Ecological Factors, Mixed Breeding System and Population

ECOLOGICAL FACTORS, MIXED BREEDING SYSTEM AND POPULATION

GENETIC STRUCTURE IN A SUBTROPICAL AND A TEMPERATE VIOLET

SPECIES

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Aurea C. Cortés-Palomec

June 2005

This dissertation entitled

ECOLOGICAL FACTORS, MIXED BREEDING SYSTEM AND POPULATION

GENETIC STRUCTURE IN A SUBTROPICAL AND A TEMPERATE VIOLET

SPECIES

AUREA C. CORTÉS-PALOMEC

has been approved

for the Department of Biological Sciences

and the College of Arts and Sciences by

Harvey E. Ballard Jr.

Associate Professor of Environmental and Plant Biology

Leslie A. Flemming

Dean, College of Arts and Sciences

CORTÉS-PALOMEC, AUREA, C. Ph.D. June 2005. Biological Sciences

Ecological factors, mixed breeding system and population genetic structure in a subtropical and a temperate violet species (186 pp.)

Director of Dissertation: Harvey E. Ballard, Jr.

A mixed breeding system involving the production of chasmogamous and cleistogamous flowers is common in most species of the genus Viola. This system can theoretically affect the patterns of reproduction and distribution of genetic variation in populations. While we understand the theoretical basis behind these expected patterns, there is little empirical evidence comparing the behavior of similar species in widely contrasting environments. To better characterize the effect that this breeding system has on reproduction and genetics a subtropical (Viola grahamii) and a temperate (V. striata) species of violets were compared. V. striata populations were studied in Ohio,

USA, and V. grahamii in Michoacán, Mexico during 2002 and 2003. Weekly observations during the reproductive season allowed for the establishment of blooming patterns of the two flower types for each species. The allocation of resources towards flowering was evaluated under natural differential light availability and soil nutrients.

The reproductive success of each of the flower types was determined and the genetic diversity in both species assessed.

Un sistema reproductivo que incluye la producción de flores casmógamas y cleistógamas en un mismo individuo es común en la mayoría de las especies del género

Viola. En teoría, el balance en la producción de estos dos tipos florales afecta dramáticamente la estrategia reproductiva de cada especie y tiene serias repercusiones

en su diversidad genética. Para caracterizar este sistema reproductivo, dos especies cercanas de violetas creciendo en ambientes contrastantes fueron escogidas para este estudio: Viola grahamii, una especie subtropical y Viola striata, una especie de climas templados. Las poblaciones de V. grahamii estudiadas se encuentran en el estado de

Michoacán en México mientras que las poblaciones de V. striata se localizan en Ohio en Estados Unidos. Censos semanales durante la época reproductiva permitieron establecer los patrones de floración de cada tipo floral para ambas especies. La cantidad de flores producidas de cada tipo fue relacionada con la cantidad de nutrientes y luz en las cuales los diferentes individuos crecían. El éxito de cada tipo floral en producir semillas fue evaluado y la diversidad genética en las distintas poblaciones fue analizada en relación con el sistema reproductivo.

Approved:

Harvey E. Ballard, Jr.

Associate Professor of Environmental and Plant Biology

Acknowledgments

This work would not have been possible without the assistance of several people. I would like to thank my advisor, Harvey E. Ballard, Jr. for introducing me to the world of violets and for all his help and support during these years. To members of my graduate committee, Kim Brown, Gar Rothwell, Royal Mapes and Kelly Johnson for valuable comments during different stages of the dissertation, in particular to Kim

Brown for her encouragement, support and comments during the development of the ecological projects. I extend a special thanks to Gene Mapes for accepting to serve on my committee in place of Gar Rothwell who was unable to attend my final defense on account of a previous foreign travel commitment. I would also like to express my gratitude to the Department of Environmental and Plant Biology, current and past students for all their assistance and advice through my time in the department.

I would like to say a special thanks to Theresa Culley from the University of

Cincinnati for sharing with me her Viola pubescens microsatellite primers, for her insightful comments about violets and most of all for instructing me in the use of allozyme techniques during two weeks spent working in her lab.

I am indebted to the Consejo Nacional de Ciencia y Tecnologia (CONACyT-

Mexico) for providing me with graduate program funding under the grant No. 128098 for most of the duration of my program, and to the Department of Environmental &

Plant Biology for taking over once my grant was over.

Funding for research was provided by grant awards from the Sigma Xi Society

(Grant-in-aid of Research), The Botanical Society of America (Karling Award), the

Office of Research and Sponsored Programs at Ohio University, and the Wilson Fund from the department of Environmental and Plant Biology. Additional funding in support of field and laboratory work was provided by my advisor Harvey E. Ballard, Jr.

Thanks to Sergio Zamudio from the Instituto de Ecología, A.C. Centro Regional del Bajío, in Pátzcuaro, Michoacán for his logistical support during my time completing field work in Mexico.

Special thanks to my very good friend José Ricardo Gonzalo Wong from the

Universidad Nacional Autónoma de México (UNAM) for his longtime friendship and for helping in the establishment my study sites in Mexico, especially for getting up early with me to go data collecting while being relentlessly pursued by Oso and other unidentified dogs during the summer of 2002.

Thanks to Judith Márquez Guzmán from UNAM for all her support and early encouragement to pursue a doctorate degree and for being my academic mentor over the years.

I would also like to thank my family for their continued support in every task I have started during my life. And finally, but not least, I would like to thank my husband, Ross A. McCauley for his help in establishing my study sites in Ohio, and in data collection during the summer of 2003 in Mexico. But most of all, I want to thank him for his unconditional love, friendship, support, and for believing in me at every moment along the way. vii

Table of Contents

Page

Abstract...... iii

Acknowledgments ...... v

List of Tables...... x

List of Figures ...... xiv

Chapter 1: Introduction...... 1 Theoretical background of a Mixed Breeding System in the genus Viola...... 1 Research site establishment and description ...... 9 Species and Sites of Study...... 9 Viola grahamii Bentham ...... 9 Viola striata Aiton...... 10 Viola grahamii Study Site ...... 11 Viola striata Study Site ...... 20 Quadrat establishment ...... 28

Chapter 2: Environmental correlates to leaf mass area and leaf nitrogen in the Neotropical violet Viola grahamii...... 30 Introduction ...... 30 Materials and Methods ...... 32 Species of Study ...... 32 Study Sites...... 33 Sampling, measurements and data handling ...... 34 Results ...... 37 Discussion...... 46

Chapter 3: Influence of annual fluctuations in nutrient availability on the mixed breeding system of the Neotropical violet species Viola grahamii, and on the chasmogamous flower production in the temperate forest violet Viola striata...... 52 Part A. Viola grahamii ...... 52 Introduction ...... 52 Materials and Methods ...... 56 Species of Study ...... 56 Study Sites...... 56 Sampling of environmental variables...... 57 Reproductive behavior...... 59 viii

Results ...... 61 Environmental factors ...... 61 Reproductive behavior...... 62 Environmental influences on reproductive responses ...... 67 Discussion...... 73 Part B. Viola striata...... 80 Introduction ...... 80 Materials and Methods ...... 80 The study species...... 80 Study Sites...... 81 Sampling strategy and ecological data ...... 81 Reproductive data...... 82 Statistical analysis ...... 82 Results ...... 83 Environmental factors ...... 83 Reproductive behavior...... 84 Environmental influences on reproductive responses ...... 86 Discussion...... 87

Chapter 4: Comparison of flower phenologies in a tropical violet (Viola grahamii) and temperate violet (Viola striata)...... 93 Introduction ...... 93 Materials and Methods ...... 98 Light environment ...... 100 Soil moisture...... 100 Climatic factors ...... 101 Results ...... 102 Climatic patterns...... 102 Phenology...... 106 Pollinators...... 119 Discussion...... 120

Chapter 5: Influence of a mixed breeding system on the population genetic structure of temperate and subtropical Viola (Violaceae)...... 127 Introduction ...... 127 Materials and Methods ...... 135 Study Species...... 135 Tissue sampling...... 136 DNA extraction and ISSR amplifications ...... 136 ISSR fragment visualization...... 138 Statistical Analysis ...... 139 Results ...... 141 Viola grahamii...... 141 ix

Viola striata...... 145 Discussion...... 149

Literature Cited...... 157

List of Tables

Table Page

1.1 Results of a vegetation survey completed at site Quiroga, Michoacán during the summer of 2003. Voucher specimens for all species are deposited in the Bartley Herbarium of Ohio University (BHO) ...... 17

1.2 Results of a vegetation survey completed at site Santa Fe, Michoacán during the summer of 2003. Voucher specimens for all species are deposited in the Bartley Herbarium of Ohio University (BHO) ...... 18

1.3 Results of a vegetation survey completed at Campground site, Ohio during spring, summer and fall of 2002 ...... 23

1.4 Results of a vegetation survey completed at Cemetery site, Ohio during the spring, summer and fall of 2002...... 26

2.1 Summary of soil chemical properties (µg/g soil), soil moisture (%) and canopy openness in the two Viola grahamii study sites in central Mexico for year 2002 (n=20; mean ± s.d.). Means with different letters between columns are significantly different (ANOVA, p<0.05)...... 38

2.2 Total number of plants and leaves per each of the 20 quadrats established in Santa Fe and Quiroga during 2002. Santa Fe shows a significantly higher number of plants per quadrat (p <0.005)...... 39

2.3 Percent of nitrogen and carbon, specific leaf area (SLA), leaf mass per area (LMA), and leaf nitrogen content (LNC) in leaves of flowering individuals of V. grahamii in two sites: Santa Fe and Quiroga. Means and standard deviations are presented. (n=40; mean ± s.d.). Significant differences between groups (p<0.05) are marked with different subscripts ...... 40

2.4 Pearson’s correlations between leaf mass area (LMA) canopy openness and soil chemical properties. *** p <0.005, ** p <0.05...... 45

2.5 Multiple regression models constructed from soil chemical data and light characteristics for Viola grahamii growing under different environmental conditions in two sites in central Mexico. All models were significant (p<0.05) ...... 46

3.1 Summary of soil chemical properties (in µg/g soil), soil moisture (%) and canopy openness in two populations of Viola grahamii (Santa Fe and Quiroga) during the year 2002 and 2003. n=20 except for Quiroga 2003, n=12. Mean ± s.d. Means with different letters between columns are significantly different (ANOVA, p<0.05)...... 63

3.2 Percentage of chasmogamous and cleistogamous flowers of individuals of Viola grahamii that successfully became fruits during 2002 and 2003. Ranges are the result of among quadrat variation. 40 quadrats for year 2002, 32 quadrats for year 2003...... 63

3.3 Number of chasmogamous and cleistogamous flowers and total number of flowers produced per individual per site based on the number of leaves that each individual produced. N=total number of plants; CH=Average number of chasmogamous flowers produced; CL= Average number of cleistogamous flowers produced. F= total average number of flowers produced ...... 65

3.4 Pearson’s correlation coefficients between chasmogamous flowers and fruits and cleistogamous flowers and fruits with soil chemical properties and light. Significant correlations (p < 0.05) are marked with asterisks...69

3.5 Multiple regression models constructed from soil chemical data and light characteristics for Viola grahamii growing under different environmental combinations. Only significant models (p<0.05) are presented. (CH=chasmogamous, CL=cleistogamous)...... 70

3.6 Pearson’s correlation coefficients between soil data from 2002 and flowering in 2003 for Viola grahamii. Significant correlations (p < 0.005) are marked with asterisks ...... 71

3.7 Multiple regression models for chasmogamous flower production in Viola grahamii in 2003 using soil data from the previous year (2002). The models were significant at p < 0.005...... 72

3.8 Summary of soil chemical properties in two populations of Viola striata (Campground and Cemetery) in southern Ohio during the years 2002 and 2003. N = 20, Mean ± s.d. Means with different letters between columns are significantly different (ANOVA, p<0.05). For light, two values are presented, top: average at the beginning of the growing season, bottom: average at the end of the study ...... 84

xii

3.9 Average number of chasmogamous flowers produced per quadrat per site and percentage of flowers of Viola striata that successfully became fruits during 2002 and 2003. Ranges are the result of among quadrat variation. n = 20...... 85

3.10 Pearson’s correlation coefficients between chasmogamous flowers and fruits and cleistogamous flowers with soil properties and light. *** Significant correlations (p < 0.05). CH=chasmogamous, CL=Cleistogamous...... 88

3.11 Multiple regression models constructed from soil chemical data and light characteristics for Viola striata growing under different environmental combinations. Only significant models (p<0.05) are presented. (CH=chasmogamous, CL=cleistogamous)...... 89

4.1 Changes in canopy openness during the flowering season of two years of observations (2002 and 2003) for two populations of Viola grahamii (Santa Fe and Quiroga) and two populations of V. striata (Campground and Cemetery). Mean % ± standard deviation are included. Repeated measures ANOVA was used to check for differences among weeks of observations. P values are included, significant differences are marked with asterisks. Superscripts indicate similar groups identified using Bonferroni multiple comparisons test ...... 105

4.2 Changes in soil moisture during the flowering season of two years of observations (2002 and 2003) for two populations of Viola grahamii (Santa Fe and Quiroga) and two populations of V. striata (Campground and Cemetery). Mean % ± standard deviation are included. Repeated measures ANOVA was used to check for differences among weeks of observations. P values are included, significant differences are marked with asterisks. Superscripts indicate similar groups identified using Bonferroni multiple comparisons test ...... 107

4.3 Correlations among climate and environmental variables and chasmogamy and cleistogamy production for Viola striata during 2002 and 2003. R2 values included. Significant correlations (p<0.05 are marked with **). CH=Chasmogamous flowers, CL=Cleistogamous flowers, DL=Daylength, SM= Soil moisture...... 113

4.4 Correlations among climate and environmental variables and chasmogamy and cleistogamy production for Viola grahamii during 2002 and 2003. R2 values included. Significant correlations (p<0.05 are marked with ***). xiii

CH=Chasmogamous flowers, CL=Cleistogamous flowers. CO=Canopy openness, DL=Daylength, SM= Soil moisture...... 119

5.1 Levels of population genetic diversity inferred from ISSR data for two populations of two violet species: Viola grahamii and Viola striata. Number of individuals in population (N), number of unique ISSR bands in population (Bands), heterozygosity (direct count) (Ho), and percentage of polymorphic loci (0.95 level) (P) are shown...... 142

5.2 Hierarchical Analysis of Molecular Variance (AMOVA) for two populations and four subpopulations of Viola grahamii in Michoacán based on the analysis of ISSR data...... 143

5.3 Hierarchical Analysis of Molecular Variance (AMOVA) for two populations and four subpopulations of Viola striata in Ohio based on the analysis of ISSR data...... 148

xiv

List of Figures

Figure Page

1.1 A. Diagram of Viola grahamii Bentham. A. Mature flowering individual B. Petals. C. Seeds. D. Open capsule. (Reproduced from Ballard, H.E. 1994. Flora del Bajio y de Regiones Adjacentes). B. Mature flowering individual of Viola striata Aiton (Reproduced from Rhodes and Block, 2000. The Plants of Pennsylvania, an illustrated manual.)...... 12

1.2 Location of Viola grahamii research sites in Michoacán, central Mexico. A: Santa Fe, B: Quiroga. Map reproduced from Carta Topográfica 1:50, 000 Pátzcuaro E14 A22, Michoacán. Instituto Nacional de Estadística e Informática, México ...... 15

1.3 Location of Viola striata research sites in Athens, Co, southern Ohio. A: Campground; B: Cemetery. Topographic map insert from USGS Athens Quadrangle 7.5 minute series, 1961/1995. Scale 1: 24,000...... 22

1.4 Map of quadrat locations for Viola striata (Ohio) and Viola grahamii (Michoacán). In Quiroga, those quadrats marked with open circles were lost between year one and two as a result of disturbance from local wood harvesting. All populations were in forested areas, however, regions depicted by trees above were dominated by conifers...... 29

2.1 Correlation between canopy openness and leaf mass area for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and the level of significance of the correlation are presented. N =80 individuals, 40 per site ...... 41

2.2 Correlations between soil nitrogen and leaf mass area for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and level of significance of the correlation are given. N=80 individuals, 40 per site...... 42

2.3 Correlation between canopy openness and leaf mass (mg) and leaf area (cm2) for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and the level of significance of the correlation are presented. N = 80 individuals, 40 per site...... 43

2.4 Correlation between leaf mass per area (LMA) and leaf nitrogen per leaf area for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). xv

Pearson’s correlation coefficient (r) and level of significance of the correlation are provided. N = 80 individuals, 40 per site ...... 44

3.1 Relationship between total number of flowers (chasmogamous and cleistogamous) per plant and number of leaves per plant. Average and standard deviations are shown. (Data shown in Table 3.3). Pearson’s correlation coefficient and level of significance of the correlation is included. Values for seven and eight leaves show a very low standard deviation due to very few (1-2) individuals in this size class...... 66

4.1 Climatic patterns of temperature (°C), rain fall (mm) and photoperiod (Daylength) during 2002. Data is presented on a weekly basis for Athens, Ohio (Viola striata) and Angamacutiro, Michoacán (Viola grahamii). Continuous line = Average Maximum temperature, Dashed line = Average Minimum temperature, Bars = Total precipitation, Black dots = Photoperiod...... 103

4.2 Climatic patterns of temperature (°C), rain fall (mm) and photoperiod (Daylength) during 2003. Data is presented on a weekly basis for Athens, Ohio (Viola striata) and Angamacutiro, Michoacán (Viola grahamii). Continuous line = Average Maximum temperature, Dashed line = Average Minimum temperature, Bars = Total precipitation, Black dots = Photoperiod...... 104

4.3 Relative production of chasmogamous and cleistogamous flowers in Viola striata for two different populations for 2002 and 2003, Campground and Cemetery. Closed squares represent chasmogamous flowers (CH), open squares represent cleistogamous flowers (CL). Means and standard deviations for each week are presented...... 108

4.4 Summary of the blooming phenology of Viola striata in two populations over two years: Campground and Cemetery. Chasmogamous flowers (closed squares), cleistogamous flowers (open squares)...... 109

4.5 Relative production of chasmogamous and cleistogamous flowers in Viola grahamii for individuals growing in two populations: Santa Fe and Quiroga during two years (2002 and 2003).Chasmogamous flowers represented by solid squares (CH), cleistogamous flowers represented by open squares (CL). Means and standard deviations for each week are presented...... 110

4.6 Summary of the blooming phenology of Viola grahamii in two populations over two years: Campground and Cemetery. xvi

Chasmogamous flowers (closed squares), cleistogamous flowers (open squares)...... 111

4.7 Viola striata blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Campground, Right panel Cemetery during the year 2002. (Daylength and rain are the same for both sites) ...... 114

4.8 Viola striata blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Campground, Right panel Cemetery during the year 2003. (Daylength and rain are the same for both sites) ...... 115

4.9 Viola grahamii blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Santa Fe, Right panel Quiroga during the year 2002. (Daylength and rain are the same for both sites)...... 117

4.10 Viola grahamii blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Santa Fe, Right panel Quiroga during the year 2003. (Daylength and rain are the same for both sites)...... 118

5.1 Range of Viola striata and Viola grahamii indicating location of study sites in Ohio and Michoacán ...... 132

5.2 Principal coordinates analysis (PCoA) using the Dice coefficient of similarity computed from ISSR data. A. Viola grahamii, Michoacán, 77 individuals in two distinct populations. B. Viola striata, Ohio, 80 individuals in two distinct populations...... 144

5.3 Partitioning of genetic variation among subgroups in two populations of Viola striata (Ohio: Cemetery and Campground) and Viola grahamii (Michoacán: Quiroga and Santa Fe). Number of plants per group (n), average heterozygosity (Ho) and percent of polymorphic loci (P) are included for each group. All populations were in forested areas, however, regions depicted by trees were dominated by conifers...... 146 1

Chapter 1: Introduction

Theoretical background of a Mixed Breeding System in the genus Viola.

The presence of a mixed breeding system with chasmogamous and cleistogamous flowers produced by the same individual has long-attracted the attention of biologists. Charles Darwin, one of the first biologists to systematically study the occurrence and function of this system of reproduction, identified 55 genera across 25 families of angiosperms exhibiting the phenomenon of cleistogamy in his 1877 treatise

The Different Forms of Flowers on Plants of the Same Species. Today we recognize it is as a phenomenon that occurs across 48 families, 199 genera and 465 species of flowering plants (Klooster and Culley, 2004). The significance of this mixed breeding system has been evaluated in many angiosperm groups by many authors (eg. Holsinger,

1992; Lloyd, 1992; Lloyd and Schoen, 1992; Masuda et al., 2001) and in particular plant groups including the Poaceae (Campbell, 1982; Clay, 1983; Schoen, 1984),

Oxalidaceae (Jasieniuk and Lechowicz, 1987; Rebdo-Torstensson and Berg, 1995),

Asteraceae (Porras and Muñoz, 1999; 2000), Polemoniaceae (Wilken, 1982),

Balsaminaceae (Stewart, 1994), Lamiaceae (Sun, 1999), Fumariaceae (Ruiz de Clavijo and Jimenez, 1993), Marantaceae (Lecorff, 1996) and Violaceae among others. These studies have suggested that the mixed breeding system can have profound consequences on the reproductive behavior and survivorship of populations and on genetic structure of both individuals and populations.

While present in a wide variety of angiosperm groups, the mixed reproductive strategy is not ubiquitous across the taxa of any one group. The strategy has apparently 2 evolved not only many times in separate angiosperms lineages, but also multiple times within families and genera in which it occurs. One of the more well-known instances of a mixed cleistogamy/chasmogamy breeding system occurs within the violet family,

Violaceae Batsch. In this worldwide family of flowering plants, consisting of approximately 25 genera and 825 species (Hekking, 1988; Ballard et al., 1999), only two of the 25 genera, Viola L. and Hybanthus Jacq. exhibit this phenomenon. It is only widespread however, in Viola, the largest genus of the family comprising between 525-

600 species extending throughout temperate regions of the Northern and Southern

Hemispheres and higher elevations of the tropics (Clausen, 1963; Ballard, 1994; Ballard et al., 1999).

Members of most infrageneric lineages of Viola produce two morphologically distinct floral types, the familiar chasmogamous flowers, which open and allow for the transfer of pollen to other flowers, and smaller cleistogamous flowers which never open and are thus self-pollinating (Calderon, 1985; Ballard, 1994; Mattila and Salonen, 1995;

Berg and Rebdo-Tortensson, 1999). The very few groups lacking cleistogamy altogether inhabit very arid or harsh exposed environments; the Nuttallineae W. Becker of the southwestern US deserts, the Pedateae Pollard represented by Viola pedata L. of dry prairie/oak savanna of North America, and the Viola pinnata – disecta complex of the Adnatae W. Becker group in Alpine scree and steppes of Europe and eastern Asia.

Peculiarly, all Eurasian members of Section Melanium, the pansies, also lack cleistogamy but the sole North American representative, Viola bicolor Pursh, produces both flower types. 3

This mixed reproductive system has been well studied in a variety of temperate woodland violets including, Viola blanda Willd. (Newell et al., 1981), V. canadensis L.

(Culley, 2000), V. cunninghamii Hook.f. (Holsworth, 1966), V. egglestonii Brainerd

(Baskin and Baskin, 1975), V. fimbriatula Sm.(Solbrig et al., 1988a, 1988b), V. hirta L.

(Rebdo-Torstensson and Berg, 1995), V. mirabilis L. (Mattila and Salonen, 1995), V. nuttallii Pursh (Turnbull and Culver, 1983), V. odorata L. (Mayers and Lord, 1983;

1984), V. pallens (Banks) Brainerd (Newell et al., 1981), V. pubescens Ait. (Culley and

Wolfe, 2001) and Viola sororia Willd. (Solbrig et al., 1980; Solbrig, 1981).

Numerous studies have suggested that chasmogamous flowers require a greater investment of energy resources than cleistogamous flowers, primarily due to the fact that chasmogamous flowers are larger in size and attract pollinators, therefore requiring greater resources for the production of petals, nectar, and general flower structure.

Cleistogamous flowers however are less “expensive”, mainly because of their cryptic nature and generally small size, the fact that they do not open, lack petals, and are obligate self-pollinators. This general trend has been observed in very different taxa of cleistogamous species such as the tropical herb Calathea micans (Maranthaceae) (Le

Corff, 1993), the grasses Danthonia spicata (Clay, 1983) and Microlaena polynoda

(Schoen, 1984), in Impatiens sp. (Balsaminaceae) (Schemske, 1978; Waller, 1979), and several violet species including, V. egglestonii (Baskin and Baskin, 1975), Viola mirabilis (Mattila and Salonen, 1995) and V. odorata (Mayers and Lord, 1983). These studies in Viola have shown that chasmogamous flowers are larger in size and require a greater investment of resources in their production (i.e., petals, nectar) than the 4 cleistogamous flowers (Beattie, 1972; Baskin and Baskin, 1975; Mayers and Lord,

1983; Le Corff, 1993; Mattila and Salonen, 1995), while cleistogamous flowers are produced with less energetic cost, principally due to the lack of nectar production

(Beattie, 1972; Mattila and Salonen, 1995).

Studies in temperate regions in several species have suggested that one factor regulating the production of differential chasmogamous/cleistogamous flowers is available solar energy and plant age. Chasmogamy has been shown to increase with greater light intensity, soil fertility and/or soil moisture, and it is present only after some time has passed following seedling establishment when the plants are large enough to invest in this type of flower. Cleistogamy seems to occur independently of environmental conditions and plant size, with younger individuals producing only this type of flower (Baskin and Baskin, 1975; Le Croff, 1993; Mattila and Salonen, 1995).

Even though in most species both floral types are produced by the same individual, they are not necessarily co-occurring during the same precise time period.

One would expect that this phenological response would be greatly influenced by environmental conditions, and in many species, this is true. These phenological differences have been widely hypothesized to be mediated by changes in photoperiod, with chasmogamous flowers produced in response to shorter days and cleistogamous flowers produced in response to longer days (Holdsworth, 1966; Baskin and Baskin,

1975; Mayers and Lord, 1983; Mattila and Salonen, 1995). This pattern in turn leads to cross-pollinated (out-crossing) flowers being produced before self-pollinated ones, at least in the temperate regions where this phenological separation has been described. It 5 has been hypothesized that this shift in time to maturity may be advantageous, allowing for the production of offspring via cleistogamy even in years of poor weather conditions or low pollinator numbers which would greatly limit reproduction via the chasmogamous “route.”

The presence of two different types of flowers in the vast majority of species of

Viola leads to the presence of two different mechanisms of fertilization. Fertilization in cleistogamous flowers takes place inside the flower, independent of insect visitation and/or environmental factors. Pollen germinates inside the anther locule and the pollen tube grows through the anther sac walls until it reaches the style and later the ovule.

Modifications in the shape of the style facilitate this process (Mayers and Lord, 1984).

Chasmogamous flowers usually require the visitation of an insect for pollination. Pollen transfer occurs primarily through interaction with insects, in particular by solitary bees of the order Hymenoptera (Beattie, 1971; 1972; Mattila and Salonen, 1995). Cross- pollination in Viola is dependent upon pollinator behavior which is closely related to environmental factors such as slope angle and aspect, the daily duration of direct sunlight, the proximity and composition of competing flowering species, and the degree of constancy of individual pollinator species (Beattie, 1971; 1972; 1976).

Chasmogamous flowers are usually self-compatible, but field observations suggest that in general the production of chasmogamous seeds is totally dependent upon pollinator visits (Beattie, 1971). In some species like Viola pubescens a phenomenon known as “delayed selfing” can occur in which unpollinated chasmogamous flowers can self pollinate (Culley and Wolf, 2001). In this later case and in cleistogamy, the genetic consequences would be identical. 6

Evolutionarily, it is hypothesized that the chasmogamous-cleistogamous mixed reproductive strategy may have evolved as a response to an environment showing high temporal and spatial variation during each season (Mattila and Salonen, 1995).

