Puma concolor in western and southern Texas, USA. We used 10 microsatellite loci and indexed population trends by estimating historical and recent genetic diversity, genetic differentiation and effective population size. Mountain lions in southern Texas exhibited a 9% decline in genetic diversity, whereas diversity remained stable in western Texas. Genetic differentiation between western and southern Texas was minimal historically , but increased 2–2.5 times in our recent sample. An index of genetic drift for southern Texas was seven to eight times that of western Texas, presumably contributing to the current differentiation between western and southern Texas. Furthermore, southern Texas exhibited a >50% temporal decline in effective population size, whereas western Texas showed no change. Our results illustrate that population declines and genetic drift have occurred in southern Texas, likely because of contemporary habitat loss and predator control. Population monitoring may be needed to ensure the persistence of mountain lions in the southern Texas region. This study highlights the utility of sampling museum collections to examine demographic histories and inform wildlife management.Elusive carnivores present a challenge to managers because traditional survey methods are not suitable. We applied a genetic approach using museum specimens to examine how historical and recent conditions influenced the demographic history of  Natural resource conservation and management relies on surveys or other data to monitor population trends . ChangesUrsus arctos (L.)    (mountain lion) has experienced severe declines in census size and geographical distribution because of habitat loss and predator management policies . For maxilloturbinates, we ground samples using a mortar and pestle and placed them in lysis buffer  set-up . Therefom of each primer and 1–1.5 μL of extracted DNA. However, for maxilloturbinate reactions, we increased primer concentration to 0.50 μm, added 0.2 mg/μL of bovine serum albumin and increased the quantity of extracted DNA to 1.5–2.5 μL. We used a touchdown PCR profile with an initial denaturation at 94°C for 10 min, 20 cycles of 94°C for 30 s, 62°C for 30 s, 61°C for 30 s, 60°C for 30 s and 72°C for 60 s, followed by 30 cycles of 94°C for 30 s, 55°C for 90 s and 72°C for 60 s, with a final extension of 60°C for 10 min. For maxilloturbinate reactions, we reduced the first set of temperature cycles to 10 and increased the second set to 50. For each individual, we combined 3 μL of PCR product from multiple loci and applied 1.5–2 μL of the PCR product mix to a denaturing formamide  and size standard mixture . We loaded the resulting mixtures onto a 3130xl genetic analyser (Applied Biosystems) for fragment separation and detection. We included a positive and negative PCR control with each run through the analyser to identify contamination and ensure consistency among runs. We inspected loci and sized alleles using GeneMapper® software v4.0 (Applied Biosystems). We reanalyzed 10% of muscle tissue samples to calculate a genotyping error rate.We used the PCR to amplify 10 microsatellite loci  described by Additional measures are required to ensure genotypes are correct for museum samples because extracted DNA is at relatively low concentrations and quality . Errors n ≥ 27 individuals, and our total data set included 299 mountain lions. The median years for the western Texas samples were 1938, 1983 and 2006, and the southern Texas medians were 1937 and 1996  and two periods for southern Texas (1934–1942 and 1985–2009). Each temporal sample had convert , expected heterozygosity  and allelic richness (ar) per locus for each temporal sample. We calculated HO, HE and A using the computer program arlequin 3.5 (ar using hp-rare 1.0 (ar within southern and western Texas using a Wilcoxon signed-rank test (one-sided). We tested ar because during demographic declines such as population bottlenecks, alleles are lost before heterozygosity changes  and that HO in southern Texas was 40% lower than in western Texas , and three estimates for western Texas . We calculated FST using the computer program arlequin 3.5  and inbreeding (NeI) effective population size for southern and western Texas to explicitly test for changes in population size over time. Effective population size is the size of an idealized population exhibiting the same rate of genetic change as the sampled population  that produced the cohorts present , n = 42 (1983) and n = 168 (2006), and n = 34 (1937) and n = 28 (1996) from southern Texas (We genotyped 10 microsatellite loci for 299 mountain lions (2% missing data) collected from Texas . Sample sizes for the median year groups from western Texas included rn Texas . PositivFIS for the recent (1985–2010) statewide sample indicating a deviation from HWE . The historical statewide sample (1934–1955) also exhibited a positive FIS, but was not statistically significant . Both temporal groups from southern Texas  and groups from western Texas  satisfied HWE. The departure from HWE in the recent statewide sample may be due to a Wahlund effect, which occurs when populations with differing allele frequencies are combined . Genetic diversity was similar in the historical samples from southern and western Texas. However, mean estimates of HO, HE and A were comparatively low in the recent sample from southern Texas. We also observed evidence for a 9% temporal reduction in ar , but approximately doubled in the recent sample , further supporting a Wahlund effect. We observed low yet significant genetic change over time within western Texas . Southern Texas, however, displayed genetic change seven to eight times greater  than western Texas over a similar temporal period (i.e. 1938–2006).Estimates of both NeV produced statistically similar means within temporal periods for western and southern Texas  and recent (1983–2006) did not overlap the means. The arithmetic mean across methods for each interval in western Texas were NeV (1938–1983) = 54, NeV (1983–2006) = 166 and NeV (1938–2006) = 109. Estimates based on the likelihood approach of NeV in western Texas, but this interval was the shortest temporal span with only four generations separating samples. The historical (1938–1983) and overall estimates (1938–2006) captured 7 and 11 generations and were more precise, reflected by narrower 95% CIs. Temporal estimates for southern Texas were precise, with an arithmetic mean of NeV (1937–1996) = 44. The mean estimate of NeV for southern Texas was 60% lower than NeV in western Texas over a similar temporal period.Estimates of rn Texas . There wNb for western Texas exhibited no statistical differences among temporal samples, suggesting the population has remained stable over time  limited the inferential power of the analyses.The demographic history of many species is poorly understood. Thus, a major challenge in conservation genetic studies is to determine whether contemporary levels of genetic diversity and differentiation are the result of historical or recent events. The initial genetic analyses of Texas mountain lions presented a similar challenge . The autar within southern Texas. Clearly, the lower diversity in contemporary mountain lions from southern Texas is a recent phenomenon.Our genetic diversity estimates in recent western and southern Texas are higher than reported by The levels of diversity we documented in our sample were comparable with other mountain lion populations. Estimates for western Texas and historical southern Texas were 20–50% lower than those observed in mountain lions from South America , but werFST = 0.13) rather than western Texas . Low levels of differentiation between historical southern Texas and the 1983 and 2006 samples from western Texas (FST = 0.04–0.05) provide further support for temporal changes occurring mainly in southern Texas. Indeed, the differentiation observed recently between mountain lions in southern and western Texas (The genetic differentiation between historical western and southern Texas was analogous to contiguous populations of mountain lions in western North America . Contemprn Texas  was not NeV and Nb substantiated our genetic diversity and differentiation findings. The temporal approach (NeV in western Texas over time, and point estimates were similar to other temporally stable populations of large carnivores (NeV for southern Texas were much lower than those for western Texas, providing evidence for a smaller average population size in southern Texas over the sampling interval. Estimates of Nb corroborated NeV results, indicating no population changes in western Texas, and a lower average population size in southern Texas. However, estimates of Nb also revealed a significant population decline in southern Texas. Moreover, we discovered a disparity when comparing the estimates of Nb and NeV for southern Texas. The recent estimate of Nb was 80% lower than the average of NeV estimates within southern Texas, and Nb was similar to a reintroduced population of brown bears (Nb and NeV estimates is likely because NeV reflects the harmonic mean over the sampled time period (Nb represents the number of breeders producing the sampled cohorts (NeV could have been influenced by larger historical population sizes, whereas recent estimates of Nb may be indicative of a small effective size in the contemporary population. This hypothesis is supported by the temporal decline we observed in genetic diversity within southern Texas.Estimates of approach  revealedrnivores . Estimatwn bears . The dife period , while N cohorts . In the Historically, mountain lions in western and southern Texas displayed high genetic diversity and low genetic differentiation indicative of a large population. Over time, western Texas exhibited essentially no change in diversity and effective population size and showed low levels of genetic drift. However, genetic diversity and effective size decreased in southern Texas, and genetic drift was extensive. Genetic differentiation has also doubled between western and southern Texas over time. Our findings highlight that mountain lions in western Texas have remained relatively stable, but population changes and declines have clearly occurred in southern Texas.The human footprint may be responsible for the population stability in western Texas and reductions or changes in southern Texas. First, urban development and sprawl have increased dramatically in southern Texas along the Mexico–USA border and in central Texas. The Rio Grande Valley region of southern Texas also supports vast areas of cropland on both sides of the border. Collectively, development and agriculture have reduced and fragmented habitat for mountain lions and increased the potential for auto-collisions and other mountain lion–human conflicts. Changes in habitat connectivity could be responsible for the increase in genetic differentiation in southern Texas, the most peripheral population we sampled. In contrast, much of western Texas remains rangeland with little urban development. The large geographical area in western Texas, lack of urbanization and proximity to adjacent mountain lion populations in New Mexico, USA, and Mexico may have maintained a large effective size in the region. Movement occurs among western Texas, New Mexico and probably Mexico , thus poAdditionally, during late 1800–mid 1900, livestock production was the dominant industry in Texas . PredatoOur results demonstrate the utility of applying a retrospective genetic approach  to evaluDeclines have occurred in genetic connectivity, genetic diversity and effective population size for mountain lions in southern Texas. In fact, the temporal decline in diversity and current effective size are outside of the ranges suggested for long-term population persistence . FurtherManagement actions are likely needed if mountain lions are to be maintained in southern Texas. First, the current population size and trend in southern Texas are unknown. Population monitoring efforts are needed to estimate occupied habitat, reproductive rates, survival and population viability without management intervention. Reporting of mountain lion harvests in southern Texas would assist monitoring efforts. If current harvest is unsustainable, regulation of harvest may be needed . A harveThis study illustrates the important role of museum collections and genetic techniques in wildlife conservation and management. Museum samples coupled with recent genetic samples allow managers to retrospectively examine evolutionary change and establish demographic baselines for data deficient or difficult to survey populations, such as Texas mountain lions. We suggest that agencies and institutions maintain or establish tissue archives to facilitate long-term monitoring. As genetic methods and analytical tools continue to advance at exceptional rates, tissue collections will become increasingly useful in natural resource conservation.


