Urbanisation versus agriculture: a comparison of local genetic diversity and gene flow between wood mouse Apodemus sylvaticus populations in human‐modified landscapes

Urbanisation and agriculture dramatically modify the landscapes available for use by wildlife, affecting key aspects of their ecology such as survival, foraging, predation, competition and reproductive success. Relatively little is known about the effects of urbanisation and agriculture on the genetic structure, gene flow and genetic diversity of wild species. Here, landscape genetic techniques were applied to compare local genetic diversity and gene flow between wood mouse populations in urban and arable landscapes. Using nine microsatellite markers, individuals were genotyped from six arable and seven urban sample sites. Inter‐population genetic differentiation was significantly greater in urban than arable habitat, while allele richness, private allele richness and heterozygosity were higher for arable sample sites, with varying degrees of significance. These suggest that urban habitat was sufficiently fragmented to limit gene flow. To test the effect of landscape features on gene flow, several cost‐distance measures were generated. Overland distance and Euclidean distance correlated best with inter‐population genetic differentiation in arable habitat, whereas distances that accommodated differences in habitat quality better explained differentiation in urban habitat. There was no evidence that margins adjacent to roads, rivers or railways facilitated gene flow. Together, the results indicate that urban landscapes expose wood mice to greater fragmentation in habitat quality than arable areas, leading to greater population isolation that is not mitigated by the presence of dispersal corridors.     

Historical Environment Is Reflected in Modern Population Genetics and Biogeography of an Island Endemic Lizard (Xantusia riversiana reticulata)

The restricted distribution and isolation of island endemics often produces unique genetic and phenotypic diversity of conservation interest to management agencies. However, these isolated species, especially those with sensitive life history traits, are at high risk for the adverse effects of genetic drift and habitat degradation by non-native wildlife. Here, we study the population genetic diversity, structure, and stability of a classic “island giant” (Xantusia riversiana, the Island Night Lizard) on San Clemente Island, California following the removal of feral goats. Using DNA microsatellites, we found that this population is reasonably genetically robust despite historical grazing, with similar effective population sizes and genetic diversity metrics across all sampling locations irrespective of habitat type and degree of degradation. However, we also found strong site-specific patterns of genetic variation and low genetic diversity compared to mainland congeners, warranting continued special management as an island endemic. We identify both high and low elevation areas that remain valuable repositories of genetic diversity and provide a case study for other low-dispersal coastal organisms in the face of future climate change.     

Gene flow connects coastal populations of a habitat specialist,the Clapper Rail Rallus crepitans

Examining population genetic structure can reveal patterns of reproductive isolation or population mixing and inform conservation management. Some avian species are predicted to exhibit minimal genetic differentiation among populations as a result of the species high mobility, with habitat specialists tending to show greater fine‐scale genetic structure. To explore the relationship between habitat specialization and gene flow, we investigated the genetic structure of a saltmarsh specialist with high potential mobility across a wide geographical range of fragmented habitat. Little variation among mitochondrial sequences (620 bp from ND2) was observed among 149 individual Clapper Rails Rallus crepitans sampled along the Atlantic coast of the USA, with the majority of individuals at all sampling sites sharing a single haplotype. Genotyping of nine microsatellite loci across 136 individuals revealed moderate genetic diversity, no evidence of bottlenecks and a weak pattern of genetic differentiation that increased with geographical distance. Multivariate analyses, Bayesian clustering and an AMOVA all suggested a lack of genetic structuring across the Atlantic coast of the USA, with all individuals grouped into a single interbreeding population. Spatial autocorrelation analyses showed evidence of weak female philopatry and a lack of male philopatry. We conclude that high gene flow connecting populations of this habitat specialist may result from the interaction of ecological and behavioural factors that promote dispersal and limit natal philopatry and breeding‐site fidelity. As climate change threatens saltmarshes, the genetic diversity and population connectivity of Clapper Rails may promote resilience of their populations. This finding helps inform about potential fates of other similarly behaving saltmarsh specialists on the Atlantic coast.     

