Genetic Variation and Inbreeding Depression in the Rare California Endemic, Astragalus Agnicidus (Leguminosae)

Genetic Variation and Inbreeding Depression in the Rare California Endemic, Astragalus agnicidus (Leguminosae)

Robin Bencie

A Thesis Presented to the Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree Master of Arts

December, 1997 GENETIC VARIATION AND INBREEDING DEPRESSION IN THE RARE CALIFORNIA ENDEMIC, ASTRAGALUS AGNICIDUS (LEGUMINOSAE)

Robin Bencie

We certify that we have read this study and that it conforms to acceptable standards of scholarly presentation and is fully acceptable, in scope and quality, as a thesis for the degree of Master of Arts.

Michael R. Mesler, Major Professor

Mich J. Bowes, Commit tee ember

Timothy E. Lawlor, Committee Member

Andrea J. Pickart, Committee Member

John O Sawyer, Committee Member

Milton J. Boyd, Graduate Coordinator

Linda A. Parker Dean for Research and Graduate Studies ABSTRACT

Predictions of low genetic variation and low levels of inbreeding depression in small plant populations were tested on Astragalus agnicidus, a species with only one known population. Loss of alleles through drift and increased inbreeding (followed by selection) may leave small populations genetically depauperate, but with negligible genetic load as well. This scenario results in low levels of inbreeding depression as selfed and outcrossed progeny become more equivalent in overall fitness (i.e., the difference in progeny fitness between pollination treatments decreases). As predicted, gel electrophoresis of isozymes indicated that A. agnicidus has low genetic variation. Five fitness variables: seed set, seed weight, germination, survival, and seedling weight were used to test for fitness differences between open-pollinated and self-pollinated progeny. Some degree of fitness reduction for selfed progeny was seen in all variables, but only in seedling survival was the difference significant. Results indicated that the effect of the maternal parent was more important than pollination treatment on determining the fitness of an individual. Therefore, there is low enzyme diversity within the population, but genetic load is still maintained in the population and varies between individuals. The role of the mating system in influencing the level of genetic variation and inbreeding was also examined. One polymorphic enzyme, IDH, was used as the genetic marker in estimating an outcrossing rate of .59, which indicates a mixed mating system. The advantages of a mixed mating system include maintenance of moderate levels of heterozygosity (and thus, genetic variation) through outcrossing and elimination of genetic load and retention of optimum genotypes through selfing. Evolutionary models predict that A. agnicidus, with a high level of self-compatability and an inbreeding depression level of <50% would evolve towards a predominantly selfing mating system. However, A. agnicidus's crossing rate remains intermediate due to an obligatory, insect mediated, tripping mechanism that virtually assures some cross- pollination in the system.

iii AKNOWLEDGMENTS

I am grateful to those who supported me during the course of this project. Andrea J. Pickart, Area Ecologist for The Nature Conservancy, was instrumental in literally bringing this project, and thus graduate school, into my world. She secured significant grant funds from The Hardman Foundation and The Department of Fish and Game's Endangered Plant Program. We visited over statistics and the history of Astragalus agnicidus. My major professor, Michael Mesler, has always been my botany mentor, and his tremendous support and encouragement throughout this project is appreciated deeply. My fellow graduate students were =hesitantly willing to listen, and always understood (even Gordon, most of the time). The Biology Department's staff and faculty were very generous with their time and expertise. The Tosten family graciously gave me unlimited access to the site of A. agnicidus. And, most of all, I give thanks to plants.

TABLE OF CONTENTS

Page No.

I. ABSTRACT iii II. ACKNOWLEDGMENTS iv III. LIST OF TABLES vi IV. LIST OF FIGURES vii V. INTRODUCTION 1 VI. THE SPECIES 4 VII. OBJECTIVES 9 VIII. METHODS 11 Genetic variation 11 Mating system 11 Inbreeding depression 13 IX. RESULTS 16 Genetic variation 16 Mating system 19 Inbreeding depression 22 X. DISCUSSION 28 XI. CONCLUSION 33 XII. RECOMMENDATIONS 34 XIII. LITERATURE CITED 36 LIST OF TABLES

Page No. Table 1. Summary of gel electrophoresis data 18 Table 2. Allele frequencies, inbreeding coefficients, and results of tests for random mating 22 Table 3. Average values for progeny fitness variables ...... 24 Table 4. Results of pollination treatment and maternal parent on progeny fitness variables 26 Table 5. Relative performance (RP) values for maternal parents 27 Table 6. Genetic diversity comparisons between Astragalus agnicidus and other species with similar life history traits 30

vi LIST OF FIGURES

Page No.

Figure 1. Vicinity map of the Astragalus agnicidus site 5 Figure 2. Map of subpopulation locations 6 Figure 3. Electrophoretic banding patterns for IDH 18 Figure 4. Electrophoretic banding patterns for 6-PGD 20

vii INTRODUCTION

The only known population of the Humboldt milk-vetch, Astragalus agnicidus Barneby, provides one example of the possible genetic and fitness scenarios that can occur in plant populations with few individuals. Small, isolated populations are vulnerable to severe reductions in fitness due to inbreeding and loss of genetic variability (Barrett and Kohn, 1991; Ellstrand and Elam, 1993). Rare plants that occur as fragmented populations can be especially vulnerable to extinction, as any single population's success may be pivotal in preservation of the species. Yet in spite of the potential negative consequences of small population size, some rare plants maintain relatively high levels of genetic variability or exhibit low levels of inbreeding depression (Karron, 1987; Ellstrand and Elam, 1993; Richter, 1994). This study investigates the degree of genetic variability and the amount of inbreeding depression present in a rare, California endemic plant. The role of the mating system in maintaining genetic variability is also examined. My intention is to use this information to formulate conservation guidelines that will sustain, and potentially improve, the health of the population. This study also provides empirical evidence for testing general predictions made by theoretical models about the relationship between inbreeding depression, the selfing rate, and the evolution of plant mating systems (Lande and Schemske, 1985; Holsinger, 1988). After suspicions of it's toxicity to livestock provoked the systematic removal of all individuals from the site, A. agnicidus was presumed extinct (Berg and Bittman, 1988). Although the species later recovered from a dormant seed bank, the progressive decrease in the existing population size has made A. agnicidus highly susceptible to loss of genetic variability via genetic drift and mating between closely related individuals (biparental inbreeding). Genetic variation is considered crucial for long-term survival as it provides the necessary genetic foundation for adaptation to changing environmental conditions (Grant, 1991). Although some rare plants benefit from reduced genetic variation in the form of highly specialized genotypes, these relatively short-term benefits

