110100011000011000110001011000000100100000000010000000001000000000100100001000000000000000000000000000 10000000000010010010000000001110110001111001011101011111111101000110111111101110101001101111111011001101110000 0010011110111000010111110000010011001101001100010111010110111010001010010110010011001101100011011100010011000 001010001100001010000010001001100001100110000010000000000101000000010001100001000000000000010001000000000101 0011000101000001100000000000000100000001001000000100000100

TU UN 17 2 01001111000001000100111011111101100111100000111110111000001101011010111100010001111000011110011010011011011011 0011000110001110011001100000110000010110011100000001010011100100111000110001011001011011000000000011101111001 011100001111001001000111001100011000000000000011000000101100000000000000000101100000110000000000000100000000 000000000000000000000010000001000100000100001000000000000110000000000000000000010000000000000000100010001000 00101110110111011011101100110110111110110110111100011011111000010110101100001011000001110011110000000110111000 0110110001110001101101100000000000110000000000000000000101100010011010101111010000000000110011011010100000011 000000101101100000000000000000000000011001100010000000001100001100110011000110000001000000000000000001000000 000000011000010000000100000000000000001000000000000000000000000000000010010000000100000011000101111110101101 10011100111001111100011111101101100110110110001011011010011110010110110011011011011110000000000000000000000011 0010011000110011101100011001110000000110001100000000000010001111000000010000000000000001000011000000000000001 100011000001100001000001000001100000010110000000000000000000100000000110010000100000000000000000000000000010 00000000001101000000000000111011000111100101110001111011111100111001111110111011100110111111101101000111000000 11011110110100010111110000110011011100001110001111000110110110001110110110011011001101100001011100010011000001 010001100001010000000000001100001100110000011000000001011000000000001100000000000000000010011000000000101001 1000100000001000000000000000100000000001000001100000100

221

TU UN 19 1 010011011010110101110111111011111101111001101110111100000111110011011000110011110010111011001101101101100110110 00110001000011001100110011011100001011100111001100001001100111110011011010111100110111100000100000111110100101 1100001101000111000011111000011000000000000011000000101101101100000000001000000000110000000010000110000000001 000000001000000000010000000000100010010001000000000000110000000000000001000000000000000000000001110001101000 10100110011111011100100110111111111000101111000101111000100111011011000011000000001110001110000011110111000110 0110001110001111110110000000010110011000000110000000100100110011001001111000000000000110000011111100011011000 000000000110000000000000000000000011000100110000000001101000100110011000011010000000000000000000001000000100 0001110000100000000000000000000000010000000000000000000000000000001100111100001100000111111110110101011011101 10001100011111010111111011011001101101100110110011011011101001101001110110000111100000000000110000110000010110 011000000011101100010111110001000110001100000000100100001101000000010100000000000001100011000000000000001100 011010001100001100011001001100000000010000000000000000000100000000010010000000010000000000000000010000100000 000000010011010000000001100110011110001111011011011110011001111111101101011011111111111110011101111101011001101 1000110000010111000001100000001101101100010100000110110110001110110110011011000100000010111101010011000011011 011100000010000000000000011001000010000011000000000011000000010000100011000010011000010000000000010000001110 1100000000100000000100000100000100001000000100010000

TU UN 19 2 01001111001011010111011111111111100111100101011101111001101001110100111100010001111010001110011010111011010011 00011001100001100111011001101110000101110011100100000100110001110001101100011110011011110000010000011111110010 111000011010001010001111010000110000000000000110000000011011011000000000001000000001100000000100001100000000 000000000010000000000100000000001000100100010000000000010100000000000000010000000000000000000001011100011000 00000001101101100111001001101011111101100011111010000111110001011101101100010100000001100011101000111101110001 1001100011000011011101100000000001100101000001100000001111000110110011011110000000000001100000111011000110110 000000000001100000000000000000000000110001001100000000011000001000100110001110100000000000000000000010000001 0000011100011000000000000000000000000100000000000000000000000000000011001100000010000001110111111101010111011 01100011100111110101111101110110011011011000101100110100111001011010011101100101111000000000001100001100001100 1001100000001110110001011111000000011000110000000010010000111000000011010000000000000110001100000000000000110 001111000110000110001100010110000000011000000000000000000010000000001001000010001000000000000000001000001000 000000001001101100000000110011011111000111101101111111100101011111111111101011111111111111011100100111001100110 1100011000001011100000110000000110110110001011000011011011000111011011001101100011000001011110101001100001101 101110000001000000000000000000100000000001100000000001100000000000010001100000001100001000000000000000000111 01100000000100000000100000100000100010000000100000000

