Bottleneck Tests Research Articles

Mid‐Holocene decline in African buffalos inferred from Bayesian coalescent‐based analyses of microsatellites and mitochondrial DNA

Genetic studies concerned with the demographic history of wildlife species can help elucidate the role of climate change and other forces such as human activity in shaping patterns of divergence and distribution. The African buffalo (Syncerus caffer) declined dramatically during the rinderpest pandemic in the late 1800s, but little is known about the earlier demographic history of the species. We analysed genetic variation at 17 microsatellite loci and a 302-bp fragment of the mitochondrial DNA control region to infer past demographic changes in buffalo populations from East Africa. Two Bayesian coalescent-based methods as well as traditional bottleneck tests were applied to infer detailed dynamics in buffalo demographic history. No clear genetic signature of population declines related to the rinderpest pandemic could be detected. However, Bayesian coalescent modelling detected a strong signal of African buffalo population declines in the order of 75-98%, starting in the mid-Holocene (approximately 3-7000 years ago). The signature of decline was remarkably consistent using two different coalescent-based methods and two types of molecular markers. Exploratory analyses involving various prior assumptions did not seriously affect the magnitude or timing of the inferred population decline. Climate data show that tropical Africa experienced a pronounced transition to a drier climate approximately 4500 years ago, concurrent with the buffalo decline. We therefore propose that the mid-Holocene aridification of East Africa caused a major decline in the effective population size of the buffalo, a species reliant on moist savannah habitat for its existence.

Evidence of recent population bottlenecks and inbreeding in British populations of Bechstein’s bat, Myotis bechsteinii

We investigated the population genetics of seven maternity roosts of Bechstein's bats widely distributed across the south of England. Across all of the populations sampled, two mitochondrial DNA microsatellite loci were fixed for single haplotypes. Genetic diversity across eight nuclear microsatellite loci was similar in all seven populations, with a mean He of 0.727. However, six of the populations showed substantial homozygote excess, with F IS estimates greater than zero, indicative of recent inbreeding. Bottleneck tests also implied that six of the populations have experienced recent declines. Genetic differentiation among the populations was low, with a mean intersite F ST estimate of 0.041. There was no significant isolation by distance using allele frequency-based criteria (F ST and genetic distances), however, a weak correlation was found using the allele size-based R ST criterion. Assignment tests were unable to distinguish the seven sampling sites as distinct clusters. Mean intra-roost relatedness (r) was 0.079, indicative of recent inbreeding relative to German populations. All but one of the bats had one or more half or full siblings in its maternity roost. In addition, family relationships of individuals within a colony were significantly commoner than family relationships among four proximal roosts <8 km apart. The results are discussed in the context of conservation requirements for this rare British bat.

Genetic structure of an isolated population of mantled howler monkeys (Alouatta palliata) on Barro Colorado Island, Panama

Habitat fragmentation may influence genetic variability through reductions in population size and the physical isolation of conspecifics. We explored the effects of these factors on genetic diversity in a population of mantled howler monkeys (Alouatta palliata) on Barro Colorado Island (BCI), Panama. The study population was established in 1914 when an unknown number of resident individuals were isolated from the surrounding mainland by damming of the Rio Chagres to create the Panama Canal. Analyses of 10 microsatellite loci indicated that, despite this isolation, the howler monkeys on BCI exhibit levels of genetic diversity (H S = 0.584 ± 0.063) among the highest reported for any species of Alouatta. These data also revealed that although relatedness among adults in a social group was significantly greater than zero, the BCI population is not highly genetically structured. Tests for genetic bottlenecks based on departures from equilibrium expectations failed to reveal evidence of a recent reduction in population size. In contrast, coalescent modeling indicated that this population has likely experienced a marked decline within the last few 100 years. These findings generate new insights into the genetic structure of A. palliata and suggest that while the formation of BCI may not have substantially reduced genetic variation in these animals, genetic diversity has been influenced by historical changes in population size.