1 2 may limit a population's ecological range and long-term genetic flexibility (Barrett and Kohn, 1991; Huenneke, 1991). Genetic drift can cause dramatic fluctuations in allele frequencies in small populations, leading to allele fixation in just a few generations. The rare and infrequent alleles that enhance genetic diversity, and that may ultimately code for traits that are necessary for survival in response to environmental change or crisis (i.e., disease or predation), face the greatest probability of extinction by virtue of their low frequency. While a single, undivided population can quickly lose alleles, A. agnicidus 's isolated subpopulations may offer some protection against loss of allele diversity as subpopulations randomly diverge (Hartl, 1987; Barrett and Kohn, 1991; Grant, 1991; Ellstrand and Elam, 1993). In small populations, inbreeding can rapidly lead to the expression of lethal and deleterious alleles in homozygous genotypes, and a concomitant reduction in fitness and allele diversity. If a population can survive this initial inbreeding depression that comes with purging detrimental alleles (genetic load), then the negative consequences of selfing are significantly diminished. Assuming A. agnicidus is a self-compatible species that has existed historically as a small population, then a low level of genetic variability, genetic load and inbreeding depression would be expected. However, if this population has only recently experienced a substantial reduction in size (bottleneck), then the level of genetic load and potential for inbreeding depression may still be high and pose a significant threat to the above ground population (Barrett and Kohn, 1991). The amount of inbreeding depression has been considered one of the primary factors influencing the evolution of plant mating systems. Genetic models have shown that when the fitness level of selfed progeny is < 50% of the fitness level of outcrossed progeny (high inbreeding depression), a predominantly outcrossing mating system will be maintained (Fisher, 1941; Lande & Schemske, 1985). Alternatively, if the fitness level of selfed and outcrossed progeny is more equivalent (little or no inbreeding depression), then there is selection for a predominantly selfmg mating system. This threshold is based on the greater number of alleles contributed to the population from 3 selling individuals and the assumption that the selfing rate evolves polygenically in small steps. In small populations, the fitness level of selfed and outcrossed progeny eventually becomes more similar over time as deleterious alleles are eliminated via inbreeding and selection. Thus, the frequency and severity of bottlenecks are highly influential in determining the rate and direction of mating system evolution, as well as, the level of genetic diversity within the population (Ledig, 1986; Charlesworth and Charlesworth, 1987). If A. agnicidus is genetically depauperate and thus exhibits little inbreeding depression, then we would expect its mating system to be, or eventually approach, predominantly selfing. However, regardless of the amount of inbreeding depression, some self-compatible, animal-pollinated legume species have morphological characters that virtually assure some degree of outcrossing, thus sustaining a mating system of both selfing and outcrossing (mixed mating) (Arroyo, 1981). The advantages of mixed mating systems include the maintenance of moderate heterozygosity levels (and thus genetic diversity) through outcrossing, in addition to, elimination of genetic load and retention of optimum genotypes through selfing (Schemske and Lande, 1985). THE SPECIES

The population of A. agnicidus is restricted to a small ridge just northwest of Bear Buttes in southern Humboldt county, California (Figure 1). According to the landowner, clumped subpopulations were originally distributed across the ridge in a mosaic pattern, interspersed amongst widely spaced old Douglas-fir trees (Tosten, personal communication). In the 1930s, death of the landowner's sheep was attributed to the toxic effects of the milk-vetch (hence the specific epithet). Consequently, the population was vigorously attacked by the family until it disappeared soon after R. C. Barneby's only site visit in 1954. Initially the milk-vetch was suspected to be a weed introduced from the Central Valley, but later Barneby determined that A. agnicidus is an endemic species closely related to A. umbracticus of the Klamath region and A. congdonii from the cismontane foothills of the southern Sierra Nevada (Barneby, 1957). Both of these species are also rare and occur as small, disjunct populations. In 1987, A. agnicidus was rediscovered at its historic site in areas that had been heavily disturbed by selective logging two years previously (Berg & Bittman, 1988; Pickart et al., 1992). The seedlings emerged from a dormant seed bank, which is common for many members of Leguminosae since their impermeable seed coats allow seeds to remain viable in the soil for many years (Baker, 1989). The initial cohort of individuals occurred in four different areas at the site (Figure 2). But due to a high mortality rate, the population currently exists as two principal subpopulations (A and C) and numerous scattered individuals. Subpopulation A is composed entirely of adult reproductive individuals from the initial cohort. The original individuals located at subpopulation C died in 1992, but in 1993, there was a flush of new seedlings that provided a new cohort of juvenile, non-reproductive individuals during the period of data collection. As these remaining subpopulations have matured, the number of individuals has decreased rapidly and there is a noticeable decrease in seedling regeneration (Pickart et al., 1992). Field observations suggest that A. agnicidus is a short-lived perennial with an estimated life span of 5-10 years. Mature plants are suffrutescent and produce many 4 5

