Sylvia atricapilla) populations with different migratory strategies across Europe. We compare the level of genetic divergence of populations migrating to southwestern (SW) or southeastern (SE) wintering areas with birds wintering in the British Isles following a recently established northwesterly (NW) migration route. The migratory divide between SW and SE wintering areas can be interpreted as a result of a re-colonization process after the last glaciation. Thus we predicted greater levels of genetic differentiation among the SW/SE populations. However, a lack of genetic differentiation was found between SW and SE populations, suggesting that interbreeding likely occurs among Blackcaps with different migratory orientations across a large area; therefore the SW/SE migratory divide can be seen as diffuse, broad band and is, at best, a weak isolating barrier. Conversely, weak, albeit significant genetic differentiation was evident between NW and SW migrants breeding sympatrically in southern Germany, suggesting a stronger isolating mechanism may be acting in this population. Populations located within/near the SW/SE contact zone were the least genetically divergent from NW migrants, confirming NW migrants likely originated from within the contact zone. Significant isolation-by-distance was found among eastern Blackcap populations (i.e. SE migrants), but not among western populations (i.e. NW and SW migrants), revealing different patterns of genetic divergence among Blackcap populations in Europe. We discuss possible explanations for the genetic structure of European Blackcaps and how gene flow influences the persistence of divergent migratory behaviors.Migratory divides are thought to facilitate behavioral, ecological, and genetic divergence among populations with different migratory routes. However, it is currently contentious how much genetic divergence is needed to maintain distinct migratory behavior across migratory divides. Here we investigate patterns of neutral genetic differentiation among Blackcap ( Patterns of geographic isolation during the Pleistocene in temperate areas have allowed behavioral, ecological, and genetic divergence among geographically isolated populations in a range of taxa ,2. HowevMigratory divides represent the locations at which populations maintain divergent migratory routes, often originating during periods of allopatry, but can also evolve into hybrid zones as a result of secondary contact and interbreeding between populations . If isolSylvia atricapilla) is a common European passerine and serves as a model species to study the evolution of migratory behavior because it spans the full spectrum of migratory distances (from sedentary to long-distance migrations) and migratory orientations. Several migratory traits have been shown to be under genetic control in Blackcaps, including timing of migration, direction, and duration/length of migration , and 10,000 randomizations were implemented. Additional Mantel tests were run for only those populations west of the SW/SE migratory divide and those populations east of the divide to investigate differential effects of isolation-by-distance on either side of the SW/SE divide.Isolation-by-distance among Blackcap populations was explored with Mantel tests using Isolation by Distance Web Service v3.23  to invesrho values determined for genetic distance vs. geographic distance were then compared to confidence intervals constructed from random distributions to determine significance of true values. Statistical analyses were conducted with R statistical computing environment [The relationship between genetic distance and longitudinal geographic distance was investigated between Blackcap populations located on opposing sides of the SW/SE migratory divide, which was approximated at 13°E ; we acknironment .K) was assessed assuming admixture and locprior of the individual populations and five iterations were run per K . Genetic structure was further investigated from genotype data using a MCMC model-based clustering method implemented with STRUCTURE software v2.3.4 . The mosδ2H was excluded from these analyses.Populations were grouped by autumn migratory orientation  and migratory distance  inferred from ring-recoveries (Q < 0.01), although patterns were not consistent across populations (Q < 0.05: Syl2/Syl6), but found to be significant only in the Kefermarkt population when populations were analyzed separately. Overall we thus can assume independence between loci used in this study. Allele frequencies at each population per locus are listed in Observed multilocus population heterozygosities were high and ranged from 0.60 - 0.73, with a mean observed heterozygosity of 0.69 and mean number of effective alleles of 4.26 . Only 18ulations . The SylK=1 (Dest = 0.029 (FST = 0.007). NW migrants breeding in Freiburg were also found to be significantly differentiated from allopatric populations in Uebersyren, Kalimok, and