Using microsatellite and MHC variation to identify species, ESUs, and MUs in the endangered Sonoran topminnow

Highly variable loci can provide insight into the recognition of species, evolutionarily significant units (ESUs) and management units (MUs). In general, the ESU and MU categories are thought to be reflective of adaptive differences between them. Here we examine this premise by presenting a comprehensive examination of genetic variation for both microsatellite loci and a major histocompatibility complex (MHC) locus, thought to be of adaptive significance, in the endangered Sonoran topminnow. The extent of variation for the microsatellite loci and the MHC gene within the 13 populations of the Gila topminnow is highly correlated, suggesting that nonselective factors have played an important role in influencing variation within and between populations for the MHC locus. Therefore, using all of these loci, we found that the eight natural populations of the Gila topminnow fell into two different ESUs, one of which had four different MUs. The source of the Boyce Thompson sample, a population that was used extensively for restocking, appeared to be Monkey Spring. The source of the Watson Wash population also appeared to be Monkey Spring (or Boyce Thompson). The newly colonized Santa Cruz River population, which had the most genetic variation of any Gila topminnow population, appeared to descend primarily from Sonoita Creek populations. The Yaqui topminnow, presently considered another subspecies of the Sonoran topminnow, was very distinct for both microsatellite (only two of 25 alleles found in the Yaqui were in any of the Gila topminnow samples) and MHC alleles (nonoverlapping sets of alleles for the two groups). As a result, it appeared that the taxonomic status of the two subspecies should be re-evaluated and that full species status for Gila and Yaqui topminnows was appropriate. There was evidence for the importance of long-term selection at the MHC locus in the higher rate of nonsynonymous than synonymous substitution. In addition, there appeared to have been a duplication of the MHC locus that was present in most of the fish in six of the natural populations of the Gila topminnow.     

Reduced MHC class II diversity in island compared to mainland populations of the black-footed rock-wallaby (<Emphasis Type="Italic">Petrogale lateralis lateralis</Emphasis>)

Many animal populations that are endangered in mainland areas exist in stable island populations, which have the potential to act as an “ark” in case of mainland population declines. Previous studies have found neutral genetic variation in such species to be up to an order of magnitude lower in island compared to mainland populations. If low genetic variation is prevalent across fitness-related loci, this would reduce the effectiveness of island populations as a source of individuals to supplement declining mainland populations or re-establish extinct mainland populations. One such species, the black-footed rock-wallaby (Petrogale lateralis lateralis), exists within fragmented mainland populations and small island populations off Western Australia. We examined sequence variation in this species within a fitness-related locus under positive selection, the MHC class II DAB β1 locus. The mainland populations displayed greater levels of allelic diversity (4–7 alleles) than the island population, despite being small and isolated, and contained at least two DAB gene copies. The island population displayed low allelic diversity (2 alleles) and fewer alleles per individual in comparison to mainland populations, and probably possesses only one DAB gene copy. The patterns of DAB diversity suggested that the island population has a markedly lower level of genetic variation than the mainland populations, in concordance with results from microsatellites (genotyped in a previous study), but preserved unique alleles which were not found in mainland populations. Where possible, conservation actions should pool individuals from multiple populations, not only island populations, for translocation programs, and focus on preventing further declines in mainland populations.     

Population fragmentation and major histocompatibility complex variation in the spotted suslik,Spermophilus suslicus

The fragmentation of populations typically enhances depletion of genetic variation, but highly polymorphic major histocompatibility complex (MHC) genes are thought to be under balancing selection and therefore retain polymorphism despite population bottlenecks. In this study, we investigate MHC DRB (class II) exon 2 variation in 14 spotted suslik populations from two regions differing in their degree of habitat fragmentation and gene flow. We found 16 alleles that segregated in a sample of 248 individuals. The alleles were highly divergent and revealed the hallmark signs of positive selection acting on them in the past, showing a significant excess of nonsynonymous substitutions. This excess was concentrated in putative antigen‐binding sites, which suggests that past selection was driven by pathogens. MHC diversity was significantly lower in fragmented western populations than in the eastern populations, characterized by significant gene flow. In contrast to neutral variation, amova did not reveal genetic differentiation between the two regions. This may indicate similar selective pressures shaping MHC variation in both regions until the recent past. However, MHC allelic richness within a population was correlated with that for microsatellites. F_ST outlier analyses have shown that population differentiation at DRB was neither higher nor lower than expected under neutrality. The results suggest that selection on MHC is not strong enough to counteract drift that results from recent fragmentation of spotted suslik populations.     

