PATTERNS OF EDAPHIC AND PHENOTYPIC VARIATION IN THE LASTHENIA CALIFORNICA SPECIES COMPLEX (ASTERACEAE)

by

Gina Choe

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

in

THE FACULTY OF GRADUATE STUDIES

(Botany)

THE UNIVERSITY OF BRITISH COLUMBIA

September 2007

© Gina Choe, 2007 ABSTRACT

Members of the Lasthenia californica species complex show extensive ecological and phenotypic diversity. This thesis investigates patterns of edaphic, physiological, flavonoid, and morphological differentiation within the Lasthenia californica species complex in two distinct studies. In the first study, patterns of ecological and physiological variation along with flavonoid polymorphism and ITS1 species type distribution were characterized. The hypothesis of parallel evolution of edaphically differentiated flavonoid races across lineages of L. californica sensu lato was also examined in this study. We found evidence refuting the predictions of this hypothesis, particularly that flavonoid type and edaphic environments are correlated across the complex. However, when major axes of ecological and physiological variation were characterized within the complex, the greatest amount of edaphic/tissue variation occurred along lines of toxicity, reaffirming prior characterization. In the second study, the basis of the phenotypic differentiation observed between inland and coastal ecotypes of Lasthenia californica sensu stricto was investigated by growing populations of each ecotype in controlled conditions under varying levels of salinity (an important environmental factor differentiating ecotypic environments). Reaction norms of populations were compared under saline conditions to test for evidence of differential fitness between ecotypes (differential salt tolerance) and differences in intrinsic and induced levels of trait expression (e.g. sodium accumulation and succulence). The hypothesis of trade-offs between salt tolerance and growth potentials was also investigated. Under experimental conditions, coastal populations demonstrated uniformly greater salt tolerance than inland populations. We found that most traits showed both intrinsic differentiation and differential responses to salinity, where the extent and direction of trait responses were ecotype specific with little within ecotype differentiation. When comparing growth rate trends between salt tolerant and non- tolerant populations across salinity treatment levels, we found evidence of trade-offs between maximum intrinsic growth rates and investments in salt tolerance. TABLE OF CONTENTS

Abstract ii Table of Contents iv List of Tables v List of Figures.. .'• vii Acknowledgements x

Chapter 1: Introduction •• 1 General introduction 1 The Lasthenia californica species complex 3

Chapter 2: Patterns of edaphic, physiological, ITS lineage and flavonoid variation across the Lasthenia californica species complex: re-evaluating the hypothesis of parallel evolution of edaphic races 12 Introduction 12 Materials and Methods 16 Results 20 Discussion 26

Chapter 3: Differentiation in morphological and physiological responses to salinity between coastal and inland populations of Lasthenia californica sensu stricto 53 Introduction 53 Materials and Methods. 58 Results ; 64 Discussion... 70

Chapter 4: Conclusion 88 Significant findings and implications 88 Hypotheses requiring confirmation and possible future studies. 89

Literature Cited 93

IV LIST OF TABLES

Table 1.1 Summary of the taxonomic history of the Lasthenia californica species complex from 1966 to present. Occurrences of flavonoid races in the members of the complex are included. The parentheses denote the flavonoid profile most common to the lineage 11

Table 2.1 Site ID, ITS species identity, locality, habitat, and flavonoid race determinations from this and previous surveys are summarized. Data from the current study is abbreviated as 2007; data from Rajakaruna and Bohm (1999) and Rajakaruna et al., 2003 surveys are abbreviated as 1999&2003; data from Desrochers and Bohm 1993 is abbreviated as 1993. Sites are roughly ordered geographically, with the northernmost sites listed at the top of the table 38

Table 2.2 Transformations used in statistical analyses. A) Transformations used in ANOVA and PC analyses for soil variables. B) Transformations used in ANOVA's for tissue variables 40

Table 2.3 ANOVA results for soil variables. Variables showing significant differences (P < 0.05) between A and C populations are bolded. Means that are significantly higher are bolded as well. Only differences between A and C were tested; mixed populations of both A and C were not included in the ANOVA. Mineral concentrations are reported in ppm; CEC in meq/lOOg 41

Table 2.4 Summary of number of A, C, or mixed A and C profile populations present on edaphically extreme sites surveyed in 2005 41

Table 2.5 PCA results of soil data. Eigenvalues, % of variance explained, cumulative % of variance explained, and Pearson's correlations (r) to axes are included 42

Table 2.6 Means and standard error of soil variables for serpentine, inland benign pastures and coastal sites. Variables showing significant differences (P < 0.05) are bolded. Significant pair-wise differences (after a Tukey HSD correction) between habitat groupings are denoted with letters where shared letters represent non-significant differences. Mineral concentrations are reported in ppm; CEC in meq/lOOg 43

Table 2.7 Means and standard error of tissue variables for A, C, and mixed A and C populations. Variables showing significant differences (P < 0.05) between A and C populations are bolded and means that are significantly higher are bolded as well. Only differences between A

V and C were tested; mixed populations of both A and C were not included in the ANOVA. All mineral concentrations are reported in ppm 44

Table 2.8 PCA results of tissue data. Eigenvalues, % of variance explained, cumulative % of variance explained, and Pearson's correlations (r) to axes are included 45

Table 2.9 Means and standard error of tissue variables for serpentine, inland benign pastures and coastal sites. Variables showing significant differences (P < 0.05) are bolded. Significant pair-wise differences (after a Tukey HSD correction) between habitat groupings are denoted with letters. Shared letters represent non-significant differences. Mineral concentrations are reported in ppm; CEC in meq/100 46

Table 2.10 Results for linear regressions of soil and tissue concentrations of P, K, Ca, S, Na, Zn, Fe, Cu, B and N (ppm) 47

Table 3.1 Summary of ecotype, population identification, localities, soil and tissue data from field sites used in this study 79

Table 3.2 Summary of ANOVA results for biomass (g), relative growth rates (In mg days"1), total leaf number, LDMC (g g"1), SLA cm2 g"1, and

2 succulence (g H20 cm" ) 80

Table 3.3 Summary of ANOVA results for ion traits: Na (ppm), K (ppm), and Na/K , 81

Table 3.4 Values and significance of Pearson product-moment correlations between leaf values of sodium and potassium, sodium and calcium, sodium and magnesium, and sodium and succulence 82

Table 3.5 Summary of ANCOVA results for sodium tissue contents and succulence 82

vi LIST OF FIGURES

Figure 2.1 PCA ordination of axes 1 and 2 for soil variables, (a) Habitat group symbols correspond to serpentine (A), coastal (X), inland benign pastures (+), and inland alkaline flats (o). Confidence ellipses represent one standard deviation from group centroids (excluding alkaline flats; N=2). (b) Symbols correspond to uniformly A profile populations (A), uniformly C profile populations (C), or populations with mixed A and C individuals (M) 48

Figure 2.2 PCA ordination of axes 1 and 2 for tissue variables, (a) Habitat group symbols correspond to serpentine (A), coastal (X), inland benign pastures (+), and inland alkaline flats (o). Confidence ellipses represent one standard deviation from group centroids (excluding alkaline flats; N=2). (b) Symbols correspond to uniformly A profile populations (A), uniformly C profile populations (C), or populations with mixed A and C individuals (M) 49

Figure 2.3 Bivariate distribution of soil (x) and tissue (y) concentrations of Ca (a) and B (b). Symbols correspond to serpentine sites (S), coastal sites (C), inland benign pastures (P), and inland alkaline flats (A). Linear regressions were fitted for the complex 50

Figure 2.4 Bivariate distribution of log soil (x) and log tissue (y) concentrations of phosphorus (a), sulfur (b), sodium (c), and zinc (d). Symbols correspond to serpentine sites (S), coastal sites (C), inland benign pastures (P), and inland alkaline flats (A). Linear regressions were fitted for the complex 51

Figure 2.5 Bivariate distribution of soil (x) and tissue (y) concentrations of magnesium (a) and potassium (b). Symbols correspond to serpentine sites (S), coastal sites (C), inland benign pastures (P), and inland alkaline flats (A). Confidence ellipses represent one standard deviation from group centroids 52

Figure 3.1 Graphical results for growth traits. Reaction norms with means and standard errors for biomass (g) and relative growth rates (In mg days" ') are shown in figure (a) and (b) respectively. Reaction norms for each population are represented by a unique line described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively, (c) Population means and standard errors for RGR values at the control treatment. Significant pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland

Vll populations and coastal populations are represented by empty bars or shaded bars, respectively 83

Figure 3.2 Reaction norm plots including means and standard errors for (a) leaf number and (b) LDMC (g g"1). Reaction norms for each population are represented by unique lines described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively, (c) LDMC (g g"1) population means and standard errors. Significant pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively 84

Figure 3.3 Reaction norm plots including means and standard errors for (a)

2 1 2 SLA (cm g" ) and (b) succulence (g H20 cm" ), Reaction norms for each population are represented by unique lines described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively. Population means and standard errors for (c) SLA (cm2 g"') and (d)

2 and succulence (g H20 cm" ). Pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively 85

Figure 3.4 Reaction norms with means and standard errors for leaf ion concentrations of (a) sodium (ppm) and (b) potassium (ppm). Reaction norms for each population are represented by a unique line described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively, (c) Population mean and standard errors for leaf ion concentrations of sodium (ppm) at control treatment. Significant pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively 86

viii Figure 3.5 Population means and standard errors for Na/K tissue ratios. Pair- wise differences between populations at each treatment level are denoted with letters, where shared letters represent non-significant differences. Note that Pair-wise tests were performed at each treatment level separately. Population 49, 54, 67, 72 are respectively represented by empty bars, lightly shaded bars, darkly shaded bars, and by black bars..'. 87

Figure 3.6 Bivariate distribution of tissue sodium contents and succulence. Linear slopes are fitted for each population 87

IX ACKNOWLEDGEMENTS

I fondly thank my supervisor, Jeannette Whitton, for her guidance, encouragement and support throughout my academic pursuits at UBC. Thanks also to my committee for their helpfulness: Joerg Bohlmann, Aine Plant (SFU), and Keith Adams. I also wish to thank my lab mates Chris Sears and Chris Lee for their help in the lab and for providing a fun and supportive work environment. Thanks to the staff and researchers at Jasper Ridge Biological Preserve for their hospitality r In particular, thanks to Nona Chiariello for her help. Thanks to Veva Stansel. I'd like to thank Sarah McKim for her comments and William Hay for his grammatical edits. Thanks to my external examiners for their comments: Sally Aitken and Mary Berbee. Thanks to the UBC Herbarium, and the UBC Botany Dept. administration and staff, especially Jin Meng for providing technical support for the growth chambers. A very special thank you to my unfailingly supportive mother, Chan Sook Choe, who has always encouraged me to follow my interests.

I gratefully acknowledge the financial assistance provided to me: The University of British Colombia, Department of Botany (Entrance Scholarship) as well from the Natural Sciences and Engineering Research Council (PGS-M award). CHAPTER 1

Introduction

General introduction

Environmental gradients and discontinuities profoundly affect community structure, niche differentiation, species diversity, and population and trait differentiation.

Ecologists and evolutionary biologists have long recognized the importance of environmental heterogeneity in promoting and maintaining diversity from the scale of communities to genotypes (Kassen 2002; Jasmin and Kassen 2007; Weeks and Hoffman

1998; Freestone and Inouye 2006; Reynolds et al. 1997; Schulter 1993, 1996; Coyne and

Orr 2004; Tilman 1988; Wellborn et al. 1996). Patterns of biological discontinuities

associated with environmental heterogeneity remains one of the most important systems

of study in ecology and evolution.

Edaphic mosaics can produce the most dramatic changes in plant distributions

over both large and small spatial scales (Anderson et al. 1999) and provide model

systems for the study of differentiation along discontinuous environmental features.

Edaphic gradients or discontinuities that include harsh substrates such as serpentine or

salinized soils are likely to exert disruptive selection pressures on neighboring

populations that inhabit contrasting sites. The hypothesis that this form of edaphically

mediated disruptive selection promotes evolutionary diversification and differentiation

(Kruckeberg 1984, 1986, 2002; Raven 1964) is well supported empirically by the

numerous cases of edaphically differentiated populations (e.g. Berglund et al. 2004;

Antfinger 1981; Bradshaw 1952), ecotypes (e.g. Nagy 1995; Sambatti and Rice 2006;

1 Reimann and Breckle 1995), and species (e.g. Kruckeberg 1984; Ellis et al. 2006;

Robichaux et al. 1990; Baldwin 2005).

Parallel edaphic selection regimes on geographically isolated populations have the potential to repeatedly generate entities with shared adaptations. Edaphically derived polyphyletic taxa are not uncommon (Levin 2001; Pepper and Norwood 2001) because

individuals growing on harsh substrates usually acquire similar sets of adaptations to tolerate their stressful conditions (e.g. Flowers et al. 1986; Kruckeberg 1984). For example, salt tolerant taxa are commonly reported as showing increased sodium accumulation and compartmentalization as well as succulence (Flowers et al. 1986). The similarity of morphology shown by coastal populations of Lasthenia californica have

historically lead to the recognition of the polyphyletic species L. macrantha sensu latd

(Chan et al. 2002; Desrochers and Bohm 2003; Rajakaruna 2002). Interestingly, the

genus Lasthenia, and members of the Lasthenia californica species complex in particular,

have a disproportionately high number of edaphically differentiated taxa and races, somej

of which are thought to have arisen in parallel in closely related lineages (Rajakaruna et

al. 2003a,b,c).

Lasthenia has approximately 21 species (according to Chan et al. 2001), many

with narrow ecological ranges. Members of the genus are reported to inhabit coastal

bluffs, guano deposits, vernal pools, alkaline flats, deserts, oak woodlands, serpentine

outcrops, grasslands, meadows, chaparral and deserts. Almost half of the species of

Lasthenia are ecological specialists, endemic to harsh or unusual habitats. As examples,

L. playcarpha and L. maritima are specialists on alkaline flats and guano laden seabird

roosting sites (respectively). In contrast, members of the Lasthenia californica complex

2 exhibit a wide range of ecological tolerances. Populations of the Lasthenia californica complex have been found in all of the habitats listed for the genus except for seabird guano deposits.

The Lasthenia californica system has complex phenotypic and genetic associations between its members, where morphological diversity is either cryptic or divergent, and is not always congruent with levels of genetic similarity or degrees of reproductive isolation. The complexity of genetic, reproductive, and phenotypic patterns within the system alongside the diversity of edaphically differentiated populations have made the Lasthenia californica complex a rich system for studying patterns and mechanisms of adaptation and differentiation to divergent ecological pressures.

The Lasthenia californica complex

The Lasthenia californica complex is the most widely distributed taxon in the genus. Members of the complex are found in abundance at low elevations throughout the

California Floristic Province. The range extends from southern Oregon to Baja

California, between the coast (including the Channel Islands) and the foothills of the

Sierra Nevada, and continues eastward into Arizona. Most of this range is Mediterranean in climate, characterized by cycles of mild, moist winters and hot dry summers; however, the coastal range is more moderate, with cooler temperatures in the summer and with rainfall that is more consistent throughout the year. Although this obligate outcrossing herbaceous plant is primarily a spring annual, year-long growth and perenniality have been recorded in coastal populations of the complex.

3 The Lasthenia californica complex shows extensive cytological, morphological, biochemical, and physiological variability. All of these characteristics have had a long history of study by various UBC researchers and those at other institutions. Three different groups of researchers have identified and confirmed the existence of diploid (n

= 8), tetraploid, and (rarely) hexaploid populations throughout the range of the complex

(Ornduff 1966; Desrochers and Bohm 1995; Chan et al. 2002). Interestingly, Desrochers and Bohm (1995) did not observe a geographical pattern to ploidy level variation.

L. californica (from here on referred to as L. californica sensu lato) was described

by Ornduff in his 1966 monograph as a morphologically variable species (note that the

name L. chrysostoma was originally used in the 1966 monograph but was later renamed

by Ornduff (1993) as L. californica). Although he attributed some of this variation to

phenotypic plasticity, he concluded that this species was composed of several genetically

distinct races that followed coastal, inland non-desert, and inland desert boundaries. The

coastal race is the most divergent of these, distinguished from the inland races primarily

by its succulent morphology. As an added complication, succulence can sometimes also

be found to a limited degree in some desert alkali sites. Desrochers and Bohm 1993

agreed with the inland versus coastal distinction among populations of Lasthenia

californica sensu lato but found that the extent of overlap in morphological variation

across the inland populations did not warrant recognition of inland desert versus inland

non-desert races.

