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Getting the dirt on Lasthenia : are edaphic factors influencing speciation in Lasthenia californica? Rajakaruna, Nishanta 1998

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"GETTING THE DIRT ON LASTHENIA" ARE EDAPHIC FACTORS INFLUENCING SPECIATION IN LASTHENIA CALIFORNIA ? by NISHANTA R A J A K A R U N A B.A. in Human Ecology, College of the Atlantic, Maine, U.S.A., 1994 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard The University of British Columbia January 1998 © Nishanta Rajakaruna In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ° T / ) ^ Y The University of British Columbia Vancouver, Canada D a t e F££ o ? J ijfff DE-6 (2/88) ABSTRACT The study of plants growing on unusual geologies has provided much insight into evolutionary biology. Examination of models of speciation shows that edaphic factors can serve as environmental triggers for models of speciation. The study presented here investigated morphological, biochemical* physiological, and ecological attributes of a species that may have evolved under edaphic influence. Lasthenia califomica is a spring annual endemic to California. Previous studies documented the existence of two races (type-A and -C) based on flavonoid pigments, achene morphology, allozyme banding patterns, and flowering time differences. These two races coexist in a population found on a serpentine outcrop at Jasper Ridge Biological Preserve, California. Studies lasting 15 years have disclosed that the two races maintain a sharply-defined boundary on this ridge. The ridge top is populated by type-C plants, and ridge bottom, by type-A plants. Studies were conducted to determine if edaphic factors play a role in maintaining this distribution pattern. Analyses of soil samples revealed significant differences in the physical and chemical features of the soils harboring each race. Analyses of plant tissue indicated that tissue concentrations of various elements in the two races are significantly different. Multivariate tests indicated that certain soil (pH, Mg, Na) as well as plant tissue (Na, Mg, Ca/Mg) characteristics are reliable in predicting each race. Examination of soil and plant tissue samples from 22 populations agreed with several patterns observed at Jasper Ridge. One of the most intriguing observations of the study was that the concentrations of sodium found in type-A plants are over three times those in type-C plants. Greenhouse studies revealed that the two races show differential responses in germination, survival, growth, and phenology to ridge top and bottom soils, indicating that edaphic factors are important in rrwintaining the distribution on this outcrop. It is suggested that type-A plants are possibly more tolerant of edaphic stresses than type-C plants. The role stress tolerance may have played in the evolution of the species is discussed. It is implied that the two races of L. californica may qualify as true edaphic races and that further studies are needed to determine i f the races deserve taxonomic recognition. TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures vii Acknowledgements viii Chapter 1: General Introduction 1 1.1. The edaphic factor in plant distribution 1 1.2. Plant life on ultramafic rocks 2 1.3. Speciation under edaphic influence 6 1.4. Lasthenia californica as a case study 10 Chapter 2: Soil and Plant Tissue Analyses 15 2.1. Introduction 15 2.2. Materials and Methods 16 2.2.1. Field Collections 16 2.2.1.1. Hants 18 2.2.1.2. Soils 18 2.2.2. Laboratory Studies 22 2.2.2.1. Flavonoid patterns of plants 22 2.2.2.2. Achenes 22 2.2.2.3. Whole Plants 23 2.2.2.4. Soil 23 2.2.2.5. Statistical Analyses 25 2.3. Results 27 2.3.1. Flavonoid data of Jasper Ridge 27 2.3.2. Flavonoid data of other populations 29 2.3.3. Achene differences ~ Jasper Ridge 31 2.3.4. Soils and plants of Jasper Ridge 34 2.3.4.1. Soils 34 2.3.4.2. Plants 40 2.3.4.3. Relationships between plant tissue and soil 44 concentrations for Jasper Ridge 2.3.5. Soils and plants of other populations 47 2.3.5.1. Soils 47 2.3.5.2. Plants 47 iv 2.3.5.3. Relationships between plant tissue and soil 51 concentrations for other populations 2.3.6. Discriminant Function Analysis - Soils and plant of Jasper Ridge 52 2.3.7. Principle Component Analysis - Soils and plants of Jasper Ridge 54 2.3.8. Discriminant Function Analysis - Other p opulations 57 2.4. Discussion 59 2.4.1. Jasper Ridge 59 2.4.2. Other Populations 65 2.5. Summary 66 Chapter 3: Greenhouse Studies 67 3.1. Introduction 67 3.2. Experiment 1: Germination and growth responses to soil treatments 67 ,3.2.1. Objective 67 3.2.2. Materials and Methods 67 3.2.3. Results 70 3.2.3.1. Germination Trial 70 3.2.3.2. Plant Survivorship 71 3.2.3.3. Growth Measures 75 3.3. Experiment 2: Germination responses to soil solutions 79 3.3.1. Objective 79 3.3.2. Materials and Methods 79 3.3.3. Results 80 3.4. Seed Bank Study 82 3.4.1. Objective 82 3.4.2. Materials and Methods 82 3.4.3. Results 82 3.5. Observations on Achene Dispersal 83 3.6. Discussion 84 3.7. Summary 90 Chapter 4: Conclusions and Future Directions 91 4.1. Is type-A a "stress tolerator" 91 4.2. Evolution under edaphic influence 94 4.3. Research Highlights 95 4.4. Summary 97 4.5 Future Directions 98 4.5.1. Edaphic Studies 99 4.5.2. Breeding Studies 100 4.5.3. Physiological Studies 100 4.5.4. Ecological Studies 102 Literature Cited 105 V LIST OF TABLES Table 2.1. Population number and locality information for samples collected 20 from the additional populations Table 2.2. Flavonoid pattern of plants collected from 22 populations 30 Table 2.3. Achene characters of plants collected from Jasper Ridge 33 Table 2.4. Soil characteristics for Jasper Ridge 36 Table 2.5. Plant tissue characteristics for Jasper Ridge 41 Table 2.6. Results of regression analyses between soil and plant tissue 45 characteristics for Jasper Ridge Table 2.7. Soil characteristics for the additional 22 populations 48 Table 2.8. Plant tissue characteristics for the additional 22 populations 50 Table 2.9. Results of regression analyses between soil and plant tissue 51 characteristics for the additional populations Table 2.10. Results of Discriminant Function Analysis - soils of Jasper Ridge 52 Table 2.11. Results of Discriminant Function Analysis - plants of Jasper Ridge 53 Table 2.12. Results of Discriminant Function Analysis - soils of additional 57 populations Table 2.13. Results of Discriminant Function Analysis - plants of additional 58 populations Table 3.1. Germination results for achenes of type-C and - A plants 70 Table 3.2. Mean height of type-C and - A plants 75 Table 3.3. Mean number of leaves of type-C and - A plants 76 Table 3.4. Mean percent of plants with inflorescences 77 Table 3.5. Mean percent of achenes with radicle growth 80 Table 3.6. Mean percent of achenes with leaf growth 81 vi LIST OF FIGURES Figure 1.1. A scheme for speciation under edaphic influence 9 Figure 1.2. A photograph of the study site at Jasper Ridge 14 Figure 2.1. A diagram showing the arrangement of Transects 17 Figure 2.2. Topographical map of the study site 19 Figure 2.3. Map showing the geographical distribution of all populations 21 Figure 2.4. Flavonoid pattern of plants collected at Jasper Ridge 28 Figure 2.5. Achene differences between type-C and - A plants 32 Figure 2.6. The difference in physical appearance of ridge top and bottom soils 35 Figure 2.7. Variation in soil characteristics along Transect 1 38 Figure 2.8. Variation in soil characteristics along Transect 1 39 Figure 2.9. Variation in plant tissue characteristics along Transect 1 42 Figure 2.10. Variation in plant tissue characteristics along Transect 1 43 Figure 2.11. Linear relationships between soil - plant tissue for both Na and M g 46 Figure 2.12. Results of P C A for soil variables at Jasper Ridge 55 Figure 2.13. Results of P C A for plant tissue variables at Jasper Ridge 56 Figure 2.14. A diagram of the ridge showing the drainage pattern 61 Figure 3.1. Survivorship of type-C plants in the three soil treatments 72 Figure 3.2. Survivorship of type-A plants in the three soil treatments 74 vii ACKNOWLEDGEMENTS A very special thank you to my research supervisor, Dr. Bruce A. Bohm, for his invaluable advice and assistance during the past two years. I thank him for his guidance, constructive comments, and financial support throughout this study. Above alL I appreciate the freedom he has given me to pursue my interests, to wherever they lead me, and collaborate with researchers from different disciplines. His continuing enthusiasm in my work has always been a source of inspiration. I have benefited much by his patient guidance. I would also like to express my gratitude to the rest of my committee, Drs. A . D . M . Glass, G E . Bradfield, and T .M. Ballard, for their support and advice during the course of the study. I am grateful to Dr. L . M . Lavkulich, Department of Soil Science, for allowing me to conduct my soil and plant tissue analyses in his laboratory. His advice during various stages of my project is greatly appreciated. I also benefited greatly from the assistance given by Drs. R. Turkington, J. Whitton, and J.R. Maze. I thank them for their comments on various aspects of my study. The administrative staff at Jasper Ridge Biological Preserve, Stanford University deserves sincere thanks for making the facilities at the Preserve available for my study. A very special thank you to my friend J.E. Page for his interest in my work. M y time here as a graduate student has been enriched from our intellectual as well as social interactions. I would also like to thank my friend J. Yang for his assistance in the field and laboratory. M y thanks to J. Dojillo-Mooney and other fellow graduate students at the Department of Botany for their friendship and support over the past two years. Friends from the Department of Soil Science, Justin Straker, Maja Krzic, Mike Shum, Susan Ames, and Anh-Toan Tran, also deserve a special thank you. Finally, I would like to thank members of the administrative staff at the Departments of Botany and Soil Science for their assistance and support during my study. viii Chapter One General Introduction 1.1. The edaphic factor in plant distribution "Within a given climatic region, growth of vegetation is mainly determined by the character of the parent material, whether limestone, igneous rock, sand deposit or clayey shale." Hans Jenny (1941) Plants with unusual or localized distribution patterns have always fascinated botanists and other natural historians. The study of such plants has provided scientists with interesting information on the history and evolution of certain regions and their floras. Further, these plants have provided opportunities to investigate aspects of evolutionary ecology and population dynamics unique to such plant populations. Unusual soil conditions give rise to localized patterns of plant distributions. The classic generalizations on the distribution of plants (Cain, 1944) place the edaphic factor second only to climate as the major environmental determinants of plant distribution. The edaphic factor pertains to the substratum upon which the plant grows and from which it derives its mineral nutrients and much of its water supply. It involves physical chemical, and biological properties of soils (Mason, 1946). Climate sets the limits for biota; however, geology enriches discontinuity and habitat diversity (Jenny, 1941). When physical and chemical properties of substrate are arrayed discontinuously, opportunities for colonization by different species as well as events leading to speciation can occur. The significance of geological phenomena in plant evolution, ecology, and geography has long been recognized (Kruckeberg, 1986). Geologic materials such as 1 limestone, dolomite, shales, gypsum, and serpentinite have been found to harbor unique plant associations, endemic and rare species, unusual distributions of taxa, as well as to foster morphological and physiological modifications in plants. Extreme edaphic conditions such as guano deposited by seabirds (Gillham, 1956; Ornduff, 1965; Vasey, 1985), vernal pools (Holland and Jain, 1977, 1981), granite outcrops (Wyatt and Fowler, 1977; Orndufl 1986), as well as soils contaminated by heavy metals (Antonovics et al., 1971, Shaw, 1990) have also been found to give rise to unique patterns of plant distributions. 1.2. Plant life on ultramafic rocks Soils derived from ultramafic rocks provide exciting opportunities to study the effects of the edaphic factor on plant distribution. Such studies have revealed many interesting facts on the ecology, evolution, and geography of plants in extreme edaphic conditions. Serpentine soils are derived from ultramafic rocks such as serpentinite. These soils are often shallow and rocky. They have exceptional physical and chemical properties that strongly reflect the elemental composition of their parent material. Iron (Fe), magnesium (Mg), and silicon (Si) are chief elements of the minerals found in serpentine soils. Nickel (Ni), cobalt (Co), and chromium (Cr) often occur in exceptional amounts. Serpentine soils have high values of exchangeable M g and exceptionally low values of exchangeable calcium (Ca). They are usually deficient in nutrients such as nitrogen (N), phosphorus (P), potassium (K), boron (B), and molybdenum (Mo). Serpentine soils tend to have lower clay content and cation exchange capacities than normal soils. Cation exchange capacities range from 5.2 - 43 C M o l ( + )/kg dry soil; p H values are often high and can range from 6.1 - 8.8 (Brooks, 1987; Kruckeberg, 1984, 1992). 2 Ultramafic rocks such as serpentinite occur on every major landmass and are found as local outcrops or extensive regional displays. Both local and massive occurrences of ultramafics are found in the three Pacific coast states of the United States and the Canadian province of British Columbia. California includes 2860 km 2 of such rocks, while Oregon and Washington include 1170 km 2 and 520 km 2 respectively (Kruckeberg, 1984). Serpentine soils are often associated with remarkable floras characterized by the paucity of species and high degree of endemicity. The earliest published record of a plant restricted to serpentine is given by A . Caesalpino in the late 16th century (Brooks, 1987). This plant was Alyssum bertolonii of the family Brassicaceae. William Brewer was one of the earliest Californian botanists to record the presence of unusual plant life in Californian serpentine soils (Brewer, 1949). Kruckeberg (1984) suggests that 215 or about 10% of the total Californian endemic vascular plants (species and infraspecific variants) are wholly or largely restricted to serpentine soils. Serpentine plants are highly specialized and are often dwarf and xeromorphic with chlorotic, narrow, and glaucescent leaves. They show strong sclerenchymatic development and possess enlarged root systems. They are often both morphologically and physiologically adapted to deal with the physical and chemical attributes of their environment. The stressful and highly selective nature of serpentine habitats is undoubtedly a consequence of the interplay of physical, chemical, and biotic factors. The ensemble of these factors, with their particular intensities, forms the feedback loop that is defined as the serpentine syndrome (Jenny, 1980). The causes of the serpentine syndrome have been traced to imbalance of Ca and M g (Vlamis and Jenny, 1948; Walker, 1954; Walker et al., 1955; Madhok and Walker, 1969; Proctor and WoodelL 1975), M g toxicity (Novak, 1928; Proctor, 1970), heavy metal toxicity (Antonovics et al., 1971; Brooks, 1987), or low levels of essential nutrients (Gordon and 3 Lipman, 1926; Proctor and WoodelL 1975; Brooks, 1987). The low nutrient status and cation imbalances, along with high temperature effects, moisture stress, soil instability, and biotic conditions, limit plant growth and survival in serpentine habitats (Tadros, 1957; Kruckeberg, 1984; Arianoutsou et al., 1993). Hence, the serpentine syndrome involves several factors ~ the interaction of stresses in chemical composition of soils, the physical features of the habitat, and the resulting biological consequences. A vast amount of research has been conducted world-wide on the ecology of serpentinized and other ultramafic areas as well as on various aspects of plant life on these extreme geologic conditions (Whittaker et al., 1954; Krause, 1958; Proctor and WoodelL 1975; Kruckeberg, 1984, 1992; Brooks, 1987; Roberts and Proctor, 1992). A n important aspect of plants growing in serpentine soils is their ability to tolerate toxic levels of heavy metals. Studies of metal tolerance by plants growing in such extreme geologic conditions have provided important information on the physiological ecology of plants. Plants growing in high-metal soils