Sevilla, while SW migrants in Freiburg were undifferentiated from all allopatric populations. Freiburg NW migrants were most genetically similar to populations within/near the contact zone at the SW/SE migratory divide , although the relationship no longer occurred when the non-breeding, short-distance migrant population in Sevilla is excluded . Among the five eastern populations (i.e. those east of the SW/SE divide), isolation-by-distance was found significant . Conversely, isolation-by-distance was non-significant among the seven western populations , even when excluding the non-breeding Sevilla population . Mantel tests revealed significant isolation-by-distance among all 12 Blackcap populations (P = 0.005). The least genetic distance was found between populations near the center of the SW/SE divide (approximated at 13°E) and genetic distance increased significantly with increased geographic distance between populations. For example, all pairwise Dest comparisons between Münsingen (Mn), Kefermarkt (Kf), and Vienna (Vn) were found to be non-significant and for the majority of these estimates (5/6 comparisons) Dest = 0, indicating a lack of genetic differentiation between these populations near the center of the zone , affirming the observed relationship between genetic and geographic distance is not a random effect. A positive relationship between pairwise genetic distance vs. geographic distance was also apparent after excluding the non-breeding Sevilla population . By exploring the relationship between genetic distance and the longitudinal geographic distance of population pairs located on opposing sides of the SW/SE divide, a strong, positive correlation was evident . No evidence of hierarchical structuring of genetic variation was found by grouping the populations by three migratory directions and three migratory distances . Such results indicate a lack of neutral genetic divergence between populations of SW and SE migrants and, in fact, many populations within/near the approximated SW/SE contact zone cannot be differentiated with our marker set. We suggest therefore that the SW/SE migratory divide is an ineffective barrier to neutral gene flow across this zone of contact. Overall, genetic differentiation across all the sampled populations was low and Bayesian clustering, pairwise comparisons and hierarchical analyses all failed to show consistent, significant genetic structure among populations corresponding to different migratory behaviors. A positive relationship was found between genetic distance and  geographic distance of population pairs of Blackcaps located on opposing sides of the SW/SE migratory divide. This result is consistent with the historical isolation of western and eastern breeding populations in Europe previously restricted to potential glacial refugia on the Iberian and Balkan peninsulas, respectively ,7,54. DeDest comparisons were found to significantly differ from SW vs. SE comparisons, confirming beliefs that different dynamics of genetic divergence occur with the NW/SW migratory polymorphism and across the SW/SE migratory divide. In southern Germany (i.e. Freiburg), weak, albeit significant, genetic differentiation was found between NW and SW migrants breeding sympatrically, corroborating results of a previous study by Rolshausen et al. [Although isolation-by-distance appears to be maintained among eastern populations, this pattern is not found among western populations and is potentially confounded by the occurrence of sympatrically-breeding populations of NW and SW migrants . Indeed, NW vs. SW n et al. , who sugAcrocephalus scirpaceus) in central Europe [Phylloscopus trochilus) in Scandinavia [Quelea quelea) migratory divide in Africa [Setophaga caerulescens) with different migratory routes in North America [Locustella luscinioides) in Iberia despite Pleistocene divergence and long periods of geographic isolation between populations [The low nuclear genetic differentiation found across the SW/SE migratory divide in the Blackcap is consistent with similar patterns found across migratory divides in Eurasian Reed Warblers . This would support the hypothesized origination of NW migrants from within the SW/SE contact zone, first proposed by Helbig [δ2H assignment method did not indicate a presence of NW migrants in the Kefermarkt population, this result may indicate that the location of the Blackcap migratory divide has shifted eastward; the low genetic divergence found between FrNW vs. Vn supports this hypothesis, potentially being driven by the expansion of SW migrants into eastern European populations, as proposed previously by Wesołowski [Potentially, NW migrant Blackcaps may also contribute to breeding populations in other areas in central Europe  located on opposing sides of their migratory divide in central Europe [Isolation-by-distance (IBD) was found to be non-significant among the 11 breeding populations (after excluding