Ornduff (1966) had also recognized a distinct coastal species, Lasthenia

macrantha (referred to here as Lasthenia macrantha sensu lato) which has been described

by other researchers as "exceedingly difficult to distinguish from L. californica [sensu

4 lato]" p. 1103 Chan et al. 2002. L. macrantha sensu lato was primarily distributed along the northern Californian and southern Oregon coast. This taxon was composed of three subspecies, subsp. macrantha, subsp. bakeri, and subsp. prisca. Typically L. macrantha sensu lato is described as an annual to perennial succulent herb with wide, strapped shaped leathery leaves. Apart from perenniality, there are no reproductive or vegetative characters that easily distinguish L. macrantha sensu lato from the coastal race of L. californica sensu lato. Even the life history traits used to distinguish the two taxa gradate. When grown in common conditions in a growth chamber, populations of coastal

L. californica sensu lato have substantially longer generation times in comparison to their inland counterparts (e.g. L. californica sensu lato individuals from coastal bluffs in Salt

Point State Park: G. Choe, personal observation). Furthermore, L. macrantha can behave as an annual, depending on water availability (Ornduff 1966; Chan 2001, Chan et al.

2002). The morphological overlap between these two formerly distinct species is reflected in their high degree of interfertility. Ornduff (1966) conducted inter- and intraspecific crosses and found that populations of L. macrantha sensu lato readily produced viable offspring in crosses with populations of inland L. californica sensu lato.

He also found that interspecific crosses tended to be as or more successful than intraspecific L. californica sensu lato crosses and that L. californica sensu lato populations with significant geographical separation had very low crossability (especially between northern and southern Californian populations).

The magnitude of variation within the Lasthenia californica complex has resulted in a complex taxonomic history. Throughout this history, its various members have been assigned to different species and subspecies across three genera and seven species.

5 Ornduff s (1966) revision of Lasthenia californica had combined previously distinct taxa into this one species and this circumscription was unchallenged until recently when phylogenetic analysis revealed that the species outlined by Ornduff actually spanned two separate phylogenetic lineages: L. gracilis and L. californica subsp. californica (Chan et al. 2002, Desrochers and Dodge 2003). Lasthenia californica sensu lato, therefore has now been recircumscribed to reflect phylogenetic relationships (Chan et al. 2002, Chan

2001). The two cryptic phylogenetic species now recognized generally follow

geographic lines: L. californica is distributed predominately in southern Oregon and

northern California, while L. gracilis is found primarily in southern California, Arizona

and Baja California (Chan et al. 2002; Desrochers and Dodge 2003). The taxonomic changes between Ornduff's (1966, 1993) and Chan's (2001) circumscriptions are

summarized in Table 1.1.

The new circumscription of L. californica sensu stricto now represents a

monophyletic lineage that not surprisingly includes subspecies of L. macrantha sensu lato

(=L. californica subsp. macrantha and L. californica subsp. bakeri) and the northern

populations of L. californica sensu lato (=L. californica subsp. californica). The third

subspecies of L. macrantha sensu lato, subspecies prisca (a coastal Southern Oregon

endemic) has been elevated to the status of species (=L. orndujfii; Table 1.1). Both DNA

sequence (Chan et al. 2001, 2002; Desrochers and Dodge 2003) and RAPD marker data

(Rajakaruna 2002) identify L. macrantha sensu lato as a polyphyletic lineage.

Populations with the 'macrantha' phenotype nest within geographic groups that include

neighboring inland populations of L. californica subsp. californica.

6 The biochemical complexity of this system has also had a long history of study

and predates the phylogenetic research. The existence of flavonoid polymorphisms was first noted in two cryptic species of L. californica sensu lato distributed in parapatry

along a serpentine grassland at Jasper Ridge Biological Preserve. Four pigment profiles

were determined, of which two predominate, profiles A and C/Bohm 1987; Bohm et al.

1989). Profile A was characterized as the more complex profile of the two and is

differentiated from C by the presence of flavonol diglycoside sulfates and a flavanone

eriodictyol 7-O-glucoside. Bohm et al. (1989) also determined that these flavonoid

profiles were genetically determined as opposed to environmentally induced. He grew

offspring of both A and C plants and found that A individuals produced exclusively A

progeny and that C plants produced progeny that were virtually all C (Bohm et al. 1989).

Desrochers and Bohm (1993, 1995) later found that these A and C profiles are distributed

throughout the range of the complex. They also determined that individuals with

flavonoid profiles A and C generally grouped into southern (recently recognized by Chan

2001 as L. gracilis) and northern clades (recently recognized by Chan 2001 as L.

californica subsp. californica), but were not restricted to these geographical zones.

Numerous polymorphic A/C populations throughout the range of the complex were also

recorded, but they found that these populations were more common in the central region

of California, where northern and southern clades broadly overlap. Table 1.1 summarizes

A/C flavonoid occurrences in each phylogenetic lineage.

Subsequent ecological and flavonoid surveys of this complex conducted by

Rajakaruna and colleagues (Rajakaruna and Bohm 1999; Rajakaruna et al. 2003a)

observed a strong correlation between flavonoid profile and soil type. Populations

7 growing on salty soils typically show flavonoid profile A (referred to as race A); while populations growing in drier, but ionically benign soils tended to show flavonoid profile

C (referred to as race C; Rajakaruna and Bohm 1999). Rajakaruna and Bohm (1999) also found that race A populations growing on salty soils tended to accumulate certain ions such as Na+ and Mg2+to a greater degree than race C populations. However, unlike

Desrochers and Bohm (1993), Rajakaruna and Bohm (1999) found that populations were fixed for either A or C flavonoid profiles.

These observations from field studies suggested that the two flavonoid races were also distinct edaphic races. Rajakaruna and colleagues (1999, 2003a,b,c) hypothesized that race A individuals had evolved greater stress tolerance and that race C individuals had evolved greater drought tolerance. To test the hypothesized differentiation of water stress tolerance between races they investigated differential responses of A and C's to water stress in a system where A and C's grow in parapatry along a gradient of increasing water stress (Rajakaruna et al. 2003b). They grew A and C individuals in controlled conditions with differing levels of water availability (Rajakaruna et al. 2003b). As predicted, they found that that C's were less affected by the negative impacts of drought stress (as indicated by number of flower heads produced). These data supported their hypothesis that race C individuals demonstrate greater drought tolerance.

To test the ionic stress tolerance component of the edaphic/flavonoid race hypothesis, Rajakaruna and colleagues (Rajakaruna et al. 2003c) grew two populations of each race from each phylogenetic lineage (four populations in total) in controlled hydroponic conditions to determine whether the ion accumulation differences observed in the field reflected genetic differences. Specific ion uptake, accumulation, and tolerance

8 indices were measured for Na+ and Mg2+. They found that race A, regardless of species membership, had higher uptake rates, accumulation, and tolerance to these ions

(Rajakaruna et al. 2003c). Rajakaruna and colleagues also proposed a potential mechanistic link between flavonoid chemistry and ionic stress resistance. They had realized that sulfated flavonoids (one of the major flavonoid fractions distinguishing A from C) were found in greater levels in salt marsh species, and from this they hypothesized that ionic stress tolerance may be conferred by or linked to the presence of sulfated flavonoids.

Rajakaruna et al. (2003a) determined phylogenetic species identities and flavonoid profiles of populations in a complex wide survey and confirmed that while the majority of L. gracilis populations were A, and that the majority of L. californica subsp. californica populations were C, both A and C's indeed occurred in both of the recently recognized phylogenetic taxa. In the survey they published in 2003, of the 16 populations of L. gracilis that they sampled, 13 were A, and 3 were C; of the 17 populations of L. californica subsp. californica sampled it was determined that 11 were race C and 6 were race A (Rajakaruna et al. 2003a). Also, note that they found only fixed

A or C populations. Field and experimental data had led Rajakaruna and colleagues

(Rajakaruna et al., 2003a,b,c) to conclude that these two races, distinct in edaphic tolerances, ion physiology, drought tolerance, and flavonoid chemistry had evolved in parallel in both phylogenetic lineages.

Objectives:

This thesis examines patterns of edaphic, physiological, and morphological

9 differentiation within and among members of the Lasthenia californica species complex,

in two distinct studies:

Chapter 2 has two primary objectives: 1) To describe patterns of edaphic and

physiological variation in populations across the species complex, as indicated by soils

and tissue data. 2) To re-examine the flavonoid/edaphic race hypothesis proposed by

Rajakaruna et al. (2003a,b,c) by comparing changes in flavonoid race distribution and the

edaphic associations of these races between various surveys published between 1993 and

this current survey.

Chapter 3 investigates the basis of the phenotypic differentiation observed

between coastal and inland ecotypes (=subspecies) of L. californica sensu stricto. The

hypothesis of adaptive ecotypic differentiation in salt tolerance and associated traits was

examined by comparing norms of reaction between populations of each ecotype grown in

common conditions with varying levels of salt. Upon detection of differential reactions

to salt stress, evidence of trade-offs between salt tolerance and growth potentials was then

investigated.

10 Table 1.1: Summary of the taxonomic history of the Lasthenia californica species complex from 1966 to present. Occurrences of flavonoid races in the members of the complex are included. The parentheses denote the flavonoid profile most common to the lineage.

Presence of Ornduff's circumscriptions flavonoid races' Chan's circumscriptions

L. californica (A)and C L. gracilis2 Aand(C) L. californica subsp. californica L. macrantha subsp. macrantha A and (C)3 L. californica subsp. macrantha L. macrantha subsp. bakeri N.A. L. californica subsp. bakeri

L. macrantha subsp. prisca N.A. ' L. ornduffii

Notes: 1) Races B and D are not included in this table. 2) L. gracilis was previously recognized as a part of L. californica sensu Ornduff (1966, 1993). 3) Determined in the current study.

11 CHAPTER 2

Patterns of edaphic, physiological, ITS lineage and flavonoid variation across the Lasthenia californica species complex: re-evaluating the hypothesis of parallel evolution

of edaphic races. INTRODUCTION

The distribution of plant species is influenced by a combination of climatic, biotic and edaphic variables. Of these factors, soil characteristics can produce the most dramatic changes in plant distributions over small spatial scales (Anderson et al. 1999).

Substrates that produce the most noticeable discontinuities in plant distributions are typically those that are unfavorable to the majority of plant species; edaphic mosaics incorporating substrates such as ultramafic rock, gypsum, limestone, and alkali flats have the potential to promote and maintain patches of divergently adapted ecotypes by means of disruptive selection. The suite of morphological and physiological adaptations that differentiate plants inhabiting these harsh soils suggests that edaphically mediated selection can be an important source of evolutionary diversification.

Numerous instances of edaphically driven differentiation have been reported

(Ellis et al. 2006; Robichaux et al. 1990; Kruckeberg 1986; Kruckeberg 2002) with particularly interesting examples from serpentine habitats (Sambatti and Rice 2006;

Gardner and MacNair 2000; Gottlieb et al. 1985; Baldwin 2005). Ultramafic soils occupy less than 1% of the land surface of the earth (Brooks 1987) but are associated with disproportionately high levels of endemic species (Kruckeberg 1984, 2002; Brooks

1987). These soils are particularly extreme because they impose both chemical (harsh

Mg/Ca ratios, low macronutrient concentrations, and high heavy metal concentrations) and physical stressors (coarsely textured with low organic matter; Brooks 1987).

12 Tolerant taxa often show similar adaptive responses; for instance, several serpentine

tolerant plants hyperaccumulate heavy metals (Reeves et al. 1999; Boyd and Martens

1998).

Tolerances to toxic substrates such as serpentine and mine tailings have been

reported as occurring repeatedly between genetically isolated populations (Berglund et al.

2004; Al-Hiyaly et al. 1988; Wu et al. 1975; Gregory and Bradshaw 1965). For instance,

Schat et al. (1999) showed that populations of Silene vulgaris from geographically

isolated sites across Europe evolved heavy metal tolerance independently and in parallel.

Another example of recurrent evolution of stress tolerant races may be found in the

Lasthenia californica complex (Rajakaruna et al. 2003a,c; Rajakaruna 2002). Two races

with distinct flavonoid and edaphic tolerances are hypothesized to have evolved in

parallel in each of the two phylogenetic lineages that compose Lasthenia californica

sensu lato. Exploring the strength of this phenomenon in the Lasthenia californica

complex is one of the major objectives of this study.

Members of Lasthenia californica grow in a wide range of habitats including

serpentine outcrops, chaparrals, grazed and ungrazed pastures, grasslands, oak

woodlands, coastal bluffs, saline and alkaline flats as well as vernal pools. This vast

ecological diversity is mirrored by extensive morphological, cytological and biochemical

complexity. Not surprisingly, the taxonomic history of the system has been equally

complex. Prior to Ornduff's monograph (1966), members of L. californica sensu lato

were assigned to different species and subspecies across three genera. Ornduff's (1966)

revision of Lasthenia californica combined previously separated taxa into one species

13 and this circumscription was unchallenged until recently. Phylogenetic analysis revealed that the species outlined by Ornduff actually spans two separate phylogenetic lineages: L. gracilis and L. californica subsp. californica (Chan et al. 2002, Desrochers and Dodge

2003). L. californica sensu lato therefore has been split to reflect phylogenetic lineages

(Chan et al. 2002; Chan 2001). These two cryptic phylogenetic species follow

geographic lines: L. californica is distributed predominately in southern Oregon and

northern California while L. gracilis is found primarily in southern California, Arizona

and Baja California (Chan et al. 2002, Desrochers and Dodge 2003). Of noteworthy

mention, this newest circumscription of L. californica now includes subsp. macrantha

and subsp. bakeri, which were considered distinct species prior to Chan's phylogenetic

work (Chan et al 2002; Chan et al. 2001).

The biochemical complexity of this system has had a long history of study, and

predates the phylogenetic research done on this system. The existence of flavonoid

polymorphisms was first noted in L. californica sensu lato into two populations

distributed in parapatry along a serpentine grassland at Jasper Ridge Biological Preserve.

Two main pigment types, A and C, were noted (Bohm 1987; Bohm et al. 1989); where A

was characterized as the more complex profile of the two. Profile A is differentiated

from C by the presence of flavonol diglycoside sulfates and a flavanone eriodictyol 7-0-

glucoside. Desrochers and Bohm (1993, 1995) later found flavonoid profiles A and C

were distributed throughout the range of the complex. They also determined that

flavonoid profiles A and C generally grouped into southern (L. gracilis) and northern

clades (L. californica subsp. californica), but were not restricted to these geographical

zones. Subsequent ecological and flavonoid surveys of this complex conducted by

14 Rajakaruna and colleagues (Rajakaruna and Bohm 1999; Rajakaruna et al. 2003a) observed a strong correlation between flavonoid profile and soil type. Populations growing on salty soils typically show flavonoid profile A (referred to as race A); while populations growing in dry, but ionically benign soils have flavonoid profile C (referred to as race C; Rajakaruna and Bohm 1999; Rajakaruna et al. 2003a). Rajakaruna and

Bohm (1999) found that race A populations growing on salty soils tended to accumulate certain ions such as Na+ and Mg2+ to a greater degree than race C populations. They also confirmed that race A and C populations occurred in both of the recently recognized phylogenetic taxa (L. gracilis and L. californica subsp. californica) that once comprised

L. californica sensu lato (Rajakaruna et al. 2003a).

The observations made in the field suggested that the two flavonoid races were also distinct edaphic races, a hypothesis that I will refer to from this point onwards as the flavonoid=edaphic race hypothesis. To test this hypothesis, Rajakaruna and colleagues grew two populations of each race in each phylogenetic lineage (four populations in total) in controlled hydroponic conditions to determine whether the ion accumulation differences observed in the field reflected genetic differences (Rajakaruna et al. 2003c).

Specific ion uptake, accumulation, and tolerance indices were measured for Na+ and

Mg2+. They found that race A, regardless of species membership, had higher uptake rates, accumulation, and tolerance to these ions (Rajakaruna et al. 2003c). Both field and experimental data led Rajakaruna and colleagues (Rajakaruna et al. 2003a,c) to conclude that these two races, distinct in edaphic tolerances, ion physiology, and flavonoid chemistry had evolved in parallel in both phylogenetic taxa.

15 In this study we re-examine the flavonoid=edaphic race hypothesis with a more in-depth sampling of the complex, increasing both the number of populations and the numbers of individual flavonoid profiles per population sampled. We also describe changes in flavonoid race distribution and frequency over time by comparing surveys published in 1993 by Desrochers and Bohm (this survey will be referred to as "1993"), in

1999 and 2003 by Rajakaruna and Bohm (1999) and Rajakaruna et al. 2003a (which will be referred to as "1999&2003") and the current work from field surveys conducted in

2005 (referred to asr"2007"). The final objective of this study is to characterize the major axes of ecological and physiological variation found across the species complex.

MATERIALS AND METHODS

Field sites

Flower heads, plant tissue and soil samples were collected from sites previously surveyed by Rajakaruna and Bohm (1999), Rajakaruna et al. 2003, and Desrochers and

Bohm (1993), as well as additional sites not previously sampled by those researchers.

Although we attempted to relocate and resample all sites, this was not always possible.

Locality and habitat information are summarized in Table 2.1.