Pristionchus pacificus in its La Réunion Island habitat, but gave no clear understanding of the role of the environment in structuring this variation. Here, we present what is to our knowledge the first study to statistically evaluate the role of the environment in shaping the structure and distribution of nematode populations. We test the hypothesis that genetic structure in P. pacificus is influenced by environmental variables, by combining population genetic analyses of microsatellite data from 18 populations and 370 strains, with multivariate statistics on environmental data, and species distribution modelling. We assess and quantify the relative importance of environmental factors  on genetic variation among populations. Despite the fact that geographic populations of P. pacificus comprise vast genetic diversity sourced from multiple ancestral lineages, we find strong evidence for local associations between environment and genetic variation. Further, we show that significantly more genetic variation in P. pacificus populations is explained by environmental variation than by geographic distances. This supports a strong role for environmental heterogeneity vs. genetic drift in the divergence of populations, which we suggest may be influenced by adaptive forces.The distribution of a species is a complex expression of its ecological and evolutionary history and integrating population genetic, environmental, and ecological data can provide new insights into the effects of the environment on the population structure of species. Previous work demonstrated strong patterns of genetic differentiation in natural populations of the hermaphroditic nematode  All organisms live in environments that vary through time and space, and the influence of environmental heterogeneity on spatial scales of population divergence and genetic isolation is a fundamental question in evolutionary biology Although much progress has been made in understanding causes and consequences of local and regional changes in genetic diversity of natural populations 2), a component of the Mascarene island chain lying east of Madagascar in the Indian Ocean, is one such biodiversity hotspot Island systems often represent diversity hotspots characterised by high species richness and endemism, resulting from local diversification of geographically or ecologically isolated populations following colonisation Pristionchus pacificus is a hermaphroditic nematode of cosmopolitan distribution that forms a non-invasive host-relationship with scarab beetle and other insect taxa P. pacificus is widely distributed across La Réunion Island where it inhabits a broad range of environments, encompassing gradients of altitude, ecozone, geography and beetle host  on genetic variation.Preliminary findings indicate a genetic delineation between drier western and wetter eastern areas of the island P. pacificus on La Réunion Island. Species distribution modelling programs identify sites with similar environments to those where a species has already been observed as potential occurrence areas.We first aimed to determine the ecological niche of P. pacificus had been successfully sampled on an annual basis between 2008 and 2012 . These data encompass biologically relevant temperature and precipitation layers created by interpolating observed climate from climate stations around the world using a thin-plate smoothing spline set to a resolution of approximately 1 km (2.5 arc minutes), over the 50-year period from 1950 to 2000 Climate data for La Réunion Island was downloaded from the WorldClim website  to generate visualisations of the association between species distribution and the hottest/driest or coldest/wettest areas of the island.In addition to generating models for ions see ,3. CombiP. pacificus in La Réunion habitats. Next, we aimed to examine genetic structure of P. pacificus and assess the effects of environmental factors on this structure. From the Sommer collection, a subset of ∼200 strains had been typed for STR markers in a previous study 10.5061/dryad.ns1stov2). An additional 176 strains were genotyped at STR markers for the current study (Dryad accession number: doi:10.5061/dryad.8883h), culminating in a final STR dataset of 370 strains from 18 geographic La Réunion Island locations  geographic sampling locality; (b) ecozone; (c) altitude; (d) host beetle species . The ecopost hoc environmental association analyses.Our final aim was to establish how local environmental effects were explicitly associated with our model of genetic structure at geographic locations. First, Pearson’s correlation analysis was performed in MINITAB ver. 14  to quantify the correlation among site-averaged climate variables (extracted from WorldClim data) - for variable pairs that were highly correlated (r≥0.75), the more biologically meaningful variable was chosen, which resulted in the selection of three uncorrelated climate variables  for Next, a Multiple Regression Model (MRM) approach was used in the program GESTE ver. 2.0 X and Y coordinates) or environmental matrix. Genetic data and population-specific climate data  were compared in a partial RDA, specifying geography (latitude and longitude) as a third ‘conditioned’ matrix so that the analysis conditions on geographic location. To examine how much of the genetic variation in P. pacificus is uniquely explained by climate, how much is uniquely explained by geography, and how much is due to the combined effect of the two, we then partitioned the variance components of our RDA by running 3 models: a full model with all important climate and geographic variables as explanatory variables; a partial model in which geography explains genetic data conditioned on important climate variables; a partial model in which important climate variables explain genetic data conditioned on geography Finally, redundancy analysis (RDA) was conducted with the vegan package in R to further disentangle the relative contribution of environmental and spatial components in driving genetic structure P. pacificus on La Réunion Island were generated in DIVA-GIS, based on presence data at 23 geographic locations and three combinations of climatic variables . Note that in P. pacificus, where dark green areas on the map are those corresponding to climate envelope outliers (in the <2.5 and >97.5 percentile). The areas of highest probabilities of occurrence largely corresponded to discrete pockets of habitat across an inner circular belt of the island, where conditions are more generally cooler and wetter . AUC values exceeded 0.70 in all analyses, indicating that MAXENT models had acceptable predictive power. Throughout the predictions, the highest contributing climate variables were always related to precipitation, regardless of the combination of variables used in the model. In the presented model  precipitation-based variables contributed 71.1% to the model   . Measurectively) . Differectively) .P. pacificus  amounts of genetic variation , and one cluster was found in nine locations , found a significant association between climate and STR genotype  and eastern (wetter) zones of the island P. pacificus habitat is partitioned across local ecozones. This was also supported by our AMOVA results, for which the dataset defined by ecozone explained approximately 45% of regional genetic variation, some 20% higher than altitude or beetle-defined datasets, and ∼6% higher than the variation explained by geographic location. The question of whether the environmental preferences borne out from our SDM analysis reflect those of the nematode or its beetle hosts is difficult to address because beetle host, ecozone and altitude cannot easily be dissociated. Therefore, host affinity among different P. pacificus genotypes may provide a stronger pressure than the environment per se. Recent work has tested this idea using chemoattraction assays to determine whether nematodes might be preferentially attracted towards beetle washes from their original host beetle species SDMs highlighted environmental distinctiveness and habitat discontinuities among P. pacificus meta-population. These distinct clusters were themselves characterised by high genetic diversity, supporting previous results at both mitochondrial and STR markers Our analysis recovered 12 genetic clusters from the La Réunion P. pacificus, suggested that precipitation-based variables were very important in determining suitable habitat for P. pacificus. To quantify the importance of environmental factors to the genetic structure of populations, we employed various post hoc association tests. These demonstrated the presence of strong correlations between environmental and genetic distances among geographic populations and suggested that patterns beyond drift in isolated populations are important determinants of genetic structure. In fact, climate was shown in redundancy analysis to explain ∼65% of the total explainable genetic variance after removing variance explained by geography.Given the strong genetic diversity within locations, it is surprising that we found signals of significant association between environmental and genetic factors at each location. This was first hinted at in the SDMs performed with MAXENT software, which, in addition to helping characterise the environmental niche of P. pacificus populations. In fact, association between putatively neutral genetic variation and environmental variation can be interpreted as evidence of diversifying selection acting over the whole genome P. pacificus genome P. pacificus, which appears to be highly diverse genetically compared to other nematode taxa (e.g. Caenorhabditis elegansP. pacificus, with its wide pool of raw genetic material and many opportunities for ecological and environmental specialisation, seems a prime target for adaptive divergence in a setting such as La Réunion Island.Significant correlations between genetic and environmental variation may arise when habitat differentiation acts as a barrier to gene flow, causing environmental isolation and genetic differentiation even when populations are spatially close Environmental heterogeneity facilitating adaptive divergence represents an important component of natural populations today, especially in the face of proposed climate changes. By imposing diverse selection pressures across geographic regions, environmental factors can affect genetic structure and distribution as shown here and in P. pacificus is influenced by environmental variables. Working with a large genome-spanning STR dataset and covering a broad geographic range of populations enabled us to evaluate the effects of environmental factors on genetic structure. Specifically, we saw how interactions between genetic diversity, multiple introductions, reproductive mode and isolation by environment are together involved in the response of P. pacificus to local environments. Environmental correlations with neutral genetic markers can help identify opportunities for natural selection (e.g. P. pacificus in the La Réunion Island setting has the potential to link genomic variation with functional studies to explore the adaptive processes and mechanisms driving present-day population genomic structure in more detail.In summary, we aimed to explicitly test the hypothesis that genetic structure in ion e.g. , but the


Hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. Eels, both from North America and Europe migrate through the Atlantic to mate in a vast, overlapping area in the Sargasso Sea. Due to the lack of direct observation, it is unknown how these species remain reproductively isolated. The detection of inter-species hybrids in Iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in North Atlantic eels.Here, we show that while mitochondrial lineages remain completely distinct on both sides of the Atlantic, limited hybridization is detectable with nuclear DNA markers. The nuclear hybridization signal peaks in the northern areas and decreases towards the southern range limits on both continents according to Bayesian assignment analyses. By simulating increasing proportions of both F1 hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the European eel. Finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism.Fluctuating degrees of introgressive hybridization between Atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the European eel. Escherichia coli, thus supporting the theoretical predictions from the Fisher-Muller model . In order to create admixed individuals, we randomly sampled the Q-proportions using multinomial sampling from the observed proportions of Q-values in each bin. More specifically, for any given admixed genotype with a certain Q-value the appropriate proportion of alleles inherited from either A. rostrata, or A. anguilla were determined for all nine loci using binomial sampling, and then, using multinomial sampling, alleles were randomly drawn from the allele frequency spectrum of each locus in each “pure species” pool. iii) Finally, we assigned linearly increasing fractions of admixed genotypes of 1%, 2%, 3% or 4%, analogous to the simplified simulation using F1 hybrids, and this again for 100 virtual individuals per population with European eels being the focal species receiving introgressed American alleles.In a second more realistic simulation approach, we created different degrees of admixed genotypes by sampling alleles randomly from the two species gene pools and this in direct proportion given by the actual admixture values apparent from the STRUCTURE output. Following this approach, we obtained virtual admixed genotypes nearly satisfying an average admixture proportion of 50% (expected for F1 hybrids), at adegenet ,120, adeadegenet , ape . To test our hypothesis, that IBD patterns can be generated in European eels by increasing levels of gene flow from South to North, the rectangular matrix of pairwise geographical distances from Wirth and Bernatchez [DCE distances among the 12 virtual populations. Thus, assuming a linear increase of gene flow, we attributed the South-Eastern-most location (River Tiber) the lowest, and the North-Western-most locality (Iceland) the highest hybridization rates, respectively. Intermediate levels were attributed in ascending order along the European coastline.rnatchez  was supeClick here for filePosterior probability distributions for the migration parameters estimated by IMa2 during the first run. (A) Posterior probability estimates for the migration rate (M/μ). (B) Posterior probability estimates for the population migration rates (2NM). Migration parameters correspond to the rate at which European populations receive genes from American populations. NA (Northern), IA (Intermediate) and SA (Southern) A. rostrata populations; NE (Northern), WE (Western) and SA (Southern) A. anguilla populations; and ICE (Icelandic population).Click here for fileDiscriminatory power of microsatellite markers to detect IBD patterns as depicted by locus-specific FST values.Click here for file


FST = 0.96). Owing to the simple and clear structure, a new graphic approach unraveled a detailed evolutionary history; regional crayfish populations were comprised of two distinct lineages that had experienced contrasting demographic processes (i.e. rapid expansion vs. slow stepwise range expansion) following differential drainage topologies and past climate events. Nuclear DNA sequences also showed deep separation between the lineages. Current ocean barriers to dispersal did not significantly affect the genetic structure of the freshwater crayfish, indicating the formation of relatively recent land bridges. This study provides one of the best examples of how phylogeographic analysis can unravel a detailed evolutionary history of a species and how this history contributes to the understanding of the past environment in the region. Ongoing local extinctions of the crayfish lead not only to loss of biodiversity but also to the loss of a significant information regarding past geological and climatic events.Intra-specific genetic diversity is important not only because it influences population persistence and evolutionary potential, but also because it contains past geological, climatic and environmental information. In this paper, we show unusually clear genetic structure of the endangered Japanese crayfish that, as a sedentary species, provides many insights into lesser-known past environments in northern Japan. Over the native range, most populations consisted of unique 16S mtDNA haplotypes, resulting in significant genetic divergence (overall  Recent human activities have threatened considerable numbers of species and populations in nature that, in turn, have significantly affected ecosystem functioning and provision of ecosystem services Another important, but not fully stressed, aspect of genetic diversity is the historical footprints of past environments. Following dynamic changes in the environment, extant species have experienced various demographic events, such as range expansion, vicariance, extinction and dispersal, over the past millions of years and especially during the Quaternary Ice Age Biological clues to our past environment are disappearing due to ongoing local extinctions. In addition, other anthropogenic activities, such as species introduction, transplantation or farming, have been shown to influence the original genetic structure Cambaroides japonicus, which is endemic to northern Japan. More detailed knowledge of past geology and climate conditions in the Japanese archipelago has been growing in the last few decades In this paper, we clearly show that a threatened freshwater crayfish contains significant paleo-geological and climatic information in the distribution of genetic variation and that such historical footprints are in danger of disappearing due to ongoing local extinctions. Our model species was the endangered Japanese crayfish, Although the Japanese crayfish has been designated an endangered species by the Ministry of Environment of Japan, there is currently no law protecting this species from handling or capture The Japanese archipelago exhibits a high endemism in fauna and flora in which speciation has occurred in isolation after colonization from the eastern Eurasian mainland Despite much research, there are still controversies as to if and when the Tsugaru and Tsushima straits became land bridges No consensus has been reached on the locations and extents of land bridges in the Tsugaru Strait. Some biologists suggest from bio- or phylogeographical analyses that bridges in the Tsugaru Strait had emerged in the middle and/or late Pleistocene Cottus nozawae) since the early Pleistocene (1.5 Myr) Lethenteron sp. N) Phylogeographic studies of mammals, insects and plants showed contrasting patterns with or without significant divergence associated with Tsugaru Strait Among more than 600 freshwater crayfish species in the world, most inhabit North America (59%) and Oceania (23%), followed by South America (10%) and Europe (5%), slow-moving crayfish that inhabits mostly small headwater streams Pacifastacus leniusculus) are the major causes of local extinction The Japanese crayfish currently suffers from various threats to extinction: habitat loss, water pollution, timber logging, overharvest and invasion by an alien crayfish  were calculated (see below) using only 51 locations where enough samples  were collected . Muscle tissue was taken from a chelipod or a pereiopod and stored in ethanol at −20°C until DNA extraction.We collected a total of 600 crayfish from 71 locations covering the entire native range, including two main islands (Hokkaido and Honshu) and five small islands near Hokkaido  and 1472 (5′-AGATAGAAACCAACCTGG-3′) 2, 10 mM Tris-HCl (pH 8.3), 0.2 mM dNTP, 0.5 µM of each primer and 0.5 units of taq DNA polymerase  with ca. 10 ng of genomic DNA. The thermal cycling parameters were as follows: 95°C/2 min for hot start, 36 cycles of dissociation (95°C/30 s), annealing (55°C/30 s) and extension (72°C/1 min), followed by a further extension (72°C/19 min). PCR products were purified with QIAquick™ PCR Purification Kit . These products were sequenced using 3100 Genetic Analizer ABI Prism  and the BigDye Terminator Cycle Sequencing kit . To investigate the congruence between different genes, the more conservative 28S rRNA gene region in nuclear DNA was also amplified for 55 subsamples from 52 locations covering the entire distributional range (5′-ACCTATTCTCAAACTTTAAATGG-3′) and rD7b1 (5′-GACTTCCCTTACCTACAT-3′) Genomic DNA was extracted using Chelex100  according to the manufacturer's instruction. A part of 16S rRNA mitochondrial DNA region was selected as a molecular marker because this appears to be the most powerful marker to identify inter- and intra-specific genetic diversity in freshwater crayfish al range . These sFST and Nei's DA, implemented in ARLEQUIN The sequences were aligned using ClustalX version 1.81 M), which is calculated from the inverse relationship with pairwise genetic divergence, i.e. M = (1/FST−1)/4, did not depend on geographic distance between the two populations. Instead, the pairwise gene flow was positively correlated with the distance between the ancestral population  and one of the populations closer to the ancestral population (i.e. older population of the pair). In other words, the level of gene flow would be higher