Chasmogamous flowers would allow for the transfer of genetic material among individuals within a population. This genetic recombination would lead to seeds and offspring that are genetically very different from the parents. Evolutionarily, this variability could impart an inherent advantage to these individuals, particularly in a variable environment or for colonization of new microsites. Alternatively, cleistogamous flowers, not allowing for the transfer of genetic material among individuals, would result in offspring genetically very similar to the parent plant. This would be advantageous for the continued persistence of a population in a stable and unchanging environment or individuals establishing themselves near the parental plant.

These different strategies of mixing population persistence and colonization have been formulated into two hypotheses regarding the adaptive advantages of a mixed breeding system, the “dual strategy” hypothesis and the “reproductive assurance” hypothesis. The dual strategy hypothesis follows the premise that equal investment should be made in maintaining a population and in colonizing new habitats. The reproductive assurance hypothesis supports the idea of assuring seed set and reproduction under any circumstance. Therefore if chasmogamous seeds fail to be produced then cleistogamous seeds will be produced thus ensuring the species persistence (reviewed by Gara, 1987). 7

Beattie (1972) has suggested that since chasmogamous flowers depend upon pollinator visits to produce seeds, they frequently fail to set seed and therefore cleistogamous flowers produce the majority of the seeds in a given season. Beattie

(1976) proposed that this would reduce the intraspecific competition that a purely cleistogamous system may produce. Mattila and Salonen (1995) found that V. mirabilis reproduced mainly via chasmogamy, as did V. pubescens (Culley and Wolfe, 2001). In contrast, Baskin and Baskin (1975) discovered that V. egglestonni produced a higher proportion of seeds from cleistogamous flowers. Thus, the proportion of chasmogamous to cleistogamous flowers seems to be species related and/or dependent on the environmental factors prevailing in the habitat of the given species.

Despite the abundance of members of the genus Viola (Violaceae) in both temperate and tropical forests, most of our understanding of their reproductive system has come solely from studies of temperate species and on the ornamental pansies.

Surveys of the literature suggest no studies have been conducted on any tropical Viola species regarding their ecology, reproduction or genetics. To this aim, I have chosen the tropical violet Viola grahamii Benth. to serve as a model to evaluate the reproductive pattern of a tropical violet in a natural setting. Like other Mesoamerican violets, V. grahamii is poorly known and little studied and thus this work will serve to increase our knowledge regarding this and related species, not only in our understanding of reproduction but also in terms of ecological relationships and patterns of growth. To make comparisons with the patterns of reproduction exhibited in the temperate zone, the northern temperate Viola striata was chosen for study alongside V. grahamii. 8

The specific goals for this work include:

1) An evaluation of the relative importance of cleistogamy and chasmogamy in

Viola grahamii and Viola striata.

2) Documentation of phenologies of chasmogamous and cleistogamous

blooming and fruiting in the two species.

3) Identification of environmental factors (light availability, soil nutrients and

soil moisture) which may be influencing reproductive components.

4) An evaluation of the mixed breeding system and the impact it has on the

reproductive strategy and genetic structure of the populations.

5) Evaluation of the dual strategy hypothesis that suggests an equal investment

of resources in both flower types.

6) Identification of patterns and levels of leaf plasticity in Viola grahamii. 9

Research site establishment and description

Species and Sites of Study

Comparisons among species are potentially more accurate and evolutionarily informative when comparing closely related species. The two species chosen for this study are members of the same generic section, section Viola which contains species in

North and South America, Europe, and Asia (Ballard et al., 1999). The species studied here are classified in different subsections, Viola grahamii in subsection Mexicanae W.

Becker of Mexico, Central America and Northern South America and Viola striata in subsection Rostratae Kupffer, a widespread group of the northern hemisphere. Both of the species occupy widely distributed ranges and are in some ways generalists, inhabiting a variety of habitats from the interior of dense forest to highly disturbed and human-mediated environments.

Viola grahamii Bentham

Viola grahamii (known in Mexico as “hoja de pasmo”, “pensamiento del cerro”,

“orejita de ratón” or “Violeta silvestre” [“Pansy of the mountain”, “Little mouse ears”,

“Wild violet”]) is a Mesoamerican violet widely distributed from northern Mexico to

Guatemala (Ballard, 1994). Viola grahamii is a perennial herb with a wide range of altitudinal habitats, ranging from 1950 to 3600 m above sea level, and it is most often associated with pine and oak forest, but can also be found in cloud forest (Ballard,

1994). Its blooming time for chasmogamous flowers has been reported between June 10 and August, corresponding to the rainy season throughout most of its range (Ballard,

1994). It is commonly found in the shade of the forest canopy, typically along streams and on small hills (Calderon de Rzedowski, 1985), but it is also common in open areas in the forest, even in places that have been disturbed (Rzedowski, et al. 2001). Viola grahamii is a stemless violet, of about 15 cm in height with a robust erect rhizome from which leaves, roots, stolons, and flowers are produced. It has large free or scarcely adnate stipules, abundant pubescence on the petioles and leaf blades, ovate to oblong leaves, ciliate calyx, 1 to 3 flowers on pedicels up to 12 cm long, and white corollas with showy purple nectar guides on the inner surfaces of the petals (Figure 1.1A). Little is known about V. grahamii or the other violets of subsection Mexicanae, in relation to reproduction and flowering. The only other research on this species is my earlier work evaluating the patterns of hybridization between V. grahamii and Viola hookeriana

Kunth. (Cortés-Palomec, 2001) and currently unpublished phylogenetic data for species of the group (Ballard, unpublished data). The first two studies showed that in areas of range overlap, hybridization was common between the two species, resulting in a hybrid swarm. Ancillary observations performed during the time spent completing that research suggested that most of the reproduction in both V. grahamii and V. hookeriana was most likely due to cleistogamy, for chasmogamous capsules and pollinators were rarely seen on that particular year.

Viola striata Aiton

Viola striata, the striped violet, is a perennial herb common in temperate forests 11 of the northern and eastern United States and southern Canada (Gleason and Cronquist,

1991). It specifically inhabits low moist places, usually around streams and floodplains and it is a pronounced calciphile and, in some cases can exhibit a weedy habit (Gleason and Cronquist, 1991). Viola striata is a stemmed violet with stems ranging from prostrate to ascending from an ascending rhizome (Strausbaugh and Core, 1972).The leaves are ovate to acuminate with crenulate margins, sparsely hairy above or less often glabrous. Stipules at the base of the petiole are lance-ovate and strongly fimbriate.

Flowering stems are weakly ascending to erect, and produce cream colored flowers with brown-purple veins (nectar guides) near the base of the petals; the spur is 3.0 to 4.8 mm long (Figure 1.1B).

Viola grahamii Study Site:

As mentioned before, Viola grahamii is a common understory species in mixed pine and oak forest. This forest type forms a very important and at one time abundant plant community in central Mexico where they covered up to 25 % of the total territory.

Today this community type is much reduced and in some places even considered threatened due to changes in land use by the local population. Due to the fertility of their soils, these communities are burned and/or cut to use in raising cattle or for other agricultural purposes (Rzedowski and McVaugh, 1966). In some cases they are also cut solely for their wood. 12

Figure 1.1: A. Diagram of Viola grahamii Bentham. A. Mature flowering individual B. Petals. C. Seeds. D. Open capsule. (Reproduced from Ballard, H.E. 1994. Flora del Bajio y de Regiones Adjacentes). B. Mature flowering individual of Viola striata Aiton (Reproduced from Rhodes and Block, 2000. The Plants of Pennsylvania, an illustrated manual.) 13

The central Mexican state of Michoacán contains some of the best remaining examples of mixed pine and oak forests in Mexico, predominantly between 1300 and

3030 m in elevation, encompassing the sixth largest area of subhumid temperate forest in Mexico with an area of 1,550,000 ha (Garcia et al., 1998). Within this region, two physiographic provinces are found, the Transvolcanic Belt and Sierra Madre del Sur

(INEGI, 1985), which together form a varied topography accounting for a high level of species diversity with over 7,000 plant species described (Rzedowski, 1993).

In this region, the climate is considered temperate humid with summer rain [type

C following Koeppen classification; type C(m)wg, based on the classification of climates in Mexico made by García (1981)]. The mean annual precipitation varies between 1500 and 2000 mm, and the mean annual temperature is 18°C (Valerio, 1994).

The soils in this region are well drained and usually not very deep, comprised principally of reddish clay.

Climatically, these communities fluctuate seasonally between a wet and dry season, however they are considered evergreen. The overstory mainly retains its leaves during all seasons though some oaks can lose their leaves in the dry season, but usually only for a few weeks before the rainy season begins (Pesman, 1962). While the overstory may be considered evergreen, the understory can fluctuate greatly in species diversity and composition from wet to dry season, even becoming almost absent in some regions during the dry season.

The pine forests in this region are predominantly composed of three species,

Pinus pseudostrobus Lindl., P. montezumae Lamb., and P. ayacahuite Schlecht., though 14 other rare species do occur, and it is mainly dependent upon the elevation and microhabitat of the region. In the case of oaks, several species have been described for the region and among them Quercus macrohylla Née, Q. aff. Aristata Hook and Arn.,

Q. planipocula Trel., Q. mexicana Humb., and Q. candicans Née often predominate

(Rzedowski and McVaugh, 1966).

The region of study was located in the mountains surrounding Lake Pátzcuaro in northeastern Michoacán, municipality of Quiroga. Two populations of Viola grahamii were chosen to the north of Lake Pátzcuaro on the southern slopes of Mt. Zirate for study (Figure 1.2). The first locality, from now on referred as site Santa Fe due to its proximity to the small town of Santa Fe de la Laguna, was located at 19º 41’ N, 101º

32’ W. The second locality, referred to as site Quiroga was located approximately 1.4 km east of Santa Fe at 19º 40’ N, 101° 33’ W above the town of Quiroga. In order to ensure similar conditions at both sites they were established at the same elevation (ca.

2200m above sea level). Additionally, both sites were established in forest with equal proximity to cleared grazing land and relatively equal abundance of V. grahamii. In general, the lower slopes of Mount Zirate, immediately below the distribution of Viola grahamii, have been cleared for livestock and consist primarily of open pasture with abundant shrubs of Bacharis conferta H.B.K. interspersed with occasional trees and cacti. 15

Figure 1.2: Location of Viola grahamii research sites in Michoacán, central Mexico. A: Santa Fe, B: Quiroga. Map reproduced from Carta Topográfica 1:50 000 Pátzcuaro E14 A22, Michoacán. Instituto Nacional de Estadística e Informática, México. 16

Despite the relative closeness of the two sites, their shared elevation and position with respect to grazing land and the lake, the two sites turned out to be quite different. The greatest difference was found in the level of disturbance, which was much greater in Quiroga where some of the forest habitat was lost to local collection of firewood between the two years of study. This forest cutting led to more open areas than in the largely undisturbed Santa Fe site.

Further differences were elucidated for the two sites through a vegetation survey completed during the summer of 2003 which indicated that while most common species were present at both sites, specific species differed significantly. While the vegetation survey was only intended to characterize the community of V. grahamii and not to be a full flora, Quiroga was seen to have 38 species of angiosperms distributed in 24 families as well as two species of gymnosperm and one pteridophyte (Table 1.1). Santa Fe contained 36 species in 22 families and two species of pteridophyte from two different families and only one species of gymnosperm (Table 1.2). Pteridophyte and gymnosperm composition was different between the two sites and angiosperm compositions diverged greatly as well. In both sites only a small number of species were dominant. Quiroga was primarily dominated by oaks, while Santa Fe was dominated by pines. 17

Table 1.1: Results of a vegetation survey completed at site Quiroga, Michoacán during the summer of 2003. Voucher specimens for all species are deposited in the Bartley Herbarium of Ohio University (BHO).

Polypodiophyta (Ferns)

Polypodiaceae Pleopeltis sp. Pinophyta (Gymnosperms)

Cupressaceae Pinaceae Cupressus sp. Pinus teocote Schlecht. & Cham.

Magnoliophyta (Angiosperms) Magnoliopsida (Dicotyledons)

Acanthaceae Fagaceae Dyschoriste microphylla (Cav.) O. Ktze. Quercus castanea Nee

Anacardiaceae Geraniaceae Rhus aromatica Ait. Geranium seemannii Peyr. Toxicodendron radicans (L.) Kuntze. Lamiaceae Asteraceae Salvia laevis Benth. Acourtia humboldtii (Less.) Turner Scutellaria caerulea Sesse & Moc. Baccharis conferta H.B.K. Guardiola mexicana Humb. & Bonpl. Lythraceae Senecio sp. Cuphea jorullensis H.B.K. Verbesina hypomalaca Rob. & Greenm. Cuphea wrightii A. Gray

Asclepiadaceae Onagraceae Pherotrichis balbisii (Decae.) A. Gray Fuchsia parviflora (Zucc.) Hemsl.

Brassicaceae Passifloraceae Eruca sativa Mill. Passiflora exsudans Zucc.

Ericaceae Polygalaceae Arbutus tessellata Sorensen. Monnina ciliolata DC. Polygala appressipilis Blake Euphorbiacae Acalypha phleoides Cav. Primulaceae Croton adspersus Benth. Anagallis arvensis L.

Fabaceae Macroptilium gibbosifolium (Ort.) A. Delgado Calliandra grandiflora (L'Her.) Benth. Cologania brossonetii (Balbis) DC. Desmodium spp.

Table 1.1 continued

Rubiaceae Ranunculaceae Galium uncinulatum DC. Ranunculus petiolaris H.B.K. ex DC. Bouvardia ternifolia (Cav.) Schlecht.

Rhamnaceae Solanaceae Ceanothus coeruleus Lag. Physalis chenopodiifolia Lam. Solanum nigrescens Mart. & Gal. Rosaceae Crataegus mexicana Moc. & Sesse ex DC. Verbenaceae Verbena carolina L.

Liliopsida (Monocotyledons)

Commelinaceae Commelina tuberosa L.

Iridaceae Sisyrinchium tolucense Peyr.

Table 1.2: Results of a vegetation survey completed at site Santa Fe, Michoacán during the summer of 2003. Voucher specimens for all species are deposited in the Bartley Herbarium of Ohio University (BHO).

Polypodiophyta (Ferns)

Dennstaedtiaceae Pteridaceae Pteridium sp. Adiantum poiretii Wikstr.

Pinophyta (Gymnosperms)

Pinaceae Pinus montezumae Lamb.

Table 1.2 Continued

Magnoliophyta (Angiosperms) Magnoliopsida (Dicotyledons)

Anacardiaceae Lamiaceae Rhus aromatica var. schimedeliodes (Schltdl.) Salvia assurgens H.B.K. Engl. Prunella vulgaris L.

Apiaceae Eryngium carlinae Delar. f. Loranthaceae Cladocolea diversifolia (Benth.) Kuijt Asclepiadaceae Pherotrichis balbisii (Decne.) A. Gray Lythraceae Cuphea jorullensis H.B.K. Asteraceae Cuphea wrightii A. Gray Acourtia humboldtii (Less.) Turner Baccharis conferta H.B.K. Oxalidaceae Guardiola mexicana Humb. & Bonpl. Oxalis tetrophylla Cav. Verbesina hypomalaca Rob. & Greenm. Senecio sp. Polygalaceae Monnina ciliolata DC. Betulaceae Polygala subulata S. Wats. Alnus jorullensis H.B.K. Polygala appressipilis Blake

Boraginaceae Primulaceae Laiarrhenum trinervium (Lehm.) Turner Anagallis arvensis L.

Convolvulaceae Rhamnaceae Ipomoea painteri House. Ceanothus coeruleus Lag.

Ericaceae Rosaceae Arbutus tessellata Sorensen. Prunus serotina Ehrh.

Fabaceae Rubiaceae Calliandra grandiflora (L’Her.) Benth. Bouvardia ternifolia (Cav.) Schlecht. Desmodium Galium uncinulatum DC. Mimosa aculeaticarpa Ort. Solanaceae Fagaceae Lycianthes moziniana (Dunal) Bitter Quecus candicans Nee Solanum lanceolatum Cav. Quecus castanea Nee

Garryaceae Garrya laurifolia Benth.

Table 1.2 continued

Liliopsida (Monocotyledons)

Orchidaceae Malaxis unifolia Michx.

Smilacaceae Smilax moranensis Mart. & Gal.

Viola striata Study Site

The eastern deciduous forests of North America once occupied about 2,560,000 km2 across the central portion of the continent from 32-48° North to 70-98° West.

Today their distribution has been reduced mainly by changes in land use (Delcourt and

Delcourt, 2000). These forests are dominated predominately by deciduous woody angiosperms with some evergreen gymnosperms like Pinus and Tsuga, and a very diverse understory of ephemeral herbs and perennials (Delcourt and Delcourt, 2000).

Within the broad range of the Eastern Deciduous Forest, Braun (1950) described nine principal subtypes of dominant vegetation. One of these, the mixed mesophytic forest encompasses the region of the west-central Appalachians, including southeastern Ohio.

Across the unglaciated portion of the Allegheny plateau region in Ohio, the forests are dominated by Fagus L., Liriodendron L., Tilia L., Acer sacharum Marsh., (at one time Castanea dentata (Marsh.) Borkh.), Aesculus flava Ait., Quercus rubra L., Q. alba L., and Tsuga candensis (L.) Carr., (Braun, 1950). While these forests are rich in species diversity they are not as complex as the forests of the same type further south 21 due to the location of southern Ohio on the border with less diverse forest types to the north in the glaciated region of the state (Braun, 1961).

The area of study for this species was Strouds Run State Park located in Canaan

Township, Athens County, Ohio, USA. The area of the park is 1033 ha, including Dow

Lake, a man made body of water of ca. 65 ha that is fed by small streams formed on the hills surrounding the park (McConnell, 1963). The forest is located eight kilometers east of the City of Athens between US Rt. 50/ OH 32 and Strouds Run Rd (Athens County

Rt. 26). The park harbors 642 plant species distributed in 362 genera and 106 families

(Harrelson, 2005). The vegetation in this region is a secondary forest community reforested with pine and hardwoods from old fields used for pasturing (McConnell,

1963). The soils in these areas correspond to sedimentary rock and receive an average of 101 cm of annual precipitation. There are four clearly distinct seasons. This environment is suitable for a variety of Viola species, among them Viola striata.

Two different populations were sampled within the park (Figure 1.3). The first site, designated as the Campground site, was located north of the campground area of the park at 39º 21’ N, 82º 02’ W. This site was situated partially in the floodplain of a small stream dominated by Acer negundo L. and on the edges of a plantation of Pinus resinosa Aiton. The second site, designated the Cemetery site, was located beyond the northwest end of Dow Lake adjacent to the Pioneer Cemetery trail at 39º 21’ N, 82º 02’

W. Most of the site was situated along the floodplain of Strouds Run and a small

Figure 1.3: Location of Viola striata research sites in Athens, Co, southern Ohio. A: Campground; B: Cemetery. Topographic map insert from USGS Athens Quadrangle 7.5 minute series, 1961/1995. Scale 1: 24,000. 23 unnamed tributary and thus much of the area was exposed to periodic flooding. Both sites were positioned at the same elevation (ca. 210 meters above sea level).

As in the Viola grahamii sites, distinct differences between the two Viola striata populations were observed, although the differences were not as marked. A floristic survey performed during spring, summer and fall of 2002, identified a total of 72 angiosperm species distributed in 39 families, three pteridophytes, Equisetum and one species of gymnosperm (Pinus) for the Campground site (Table 1.3). In the case of the

Cemetery Site (Table 1.4) two ferns and one Pinus were found as well as 69 angiosperm species in 35 families. In general the two sites were very similar to each other although some species were only present in one site. The sites differed particularly in abundance of Pinus (mainly in Campground site) and a greater disturbance by flooding in the

Cemetery site.

Table 1.3: Results of a vegetation survey completed at Campground site, Ohio during spring, summer and fall of 2002.

Equisetophyta

Equisetaceae Equisetum sp.

Polypodiophyta (Ferns)

Aspleniaceae Onocleaceae Dryopteris carthusiana (Villars) H. P. Onoclea sensibilis L. Fuchs. Polystichum acrostichoides (Michx.) Schott.

Table 1.3 Continued

Pinophyta (Gymnosperms) Pinaceae Pinus resinosa Aiton. Magnoliophyta (Angiosperms) Magnoliopsida (Dicotyledons)

Aceraceae Caryophyllaceae Acer rubrum L. Stellaria media (L.) Villars. Acer saccharum Marshall. Acer negundo L. Celastraceae Euonymus atropurpureus Jacq. Adoxaceae Viburnum prunifolium L. Clusiaceae Hypericum sp. Anacardiaceae Toxicodendron radicans (L.) Kuntze. Cornaceae Cornus florida L. Annonaceae Asimina triloba (L.) Dunal. Fabaceae Cercis canadensis L. Apiaceae Robinia pseudoacacia L. Chaerophyllum procumbens (L.) Crantz. Gleditsia triacanthos L. Osmorhiza longistylis (Torr.) DC. Fagaceae Asteraceae Fagus grandifolia Ehrh. Ambrosia trifida L. Quercus alba L. Aster simplex Willd. Quercus rubra L. Eupatorium rugosum Houttuyn. Quercus muehlenbergii Engelm. Verbesina alternifolia (L.) Britton. Quercus velutina Lam.

Berberidaceae Hippocastanaceae Berberis thunbergii DC. Aesculus flava Aiton.

Betulaceae Juglandaceae Corylus americana Walter. Juglans nigra L. Ostrya virginiana (Miller) K. Koch. Carya cordiformis (Wangenh.) K. Koch. Carpinus caroliniana Walter. Carya laciniosa (Michx. f.) Loudon

Brassicaceae Lamiaceae Alliaria petiolata (Bieb.) Cavara & Grande. Glechoma hederacea L. Lamium purpureum L. Caprifoliaceae Lonicera maackii (Rupr.) Maxim. Lauraceae Lonicera japonica Thunb. Lindera benzoin (L.) Blume.

Magnoliaceae Liriodendron tulipifera L.

Table 1.3 Continued

Menispermaceae Rubiaceae Menispermum canadense L. Galium sp.

Oleaceae Scrophulariaceae Fraxinus americana L. Collinsia verna Nutt. Fraxinus pennsylvanica Marsh. Thymelaeaceae Oxalidaceae Dirca palustris L. Oxalis sp. Ulmaceae Platanaceae Ulmus americana L. Platanus occidentalis L. Ulmus rubra Muhl. Celtis occidentalis L. Polygonaceae Polygonum virginianum L. Urticaceae Boehmeria cylindrica (L.) Swartz. Primulaceae Pilea pumila (L.) A. Gray Lysimachia nummularia L. Valerianaceae Rosaceae Valerianella locusta (L.) Betcke. Agrimonia gryposepala Wallr. Duchesnea indica (Andrews) Focke. Violaceae Geum canadense Jacq. Viola striata Aiton. Geum vernum (Raf.) T. & G. Viola sororia Willd. Prunus serotina Ehrh. Rosa multiflora Thunb. Vitaceae Rubus occidentalis L. Parthenocissus quinquefolia (L.) Planchon. Vitis sp.

Liliopsida (Monocotyledons)

Cyperaceae Carex sp.

Poaceae Elymus hystrix L. Poa sp.

Smilacaceae Smilax rotundifolia L. Smilax glauca Walter.

Table 1.4: Results of a vegetation survey completed at Cemetery site, Ohio during the spring, summer and fall of 2002.

Polypodiophyta (Ferns) Aspleniaceae Dryopteris sp. Polystichum acrostichoides (Michx.) Schott.

Pinophyta (Gymnosperms) Pinaceae Pinus strobus L.

Magnoliophyta (Angiosperms) Magnoliopsida (Dicotyledons)

Aceraceae Cardamine hirsuta L. Acer saccharum Marshall. Acer negundo L. Caryophyllaceae Stellaria media (L.) Villars. Adoxaceae Viburnum prunifolium L. Caprifoliaceae Lonicera maackii (Rupr.) Maxim. Anacardiaceae Toxicodendron radicans (L.) Kuntze. Celastraceae Celastrus scandens L. Annonaceae Asimina triloba (L.) Dunal. Fabaceae Cercis canadensis L. Apiaceae Robinia pseudoacacia L. Chaerophyllum procumbens (L.) Crantz. Amphicarpaea bracteata (L.) Fern. Cryptotaenia canadensis (L.) DC. Osmorhiza longistylis (Torr.) DC. Geraniaceae Geranium maculatum L. Asteraceae Ambrosia trifida L. Hippocastanaceae Aster prenanthoides Muhl. Aesculus flava Aiton. Aster shortii Lindley. Helianthus tuberosus L. Hydrophyllaceae Verbesina alternifolia (L.) Britton. Hydrophyllum canadense L. Vernonia gigantea (Walter) Trel. Juglandaceae Balsaminaceae Juglans nigra L. Impatiens capensis Meerb. Impatiens pallida Nutt.

Berberidaceae Berberis thunbergii DC.

Brassicaceae Cardamine douglassii Britton.

Table 1.4 Continued

Lamiaceae Ranunculaceae Glechoma hederacea L. Clematis virginiana L. Ranunclus abortivus L. Lamium purpureum L. Ranunclus recurvatus Poiret. Monarda sp. Rosaceae Lauraceae Agrimonia gryposepala Wallr. Lindera benzoin (L.) Blume. Crataegus sp. Duchesnea indica (Andrews) Focke. Limnanthaceae Geum vernum (Raf.) T. & G. Floerkea proserpinacoides Willd. Prunus serotina Ehrh. Rosa multiflora Thunb. Magnoliaceae Liriodendron tulipifera L. Rubiaceae Galium aparine L. Oleaceae Fraxinus americana L. Scrophulariaceae Ligustrum vulgare L. Mimulus alatus Aiton.

Platanaceae Ulmaceae Platanus occidentalis L. Ulmus americana L. Ulmus rubra Muhl. Polemoniaceae Phlox divaricata L. Urticaceae Phlox paniculata L. Pilea pumila (L.) A. Gray. Urtica dioica L. Polygonaceae Polygonum sp. Violaceae Polygonum cespitosum Blume. Viola striata Aiton. Polygonum scandens L. Viola sororia Willd. Polygonum virginianum L. Valerianaceae Primulaceae Valerianella locusta (L.) Betcke. Lysimachia nummularia L. Vitaceae Parthenocissus quinquefolia (L.) Planchon. Vitis sp.

Table 1.4 Continued

Liliopsida (Monocotyledons) Cyperaceae Carex amphibola Steudel.

Smilacaceae Smilax hispida Muhl. Smilax rotundifolia L.

Quadrat establishment

2 In each site, twenty quadrats of 0.5 m were established using a modified stratified random design (Barbour et al., 1999). Due to the patchy distribution of violets both in Michoacán and Ohio the stratified random design was modified to focus only on regions with patches of Viola. Within these large patches plastic quadrats were thrown in a random pattern. A random quadrat placement was finalized if it contained more than three individuals. These final quadrats were marked with flags at the corners corresponding to quadrat number and left in the site for the duration of the study. Exact coordinates were established for each quadrat using a handheld GPS. These latitude and longitude values were used to produce maps of each site and determine the distance among quadrats for later investigation of correlation between geographic and genetic distance within populations (Figure 1.4).

Figure 1.4: Map of quadrat location for Viola striata (Ohio) and Viola grahamii (Michoacán). In Quiroga, those quadrats marked with open circles were lost between year one and two as a result of disturbance from local wood harvesting. All populations were in forested areas however regions depicted by trees above were dominated by conifers.