Habitat fragmentation and genetic diversity of an endangered, migratory songbird, the golden-cheeked warbler (Dendroica chrysoparia)

Landscape genetic approaches offer the promise of increasing our understanding of the influence of habitat features on genetic structure. We assessed the genetic diversity of the endangered golden-cheeked warbler (Dendroica chrysoparia) across their breeding range in central Texas and evaluated the role of habitat loss and fragmentation in shaping the population structure of the species. We determined genotypes across nine microsatellite loci of 109 individuals from seven sites representing the major breeding concentrations of the species. No evidence of a recent population bottleneck was found. Differences in allele frequencies were highly significant among sites. The sampled sites do not appear to represent isolated lineages requiring protection as separate management units, although the amount of current gene flow is insufficient to prevent genetic differentiation. Measures of genetic differentiation were negatively associated with habitat connectivity and the percentage of forest cover between sites, and positively associated with geographic distance and the percentage of agricultural land between sites. The northernmost site was the most genetically differentiated and was isolated from other sites by agricultural lands. Fragmentation of breeding habitat may represent barriers to dispersal of birds which would pose no barrier to movement during other activities such as migration.     

Does fragmentation of wetlands affect gene flow in sympatric Acrocephalus warblers with different migration strategies?

Wetlands are naturally patchy habitats, but patchiness has been accentuated by the extensive wetlands loss due to human activities. In such a fragmented habitat, dispersal ability is especially important to maintain gene flow between populations. Here we studied population structure, genetic diversity and demographic history of Iberian and North African populations of two wetland passerines, the Eurasian reed warbler Acrocephalus scirpaceus and the moustached warbler Acrocephalus melanopogon. These species are closely related and sympatric in our study sites, but the reed warbler is a widespread long‐distance migrant while the moustached warbler's breeding range is patchier and it is resident or migrates over short distances. Using microsatellite and mtDNA data, we found higher differentiation in moustached than in reed warblers, indicating higher dispersal capability of the latter species. Our results also suggest that the sea limits dispersal in the moustached warbler. However, we found evidence of gene flow between the study sites in both species, indicating a capability to compensate for habitat fragmentation. In most cases, the gene flow was restricted, possibly because of the large distances between study sites (from ca 290 to 960 km) or breeding site fidelity. The reed warbler had higher haplotype diversity, likely due to dispersal from different populations, past admixture event and a larger population size. We found also signs of postglacial population growth for both species and evidence of a recent colonization or re‐colonization of the Mallorca Island by the moustached warbler.     

Population structure on breeding grounds of Lake Malawi’s ‘twilight zone’ cichlid fishes

Aim Free‐ranging benthopelagic fishes often have large population sizes and high rates of dispersal. These traits can act to homogenize population structure across the distributional range of a species and to reduce the likelihood of allopatric speciation. The apparent absence of any barriers to gene flow among populations, together with prior molecular evidence for panmixia across the ranges of three species, has resulted in Diplotaxodon, a genus of benthopelagic cichlid fishes of Lake Malawi, being proposed as a candidate case of sympatric speciation. Our aim was to further investigate this possibility by testing for intraspecific genetic subdivision among breeding populations, and intraspecific differences in breeding habitat. Location Lake Malawi, central‐east Africa. Methods We analysed eight microsatellite DNA loci to test for spatial genetic differences among populations on breeding grounds of eight Diplotaxodon species. We also tested for temporal population genetic differences within breeding grounds of three species. Records of ripe Diplotaxodon encountered during sampling were analysed to test if spatial variation in assemblage structure was linked to nearshore water depth and geographic proximity of sampling sites. Results Consistent with previous molecular evidence, within four of the eight species tested we found no evidence of spatial genetic structuring among breeding populations. However, within the other four species we found slight yet significant spatial genetic differences, indicating restricted gene flow among breeding grounds. There was no evidence of temporal genetic differences within sites. Analyses of the distributions of ripe Diplotaxodon revealed differences in assemblage structure linked to nearshore water depth. Main conclusions Together, these results demonstrate both the evolution of fidelity to deep‐water breeding locations in some Diplotaxodon species, and differences in breeding habitat among species. These findings are consistent with a role for divergence of breeding habitat in speciation of these cichlids, possibly promoted by dispersal limitation among geographically segregated spawning aggregations.     