when both populations are more distant from the ancestor (i.e. more recent). In this stepwise colonization process, therefore, genetic divergence should be negatively correlated with the distance from the ancestral population DA, instead of M or FST, because M cannot be calculated when FST is zero and because FST ranges only from zero to one. Similarly, gene diversity should be negatively correlated with distance from the ancestral population, because intra-population genetic diversity should be higher in older populations than in more recently founded ones Patterns of colonization can be inferred once an ancestral population has been determined We also developed a new graphical approach to investigate the colonization process in the Japanese crayfish. Based on coalescent theory, the relative ages of each haplotype can be compared within a haplotype network: interior haplotypes in a network should be older and tip haplotypes should be younger FST = 0.96 for the 51 locations; FST values for each lineage separately. Again, populations were highly diverged . Weak, but significant, positive correlations were observed between pairwise FST and geographic distance .Based on a 490 bp of 16S mtDNA, 46 haplotypes were detected among 600 individuals from 71 locations . All theThe minimum spanning network showed these two divergent lineages were separated by a minimum of seven mutation steps, with only a few missing haplotypes within each lineage . Mean seGeographic distributions of haplotypes and nested haplotype clades were concordant with the haplotype network . HaplotyDA; r2 = −0.0004, Mantel's P>0.05) nor the intra-population genetic diversity  was correlated with geographic distance from the ancestral area  reported for any organism Japanese crayfish showed one of the highest levels of genetic differentiation (Because of prominent divergence in both mtDNA (16S rRNA) and nuclear DNA (28S rRNA) and of geographically separated distributions, the western and eastern lineages may possibly represent two cryptic species or sub-species. In other countries, some crayfish lineages that showed equivalent divergence to the western and eastern Japanese crayfish lineages presented here were treated as separate species or sub-species The pronounced genetic structure of the Japanese crayfish is likely due to its patchy distribution, low dispersal ability and small effective population sizes. High levels of genetic differentiation at each locality  indicated highly localized populations with almost no gene flow with other river drainages (at least in the western lineage). The crayfish can walk on the land, but such movements may rarely contribute to gene flow to other river systems. Colonization or gene flow to other drainages may be achieved by terrestrial dispersal on extremely rare occasions or, more likely, through drainage rearrangements, such as stream capture or drainage diversion The Japanese crayfish represents a paradox with regard to genetic history. Extinction of local populations could naturally occur due to biological characteristics such as small population size and low dispersal ability. However, patterns of genetic variation have been maintained for a long time, possibly several million years. Supporting evidence for the stability of genetic structure is the low frequencies of missing haplotypes within lineages. This is surprising, considering that each local population may be ephemeral and has a unique haplotype. There may be some mechanisms for long-term persistence even in small populations The evolutionary history of the Japanese crayfish provided many insights into the past geography and climate in northern Japan. The only significant barrier to the crayfish was the Hidaka Mountains, separating the western and eastern lineages. Surprisingly, current ocean barriers have not significantly affected the genetic structure of the crayfish. All small islands shared the haplotypes with the nearest areas of Hokkaido Island, indicating land connections existed in the recent past, possibly in the LGM . Although crayfish had once been distributed to different areas as a traditional medicine 200 years ago et al.'s Our results suggest the existence of a land bridge connection across the Tsugaru Strait. Genetic divergence was not pronounced among the crayfish populations in southern Hokkaido and northern Honshu. In addition, the closest populations in Hokkaido and Honshu, in terms of linear geographic distance, shared the same common haplotype. Because there is no strictly freshwater fish species distributed both in Hokkaido and Honshu , the genetic structure of Japanese crayfish provides the first strong biological indication of the existence of a land bridge in the Tsugaru Strait. Dating of a putative land bridge has been controversial, but if we use the Schubart et al.Lethenteron sp. N) and sculpin (Cottus nozawae) populations displayed unique haplotypes in eastern Hokkaido The spread of a single haplotype (Y) over eastern Hokkaido may be due to rapid habitat shifts during the ice ages. Eastern Hokkaido is characterized by flat topology and colder temperature with large areas of wetlands compared to other parts of northern Japan. Yonekura The genetic structure observed in Japanese crayfish may add new phylogeographic perspectives on highly sedentary species. In the Northern Hemisphere, temperate species generally have glacial refuges in the southern range of the distribution, whereas arctic species have interglacial refuges in northern range Another unexpected pattern is the equivalent pace of the northward and southward colonization, regardless of geography and climate. We hypothesize that colonization events might have occurred along coastlines. When sea levels decreased in the glacial periods, neighboring rivers might have been connected or closer to each other downstream Although the stepping-stone colonization process of Japanese crayfish appears to be simple , the obsThe reason(s) that mutations were fixed only in newly established populations is unclear, but may be explained by either or both of the following two reasons. First, mutations may be more likely to occur in newly established populations if new habitats are less suitable (more stressful) and mutations occur in response to harsher conditions .(TIF)Click here for additional data file.Table S1Information on sampling location, sample size and haplotype frequency in the Japanese crayfish (Cambaroides japonicus).(TIF)Click here for additional data file.


Species formation during adaptive radiation often occurs in the context of a changing environment. The establishment and arrangement of populations, in space and time, sets up ecological and genetic processes that dictate the rate and pattern of differentiation. Here, we focus on how a dynamic habitat can affect genetic structure, and ultimately, differentiation among populations. We make use of the chronology and geographical history provided by the Hawaiian archipelago to examine the initial stages of population establishment and genetic divergence. We use data from a set of 6 spider lineages that differ in habitat affinities, some preferring low elevation habitats with a longer history of connection, others being more specialized for high elevation and/or wet forest, some with more general habitat affinities. We show that habitat preferences associated with lineages are important in ecological and genetic structuring. Lineages that have more restricted habitat preferences are subject to repeated episodes of isolation and fragmentation as a result of lava flows and vegetation succession. The initial dynamic set up by the landscape translates over time into discrete lineages. Further work is needed to understand how genetic changes interact with a changing set of ecological interactions amongst a shifting mosaic of landscapes to achieve species formation. The structuring of populations is critical to an understanding of the process of speciation. While evolutionary studies typically focus on longer time scales, the rapidity of adaptation has highlighted the importance of integrating ecological and evolutionary approaches to understand the initial stages of differentiation Schoener . The estMigration, or gene flow, and population size are important factors dictating population establishment and persistence. Gene flow allows populations to become established, and may promote evolution by spreading new genes and combinations of genes throughout a species’ range; alternatively, gene flow can constrain evolution by preventing adaptation to local conditions Slatkin . The impThe genetics of speciation has a long history associated with founder events Provine . During Carson et al.  were theA clearer understanding of how aspects of early-stage colonization history may affect the genetic capacity for adaptive responses is required  when lava flows over or around them. After a lava flow cools, the forest gradually regenerates, receiving founders from other intact areas. A mature, closed canopy forest has been estimated to develop in 300–3,000 years on new flows, with temporal differences depending on abiotic conditions  and biotic factors. Detailed work on dating of island ages, the soils and lava flows by the USGS  level in the chronosequence, is Maui Nui, a composite of 4 separate volcanic islands, Maui, Molokai, Lanai, and Kahoolawe Fig. . It was Active volcanism fosters genetic diversity. Studies have shown that lava flows divide habitats, causing isolation, yet this isolation is transient and the open habitat subsequently becomes available for further colonization. Our hypothesis is that this dynamic creates the ideal setting for promoting and maintaining genetic diversity because it provides for isolation and local selection, but also an avenue for repeated colonization. Therefore, we predict (a) that population structure in the Big Island region of more recent volcanic activity should reflect significant dispersal that can foster genetic diversity across populations. Also, (b) populations of species that are habitat specialists in subdivided forest habitats should be more structured than populations of habitat generalists  on Maui Nui and the Big Island. T. grallator is a resident of the wet and mesic forests on all high islands of the archipelago except Kauai and has a web that consists of a scanty, two-dimensional layer of silk covering the entire underside of a leaf  that volcanic maturation promotes opportunity for isolation we estimated standardized genetic variance, Nem, among populations. Calculations are described below. If active volcanism fosters genetic diversity, we predict (a) that in the kīpuka region characterized by a metapopulation