Flavonoid identification

Flavonoid profiles were determined as described in Desrochers and Bohm (1993) except that Machery-Nagel TLC plates were used instead of "home-made" plates (p 449,

Desrochers and Bohm 1993). Ray florets were soaked in MeOH for several days. The extracts were spotted on 20X20cm Polyamide 6 thin layer chromatography plates (0.1

16 mm Polyamide TLC 6 UV254; Machery-Nagel, Gutenberg, Germany). Plates were developed one-dimensionally in a water, n-butanol, acetone, and dioxane (70:15:10:5) solution. Plates were air dried, then sprayed with aminoethyl-diphenylborate, visualized under UV light (Alpha-Imager 1200 gel documentation system; Alphalnanotech

Corporation, CA, USA), and scored.

A total of 670 individual flower heads was tested for flavonoid profiles. 15 to 30 individuals per population were initially sampled. When initial surveys showed fixed flavonoid profiles, no further sampling for these populations was done. Populations showing mixtures of race profiles, or differences between surveys, were sampled in greater depth. In addition, flowers from Desrochers' and Rajakaruna's herbarium vouchers with known flavonoid status were re-sampled in this study to check for consistency in flavonoid profile interpretation. Questionable samples were re-run twice and excluded if unreliable.

ITS species identity

Phylogenetic species L. gracilis and L. californica were identified for populations not previously determined by Rajakaruna et al. 2003a. These two phylogenetic lineages are reliably separated by a llbp indel in the rDNA internal transcribed spacerl (ITS1) region. ITS1 size differences were determined following Rajakaruna et al. 2003 except that DNA was extracted with a modified CTAB (Doyle and Doyle, 1987) extraction using QIAquick purification columns (Qiagen, Mississauga, Ontario, Canada) to purify

DNA extracts. The ITS1 region was amplified using primers ITS2 and ITS5 (White et al. i990). 25 pi reactions of 10 ng template DNA, 30 mM Tris-Hcl, 50 pM KC1, 2mM

17 MgCl2, 0-1 mM each dNTPs, 10 pmoles primer, and 1.5 units DNA polymerase were amplified in a PT-100 thermal cycler (MJ Research, Waltham, MA, USA) with an initial

3 minute denaturation at 94°C, followed by 35 cycles of (1 minute 94°C, 1 minute 50°C,

1 minute 72°G), and a final extension of 7 minutes at 72°C. 2 ]A aliquots of each PCR sample were separated by electrophoresis in 2% SeaPlaque agarose (BMA, Rockland,

ME, USA) with 0.5X TBE buffer. The gels were then visualized by UV light after an ethidium bromide stain and scored against samples of known ITS size as standards.

Soil and tissue analyses

Soil samples were collected from rooting zone depths (5-15cm) using a hand trowel. Subsamples were taken haphazardly within each site and pooled into one sample

per population. All soil samples were air-dried in open plastic bags then rocks and gravel

were removed. Major mineral concentrations of soil samples were determined by ICP

spectrometry following a Mehlich III extraction. Heavy metals were extracted by

HN03:HC1 total digestions. Extractable phosphorus and NO3" concentrations were

determined following Bray PI and colorometric CaCl2 Cd reduction procedures,

respectively. Organic material concentrations were determined by loss on ignition at

360°C. PH was determined in 1:1 soil to H20 mixtures.

Individuals collected within a population were pooled into one representative

tissue sample. These samples were first air-dried at room temperature, and processed by

removing adhering environmental particles and by separating organ types (roots, shoots,

flower heads). Shoot tissue was analyzed for mineral concentrations by ICP

18 spectrometric analysis after a total acid digestion. All soil and tissue mineral quantification were conducted at A&L laboratories inc., London, Ontario, Canada.

Data analysis

Flavonoid profiles were summarized as simple tallies and are shown in Table 2.1.

Univariate ANOVAs were used to determine whether soil and plant tissue characteristics

between A and C populations differed significantly for any single mineral. The residuals

of these analyses were inspected for normality and homogeneity of variance. Variables

not adhering to the assumptions of ANOVA were transformed. Transformed variables

are listed in Table 2.2. Note that populations with mixtures of A and C were excluded from the analysis. We decided that excluding these populations would be more prudent

than including these points because inclusion would require points to be entered twice in

both A and C categories (resulting in pseudoreplication). An important caveat is that

differences between groups using tests excluding mixed populations may exaggerate

biological differences between races.

Multivariate patterns in soils and tissue data were explored with principle

component analyses (PCA). PCA is an eigenvector-based analysis that summarizes data

from multiple variables by reorganizing their variance components into new orthogonal

axes. The axes are extracted so that the first axis explains the greatest amount of the

variance in the original variables, the second axis explains much of the remaining

variance, and so on. The assumptions of PCA (linear association between variables and

multivariate normality) were checked and deviations were remedied by transformations.

For soil analyses, transformations used were those listed in Table 2.2a. For plant tissue

19 analyses, variables were square root transformed. Note that strict adherence to the assumptions of PCA is not as critical as these are only conditions that improve the explanatory power of the analysis (Tabachnick and Fidell 2001). PC analyses were performed using correlation matrices to standardize the distribution of variance between

variables.

From the PCA ordinations, we confirmed the distinctiveness of a priori ecological

groupings of coastal, serpentine, alkaline flat, and inland grasslands/pastures: We then

determined whether there were significant differences between ecological groupings for

soil and tissue variables using univariate ANOVAs. Pair-wise differences between

groups were determined for significant ANOVA tests. Family-wise type I error inflation

rates were controlled for each pairwise analysis by a Bonferroni adjustment.

Relationships between soil and tissue characteristics were explored with linear

regressions. Residuals were inspected for normality. Significant regressions from

misleading relationships (driven by the presence of numerous outliers) were not included.

Single outliers with extraordinary leverage unmanageable by transformations were also

excluded from a few analyses.

RESULTS

Flavonoid and ITS analyses

The results of flavonoid analyses (Table 2.1) show that the distribution of A and

C profiles usually adhered to geographical patterns. The A profile was most common in

southern California, while C profile individuals were found mostly in northern California

and southern Oregon. These results are largely consistent with the patterns described by

20 Desrochers and Bohm (summarized under the heading "1993" in Table 2.1) and

Rajakaruna and Bohm (summarized under the heading "1999&2003" in Table 2.1). We confirmed that flavonoid profiles A and C were most prevalent but not restricted to ITS species L. gracilis and L. californica, respectively.

Flavonoid composition in central California populations showed the most variation between surveys. Whereas Rajakaruna and colleagues (1999&2003) reported the coastal bluff site SPS (Table 2.1) as homogenously A, we found that this same population consisted mostly of C's with only a few A individuals present. The 1993 survey also found a mixed population but with a greater proportion of A's to C's than was shown by our survey. A similar trend was observed at Mt. Tamalpais (21-41, Table

2.1). At this serpentine site, mixtures of mostly A and some C's were reported in 1993 and this was also the case in our survey. Only individuals with A profiles were reported at this site by Rajakaruna and colleagues (1999&2003). In contrast, similarities between

1999&2003 and the current survey (2007) were found in the pasture lands of interior central California. Both 1999&2003 and 2007 survey authors found homogenous populations of C's where as Desrochers and Bohm (1993) reported mixed populations of

A and C's (e.g. site 26, Table 2.1).

An alkali flat in Byron (22, Table 2.1) showed a striking difference between flavonoid composition between the 1999&2003 and 2007 surveys. While Rajakaruna

(1999&2003) reported a uniformly A population, this same site was found to be completely C in our survey. The populations that changed most in flavonoid composition between the current survey and 1999&2003 were those central California populations inhabiting the most extreme habitats, such as alkali flats and serpentine sites.

21 Soil analyses

Race A and C differences

Univariate ANOVA's indicate that organic matter, pH, P, Na/K, K, Na, S, Fe, and

Al concentrations are significantly different between C and A sites (Table 2.3). In comparison to Rajakaruna and Bohm (1999), we found greater numbers of significantly different soil variables between A and C sites in our survey. An important difference between years is that sodium and sulfur levels are significantly higher in C than A sites in our survey.

After verifying increased toxicity of soils found in serpentine, alkali flat, and coastal areas, we tallied the number of A, C, and mixed flavonoid race populations observed in edaphically harsh habitats in our sample year. As shown in Table 2.4, no homogenously A populations grew on salinized soils. Also, while some fixed A populations were found on serpentine soils, both C and mixed populations were well represented in these habitats as well.

Patterns of edaphic diversity over the complex

Much of the soil chemistry variation found across the Lasthenia californica complex follows habitat differences. Principal component analyses (PCA) show that over half of the variation between sites lies along axes that can be interpreted as serpentine and coastal. Table 2.5 describes the correlations found between the first two

PC axes and the original soil variables. The first axis is correlated to variables contributing to the "serpentine syndrome": concentrations of magnesium and heavy

22 metals, chromium, cobalt and nickel. Axis 2 represents a gradient of saline factors, indicated by the strong correlation to Na and S. PC axis 3 does not account for much of the original variation, but showed the greatest amount of separation between pasture sites

(data not shown), suggesting that it represents an axis of ionically benign inland pasture site variation. There are no strong correlations between soil characteristics and this axis

(Table 2.5).

A PCA ordination of axes 1 and 2 (Figure 2.1a) shows that sites from similar habitats cluster into discrete groupings: serpentine sites cluster in the upper right quadrant, the saline sites in the lower regions, and inland pastures in the upper left quadrant. When populations with profile A, C and mixed A and C populations were used as symbols to visualize the distribution of flavonoid races in this ordination, it then appeared that much of the variation within A and C groupings lie on axes 1 and 2, respectively (Figure 2.1b). Figure 2.1b also makes it apparent that the edaphic extremes

(increasingly positive x axis values and increasingly negative y axis values) are not associated with profile A, as predicted under the flavonoid=edaphic race hypothesis.

Univariate ANOVA's on the following habitat groupings: coastal, inland pastures, and serpentine, corroborate the differences in soil characteristics illustrated by the PCA

(Table 2.6). Coastal habitats are significantly higher in sodium and sulfur. Serpentine sites are significantly higher in heavy metal concentrations (nickel, chromium and cobalt), and magnesium (Table 2.6). Inland pastures and grasslands are typically lower in toxic minerals and higher in beneficial elements like potassium (marginally significant), phosphorus and calcium than the edaphically extreme habitats (Table 2.6). Contrary to the literature, nutrient concentrations of potassium, calcium, nitrogen and organic matter

23 content were not significantly lower in serpentine areas (Table 2.6).

Plant tissue analyses

Differences between A and C

Univariate ANOVAs found few significant differences in tissue variables between

A and C populations (Table 2.7). The only significant differences found between A and

C populations were for phosphorus, sodium, and manganese. Differences in sodium

between A and C most likely reflect the greater number of coastal C representatives

present in the data set than coastal A representatives.

Patterns of tissue differentiation between populations

Similar to the soil trends, differences in tissue characteristics between populations

of the Lasthenia californica complex follow habitat differences. PC analyses suggest that

tissue ion characteristics of populations have the greatest variation along lines of coastal

and serpentine gradients. PC axis 1 represents a coastal gradient and is strongly

correlated to sodium concentrations in tissues (Table 2.8). PC axis 2 is strongly

correlated to the serpentine variable nickel, but not to other serpentine variables such as

magnesium (Table 2.8). Magnesium shows a split loading between axis 1 and 2, and thus

is not strongly correlated to either axis (Table 2.8). Despite this, PC axis 2 separates

populations based on serpentine and non-serpentine characteristics (Figure 2.2a).

A PCA ordination of axes 1 and 2 (Figure 2.2a) clearly show that populations

cluster based on habitat origin. When flavonoid information was added to the PCA

ordination, it became apparent that much of the variation within A and C groupings lay

24 on separate axes (Figure 2.2b). Tissue characteristics suggesting increased ionic stress tolerance (increasingly positive PC 1 values) are not associated with .profile A, as earlier

hypothesized by Rajakaruna and colleagues (Rajakaruna and Bohm 1999; Rajakaruna et

al. 2003a,c).

Univariate ANOVAs using habitat types as groups found significant differences

between habitats for many of the elements analyzed (Table 2.9). Coastal sites showed

significantly higher levels of sodium tissue accumulation than in other habitat groups

(pastures and serpentine; see Table 2.9). Also sulfur levels were higher in coastal groups

(Table 2.9). Interestingly, plants inhabiting coastal sites are significantly higher in most

other ions, such as copper, zinc, aluminum, and manganese (Table 2.9). Plants in

serpentine habitats are differentiated by their significantly higher tissue levels of

magnesium and nickel (Table 2.9). Tissues of plants from inland pastures are higher in

macronutrients such as calcium and potassium (Table 2.9).

Relationships between edaphic and tissue characteristics across the complex

Relationships between soil and tissue ion concentrations across the Lasthenia

californica system are complex and rarely linear without transformation. Exceptions to

this trend include the relationships between soil and tissue levels of calcium and boron

(Table 2.10 and Figure 2.3a,b). As shown in Table 2.10, many scatter plots were cloud•

like, lacking any obvious relationship between soil and tissue element concentrations

(e.g. potassium, iron, copper, nitrogen). The relationships between tissue and soil

concentrations of other minerals such as phosphorus, sulfur, sodium and zinc are

moderately or weakly defined by a linear relationship on logarithmic scales (Table 2.10;

25 Figure 2.4a,b,c,d).

Relationships between tissue and soil concentrations of cations across the L. californica complex were not always homogenous between habitat groupings. This caused difficulties when modeling the relationship between the two axes. For example, the relationship between magnesium levels in tissues and soils seemed to be split between

coastal groupings and the other habitat types (Figure 2.5a). This finding is consistent

with the split loading of magnesium in the PC analysis of tissues. Although it would

have been more accurate to model the soil-tissue relationship of certain ions separately for each habitat type, all groups other than "inland pastures" lacked sufficient N. Besides

a few elements such as potassium, seemingly random associations (in regards to linear

correlations) between soil and tissue levels showed discernable patterns of differentiation

when grouped by habitat (Figure 2.5b).

DISCUSSION

Re-evaluating the flavonoid=edaphic race hypothesis.

The flavonoid=edaphic race hypothesis proposes that salt tolerance is partly

conferred by or is at least linked to the presence of sulfated flavonoids. Individuals with

flavonoid profile A are differentiated from profile C in part by the presence of sulfated

flavonoids, and these A's are thought to be salt tolerant whereas individuals with

flavonoid profile C are thought to lack salt tolerance. If this is true, one would expect

that ionically extreme habitats across the range of the complex would be occupied most

often by race A individuals. We have shown that this is not the case in the current survey

by the following results: simple tallies show that the number of edaphically extreme sites

26 colonized by C is higher than the number of sites inhabited by A; soil ANOVAs indicate significantly higher levels of sodium in C than A sites; PCA ordinations show that sites on the ionically harsher ends of PC 1 and PC 2 are not associated with profile A populations. These data do not support predictions made under the flavonoid=edaphic race hypothesis;

Another clear refutation of the flavonoid=edaphic race hypothesis is shown by the fact that individuals with profile C increased in frequency in the majority of edaphically extreme sites initially reported as polymorphic in 1993. Under the predictions of the hypothesis, we should find that selection increases the frequency of A individuals over time in edaphically extreme sites with initial A/C polymorphism. Instead, we found that the two serpentine sites on Mt. Tamalpais (21-41,42), and the coastal bluff site (SPS) in

Sonoma Co. increased substantially in C's relative to A's between 1993 and our current study. Interestingly, the frequencies of A and C changed even more dramatically between the Rajakaruna and colleagues surveys of 1999&2003 and this current survey.

In the 1999&2003 surveys, the authors report fixed A populations on the Mt. Tamalpais site and the coastal bluff site SPS.

The geographic location of the occurrence of A/C frequency shifts also weakens the flavonoid=edaphic race hypothesis. We found that only populations in central

California (the interface between the mostly southern A and northern C) shifted in A/C frequency between 1999&2003 and our survey. This suggests that gene flow affects flavonoid frequency as much as or more than selection for flavonoid/ionic tolerance trait complexes. If individuals with favorable flavonoid/ionic tolerance trait complexes were sufficiently fitter than maladapted migrants in ionically harsh environments, selection

27 should sustain increases in A frequency despite gene flow to those sites. The marked increases of C's in polymorphic populations in central California inhabiting ionically extreme sites as well as the complete shift to all C's from all A's at the alkali site at

Byron (also in central California) suggest that this is not the case. In light of recent evidence suggesting that levels of introgression and gene flow are thought to occur at higher rates and distances than traditionally expected (Ellstrand 2003; Morjan and

Rieseberg 2004), the spread and maintenance of flavonoid polymorphisms by gene flow is a plausible hypothesis.