TU UN 20 1 01001001000000010010111111111101101111100000111110111000010011011000111100010001111000111110010110011011011011 0011010110000111011010110010110000000110001100000100010011001111001000110000101000011111000000000000100011011 011100000101101001100011101000011000000000000000000101100000111100000001000100000000110000000000000100000000 00000000000000000000001000000000000000010000100000000000100100000000001000010000000000000000000001001000100 000110110111111011011111110110110111110110001111100011011111000111101101100001011001100110011100100110110111000 1100110000100111111110100000000000110001000000011000000110100011011000101111100000000000110011011001100001011 000000001100110000000000000000000000111001100000000000001100000100010011000100000010000000000110000011000001 100000011000000000000100000000000000000000000000000000000000000000100110000100010100000101111111010110111010 00011100110001111100011101101101110110100110100011011110111110100110100111011101100110000000001100000011000011 0110011000110011101100001001110000110110111100000000000100001101010000000001000000000001000011000000000000001 100011000001100000100001000100100000000010000000000000000000010000000010010000000000000000000000000000000000 00000000001001101000000000000011011101000111101101101011101100111111111110101101110111111011101101010111000000 1001111011000000111101111110000001110001011001011101011011011100001011011001101000010010000101010001001100000 100100000000101001100000000110000000001000001000001100011100000001000010000100000001100001000000000001000000 10000000000000000000000000000100000001000000000100000000

TU UN 20 2 01001101100110000000111111111101101110100000111101111000001101011000111100010011111000011110010110011011011011 0011010110000111011010101010110000000110000100000000010011001111011000110001011000011111000000000001100011011 011100000101000111000001101000011000000000000000000101100000111100000010000100000000110000000000000100000000 000000000000000000000010000000000000000100001000000000001010000000000010000100000000000000000000100010001110 101011101111110100111111101100111111101100011111001010111110000111011011000010100011001110110001001101101110001 1101100010001111000100100000000001110110000100110000001011000100110011011110000000000001100110110111000010110 000000011011000000000000000000010011010011000100000000011000001000110110001000001100000000001100000110000011 000000110000000000001000000000000000000000000000000000000000000000001100000000001000010111111110101101110100 00101001100011111010111010111011101101101110000110111101111101001101001100110011001100000000011000010110000110 0000110001100111011000110011100001100101011000001000001000010110000000110010000000000110000110000000000000011 010110100011000011000110001011000000101100000000010000000001000000000100100001000000000000000000000100000000 00000000010011010000000100000110111010011111011101010110111001111111111100111111101111110110111010101110010001 1011110110000001111000000100000011100001110010111000110110111001010110110010010001100100001011100010011000011 101100000001010011000000001100000000010000011000011000011000000000001100001000000011000010000000000010000001 1001010000001000000000100000100000001000000000000000000

(End of data)

222

VITA

Young Jin Chun Department of Ecology, Evolution, and Organismal Biology Phone: (515) 450-4270 Iowa State University FAX: (515) 294-1337 Ames, IA 50011 e-mail: [email protected]

Education

Ph. D. 2007 – Dept. of Ecology, Evolution, and Organismal Biology, Iowa State University Advisors: John D. Nason and Kirk A. Moloney; Dissertation: The role of adaptive evolution of phenotypic plasticity and historical population genetic processes in purple loosestrife (Lythrum salicaria L.) invasion in North America M. S. 2002 – School of Biological Sciences, Seoul National University Advisor: Eun Ju Lee; Thesis: Population structure and dynamics of Ageratina altissima (white snakeroot). B. S. 1996 – Dept. of Biology, Seoul National University Advisor: Jae-Chun Choe; Thesis: Oviposition and Branch-cutting Behavior of Mechoris ursulus Roelofs (Coleoptera: Attelabidae).

Experience

Dr. John Nason, Department of Ecology, Evolution, and Organismal Biology. 05/2004 – 05/2007. Teaching Assistant in Evolution lecture class and Ecology laboratory. Dr. John Nason, Department of Botany. Iowa State University. 08/2003 – 05/2004. Research Assistant as a departmental IT person. Dr. Eun Ju Lee, School of Biological sciences, Seoul National University, Korea. 03/2001 – 08/2001. Teaching Assistant in General Biology Laboratory. Dr. Eun Ju Lee, School of Biological sciences, Seoul National University, Korea. 05/2000 – 11/2000. Research Assistant in a project entitled “A study on the environmental and biological characteristics and management plans of reclaimed wetlands” supported by Korea Agricultural & Rural Infrastructure Corporation (KARICO).

Publications

Chun, Y. J., M. L. Collyer, K. A. Moloney, and J. D. Nason. 2007. A novel multivariate analysis of phenotypic plasticity applied to the invasive plant purple loosestrife (Lythrum salicaria L., Lythraceae). Ecology 88:1499-1512. Chun, Y. J., J. D. Nason, and K. A. Moloney. In preparation. Are invasive species “made”? : A summary of mechanistic approaches to the study of invasiveness (review paper). Chun, Y. J., and J. D. Nason. In prepatation. A method for extracting genomic DNA from purple loosestrife (Lythrum salicaria L.) containing high levels of secondary metabolites (technical paper).

223

Chun, Y. J., H.-W. Lee, and E. J. Lee. 2001. Allozyme Variation and Population Structure of an Invasive Plant, Ageratina altissima (White Snakeroot) in Seoul. Korean Journal of Biological Sciences 5: 309 – 312. You, Y.-H., Y. J. Chun, C.-S. Lee, and H.-S. Lee. 2001. Distribution of damaged oaks and annual oak biomass removal by oak nut weevil (Mechoris ursulus) in Korea. Korean Journal of Ecology 24(6): 377 – 380.