Figure 1. Vicinity map for the single population of A. agnicidus. `*' at bottom middle is approximate location. Source: Humboldt, 1974. Figure 2. Map of A. agnicidus subpopulation locations at historical site. Capital letters are names of subpopulations; `__` = access routes. Map adapted from Steele, 1991. 6 7 long, often decumbent stems in a basal rosette fashion. Each stem produces several densely compacted inflorescences that arise singly from the leaf arils and range in flower number from 10-40. Flower morphology is typical of papilionoid legumes with each averaging 13 mm in length and 3 mm in width. Flowering begins in early May and fruits are mature by late August. Fruits contain from 1-8 seeds each and are vulnerable to predation by caching rodents. A specimen voucher is deposited at the Humboldt State University Vascular Plant Herbarium (HSC). Since the reappearance of A. agnicidus, the landowners and The Nature Conservancy of California (TNCC) have cooperated to protect this new population by constructing exclosures to prevent herbivory and facilitate research aimed at characterizing the milk-vetch's life history and habitat requirements. Laboratory work by Hiss (1991) concluded that seeds require a strict regime of scarification followed by stratification in order to germinate. In comparative growth experiments, seedlings exhibited optimum development when grown under maximum light conditions (Enberg, 1990). These results suggest that A. agnicidus is an early successional species that survives periods of inadequate growing conditions by remaining dormant in the soil seed bank until disturbance creates the necessary habitat conditions for population re- establishment (Baker, 1989). Apparently, the logging activities brought seeds to the soil surface, causing scarification of the seed coat and possibly a dramatic temperature change in the seeds' microclimate (Pickart et al., 1992). The gaps created in the canopy allowed the necessary light exposure for successful growth of the new population. Historical population dynamics are important in determining current genetic composition and fitness levels (Beardmore, 1983; Ledig, 1986). The ecology of A. agnicidus suggests that the population would tend to maintain itself at a small size through a recurring scenario of: 1) above ground disappearance due to deterioration of suitable habitat from tree encroachment, followed by 2) regeneration of seedlings and suitable habitat by disturbance. If this scenario is accurate and A. agnicidus has had a long history of bottlenecks, the population may indeed have low genetic variation, a minimal amount of inbreeding depression, and a predominantly selfing mating system. This proposed historical pattern also suggests that these current transient subpopulations 8 have genetic and fitness characteristics that are typical for previous and future generations. However, if this population has only recently become small and isolated, then high levels of genetic load, inbreeding depression, and outcrossing may exist. OBJECTIVES

The natural decline of the current population of A. agnicidus provokes the conservation question of whether or not to pursue an active role in sustaining the above ground, actively breeding population. Aggressive management plans could include the enlargement of the existing subpopulations and/or the establishment of new subpopulations with the goal of merging them into one interbreeding population. Or, separate subpopulations could be continually established and managed individually to increase genetic variation between subpopulations (Namkoong, 1983; Pickart et al., 1992). Alternatively, a passive management role would allow the dwindling population to revert back to a dormant seed bank until the next natural regeneration. Since rare species vary in their life histories, universally successful practices for conserving genetic diversity and reducing inbreeding depression are difficult to conceive (Holsinger and Gottlieb, 1991). Results of this study can be used as a model for species that possess a parallel history and similar reproductive characteristics to A. agnicidus, such as the many other rare Astragalus species. The emphasis for any management plan should be to prevent population decline and habitat loss, and to promote genetic diversity by counteracting the ultimate effects of drift and inbreeding (Barrett and Kohn, 1991). To develop a successful management plan that incorporates A. agnicidus's natural history, the following five objectives were pursued in this study: 1)Estimate the amount and distribution of genetic variation in the population. 2) Describe the mating system, including the degree of self-compatibility and the outcrossing rate. 3) Determine whether inbreeding depression is present at various stages in the life cycle. 4) Examine the change in frequency of genotypes from the seedling to adult stages in order to estimate the total amount of inbreeding depression in the field.

9 10 5) Compare the observed selfing rate, the level of inbreeding depression, and the mating system with predictions made by evolutionary models. METHODS

Genetic variation The amount of genetic variation in A. agnicidus was estimated using starch-gel electrophoresis. I used standard methods to screen 17 enzymes for scorable banding patterns. Extracts were prepared by grinding young leaflets in a phosphate extraction buffer (pH 7.5) intended for plants with moderate levels of phenols. Gels (12.4% potato starch by volume) were run for a minimum of 8 hours to clearly separate allozymes. With the exception of one buffer system, all the recipes for grinding, gel, and electrode buffers and enzyme stains were taken from Soltis et al. (1983). An electrophoretic survey of all individuals in the population was conducted in June 1992. Genetic interpretations of banding patterns were made based on the quaternary structure, subcellular compartmentalization, and the frequency of banding patterns for each enzyme (Weeden and Wendel, 1989; Wendel and Weeden, 1989). At each putative locus, the most anodal allele was designated "a", with successively slower alleles labeled in alphabetical order. Loci were considered polymorphic if the frequency of the dominant allele was less than 99%. For enzymes with more than one isozyme (variant forms of an enzyme found at separate loci), the most anodal isozyme was labeled "1" and successively slower isozymes were labeled in numerical order. The proportion of polymorphic loci (P), the average number of alleles per locus (A), and the observed heterozygosity (H) were estimated as standard measures of genetic diversity (Brown and Weir, 1983). Wright's hierarchical F-statistics were used to estimate the degree of genetic differentiation between subpopulations (Fst), and the excess in homozygosity within the entire population that is attributable to non-random mating and population substructure (Fit) (Wright, 1965; Hartl, 1987).

Mating system The initial steps in examining the mating system were to determine: 1) whether A. agnicidus is self-compatible, and 2) whether flowers can self-pollinate without insect visitation. In many papilionoid legume flowers, the stigmatic cuticle must be broken by

11 12 a foraging insect so that pollen can adhere and germinate on the stigma (Heslop and Harrison, 1983). The cuticle is ruptured when an insect depresses ("trips") the bottom keel petal, causing the stigma to strike its abdomen. To evaluate the degree of self- compatibility and obligatory tripping, I compared the fruit set of hand-tripped and unmanipulated flowers. On 15 individuals, one inflorescence was chosen to receive each treatment. Inflorescences were kept enclosed in lightweight, netted bags to exclude pollinators. On the manipulated inflorescences, all open flowers were tripped every other day. Fruits were collected at maturity, before dehiscence. The outcrossing rate (t) was estimated using open-pollinated array analysis and the joint maximum likelihood estimator assuming a mixed mating model (Brown et al., 1975; Ritland, 1983). All eight reproductive homozygous individuals from subpopulation A were selected as the maternal parents for the analysis. Three inflorescences from each mother were randomly selected during the early flowering period of mid May. In late August, ten mature pods were collected from the basal end of each inflorescence and all the seeds from a single mother were combined into one bulk sample. From each of these eight samples, 40 seeds were germinated using the methodology of Hiss (1991). Scorable banding patterns were obtained for 291 seedlings. All heterozygous progeny were undisputedly a result of cross-pollination and provide a minimum estimation of the outcrossing rate. Theoretically, an estimated outcrossing rate of < 1.0 indicates a departure from random mating. To determine whether t is significantly less than 1.0, I used the following two tests: 1) a standard one-tailed t-test to compare the deviation of a sample statistic from a parametric value, and 2) a Chi-square goodness of fit test to compare the observed progeny genotypes to the expected (Sokal and Rohlf, 1981). The expected progeny genotype proportions were determined using the mixed mating model with complete outcrossing (t=1.0) and pollen pool allele frequencies estimated directly from the progeny (Clegg, 1981). Additionally, the inbreeding coefficient (F=1-H/2pq) was used to estimate the population's deviation from the heterozygosity level expected under random mating (F=0). A deviation generally indicates either inbreeding (F0) or natural selection 13 favoring heterozygotes (F<0) (Ritland, 1990; Barrett and Kohn, 1991). To determine whether the F within each subpopulation represents a significant departure from the Hardy-Weinberg random mating model, I used a Chi-square test to compare observed genotypes with those expected and a standard one-tailed t-test to compare a sample statistic to a parametric value (Hard, 1987; Sokal and Rohlf, 1981).