Although the above results provide strong evidence against the flavonoid=edaphic race hypothesis, differences between surveys may also be due to differences in methodology. For example, differences in sampling procedures within sites can produce apparent shifts in A/C frequencies if between years the different researchers collect from different locations within sites. This would then overlook potential within-site covariation in flavonoid/substrate patterns. Another obvious methodological difference that may contribute to the differences found between years lies in the number of individuals sampled. Surveys in 1999&2003 only tested 3-6 individuals per population compared to the larger number of individuals tested in 1993 and in this survey. Because larger sample sizes are more likely to incorporate low levels of flavonoid diversity within populations, we believe that the most obvious explanation for the result that only the recent and 1993 surveys had reported mixed populations (with the exception of Jasper ridge) is due to the depth of sampling.

Other than methodological differences, temporal heterogeneity in opposing selection pressures can also drive the observed changes in flavonoid frequencies.

28 Theoretical and empirical evidence support the hypothesis that polymorphisms can be

maintained by temporal variation in antagonistic selection pressures (Reimchen and Nosil

2002; Borash et al. 1998; Schemske and Bierzychudek 2001). It is possible that an

opposing selection pressure favoring C's exists and fluctuates in strength between years.

If the opposing selection pressure overcomes the ionic stress pressure imposed in

edaphically extreme sites, C individuals could then rise in frequency in spite of their

hypothesized inefficient ionic stress tolerance physiology. It is enticing to present

drought stress as a counter selection pressure favoring C individuals over A's because

another facet of the flavonoid=edaphic race hypothesis proposes that race C individuals

are greater drought tolerators than A's. In light of this, it is worth noting that drought

stress was unlikely to have occurred in the 2005 growth season, as much of California

had recorded higher than average rainfall that year (Western U.S. Climate Historical

Summaries, http://www.wrcc.dri.edu/climsum.html).

Evaluating the adaptiveness of sulfated flavonoids in saline environments

The link between increased sulfate conjugation with phenolic compounds such as

flavonoids in plants subjected to waterlogging and high salinity has been well

documented (Harborne 1975, 1993; Barron et al. 1988). The toxicity of elevated sulfate

concentrations in these environments may be ameliorated by the conjugation of inorganic

sulfates with flavonoids (Harborne 1975, 1993; Barron et al. 1988). From the results of

Tomas-Barberan et al.'s 1987 study, Rajakaruna et al. 2003 c suggested that sulfated

flavonoids may provide a counter ion to ameliorate the negative effects of increasing salt

accumulation in plants growing in saline areas. Although often cited in the literature, the

29 adaptiveness of sulfated flavonoids in waterlogged and saline environments is largely

untested and remains mostly conjectural. While sulfated flavonoids have been identified

in several halophytes such as Suaeda maritima (Harborne 1993), and Helianthus paradoxus (Karrenburg et al. 2006) they are absent in others such as Plantago maritima

(Harborne 1993). Furthermore, the presence of sulfated flavonoids is not correlated to

salt tolerance across the genus Lasthenia. While many species of Lasthenia inhabit saline

areas (eg. L. chrysantha, L.ferrisiae, L. glabrata, L. maritima, L. playcarpha) none of

these taxa produce sulfated flavonoids. The two species other than L. californica sensu

lato that produce sulfated flavonoids are L. conjugens and L. fremontii and they inhabit

waterlogged but ionically benign environments (vernal pools and wet meadows; Ornduff

1993).

To conclude, the results from our survey show that the correlation between ionic

tolerance and sulfated flavonoids is not as strong as previously hypothesized by

Rajakaruna et al. 2003a,c. However, the evidence provided in this study is correlative,

and experimental evidence providing a mechanistic link between sulfated flavonoid

production and enhanced salt tolerance is necessary to fully address this issue.

Patterns of ecological and physiological differentiation across the complex

Results from PCA and univariate ANOVAs of soil and tissue characteristics

indicate that the major axes of edaphic variation in the complex occur along lines of

increasing edaphic toxicity. Although the majority of sampled sites were ionically

benign inland sites (e.g. grazed pastures), our PC analyses failed to detect strong

30 correlations between original tissue/soil variables and the major axis of variation that described this inland group. The patterns of variation in soil and tissue characteristics found between serpentine, coastal, and pasture habitats are fairly congruent with findings

reported in the literature. The following sections will assess the similarities and

differences of the edaphic and physiological differentiation found across the Lasthenia

californica complex with other findings reported in the literature.

Patterns of edaphic variation

Edaphic patterns for serpentine sites sampled

PC analyses and ANOVAs show that levels of magnesium, nickel, chromium and

cobalt are quite divergent between, serpentine sites and the other habitats groups

analyzed. In our survey, mean magnesium concentrations from serpentine sites were

almost 5 times higher than soils from non-serpentine sites. Elevated magnesium

concentrations are common to serpentine soils (Brooks 1987). Concentrations of up to

36% MgO in soils from Mt. Tamalpais have been reported, whereas the percentage

composition of MgO in non-ultramafic soils are generally less than 1% (Robinson et al.

1935, in Brooks 1987). High concentrations of heavy metals such as chromium, nickel

and cobalt have been reported in soils derived from ultramafic rocks across the globe,

including Mt. Tamalpais (Robinson et al. 1935, in Brooks 1987). Although ANOVA's

show significant increases in chromium and cobalt concentrations in serpentine soils, the

actual availability of these heavy metals can be quite low (Brooks 1987), and so may not

exert a stress on plant growth (Halstead 1968; Crooke and Inkson 1955). This point will

be re-examined below in light of tissue data findings.

31 The chemistry of serpentine soils challenges plant growth with a combination of increased magnesium and heavy metal levels and decreased nutrient concentrations, in particular, that of calcium (Walker 1954). Although some researchers have shown that calcium availability is not limiting in serpentine environments (eg. Proctor et al. 1971;

Proctor et al. 1981), most researchers support the hypothesis that the negative effects of

lowered calcium levels is exacerbated by the high levels of magnesium also found in

these soils (Kruckeberg 1954, 1989; Proctor and Woodell 1975) because plants generally

require magnesium/calcium ratios of less than one for optimal growth. Of the habitat

groupings we sampled, mean calcium levels were lowest in serpentine soil; however, the

difference between serpentine and coastal groupings was not significant. The overall

magnesium/calcium ratio did prove to be significantly higher in serpentine soils. It is

suspected that other nutrients such as nitrogen have lowered concentrations in serpentine

soils due to the sparse vegetation associated with these habitats (Kruckeberg 1984;

Brooks 1987; Nagy and Proctor 1997; Proctor 1971a). We found that on average,

nitrogen concentrations were not significantly lower in serpentine sites. Potassium

deficiencies are not commonly associated with serpentine soils, but deficiencies have

been reported in some sites in Western North America because of low mineralogical

concentrations in ultramafic parent material (Burt et al. 2001). We found no significant

differences in potassium concentrations in serpentine soils. These results when

considered together suggest that the sampled serpentine populations of Lasthenia

californica sensu Ornduff are generally not suffering from nutrient deficiencies.

The chemical adversities of serpentine soils are often coupled with unfavorable

physical properties such as low organic matter percentage, low water retention, and

32 coarse texture (Brooks 1987). These characteristics can then induce drought stress in

serpentine dwelling plants. This stress is hypothesized to be an important component to

the low productivity shown by serpentine vegetation in dry climates such as California

and South Africa (Kruckeberg 1954; Gardner and Macnair 2000; Hughes et al. 2001).

Although we did not directly measure particle size and water content, we did measure

levels of organic matter content, which affects soil texture and water retention capacity

(Brooks 1987). We found that the sampled serpentine sites did not have lower than

average organic matter content. This result is consistent with the moderate levels of

nutrients found in our serpentine sites. The overall conclusion from our results suggests

that the stress imposed on populations of Lasthenia californica sensu Orduff from

serpentine sites is due primarily to chemical toxicity (elevated magnesium and heavy

metal concentrations) and not due to nutrient deficiency or physical harshness.

Edaphic patterns for coastal sites sampled

As mentioned, the PC 2 axis was tightly correlated to concentrations of sodium

and to a lesser extent sulfur, which agrees with other surveys showing increased substrate

salinities found in coastal sea spray communities (Boyce 1954). The levels of substrate

salinity can be variable at coastal sites, and is dependent on the region and distance from

the sea (Boyce 1954; Barbour 1978). MgCl is another salt in sea spray that may exert a

potential stress on plants growing in coastal communities (Wu 1981; Ashraf et al 1981;

Hodson et al. 1981). However, we did not find a significantly increased level of

magnesium in the coastal sites surveyed compared to ionically benign pastures.

33 Relationships between ecological and physiological variation

Serpentine environments impose a diverse ensemble of chemical and physical stresses. Hypothesized physiological adaptations are also diverse and include drought tolerance (Gardner and Macnair 2000; Hughes et al. 2001), resilience to low nutrient levels (Proctor 1971a), mechanisms of tolerating adverse magnesium/calcium ratios

(O'Dell et al. 2006; O'Dell and Claassen 2006; Main 1974; Madhok and walker 1969;

Marrs and Proctor 1976) and resistance to heavy metal toxicity (Reeves et al. 1999; Boyd and Martens 1998). As mentioned, the most likely stress imposed by serpentine soils onto the sampled L. californica populations is one of excess magnesium and perhaps heavy metals. ANOVA's performed on tissue samples from these sites show that magnesium levels are significantly higher in tissues from serpentine sites than compared to benign inland pasture habitats. These results suggest that serpentine plants have acquired tolerances to high shoot concentrations of magnesium. It may also suggest that these plants cope with high magnesium by accumulating this ion in the shoot (Marrs and

Proctor 1976; Main 1981).

Soils data show elevated heavy metal concentrations of nickel, chromium, and cobalt from serpentine soils, suggesting that these ions may impose a stress in serpentine sites. Interestingly, plant tissues from serpentine sites do not show elevated concentrations of cobalt and chromium. Low concentrations of chromium and cobalt in the tissues of plants from serpentine habitats suggests two possibilities: that these populations have adapted efficient mechanisms of heavy metal exclusion; or more likely, that these heavy metals are not present in an available form. Amelioration of the potential toxicity of heavy metals occurs when serpentine soils are sufficiently high in pH

34 and organic matter content (Brooks 1987). In particular, chromium becomes increasingly insoluble at higher pH, a condition typical of serpentine soils. For example, it has been shown that chromium concentrations in plant-available forms are very low in serpentine soils with a pH over 6.8 (Brooks 1987). ANOVA's of soil data confirm that serpentine soils from the sites we sampled have a mean pH (6.8) that is significantly higher than in other habitats. As previously stated, increased organic matter content has been shown to reduce plant available heavy metal concentrations by chelating the ionic forms of these metals (Crooke 1956; Halstead et al. 1969). Given that the organic composition of these soils is not lower than in other habitats, there is likely sufficient organic mass to reduce available heavy metals in these serpentine soils. Finally, competition between heavy metal ions and other abundant divalent cations during uptake may contribute to heavy metal stress resistance (Proctor 1971b). For instance, Proctor et al. 1971b and Robertson et al. 1985 demonstrated that increasing divalent ions in soil solutions with high Ni2+ or

Cr2* decreased the toxicity of these heavy metals. Thus, for the serpentine populations of

Lasthenia californica studied, high magnesium in these soils may mitigate the toxicity of heavy metals.

Hyperaccumulation is another commonly reported adaptation acquired by serpentine adapted taxa (Boyd and Martens 1998). Tissue data show that hyperaccumulation is not occurring in serpentine tolerant plants in Lasthenia californica sensu lato. Hyperaccumulators such as Thlaspi montanum var. siskiyouense and

Streptanthus polygaloides have been shown to accumulate as much as 10,000 ppm of nickel (Reeves et al. 1983). Our tissue data show that nickel hyperaccumulation is not occurring in serpentine tolerant plants tested; the mean nickel concentration in tissues

35 from serpentine tolerant Lasthenia californica sensu lato sites tested is 17 ppm. This finding is corroborated by another tissue analysis performed on individuals at a serpentine grassland at Jasper Ridge which showed similarly low levels of nickel accumulation (Rajakaruna and Bohm, 1999).

Other than the inconspicuous physiological mechanisms acquired to cope with the challenges of serpentine, serpentine ecotypes also evolved putatively adaptive morphological traits such as xeromorphy or increased root to shoot ratios (Kruckeberg

1984). Obvious above-ground morphological differentiation by serpentine tolerant populations of Lasthenia californica sensu lato is noticeably absent. In contrast, individuals from saline areas, in particular coastal areas, have noticeably divergent morphologies. For example, specimens from salt-deserts from southern California are noticeably stunted and succulent. Moreover, coastal populations of Lasthenia californica are very morphologically diverse. Although usually succulent, this trait varies in magnitude. Plants from the coast can be annual or perennial and large or stunted. Some of these morphological changes are analogous to adaptations reported in the majority of halophytes. These adaptations include: succulence, increased cell volume, increased epidermal thickness, and increased root to shoot ratios (Flowers et al. 1986).

Morphological changes such as succulence are correlated with physiological adaptations such as sodium accumulation, vacuolar compartmentalization of sodium, and the ability to replace sodium for potassium as a vacuolar osmoticum (Reimann and

Breckle 1995; Greenway and Munns 1980; Flowers et al. 1986). Tissue data suggest that populations of saline inhabiting populations of Lasthenia californica have adapted to salt stress by increased sodium accumulation. This may be evident in the significantly higher

36 tissue levels of sodium in coastal areas. Potassium tissue levels did not correlate significantly to potassium soil levels across the complex, but rather, appeared to group based on ecology. Although soil potassium means were almost identical between coastal and inland sites, concentrations in tissues were significantly lower in coastal versus inland populations as determined by pairwise contrasts. This may mean that coastal

individuals differentially accumulate sodium and use it to replace potassium as a vacuolar osmoticum. While potassium tissue levels seem differentiated based on ecological

origins, levels of sodium accumulation generally respond to levels of substrate sodium

across sites fairly consistently (indicated by the significant regression). This suggests that across the complex, plants respond to increased sodium levels by increasing sodium

uptake and accumulation.

Members of the Lasthenia californica complex display a wide range of

ecological, morphological, and physiological diversity. Soil, tissue and morphological

data collected based on field sampling all suggest that salinity is associated with the most

striking patterns of variation in the complex. Although the morphological distinctiveness

of L. californica coastal populations has sparked the curiosity of several botanists

(Ornduff 1966, Rajakaruna et al. 2003a), the putative adaptiveness of coastal traits has

not been tested. In Chapter 3, we explore whether differentiation in coastal traits is

genetically determined and whether these traits are responsive to the major environmental

variable differentiating its environment: salinity.

37 Table 2.1: Site ID, ITS species identity, locality, habitat, and flavonoid race determinations from this and previous surveys are summarized. Data from the current study is abbreviated as 2007; data from Rajakaruna and Bohm (1999) and Rajakaruna et al., 2003 surveys are abbreviated as 1999&2003; data from Desrochers and Bohm 1993 is abbreviated as 1993. Sites are roughly ordered geographically, with the northernmost sites listed at the top of the table.

Site ID ITS species 19 93 1999&2003* 20 07 Location and habitat A C A C State County 55 L. californica C 15 Medford, across road from a water treatment plant, grassy pasture OR Jackson 56 L. californica C 15 Medford, near Kodac plant, grassy pasture OR Jackson 54 L. californica .30 C 20 Table rock summit, meadow and vernal pool. OR Jackson 73-75 L. orduffii**** Otter Point, coastal bluff OR Curry 74 L. orduffii**** Cape Sebastian, coastal bluff OR Curry 76 L. orduffii**** Cape Blanco, coastal OR Curry 27 L. californica 33 C 15 Near Red Bluff, pasture CA Tehama 72 L. californica 32 Pudding, coastal bluff CA Mendocino * 67 L. californica 20 Near Corlevaro way and Grill way, coastal bluff CA Sonoma SPS L. californica 25 5 A 8 37 Salt Point State Park, coastal bluff CA Sonoma 26 L. californica 22 5*** C 20 Near Clay, pasture CA Sacramento 25-36 L. californica C 30 Near Clay, pasture CA Sacramento 24-35 L. californica 25 Outside Clay, pasture CA Amador

51 L. californica v 15 Just past Lake Co. and Colusa Co. boundary, pasture CA Colusa 48 L. californica 20 Near Middletown, pasture CA Lake 49 L. californica 20 Junction of Rte. 53 and Rte. 20, pasture CA Lake 43 L. californica 30** C 20 Point Reyes, near coastal lighthouse CA Marin 58 L. californica 18 Steep Ravine Campground, serpetine till CA Marin 62 L. californica 15 Dillion Beach, coastal CA Marin 63 L. californica 30 18 Near Dillion Beach, rocky roadside CA Marin 42 L. californica 11 15 Mt. Tamalpais, near O'roukes, serpentine CA Marin 21-41 L. californica 54 6 A 18 5 Mt. Tamalpais, near old mine trail, serpentine CA Marin 59-44 L. californica 21 Chimney Rock, coastal CA Marin JRA L. californica mostly A 12 Jasper ridge lower reaches of serpentine ridge CA San Mateo JRC L. gracilis mostly C 2 10 Jasper ridge upper reaches of serpentine ridge CA San Mateo 40 L. californica A 13 Kirby, serpentine CA Santa Clara 22 L. gracilis A 24 Byron, alkali flat CA Contra Costa 3 L. gracilis A 15 Coalinga, sloping pasture CA Fresno 4 L. gracilis A 15 - Coalinga, pasture CA Fresno 30 L. californica A 15 Arroyo Seco, sloping meadow CA Monterey

1 Front gate at Palmer Ranch, salinized pasture CA Monterey P L. gracilis C 2 18 p2 L. gracilis A 8 33 Palmer Ranch near a swampy area, salinized pasture CA Monterey 17-34 L. gracilis 36 A 20 Priest Valley, pasture CA Monterey 5 L. gracilis 65 A 15 Near Tehachipi, road side embankment CA Kern 10 L. gracilis A 20 Outside Motte Rimrock, grassy yard near houses CA Riverside 7 L. gracilis 32 A 15 Palmdale, meadow CA Los Angeles

Notes: * Rajakaruna and colleagues (Rajakaruna and Bohm 1999; Rajakaruna et al. 2003) reported only uniformly A or C populations (except at Jasper ridge). The number of plants surveyed per site was between 3-6. ** Originally reported as "type B" and described as a modified profile of C (Desrochers and Bohm 1993), this profile waslater interpreted as C by Rajakaruna et al. 2003. This interpretation is followed here (2007 data). *** One of these C's was originally reported as profile B (Desrochers and Bohm 1993).