Sevilla). However, patterns of IBD differed on either side of the SW/SE migratory divide: significant IBD was evident among eastern populations (with greater levels pairwise genetic divergence), but not among western populations (with lower levels of pairwise genetic divergence); this result is consistent with the genetic structure of Blackcap mtDNA by Pérez-Tris et al. , who foul Europe . Such coper se, but a gradual transition between migratory orientations across central Europe. Our results indicate an absence of NW migrants in the Białowieża population , but cannot exclude the occurrence of SW migrants occurring at this location. In fact, low levels of genetic divergence are found between Białowieża Blackcaps and several SW migrant populations  as well as SE migrant populations , suggesting Białowieża birds are genetically similar to many other European population due to introgression. Additionally, the distribution of claw δ2H from Białowieża birds is roughly consistent with the δ2H distribution of Freiburg SW migrants . Our results, together with those of Wesołowski [Our results provide little evidence that the historical migratory divide in central Europe provides sufficient reproductive isolation to influence mating preferences based on migratory orientation in the Blackcap. Rather, it appears that interbreeding between SW and SE migrants  results in high levels of gene flow across the migratory divide where populations come into contact. Asymmetrical gene flow across the SW/SE migratory divide (i.e. SW migrant Blackcaps outcompeting SE migrants) has been argued to be responsible for recent demographic changes at Polish breeding grounds ; interbrsołowski , may prosołowski ,60. WhilCatharus ustulatus) where the existence of a migratory divide in North America has been found [Recent studies are now utilizing geolocators to record the annual cycle of long-distance passerine migrants, including migratory routes, durations, and strategies ,62, as fen found . At thisen found ,19. Simien found , but wilen found . Still, en found . Neverthen found .Rapid genetic divergence between sympatric NW and SW migrant Blackcaps may be promoted by pre-mating isolation. Temporal isolation in spring arrival to breeding grounds in southern Germany has been documented between NW and SW migrants ,18,30 anNW and SW migrants (i.e. Sv and Ub) may indicate that such divergence is not locally adaptive, but that this polymorphism has evolved repeatedly, independent of adaptive pressures on the breeding ground. The behavioral, morphological, and genetic traits observed to be diverging in the Blackcap may be part of a ‘migratory syndrome’ or ‘migratory gene package’ in this species whereby a suite of correlated traits that evolve together are targeted by correlational selection [We not only find evidence of significant genetic differentiation between NW and SW migrant Blackcaps in sympatry, but NW migrants were significantly differentiated from other populations of SW migrants in western Europe. The observed divergence between NW and SW migrants may be the consequence of a founder effect resulting from a small number of genetically distinct individuals initially establishing the NW migratory route. Allopatric genetic differentiation demonstrated between Frelection –69. Whetelection , but proAlthough our Bayesian clustering and hierarchical analyses failed to find significant genetic structure among populations corresponding to the different migratory behaviors investigated, significant (but weak) genetic structure in mtDNA was found among eastern (including SE migrants) and western Blackcap populations (including SW and NW migrants) in a previous study . MaternaFigure S1Stable isotope assignments. Claw δ2H was used to categorize Spring 2011 Blackcaps caught at four locales  as inhabiting northern wintering grounds on the British Isles (i.e. NW migrants) or southern wintering grounds on Iberian or Balkan Peninsulas (i.e. SW or SE migrants) based on reference claw δ2H from British Isles and Spain, respectively (see Methods). (A) Isotope assignment results per population. Uebersyren, LU (Ub): NW = 1, SW = 42; Freiburg, DE (Fr): NW = 23, SW = 176, Unassigned (?) = 10; Kefermarkt, AT (Kf): South = 105; Białowieża, PL (Bw): South = 61. (B) Distribution of raw claw δ2H isotopes from each population, Ub = 61; Fr = 209; Kf = 105; Bw = 61. Claw δ2H from Ub, Kf, and Bw was found to significantly differ from Frδ2H distribution (Wilcoxon Rank Sum Test P < 0.05), while only Kf and Bw were found to significantly differ among remaining comparisons. (C) δ2H Distribution of N = 23 NW migrants and N = 82 SW migrants selected from Freiburg, Fr population for genetic analyses Width of bars are proportional to sample size.(TIF)Click here for additional data file.Table S1Characteristics of 14 microsatellite markers. Accession No.: GenBank accession numbers; N alleles: number of alleles per marker; Ho / He: Observed / Expected Heterozygosities; HWE by pop: populations that were found to deviate significantly for HWE; Null Allele Freq: mean frequency of null alleles per marker.