have both external and internal mechanisms to tolerate high metal concentrations. By avoiding excessive uptake of toxic ions, adopting excretory mechanisms, and developing storage devices, metal-tolerant plants are able to survive in soils toxic to most vegetation. Studies show that mechanisms of metal tolerance may be different in different species and for different metals. The picture so far suggests that cell wall binding, active storage in vacuoles, complexing by organic acids and metal-binding proteins, all play their part (Bradshaw et al., 1990). There is recent evidence that novel low-molecular weight compounds, phytochelatins, may be involved in metal tolerance (Salt et al., 1989). Plants exhibit several patterns of distribution on serpentine soils. Firstly, there are species that are strictly endemic to these soils. Serpentine endemism can be very local - a single outcrop - to regional and widespread occurrences. Species of the genus Streptanthus Nutt. 4 (Brassicaceae) in California illustrate each of these three levels of endemism: S. niger (Greene) Munz., S. batrachopus Morrison, and S. brachiatus Hoffman are striking examples of very local, narrow endemics found on only one or a few outcrops; S. hesperides Jeps. and S. polygaloides Gray represent the intermediate level where restriction to serpentine occurs over a broader regional area; S. glandulosus Hook., on the other hand, is widespread in occurrence on serpentine outcrops in California. Secondly, there are species whose main range of distribution is on nonserpentine substrates but which are clearly faithful to serpentine in areas beyond their main range. Two conifers, Pinus jeffreyi Grev. & Balf. and Calocedrus decurrens (Torr.), portray features of this pattern. Thirdly, there are species that are widespread on serpentine and nonserpentine soils but show regional prominence on serpentine. Here, it is not a matter of exceptional range extensions onto serpentine, but rather that these plants, come to assume a dominant or indicator status on serpentine. Pinus sabiniana Dougl. and Pinus attenuata Lemmon. typify this indicator role. Finally, some taxa occur on and off of serpentine repeatedly throughout their ranges. In any given locality, such taxa are found on adjacent nonserpentine substrates as well as on serpentine (Kruckeberg, 1984). Such plants are often categorized as indifferent/ubiquist (Krause, 1958) or bodenvag (Unger, 1836; Kruckeberg, 1969) species. Bodenvag species appear on both sides of an edaphic boundary, seemingly indifferent to substrate differences. A number of introduced annual grasses such as Bromus mollis L . , Avena fatua L . as well as composites such as Hypochoeris radicata L . and Lactuca serriola L . can be categorized as bodenvag species. 5 1.3. Speciation under edaphic influence "The red-rock forest may seem hellish to us, but it is a refuge to its flora....it is the obdurate physical (and chemical) adversity of things such as peridotite (ultramafic) bedrock which often drives life to its most surprising transformations." David Rains Wallace (1983) Patchiness or discontinuity of edaphic phenomena leads to biological discontinuity. The most significant causes of localized or unusual distribution patterns of plants are traceable to isolating effects of discontinuities in geology and edaphics ~ geoedaphics (Kruckeberg, 1986). Geoedaphic isolates may range from distinct genotypes within species, through detectable local populations or races, to uniquely edaphically endemic species or even higher categories. Plant populations can either have the potential to tolerate unusual edaphic situations (preadaptedness) or evolve tolerance to them Studies on heavy metal tolerance on mine areas (Antonovics et al., 1971) and ecotypic variation in tolerance to serpentine soils (Kruckeberg, 1951, 1967) provide evidence for both scenarios. Research has shown that some species or populations of certain species may have the genetic preadaptedness to venture successfully onto soils that are edaphically extreme: a few preadapted genotypes could become founders of a tolerant population. Populations that evolve tolerance to heavy metals on recently contaminated soils come closest to revealing natural selection in action, since their origins are so recent. Genetic accommodation to a heavy metal environment can take place quite rapidly, even within a few generations (Antonovics et al., 1971; Bradshaw and McNeilly, 1981; Liu and Godt, 1983; Shaw, 1990). Hence, edaphic conditions, when manifested in extreme form, can be potent agents of natural selection. 6 Several modes of origin of edaphic endemics have been proposed (Stebbins, 1942, 1980; Stebbins and Major, 1965; Proctor and Woodell, 1975; Raven and Axelrod, 1978; Kruckeberg, 1984). Biotype depletion, drift, catastrophic selection and saltational speciation, standard allopatric speciation with ecogeographic specialization, ecotypic differentiation, and hybridization with or without allopolyploidy are some modes of origin presented in explaining edaphic endemism Using serpentine soils as an example of a challenging edaphic situation, Kruckeberg (1984, 1986) describes a set of stages that may lead to the establishment of an edaphically endemic species (Figure 1.1). Firstly, there exists preadaptedness for serpentine tolerance in nonserpentine populations. Then, disruptive selection, catastrophic selection (Lewis, 1962; Raven, 1964), or gradual divergence effectively separates a species into serpentine-tolerant and -intolerant gene pools. Further, genetic divergence in structural and functional traits occurs within the serpentine-tolerant part of the effectively discontinuous populations. As a result, isolation between serpentine- tolerant and -intolerant segments of the species becomes genetically fixed and the two populations are unable to exchange genes. Further reinforcement of genetic-ecologic isolation leads to consequent divergence of the serpentine population. This sequence encompasses an evolutionary history from the initial compatibility between habitat and certain preadapted variants to clear-cut species formation. These stages can be appropriately applied to other forms of geoedaphic challenges ~ mine soils with heavy metals (Bradshaw et al., 1990) and guano (OrndufE 1965) ~ also leading to the formation of edaphically endemic taxa. The significance of geoedaphic phenomena in deterrnining plant distribution is apparent from studies done on plant life on extreme and unusual geologic situations. Close examination of models of speciation (Grant, 1981) shows that the edaphic factor can serve as the 7 environmental trigger for most every model of diversification and speciation. Hence, the edaphic factor, singly or in combination with climatic and biotic factors, is an important determinant of localized distribution patterns found in the world of plants. The study of plants growing on unusual geologies has provided much information on and insight into plant population biology and eco-physiology and served as a base for understanding many mysteries of evolutionary biology. 8 Acquisition of edaphic or site specific tolerance Distinct race by disruptive or catastrophic selection Distinct race by gradual divergence Self-perpetuating race Gene flow between adjacent populations reduced by intrinsic (genetic) and extrinsic (edaphic) factors = Incipient Isolation Isolation yields further divergence in morphological-physiological traits as well reproductive traits = Speciation Figure 1.1. A scheme for speciation under edaphic influence (Kruckeberg, 1986). 1.4. Lasthenia californica as a case study Lasthenia californica DC ex. Lindley (Asteraceae: Heliantheae) is the most widely distributed of the 17 species included in this mostly Californian genus. This obligatory outcrossing, spring annual ranges from south-central Oregon throughout California, from the foothills of the Sierra Nevada to the coast, east into Arizona and in northern Baja California. The distribution of the species may be limited by its preference for a Mediterranean-type climate, characterized by mild, wet winters and long, hot, dry, summers. Lasthenia californica shows a high degree of morphological, cytological (Ornduff, 1966), and biochemical diversity (Bohm et al., 1974, 1989; Desrochers and Bohm, 1993, 1995) and is probably the most variable taxon in the genus. Lasthenia californica has wide ecological tolerance ~ it occurs on coastal bluffs, in open grasslands, oak woodlands, alkali flats, chaparral, pastures along roadsides, serpentine outcrops, and in the desert. Serpentine outcrops possibly provide the most extreme growth conditions for this species. However, in these environments, L. californica achieves dominance and is often restricted to the serpentine side of the serpentine and nonserpentine boundary. Thus, L. californica fits the previously described third category (Kruckeberg, 1984) of plant distribution patterns on serpentine soils ~ species that are widespread on serpentine and nonserpentine soils but show regional prominence on serpentine. Morphological and chemical studies conducted on plants collected from the entire range of L. californica have demonstrated the existence of two genetically distinct geographical races based on pappus shape, allozymes, and flavonoid patterns (Desrochers and Bohm, 1993, 1995). One race is characterized by a linear pappus, flavonoid pattern A (consisting of glucoronides, anthochlors, sulfated diglycosides, and eriodictyol 7-glucosides), b allele at NADHdh, and 10 faster allozymes encoded by 6Pgd-l, while the other race is characterized by a lanceolate pappus, flavonoid pattern C (glucuronides, anthochlors) or B (glucuronides, anthochlors, and luteolin 7-glucosides), a allele at NADHdh, and slower allozymes encoded by 6Pgd-l (Desrochers and Bohm, 1995). Crossing experiments between the two geographical races indicate a low level of crossability (Desrochers, 1992). Observations in the field have also revealed differences in the flowering times of the two races (Desrochers and Bohm, 1995). Greenhouse experiments have indicated that the pattern of flavonoids seen within the populations of L. californica is controlled genetically. The flavonoid chemistry of an offspring almost always matches that of its female parent (Bohm et al., 1989). Flavonoid pattern does not seem to respond to edaphic conditions, since cultivation of plants from seed in greenhouse potting soils did not affect the flavonoid pattern expressed in the offspring. Variation in flavonoid patterns between and within populations of plants is a well documented phenomenon (Mears, 1980; Waser and Price, 1981; Levy, 1983; Bohm et al., 1984, 1987). However, no explanations have been provided for the variability seen in the flavonoid patterns in L. calif ornica. The existence of different flavonoid and floral pigmentation patterns in plants seems to be influenced by soil type (Horovitz, 1976; Mears, 1980; Menadue and Crowden, 1983; Heywood, 1986; Reid, 1995; Rosenthal and Human, 1997). It is uncertain, however, i f this reflects a direct cause and effect relationship or a mere correlation. Any potential relationships between the flavonoid pattern of L. californica and its soil type have not been examined. The two races of L. californica show a north-south distribution pattern within its range. The race with the flavonoid pattern C (Type-C) is found predominantly from southern Oregon to northern and north-central California while the race with flavonoid pattern A (Type-A) is 11 found predominantly in the southern parts of California (Desrochers, 1992). In the central parts of California, there exist several populations with both races. The largest of the mixed populations so far encountered occurs in the Jasper Ridge Biological Preserve of Stanford University, San Mateo County, in central California. The preserve is located west of Palo Alto, California, in the Santa Cruz Mountains. Approximate coordinates of the preserve are 37°25' north and 122°2.5' west. Here, both races of the species coexist in a population found on an extensive serpentine outcrop that runs in a west-northwesterly to east-southeasterly direction. This population lies at an elevation of approximately 180 meters. The climate at Jasper Ridge is Mediterranean, with a mean annual rainfall of 480 mm (Hobbs and Mooney, 1991). Lasthenia plants germinate after the first significant rains in the late fall and flower and set seed mostly by April or early May. The distribution pattern of the two races of this species has been studied for over ten years on this serpentine ridge. Ridge top was always populated by type-C plants, and ridge bottom, by type-A plants. Year after year, the two races have maintained distinct boundaries (Bohm et a l , 1989; Desrochers and Bohm 1993, 1995), with a sharply-defined transition zone which varies by no more than a meter, at most (Figure 1.2). This distinct pattern is the first case study of a sharp boundary between two races of an annual within serpentine soils. Previous studies all deal with boundaries between species or genetically distinct races of the same species on two geologically very different substrates ~ serpentine and nonserpentine (Kruckeberg, 1951, 1954, 1967, 1992; Proctor and WoodeH, 1975; Brooks, 1987; Mayer and Soltis, 1994) and calcareous and noncalcareous (Snaydon, 1962; Kruckeberg, 1969; Heywood, 1986). Another well-studied example of sharply demarcated boundaries in nature involve areas of heavy metal contamination (Antonovics et al., 1971; Bradshaw, 1972; Shaw, 1990). Here, 12 the vegetation boundary possibly exists on the same geologic substrate and is due to obvious contamination of one side of the boundary with heavy metals. Studies have shown that distinct populations, with different levels of metal tolerance, can be found within distances as small as a meter (Snaydon, 1963, 1970; Jain and Bradshaw, 1966; Antonovics, 1978; Snaydon and Davies, 1976; Al-Hiyaly et al., 1993) to several meters (McNeilly, 1968; Antonovics and Bradshaw, 1970; Watson, 1970). The distinct distribution pattern observed at Jasper Ridge is yet another example of distinct populations capable of maintaining sharply defined boundaries over a period of years. The microhabitats occupied by these two races have not been exarnined to determine if there are any edaphic features involved in iraintaining this distinct spatial distribution. The objective of the thesis was to determine if edaphic attributes contribute to the pattern of distribution observed at Jasper Ridge and to determine i f edaphic factors are important in the distribution of the two races throughout their range. A close examination of edaphic characteristics at Jasper Ridge and other populations, along with greenhouse studies, will reveal if edaphic factors may have contributed to racial differentiation in this species. 13 Figure 1.2. A photograph of the study site at Jasper Ridge Biological Preserve indicating the distinct boundary between type-C and type-A plants (Photograph by Bruce A. Bohm). Chapter Two Soil and Plant Tissue Analyses 2.1. Introduction In this chapter, attempts to reconfirm the distinct distribution pattern of the two races observed for over 10 years at Jasper Ridge are described. Then edaphic attributes of the microhabitats occupied by the two races are described in order to determine i f edaphic factors vary across the type-C and - A plant boundary. Total plant tissue elemental composition was examined to determine i f plants of the two races were responding differentially to any differences in the edaphic features. This close examination will be essential in understanding if edaphic factors play any role in the distribution pattern observed at Jasper Ridge. Regression analyses between soil and plant tissue concentrations of various elements were conducted with a view to identifying possible physiological differences that may exist between the two races. In addition to the detailed study at Jasper Ridge, edaphic and plant tissue characteristics were examined for samples collected from 22 populations, representing the entire range of the species. 15 2.2. Materials and Methods 2.2.1. Field Collections In April 1996, plant and soil samples were collected along five line transects on the serpentine outcrop at Jasper Ridge Biological Preserve; transects 2, 3, and 5 correspond to transects I, n , IV-90 in Desrochers and Bohm (1993). The length of these transects ranged from 44 - 65 meters. A Total of 123 plant and soil samples were collected at two meter (2 m) intervals along all of the transects. In April 1997, plant samples were collected at 2 m intervals along a new transect (transect 6), a 50 meter transect running along the oak-grassland boundary from the bottom of transect 2 to the bottom of transect 5 (Figure 2.1). 