Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8127-8131, August 1994 Population Biology Contrasting the distribution of chloroplast DNA and allozyme polymorphism among local populations of Silene alba: Implications for studies of gene flow in plants DAVID E. MCCAULEY Department of Biology, Vanderbilt University, Nashville, TN 37235 Communicated by Michael T. Clegg, May 2, 1994

ABSTRACT The distribution of chloroplast DNA (cp- literature showing that intraspecific cpDNA variation can be DNA) length variants was analyzed within and among 10 local detected in a variety of species and that such variation populations of Sikne alba, a dioecious angiosperm. The pop- generally shows considerable spatial structure (1, 2, 4, 9-12). ulations displayed considerable allele frequency variation, re- However, comparisons to nuclear variation are limited. Since sulting in an estimate of Wright's Ft of 0.67 over a 25 x 25 km most of the published studies have been conducted on a portion ofthe species' range. By contrast, a concurrent analysis spatial scale that encompasses a major portion ofthe species' of the genetic structure of these same populations based on range, the questions ofwhether cpDNA variation can be used seven polymorphic allozyme loci yielded an estimate of Fat of to document genetic structure at relatively fine geographic 0.13. The two Ft estimates are significantly different from one scales and how the relative magnitudes of the within- and another when their respective confidence limits are estimated among-population components of that variation compare to by jackknifing. The results of a breeding study were consistent the local distribution of nuclear variants remain open. This with maternal inheritance of the cpDNA variants. With ma- paper represents one of the first attempts to address these ternal inheritance the genetic structure of the cpDNA should issues in natural populations of a plant species. reflect seed movement, whereas the genetic structure of the Here I report on the use of PCR-based cpDNA variants in nuclear-encoded allozyme loci should reflect the movement of the analysis of the genetic structure of local populations of a both seeds and pollen. Comparison of the two markedly dioecious angiosperm, Silene alba (= Silene latifolia, Caryo- different Fst estimates in the context of recent models of the phyllaceae). I show that the distribution of chloroplast DNA population genetics oforganelles suggests that the movement of polymorphisms among populations separated by only a few both seeds and pollen contributes significantly to gene flow. kilometers displays markedly more spatial structure than does the distribution of nuclear-encoded allozyme markers Recently it has been suggested that the study of the genetic when compared using Fq, Wright's (13) measure of genetic structure of plant populations could be greatly facilitated by structure. I further discuss the inferences about the contri- employing markers encoded by chloroplast DNA (cpDNA) bution of seed and pollen movement to gene flow that can be (1-4). One reason is that in species in which cpDNA displays drawn by the joint consideration of these Fst estimates. maternal or maternal-biased inheritance, as is often the case in angiosperms (5, 6), gene flow in cpDNA is restricted, or MATERIALS AND METHODS largely restricted, to incidents of seed movement. Since gene flow in nuclear DNA can occur via the dispersal of either S. alba (white campion) is a patchily distributed roadside seeds or pollen, information about the relative influence of weed in the area of southwestern Virginia in which this study the two sources of gene flow on genetic structure might be was conducted. Long-term studies (14) have identified nu- contained in the comparative behavior of genetic markers merous populations of S. alba, ranging in size from a few to derived from the two genomes. Furthermore, in plant meta- >200 individuals along roadsides within a 15-km radius ofthe populations in which the local demes are subject to frequent Mountain Lake Biological Station in Giles County, VA. Ten extinction and recolonization, the imprint of recent founding representative populations, each consisting of >30 individ- events on genetic structure should be particularly evident in uals, were selected from this area for genetic sampling. the distribution of cpDNA variants, since local demes are Between 20 and 50 individuals were sampled from each site founded by the movement of seeds into empty patches of by removing single leaves. Collection sites were spaced such favorable habitat (7). Thus, chloroplast and nuclear genes that nearest neighbors were separated from one another by could display markedly different patterns of spatial genetic 5-10 km; the greatest distance separating any ofthe sampled structure, particularly in those species in which pollen and sites was -25 km. Note that a number of unsampled popu- seed dispersal patterns differ. Taken together, the two classes lations were distributed along the intervals of roadside that of genetic markers have the potential to be quite useful for separated the sampled sites. inferring the demographic processes that underlie genetic Because the goal of this study was to use cpDNA poly- structure, especially when evaluated in the added context of morphisms to estimate the among-population component of recent population genetic models of organelles (3, 4, 8). gene frequency variation, rather than to estimate total nu- Despite the potential value of such studies ofcpDNA, they cleotide diversity, the initial survey for markers focused on are still relatively few. The chloroplast genome is generally a region of the chloroplast genome most likely to vary. To highly conserved and it is not yet clear whether intraspecific that end, a PCR-based approach employed a nested set of variation of the sort needed for studies of population struc- primers expected to anneal within tRNA coding regions, but ture is sufficiently common for cpDNA to be a widely used flanking noncoding regions, either intergenic spacers or an tool in plant population biology. There is a small but growing intron. These primers were designed to amplify regions of DNA that would be expected to be less well conserved than The publication costs of this article were defrayed in part by page charge coding regions (15) (see Fig. 1). Genomic DNA was collected payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: cpDNA, chloroplast DNA.