Genetic impoverishment of the last black grouse (Tetrao tetrix) population in the Netherlands: detectable only with a reference from the past

We have studied a small isolated population of black grouse (Tetrao tetrix) in the Netherlands to examine the impact of isolation and reduction in numbers on genetic diversity. We compared the genetic diversity in the last extant Dutch population with Dutch museum samples and three other black grouse populations (from England, Austria and Norway, respectively) representing isolated and continuous populations. We found significantly lower allelic richness, observed and expected heterozygosities in the present Dutch population compared to the continuous populations (Austria and Norway) and also to the historical Dutch population. However, using a bottleneck test on each population, signs of heterozygosity excess were only found in the likewise isolated English population despite that strong genetic drift was evident in the present Dutch population in comparison to the reference populations, as assessed both in pairwise F(ST)and STRUCTURE analyses. Simulating the effect of a population reduction on the Dutch population from 1948 onwards, using census data and with the Dutch museum samples as a model for the genetic diversity in the initial population, revealed that the loss in number of alleles and observed heterozygosity was according to genetic drift expectations and within the standard error range of the present Dutch population. Thus, the effect of the strong decline in the number of grouse on genetic diversity was only detectable when using a reference from the past. The lack of evidence for a population reduction in the present Dutch population by using the program bottleneck was attributed to a rapidly found new equilibrium as a consequence of a very small effective population size.

INFERRING POPULATION HISTORY AND DEMOGRAPHY USING MICROSATELLITES, MITOCHONDRIAL DNA, AND MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) GENES

Microsatellites and mitochondrial DNA (mtDNA) have traditionally been used in population genetics because of their variability and presumed neutrality, whereas genes of the major histocompatibility complex (MHC) are increasingly of interest because strong selective pressures shape their standing variation. Despite the potential for MHC genes, microsatellites, and mtDNA sequences to complement one another in deciphering population history and demography, the three are rarely used in tandem. Here we report on MHC, microsatellite, and mtDNA variability in a single large population of the eastern tiger salamander (Ambystoma tigrinum tigrinum). We use the mtDNA mismatch distribution and, on microsatellite data, the imbalance index and bottleneck tests to infer aspects of population history and demography. Haplotype and allelic variation was high at all loci surveyed, and heterozygosity was high at the nuclear loci. We find concordance among neutral molecular markers that suggests our study population originated from post-Pleistocene expansions of multiple, fragmented sources that shared few migrants. Differences in N(e) estimates derived from haploid and diploid genetic markers are potentially attributable to secondary contact among source populations that experienced rapid mtDNA divergence and comparatively low levels of nuclear DNA divergence. We find strong evidence of natural selection acting on MHC genes and estimate long-term effective population sizes (N(e)) that are very large, making small selection intensities significant evolutionary forces in this population.

Demographic bottlenecks and low gene flow in remnant populations of the critically endangered Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) inferred from microsatellite markers

Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is an endangered plant with only four remnant populations in eastern China. Population genetic information is essential for understanding population history and formulating conservation strategies for this species. Thirteen microsatellite loci were used to investigate genetic variation and population structure of the four remnant populations. Moderate levels of expected heterozygosity (HE = 0.466–0.543) and low allelic diversity (A = 3.1–3.6 and AR = 2.2–2.4, respectively) were observed within populations. Bottleneck tests found three out of four populations to deviate from mutation-drift equilibrium under the two-phase model (TPM), suggesting a recent population decline, which is congruent with known demographic history. The evolutionary history of the species seems dominated by genetic drift rather than gene flow. Low historical gene flow was inferred from several different approaches and Nm ranged from 0.582 by the private allele method to 0.783 by the coalescent method. Contemporary gene flow was also found to be even lower for only one first generation migrant was detected with individual-based assignment analysis. Restricted pollen and seed dispersal as well as a recent decline in population size associated with habitat fragmentation may have contributed to low levels of historical and contemporary gene flow, and resulted in a high genetic differentiation. Under this scenario, Berchemiella wilsonii var. pubipetiolata populations are expected to display more pronounced population genetic structure in the future as a result of increased inbreeding and genetic drift.

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf (Canis lupus) population

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central-southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise F(ST) values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first-generation migrants indicates that migration has been unidirectional and male-biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long-range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25-2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8-16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.

