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a Department of Agronomy and Range Science and Center for Population Biology, University of California Davis, One Shields Avenue, Davis, California 95616-8515; and b University of California Bodega Marine Laboratory, Bodega Bay, California 94923
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: endangered species • Fabaceae • genetic variation • population bottleneck • Trifolium albopurpureum • Trifolium amoenum • Trifolium macraei
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flower bagging experiments have demonstrated that T. amoenum is capable of self-pollination (Connors, unpublished data), and we conclude that the single wild plant discovered in 1993 must have produced seed through self-pollination. Seeds collected from plants in subsequent generations of seed multiplication may be the outcome of either self- or cross-pollination, as they were produced in groups of several plants that were visited by bumble bees. The resultant collection of seeds is exceptional in one genetic respect, however: the entire population has gone through a single-founder bottleneck. This raises the concern that genetic variation might be severely reduced compared with former levels, a condition that could adversely affect future attempts to reestablish wild populations.
To assess genetic consequences of the bottleneck, we evaluated allozyme variation within the Occidental T. amoenum population, intending initially to compare it with allozyme variation in natural populations of two closely related native annual species, T. macraei and T. albopurpureum var. dichotomum. Trifolium macraei is a primarily coastal species of northern and central California, but also grows in South America. Trifolium albopurpureum var. dichotomum occupies coastal to interior grasslands from central California north to Washington. In the process of collecting material of these two species, we discovered a previously unknown natural T. amoenum population of ~225 plants near Dillon Beach in Marin County, California, 16 km distant from the single individual found in 1993. This presented the unexpected opportunity to extend our planned among-species comparisons to include a within-species comparison of the single-founder population and the larger Dillon Beach population.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We resolved loci with standard starch gel electrophoresis techniques, using histidine-citrate, tris-citrate, or sodium-borate buffer systems (Table 1). Gels were made with 12% w/v Connaught brand starch. Staining followed recipes from Wendel and Weeden (1989), with the exception of ACO, which was stained according to Morden, Doebley, and Schertz (1987), and IDH, which was stained according to Soltis et al. (1983). Amylase (AMY) appeared as translucent bands on the gel stained for LAP, and genotypes at this locus were scored after storing the gel slice in a refrigerator at 4°C for 3 d. We inferred the genetic basis of banding patterns from known enzyme subunit structure and intercellular compartmentalization for these enzymes (Kephart, 1990). Loci for each stain and alleles at each locus were numbered sequentially, from fastest to slowest migrating. Using BIOSYS-1 (Swofford and Selander, 1989), we calculated genetic variability statistics and performed a cluster analysis on the matrix of genetic distances (Nei, 1978) between populations with the unweighted pair-group method using arithmetic averages (UPGMA). We considered a locus polymorphic if the frequency of the most common allele did not exceed 0.99.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.001) that any additional scorable variation at these loci went undetected.
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Allozyme variation among populations
Allozyme alleles found in the Occidental T. amoenum population were a subset of alleles present in the Dillon Beach T. amoenum population. The considerable allozyme differentiation between these two populations (FST = 0.225) was due mainly to fixation in the Occidental T. amoenum population of a PGI allele which was rare in the Dillon Beach T. amoenum population. (For comparison, the FST between T. albopurpureum var. dichotomum populations was 0.047, and the FST among T. macraei populations was 0.483.)
Allozyme variation among species
Cluster analysis by the UPGMA method demonstrated that all three species possessed similar allozymes (Fig. 2), suggesting that the species are closely related to each other. The average Nei's genetic distance between T. amoenum and T. macraei populations was 0.080, while an average Nei's genetic distance of only 0.030 separated populations of T. amoenum from populations of T. albopurpureum var. dichotomum. The mating system similarities indicated by the observed:expected heterozygosity ratios also suggest that these latter two species are the most closely related of the three.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Measures of overall genetic variation may not change appreciably unless a bottleneck is severe because the alleles most likely to be lost are generally in low frequency and therefore contribute comparatively little to measures of population variance (Frankel and Soulé, 1981). However, even alleles that are currently rare in a population may be of considerable evolutionary importance in the long term, particularly in a variable and changing selective landscape (Millar and Libby, 1991; Lesica and Allendorf, 1995).