8127 Downloaded by guest on September 29, 2021 8128 Population Biology: McCauley Proc. Natl. Acad. Sci. USA 91 (1994) 700 bp 650 bp 450 bp region, respectively. The frequency of these haplotypes was then estimated for samples of 10-15 individuals from each of I1I the 10 study populations. In addition, cpDNA haplotypes trnT trnL trnL trnF were determined for the parents and offspring drawn from three laboratory crosses in which the parents differed at the variable trnL intron site. For comparative purposes, genetic structure was also FIG. 1. Location and alignment of primers designed to anneal to estimated for nuclear loci by the analysis of electrophoretic tRNA gene T, L, or F within the chloroplast genome, as redrawn protein polymorphisms. Population-speciflic allele frequen- from ref. 15. See that paper for primer sequences and positions relative to the published tobacco cpDNA sequence. Cross hatches cies at each of seven polymorphic allozyme loci were esti- represent the approximate location of insertion/deletion polymor- mated from samples of 20-50 individuals taken from the 10 phisms. study populations. The enzymes studied and their EC num- bers are phosphoglucomutase (PGM; 5.4.2.2), glucose-6- from leaf material by a CTAB/chloroform extraction method phosphate isomerase (GPI; 5.3.1.9), shikimate 5-dehydroge- (16) and then used as a template in the PCR. The PCR cycle nase (SKDH; 1.1.1.25), isocitrate dehydrogenase (IDH; was as follows: 1 min at 940C followed by 1 min at 450C and 1.1.1.42), phosphogluconate dehydrogenase (6-PGD; 2 min at 720C. This was repeated 35 times. In the original 1.1.1.44), malate dehydrogenase (MDH; 1.1.1.37), and leucyl survey for variation the outside pair of primers (Fig. 1) was aminopeptidase (LAP; 3.4.11.1), for which a total of 26 used to amplify the entire z1800-bp sequence from several putative alleles could be scored. Allozyme variation was individuals. Separate aliquots (10 1.l) of the 50-Al PCR assayed by starch gel electrophoresis. Tissue preparation and products were then digested with one of several restriction enzyme staining followed standard methods (17). using enzymes. The digestion products were electrophoresed on Genetic structure was quantified by calculating F~t algorithms developed for both haploid and diploid data (18). 4% NuSieve (FMC) agarose minigels and visualized by Fq was selected as a measure of genetic structure because it ethidium bromide staining. While no interindividual variation describes the relative contribution ofamong-population allele in restriction site number was detected, the size of the frequency variation to total genetic variance. This approach restriction fragments varied among individuals in several of facilitates comparing or averaging loci with varying degrees the digests, suggesting length variation owing to insertion/ of absolute polymorphism. Furthermore, the algorithm in- deletion polymorphism. By employing nested combinations cludes sample size corrections that allow comparisons based of primer pairs illustrated in Fig. 1, two variable regions were on samples of different numbers of individuals. Finally, Fq identified, one within the trnL (UAA) intron and one in the (or the related statistic, Gs,) is a variable often used in region that separates the trnL (UAA) and trnF (GAA) coding population genetic studies that relate genetic structure to regions (15). A slight size variation was apparent by inspec- various demographic processes (13, 19, 20). tion after electrophoresis of the uncut PCR products. Visu- For the allozyme data, Fst was first calculated for each alization of the length variants was facilitated by digestion of allele. Information was then combined across alleles to give the PCR product with Hinfl prior to electrophoresis (Fig. 2). locus-specific values and finally combined across loci in Four haplotypes were identified by considering length vari- order to provide a summary statistic for the entire allozyme ation within each of the two regions. These haplotypes will be data set. Similarly, the four cpDNA haplotypes were treated referred to as +/+, +/-, -/+, and -/-, where + or as alleles, with the information from the four alleles then refers to a longer or shorter fragment in the intron or spacer combined to give a summary statistic for the cpDNA data set. Confidence limits were set to the allozyme and cpDNA estimates of Fst (18). It was assumed that the allozyme loci represent seven independent estimates of the true Fst that describes the genetic structure of some number of nuclear bp loci subjected to similar demographic forces. Under this assumption, the 95% confidence limits for this value were 2 / / estimated by jackknifing across the seven loci. Confidence limits for the cpDNA estimate were set by jackknifing across the 10 populations since in the absence of recombination the 2 I f) entire cpDNA genome would be inherited as a "locus." In addition, Fi, a measure of the deviation ofgenotype frequen- cies from Hardy-Weinberg equilibrium within populations, 1 94 was calculated for the allozyme data. Fis is undefined for cpDNA since it is being treated as haploid.