Chapter 2:

Environmental correlates to leaf mass area and leaf nitrogen in the Neotropical

violet Viola grahamii

Introduction

Plant growth and reproduction depend in part on resource availability and the physical environment present in a given site. Small-scale differences in environmental norms may trigger morphological and physiological changes in an individual, broadly defined as phenotypic plasticity. Due to their importance in energy capture, leaf characteristics are commonly highly plastic due to heterogeneous or stochastic environments. One of the most direct methods of evaluating species level leaf responses to heterogeneous environments is to test for correlations between leaf traits and environmental variables. Leaf Mass per unit Area (LMA) is the ratio of leaf blade mass to area (kg m-2) and is a measure of leaf toughness and thickness (Larcher, 2003). This index has been shown to vary in response to fine-scale environmental differences across a species range, across a population or even within an individual exposed to heterogeneous conditions. In particular, LMA has been shown to be sensitive to light conditions (Ballaré, 1994), with expanding leaves in the sun commonly becoming thicker, and leaves in the shade becoming thinner and larger to maximize light capture

(Arens, 1997; Cramer et al., 2000; Givinish, 1988; Pearcy and Sims, 1994). LMA is also sensitive to level of nutrients in the soil, with higher values of LMA under low

31 nutrient environments (Jurik et al., 1982; Jurik, 1986) and water stress (Jurik, 1986).

These differences in LMA under varied ecological conditions are the result of differential mesophyll development which in turn determines the photosynthetic capacity of the leaf (Jurik, 1986). Besides LMA, leaf nitrogen content has also been shown to correlate highly with photosynthetic capacity, and LMA and leaf nitrogen content have also been used as indicators of leaf gas exchange capacity, plant growth rate, and longevity (Reich et al.,1988; Garnier et al., 1997), because they express a positive correlation in perennial species (Garnier et al., 1997).

Understory herbs from temperate deciduous forests encounter dramatic changes in light availability due to an increase in canopy cover as the growing season progresses

(Rothstein and Zak, 2001). Photoperiod is not constant and precipitation is not evenly distributed across the growing season and other factors such as air and soil temperature may play a role in plant responses to their environment that would not occur in regions near the equator. In subtropical climates there is generally less environmental variation, often driven solely by changes in precipitation marking the dry and the wet seasons.

Viola L. species growing in these forests have adapted to these environments by developing two divergent leaf phenotypes with different LMA throughout the growing season (Rothstein and Zak, 2001). In V. pubescens Ait., of the deciduous forest of

Eastern North America, leaves with higher LMA values are produced in the spring when plants are growing under an open canopy, and lower LMA leaves are produced in the summer after all the trees in the canopy have leafed out (Rothstein and Zak, 2001).

Similar leaf plasticity has also been described in the Eastern deciduous understory

32 species V. fimbriatula Sm., other species like V. blanda Willd. have been shown to be totally adapted to the low light levels in its habitat and does not perform very well when transplanted to a different light environment (Curtis, 1984).

Despite the abundance of Viola species in forests worldwide, most of our understanding of the biology of the genus comes from work on temperate species and on the ornamental pansies. To the best of my knowledge, no studies have been conducted on any tropical or subtropical Viola species regarding their ecology, reproduction or genetics. One would predict that violets growing under a continuous growing season might behave ecologically in quite different patterns to temperate violets constrained by dramatically different seasons. My expectation is that subtropical violets increase their LMA to increases in light within patchy forest, and that the light conditions within a growing season are stable. The objectives of this study are to evaluate the influence of canopy openness, soil moisture and soil nutrients on the leaf structure and nutritional content of the subtropical violet species Viola grahamii

Bentham and compare patterns of correlation with expectations and results from temperate violets.

Materials and Methods

Species of Study

Viola grahamii (known in Mexico as “hoja de pasmo”, “pensamiento del cerro”,

“orejita de ratón” or “Violeta silvestre”) is a Mesoamerican violet widely distributed from northern Mexico to Guatemala (Ballard, 1994). Viola grahamii is a stemless

33 perennial herb with a wide range of altitudinal habitats, ranging from 1950 to 3600 m above sea level, and is most often associated with pine and oak forest, but can also be found in cloud forest (Ballard, 1994). Its blooming time for chasmogamous flowers has been reported between June and August, corresponding to the regional rainy season throughout most of its range. It is commonly found in the shade of the forest canopy, typically along streams and on small hills (Calderon de Rzedowski, 1985), but it is also common in open areas in the forest, even in places that are perturbated (Rzedowski, et al. 2001).

Study Sites

The study was conducted during the summer of 2002 in the mountains surrounding Lake Pátzcuaro in the state of Michoacán, municipality of Quiroga, Mexico in a forest community dominated by a mix of pine and oak. Climatically, these communities fluctuate seasonally between a wet (June-October) and dry (November-

May) season, however they are considered evergreen. For the year of the study the total annual precipitation was 725.7 mm. The minimum temperature was 12.5°C, and the maximum was 27.04°C (Comisión Nacional del Agua, Mexico). The soils in this region are well drained and usually not very deep, being comprised principally of reddish clay.

The overstory mainly retains its leaves during all seasons though some oaks can lose their leaves in the dry season, but usually only for a few weeks before the rainy season begins (Pesman, 1962). While the overstory may be considered evergreen, the understory and herbaceous layer can fluctuate greatly in species diversity and

34 composition from wet to dry season, even becoming depauperate in some regions during the dry season.

Two sites with Viola grahamii were identified to the north of Lake Pátzcuaro on the southern slopes of Mt. Zirate. The first site, Santa Fe [19º41’N, 101º32’W] was above the town of Santa Fe de la Laguna. The second, Quiroga [19º40’N, 101° 33’W] was located above the town of Quiroga. Both sites were established at the same elevation (ca. 2200m above sea level). The floristic composition for both sites is different in terms of their overstory species, Santa Fe was richer in Pinus, while

Quiroga had a larger composition of Quercus. Due to the clustered nature of Viola populations, quadrats were established using a stratified random design (Barbour et al.,

1999). Twenty quadrats of 0.25 m2 were placed in each population by throwing plastic quadrats in an area inhabited by Viola, and marking the corners of each quadrat with flags (For additional information see Chapter 1).

Sampling, measurements and data handling

To characterize the light environment of each quadrat, hemispherical photographs were taken above each quadrat on overcast days or very early in the morning, using a digital Nikon Coolpix 955 camera equipped with a 180° Nikon FC-E8

Fish-eye converter lens. Images were saved using the FINE format (1:4 compression

JPEG, 1600 x 1200 pix) (Frazer et al., 2001). To verify potential changes of light availability through the growing season, weekly pictures were taken from early June to late August. A total of 240 images (40 quadrat images over 6 weeks) were analyzed for

35 canopy openness using Gap Light Analyzer (GLA version 2.0.4) image processing software (Frazer et al., 2001). Canopy openness data were arcsine transformed, and an analysis of variance (ANOVA) with weekly measurements as a random effect variable was performed to test for significant changes in light availability in each quadrat throughout the growing season. A one-way ANOVA was performed to check for differences in light between the two study sites using NCSS 6.0 (Hintze, 1996).

Soil chemical properties for each of the 40 quadrats were determined from soil cores collected from the center of each plot and stored in Ziploc bags. Soil was collected at the peak of the flowering season (chasmogamous peak). Soil moisture was determined using the gravimetric method (Hadley and Levin, 1967), on samples collected on a weekly basis through the growing season, and the average for each quadrat was used. All other analyses were performed on one individual set of air-dried soil samples. Soil pH was determined by dissolving 25g of soil in 100 ml of distilled, de-ionized water and testing using a Corning 430 pH/ion meter w/pH electrode. Soil nitrogen was measured using the Cadmium Reduction Method, using the Hach Pocket colorimeter analysis system [Nitrate (NO3-N), High Range 0 to 30 mg/L NO3-] (Hach

Co. USA). Calcium, magnesium and phosphorous were extracted using the Mehlich III method (Mehlich, 1984). Calcium and magnesium were determined using atomic absorption (Varian SpectrAA instrument. Varian Instruments, Australia), while phosphorous was determined using a Cary 50 Bio UV-visible spectrophotometer

(Varian Instruments, Australia). All soil analyses were conducted in the Department of

Environmental and Plant Biology at Ohio University. All raw data were corrected for

36 dilution factors and converted into µg of element g-1 of soil (Robertson et al., 1999).

Soil chemical data (Ca, Mg, P, K and N) were log10 transformed, while soil moisture data were arcsine transformed, to meet the assumptions of multivariate normal distribution. One value for potassium and one value for magnesium were winsorized

(Sokal and Rohlf, 1995), since both were outliers.

For each quadrat, at the peak of blooming of chasmogamous flowers, the total number of plants as well as the total number of leaves that each plant presented was recorded. To compare the total number of plants per quadrat a two sample t-test was performed (Sokal and Rohlf, 1995). The average number of leaves per plant per quadrat was compared between the two sites using a Mann-Whitney U test due to the non- normality of the data (Sokal and Rohlf, 1995). To measure leaf variables of individuals growing under different environmental conditions, a total of 80 leaves, each from a different individual, were collected. The largest leaf was removed from each of two individuals randomly selected within each quadrat at the peak of blooming (40 quadrats x 2 plants). Immediately after collection and before the leaves were dehydrated, the silhouettes of leaves were traced on paper; later the area of these leaf silhouettes was calculated using an LI-3000A Portable Area Meter, LI-3050A Belt Converter (LI-COR,

Nebraska, USA). The dry mass of the leaves was measured in milligrams using a digital balance. These values of leaf area and dry mass were used to calculate Leaf Mass Area

(LMA= dry matter leaf area-1) (Larcher, 2003). Following calculation of LMA, leaf nitrogen and carbon content, on a dry mass basis, were obtained using an Elementar

Vario EL CN elementar combustion analyzer (Elementar, Hanau, Germany).

To examine interdependence between these leaf traits and environmental variables (soil characteristics, light) under which plants were growing, and to test for correlations between leaf nitrogen content and LMA, Pearson’s correlation analyses were performed (Zar, 1999). Additionally leaf area and leaf mass were correlated independently to canopy openness. Leaf area and mass were log transformed to meet the normality criteria. The data from both sites were combined since the two sites behave as a continuum of each other, and stronger correlations were found between the factors when all the data were analyzed together. A multiple regression was applied to examine the combined effects of all the environmental variables on the leaf characteristics. This analysis was performed twice, once with the data from the two sites separate and once more treating the data as one sample. Initial selection of variables was performed using the multivariate variable selection procedure of NCSS (Hintze,

1996) to reduce the number of variables to the ones that provided the maximum value of R2. The selected variables were then used to run all possible regressions to create the best model with the least number of independent variables explaining the data.

Correlations and regressions were performed using NCSS (Hintze, 1996).

Results

Canopy openness values for quadrats at site Santa Fe (mean = 24.72%) were significantly lower than those at Quiroga (mean = 38.86%) (Table 2.1). Within each quadrat, light availability did not change through the growing season in either site

(Santa Fe: ANOVA: d.f.=5,114; F=1.08; P =0.37; Quiroga: ANOVA: d.f.=5,111;

F=0.52; P=0.75). Quiroga had higher soil magnesium and potassium, and less nitrogen than Santa Fe (Table 2.1). No significant differences were found between the two sites in soil calcium, phosphorous, moisture, and pH. In some cases like phosphorous and calcium, a great deal of variation was found among individual quadrats (Table 2.1).

Table 2.1: Summary of soil chemical properties (µg/g soil), soil moisture (%) and canopy openness in the two Viola grahamii study sites in central Mexico for year 2002 (n=20; mean ± s.d.). Means with different letters between columns are significantly different (ANOVA, p<0.05).

Parameter Santa Fe Quiroga Calcium (µg/g soil) 3164.90 ±2186.80 2166.90 ± 2004.90

Phosphorous (µg/g soil) 3.01 ±1.43 3.95 ± 3.19

Magnesium (µg/g soil) 805.00 ± 323.84 a 1035.06 ± 355.77 b

Nitrogen (µg/g soil) 37.48 ± 15.01 a 17.70 ± 12.18 b

a b Potassium (µg/g soil) 1359.56 ± 377.40 1914.29 ± 424.43 pH 6.35 ± 0.21 6.48 ± 0.27

Soil Moisture (%) 31.96 ± 2.86 30.94 ± 4.03

a b Light (% openness) 24.72 ± 5.61 38.86 ± 10.24

In Santa Fe, each plot had an average of 14.5 plants per quadrat (± 6.56), while

Quiroga quadrats had a mean of 10.9 plants (± 3.60). Thus Santa Fe had significantly more plants growing per quadrat than Quiroga (T test: T=2.05, p =0.02). In terms of the number of leaves that each plant presented on each site, there were no statistically

39 significant differences (Mann-Whitney U: Z=1.32, p=0.09). On average each plant had

3.84 (±2.06) leaves in Santa Fe and 2.83 (±1.04) leaves in Quiroga (Table 2.2).

Table 2.2: Total number of plants and leaves per each of the 20 quadrats established in Santa Fe and Quiroga during 2002. Santa Fe shows a significantly higher number of plants per quadrat (p <0.005).

Santa Fe Quiroga Quadrat Total number Number Ave. number of Total Number Ave. number of plants of leaves leaves per plant number of of leaves of leaves per plants plant I 9 27 3.00 8 17 2.12 II 21 81 3.80 8 23 2.80 III 13 40 3.00 9 27 3.00 IV 13 22 1.60 5 17 3.40 V 20 78 3.90 14 30 2.14 VI 30 120 4.00 10 34 3.40 VII 13 48 3.60 5 10 2.00 VIII 16 41 2.50 16 46 2.80 IX 21 78 3.70 10 20 2.00 X 12 65 5.40 10 21 2.10 XI 11 22 2.00 16 32 2.00 XII 7 52 7.40 10 31 3.10 XIII 4 33 8.25 14 82 5.80 XIV 9 24 2.66 8 25 3.10 XV 9 13 1.40 12 22 1.83 XVI 16 21 1.30 7 12 1.71 XVII 6 50 8.30 17 35 2.05 XVIII 13 22 1.60 14 62 4.42 XIX 22 110 5.00 9 40 4.44 XX 24 95 3.90 16 36 2.25 Total 289 218 Average 14.45 3.80 10.90 2.83 S.D. 6.5 2.06 3.60 1.04

Leaf Mass per Area showed a strong positive correlation with canopy openness

(Figure 2.1), and soil magnesium, but a negative correlation with soil nitrogen (Figure

2.2). Quiroga was in general more open than Santa Fe and the plants at Quiroga had higher values of LMA (Table 2.3). LMA did not correlate with soil pH, calcium, phosphorous or moisture (Table 2.4). Leaf area did not correlate with canopy openness, but leaf mass did (r= 0.33, p=0.002) (Figure 2.3), therefore the LMA values were highly influenced by leaf mass. Surprisingly, leaf nitrogen content was not correlated with soil nitrogen (r= -0.09; P=0.57), although leaf nitrogen per area was positively correlated to

LMA (r=0.85; p< 0.005) (Figure 2.4).

Table 2.3: Percent of nitrogen and carbon, specific leaf area (SLA), leaf mass per area (LMA), and leaf nitrogen content (LNC) in leaves of flowering individuals of V. grahamii in two sites: Santa Fe and Quiroga. Means and standard deviations are presented. (n=40; mean ± s.d.). Significant differences between groups (p<0.05) are marked with different subscripts.

Santa Fe Quiroga % Nitrogen 2.25 (± 0.14) 2.40 (± 0.20) % Carbon 44.18 (±1.27) 43.96 (± 1.01) Ratio C:N 19.73 (± 1.51) 18.80 (± 1.50) SLA (m2 g-1) 0.03 (± 0.004) a 0.02 (± 0.002) b LMA (g m-2) 36.60 (± 5.78) a 52.80 (± 8.12) b LNC area (g m-2) 0.82 (± 0.17) 1.26 (± 0.28)

60 ) 2 - m

g 50 A ( LM 40

30 r = 0.66, p < 0.005

20 0 10203040506070 Canopy openness (%)

Figure 2.1: Correlation between canopy openness and leaf mass area for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and the level of significance of the correlation are presented. N =80 individuals, 40 per site.

60 ) -2

50 LMA (g m 40

30 r= -0.47, p <0.005

20 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

-1 Soil Nitrogen (log µg g soil )

Figure 2.2: Correlations between soil nitrogen and leaf mass area for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and level of significance of the correlation are given. N=80 individuals, 40 per site.

43 25

20 ) 2

m 15 c (

af ar 10 Le

r = 0.09, p = 0.39

0 0 10203040506070 Canopy openness (%) 90

g) 60 (m ss

a 50

40 Leaf m

20 r = 0.33, p = 0.003

10 0 10203040506070 Canopy openness (%)

Figure 2.3: Correlation between canopy openness and leaf mass (mg) and leaf area (cm2) for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and the level of significance of the correlation are presented. N = 80 individuals, 40 per site.

0.20

) 0.18 -2 cm

0.16 gN m ( 0.14

entration 0.12 onc

C 0.10 en g tro

i 0.08 N f a e 0.06 r = 0.85, p <0.005 L

0.04 2345678

LMA (mg cm-2 )

Figure 2.4: Correlation between leaf mass per area (LMA) and leaf nitrogen per leaf area for Viola grahamii in Santa Fe (Black dots) and Quiroga (White dots). Pearson’s correlation coefficient (r) and level of significance of the correlation are provided. N = 80 individuals, 40 per site.

Table 2.4: Pearson’s correlations between leaf mass area (LMA) canopy openness and soil chemical properties. *** p <0.005, ** p <0.05.

LMA Canopy openness 0.66 *** Soil Magnesium 0.35 ** Soil Nitrogen -0.47 *** Soil Calcium 0.16 Soil Phosphorous 0.12 Soil pH 0.14 Soil moisture 0.05

The best model generated by the multiple regression analysis included different numbers of variables depending on the variable being tested. For LMA, nitrogen, magnesium, calcium and light were important (F=26.03; P<0.005; R2=0.58), for N content calcium, phosphorus and light were significant in the model (F=3.57; P =0.017;

R2=0.12), while for N/leaf area nitrogen, calcium and light were important (F=30.19; P

<0.005; R2=0.54) (Full models are presented in Table 2.5).

Table 2.5: Multiple regression models constructed from soil chemical data and light characteristics for Viola grahamii growing under different environmental conditions in two sites in central Mexico. All models were significant (p<0.05).

Regression MODEL Coefficient ± SE LMA (R2=0.58) Intercept 5.45 18.85 nitrogen -.9.75 2.92 magnesium 6.92 4.58 calcium -9.03 0.13 Light (Canopy openness) 15.11 2.66 Nitrogen content (R2=0.12) Intercept 0.28 0.66 calcium 0.18 0.11 phosphorous -0.12 0.18 Light (Canopy openness) 0.37 0.11 Nitrogen/area (R2=0.54) Intercept -0.44 0.49 nitrogen -0.23 0.01 calcium -0.13 0.01 Light (Canopy openness) 0.58 0.08

Discussion

Both sites were initially established in what appeared to be ecologically equivalent settings and would thus serve as replicate sites. Upon further examination, however, it became evident that the sites were divergent in a number of respects.

Quiroga was richer in Quercus species while Santa Fe presented a higher proportion of

Pinus species. Quiroga was more disturbed than Santa Fe, with strong differences in canopy openness (greater light availability in the understory in Quiroga than in Santa

Fe). Due to the evergreen nature of the forest, no changes in canopy openness were found through the growing season at either site, and mainly the light available to each

47 quadrat remained the same for the duration of the study. Some changes in light were recorded in a few quadrats, but those changes were mainly due to human activity rather than intrinsic changes of the forest canopy itself and were not significant. In terms of soil properties, most of the variables were variable among the different quadrats, indicating a highly heterogeneous soil environment. As the study was conducted during the rainy season when afternoon rain is common, some changes in moisture were recorded from week to week, but did not demonstrate a clear pattern. Interestingly, pH was similar in both sites even though the overstory vegetation was quite different. Other soil chemical differences might be attributed to these differences in the overstory and overall species composition since the sites share the same regional geological history.

There were more individual V. grahamii plants in Santa Fe than in Quiroga, most likely related to the fact that this site was less disturbed than the latter. However, when comparing the number of leaves that each plant presented, as an indicator of overall plant size, no significant differences were found, so in terms of size, plants from both sites were similar. There was a clear relationship in Viola grahamii between light and values of LMA, with plants growing under more shaded conditions expressing smaller LMA than those in higher light. Leaf mass was the parameter that changed from site to site driving the changes in LMA. This change in LMA was likely related to developmental adaptations to maximize light capture in response to environmental conditions such as sun flecks, with thin and broad leaves most commonly produced in shaded environments (Jurik et al., 1982). In understory herbs like Viola blanda, Curtis and Kincad (1984) have shown that sun flecks can represent up to 25% of the light that the species uses for growth.

Leaf mass area values for fully differentiated leaves adapted to high light conditions have been reported in the range of 40 to 80 g m-2 for a diversity of herbaceous plants (Larcher, 2003) and between 19.8 to 30.2 g m-2 in other violet species

(Rothstein and Zak, 2001). Values of LMA in V. grahamii range between 36 and 52 g m-2. One factor that has been suggested to affect LMA is water stress, which produces an increase in LMA due to a reduction in cell expansion (Jurik, 1986). Viola grahamii produces its leaves during the advent of the main rainy season, and the soil remains saturated from almost daily precipitation during the growing season. Therefore moisture most likely was not a stress factor affecting LMA in V. grahamii.

In the regional subtropical climate of V. grahamii, many ecological parameters such as canopy openness and soil moisture remain generally consistent through the growing season. In contrast, temperate violets are subject to very dramatic changes in light availability and soil moisture in the understory of the seasonal forests in which they live. Temperate violets are considered summer green species, producing their leaves before the forest canopy closes, keeping their leaves during the summer and senescing before the canopy opens again in fall (Rothstein and Zak, 2001). Photoperiod is not constant and precipitation is not evenly distributed across the growing season and other factors such as air and soil temperature may play a role in plant responses to their environment that would not occur in regions near the equator. Rothstein and Zak (2001) showed that V. pubescens changes its leaves, altering their structure from sun leaves to shade leaves as the canopy closed, a change related to a decrease in LMA. In contrast,

49 large changes in light availability do not occur during the growing season of V. grahamii. Since most of the leaves expanded earlier in the growing season and all were exposed to similar light levels, leaves collected and analyzed in this study are considered to be representative of the LMA trends in the species; it is unlikely that a change in leaf shape occurs. Jurik (1996) has showed that understory leaves growing under stable conditions will maintain a constant developmental trajectory to produce the same leaf morphology.

Decreased nutrient availability usually increases LMA, although a few exceptions have been found for example in Oryza (Jurik, 1986). My results indicate that an increase in nitrogen content in the soils corresponded to a reduction in LMA, and this matches well with published results by other investigators. In the case of magnesium the opposite correlation was observed, increased magnesium associated with larger LMA values. From these results it is evident that light, soil nitrogen and soil magnesium were important factors regulating LMA. The combined effect of these factors, as evaluated by multiple regression, revealed the same pattern with light having the strongest effect on LMA, followed by nitrogen (with a negative effect), magnesium (with a positive effect) and finally calcium (negative effect). Interestingly, calcium by itself was not significantly correlated with LMA, probably related to its high variance in the area. In the case of nitrogen concentration per unit area, light and nitrogen again played an important role; however, magnesium did not affect it. Calcium again showed a significant combined effect but not one by itself. A study on Viola blanda and its ecological distribution showed a higher incidence of plants in areas richer in

50 phosphorous (Griffith, 1996), a pattern not seen in V. grahamii. In general, though, the documented correlations of ecological factors with LMA and leaf N/ area conformed closely to expectation and patterns from other studies.

The positive relationship between LMA and nitrogen concentration in leaves showed the same pattern found by Garnier et al. (1997) in several perennial species of grasses. Nitrogen content per area of leaf has been reported of 0.61 to 1.51% in V. pubescens (spring-summer values respectively) (Rothstein and Zak, 2001), while values of 2.31 to 2.75 % nitrogen have been noted in V. rotundifolia in forests of West

Virginia, (Gilliam et al., 1996). These levels of leaf nitrogen per area are comparable with those in V. grahamii (2.25-2.4%). In V. sylvatica, leaf area has been reported to be between 7 and 11.4 cm2 by Elias (1985). In V. grahamii those values ranged between 7 and 22 cm2. Overall, measurements of leaf traits in V. grahamii were comparable with those from temperate violet species.

The data indicates that V. grahamii shows substantial plasticity in leaf traits represented by LMA, nitrogen content and nitrogen/leaf area, and that these correlate strongly (positively in some cases, negatively in others) with particular ecological variables, namely light, soil nitrogen, magnesium and calcium. Considering that most ecological factors vary extensively across sites and at lesser extent even within quadrats, it is clear that Viola grahamii is responding to heterogeneous conditions over a range of microsites.

Not every violet species that has been studied shows such broad leaf plasticity.

Viola blanda grows strictly as an understory herb in closed canopy forest and swamps

51 and is well adapted to shade. Individuals grown under high levels of light experience substantial leaf death (Curtis, 1984). In contrast, Viola fimbriatula, a prairie, savanna and meadow species exhibits substantial leaf plasticity and retains the ability to adapt to both shade and light environments (Curtis, 1984).

This leaf plasticity in Viola grahamii as reflected by changes in LMA under different light environments, can be highly advantageous since it allows the species to colonize different light environments, and may explain in part its wide distribution and successful establishment across montane areas of Mexico and regions southward

(Ballard, 1994). Additionally it may help the species to cope with human disturbances impacting light levels in the area. Forests of pine and oak in central Mexico are widely used as a source of wood for local communities; therefore the amount of light reaching violet plants in the ground layer can change dramatically throughout the season or over time due to selective harvests of trees.

Chapter 3:

Influence of annual fluctuations in nutrient availability on the mixed breeding system of the Neotropical violet species Viola grahamii, and on the chasmogamous

flower production in the temperate forest violet Viola striata

Part A. Viola grahamii

Introduction The presence of a mixed breeding system with chasmogamous and cleistogamous flowers produced by the same individual has long-attracted the attention of biologists.

Darwin in his book The Different Forms of Flowers on Plants of the Same Species

(1877) provides a good compilation of what was known at the time about this breeding system. With some exceptions, chasmogamous flowers depending solely upon pollinator visits to produce seeds may frequently fail to set seed, a perilous condition if reproduction is necessary for population survival. Cleistogamous flowers, allow for the production of seeds whether or not pollinators are available and thus ensure reproduction, due to the obligately self-pollinating nature of these flowers. This

“mixed” chasmogamous-cleistogamous reproductive strategy may have evolved as a response to high temporal and spatial variation in the environmental conditions that a plant encounters during each season (Mattila and Salonen, 1995), allowing for the production of seeds despite varied conditions. Symonides (1987) considers this presence of cleistogamous flowers a “pessimistic” approach to reproduction by the plant. Rebdo-Torstensson and Berg (1995) suggest that the allocation of resources to

53 cleistogamous flowers is related to the success of chasmogamous flowers during the season. Thus cleistogamy in this second view point represents a back-up, self- pollination strategy for survival (Schoen and Loyd, 1984). If that is true, it seems to be species related and/or dependent on the environmental factors prevailing in the habitat of the species.