Genetic wealth,population health: Major histocompatibility complex variation in captive and wild ring‐tailed lemurs (Lemur catta)

Across species, diversity at the major histocompatibility complex (MHC) is critical to individual disease resistance and, hence, to population health; however, MHC diversity can be reduced in small, fragmented, or isolated populations. Given the need for comparative studies of functional genetic diversity, we investigated whether MHC diversity differs between populations which are open, that is experiencing gene flow, versus populations which are closed, that is isolated from other populations. Using the endangered ring‐tailed lemur (Lemur catta) as a model, we compared two populations under long‐term study: a relatively “open,” wild population (n = 180) derived from Bezà Mahafaly Special Reserve, Madagascar (2003–2013) and a “closed,” captive population (n = 121) derived from the Duke Lemur Center (DLC, 1980–2013) and from the Indianapolis and Cincinnati Zoos (2012). For all animals, we assessed MHC‐DRB diversity and, across populations, we compared the number of unique MHC‐DRB alleles and their distributions. Wild individuals possessed more MHC‐DRB alleles than did captive individuals, and overall, the wild population had more unique MHC‐DRB alleles that were more evenly distributed than did the captive population. Despite management efforts to maintain or increase genetic diversity in the DLC population, MHC diversity remained static from 1980 to 2010. Since 2010, however, captive‐breeding efforts resulted in the MHC diversity of offspring increasing to a level commensurate with that found in wild individuals. Therefore, loss of genetic diversity in lemurs, owing to small founder populations or reduced gene flow, can be mitigated by managed breeding efforts. Quantifying MHC diversity within individuals and between populations is the necessary first step to identifying potential improvements to captive management and conservation plans.     

Population structure and genetic diversity of trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) above and below natural and man-made barriers in the Russian River,California

The effects of landscape features on gene flow in threatened and endangered species play an important role in influencing the genetic structure of populations. We examined genetic variation of trout in the species Oncorhynchus mykiss at 22 microsatellite loci from 20 sites in the Russian River basin in central California. We assessed relative patterns of genetic structure and variation in fish from above and below both natural (waterfalls) and man-made (dams) barriers. Additionally, we compared sites sampled in the Russian River with sites from 16 other coastal watersheds in California. Genetic variation among the 20 sites sampled within the Russian River was significantly partitioned into six groups above natural barriers and one group consisting of all below barrier and above dam sites. Although the below-barrier sites showed moderate gene flow, we found some support for sub-population differentiation of individual tributaries in the watershed. Genetic variation at all below-barrier sites was high compared to above-barrier sites. Fish above dams were similar to those from below-barrier sites and had similar levels of genetic diversity, indicating they have not been isolated very long from below-barrier populations. Population samples from above natural barriers were highly divergent, with large F _st values, and had significantly lower genetic diversity, indicating relatively small population sizes. The origins of populations above natural barriers could not be ascertained by comparing microsatellite diversity to other California rivers. Finally, below-barrier sites farther inland were more genetically differentiated from other watersheds than below-barrier sites nearer the river’s mouth.     

Major histocompatibility complex class II genetic variation in forest musk deer (Moschus berezovskii) in China

The major histocompatibility complex (MHC) plays an important role in the immune system of vertebrates. We used the second exon of four MHC class II genes (DRA, DQA1, DQA2 and DRB3) to assess the overall MHC variation in forest musk deer (Moschus berezovskii). We also compared the MHC variation in captive and wild populations. We observed 22 alleles at four loci (four at DRA, four at DQA1, four at DQA2 and 10 at DRB3), 15 of which were newly identified alleles. Results suggest that forest musk deer maintain relatively high MHC variation, which may result from balancing selection. Moreover, considerable diversity was observed at the DRA locus. We found a high frequency of Mobe‐DRA*02, Mobe‐DQA1*01 and Mobe‐DQA2*05 alleles, which may be important for pathogen resistance. A Ewens–Watterson test showed that the DRB3 locus in the wild population had experienced recent balancing selection. We detected a small divergence at the DRA locus, suggesting the effect of weak positive selection on the DRA gene. Alternatively, this locus may be young and not yet adapted a wide spectrum of alleles for pathogen resistance. The significant heterozygosity deficit observed at the DQA1 and DRB3 loci in the captive population and at all four loci in the wild population may be the result of a population bottleneck. Additionally, MHC genetic diversity was higher in the wild population than in the captive, suggesting that the wild population may have the ability to respond to a wider range of pathogens.     

