Geographic patterns of microsatellite variation in Boechera stricta, a close relative of Arabidopsis

The genus Boechera is a widespread North American group with great potential for studies of ecology and evolution: Boechera is closely related to Arabidopsis and exhibits different ecological and reproductive strategies. Boechera stricta (previously Arabis drummondii) is a morphologically and genetically well-defined, perennial crucifer species. Fifteen natural populations of diploid individuals from the Rocky Mountains were analysed using 21 microsatellite loci. In accordance with our expectation for this predominately inbreeding species, a high F IS value (0.89) was observed. Furthermore, populations of B. stricta were highly differentiated, as indicated by F ST = 0.56. Three clusters were identified using structure- the majority of populations belonged to either the Northern or Southern cluster. Together, the north-south partitioning and evenness of genetic variation across the two clusters suggested multiple refugia for this perennial herb in the Rocky Mountains. Pleistocene glaciation, together with the topographically and climatologically heterogeneous cordillera, has profoundly influenced the genetic architecture of B. stricta. Genetic population structure was also influenced by relatively recent genome admixture at two levels: within species (involving individuals from the Northern and Southern clusters) and between species (with the hybridization of B. stricta and Boechera holboellii). This complexity of population structure at presumably neutral microsatellite loci located throughout the genome in B. stricta provides a baseline against which to test whether functional genetic variation is undergoing local adaptive evolution throughout the natural species range.     

Sexual reproduction, hybridization, apomixis, and polyploidization in the genus Boechera (Brassicaceae)

Of the 340 genera in the Brassicaceae, apomictic reproduction is found only in the North American genus Boechera. We investigated phylogenetic relationships, ability to hybridize, mating system, and ploidy levels of 92 lines sampled from 85 populations and representing 19 Boechera species. Phylogenetic analyses based on chloroplast DNA sequences identified three lineages in the genus. Reciprocal crosses of each line were made to a common sexual diploid B. stricta tester. The resulting F(1) progeny were analyzed for the inheritance of polymorphic microsatellite loci, genome size, and seed production. Intraspecific B. stricta crosses confirmed that this species is mostly diploid and sexual. Interspecific crosses revealed many other species were diploid and sexual and could be successfully hybridized with the tester. We also found obligate and facultative apomictic diploid and triploid lines. De novo F(1) polyploids (either triploids or tetraploids) were derived from the union of nonreduced (from an apomictic parent) and reduced (from the tester) gametes. However, seed production of these F(1) plants was generally low, suggesting a failure in the transmission of apomixis. The creation of a wide array of segregating genetic populations will facilitate future research on the evolution and inheritance of quantitative variation in Boechera.     

Intraspecific diversification in North American Boechera stricta (= Arabis drummondii), Boechera xdivaricarpa, and Boechera holboellii (Brassicaceae) inferred from nuclear and chloroplast molecular markers--an integrative approach

We performed a combined evolutionary analysis of North American Boechera stricta, Boechera holboellii, and their hybrid Boechera ×divaricarpa using information on ploidy level estimators, allelic microsatellite variation, noncoding regions of the plastidic genome (cpDNA), and sequences of the internal transcribed spacers 1 and 2 of the nuclear ribosomal DNA (ITS). Somatic ploidy levels of herbarium specimens were estimated based on comparison of pollen size and the number of alleles per locus at seven microsatellites. Results indicate that B. stricta and B. holboellii are genetically distinct from each other, although we also find evidence for occasional introgression between both parental species. Microsatellite patterns for B. stricta from northeastern North America are genetically distinct from western populations, suggesting isolation in glacial refugia along the southeastern margin of the continuous ice shield. Microsatellites supported recent origin of B. ×divaricarpa. Correspondence of nrDNA with cpDNA genetic variation for the majority of diploid B. holboellii accessions suggests a basal, sexual evolutionary unit within a polymorphic B. holboellii group. Hybridization of genetically distinct lineage(s) evidently played an important role in the establishment of polyploid B. holboellii. Frequency of polyploid B. holboellii is substantially higher in the southern United States. This trend corresponds to a southerly distribution of derived chloroplast haplotypes, suggesting an evolutionary advantage of polyploidy and associated apomixis in the colonization of the Sierra Nevada and the Southern Rocky Mountains.     