16 \ N Ridge Top T R A N S E C T T R A N S E C T T R A N S E C T T R A N S E C T T R A N S E C T 6 T R A N S E C T Fire Road Oak-Grassland Boundary Figure 2.1. A diagram showing the arrangement of transects (1996/1997). 10 m 17 2.2.1.1. Plants Four to 10 plants were collected at each 2 m interval along all transects. Plants were cleaned of adhering soil/dust and then placed separately in paper bags for later determination of elemental composition of plant tissue. Several flower heads from each individual plant were collected to obtain achenes as well as to analyze the flavonoid pattern. Flower heads for analysis of flavonoids were placed in small vials while those for obtaining achenes were placed in small envelopes. 2.2.1.2. Soils Approximately 300 g of soil from the rooting zone at each of the 2 m intervals was dug and placed in plastic bags. A l l soil samples were collected at 0 - 10 cm depth by using a plastic hand trowel. (A stainless steel trowel was not used primarily to avoid the possible contamination of soil samples with Cr or Ni). At several points on each transect, soil samples were collected in air-tight soil cans for determining the relative water content in soil. Topographies along transects were examined by use of a clinometer to gain a better understanding of the elevational gradients along which the plants were distributed. Figure 2.2, based on the slope information gathered in April 1996, shows a topographical map of the study site. In addition to the detailed survey at Jasper Ridge, plant and soil samples were collected from 22 populations of L. californica throughout its range (Desrochers, 1992). Three samples were randomly collected from each population. Each sample consisted of 4 -10 plants and about 300 g of soil from the rooting zones of those plants. Table 2.1 and Figure 2.3 document the populations from which samples were collected in April, 1996. 18 Table 2.1. Population number and locality information for samples collected from populations ranging from southern Oregon to southern California. Sample ID Population Location Information Number O R - 1 1 Near Gold Hill, Jackson County, Oregon O R - 2 2 Across from Water Treatment Plant, Jackson County, Oregon OR - 3 3 Across Road from Pop. 2, Jackson County, Oregon O R - 4 4 Trail Head of Table Rock, Jackson County, Oregon OR - 5 5 Far Trail of Table Rock, Jackson County, Oregon C A - 1 6 Near Red Bluff, Tehama County, California C A - 2 7 Near Paskenta, Tehama County, California C A - 3 8 East of Paskenta Bridge, Tehama County, California (Serpentine Site) C L A Y - 1 9 Clay, Sacramento County, California C L A Y - 2 10 Clay, Sacramento County, California RS 11 Rattle Snake Rock, Jasper Ridge Bio. Preserve, San Mateo (Serpentine Site) County, California C A - D 1 12 Route 104, Sacramento County, California C A - D 2 13 Route 104, Sacramento County, California C A - C2 14 Near Priest Valley, Monterey County, California C A - C 1 15 Near Priest Valley, Monterey County, California C A - B 1 16 Palmer Ranch, Priest Valley, Monterey County, California C A - B 2 17 Palmer Ranch, Priest Valley, Monterey County, California C O A 1 18 Coalinga, Fresno County, California C O A 2 19 Coalinga, Fresno County, California A S E C C O 20 Arroyo Secco, San Luis Obispo County, California T E H A 1 21 Tehachapi, Kern County, California T E H A 2 22 Tehachapi, Kern County, California 20 Figure 2.3. Map showing the geographical distribution of all populations surveyed during 1996. Population 11 indicates location of Jasper Ridge Biological Preserve. Here, Lasthenia grows on the primary study site as well as on Rattle Snake Rock, another serpentine outcrop. 21 2.2.2. Laboratory Studies 2.2.2.1. Flavonoid Pattern of Plants Flavonoid patterns of the plants were examined to confirm the previously observed distribution pattern of the two geographical races on the serpentine outcrop at Jasper Ridge. Flower heads from four plants were separately tested to determine flavonoid pattern at each 2 m interval along all transects. Approximately 4 - 6 plants per population were also used to characterize plants from each of the additional populations. A few drops of MeOH (methanol) were added to vials that contained flower heads which were then allowed to stand at room temperature for one day. This was sufficient time to complete the extraction of the floral flavonoids. Samples for chromatography were taken by means of capillary spotting tubes and spotted on 20 x 20 cm home-made Polyamid 6.6 plates. Approximately 15 samples were spotted on each plate which was then developed one-dimensionally in a mixture of water-n-butanol-acetone-dioxane (70:15:10:5). After drying, the plates were sprayed with aminoethyl-diphenylborate, again allowed to dry in the ah, and examined under 366 tun U V to determine the flavonoid pattern. 2.2.2.2. Achenes Achenes were stored in a warm, dark place for approximately three months (for after-ripening of achenes) and then transferred to a laboratory refrigerator (5-6° C). To corifirm an observed difference between the two races in achene size and shape, achenes from the Jasper Ridge population were examined to determine i f achene length, width, and weight were different for the two races of this population. 22 2.2.2.3. Whole Plants Plants from each sample were shaken to free the plants of adhering soil. The samples were then dried for 24 hours at 80° C in a forced draft oven. Each sample was then ground using a coffee grinder {Sunbeam Cafe'Mill Model C G 150). Prior to grinding actual samples, trials were conducted to determine if the stainless steel coffee grinder would introduce any metals to the sample. Metal content was detennined for Pine needle samples (collected from U.B.C campus) ground using the grinder as well as in liquid Nitrogen. I consistently observed approximately 1% increase in Cr in Pine needle tissue ground using the coffee grinder. Nevertheless, due to the efficiency of the coffee grinder, I decided to retain this method to grind the Lasthenia samples, keeping in mind that the reading for Cr would be approximately 1% higher than the actual concentration. Plants collected at each 2 m interval, were analyzed to determine the total concentrations of Ca, Mg , Na, K, AL Fe, N i , Cr, Mn, and Zn in the plant tissue. Elements were determined by the modified dry ashing method using 1M HNO3 and 2 M HC1 (UBC Soil Science Laboratory Manual, 1981). 2.2.2.4. Soil A l l soil samples were placed in plastic trays and air-dried at room temperature for approximately one week. Each sample was then sieved in a fumehood through a home-made wooden sieve with a nylon (stainless steel was not used to avoid the possible contamination of the sample with Cr/Ni) mesh of 2 mm to obtain the less than 2 mm soil particles for analysis. Gravel was removed from the remaining aggregates of soils. The remaining aggregates were then placed on a sheet of brown paper and crushed using a wooden rolling pin. The soil was 23 again passed through the sieve, and the entire process repeated until all soil aggregates were crushed. Cleaning, crashing, and sieving were all conducted in a fumehood. A SurvivAir air-purifying respirator was worn at all times to avoid the possible harmful effects of asbestos (chrysotile) dust, a commonly found mineral in serpentine soils. Processed soil from each sample was then placed in plastic bags and stored for future analysis. Moisture content (relative water content), soil color, texture class, pH, and cation exchange capacity of the soil samples were evaluated. Exchangeable Na, K , Ca, and M g as well as extractable Fe, N i , Cr, Mn, Co, Cu, Zn, and Cd were measured. Total percent N and exchangeable A l were measured for a few randomly collected soil samples from the serpentine outcrop at Jasper Ridge. The relative water content (RWC) in soil was determined by the Moisture Can Method (Black et a l , 1965; U B C Soil Science Laboratory Manual, 1981). Soil samples, which were collected in small sealed metal cans, were weighed immediately to determine wet weight. Relative water content per 100 g of dry soil was then determined by oven-drying soil at 105° C for 16 hours. Soil color was determined for soils characteristic of type-A and -C plants at Jasper Ridge using Munsell Soil Color Index (US Department of Agriculture Handbook 18). Six soil samples from each plant type were exposed to the color test under both dry and wet conditions. Soil texture was determined by particle size analysis using the Hydrometer Method and the computer program M E T L A B (UBC Soil Science Laboratory Manual, 1981). p H was determined by the Dixie Cup Method using both water and 0.01 M CaCl 2 solutions (Black et al., 1965; U B C Soil Science Laboratory Manual, 1981). Ten grams of soil were mixed separately with 20 ml of water and 20 ml of 0.01 M CaCl 2 solutions (1:2). The 24 suspensions were stirred several times for half hour and allowed to stand for one hour. Once the suspended soil particles had settled, p H was measured using an ORION Model 420A pH meter. Cation exchange capacity (CEC) and exchangeable cations (Na +, K + , Ca 2 + , Mg 2 + ) were determined by the Ammonium Acetate Method at p H 7.0 using 1M N H 4 O A C and 1M KC1 (Black etal., 1965; U B C Soil Science Laboratory Manual, 1981). Exchangeable A l was determined by 1M KC1 extraction (Page et al., 1982). Total N was determined by the Kjeldhal Method (UBC Soil Science Laboratory Manual, 1981). Concentrations of exchangeable cations were then determined by Atomic Absorption Spectroscopy using a Perkin Elmer Model 306 Spectrophotometer. Total CEC and total N were determined using a LACHAT Quick Chem Automated Ion Analyzer (Model 2300-000). Micronutrients and metals (Mn, Fe, Zn, Cu, N i , Cr, Co, Cd) were extracted with dimethylenetriarninepentaacetic acid in the DTPA soil test using 0.005M DTPA, 0.01M CaCl 2 , and 0.1M T E A adjusted to p H 7.3 with dilute HC1 acid (Lindsay and NorvelL 1978). Concentrations of extracted elements were determined by Inductively Coupled Plasmaspectroscopy (ICP), using a Thermo Jarrell Ash ICAP 61 Spectrometer. A l l soil and plant tissue analyses were conducted at the Department of Soil Science, U.B.C. 2.2.2.5. Statistical Analyses The soil and plant data sets were tested for normality and homogeneity of variance by using Shapiro Wilks' Test and the Levene Test respectively. Once the assumptions of both normal distribution and equal variances were found to be applicable to the data sets, one-way analysis of variance (ANOVA) was used to determine whether means of soil and plant tissue 25 variables differed for type-A and -C plants. Regression Analyses were used to examine functional relationships between soil concentrations of various elements and those element concentrations in plants. Discriminant function analyses (DFA) and principal component analyses (PCA) were conducted to determine i f soil and plant tissue characteristics were reliable in predicting the race of plants. The obvious advantage of these two multivariate tests over one-way A N O V A and regression analysis is that they permit an integrated assessment of variation in the system tested in which due regards is given to covariation between variables (Rayment et a l , 1984). Principal component analysis is used for the analysis of the structure of multivariate observations, and in particular investigating the dependence structure occurring in a suite of observations when no a priori patterns of interrelationships can be suggested or are suspected. P C A allows one to reduce an original set of variables into a smaller set of uncorrelated variables while accounting for as much of the variation as possible in the original set of variables (factors are determined so as to account for maximum variance of all the observed variables). Thus, P C A is variance-oriented. On the other hand, D F A is designed to maximize the separation between two (or more) populations. The ideas underlying this analysis can be discussed in terms of two universes. Samples are assumed to be available from each of the two universes and a linear discriminant function is constructed on the basis of these samples. A new sample, on which the same measurements are available, can then be assigned to one of the universes. Thus, D F A is correlation-oriented. A l l tests were conducted using statistical packages SPSS (Norusis, 1993) and SYSTAT: Statistics Version 5.2 (Wlikinson, 1992) on an I B M Compatible PC at the Department of Botany, U.B.C. 26 2.3. Results 2.3.1. Flavonoid Data of Jasper Ridge The patterns of flavonoid data determined for plants collected along each transect at Jasper Ridge confirmed those of previous years (Bohm et al., 1989; Desrochers, 1992; Desrochers and Bohm, 1993, 1995). The plants along the oak-grassland boundary were consistently type-A, while those of the upper portion of the ridge were almost always type-C (Figure 2.4). Collections along transect 6 confirmed this observation, since no type-C individuals were found along the type-A dominated oak-grassland boundary. 27 • Transect 1 cccccccccccccccccccccccccc 0 10 20 30 40 50 Meters • Transect 2 C C C C / B C C C C C C C C C C C / A A A C / B C / B C C C A A A A A A A A 0 10 20 30 40 50 60 Meters • Transect 3 C C C / B C C C C C C C C C C C C C C C C C C C C C C A A A A A A A A A 0 10 20 30 40 50 60 Meters • Transect 4 C C C C A A 0 10 20 30 40 50 60 Meters • Transect 5 C C C C C C C C C / B C C C / B C C C C C C A C C C C C C A A A A A A A A A 0 10 20 30 40 50 60 Meters • Transect 6 A A A A A A A A A A A A A A A A A A A A A A A A A A A 0 10 20 30 40 50 Meters Figure 2.4. Flavonoid pattern of plants collected along 6 transects at Jasper Ridge. Distance 0 is at Fire Road. Four plants were tested for flavonoid pattern at each 2 meter interval. At some locations more than one type was present (i.e. C / B ) . 28 2.3.2. Flavonoid Data of Other Populations In addition to those of Jasper Ridge, the flavonoid patterns observed by Desrochers (1992) for several populations from the range of L. californica were confirmed. In general, the data agree with previous observations — type-C was found predominantly from southern Oregon to northern and north-central California while type-A was found predominantly in southern parts of California (Table 2.2). 29 Table 2.2. Flavonoid pattern of plants collected from 22 populations representing the entire range of the species. Old population number and flavonoid pattern (Desrochers, 1992) listed where applicable. Sample 11) Population Number Type Population Number (Desrochers, 1992) Type (Desrochers, 1992) OR - 1 1 C 453 C OR - 2 2 C 453 C OR - 3 3 C 453 C OR-4 4 C 454 C OR - 5 5 C 454 C C A - 1 6 C 425 C C A - 2 7 A 428 CA - 3 8 A (Serpentine Site) C L A Y - 1 9 C 445 C/A CLAY - 2 10 C 445 C/A RS 11 A (Serpentine Site) CA-D1 12 A CA-D2 13 A C A - C 2 14 A 325 A C A - C l 15 A 325 A CA-B1 16 C CA-B2 17 A COA1 18 A 464 A COA2 19 A 464 A A.SECCO 20 A TEHA 1 21 A 328/432 A TEHA2 22 A 328/432 A 30 2.3.3. Achene Differences - Jasper Ridge Achene characteristics revealed a morphological difference between the two races. Type-A plants had lanceolate (wedge-shaped) and heavier achenes while type-C plants had linear and lighter achenes ~ type-A achenes were significantly shorter, wider, and heavier than achenes of type-C plants (Figure 2.5; Table 2.3). This difference adds to the already known array of morphological and biochemical differences between the two races. 31 Figure 2.5. Achene differences between type-A and type-C plants of Jasper Ridge. Table 2.3. Mean length, width, and weight of achenes of type-A and -C plants collected from Jasper Ridge. Achene Character Type-A Type-C F Probability Value (One-Way-Anaysis of Variance) Length (mm) N = 127 1.84 (SE ± 0.012) 2.16 (SE + 0.018) P < 0.0001 Width (mm) N = 5 0 0.41 (SE ± 0.004) 0.31 (SE ± 0.004) P < 0.0001 Weight of 20 achenes (mg) 1.78 (SE ± 0.03) 1.34 (SE + 0.03) P < 0.0001 N = 25 33 2.3.4. Soils and Plants of Jasper Ridge One-way A N O V A s conducted on 123 soil and plant samples (93 type-C; 30 type-A) revealed several significant differences in the means of soil and plant tissue characteristics of type-A and -C plants (Tables 2.4 and 2.5). 2.3.4.1. Soils A color photograph of soils collected from the two extreme ends of transect 3 clearly indicates the difference in the physical appearance of the soils on which type-C and - A plants grow (Figure 2.6). Percent clay content, relative water content (RWC), cation exchange capacity (CEC), pFL and exchangeable M g and Na were significantly higher (P < 0.0001) in ridge bottom soil (type-A soil) than in ridge top soil (type-C soil). Type-C soil had significantly higher means for exchangeable Ca and K , extractable Ni , and Ca/Mg ratios than type-A soils (P < 0.01). A detailed examination of soil characteristics along transects provides important information on the mobility and distribution of elements on the serpentine outcrop. In general, there was a tendency for pH, CEC, percent clay, RWC, exchangeable M g and Na, and extractable Cr to increase along transects from ridge top to bottom (Figure 2.7). By contrast, Ca, K, N i , as well as Ca/Mg ratios were relatively higher at the top of the ridge. These soil features showed a tendency to decrease as the distance increased from ridge top to bottom along each transect (Figure 2.8). The other tested cations (Zn, Fe, Mn, Co, Cu) showed no apparent trend along transects. 