The amount of genetic variation remaining following a bottleneck is a function of not just the severity of the bottleneck, but how rapidly the population size rebounds (Nei, Maruyama, and Chakraborty, 1975). However, population growth will do little for restoring alleles lost during a bottleneck (Nei, Maruyama, and Chakraborty, 1975; Barrett and Kohn, 1991). Over the longer term, rates of recovery of quantitative variation following a bottleneck may be much faster than recovery of allozyme variation, due to higher rates of spontaneous mutation for quantitative traits (Lande, 1980; Barrett and Kohn, 1991). However, in this extreme case of a single-founder population, loss of even the most common allozyme and quantitative variation is a concern.
Genetic variation within the Occidental T. amoenum population
Considering the severity of the bottleneck in the Occidental T. amoenum population, it is surprising that the amount of allozyme variation remains as high as shown in this survey. We can infer that the single plant from which this population originated was heterozygous at three allozyme loci. (By comparison, only one of the 20 plants sampled from the Dillon beach T. amoenum population B was heterozygous at as many as three loci.) Genetic variability for morphological traits has apparently persisted in the Occidental T. amoenum population as well. For example, first-generation offspring grown together in a common garden environment had seed color and seed markings that varied among individuals but were consistent within individuals (Connors, unpublished observations).
Moderate levels of allozyme variation remaining in the Occidental T. amoenum population provide empirical evidence that rapid population growth following a bottleneck may allow a considerable portion of the genetic variation to be maintained. It is also likely that the bottleneck in this case was not long in duration. Due to the cumulative nature of the effects of genetic drift in small populations, bottlenecks of this severity, if maintained for several generations, would likely result in complete or nearly complete loss of genetic variation. Our results are consistent with a suggestion by Connors (1994) that the seed from which the 1993 wild plant grew may have been produced many years earlier when the population size was larger. Clovers produce hard-coated seeds capable of remaining viable for decades (Hull, 1973). Thus the 1993 seed may have germinated from a long-dormant seed bank when disturbed by road construction during the previous year.
Comparisons with other populations
A single individual is unlikely to harbor all of the genetic variation that existed in the original population prior to the bottleneck. Questions remain about how much variation was lost, and how this loss of variation will impact strategies for reintroduction. The best estimates of natural levels of background genetic variation come from comparisons with the Dillon Beach T. amoenum population, and from comparisons with populations of closely related Trifolium species. We chose populations of T. albopurpureum var. dichotomum and T. macraei, two species with morphologies, life histories, and geographic ranges similar to T. amoenum for the later comparisons. The allozyme data indicate a strong genetic similarity among these three species. While reviews of the literature by Gottlieb (1981b) and Crawford (1983) have shown that, on average, congeneric species are separated by a Nei's genetic distance of ~0.33, the greatest Nei's distance among these three species, averaged across populations, was 0.08 (T. amoenum and T. macraei). The average Nei's genetic distance between populations of T. amoenum and T. albopurpureum var. dichotomum was only 0.03.
Levels of within-population genetic variation are also best compared among species that share the same mating system (Barrett and Kohn, 1991). Species in which seeds are produced primarily through self-pollination tend to contain less allozyme variation at the within-population level than outcrossers. For example, data from previous studies on plants, compiled by Hamrick and Godt (1990), showed that an average of 20.0% of loci were polymorphic in populations of selfing species, while 38.7% of loci were polymorphic in populations of species with mixed or outcrossing mating systems. Hamrick and Godt (1990) also calculated that populations of selfing species contained an average of 1.31 alleles per locus, whereas populations of species with mixed or outcrossing mating systems contained an average of 1.60 alleles per locus.