Comparative genetic mapping in Boechera stricta, a close relative of Arabidopsis

The angiosperm family Brassicaceae contains both the research model Arabidopsis (Arabidopsis thaliana) and the agricultural genus Brassica. Comparative genomics in the Brassicaceae has largely focused on direct comparisons between Arabidopsis and the species of interest. However, the reduced genome size and chromosome number (n = 5) of Arabidopsis complicates comparisons. Arabidopsis shows extensive genome and chromosome reshuffling compared to its close relatives Arabidopsis lyrata and Capsella rubella, both with n = 8. To facilitate comparative genomics across the Brassicaceae we recently outlined a system of 24 conserved chromosomal blocks based on their positions in an ancestral karyotype of n = 8, rather than by their position in Arabidopsis. In this report we use this system as a tool to understand genome structure and evolution in Boechera stricta (n = 7). B. stricta is a diploid, sexual, and highly self-fertilizing species occurring in mostly montane regions of western North America. We have created an F(2) genetic map of B. stricta based on 192 individuals scored at 196 microsatellite and candidate gene loci. Single-nucleotide polymorphism genotyping of 94 of the loci was done simultaneously using an Illumina bead array. The total map length is 725.8 cM, with an average marker spacing of 3.9 cM. There are no gaps greater than 19.3 cM. The chromosomal reduction from n = 8 to n = 7 and other genomic changes in B. stricta likely involved a pericentric inversion, a chromosomal fusion, and two reciprocal translocations that are easily visualized using the genomic blocks. Our genetic map will facilitate the analysis of ecologically relevant quantitative variation in Boechera. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DU 667459 to DU 708532.     

Local adaptation across a climatic gradient despite small effective population size in the rare sapphire rockcress

When assigning conservation priorities in endangered species, two common management strategies seek to protect remnant populations that (i) are the most genetically divergent or (ii) possess the highest diversity at neutral genetic markers. These two approaches assume that variation in molecular markers reflects variation in ecologically important traits and ignore the possibility of local adaptation among populations that show little divergence or variation at marker loci. Using common garden experiments, we demonstrate that populations of the rare endemic plant Arabis fecunda are physiologically adapted to the local microclimate. Local adaptation occurs despite (i) the absence of divergence at almost all marker loci and (ii) very small effective population sizes, as evidenced by extremely low levels of allozyme and DNA sequence polymorphism. Our results provide empirical evidence that setting conservation priorities based exclusively on molecular marker diversity may lead to the loss of locally adapted populations.     

Genetic variation in Pueraria lobata (Fabaceae), an introduced, clonal, invasive plant of the southeastern United States

Pueraria lobata (kudzu), a clonal, leguminous vine, is invading the southeastern United States at a rate of 50 000 ha per year. Genetic variability and clonal diversity were measured in 20 southeastern U.S. populations using 14 allozyme loci. Within its U.S. range, 92.9% of the loci were polymorphic and overall genetic diversity was 0.290. Such high levels of genetic diversity are consistent with its history of multiple introductions over an extended period of time. The average proportions of polymorphic loci and genetic diversity within populations were 55.7% (range = 28.6–85.7%) and 0.213 (range = 0.114–0.317), respectively. The proportion of total genetic diversity found among populations was similar to species with equivalent life history characters (G_ST = 0.199). No regional patterns of variation were seen. The number of putative genotypes in each population ranged from 2 to 26. Mean genotypic diversity was 0.694, ranging from 0.223 to 0.955. Such high levels of genotypic diversity indicate that local sites are often colonized by several propagules (most likely seeds) and/or that sexual reproduction occurs within populations after establishment. An excess of heterozygosity was observed in populations with few unique genets, implying that selection for highly heterozygous individuals may occur in populations of P. lobata.     