(DOCX)Click here for additional data file.Table S2Allele frequency comparison over populations.(XLS)Click here for additional data file.Table S3Summary of STRUCTURE results.#K: number of clusters; K)Mean LnPStdev LnP(: standard deviation of mean posterior probability of given K.(DOCX)Click here for additional data file.Table S4F-statistics of hierarchical structuring of genetic variance. Relationship between hierarchy levels are indicated in rows with ‘/’ indicating ‘within’; Hierarchy A: 3 migratory directions  and 3 migratory distances  with the Os population characterized having SW migratory direction (dir); Hierarchy B: 3 migratory directions  and 3 migratory distances  with the Os population characterized having SE migratory direction (dir).(DOCX)Click here for additional data file.


Ranodon sibiricus), distributed in geographically isolated areas of Central Asia, is an ideal alpine species for studies of conservation and phylogeography. However, there are few data regarding the genetic diversity in R. sibiricus populations.The Siberian salamander  method to study the demographic history of the populations. This analysis suggested that the extant populations diverged from the ancestral population approximately 120 years ago and that the historical population size was much larger than the present population size; i.e., R. sibiricus has experienced dramatic population declines.We used two genetic markers (mtDNA and microsatellites) to survey all six populations of R. sibiricus populations is the lowest among all investigated amphibians. We conclude that the isolation of R. sibiricus populations occurred recently and was a result of recent human activity and/or climatic changes. The Pleistocene glaciation oscillations may have facilitated intraspecies genetic homogeneity rather than enhanced divergence. A low genomic evolutionary rate and elevated inbreeding frequency may have also contributed to the low genetic variation observed in this species. Our findings indicate the urgency of implementing a protection plan for this endangered species.Our findings suggest that the genetic diversity in the  Ne) based on extant samples. And it should be stressed that molecular markers with different rates of substitution can capture signatures of evolutionary processes at different timescales and can therefore greatly influence the interpretations of historical demographic patterns Loss of genetic diversity has been considered a crucial genetic factor that tends to produce inbreeding depression, reduced adaptation and fitness and a decrease in the long-term species survival Ranodon sibiricus; Amphibia: Caudata) is a montane species that is well suited for such studies. This species is endemic to two mountains of Central Asia (the Junggarian Alatau and the Northern Tian Shan) and is restricted to geographically small areas in Wenquan County, northwestern Xinjiang of China and southern Kazakhstan. Only six small and isolated populations have been detected in China 's red list of threatened species for Kazakhstan and as “Critically Endangered” on the red list of threatened species for China (2005). Although various aspects of this species' ecology, morphology, physiology, cytogenetics and phylogenetic position have been well documented, there are few data on the genetic variation within and among populations in China The Siberian salamander  at altitudes of approximately 2100–3200 m above sea level, where the habitat is at almost the extreme limit of tolerance for cold-blooded animals. To adapt to its unique environment, R. sibiricus has developed some behavioral and ecological traits that have presumably been caused by selection pressures. For example, its egg sacs favor cold stream water; its larvae develop slowly; and its hibernation time is as long as half a year R. sibiricus in China were located at different altitudes and isolated far from each other. An interesting question is whether these six populations survived the Quaternary ice ages in situ or responded to climatic oscillations in a similar manner to other alpine species.Many recent studies have focused on the influence of topography and Pleistocene climatic changes on regional species R. sibiricus in China. We then attempted to determine whether those patterns are due to recent environmental impacts or due to much older historical events , using statistical evolutionary hypotheses under the approximate Bayesian computation (ABC) method. The ABC method has exhibited powerful potential in phylogeography In this study, we utilized two types of markers, mitochondrial DNA (D-loop region) and microsatellites, to characterize the patterns of genetic diversity in all the populations of The tissue samples were collected under the permission of the