Genetic structure of the European polecat (Mustela putorius) and its implication for conservation strategies

AbstractDuring the last century, the European polecat Mustela putorius populations in most of Europe declined and survived in fragmented patches, because of habitat alterations and direct persecution. To assess the genetic consequences of the demographic decline and to describe the spatial pattern of genetic diversity, 250 polecats sampled at seven localities from five European countries – Poland, Denmark (southern Denmark and northern Denmark), Spain, Belgium (eastern and western) and the Netherlands – were screened by means of nine microsatellite loci. Genetic diversity estimated by mean expected heterozygosity (HE) and allelic richness (AR) were moderately high within populations [range: 0.50 (northern Denmark) ≤HE≤0.64 (Poland) and 1.33≤AR≤7.80] as compared with other carnivores and mustelids. Bottleneck tests suggested that polecat populations in southern Denmark and Poland have declined recently and populations from northern Denmark and the Netherlands have expanded recently, whereas the remaining populations did not show any sign of demographic change. Recent demographic changes could suggest that some of the populations are still not in equilibrium, which could partly explain the relatively high genetic variability observed in polecat populations despite the drastic decline in population size observed in several European countries. A significant heterozygote deficiency [FIS=0.19; 0.01≤95% confidence interval (CI)≤0.32] suggests substructuring within the total European sample. Partitioning of the genetic variation among sampling locations (FST=0.14; 0.06≤95% CI≤0.23) and pairwise FST between localities (range: 0.01≤FST≤0.37) without any correlation with the geographic distances between localities were found, suggesting a recent divergence and a restriction of gene flow between populations and the action of genetic drift. An assignment test showed that the Polish and the northern Danish populations were the most unique, whereas the other populations were partially admixed. Factorial component analysis tests indicate a subdivision of the total sample into two distinct groups: one including the samples from Poland and the two Danish localities and the second group comprising the remaining localities investigated. The observed pattern of genetic differentiation is suggested to be due to two main routes of recolonization after the last glacial period. To compare the results obtained with microsatellite data, the most variable region of the mitochondrial DNA (d‐loop) was sequenced and different phylogenetic reconstructions and genetic diversity analyses based on nucleotide (π) and haplotype diversity (h) measures within populations were performed using a subsample of populations. The lack of well‐defined geographical structure, as well as the reduced level of mitochondrial DNA variability (π: 0.00274±0.00038; h: 0.876±0.028) that was found, has been previously reported in several studies on different carnivores and supports the hypothesis of post‐glacial recolonization from southern or eastern refugees of Europe as suggested by the microsatellite data. Implications for conservation strategies of the polecat at the European level are discussed.

Underwater but not out of sight: genetic monitoring of effective population size in the endangered North Sea houting (Coregonus oxyrhynchus)

We analysed 12 microsatellite DNA loci in temporal samples (1980, 1994, and 2002) from the only remaining indigenous population of the North Sea houting (Coregonus oxyrhynchus) in the Vidaa River, Denmark. Using a novel temporal method, we estimated effective population size (Ne) to be 577.4 (90% highest posterior density limits 297.2–3719.8). The same method was used to estimate Neat the beginning and end of the sampled time interval, and the results were indicative of a relatively stable population. In contrast, tests for recent bottlenecks suggested population declines in the 1980 and 1994 samples, possibly reflecting declines prior to 1980 in the total North Sea houting population. To evaluate the usefulness of the two methods for routine genetic monitoring, we simulated population declines corresponding to reproduction by only 20 or 50 parents in 2002. For both simulated samples, the temporal method provided evidence for a population decline, whereas the test for bottlenecks did not suggest population decline. We conclude that the North Sea houting in the Vidaa River is not immediately threatened by inbreeding or loss of evolutionary potential, and the applied temporal method appears very useful for genetic monitoring of effective population size in endangered, isolated fish populations.

Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park

Population declines caused by natural and anthropogenic factors can quickly erode genetic diversity in natural populations. In this study, we examined genetic variation within 10 tiger salamander populations across northern Yellowstone National Park in Wyoming and Montana, USA using eight microsatellite loci. We tested for the genetic signature of population decline using heterozygosity excess, shifts in allele frequencies, and low ratios of allelic number to allelic size range (M-ratios). We found different results among the three tests. All 10 populations had low M-ratios, five had shifts in allele frequencies and only two had significant heterozygosity excesses. These results support theoretical expectations of different temporal signatures among bottleneck tests and suggest that both historical fish stocking, recent, sustained drought, and possibly an emerging amphibian disease have contributed to declines in effective population size.

Stocking impact and temporal stability of genetic composition in a brackish northern pike population (Esox lucius L.), assessed using microsatellite DNA analysis of historical and contemporary samples

During the last decade, brackish northern pike populations in Denmark have been subject to stocking programmes, using nonindigenous pike from freshwater lakes, in order to compensate for drastic population declines. The present study was designed to investigate the genetic impact of stocking freshwater pike into a brackish pike population in Stege Nor, Denmark. We analysed polymorphism at eight microsatellite loci in samples representing the indigenous Stege Nor population prior to stocking (ie from 1956 to 1957), along with a sample of the contemporary Stege Nor population and samples from the three populations used for stocking. Despite large numbers of stocked fry, the results from both individual and population level admixture analyses demonstrated extremely poor performance and <1% introgression of stocked freshwater pike into the brackish pike population. Furthermore, pairwise F(ST) estimates between samples demonstrated close genetic relationship among temporal samples from Stege Nor, indicating temporal stability over the last 45 years. We also estimated the effective population size (N(e)) of pike in Stege Nor and applied a test for recent population bottlenecks. The harmonic mean of N(e) was relatively high (>250), but there were indications of bottlenecks in all samples and populations. We ascribe this finding to historical rather than recent bottlenecks, possibly dating back to founder events associated with postglacial recolonisation.

Genetic analysis of four wild chum salmonOncorhynchus keta populations in China based on microsatellite markers

To assess the genetic variation and population structure of wild chum salmon in China, we analyzed microsatellite loci for populations in the Amur, Wusuli, Suifen Current and the Tumen rivers. We evaluated expected heterozygosity with two estimators of genetic differentiation (FST and GST) and Nei’s standard genetic distance. The average expected heterozygosity across the 10 loci was 0.65 in the Wusuli River and the Suifen Current River, 0.64 in the Amur River and 0.66 in the Tumen River, The results of this study show that the recent declines in chum salmon have not led to low levels of genetic variability in China. The proportion of inter-population subdivision among chum salmon was between 5.7 and 6.8%. According to the estimator used, the NJ tree based on Nei’s standard genetic distance indicated that there were two different branches (the Sea of Okhotsk branch and the Sea of Japan branch), the Amur River and the Wusuli River populations were closer, while the Suifen Current River and the Tumen River clustered together. The genetic test for population bottlenecks provided no evidence for a significant genetic signature of population decline, which is consistent with the record of the four populations we have in the last few years.

A method for adjusting allele frequencies in the case of microsatellite allele drop‐out

AbstractA new method is proposed to adjust allele frequencies when allelic drop‐out is common. This method assumes Hardy–Weinberg equilibrium (HWE), and treats the problematic alleles as a one‐locus two‐allele system with dominance. By assuming that the homozygote frequency of the ‘recessive’ allele is measured correctly, we can back calculate the allele frequency of the ‘dominant’ allele, and adjust the heterozygote frequency accordingly. The drawback is that multilocus genotypes cannot be constructed and tests that use deviations from Hardy–Weinberg such as tests for bottlenecks become impossible. An example is given where a large homozygote excess (FIS = 0.44) is adjusted to a reasonable level (FIS = 0.046). The effect of scoring error was set in relation to sampling error and while FIS values can be seriously biased, FST values are not necessarily so, if scoring error and sample size are both low. As sample size increases, the effect of scoring error increases.