structure of population extinction and recolonization, genetic variance among populations should be low and migration high. Because only the Big Island is active volcanically, there is no ideal geographic comparison of the same scale elsewhere in the Hawaiian Islands; a previous study compared populations in the forest patches with neighboring continuous forest  found in the kīpuka region.To test the hypotheses (1) that volcanic maturation promotes opportunity for isolation, we predict (a) more genetic variance and less migration among populations on the older Maui Nui than on the younger Big Island. Further, because the lower elevation habitats of Maui Nui have been continuous for longer than the higher elevation habitats, we predict more subdivision in mid-high elevation species  than species that also inhabit lower elevations . We also predict increasing isolation by distance with volcanic maturation, from populations near active volcanism on the Big Island, to between volcanoes on the Big Island and Maui Nui, to between islands.If rlequin , where Ne is the effective population size, m is the migration rate between two populations. For each group of populations examined, a full migration model was examined, allowing unrestricted migration among all populations. Migrate-N runs for both the mtDNA and allozyme data consisted of a burn-in discarding 50,000 genealogies per chain. Five replicates were run, each sampling every 200 steps and recording 20,000 genealogies for the mtDNA data and 10,000 genealogies for the allozyme data such that 20,000,000 (mtDNA) and 10,000,000  genealogies were visited. An adaptive heating scheme, spread across four MCMC chains was employed and the mutation rate among allozyme loci was allowed to vary (as estimated from the data by the software). For each species at a given geographical scale the range of the median posterior values of Nem among all pairs of populations is presented. We also calculated an estimate of relative effective population size, θ = Neμ, where Ne and m are defined as above and μ is the mutation rate per generation at the locus considered.For species collected in at least 2 localities Table , pair-wiactive volcanism fosters genetic diversity. Populations were found to be highly structured overall, including populations of species found in the kīpuka region , than among populations of the habitat generalist . However, previous analyses of a subset of these species  found that the amount of genetic differentiation among fragments was greater than that predicted by distance alone in nearby continuous mature wet forest  and a habitat generalist (L5 T. quasimodo), the results are consistent with the idea that habitat dynamics imposed by volcanic activity contributes to the maintenance of genetic variation.In this study of genetic structure of 6 spider lineages we found evidence that dynamic habitats can contribute to population structuring, even with substantial gene flow. Our results from both mitochondrial and nuclear markers show that genetic structure was significant at nearly all scales, including among populations in the on Table A. For thkīpuka separated by <100 m and that differentiation among fragments is greater than that found by distance alone in nearby continuous mature wet forest . These estimates likely reflect ongoing gene flow, but possibly also the relatively small amount of time since geographic isolation. Theoretical work suggests that estimates of gene flow from measurements of genetic divergence are valid only after time sufficient for drift and migration to reach equilibrium, which may be on the order of 4 × Ne generations, where Ne = effective population size , disjunction should increase with island age, as we find. Alternatively, because of its geological history, the lowland areas separating the volcanoes of Maui Nui have always been more expansive than those on the Big Island  was greater between the islands of Maui Nui and the Big Island, than among volcanoes within Maui Nui or the Big Island Table B; Fig. 3kīpuka area, genetic isolation in the absence of diversifying selection is unlikely to result in speciation, given the timeframes of habitat regeneration that occurs on new flows. Populations that expand in the maturing habitat will reconnect and re-establish gene flow in the absence of other isolating factors. However, population growth can increase the relative importance of even weak directional selection  active volcanism maintains and fosters genetic diversity, although through a complicated process involving population subdivision in the context of migration and admixture; and (2) volcanic maturation results in greater isolation of populations over time, thus promoting and increasing opportunities for isolation. Future studies must focus explicitly at the population level to gain more precise insights into the temporal framework of connectivity and isolation under which differentiation can occur (see Vuilleumier et al.


Carcharhinus brevipinna), a large cosmopolitan carcharhinid, sampled from eastern and northern Australia and South Africa.Quantifying genetic diversity and metapopulation structure provides insights into the evolutionary history of a species and helps develop appropriate management strategies. We provide the first assessment of genetic structure in spinner sharks (h = 0.6770 ±0.025). While two metrics of genetic divergence (ΦST and FST) revealed somewhat different results, subdivision was detected between South Africa and all Australian locations . Evidence for fine-scale genetic structuring was also detected along Australia’s east coast , and between south-eastern and northern locations .Sequencing of the mitochondrial DNA NADH dehydrogenase subunit 4 gene for 430 individuals revealed 37 haplotypes and moderately high haplotype diversity (C. brevipinna between Australia and South Africa. Gene flow also appears restricted along a continuous continental margin in this species, with data tentatively suggesting the delineation of two management units within Australian waters. Further sampling, however, is required for a more robust evaluation of the latter finding. Evidence indicates that all sampled populations were shaped by a substantial demographic expansion event, with the resultant high genetic diversity being cause for optimism when considering conservation of this commercially-targeted species in the southern Indo-Pacific.The Indian Ocean represents a robust barrier to contemporary gene flow in  Patterns of genetic variability in extant taxa have been generated by events and processes occurring over evolutionary time scales. Genetic bottlenecks and demographic expansions, coupled with associated fluctuations in effective population size, are examples of such events, respectively manifesting as low and, eventually, high levels of genetic diversity -8. EvoluCarcharhinidae represent large, cosmopolitan shark species occupying predominantly continental-shelf waters  by incubation overnight at 55 °C. Proteins were precipitated by adding 170 µl of 5 M NaCl followed by microcentrifugation at 14,000 rpm for 5 min. Supernatant (600 µl) was recovered into a fresh tube and the DNA precipitated by adding 600 µl of ice-cold 100% ethanol. Tubes were stored at −20 °C for approximately 1 h. DNA was then recovered by microcentrifugation at 14,000 rpm for 15 min, and the ethanol decanted. The resulting DNA pellet was washed with 200 µl of 70% ethanol, 100 mM sodium acetate solution, and microcentrifuged at 14,000 rpm for 3 min. Following decanting, all remaining ethanol was removed using a micropipette. DNA was air-dried, resuspended in 100 µl of TE buffer [10 mM Tris.HCl (pH 7.6) and 1 mM EDTA] and stored at −20 °C. DNA yield was checked on a 1.0% agarose TBE (90 mM TRIS-borate and 2 mM EDTA) gel run at 110 V.To obtain mtDNA sequence data, total genomic DNA was extracted from 5 mg of tissue using a modified salting-out protocol . Samples2 and 200 µM deoxynucleoside triphosphates (dNTPs)] , 0.5 µl of RNase (1 mg·ml-1), and 0.5 µM of each of the primers ND4 (5’ CAC CTA TGA CTA CCA AAA GCT CAT GTA GAA GC) [TTG CAC CAA GAG TTT TTG GTT CCT AAG ACC) [Polymerase Chain Reaction (PCR) was used to amplify the mitochondrial DNA NADH dehydrogenase subunit 4 (ND4) gene from all tissue samples. Reactions were carried out in 50 µl volumes containing 1 µl of DNA template, 1× GoTaq Colourless reaction buffer [consisting of 1.5 mM MgCl GAA GC)  and H122AAG ACC) . Amplifihttps://biomanager.info) and aligned using the ClustalW (accurate) algorithm [Cbrevipinna.  were available for the cytochrome oxidase I (CO1) gene, but not for ND4, prior to this study. Therefore, to validate that the study species had been correctly identified and also to determine the species identity of any misidentified individuals, randomly-selected representatives from each separate haplotype determined from the alignment output were amplified for the CO1 gene using the primers Fish F1 (5’ TCA ACC AAC CAC AAA GAC ATT GGC AC) and Fish R1 (5’ TAG ACT TCT GGG TGG CCA AAG AAT CA) [Sequences were trimmed and edited manually. Edited sequences were entered into Biomanager (lgorithm . GenBank AAT CA) . PCRs weCbrevipinna.  