Inbreeding depression To assess the level of genetic load, I compared the fitness levels of open- and self-pollinated progeny at several stages of the plant's life cycle. Six inflorescences from each of 25 individuals in subpopulation A were randomly chosen to receive open- and self-pollination treatments. Three inflorescences were tagged but left unmanipulated as the open-pollinated treatment. The remaining three inflorescences were bagged and the flowers tripped by hand for the self-pollinated treatment. This sampling design allowed comparisons to be made both between treatments and between mothers. When all flowers on an open-pollinated inflorescence had senesced, the inflorescence was bagged to prevent herbivory. Inflorescences were collected after seed maturity in late August 1992. I attempted a third treatment of cross-pollination only, but this was forfeited due to the abortion of fruits in emasculated flowers, the likelihood of self-pollen contamination within the protandrous flowers, and uncertainty in the timing of stigma receptivity. The fitness variables used to quantify the amount of inbreeding depression were: percent seed set, seed weight, percent germination, percent survival, and seedling biomass after 13 weeks. For percent seed set, I tallied the number of aborted ovules and mature seeds per fruit, resulting in a sample of 383 pods distributed across 13 mothers. All seeds were then placed into bulk samples according to treatment and mother. For each of the remaining fitness experiments, seeds were randomly chosen from these bulk samples. To test for a difference in seed weight, 256 seeds representing 13 mothers were weighed individually to the nearest tenth of a milligram with a Mettler H80 scale. A single replicate germination test was conducted using a total of 932 seeds from 22 mothers. To induce germination, seeds were first scarified in sulfuric acid until the seed 14 coat was sufficiently eroded (30-50 min) and then placed in a cold room at 2 C° (35 F) for 3 weeks. A separate sample of 260 seeds was grown out in a greenhouse during the spring of 1993 for use in the percent survival and seedling biomass test. In February, seeds were inoculated with nitrogen fixing bacteria and planted in a standard Cornell soil mix consisting of 1/2 peat moss, 1/4 vermiculite, and 1/4 perlite. Two seeds per treatment were planted in 10.2 cm (4 in) pots to mimic competitive conditions encountered in the field. Plants were fertilized every other week with a diluted solution of Schulz-Instant Liquid Plant Food. A plant was considered a survivor if it appeared robust and capable of continued growth on July 1. These individuals ranged from 9-13 weeks of age depending on the date of germination. All pots with four surviving individuals at 13 weeks of age were used in the seedling biomass comparison. These plants were dried and the aboveground biomass weighed to the nearest tenth of a milligram. I attempted to collect data for percent fruit set, however, the apparent lower fruit set in the self-pollinated treatment was confounded by the possibility that some fruit abortions resulted from errors in the timing of flower tripping rather than from genetic load. A second trial was conducted the following field season, but nearly all the open- pollinated inflorescences were taken by rodents. Differences in percent seed set, seed weight, and seedling biomass were analyzed using the NCSS 5.03 program for a two-way, mixed model ANOVA. Open- and self- pollination were the fixed treatments, while maternal parent was the random effect. A one-way ANOVA was used for the percent germination test since the maternal effect could not be evaluated with only a single data point per mother. Percent survival was analyzed using a Chi-square test for independence of attributes. Cumulative fitness (W) of the progeny from each pollination treatment was estimated by the product of the average values for each fitness variable (Montalvo, 1994). The ratio of W for the self- pollinated progeny to the open-pollinated progeny was subtracted from 1.0 (equal fitness between treaments) to determine the total amount of inbreeding depression. To further evaluate maternal effects, I followed a method by Agren and Schemske (1993) that compares the relative fitness of self- to open-pollinated progeny 15 between mothers. First, W was separately estimated for the self- and for the open- pollinated progeny from each mother that was sampled for all five fitness variables (n=9). For each mother, the cumulative relative performance (RP) of progeny was determined by either: 1) subtracting the ratio of W for the self- to the open-pollinated progeny from 1.0 when the fitness of the selfed progeny was less than the open- pollinated progeny, or 2) subtracting 1.0 from the ratio of W for the open- to the self- pollinated progeny when the fitness of the selfed progeny was greater than the open- pollinated progeny. Thus, RP values ranged between +1 (open-pollinated progeny outperform self-) and -1 (self-pollinated progeny outperform open-). A one-sample t-test was then used to evaluate whether this sample of cumulative RP values was significantly different than 0, which would indicate equal fitness between treatments. I also calculated RP values for each fitness variable per mother and examined the change in progeny performance between the stages of maturation. The level of inbreeding depression within subpopulation A was separately estimated using a method that utilizes the selling rate (s) and the adult inbreeding coefficient (F) to determine the relative fitness of self- to open-pollinated progeny (w) (Ritland, 1990). An assumption in this analysis is that subpopulation A is in inbreeding equilibrium, i.e., the adult F eventually returns to a constant value, typically due to the opposing effects of the selfing rate (increases F) and selection against self-pollinated progeny (decreases F). Finally, I examined the change in F between the progeny of subpopulation A and each of the subpopulations (A and C) to estimate the total amount of inbreeding depression for a single cohort over time. Presumably, the genotype compositions found in the seedlings from subpopulation A, the juvenile subpopulation C, and the adult subpopulation A are representative of an average cohort in three distinct life cycle stages. For this evaluation to be reasonable, it is assumed that each of the mating populations that gave rise to these factions had equivalent outcrossing rates and allele frequencies. RESULTS