34 Ridge Top Soil Ridge Bottom Soil Figure 2.6. The difference in the physical appearance of ridge top and bottom soils. 35 Table 2.4. Soil characteristics for Jasper Ridge. Concentration of elements given in ppm (p.g/g soil). Standard errors within parentheses. Higher mean value in bold font. Soil Variable Plant Type Mean F Prob. Value Range (min. - max.) Soil Color C Dark Brown A V. Dark Greyish Brown RWC/lOOgSoil C 25.17 (± 0.81) P < 0.0001 17.62- 32.53 A 38.83 (±2.92) 26.57 - 49.63 CEC CMol(+)/kg Sou C 43.99 (±0.52) P < 0.0001 30.14- 55.64 A 51.47 (±1.64) 32.48- 71.96 Percent Clay C 32.08 (±1.01) P < 0.0001 21.32- 38.39 A 40.3 (±1.41) 33.51 -45.83 pH in H z O (1:2) C 6.63 (± 0.02) P < 0.0001 6.16-6.99 A 6.85 (± 0.03) 6.53-7.14 pHinCaCl 2(l:2) C 5.85 (±0.02) P < 0.0001 5.47-6.21 A 6.16 (± 0.04) 5.84 - 6.6 Ca C 610.74 (±13.28) P < 0.0001 382.5 - 982.5 A 507.75 (± 20.38) 252.5 - 807.5 Ca/Mg C 0.25 (±0.01) P < 0.0001 0.15 - 0.49 A 0.17 (±0.01) 0.06-0.36 K C 164.97 (±6.71) P < 0.0065 65.0- 547.5 A 130.5 (±6.74) 47.5 - 207.5 Mg C 2485.22 (±46.52) P < 0.0001 1300.0 - 3950.0 A 3316.67 (± 142.72) 1750.0 - 4600.0 Na C 23.34 (±0.49) P < 0.0001 16.25 - 53.0 A 32.02 (±1.71) 18.75 - 52.5 36 Table 2.4. (cont'd.) Soil Variable Plant Type Mean F Prob. Value Range (min. -max.) Total N % C 0.182 (± 0.001) P>0.05 (4 Samples Each) A 0.162 (+0.001) Ni C 127.96 (± 2.7) P < 0.0001 34.71 - 190.79 A 104.87 (±5.16) 56.96 - 186.74 Al C 0.25 (±0.12) P>0.05 (4 Samples Each) A 0.47 (± 0.22) Zn C 3.36 (± 0.2) P>0.05 1.18-4.76 A 4.1 (± 0.45) 1.05 - 9.94 Fe C 71.93 (±1.98) P > 0.05 43.51 - 127.01 A 76.2 (±5.05) 46.57- 158.89 Mn C 34.22 (±1.02) P>0.05 5.02 - 55.67 A 33.52 (±2.21) 14.32 - 70.69 Cr C 0.06 (±0.00) P > 0.05 0.02-0.124 A 0.12 (±0.05) 0.03 - 1.66 Co C 1.55 (±0.07) P>0.05 0.21-4.59 A 1.3 (±0.13) 0.19-3.86 Cu C 1.61 (±0.04) P>0.05 0.22 - 2.34 A 1.77 (±0.08) 0.9-3.14 Cd C Below Detection A 37 CO 0 10 20X40S06070 5000 4000 2000 1000 0 10 203043906070 Plant Type •Type-A A Type-C Total Population Distance (m) Figure 2.7. Variation in soil characteristics along Transect 3 at Jasper Ridge. Distance 0 is at fire road and distance 70 is at the oak-grassland boundary. 38 0 10 20 3D 40 50 63 70 Plant Type ATyp*C •Type* Total Population •10 0 10 20 30 40 50 60 70 Ui O 900 0 10 20 30 40 50 60 70 Plant Type AType-C •Type-A Total Population Distance (m) Figure 2.8. Variation in soil characteristics along Transect 3 at Jasper Ridge. Distance 0 is at fire road and distance 70 is at the oak-grassland boundary. 2.3.4.2. Plants There are several differences in total plant tissue elemental composition between the two races (Table 2.5). Means for total Mg, Na, AL and Zn were significantly greater in plant tissue of type-A plants (P < 0.01), while means for total Ca, K , and Ca/Mg ratio were significantly greater in plant tissue of type-C plants (P < 0.02). Plant tissue elemental concentrations were often closely related to and a good indication of those concentrations in soils. In general, total Mg , Na, A l , and Zn increased in plants collected along transects from mid-ridge to ridge bottom (Figure 2.9). Total Ca, K , Ca/Mg ratio, and N i decreased in plants collected along transects from mid-ridge to ridge bottom (Figure 2.10). Cations such as Fe, Mn, Cr, and Cu showed no obvious patterns in their distribution on the outcrop. 40 Table 2.5. Plant tissue characteristics for samples collected at Jasper Ridge. Concentration of elements given in ppm (ug/g plant tissue). Standard errors within parentheses. Higher mean value in bold font. Plant Tissue Variable Plant Type Mean F Prob. Value Range (min. - max.) Ca C 5445.3 (± 90.54) P < 0.0135 3811.69- 7934.59 A 4965.82 (+186.28) 3538.96- 7819.67 Ca/Mg C 0.83 (±0.02) P < 0.0001 0.37- 1.28 A 0.6 (±0.04) 0.28- 1.14 K C 15057.36 (±247.95) P < 0.0157 6750.0 - 20683.23 A 13636.84 (±675.21) 4090.91 - 19866.67 Mg C 6778.35 (± 121.23) P < 0.0001 4431.51 - 11292.33 A 8887.06 (± 332.69) 5863.24 - 12576.97 Na C 672.43 (±33.13) P < 0.0001 98.36 - 1970.59 A 2303.95 (± 232.02) 619.12- 5268.29 Ni C 86.6 (±4.27) P < 0.0001 28.46 - 243.43 A 83.9 (±8.45) 20.98 - 198.93 A l C 111.0 (± 6.06) P>0.05 29.75 -400.13 (4 Samples Each) A 179.25 (±21.64) 52.0-491.6 Zn C 62.69 (± 0.75) P>0.05 48.03 - 80.89 A 67.02 (±1.98) 47.09 - 97.9 Fe C 1807.54 (±109.81) P>0.05 357.31 - 6049.74 A 1950.74 (±236.94) 530.19-4984.06 Mn C 81.38 (±2.78) P > 0.05 29.12- 182.37 A 75.65 (±6.43) 34.14- 183.87 Cr C 18.19 (±119) P>0.05 4.05 - 69.04 A 21.48 (± 2.92) 3.97-61.54 Cu C 9.5 (±0.35) P>0.05 1.87- 22.77 A 7.82 (±1.17) 0.0 - 36.94 41 5D00 4XD 3000 1C0D Plant Type * Type-C • Type-A Total Population 503 400 300 c N 0 10 203040508)70 Plant Type ATyp»C •Type-A Total Population 0 10 20 30 40 50 60 70 Distance(m) Figure 2.9. Variation in plant tissue characteristics along Transect 3 at Jasper Ridge. Distance 0 is at fire road and distance 70 is at the oak-grassland boundary. 42 0 10 20 30 40 50 60 70 0 10203040506070 200 180 160 140 120 100 80 60 40 0 10 20 30 40 50 60 70 Plant Type AType-C •Type-A Total Population Distance(m) Figure 2.10. Variation in plant tissue characteristics along Transect 3 at Jasper Ridge. Distance 0 is at fire road and distance 70 is at the oak-grassland boundary. 43 2.3.4.3. Relationships between plant tissue and soil concentrations for Jasper Ridge Both plant races showed significant correlations between soil and plant tissue concentrations of Ca, Na, and Ca/Mg ratios (Table 2.6). For type-A plants the correlations for Ca, Ca/Mg, and Na are much higher than those of type-C plants. Certain elements showed significant correlations in one race but not in the other. For example, M g is significantly correlated for type-A plants while non-significant for type-C plants, whereas K is significantly correlated for type-C plants while non-significant for type-A plants. For heavy metals, the correlations were non-significant for both races. Figure 2.11 indicates the linear relationship between soil and plant tissue concentrations for M g and Na. 44 Table 2.6. Significance of regression analyses between soil and plant tissue concentrations of various elements for type-A and -C plants, according to Power Model (y = ci x c 2); or Linear Model * (y = mx + c). Significant correlations in bold. Element Plant Type r Value F Prob. Value Ca C 0.32 P < 0.002 A 0.45 P < 0.027 M g C 0.005 P>0.05 A 0.49 P < 0.01 Ca/Mg C 0.44 P < 0.0001 A 0.47 P < 0.01 Na C 0.253 P < 0.015 A 0.735 P < 0.0001 K C 0.229 P < 0.03 A 0.186 P>0.05 Fe C 0.119 P>0.05 A 0.005 33 C u * C 0.008 P>0.05 A 0.077 55 M n C 0.031 P>0.05 A 0.155 33 Cr * C 0.065 P > 0.05 A 0.207 N i C 0.014 P>0.05 A 0.078 55 Zn C 0.08 P>0.05 A 0.076 33 45 Plant Type/ r Value • Type-C: r = 0.384* A Type-A r = 0.683* 10 20 30 40 50 60 Na (ppm in Soil) AType: y= 92.52 X+658.23 C Type: y=26.05 X+64.21 14000 1000 2000 3000 4000 5000 Mg (ppm in Soil) AType: y = 1.014 x+6524.55 C Type: y=- 0.044 x+ 6888.32 Figure 2.11. Linear relationship between soil and plant tissue concentrations of Na and Mg for type-A and -C plants of Jasper Ridge. Significant (P < 0.05) correlations are indicated by * after r value. 46 2.3.5. Soils and plants of other populations One-way A N O V A s were conducted on 66 soil and plant samples collected from the other 22 populations. Results of soil and plant tissue analysis from these additional populations - 8 type-C, 14 type-A - agreed with most other patterns observed at Jasper Ridge. 2.3.5.1. Soils Soil p H and exchangeable M g were still significantly greater (P < 0.02) in type-A soils than in type-C soils. Percent clay content, CEC, and exchangeable Na were also greater for type-A soils, although the means were not significantly different (P > 0.05). Type-C soils, similar to those in Jasper Ridge, had a higher mean for Ca/Mg ratio (P> 0.05). Several differences were found between the patterns seen at Jasper Ridge and those at these other sites. For example, exchangeable K was significantly higher in type-A soils in contrast to the pattern observed at Jasper Ridge. Likewise, exchangeable Ca and N i were also higher for type-A soils, although the means were not significantly different (P> 0.05). Table 2.7 documents the means of all soil characteristics for samples collected from the 22 populations. 2.3.5.2 Plants Results of plant tissue analysis from 22 additional populations (Table 2.8) strongly agreed with most other patterns observed at Jasper Ridge, although none of the means found to be significantly different for the two races at Jasper Ridge were significantly different at P < 0.05 at these additional sites. However, the significantly greater means of AL Fe, and M n found in the plant tissue of type-C plants are noteworthy. 47 Table 2.7. Soil characteristics for samples collected from 22 populations representing the entire range of the species. Concentration of elements given in ppm (ug/g soil). Standard errors within parentheses. Higher mean value in bold font. Soil Variable Plant Mean F Prob. Value Range Type (min. -max.) CEC CMol(+)/kg SoU C 21.05 (±2.4) P > 0.05 11.11 - 29.65 A 29.46 (±3.48) 9.18-52.53 Percent Clay C 16.12 (±1.96) P>0.05 5.18-23.27 A 24.02 (±2.83) 7.42-41.32 p H i n H 2 0 (1:2) C 5.64 (±0.16) P < 0.041 4.81 - 6.16 A 6.42(±0.15) 5.26-7.12 pHinCaCl 2(l:2) C 4.63 (±0.13) P < 0.0008 4.17-5.29 A 5.48 (±0.15) 4.51-6.17 Ca C 1275.31 (± 290.61) P>0.05 202.5 - 2750.0 A 1233.93 (± 185.39) 442.5 - 2850.0 Ca/Mg C 5.23 (±0.56) P > 0.05 2.62-8.1 A 3.97 (±1.39) 0.24 - 17.0 K C 175.0 (± 18.29) P < 0.0116 112.5 -260.0 A 385.96 (± 56.63) 170.0 - 757.5 Mg C 280.63 (±78.42) P < 0.0237 25.0- 650.0 A 1146.96 (± 260.99) 52.0 - 2800.0 Na C 19.88 (±3.58) P > 0.05 11.25-42.0 A 60.82 (± 39.59) 1.75 - 552.5 Ni C 0.69 (± 0.23) P>0.05 0.158-2.17 A 16.17 (± 7.56) 0.066 - 102.03 Zn C 2.49 (± 0.55) P>0.05 0.79-5.8 A 3.43 (±1.33) 0.42 - 17.79 48 Table 2.7. (cont'd.) Soil Variable Plant Type Mean F Prob. Value Range (min. -max.) Fe C 90.08 (+ 9.5) P > 0.05 5.57 - 120.47 A 71.28 (+13.59) 4.88 - 172.22 Mn C 45.78 (± 7.53) P>0.05 12.7-77.05 A 36.17 (±3.99) 16.54- 68.72 Cr C 0.002 (±0.001) P > 0.05 0 - 0.0084 A 0.015 (± 0.0057) 0-0.0716 Co C 0.1001 (±0.0422) P>0.05 0 - 1.26 A 0.2841 (±0.1033) 0-0.261 Cu C 2.09 (±0.25) P > 0.05 0.89 - 3.42 A 2.11 (±0.34) 0.33 - 4.83 Cd C 0.03 (±0.004) P>0.05 0.015 - 0.052 A 0.067 (±0.014) 0 - 0.214 49 Table 2.8. Plant tissue characteristics for samples collected from 22 populations representing the entire range of the species. Concentration of elements given in ppm (ug/g soil). Standard errors within parentheses. Higher mean value in bold font. Plant Tissue Variable Plant Type Mean F Prob. Value Range (min. - max.) Ca C 12473.27 (+ 526.7) P>0.05 1389.86 - 15887.86 A 10843.91 (±1146.97) 5370.93 - 17601.64 Ca/Mg C 4.62 (±0.19) P>0.05 3.8-5.37 A 3.86 (±0.73) 0.7-8.03 K C 20424.48 (± 1098.12) P>0.05 14642.26 - 24642.9 A 17958.98 (± 1627.82) 226.63 - 29563.64 Mg C 2714.56 (±107.74) P > 0.05 2337.55 - 3257.77 A 3707.27 (± 267.49) 2018.81 - 7651.89 Na C 357.93 (±21.92) P>0.05 269.23 - 460.83 A 547.46 (± 232.02) 120- 3831.85 Ni C 5.72 (±1.02) P>0.05 3.18- 12.39 A 11.38 (±3.76) 1.66- 56.56 A l C 2425.99 (±396.64) P < 0.0001 73.37- 2116.6 A 584.68 (±133.88) 526.55 - 3550.87 Zn C 71.77 (± 9.34) P>0.05 40.62- 112.47 A 61.09 (±3.53) 35.4- 83.7 Fe C 1828.89 (±333.47) P < 0.0107 56.99- 2991.88 A 876.37 (±172.59) 27.97- 2843.17 Mn C 282.91 (±49.11) P < 0.0001 117.72- 555.95 A 74.08 (± 10.39) 28.85 - 157.2 Cr C 8.18 (±1.66) P>0.05 2.15-21.16 A 7.05 (±2.14) 0.92-21.67 Cu C 11.06 (±0.48) P < 0.0334 9.35- 13.35 A 9.34 (±0.5) 6.71 - 14.15 50 2.3.5.3. Relationships between plant tissue and soil concentrations for other populations Correlation analyses were computed to determine any relationships between soil and plant tissue concentration of various elements. Results agreed with most patterns observed at Jasper Ridge. Table 2.9 documents results of regression analysis for type-A and -C plants from the other populations. Table 2.9. Significance of regression analyses between means of soil and plant tissue concentrations of various elements for type-A plants, according to Power Model (y = ciX c 2 ); or Linear Model * (y = mi + c). Significant correlations in bold. Flpment Plant F Prob. Vfll"? C a * C 0.36 P>0.05 A 0.78 P < 0.024 M g C 0.31 P>0.05 A 0.81 P < 0.0004 Ca/Mg c 0.91 P < 0.0001 A 0.94 P < 0.0001 Na c 0.23 P>0.05 A 0.87 P < 0.0001 K C 0.35 P>0.05 A 0.23 P>0.05 Fe C 0.22 P>0.05 A 0.13 Cu C 0.008 P>0.05 A 0.077 M n C 0.53 P>0.05 A 0.49 C r * C 0.69 P = 0.058 A 0.17 P>0.05 N i * C 0.96 P < 0.0001 A 0.92 P < 0.0001 Zn C 0.16 P > 0.05 A 0.57 P < 0.04 51 2.3.6. Discriminant Function Analysis - soils and plants of Jasper Ridge Discriminant function analyses indicated that both soil and plant tissue characteristics are highly reliable in predicting the two races of L. californica. Analysis of soil samples indicated significantly different (P < 0.0001) group means (centroids) for type-A and -C soils, indicating that soil characteristics are highly reliable in predicting the plant race. The soil variables were correlated with the (hscriminant function in the following order: p H (rvalue = 0.62), M g (0.51), Na (0.47), Ca/Mg ratio (0.44), N i (0.29), Ca (0.28), K (0.2), Cu (0.14), Cr (0.13), Zn (0.12), Co (0.11), Fe (0.07), and M n (0.02). Table 2.10 provides a summary of the classification results of cuscriminant function analysis for soils of Jasper Ridge. Table 2.10. Classification results from Discriminant Function Analysis for soil samples of Jasper Ridge. Average percent of "grouped" cases correctly classified: 92.7 %. Actual Group Number of Cases Predicted Group Membership Type-A Type-C Type-A 30 24 (80%) 6 (20%) Type-C 93 3 (3.2%) 90 (96.8%) 52 Analysis of plant tissue samples indicated significantly different (P < 0.0001) group means (centroids) for plant tissue of type-A and -C plants, revealing that tissue characteristics are also highly reliable in predicting the race. The plant tissue variables were correlated with the discriminant function in the following order: Na (r value = 0.77), M g (0.5), Ca/Mg (0.37), A l (0.29), Ca (0.17), Zn (0.17), K (0.17), Cu (0.13), Cr (0.08), M n (0.06), Fe (0.04), and N i (0.02). Table 2.11 provides a summary of the classification results of discriminant function analysis for plant tissue of Jasper Ridge. Table 2.11. Classification results from Discriminant Function Analysis for plant tissue samples of Jasper Ridge. Average percent of "grouped" cases correctly classified: 93.5 %. Actual Group Number of Cases Predicted Group Membership Type-A Type-C Type-A 30 24 (80%) 6 (20%) Type-C 93 2 (2.2%) 91 (97.8%) 53 2.3.7. Principal Component Analysis - soils and plants of Jasper Ridge Figure 2.12 documents P C A results for soil analysis. Here, the two P C A axes explain 54 % of the total variance. Figure 2.13 indicates results for plant tissue analysis. The two PCA axes explain 61 % of the total variance. It is interesting to note the difference in the results between P C A and D F A ~ although DFA indicated that the centroids of type-A and -C differed significantly for both soil and plant tissue analyses, PCA shows considerable overlap in the variation between the two groups. 