Cleistogamous flowers have the advantage of requiring fewer resources to produce and ensure the production of seeds; however, chasmogamous seeds with theoretically greater gene diversity have the potential to respond to rapid changes in the habitat (Clausen, 1963), as well as reduce intraspecific competition among very closely related plants that a purely cleistogamous system may produce, and counterbalance the theoretically deleterious effects of high inbreeding rates (Beattie, 1976). Therefore, plants with a mixed breeding system would have the ability to produce seeds with two different genotypic variances and genetic makeup, exercising a potentially highly advantageous system that assures both population persistence under more stable ecological conditions and colonization success under variable ones. Little research has been done on wild populations in relating reproductive output in the chasmogamous- cleistogamous mixed breeding system to environmental variables. Although this particular mixed breeding system is represented across many lineages of flowering plants (ca. 48 families and 465 species (Klooster and Culley, 2004)), it is sporadic even within lineages. This is well illustrated in the family Violaceae, where only two of the

25 genera, Viola L. and Hybanthus Jacq., exhibit this intriguing evolutionary strategy. It is, however, most widespread and best known in Viola, in which nearly every infrageneric group and over 95% of species utilize this breeding system.

In Viola mirabilis L. and Viola pubescens Ait., reproduction has been shown to be mainly chasmogamous (Mattila and Salonen, 1995; Culley and Wolfe, 2001), while in

Viola egglestonii Brainerd and some Siberian violet species reproduction is primarily cleistogamous (Baskin and Baskin, 1975; Elisafenko, 2001). In numerous temperate species of Viola, as with other angiosperm lineages with the mixed breeding system, it has been shown that chasmogamous flowers are much larger in size and represent a greater investment of resources in their production (i.e., petals, nectar, flower structure in general) than the cleistogamous flowers (Beattie, 1972; Baskin and Baskin, 1975;

Mayers and Lord, 1983; Le Corff, 1993; Mattila and Salonen, 1995). Chasmogamy has been shown to increase in Viola and other angiosperm groups with increasing light intensity, soil fertility, and/or soil moisture, depending on the species. Chasmogamous flowers are produced only after some time has passed—usually two years or more following seedling establishment, when plants are large enough to invest in these flowers. Cleistogamous flower production in previously studied examples seems to be independent of environmental conditions, plant size or age (Baskin and Baskin, 1975;

Solbrig et al., 1980a; Wilken, 1982; Schoen, 1984; Le Croff, 1993; Diaz and Macnair

1998).

Despite great species diversity and abundance of members of the genus Viola

(Violaceae) in temperate and montane tropical forests around the world, much of the focus on ecological factors and breeding system responses has been primarily on temperate species of North America and Europe, including V. canadensis L. (Culley,

2000); V. cunninghamii Hook.f. (Holswort, 1966); V. egglestonii (Baskin and Baskin,

1975); Viola hirta L.(Rebdo-Torstensson and Berg, 1995); V. mirabilis (Mattila and

Salonen, 1995); V. nuttallii Pursh (Turnbull et al., 1983); V. odorata L. (Mayers and

Lord, 1983; 1984), and V. pubescens (Culley and Wolfe, 2001). To the best of my knowledge, no investigations of ecology, reproduction and genetics have ever been conducted on any montane tropical or subtropical Viola species.

In order to increase our knowledge of breeding systems and ecological influences in the genus Viola worldwide, Viola grahamii, a Mesoamerican species ranging from Northern Mexico to Guatemala in a diversity of montane forest communities, has been chosen as a focus for study. These investigations are part of a series on Viola grahamii to elucidate reproductive biology, population ecology, genetic diversity and hybridization (Cortés-Palomec, 2001). Previous field observations of

Viola grahamii revealed low pollinator visitation and very low chasmogamous fruit set but high cleistogamous fruit set. Rigorous investigations were therefore expected to confirm that most of the seed set would be the result of an extended cleistogamous flowering period, with cleistogamous flower and fruit production largely independent of site-specific environmental factors. Based on other studies, chasmogamous flower production was expected to be regulated at least somewhat by site-specific environmental parameters. To test these predictions, a range of light and soil parameters were measured and compared analytically to measures of chasmogamous and cleistogamous flower and fruit production.

Materials and Methods

Species of Study

Viola grahamii Benth. (Violaceae), known in Mexico as “hoja de pasmo”,

“pensamiento del cerro”, “orejita de ratón” or “Violeta silvestre”, is a Mesoamerican violet that is widely distributed from Northern Mexico to Guatemala (Ballard, 1994). It is a perennial herb thriving across a broad altitudinal range from 1950 to 3600 m above sea level, and occupying a diversity of forest communities including pine and oak forest and cloud forest (Ballard, 1994). Its blooming time has been reported between June and

August, just after the rainy season begins. Chasmogamous flowers are produced during a relatively short period of time while cleistogamous flowers are produced for a much longer period, sometimes to December if conditions are favorable. It is typically found in the shade of the forest canopy and commonly along streams and on small hills

(Calderon de Rzedowski, 1985), but it is also common in natural openings in forests, and persists for at least some time in places that are perturbated (Rzedowski, et al.

2001).

Study Sites

Field observations were conducted during the summers of 2002 and 2003 in the mountains surrounding Lake Pátzcuaro in the state of Michoacán, municipality of

Quiroga, Mexico in a forest community dominated mainly by a mix of pine and oak.

Climatically, these communities fluctuate seasonally between a wet and dry season, however they are considered evergreen. The mean annual precipitation varies between

1500 and 2000 mm, and the mean annual temperature is 18°C (Valerio, 1994). The overstory mainly retains its leaves during all seasons though some oaks can lose their leaves in the dry season, but usually only for a few weeks before the rainy season begins (Pesman, 1962). While the overstory may be considered evergreen, the understory can fluctuate greatly in species diversity and composition from wet to dry season even becoming almost absent in some regions during the dry season.

Two sites corresponding to two distinct Viola populations were established to the north of Lake Pátzcuaro on the southern slopes of Mt. Zirate. The first locality, Site

Santa Fe, [19º41’ N, 101º32’ W] was near the town of Santa Fe de la Laguna. The second locality, Site Quiroga, [19º40’ N, 101° 33’W] was located near the town of

Quiroga. Both sites were established at the same elevation (ca. 2200m above sea level) to minimize effects from differences in community structure due to elevation. Due to the clustered nature of Viola populations, quadrats were randomly established using a stratified random design (Barbour et al., 1999). Twenty quadrats of 0.25 m2 were located in each population by throwing plastic quadrats in an area inhabited by Viola, and marking quadrat corners with flags. For more information on specifics of quadrat establishment see the general introduction, Chapter 1.

Sampling of environmental variables

To characterize the light environment above each quadrat, hemispherical photographs were taken on overcast days or in early morning before the sun came over the mountain using a Nikon Coolpix 955 digital camera equipped with a 180° Nikon

FC-E8 Fish-eye converter lens. Images were saved using the format FINE (1:4 compression JPEG, 1600 x 1200 pix) (Frazer et al., 2001). To check for any potential changes of light availability through the growing season, weekly pictures were taken from early June to late August. Images were analyzed to obtain light availability values as percent canopy openness, using the gap light analyzer (GLA) version 2.0.4. image processing software (Frazer et al., 2001). Light availability data were arcsine transformed, and an analysis of variance with weekly measurements as a random effect variable was performed to test for significant changes in light availability per quadrat throughout the growing season. A one-way ANOVA was performed to check for differences in light availability between the two study sites.

Soil properties for each of the 40 quadrats were determined from soil cores immediately stored in Ziploc bags. Soil moisture was determined using the gravimetric method (Hadley and Levin, 1967), weighing soil samples first upon collection in the field and later, after drying. Soil pH, magnesium, nitrogen, potassium, calcium and phosphorous were determined from fully air-dried soil in the laboratory at Ohio

University. Soil pH was determined by dissolving 25g of air-dried soil in 100 ml of distilled, de-ionized water and testing using a Corning 430 pH/ion meter w/pH electrode. Soil nitrogen was measured using the Cadmium Reduction Method, with the

Hach Pocket colorimeter Analysis system [Nitrate (NO3-N), High Range 0 to 30 mg/L

NO3-] (Hach Co. USA). Calcium, magnesium and phosphorous were extracted using the

Mehlich III method (Mehlich, 1984). Calcium and magnesium were determined using atomic absorption (Varian SpectrAA instrument. Varian Instruments, Australia), while

59 phosphorous was determined using a Cary 50 Bio UV-visible spectrophotometer

(Varian, Australia). All raw soil chemical data were corrected for dilution factors used in the different analyses, converted into µg of element g-1 of soil (Robertson et al.,

1999), and log10 transformed to meet assumptions of a normal distribution. One value for potassium and one value for magnesium had to be winsorized (Sokal and Rohlf,

1995). Soil moisture data were arcsine transformed to approximate a normal distribution.

Reproductive behavior

During the blooming season of 2002 and 2003 (June-August), weekly visits were made to the study sites to record the phenology of chasmogamous and cleistogamous flowers (Chapter 4). The total number of chasmogamous and cleistogamous flowers and fruits produced by marked individuals was recorded. To evaluate differences in flower and fruit production between chasmogamous and cleistogamous types, a Mann-Whitney U-test was performed (Sokal and Rohlf, 1995), with arcsine transformation of percentages where appropriate to meet the assumption of univariate normality. Seed number between the two types was determined from sixty randomly selected mature capsules of each type (totaling 60 chasmogamous and 60 cleistogamous fruits), and was compared after log10-transformation using a t-test (Sokal and Rohlf, 1995). To test for potential biomass differences between chasmogamous and cleistogamous seeds, 368 mature seeds from 20 randomly selected capsules of each type were weighed individually on an analytical balance, with weight recorded to the nearest

60 milligram. The biomass values for the two types of seeds were compared using a t-test.

The mass of each seed was determined independently to determine any possible correlation (Pearson’s correlation) between the number of seeds per capsule and mass.

To obtain a measure of plant vigor, the numbers of leaves produced by 85 reproductive individuals were counted at the peak of blooming during 2002. These values were correlated statistically with the total number of flowers from the same individuals through the entire reproductive season using Pearson’s correlation coefficient (Zar,

1999).

To examine potential influences of ecological factors on chasmogamous and cleistogamous flower production, the relative number of chasmogamous and cleistogamous flowers produced per individual per quadrat during the blooming season was correlated statistically with measured environmental factors (i.e. light availability, soil moisture, soil Ca, Mg, N, P, and K) using Pearson’s correlation coefficient (Zar,

1999). These correlations were performed for the two years, 2002 and 2003, independently to look for recurrent or divergent patterns. Additionally, the reproductive data collected from 2003 was correlated with soil characteristics during 2002 to evaluate the possible cumulative effect of nutrients in reproduction due to the perennial nature of V. grahamii.

A multiple regression approach was used to examine the combined effect of all the independent variables on reproduction. Initial selection of variables was performed using the multivariate variable selection and the “all possible regression” procedure of

NCSS (Hintze, 1996). The regression model was constructed using the selected

61 variables to best explain the production of chasmogamous flowers, cleistogamous flowers, chasmogamous fruits, and cleistogamous fruits. All statistical analyses described above were performed using NCSS (Hintze, 1996).

Results

Environmental factors

For the year 2002 Quiroga and Santa Fe differed significantly in a number of environmental parameters, with quadrats at Santa Fe experiencing more shade, less soil magnesium and potassium and more nitrogen. In the year 2003 a similar pattern was observed between the two sites, except that nitrogen did not differ (ANOVA: d.f. =1,30;

F=0.12; p = 0.72). Some differences in the pattern of environmental changes were observed in each site between the two years. Santa Fe showed a significant reduction in calcium (ANOVA: d.f. =1,39; F=9.25; p = 0.004) and magnesium (ANOVA: d.f. =1,39;

F=10.31; p = 0.003), but an increase in pH (ANOVA: d.f. =1,39; F=8.99; p =0.004) and moisture (ANOVA: d.f. =1,39; F=22.16; p <0.005) (Table 3.1). Quiroga expressed a significant reduction in magnesium (ANOVA: d.f. =1,22; F=5.63; p =0.03) and potassium (ANOVA: d.f. =1,22; F=4.68; p =0.04), but an increase in nitrogen

(ANOVA: d.f. =1,22; F=6.39; p = 0.01) and moisture (ANOVA: d.f. =1,22; F=10.92; p=0.003) (Table 3.1). The overall results indicated a surprising degree of microhabitat heterogeneity from year to year. At Quiroga 12 quadrats out of the 20 sampled were lost due to the activity of local wood collectors between years.

Reproductive behavior

A total of 506 flowering Viola grahamii individuals were tracked for reproductive behavior during the year 2002, from which a total of 261 chasmogamous and 382 cleistogamous flowers were produced [Santa Fe: chasmogamous = 141; cleistogamous = 192. Quiroga: chasmogamous=130; cleistogamous= 190]. Both sites produced more cleistogamous flowers at statistically significant levels across quadrats

(One way ANOVA: d.f.= 1,75; F= 6.46; p=0.013). During the year 2003, 477 individuals of V. grahamii were tracked, from which a total of 165 chasmogamous and

251 cleistogamous flowers were produced. These differences were again statistically significant across quadrats (One way ANOVA: d.f. = 1, 56; F=6.96; p=0.015). Thus, in both years cleistogamy was the most common flower type produced.

Considering fruiting success across quadrats at both sites together in 2002, ca.

68 % of chasmogamous flowers yielded fruits, while ca. 81% of cleistogamous flowers did so. The same general proportion of fruiting success was observed in 2003, where ca.

60% of the chasmogamous flowers set fruit as compared with ca. 78% of cleistogamous flowers (Table 3.2). A Mann-Whitney U-test of arsine-transformed data showed statistically greater fruiting success by cleistogamous over chasmogamous flowers during both 2002 (Z-value: 2.73; p = 0.006) and 2003 (Z-value: 2.44; p =0.01). Results from the previous analysis and the present one indicate that cleistogamous flowers were not only the most abundant flower type but also the most successful in leading to fruit set.

Table 3.1: Summary of soil chemical properties (in µg/g soil), soil moisture (%) and canopy openness in two populations of Viola grahamii (Santa Fe and Quiroga) during the year 2002 and 2003. n=20 except for Quiroga 2003, n=12. Mean ± s.d. Means with different letters between columns are significantly different (ANOVA, p<0.05).

Parameter Santa Fe Quiroga 2002 2003 2002 2003 Calcium 3164.90 ± 2186.80 a 1573.40 ±1110.40 b 2166.90 ± 2004.90 2093.40 ± 1145.12 (µg/g soil) Phosphorous 3.01 ± 1.43 2.60 ± 1.04 3.95 ± 3.19 4.11 ± 3.17 (µg/g soil) Magnesium 805.00 ± 323.84 a 482 .99 ± 156.10 b 1035.06 ± 355.77 a 757.00 ± 302.32 b (µg/g soil) Nitrogen 37.48 ± 15.01 78.84 ± 73.00 17.70 ± 12.18 a 67.02 ± 54.90 b (µg/g soil) Potassium 1359.56 ± 377.40 1322.34 ± 301.44 1914.29 ± 424.43 a 1755.00 ± 229.59 b (µg/g soil) pH 6.35 ± 0.21 a 6.52 ± 0.12 b 6.48 ± 0.27 6.55 ± 0.13 Soil Moisture 31.96 ± 2.86 a 35.91 ± 2.34 b 30.94 ± 4.03 a 35.94 ± 3.92 b (%) Light 24.72 ± 5.61 25.17 ± 6.55 38.86 ± 10.24 42.85 ± 8.10 (% openness)

Table 3.2: Percentage of chasmogamous and cleistogamous flowers of individuals of Viola grahamii that successfully became fruits during 2002 and 2003. Ranges are the result of among quadrat variation. 40 quadrats for year 2002, 32 quadrats for year 2003.

Santa Fe Quiroga Chasmogamous Cleistogamous Chasmogamous Cleistogamous Year 2002 Mean 61. 75 % 85.40 % 76.94 % 91.20 % Standard deviation ± 25.72 ± 14.40 ± 22.08 ± 11.26 Range 9-100 62-100 20-100 60-100

Year 2003 Mean 68.02 % 86.02 % 48.00 % 64.53 % Standard deviation ± 26.50 ±16.24 ± 15.20 ± 17.60 Range 33-100 57-100 25-77 40-100

No statistical differences were revealed between seed number in chasmogamous and cleistogamous fruits (T-test: t-value=0.54; p = 0.59). On average, each chasmogamous capsule produced 17.93 (± 7.37) seeds, and each cleistogamous capsule produced 18.68 (± 7.68) seeds.

Seed biomass ranged from 0.20 to 1.70 mg. On average each chasmogamous seed weighed 1.03 (±0.28) mg, and each cleistogamous seed, 0.92 (±0.29) mg.

Chasmogamous seed biomass was statistically higher (t-test: t-value = 3.95, p<0.05) than that of cleistogamous seeds. Seed biomass and number of seeds per capsule showed a strong correlation in both chasmogamous capsules (r = 0.36, p <0.005) and cleistogamous capsules (r = 0.29, p <0.05) when each of the seeds was considered independently. When the biomass of all seeds per capsule was correlated against total number of seeds per chasmogamous or cleistogamous capsule, the correlation was still significant (chasmogamous: r = 0.88, p<0.005) (cleistogamous: r = 0.74, p =0.021).

The number of leaves per individual was statistically correlated to the number of chasmogamous and cleistogamous flowers produced and to total flower production.

From the 85 individuals analyzed, most individuals had an average of four leaves (Table

3.3). There was a significant positive correlation between number of leaves and number of flowers per individual, in particular when looking at total number of flowers produced: CH: r = 0.94, p= 0.003; CL: r = 0.89, p = 0.002; total flowers: r = 0.94, p =

0.003 (Figure 3.1). The results suggest a tendency for larger (more vigorous or older) individuals to produce more flowers than smaller ones. When the two different types of flowers are considered independently the observed pattern is the same.

Table 3.3: Number of chasmogamous and cleistogamous flowers and total number of flowers produced per individual per site based on the number of leaves that each individual produced. N=total number of plants; CH=Average number of chasmogamous flowers produced; CL= Average number of cleistogamous flowers produced. F= total average number of flowers produced.

Leaves per N CH CL F plant 1 7 0.42 0.28 0.71 2 10 1.10 1.20 2.30 3 18 0.77 0.88 1.66 4 27 1.48 1.88 3.37 5 17 2.05 2.53 4.58 6 3 2.33 2.00 4.33 7 1 3.00 4.00 7.00 8 2 4.50 3.00 7.50 total 85

ers 5 r = 0.947, p = 0.003 w o l 4

3 gamous f o 2 chasm

1 er of b

m 0 Nu -1 0246810 Number of leaves

Figure 3.1: Relationship between total number of flowers (chasmogamous and cleistogamous) per plant and number of leaves per plant. Average and standard deviations are shown. (Data shown in Table 3.3). Pearson’s correlation coefficient and level of significance of the correlation is included. Values for seven and eight leaves show a very low standard deviation due to very few (1-2) individuals in this size class.

Environmental influences on reproductive responses

Tests of influence by different environmental factors on reproductive output in

2002 at Santa Fe revealed only one significant correlation, between the number of chasmogamous fruits produced and light availability across quadrats (Table 3.4). The same positive correlation was found for Quiroga, in addition to other significant correlations between chasmogamous flowers and nitrogen, calcium, and potassium; cleistogamous flowers and magnesium; and cleistogamous fruits and magnesium (Table

3.4). Significant correlations were found in plants in 2003 at Santa Fe between chasmogamous flowers and pH and light, cleistogamous flowers and pH and magnesium, chasmogamous fruits and pH and light, and cleistogamous fruits and magnesium. For Quiroga, only two significant correlations were present in 2003 between chasmogamous flowers and pH and chasmogamous fruits and pH (Table 3.4).

Most multiple regression models generated were not significant for Santa Fe in

2002 [chasmogamous flowers: F=1.51, p=0.25, power=0.10, R2=0.35, R2 Adj. = 0.12; cleistogamous flowers: F=1.45, p=0.27, power=0.11, R2=0.28, Adj. R2=0.09; cleistogamous fruits: F=1.13, p=0.34, power= 0.09, R2= 0.23, Adj. R2=0.02], except for chasmogamous fruits [chasmogamous fruits: F= 3.97, p=0.02, power=0.24, R2=0.52,

Adj. R2 = 0.39, (Table 3.5)]. For Quiroga in 2002, a significant model was generated for chasmogamous flowers (F=5.98, P = 0.004, power =0.35, R2 = 0.61, Adj. R2 = 0.51), chasmogamous fruits (F= 6.99, P =0.002, power = 0.41, R2=0.65, Adj. R2 = 0.56) and cleistogamous fruits (F= 7.85, P = 0.002, power = 0.53, R2=0.60, Adj. R2 = 0.52 (Table

3.5). No significant models were generated for cleistogamous flowers (F=2.68, P=0.07,

68 power = 0.17, R2=0.42, Adj. R2 = 0.26). Significant multiple regression models were generated for Santa Fe in 2003, for all the reproductive variables evaluated: chasmogamous flowers (F= 10.55, P = 0.0002, power = 0.59, R2=0.73, Adj. R2 = 0.66), cleistogamous flowers (F= 3.99, P = 0.02, power = 0.24, R2=0.52, Adj. R2 = 0.39), chasmogamous fruits (F= 7.49, P = 0.0015, power =0.44, R2=0.66, Adj. R2 = 0.57) and cleistogamous fruits ( F = 4.65, P = 0.012, power = 0.28, R2=0.55, Adj. R2 = 0.43)

(Table 3.5). Significant models for Quiroga in 2003were generated for chasmogamous flowers (F =5.49, P =0.03, power = 0.19, R2=0.82, Adj. R2 = 0.67) and chasmogamous fruits (F = 5.43, P = 0.026, power = 0.24, R2=0.76, Adj. R2 = 0.62). Non-significant models were generated for cleistogamous flowers (F = 2.03, P = 0.23, power = 0.08,

R2=0.70, Adj. R2 = 0.36) and cleistogamous fruits (F = 1.63, P = 0.28, power =0.08,

R2=0.57, Adj. R2 = 0.22) (Table 3.5).

When the data from 2002 was considered in evaluating flower production during

2003, significant correlations were found only for chasmogamous flower production.

Phosphorous was negatively correlated, and light was positively correlated with chasmogamous flower production in Santa Fe, while calcium was negatively correlated in Quiroga (Table 3.6). In the multiple regression model chasmogamous flower production was significant with phosphorous and calcium in both sites and nitrogen and light for Santa Fe and magnesium for Quiroga (Table 3.7).

Table 3.4: Pearson’s correlation coefficients between chasmogamous flowers and fruits and cleistogamous flowers and fruits with soil chemical properties and light. Significant correlations (p < 0.05) are marked with asterisks.

Chasmogamous Cleistogamous Chasmogamous Cleistogamous flowers flowers fruits fruits Santa Fe 2002 pH -0.34 -0.35 -0.33 -0.27 Nitrogen -0.35 -0.28 -0.25 -0.21 Magnesium -0.04 0.01 -0.02 -0.12 Calcium -0.05 0.10 -0.01 -0.06 Phosphorous -0.18 -0.11 -0.10 -0.22 Potassium 0.19 0.04 -0.02 0.06 Light 0.13 0.06 0.47*** 0.09 Moisture -0.13 -0.06 -0.22 -0.17

Quiroga 2002 pH -0.08 0.22 -0.23 0.20 Nitrogen -0.44 *** -0.05 -0.14 -0.11 Magnesium 0.18 -0.45 *** -0.22 -0.50 *** Calcium 0.46 *** 0.10 -0.41 0.09 Phosphorous 0.05 0.22 0.01 0.26 Potassium -0.45 *** 0.39 0.39 0.41 Light 0.25 -0.26 0.49 *** -0.19 Moisture 0.05 -0.01 0.14 -0.03

Santa Fe 2003 pH -0.67 *** -0.45 *** -0.72 *** -0.32 Nitrogen 0.16 0.10 -0.01 0.24 Magnesium -0.36 -0.45 *** -0.25 -0.54 *** Calcium -0.07 -0.42 -0.21 -0.42 Phosphorous -0.01 -0.13 -0.13 -0.02 Potassium -0.33 -0.21 -0.28 -0.30 Light 0.69 *** 0.35 0.61 *** 0.32 Moisture -0.21 -0.24 -0.39 -0.17

Quiroga 2003 pH -0.69 *** -0.47 -0.64 *** 0.08 Nitrogen 0.15 0.30 0.14 0.17 Magnesium 0.11 0.29 0.17 0.38 Calcium -0.38 -0.31 -0.46 -0.10 Phosphorous -0.41 -0.27 -0.42 -0.14 Potassium -0.16 0.06 -0.04 0.26 Light -0.18 0.14 -0.27 0.27 Moisture -0.55 -0.38 -0.35 -0.24

Table 3.5: Multiple regression models constructed from soil chemical data and light characteristics for Viola grahamii growing under different environmental combinations. Only significant models (p<0.05) are presented. (CH=chasmogamous, CL=cleistogamous).

Regression Model Coefficient ± SE CH fruits Santa Fe 2002 (R2=0.51, Adj. R2 = 0.38) Intercept 4.44 3.04 pH -9.33 3.98 Nitrogen -0.53 0.36 Magnesium 0.46 0.27 Light 0.78 0.23

CH flowers Quiroga 2002 (R2=0.61, Adj. R2 = 0.51) Intercept 13.36 4.04 Nitrogen -0.55 0.31 Magnesium -1.95 0.68 Potassium -3.25 1.02 Light 1.07 0.41

CH fruits Quiroga 2002 (R2=0.65, Adj. R2 = 0.55). Intercept 4.81 2.66 Nitrogen -0.49 0.25 Calcium -0.63 0.24 Potassium -1.86 0.77 Light 1.07 0.30

CL fruits Quiroga 2002 (R2=0.60, Adj. R2 = 0.52) Intercept -2.50 5.34 Magnesium -3.34 0.94 Phosphorous 1.47 0.57 Potassium 4.01 1.40

CH flowers Santa Fe 2003 (R2=0.73 Adj. R2=0.66) Intercept 24.63 7.86 pH -29.35 8.97 Nitrogen 0.28 0.16 Potassium -0.94 0.56 Light 0.58 0.26

CL flowers Santa Fe 2003 (R2=0.515, Adj. R2=0.38) Intercept 34.18 11.74 pH -34.98 14.74 Nitrogen 0.36 0.30 Magnesium -1.28 0.67 Calcium -0.63 0.41

Table 3.5 Continued

CH fruits Santa Fe 2003 (R2=0.666, Adj. R2=0.577) Intercept 22.08 6.90 pH -22.84 7.07 Nitrogen 0.16 0.13 Light 0.24 0.21 Moisture -1.02 0.79

CL fruits Santa Fe 2003 (R2=0.5539, Adj. R2 = 0.4350) Intercept 25.65 10.35 pH -24.55 12.99 Nitrogen 0.47 0.26 Magnesium -1.42 0.59 Calcium -0.61 0.36

CH flowers Quiroga 2003 (R2=0.82, Adj. R2=0. 67) Intercept 58.83 11.90 pH -61.91 15.05 Nitrogen -0.50 0.38 Potassium 7.76 3.10 Light 0.34 0.63 Moisture -7.79 2.73

CH fruits Quiroga 2003 (R2=0.756, Adj. R2=0.617) Intercept 17.51 6.10 pH -36.96 9.69 Nitrogen -0.57 0.29 Calcium -0.83 0.38 Potassium 5.20 1.88

Table 3.6: Pearson’s correlation coefficients between soil data from 2002 and flowering in 2003 for Viola grahamii. Significant correlations (p < 0.005) are marked with asterisks.

Santa Fe Quiroga CH flowers CL flowers CH flowers CL flowers Nitrogen 2002 0.21 -0.18 -0.30 -0.27 Magnesium 2002 -0.36 -0.28 -0.24 -0.01 Calcium 2002 -0.40 0.01 -0.80 *** 0.22 Phosphorous 2002 -0.53 *** -0.22 -0.09 -0.43 Potassium 2002 -0.05 -0.06 -0.53 -0.25 Light 2002 0.58 *** 0.31 -0.19 0.28

Thus, substantial changes in chasmogamous and cleistogamous flower and fruit production have been documented at each site from year to year and between sites in a given year. Moreover, individual reproductive responses were not always clearly correlated with substantial site differences or within-site changes in environmental parameters from year to year, although chasmogamous flower and fruit production seemed to show a more consistent relation with light availability and certain other soil properties. In general, results suggest a surprisingly dynamic microenvironment across each site from year to year, significant differences between sites, and substantial decoupling of reproductive behavior from seasonal, annual and site-specific changes.