Sibship within samples of brown trout (Salmo trutta) and implications for supportive breeding

We analysed family relationships among brown trout from two small tributary populations that have been suggested as a source of individuals for supportive breeding, using variation at eight microsatellite loci. As a control, we analysed a sample of supposedly unrelated individuals representing a large anadromous population, and we simulated unrelated individuals based on the allelic distributions in all three samples. Two different approaches were used: (1) pairwise estimates of relatedness between individuals and (2) a method for partitioning individuals into half-sib and full-sib families. The anadromous population did not show evidence of a significant number of closely related individuals. In both tributary populations, however, the distributions of pairwise relatedness estimates suggested the presence of several related individuals, and sibship reconstruction suggested fewer families consisting of more individuals than were observed for the simulated individuals. The expected increase of inbreeding coefficient in the two samples due to family structure was 0.026 and 0.030 respectively. Moreover, tests for recent bottlenecks yielded significant outcomes in both populations suggesting a history of low effective population sizes. Depending on the effective population size of captive spawners and past effective population sizes in the populations it could be beneficial to conduct sib-avoidance matings, though this cannot eliminate inbreeding but only delay it. Alternatively, individuals from different populations could be crossed. Sibship reconstruction provided the clearest evidence for family structure, but pairwise relatedness is the best measure for designing mating schemes, as it allows for mating as unrelated individuals as possible rather than just avoiding mating between sibs.

Genetic variability in Danish polecats Mustela putorius as assessed by microsatellites

Genetic variability and population structure was investigated in 83 European polecats Mustela putorius by means of six microsatellite markers. The samples came from two areas in Denmark, Østjylland and Thy, which are separated by the Limfjord. The genetic diversity (He = 0.583) found in the total sample was similar to those found in other mustelid species and carnivores in general. A heterozygote deficiency, probably due to a Wahlund effect, suggested a further substructuring of the Danish sample. Population genetic substructuring was investigated in three different ways: by means of the program STRUCTURE, Wright's F‐statistics and by an assignment test. All the tests indicate a subdivision of the sample into two distinct groups, which is concordant with the two sampling locations, with an average genetic divergence of FST = 0.126 and RST = 0.1692. The higher genetic diversity found in the Thy population (He = 0.578), as compared to the Østjylland population (He = 0.420), could be explained by assuming two ancient waves of colonisation of the Danish peninsula. Tests for recent bottlenecks were conducted, and the results suggest no evidence of neither population decline nor expansion. Our study is the first one in which microsatellite markers are used on polecat samples, and one locus (mv54) was found to be diagnostic in distinguishing between American mink Mustela vison and European polecat.

Genetic structure of Asian populations of Bombus ignitus (Hymenoptera: Apidae).

The genetic structure of seven mainland and island Asian populations of Bombus ignitus was investigated using nine microsatellite markers and the sequences of part of the mitochondrial cytochrome b (cytb) gene. While microsatellite markers showed high genetic variability, no sequence variation was found in the cytb gene fragment analyzed. The number of microsatellite alleles ranged from 9 to 24. Gene diversities per locus per population ranged from 0.378 to 0.992. Analysis of molecular variance (AMOVA) and most pairwise F(ST) values showed significant genetic differentiation between mainland and island populations. Cytb sequences data and microsatellite bottleneck tests indicated that almost all populations were subjected to recent bottlenecks. Our results suggest that B. ignitus populations diverged due to recent bottlenecks and geographic isolation.

Relationship between population size and genetic diversity in endangered populations of the European bullhead ( Cottus gobio): implications for conservation

This study investigated the relationship between the current size of endangered bullhead ( Cottus gobio) populations and microsatellite genetic variability. Additionally, the microsatellite data were used to evaluate whether a genetic test for population bottlenecks was able to provide evidence of recent severe population declines. Finally, our results were used to develop conservation priorities and measures. Population size appears to be a crucial parameter in determining the amount of genetic diversity that can be preserved in bullheads, since a significant positive correlation was observed between both variables. Furthermore, in some populations we were able to detect genetic signatures of the documented decline in population size. We suggest that the most immediate goal for bullhead conservation should be to increase the size and the range of the populations, and in doing so minimise or even reverse further genetic erosion. Potential management actions like habitat quality improvement, reduction of river fragmentation and supplementation programmes (translocation, supportive breeding) are discussed.