administrative institution of the Forest Department of Xinjiang Province, which is responsible for the regulation of local animals in Xinjiang Province. Our project was also approved by the National Natural Science Foundation of China (No. 30771151) and supported by Sun Yat-sen University.R. sibiricus in China, from May to August 2006  and found that the D-loop region was the most variable region. We therefore chose a segment of mtDNA that encompassed the D-loop region to investigate the genetic variability within and among populations of R. sibiricus. A 1323 bp segment of the mitochondrial genome was selected that spans the tRNA-Thr, tRNA-Pro, tRNA-Phe, D-loop region and partial Cytb and 12S rRNA sequences and has been found to show considerable variation b A We aligned the entire mtDNA genome of seven genera of the family Hynobiidae (13 and (GATA)8, were used to construct the libraries. The colonies were screened for inserts using a technique similar to the PIMA method 10 or (GATA)5 as primers. The clones produced one clear band in agarose electrophoresis that was likely to contain microsatellite repeats. The positive clones with distinct amplification bands were obtained and sequenced. The sequences were proofread by eye with the aid of the Tandem Repeats Finder software Microsatellites are an ideal biparentally inherited nuclear marker with high resolution for estimating male-mediated gene flow and population structure DFsD or Fu's Fs indicate that the sequences deviate from neutrality or that populations were previously subdivided and/or have experienced past fluctuations . The genetic differentiation and gene flow between populations were also assessed, by calculating pairwise FST values using the same software. Contrasting plots of observed vs. theoretical distributions of site differences (mismatch) gave insight into past population demographics www.fluxus-engineering.com). The TCS program, version 1.21 HO), expected heterozygosity (HE) and F-statistics were performed using the POPGENE 32 program, version 1.32 The sequences obtained from the two markers were edited and aligned by eye using the DNAStar 5.0 program. For comparisons of within-region genetic diversity, DnaSP4.0 was used to estimate population haplotype diversity, mean number of pairwise differences and nucleotide diversity. Neutrality tests of Tajima's R. sibiricus, we applied the approximate Bayesian computation (ABC) method using the DIYABC program (version 1.0.4.39) R. sibiricus populations was caused by recent habitat deterioration and anthropogenic activities; (2) R. sibiricus populations have been separated for a long time; individual populations merged during the Pleistocene glaciations and became separated again following the last glaciation; (3) R. sibiricus populations became separated before the Pleistocene glaciation oscillations, and the glaciations caused a bottleneck effect on individual populations rather than facilitating the mixing of populations.To reconstruct the evolutionary history among these six populations of Ne value . According to the recently observed population sizes  than after the bottleneck occurred . The six populations were considered to have diverged from an ancestral population at a much earlier time (t3), which was set between 10000 to 50000 generations. The NA values of all three scenarios were considered to be identical, between 10 to 10000, based on the assumption that historical population sizes were much larger than present population sizes. This is according to the fact that the population sizes observed at the time the populations were first discovered were larger than today . For the three scenarios, we ordered the times as t1<t2<t3. The other parameters are listed in R. sibiricus was caused by recent human and environmental factors (Scenario 1) or by historical climatic oscillations (Scenario 2 and 3). If the latter was the case, we wished to test whether the effect of glaciations on population dynamics is to increase the gene flow among populations (Scenario 2) or to reduce the size of separated populations (Scenario 3).In view of the limited dispersal ability and isolated geographic localities of the six populations, each population was treated as having evolved independently. In all of our scenarios, the six populations  had potentially different contemporary on sizes , the priOver 3000 clones of the CA and GATA libraries were screened in a search for microsatellites, and 130 positive clones having one distinct amplification band were obtained and sequenced. Of these 130 sequenced clones, 70 contained unique repeat sequences, of which the numbers of mono-, di-, tri-, tetra-, and pentanucleotide repeat sequences were 7, 50, 7, 5 and 1, respectively. We designed 66 primer sets from the flanking regions of 59 loci , of which 50 contained expected-size bands and repeatable banding patterns and 9 were not amplified consistently. Initially, these 50 loci