Genetic variation Nine enzymes were resolved using the following four buffer systems: 1) sodium- borate/Tris-citrate: glutamate oxaloacetic transaminase (GOT) and phosphogluco- isomerase (PGI); 2) lithium-boratearis-citrate: aldolase (ALD), colorimetric esterase (CE), leucine aminopeptidase (LAP), and triose-phosphate isomerase (TPI); 3) histidine- citrate: isocitrate dehydrogenase (IDH) and 6-phosphogluconate dehydrogenase (6- PGD); and 4) discontinuous Tris-citric acid (Mitton et al., 1977): malate dehydrogenase (ME). Banding patterns were consistently unresolvable for aconitase (ACO), alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), peroxidase (PRX), phosphoglucomutase (PGM), shikimate dehydrogenase (SKDH), and superoxide dismutase (SOD). A total of 16 putative gene loci and 19 alleles were identified (Table 1). Single- banded patterns for GOT, ALD, and CE indicate enzymes that are fixed for a single allele (monomorphic). The banding patterns for the remaining six enzymes can be most parsimoniously explained if I assume that each enzyme has more than one gene locus. A consistent two-banded pattern for LAP, PGI, and ME indicates two monomorphic isozymes. The invariant five-banded pattern exhibited by TPI suggests three monomorphic isozymes in which the two homozygous alleles at each locus, in addition to producing a single band, form a hybrid band (intergenic heterodimer) of intermediate mobility with the homozygous alleles at the other loci. The hybrid band formed by TPI-1 and TPI-3 has the same mobility as the allele at TPI-2 (Wendel and Weeden, 1989). For IDH, analysis of progeny arrays from open- and self-pollination treatments confirmed the presence of two isozymes, one with two alleles (polymorphic, IDH-1) and one monomorphic (IDH-2). Genetic interpretations of the banding patterns for this dimeric enzyme are similar to those given by Odrzykoski and Gottlieb (1984, Fig. 1D) and are presented here in Figure 3. An individual homozygous at both loci had a three- banded pattern, with the intermediate band representing an intergenic hybrid (1a2a or 1b2a). Individuals heterozygous at IDH-1 exhibited a five-banded pattern due to: 1) the

16 17 Table 1. Summary of gel electrophoresis data for Astragalus agnicidus. Buffer systems are encoded as follows (electrode/gel): A: sodium-borate/Tris-citrate, B: lithium- borate/Tris-citrate, C: Trisodium citrate/histidine, D: discontinuous Tris-citric acid.

Enzyme System No. plants assayed* # loci # alleles AID B 50 1 1 CE B 51 1 1 GOT A 54 1 1 IDH C 110 2 3 LAP B 72 2 2

ME D 66 2 2 PGI A 60 2 2 TPI B 72 3 3

6-PGD C 47 _2_ 4 Totals 16 19 *Sample sizes varied due to poor resolving of some individual banding patterns and the appearance of many new seedlings when the IDH enzyme was surveyed in 1993. 18

Figure 3. The three electrophoretic banding patterns resolved for IDH. 19 single band produced by each of the three alleles (la, lb, 2a), 2) the intragenic hybrid band formed by the alleles at the IDH-1 locus (1a1b), and 3) the intergenic hybrid band formed by the allele at IDH-2 and the slow allele at IDH-1 (1b2a). The slow allele at IDH-1 (lb) and the hybrid band formed between IDH-1 and IDH-2 (1a2a) have identical mobility. This enzyme provided an easily interpretable marker locus for evaluation of the population genetic structure and the outcrossing rate. The complex multi-banded patterns exhibited by 6-PGD could not be interpreted unequivocally. For the purposes of estimating genetic variation, a conservative approach was adopted and 6-PGD was assigned two loci with two alleles each. An alternative interpretation of two loci with five alleles may be conceivable, but two specific intragenic hybrid bands expected under this interpretation were not observed. Poor resolving of intermediate bands located between closely adjacent parental bands is one explanation for the absence of these phenotypes. The 11 variant electromorphs of 6-PGD and the possible genotypes based on an enzyme system with two polymorphic loci and five alleles are shown in Figure 4. Three of the 16 putative loci were polymorphic, yielding P = .188. The average number of alleles per locus was 1.19. Allele frequencies for each of the population factions are given in Table 2. Although 19% of these loci are polymorphic, an H = .031 suggests that the average individual is heterozygous at only 3% of its loci. Since I could not include the 6-PGD loci in the calculation of H due to the uncertainty of genotypes, the actual level of heterozygosity in the population is possibly much higher than my estimate. Considering only IDH-1, the genetic divergence between subpopulations is negligible (Fst = 0.02) and the total excess in homozygosity within the population is low (Fit = 0.14).

Mating system All fruits from the unmanipulated flowers were consistently aborted, whereas fruit set for hand-tripped flowers was > 90%. This demonstrates that insect visitation is necessary for fruit set in A. agnicidus, even though plants have a high degree of self- compatibility. Tripped flowers generally senesced within two days after manipulation, Figure 4. The 11 electrophoretic banding patterns resolved for 6-PGD. The genotypes given are based on a putative two loci, five allele system. In estimating genetic variation for A. agnicidus, 6-PGD was conservatively assigned two loci with two alleles each. 2 0 Table 2. Allele frequencies, inbreeding coefficients, and the results of tests for random mating at IDH-1 for A. agnicidus. P-value is the probability of obtaining this sample if the null hypothesis is true. `*' = rejection of the null hypothesis of random mating (F = 0).