High MHC DQB Variation and Asymmetric Allelic Distribution in the Endangered Yangtze Finless Porpoise, Neophocaena phocaenoides asiaeorientalis

The endangered Yangtze finless porpoise is found in the middle and lower reaches of the Yangtze River and its adjoining big lakes. To explore the major histocompatibility complex (MHC) genetic diversity and allelic distribution patterns across its range, we investigated variation at DQB exon 2. From 76 porpoises, we identified 18 DQB sequences. The freshwater Yangtze populations had much higher allelic diversity than marine populations. Among these freshwater populations, the middle-reach population had higher allelic diversity than the lower-reach population. The high DQB diversity level, relative to that of a neutral mtDNA locus, suggests that balancing selection is acting at the DQB gene and that rapid evolution and local positive selection play critical roles in generating and retaining high MHC diversity in the freshwater population. As the balancing selection might be driven by environmental pathogens, we suggest that maintaining MHC variation should be a high priority in the conservation and management of this endangered population, especially as an ex situ conservation strategy.     

Major histocompatibility complex (MHC) class II polymorphism and paternity in the monogamous Hypogeomys antimena, the endangered, largest endemic Malagasy rodent

Sequence variation at a major histocompatibility complex (MHC) class II gene was examined in Hypogeomys antimena, a monogamous endemic rodent of Madagascar. The study was conducted throughout its remaining geographical range (20 x 40 km) by direct sequencing and single-strand conformation polymorphism (SSCP). The objectives of the study were: (i) to investigate levels of polymorphism in the MHC complex of a highly endangered species that experienced a severe reduction in population size; and (ii) to investigate the genetic mating system by assessing the frequency of extra-pair paternity (EPP) as EPP might have important consequences to increase gene flow and, therefore, genetic variability within a population. The amplified gene segment had a very low variability (only two alleles) in H. antimena compared with other mammalian species. The alleles segregated consistently with Mendelian expectations in families. No case of EPP was found. The present data suggest no difference between the social and the genetic mating system.     

MHC Variability of a Small Lemur in the Littoral Forest Fragments of Southeastern Madagascar

Habitat fragmentation inhibits gene flow between populations often resulting in a loss of genetic diversity with possible negative effects on fitness parameters. In vertebrates, growing evidence suggests that such genetic diversity is particularly important at the level of the major histocompatibility complex (MHC) because its gene products play an important role in immune functions. Diversity in the MHC is assumed to improve population viability. Here, we investigated the impact of forest fragmentation on the genetic variability of one of the functionally important parts of the MHC, DRB exon 2, of the endemic mouse lemur Microcebus murinus by comparing populations inhabiting two littoral forest fragments of different size in southeastern Madagascar. Twelve different alleles of DRB exon 2 were found in 145 individuals of M. murinus with high levels of sequence divergence between alleles. In both subpopulations, levels of genetic diversity were high, and the genetic analyses revealed only limited effects of fragmentation. Significantly more non-synonymous than synonymous substitutions were found in the functionally important antigen recognition and binding sites indicating selection processes maintaining MHC polymorphism. This is the first study on MHC variation in a free-ranging Malagasy lemur population.     

Small edge populations at risk: genetic diversity of the green lizard (<Emphasis Type="Italic">Lacerta viridis viridis</Emphasis>) in Germany and implications for conservation management

Edge and central populations can show great differences regarding their genetic variation and thereby also in their probability of extinction. This fact might be of great importance for the conservation strategies of endangered species. In this study we examine the level of microsatellite variability within three threatened edge populations of the green lizard subspecies Lacerta viridis viridis (Laur.) in Brandenburg (Germany) and compare the observed variation to other edge and central populations within the northern species range. We demonstrate that the northernmost edge populations contain less genetic variation in comparison to the central population. However, there were no observable significant differences to the other edge population included in this study. Surprisingly, we observed a high genetic differentiation in a small geographical range between the three endangered populations in Brandenburg, which can be explained by processes like fragmentation, isolation, genetic drift and small individual numbers within these populations. We also detected unique genetic variants (alleles), which only occurred in these populations, despite a low overall genetic variation. This study demonstrates the potential of fast evolving markers assessing the genetic status of endangered populations with a high resolution. It also illustrates the need for a comparative analysis of different regions within the species range, achieving a more exact interpretation of the genetic variation in endangered populations. This will aid future management decisions in the conservation of genetic diversity in threatened species.     