Table 3.7: Multiple regression models for chasmogamous flower production in Viola grahamii in 2003 using soil data from the previous year (2002). The models were significant at p < 0.005.

CH flowers 2003 Santa Fe (R2 = 0.65; Adj. R2 =0.55) Variable Regression coefficient Standard error Intercept -2.16 1.35 Nitrogen 2002 0.63 0.42 Calcium 2002 -0.49 0.23 Phosphorous 2002 -0.55 0.41 Light 2002 94.98 0.28

CH flowers in Quiroga (R2 = 0.78; R2 = 0.68) Intercept -0.30 1.01 Magnesium 2002 -0.31 0.29 Calcium 2002 0.70 0.14 Phosphorous 2002 -0.37 0.20

Discussion

Due to the evergreen nature of the forest sites inhabited by Viola grahamii, no changes in canopy openness were found throughout the blooming season. Some changes in light were recorded in individual quadrats, but those changes were not significant and mainly due to human activity rather than intrinsic changes in the overstory itself. Concerning soil properties, most were surprisingly variable, changing from year to year. Changing soil conditions yield a heterogeneous environment in which V. grahamii must survive and reproduce. The study was conducted during the rainy season, and therefore no changes in moisture were recorded during a particular year in either site; however, an increase in the amount of soil moisture in 2003 was evident in both sites, probably the result of different yearly patterns of precipitation.

There were no significant differences between the two sites in pH, even though the overstory vegetation is different. Quiroga is richer in Quercus species while Santa Fe has a higher proportion of Pinus species, so a more acidic soil would be expected at

Santa Fe; this was not observed. Interestingly, pH at Santa Fe became more basic between 2002 and 2003. Changes in most of the soil chemical properties (nitrogen, calcium, magnesium, and potassium) from year to year speak of soils that are changing due to natural processes, or to human activities in the area such as livestock grazing or collecting of wood for local consumption, although any discussion in this regard would be speculative.

Results clearly show that V. grahamii reproduces at a modest level through chasmogamy but mainly through cleistogamy, the latter the most abundant flower type

74 and evidently the more successful in producing seeds. This conclusion was upheld in both years of the present study, and from a different study conducted in 2000 (Cortés-

Palomec, 2001). Chasmogamous flower production was not correlated with light, however chasmogamous fruit production was correlated with the amount of light reaching the plant with greater numbers of chasmogamous flowers successfully producing seeds under higher light conditions. This can be explained by the dependence of these flowers to pollinator visits in order to produce seeds. As part of a related study, fifteen chasmogamous flowers of Viola grahamii were bagged before anthesis to prevent insect visitations and observed over a period of time to determine whether self- pollination was possible in the absence of pollinators; these bagged flowers failed to set seed. This result suggests that selfing in chasmogamous flowers does not occur and therefore all chasmogamous seeds must be the result of cross-pollination and therefore their correlation with higher levels of light. Several floral visitors were observed in V. grahamii, but only on sunny, clear days, and on flowers in partial or full sunlight. The narrow and well-defined blooming period corresponds mainly to the rainy season.

Under these conditions, mainly or mostly cloudy days were common, reducing the periods of pollinator activity and consequent pollinations of the species. Plants exposed to more sunflecks or direct light would receive more visitors and therefore produce more fruits. Beattie (1972) suggested that the dependence of chasmogamous flowers on pollinator visits to produce seeds sometimes leads to frequent failure in seed set under adverse conditions, and could explain why cleistogamous flowers produce the majority of the seeds in any given season. The failure to set seed in chasmogamous flowers, in

75 the absence of pollinators, has been observed in V. pedata L., V. rostrata Pursh, V. pensylvanica Michx. [=V. pubescens], V. blanda Willd. (Beattie and Culver, 1979), V. riviniana and V. hirta (Berg, 2003). These results contrast with those of Culley (2002) and Culley and Grubb (2003) for Viola pubescens in which chasmogamous flowers had the ability to self-pollinate if cross-pollination did not take place, a phenomenon they term “delayed selfing”, and which has been reported in other cleistogamous species

(Schemske, 1978; Stewart, 1994; Porras and Munoz Alvarez, 1999).

Cleistogamous fruits seem to be considerably more predated on than chasmogamous ones. Cleistogamous flowers and fruits are produced on shorter pedicels and are therefore closer to the ground than chasmogamous capsules. A good number of developing cleistogamous capsules would disappear from visit to visit (hence not all the cleistogamous flowers successfully produced seed, the reason why cleistogamous success was not close to 100%). It is unknown which animals could be feeding on these capsules, but it has been suggested that seed predation in violets in temperate regions is due to rodents such as voles (Mattila and Salonen, 1995).

In terms of the number of seeds produced by each flower type, we observed that

V. grahamii produces an equal number of seeds in both chasmogamous and cleistogamous capsules, the same pattern described in other violet species (Culley,

2002). Evidently, differential investment in production of the two flower types does not manifest itself in different amounts of seed production at the level of the capsule but in a substantially larger proportion of cleistogamous capsules in the total capsule output.

In other cleistogamous species such as Oxalis acetosella L., a higher number of seeds

76 from cleistogamous capsules has been reported (Rebdo-Torstensson and Berg, 1995).

The mean number of seeds produced in V. grahamii is 18 seeds per capsule, a value similar to that in other violet species. Similar numbers of seeds (19.93-17.01) have been recorded in Viola mirabilis (Mattila and Salonen, 1995), fewer (8-11) have been noted in V. pubescences (Culley, 2002) and more (45) in V. fimbriatula Sm. (Solbrig et al.,

1988). The largest number of seed produced by any fruit in V. grahamii was 27, very low when compared to 76, the largest number in V. fimbriatula. There may be fewer V. grahamii seeds than V. fimbriatula because the former are larger/heavier and therefore there are fewer of them in each capsule. Since there were no differences in the number of seeds per capsule between the chasmogamous and cleistogamous capsules, looking at the number of capsules that were produced in a population was a good indicator of the number of seeds being produced. We can therefore say that cleistogamous seeds are the most abundant type of seed produced by Viola grahamii.

Viola grahamii seed biomass is on average 1 mg for chasmogamous seeds and

0.9 mg for cleistogamous seeds. The average biomass of seeds in other violet species is

0.831 mg for V. fimbriatula, 0.656 mg for V. sororia Willd., and 0.733 for V. blanda

(Solbrig et al., 1988, who unfortunately did not distinguish between chasmogamous and cleistogamous seeds) and 2.56 mg for chasmogamous and 2.48 mg for cleistogamous seeds of V. mirabilis (Mattila and Salonen, 1995). In V. grahamii, chasmogamous seeds had larger biomass than cleistogamous seeds. The larger biomass of chasmogamous seeds suggests a greater investment in these seeds on a per capsule basis.

The number of leaves per plant showed a significant correlation with the number

77 of flowers produced per individual; the larger the number of leaves, the older or more vigorous the individual, and the more flowers it produced (Figure 1). The same phenomenon has been described in Viola blanda and V. pallens (Banks ex Ging.)

Brainerd (Newell et al., 1981), and Viola sororia (Solbrig et al., 1980). In those species the number of fruits per plant was exponentially related to the number of leaves. In this study we looked at the number of flowers produced, rather than the number of fruits, since we considered flowers to be a better indicator of the reproductive investment by the plant. Most flowering individuals had between two and five leaves and an average of four, with the largest having eight leaves.

Regarding environmental factors that may influence one or more aspects of the breeding system, chasmogamous fruit production was positively correlated with light availability, except for Quiroga in 2003. At both sites in 2002 and at Santa Fe (which was undisturbed) in 2003, quadrats in partial or full sun successfully set more fruits than quadrats in shade. Quiroga was not significant in 2003, perhaps due to a higher canopy openness beyond the threshold at which it could be significant.

The negative effect of nitrogen and potassium on production of chasmogamous flowers at Quiroga in 2002 was unexpected. Chasmogamy is suggested by theory and previously published observations to be more energetically expensive, and therefore more chasmogamous flowers should be produced under more favorable growing conditions. Nevertheless, our findings contradict this theory. Matila and Salonen (1995) showed that in the perennial V. mirabilis increased nutrient availability due to fertilization did not increase the production of chasmogamous flowers either, suggesting

78 perhaps a genetic regulatory mechanism. In Sanguinaria canadensis Marino et al.

(1997) found that plants growing in high levels of nutrients at low light conditions could not cope with the extra nutrients and died due to nutrient burn. The data express substantial variance indicative of moderate to high levels of heterogeneity in several environmental parameters, among quadrats, between sites and between years. A great deal of heterogeneity has been reported in mixed mesophytic forest sites in longer-term studies (Crozier and Boerner, 1984), and the same site heterogeneity over time and space is evidently true for the V. grahamii sites in Mexico.

The soil characteristics from 2002 showed a significant influence in chasmogamous reproduction during 2003, although a clear, repeatable pattern was not observed. Perhaps looking at several years worth of data could clarify some of the reproductive patterns. The perennial nature of this species can also be masking the effects of nutrients and light availability in reproduction since nutrients can be stored and past conditions can reflect the reproductive outcome in a particular year.

Despite this multi-scale site heterogeneity, however, chasmogamous flower output continues in a relatively predictable manner, showing a highly predictable phenological pattern that seems very tightly tied to seasonal climatic variables (Chapter

5). Fruit set is probably closely tied with levels of pollinator visitation. Cleistogamous flower and fruit production, conversely, seems to be independent of all measured or inferred site-specific or regional environmental conditions. We interpret the temporally and regionally inconsistent “correlations” between chasmogamous and cleistogamous flower and fruit production on the one hand, with various environmental variables on

79 the other, as largely coincidental correlations manifested by a highly heterogeneous environment from year to year and site to site. The best explanation for the evident patterns of flower production invokes a process largely independent of environmental heterogeneity, with fruit set primarily based on pollinator behavior. It seems from the data that plant size is the main factor regulating flower production, both chasmogamous and cleistogamous, and being more or less independent of environmental factors.

Even though there were a lot of differences in soil properties from one year to another and a lot of heterogeneity in the area for the two years that the species was followed, the same reproductive trends in the population as a whole were observed.

More cleistogamous flowers and fruits were produced independently of localized changes in the area. The area of distribution of Viola grahamii is very large and future studies should involve other populations perhaps growing in more contrasting habitats to look at their effect in species reproduction. Other montane neotropical species should also be examined to see if the pattern holds true for them.

Part B. Viola striata

Introduction

In order to test the hypothesis that greater nutrient availability will lead to an increase in chasmogamous flower production in a natural setting, Viola striata Aiton was chosen as model species. Viola striata belongs to the section Viola of the genus

Viola, one of the largest of several sections into which the genus has been subdivided

(Becker, 1925). Within section Viola the species belongs to subsection Rostratae, a widespread northern hemisphere group (Ballard et al., 1999). Chasmogamous flower production was documented over two years in two populations of the species. Soil data and light availability were recorded during this time and analyzed to test the hypothesis.

Due to the perennial nature of V. striata and the fact that sexual reproduction may not respond to nutrient increases over only one growing season (Marino et al., 1997), reproduction from year two was analyzed in two separate data sets, the first with environmental data from year one and the second data set with environmental data from year two. I hypothesized that chasmogamous flower reproduction would increase with natural nutrient availability.

Materials and Methods

The study species

Viola striata, the striped violet, is a stemmed perennial herb that is common in temperate forests of the northern and eastern United States and southern Canada

(Gleason and Cronquist, 1991). It specifically inhabits low moist places, usually along streams and in floodplains with alkaline substrates, where it sometimes can exhibit a weedy habit (Gleason and Cronquist, 1991). It produces chasmogamous flowers early in the growing season, before the canopy closes, and cleistogamous flowers once the canopy has closed, throughout late spring to late summer or fall (Chapter 4).

Study Sites

Field observations were conducted during the summers of 2002 and 2003 in two populations located in Strouds Run State Park, Canaan Township, Athens County, Ohio in a mixed mesophytic forest. The first site, designated as Campground, was located immediately north of the park campground area at 39º21’N, 82º02’W. This site was situated partially in the floodplain of a small stream dominated by Acer negundo L.

(Box Elder) and on the edges of a plantation of Pinus resinosa Aiton (Red Pine). The second site, designated Cemetery, was located beyond the northwest end of Dow Lake adjacent to the Pioneer Cemetery trail at 39º21’N, 82º02’W. Most of the site was situated along the floodplain of Strouds Run and Gillette Run and thus was exposed to periodic flooding. Both sites were positioned at the same elevation (ca. 210 meters above sea level).

Sampling strategy and ecological data

Quadrat establishment and sampling of environmental variables followed that of

Viola grahamii described above.

Reproductive data

During the blooming season (April-June), weekly visits were made to the study sites to record the phenology of chasmogamous and cleistogamous flowers. The total number of chasmogamous flowers and fruits produced by marked individuals was recorded for each week.

Statistical analyses

Due to the non-normality of the data, a Mann-Whitney U test was performed to check for differences in chasmogamous flower production between the two sites. An

ANOVA was performed with the year as a fixed variable to check for differences in flower production between years.

To examine potential influences of ecological factors on chasmogamous flower production, the relative number of chasmogamous flowers per individual per quadrat during the blooming season was correlated statistically with environmental factors (i.e. light availability, soil moisture, soil pH, soil calcium, magnesium, nitrogen, phosphorous, and potassium) using Pearson’s correlation coefficient (Zar, 1999). To meet the assumptions of the analysis, one value for pH at Cemetery in 2002, and one value for magnesium in Campground in 2002 and at Cemetery in 2003 were winsorized

(Sokal and Rohlf, 1995).

These correlations were performed for years 2002 and 2003 independently to look for recurrent or divergent patterns. Since canopy openness (reflecting light availability) changed considerably from the first week to the fourth week as the canopy closed, the values for the first four weeks were included individually in the correlation.

A multiple regression approach was used to look at the combined effect of all independent variables on reproduction. Initial selection of variables was performed using the multivariate variable selection and the “all possible regression” procedures of

NCSS (Hintze, 1996). The regression model was constructed using the selected variables to best explain the production of chasmogamous flowers and chasmogamous fruits. Due to the perennial nature of V. striata, an additional analysis for chasmogamous flower and fruit production for 2003 including all variables for 2003 plus nitrogen, calcium, phosphorous, potassium, and magnesium from the previous year

(2002) was performed to look for possible past influences on subsequent reproductive output. Only potassium and phosphorous were not correlated between years, so only these two variables were evaluated together, to avoid problems of multicollinearity.

Results

Environmental factors

Comparison of average values for environmental factors at the quadrat level among sites indicated only one significant difference in magnesium during 2002.

Among all variables the standard deviation was high, suggesting a highly heterogeneous soil environment across quadrats (Table 3.8).

Table 3.8: Summary of soil chemical properties in two populations of Viola striata (Campground and Cemetery) in southern Ohio during the years 2002 and 2003. N = 20, Mean ± s.d. Means with different letters between columns are significantly different (ANOVA, p<0.05). For light, two values are presented, top: average at the beginning of the growing season, bottom: average at the end of the study.

Parameter Campground Cemetery

2002 2003 2002 2003 Calcium 1041.00 ± 640.00 854.81 ± 375.86 1600.00 ± 931.00 1150.72 ± 527.90 (µg/g soil) Phosphorous 17.64 ± 6.03 14.75 ± 2.77 34.24 ± 28.50 29.73 ± 34.96 (µg/g soil) Magnesium 291.30 ± 52.54 a 232.29 ± 55.81 296.06 ± 117.16 b 270.66 ± 120.30 (µg/g soil) Nitrogen 21.64 ± 15.46 24.46 ± 13.24 22.00 ± 10.89 27.45 ± 16.95 (µg/g soil) Potassium 409.69 ± 98.65 473.83 ± 137.06 396.21 ± 162.38 421.23 ± 142.36 (µg/g soil) pH 5.76 ± 0.38 5.82 ± 0.35 6.34 ±0.37 6.36 ± 0.33

Soil Moisture 27.41 ± 4.31 24.96 ± 6.38 27.74 ± 6.42 24.89 ± 6.83 (%) Light (% openness)

Early in the season 18.50 ± 3.30 25.80 ± 2.60 19.70 ± 3.40 25.30 ± 3.00 Late in the season 8.00 ± 1.20 6.20 ± 1.30 4.50 ± 1.60 7.10 ± 0.90

Reproductive behavior

A total of 164 flowering Viola striata individuals were tracked for reproductive behavior during the year 2002, from which a total of 1495 chasmogamous flowers were produced [Campground: n = 80 reproductive individuals, 653 flowers; Cemetery: n = 84 reproductive individuals, 842 flowers]. No significant differences in flower production were found between the two sites (Mann Whitney: Z = -1.25, p = 0.2), although

Cemetery showed a greater number of flowers during 2002. During the year 2003, 195 individuals of V. striata were tracked, from which a total of 1144 chasmogamous

85 flowers were produced [Campground: n = 96 reproductive individuals, 431 flowers;

Cemetery: n = 99 reproductive individuals, 713 flowers]. There was a significantly greater proportion of flowers at the Cemetery (Mann Whitney: Z = -2.31, p = 0.02). At the Campground the difference in chasmogamous flower production between years was significant (ANOVA: df = 1,39; F = 4.31; p = 0.04), with a reduction in the number of flowers in 2003 from 2002. At the Cemetery no differences were found between years

(ANOVA: df = 1,39; F = 0.73; p = 0.39). Across both sites each individual produced an average of 9.1 chasmogamous flowers in 2002 and 5.8 chasmogamous flowers during

2003. The success of chasmogamous flowers in becoming fruits and producing seed was highly variable, both between sites and between years, with ranges between 37.19 to 65.33 % (Table 3.9).

Table 3.9: Average number of chasmogamous flowers produced per quadrat per site and percentage of flowers of Viola striata that successfully became fruits during 2002 and 2003. Ranges are the result of among quadrat variation. n = 20

Campground CH Cemetery CH Campground, Cemetery, flowers produced flowers produced Percent of Percent of successful flowers successful flowers Year 2002 Mean 32.65 42.10 37.19 % 49.48 % Standard deviation ± 19.90 ± 25.90 ± 25.80 ± 20.15 Range 5-81 9-131 6.8-100 14.1-100

Year 2003 Mean 21.50 35.60 65.33 % 61.33 % Standard deviation ± 12.04 ± 20.39 ± 21.16 ± 27.90 Range 2-51 12-88 31.70-100 1.42-100

Environmental influences on reproductive responses

No significant correlations were revealed between any of the independent environmental variables and chasmogamous flower production, for data from the

Campground in 2002. Data for light availability at this site in 2003, however, was significantly correlated with chasmogamous flower production during the second week

(just one week before the peak of chasmogamous flowering). A different pattern of light availability and chasmogamous flowering was suggested in data from the

Cemetery in 2002 by a significant correlation between light during week three (the peak of chasmogamous flower production) and chasmogamous flowering; no significant correlations were found for the site in data from 2003 (Table 3.10).

For chasmogamous fruit production, a significant correlation was found with light availability during weeks two and three in 2003 at the Campground.

Chasmogamous fruiting was also correlated with phosphorous during the same time period and year at the Cemetery. As expected, chasmogamous fruit production was highly correlated with the number of chasmogamous flowers produced.

In the Campground data, none of the multiple regression models generated were significant for chasmogamous flower production [2002: F= 1.59, p= 0.22, R2 = 0.36, R2 adj. = 0.13; 2003: F = 2.41, p = 0.088, R2 = 0.46, R2 adj. = 0.27]. In contrast, in

Cemetery, models for both years were significant. During 2002, soil pH, calcium, phosphorous, and light availability during weeks two and three influenced chasmogamous flowering in the multiple regression model (F = 3.6, p = 0.02) (Table

3.11). In 2003, calcium, magnesium, nitrogen, and phosphorous were significant for

87 chasmogamous flower production (F = 3.9, p = 0.02) (Table 3.11). When chasmogamous flower production in 2003 was analyzed with the soil data from the previous year (2002), the model generated for the Campground was significant (F = 4.0, p = 0.01) and included magnesium in 2002, and calcium, potassium, phosphorous and light availability from weeks one, three, and four in 2003. For the Cemetery, the best model included calcium and potassium from the 2002, and potassium, magnesium, and light availability from week four, and soil moisture in 2003 (F = 3.8, p = 0.02) (Table

3.11).

Discussion

Soil properties were highly heterogeneous across the study area. The data from quadrats over the two populations expressed substantial variation in most environmental parameters among quadrats, sites and years. This heterogeneity corresponds to the pattern typically observed in mixed mesophytic forests (Crozier and Boerner, 1984).

Light availability changed dramatically—up to 19% in some quadrats—during the flowering season as the canopy of the forest closed. Significant weekly differences were found in soil moisture during both years. However, this pattern was complex, varying from week to week without a clear tendency towards increased or decreased moisture during the season.

Table 3.10: Pearson’s correlation coefficients between chasmogamous flowers and fruits and cleistogamous flowers with soil properties and light. *** Significant correlations (p < 0.05). CH=chasmogamous, CL=Cleistogamous

CH flowers CH fruits CH flowers CH fruits Campground 2002 Cemetery 2002 pH 0.24 0.03 pH 0.18 0.07 Nitrogen -0.03 0.14 Nitrogen 0.30 -0.04 Magnesium 0.33 0.32 Magnesium 0.23 -0.08 Calcium -0.29 -0.43 Calcium 0.21 -0.03 Phosphorous 0.09 0.21 Phosphorous 0.25 0.43 Potassium 0.08 -0.14 Potassium 0.05 0.08 Light week 1 0.14 0.02 Light week 1 0.28 -0.05 Light week 2 0.04 -0.09 Light week 2 0.16 -0.37 Light week 3 0.06 -0.24 Light week 3 0.53*** 0.19 Light week 4 0.05 -0.20 Light week 4 -0.13 0.24 Moisture 0.22 0.02 Moisture -0.33 -0.04 CL flowers 0.34 0.36 CH flowers 0.05 0.26 CH fruits 0.64*** 1.00 CH fruits 0.55*** 1.00 Campground 2003 Cemetery 2003 pH 0.16 0.09 pH 0.25 0.08 Nitrogen 0.02 0.07 Nitrogen 0.28 -0.13 Magnesium 0.32 0.22 Magnesium 0.41 0.17 Calcium 0.38 0.28 Calcium 0.18 0.36 Phosphorous 0.08 0.25 Phosphorous 0.41 0.57*** Potassium 0.04 0.02 Potassium 0.23 0.37 Light week 1 0.20 0.29 Light week 1 0.02 0.35 Light week 2 0.45*** 0.51*** Light week 2 0.23 0.27 Light week 3 0.41 0.50*** Light week 3 0.24 0.32 Light week 4 0.13 0.30 Light week 4 0.19 0.25 Moisture 0.29 0.18 Moisture 0.09 0.11 CL flowers 0.20 0.09 CL flowers 0.06 0.39 CH fruits 0.71*** 1.00 CH fruits 0.61*** 1.00

Table 3.11: Multiple regression models constructed from soil chemical data and light characteristics for Viola striata growing under different environmental combinations. Only significant models (p<0.05) are presented. (CH=chasmogamous, CL=cleistogamous).

Regression ± SE Model Coefficient CH flowers Cemetery 2003 (R2=0.51, Adj. R2 = 0.38) Intercept -25.05 21.18 Nitrogen -4.19 3.24 Magnesium 18.10 7.39 Calcium -4.62 3.20 Phosphorous 6.80 2.46

CH flowers Cemetery 2002 (R2=0.56, Adj. R2 = 0.4) Intercept -78.27 51.00 pH 127.97 57.09 Calcium -9.90 6.26 Phosphorous 7.11 4.03 Light week 2 -13.59 6.08 Light week 3 18.31 8.69

CH flowers campground 2003, including data from 2002 (R2 =0.70, R2 = 0.52) Intercept -84.60 39.79 Magnesium content from 2002 31.80 9.70 Calcium 7.61 2.60 Phosphorous 21.09 9.13 Potassium -8.33 5.70 Light week 1 -9.85 6.11 Light week 3 -12.54 3.88 Light week 4 22.06 8.69

CH flower production 2003 Cemetery including 2002 data (R2 =0.63, R2 = 0.46) Intercept 12.70 40.30 Calcium data from 2002 -10.24 4.11 Potassium data from 2002 -12.12 6.80 Magnesium 21.48 7.10 Phosphorous 10.35 2.80 Light week 4 -16.07 8.40 Moisture 9.67 6.70

Cemetery supported a higher level of chasmogamous flower production over both years in comparison with Campground, even though the difference between sites was only significant during the second year. In a separate study dealing with the population genetics of this species, it was clear that the Cemetery also maintains a higher level of genetic diversity than the Campground (Chapter 5), interpreted as the likely result of lower population subdivision based on higher levels of chasmogamous reproduction. The higher level of chasmogamous flower production in 2002 over 2003 might be attributed to damage to some plants affected by a two-day flooding event during the first week of May. This resulted in a dramatic decrease in chasmogamous flowers on plants in those submerged quadrats. The success of flowers maturing to fruits was variable, 1.2-100%, depending on the quadrat position and site, presumably simply a function of differential pollinator activity across quadrats and sites.

Chasmogamous flower production in this study was found to correlate significantly with light availability during the second or third week but not with other environmental variables (including nutrients); however, this correlation was not consistent in all cases. The peak of chasmogamous flowering time was achieved within the first four weeks of measurements (on or before May 2nd). If light availability has an enduring major impact on chasmogamous reproduction in Viola striata, as shown in

Impatiens (Schemske, 1978), this influence would be expected to operate particularly strongly just before peak chasmogamous flowering. The only significant correlation of chasmogamous flower production, with light, is precisely during this time period, supporting light availability as the most important effect on chasmogamous flower

91 production and fruit set. This effect probably manifests in canopy closure with decreasing pollinator visitations to chasmogamous flowers, and in sun flecks influencing local pollinator movements in locating flowers.

The combined effect of the measured parameters was important for Cemetery. In

2002, soil pH and light in the second and third week were important, while in 2003 magnesium and nitrogen were also important. When the effects of nutrients from 2002 and 2003 were combined, significant models were generated for both sites during 2003.

Calcium was important again in both models, while phosphorous was only important in

Campground.

An evaluation of the macronutrient accumulation in different organs of the ornamental Viola parmensis Vilm. [as a variety of V. odorata L.] determined that the corolla was the richest organ of the plant in nitrogen, phosphorous and sulfur, and equal with the pedicels in carbon and potassium (Shan Sei Fan and Morard, 1993). Fitter and

Setters (1988) demonstrated that sexual reproductive structures accumulate greater potassium and phosphorous than vegetative structures in six species of Viola (Viola hirta L., V. lutea Huds., V. odorata L., V. palustris L., V. riviniana Rchb. and V. tricolor

L.). This differential allocation of resources, in particular phosphorous, to reproductive structures in other violet species corresponds to the positive effect that phosphorous has on V. striata reproduction; the effect was evident only in combination with calcium and nitrogen, which also have been implicated in allocation to reproductive structures (Fitter and Setters, 1988; Shan Sei Fan and Morard, 1993).