Conservation genetics of endangeredBrasenia schreberi based on RAPD and AFLP markers

Brasenia schreberi J.F. Gmelin is a declared endangered species found in the lakes and ponds of South Korea. For planning its conservation strategy, we examined the genetic diversity within and among six populations, using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High genetic diversity was recognized within populations: polymorphic loci (PPL) values ranged from 36.3% in the CJM population to 74.5% in the GGT population, with a mean value of 47.8% based on AFLP markers. Great genetic differentiation (θ^B) was detected among the six populations (0.670 on RAPD and 0.196 on AFLP), and we calculated a low rate of gene flow (N_em), i.e., 0.116 on RAPD and 0.977 on AFLP. Furthermore, a Mantel test revealed that no correlation existed between genetic distances and geographical distances among the six local populations, based on RAPD or AFLP markers. These results are attributed to a number of factors, including an insufficient length of time for genetic diversity to be reduced following a natural decline in population size and isolation, adaptation of the genetic system to small population conditions, and a restricted gene flow rate. Based on both its genetic diversity and population structure, we suggest that a strategy for conserving and restoringB. schreberi must focus on maintaining historical processes, such as high levels of outbreeding, while monitoring increased gene flow among populations. This is because a reduction in genetic diversity as a result of genetic drift is undesirable.     

Genetic diversity and gene flow in the endangered dwarf bear poppy, Arctomecon humilis (Papaveraceae)

Arctomecon humilis is a critically endangered species endemic to the Moenkopi shale of Washington County, Utah. Recovery plans for the species would be improved by an understanding of genetic diversity and gene flow among its remaining populations. Ten variable isozyme loci were used to calculate genetic diversity statistics for study populations. Westerly populations possessed higher levels of genetic variability than other populations at the same isozyme loci. Three of the populations exhibited significant deviations from Hardy-Weinberg expectations. No correlation existed between genetic distance and geographic distance. Most of the genetic diversity was distributed among populations with little gene flow between populations, suggesting that observed genetic differences may arise from genetic drift. For the westerly populations, similar genotypes were observed in the seedling and old age classes, while intermediate age classes typically possessed an alternate set of genotypes at Pgi-2. Mean heterozygosity increased with age class across populations. Westerly populations of A. humilis shared more alleles with the nearest geographic population of A. californica than other populations. Since the westerly populations contained more genetic variability and more alleles in common with a near relative, they may be relictual. Other populations may contain less genetic diversity due to founder effects and/or genetic drift.     

Disjunct habitats as islands: genetic variability in the Caucasian rock lizard Lacerta portschinskii

Genetic diversity at 37 allozyme loci was surveyed in Lacerta portschinskii from contiguous populations and from a disjunct population. Indices of genetic diversity (heterozygosity, number of alleles per locus, and percentage of loci polymorphic) were greater in contiguous populations than in the smaller disjunct population. In this regard, disjunct populations appear to be similar to island populations. Indices of genetic diversity in Caucasian Lacerta are less than those reported from vagile lizard taxa and more similar to those of sit-and-wait predators. This revised version was published online in July 2006 with corrections to the Cover Date.     

Asexual reproduction in a close relative of Arabidopsis: a genetic investigation of apomixis in Boechera (Brassicaceae)

Understanding apomixis (asexual reproduction through seeds) is of great interest to both plant breeders and evolutionary biologists. The genus Boechera is an excellent system for studying apomixis because of its close relationship to Arabidopsis, the occurrence of apomixis at the diploid level, and its potentially simple inheritance by transmission of a heterochromatic (Het) chromosome. Diploid sexual Boechera stricta and diploid apomictic Boechera divaricarpa (carrying a Het chromosome) were crossed. Flow cytometry, karyotype analysis, genomic in situ hybridization, pollen staining and seed-production measurements were used to analyse the parents and resulting F1, F2 and selected F3 and test-cross (TC) generations. The F1 plant was a low-fertility triploid that produced a swarm of aneuploid and polyploid F2 progeny. Two of the F2 plants were fertile near-tetraploids, and analysis of their F3 and TC progeny revealed that they were sexual and genomically stabilized. The apomictic phenotype was not transmitted by genetic crossing as a single dominant locus on the Het chromosome, suggesting a complex genetic control of apomixis that has implications for future genetic and evolutionary analyses in this group.     