54 -4 -3 • 1 0 1 2 PCA 1 (33%) Figure 2.12. Results of Principal Component Analysis for all soil variables at Jasper Ridge. Ellipses indicate 95 % confidence intervals. Value within parenthesis indicate the variance explained by each PCA axis. Cvl < O CL 0 -3 0 2 4 PCA 1 (41%) Figure 2.13. Results of Principal Component Analysis for all plant tissue variables at Jasper Ridge. Ellipses indicate 95 % confidence intervals. Value within parenthesis indicate the variance explained by each PCA axis. 56 2.3.8. Discriminant Function Analysis — Other populations Discriminant function analysis of soil and plant tissue variables from the 22 populations also indicated that both soils (Table 2.12) and plant tissue (Table 2.13) are highly reliable predictors of the two races of this species. Group means from type-A and -C soil and plant tissue analysis differed significantly (P < 0.006). However, the size of correlation of some discriminating variables differed between this analysis and the analysis for Jasper Ridge. The soil variables were correlated with the (hscriminant function in the following order: p H (r value = 0.12), K (0.08), M g (0.07), Cd (0.06), % Clay (0.06), Cr (0.05), CEC (0.05), N i (0.04), Co (0.04), M n (0.04), Fe (0.03), Na (0.02), Ca/Mg (0.02), Zn (0.02), Ca (0.01), and Cu (0.001). The plant variables were correlated with the discriminant function in the following order: Mn (0.37), A l (0.37), Fe (0.19), Cu (0.16), M g (0.12), Zn (0.09), N i (0.08), K (0.07), Ca (0.07), Ca/Mg (0.05), Na (0.04), and Cr (0.03). Table 2.12. Classification results from Discriminant Function Analysis for soil samples from 22 populations. Average percent of "grouped" cases correctly classified: 100 %. Actual Group Number of Cases Predicted Group Membership Type-A Type-C Type-A 14 14 (100%) 0 (0%) Type-C 8 0 (0%) 8 (100%) 57 Table 2.13. Classification results from Discriminant Function Analysis for plant tissue samples from 22 populations. Average percent of "grouped" cases correctly classified: 100 %. Actual Group Number of Cases Predicted Group Membership Type-A Type-C Type-A 14 14 (100%) 0 (0%) Type-C 8 0 (0%) 8 (100%) 58 2.4. Discussion 2.4.1. Jasper Ridge Results from flavonoid analyses documented that the pattern of distribution of the two races of L. californica on the serpentine outcrop at Jasper Ridge has remained essentially constant for almost 15 years. This indicates that strong selective forces must be mamtaining the boundary of the races on this outcrop. The achene shape difference displayed by this population adds to the already known list of morphological and biochemical differences in these two races, estabhshing that the two races have diverged in at least one entire morphological character. Other studies of closely related taxa indicated that achene characters are possibly the earliest detectable morphological differences in closely related taxa (Vasey, 1985; Crawford et al., 1985; Heywood, 1986). In some of these case studies, the achene differences may have contributed to effective isolation of the distinct taxa via long-distance dispersal (Vasey, 1985). The role that achene shape differences may play in the case of the two races of L. californica is unclear. Our data on soil chemical and physical features for Jasper Ridge generally agree with data previously collected from Jasper Ridge (Proctor and Whitten, 1971: Turitzin, 1982; Streit et al., 1993) and other serpentine sites in California (Woodell et al., 1975; Fiedler, 1985; Kruckeberg, 1951, 1984, 1992; Arianoutsou et al., 1993), except for heavy metal concentrations which are slightly lower. Soil concentrations of various elements can often vary from study to study, depending on time and location of sampling, method of soil collection and preparation, as well as method of analysis. The low heavy metal concentrations of serpentine soils at Jasper Ridge may also be an indication of the poorly 59 developed nature of these soils, reflecting the slow rate of weathering in this region. Rainfall is low and is practically absent in the summer. The low rainfall reduces the leaching of soluble ions and slows the decay of organic matter (Streit et al., 1993). Analyses indicated several differences in the physical and chemical features of type-A and -C soils. The ridge top and bottom soils clearly differed in their physical appearances. The ridge bottom soils, populated by type-A plants, are much darker, highly aggregated, and clayey in texture. The dark color possibly indicates a higher organic matter content, while the high aggregation is a result of the presence of high concentration of clay. The clay fraction of these soils is possibly montmorillonite, characterized by large cracks that are often visible on the drier surfaces of these soils. Montmorillonite clays are composed of one octahedral layer of AL Mg, and O (or O H " ) sandwiched between two tetrahedral layers of Si and O (2:1 clays). This layered structure allows the soils to shrink when dry and swell when wet, making these soils physically unstable and quite unfavourable for root growth. The ridge bottom soils are often wet and at times, water-logged. In wet years, type-A plants have been observed growing in standing water (Bohm et al., 1989). In contrast, the ridge top soils, populated by type-C plants, are much lighter in color, loosely aggregated, and often quite dry. Close examination of changes in soil characteristics along transects from the mid-ridge to ridge bottom indicated that ridge bottom soils have a higher RWC, pH, and CEC than ridge top soils. Concentrations of several elements gradually increased with distance increasing from ridge top to bottom. This gradual increase of certain soil features may result from the drainage pattern on the outcrop where most of the leachate is ending up in the swale occupied by type-A plants, making ridge bottom soils more concentrated than the soils above (Figure 2.14). 60 C Type leaching of ions and other substances High pH, Clay, M g 2 + , Na + , Cr 3*, Fe 3 + , organic acids, and low Ca/Mg Figure 2.14. A diagram of the ridge showing the drainage pattern on the outcrop. A highly concentrated leachate may accumulate in the swale occupied by type-A plants. 61 Values of plant tissue concentrations of various elements at Jasper Ridge generally agreed with the range of values recorded for serpentine plants (Proctor and Whitten, 1971; Woodell et al., 1975; Fiedler, 1985; Arianoutsou et al., 1993; Kruckeberg and Reeves, 1995). A close examination of changes in plant tissue elemental composition along transects from mid-ridge to ridge bottom indicated that tissue concentrations are often a good indication of the concentrations of those elements in soils. Regression analyses between soil and plant tissue concentrations of various elements indicated that neither races take up certain heavy metal cations in proportion to their concentrations in the soil. This behaviour suggests that the two races may have exclusion mechanisms to deal with potentially toxic levels of heavy metals found in serpentine soils. However, several serpentine plants deal with such high metal environments by hyperaccumulating the metals (Brooks, 1987; Kruckberg and Reeves, 1995). Regression analysis also suggested that the two races may have differential responses to certain soil elements. Of particular interest are Mg, Na, and K Results indicated that type-C plants maintained a relatively constant M g and Na concentration in their tissues while type-A preferentially accumulated both Mg and Na in proportion to the concentrations found in the soils. In contrast, type-C plants showed a significant positive correlation for K , while the correlation for K in type-A plants was non-significant. Sodium was of interest for it was the only cation that showed differences between the two races in concentrations possibly that had biological significance. Although both type-A and -C plants grew in soils of more-or-less similar Na levels, type-A accumulated Na in its plant tissue to at least three times the concentration of type-C plants. The levels of Na found in type-A plants was equivalent to 62 levels found in halophytes and desert plants exposed to extreme water stress (Ravetta et al., 1997). The value of accumulating Na for type-A plants is unclear, even for purposes of osmotic and ionic balance, especially because type-A plants seemed to grow in much wetter soils at Jasper Ridge. However, it is possible that the water is not as readily available to these plants, because of the highly aggregated nature of the ridge bottom soils. Further, type-A plants may have a much higher requirement for water than type-C plants, and the higher Na concentration may be used to maintain an adequate amount of water in the plant. The Na-accumulating nature may also explain how type-A plants often successfully colonize coastal and desert areas of southern California, western Arizona, and northern Baja California, where the climate is much drier than the type-C-dominating northern parts of California or southern Oregon. The physiology of Na-uptake is clearly an important aspect to investigate in the future. The detailed soil chemical analyses of Jasper Ridge indicated that, despite the sharp boundary seen between the two plant races, there are no abrupt changes in soil chemistry along this gradient. The changes are gradual, with certain elements and soil features increasing or decreasing gradually along each transect. Even though no sharply defined changes were present in the separately tested soil characteristics, both discriminant function analysis and principal component analysis recognized the soils of type-A and -C plants as separate groups, indicating that soil characteristics are reliable in predicting the occurrence of the two races of L. californica. 63 It is important to understand that ecosystems function as multi-factorial, holocoenotic systems. Single factor dependence is merely a simplifying device for the scientist studying the system Further, in addition to the already known array of soil differences, it is possible that other inorganic, organic, or biotic (Tadros, 1957; Hopkins, 1987; Turkington and Aarssen, 1984) factors may differ between ridge top and bottom It is often the ensemble of soil chemical, physical, and biotic factors that result in the highly selective nature of an edaphic environment, leading to biological consequences such as the distribution pattern at Jasper Ridge. The study has indicated that gradual changes of soil characteristics can give rise to different edaphic conditions along an elevational gradient. This adds a new dimension to studies of vegetation boundaries. Many studies in the past have documented sharp boundaries in single elements (especially heavy metals) leading to vegetation boundaries over short distances (reviewed by Linhart and Grant, 1996). However, the picture at Jasper Ridge suggests that gradual increases of a number of soil characteristics (organic substances, RWC, pH, CEC, Clay, Mg, Na, Fe, Zn, Cr) over a short distance may have resulted in an extreme edaphic condition that is inhospitable for type-C plants. The lack of type-C plants in soils with type-A plants adds credence to this hypothesis. Type-A plants may be more tolerant ("stress tolerator") of this extreme edaphic condition at the ridge bottom, thus colonizing the habitat left untouched by the more dominant type-C plants. The lack of type-A plants on soils with type-C plants is possibly a result of the more vigorous or dominant type-C plants "outcompeting" type-A in the ridge top soil. Further, the lower water-holding capacity of ridge top soils may also play a role in preventing type-A plants from colonizing the ridge top. 64 2.4.2. Other Populations Flavonoid analyses of plants collected from 22 populations agreed with flavonoid patterns observed for those populations by Desrochers (1992). Type-A plants were found mostly in southern parts of the species' range while type-C plants dominated the northern parts, fteliminary observations of achene shape (from populations number 1 [type-C] and 22 [type-A]) confirmed that the achene differences observed for Jasper Ridge also held true for other populations, indicating that this character has diverged in the two races throughout their range. Soil and plant tissue analyses indicated patterns that agree with most patterns observed for Jasper Ridge. Similar to Jasper Ridge, type-A plants were often found in soils with higher pH, CEC, % Clay, Mg , and Na concentrations than type-C plants. Further, the Ca/Mg ratio and K was higher for soils of type-C plants. Plant tissue analyses indicated that type-A plants contained higher concentrations of Na and Mg, showing a similar pattern to type-A plants found at Jasper Ridge. However, unlike the situation at Jasper Ridge, most means for the two races were not significantly different. This was possibly a result of the smaller sample size and the high variation in the range of values found within each race (for example, the wide range of values observed for soil and tissue Na of type-A plants). The high within-group variation may be partly due to comparing samples growing on a variety of geologic substrates, from serpentine and coastal to chapparal and pastures. Sodium, however, was of special interest because regression analysis again indicated that type-A plants had a much higher positive correlation for soil and plant tissue Na than type-C plants. This pattern once again indicates the value of pursuing investigations into the physiology of Na uptake within these two races. A more efficient Na-uptake system in type-A plants may explain i f racial differentiation in this species resulted from the ability of type-A plants to colonize areas subject to water stress (or 65 other forms of edaphic stress), be they coastal, desert, or highly concentrated soils such as those found at the ridge bottom of the serpentine outcrop at Jasper Ridge. 2.5. Summary Detailed analyses of soils and plant tissue from Jasper Ridge and 22 additional sites confirmed that type-A and -C individuals of L. californica tend to grow in soils that are chemically and physically different. Regression analyses indicated that the two races may be physiologically different. It is reasonable to assume then that the two races may have differential responses to ridge top and bottom soils in terms of their germination, growth, and reproduction. It is of interest to determine experimentally, via field transplant or greenhouse experiments, i f such differential responses exist between the two races. The next chapter documents results of greenhouse experiments conducted to confirm i f the differences shown in the edaphic features of ridge top and bottom play a selective role in maintaining the distinct pattern of distribution of the two races observed at Jasper Ridge. 66 Chapter Three Greenhouse Studies 3.1. Introduction In this chapter, I describe greenhouse studies of the two races of L. californica with respect to their responses in germination, survival, growth, and phenology to soils characteristic of type-C and - A plants. These studies were essential to affirm i f those correlations established in the previous chapter may have any impacts on the actual distribution pattern exhibited at Jasper Ridge. In addition to conducting two experiments aimed at understanding any effects the edaphic features may have on the distribution pattern, I conducted a study that looked at the importance of a seed bank in the establishment of a new population and made observations in the field of potential distances of achene dispersal. 3.2. Experiment 1 : Germination and Growth Responses to Soil Treatments 3.2.1. Objective The objective of this experiment was to determine if soils of ridge top (type-C soil) and ridge bottom (type-A soil) of the serpentine outcrop at Jasper Ridge differentially affect germination, growth, survival, and phenology of type-C and - A plants. 3.2.2. Materials and Methods The experiment was conducted in a greenhouse at the Department of Botany, University of British Columbia (UBC). 