Presented talks

Chun, Y. J., M. L. Collyer, K. A. Moloney, and J. D. Nason. A novel multivariate analysis of phenotypic plasticity applied to the invasive plant purple loosestrife (Lythrum salicaria L., Lythraceae). 91th Ecological Society of America Annual Meeting. Aug. 10, 2006. Chun, Y. J., and E. J. Lee. Responses of Ageratina altissima (white snakeroot) to variation in light availability. Ecological Society of Korea. Joongang University, Seoul, Korea, Oct. 27, 2001. Chun, Y. J., and E. J. Lee. Distribution and biomass allocation strategy of Ageratina altissima (white snakeroot) in the forest edge and forest interior. 19th International Symposium on Plant Biology. The Catholic University of Korea, July 24, 2001. Chun, Y. J., H.-W. Lee, and E. J. Lee. Allozyme variation and population structure of an invasive plant, Ageratina altissima (white snakeroot) in Seoul. 19th International Symposium on Plant Biology. The Catholic University of Korea, July 24, 2001. Chun, Y. J., and E. J. Lee. A phenological study of Ageratina altissima (white snakeroot). Ecological Society of Korea. Paichai University, Daejeon, Korea, Oct. 28, 2000 (in Korean).

Workshops Attended

2006 – 91st ESA annual meeting: Icons and Upstarts in Ecology, Memphis, TN. 2003 – Ecological Genomics Symposium: Genes in Ecology, Ecology in Genes, Kansas NSF EPSCoR, KS. 2003 – VIGRE Minicourse: The Mathematics behind Biological Invasions, University of Utah, Salt Lake City, UT. 2002 – 49th Annual Systemaics Symposium, Missouri Botanical Garden, St. Louis, MO.

Scholarships and Awards

2005. Scholarship, SNUAA (Seoul National University Alumni Association) Heartland Branch. 2005. Scholarship, KUSCO (Korean-U.S. Science Cooperation Center) / KSEA (Korean- American Scientists and Engineers Association). 2005. Outstanding Student Leadership Award, LINC (Leaders Inspiring Connections), Iowa State University. 2004. Research travel grant, CGRER (Center for Global and Regional Environmental Research), University of Iowa. 224

2002 – 2006. Fellowship, Plant Sciences Institute, Iowa State University. 2001. Scholarship, Seoul National University Foundation. 2001. Outstanding research prize, BK (Brain Korea) 21-Division of Life Science. 1994. Scholarship, Oi-Su Scholarship Foundation. 1994. Scholarship, Seoul National University Foundation. 1992. Scholarship, Han-Saem Scholarship Foundation.

Laboratory Skills

Allozyme extraction and electrophoresis, genomic DNA extraction, restriction endonuclease digestions, Polymerase Chain Reaction (PCR), electrophoresis, Amplified Fragment Length Polymorphism (AFLP), flow cytometry.

Computer Skills

General: network administration, computer system buildup and troubleshooting, web programming and publication, Microsoft office, Adobe photoshop and illustrator Statistical: SAS, JMP, AMOS, NTSYS, Sigmaplot, R, S-plus, PopTools, GGobi, Canoco, PC-ORD Molecular population genetic analysis: Genescan, Popgene, Genographer, Arlequin, Paup, GenAlEx

Social Activity

President of Korean Student Association at Iowa State University. 12/2004 – 08/2005. Chair of web-based club “iastate 2002-2005” for prospective Korean students at ISU. 03/2002 – 05/2005.

Military

ROK (Republic of Korea) Army, First Lieutenant, 10/1997 – 09/1999. ROK (Republic of Korea) Army, Second Lieutenant, 10/1996 – 09/1997. Honorable discharge.

Referees

(1) Dr. John D. Nason (2) Dr. Kirk A. Moloney Associate Professor Associate Professor Department of Ecology, Evolution, Department of Ecology, Evolution, and Organismal Biology and Organismal Biology 341 Bessey Hall 143 Bessey Hall Iowa State University Iowa State University Ames, IA 50011, USA Ames, IA 50011, USA TEL: (1) 515-294-2268 TEL: (1) 515-294-6415 FAX: (1) 515-294-1337 FAX: (1) 515-294-1337 e-mail: [email protected] e-mail: [email protected] 225

(3) Dr. Jonathan Wendel (4) Dr. Eun Ju Lee Professor, Chair of EEOB Assistant Professor Department of Ecology, Evolution, School of Biological Sciences and Organismal Biology Seoul National University 251B Bessey Hall 56-1, Shillim-Dong, Kwanak-Gu Iowa State University 151-742, Seoul, Republic of Korea TEL: (1) 515-294-7172 TEL: (82) 2-880-6673 FAX: (1) 515-294-1337 FAX: (82) 2-872-6881 e-mail: [email protected] e-mail: [email protected]

I hereby declare that the above statements are true and correct in every respect to the best of my knowledge.

Chun, Young Jin