Genetic Diversity in Cymophyllus fraserianus (Cyperaceae), a Rare Monotypic Genus

Cymophyllus fraserianus (Fraser's sedge) is a rare perennial evergreen herb found in late-successional forest communities in the Appalachian Mountains of the U.S. Genetic diversity was assessed at 19 allozyme loci for 12 populations sampled from the southern and central portions of the range of this primitive monotypic genus. Species variation was comparable to that found for other narrowly distributed species, although mean population genetic diversity was somewhat higher. Sixteen of the 19 loci (84%) were polymorphic, with a mean of 46% polymorphic within populations. On average, 2.7 alleles were detected per polymorphic locus. Genetic diversity within populations (Hep) averaged 0.113, whereas species' genetic diversity (Hes) was 0.131. Although a twofold range of within-population genetic diversity (Hep = 0.075-0.158) was found, less than 13% of the genetic variation was distributed among populations (G(ST) = 0.129). An indirect estimate of historical levels of gene flow (Nm = 1.69) was consistent with the high mean genetic identity (mean I = 0.96) found between populations. Despite the relictual nature of C. fraserianus, its rarity and fragmented distribution, genetic diversity within the genus is typical of herbs with similar life-history characteristics. No association was found between geographic and genetic distances between populations (r = -0.01; p > 0.9), suggesting that present day populations are relatively isolated.     

Variation in circadian rhythms is maintained among and within populations in Boechera stricta

Circadian clocks have evolved independently in all three domains of life, and fitness benefits of a functional clock have been demonstrated in experimental genotypes in controlled conditions. Still, little is known about genetic variation in the clock and its fitness consequences in natural populations from heterogeneous environments. Using Wyoming populations of the Arabidopsis relative Boechera stricta as our study system, we demonstrate that genetic variation in the clock can occur at multiple levels: means of circadian period among populations sampled at different elevations differed by less than 1 h, but means among families sampled within populations varied by as much as 3.5 h. Growth traits also varied among and within populations. Within the population with the most circadian variation, we observed evidence for a positive correlation between period and growth and a negative correlation between period and root‐to‐shoot ratio. We then tested whether performance tradeoffs existed among families of this population across simulated seasonal settings. Growth rankings of families were similar across seasonal environments, but for root‐to‐shoot ratio, genotype × environment interactions contributed significantly to total variation. Therefore, further experiments are needed to identify evolutionary mechanisms that preserve substantial quantitative genetic diversity in the clock in this and other species.     

Multilocus Patterns of Nucleotide Diversity, Population Structure and Linkage Disequilibrium in Boechera stricta, a Wild Relative of Arabidopsis

Information about polymorphism, population structure, and linkage disequilibrium (LD) is crucial for association studies of complex trait variation. However, most genomewide studies have focused on model systems, with very few analyses of undisturbed natural populations. Here, we sequenced 86 mapped nuclear loci for a sample of 46 genotypes of Boechera stricta and two individuals of B. holboellii, both wild relatives of Arabidopsis. Isolation by distance was significant across the species range of B. stricta, and three geographic groups were identified by structure analysis, principal coordinates analysis, and distance-based phylogeny analyses. The allele frequency spectrum indicated a genomewide deviation from an equilibrium neutral model, with silent nucleotide diversity averaging 0.004. LD decayed rapidly, declining to background levels in ~10 kb or less. For tightly linked SNPs separated by <1 kb, LD was dependent on the reference population. LD was lower in the specieswide sample than within populations, suggesting that low levels of LD found in inbreeding species such as B. stricta, Arabidopsis thaliana, and barley may result from broad geographic sampling that spans heterogeneous genetic groups. Finally, analyses also showed that inbreeding B. stricta and A. thaliana have ~45% higher recombination per kilobase than outcrossing A. lyrata.     