67 Achenes for the experiment were collected from type-C and - A plants from the population at Jasper Ridge Biological Preserve. These achenes were collected from plants found growing along the extreme 5 m ends of transects 2, 3, and 5 (see Figure 2.1). Achenes of type-C and - A plants were separated by using achene features (pappus and seed shape). Achenes of each plant type were pooled together, placed in paper envelopes, and stored in the dark. Achene collections were made the previous year (ApriL 1996) in order for the achenes. to go through an after-ripening period (preliminary germination trials indicated that achenes need at least a three month period of after-ripening for optimal germination). Approximately 3 kg of soil was collected from the fire road and ridge bottom ends of transect 3. Soils were collected along the first and the last five meters of the transect, where type-C and - A plants, respectively, were found to predominate. Soils were collected from the rooting zones of plants (2-10 cm depth) and placed in large plastic bags. Soils collected from Jasper Ridge were brought to U B C , air-dried for one week, and crushed and sieved through a stainless steel standard mesh No. 4 sieve to obtain the < 4.75 mm fraction of soil. Two hundred grams of ridge top (type-C soil) and ridge bottom (type-A soil) soils were then placed separately in plastic dishes (12.5 x 12.5 x 2.5 cm) and thoroughly soaked (with 100 ml of tap water) to bring soil to saturation. Dishes were placed in a cold room at 5 °C for one week to stimulate germination of Lasthenia or other seed from the natural seed bank. Dishes were then moved to the greenhouse and observed for one week. A l l genninants (mostly grasses) were removed. No Lasthenia seedlings were observed. Soils were thoroughly watered once again. Thirty achenes from type-C and - A plants were sown separately on four dishes each, containing the three treatments of type-C, -A , and potting soils (P Soil - Terra Lite Soil Mix). Dishes were returned to the cold room, arranged 68 randomly on a bench, and left in the dark at 5 °C for one week. After the week-long cold treatment, the dishes were arranged randomly on a table in the greenhouse. Germinants were counted on the 8th day after sowing. Germination was scored on the basis of achenes showing emergence of a radicle. From days 9-36, plant survival was scored daily on the basis of the percent standing plants with healthy foliage. The final count was done 60 days after sowing. Plants that were either wilted or dead were not taken into the daily count. However, i f a wilted plant revived by the following day it was included in the count of standing plants. After each count, plants were watered with 50 ml of tap water. Watering was done daily except between days 12-14, 17-19, 21-23, 25-27, and 30-34. This watering system was designed to provide periods of ,,mini-droughts,, characteristic of the natural conditions under which these plants are often found. The temperature in the greenhouse ranged between 70-90 °F during the growth period. Approximately 12-14 hours of daylight were provided every day. To determine any differential effects the soil treatments may have on the growth of type-C and - A plants, I measured plant height, number of leaves per plant, and percent of individual plants with inflorescences during the growth period. Plant height and the number of leaves per plant were measured on three-week-old plants. Height was measured from base of stem to the tip of the tallest leaf. The date of first inflorescences opening as well as the number of individuals with inflorescence were recorded 45 days after germination. One-way A N O V A s were conducted to determine i f means of various growth measures of type-A and -C plants differed among the three soil treatments. Paired-samples t tests were used when means of various growth measures of each plant type in two soil treatments had to 69 be compared in a pairwise manner. A l l statistical tests were conducted using SPSS (Norusis, 1993) on an I B M Compatible PC at the Department of Botany, U.B.C. 3.2.3. Results 3.2.3.1. Germination Trial Both type-C and - A achenes were about equally capable of germination in all three types of soils. One-way A N O V A s conducted on the mean percent germination indicated that soil treatments have no significant effects on germination (P > 0.05). However, results indicated that type-C plants, regardless of soil treatment, have much higher mean germination rates than type-A plants. Table 3.1 documents the one-way A N O V A results of the germination trial. Table 3.1. Germination results for achenes of type-C and -A plants 8 days after sowing seed in ridge top (type-C soil), bottom (type-A soil), and potting soils (P Soil). Percent values based on 30 achenes per dish x 4 dishes per treatment. Plant Type Soil Treatment Mean % Gemiination (± SE) F Prob. Value % Range (min.- max.) Type-C Type-C soil 80 (±3.15) P>0.05 76.67 - 90.0 Type-A soil 68 (± 7.52) 56.67- 90.0 P S o i l 83 (± 2.36) 76.67 - 86.67 Type-A Type-C soil 34 (± 4.59) P>0.05 23.33 -43.33 Type-A soil 28 (±4.41) 16.67 - 36.67 P S o i l 38 (±3.19) 33.33 - 46.67 70 3.2.3.2. Plant Survivorship Survivorship of both type-C and - A plants in the three soil treatments was observed daily from 9 - 36 days after sowing. The final count was taken 60 days after sowing. Paired-samples t tests indicated that type-C plant survivorship (Figure 3.1) in ridge top (type-C soil) and potting soils (P Soil) was not significantly different (P > 0.05) throughout the growth period. However, type-C survivorship in type-C soil and ridge bottom soils (type-A soil) was significantly different (P < 0.001) throughout the growth period. Type-C plants maintained an approximately 80 % survivorship in their own type-C soil while showing significant mortality in type-A soils. On day 60, when the final count was taken, type-C plants showed 78 % survivorship in type-C soil while showing only 6 % survivorship in type-A soils. Results clearly indicate that type-C and - A soils have differential effects on the survival of type-C plants — type-C plants are able to maintain a significantly higher percent survivorship in their own type-C soil while showing high mortality in type-A soils. 71 Figure 3.2 documents results of type-A plant survivorship in the three soil treatments. On day 60, when the final count was taken, type-A showed 10% survivorship in type-A soil and 8% survivorship in type-C soil. Type-A survivorship in the P Soil treatment was significantly higher (P < 0.001) than in the other two soils during the entire growth period. Paired-samples t tests indicated that type-A plant survivorship in type-C and - A soil was not significantly different (P > 0.05). Results clearly document that type-C and - A soils do not differentially affect the survival of type-A plants — type-A plants are capable of mamtaining a low but similar percent survivorship value in both their own type-A soil as well as in type-C soil. 73 3.2.3.3. Growth Measures Height One-way A N O V A s indicated that both type-A and -C plants were significantly taller in potting soils than when grown in either type-C or - A soils. Type-C soil was the next preferred media by both plant types. Life on type-A soil seem harsh for both type-C and - A plants, well indicated by their stunted growth forms. Table 3.2 documents results of height measures taken three weeks after germination. Table 3.2. Mean height (± SE ) of three-week-old type-C and -A plants growing in type-C soil, -A soil, and P Soil treatments. Plant Type Soil Treatment (Sample Size) Mean Height (mm) F Prob. Value Type-C Type-C soil 21.24 (±0.62) P < 0.0001 (102) Type-A soil 8.92 (± 0.45) (36) P S o i l 73.29 (± 2.01) (101) Type-A Type-C soil 12.6 (± 1.21) P < 0.0001 (20) Type-A soil 8.58 (± 0.47) (19) P S o i l 53.7 (±2.55) (44) 75 Results from paired-samples t tests indicated that both type-C plants (P< 0.0001) and type-A plants (P< 0.01) are significantly taller in type-C than in - A soils. Number of Leaves Table 3.3 documents results of a one-way A N O V A for the number of leaves of three-week-old type-C and - A plants grown in the three soil treatments. Table 3.3. Mean number (± SE) of leaves of three-week-old type-C and - A plants grown in type-C soil, - A soil, and P Soil treatments. Plant Type Soil Treatment (Sample Size) Mean Number Leaves F Prob. Value Type-C Type-C soil 7.08 (± 0.59) P < 0.0001 (102) Type-A soil 4.28 (±0.18) (36) P S o i l 18.87 (±7.93) (101) Type-A Type-C soil 4.9 (± 0.34) P < 0.0001 (20) Type-A soil 4.42 (± 0.25) (19) P S o i l 10.09 (± 0.28) (44) 76 Results indicate that both plant types exhibit the highest number of leaves when grown in P Soil treatment. Results from paired-samples t tests indicated that type-C plants have significantly more leaves when found in type-C soil than when found in - A soil (P< 0.0001). Type-A plants, however, produced about the same number of leaves in both type-C and - A soils (P> 0.05). Phenology Observations on the flowering times of the two races indicated that type-C plants flower approximately eight days before type-A plants. Table 3.4 compares the percentage of individuals with inflorescences counted 45 days after germination. Table 3.4. Mean percent of plants (±SE) with inflorescences 45 days after germination in type-C soil, - A soil, and P Soil treatments. Percent values based on percent of surviving individuals per dish x 4 dishes per treatment. Plant Type Soil Treatment Mean % with Inflorescences F Prob. Value Type-C Type-C soil 72.04 (± 5.46) P < 0.0001 Type-A soil 0 P S o i l 94.9 (± 1.91) Type-A Type-C soil 12.5 (± 1.94) P < 0.0001 Type-A soil 14.29 (± 1.63) P S o i l 63.04 (± 5.4) 77 Results from paired-samples t tests indicate that the number of flowering individuals of type-C plants in type-C and - A soils was significantly different (P< 0.001). However, the number of flowering mdividuals of type-A plants in type-C and - A soils was not significantly different (P> 0.05). Results once again indicated the pronounced inhibitory effect of type-A soil on type-C plants. Type-A plants seem to be able to tolerate both type-C and - A soils with a similar percent of mdividuals reaching flowering in both treatments. Another count was made 53 days from germination. This was necessary because type-A plants reached full flowering about a week after this initial count was made (the initial count was made when type-C plants were in full flower). However, the results still showed a similar trend. Fifty-three days after germination, 31% of type-A individuals had inflorescences in type-C soil, 35 % in type-A soil, and 96 % in the P Soil treatment. Results from paired-samples t tests indicated that the differences between the means for percent of type-A plants with inflorescences in type-C and -A soils were not significant (P> 0.05). Measures of plant survival, growth, and phenology all clearly indicate the differential effects of type-C and - A soil treatments on type-C plants and the ability of type-A plants to be almost indifferent to the two soil treatments. 78 3.3. Experiment 2 : Germination Responses to Soil Solutions 3.3.1. Objective Results from experiment 1 clearly indicate the hostile nature of ridge bottom soils. The adverse effects of ridge bottom soils were more pronounced on type-C plants than on type-A plants. The second experiment was conducted to determine i f the hostile nature of ridge bottom soils was a result of the chemical components of the soil solution or the physical attributes of the soil. Emergence of radicle as well as the development of leaves were observed on type-C and - A achenes sown on filter paper soaked with soil solutions extracted from ridge top and bottom soils. 3.3.2. Materials and Methods Three 50 g samples of each ridge top (type-C soil) and bottom (type-A soil) soils were each mixed with 50 g of distilled water (1:1), shaken for 0.5 hour, and then centrifuged at 10,000 rpm for five minutes. The supernatant was then filtered through Whatman 42 filter paper. The p H of the solution of type-C soil was 7.06, whereas the p H of the solution of type-A soil was 7.2. These two solutions, along with distilled water (pH 6.09), were used to test the effects of these solutions on aspects of germination of the type-A and -C achenes. Fifteen achenes each of type-A and -C plants were sown separately on Whatman 42 filter paper placed on standard Fisher brand plastic petri dishes (100 x 15 mm). Each treatment was replicated four times and then soaked with the appropriate solution. Petri dishes were then stored in the dark at 5 °C for one week. After the week-long cold treatment, dishes were moved to a growth chamber and arranged randomly (temperature: 25 °C by day and 20 °C by 79 night; daylight: 12 hours). A few drops of the appropriate solution were added to each petri dish daily. On day eight, achenes showing emergence of a radicle were counted. 3.3.3. Results Radicle Growth The one-way A N O V A results shown in Table 3.5 indicate a very similar pattern to that observed in the germination trial of experiment 1. However, the results are significant in this experiment, clearly indicating the hostile nature of type-A soil solution on type-C achenes. Paired-samples t tests indicated that the number of type-C individuals with radicle growth was significantly higher in type-C soil solution than in - A soil solution (P< 0.03). However, the number of type-A individuals with radicle growth was not significantly different between the type-C and - A soil solutions (P> 0.05). Table 3.5. Mean percent achenes (± SE) with radicle growth 8 days after sowing on filter paper soaked with ridge top (type-C) and bottom (type-A) soil solutions and distilled water (DW). Percent values based on 15 achenes per dish x 4 dishes per treatment. Plant Type Solution Treatment Mean % with Radicle F Prob. Value Type-C Type-C Solution 80 (± 2.72) P < 0.005 Type-A Solution 61.67 (± 1.67) D W 76.67 (± 4.3) Type-A Type-C Solution 57 (±3.33) P > 0.05 Type-A Solution 47 (± 4.71) D W 43 (± 4.3) 80 Leaf Growth One-way A N O V A results of the percent of achenes showing development of leaves two weeks after sowing are presented in Table 3.6. Table 3.6. Mean percent achenes (± SE) with leaf growth 14 days from sowing on filter paper soaked with ridge top (type-C) and bottom (type-A) soil solutions and distilled water (DW). Percent values based on 15 achenes per dish x 4 dishes per treatment. Plant Type Solution Treatment Mean % with Leaves F Prob. Value Type-C Type-C Solution 66.67 (± 2.72) P < 0.002 Type-A Solution 41.67 (±3.19) D W 66.67 (± 2.72) Type-A Type-C Solution 28.34 (±4.19) P > 0.05 Type-A Solution 31.67 (±3.19) D W 30 (± 4.3) Once again, type-A soil solution had a pronounced effect on type-C plants. Type-A plants, however, developed about the same number of leaves in both type-C and - A solutions, clearly indicating the ^difference of type-A plants to ridge top and bottom soils. These results are supported by paired-samples t tests: the number of type-C achenes developing leaves was significantly higher in type-C solution than in - A solution (P< 0.005), while the number of type-A achenes developing leaves was not significantly different in the two soil solutions (P> 0.05). 81 3.4. Seed Bank Study 3.4.1. Objective The objective of this study was to document the importance of a seed bank in the establishment of each years' population. 3.4.2. Materials and Methods Soils were collected from the soil surface to about 5 cm depth at the extreme five-meter ends of transects 2, 3, and 5 (soils were collected from a surface area of 5 x 1 meters). These extreme ends were dominated by either type-C or - A plants. Soils were collected just prior to seed fall and placed in large plastic bags. At U.B.C. , these soils were placed in large plastic trays (50 x 20 x 5 cm). Soils from each end of the transects were placed in two trays each. Trays were watered thoroughly and placed in the dark at 5 °C for one week. Trays were then moved to a greenhouse and arranged randomly. Observations were made on the composition of germinants for a period six months. Trays were watered a few times every week. 3.4.3. Results Many species characteristic of the serpentine outcrop at Jasper Ridge emerged during the first month of the study. Trays containing ridge bottom soils, however, showed a much lower species composition than trays with ridge top soils. Trays with soils of the ridge bottom were dominated by a species oiTrifolium L . (Fabaceae). Two species of the Poaceae were also recorded. No Lasthenia seedlings were observed. Trays with soils from the ridge top showed a higher diversity of species. Plantago erecta Morris., Layia platyglossa (F. & M.) 82 Gray, Brodiaea spp., Eschscholizia californica Cham., Castilleja spp., and three grasses were the dominant species. Only one Lasthenia californica seedling emerged during the study. This individual, found in a tray containing ridge top soils, was a type-C plant. The lack of viable achenes of L. californica in the seed bank indicates the importance of each years' seed fall in the estabhshment of a new population. 