RESULTS Among the 123 individuals collected from natural populations the frequencies of the +/+, +/-, - /+, and - /- cpDNA C98 haplotypes were 0.033, 0.225, 0.516, and 0.225, respectively. The population-to-population distribution of the four haplo- -- 8 7 types is illustrated in Fig. 3. Note that despite the relatively high degree of overall polymorphism, most of the local populations consisted of predominantly one type and that all four of the haplotypes co-occur at just one locality. This FIG. 2. Pattern displayed when HinfI digests of three PCR frequency distribution yields an estimate of Fst of 0.674. products, obtained using primers flanking the trnL (UAA) intron, are The results of the laboratory crosses are shown in Table 1. electrophoresed on a 4% NuSieve (FMC) agarose gel. Note the All 37 offspring, representing three progeny arrays, displayed interindividual size variation in the largest fragment-i.e., that two the maternal haplotype. While there is no evidence of pater- + individuals flank a - individual. nal leakage in this sample, a much larger study would be Downloaded by guest on September 29, 2021 Population Biology: McCauley Proc. Natl. Acad. Sci. USA 91 (1994) 8129 Table 2. Ft estimated from 10 populations of S. alba using allozyme and cpDNA genetic markers Si~~~~~g Genetic marker Ft value Individual allozyme PGM 0.145 GPI 0.125 MDH 0.230 6-PGD 0.042 SKDH 0.114 IDH 0.083 LAP 0.172 Allozymes combined 0.134 (0.195-0.073) .+/- '-j't cpDNA 0.674 (0.941-0.407) The 95% confidence limits are also given for the F5t estimates m/ j combined across allozymes and for the cpDNA. Enzyme abbrevia- tions are identified in the text. FIG. 3. Relative frequencies of four cpDNA haplotypes in sam- structure, especially when used in conjunction with nuclear ples taken from 10 natural populations of S. alba. markers. Evaluation ofthe factors likely to be responsible for required to determine whether the inheritance is indeed the disparity in observed Ft values is facilitated by the joint exclusively maternal. The equation /3 = 1 - (1 - p)N can be consideration of the natural history of S. alba and the used to estimate the power, 3, of a sample of N individuals comparative predictions of population genetic models of to detect paternal leakage, if it occurs in a fraction, P, of all organelle and nuclear genes. fertilizations (21). By setting 13 arbitrarily at 0.95 and letting It is necessary to first consider the nature of the markers N = 37 one can solve for P = 0.077-that is, the results of themselves. To date, length variants comprise a relatively these crosses suggest that there is a 95% probability that the large proportion of the intraspecific cpDNA polymorphisms true proportion of S. alba cpDNA genotypes displaying a that have been documented (2, 11, 12) and were the subject detectable paternal contribution is <7.7%. of this study. It has been suggested that cpDNA length The estimate of Fis, combined across the seven allozyme polymorphisms have limited utility for the study of popula- loci, was 0.031, indicating nearly random mating within tion genetic processes (11). This is primarily because muta- patches. The allozyme locus-specific estimates of Ft ranged tion rates at such sites may be atypical for the chloroplast from 0.230 to 0.042 (Table 2). Because of this relatively large genome as a whole and because it is difficult to establish range in locus-specific F~g estimates, a simple test for heter- allelic homology among apparently similar electrophoretic was conducted the patterns. This particularly compromises their use in studies ogeneity by using across-population jack- of rates of molecular evolution and of the phylogenetic knife procedure to estimate locus-specific confidence limits relationships among geographic populations. These problems for the loci displaying the largest and the smallest F~t values may not be as severe in this study of local populations of S. (MDH and 6-PGD, respectively). Since there was overlap in alba as they might be in a case in which populations are these two 95% confidence limits the entire set of locus- isolated from one another by greater distances and in which specific F,, values was considered to be sufficiently homo- that isolation has been of longer duration. In that case the geneous to be combined into a joint estimate. The Ft value mutation rate is more clearly an important factor in the obtained by combining information from all seven allozyme process of population differentiation. loci was 0.134. The 95% confidence limits developed for the In this study the cpDNA variants are being used as markers cpDNA and thejoint allozyme Fst estimates, 0.941-0.407 and subject to the influence of very recent demographic pro- 0.195-0.073, respectively, do not overlap with either zero or cesses. S. alba is not native to North America and has one another. probably been found in southwest Virginia for <150 years (fewer generations) (22). Further, local populations within DISCUSSION this area are known to turn over on an even shorter time scale Clearly, cpDNA variation in S. alba displays considerable owing to human disturbance and