**** us fype similar to L. californica. This ecotype is now recognized as L. Ornduffii.

\ Table 2.2: Transformations used in statistical analyses. A) Transformations used in ANOVA and PC analyses for soil variables. B) Transformations used in ANOVA's for tissue variables.

Soil variables Tissue variables Variable Transformation Variable Transformation Organic matter Square root N LoglO CEC Square root S Square root pH None P LoglO N Loglf/ Mg None P Fourth root Ca None K Square root K None Ca none Na Fourth root Mg LoglO Fe Fourth root S LoglO Mn LoglO Na LoglO B LoglO Zn LoglO Cu LoglO Mn LoglO Zn Square root Fe None Al Fourth root Cu LoglO Cr LoglO (1 +Cr) B Square root Ni LoglO (1 + Ni) Al LoglO Cr LoglO Co LoglO Ni LoglO

40 Table 2.3: ANOVA results for soil variables. Variables showing significant differences (P < 0.05) between A and C populations are bolded. Means that are significantly higher are bolded as well. Only differences between A and C were tested; mixed populations of both A and C were not included in the ANOVA. Mineral concentrations are reported in ppm; CEC in meq/lOOg. A C AC mixed Variable Mean S.E. Mean S.E. Mean S.E. Organic matter (%) 3.0 0.5 4.5 0.4 5.0 1.8 CEC 14.6 2.5 13.5 0.8 14.3 1.8 pH 6.7 0.1 5.9 0.1 6.3 0.3 Nitrate 16.5 9.0 10.4 3.4 8.9 1.7 P 37.7 7.4 15.5 2.3 18.9 7.5 Na/K 0.112 0.046 0.902 0.296 0.823 0.294 Mg/Ca 1.864 0.881 0.761 0.218 1.718 0.650 K 193.5 26.6 128.8 12.3 114.1 35.2 Ca 937.3 143.1 747.1 77.4 518.8 110.5 Mg 913.6 312.0 458.3 89.4 746.3 195.7 S 6.7 0.6 10.3 I. 1 19.5 9.2 Na 14.6 5.1 106.4 29.0 119.9 59.6 Zn 3.4 0.5 3.3 0.4 2.4 0.3 Mn 44.5 4.5 51.4 II. 0 27.3 6.3 Fe 64.8 5.9 85.3 4.3 76.4 9.0 Cu 1.0 0.2 1.4 0.2 0.8 0.2 B 0.5 0.1 0.7 0.3 0.5 0.1 Al 471.6 47.2 753.9 83.4 749.4 301.6 Cr 204.5 97.9 117.8 53.1 557.0 294.8 Co 44.7 18.6 25.0 9.3 61.6 29.3 Ni 689.3 351.6 248.2 189.8 940.3 522.5

Table 2.4: Summary of number of A, C, or mixed A and C profile populations present on

Edaphic type A C Mixed Salinized (coastal bluffs and alkaline flats) 0 . 5 2 Serpentine 3 1 2

41 Table 2.5: PCA results of soil data. Eigenvalues, % of variance explained, cumulative % of variance explained, and Pearson's correlations (r) to axes are included.

PC axis 1 2 3 Eigenvalues 5.494 4.132 2.041 % of variance 28.918 21.75 10.742 Cumulative % of variance 28.918 50.668 61.41

Variables Organic matter (%) 0.311 -0.711 0.451 CEC 0.655 -0.282 0.608 pH 0.4 0.617 0.177 Nirate 0.146 0.002 0.124 P -0.761 0.059 0.413 K -0.529 -0.025 0.627 Ca -0.436 0.557 0.474 Mg 0.875 0.134 0.378 S -0.11 -0.829 0.252 Na 0.119 -0.845 -0.052 Fe 0.26 -0.377 -0.24 Zn -0.411 0.273 0.549 Mn -0.023 0.76 -0.031 Cu 0.298 0.395 0.051 B 0 -0.348 -0.102 Al -0.281 -0.513 0.154 Cr 0.929 -0.044 0.108 Co 0.894 0.302 0.108 Ni . 0.938 0.065 0.048

42 Table 2.6: Means and standard error of soil variables for serpentine, inland benign pastures and coastal sites. Variables showing significant differences (P < 0.05) are bolded. Significant pair-wise differences (after a Tukey HSD correction) between habitat groupings are denoted with letters where shared letters represent non-significant differences. Mineral concentrations are reported in ppm; CEC in meq/lOOg.

Coastal bluffs Inland pastures ** Serpentine Variables Mean S.E. Mean S.E. Mean S.E. Organic matter (%) 6.5 0.8 a 3.0 0.3 b 4.1 0.5 ab CEC 16.0 1.2 a 11.4 0.8 b 19.9 2.9 a PH 5.8 0.2 a 6.2 0.1 a 6.9 0.1 b Nitrate 8.1 3.0 14.5 5.8 11.2 2.8 P 20.2 4.3 * 28.4 4.9 * 7.2 1.0 Na/K 1.444 0.468 a 0.143 0.028 b 0.319 0.049 b Mg/Ca 0.653 0.044 b 0.418 0.099 b 4.178 0.949 a Kt 159.8 22.2 160.0 17.7 88.2 17.3 Ca 525.6 67.5 a 1049.0 79.7 b 406.7 30.5 a Mg 335.0 40.1 a 403.8 77.6 a 1850.8 302.8 b S 16.1 1.8 b 6.9 0.3 a 6.5 0.9 a Na 180.8 32.2 a 17:5 3.8 b 27.2 6.8 c Zn 2.9 0.5 3.6 0.5 2.2 0.1 Mn 16.0 3.0 a 64.1 10.0 b 35.8 6.7 b Fe 78.0 5.4 72.8 4.6 77.8 7.9 Cu 1.0 0.3 1.5 0.2 1.1 0.1 B 0.5 0:1 a 0.3 0.0 b 0.6 0.1 a Al 1105.2 153.7 a 593.8 37.3 b 376.3 45.8 c Cr 69.9 12.4 a 53.3 11.3 a 866.7 141.6 b Co 9.9 1.2 a 1.6.8 1.8 a 139.6 18.3 b .Ni 48.7 8.2 a 58.7 19.8 a 2480.2 404.4 b

* Pair-wise differences are not shown because nonsensical differences were found due to insufficient power after Tukey HSD corrections. fP=0.053. ** The saline/alkaline pools were excluded from the inland pasture grouping, and not included as a separate habitat category because of the low N of 2.

43 Table 2.7: Means and standard error of tissue variables for A, C, and mixed A and C populations. Variables showing significant differences (P < 0.05) between A and C populations are bolded and means that are significantly higher are bolded as well. Only differences between A and C were tested; mixed populations of both A and C were not included in the ANOVA. All mineral concentrations are reported in ppm.

A C AC mixed Variable Mean S.E. Mean S.E. Mean S;E. N 12262.0 3319.3 10182.6 666.9 9477.5 2356.9 S 1355.7 219.4 2204.6 299.1 2628.6 778.5 P 3258.4 568.7 1685.8 120.6 1872.8 382.2 Mg/Ca 3.599 0.139 0.373 0.052 0.635 0.137 Na/K 0.091 0.078 0.455 0.154 0.378 0.190 K 20055.5 1821.0 20688.6 1339.2 15159.3 1527.0 Na 854.5 612.5 6616.7 2000.6 5487.7 2669.8 Mg 2878.8 672.3 3559.5 362.3 5401.3 624.5 Ca 11978.9 1308.3 10749.1 556.5 9426.4 1874.2 Fe 225.2 36.3 288.1 53.9 150.9 39.5 Mn 54.3 9.5 154.3 20.7 97.7 48.4 B 24.0 1.6 29.9 4.7 25.4 3.4 Cu 5.9 0.3 7.2 0.5 5.2 L0 Zn 48.6 5.0 52.0 4.1 33.5 2.1 Al 37.5 8.8 107.2 30.7 71.8 30.5 Cr 1.8 0.4 2.5 0.4 1.9 0.2 Ni 5.1 2.3 3.1 1.2 8.2 3.8

44 Table 2.8: PCA results of tissue data. Eigenvalues, % of variance explained, cumulative % of variance explained, and Pearson's correlations (r) to axes are included.

PC axis 1 2 3 Eigenvalues 5.264 3.394 1.22 % variance 35.094 22.624 8.132 Cumulative % variance ; 35.094 57.718 65.85

Variables N 0.28 0.546 0.027 • S 0.877 0.183 0.108 P -0.21 0.534 -0.316 Mg 0.705 -0.57 0.1.28 Ca -0.215 0.82 0.02 K -0.58 0.451 0.444 Na 0.95 -0.021 -0.186 Fe 0.692 0.045 0.313 Mn 0.479 0.56 0.327 B 0.397 0.251 -0.656 Cu 0.649 0.535 -0.094 Zn 0.131 0.247 0.446 Al 0.857 0.223 0.018 Cr 0.721 -0.222 0.046 Ni 0.175 -0.866 0.115

45 Table 2.9: Means and standard error of tissue variables for serpentine, inland benign pastures and coastal sites. Variables showing significant differences (P < 0.05) are bolded. Significant pair-wise differences (after a Tukey HSD correction) between habitat groupings are denoted with letters. Shared letters represent non-significant differences. Mineral concentrations are reported in ppm; CEC in meq/lOOg.

Coastal Inland pastures** Serpentine Variable Mean S.E. Mean S.E. Mean S.E. •N 13140.0 694.1 a 10781.1 1867.9 ab 6964.0 1267.9 b S 38.82.3 217.2 a 1269.5 127.9 b 1555.8 267.9 b P 1799.0 188.2 * 2593.0 361.8 * 1212.3 210.0 * Mg/Ca 0.484 0.039 a 0.196 0.027 b 1.048 0.089 c Na/K 1.106 0.224 a 0.006 0.001 b 0.269 0.135 b K 15609.2 1281.1 a 23707.0 925.6 b 13767.0 1761.2 a Na 15952.0 2183.7 a 148.1 12.7 b 2813.6 940.0 c Mg 5236.9 411.9 a 2265.1 173.7 b 6032.1 369.7 a Ca 10991.4 740.0 a 12588.1 575.3 a 5917.0 570.2 b Fe 444.3 91.9 a 196.1 29.6 b 188.4 51.4 ab Al 245.7 35.9 a 27.8 5.4 b 24.1 10.7 b Mn 197.4 22.1 a 107.0 20.9 b 36.1 3.9 c B 31.9 1.4 a 22.7 1.0 b 21.0 1.2 b Cu 8.9 0.9 a 6.0 0.2 b 4.7 0.5 c Zn 54.5 9.3 48.8 3.0 45.1 6.2 Cr 3.5 0.6 a 1.5 0.2 b 3.0 0.7 a Ni 2.4 0.9 a 1.8 0.6 a 17.5 1-9 b

* Pair-wise differences are not shown. Nonsensical differences were found due to insufficient power after Tukey HSD corrections. ** The saline/alkaline pools were excluded from the inland pasture grouping, and not included as a separate habitat category because of the low N of 2.

46 Table 2.10: Results for linear regressions of soil and tissue concentrations of P, K, Ca, S, Na, Zn, Fe, Cu, B and N (ppm).

Element X Transformation Y Transformation Constant Slope R2 P P log(base 10) log(base 10) 2.655 0.506 0.642 0.0001 K none none 0.289 Ca* none none 3.227 0.067 0.449 0.0001 S * log(base 10) log(base 10) 2.285 1.021 0.584 0.0001 Na log(base 10) log(base 10) 1.041 1.247 0.712 0.0001 Zn log(base 10) log(base 10) 1.518 0.335 0.185 0.012 Fe none none 0.496 Cu log(base 10) log(base 10) 0.1058 B * none none 17.291 18.556 0.348 0.0001 N * . none none 0.441

>.

*in all these cases, 1 outlier of exceptional leverage or influence was removed from the analysis

47 a) 3 3 + I I I —I—c—I— ~l 1

4- X. A A C 2 - 4- 4- 2 I- A A A H A A A ^ M M A 4- 4- * + 1 ] ' 14- C V- 4- c 0 \ A 4^ .0 A -4* — X 4- CN CN o -1 o -1 CL /o CL M — /xr * J — -2 O -2

-3 / x/ -3 — c c c -4 •xf -4 M I /I I I _L I I I -5 -2 0 -5- 6 -4 -2 0 2 4 PC 1 PC 1

Figure 2.1: PCA ordination of axes 1 and 2 for soil variables, (a) Habitat group symbols correspond to serpentine (A), coastal (X), inland benign pastures (+)> and inland alkaline flats (o). Confidence ellipses represent one standard deviation from group centroids (excluding alkaline flats; N=2). (b) Symbols correspond to uniformly A profile populations (A), uniformly C profile populations (C), or populations with mixed A and C individuals (M). a)

I I A A

c c c M — A & . c c A C ^ M C - A - - C ~ . A - MM

c A

I I -5 0 5 10 -5 0 5 10 PC 1 PC 1

Figure 2.2: PCA ordination of axes 1 and 2 for tissue variables, (a) Habitat group symbols correspond to serpentine (A), coastal (X), inland benign pastures (+), and inland alkaline flats (o). Confidence ellipses represent one standard deviation from group centroids (excluding alkaline flats; N=2). (b) Symbols correspond to uniformly A profile populations (A), uniformly C profile populations (C), or populations with mixed A and C individuals (M).

V Figure 2.3. Bivariate distribution of soil (x-axis) and tissue (y-axis) concentrations of Ca (a) and B (b). Symbols correspond to serpentine sites (S), coastal sites (C), inland benign pastures (P), and inland alkaline flats (A). Linear regressions were fitted for the complex. a) b)

E CL -S 00 ID

O

1.0 1,5 ' 0.6 0.7 0.8 0:9 1.0 1.1 1:2 1.3 1.4 Log [Soil P (ppm)], Log [Soil S (ppm)] .

c) d)

J I J I I I J L 1 2 3 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Log [Soil Na (ppm)] Log [Soil Zn (ppm)]

Figure 2.4. Bivariate distribution of log soil (x-axis) and log tissue (y-axis) concentrations of phosphorus (a), sulfur (b), sodium (c), and zinc (d). Symbols correspond to serpentine sites (S), coastal sites (C), inland benign pastures (P), and inland alkaline flats (A). Linear regressions were fitted for the complex.

S1 [Soil Mg] ppm [K soil] (ppm)

Figure 2.5. Bivariate distribution of soil (x-axis) and tissue (y-axis) concentrations of magnesium (a) and potassium (b). Symbols correspond to serpentine sites (S), coastal sites (C), inland benign pastures (P), and inland alkaline flats (A). Confidence ellipses represent one standard deviation from group centroids. CHAPTER 3

Differentiation in morphological and physiological responses to salinity between coastal and inland populations of Lasthenia californica sensu stricto

INTRODUCTION

Plants can exhibit a large amount of phenotypic variation within species. Patterns of ecotypic differentiation that are associated with environmental gradients demonstrate the importance of selection in generating morphological diversity and have inspired a rich history of botanical inquiry (e.g. Turesson 1922a,b; Clausen et al. 1948; Grant and

Hunter 1962). Divergent selection pressures such as those imposed by coastal versus inland environments produce particularly interesting lines of phenotypic - ecological covariation and remain relevant systems for studying patterns and mechanisms of local adaptation and trait differentiation (Antfinger 1981; Nagy 1995).