3.5. Observations on Achene Dispersal Observations in the field suggest that achenes of L. californica possibly do not disperse very far. Mature inflorescences usually snap at the end of the pedicel, just below the receptacle. This brings about an approximately 180° twist, turning the inflorescence face down. A gentle tap or a blow dispersed achenes about 5-8 cm from the base of the mother plant, with most achenes dropping right at the base. The achenes that do get away get trapped in tufts of grass found adjacent to the Lasthenia plants. During this latter stage of the life cycle, the serpentine outcrop is usually taken over by an abundance of perennial grasses making it difficult for the achenes to disperse very far. 83 3.6. Discussion Both experiments discussed in this chapter support the claim that edaphic factors may be contributing to the unique distribution pattern observed year-after-year on the serpentine outcrop at Jasper Ridge. Germination trials of experiment 1, however, indicated that both plant types germinate about equally well in both ridge top and bottom soils. This suggests that if edaphic factors are contributing to maintenance of the distribution, selection is possibly not strongest at the germination stage. Selection is gradual and is possibly taking place during the entire growth season. Experiment 1 clearly documented that type-C plants are unable to tolerate ridge bottom soils. There was significant mortality of type-C plants in ridge bottom soils throughout the growth season. Measurements on aspects of growth also indicated the adverse effects of ridge bottom soils on type-C plants. In contrast, type-A plants were able to tolerate both ridge top and bottom soils. Measures of survival and growth support the claim that type-A plants are possibly mdifferent to the edaphic differences between ridge top and bottom Observations on phenology documented a similar trend. No type-C mdividuals reached the flowering stage in ridge bottom soils, whereas, a large proportion of type-C individuals produced inflorescence in ridge top soils. Type-A plants, however, reached flowering in both soil treatments. Experiment 2 indicated that the soil solution of ridge bottom may be responsible for the adverse effects ridge bottom soils have on type-C plants. The experiment indicated that differential responses can exist at the germination stage, especially when the achenes are sown on soil solutions extracted from the respective soils. Ridge bottom soil solution had an adverse effect on aspects of germination of type-C plants while type-A plants, once again, were indifferent to the soil solutions. It is uncertain, however, how well this extracted solution 84 represents the actual strength of the solution an achene would experience in the field. Due to lack of buffering from soils, the solution used in this experiment is likely to be somewhat more concentrated than the actual soil solution. It will be of interest to determine what inorganic or organic substance or a combination of substances are possibly responsible for the pronounced inhibitory effects the ridge bottom soils have on type-C plants. Both experiments indicate higher vigour of type-C plants. This was evident when comparing germination and aspects of growth of the two plant types in potting soils, the media preferred most by both types. Here, type-C plants showed an overall higher percent gennination rates, survivorship, and growth than type-A plants. This may be due to naturally low vigour of type-A plants or due to the fact that it was not possible to provide the best conditions in the greenhouse for optimal germination and growth of type-A plants. Further studies are needed to determine i f type-C and - A plants show differences in vigour under natural conditions. Studies by Harper (1977) document that seed and dormancy polymorphisms are common within species and that it is dangerous to ascribe to a species any particular germination regime. Studies on Chenopodium album L . demonstrated that different phenotypes within this species require different conditions for optimal germination (Williams and Harper, 1965). Seeds with different sizes, thicknesses of seed wall, and reticulations of seed coat required different set of conditions for optimal germination. Hence, seed variability must be considered when attempting to stimulate maximum germination in a species. If this is not considered, earlier or later germinating phenotypes may be inadvertently favoured. The role that achene variability of the two races of L. californica may play in terms of their germination must be examined in more detail. 85 Experiment 1 also documented that both races of L. californica grow better in potting soils than in soils from the study site. This is not surprising because potting soils have a much higher water-holding capacity and are designed to provide optimal nutritional conditions for plant growth. Vigorous growth in potting soils show that neither race may be dependent on any unique substance found in serpentine soils for successful survival and growth. Ridge top and bottom soils, due to their serpentinite origin, provide chemical adversities (Walker, 1954; Walker et al., 1955; Proctor and WoodelL 1975; Kruckeberg, 1984). These nutritional imbalances are evident by the relatively stunted growth forms of both plant types in the two serpentine soils. Our studies of the edaphic features of the ridge (presented in Chapter Two) documented the highly concentrated nature of ridge bottom soils. Studies of this chapter provide evidence suggesting that type-C plants are sensitive to those edaphically extreme conditions at the ridge bottom while type-A plants are indifferent to those conditions. The study presented here of differential responses of the two races of L. caifornica to edaphic conditions along a distance of only 60 meters is unique ~ the study has documented differential responses of two races of an annual to two naturally occurring edaphic conditions within a geologic substrate. In the past, most studies have looked at differential responses of races of a species to geologically different substrates. A well studied example is the study of responses of serpentine-tolerant and -intolerant races within a species to serpentine and non-serpentine soils. These studies have shown that for a serpentine intolerant ("stress intolerant") race of a species, germination and growth rates are almost always significantly higher in non-serpentine soils (Kruckeberg, 1951, 1954, 1967). In contrast, germination and growth rates of individuals of the serpentine tolerant ("stress tolerant") race are more-or-less similar in both serpentine and non-serpentine soils ~ serpentine-tolerant individuals are indifferent to 86 serpentine and non-serpentine soils. Studies have also indicated that when serpentine-tolerant ("stress tolerant") and -intolerant ("stress-intolerant") individuals of a species are grown together in non-serpentine soils, the "stress tolerant" race is competitively inferior to the "stress intolerant" race (Kruckeberg, 1954). This pattern also holds true for plants of calcareous soils (Snaydon, 1962) and of metal-contaminated sites (Cook et al., 1972; Hickey and McNeilly, 1975). The patterns we have documented with type-C and - A plants of L. californica are in some ways comparable to certain patterns observed in these earlier studies. Type-A plants, growing in the ridge bottom soils, are more like the "stress tolerant" races of these previous studies, showing indifference to both ridge top and bottom soils. Type-C plants best fit the "stress intolerant" category, growing best in their own ridge top soil and very poorly in ridge bottom soils. Both experiments presented in this chapter indicate that type-C plants are physiologically unable to deal with possibly severe chemical conditions found in the ridge bottom soils. Type-C plants may survive in very low frequencies in the ridge bottom. It is unlikely, however, that many type-C individuals will reach the flowering stage in ridge bottom soils. In contrast, this study indicated that type-A plants are able to grow in both ridge top and bottom soils and will reach flowering in both soils. Both results are strongly supported by field observations. During the past 15 years of sampling at Jasper Ridge, not a single type-C plant has been collected in the ridge bottom. This is possibly due to the fact that all field collections were done in the late flowering stage of this population. Further, only flowering individuals are collected for flavonoid analysis. Since type-C individuals do not flower in the ridge bottom it is unlikely that any type-C plants would have been collected. On the other hand, a few type-A plants have been found near the ridge top, almost every year this population was sampled. The 87 low frequency of type-A plants near the ridge top may be due to the fact that type-A plants are competitively inferior to the more vigorous type-C plants. During the growing season, type-C plants may outcompete type-A plants from ridge top soils. This is usually the case with most stress tolerant races ~ when grown in stress-free soils, they are competitively inferior to the stress-intolerant race (Kruckeberg, 1954). It will be of interest to sample this population a few weeks after germination to see i f the distribution patterns of the two races are not as distinctly demarcated as they are later in the growing season. Experiment 1 strongly supports field observations on flowering time differences between the two races (Desrochers and Bohm 1995; personal observations). At Jasper Ridge, type-C individuals always flower 1-2 weeks prior to type-A plants. The greenhouse study documented that type-C plants flower 7-10 days prior to type-A plants: type-C is always at or past mid-flowering when type-A has begun to flower. The study looking at the importance of a seed bank in the establishment of a population indicated that a new population is primarily dependent on each years' seed fall. Only one achene germinated in 12 trays filled with soils collected just prior to the years' seed fall. This strongly supports the claim made by Ornduff (1966) that all viable seeds may germinate after the first significant rains in the late fall. Ornduff indicated that, in the field, seed storage beyond a season may not be significant and that nearly all seed present at a site will germinate after sufficient rain has fallen. Species vary considerably with respect to the longevity of their seeds in the soil. Archaeological sites have revealed viable seeds of Chenopodium album L . and Spergula 88 arvensis L . believed to be about 1,700 years old (Odum, 1965, 1974). Agricultural studies have shown that, in general, seeds of Poaceae and crops generally succumb early, whereas seeds of weeds and Fabaceae remain viable for a much longer time (Lewis, 1973). The seed bank, or the soil seed reservoir, is an important means by which species are able to ensure survival and success, especially in environments where frequent disturbances prevail. Seed banks are especially critical for annual plants in contrast to perennials, because the seeds of annuals represent the only link between generations in those species. In the case of the annual L. californica, all achenes seem to germinate after a few months in the soil. It would be of interest to determine i f the chemically adverse nature of serpentine soils as well as seed predation may also be responsible for the short life span of achenes in these soils. The observations made on distances of achene dispersal indicates that achenes may not disperse far; in most cases the achenes land very close to the mother plant. This observation is strongly supported by genetic diversity studies, conducted by Desrochers and Bohm (1995), that suggest very low levels of pollen and seed movement in this population. Hence, i f seeds land close to the mother plant, and i f the composition of a new population is solely dependent on each years' seed fall, it is likely that the composition of the population at Jasper Ridge will remain relatively constant. 89 3.7. Summary Experiments described in this chapter demonstrated that the two races of L. californica respond differentially to soil conditions of ridge top and bottom Type-C plants grow best in ridge top soils and are unable to tolerate ridge bottom soils. Observations in the field support these experimental results ~ type-C plants occupy most parts of the ridge except the bottom swale. Type-A plants are found solely in this bottom swale and appear to be able to grow in these edaphically extreme soils as well as in ridge top soils. Type-A plants may be unable to colonize further up the ridge owing to their inabihty to compete with the more dominant and vigorous type-C plants. Observations made on viability of seed in soil as well as of potential distances of seed dispersal indicate that achenes of L. californica do not disperse far, either in time or space. These observations are supported by results from genetic diversity studies indicating low levels of gene flow between the two races of this population. Hence, differential responses to edaphic conditions, low distances of achene dispersal, as well as flowering time differences may be important factors contributing to mamtaining the sharply demarcated boundary between the two races observed on this serpentine outcrop. Further studies are needed to determine i f the patterns shown for the two races at Jasper Ridge hold true for the two races from the entire range of the species, and thus i f racial differentiation in L. californica is a prelude to edaphically induced speciation. 90 Chapter Four Conclusions and Future Directions 4.1. Is type-A a "stress tolerator" ? The detailed study of L. californica has suggested that type-A plants are possibly able to tolerate a more stressful edaphic condition than type-C plants. There are several lines of evidence lending support to this suggestion. Firstly, there is ecological evidence. The study at Jasper Ridge has shown that type-A plants dominate ridge bottom soils while type-C plants are never found in the ridge bottom. Soil analyses clearly documented the extreme chemical and physical features of ridge bottom soils. These extreme edaphic features can bring about unfavourable growth conditions to plants. The experimental data strengthen the observations from the field: type-C plants are not as tolerant as type-A plants to ridge bottom soils. Hence, at Jasper Ridge, type-A plants may have taken the role of a typical "stress tolerant" race, growing in a habitat left uncolonized by the more dominant, but less adapted, race. In addition to Jasper Ridge, the other serpentine sites (populations 8, 11) sampled indicated the presence of just type-A plants. Serpentine soils are chemically imbalanced and can create highly stressful conditions for plant growth. It is interesting to find that both these sites consisted only of type-A plants. Further, populations found along the coast from central to southern California, in inland parts of southern California, in northern Baja California, as well as in deserts of western Arizona consisted predominantly of type-A plants. A l l these habitats provide stressful conditions for plant growth, either in terms of ionic imbalances or water stress. Hence, it is 91 reasonable to suggest that a correlation can be established between type-A plants and then-habitats : type-A plants are generally found in habitats that cause "stressful" conditions for plant life. Secondly, there is physiological evidence. Plant tissue analyses revealed that type-A plants contain in their plant tissues significantly higher levels of Na. Regression analyses indicate a significantly higher positive correlation value for soil versus plant tissue Na in type-A plants. These patterns were observed at both Jasper Ridge and the other sites, indicating that type-A plants seem to accumulate Na preferentially in their tissues. The levels of Na found in some type-A plants are even higher than levels found in certain halophytes and other plants growing under extreme water stress (Ravetta et al., 1997). This was an exciting discovery because accumulation of Na, once again, adds credence to the claim that type-A plants may be better adapted to grow in stressful environments. High concentrations of Na are usually found in plants faced with either ionic imbalances or water-stress. Accumulation of Na in the vacuole allows these plants to alter their internal osmotic pressure, which lowers the water potential, and allows the plant to maintain a positive water potential gradient between the soil and plant. Such adjustments can therefore allow plants to grow at high external salt concentrations, such as those found in saline and other environments characterized by high ion concentrations. It seems as though most environments where type-A plants occur have soils with extreme ionic imbalances or water stress. It is possible that the capacity to accumulate high concentrations of Na provides type-A plants a means by which they can survive in these stressful soils. Another line of physiological evidence comes from a recent study I conducted that looked at the effects of three water treatments on total biomass and root/shoot ratios of type- C and - A plants. This experiment was concluded recently and has not been included in this thesis. 92 A summary of this work follows. The experiment clearly demonstrated that both races of L. californica grew best in extremely wet soils. The study documented that total shoot biomass in both types was significantly increased in the high water treatment where soils were brought to saturation every third day. This is not surprising because Lasthenia plants are usually known to occupy habitats which are often wet. interestingly, type-A plants, regardless of water treatment, had a significantly higher root biomass and root/shoot ratios than type-C plants. It seems as though type-A plants are genetically programmed to produce more root tissue ~ another indication of a possible adaptation for growing under water stress. The final line of evidence supporting the claim that type-A plants are "stress tolerators" comes from the literature of flavonoid chemistry. It is suggested that sulfated flavonoids in plants seem to have some relationship to the type of ecological habitat in which they grow (Harbourne, 1975; Barron et al., 1988). For example, plants growing in aquatic habitats, especially those of saline environments, contain sulfated flavonoids. The role(s) played by sulfated compounds in these environments is unknown. Experiments done on Zostera, a salt-tolerant plant, suggested that flavonoid sulfates may have an active function in salt uptake and metabolism (Nissen and Benson, 1964). In a feeding experiment supplying 3 5 SO42" to Zostera, the researchers found that, after 36 hours, 50% of the accumulated radioactivity was present in the flavonoid fraction. The results of this experiment suggest that flavonoids may play an active role in ion balance, in the incorporation of inorganic sulfate or in the transfer of sulfate to other organic substances. It is interesting to note that type-A plants found in saline, arid, or extreme serpentine environments also contain sulfated flavonoids, the main chemical difference that separates them from type-C plants. 