ND4 sequences were imported to Arlequin 3.5.1.2 [To identify and characterise mitochondrial haplotypes, aligned  3.5.1.2 . A seque 3.5.1.2 . Phyloge 3.5.1.2 , and gen 3.5.1.2  with 1,0 3.5.1.2 . To asseπ). Harpending’s raggedness index (HRI) was estimated from nucleotide mismatch distributions constructed in Arlequin under the sudden demographic expansion model with 20,000 bootstrap replicates [D and Fu’s F neutrality indices were also estimated in Arlequin, and are indicative of departures from mutation-drift equilibrium or patterns of selection [HRI, the latter two analyses can be used to determine if a population has undergone an expansion event (possibly following a genetic bottleneck). Mismatch distributions will be multi-modal (or ragged) in a stable population, where the generation of new mutations is offset by random drift, and uni-modal for expanding populations, where new mutations accumulate faster than their loss due to drift [D and Fu’s F, signals of population expansion are denoted by significant negative test statistic values. Statistical significance was assessed here, following 20,000 simulated samples, at α = 0.05 and α = 0.02 for D and F values respectively [Genetic diversity indices were obtained with Arlequin using the Tamura-Nei substitution model , and incplicates . Tajima’election ,83. In cto drift . For TajCbrevipinna. , an analysis of molecular variance (AMOVA) [F-statistic metrics of genetic divergence: ΦST [FST [ST has been regarded as the superior metric on the basis of its incorporation of a measure of genetic distance between haplotypes, frequency-based FST has been proposed as potentially a more appropriate measure of genetic differentiation among locations where migration is theoretically occurring at a faster rate than mutation [ST was calculated via the computing of a distance matrix using the Tamura-Nei model [FST used haplotype frequencies only. AMOVA partitioned genetic variance among, and within, sample locations, and calculated overall ΦST and FST fixation indices. Genetic differentiation between each pair of locations was also measured by calculating pairwise ΦST and FST estimates. Statistical significance was determined following 20,000 permutations of the sequence data and, in the case of pairwise ΦST and FST, assessed at an initial critical significance level of α = 0.0083 (adjusted from α = 0.05) following sequential Bonferroni correction for six simultaneous comparisons [n = 208), QLD (n = 63), NT (n = 97) and South Africa (n = 62) (n = 43) and north-western NT (n = 54) waters. This analysis indicated genetic homogeneity , hence providing justification for pooling GoC and north-western NT samples to create one northern population termed ‘NT’.To test the null hypothesis of panmixia (genetic homogeneity) in Australian and South African waters for  (AMOVA)  was implnce: ΦST  and FSTST [FST . While Φmutation . ΦST wasei model  for estiparisons ,88. The (n = 62) . The anaCarcharhinusbrevipinna sample sizes were clearly biased towards NSW , n = 100 and n = 150 were selected without replacement from the NSW population, while QLD, NT and South African sample sizes were kept unchanged. Population pairwise ΦST and associated p values were generated for each replicate random sample-set in Arlequin using the batch processing function and permutation settings as outlined above. Resultant ΦST and p value distributions were plotted, and the likelihood of producing a result contradictory to that of the original analysis was calculated as either the proportion of p values ≤ 0.05 or > 0.05, depending on the result of the original analysis. That is, if the original pairwise p value was significant (p ≤ 0.05), the likelihood of a contradictory result equals the absolute number of p values > 0.05/10,000.ards NSW , where 2ST) and geographic  distances between the four putative populations were calculated in GenAlEx [The ‘Isolation by Distance’ (IBD) hypothesis was also tested to determine if inter-population genetic distances increased linearly with geographic distance. Genetic  was calculated using the rarefaction formula of Hurlbert , and exeCbrevipinna.  in the NSW OTLF from 2007–2010. Of these, 187 were genetically confirmed to be Cbrevipinna. , translating to an observer-accuracy estimate of 98.4% for the identification of this species in the fishery (n = 3) comprised two Climbatus.  and one Cobscurus.   was found in NSW and NT waters, despite NSW having over double the sample size (π) diversities were very similar, and high in the case of the former and low in the case of the latter, across three of the four putative populations  . All mismatch distributions were consistent with the sudden population expansion model, with no significant deviation from a uni-modal distribution    . Compara4–0.099) . In supp -4.464) .Cbrevipinna.  population appears to be site dependent.Rarefaction exact curves indicated trends towards asymptotic relationships for both the NSW and South African locations , despiten = 21 of n = 36) of lower frequency haplotypes being shared between two or more locations  surrounded by an array of low, or lower, frequency variants (SP2-SP27) . A high ocations , Table 2F-statistic metrics  , with all three comparisons significant after Bonferroni correction  which was also significant after sequential Bonferroni adjustment, as well as some evidence for genetic subdivision between NSW and NT (pairwise ΦST = 0.00669) which was significant at p < 0.05 but not after Bonferroni correction  . The finding of genetic homogeneity between QLD and NT was concordant between both F-statistics. A strong positive relationship, with high goodness-of-fit (r2 = 0.86), was observed between pairwise genetic and geographic distances for Cbrevipinna. . This relationship, being driven entirely by differences between Australian locations and South Africa, was not statistically supported by a mantel test (p = 0.091).Despite this, AMOVA fixation indices detected significant levels of genetic differentiation between the four putative populations for both  0.0035) . We therrrection . ΦST alsrrection . In cont 0.0008) . All othCbrevipinna.  sampled from NSW on the F-statistics analysis of pairwise population comparisons. Random re-samplings demonstrated an increasing likelihood of finding a non-significant result between NSW and QLD, and between NSW and NT, with decreasing NSW sample size . Given the fundamental importance of accurate catch-composition data in fisheries (and species) management [Observer accuracy was high (98.4%) in the identification of nagement ,92,93, tnagement  confirmsCbrevipinna.  in the NSW OTLF was higher than that reported for the same species by Tillett et al. [Cbrevipinna.  by observers in the latter fishery were ≤ 1.2m TL [Cbrevipinna.  is characterised by diagnostic traits that become increasingly discernible as an individual grows larger, most notably tooth shape and fin pigmentation [Cbrevipinna.  can be difficult to distinguish from a range of other morphologically-similar carcharhinid species [Our measure of observer accuracy (98.4%) for t et al.  in the Nt et al. . In cont 1.2m TL . Size-atentation . At smal species .Cbrevipinna.  haplotype network was distinctly star-shaped, characterised by a single dominant haplotype surrounded by a high number of low, or lower, frequency variants. This central, and presumably ancestral, haplotype was prominent in all three Australian sample locations, as well as off the coast of South Africa - evidence that Australian and South African waters share common ancestry in this species.The Cbrevipinna.  is indicative of a contemporary demographic expansion event having occurred throughout the southern Indo-Pacific. This hypothesis is supported by a range of evidence: the distinctly ‘star-burst’ haplotype network denoted by numerous low-frequency mutations, mismatch distributions and neutrality test statistics suggesting strong departures from mutation-drift equilibrium for all four putative populations and the observed combination of generally high haplotype and low nucleotide diversities [The pattern of genetic diversity observed here in ersities ,83,94-96It must be noted here, however, that spatial sample coverage in the present study is limited to only a very small area of this species’ global distribution range, which includes much of the world’s tropical and warm-temperate continental shelf waters . TherefoCbrevipinna.  is unprecedented among sharks, with comparable signals more commonly associated with taxa such as humans [The strong signal of population expansion reported here in s humans  and teles humans ,97,98. Es humans , star-lis humans ,100,101,s humans ,103.Cbrevipinna. . The application of two metrics of genetic divergence (ΦST and FST) demonstrated that population genetic findings can be dependent on the F-statistic employed - especially pertinent where subdivision is at the margins of statistical significance [This study marks the first dedicated assessment of genetic structure in ificance . We therWith this in mind, genetic differentiation was detected over a broad spatial scale between Australian and South African waters. This finding based on mtDNA was not unexpected and, being consistent with a range of other shark population genetic studies ,104-107,Cbrevipinna. ’s potential for active dispersal. That said, however, genetic differentiation has previously been detected in similar and related shark species over comparable geographic scales in Australian waters [Evidence for genetic subdivision, albeit weak, was also detected over finer spatial scales within Australian waters, i.e. between NSW and both QLD and, to a lesser degree, NT. Genetic homogeneity was observed between QLD and NT waters. These results tentatively suggest that gene flow is restricted to some degree along Australia’s eastern continental margin as well as between the south-eastern and northern coastlines, and that gene flow is unencumbered between north and north-eastern Australian waters. These findings were somewhat unexpected given n waters ,108,109,n waters ,30.Reproductive philopatry, or the fidelity of gravid females to nursery areas, is typically invoked to explain fine-scale genetic structuring  in the absence of barriers to dispersal for highly-vagile sharks ,41,109. Cbrevipinna.  has influenced our findings of fine-scale genetic differentiation to some degree. In their study, Ovenden et al. [Rhizoprionodonacutus) using ND4 sequence data. Rhizoprionodonacutus, a considerably smaller-bodied and presumably less-vagile species than Cbrevipinna. , conforms to a population model characterised by permanent habitation of nearshore waters without the use of discrete nursery areas [Cbrevipinna.  