stochastic processes (14). At spatial structure and over a more limited geographic scale a spatial scale at which the relatively frequent exchange of than has been considered previously. Most important for the migrants is possible, population genetic processes are likely at there is a 5-fold difference in to be driven primarily by the joint influence of finite local discussion hand, the magni- population size and migration on standing variation rather tude of genetic structure as measured by Fst, depending on than by the mutation rate, especially when there has been whether the genetic polymorphism employed in the analysis relatively little time for the accumulation of new mutations is derived from the nuclear DNA or cpDNA. One motivation within the local populations (23). The question of homology for studying the spatial distribution of cpDNA polymor- is more problematic but is less of an issue when small PCR phisms is that they have the potential to contribute to our products specific to a known region of the genome, rather understanding ofthe evolutionary forces that underlie genetic than restriction digests ofthe whole chloroplast genome, are Table 1. Results of three crosses between S. alba parents the subject of study and when phylogenetic relationships are differing with regard to a cpDNA insertion/deletion (+ or -) not inferred. within the trnL (UAA) intron Most of the previous studies of cpDNA polymorphism have employed a sampling design that encompasses a major Parent Offspring portion ofthe species' range rather than addressing structure & Y + _ at the level of local populations. In these studies marked _ + 9 0 structuring of cpDNA variation has been the rule (1, 2, 4, 11) + - 0 12 with the clover Trifoliumpratense being a notable exception. - These results are in keeping with the theoretical expectation + 16 0 that organelle DNA polymorphism should generally display Downloaded by guest on September 29, 2021 8130 Population Biology: McCauley Proc. Natl. Acad. Sci. USA 91 (1994) considerably more spatial structure than nuclear polymor- and that there is no sex bias in seed movement (i.e., letting phisms (8), a prediction that has also held in comparisons of m = me), and recalling that Ne 2N,,, one would predict mitochondrial and nuclear genes in both conifers (24, 25) and from the cpDNA results that Fq for nuclear markers should invertebrates (26, 27). In angiosperms, nuclear-chloroplast be =0.33. This is more than twice the observed value of0. 134 F~t differences at the local population level are most likely and well outside the 95% confidence limits calculated for that due to two demographic effects when inheritance is maternal estimate. One simple explanation for the difference between and haploid. First, this mode of inheritance would reduce the the observed and expected nuclear Fst values is that m and m, local effective population size, relative to nuclear genes, the are quite different owing to a large added component of gene exact amount depending largely on the sex ratio. Second, it flow in the allozymes as a result of pollen movement (m >> could reduce the rate of gene flow because, with maternal me). inheritance, gene flow in cpDNA would be restricted to Recall, however, that the simple approximations used here incidents of seed movement, where as in outcrossing plants assume an equilibrium between the effects ofgenetic drift and nuclear gene flow can also occur via the movement of pollen. gene flow that is unlikely to be strictly met in S. alba owing In that regard nuclear-chloroplast comparisons have the to recurrent founder events and frequent extinctions that can potential to help determine the relative contributions of seed perturb this equilibrium (31, 32). With extremely rapid turn- and pollen movement to overall genetic structure; an issue over, the genetic differentiation of local populations can be that is not easily resolved (28, 29). Unfortunately, both the determined primarily by sampling events associated with effective population size and gene flow effects would act to colonization, rather than the long-term effects of gene flow, increase F5t in cpDNA, relative to the value expected for because all populations are just a few generations removed nuclear polymorphisms, and these effects are therefore dif- from their respective founding events. Since some modes of ficult to disentangle. colonization by seeds would generate much greater sampling With regard to differences in effective population size, the variance in the frequency of alleles encoded in organelle organelle effective population size (Neo) is influenced by the nuclear genes (7), the sex ratio, the relative maternal and paternal transmission and DNA, when compared to that of migration rates, and the frequency of heteroplasmy (8). A disparity in F~t values might also be influenced by the recent study of sex ratio of S. alba in this area of Virginia has differential effects of founding events on the two genomes. shown only a slight female bias (30). The results presented Further, the approach to equilibrium following a perturba- here show that cpDNA is largely, if not entirely, maternally