93 Evidence stated above from ecological observations, greenhouse studies, as well as from the flavonoid literature, strengthens the claim that type-A plants may be better adapted to grow in environments of stress, be they saline, arid, or serpentine. 4.2. Evolution under edaphic influence The detailed study of L. californica has documented the existence of two distinct races. Studies of edaphic and plant tissue attributes of these two races have documented that they may be adapted to grow in different soil environments. The discussion above suggested that type-A plants may be preadapted or have evolved tolerance to grow in environments of stress. The capacity to survive in stressful environments may have set the stage, at some point in the evolutionary history of this species, for type-A plants to become effectively isolated from type-C plants. Patterns of variation now observed between the two races indicate that speciation within the species has been gradual and conservative. Different selection pressures on allopatric populations, perhaps combined with drift, may have caused gradual divergence in chemical, morphological, and physiological features over a long period of time, resulting in the formation of distinct races. It is evident from field observations as well as from greenhouse studies that gene flow between sympatric populations is reduced due to edaphic as well as intrinsic factors. The flowering time differences provide an effective barrier to gene flow between the races. Genetic identity studies reveal that gene flow between the races is reduced, fteliminary studies have also indicated naturally low levels of inter-racial fertility. These genetic factors, combined with their abilities to colonize different edaphic environments, may be contributing to maintaining an effective isolation of the two races. 94 The two races have diverged in at least one apparent morphological character as well as in several chemical and possibly physiological features. Achene shape, flavonoid pigments, allozymes, as well as possible differences in the Na ion-uptake physiology effectively distinguish the two races of L. californica. Reproductive traits appear to have diverged only in terms of flowering time differences. Both races are thought to be obligatorily outcrossing. Extensive breeding programs are needed to understand fully the extent of breeding between and within races of this species. Such studies should reveal if the two races are in fact two biological entities deserving their own taxonomic ranks. 4.3. Research Highlights The research conducted for this Master's thesis was an extension of a series of studies that were done on L. californica over the past several decades. Following is a brief list of important observations made and studies done from the time the genus was first named to the present. 1834 H . Cassini proposed Lasthenia as a genus and decided to name it after a Greek girl who attended the lectures of Plato in the garb of a man (Cassini, 1834 in Ornduff, 1966; Munz, 1968; Hickman, 1993) 1836 F.E.L. Fischer & C. A. Meyer described Baeria chrysostoma as a species (Fischer and Meyer, 1836 in Ornduff, 1966) 1876 John Lindley and Thomas Moore, referring to the genus, stated that "they grow in wet places and appear to be uninteresting weeds" (cited by Ornduff, 1966) 95 1894 E . Greene renamed B. chtysostoma as Lasthenia chrysostoma (Fisch. & Mey.) Greene (Greene, 1894 in Ornduff, 1966) 1966 Robert Ornduff wrote a monograph for the genus. He identified L. chrysostoma as the most variable taxon in the genus and recognized three morphologically distinct ecological races ~ coastal, inland non-desert, and inland desert (Ornduff, 1966) 1974 Flavonoid studies revealed chemical races within L. chrysostoma (Bohmetal., 1974) 1970's or 1980's L. californica DC ex. Lindley was recognized as the correct name for L. chrysostoma 1982 The unique distribution pattern of type - A and -C plants on the serpentine outcrop at Jasper Ridge Biological Preserve was discovered (Bohm et. aL 1989) 1990-1995 Biosystematic study of L. californica revealed two geographical races. The distribution pattern of these two races at Jasper Ridge maintained itself year-after-year (Desrochers, 1992; Desrochers and Bohm, 1993; Desrochers and Bohm, 1995) 1995-1997 "Getting the dirt on Lasthenia" Are edaphic factors contributing to speciation within L. californica ? (M. Sc. Thesis Research) 96 4.4. Summary The work done over the past two years has led to significant findings that corroborate previous research and add several new dimensions to the study of this species. The following is a list of discoveries and observations made during the course of this M.Sc. thesis. * Achene shape differences are documented. This morphological character adds to the other known morphological and chemical differences between the two races * * * * * Detailed analyses of soil samples revealed edaphic differences between ridge top and bottom of the serpentine outcrop at Jasper Ridge Type-A plants are generally found in soils characterized by significantly higher pH, cation exchange capacities, Na, Mg, Cr, percent clay, percent moisture, and organic substances Type-C plants are generally found in soils having significantly higher Ca, K , Ca/Mg, and N i Plant tissue analyses revealed that tissue concentrations of various elements are often good indications of those concentrations in soil. Levels of Na found in plant tissue of type-A plants, however, are over three times those in type-C plants Discriminant function analyses as well as principal component analyses revealed that soil and plant tissue characteristics are reliable in predicting the race Regression analysis indicated possible physiological differences between the two races. Type-A plants seem to accumulate Na as well as Mg preferentially 97 Patterns seen for soil and plant tissue analyses from Jasper Ridge agreed with those patterns found in analysis of 22 additional populations Greenhouse studies revealed that the two races show differential responses in terms of germination, survival, growth, and phenology to soils of ridge top and bottom Flowering time differences of the two races observed in the field are supported by greenhouse studies Ecological, physiological, and biochemical information suggested that type-A plants are possibly more tolerant of edaphic stresses than type-C plants The role that stress tolerance may have played in the evolutionary history of the species is discussed It is suggested that the two races of L. californica may qualify as true edaphic races but that further studies are needed to determine i f the two races can be recognized as true species 4.5. Future Directions The work done during the M.Sc thesis research has set the stage for several areas of extensive research. The multi-m^ciplinary work I propose to do during the next few years will help gain a better understanding of the evolutionary ecology of this species. Several areas of research are proposed. 98 4.5.1. Edaphic Studies I will take a closer look at the edaphic conditions of Jasper Ridge hoping to isolate a factor or factors possibly responsible for the inhibitory effects the ridge bottom soils have on type-C plants. I will also determine the organic substances as well as the clay fraction found in these soils. Anion concentrations, especially sulfates, will be determined. I will attempt to identify the minerals found in these soils to gain a better understanding of aspects of availability of the inorganic substances. A recent study demonstrated that roots of L. californica are highly infected by two species of mycorrhizal fungi (Hopkins, 1987). The study investigated the extent of root colonization by vesicular - arbuscular mycorrhizal (VAM) fungi of annual plant species found on a serpentine grassland in central California. The two main species of fungi found were Glomus tenue (Greenhall) Hall and Glomus fasciculatum (Thaxter sensu Gerd.) Gerdemann & Trappe. Lasthenia californica roots were heavily colonized by both species. The researchers recognized L. californica as one of a few species that had 76 -100 % of the root sohdly colonized by these fungi. The heavy colonization of roots of these plants may explain how they often successfully colonize nutritionally poor soils such as those found on serpentine habitats. It will be of interest to determine i f the two races are colonized by different species of mycorrhizal fungi and i f the extent of colonization is different in the two races. Other populations will be revisited in order to conduct a more detailed analyses of the soils. Populations that were of mixed races are of special interest. Extensive sampling of such populations may help gain a better understanding of the role edaphic factors may play in the 99 distribution of the two races. Long-term observations of these populations may reflect i f boundaries such as those found between type-A and -C plants at Jasper Ridge also exist in other mixed populations. 4.5.2. Breeding Studies Breeding studies are necessary to determine the extent of intra- and inter-racial crossing of L. californica. fteliminary studies done by Desrochers (1992) showed that the two races do not appear to interbreed. The number of crosses attempted in that work was small however. I intend to undertake a more extensive breeding programme, using achenes collected from populations from the entire range of the species. Should the lack of gene exchange between these two races prove to be real, one could argue that racial differentiation has progressed to a point where a significant breeding barrier now exists. 4.5.3. Physiological Studies The detailed soil and plant tissue analysis from Jasper Ridge and 22 other populations indicated that the two races may be different in physiological mechanisms such as ion-uptake. It was evident from these analyses that type-A plants have the capacity to accumulate significant amounts of Na in their plant tissues. Experiments will be conducted to ascertain the relative Na ion-uptake capacities of the two races under controlled conditions. If it should prove that type-A plants have a unique capacity to accumulate Na, relative to type-C plants, it will be of interest to pursue the possibility that an efficient Na transport system is at work in these plants. The studies have also indicated differential responses to K + . A series of experiments, such as those reviewed by Flowers et al. (1977, 1986) and described by Glenn and 100 O'Leary (1984) and Ravetta et al. (1997), will be conducted looking at the relative uptake of Na and K by the two races. A recent study has identified a high affinity K uptake transporter to function as a high-+ + . + affinity K - N a cotransporter (Rubio et al., 1995). SuchNa coupled transporters had been + + thought not to occur in higher plants (Sussman, 1994). Coupling of K uptake to the Na gradient can provide plant cells with sufficient K for plant nutrition from soil solutions with extremely low K concentrations. The results of that study have, for the first time, provided insight into a molecular pathway of Na uptake in higher plants. The study of Na and K ion-uptake in L. californica may shed light on additional Na ion-uptake pathways and is an important area to pursue. The studies on L. californica demonstrated differential responses of the two races to soil M g as well. Type-A plants seem to accumulate higher concentrations of M g in their plant tissues, compared to type-C plants. The studies also documented significant negative correlations between soil M g versus plant tissue Ca and soil Ca versus plant tissue M g for both plant races. These results strongly agree with those of Main (1974) indicating possible depression of plant Ca by high soil M g and a possible depression of plant M g by high concentrations of soil Ca. It has been demonstrated that M g and Ca compete for the same absorption sites on the exchange surface and that Mg competitively inhibits the uptake of Ca (Grover, 1960). Similar results have been documented by Madhok (1965) and Madhok and Walker (1969). The results from this study show that both type-C and - A plants sequester Ca at low soil M g concentrations while an increase in soil M g decreases plant Ca. Differential responses to soil Ca have previously been recorded for ecotypes and races within the same 101 species (Snaydon and Bradshaw, 1961, 1969; Snaydon, 1962; Ramakrislinan and Singh, 1966). Differential responses to soil Mg have also been documented for two closely related species (Madhok and Walker, 1969) and for races within the same species (Main, 1974). Any differential responses the two races of L. californica may have to M g and Ca have not been tested under laboratory conditions. The study looking at the effects of water on aspects of plant biomass showed that type-A plants, regardless of the water treatment, have significantly higher root mass than type-C plants. Growth experiments will be conducted to observe root growth and measure root biomass of the two races under controlled conditions. These studies will be essential in order to establish whether the larger root growth of type-A plants is a normal condition. 4.5.4. Ecological Studies Field studies are necessary to support the findings from experiments conducted in the greenhouse. Reciprocal transplant experiments will be conducted at Jasper Ridge. Such studies will demonstrate i f the patterns observed in greenhouse studies can also be shown under natural conditions. Further, a detailed sampling of the population will be done a few weeks from germination in order to determine i f the distribution of the two races is not as sharply demarcated as later in the growing season. This would be easy to do since the flavonoid chemistry of the plants can also be determined by using juvenile leaf tissue. Inter-racial competition studies will be conducted in the field as well as in the greenhouse to observe aspects of vigour of type-C and - A plants. This type of study is necessary to confirm i f competition, either for space or resources, is contributing to preventing 102 type-A plants from moving into ridge top areas. The distribution pattern at Jasper Ridge may also provide a good model system to test theories of competition of both Grime and Tilman. Grime's theory of life histories (Grime, 1979) and Tihnan's resource-based theory of competition (1977, 1985, 1988) make certain predictions on and assumptions of competition. Considerable debate, however, has developed concerning the vahdity of both these theories (Thompson, 1987; Tilman, 1987, 1989; Grime, 1988; Thompson and Grime, 1988; Grace, 1990, 1991). A series of experiments can be conducted using type-C and - A plants to test certain predictions on competition made by both Grime and Tilman. Ornduff (1966) recognized the existence of at least three morphological races within L. californica. Greenhouse studies done over the past two years have demonstrated that both races are extremely plastic. Plants growing under artificial light in growth chambers were much shorter, more highly branched, and more robust than plants growing in either the greenhouse or in the field. Greenhouse plants are phenotypicalfy similar to ones in the field, even though they tend to be somewhat taller with fewer branches and leaves. When plants from one environment are moved to another, the growth habit changes appreciably within a few weeks. It will be of interest to determine if the two races are different with respect to their plasticities to different environmental conditions. Such studies can shed light on the role phenotypic plasticity may have played in the racial differentiation of this species. 103 The studies proposed to be undertaken in areas of soil chemistry, soil-plant interactions, reproductive biology, ion-uptake physiology, and ecology will undoubtedly shed light on many unresolved mysteries of L. californica. 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