were tested for polymorphism with a small sample pool. Only 2 loci, Rsi-5 and Rsi-17, showed microsatellite polymorphism. To confirm this result, of the 48 loci without polymorphism, 15 loci with more than 10 dinucleotide repeats and 5 loci with more than 5 tetranucleotide repeats  to 0.00091 (SLBZ)  . There w1 (SLBZ) .HO and HE values of these loci ranged from 0.080 to 0.090 and from 0.077 to 0.086, respectively  are less common than dinucleotides For the two polymorphic loci of the microsatellites, the number of alleles was two per locus. The ectively . One allD and Fu's Fs test for most of the populations, indicating that the populations had experienced past fluctuations and were not in migration-drift equilibrium. Only AKS and SAR, which contained mainly the haplotype Hap-1, showed low negative values of Tajima's D without obvious statistical significance  for the six populations were 278, 54, 60, 108, 68 and 64, respectively. The NA value of the common ancestral population is roughly two orders of magnitude higher than that of the extant populations (NA = 2640), suggesting the occurrence of a dramatic historical decline in the number of individuals.Neutrality tests and mismatch analysis are not effective tools for revealing the historical demography of populations R. sibiricus was deeply differentiated as a result of the fragmented habitat, mountain barriers to gene flow, small population sizes and low dispersal capacity R. sibiricus populations.We expected to find that the population structure of R. sibiricus by Zhang & Wu R. sibiricus showed the lowest genetic diversity for these two markers.This finding was consistent with a preliminary survey of R. sibiricus appeared to be inconsistent with the morphological differences observed in some individuals; this inconsistency has also been observed in other amphibians R. sibiricus, it seems reasonable that the extra digit is a result of inbreeding, rather than a local adaptation to the surrounding environment . Although some species display a low level of genetic diversity among populations, they display a strong phylogeographic structure R. sibiricus in China. Future studies should include genetic data from the populations in Kazakhstan to determine whether those populations exhibit greater differentiation (relative to each other or to the China populations) or low genetic diversity similar to that of the China populations.The uniformity of genetic markers for Extremely low genetic diversity accompanied by shallow population differentiation was observed in this study. These findings can be attributed to several factors, including: (a) special population history, (b) a slow evolutionary rate at the genomic level and (c) a high frequency of inbreeding resulting from a small population size.R. sibiricus, the glaciations may have had the opposite effect of enhancing the interactions among populations. This hypothesis is supported by the fact that R. sibiricus has evolved several traits to adapt to high-elevation habitats: (i) the egg sacs favor cold water in the headwaters of small mountain streams, brooks and lakes; (ii) the larvae develop slowly and feed mainly on stream invertebrates; and (iii) the hibernation time is as long as half a year R. sibiricus populations to be scattered at altitudes approximately 2100–3200 m above sea level on different mountains, thus isolating the populations from one another. When glaciation occurred, the montane species expanded and contracted along an elevation gradient in response to the descending snow limit R. sibiricus to reach the feet of mountains, and the gene flow among previously isolated populations could be unblocked.The Pleistocene glaciation cycles are considered to have been critical in shaping the distributions and genetic attributes of species R. sibiricus therefore had to move down ∼1000 m to the feet of mountains along streams, which facilitated the interactions between populations. The multiple glaciations may also have caused frequent gene flow among the populations.At least four main Quaternary glaciations occurred in the Tian Shan R. sibiricus, as also evidenced by the shallow genetic structure among the populations. In contrast, intraspecific gene flow may have been facilitated during this period, as assumed in Scenario 2. A possible reason for the statistical rejection of Scenario 2 is our assumption that the separation of the R. sibiricus populations occurred shortly after the Last Glacial Maximum. Scenario 1, which assumed that the divergence happened within 1000 generations, was strongly supported by the ABC method, suggesting that the divergence occurred only 120 years ago, when the populations were under the strong influence of human disturbances and/or recent climatic changes . In this scenario, we proposed that the historical distribution of R. sibiricus was continuous and that the species may have consisted of a single large population until recently . This scenario would imply that the distribution range of R. sibiricus was much wider at that time than at present. Recent human activities and climatic changes may have caused habitat fragmentation as well as population size decline. Recent grazing has been reported to cause great harm to R. sibiricus populations during the time of reproduction. In view of its limited motility, R. sibiricus is extremely vulnerable to the damage caused by herds of cattle; e.g., egg sacs in the streams are likely to be destroyed by the cattle. Recent studies have shown that global warming may have driven the extinction of amphibians by facilitating epidemic diseases R. sibiricus was caused by a particular pathogen, but this species is certainly vulnerable to pathogenic infection. In view of the cold-resistant adaptations of R. sibiricus, climatic warming may have cause shifts of the populations toward higher-altitude habitats, which could aggravate the fragmentation of populations.The ABC method applied in this study gave no statistical support to Scenario 3, which assumed that the six populations diverged independently from a common ancestral population before the Pleistocene period and underwent a bottleneck process during the glaciations. This finding suggests that a glaciation effect leading to greater intraspecies divergence did not occur in R. sibiricus. There is no direct evidence to support this hypothesis because we could not find ancient samples to infer the genomic evolutionary rate of this species. However, a study by Allen et al. indicated that individual metabolic rate is a primary determinant of evolutionary rates: 1013 J of energy flux per gram tissue generates one substitution per nucleotide in the nuclear genome R. sibiricus is distributed at altitudes approximately 2100–3200 m above sea level, where the habitat is at almost the extreme limit of tolerance for cold-blooded animals, and the metabolism level of this species is low. And some studies suggest that R. sibiricus is a relatively primitive species among the Hynobiidae −8 to 10−7 per site per generation), as also used in some studies of other Hynobiidae species R. sibiricus mtDNA to a lower order of magnitude, i.e., 10−9 to 10−8 per site per generation, the estimated t1 turns out to be greater than 100 generations; however, this estimation does not bias our main conclusion.Genomic evolutionary rates have been reported to vary depending on the species. For instance, mtDNA exhibits a low evolutionary rate in plants and some turtles R. sibiricus have declined dramatically during the past three decades, and this sharp decline has clearly had some impact on genetic diversity and population structure, e.g., in the AKS and JMK populations. Under the ABC method, the Ne of the common ancestral population was roughly two orders of magnitude higher than that of the modern populations, suggesting a relatively large historical population size of this species. The reduction in population size has increased the possibility of inbreeding, as exemplified by the AKS individuals with six toes on the hind limbs. In summary, our results suggest that the R. sibiricus populations separated fairly recently (∼120 YBP) as a result of human activities and/or climatic change and that Pleistocene glaciation oscillation may have facilitated intraspecies gene flow rather than enhanced divergence. A low genomic evolutionary rate and elevated inbreeding frequency may also have contributed to the low genetic diversity observed in this species.Field populations of R. sibiricus was much larger than the present population, and the causes of extreme genetic uniformity may be a combination of recent and historical factors, including the species' unique population history, slow evolutionary rate at the genomic level and the recent dramatic population decline. The recent decline caused by climatic changes has obviously affected some small populations (such as AKS and JMK), and the trend that “population reduction is the cause of loss of genetic diversity” has become increasingly evident. We should also pay attention to the fact that the geographic range and the population size of R. sibiricus have decreased dramatically during the past decade. Because of the high level of genetic homogeneity, R. sibiricus populations are prone to reduction of fitness when the environment changes dramatically. The species' adaptations to cold water also make R. sibiricus vulnerable to unfavorable environments, particularly the desiccation of wetlands. The rapid extinction of many amphibian species has received great attention in recent years and is attributed mainly to the deterioration of the global climate R. sibiricus suggests that programs to conserve living amphibian species should be accelerated.Although loss of genetic diversity is recognized as being directly related to population reduction Two goals of any conservation program should be to ensure the survival of a species and to maintain its genetic diversity for long-term evolutionary success Text S1Approximate Bayesian computation analysis.