Several lines of evidence point to significant outcrossing within populations of T. amoenum. The high level of heterozygosity that apparently existed in the original Occidental T. amoenum plant would not have been expected in a predominantly self-pollinating species. In addition, a mixed (mixture of selfing and outcrossing) mating system is suggested by the ratio of observed heterozygosity to expected heterozygosity within both T. amoenum populations (Table 3). Populations of T. albopurpureum var. dichotomum showed ratios of observed heterozygosity to expected heterozygosity which were similar to those in T. amoenum. In contrast, populations of T. macraei were nearly devoid of heterozygosity, indicating a mating system of a high degree of selfing. Amount of prebottleneck allozyme variation in the Occidental T. amoenum population is therefore best estimated through comparisons to the Dillon Beach T. amoenum population and the two populations of T. albopurpureum var. dichotomum. Levels of allozyme variation within these latter three populations, B, C, and D, were very similar (Table 3). The Occidental T. amoenum population A contained half the number of polymorphic loci and 21% fewer alleles per locus compared with the Dillon Beach T. amoenum population B. Considering populations B, C, and D together, the bottlenecked Occidental T. amoenum population contained 53% fewer polymorphic loci and 20% fewer alleles per locus. Still, because we do not know how much allozyme variation occurred in the Occidental T. amoenum population (A) prior to the bottleneck, or the evolutionary history of populations B, C, and D, such comparisons can provide only a rough estimate of the amount of variation that was lost.
An examination of leaf markings in the two T. amoenum populations suggests that genetic variation for traits other than allozymes may have been affected by the population bottleneck as well. Different leaf markings (light or dark bands, spots, or chevrons) are common in clovers, and the genetic basis of this variation has been demonstrated in crossing experiments with T. repens (Davies, 1963). At least four distinct leaf marking patterns occurred among 34 plants grown in 1997 at the University of California Bodega Marine Reserve from seeds collected from the Dillon Beach T. amoenum population. Uniform green (with no red or white markings) was the most common pattern, but even the rarest of the four patterns occurred at a frequency of 0.09. In contrast, leaves of 48 T. amoenum plants grown at the same site from seed of the Occidental population showed only the uniform green color pattern (Connors, unpublished data). It is possible that the less common marking patterns were also present in the Occidental T. amoenum population prior to the bottleneck and have been lost.
Implications for reintroducing T. amoenum
Populations with limited genetic variation are most vulnerable to extinction, due to reduced potential for evolution in response to environmental changes (Beardmore, 1983; Huenneke, 1991). The presence of adequate genetic variation might be especially critical to T. amoenum populations used for reintroduction, because these populations will likely need to evolve and adapt to the major changes that have occurred within California grassland plant communities. The biotic environment has, in many cases, been greatly altered by competition with exotic species as well as herbivory by cattle and other grazers.
We demonstrated that the Occidental T. amoenum population contains less allozyme variation than a larger population of the same species and populations of a closely related species. Assuming that this loss of allozyme variation resulted from the documented population bottleneck, a reduction in genetic variation for other traits of potentially greater adaptive importance would be expected as well. Losses of this often quantitative variation should be proportional to losses of allozyme variation, because genetic drift is the dominant evolutionary force in small populations (Barrett and Kohn, 1991), and genetic drift affects all genetic variation, allozyme or quantitative, in a similar fashion. This has been shown experimentally by Polans and Allard (1989) in populations of ryegrass (Lolium multiflorum), where restrictions in size of populations led to reductions in levels of allozyme variation and also to deleterious effects for quantitative traits, thought to be due to this loss of genetic variation.
However, low levels of genetic variation do not necessarily mean that reintroduction attempts could not succeed. For example, Schwaegerle and Schaal (1979) described a thriving population of over 100 000 pitcher plants (Sarracenia purpurea) originating from a single translocated individual. Success of this introduction occurred in spite of the apparently reduced allozyme variation that accompanied the population bottleneck.
In addition to loss of evolutionary potential, inbreeding depression may be a factor within the Occidental T. amoenum population. Inbreeding increases the probability that deleterious recessive alleles will become fixed, and it is often most severe in populations of plants that normally outcross (Barrett and Kohn, 1991). However, even relatively low rates of self-pollination may purge much of the genetic load from a population (Lande and Schemske, 1985; Charlesworth and Charlesworth, 1987). Therefore, if the natural mating system of the Occidental T. amoenum population included some degree of self-pollination, the likelihood that inbreeding will have significant negative effects is reduced. Indeed, plants produced from these seeds so far appear quite robust, but no formal tests of inbreeding depression have been conducted.