MHC diversity in bottlenecked populations: a simulation model

The depletion of variation at MHC loci, which play a crucial role in pathogen recognition, has been postulated to be one of important extinction risk factors for endangered populations. Thus, it is important to understand how selection affects the level of polymorphism in these genes when populations undergo a reduction in size. We followed MHC diversity in computer simulations of population bottlenecks. The fates of MHC alleles in the simulations were determined either by drift, or by balancing selection resulting from host–parasite coevolution. We found that the impact of selection on MHC polymorphism in bottlenecked populations was dependent upon the timescales involved. Initially, selection maintained lower number of alleles than drift, but after ~40 generations of hosts selection maintained higher MHC diversity, as compared to drift. The adverse effects of decreased MHC polymorphism on population viability may be, to some extent, compensated for if selection helps to retain MHC alleles which show high functional diversity, which should allow protection against a broader range of pathogens. Our simulation shows, however, that the mean divergence of alleles retained under selection in bottlenecked populations is not, on average, significantly higher than the divergence due to drift.     

Signals of heterogeneous selection at an MHC locus in geographically proximate ecotypes of sockeye salmon

The genes of the major histocompatibility complex (MHC) are an important component of the vertebrate immune system and can provide insights into the role of pathogen‐mediated selection in wild populations. Here, we examined variation at the MHC class II peptide‐binding region in 27 populations of sockeye salmon (Oncorhynchus nerka), distributed among three distinct spawning ecotypes, from a complex of interconnected rivers and lakes in south‐western Alaska. We also obtained genotypes from 90 putatively neutral single nucleotide polymorphisms for each population to compare the relative roles of demography and selection in shaping the observed MHC variation. We found that MHC divergence was generally partitioned by spawning ecotype (lake beaches, rivers and streams) and was 30 times greater than variation at neutral markers. Additionally, we observed substantial differences in modes of selection and diversity among ecotypes, with beach populations displaying higher levels of directional selection and lower MHC diversity than the other two ecotypes. Finally, the level of MHC differentiation in our study system was comparable to that observed over much larger geographic ranges, suggesting that MHC variation does not necessarily increase with increasing spatial scale and may instead be driven by fine‐scale differences in pathogen communities or pathogen virulence. The low levels of neutral structure and spatial proximity of populations in our study system indicate that MHC differentiation can be maintained through strong selective pressure even when ample opportunities for gene flow exist.     

Contrasting patterns of variation in MHC loci in the Alpine newt

Major histocompatibility complex (MHC) genes are essential in pathogen recognition and triggering an adaptive immune response. Although they are the most polymorphic genes in vertebrates, very little information on MHC variation and patterns of evolution are available for amphibians, a group known to be declining rapidly worldwide. As infectious diseases are invoked in the declines, information on MHC variation should contribute to devising appropriate conservation strategies. In this study, we examined MHC variation in 149 Alpine newts ( Mesotriton alpestris ) from three allopatric population groups in Poland at the northeastern margin of the distribution of this species. The genetic distinctiveness of the population groups has previously been shown by studies of skin graft rejection, allozymes and microsatellites. Two putative expressed MHC II loci with contrasting levels of variation and clear evidence of gene conversion/recombination between them were detected. The Meal-DAB locus is highly polymorphic (37 alleles), and shows evidence of historical positive selection for amino acid replacements and substantial geographical differentiation in allelic richness. On the contrary, the Meal-DBB locus exhibits low polymorphism (three alleles differing by up to two synonymous substitutions) and a uniform distribution of three alleles among geographical regions. The uniform frequencies of the presumptively neutral Meal-DBB alleles may be explained by linkage to Meal-DAB . We found differences in allelic richness in Meal-DAB between regions, consistent with the hypothesis that genetic drift prevails with increasing distance from glacial refugia. Pseudogene loci appear to have evolved neutrally. The level of DAB variation correlated with variation in microsatellite loci, implying that selection and drift interplayed to produce the pattern of MHC variation observed in marginal populations of the Alpine newt.     