for parturition and juvenile development is well documented . Differing life-cycles denoted by varying usage of nearshore habitat, therefore, may account for these contrasting genetic structures observed along Australia’s east coast.Consideration of our results in light of those by Ovenden et al. , howevern et al.  failed try areas . In contCarcharhinusamboinensis) [Cbrevipinna. , have a distribution restricted to northern regions in Australian waters [Alternatively, genetic differentiation between NSW and NT may be a relict signature of repeated periods of temporary isolation due to the rise and fall of the Torres Strait land bridge caused by fluctuating sea levels during the Pleistocene epoch ,112. Thinensis)  which, ln waters . Under tSimilarly, a marked change in marine environment coinciding with the Tropic of Capricorn  represenST and FST metrics  is suggestive of a slow rate of mutation in the ND4 gene region. This raises considerable doubt as to the ability of ND4 to effectively discriminate population structure in Cbrevipinna. . For example, pairwise F-statistic estimates involving the South African population were demonstrably low in the present study  compared to others reporting genetic differentiation in sharks over comparable spatial scales   and zebra (Stegostomafasciatum) sharks, ND4 was the most polymorphic of a range of mtDNA markers, including the control region. Alternatively, therefore, low F-statistic values associated with observed genetic structuring between Australia and South Africa, as well as within Australian waters, may reflect continued low-level gene flow, or a recent cessation of gene exchange, between subdivided locations. Until the relative mutation rates of ND4 and CR are determined for Cbrevipinna. , however, or this study is reassessed via sequencing of CR, it is impossible to confidently support or refute the abovementioned hypotheses. Moreover, this issue emphasises the limitations inherent in the analysis of only one mtDNA locus.This study was subject to a range of limitations requiring careful consideration. To begin with, very low values for both Φ8–0.991) . Given tn et al.  and Ovenn et al.  who demoThe clear bias in sample sizes weighted towards the NSW population represents another major limitation of this study. Random-resampling simulations provided some evidence that the detections of significant genetic differentiation within Australian waters  were driven in large part by this bias. Replicate pairwise comparisons for both sets of locations indicated an increasing likelihood of finding a non-significant result as the NSW sample size decreased towards a more balanced analysis. This either serves to emphasise the weak nature of the observed fine-scale genetic subdivisions within Australian waters, or draw their actual existence into question. Conversely, replicate pairwise comparison between NSW and South Africa returned a significant difference independent of the NSW sample size, hence reinforcing the strength of the genetic subdivision between the latter two regions, and indicating that the original analysis was robust to the bias in sample size in this instance.Cbrevipinna.  population is site dependent. For Australian locations, sample sizes in excess of 100 were required for robust comparisons, whereas a sample size of ~60 appeared sufficient for South African waters.Rarefaction analysis added further uncertainty regarding the reliability of our fine-scale findings reported in the present study. NSW and South Africa were the only two locations at which adequate levels of the available genetic diversities were likely sampled, hence confirming the robustness of the comparison between these two putative populations. In contrast, a proportion of the available diversity appeared to have remained unsampled from QLD and NT, suggesting that findings emanating from comparisons involving the latter two locations should be treated with some degree of caution. Rarefaction curves demonstrated that the optimum sample size required to accurately represent levels of haplotypic variation, and in turn to confidently discern haplotype relative frequencies, within any given Cbrevipinna.  is cause for optimism when considering the management and conservation of this commercially-targeted species in southern Indo-Pacific waters. Carcharhinusbrevipinna exhibited high haplotype numbers and similar or high haplotypic diversity  compared to Cobscurus.   and Cplumbeus.  , two closely-related species, off Australia’s east coast . Comparatively high haplotype numbers implies that Cbrevipinna.  may display a greater resilience to a loss of genetic diversity, as a result of high-intensity fishing pressure, than these other commercially-targeted shark species in Australian waters.The generally high genetic diversity reported here in Cbrevipinna.  from the south-eastern zone (h = 0.5984), compared to QLD (h = 0.7424) and NT (h = 0.7279), may be accounted for by NSW representing sampling of the species’ southern-most distribution limit [Cbrevipinna.  between NSW, QLD and NT, as well as a lack of knowledge pertaining to original population sizes and periods of time required to affect quantifiable reductions in genetic diversity.The lower genetic diversity observed in on limit . Range lon limit ,116. AltCbrevipinna.  in the southern Indo-Pacific – Australia and South Africa. The most appropriate boundary between these management units, however, is unknown and would require more detailed spatial sampling within the Indian Ocean basin. Our data also suggest, albeit tentatively, two management units within Australian waters – south-eastern (NSW) and northern (QLD and NT) Australia. This implies that, in the event of a population collapse in south-eastern Australia, recovery of genetic diversity would rely largely on reproduction by surviving local individuals in NSW waters. Currently, each Australian state is independently responsible for the management of shark fishing operations occurring within its respective waters, with little to no collaboration across jurisdictional borders. Our results suggest that the independent management of NSW and QLD Cbrevipinna.  populations is perhaps appropriate, but that cooperation between QLD and NT would be prudent.Our genetic structure results indicate the delineation of two management units for In light of the limitations of the present study, however, we recommend this work be considered as a starting point for evaluations of genetic structure in this commercially-important species, rather than a study upon which definitive management decisions are made. Moreover, we strongly urge future studies to focus on achieving greater population structure resolution via more extensive sampling within Australian waters, as well as throughout this species’ global distribution range, in conjunction with analysis of nuclear and/or additional mitochondrial markers. Such studies, conducted in association with active tagging and tracking, would assist with more robust allocations of management units, and hence the sustainable exploitation of this target species.Table S1Carcharhinusbrevipinna.Polymorphic sites for mitochondrial DNA ND4 haplotypes defined for (DOC)Click here for additional data file.


Oligoryzomys longicaudatus (Sigmodontinae), the major reservoir of Hantavirus in Chile and Patagonian Argentina, is widely distributed in the Mediterranean, Temperate and Patagonian Forests of Chile, as well as in adjacent areas in southern Argentina. We used molecular data to evaluate the effects of the last glacial event on the phylogeographic structure of this species. We examined if historical Pleistocene events had affected genetic variation and spatial distribution of this species along its distributional range. We sampled 223 individuals representing 47 localities along the species range, and sequenced the hypervariable domain I of the mtDNA control region. Aligned sequences were analyzed using haplotype network, Bayesian population structure and demographic analyses. Analysis of population structure and the haplotype network inferred three genetic clusters along the distribution of O. longicaudatus that mostly agreed with the three major ecogeographic regions in Chile: Mediterranean, Temperate Forests and Patagonian Forests. Bayesian Skyline Plots showed constant population sizes through time in all three clusters followed by an increase after and during the Last Glacial Maximum . Neutrality tests and the “g” parameter also suggest that populations of O. longicaudatus experienced demographic expansion across the species entire range. Past climate shifts have influenced population structure and lineage variation of O. longicaudatus. This species remained in refugia areas during Pleistocene times in southern Temperate Forests (and adjacent areas in Patagonia). From these refugia, O. longicaudatus experienced demographic expansions into Patagonian Forests and central Mediterranean Chile using glacial retreats.The long-tailed pygmy rice rat  Glacial cycles that have affected the distributional range of species during the Quaternary have been the focus of multiple phylogeographic studies intending to evaluate the occurrence of single or multiple gene genealogies The southern Andes were repeatedly glaciated during the Late Cenozoic. In particular, the Patagonian Andes were glacier-covered prior to 4.6 mya (Late Miocene) and icefields repeatedly expanded between 2.4 and 1.2 mya Nothofagus forests, the “southern beeches”) were strongly affected by glacial cycles of the Pleistocene Throughout the Quaternary, the mountain range of the Andes, the “Cordillera de los Andes” (the eastern geographic limit of Chile) experienced strong volcanic activity and glaciation events in the Temperate region Oligoryzomys longicaudatus . Latitudinally, it ranges between 27–54°S, encompassing three of the major ecogeographic regions in the southcentral portion of Chile: the Mediterranean, the Temperate, and the Patagonian Forests . Knowledge of phylogeographic patterns of reservoir populations can provide insight into factors responsible for the occurrence and spread of the virus in natural populations An important component of the small mammal fauna of southern forests in Chile and adjacent areas in Argentina is the “long-tailed pygmy rice rat” (“colilargo”)  Forests ; Fig. 1.Oligoryzomys longicaudatus occurs in areas that were glaciated during the Pleistocene, and in formerly non-glaciated zones that presumably constituted refugia  control region, and aligned sequences were analyzed using Bayesian approaches.This study evaluates the genetic consequences of Pleistocene glaciations on the phylogeographic structure of the “long-tailed pygmy rice rat” hypervariable region I (HVI), were sequenced from 223 specimens representing 47 localities in Chile and Argentina , rgentina . We recorgentina .O. longicaudatus was evaluated using the GENELAND v. 1.0.7 program The population structure for Regarding segregating sites per cluster, we found 51 in Cluster 1 Mediterranean, 46 in Cluster 2 Temperate Forests, and 47 in Cluster 3 Patagonia. The three groups showed similar haplotype diversity although Patagonia was slightly higher, whereas overall nucleotide diversity was low especially for Mediterranean Chile. The haplotype diversity (Hd) was 0.967 for Cluster 1 Mediterranean, 0.968 for Cluster 2 Temperate Forests, and 0.991 for Cluster 3 Patagonia . The nucP = 0.000), Temperate Forests , and Patagonia , indicating that the null hypothesis of population equilibrium is rejected in favor of a population expansion. Maximum likelihood estimations of the ‘g’ parameter were positive and significant  for each of the clusters along the O. longicaudatus's range . After a period of constant population size, analyses showed an increase in the effective population size starting about 12,000 years ago for the Mediterranean cluster , about 2's range : Mediter's range .Oligoryzomys longicaudatus has high haplotype diversity , but low nucleotide diversity for each of the clusters, suggesting recent differentiation with rapid population growth b mitochondrial sequences in a previous study O. longicaudatus between 