Coastal salt spray communities are subject to the stresses imposed by salt exposure (Oosting 1945; Boyce 1954; Barbour 1978; Cartica and Quinn, 1980; Sykes and

Wilson 1988, 1999) along with other stresses that may include sand accretion (Van der

Valk 1974; Sykes and Wilson 1990, 1999), wind exposure, and low soil water-holding capacity due to coarse, sandy substrates (Boyce, 1954). Salt loads from sea spray induce stress on vegetation by both increasing substrate salinity (Barbour 1978; Sykes and

Wilson 1999) and causing leaf necrosis because of salt accumulation on leaf surfaces

(Griffiths and Orians 2004). While coastal habitats, including those in California, are under constant levels of salinity stress, the climate along the coast is generally milder than in associated inland areas, which receive sufficient year-round precipitation. In contrast, the interior of California alternates between moist winters and hot summers which exert the predictable droughts that characterize Mediterranean climates (Barbour et

53 al. 2000; Tenhunen et al. 1985). In habitats such as disturbed roadsides and grasslands, the success of many annuals depends on rapid and competitive growth during the wet season before the onset of summer droughts (Grime 1979; Aronson et al. 1992; Fitter and

Hay 2002).

Although the extent of sodium stress varies between and within coastal sites - salt loads change with proximity to the source of exposure and with the amount of wind force available to propel sea spray (Barbour et al. 1985) - sodium has been suspected or indicated as being the primary factor affecting species distributions or morphological variation of plants in these areas (Wells and Shunk 1938; Sykes and Wilson 1999;

Griffiths and Orians 2004; Goldstein et al. 1996). For example, an ecological survey at

Point Reyes, in central California, has shown that salt amounts in collection traps correlates with plant species distribution (Barbour 1978). The importance of salt as a selection pressure in coastal environments is also demonstrated by detection of genetic differentiation in salt tolerance in coastal populations of a number of unrelated taxa that span inland and coastal habitats (Reimann and Breckle 1995; Kohl 1997; Tiku and

Snyadon 1971; Rozema et al. 1978; Ab-shukor 1988; Watt 1983).

Studies of coastal ecotypes further reveal that populations growing in salt exposed environments have independently acquired similar sets of morphological and physiological adaptations to cope with the stresses of salinity. Most of these adaptations tackle aspects of the ionic and osmotic stresses induced by increased salinity (Flowers et al. 1986; Greenway and Munns 1980). Enhanced ionic stress tolerance through increased sodium accumulation and vacuolar compartmentalization of sodium has been reported in many coastal taxa (Flowers et al. 1986; Tester and Davenport 2003). Osmotic regulatory

54 adaptations such as vacuolar sodium/potassium osmoticum replacement and the production of organic osmotic regulating compounds (eg. proline and mannitol) are also commonly reported adaptations in coastal plants (Maathuis and Amtmann 1999; Munns

2002; Harborne 1993; Yeo 1998; Harborne 1993). These physiological and morphological investments in stress regulatory mechanisms are empirically and theoretically linked to trade-offs in lowered intrinsic growth potentials (RGRmax) when these stresses are relaxed (Grime and Hunt 1975; Arendt 1997; Chapin 1980, 1991;

Orians and Solbrig 1977; Fernandez and Reynolds 2000). While broadly advantageous alleles can spread rapidly across populations under low levels of gene flow and promote cohesiveness within species (Rieseberg and Burke 2001; Morjan and Rieseberg 2004), tradeoffs have the potential to maintain variation between neighboring populations by limiting the spread of alleles underlying traits that differ in net fitness in contrasting environments.

In this study we focus on patterns of differentiation in stress tolerance and growth as well as potential trade-offs between these in coastal and inland ecotypes of Lasthenia californica (Asteraceae). The taxonomic history of this species has been complicated by complex patterns of cryptic genetic and morphologically divergent variation within members of the L. californica species complex. The new circumscription of L. californica sensu stricto (henceforth referred to as L. californica) now reflects a monophyletic lineage that includes two formerly distinct species: L. macrantha and the northern populations of L. californica sensu lato. L. macrantha (as it was formerly known) was a mostly coastal assemblage of three subspecies all showing distinct morphology from interior populations of L. californica sensu lato. Lasthenia macrantha

55 was distinguished by larger strap shaped, succulent leaves and facultative perenniality

(Ornduff 1966; Chan et al. 2002). Both DNA sequence (Chan et al. 2001, 2002) and

RAPD marker data (Rajakaruna 2002) have shown that L. macrantha is polyphyletic in origin, with populations tending to nest within geographically neighboring inland populations of L. californica sensu lato. While easily distinguishable in overall shape and form, there is little reproductive differentiation between the two formerly distinct species (Ornduff 1966; Chan et al. 2002). The high level of interfertility between these taxa (Ornduff 1966) reflects the general genetic cohesiveness of L. californica shown by molecular data.

Recent field surveys of populations of L. California have confirmed the ecological, physiological and morphological distinctiveness of coastal versus inland populations. Soil samples show that coastal sites have markedly higher levels substrate salinity (>25 fold) than inland sites. These environmental differences are amplified in levels of tissue sodium (>130 fold higher sodium in coastal tissues). Substrate potassium levels were also higher in coastal sites (almost doubled), however, coastal tissue potassium contents was only 2/3 that of inland sties (a result generally shown by halophytes; Flowers et al. 1986). Observations of succulence found that while differentiation is greatest between inland and coastal sites, significant variation can also be found within coastal populations. At coastal sites, leaves of individuals growing on edges of sea exposed bluffs tended to be the most succulent with succulence decreasing with distance from the sea spray exposure line (G. Choe, personal observation). The morphological and physiological observations in the field suggest that phenotypic

56 divergence between populations of Lasthenia californica may be driven by divergent responses to environmental sodium.

Here we investigate the basis of the phenotypic differentiation observed between ecotypes of Lasthenia californica by growing two populations of each ecotype in common conditions with varying levels of the environmental factor we hypothesize as the most likely to promote the phenotypic divergence shown between ecotypes: salinity.

Phenotypic divergence in ecologically relevant traits can be attributed to: 1) adaptive genetic differentiation; 2) phenotypic plasticity, in which different phenotypes are induced by environmental differences; or 3) by a combination of these components where the environment induces genetically differentiated responses between ecotypes. Given the body of evidence indicating similar trait differentiation in a number of coastal taxa, it is unlikely that the patterns under study reflect random genetic differentiation due to drift.

We take the classical approach of comparing norms of reaction between populations across salinities to investigate the possibility that coastal populations are adaptively differentiated in salt tolerance and associated tolerance traits. We first tested differentiation in performance (i.e. salt tolerance) between populations under increasing salinity. Adaptive differentiation was inferred when coastal populations showed uniformly higher fitness under high salinity. We then tested for differences in intrinsic and induced stress tolerance expressed in morphological and physiological traits. Upon detection of differentiation in both salt tolerance and related traits, we looked for evidence of ecotypic differences in trade-offs between stress tolerance and growth potentials.

57 MATERIALS AND METHODS

Seed collection

The populations used in this study were selected from sites widely distributed within the California floristic province. This sampling strategy was implemented to minimize possible trait similarities between populations due to shared population histories or gene flow. Four populations, two inland and two coastal, were tested (Table

3.1). Between April and July of 2005 seeds were collected from these sites from one mature flower head for each of >30 individuals sampled haphazardly at each site. Each

> head contains approximately 50-200 full or half-siblings. These sites were also part of a broader survey of soil and plant tissue variables (Chapter 2).

Inland and coastal sites differ in numerous environmental variables. Inland habitats are Mediterranean in climate, with cool, wet winters and hot, dry summers. The mean annual precipitation at the two sites inland sites ranges between 48 - 63 cm

(National Climatic Data Center, 1948-2006; http://www.ncolc.noaa.gov). Coastal sites are cooler and consistently moist year round, with mean annual precipitation at these two sites between 96 - 104 cm (National Climatic Data Center, 1948-2006; http://www.ncolc.noaa.gov). Sites also differ in various soil characteristics. Most notably, sodium concentrations in the soil are substantially higher in coastal populations than inland populations (Table 3.1). Field data also show that the average level of tissue sodium and potassium concentrations of individuals from these sites differ dramatically in inland versus coastal populations (Table 3.1).

58 Germination and experimental design

Seeds were ripened at room temperature for several months and then pooled within populations. These seeds were sown in Petri-dishes filled with nutrient medium

composed of these following ingredients: macronutrients: 0.25mM KH2P04, 0.5mM

KN03, 0.5mM Ca(N03)2.4H20, 0.5mM MgS04.7H20, O.lmM NH3N03; micronutrients:

20/

l^MCuS04.5H20, 0.2^M Na2Mo04.2H20, 50^M NaCl. The nutrient solution was

neutralized to a final pH of 6 using Ca(OH)2. 0.7% agar was added to the solution to create a semi-solid medium.

The sown seeds were placed in a growth chamber with initial conditions set at

17°C days and 14°C nights, cycling at 12 hour photoperiods. Germination and initial seedling growth occurred over 7 days. A total of 512 pots (2 by 7 inch Cone-tainers,

Stuewe & Sons, Inc., Corvallis, OR, USA) were filled with 6 parts soil-less potting medium (Terra Lite Soil Mix, W. R. Grace, Ajax, Ontario, Canada) and 1 part inert fritted clay (Turface, Profile Products LLC, Buffalo Grove, IL, USA). Two seedlings were planted in each pot. These pots were watered with a nutrient solution similar in composition to the agar medium described above, with the two exceptions that macronutrient concentrations were doubled and no agar was added. Pots were randomly arranged in a growth chamber set to provide a 14 hour photoperiod with 22°C day and

18°C night cycles. Photosynthetic photon flux of approximately 290/

Utah, USA). After 14 days of growth, seedlings were inspected for vigor, and then pots with healthy seedlings were grouped into fours. Groups were arranged by size. One

59 member of each group was used for an initial harvest to obtain initial dry weights for relative growth rate calculations (Evans 1972). The remaining unharvested pots were re- randomized within the chamber where they grew until the final harvest. NaCl treatment commenced after the initial harvest. NaCl treatments were applied in a randomized arrangement to the pots. Given both population and treatment application were both randomized without restriction, the experiment followed a completely randomized design.

Treatment solutions were composed of the aforementioned nutrient solution plus either a OmM, 70mM, 120mM addition of NaCl for the control, medium stress, and high stress treatment, respectively. Medium and high NaCl treatment concentrations were determined based on trial experiments which suggested that NaCl concentration causing

50% decline in biomass for coastal population was within the range of 120 mM NaCl, which was not overly toxic to inland populations. NaCl stress was increased

Incrementally with each watering. This slow ramping of stress was implemented in order to avoid sudden NaCl shock. The initial treatment for both medium and high stress received only 35mM NaCl. For the second treatment, 70mM NaCl was applied for both the medium and high stress treatments. The final 120mM NaCl concentration for the high NaCl stress treatment was applied on the fourth watering after an intermediary

90mM NaCl watering step. Watering treatments were continued as needed until the final harvest.

To ensure that tissue analyses reflected uptake amounts, all plants were sprinkled from above with distilled water several days prior to the final harvest. This was done to rinse off salts that may have accumulated on the leaves during treatment applications.

60 The plants were harvested over two days, 54-55 days after the initial planting. Harvest

began before the onset of flowering to ensure that most individuals were at similar

developmental stages (pre-bolting).

Plants were cut at the root-shoot transition zone. Previous attempts to reliably

record root mass were unsuccessful as plants could not grow robustly in media where

their roots could be easily separated from the matrix. Leaves were separated and their

fresh weight was determined. The leaves were arranged in a flatbed scanner; leaf number

and leaf area of the scanned images were then determined using Sigmascan Pro (SPSS

1999). Leaves were then dried in a forced draft oven at 60°C for 2-3 days until a constant

weight was reached. Above ground dry mass (biomass) was then determined. This was

used as a measure of fitness. The concentrations of Na+, K+, Mg2+ and Ca2+ in dried leaf

tissues were determined by ICP detection. This procedure involved an initial VHP closed

vessel microwave acid digestion and a subsequent ICP spectrometer analysis of digested

solutions (Analytical Chemistry Laboratories, Ministry of Forestry, Victoria, BC,

Canada). The dry matter of three pots was randomly pooled to generate sufficient mass

required for the procedure. In total, 96 groups were created (8 groups of 3 pots per

population X 4 populations X 3treatments). Due to equipment malfunction, the leaf ion

data of only 93 samples are reported here. The data set was trimmed to 288 pots (24 per

population X 4 populations X 3 treatments) to maintain a balanced design (all crossed

levels having 24 cases) in spite of unequal mortality and additional losses due to

harvesting errors. Analyses that explored the relationship between ion content and

growth and morphology traits used averaged growth and morphology values within

pooled ion samples.

61 From the traits measured during the two harvests, the following derived traits were determined: Relative growth rate (RGR), leaf dry matter content (LDMC), specific leaf area (SLA) and succulence. Relative growth rates were calculated from the following formula (Evans 1972): [In dryweight (final harvest) - In dryweight (initial harvest)] / number of days. Succulence was calculated with the following equation

(Debez et al. 2004; Karrenberg et al. 2006): (fresh weight - dry weight) / surface area.

Specific leaf area (SLA) and leaf dry matter content (LDMC) were calculated as (leaf dry weight / area) and (fresh weight /dry weight), respectively.

Data analysis

Differences in growth, morphology and leaf ion traits were analyzed with an analysis of variance using the GLM packages in SYSTAT 11 (SYSTAT, 2005) and JMP

IN 4.04 (SAS Institute, 2000). Significant interaction terms were first tested for each trait. We interpreted significant interaction terms for biomass as evidence for differentiation in salt resistance, and significant population by salt treatment interactions of RGR (specifically, crossed reaction norms) as evidence of trade-offs in growth rates and salt tolerance. When significant interaction terms were detected, the nature of the interaction was further explored by decomposing the interaction sum of squares into the following treatment X population contrasts testing 1) whether the response to salinity differed between inland versus coastal ecotypes 2) whether the response to salinity was consistent between populations within each ecotype (i.e: inland 49 vs. 54 or coastal 67 vs.

72). These essentially test for differences of reaction norm slopes between assigned groups. These contrasts were tested as a single df contrast between the linear trend of 1)

62 \

averaged response of coastal populations versus inland populations and 2) populations from the same habitat. Before linear contrasts were investigated, an initial null of no

linear trend was tested to confirm that reaction trends could be sufficiently captured by a

linear model. Sets of orthogonal quadratic polynomials were examined, but in all tests,

linear trends provided a better fit (data not shown) and so quadratic models were not

included. Because contrasts were pre-planned and orthogonal, their significance was

tested against an alpha of 0.05 without adjustments (Sokal and Rohlf 1995).

Along with interaction terms, significant main effects were determined. Significant

simple effects of populations within treatment levels were determined for a few traits; of

particular importance, simple population effects for RGR's at the control treatment were

determined to test for differences in RGRmax. Pair-wise tests between means were

tested when significant terms were determined for a few traits of interest (such as RGR).

Post-hoc, pair-wise contrasts were controlled for inflated family-wise type I error rates

with a Bonferroni adjustment.

The residuals of ANOVA tests were visually inspected and tested with

Wilk-Shapiro's test (a test of normality) and Levene's test (a test of unequal variances) to

verify their adherence to the assumptions of ANOVA. Na+ leaf concentrations and

Na+/K+ content of leaves were log or log(l + x) transformed to improve variance

differences between groups. In general, non-extreme deviations from ANOVA

assumptions were generally ignored as ANOVAs with balanced cells tend to be fairly

robust against minor deviations (Quinn and Keough 2002).

We used ANCOVAs to determine whether the relationship between sodium

accumulation and succulence differs between ecotypes. The ANCOVA tests whether the

63 linear relationship between sodium leaf content and succulence is similar or divergent between ecotypes by testing whether the ecotype slopes are homogenous.

The relationships between leaf ion accumulation between Na+ and K+ and Ca2+ and Mg2+ were determined by calculating Pearson product-moment correlations. For these multiple pair-wise correlations, a false discovery rate was controlled using a

Bonferroni adjustment on the family of tests.

RESULTS

Growth measures: biomass and relative growth rates

Both population and treatment main effects were significant for biomass (Table

3.2). Populations strongly differed in biomass production in response to salinity

(indicated by the highly significant population by treatment interaction; Table 3.2 and

Figure 3.1a), which provided evidence of population differentiation in salt tolerance.

When this interaction was further explored, we found that the linear trend between inland and coastal ecotypes showed a significant difference but the trend between the populations within each ecotype did not differ (Table 3.2). This suggests that while on the ecotypic level there is clear differentiation in salt tolerance, there is little difference in tolerance to salinity between populations from similar habitats. Coastal populations were uniformly less hindered by the negative effects of salinity than were inland populations.