The influence of mating system, demography, parasites and colonization on the population structure of Biomphalaria pfeifferi in Madagascar.

Current evolutionary forces and historical processes interact to shape the distribution of neutral genetic variability within and among populations. Focusing on the genetics of recently introduced organisms offers a good opportunity to understand the relative importance of these factors. This study concerns variation at 8 polymorphic microsatellite loci in 30 populations of Biomphalaria pfeifferi. The sampling area spans most of the species' range in Madagascar where it was probably introduced recently. Extremely low variation was found within all populations studied, which may partly result from high selfing rates. However, this cannot account for the variance of variation across populations, which is better explained by habitat openness (that reflects environmental stochasticity), the prevalence of the parasitic trematode Schistosoma mansoni and historical demography (colonization and subsequent bottlenecks). Large global differentiation was also observed, suggesting that current gene flow among populations is limited to small distances, within watersheds and to few individuals. Our data set also allowed us to test several hypotheses regarding colonization, based on bottleneck and admixture tests. The observed pattern requires at least two independent introductions from slightly differentiated genetic sources in the western part of Madagascar. Another introduction, from a very different genetic origin, should also be postulated to explain the genetic composition of eastern populations. That this introduction occurred recently suggests that the colonization of Madagascar by B. pfeifferi is an ongoing process.

Genetic monitoring of brackish-water populations: the Mediterranean toothcarp Aphanius fasciatus (Cyprinodontidae) as a model

The measurement of genetic variability and assessment of population genetic losses are important components of environmental management programs. Twenty-three natural populations of the Mediterranean brackish-water toothcarp Aphanius fasciatus were investigated using different statistical approaches based on genetic data at 13 polymorphic allozyme loci. In general, no differ- ences between values of within-population genetic variability estimates occurred. The Wilcoxon sign-rank test for heterozygosity excess due to a recent bottleneck was conducted on the array of populations. In addition, a qualitative descriptor of allele frequency distribution was used to infer bottlenecks. Only populations from the Orbetello lagoon and La Salina at Elba Island revealed sig- nificant heterozygosity excess under both the infinite allele model (IAM) and stepwise mutation model (SMM). A recent dystrophic crisis may account for the genetic loss detected in the population of A. fasciatus from the Orbetello lagoon, whereas exceptionally high predation pressure and/or the increased mortality rate created by local hydrological conditions could be responsible for the bottle- neck in the population at La Salina. Tests for bottlenecks have proved effective tools for genetic monitoring of A. fasciatus populations.

Genetic diversity, mating system, and conservation of a Mexican subalpine relict, Picea mexicana Martínez

Mexican spruce (Picea mexicana Martinez), an endangered species of the highest sky islands in Mexico's Sierra Madre Oriental and Sierra Madre Occidental, is threatened by fire, grazing, and global warming. Its conservation depends on whether it also is threatened by inbreeding and loss of genic diversity. We used 18 isozyme markers in 12 enzyme systems to assay genic diversity, characterize the mating system, and test for recent bottlenecks in three known populations. Unbiased, expected heterozygosity (H e ) averaged 0.125. Despite a separation of 676 km between populations in the Sierra Madre Oriental and the Sierra Madre Occidental, Wright's F ST , the proportion of total genic diversity among populations, was only 6.9%. Nei's genetic distance was 0.001 between the populations in the Sierra Madre Oriental and more than an order of magnitude greater, 0.019, between the Sierra Madre Oriental and Sierra Madre Occidental. However, both values point to relatively recent divergence. Mating systems were predominantly outcrossing, but with significant selfing. Multilocus estimates of selfing varied from 19% to 41%, and the means of single-locus estimates were higher, suggesting that additional inbreeding occurred by mating among relatives. Despite significant inbreeding, observed heterozygosity was as high as or higher than H e ; Wright's fixation index, F IS , was −0.107. Under the observed level of selfing, positive values of F IS were expected. Therefore, selection against inbreds and homozygotes must be intense. Cornuet-Luikart tests indicate recent bottlenecks in at least two of the three populations. The results suggest that Mexican spruce is a genetically viable species, and threats are primarily environmental.