Both the loss of genetic variation and inbreeding depression might be ameliorated by expanding the genetic base of the Occidental T. amoenum population before or during the process of reintroduction. Numerous searches for additional remnant plants in the wild have been conducted since 1993 without success until our 1996 discovery at the Dillon Beach site. Using seeds from this more variable coastal T. amoenum population to expand the genetic base of the inland Occidental population might now be an option, particularly if reintroductions of the inland population are unsuccessful, and lack of population persistence or reductions in the fitness of individual plants can be attributed to genetic limitations associated with reduced variation.
However, genetic mixtures may be problematic because plants from the two T. amoenum populations differ in aspects of plant architecture that may indicate local adaptation. The original wild Occidental plant and its offspring all have an erect growth form, generally taller than broad. This matches the architecture of 28 herbarium specimens we have examined from historic populations at other inland sites. Before discovery of the Dillon Beach T. amoenum population, all reported populations and all herbarium specimens of this species, like the Occidental plant, occurred at inland locations. In contrast, plants of the Dillon Beach population, growing in the windy environment of a coastal bluff, have an almost prostrate growth form. These phenotypic differences have a genetic basis. When plants from both populations were grown in a common garden at an inland location near Occidental, the differences in plant architecture were maintained (ratio of maximum height to maximum width was 1.13 ± 0.19 [N = 7] for plants from the Occidental population and 0.37 ± 0.10 [N = 10] for plants from the Dillon Beach population) (Connors and J. L. Maron, unpublished data).
Coastal bluff environments are cooler, moister, and much windier than inland grasslands. The prostrate growth form of plants from the Dillon Beach T. amoenum population may be an adaptation to strong winds and is a characteristic shared by most members of the coastal bluff plant community. Among other species growing in both communities, grasses such as Bromus carinatus and Hordeum brachyantherum also have a low, decumbent form on the coastal bluffs and a tall, erect form in inland grasslands.
This suggestion of local adaptation argues for caution in mixing inland (Occidental) and coastal (Dillon Beach) genetic stocks and leaves us with the question of how best to reestablish the inland form. A focus on reestablishing this form, represented by the single-founder Occidental population, is appropriate because it is apparently more similar to historical populations (as indicated by comparisons with herbarium specimens) and thus has the potential for reintroduction over a much wider geographic range than the coastal form.
A strategy of controlled introgression might provide a compromise between maintaining local adaptive variation and promoting adequate levels of within-population genetic variation. Introgression would involve mixing a small proportion of the nonlocal Dillon Beach source into the Occidental T. amoenum population over time. Reintroducing T. amoenum populations varying in the proportion of the local source and thus presumably differing in amount of genetic variation could be conducted in an experimental context (Barrett and Kohn, 1991; Guerrant, 1996). This would increase our understanding of the trade-offs involved. Mixing populations would not be the method of choice if cross-pollinations between populations result in progeny exhibiting outbreeding depression. Although outbreeding depression has been reported in several plant species, how widespread this phenomenon is and the degree to which it should be considered when making decisions about whether to mix populations for conservation purposes, are still subject to debate (Fenster and Dudash, 1994).
Two generations of seed increase have resulted in the production of >50 000 seeds of the Occidental T. amoenum population. These seeds remain in storage at various locations since suitable sites for reintroduction have not yet been identified. This is partially due to the lack of knowledge about interspecific competition and herbivory and how these variables impact the species, which may make certain sites better candidates than others. Efforts are currently underway to investigate more completely the adaptive differences between the two T. amoenum populations and to determine reasons for the species' decline, in order to better understand site requirements for future reintroduction attempts.
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FOOTNOTES |
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4 Author for correspondence (Tel.: 530-752-1701; e-mail: eeknapp{at}ucdavis.edu
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