This is illustrated by the decreased steepness of the reaction norms displayed by coastal populations versus those of inland populations (Figure 3.1a). Interestingly, the population least affected by salinity stress was also from the saltiest coastal site

(population 67, Table 3.1 and Figure 3.1a); but as mentioned earlier, the difference in

64 reaction norms between populations of the same ecotype did not prove to be significant.

We found that biomass decreased in fairly equal increments across salt treatments for coastal populations. In contrast, the decline in biomass between the control and medium stress treatments for inland populations was noticeably more severe than the decrease in biomass between medium and high stress treatments. This could be due to population differences in the response to the slower ramping of stress levels in the high versus medium stress treatment.

There was a significant population by treatment interaction term for relative growth rates (Table 3.2; see Figure 3.1b). The significant crossing of reaction norms

(Figure 3.1b) suggests trade-offs in RGRs' and salt tolerance. The decline of RGRs' for the inland ecotype under increasing salinity was more severe than the decline of RGRs' for the coastal ecotype, a result supported by the significant ecotype X treatment contrast

(Table 3.2). As found for biomass, populations from the same habitat were not differentiated in their response to salinity; the linear trends between populations within ecotype groupings were not significantly different from each other (Table 3.2; Figure

3.1b). Both population and treatment main effects were significant (Table 3.2). We tested for difference in RGRmax (simple population effects at control treatment level), and found significant differences. Pair-wise tests between populations at the control treatment showed that inland populations have significantly higher RGRmax than coastal populations (Figure 3.1c).

65 Leaf morphology

Most morphological traits showed significant population by treatment interaction terms. Specifically, leaf number, LDMC, and succulence showed significant interaction terms. SLA is the only trait lacking significant interactions between factors. Generally, population differences in morphological responses to environmental sodium were muted compared to differences in responses shown by growth traits (biomass and RGR). This result can be intuitively appreciated by the lack of crossed reaction norms for morphological traits, as compared to biomass and RGR, which both showed crossed trends between ecotypes. Most traits also showed significant main effects of both treatment and population. These results suggest that the majority of morphological traits are genetically differentiated between populations and that environmental sodium levels have an effect on morphology.

Leaf number decreased across sodium treatments for all populations tested

(significant treatment effect; Table 3.2 and Figure 3.2a). However, the extent of the decline in leaf number is dependent on ecotype. When interaction terms were partitioned into a contrast between ecotype, the contrast in ecotype trends proved significant (Table

3.2). Populations within ecotypes showed similar reactions to one another as indicated by non-significant contrasts of trends between populations 49 and 54, as well as between

67 and 72. Generally, coastal populations had more leaves across all sodium treatments

(indicated by the significant population effect; Table 3.2 and Figure 3.2a).

Population by salt treatment interaction for LDMC was significant (Table 3.2).

Like most other traits, the reaction norms were significantly different between ecotypes but were not significantly different between populations of the same ecotype (tested by

66 linear contrasts; see Table 2 and Figure 3.2b). In general, coastal leaves had a higher water component to their leaves (indicated by the lower ratio of dry over fresh weight) regardless of salt treatment. This result is demonstrated by the pair-wise contrasts of marginal (pooled across salt treatments) population means (Figure 3.2c).

Figure 3.3a illustrates that while SLA is both affected by salt treatments and is intrinsically different between populations, the way in which SLA changes in response to environmental sodium is fairly consistent between populations. ANOVA results show that while treatment and population main effects were highly significant (P < 0.0001 in both cases), SLA did not show a significant population X treatment interaction (Table

3.2). Pair-wise tests between populations show that coastal populations have significantly thicker/denser leaves than inland populations as indicated by their lower

SLA values (Figure 3.3c).

Succulence can be interpreted as a measure that combines the water content component of LDMC and the leaf thickness component of SLA. Whereas the responses of SLA and LDMC to salt treatments are either non-significant (SLA) or similar in response between populations (LDMC), succulence has a clear difference in the direction of response to salinity between populations (population X treatment interaction with P

<0.0001; Table 3.2). When main effects were investigated, a significant population and a non-significant treatment term were found. Note that the significance of the salt treatment effect was masked by the nature of the population X treatment interaction. The salt treatment main effect tests for significant differences by comparing the pooled means

(across all populations) at each treatment level. Because salt affected populations in a way that either increased or decreased succulence values fairly symmetrically, the

67 resulting means were therefore fairly equal across treatment levels. We tested for differences in succulence values between the control and high stress treatment for each population separately (a test of simple treatment effects within populations) and found that succulence differed significantly between sodium treatments for each population tested (tested at an alpha of 0.05, consistent with contrasts using sets of orthogonal tests).

This confirms that environmental salt levels do indeed impose a biological effect on succulence. Figure 3.3b graphically illustrates this finding showing that while coastal populations tend to gain in succulence under increasing salinity, inland populations tend to lose succulence under saltier conditions. In general, much of the variation in response to salinity can be attributed to ecotypes. Whereas the reaction norms between ecotypes differ significantly (P<0.0001), the responses between populations of the same ecotype do not differ significantly (Table 3.2). Coastal populations had significantly higher values of succulence compared to inland populations across all salt treatments (as indicated by pairwise comparisons of means; Figure 3.3d).

Leaf ion content

All populations dramatically increased in sodium tissue content under increasing salt stress. However, the magnitude of the response differs between ecotypes (indicated by the significant treatment X population interaction and the significant inland versus coastal trend contrast; Table 3.3, Figure 3.4a). The responses between populations of the same ecotype did not differ significantly (Table 3.3). Coastal populations tended to accumulate greater levels of sodium over all treatments including the control (population effect < 0.0001; pair-wise differences at control are shown in Figure 3.4c). Populations

68 thus appear both genetically differentiated in constitutive levels of sodium accumulation as well as in their ability to increase sodium accumulation with increasing environmental salinity. Potassium levels in tissues also responded to salt exposure. Potassium levels decreased demonstrably with increasing salinity (Figure 3.4b) and showed significant treatment by population interactions (Table 3.3). Leaf potassium concentration responses showed a greater differentiation between populations and a greater complexity in the shape of the response than did sodium concentrations. This is demonstrated by the following results: 1) leaf potassium trends were significantly different between the two inland populations, which is indicated by the 49 vs. 54 X treatment contrasts (Table 3.3) and 2) the reaction norms of 67 crossed both 49 and 54. Generally, coastal populations tended to have lower potassium contents in their leaves in conditions of high salinity (see pair-wise differences at medium and high NaCl stress, Figure 3.4b); but interestingly, coastal population 67 showed the highest level of leaf potassium concentration at the control (Figure 3.4b). Results for sodium/potassium ratios of leaves logically followed the trends shown by leaf sodium and potassium concentration results already described.

Sodium/potassium ratios increase greatly under increasing salinity for all populations

(indicated by the highly significant treatment effect, Table 3.3), but the magnitude of sodium/potassium replacement differed between populations (significant interaction term,

Table 3), ecotypes (significant ecotype trend contrast, Table 3.3), and populations of each ecotype (significant population within ecotype trend contrast, Table 3.3). Coastal populations tended to have the highest levels of sodium to potassium (Figure 3.5).

69 Correlations between traits

Correlations between Na+ and K+ leaf contents are very strong for all populations, with P<0.0001 (Table 3.4). In comparison, correlations between Na+ and divalent cations, Mg2+ and Ca2+, are mostly non-significant (Table 3.4). This suggests that the accumulation of Na+ and Mg2+ and Ca2+ are generally independent. Coastal population 72 is the one exception to this trend. Na+ accumulation is negatively correlated to both Mg2+ and Ca2+ levels in leaf tissues (P<0.0001; Table 3.4).

Although all populations showed significant correlations between sodium accumulation and leaf succulence (Table 3.4), the direction of the correlation differed between ecotypes. Inland populations showed a negative correlation between succulence and sodium accumulation while coastal populations showed a positive correlation between sodium content and succulence (Figure 3.6). When homogeneity of slopes was tested between ecotypes using an ANCOVA, a significant sodium slope X ecotype interaction confirmed that the relationship between sodium accumulation and succulence did indeed differ between populations (Table 3.5).

DISCUSSION

Local adaptation to an environmental pressure is interpreted from norms of reaction data when two criteria are met (Murren et al. 2006; Schmitt 1993; Silander and

Antonovics 1979): 1) when significant habitat origin X treatment interactions in plant performance is present 2) when the populations showing highest performance in experimental conditions are from similar historical conditions in the field. When we examined the performance (biomass) of Lasthenia californica populations in differing

70 levels of salinity we found a significant population by treatment interaction; secondly, we found that coastal populations were more fit under increasing salinity. The result that both populations from habitats with greater substrate salinity were less impacted by the negative effects of salt (a result supported by the ecotype trend contrast) strengthens the argument that coastal populations have evolved greater salt tolerance due to historical environmental pressures. But note that because the seeds used in this experiment were field collected, differences shown between populations could be a combined result of genetic and maternal effects (Rossiter 1996; Roach and Wulff 1987). However, maternal effects have been shown to diminish with time (Schmitt and Antonovics 1986) and so have less influence in mature plants, such as those used in this experiment. In general, our results are supported by a large body of literature reporting genetic differentiation in salt tolerance between coastal and non-coastal populations (Reimann and Breckle 1995;

Kohl 1997; Tiku and Snyadon 1971; Rozema et al. 1978; Ab-shukor 1988; Watt 1983;

Kik 1989) and imply that accompanying morphological and physiological adaptations that accommodate greater salt tolerance are likely to be found in coastal populations of

Lasthenia californica.

Field observations suggest that increased succulence, sodium accumulation and compartmentalization, and vacuolar osmoticum replacement of potassium with sodium are all possible morphological and physiological investments made to enhance salt tolerance in coastal environments. Our results suggest that the phenotypic divergence in these traits in the field reflects both genetic differentiation in traits and in trait responses to salt. When we analyzed the response of population trait means across sodium levels, we found a clear differentiation between populations in the direction or magnitude of trait responses to environmental sodium. Like the results for biomass, population differences in trait expression under experimental conditions followed predictions made from observed differentiation in the field. Succulence, sodium accumulation and sodium/potassium ratios were expressed at higher levels in coastal sites, and for the most part, these coastal individuals showed greater intrinsic and induced levels of expression in controlled conditions. We also found surprisingly convergent intrinsic and induced levels of expression from populations of the same habitat type, which further supports the argument that salinity pressure, in part, shaped the divergent morphology of coastal versus inland ecotypes of Lasthenia californica. The following paragraphs will outline in greater detail our hypothesis for the ecological relevance of the morphological and physiological trait differences shown in controlled conditions.

Field collected coastal plants contain over 140 times the sodium as those in non- saline sites. The results presented here suggest that these differences reflect both genetic differentiation in constitutive levels of sodium accumulation (as indicated by the differences at the control) and differences in their ability to increase sodium accumulation with increasing environmental salinity. In general, increased levels of sodium accumulation indicate increased efficiencies in sodium compartmentalization (Flowers et al. 1986). Efficient sodium compartmentalization in the vacuole is thought to be adaptive because it alleviates both the ionic and osmotic stress components of sodium toxicity

(Chinnusamy et al. 2005). Vacuolar compartmentalization of Na+ decreases ionic stress by reducing cytosolic Na+, where it competes with K+ in binding sites of important enzymatic reactions (Yeo 1998; Tester and Davenport 2003). Osmotic stress is partly

72 relieved by vacuolar accumulation of sodium by lowering solute potential in the vacuole, which in turn promotes turgor (Chinnusamy et al. 2005).

Typically most glycophytic plants use potassium, and to a lesser degree, calcium and magnesium as vacuolar osmotica (Chinnusamy et al. 2005; Greenway and Munns

1980; Tester and Davenport 2003; Niu et al. 1995). Salt tolerant species are commonly found to replace sodium for potassium (Flowers et al. 1986, Chinnusamy et al. 2005).

This is adaptive because it allows the remaining potassium to localize in the cytosol, and removes toxic sodium from the cytosol to the vacuole, where it is then used as a 'cheap osmoticum' (Tester and Davenport 2003; Chinnusamy et al. 2005). As mentioned, field data suggest that this type of osmoticum switching is employed by coastal individuals as a way of achieving salinity resistance; potassium levels in coastal tissues were found to be two-thirds the levels of inland tissues despite higher substrate potassium levels in coastal areas. Our experimental results support the hypothesis of enhanced osmoticum replacement by coastal individuals. When grown under common conditions, potassium levels tended to decrease more dramatically in coastal populations with increasing environmental sodium. The significant ecotype trend contrast for leaf sodium/potassium reinforces the result that potassium is replaced by sodium under increasing salinity to a higher degree in coastal than inland ecotypes.

Osmoticum replacement not only occurs between sodium and potassium, but sodium can also replace magnesium and calcium to a limited degree in the vacuoles of some halophytes (Greenway and Munns 1980; Karrenberg et al. 2006). Only Coastal population 72 showed a significant negative correlation between tissue levels of sodium and magnesium or sodium and calcium. Because these correlations were absent in an

73 equally salt tolerant population (coastal 67), it suggests that salinity resistance in L. californica can be achieved by different pathways.

Succulence is the most obvious morphological difference between these two ecotypes of L. californica in the field (Ornduff 1966). In coastal areas, succulence levels can differ between and within populations. In contrast, the level of succulence in inland populations (excluding alkali flats) shows little variation. Our experimental results indicate genetic differentiation in intrinsic and salt induced levels of succulence. At every treatment level, coastal individuals are at least twice as succulent as inland individuals. Furthermore, coastal populations show a slight but significant increase in succulence under increasing salinity while inland populations decrease in succulence with increasing sodium stress. The changes in succulence shown by coastal populations in experimental conditions partly explains the observed differences in coastal succulence in the field. At coastal sites, individuals tend to become more succulent with increasing salt spray exposure, our experimental results suggest that these differences are in part due to plastic responses to sodium. The minor decreases in succulence shown by inland individuals under increasing experimental sodium levels most likely reflects losses of turgor, presumably due to an inability to overcome the osmotic stress (water potential) exerted by high salt concentrations. The suggestion that water content is higher for coastal versus inland leaves is supported by the results that show that LDMC (leaf dry mass/leaf fresh mass) values are lowest for coastal populations in all treatment levels.

Succulence is associated with increases in cell and vacuolar volume (Flowers et al. 1986; Yeo and Flowers 1980; Hajibagheri et al. 1984). Increased vacuole volume can lead to increased sodium accumulation, and in this way, succulence is thought to be

74 adaptive in saline environments (Greenway and Munns 1980). Since succulence is correlated to a greater capacity to regulate sodium, the amount of sodium in leaves should then be positively correlated with succulence in salt tolerant genotypes. Under

experimental conditions, coastal populations show a positive correlation between

succulence and sodium tissue levels whereas inland populations show a negative

correlation between succulence and leaf sodium levels. The difference in trait association

between ecotypes is supported by the significant sodium by ecotype interaction in an

ANCOVA test. This result reinforces the argument that coastal populations attain greater

sodium tolerance by a suite of composite adaptations involved in enhancing sodium

regulation via sodium accumulation and compartmentalization.

Correlations between traits can arise by linkage disequilibrium (nonrandomly

associated alleles tied by the influence of evolutionary action) or pleiotropy (where one

locus contributes to the expression of both traits). Under linkage disequilibrium, strong

selection can maintain trait associations, where the fitness benefit of joint trait expression

overcomes recombination (Lynch and Walsh 1998). An association between sodium

accumulation and succulence is broadly reported across taxa (Flowers et al. 1986), and

our results add to this pattern, as these two traits are correlated in coastal ecotypes in L.

californica. Given that only coastal ecotypes show a positive correlation between these

two traits, selection rather than pleiotropy is the likely cause of the correlation.

f

Negative genetic correlations in the form of constraints have important impacts on

the direction of response to selection, where the response of one trait to selection is

limited by the fitness of the correlated trait (Lynch and Walsh 1998; Maynard Smith et al.

75 1985; Mitchell-Olds 1996). When selection favors alternate traits in neighboring populations, trade-offs have the potential to enhance or maintain trait variation between these populations (Fry 2003). We asked whether there is evidence of trade-offs between increased salt tolerance and lowered intrinsic growth potentials for populations of

Lasthenia californica.

To test for evidence trade-offs between growth potentials and stress tolerance, we first established that coastal populations are more salt tolerant. We then compared relative growth rates of populations under control levels of salt, as well as their changing growth rates under increasing salinity. Our results show that growth rates are uniformly lower for coastal types than inland populations when grown under non-stressful conditions (RGRmax), and that with increasing salinity, growth rate trends cross between ecotypes so that coastal populations show higher relative growth rates than inland populations under conditions of high salinity stress. These results support the existence of trade-offs between stress tolerance and relative growth rates in coastal versus inland populations of L. californica.