Habitat islands, genetic diversity, and gene flow in a Patagonian rodent

The effects of terrestrial habitat islands on gene flow and genetic diversity in animal populations have been predicted and discussed in theoretical terms, but empirical data are needed to test these predictions and provide an understanding of the relationships of life-history characteristics to genetics of insular species. We studied saxicolous mice ( Phyllotis xanthopygus ) in Patagonia to explore genetic structure, phylogeography, and gene flow in a species inhabiting natural habitat islands. Phylogeographic analyses based on mtDNA sequences revealed two haplotype clades, which presumably reflect early Pleistocene factors that temporarily separated the mice into two geographically isolated groups. The Río Chubut, which lies within a glacial drainage basin bisecting northern Patagonia, might have affected gene flow in the species. Although we anticipated isolation by distance and founder phenomena associated with habitat islands, in some habitat patches we found evidence of high local genetic diversity. The amount of divergence in the mitochondrial cytochrome b gene (≈ 3.4%) in animals at a single locality could best be explained through a combination of historical factors and metapopulation source–sink theory. Demographic shifts, dispersal, and episodic recolonization are important in the life history and genetic population structure of P. xanthopygus .     

Genetic diversity and population structure of Tasmanian devils, the largest marsupial carnivore

Genetic diversity and population structure were investigated across the core range of Tasmanian devils (Sarcophilus laniarius; Dasyuridae), a wide-ranging marsupial carnivore restricted to the island of Tasmania. Heterozygosity (0.386-0.467) and allelic diversity (2.7-3.3) were low in all subpopulations and allelic size ranges were small and almost continuous, consistent with a founder effect. Island effects and repeated periods of low population density may also have contributed to the low variation. Within continuous habitat, gene flow appears extensive up to 50 km (high assignment rates to source or close neighbour populations; nonsignificant values of pairwise FST), in agreement with movement data. At larger scales (150-250 km), gene flow is reduced (significant pairwise FST) but there is no evidence for isolation by distance. The most substantial genetic structuring was observed for comparisons spanning unsuitable habitat, implying limited dispersal of devils between the well-connected, eastern populations and a smaller northwestern population. The genetic distinctiveness of the northwestern population was reflected in all analyses: unique alleles; multivariate analyses of gene frequency (multidimensional scaling, minimum spanning tree, nearest neighbour); high self-assignment (95%); two distinct populations for Tasmania were detected in isolation by distance and in Bayesian model-based clustering analyses. Marsupial carnivores appear to have stronger population subdivisions than their placental counterparts.     

Influence of geomorphology and surface features on the genetic structure of an important trogloxene,the secret cave cricket (<Emphasis Type="Italic">Ceuthophilus secretus</Emphasis>)

Cave ecosystems supporting a variety of endemics depend on the carbon, nitrogen, and nutrients brought into caves by trogloxenic species, such as the secret cave cricket (Ceuthophilus secretus). Surface movements of trogloxenes may comprise the strongest ecological connections among caves. Our objective was to better understand dispersal patterns in C. secretus in order to inform management of this species and the cave endemics that depend upon them. We used microsatellite loci to estimate gene flow and genetic diversity among 42 karst features supporting C. secretus on the Fort Hood Military Reserve, Texas, USA. This sampling was used to assess the influences of karst topography and other landscape features on genetic diversity and population structure. Cave populations did not exhibit evidence of recent bottlenecks and genetic diversity was similar among sites, with the exception of one sample from an isolated cave. Samples exhibited a strong pattern of isolation by distance, but karst topology was also influential, with genetic differentiation being much higher between samples from separate ridges than among those on the same ridge. It appears that co-location on a ridge was an important factor facilitating dispersal among karst features. There was little evidence that other surface features such as forest cover, roads or streams influenced gene flow and genetic differentiation. The low genetic connectivity among ridges suggests that isolated caves on ridges where cricket habitat is uncommon or degraded might not be easily recolonized after extinction events, with potentially negative consequences for associated cave communities.     