Chasmogamous fruit production was correlated with light during the main peak

92 of blooming at the Campground during 2003, while it was correlated with phosphorous at Cemetery in the same year. Light is likely influencing pollinator behavior by favoring pollinator visitation in plants growing in more light-filled microsites (Beattie, 1976), although this correlation of chasmogamous fruits with light availability was only significant for one year at one site. Different combinations of soil nutrients and light availability have been demonstrated to correlate with chasmogamous reproduction in V. striata, depending on the year, site characteristics and, probably, quadrat-level microsite conditions. In some years for a given site, increased nutrients and increased light availability together were correlated with increased chasmogamous flowering and fruiting (e.g, at the Cemetery), but this was not the situation in other years or at the

Campground. In general, calcium and phosphorous were important contributors toward chasmogamous flowering, in combination with other factors. Light availability also played a significant role at times, depending on various factors. The finding of significant explanatory models for chasmogamous flower production using two years of data emphasizes the importance of accounting for previous ecological and climatic factors in the current year’s reproduction for perennial species. The present study had only a two-year duration. Long-term investigations would surely provide additional insights into the various factors contributing (or not contributing) to chasmogamous reproduction in Viola striata.

Chapter 4:

Comparison of flower phenologies in a tropical violet (Viola grahamii) and

temperate violet (Viola striata)

Introduction

Phenology refers to the study of periodic biological events as influenced by the environment (Schwartz, 2003). Thus, flowering phenology refers to the timing of plant reproductive cycles, which affect both plant populations and the animals depending on those plants (Newstrom et al., 1994). Different flowering strategies are exhibited by different plant species. Some species experience a massive flowering period, producing large quantities of flowers over a short time; others participate in an extended flowering period in which a few flowers at a time are open and ready for reproduction over a much longer time span; and others fall within the endpoints of this continuum

(Powlesland et al., 1985). Several factors, including climatic (photoperiod, temperature and precipitation) and biotic (competition for pollinators and seed dispersers, selection against interspecific pollen flow, plant breeding systems, predation on flowers and/or fruits, phylogenetic relationships, life form and fruit size), have been shown to have an influence on plant flowering phenology (Smith-Ramirez and Armesto, 1994; Marques et al., 2004).

The effect of different climatic factors on plant reproduction is highly dependent upon the latitude where the plants grow. In the tropics, dry and wet periods have shown clear influences with phenological responses while in regions removed from the

94 equator, other factors including daylength and temperature become more important at regulating these responses (Marques et al., 2004). In some herbaceous species it has been observed that populations of the same species growing at higher latitudes flower sooner than populations growing closer to the equator (Rivera and Borchet, 2001). Over time these phenological differences in flowering time can eventually become genetically fixed and lead to reproductive isolation and evolutionary diversification

(Gentry, 1974).

A special case of flowering phenology involves species with a mixed breeding system, with reproduction by both chasmogamous and cleistogamous flowers. In this system blooming time is not a single event but rather two, corresponding to the production of both flower types. Flower production of the two types may occur simultaneously; or each type may predominate at a different time of year with strong temporal separation between them; or the two flower types may heavily overlap but express modally different peaks. All these have been reported on species with a mixed breeding system. The three major environmental factors that have previously been shown to correlate with such phenological patterns are photoperiod, temperature and precipitation, which will be discuss below.

When photoperiod acts as a regulator, chasmogamous flowers are produced earlier in the growing season, during short days, and cleistogamous flowers are produced later on, mainly during long days. Thus cross-pollinated (out-crossing) flowers are produced before self-pollinated ones. This is the pattern in all Viola species that have been studied in the temperate region: V. blanda Willd. (Newell et al.,1981;

Schellner et al., 1982), V. canadensis L. (Culley, 2000), V. cunninghamii Hook.f.

(Holswort, 1966), V. egglestonii Brainerd (Baskin and Baskin, 1972, 1975), V. fimbriatula Sm. (Solbrig et al., 1988a, 1988b), V. hirta L. (Rebdo-Torstensson and

Berg, 1995), V. incognita Brainerd (Schellner et al., 1982), V. lanceolata (Solbrig et al.,

1988), V. mirabilis L. (Mattila and Salonen, 1995), V. nuttallii Pursh (Turnbull et al.,

1983), V. odorata L. (Mayers and Lord, 1983; 1984), V. pallens (Banks) Brainerd

(Newell et al., 1981; Schellner et al., 1982), V. palustris L. (Jensen and Meyer, 2001),

V. pensylvanica Michx. [=V. pubescens Ait.] (Beattie and Culver, 1979), V. pedata L.

(Beattie and Culver, 1979), V. pubescens Ait. (Culley and Wolfe, 2001), V. rostrata

Pursh (Beattie and Culver, 1979), and V. sororia Willd. (Solbrig et al., 1980, 1981,

1988; Antfinger et al., 1985).

The hypothesis of photoperiod control in violets has been tested in Viola odorata by Mayers and Lord (1983). In that species, chasmogamous flowers are produced when individuals are exposed to 11 hr or less of daylight, and cleistogamous flowers are produced under a 14-hour daylight regime (Mayers and Lord, 1983). The same is true for the New Zealand species Viola cunninghamii in which the production of cleistogamous flowers was also shown to correspond to long days (Holdsworth, 1966).

Therefore, in both the North and the South temperate regions, which experience distinct changes in photoperiod throughout the growing season, daylength changes affect chasmogamous flower production. The regulation of chasmogamous flowering by photoperiod is apparently very precise in most temperate species of Viola. Viola cucullata was reported to have a second flowering season in addition to its spring one,

96 in Nova Scotia during the fall of 2001 when temperatures were abnormally high and the growing season extended late into fall (Taylor and Garbary, 2003). The authors do not mention whether they refer specifically to chasmogamous flowers although it is implied. Chasmogamous flowers produced in response to the short days during early spring were produced again in response to the shorter days occurring again in the fall.

Usually the unfavorable temperature would have prevented continual plant growth.

Such anomalous blooms have also been noted on occasion in Viola canadensis and other species (Ballard, unpublished data).

In V. cunninghamii temperature has also been demonstrated to be an important factor regulating cleistogamy, and this factor could mask the effects of daylength which is generally correlated with it. During short days a higher-than-typical temperature was shown to counteract the effect of daylength and induce production of cleistogamous flowers; whereas lower-than-typical temperatures during long days could have the opposite effect of producing chasmogamous flowers (Holdsworth, 1966). In

Ceratocapnos heterocarpa (Fumariaceae), Ruiz de Clavijo and Jimenez (1993) reported that cleistogamous flowers were produced in the winter when temperatures are lower, and chasmogamous flowers are produced in the spring as soon as temperatures increase; but this could potentially be related to photoperiod, since these two factors had been shown to be highly correlated (Marques et al., 2004). Contrary to the situation with other violet species, Solbrig et al. (1980) concluded that temperature rather than photoperiod may be influencing reproduction in Viola sororia. This was based on the observation that chasmogamous flowers are produced soon after the first leaves emerge,

97 when the soil temperature reaches 100C and air temperature is above 00C, but there can be a delay of up to a month in leaf emergence depending on the temperature of a particular year. If flowering production was actually regulated by photoperiod, this delay could trigger the production of cleistogamous flowers as the first flower type.

Consequently, the difference is presumably related to temperature rather than to daylength. Other investigations should test this hypothesis in order to tease apart the cryptic and potentially confounding effects of temperate and daylength; growth chamber experiments could facilitate this.

Precipitation has not been invoked as a factor regulating chasmogamous- cleistogamous flowering in Viola, presumably because the species previously studied grow in the temperate zone where photoperiod and temperature have already been demonstrated as major factors influencing the phenological patterns. In other Violaceae such as Hybanthus prunifolius, multiple blooming episodes in response to rain have been reported (Auspurger, 1981), although direct comparisons are difficult since this species does not have cleistogamous flowers. In the tropical species Ruellia brevifolia

(Acanthaceae) and other species, chasmogamous flowers are produced during the rainy season, while cleistogamous flowers are more abundant in the dry season (Sigrist and

Sazima, 2002). In Calathea micans (Maranthaceae), chasmogamous reproduction is affected by rainfall while cleistogamous reproduction is not (Le Corff, 1993).

The dearth of investigations on mixed breeding systems in tropical violets, or tropical species generally, permits very interesting hypotheses regarding the flowering phenology of tropical species. Photoperiod would certainly be expected to regulate

98 blooming in the temperate species V. striata based on studies of other temperate violets.

Changes of as little as 30 min in daylength have been found to trigger flowering in tree species of Costa Rica (Rivera and Borchet, 2001), suggesting that daylength may also serve an important function in induction of flowering in other tropical species. If the phenological pattern of Viola species in temperate regions also holds for violet species in the Tropics, it would be expected that cleistogamous flowers will be produced during a second, non-overlapping blooming peak following that of the chasmogamous flowers.

The switch from chasmogamous to cleistogamous flower production would perhaps be regulated by smaller changes in photoperiod than those regulating violets in the temperate regions, given less dramatic seasonal changes in ecological conditions

(including uniform light availability due to constant canopy). The alternative mechanism hypothesized for phenological regulation of flowering in some tropical plants is precipitation (Le Corff, 1993; Sigrist and Sazima, 2002). The present study investigates these two previously proposed mechanisms as hypotheses for explaining patterns of phenology in chasmogamous and cleistogamous flower types, in a temperate and a tropical violet species.

Materials and Methods

Two populations of Viola striata in Strouds Run State Park, Ohio and two populations of V. grahamii in Michoacán’s forests in central Mexico were studied over a two year period. (For detailed descriptions of the study sites and site establishment refer to Chapter 1).

In order to record phenology, the sides of each quadrat were marked every 10 cm to allow for establishment of coordinates for individual plants inside each quadrat.

Each plant was then marked with a plastic toothpick and assigned a letter in order to follow its phenology throughout the reproductive season. Phenological observations at each site consisted of a weekly census of the number of flowers of each type on marked plants in each quadrat, during the flowering season over the two years. These numbers were used to produce phenological histograms for the two flower types and analyze influences on the onset and offset of flowering time, and peak blooming.

For Viola striata data were collected during 14 April-9 June 2002 and 16 April-

8 June 2003. For Viola grahamii data collection was conducted during 17 June-29 July

2002 and 25 June-8 August 2003.

For an analysis of flowering phenology per plant per week, the total number of flowers recorded at a site for a given week was divided by the number of reproductive individuals to get the average number of flowers per individual for that week. To compare phenologies of the two flower types, the total number of flowers recorded for a given week was summed, and values were plotted for all weeks to generate a phenological histogram. Flowering phenologies of the two flower types were considered separately for sites, species and years to interpret similarities and differences.

To examine different environmental factors that could be regulating blooming phenologies the following data were collected.

100

Light environment

To characterize the light environment, hemispherical photographs were taken above each quadrat on overcast days or in early morning before the sun rose over the mountain, using a digital Nikon Coolpix 955 camera equipped with a 180° Nikon FC-

E8 Fish-eye converter lens. Images were saved using the FINE format (1:4 compression

JPEG, 1600 x 1200 pix) (Frazer et al., 2001). To verify changes of light availability through the growing season, weekly pictures were taken during the time data were being recorded for the two species. Images were analyzed for canopy openness using

Gap Light Analyzer (GLA version 2.0.4) image processing software (Frazer et al.,

2001). In V. striata, differences were expected in the same quadrat throughout the reproductive period due to the deciduous nature of the canopy trees that shade them. As the growing season progressed a dramatic increase in canopy closure was expected. In the case of the tropical forest where V. grahamii grows, differences in the light environment were not expected to vary much over time. Data were arcsine-transformed to meet the assumptions of the repeated measures ANOVA, and differences among the different weeks of study for each site were tested using Bonferroni all pair-wise multiple comparisons.

Soil moisture

Soil moisture was determined for each quadrat on a weekly basis using the gravimetric method (Hadley and Levin, 1967). This permitted tests of potential changes in soil moisture during the growing season that could affect phenological patterns

101 through drought- or flooding plant stress. Data were arcsine-transformed and a repeated measures ANOVA was performed to test for differences between weekly observations at each site. Bonferroni all pair-wise multiple comparisons were conducted.

Climatic factors

Precipitation and temperature values for the region during the two study seasons were obtained from a meteorological station located near each study site. For Viola grahamii data were obtained from the Estación Metereológica Automatizada de

Angamacutiro, Michoacán, maintained by the Comisión Nacional del Agua, Servicio

Metereológico Nacional, México D.F. México. For Viola striata data were obtained for

Meteorological Station Athens 1 N from the National Climatic Data Center (NCDC).

Daylength for both sites was obtained from the Astronomical Division of the United

States Naval Observatory. Spearman’s correlations between the different climatic factors and chasmogamous and cleistogamous flower production were performed. All statistical tests were performed using NCSS software (Hintze, 1996).

Observations on flower visitors and potential pollinators were made during peak chasmogamous blooming. About 15 hr of observations were made for each year for

Viola striata; around 10 hr were accumulated for V. grahamii during 2002 and 18 hr during 2003.

102

Results

Climatic patterns

The habitats of the two species are highly contrasting in vegetation (see Chapter

1) and climate, dictated by their 20° difference in latitude. The forests of northern

Michoacán in Mexico exhibit a sharply demarcated wet and dry season, with rain occurring primarily between June and October. Conversely, the forests of Ohio receive rain over much of the year, with much of it occurring in early spring; the spring rainy season and summer dry period is much less pronounced than in Mexico (Figures 4.1 and 4.2). Temperature and photoperiod differ markedly between the two regions. There is greater seasonal variation in Ohio with lower temperatures and greater variability in photoperiod (332 min of difference between the longest and shortest day), and a higher level of total precipitation. Michoacán maintains an almost constant temperature across the seasons and expresses lesser variability in photoperiod (158 min of difference).

The canopies of deciduous mixed mesophytic forests in Ohio and oak-pine evergreen forests in Mexico behave as expected (Table 4.1). During the growing season dramatic changes in light availability occur during spring in Ohio’s forests, with significant weekly reductions during the first weeks in light availability (as a function of canopy openness) as great as 19%. Leafout proceeded throughout spring and the chasmogamous flowering period of the violets, until the canopy become totally closed by late summer. In Michoacán’s forests the canopy cover remained essentially the same throughout the season (ca. 25% canopy openness for Santa Fe and 40% for Quiroga).

103

950 40 120 Ohio 900 30 100 850 20

800 80 m) in) 10 m 750 ion (m

ture (°C) 0 60 at 700 t ipi ylength ( -10 c

Da 650 40 Pre Tempera -20 600 20 550 -30

500 -40 0 6 5 2 t c 1 20 v 3 17 14 28 13 27 11 25 16 30 19 n 22 10 24 t r 21 r 10 r 24 c 15 c 29 c 30 n n p b b Ju Oc Sep 8 Apr 7 De Jul Jul No May Ja Ja Jun Jun Oc Se Fe Fe Ap De De De Ma Ma Nov Aug Aug May 950 40 120 Michoacán 900 30 100 850 20 ) 800 80 10

750 n (mm h (min) io ture (°C) t 0 60 t 700 -10 ipita rec Dayleng 650 40 Tempera P -20 600 20 550 -30

500 -40 0 7 1 5 6 0 5 9 2 0 0 4 0 4 9 3 7 1 0 4 8 5 t 6 y 1 2 3 1 p 8 r1 r 2 ec 1 y n 1 n 2 p 2 b 1 b 2 Jun 2 Oc Se Apr 7 D Jul 1 Jul 2 Nov 3 Ma Ja Ja Jun 1 Jun 3 Ma Oct 2 Se Fe Fe Apr 2 Dec Dec Dec Ma Nov 1 Aug 1 Aug 2 Ma

First day of week (2002) Figure 4.1: Climatic patterns of temperature (°C), rain fall (mm) and photoperiod (Daylength) during 2002. Data is presented on a weekly basis for Athens, Ohio (Viola striata) and Angamacutiro, Michoacán (Viola grahamii). Continuous line = Average Maximum temperature, Dashed line = Average Minimum temperature, Bars = Total precipitation, Black dots = Photoperiod.

104 950 40 120 Ohio 900 30 100 850 20

800 80 m) in) 10 m (°C) (

750 n io ture t

gth (m 0 60 a a r n it

700 e p p i

-10 c m e

Dayle 650 40 T Pre -20 600 20 550 -30

500 -40 0 9 3 7 0 5 9 4 9 2 1 r 6 r y 1 18 23 14 28 p 7 29 10 24 16 30 l 1 l 2 r r 2 c c c y b 16 b 23 p 21 n 12 n 26 n 1 n 2 Jun Oct 5 a a Se Ap Ma Ju Ju Nov Ma J J Ju Ju Oct Se Fe Fe Ap De De De Ma Aug Aug Nov Nov Ma 950 40 120 Michoacán 900 30 100 850 20

800 80 m) in) 10 m m e (°C) (

750 n io t atur 0 60 a er it ength ( 700 p p i

-10 c e r Dayl 650 40 Tem P -20 600 20 550 -30

500 -40 0 9 4 8 9 3 7 0 9 4 9 7 r 6 r y 23 18 l 1 l 2 r r 2 y n 12 n 26 b 23 p 21 un 1 ov 2 J Oct 5 a a e e Feb Sep un 15 un 29 ug 10 ug 24 ov 16 ov 30 Ma Ju Ju N Ap Ma J J J J Oct 1 F S Ap Dec 1 Dec 2 Dec 2 Ma A A N N Ma First day of week (2003) Figure 4.2: Climatic patterns of temperature (°C), rain fall (mm) and photoperiod (Daylength) during 2003. Data is presented on a weekly basis for Athens, Ohio (Viola striata) and Angamacutiro, Michoacán (Viola grahamii). Continuous line = Average Maximum temperature, Dashed line = Average Minimum temperature, Bars = Total precipitation, Black dots = Photoperiod.

105

Table 4.1: Changes in canopy openness during the flowering season of two years of observations (2002 and 2003) for two populations of Viola grahamii (Santa Fe and Quiroga) and two populations of V. striata (Campground and Cemetery). Mean % ± standard deviation are included. Repeated measures ANOVA was used to check for differences among the different weeks of observations. P values are included, significant differences are marked with asterisks. Superscripts indicate similar groups identified using Bonferroni multiple comparisons test.

Viola striata (Ohio) Viola grahamii (Michoacán) 2002 2003 2002 2003 Campground Cemetery Campground Cemetery Santa Fe Quiroga Santa Fe Quiroga Week 1 18.50 ± 3.30 a 19.70 ± 3.40 a 25.80 ± 2.60 a 25.30 ± 3.00 a 25.00 ± 6.50 36.20 ± 8.80 27.00 ± 6.10 45.20 ± 7.50 Week 2 14.00 ± 2.40 b 13.70 ± 2.60 b 17.70 ± 2.60 bc 15.70 ± 2.40 bc 25.00 ± 4.50 40.20 ± 11.60 23.90 ± 6.90 43.20 ± 7.60 Week 3 8.10 ± 1.20 c 8.30 ± 1.30 c 11.00 ± 1.70 bcd 10.6 ± 2.10 bcd 27.00 ± 6.20 39.70 ± 11.00 24.30 ± 6.90 43.00 ± 7.60 Week 4 8.60 ± 0.90 c 7.80 ± 1.80 c 8.00 ± 0.80 cde 9.30 ± 0.90 cde 22.70 ± 5.10 38.10 ± 10.90 24.70 ± 6.90 43.40 ± 8.70 Week 5 10.40 ± 1.60 d 7.60 ± 1.60 c 6.90 ± 0.90 def 7.50 ± 0.90 de 23.60 ± 5.70 40.40 ± 10.80 25.70 ± 7.40 41.20 ± 9.30 Week 6 7.80 ± 1.60 c 4.40 ± 1.70 d 5.80 ± 1.00 ef 5.90 ± 0.90 e 24.49 ± 6.70 38.50 ± 12.20 25.10 ± 6.50 42.85 ± 8.10 Week 7 8.00 ± 1.20 c 4.50 ± 1.60 d 6.20 ± 1.30 def 7.10 ± .9.00 de 24.70 ± 5.60 42.85 ± 8.00

ANOVA P < 0.05 *** P < 0.05*** P < 0.05 *** P < 0.05 *** P = 0.48 P = 0.94 P =0.71 P =0.93

106

Significant weekly differences were found in soil moisture for both species at all sites during both years. However the pattern of variation was complex, varying from week to week without a clear tendency towards increased or decreased moisture during the season (Bonferroni multiple comparisons, Table 4.2).

Phenology

A discrete blooming period for chasmogamous flowers was recorded in Viola striata during late April and early May. By mid-May, chasmogamous flower production had ceased and cleistogamous flowers were being produced. There was a sharp temporal separation between the blooming peaks of the two flower types, with essentially no overlap between them in both study seasons (Figures 4.3 and 4.4). The flowering of individual plants during the season renders the peak blooming times for chasmogamous and cleistogamous flowers even more apparent (Figure 4.3). When examined over all populations for the two years of study, two blooming peaks for both sites for both years are evident (Figure 4.4). Chasmogamous flowering during both years started at approximately the same time, during the second week of April; the onset of flowering was generally synchronous among many individuals.

In Viola grahamii both chasmogamous and cleistogamous flowers were produced by individuals simultaneously, at the beginning of the flowering season, expressing co-extensive overlap in phenologies (Figures 4.5 and 4.6).

107

Table 4.2: Changes in soil moisture during the flowering season of two years of observations (2002 and 2003) for two populations of Viola grahamii (Santa Fe and Quiroga) and two populations of V. striata (Campground and Cemetery). Mean % ± standard deviation are included. Repeated measures ANOVA was used to check for differences among the different weeks of observations. P values are included, significant differences are marked with asterisks. Superscripts indicate similar groups identified using Bonferroni multiple comparisons test.

Viola striata (Ohio) Viola grahamii (Michoacán) 2002 2003 2002 2003 Campground Cemetery Campground Cemetery Santa Fe Quiroga Santa Fe Quiroga Week 1 28.1 ± 4.0 ab 26.7 ± 5.5 ab 21.3 ± 3.8 abc 19.6 ± 4.4 abcd 33.4 ± 9.8 a 25.2 ± 6.9 a 34.4 ± 3.6 a 34.4 ± 3.4 Week 2 27.7 ± 3.7 ab 26.7 ± 5.7 ab 20.7 ± 4.6 ac 22.0 ± 4.2 abcd 27.2 ± 3.1 b 28.1 ± 6.3 ab 37.6 ± 2.5 b 34.3 ± 2.2 Week 3 25.1 ± 3.53 b 29.1 ± 5.2 ab 19.9 ± 4.2 ac 18.7 ± 4.6 ab 31.8 ± 2.6 ab 31.9 ± 8.9 ab 35. 8 ± 4.6 ab 36.25 ± 5.4 Week 4 29.5 ± 4.1 a 31.9 ± 5.6 a 25.6 ± 4.4 abd 23.7 ± 4.5 acde 32.4 ± 3.4 a 33.03 ± 5.5 b 34.2 ± 3.1 a 33.97 ± 3.2 Week 5 27.0 ± 5.0 ab 25.4 ± 8.1 b 19.1 ± 4.8 ac 22.6 ± 4.7 abcd 32.8 ± 8.7 ab 32.9 ± 8.6 b 37.0 ± 2.8 ab 36.1 ± 2.6 Week 6 26.8 ± 3.9 ab 26.3 ± 5.3 ab 29.5 ± 4 bde 28.3 ± 4.3 cef 33.8 ± 5.2 a 34.2 ±7.9 b 36.1 ± 2.7 ab 36.9 ± 4.6 Week 7 27.4 ± 2.3 ab 27.7 ± 4.5 ab 30.3 ± 1.5 bde 31.3 ± 5.4 ef 35.9 ± 2.3 ab 35.3 ± 2.1 Week 8 33.0 ± 2.0 d 32.4 ± 5.7 ef

ANOVA P < 0.05 *** P < 0.05 *** P < 0.05 *** P < 0.05 *** P < 0.05 *** P < 0.05 *** P < 0.05 *** P =0.415

108

8 Campground 2002 Campgrmpgroundound 2003 6 S R E 4 W O L

F 2 CH

8 April16 April 25 May2 May 9 May 17 May 25 May 31 June 8 Campground 2002 Campground 2003 6 S

ER 4

2 CL FLOW

8 Cemetery 2002 Cemetery 2003 6 S R E 4 OW

2 CH FL

8 Cemetery 2002 Cemetery 2003 6

ERS 4 W O L 2 F CL 0

-2 4 1 8 6 9 7 6 3 8 1 2 5 1 2 1 1 31 8 l y 25 ne y 10 26 ril ril 2 i y y une ril ril 2 y y y ay p Ma J Ju p Ma June Ap A Apr Ma Ma Ap A Ma Ma Ma M Figure 4.3: Relative production of chasmogamous and cleistogamous flowers in Viola striata for two different populations for 2002 and 2003, Campground and Cemetery. Closed squares represent chasmogamous flowers (CH), open squares represent cleistogamous flowers (CL). Means and standard deviations for each week are presented.

109

Campground 2002 Campground 2003 300 owers

200 ber of fl

num 100 l a t o T

4 9 7 5 4 9 1 1 5 2 9 5 8 y e e 16 2 ay 2 e ril1 ril 20 a y n n ril y n p M Ju Ju ril M May ay 17 Ju A Ap April 2 Ma May 2 Ap Ap M Ma May 31

400 Cemetery 2002 CemCemetereteryy 2003

300 owers

200 ber of fl

l num 100 a t o T

4 1 8 6 5 2 7 6 8 6 2 9 3 1 2 31 8 ay 5 e e y 10 e ril 1 ril 2 ay 1 ay n ril 2 y ay y ay n p M Ju Jun Ma Ju Ap April 2A M M April 1Ap Ma M Ma M

Figure 4.4: Summary of the blooming phenology of Viola striata in two populations over two years: Campground and Cemetery. Chasmogamous flowers (closed squares), cleistogamous flowers (open squares).

110 2.0 Santa Fe 2002 Santa Fe 2003 1.5

ers 1.0 ow l f 0.5 CH

0.0

2.0 Santa Fe 2002 Santa Fe 2003 1.5 s

er 1.0 ow l

0.5 CL f

0.0

2.0 QuiJuneroga 17June 26July 4July 12July 20July2 00229 QuiJuneroga 25July 4July 1Ju1 ly 1Ju8 ly Augus25 August 20031 t 8 1.5 ers

ow 1.0 l f

CH 0.5

0.0

2.0 Quiroga Quiroga June 17June 26July 4July 12July 20July2 00229 June 2July5 Ju4 ly 1Ju1 ly 1July8 Au25gusAugut 20031 st 8 1.5

ers 1.0 ow l

0.5 CL f

0.0

4 4 8 1 8 26 y 12 20 29 25 y 11 1 25 st y y ly y u ust Jul ne Jul u June 17 June July Jul Jul Ju July J Jul Aug Aug

Figure 4.5: Relative production chasmogamous and cleistogamous flowers in Viola grahamii for individuals growing in two populations: Santa Fe and Quiroga during two years (2002 and 2003).Chasmogamous flowers represented by solid squares (CH), cleistogamous flowers represented by open squares (CL). Means and standard deviations for each week are presented.

111

140 Santa Fe 2002 Santa Fe 2003 120 s r 100

ber of flowe 60

num 40 l a t o

T 20

7 6 4 2 0 9 1 2 3 4 5 6 7 1 2 1 2 2 e y ly ly ne n July ul u Ju Ju J Ju J 120 Quiroga 2002 Quiroga 2003 100 s r

60 ber of flowe 40 num l a t

o 20 T

6 4 2 0 5 4 8 5 1 8 29 11 17 2 ly 1 2 ly 1 2 st st e ly ly 2 y e ly ly ly u u ne n Ju ul n Ju g g Ju Ju Ju Ju J Ju Ju Ju Ju Au Au

Figure 4.6: Summary of the blooming phenology of Viola grahamii in two populations over two years: Campground and Cemetery. Chasmogamous flowers (closed squares), cleistogamous flowers (open squares).