The hypothesis of trade-offs between growth potential and stress tolerance has a wealth of empirical support from studies of various harsh environments, in particular nutrient stressed habitats (Arendt 1997; Chapin et al. 1993; Chapin 1980, 1991; Orians and Solbrig 1977; Fernandez and Reynolds 2000; Grime and Hunt 1975). The trade-off between tolerance and growth is thought to arise because of genetically determined constraints on resource allocation, where investments to stress, tolerance adaptations

(such as succulence) come at the expense of resource expenditure on growth (Arendt

1997; Chapin et al. 1993). Correlations between leaf investment indices such as SLA (a

76 measure of leaf density) and growth potentials have been studied across numerous taxa.

It is hypothesized that leaves of high RGR species maximize growth potentials by increasing their photosynthetic capacity (leaf area) while minimizing their investments in structural or protective investments (leaf area/mass), which then results in higher SLA values (Lambers and Poorter 1992; Chapin et al. 1993; Fitter and Hay 2002). These studies have generally shown that SLA and growth rates are widely correlated over numerous taxa (Poorter and Remkes 1990; Lambers and Poorter 1992), with lower values of SLA associated with slow growing stress tolerant genotypes, and with higher SLA values associated with ruderal or highly competitive plants with rapid growth rates (Fitter and Hay 2002; Poorter and Remkes 1990; Lambers and Poorter 1992). Our results are consistent with overall trends in SLA values shown by other stress tolerant genotypes and non-stress tolerant genotypes. SLA values were found to be uniformly lower in coastal than inland populations of L. californica.

In life-history strategy theories proposed by Grime (1977, 1979), environments with high abiotic stress and low disturbance are predicted to favor genotypes that allocate more resources to stress tolerance adaptations and less resources to rapid growth (i.e.

'stress tolerators'). In environments with low stress but high disturbance or in environments of overall low stress, genotypes with rapid and/or competitive growth are thought to be favored (i.e. 'ruderals' or 'competitors'; Grime 1977, 1979). Inland populations of Lasthenia californica grow in areas that are either disturbed (eg. roadsides) or relatively competitive (eg. grasslands; MacDougall and Turkington 2004) and are likely to exert selection favoring ruderal or competitive strategies (Grime 1979).

Moreover, the climate of interior California may select for an accelerated life-history

77 strategy to avoid the predictable droughts of the hot and dry Mediterranean summers

(Aronson et al. 1992; Fitter and Hay 2002). In comparison, coastal ecotypes grow in relatively moist and ocean-moderated climates that can support slower, year-round growth (evident in the later flowering times or perenniality of coastal L. californica) provided that the plants allocate sufficient resources to tolerate the constant abiotic stress imposed by their environment. In these contrasting environments, the relative importance of abiotic versus biotic pressures on fitness are thought to shift (Weldon and

Slauson 1986; Greiner La Peyre 2001; Crain et al. 2004), where traits related to tolerance are more intensely selected for in stressed environments while traits related to rapid growth or competitive ability are favored in non-abiotically stressed environments

(Chapin et al. 1993). Because trade-offs act to magnify divergence in traits when contrasting environments favor opposing traits (Futuyma and Moreno 1988; Rice and

Salt 1990), potential trade-offs between allocation to growth and stress tolerance may help to explain why the morphologies of coastal versus inland populations of Lasthenia californica are so divergent in the field.

78 Table 3.1: Summary of ecotype, population identification, localities, soil and tissue data from field sites used in this study. Ecotype Site Locality Soil Soil Tissue Tissue ID Na+ K+ Na+ K+ (ppm) (ppm) (ppm) (ppm) Inland 49 Junction of Rte. 53 and Rte. 20, 10 120 112 22879 grassy pasture. Elevation: 501m. Lake Co. CA Inland 54 Table rock summit, andesite deposit. 17 80 142 26264 Along meadows near vernal pools. Elevation: 610m. Jackson Co. OR Coastal 67 Near Corlevaro way and Grill way, 237 269 17688 16975 coastal bluff. Elevation 15m. Sonoma Co. CA Coastal 72 Pudding, coastal bluff. Elevation 159 136 16750 13911 4m. Mendocino Co. CA

79 Table 3.2. Summary of ANOVA results for biomass (g), relative growth rates (In mg days"1), total leaf number, LDMC (g g'), SLA

2 2 cm g \ and succulence (g H20 cm' ).

Traits

Source of variation df Biomass RGR Leaf number LDMC SLA Succulence

Population 3 SS 0.1636969 0.0005638 87982 0.021361 1643900 0.0709201 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Treatment 2 SS 0.2854945 0.05135924 74395 0.010429 103559 0.0000074 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 N.S

Population X Treatment 6 SS 0.0452703 0.01302682 6831 0.001091 17276 0.0012794 P <0.0001 <0.0001 0.0003 0.0001 N.S <0.000L

*(49,S2) vs (67,72) X Treatment 1 SS 0.0331827 0.0115853 4681 0.000378 12044 0.0010371 P <0.0001 <0.0001 <0.0001 0.0017 <0.0001

*49 vs 54 X Treatment 1 SS 0.0007889 0.0000045 284 0.000001 2 0.0000045 P N.S N.S N.S N.S N.S

*67 vs 72 X Treatment SS 0.0014797 0.0000252 1 0.000072 2494 0.0000339 P N.S N.S N.S N.S N.S

Error 276 SS 0.1168866- 0.00321575 71133 0.010386 436727 0.0078214

*above are contrasts of linear trends

/ Table 3.3. Summary of ANOVA results for ion traits: Na (ppm), K (ppm), and Na/K.

Ion trait

Source of variation df Log (Na) K Log(l+Na/K)

Population 3 SS 3.37257 . 7134348375 0.625194 P :

Treatment 2 SS 63.27856 35150000000 1.701708 P <0.0001 <0.0001 <0.0001

Population X Treatment 6 SS 0.50520 1500246175 0.294249 P <0.0001 <0.0001 <0.0001

*(49,52) vs (67,72) X Treatment 1 SS 0.46119 925502722 0.201967 P <0.0001 <0.0001 <0.0001

*49 vs 54 X Treatment 1 SS 0.00014 99341598 0.009832 P N.S 0.0133 0.0123

*67 vs 72 X Treatment 1 SS 0.00027 38852113 0.019300 P N.S N.S 0.0005

Error 81 0.26854 1255021012 0.126200

above are contrasts of linear trends

81 Table 3.4. Values and significance of Pearson product-moment correlations between leaf values of sodium and potassium, sodium and calcium, sodium and magnesium, and sodium and succulence.

Population Na+- K+ Na+ - Mg2+ Na+- Ca2+ Na+- Succulence

49 r (-) .0.940 - - (-) 0.701 P <0.0001 N.S. N.S. <0.0001

54 r (-) 0.941 _ _ (-) 0.759 P <0.0001 N.S. N.S. <0.0001

67 r (-) 0.986 _ _ 0.436 P <0.0001 N.S. N.S. 0.033

72 r (-) 0.992 (-) 0.875 (-) 0.936 0.745 P <0.0001 <0.0001 <0.0001 <0.0001

Table 3.5. Summary of ANCOVA results for sodium tissue contents and succulence.

Source of variation df SS

Ecotype 1 0.01925744 <0.0001 Na ppm 1 0.00002102 N.S. Ecotype X Na ppm 1 0.00035386 <0.0001 Error 89 0.00073502

82 a) b)

0.25 0.14

C M C M Treatment Treatment

49 - 49 Legend 54 Legend - 54 67 - 67 72 72 c)

0.14

0.13 -

trt 0.12

0.11 CD E c 0.10 I- n 0.09 k O

11 0.08 |-

0.07

0.06 49 54 67 72 Population

Figure 3.1. Graphical results for growth traits. Reaction norms with means and standard errors for biomass (g) and relative growth rates (In mg days"1) are shown in figure (a) and (b) respectively. Reaction norms for each population are represented by a unique line described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively, (c) Population means and standard errors for RGR values at the control treatment. Significant pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively.

83 a) b)

150 0.110 I i I

0.095 I 100 i1

y 0.080 ..-IC Q _i ....-I--""' „„IC I ' - 0.065

I i I M 0.050 C M H Treatment Treatment

49 49 54 Legend Legend 54 67 — 67 72 72

C)

0.130

0.060 49 54 67 72 Population

Figure 3.2. Reaction norm plots including means and standard errors for (a) leaf number and (b) LDMC (g g '). Reaction norms for each population are represented by unique lines described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively, (c) LDMC (g g"1) population means and standard errors. Significant pair- wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively.

84 a) b)

550 0.080

0.015 C M H M

Treatment Treatment

49 49 54 Legend Legend 54 67 67 72 72

C) d)

0.080

0.010 49 54 67 72 54 67 72 Population Population

Figure 3.3. Reaction norm plots including means and standard errors for (a) SLA (cm2 g

2 ') and (b) succulence (g H20 cm" ). Reaction norms for each population are represented by unique lines described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively. Population means and standard errors for (c) SLA (cm2 g"1) and (d) and succulence (g

2 H20 cm" ). Pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively.

85 a) b)

49 49 54 Legend Legend 54 67 67 72 72

C)

2000

1500 ? Q. £ 1000 1 F Z 500

Ql I I « I MMII I 49 54 67 72 Population

Figure 3.4. Reaction norms with means and standard errors for leaf ion concentrations of (a) sodium (ppm) and (b) potassium (ppm). Reaction norms for each population are represented by a unique line described in the legend. Coastal and inland populations are labeled with a C or I, respectively. Control, medium and high stress treatments are denoted with a C, M and H, respectively, (c) Population mean and standard errors for leaf ion concentrations of sodium (ppm) at control treatment. Significant pair-wise differences between populations are denoted with letters, where shared letters represent non-significant differences. Inland populations and coastal populations are represented by empty bars or shaded bars, respectively.

86 C M H Treatment

Figure 3.5. Population means and standard errors for Na/K tissue ratios. Pair-wise differences between populations at each treatment level are denoted with letters, where shared letters represent non-significant differences. Note that Pair-wise tests were performed at each treatment level separately. Population 49, 54,67,72 are respectively represented by empty bars, lightly shaded bars, darkly shaded bars, and by black bars.

0.07

E o

X Legend

CD o 49 (f) x 54 + 67 A 72 0 • X a? J> it [Na Tissue] (ppm)

Figure 3.6. Bivariate distribution of tissue sodium contents and succulence. Linear slopes are fitted for each population.

87 CHAPTER 4

Conclusion

Significant findings and implications

The complex patterns of ecological, morphological, physiological, biochemical and genetic variability shown by members of the Lasthenia californica complex offer both an interesting and challenging study system regarding questions related to ecological differentiation and adaptation. This complexity has contributed to the recognition of polyphyletic taxa (particularly L. macrantha sensu lato) and to the hypothesis of parallel evolution of flavonoid/edaphic races in closely related but phylogenetically distinct lineages. While in the first example, the polyphyletic assemblage of L. macrantha sensu lato has been confirmed independently by different researchers (Chan et al. 2002;

Desrochers and Dodge 2003), the results presented in this thesis suggest that the second case of recurrent evolution proposed for L. californica should be re-examined. Our results suggest that the association between edaphic tolerances and flavonoid profile is not as strong as previously hypothesized by Rajakaruna and colleagues (Rajakaruna and

Bohm 1999; Rajakaruna 2002, Rajakaruna et al. 2003a,b,c). We found that populations of A's did not show a greater affinity to ionically stressed habitats than did C's. It is also likely that the presence of A/C flavonoid polymorphisms did not arise in these phylogenetic lineages by means of parallel evolution. The localized distribution of populations showing polymorphisms and/or shifts in A/C frequency suggests that gene flow is a plausible mechanism of introducing and/or maintaining this polymorphism.

88 In chapter 3, our results suggest that coastal populations of L. californica sensu stricto are more salt tolerant than the inland populations and that these two ecotypes are genetically differentiated in a number of ecologically relevant traits. One such trait, relative growth rate, was found to be significantly lower in coastal populations under non-stressful growth conditions, but then showed greater growth rates than inland populations under stressful conditions. These results support the hypothesis of trade-offs between growth potential and tolerance, as predicted by Grime (1977). Trade-offs between growth potentials and stress tolerance are most widely studied in nutrient gradients (Grime and Hunt 1975; Chapin 1980, 1991; Arendt 1997) and to a lesser degree in aridity gradients (Orians and Solbrig 1977; Fernandez and Reynolds 2000). This hypothesis has relatively less empirical support for saline systems although it is a natural extension of the theory and has been generally hypothesized as occurring (e.g. Chapin et al. 1993). It is generally thought that investigations of traits in intraspecific systems are particularly useful (e.g. Freemon and Herron 2001) because the confounding effects of phylogeny are not a concern as they are in cross species comparisons commonly used for the study of trade-offs in the ecological literature (e.g. Fernandez and Reynolds 2000).

Hypotheses requiring confirmation and possible future studies

As mentioned, the results presented in Chapter 2 provide evidence refuting the hypothesis of parallel evolution of edaphic/flavonoid races across L. californica sensu ( lato. Even in light of these findings, the possible link between flavonoid production and ionic stress physiology has not been definitively answered. Specifically, the strength of genetic linkage between flavonoid production and physiological traits conferring ionic

89 stress tolerance should be determined. Flavonoid profiles A and C are differentiated in a number of compounds, and it could be that only the genes producing sulfated flavonoids co-segregate with tolerance conferring traits. In this way, the sulfated flavonoid/tolerance affinity aspect of the flavonoid=edaphic race hypothesis could be confirmed in the lab, even though in the field, associations between A and ionically stressed habitats seem not to exist.

The flavonoid polymorphism found within edaphically harsh sites (e.g. serpentine populations at Mt. Tamalpais and the coastal bluff site at Salt Point State) hold interesting possibilities for future study. In the Lasthenia californica system, environmental heterogeneity along a serpentine grassland at Jasper Ridge has been shown to support the coexistence of two parapatric populations of L. californica subsp. californcia and L. gracilis that also differ in flavonoid composition. Generally, environmental heterogeneity has the potential to promote and maintain diversity in genotypes and species across landscapes (Kassen 2002; Jasmin and Kassen 2007; Weeks and Hoffman

1998; Freestone and Inouye 2006). It is possible that sites such as SPS and Mt.

Tamalpais have established similar covarying distributions of soil' chemistry and flavonoid type. There is some observational data from our current work that suggest that increased salt exposure is linked to colonization of A flavonoid types at Salt Point State coastal bluffs (SPS). We have found that in one area of the SPS population, locations closer to the bluff edge had higher proportions of A than C individuals than did the less spray exposed areas further from the bluff's edge. If microsite co-variation between flavonoid profile and soil variation is found at these polymorphic sites, this would then

90 support the link between edaphic profiles and stress physiology (but only if A types tend to inhabit saltier areas).

The polyphyletic assemblage L. macrantha sensu lato, provides an interesting system to the study of salinity adaptation. We provided evidence supporting differentiation of salt tolerance traits between populations. However, to confirm that the traits examined in our experiment are correlated to salt tolerance it is necessary to analyze the correlation between tolerance traits and salt resistance within populations. This could be done using standard quantitative genetic methods with replicated families of full or half sibling. We have also provided evidence of local adaptation of coastal populations to salt exposure. To confirm the hypothesis of local adaptation, it would be ideal to analyze the fitness of these populations in the field, in the form of a reciprocal transplant study. It would then also be possible to analyze the correlation of the traits that differentiate ecotypes to their fitness in the field.

As mentioned, Lasthenia macrantha sensu lato provides an interesting system for the study of salinity adaptation, but it also has the potential to provide insights into the study of stress adaptation in general. In our study we provided evidence of potential trade-offs between increased stress tolerance and lowered intrinsic growth rates. To explore this hypothesis in greater depth, the extent of heritable variation for alternative combinations of traits, particularly between tolerance traits and growth, should be determined. Also in Chapter 3, we proposed a potential counter selection pressure that could be associated with inland sites (namely competition) that would favor increased growth rates. For a trade-off to occur, competitive interactions should be at a greater

91 intensity in grasslands and pastures sites compared to the intensity of biotic interactions

at coastal sites. Also, competition at coastal sites should be relatively less important than

. abiotic selection pressures. These proposed counter selection pressures should be

verified in the field by measuring the relative intensity of biotic stress on fitness at these

contrasting sites.

In closing, the Lasthenia macrantha sensu lato system can also be useful to study

the mechanisms of recurrent phenotypic evolution. The 'macrantha' phenotype is

expressed in other phylogenetically distinct lineages in addition to the L. californica

lineage, namely, L. gracilis (the southerly distributed member of L. californica sensu

lato) and L. ornduffii (which was a former subspecies of L. macrantha sensu lato). It

would be of interest to examine whether the recurrent evolution of this phenotype follows

lines of greatest genetic variation and covariance in quantitative traits (e.g. Schluter

1996a) and whether the biased use of a few key genes universally present in these closely

related lineages produced the similarity in phenotypes shown (e.g. Schluter et al. 2004).

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