Genetic diversity and population structure of the northern snakehead (<Emphasis Type="Italic">Channa argus</Emphasis> Channidae: Teleostei) in central China: implications for conservation and management

Major threats to freshwater fish diversity now include loss of native genetic diversity as a consequence of translocations of fishes between sites and from hatcheries to sites, and small effective population sizes resulting from overfishing and/or habitat loss. Ten polymorphic microsatellite markers were employed to evaluate genetic diversity, population genetic structure and gene flow amongst nine populations of the ecologically and economically important fish, the northern snakehead (Channa argus), in three river systems in central China. Multiple analyses revealed evidence of high genetic diversity and pronounced subdivision based on both regional separation and on river systems. A lack of evidence of genetic bottleneck over recent generations was consistent with the long-term stability of population size and contemporary distribution. The effective population sizes for most C. argus populations were small, suggesting the need for future conservation efforts focusing on these populations. Different lines of evidence point to the local enhancement of stocks by both aquaculture-reared fish and the transfer of wild fish. This study illustrates how human activities may affect genetic diversity and population genetic structure of C. argus populations, and highlights the need for new management regimes to protect native freshwater fish genetic diversity.     

Low genetic diversity and local adaptive divergence of Dracaena cambodiana (Liliaceae) populations associated with historical population bottlenecks and natural selection: an endangered long‐lived tree endemic to Hainan Island,China

Historical population bottlenecks and natural selection have important effects on the current genetic diversity and structure of long‐lived trees. Dracaena cambodiana is an endangered, long‐lived tree endemic to Hainan Island, China. Our field investigations showed that only 10 populations remain on Hainan Island and that almost all have been seriously isolated and grow in distinct habitats. A considerable amount of genetic variation at the species level, but little variation at the population level, and a high level of genetic differentiation among the populations with limited gene flow in D. cambodiana were detected using inter‐simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analyses. No significant correlation was found between genetic diversity and actual population size, as the genetic diversities were similar regardless of population size. The Mantel test revealed that there was no correlation between genetic and geographic distances among the 10 populations. The UPGMA, PCoA and Bayesian analyses showed that local adaptive divergence has occurred among the D. cambodiana populations, which was further supported by habitat‐private fragments. We suggest that the current genetic diversity and population differentiation of D. cambodiana resulted from historical population bottlenecks and natural selection followed by historical isolation. However, the lack of natural regeneration of D. cambodiana indicates that former local adaptations with low genetic diversity may have been genetically weak and are unable to adapt to the current ecological environments.     

Response of a southern temperate marsupial, the Tasmanian pademelon (Thylogale billardierii), to historical and contemporary forest fragmentation

Despite only limited Pleistocene glacial activity in the southern hemisphere, temperate forest species experienced complex distributional changes resulting from the combined effects of glaciation, sea level change and increased aridity. The effects of these historical processes on population genetic structure are now overlain by the effects of contemporary habitat modification. In this study, 10 microsatellites and 629 bp of the mitochondrial control region were used to assess the effects of historical forest fragmentation and recent anthropogenic habitat change on the broad-scale population genetic structuring of a southern temperate marsupial, the Tasmanian pademelon. A total of 200 individuals were sampled from seven sites across Tasmania and two islands in Bass Strait. High mitochondrial and nuclear genetic diversity indicated the maintenance of large historical population sizes. There was weak phylogeographical structuring of haplotypes, although all King Island haplotypes and three Tasmanian haplotypes formed a divergent clade implying the mid-Pleistocene isolation of a far northwestern population. Both the mitochondrial and nuclear data indicated a division of Tasmanian populations into eastern and western regions. This was consistent with a historical barrier resulting from increased aridity in the lowland 'midlands' region during glacial periods, and with a contemporary barrier resulting from recent habitat modification in that region. In Tasmania, gene flow appears to have been relatively unrestricted during glacial maxima in the west, while in the east there was evidence for historical expansion from at least one large glacial refuge and recolonization of Flinders Island.     