Isolation and characterization of microsatellite loci in the blue manakin,Chiroxiphia caudata (Aves,Pipridae)

We designed primers for amplifying 11 polymorphic microsatellite loci in the blue manakin, Chiroxiphia caudata, a neotropical passerine bird which inhabits a critically endangered tropical ecosystem, the Brazilian Atlantic forest. Based on genotypes from 24 individuals from a single population, we detected between four and 22 alleles per locus with observed heterozygosities ranging from 0.54 to 0.92. These highly variable loci will be useful for determining levels of population differentiation and assessing the impact of habitat fragmentation on levels of genetic variation in isolated populations of these birds.     

Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure

Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre‐eminent system for the study of selective pressures that arise from host–pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population‐genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.     

Low MHC DRB class II diversity in the mountain goat: past bottlenecks and possible role of pathogens and parasites

Major histocompatibility complex (MHC) genes are the most polymorphic in vertebrates and code for molecules playing a central role in pathogen resistance. We studied levels of MHC DRB class II diversity in a long-term study population of mountain goats (Oreamnos americanus) at Caw Ridge, Alberta, and two other populations from British Columbia, Canada. Only two alleles were found among the three populations sampled. The Caw Ridge population was fixed for one of the two MHC DRB alleles, but this lack of variation did not appear to have affected it negatively because the population doubled over two decades and had no history of any apparent infectious diseases. Past population bottlenecks during Pleistocene glaciations are thought to have been the main factor contributing to the low levels of MHC diversity in mountain goats, a hypothesis supported by our previous work reporting low polymorphism at neutral loci. Additionally, the limited MHC variability in mountain goats may be related to its northern distribution as we found that allelic diversity at MHC DRB class II in wild ungulates decreases with increasing latitude, possibly as a result of low parasite diversity at high latitudes. The low MHC variation in mountain goats and other northern ungulates such as muskoxen (Ovibos moschatus) may expose these species to population outbreaks that could be generated by introduced pathogens or northward shifts in the distribution of pathogens with global climate warming.     

GENETIC POPULATION STRUCTURE AND LEVELS OF GENE FLOW IN THE STREAM DWELLING WATERSTRIDER,AQUARIUS (=GERRIS) REMIGIS (HEMIPTERA: GERRIDAE)

Gene flow, in combination with selection and drift, determines levels of differentiation among local populations. In this study we estimate gene flow in a stream dwelling, flightless waterstrider, Aquarius remigis. Twenty-eight Aquarius remigis populations from Quebec, Ontario, New Brunswick, Iowa, North Carolina, and California were genetically characterized at 15 loci using starch gel electrophoresis. Sampling over two years was designed for a hierarchical analysis of population structure incorporating variation among sites within streams, streams within watersheds, watersheds within regions, and regions within North America. Hierarchical F statistics indicated that only sites within streams maintained enough gene flow to prevent differentiation through drift (Nm = 27.5). Above the level of sites within streams gene flow is highly restricted (Nm ≤ 0.5) and no correlation is found between genetic and geographic distances. This agrees well with direct estimates of gene flow based on mark and recapture data, yielding an N_e of approximately 170 individuals. Previous assignment of subspecific status to Californian A. remigis is not supported by genetic distances between those populations and other populations in North America. Previous suggestion of specific status for south-eastern A. remigis is supported by genetic distances between North Carolina populations and other populations in North America, and a high proportion of region specific alleles in the North Carolina populations. However, because of the high degree of morphological and genetic variability throughout the range of this species, the assignment of specific or subspecific status to parts of the range may be premature.     

Are there Ubiquitous Parasite-driven Major Histocompatibility Complex Selection Mechanisms in Gray Mouse Lemurs?