Fine-scale population structure and dispersal in Biomphalaria glabrata, the intermediate snail host of Schistosoma mansoni, in Venezuela

Biomphalaria glabrata is the main intermediate host of Schistosoma mansoni in America and one of the most intensely studied species of freshwater snails, yet very little is known about its population biology. Here, we used seven highly polymorphic microsatellite loci to analyse genetic diversity in the Valencia lake basin, which represents the core of the endemic area for schistosomiasis in Venezuela. Populations were sampled at short spatial scale (a few kilometres), both inside the lake and in ponds or rivers near the lake. Our results indicate that B. glabrata essentially cross-fertilizes, with little variation in selfing rates among populations. Our markers detected considerable genetic variation, with an average heterozygosity of 0.60. More diversity per population was found within than outside the lake, suggesting an influence of connectivity among populations on the levels of genetic diversity. A marked population structure was detected and lake populations were less structured than other populations. Most individuals were assigned to their population of origin using an assignment test. No strong demographic signal (e.g. bottleneck) was detected, though lake populations are likely to experience bottlenecks more frequently than the other populations analysed. Differences in gene flow therefore seem to play an important role in population differentiation and in the restoring of genetic diversity in demographically unstable populations.     

Conservation genetics of an endemic and endangered epiphytic Laelia speciosa (Orchidaceae)

We used isozymes (16 loci in 11 enzymatic systems) from Laelia speciosa, an endemic and endangered epiphytic orchid of Mexico, to assess the genetic diversity and population genetic structure in nine populations distributed along its geographic range, as well as to detect those populations that are genetically unique and therefore deserve high-priority protection. On average, the genetic diversity was high (percentage of polymorphic loci, P(p) = 76%, mean number of alleles per locus, A = 3.34, the average observed heterozygosity H(O) = 0.302, the average expected heterozygosity H(E) = 0.382). Moderate levels of inbreeding (?f = 0.216, 95% confidence interval = 0.029-0.381) were found. Low levels of genetic differentiation were observed among populations ((p) = 0.040); however, there was a significant correlation between geographic and genetic distances among the populations (Mantel test: r(2) = 0.43, P < 0.05). Populations located within the same mountain range were genetically more similar. Private alleles were found, so proper management requires protection and maintenance of genetic diversity throughout its range. In case of reintroduction, we suggest using individuals propagated from seeds from as many capsules as possible, from close populations. An ex situ conservation strategy also is proposed.     

Comparison of genetic diversity estimates within and among populations of maritime pine using chloroplast simple-sequence repeat and amplified fragment length polymorphism data

We compared the genetic variation of Pinus pinaster populations using amplified fragment length polymorphism (AFLP) and chloroplast simple-sequence repeat (cpSSR) loci. Populations' levels of diversity within groups were found to be similar with AFLPs, but not with cpSSRs. The high interlocus variance associated with the AFLP loci could account for the lack of differences in the former. Although AFLPs revealed much lower genetic diversity than cpSSRs, the levels of among-population differentiation found with the two types of marker were similar, provided that loci showing fewer than four null-homozygotes, in any population, were pruned from the AFLP data. Moreover, the French and Portuguese populations were clearly differentiated from each other, with both markers. The Mantel test showed that the genetic distance matrix calculated using the AFLP data was correlated with the matrix derived from the cpSSRs. Because of the concordance found between markers we conclude that gene flow was indeed the predominant force shaping nuclear and chloroplastic genetic variation of the populations within regions, at the geographical scale studied.     

High genetic diversity in Sarracenia leucophylla(Sarraceniaceae), a carnivorous wetland herb