tion, such as a founding event, might occur at different rates inherited in S. alba. While it is not clear how sensitive the in nuclear and organellar genes (8). PCR method would be to heteroplasmy, no heterogeneous In all likelihood the S. alba populations used in this study PCR products were detected, indicating that cpDNA is are neither at equilibrium with regard to drift and gene flow effectively haploid. In the ideal case, with equal sex ratio and nor just established. While earlier work has documented a complete maternal inheritance, the nuclear effective popu- high rate of turnover of local demes (14), the particular lation size (Ne) is approximately twice that of the organelle populations used in this study must have become established effective population size. If females are homoplasmic (hap- sometime prior to 1988, since they were known to exist at the loid) then the effective number of organellar DNA gene start of a census oflocal populations that has been conducted copies is further reduced by half relative to diploid inheri- yearly since that time. Since S. alba is a short-lived perennial, tance. It appears that these conditions must hold at least each of the 10 populations used in this study must then be a approximately in S. alba, and effective population size ef- minimum of three or four generations removed from its fects are likely to contribute significantly to the marked respective colonization event and subject to some influence difference in observed Ft values. of pollen flow. What of the relative influences of seed and pollen move- Clearly, a better understanding ofthe demographic basis for ment on gene flow? Clearly, seeds must be transported out of the observed wide disparity in the Fq values would require established populations with some regularity, since a recent combining more detailed knowledge of the population and study of the dynamics of local patches has documented pollination ecology of the species with insight gained from frequent colonization events, which require seed movement models such as those described above. Further, this study has (14). Some contribution of seed movement to gene flow is not considered the influence of selection on genetic structure, also evident in the fact that, while Fst for cpDNA is much especially the possibility that the two classes ofgenetic mark- higher than that seen for the allozymes, some within- ers could be under rather different selective regimes. For population cpDNA polymorphism is maintained in this sys- example, strong balancing selection could limit the potential tem. The natural history also argues for some role for pollen for the allozyme loci to undergo population differentiation, movement in nuclear gene flow. As a dioecious species S. even with limited gene flow (27). Still, this study has shown alba is an obligate outcrosser, pollinated by several species that cpDNA markers can provide considerable insight into the ofbees and moths. The very low Fjs value suggests that pollen demographic processes controlling plant population genetic moves freely within populations. It follows that at least some structure. If intraspecific cpDNA polymorphism is wide- between-population movement of pollen should also occur. spread within the angiosperms, contrasts with nuclear poly- Thus, it is likely that the rate ofgene flow in nuclear genes (m) morphisms should prove to be a generally useful tool for is somewhat greater than the effective rate oforganellar gene studying the relative contributions of seed and pollen move- flow (me), owing to the extra opportunity for nuclear genes to ment to genetic structure, especially when evaluated in the move in pollen. This should be reflected in the difference in context of recent theoretical developments (3, 4). the magnitude of the two F~t values. The potential impact of pollen flow on the comparative F~t I thank A. Abbott, H. Alexander, J. Antonovics, C. dePamphilis, values is illustrated by considering the empirical results T. Meagher, L. Oh, J. Raveill, M. Rausher, C. Richards, C. Shaw, a D. Taylor, P. Thrall, and A. Wolfe for contributing to various aspects presented here in the context of model of organelle popu- of this project. Logistical support was provided by the Mountain lation genetics (8). Given an equilibrium between drift and Lake Biological Station and the Department of Botany, Duke Uni- gene flow (and ignoring mutation), Ft for organelle genes is versity. Financial support was provided by National Science Foun- approximately 1/(1 + 2Neome). Using the observed F~t of dation Award DEB-9221175. 0.674 to solve for Ne0m yields an estimate of Neome - 0.25. For nuclear genes the approximate equilibrium is the familiar 1. Milligan, B. G. (1991) Curr. Genet. 19, 411-416. 1/(1 + 4Nem). Assuming first that all gene flow is via seeds 2. Soltis, D. E., Soltis, P. S. & Milligan, B. G. (1992) in Molecular Downloaded by guest on September 29, 2021 Population Biology: McCauley Proc. Natl. Acad. Sci. USA 91 (1994) 8131 Systematics ofPlants, eds. Soltis, P. S., Soltis, D. E. & Doyle, 16. Doyle, J. 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