Population Size Allele frequencies Inbreeding coefficient Chi-square i-t a b F = 1 - (HI2pq) P-value t-testvalue

Entire N = 110 .53 .47 .14 .187 .142 (Subpop. A and C) Subpop. A N = 58 .46 .54 .06 .710 .657 (adults) Subpop. C N = 52 .62 .38 .18 .250 .206 (juveniles) Progeny N = 291 .46 .54 .40 < .001 * < .001* (of Subpop. A) 21 22 while unmanipulated flowers remained open for up to ten days. This delayed senescence appears to be a mechanism for facilitating cross-pollination, as flowers apparently "wait" for a pollen vector. The most common visitor to A. agnicidus flowers is a native Bombus species that forages for both nectar and pollen. The bees approach an inflorescence from the base and systematically visit all open flowers in an upward spiral before traveling to the next adjacent inflorescence, which is frequently on the same plant. This visitation pattern presumably results in a high level of geitonogamy, or self-pollination via transfer of pollen between flowers on the same plant (de Jong et al., 1993). If a maturing flower has not quite reached anthesis, these bumblebees will pry the banner petal open to gain access. Occasional visits from honeybees and syrphid flies were also observed. Progeny of subpopulation A had a high deficiency of heterozygotes relative to random mating expectations (F=.40), which resulted in an outcrossing rate of t = 0.59 ± .05 (s.e.). Both the Chi-square test and the t-test confirmed that the outcrossing rate is significantly less than 1.0 (P<.001, Table 2). This data, provided by the progeny at the seedling stage of development, indicate non-random mating and a relatively high selfing rate (s = .41). However, the genotype composition of both subpopulations is consistent with predictions from the random mating model (Table 2). The degree of fit to the model is better for subpopulation A than for subpopulation C (P=.710 vs P=.250). The juvenile subpopulation C has a greater excess of homozygotes (F=.18) compared to the adult subpopulation A (F=.06), given the allele frequencies observed within each subpopulation.

Inbreeding depression For each fitness variable, the mean for the open-pollinated treatment was greater than the mean of the self-pollinated treatment (Table 3). However, percent survival was the only variable that indicated selfed progeny suffer a significant reduction in fitness (Table 4). The mortality in both treatments was greatest during the first two weeks of seedling development, with the majority of deaths occurring before seedling emergence. Since the difference in percent germination between treatments was not significant 23

Table 3. Average values for progeny fitness variables (standard error) from pollination treatments.

Fitness Variable Open-pollination Self-pollination Seed set (%) 84.9 (2.1) 82.1 (2.9) Seed weight (mg) 2.0 (.04) 1.8 (.04) Germination (%) 94.3 (2.0) 88.9 (3.5) Survival (%) 83.1 (3.1) 69.5 (4.2) Seedling biomass 33.7 (3.3) 29.1 (3.3) (mg) 24

Table 4. Results of the effects of pollination treatment and maternal parent on progeny fitness variables for A. agnicidus. For Treatment, total progeny sample sizes are given as 'open-pollinated, self-pollinated'. For Mother, sample sizes are given as the total number of mothers (N) and the range in number of progeny sampled per mother. `*' = Rejection of the null hypothesis (no difference in fitness effects) with P< .10; `**' = rejection with P< .01; '***' = rejection with P< .001.

Variable Effect Sample size F value P-value

Seed set (%) Treatment 249, 134 .52 .487 Mother (13) 18 - 44 1.73 .058 * Interaction 2.71 .002 ** Seed weight Treatment 162, 162 2.85 .117 (mg) Mother (13) 12 - 32 49.91 .000 *** Interaction 8.83 .000 *** Germination Treatment 469, 463 1.76 .192 (mg) (22 mothers) (Total # seeds) Survival (%) Treatment 142, 118 6.72 .009 *** (18 mothers) (Total # seedlings) (X2 value) Seedling Treatment 38, 38 .74 .411 biomass Mother (10) 4 - 20 1.34 .236 (mg) Interaction .52 .853 25 (P=.19), seedlings appear most susceptible to death after germination, but before emergence. Cumulative fitness estimates (W) for self- and open-pollinated progeny indicate a 41% reduction in overall fitness for self-pollinated progeny. The effect of pollination treatment on progeny fitness is dependent upon maternal parent. The ANOVA results show significant maternal and interaction effects for percent seed set and seed weight (Table 4). Self-pollinated progeny display varying degrees of fitness reduction dependent upon the maternal parent and the fitness variable examined (RP value is positive), but, self-pollinated progeny also outperform open-pollinated progeny to varying degrees dependent, again, upon mother and fitness variable (RP value is negative) (Table 5). Furthermore, within a single mother, RP values fluctuate between positive and negative depending on the fitness variable examined. The progressive increase in the range of RP values with each fitness variable indicates that maternal effects become more pronounced with progeny age (Table 5). Thus, whether inbreeding depression is experienced in a particular stage of progeny development depends upon the maternal parent. Three mothers (A14, B7, and B4) had open-pollinated progeny with cumulative fitness levels greater than twice that of their self-pollinated progeny, whereas two mothers (A2 and A24) had self-pollinated progeny that were nearly twice as fit as their open-pollinated progeny. The remaining four mothers had self-pollinated progeny with cumulative fitness levels that were within 25% of their open-pollinated progeny (Table 5). Oddly, cumulative RP values were not significantly different than 0 (P=.19), although the wide range (+ .75 to - .49) suggests a strong maternal and interaction effect. Generally, the RP values for the separate fitness variables do not reflect the magnitude of the results seen in the ANOVA analyses. The wide range of RP values for seedling biomass suggests that maternal effects are most severe in the latter stages of progeny development, which is contradictory to the insignificant effects of the maternal and interaction factors from the ANOVA analysis. The RP values tended to be lowest for the seed set and seed weight variables, which also contradicts the significant random effcts from the ANOVA analyses. Although the Chi-square test for percent survival Table 5. Relative performance (RP) values for the nine mothers sampled for all five fitness variables. Positive values indicate open-pollinated progeny outperform self-pollinated progeny; negative values indicate self-pollinated progeny outperform open-pollinated progeny.