13,000  and 25,000–26,000 years ago , which is in the range of the LGM event g’ parameter. According to our GENELAND results that recovered localities in glaciated and non-glaciated areas of southern Chile, we hypothesize that populations of the long-tailed mouse may have dispersed from these areas that might have constituted refugia in the Temperate Forests and Patagonia.O. longicaudatusFitzroya cuppresoidesHypochaeris palustrisNothofagus pumilioLiolaemusSamastacus spinifrons , and the sigmodontine mice Abrothrix olivaceusA. longipilisO. longicaudatus along its distribution in Chile and adjacent Argentina. Glacial cycles strongly affected Temperate Forests, the primary habitat of Oligoryzomys, which may have disturbed local populations of this species. Our results support the hypothesis that postglacial colonization of O. longicaudatus probably occurred from multiple refugia in southern Chile between 26,000 to about 13,000 ya, which is in agreement with palynological data At least three events of glacial cycles have been reported for southern Patagonian Forests that affected the biota of the area Oligoryzomys longicaudatus that closely correspond to the Mediterranean, the Temperate Forests and Patagonian steppes and forests. Since our results suggest that Temperate Forests samples constitutes the ancestral haplotypes because they are scattered through the network, we hypothesize that expansion of populations might have occurred from southern refugia, such as the localities assigned to the Mediterranean cluster within and outside the limits of the LGM  due to global cooling in the Pleistocene. For example, one hypothesis to explain the current relictual distribution of Temperate Forests in the coast of central Chile , which are currently separated by more than 1,000 km from southern forests, is that they are remnants of southern vegetation that expanded during glaciations In temperate areas of North America, dispersal of mammals to lower latitudes during glacial periods followed by demographic expansion during warm interglacials is well documented O. longicaudatus west of the Andes, former studies have suggested a colonization from the south (Temperate Forests) to Patagonia and to the central-north of Chile O. longicaudatus through the Andes in a west-east direction O. longicaudatus is part of an Andean-Chacoean clade O. andinus from the highlands Andes of Peru and Bolivia, O. fornesi from Bolivia, northern Argentina, southern Brazil, and the Paraguayan Chaco O. andinus and O. spB in the highlands of the central Andes, O. fornesi in the foothills of the Andes and Chaco region, and O. longicaudatus in the southern lowland that ranges from the Andes mountains of Argentina and Chile, southward to Patagonia et al.O. longicaudatus on populations west of the Andes, but they only included 4 localities  from the Chilean side. Their hypothesis is also based on the biogeography of another sigmodontine rodent, Abrothrix olivaceus, whose populations may have crossed the lower elevations of the Andes through the Nothofagus forests located in southern Chile and Argentina (about 40°S) in an west-east direction. The biogeographic history of O. longicaudatus, however, is different to that of A. olivaceus. While A. olivaceus ranges from southern Peru southward to Patagonia, Oligoryzomys longicaudatus ranges from the southern limit of the Atacama Desert  southward to Patagonia. Thus, we believe that the most parsimonious hypothesis for the entrance of O. longicaudatus into the Chilean Andes is from the east (Argentina), entering through the lower elevations of the Andes at about 40°S. Following predictions from the coalescent theory O. longicaudatus are ancestral to central Chile and Patagonian populations, and that the entrance of the species into the Chilean side could have been from the south.Regarding the biogeographic history of O. longicaudatus through GENELAND and Neighbor-net analyses are in agreement with the major ecogeographic regions recognized in Chile, the Mediterranean, the Temperate Forests and the Patagonian Forests. These three groupings concur with the three subspecies originally proposed by Osgood O. longicaudatus in each of these ecoregions, respectively: O. l. longicaudatus, O. l. philippii and O. l. magellanicus. An earlier study discarded the occurrence of subspecies within O. longicaudatuslongicaudatus and philippii as a synonym of the former and recognizing the species O. longicaudatus (Mediterranean and Temperate Forests) and O. magellanicus (Patagonia). However, we suggest that the latter proposition should be revised. Oligoryzomys longicaudatus has a broad distribution in Chile, south of the Atacama Desert (about 27°S) downwards to the Patagonia including Tierra del Fuego (Chile and Argentina), and the Navarino Island , and historical effects  O. longicaudatusO. l. longicaudatus to the Mediterranean ecoregion between Atacama and Biobio (27–38°S), O. l. philippii to the Temperate Forests (38–45°S), and O. l. magellanicus to the Patagonian Forests (45–55°S). As of O. l. pampanus from Bahía San Blas, Buenos Aires, Argentina, this taxon was recovered within the Patagonian cluster, but further analyses will be necessary to uncover the taxonomic status of this form.The three genetic clusters recovered within Oligoryzomys longicaudatus remained in multiple refugia within the Temperate Forests and Patagonia ecoregions of Chile and Argentina, and inside and outside of LGM icesheet. Subsequent dispersal to the north and south of previously proposed refugia and non-glaciated areas may have occurred during and after the LGM. Our results supported a population expansion scenario in the time period of glacial retreats, which may have been facilitated by the dispersal capabilities of the species. In addition, the low nucleotide diversity found among Oligoryzomys haplotypes suggests a recent history of expansion with high admixture between populations. A hypothesis of expansion from multiple refugia in the southern range of the species, and northern expansion due to global cooling during Pleistocene glaciation, with further retraction towards the south, was supported.Our analyses suggest that populations of Voucher specimens sequenced in this study were deposited in the Colección de Flora y Fauna Profesor Patricio Sánchez Reyes (SSUC), Departamento de Ecología, Pontificia Universidad Católica de Chile, Santiago, Chile; Colección de Mamíferos del Centro Nacional Patagónico (CENPAT), Puerto Madryn, Argentina (field numbers UP and LB); the Museum of Southwestern Biology (MSB), Department of Biology, University of New Mexico; and the Instituto de la Patagonia (CZIP), Universidad de Magallanes, Chile.O. longicaudatus, representing 42 localities in Chile and five in Argentina; O. longicaudatus is available O. longicaudatus in Chile and Argentina are given in We analyzed 223 samples from 47 localities throughout the range of We followed the guidelines of the American Society of Mammalogists during the collection and handling of the animals used in this work CGGAGGCCAACCAGTAGA-3)′ and DLO-H (TAAGGCCAGGACCAAACC-3′). The PCR program consisted of an initial denaturation time of 3 min at 94°C followed by 25 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 57°C, and extension for 1 min 30 s at 72°C. Double-stranded PCR products were purified with QIAquick PCR Purification Kit (Qiagen). Cycle sequencing was performed using primers DLO-L and DLO-L2 (CACTTGGGGGTGTCTAACC-3′) labeled with the Big Dye Terminator kit of Perkin Elmer . Sequencing reactions were analyzed using an Applied Biosystems Prism 310  automated sequencer, and sequences were aligned using the ClustalW program DNA was extracted from frozen liver samples using QIAamp Mini Kit . The HVI mtDNA control region was amplified via polymerase chain reaction (PCR) using primers DLO-L . This model's objective is to infer and locate the genetic discontinuities between populations of geo-referenced genotypes, considering the uncertain localization of the sampled individuals. The number of clusters was determined by running MCMC (Markov chain Monte Carlo) iterations five times, allowing K  to vary, with the following parameters: 5×106 MCMC iterations, maximum rate of the Poisson process fixed to 100 , uncertainty attached to the spatial coordinates fixed at 5 km to account for the possibility that inaccurate coordinates were provided, and considering that the maximum home range reported for O. longicaudatus is 4.5 km K fixed to the inferred number of clusters, with the other parameters the same as above. The mean logarithm of the posterior probability was calculated for each of the 30 runs and the posterior probability of population membership for each pixel of the spatial domain was then computed for the three runs with the highest values.We used the DnaSP v 5.10.01 software O. longicaudatus we used a Bayesian coalescent-based framework. Coalescence explains how population genetic process through time have shaped an allelic or haplotypic genealogy To establish the relationships between haplotypes, we constructed a haplotype network using the Neighbor-Net BF = 3.073, cluster 2 log BF = 6.82 and cluster 3 log BF = 6.03). The Bayes factor compares two posterior probability distributions, by asking which is larger after having adjusted differences between the models (Mi vs Mj) in terms of the numbers of parameters that they use. Given marginal likelihoods for two different models, the log-Bayes factor is defined as log BF = −2 log [p (D|Mi)/p ]. We followed previously reported criteria to determine the support for the favored model j. The marginal likelihood was estimated using the method previously described Mus musculusWe used the coalescent-based Bayesian Skyline Plot approach BSP;  as impleeNμ  jointly with the population growth rate ‘g’, assuming an exponential model of growth, and using the Bayesian MCMC sampling. The ‘g’ parameter reflects the exponential growth model; positive values of ‘g’ indicate (population) growth, and negative values indicate population shrinkage. We set the program to compute the Watterson estimate of Θ, and allowed the population to change in size, with an initial value for ‘g’ set to 0.1, a random starting tree, and the transition/transversion ratio obtained from jModelTest 2The program LAMARC 2.1.3 Table S1List of geographic localities analyzed in this study. Refer to numbers depicted in the map for localities Click here for additional data file.Table S2List of haplotypes recovered through the mitochondrial control region (hypervariable I domain) for Oligoryzomys longicaudatus. In parenthesis the number of haplotypes. The localities (see (DOC)Click here for additional data file.