112

In 2002, blooming had commenced at or before the time of the first census

(second week of June), with Quiroga apparently beginning one or more weeks earlier than Santa Fe (Figure 4.5). During 2003 the reproductive season started approximately one week later than in 2002, when violets at both sites started blooming at the same time. During 2003 cleistogamous flower production did not initiate on quite the same massive scale during the first couple of weeks as in the previous year (Figure 4.6). At each site both flower types were produced simultaneously and reached peak blooming at more or less the same time. Cleistogamous flower production continued for an unspecified period of time after the census was concluded, and may well have ended only with the beginning of the next dry season (Figure 4.6).

During chasmogamous blooming time in Viola striata and during the transition to cleistogamy, soil moisture remained more or less constant; precipitation was low for the first few weeks then increased (Figures 4.7 and 4.8). By the fourth week the canopy had completely closed, at a time corresponding to the switch from chasmogamy to cleistogamy. Peak chasmogamy had passed and chasmogamous flower production was declining in both 2002 and 2003 (Figures 4.7 and 4.8). Daylength increased at this time, changing from765 to 790 min (a difference of 25 min during the 8 week study period).

Cleistogamous flowers were found only when daylength reached ca. 790 min (ca. 13 hr).

Significant correlations were revealed between daylength and canopy openness

(R2 = 0.84, p < 0.005) in Ohio. Therefore, only daylength was used for subsequent correlations with chasmogamous or cleistogamous flower production. Daylength and

113 cleistogamous flowers were strongly correlated for both years and both populations

(Table 4.3). Other statistically significant correlations were noted but were inconsistent across years and sites; these were interpreted as coincidental and not explanatory or causal.

Table 4.3: Correlations among climate and environmental variables and chasmogamy and cleistogamy production for Viola striata during 2002 and 2003. R2 values included. Significant correlations (p<0.05 are marked with **). CH=Chasmogamous flowers, CL=Cleistogamous flowers, DL=Daylength, SM= Soil moisture.

Campground, 2002 Cemetery, 2002 CH CL DL SM CH CL DL SM CH 1 CH 1 CL 0.48 1 CL 0.36 1 DL 0.45 0.55** 1 DL 0.42 0.54** 1 SM 0.53** 0.72** 0.53** 1 SM 0.51** 0.78** 0.73** 1 Campground, 2003 Cemetery, 2003 CH CL DL SM CH CL DL SM CH 1 CH 1 CL 0.25 1 CL 0.30 1 DL 0.11 0.64** 1 DL 0.28 0.68** 1 SM 0.22 0.67** 0.57** 1 SM 0.45 0.90** 0.23 1

In the case of Viola grahamii, soil moisture changed significantly. Statistically significant correlations were found between chasmogamous flower production and soil moisture for 2002 but not 2003. Thus, soil moisture varied too much to provide a consistent pattern of influence on phenological patterns.

114 s Campground Cemetery s

300 300 er ower l ow l f f f o

200 200 of er b ber m 100 100 Total nu 0 0 Total num

50 50 e e r r u u . .

t 40 40 t is is o o

S.D 30 30 S.D il m il m o 20 20 o % s Mean ± % s Mean ± 10 10

30 30 Apr14 Apr 20 Apr 29 May 7 May 15May 24 June 1 June 9 Apr14 Apr 20 Apr 29 May 7 May 15May 24 June 1 June 9 25 25

20 20 . . openness openness D D

. 15 15 .

10 10

% canopy Mean ± S 5 5 % canopy Mean ± S

900 900 Apr14 Apr 20 Apr 29 May 7 May 15May 24 June 1 June 9 Apr14 Apr 20 Apr 29 May 7 May 15May 24 June 1 June 9 )

880 880 ) n i m (min

860 860 ( h t g

n 840 840 ength l yle y a Da 820 820 D

80025 80025 Apr14 Apr 20 Apr 29 May 7 May 15 May 24 June 1 June 9 ) ) 20 20 m m m m 15 15 n ( on (

10 10 itatio p i

c 5 5 ecipitati e r r P P 0 0

7 1 9 7 1 9 20 y 15 24 y 15 24 a pr14 Apr14 M ay une une A pr 29 Ma ay une une Apr Apr 29 M May J J Apr 20A M May J J Figure 4.7: Viola striata blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Campground, Right panel Cemetery during the year 2002. (Daylength and rain are the same for both sites).

115

400 400 s Campground Cemetery s

ower 300 300 ower l l f f f f o o 200 200 er er b b m m

100 100

Total nu 0 0 Total nu

50 50

40 40 .) .) isture isture D D o o . .

S 30 30 S m m ( ( n n

a 20 20 a % soil Me % soil Me 10 10

30 April 17April 26May 3 May 10May 18May 26May 31 June 8 April 17April 26May 3 May 10May 18May 26May 31 June 8 30 s s 25 25 enes enes

p 20 20 p o o .D.) .D.) 15 15

10 10

% canopy Mean (S 5 5 % canopy Mean (S

0 0 April 17April 26May 3 May 10May 18May 26May 31 June 8 April 17April 26May 3 May 10May 18May 26May 31 June 8 880 880 ) in) in m

860 860 m ( h (

840 840 engt l ength l y y a a

D 820 820 D

80060 60800 April 17 April 26 May 3 May 10 May 18 May 26 May 31 June 8 April 17 April 26 May 3 May 10 May 18 May 26 May 31 June 8

) 50 50 ) m m m 40 40 on (

30 30 tation (m

20 20 ecipitati r ecipi P 10 10 Pr

0 0 6 3 3 1 10 18 10 18 26 3 l 17 l 2 ay 26 31 l 17 l 26 ay i i ay ay ay i i ay ay M June 8 M June 8 Apr Apr May M M M Apr Apr May May M M Figure 4.8: Viola striata blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Campground, Right panel Cemetery during the year 2003. (Daylength and rain are the same for both sites).

116

Canopy openness remained constant through the season and was presumed to have no influence on phenology of either flower type. Rainfall fluctuated during the observation period and showed no clear trend that would coincide strongly with either flower type, except that the advent of the spring rains corresponded closely with initiation of both chasmogamous and cleistogamous flowering time. Daylength changed during this time period from about 804 min in length to 780 min after 6 weeks, a difference of around 24 min (Figures 4.9 and 4.10). Both flower types started when days were 804 min long (ca. 13 hr and 14 min). The relatively small time difference in daylength between the beginning and end of chasmogamous blooming time does not seem especially dramatic, and would still not explain the coincident blooming of the two flower types.

Cleistogamous flower production was significantly correlated with daylength at

Quiroga in both years, but not for Santa Fe (Table 4.4) and with soil moisture during

2002; the importance of this site-specific correlation is not clear.

117 140 140 Santa Fe Quiroga s er ers 120 120 ow ow l l 100 100 f f

of 80 80 ber of ber 60 60

num 40 40 tal num tal

20 20 o o T T 60 60

50 50 e 40 40 .) .) isture istur o o .D .D S 30 30 S m ( il m n o 20 20 % s Mean ( % soil Mea 10 10

70 70 June 17 June 26 July 4 July 12 July 20 July 29 June 17 June 26 July 4 July 12 July 20 July 29 60 60 ss ss e e n n

n 50 50 n e e

op 40 40 op .D.) .D.)

30 30

% canopy Mean (S 20 20 % canopy Mean (S

805 805 June 17 June 26 July 4 July 12 July 20 July 29 June 17 June 26 July 4 July 12 July 20 July 29

) 800 800 ) n n i i 795 795 (m (m h h t t

g 790 790 g n n

yle 785 785 yle Da Da 780 780

120 120 June 17 June 26 July 4 July 12 July 20 July 29 June 17 June 26 July 4 July 12 July 20 July 29 100 100 ) ) m m 80 80 m m

60 60 on ( on ( i i t t a 40 40 a

20 20 ecipit ecipit r r

P 0 0 P

4 y 12 20 29 y 4 l y y y l y 12 y 20 y 29 Ju ul Ju June 17 June 26 Jul Jul J June 17 June 26 Jul Jul Jul Figure 4.9: Viola grahamii blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Santa Fe, Right panel Quiroga during the year 2002. (Daylength and rain are the same for both sites).

118 100 100 s s

er Santa Fe Quiroga er 80 80 ow ow l l

f f 60 60 of of

ber 40 40 ber

20 20

0 0 Total num Total num 50 50

45 45

40 40 isture isture o o

m 35 35 m (S.D.) (S.D.) n n a a

e 30 30 e % soil M % soil M 25 25

June 25 July 4 July 11 July 18 July 25 August 1August 8 June 25 July 4 July 11 July 18 July 25 August 1August 8 50 50 ss ss e e

enn 40 40 enn op op

py py

(S.D.) 30 30 (S.D.) n n a a e e

% Cano M 20 20 % Cano M

805 805 June 25 July 4 July 11 July 18 July 25 August 1August 8 June 25 July 4 July 11 July 18 July 25 August 1August 8

) 800 800 in) n i m

m 795 795 h (

gt 790 790 ength ( n l e l

y 785 785 a Day D 780 780

June 17 June 26 July 4 July 12 July 20 July 29 June 17 June 26 July 4 July 12 July 20 July 29 100 100 ) ) m m 80 80 m m

60 60

40 40

20 20 ecipitation ( ecipitation ( r r P P 0 0

1 8 4 1 25 y 4 y 8 y 11 y 18 y 25 y 11 y 18 y 25 Jul Jul gust June Jul Jul Jul June 25 Jul Jul Jul u August August August A

Figure 4.10: Viola grahamii blooming phenology of chasmogamous and cleistogamous flowers compared with soil moisture, canopy openness, photoperiod and rainfall during the blooming season. Each point represents a week of measurements. Left panel Santa Fe, Right panel Quiroga during the year 2003. (Daylength and rain are the same for both sites).

119

Table 4.4: Correlations among climate and environmental variables and chasmogamy and cleistogamy production for Viola grahamii during 2002 and 2003. R2 values included. Significant correlations (p<0.05 are marked with ***). CH=Chasmogamous flowers, CL=Cleistogamous flowers. CO=Canopy openness, DL=Daylength, SM= Soil moisture

Santa Fe, 2002 Quiroga, 2002 CH CL CO DL SM CH CL CO DL SM CH 1 CH 1 CL 0.29 1 CL 0.65** 1 CO 0.38 0.01 1 CO 0.04 0.00 1 DL 0.29 0.00 0.17 1 DL 0.70** 0.90** 0.09 1 SM 0.73** 0.14 0.05 0.19 1 SM 0.80** 0.61 0.29 0.71** 1 Santa Fe, 2003 Quiroga, 2003 CH CL CO DL SM CH CL CO DL SM CH 1 CH 1 CL 0.00 1 CL 0.38 1 CO 0.25 0.31 1 CO 0.03 0.20 1 DL 0.50 0.17 0.00 1 DL 0.31 0.65** 0.61** 1 SM 0.04 0.00 0.21 0.01 1 SM 0.00 0.14 0.76** 0.26 1

Pollinators

For V. striata the main pollinator observed is the solitary bee Andrena violae

Robertson (Andrenidae). Other flower visitors included the carpenter bee Ceratina dupla dupla Say (Xylocopidae) and the bee fly (Bombylius major L. (Bombiliidae). In the case of V. grahamii, the golden banded skipper (Autochton cellus Boisduval and Le

Conte, Hesperiidae) was observed to visit the violet flowers and also Salvia assurgens

H.K.B. (Lamiaceae), Croton adspersus Benth (Euphorbiaceae), Verbena carolina

L.(Verbenaceae) and Geranium seemannii Peyr. (Geraniaceae) in the area. Due to broad flower visitation it is unlikely that the skipper is a highly effective pollinator, although low visitation rates by such generalists may contribute to pollination success. A species

120 of bee was repeatedly observed visiting and apparently pollinating V. grahamii and no other species in the area; it is therefore a primary pollinator of the violet. It is an undescribed species of the genus Dianthidium (Adanthidium) in the Megachilidae

(Griswold, T. personal communication).

Discussion

Application of a specific classification term to describe the blooming phenology of these species is difficult because there is no classification for situations in mixed breeding systems. If we consider only chasmogamous blooming time, then both species exhibit the “very short massive” blooming syndrome (Newstrom et al., 1994).

However, if we consider both chasmogamous and cleistogamous flower production as a combined reproductive event, then both species show a nearly annual pattern of flowering; that is, they only reproduce in one major cycle per year with an extended flowering time (more than 5 months) (Newstrom et al., 1994). Chasmogamous flowering is synchronized in both species for a particular season and given geographic region. This pattern fosters gene flow among different flowering individuals within a population.

In Viola striata, chasmogamous flowers are produced during short days, at a time when the canopy is very open. Cleistogamous flowers are produced later in the season, after chasmogamous flowering has essentially ceased, during longer days when the canopy has closed. However, cleistogamous flower production extends far beyond

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the longest day of the year and into fall, when days are shortened again. Canopy closure resulted in a drop in canopy openness from 19% to 6% from the first to the last week of observations in 2002, and from 25% to 6% in 2003. Culley (2000) reported changes in the amount of photosynthetically active radiation (PAR) during the chasmogamous flowering season of V. pubescens, showing that maximum PAR was highest during the peak of chasmogamous production. A reduction to 7% was noticed in PAR after the canopy had closed. This pattern involving two peaks of blooming, the chasmogamous peak in early spring followed by the cleistogamous peak in late spring and summer through fall, seems to be a common one in all species with a mixed breeding system in the temperate region (Jolls, 2003). Cleistogamy in the temperate violet V. odorata is regulated by changes in photoperiod, with cleistogamous flowers produced upon reaching daylengths of 14 hr, compared with 11 hr days for chasmogamous flowers

(Mayers and Lord, 1983).

Results for Viola striata follow these same phenological patterns; strong and consistent correlations with daylength in the two flower types also support photoperiod as a major factor regulating both. The only consistently strong correlation involving cleistogamy was that with daylength (using canopy openness as the surrogate), supporting the hypothesis of photoperiodic regulation. This phenological pattern governed by photoperiod and related to light availability can be viewed as a genetically programmed adaptive response. Production of chasmogamous flowers coincides with the peak time of pollinator availability and the optimal light conditions which foster

122 detection of flowers by insect visitors. Under the closed canopy and additional overgrowth of elongating herbaceous and shrubby species that are characteristic of late spring and early summer, violets grow under highly shaded conditions shunned by potential pollinators such as bees. Therefore, high spring light conditions favor chasmogamous flower production because of optimal pollinator availability and high pollination success, whereas low summer light conditions favor cleistogamous flower production in the absence of pollinators.

If the hypothesis that chasmogamous flowers are more expensive to produce than cleistogamous flowers is true, then chasmogamous flowering should be dependent on environmental conditions or resource availability, whereas the cheaper cleistogamous flowers should be produced independent of these factors. This situation has been reported in Calathea micans (Marantaceae) and Viola egglestonii, where cleistogamy is indeed independent of environmental conditions and plant size (Le Croff,

1993; Baskin and Baskin, 1975). Conversely, in Viola mirabilis and V. palustris, when competition for light is high, both chasmogamous and cleistogamous flower production is reduced (Jensen and Meyer, 2001; Mattila and Salonen, 1995). The same phenomenon has been observed in other cleistogamous species such as Dicanthelium clandestinum in which light reduces the production of both flower types (Bell and

Quinn, 1987). In V. striata cleistogamous flowers are produced only after the canopy has closed and other vegetation overtops the violets to increase the shade regime, suggesting that their production is independent of light availability.

Evolutionary, chasmogamous fruit production requires pollinator visitation to

123 produce fruits and therefore seeds (field observations suggest that delayed selfing, which is present in Viola pubescens, appears to be absent in this species). Beattie (1971,

1972, 1976) suggested that pollinators will preferentially visit those flowers that are displayed in sunflecks or canopy gaps. Since insects depend on thermoregulation, their activity has been shown to relate to the presence of open areas and sunflecks in the understory floor with higher ambient temperatures (Schultz, 1998). Therefore, the daily duration and intensity of direct sunlight for a colony of violet plants would play a primary role in the pollination of those flowers (Beattie, 1971, 1972, 1976). Visual observations of pollinator behavior also support insect preferences toward flowers displayed in sun-lit areas. Therefore, even if flowers were produced later in summer they would probably not be pollinated, because heavy shade would hinder potential pollinators from finding the flowers to pollinate them. The massive blooming strategy with chasmogamous flowers in temperate violets during early spring, before leaf-out and eventual canopy closure, therefore represents a necessary adaptation to ensure at least some outcrossing success.

The simultaneous chasmogamous and cleistogamous phenologies of Viola grahamii represents a unique pattern never before described in Viola. The onset of the coincident phenologies corresponds to the beginning of the rainy season for the montane tropical region inhabited by the species. Flowering time associated with the end of the dry season and beginning of the rainy season is a very common and predictable phenological pattern for the Tropics (Le Corff, 1993; Sigrist and Sazima,

2002) and is clearly driven by seasonal climate rather than photoperiod. The forests

124 harboring V. grahamii are considered evergreen, with an overstory that retains its leaves through the entire year. Some oaks can lose their leaves in the dry season, this usually spans only a few weeks, during the time immediately before the rainy season (Pesman,

1962). Although the overstory is considered evergreen, understory diversity and composition can fluctuate greatly from the rainy to the dry season, with substantial loss of species during extreme drought in some regions. The climatically driven flowering phenological pattern in this region is classified as Seasonal Flowering (Augspurger,

1983; Newstrom et al., 1994).

The fully overlapping summer flowering phenology for the two flower types was consistent over the two years of the study, as well as a previous year of observations during another research project involving V. grahamii and a related species. In the same area, Viola hookeriana Kunth follows the same phenology pattern, with both chasmogamous and cleistogamous flowers produced over an overlapping period and beginning with the advent of the region’s rainy season (Cortes-Palomec,

2001). Although a sample size of two species, both in the same genus and species complex, is too low to make a generalization for tropical montane plants, there is a clear need for reproductive studies of other violets with mixed chasmogamous-cleistogamous breeding systems in tropical regions to determine if seasonality is the major regulatory factor in flower phenologies.

Beyond the conclusion of a seasonal flowering pattern for the onset of both chasmogamous and cleistogamous flowering in V. grahamii it is difficult to identify any other contributing factors in the phenological pattern. Data suggest that soil moisture

125 could be play some kind of role in chasmogamous flowering, but correlations between chasmogamous reproduction and soil moisture were not consistent across both years.

The same can be said for daylength. These “false correlations” are interpreted here as indicators of considerable within-site and annual heterogeneity in ecological characteristics, and may sometimes coincidentally correlate with increased reproductive output in one or the other flowering type without a true causal or contributory relationship.

While chasmogamous flowering phenology in Viola grahamii is restricted to the first month of the rainy season and is fully coincident with the onset of cleistogamous flowering, the latter continues well into summer (after which field studies were terminated each year) and probably into late fall as well. There is evidence that the cleistogamous flowering ceases as the dry season progresses. If true, then the cessation of cleistogamy is controlled in the same fashion as the induction of flowering: by a seasonal (climatic) cue provided by the transition from the rainy to dry season.

Cleistogamy has generally been viewed as a backup strategy to ensure the production of seeds under any conditions, usually unfavorable ones. This theory implicates intrinsic or extrinsic cues to the plant when chasmogamous flowers have failed to attract pollinators and set fruit. Such a mechanism would probably be operative in temperate species that experience non-overlapping chasmogamous and cleistogamous flowering phenology, as is the case with Viola striata. In the Tropics, however, plants may produce the two flower types simultaneously with the onset of the rainy season, as does tropical montane V. grahamii. The plants therefore cannot receive cues regarding

126 pollinator success with chasmogamous flowers in advance of cleistogamous flower production. Furthermore, cleistogamous seed production in both the temperate and the tropical montane species contributed the majority of reproduction in both study years; and discontinuation of field observations at the end of summer poses the larger percentages of cleistogamous vs. chasmogamous seeds as substantial underestimates for cleistogamy. Rather than a “backup” strategy, cleistogamy greatly extends the reproductive power of the species far beyond that allowed by chasmogamy alone in these spatially and temporally heterogeneous forest habitats. Indeed, given the much larger role by cleistogamy in survival and continuation of the species than that played by chasmogamy, one could legitimately view chasmogamy as the “backup” strategy that provides a few additional seeds and maintains inter-population gene flow through occasional outcrossing.

This new climatically regulated and coincident pattern of flowering in V. grahamii deserves further study to fully understand the mechanisms involved in its maintenance. Studies of other subtropical and tropical plant groups are needed to seek additional examples of this new phenological pattern in chasmogamous-cleistogamous mixed breeding systems and to assess its importance in tropical plant communities.

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Chapter 5:

Influence of a mixed breeding system on the population genetic structure of

temperate and subtropical Viola (Violaceae)

Introduction

The presence of a mixed reproductive strategy in angiosperms, that combines the two extremes of outcrossing and selfing in the same individual, is a phenomenon that occurs in 48 families, 199 genera and 465 species (Klooster and Culley, 2004).

Such strategies involve the presence of morphologically distinct chasmogamous flowers which typically outcross, on the one hand (though in some cases they may be self-fertile or capable of “delayed selfing” in the absence of pollinators [Porras and Munoz

Alvarez, 1999; Schemske, 1978, Culley, 2002]), and cleistogamous flowers which are obligate selfers, on the other. While present in diverse angiosperm groups, this mixed chasmogamous-cleistogamous breeding system is not necessarily ubiquitous among taxa of any single group. The strategy has apparently evolved many times in higher angiosperm lineages and also multiple times within families and genera in which it occurs (Klooster and Culley, 2004; Lord, 1981).

The presence of both chasmogamous (outcrossing) and cleistogamous (selfing) reproduction in the same individuals theoretically imparts an advantage to populations and species by ensuring the production of offspring under any environmental condition

(Clausen, 1963; Elisafenko, 2001). Rebdo-Torstensson and Berg (1995) have suggested that the allocation of resources to cleistogamous flowers would be dependent on the

128 success of chasmogamous flowers earlier during the chasmogamous flowering season, and therefore would represent a backup self-pollination mechanism if the chasmogamous flowers failed to set seed (Schoen and Loyd, 1984). Cleistogamous offspring would encompass reduced genetic variability, but because cleistogamous seed production would augment output from chasmogamous flowers and would serve primarily for population persistence, the consequences of this reduction would probably be inconsequential. In some instances cleistogamy has been shown to be advantageous.

In the invasive species Microstegium vimineum (Poaceae), cleistogamy allows for the production of a larger number of seeds, thereby increasing colonization success of the species in new habitats (Barden, 1987; Ehrenfeld, 1999). The relative proportion of reproduction through chasmogamy vs. cleistogamy is usually species related, and the genetic consequences of it depend on the species.

In species where chasmogamy is the main source of seed set, patterns of genetic variability and relatedness should be very similar to those in obligate outcrossers. In

Viola pubescens, which reproduces primarily through chasmogamy, studies have shown populations to have high levels of genetic variation, presumably the result of extensive outcrossing (Culley and Wolfe, 2001). However, Viola pubescens exhibited greater variation than expected, given that delayed selfing operates when chasmogamous flowers have not been pollinated by insect visitors.

In species where reproduction is mainly due to cleistogamy, studies have generally concluded that high levels of selfing lead to potential problems stemming from inbreeding depression, erosion of genetic diversity within populations and

129 increased genetic isolation between populations. This has been shown in Scutellaria indica (Lamiacae), in which a reduction in the amount of polymorphic loci has been verified by the use of allozymes and RAPD data (Sun, 1999). The same holds true for

Impatiens pallida (Balsaminaceae), in which geitonogamous selfing of chasmogamous flowers accounts for more than a third of chasmogamous seed production and, when considered with cleistogamy, results in high levels of inbreeding (Steward, 1994). A breeding system that involves mainly cleistogamous reproduction could additionally lead to a pattern of population substructuring analogous to “isolation by distance”

(Wright, 1943) between populations. In Polygonum thunbergii (Polygonaceae) cleistogamous and chasmogamous seeds lack dispersal mechanisms, enforcing a clear clumped population substructure with genetically closely related individuals as close as three meters; in such situations one finds a significant negative correlation between genetic relatedness and geographic distance (Konuma and Terauchi, 2001).

The genus Viola includes a great many species that reproduce via a mixed chasmogamous-cleistogamous breeding system. Additionally, seed dispersal is generally limited to a few meters, resulting from either an active or passive mechanism.

Two dispersal strategies are employed by violets. The active mechanism, diplochory or ballistic dispersal, takes place in fruits borne on erect peduncles well above the ground.

At maturity the capsule dehisces along three sutures spreads the valves outward to display the seeds. As the elastic valves dry and shrink inward laterally, they forcibly eject seeds a short distance from the parent plant. The seeds may be subsequently found by itinerant ants and carried off an additional short distance. The passive mechanism,

130 myrmecochory, involves gradual decomposition of the capsule wall and passive spilling of the seeds onto the ground. As in diplochory, ants may occasionally find the seeds and carry them short distances from the original site. One published measure of dispersal for both mechanisms suggest an average of 200 cm (Culver and Beattie, 1978), although some reports extend this to 510 cm in some species (i.e. V. pedata; Jolls, 2003). In

Japanese populations of Viola selkirkii and Viola verecunda, ant dispersal distances have been reported as 28 and 36 cm respectively (Ohkawara and Higashi, 1994).

The mixed chasmogamous-cleistogamous breeding system of Viola has been well-documented in terms of its ecological patterns, pollinator behavior and embryological patterns for temperate species (Holdsworth, 1966; Beattie, 1971, 1972,

1976; Baskin and Baskin, 1975; Newell et al., 1981; Mayers and Lord, 1983; Solbrig et al., 1988a; Mattila and Salonen, 1995). However, only a few studies have addressed the evolutionary or population genetic consequences of this breeding system (Auge et al.,

2001; Culley and Wolf, 2001; Batista and Sosa, 2002; Culley and Grobb, 2003). A handful of other studies have indirectly investigated population diversity involving the breeding system while addressing principally taxonomic questions (Kim et al., 1991;

Ko et al., 1998; Marcusen and Borgen, 2000; Marcusen, 2003).

To further examine population genetic diversity of Viola with a mixed chasmogamy-cleistogamy breeding system, and to extend these studies to tropical regions lacking such information, I chose to compare a species from temperate forests and another from subtropical forests. To reduce the effect of phylogeny on reproductive behavior I selected two species from Viola section Viola, acaulescent V. grahamii

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Bentham (subsection Mexicanae W. Becker) of evergreen forests of Mexico and northern Guatemala, and caulescent Viola striata Aiton (subsection Rostratae Kupffer) of seasonal mesophytic forests of the eastern United States and southern Canada

(Ballard et al., 1999) (Figure 5.1). Both species exhibit the typical mixed breeding system that is very common in the genus.

Viola grahamii is a perennial herb inhabiting a wide range of altitudes, from

1950 to 3600 m above sea level, and diverse habitats including oak-pine forest and cloud forest (Ballard, 1994). It produces both chasmogamous and cleistogamous flowers at the same time, usually beginning in June at the start of the rainy season.

Chasmogamous flowers are produced for only a few weeks, whereas cleistogamous flowers continue for as long as six additional months, into the dry season if conditions are favorable (Chapter 4). Most seed set is the result of cleistogamous reproduction

(Chapter 3). Additionally, asexual reproduction via surficial stolons is common in V. grahamii, with these disintegrating after the successful establishment of plantlets; loss of this temporary connection makes the later discrimination of ramets from genets all but impossible. The combination of selfing (through cleistogamy) and clonality

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Figure 5.1: Range of Viola striata and Viola grahamii indicating location of study sites in Ohio and Michoacán.

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(through stolons) could theoretically lead to a significant reduction in genetic diversity within populations and heightened isolation between populations, as predicted from studies of other substantially inbred plant taxa.