Maternal Parent A2 A13 A14 A24 B4 B5 B7 B9 Range Variable

Seed set +.13 +.07 +.32 +.04 -.02 +.03 -.09 +.29 +.14 -.09 to +.32 Seed weight +.10 -.22 +.05 -.15 +.18 +.25 -.22 29 +.14 -.22 to +.29 Germination -.21 -.07 0.00 0.00 -.05 +.13 +.14 -.20 +.53 -.21 to +.53 Survival -.21 -.17 -.02 +.10 -.13 -.03 +.31 +.60 -.17 -.21 to +.60 Seedling biomass -.37 +.46 +.62 -.42 +.19 +.32 -.25 +.29 +.25 -.42 to +.62 Cumulative -.49 +.17 +.75 -.44 +.17 +.55 +.10 +.72 +.25 -.49 to +.75 26 27 could not separate the effect of maternal parent from treatment, the RP values indicate a strong maternal influence, particularly for mother B7. Utilizing s and F from the adult subpopulation A, the relative fitness of self- pollinated progeny is w = .18. This estimate suggests that over the life of one generation, selfed progeny suffer an 82% reduction in cumulative fitness (Ritland, 1990). A high level of inbreeding depression is also reflected in the progressive decrease in F from the progeny (F=.40), to the juvenile subpopulation C (F=.18), to the adult subpopulation A (F=.06). DISCUSSION

A. agnicidus has less genetic variation (P, A, and H) than the average for species with similar life-history traits (Table 6) (Hamrick and Godt, 1990). As expected, its level of genetic diversity is also less than some widespread species of Astragalus (Karron, 1988). The genetic diversity of A. agnicidus is comparable to rare A. osterhouti, but is considerably lower than rare A. linifolius (Karron, 1988). Although H is relatively low for A. agnicidus, I suspect that the actual heterozygosity level is greater than my estimate given the number of different banding patterns shown at the 6-PGD loci. The disparites amongst these rare species may reflect differences in population histories, or, that some factor promotes genetic diversity despite the consequences of small population size. Genetic divergence between the subpopulations of A. agnicidus is negligible and considerably lower than the averages for species with similar life-history traits (Table 6). Unexpectedly, this low Fst value is similar to that of widespread congeners and is less than that of other rare Astragalus species (Karron, 1988). Low Fst values are predicted for widespread, interbreeding populations, but not for small, fragmented populations that presumably should experience random and rapid changes in allele frequencies. The recent and evolutionary history of A. agnicidus is reflected in the amount and distribution of its genetic variation (Beardmore, 1983; Ledig, 1986; Barrett and Kohn, 1991). The low genetic diversity within the entire population suggests that the species has existed historically as a small, isolated population. A long history of genetic drift and increased inbreeding has resulted in a high level of allele fixation at enzyme loci. Low genetic diversity between subpopulations indicates that the segregation of subpopulations is recent, the isolation time being insufficient to generate divergence in allele frequencies. These extant subpopulations were once part of a single, interbreeding population in which there was an absence of large genetic neighborhoods. Given the low genetic diversity and long history of isolation, the level of genetic load is expected to be negligible in this population that presumably has already purged unfavorable alleles through increased inbreeding (Barrett and Kohn, 1991). However,

28 29

Table 6. Genetic diversity comparisons between A. agnicidus, rare congeners, and species with similar life history traits. P = % polymorphism, A = # alleles per locus, H = heterozygosity, Fst = fixation index.

P A H Fst

A. agnicidus 18.8 1.19 0.031 0.02 A. HhoutiFst 16.7 1.25 no data 0.14 A. linifolius 33.3 1.42 no data 0.09 Endemic 26.3 1.39 0.063 0.248 Animal- 29.2 1.43 0.09 0.216 pollinated Early 29.6 1.39 0.107 0.289 successional

Sources: Karron, 1988; Hamrick and Godt, 1990. 30 the lower cumulative fitness for inbred progeny and the variable progeny fitness levels amongst maternal parents suggests that, despite the low isozyme variation, there is genetic load at loci directly affecting fitness. In fact, the actual level of genetic load in the population is likely to be greater than the results of my progeny array analyses given the presence of selfed progeny in the open-pollinated treatment. The comparison between open- and self-pollinated progeny gives a lower estimate of inbreeding depression than would be observed between selfed progeny and strictly outcrossed progeny (Holtsford, 1990). Presence of genetic load also explains the decrease in homozygosity levels from the seedling to the adult stages (Ritland, 1990). The wide range of inbreeding depression estimates and some possible bias in the analyses prevent a precise estimate of the average loss of fitness within a generation. If the proportion of heterozygotes and the selfing rate remain constant across adult generations, a severe level of inbreeding depression is expected (82%). Presumably, this high level of genetic load would have been identified by significant treatment effects. Thus, this inbreeding depression estimate seems inaccurate, probably due to fluctuations in the selfing rate between generations (Ritland, 1990). Fitness variable means indicate a cumulative reduction in fitness for selfed progeny (41%) that is only half the intensity that Ritland's equation predicts. However, favorable environmental conditions in the greenhouse and lab could have resulted in higher mean values for survival, biomass, and germination variables than would have been observed under natural habitat conditions (Montalvo, 1994). A more accurate measure of inbreeding depression could be obtained with field studies that track individuals and their progeny through development and reproduction. A significant amount of the variation in progeny fitness is attributable to maternal and interaction effects. If differences in progeny performance are due to heritable genetic traits, instead of the maternal parent's environment, then adults must possess a considerable variety of alleles with a wide range of adaptive value (Roach and Wulff, 1987). Maternal parents that harbor high levels of genetic load produced selfed progeny with lower fitness than their outcrossed progeny (positive RP). Mothers with little genetic load had selfed progeny that outperformed their outcrossed progeny (negative 31 RP). Negative RP values may indicate genetic structure, as progeny out-crossed with pollen from outside a genetic neighborhood may have lower fitness than progeny that are inbred (Price and Waser, 1979). Limited dispersal of seeds via the caching behavior of rodents supports the potential for small scale genetic structure representing family groups within the subpopulations. The differing RP values within a single mother, across fitness variables, reflect the degree of genetic load at separate loci. A similar combination of high selfing rate (and presumably low genetic diversity), inbreeding depression, and significant maternal and interaction effects has been found in other small plant populations (Holtsford, 1990; Agren and Schemske, 1993; Hamilton, 1994). As with A. agnicidus, these unexpected high levels of genetic load existing within small, inbreeding populations are consistent with hypotheses of polygenic mutation or high rates of mildly deleterious mutations (Lande and Schemske, 1985; Hamilton, 1994). If a high rate of gene mutation is the principal factor influencing genetic load, then every generation is vulnerable to significant detrimental effects from inbreeding. By maintaining heterozygosity levels, the amount of inbreeding depression experienced by each generation would be reduced as deleterious alleles remain unexpressed. The pollinator tripping mechanism virtually guarantees a minimum level of cross- pollination, and thus, a high probability of heterozygotes in all developing fruits. However, the realized outcrossing rate, and thus the level of heterozygosity, will fluctuate depending on pollinator behavior and abundance, and changes in the plant's population size and density (Brown et al., 1989). As the population size of A. agnicidus has decreased, the corresponding increase in each plant's size and number of inflorescences has likely increased the selfing rate through elevated levels of geitonogamy (de Jong et al., 1993). Thus, the subsequent increase in homozygosity has the potential to result in maximum inbreeding depression as accumulated mutations are purged. The tripping mechanism acts as a safeguard for minimum levels of heterozygosity in this highly self-compatible plant, but the success of the mechanism in promoting heterozygosity and reducing inbreeding depression is dependent on the density of individuals and inflorescences. 32 The tripping mechanism ensures that despite the relative fitness of inbred and outcrossed progeny, A. agnicidus maintains a mixed mating system. The cumulative fitness reduction for self-pollinated progeny (41%) is less than the theorized 50% threshold that is required to maintain outcrossing and prevent a mating system from evolving towards selfing. Theoretically, the mating system of A. agnicidus should evolve towards selfing. However, the obligatory nature of the tripping mechanism prevents the mating system from becoming predominantly selfing. Thus, the level of inbreeding depression is not a reliable predictor for the type of mating system that will evolve within a population. If mandatory tripping was lost in A. agnicidus, then the mating system may evolve toward predominantly selfing given: 1) the high degree of self-compatability, and 2) an equilibrium between the mutation rate and the inbreeding level that would result in inbreeding depression levels that are < 50%. Some Lotus species have bypassed the reproductive constraint of tripping by evolving an automatic rupturing of the stigmatic membrane without insect visitation (Arroyo, 1981). CONCLUSION