Assessing the genetic consequences of habitat fragmentation on the federally threatened cheat mountain salamander (Plethodon nettingi): a comparative,multi-locus approach

Comparative population genetic studies of closely related taxa provide a powerful framework for evaluating if and to what degree a species of conservation concern has been negatively impacted by factors such as habitat fragmentation, decreased population connectivity, inbreeding and genetic drift. In this study, we take advantage of a paired sampling strategy to compare the population genetics of the geographically restricted, federally threatened Cheat Mountain salamander (Plethodon nettingi) to those of its partially sympatric, but much more widely distributed congener, the red-backed salamander (P. cinereus), where the two species overlap in the Appalachian mountains of West Virginia. Mitochondrial DNA haplotype and nucleotide diversity were lower in P. nettingi, as were a variety of metrics of nuclear genetic diversity estimated from microsatellite data. Population differentiation and structuring were greater in P. nettingi, suggesting reduced gene flow following fragmentation. Significant inbreeding and evidence of recent population bottlenecks were also seen in P. nettingi and estimated population sizes were smaller. Estimates of contemporary gene flow, as measured through kinship, also showed more restricted gene flow in P. nettingi. Overall, our comparative study provides strong evidence that the small and highly fragmented nature of its geographic distribution has resulted in a suite of negative genetic consequences for the federally threatened Cheat Mountain salamander. Management efforts aimed at enhancing the genetic health and long-term viability of this species should focus on increasing population connectivity through establishment of forest habitat corridors where possible and exploring the potential merits of translocations.

     

Genetic connectivity and diversity in inselberg populations of Acacia woodmaniorum,a rare endemic of the Yilgarn Craton banded iron formations

Historically rare plant species with disjunct population distributions and small population sizes might be expected to show significant genetic structure and low levels of genetic diversity because of the effects of inbreeding and genetic drift. Across the globe, terrestrial inselbergs are habitat for rich, often rare and endemic flora and are valuable systems for investigating evolutionary processes that shape patterns of genetic structure and levels of genetic diversity at the landscape scale. We assessed genetic structure and levels of genetic diversity across the range of the historically rare inselberg endemic Acacia woodmaniorum. Phylogeographic and genetic structure indicates that connectivity is not sufficient to produce a panmictic population across the limited geographic range of the species. However, historical levels of gene flow are sufficient to maintain a high degree of adaptive connectivity across the landscape. Genetic diversity indicates gene flow is sufficient to largely counteract any negative genetic effects of inbreeding and random genetic drift in even the most disjunct or smallest populations. Phylogeographic and genetic structure, a signal of isolation by distance and a lack of evidence of recent genetic bottlenecks suggest long-term stability of contemporary population distributions and population sizes. There is some evidence that genetic connectivity among disjunct outcrops may be facilitated by the occasional long distance dispersal of Acacia polyads carried by insect pollinators moved by prevailing winds.     

Conservation genetics of the endangered terrestrial orchid Liparis japonica in Northeast China based on AFLP markers

Levels of genetic diversity and population genetic structure of the rare, endangered terrestrial orchid Liparis japonica were examined for eight natural populations (n = 185) in Northeast China using six AFLP primer pairs, where this species has experienced severe habitat loss and fragmentation. Based on 406 DNA bands, a high level of genetic diversity was found at the species level with the PPB of 85.47 %, while the genetic diversity at the population level was low (PPB = 47.48 %). A significantly high degree of population differentiation was found with 42.69 % variation existed among populations as measured by AMOVA, indicating potential restricted gene flow. The genetic distances between populations were independent of the corresponding geographic distances, and the genetic relationship of individuals had no significant correlation with their spatial distribution. The restricted gene flow might be impacted by reduced population size, habitat destruction and fragmentation. The results in this study suggested that habitat protection and keeping a stable environment are critical for the conservation of L. japonica species.     

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as ‘endangered’ under the Environment Protection and Biodiversity Conservation Act 1999, and ‘vulnerable’ under the International Union for Conservation of Nature’s Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy–Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F _ST  = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.     

Multilocus approach reveals an incipient differentiation process in the Stone-curlew, <Emphasis Type="Italic">Burhinus oedicnemus</Emphasis> around the Mediterranean basin

The Stone-curlew Burhinus oedicnemus, a steppe bird species, is mainly distributed in the Mediterranean and Macaronesian regions, which are considered hotspots of biodiversity with priority for animal and plant species richness conservation. In this study, we investigated the genetic diversity of the Stone-curlew in the Mediterranean basin and in the Canary Islands by applying a multilocus approach. We analysed mitochondrial and nuclear markers in order to evaluate the genetic structure and the congruence between morphological subspecies and geographic samples. We found a significant level of genetic differentiation between Mediterranean and Canary Island populations with all markers. Both in the Mediterranean basin and in the Canary Islands, we found a significant level of genetic diversity with nuclear markers only. We identified seven population groups, including insular populations. The four subspecies described for the Western Palaearctic were confirmed with some changes in distribution range. In spite of habitat fragmentation and negative population trend, the Stone-curlew showed a significant level of genetic diversity and gene flow among continental populations. However, islands constitute important reservoirs of genetic diversity and a potential for the evolution of the species.     