Viola striata inhabits low moist places, usually around streams and floodplains, and is associated with a wide range of forest types in eastern North America where it can establish large populations and even become weedy (Gleason and Cronquist, 1991).

It reproduces via chasmogamous flowers early in the spring and by cleistogamous flowers produced afterwards to the end of the growing season, usually until mid-

October. Unlike V. grahamii, V. striata produces the two flower types in two non- overlapping time periods, with chasmogamous flowers first. Like V. grahamii, however, it produces the majority of its seeds through cleistogamy (Chapter 4). This species is not known to reproduce asexually; therefore, each individual in a population represents a genet.

I would expect reduction of the genetic diversity in populations of both species due to the high levels of selfing through cleistogamy (and in V. grahamii, additionally through clonality), although this might be lessened in one or the other due to differences in their contrasting environments or reproductive biology. I compared estimates of population genetic diversity, local population differentiation and population subdivision in Viola striata of temperate deciduous forests and V. grahamii of evergreen tropical montane forests to ascertain patterns of genetic variation and to determine the roles of geography, ecology or site conditions in those patterns.

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To evaluate these questions, I utilized variation in Inter-Simple Sequence Repeat

(ISSR) markers, PCR-generated DNA fragments representing the intervening region between nearby microsatellites (tandem repeats, or SSRs) on complementary strands.

ISSR fragment generation utilizes a single primer with a microsatellite motif embedded within the primer (Nagaoka and Ogihara, 1997). These markers provide a straightforward and relatively inexpensive estimate of population genetic variation similar to those derived from AFLPs (amplified fragment length polymorphisms) and

RAPDs (randomly amplified polymorphic DNA). Since their first use with the identification of cultivars (Fang and Roose, 1997), ISSRs have seen wide application in studies of plant genetic diversity (McCauley and Ballard, 2002; Chapman et al., 2004), hybridization (Wolfe et al., 1998; Archibald et al., 2004), and studies of plant taxonomy

(Wolfe and Randle, 2001; Yockteng et al., 2003) largely due to their ready application to a large variety of plant groups.

Unlike co-dominant marker systems such as allozymes or microsatellites, ISSRs are dominant markers and, therefore, require certain assumptions invoking Hardy-

Weinberg equilibrium in order to estimate the homozygous recessive (non-amplifying) fraction. Presence of an ISSR fragment (= “locus”) indicates either a dominant homozygote or heterozygote, leading to an inability to distinguish these two genotype categories. The absence of a locus may also not represent the presence of a homozygous recessive genotype but may in fact be the result of nucleotide changes at the priming site. Consequently, this may give inaccurate estimations of relationship or ancestry where the absence of an amplification product is used to indicate relationship

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(Culley and Wolfe, 2001). Despite the disadvantage of such a dominant marker technique, Culley and Wolf (2001) have shown that ISSRs are capable of providing similar results to allozymes in the amount of genetic variation revealed in V. pubescens.

Materials and Methods

Study Sites

Viola grahamii: Two replicate sites representing separate populations of V. grahamii were established in the primary evergreen forest to the north of Lake

Pátzcuaro on the southern slopes of Mt. Zirate, in the municipality of Quiroga, state of

Michoacán, Mexico. The first locality, Santa Fe, was approximately 1.5 km walking distance above the town of Santa Fe de la Laguna at 19º41’N, 101º32’W. At this point the mixed scrub pasture land of the lower slopes of Mt. Zirate gives way to uninterrupted forest dominated principally by a mix of Quercus and Pinus. The second locality, Quiroga, was approximately 1.4 km east of the first site above the town of

Quiroga in an area of similar vegetation at 19º40’N, 101º33’W.

Viola striata: As in central Mexico, two replicate and apparently unconnected populations were chosen within Strouds Run State Park in Canaan Township, Athens

County, Ohio, USA. The first site, Campground, was adjacent to the north end of the park campground at 39º21’N, 82º02’W. The second site, designated Cemetery, was immediately beyond the northwest end of Dow Lake adjacent to the Pioneer Cemetery trail at 39º21’N, 82º02’W. Both sites were physiographically similar in being adjacent to small streams in continuous deciduous forest dominated by Acer and Platanus. The

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Campground differed floristically from the Cemtery in containing a higher proportion of conifers from an adjacent plantation of Pinus resinosa.

Tissue sampling

In each site 20 quadrats were established randomly within each population for genetic sampling and ecological studies (Chapter 3). Two individuals from each quadrat were randomly selected for genetic analysis. A young leaf from each individual was collected and preserved in a marked bag with silica gel desiccant (28-200 mesh).

These tissue samples were stored in a larger bag of silica gel at -20°C until DNA extraction. At the time of collection, geographical distances in meters among quadrats were determined for later comparison of genetic and geographic distance. Following the field studies and prior to statistical analyses, the 20 quadrats were classified into four apparent groups corresponding to microtopographical “subpopulations”. This permitted testing of potential small-scale ecological or distributional patterns that might relate to genetic relatedness among individuals or groups of individuals.

DNA extraction and ISSR amplification

Viola striata: Extraction of DNA was performed using a chloroform-isoamyl alcohol extraction procedure (Ballard et al., 1999) modified from the SDS buffer protocol of Edwards et al. (1991). Approximately 40 mg of silica gel dried plant tissue was used in each extraction. Following extraction, DNA extracts were stored at -20°C until PCR amplification. ISSR primers were screened among those that had successfully

137 produced consistent patterns of variation in a previous study with Viola grahamii

(Cortés-Palomec and Ballard, in review) and for V. pubescens (Culley and Wolfe,

2001). Four primers, two with di-nucleotide repeat and two with tri-nucleotide repeat motifs were chosen based on desired levels of variation and ease of scoring due to band sharpness. ISSR amplifications were performed in replicated single-primer 25µL reactions and were optimized for each individual primer by altering concentrations of

MgCl2 (25 mM, Applied Biosystems), and sample DNA and by adjusting the primer annealing temperature. The primer HB 15 [(GTG)3GC] was optimized using 6 mM of

MgCl2, a sample DNA dilution of 1:30, and an annealing temperature of 43ºC. Primer

MAOac [(CTC)4AC] was optimized using 4 mM of MgCl2, a sample DNA dilution of

1:30 and an annealing temperature of 45ºC. For the two dinucleotide primers 17899b

[(CA)6GG] and 17898a [(CA)6AC] the optimal running conditions included 6 mM of

MgCl2 and a sample DNA dilution of 1:10; the annealing temperature varied slightly,

43ºC for primer 17899b and 42ºC for 17898a. For all reactions and primers the remaining cocktail contained 10x PCR buffer (Applied Biosystems), 0.8 mM dNTP mix

(10mM, Fisher), 2 µg BSA (bovine serum albumin, Fisher), 0.2 mM primer (produced by Operon Technologies), 1.25 U (Taq polymerase (AmpliTaq®, Applied Biosystems),

1µL DNA sample (adjusted for concentration through dilution), and autoclaved distilled water to bring the reaction to volume. The polymerase chain reaction was performed in a Stratagene RoboCycler 96 with hot-top (Stratagene Inc, La Jolla, CA ) and programmed for 2 min. at 94ºC; 44 × 30 sec. at 94ºC, 45 sec. at 42ºC, 43ºC, or 45ºC, 1 min. 30 sec. at 72ºC; 20 min at 72ºC.

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Viola grahamii: Genomic DNA was extracted from the silica gel-dried leaves using a Wizard Genomic DNA purification kit (Promega) following the protocol for isolation of genomic DNA from plant tissues. Due to the high concentrations of mucilage in the leaves, an additional 200 µL of Protein Precipitation solution was added during the extraction procedure. ISSR primers were screened as described before for V. striata. The primer HB 15 [(GTG)3GC], was optimized using 4 mM of MgCl2, a sample

DNA dilution of 1:60, and an annealing temperature of 42ºC. For the two dinucleotide primers 17899b [(CA)6GG] and 17898a [(CA)6AC] the optimal running conditions included 6 mM of MgCl2 and a sample DNA dilution of 1:10; and an annealing temperature of 42ºC. The remainder of the reaction cocktail and PCR program was the same as for V. striata.

ISSR fragment visualization

Following PCR amplification, 1 µL loading dye was added to the reactions and the samples loaded into a 1.3% agarose gel in 0.5 x TBE buffer. Multiple 250 bp DNA ladders (Fisher) were run in each gel for later size comparison. Gels were run at constant voltage (= 85 V) for 2 hours. Gels were stained with a solution of ethidium bromide in 0.5 x TBE buffer for 20 minutes and imaged under UV light using a Flor-

S™ MultiImager (Bio-Rad, Hercules, CA, USA). Analysis of images and identification of fragment patterns was performed with the Biomax 1-D image analysis software

(Version 2.0.3, Eastman Kodak Company, Rochester, NY). Fragment size was determined through comparison with the included size standards. Loci were compiled

139 into a binary data matrix of 0s (locus absent) and 1s (locus present) prior to statistical analysis.

Statistical Analysis

Genetic diversity within and between populations: Because of the probability of violating assumptions of Hardy-Weinberg equilibrium in estimating the recessive homozygote fraction from the dominant data, analyses and subsequent interpretation emphasized broader issues of genetic diversity and deemphasized results from F- statistic analogs. Fixed loci and population-specific loci were determined through visual inspection of the data; heterozygosity (Ho; direct count) and percent polymorphic loci

(P; 0.95 level) were determined for each population and groups within populations using Tools for Population Genetic Analysis software (TFPGA) 1.3 (Miller, 1997) with the dominant marker estimation procedure of Weir (1990).

Genetic differentiation: Genetic differentiation was determined through an

Analysis of Molecular Variance (AMOVA) performed using AMOVA 1.55 (Excoffier,

1993). This statistical method was used to quantify the level of genetic variability among individual populations (sites), and the assigned quadrat group within each population. The analysis partitioned genetic variance between and within populations

(groups), tested the significance of this variation within and among populations

(groups), calculated ΦST (equivalent to FST or GST for co-dominant data) and tested the homogeneity of molecular variance (HOMOVA) using the Bartlett statistic (Stewart and

Excoffier, 1996)..Modified input files were created with AMOVA-PREP 1.01 (Miller,

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1998) using the Euclidean distance metric of Excoffier et al. (1992) a procedure amounting to a tally of fragment differences among individuals (Huff et al., 1993).

Visualization of relationship: Principal Coordinates Analysis (PCoA) was used to explore patterns of relationship among individuals among and within each population for each species. Analyses were performed in NTSYS-pc 2.10t (Rohlf, 1999) and utilized a similarity matrix derived from the Dice coefficient (Dice, 1945). This coefficient is equivalent to that of Nei and Li (1979) and has been shown to provide superior performance in recent simulation experiments using dominant marker genetic data (Meyer et al., 2004). Its primary advantage for dominant data stems from the mathematical behavior of downweighting “shared absence” of a fragment for a given locus between samples and upweighting “shared presence” for one, because absence of a fragment cannot be construed as evidence for a close relationship.

Genetic/geographic distance correlation: A Mantel test (Mantel, 1967) was performed to test for correlations between genetic and geographic distances among individuals within each population. Since two individuals were collected per quadrat, values in the geographic distance matrix were duplicated for those individuals sharing the same quadrat. The Mantel test was performed using the MXCOMP procedure in

NTSYS-pc 2.10t (Rohlf, 1999).

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Results

Viola grahamii

A total of 43 scorable bands were generated using three primers in 77 individuals for the two populations. Three individuals were dropped from the analysis due to the inability to generate amplification products. Of the 43 bands, five occurred in only one individual each in the two populations. Across the two populations variation was seen in all loci, resulting in 100% polymorphic loci. Seven bands were exclusive to

Santa Fe but only three were exclusive to Quiroga. The larger number of unique bands in plants at Santa Fe also corresponded to a slightly higher (63.4%) percentage of polymorphic loci than at Quiroga (60.97%) (Table 5.1). Although V. grahamii reproduces clonally and one would expect to encounter the same genotype in multiple individuals, no two individuals in either population possessed the same composite ISSR genotype.

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Table 5.1: Levels of population genetic diversity inferred from ISSR data for two populations of two violet species: Viola grahamii and Viola striata. Number of individuals in population (N), number of unique ISSR bands in population (Bands), heterozygosity (direct count) (Ho), and percentage of polymorphic loci (0.95 level) (P) are shown.

Species Population N Bands Ho P V. grahamii Santa Fe 38 7 0.167 63.41 Quiroga 39 3 0.169 60.97 Total 77 0.213 100.00 V. striata Campground 40 6 0.139 46.15 Cemetery 40 14 0.169 60.25 Total 80 0.164 58.97

Analysis of genetic differentiation with AMOVA indicated that most of the variance was accounted for by variation within each population. Only 9.48% of the variation was explained by differences between the two populations (Table 5.2).

Additionally the very low ΦST indicated little if any fixation of difference between the sites and the non-significant Bartlett’s statistic indicated an equal level of genetic variability within each of the two sites. The pattern of very low differentiation between the two populations was additionally shown in the PCoA by the high overlap of individuals from the two sites; however, some individuals from both populations were genetically distinct overall from those at the other site (Figure 5.2A).

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Table 5.2: Hierarchical Analysis of Molecular Variance (AMOVA) for two populations and four subpopulations of Viola grahamii in Michoacán based on the analysis of ISSR data.

Bartlett’s Source df Variance % P-value* ФST Statistic P-value* Santa Fe vs. Quiroga Between pops. 1 0.4877 9.48% <0.005 0.095 0.021 0.492 Within pops. 75 4.658 90.52% Subpopulations in Santa Fe Among groups 3 0.4115 8.45% <0.0005 0.084 0.069 0.744 Within groups 34 4.4594 91.55% Subpopulations in Quiroga Among groups 3 0.4750 10.19% <0.0005 0.102 0.671 0.001 Within groups 35 4.1864 89.81% *nonparametric randomization test (2000 permutations)

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0.6 A. V. grahamii, Michoacán 0.4

0.2

0.0

-0.2

Axis 2 (9.21 %) -0.4

-0.6 Santa Fe Quiroga -0.8 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Axis 1 (14.21%)

0.6 B. V. striata, Ohio 0.4

0.2

3 %) 0.0

-0.2 is 2 (8.4

Ax -0.4

-0.6 Campground Cemetery -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

Axis 1 (9.98%)

Figure 5.2: Principal coordinates analysis (PCoA) using the Dice coefficient of similarity computed from ISSR data. A. Viola grahamii, Michoacán, 77 individuals in two distinct populations. B. Viola striata, Ohio, 80 individuals in two distinct populations.

145

Although most of the variation was partitioned within populations, the Mantel test gave non-significant results for population substructure; genetic distance did not correlate with geographic distance within either population (data not shown). Within the Santa Fe population the four subgroups showed varying levels of polymorphic loci between 46 and 60 %, with the highest level in quadrats outside the area of conifers in broadleaf evergreen forest (Figure 5.3). There was low differentiation among the groups of quadrats, indicating consistent (albeit periodic) gene flow (Table 5.2). Within

Quiroga greater divergence was seen among the four quadrat groups, indicated by a significant Bartlett’s statistic. The two groups of quadrats closest to the semi-open pasture (II and IV) showed lower levels of polymorphic loci (36-41%), while the other two groups (I and III) showed increased levels of heterozygosity and polymorphic loci, perhaps resulting from their positions in less disturbed forest (Figure 5.3). The AMOVA additionally indicated a slightly greater differentiation among the quadrat groups at

Quiroga, with 10.19% of the total within-site variation resulting from differences among the groups.

Viola striata

A total of 78 bands were recorded from four primers applied to 80 individuals across two populations. Of the 78 bands, five occurred in only one individual each in both populations, and one was present in all individuals in both populations. Variation across the two sites was lower than in V. grahamii (58% polymorphic loci, Table 5.2).

146

Figure 5.3: Partitioning of genetic variation among subgroups in two populations of Viola striata (Ohio: Cemetery and Campground) and Viola grahamii (Michoacán: Quiroga and Santa Fe). Number of plants per group (n), average heterozygosity (Ho) and percent of polymorphic loci (P) are included for each group. All populations were in forested areas however regions depicted by trees were dominated by conifers.

147

Fourteen bands were unique to Cemetery while only six were exclusive to Campground.

Correspondingly, polymorphic loci were higher in Cemetery (60.25%) than in

Campground (46.15%), a difference apparently driven by individual quadrats and not generalized across the population as a whole.

AMOVA revealed that, like V. grahamii, V. striata partitioned most genetic variation within individual populations (88.3%) and showed very little differentiation between populations (Table 5.3). Genetic diversity differed between the two sites, as indicated by the significant Bartlett’s statistic. Overlap in genotypes, heavy but not complete between the two sites, was clearly evident in the PCoA (Figure 5.2B).

148

Table 5.3: Hierarchical Analysis of Molecular Variance (AMOVA) for two populations and four subpopulations of Viola striata in Ohio based on the analysis of ISSR data.

Bartlett’s Source df Variance % P-value* ФST Statistic P-value* Campground vs. Cemetery Between pops. 1 1.107 11.7% <0.005 0.117 0.271 0.001 Within pops. 78 8.361 88.3% Subpopulations in Campground Among groups 3 0.598 7.64% <0.0005 0.076 0.199 0.05 Within groups 36 7.238 92.36% Subpopulations in Cemetery Among groups 3 1.378 14.69% <0.0005 0.147 0.540 0.001 Within groups 36 8.008 85.31% *nonparametric randomization test (2000 permutations)

149

Discussion

Both V. grahamii and V. striata exhibited higher levels of genetic polymorphism than expected from high selfing rates via cleistogamy. In the case of V. grahamii, which is also clonal through stolons, no identical genets were identified in either population; the levels of genetic diversity were actually higher than those recorded for V. striata

(which is not clonal). It is highly unlikely that clonally derived ramets of V. grahamii simply do not persist or survive. A more exhaustive, long-term demographic study is needed to determine how vegetative reproduction contributes to recruitment (or does not) in this species. The clonal species Calamagrostis porteri (Poaceae) had only 10-20

% polymorphic loci assayed using RAPDs and ISSRs (Esselman et al., 1999), and the primarily cleistogamous Scutellaria indica (Labiatae) exhibited an average of 8.9 % in studies using RAPD data (Sun 1999). These values are much lower than those for V. grahamii (60-63%) and V. striata (46-60%).

While my results appear anomalous in comparison with other highly selfing or clonal species, high levels of population variation have been identified in other violet species with mixed breeding systems. In the mainly chasmogamous species Viola pubescens, Culley and Wolf (2001), using allozymes, reported a mean polymorphic loci value of 77.1%. Culley and Grubb (2003) reported that fragmented populations showed unexpectedly high levels of genetic variation, high outcrossing rates and no correlation between genetic and geographic distances, in the same species.

150

My results from both V. grahamii and V. striata suggest that these species maintain genetically heterogeneous populations with their mixed breeding system despite high levels of selfing from cleistogamous flowers. Selfing via cleistogamy is still a mode of sexual recombination; therefore, additional (albeit lower) genetic recombination from substantial levels of cleistogamy will nevertheless augment that derived from lower rates of recombination through outcrossing chasmogamous flowers.

Wright (1931) has also suggested that if at least one successful outcrossing event (or migration) occurs between populations once every other generation, the genetic recombination would be sufficient to maintain gene flow between the populations. In addition to genetic recombination from the breeding system, ecological, abiotic and other factors may operate to influence the genetic substructure within populations and differentiation between them. The uneven age structure of these perennial forest herbs is clear and the individuals themselves may be acting as a storehouse of genetic diversity.

Many generations of plants are present during any growing season and their genetic composition is a reflection of growth conditions and pollinator visitation in the past.

The geographic location of these and other individuals in a given population, their reproductive behavior, and their contribution to current and future genetic diversity in the population or differentiation from other nearby populations, may be influenced by microtopography, local and regional climate, disturbance regimes and other abiotic variables; by pollinators, herbivores, pathogens and potential seed dispersers; and by spontaneous mutations or immigration from other populations. Thus, the breeding

151 system, site characteristics, regional landscape and climate inhabited by the species contribute to the long-term genetic history of each population and into the species.

Geographic and genetic distance did not correlate among populations in either V. grahamii or V. striata. This is probably due in part to the low genetic differentiation

(suggesting high levels of gene flow) between populations in each species.

Differentiation among sites expressed minimal fixation (V. grahamii ΦST = 0.09; V. striata ΦST = 0.12)--much lower than values generally found in other species with mixed breeding system. An average AMOVA-derived ΦST value of 0.28 has been reported in mixed breeding species based on RAPDSs (Nybom and Bartish, 2000 as cited in Auge et al.,2001), and 0.21 for GST using allozymes (Hamrick and Godt, 1990).

These values are larger than those reported here. Similar findings have also been reported for V. palmensis, an endemic violet of the Canary Islands, with most variation within populations (90%) and no correlation between genetic and geographic distances

(Batista and Sosa, 2002). This unexpected result defied the locally restricted locations, small population sizes and confinement to an oceanic island (presumably originating from long-distance dispersal from continental Europe). Nevertheless, high levels of polymorphism and low differentiation suggested either recurrent colonization and establishment from different continental source populations, with subsequent remixing; or high levels of gene flow by seeds, pollen or both.

The pattern of low population differentiation and apparently high gene flow, in the violets studied here and in the Canary Island endemic, cannot be generalized across

152 the genus Viola. In the clonal central European violet V. riviniana, variation was concentrated in differences among sites (41.2%) rather than within sites. However, since seedling recruitment was as important as clonality in the population, higher values than those of mainly clonal species were found (Auge et al., 2001) in the species (ΦST =

0.41) (Auge et al., 2001).

My low rates of population differentiation may be attributable to the geographic sampling scale of my populations. While the several-kilometer distances separating the study sites for each species were deemed far greater than predicted yearly pollinator movements, perhaps the distances would not preclude longer-term gene flow. It is also possible that intervening populations serve as a genetic “bridge” between my study sites over a moderate length of time, and that genetic sampling of these proximal populations might reveal the pattern of gene flow more fully. It is also conceivable that recognizable patterns of population differentiation may not become evident with distances less than tens or hundreds of kilometers. Genetic results may also manifest founder effects of recolonization from localized source areas following past human disturbance or ecological perturbation, or reflect shared ancestral polymorphisms from the recent past when populations were continuous throughout the region. Without strong selection pressures acting differentially upon the populations, they have been very slow to differentiate.

Aside from intermittent visitors such as the Golden Banded Skipper Autochton cellus Boisduval and Le Conte on V. grahamii, an as yet undescribed species of

153

Dianthidium is reported here to be the primary pollinator (Chapter 4). Nothing is known about the biology of the bee, or whether it travels over long distances (thus serving to enhance gene flow and counteract population differentiation. In V. striata solitary bees (Andrena violae Robertson), carpenter bees (Ceratina dupla dupla Say) and the bee fly (Bombylius major L.) have been observed visiting the flowers, and as in

V. grahamii they may move pollen further than has previously been proposed.

At the subpopulation level in both V. grahamii and V. striata, site-specific characters are apparently influencing the genetic patterns observed within each population. Within V. grahamii differences between the two sites may explain the different patterns of group differentiation and heterogeneity of variances observed within each population (Figure 5.3). In Quiroga, a high level of disturbance is found, especially in the area close to the semi-open pastures. This land was originally forested in mixed oak and pine forest and has likely only recently been cleared, however I have no precise estimation for the length of time this land has been in pasture. Currently the semi-open pasture is dominated by Baccharis with a few species of trees and large

Opuntia cacti widely scattered throughout the area. My sampled population of V. grahamii begins near the edge of the pasture in highly perturbed pine-oak forest. Local woodcutting is very common in the ecotone region bordering the semi-open pasture and wood harvesting is evident in the site, and substantial foot traffic and local disturbance is apparent on the small footpath frequently used by woodcutters and their pack animals. I suspect that this disturbance is responsible for the reduced genetic diversity documented in this area of the population. The two groups of quadrats closest to the

154 pasture land, and therefore presumably subjected to greatest degree of direct or indirect disturbance from timbering, livestock and foot traffic, expressed reduced genetic polymorphism, perhaps as the result of plant damage or cattle grazing. The group with the greatest genetic diversity was group I, which was farthest from the pasture in the least disturbed portions of the forest.

Santa Fe site was generally more topographically and ecologically homogeneous, and the values of genetic differentiation and variance homogeneity parallel these site conditions. Much of the site is dominated by coniferous forest of

Pinus montezumae and, although it is near pasture land, there is very little disturbance from wood cutting or grazing cattle. Although there was no strong statistical support for differentiation or variance heterogeneity within the population, there was a clear pattern of higher genetic diversity in the region characterized by greater canopy openness. The higher light availability may be leading to more outcrossing of the chasmogamous flowers, since it has been shown that chasmogamous fruit production is positively correlated with greater light reaching the plants (Chapter 3), presumably stimulating increased pollinator activity.

In Viola striata differentiation among the two populations was slightly higher than that seen in V. grahamii and there was a significant difference in genetic variation between the two sites, suggesting different microsite conditions maintaining genetic diversity at different levels. Ecologically there were few differences between the two sites except for the much higher proportion of conifers at the Campground, which had correspondingly lower values of genetic diversity (Figure 5.3). The major difference

155 accounting for unequal genetic diversity between the two sites may be related to the total area encompassed by the populations. The Campground population was smaller in area extent than Cemetery population. While the numbers of individuals sampled were identical and their spatial distribution essentially equal, the proportion of the population extending beyond the immediate sampled area was much smaller in the Campground, bounded on the south by the campground and on the east by the road and inhospitable habitat beyond that.

In Cemetery differentiation and diversity are likely influenced by dynamics of the floodplain. Group II, found immediately adjacent to the stream, contained a much higher level of genetic diversity than other quadrat groups. As previously mentioned, this group has been frequently inundated by flood waters, at least once during each growing season between 2002 and 2004. The inundation generally takes place in response to heavy summer rains and usually occurs after chasmogamous flowering.

During this time there is a reduction in herbaceous vegetation in the area, although violets generally survive and quickly recover. As flooding occurs generally outside of the time to directly influence pollen movement via insects, it may be indirectly affecting pollinator movement in subsequent years by creating a region of more cleared forest adjacent to the stream which they may be using as a corridor. Thus this streamside microsite may be favoring increased pollinator activity ultimately resulting in greater genetic diversity of plants residing within this microsite.

In conclusion, I have shown that not all species utilizing substantial levels of selfing (in this case, cleistogamy) in their breeding system will inevitably suffer from

156 high population subdivision, high population differentiation and low genetic diversity within populations. My studies on a temperate and a montane tropical violet species,

Viola striata and V. grahamii, respectively, show that none of these scenarios hold despite the fact that the majority of seed production in both is due to cleistogamy. Both species maintain a mixed chasmogamous-cleistogamous breeding system, and V. grahamii is also clonal through production of surficial stolons. Nevertheless, they are exceptions to the theoretical outcomes proposed for the majority of other plant species that argue against selfing as an advantageous evolutionary mechanism. These species and others, including V. pubescens, are capable of maintaining levels of diversity as high and higher than that seen in purely outcrossing species. The mixed chasmogamous-cleistogamous breeding system is clearly a successfully balanced and robust system in maintaining populations, and could be construed evolutionarily to be highly advantageous for the species utilizing it, both in terms of demographic maintenance and propagation of high genetic diversity for establishment of new populations. Apparently few cross-pollination events are necessary to introduce genetic novelty into the population or to maintain gene flow between nearby populations. The genetic diversity is maintained through selfing throughout much of the year in the age- structured populations. Additional comprehensive studies are encouraged with other species that rely on mixed breeding systems. These are essential to assess whether the theoretical principles of selfing disadvantage are the rule, especially with plants outside of the temperate region and in non-forest biomes.

157

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