A. agnicidus is apparently able to maintain a certain level of genetic diversity, evident in the estimates of genetic load, even though its small population size and history of bottlenecks have resulted in low isozyme variation. Contrary to expectations, the low level of isozyme diversity does not predict the high levels of genetic load and inbreeding depression in the population. I suspect that A. agnicidus maintains its genetic diversity through an equilibrium between the rate of mutation, which generates diversity and effectively increases population size, and the rate of inbreeding, which purges genetic load through inbreeding depression. In turn, the level of inbreeding is controlled by the pollination system and the demographics of the population. The pollinator mechanism, (virtually mandating a mixed mating system) reduces inbreeding, maintains minimum levels of heterozygosity, and thus, reduces inbreeding depression. The mixed mating system protects the population from the complete expression of its genetic load.

33 RECOMMENDATIONS

Historically, A. agnicidus's population size was probably kept relatively constant by disturbances and environmental factors that would affect the entire small population and lead to either the growth or decline of the population (e.g., fire followed by tree encroachment). Patches of individuals were once widespread across the ridge, but close enough so that pollinators could easily facilitate inter-breeding within the entire population. A high realized outcrossing rate and a corresponding high level of heterozygosity would have reduced the expression of genetic load in previous generations. The significant reduction in population size, via the attempted eradication effort, likely disrupted a historical relationship between population size and inbreeding depression. That previous population's likely reduction in outcrossing rate and progeny heterozygosity, as compared to the many generations before, may have produced a seed bank that has now generated a population with an overall fitness that is lower than fitness levels of previous generations. Thus, the subpopulations may be experiencing a much greater rate of mortality than would be expected from cohorts derived from a large, interbreeding population. If the conservation goal is to ensure that the population can adequately maintain high levels of diversity and fitness, then increasing the population size is required. A. agnicidus's mixed mating system can most optimumly maintain heterozygosity, and the corresponding reduced level of inbreeding depression, with a large number of individuals. My suggestion is to increase the population size with the intent to merge subpopulations, and then allow the new, continuous population to reproduce at an increased outcrossing rate and decline through natural habitat deterioration. The timing of regeneration of future populations could be left to natural causes, or imposed at regular intervals depending on management resources. The interval between regenerations is less important than ensuring that each new population is large enough to promote high outcrossing rates. I do not suggest actively maintaining separate small

34 35 subpopulations since there is a high risk of allele fixation through drift, and intensive monitoring for inbreeding depression may be required. Obviously, each above ground population will cycle through increased inbreeding and inbreeding depression as it gradually decreases in size from loss of suitable habitat. However, genetic diversity and heterozygosity will have already been enhanced through many seasons of seeds deposited to the seed bank (Levin, 1990). Seed banks accumulate diversity not only by retaining individual genotypes from all generations, but also by generating novel genotypes via mutations that occur as seeds age (Levin, 1990). Seed bank studies have shown that allele frequencies can differ significantly between dormant seeds and adult plants (Tonsor et al., 1993). When increasing population size, consideration must be given to balancing: 1) the degree of retrieval of the dormant gene pool (seed bank) to augment current genetic diversity, with 2) preserving the seed bank to offer protection against future severe fluctuations in population size or environmental catastrophe. Before any population manipulation is conducted, the density and boundary of the seed bank should be examined further. Allowing the current population to naturally senesce back to a dormant seed bank will simply return A. agnicidus to an inactive state and poses no immediate threat to the species. However, recurring regenerations of small, isolated subpopulations could slowly erode the overall level of diversity and population fitness. Protection of pollinators and their habitat is necessary to ensure reproduction in A. agnicidus. In the event of pollinator scarcity, manual pollinations could be required to provide minimal fruit set. The importance of bee pollination in maintaining genetic diversity and heterozygosity has also been noted in small populations of a rare Delphinium (Richter et al., 1994). A. agnicidus provides a model population for exploring management techniques that increase genetic diversity in small populations of perennial species, which are both self-compatible and possess insect-mediated mixed mating systems. Additionally, this study may provide insight for conservation strategies involving the many rare, threatened, and/or endangered Astragalus species. LITERATURE CITED

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