A major goal of evolutionary biology is to understand how selection drives local adaptation. For example, the major histocompatibility complex (MHC) plays an important role in the immune system, and high levels of MHC variation are thought to be a form of adaptation in natural populations. Individual MHC composition may influence parasite resistance via advantages associated with 1) heterozygosity, because heterozygotes recognize a broader range of different antigens than homozygotes (heterozygote advantage); 2) highly variable amino acid sequences in MHC alleles, allowing individuals to bind a broader spectrum of parasite-derived peptides (divergent-alleles advantage, a mechanistic variant of the heterozygote advantage model); or 3) specific MHC alleles (rare allele advantage or frequency dependent selection). We investigated relationships between gastrointestinal nematode burden and both adaptive immune gene variability (MHC class II DRB) and neutral microsatellites in free-living gray mouse lemurs (Microcebus murinus) native to a dry deciduous forest population in western Madagascar to test these hypotheses. The individual MHC composition was related to parasite infestation. Specific MHC alleles were involved in parasite resistance and the presence of common alleles negatively influenced infestation intensity. We found no support for the heterozygote advantage hypothesis, but we did find support for the divergent-MHC allele advantage hypothesis: Individuals with very divergent MHC alleles carried fewer and less intense nematode infestations than individuals with more similar alleles in the more variable dry deciduous forest population. These results indicate that intestinal parasites are important selection pressures under natural conditions and suggest that different selection mechanisms are not mutually exclusive. In contrast, we detected no association between neutral overall individual genetic diversity (measured via 17 microsatellites) and parasite load. Finally, we investigated the ubiquity of parasite-driven selection mechanisms by comparing our results with a previous study of a mouse lemur population from the climatically different littoral forest in southeastern Madagascar, ca. 500 km away. This revealed that different specific MHC alleles were involved in parasite resistance in the 2 habitats, showing that gene-parasite associations are not consistent between populations.     

Specific alleles at immune genes,rather than genome‐wide heterozygosity,are related to immunity and survival in the critically endangered Attwater's prairie‐chicken

The negative effects of inbreeding on fitness are serious concerns for populations of endangered species. Reduced fitness has been associated with lower genome‐wide heterozygosity and immune gene diversity in the wild; however, it is rare that both types of genetic measures are included in the same study. Thus, it is often unclear whether the variation in fitness is due to the general effects of inbreeding, immunity‐related genes or both. Here, we tested whether genome‐wide heterozygosity (20 990 SNPs) and diversity at nine immune genes were better predictors of two measures of fitness (immune response and survival) in the endangered Attwater's prairie‐chicken (Tympanuchus cupido attwateri). We found that postrelease survival of captive‐bred birds was related to alleles of the innate (Toll‐like receptors, TLRs) and adaptive (major histocompatibility complex, MHC) immune systems, but not to genome‐wide heterozygosity. Likewise, we found that the immune response at the time of release was related to TLR and MHC alleles, and not to genome‐wide heterozygosity. Overall, this study demonstrates that immune genes may serve as important genetic markers when monitoring fitness in inbred populations and that in some populations specific functional genes may be better predictors of fitness than genome‐wide heterozygosity.     

Genetic diversity in fragmented populations of Berchemiella wilsonii var. pubipetiolata (Rhamnaceae)

BACKGROUD AND AIMS: Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is distributed in fragmented habitat patches in eastern China. It is highly endangered because of severe disturbance by anthropogenic activities. Information on genetic variation and structure is critical for developing successful conservation strategies for this species. METHODS: Allozyme variation of population genetic diversity and structure was investigated for a total of 98 individuals sampled from four extant populations using isoelectric focusing in thin-layer polyacrylamide slab gels. KEY RESULTS: Based on 20 loci scored from the nine enzymes examined, a high genetic diversity was detected at both the species and population level, while there was a loss of low frequency alleles (<0.1) in all populations. Most loci showed deviation from Hardy-Weinberg equilibrium due to excess of heterozygotes in all populations, suggesting that selection for heterozygotes has occurred in this species. The genetic diversity was mainly found within populations with a moderate genetic differentiation (F(ST) = 0.13), but the two geographically discontinuous population groups showed significant differences, with F-statistic values of 0.078 for the Zhejiang populations and 0.014 for the Anhui populations, respectively. CONCLUSIONS: It appears most likely that this species has experienced a recent decrease in population size, and genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. The differentiation into two population groups reflects a population genetic consequence that has been influenced by the different land-use in the two regions. Some conservation concerns are discussed together with possible strategies for implementing in situ and ex situ conservation.