Eighteen allozyme loci were used to examine genetic diversity in 10 natural populations of Sarracenia leucophylla Raf., a pitcher plant restricted to the southeastern United States. One ex situ population propagated for restoration in Georgia was also analyzed. S. leucophylla is an insect-pollinated, outcrossing perennial wetland herb that is threatened over much of its geographic range. Fifteen loci (83.3%) were polymorphic, with a mean number of alleles of 3.33. Compared to species having similar life-history traits and to previously analyzed Sarracenia species, S. leucophylla displayed unexpectedly high genetic diversity. For example, genetic diversity within the species (Hes) was 0.224 and mean population genetic diversity (Hep) was 0.183. Although small S. leucophylla populations maintained less genetic diversity than larger ones, these differences were not statistically significant. Nonetheless, this suggests that small populations may have lost rare alleles. Statistically significant genetic differentiation among populations was found (theta = 0.192, P < .01), although it was not atypical considering the species' life-history characteristics. A significant correlation (P < .01) between genetic and geographic distance was found, indicating an isolation-by-distance effect. However, the correlation coefficient for this relationship was low (r = 0.46), suggesting that factors other than gene flow play a prominent role in the geographic distribution of genetic diversity within the species. The ex situ population captured most of the allozyme variation found in its source population.     

Population genetic structure of Bellamya aeruginosa (Mollusca: Gastropoda: Viviparidae) in China: weak divergence across large geographic distances

Bellamya aeruginosa is a widely distributed Chinese freshwater snail that is heavily harvested, and its natural habitats are under severe threat due to fragmentation and loss. We were interested whether the large geographic distances between populations and habitat fragmentation have led to population differentiation and reduced genetic diversity in the species. To estimate the genetic diversity and population structure of B. aeruginosa, 277 individuals from 12 populations throughout its distribution range across China were sampled: two populations were sampled from the Yellow River system, eight populations from the Yangtze River system, and two populations from isolated plateau lakes. We used seven microsatellite loci and mitochondrial cytochrome oxidase I sequences to estimate population genetic parameters and test for demographic fluctuations. Our results showed that (1) the genetic diversity of B. aeruginosa was high for both markers in most of the studied populations and effective population sizes appear to be large, (2) only very low and mostly nonsignificant levels of genetic differentiation existed among the 12 populations, gene flow was generally high, and (3) relatively weak geographic structure was detected despite large geographic distances between populations. Further, no isolation by linear or stream distance was found among populations within the Yangtze River system and no signs of population bottlenecks were detected. Gene flow occurred even between far distant populations, possibly as a result of passive dispersal during flooding events, zoochoric dispersal, and/or anthropogenic translocations explaining the lack of stronger differentiation across large geographic distances. The high genetic diversity of B. aeruginosa and the weak population differentiation are likely the results of strong gene flow facilitated by passive dispersal and large population sizes suggesting that the species currently is not of conservation concern.     

Balancing selection, random genetic drift, and genetic variation at the major histocompatibility complex in two wild populations of guppies (Poecilia reticulata)

Our understanding of the evolution of genes of the major histocompatibility complex (MHC) is rapidly increasing, but there are still enigmatic questions remaining, particularly regarding the maintenance of high levels of MHC polymorphisms in small, isolated populations. Here, we analyze the genetic variation at eight microsatellite loci and sequence variation at exon 2 of the MHC class IIB (DAB) genes in two wild populations of the Trinidadian guppy, Poecilia reticulata. We compare the genetic variation of a small (Ne, 100) and relatively isolated upland population to that of its much larger (Ne approximately 2400) downstream counterpart. As predicted, microsatellite diversity in the upland population is significantly lower and highly differentiated from the population further downstream. Surprisingly, however, these guppy populations are not differentiated by MHC genetic variation and show very similar levels of allelic richness. Computer simulations indicate that the observed level of genetic variation can be maintained with overdominant selection acting at three DAB loci. The selection coefficients differ dramatically between the upland (s > or = 0.2) and lowland (s < or = 0.01) populations. Parasitological analysis on wild-caught fish shows that parasite load is significantly higher on upland than on lowland fish, which suggests that large differences in selection intensity may indeed exist between populations. Based on the infection intensity, a substantial proportion of the upland fish would have suffered direct or indirect fitness consequences as a result of their high parasite loads. Selection by parasites plays a particularly important role in the evolution of guppies in the upland habitat, which has resulted in high levels of MHC diversity being maintained in this population despite considerable genetic drift.     

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake,the Eastern Massasauga (Sistrurus catenatus)

Genetic diversity is fundamental to maintaining the long‐term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.