Genetic population structure of the sagebrush Brewer’s sparrow, <Emphasis Type="Italic">Spizella breweri breweri</Emphasis>, in a fragmented landscape at the northern range periphery

Assessing the genetic consequences of habitat fragmentation is a crucial step in conservation planning for species in endangered habitats. We tested for the impact of natural habitat fragmentation on gene flow and genetic diversity in seven northern breeding locations of the sagebrush Brewer’s sparrow, Spizella breweri breweri. Genetic analyses using five highly variable DNA microsatellite loci suggested that individuals sampled within a sagebrush landscape fragmented by natural elements such as coniferous forest, comprise a single genetic population and that gene flow among them is unimpeded. We posit that juvenile dispersal links seemingly isolated breeding locales of this species, and discuss implications of our findings for conservation of migratory songbirds in the northern portion of their ranges in light of potential shifts in distribution due to climate change.     

Effective population size is strongly correlated with breeding pond size in the endangered California tiger salamander, <Emphasis Type="Italic">Ambystoma californiense</Emphasis>

Maintaining genetic diversity and population viability in endangered and threatened species is a primary concern of conservation biology. Genetic diversity depends on population connectivity and effective population size (N_e), both of which are often compromised in endangered taxa. While the importance of population connectivity and gene flow has been well studied, investigating effective population sizes in natural systems has received far less attention. However, N_e plays a prominent role in the maintenance of genetic diversity, the prevention of inbreeding depression, and in determining the probability of population persistence. In this study, we examined the relationship between breeding pond characteristics and N_e in the endangered California tiger salamander, Ambystoma californiense. We sampled 203 individuals from 10 breeding ponds on a local landscape, and used 11 polymorphic microsatellite loci to quantify genetic structure, gene flow, and effective population sizes. We also measured the areas of each pond using satellite imagery and classified ponds as either hydrologically-modified perennial ponds or naturally occurring vernal pools, the latter of which constitute the natural breeding habitat for A. californiense. We found no correlation between pond area and heterozygosity or allelic diversity, but we identified a strong positive relationship between breeding pond area and N_e, particularly for vernal pools. Our results provide some of the first empirical evidence that variation in breeding habitat can be associated with differences in N_e and suggest that a more complete understanding of the environmental features that influence N_e is an important component of conservation genetics and management.     

Effects of roads on patterns of genetic differentiation in red-backed salamanders, <Emphasis Type="Italic">Plethodon cinereus</Emphasis>

Roads can fragment animal populations by reducing gene flow, which can lead to drift and the loss of genetic diversity. One of the principle signatures of reduced gene flow is increased genetic differentiation in isolated populations, and evidence that roads contribute to such differentiation has been reported for several species. We used microsatellites to examine whether six roads led to increased genetic differentiation in red-backed salamanders (Plethodon cinereus). These six roads included one divided interstate highway, one undivided four-lane highway, and four secondary roads. We found that the genetic distance between plots that were bisected by the interstate highway was significantly greater than the genetic distance between equidistant plots on the same side of the highway. However, for the five smaller roads, plots across the road were no more genetically distinct than were plots on the same side of the road. Bayesian clustering methods also supported both of these findings. The optimal clustering of plots for the interstate highway consisted of two clusters that corresponded to the two sides of highway. For the other five sites, the optimal grouping consisted of a single cluster containing all of the plots. Our findings suggest that gene flow across very large roads is rare and that bisected red-backed salamander populations are likely to diverge from one another. For smaller roads, our results imply that the indirect effects of roads on genetic population structure are probably less of a pressing concern for terrestrial salamanders than are the direct effects of mortality and habitat alteration.