(DOC)Click here for additional data file.Text S2The references of species listed in  were listed as follows.(DOC)Click here for additional data file.Table S1Primers used in the study of mtDNA and microsatellites.(DOC)Click here for additional data file.Table S2The prior distributions of parameters used for description of the three scenarios analyzed via the ABC method.(DOC)Click here for additional data file.Table S3Characterization of two polymorphic loci and twenty microsatellite loci with good repeats but no polymorphism in Ranodon sibiricus.(DOC)Click here for additional data file.Table S4Bias and precision of parameter estimation under Scenario 1.(DOC)Click here for additional data file.


Thuja occidentalis) as a model species for declining to endangered long-lived tree species with mixed-mating system.Fine-scale or spatial genetic structure (SGS) is one of the key genetic characteristics of plant populations. Several evolutionary and ecological processes and population characteristics influence the level of SGS within plant populations. Higher fine-scale genetic structure may be expected in peripheral than core populations of long-lived forest trees, owing to the differences in the magnitude of operating evolutionary and ecological forces such as gene flow, genetic drift, effective population size and founder effects. We addressed this question using eastern white cedar  than in core  populations. However, the mean neighborhood size was higher in the core (Nb = 82) than in the peripheral (Nb = 48) populations.We determined the SGS in two core and two peripheral populations of eastern white cedar from its Maritime Canadian eastern range using six nuclear microsatellite DNA markers. Significant SGS ranging from 15 m to 75 m distance classes was observed in the four studied populations. An analysis of combined four populations revealed significant positive SGS up to the 45 m distance class. The mean positive significant SGS observed in the peripheral populations was up to six times (up to 90 m) of that observed in the core populations (15 m). Spatial autocorrelation coefficients and correlograms of single and sub-sets of populations were statistically significant. The extent of within-population SGS was significantly negatively correlated with all genetic diversity parameters. Significant heterogeneity of within-population SGS was observed for 0-15 m and 61-90 m between core and peripheral populations. Average Eastern white cedar populations have significant fine-scale genetic structure at short distances. Peripheral populations have several-folds higher within-population fine-scale genetic structure than core populations. Anthropogenic disturbances and population fragmentation presumably have significant effects on fine-scale genetic structure in eastern white cedar. Core populations have higher neighborhood size than peripheral populations, whereas gene flow distances are higher in peripheral than in core populations. The results of our study contribute to the knowledge of poorly-understood spatial genetic structure of core versus peripheral populations in plants. As well, the information is of significance for conservation of genetic resources of eastern white cedar and perhaps of other long-lived forest trees with mixed-mating system. Picea sitchensis). The level of SGS within plant populations depends on seed and pollen dispersal, mating system, mode of reproduction, size of the founder population, species longevity, adult population density, effective population size, and micro environmental selection [Recently, in addition to core, peripheral populations are also gaining importance for gene conservation, because under global climate change conditions they may possess genotypes of future adaptive potential. Plant populations tend to migrate further from their range periphery to ensure their survival by developing adaptability in a changing environmental condition due to global warming . Geneticelection -13. Anthelection ,15. Infoelection ,17. FurteN), and disturbance and selection regimes. Restricted gene flow due to limited seed and pollen dispersal can promote mating among relatives which can result in isolation by distance within a population [e N[e Nand decreased genetic drift over time [Higher SGS may be expected in peripheral than in core populations of long-lived plants, such as forest trees, owing to the differences in founding population, seed and pollen dispersal, adult plant density, inbreeding levels, effective population size  outcrossing mating system. Studies in conifers reported either no or weak levels of SGS within populations [Pinus or Picea species. Furthermore, with the exception of two [Conifers are generally long-lived plants that are ecologically and economically important globally. They constitute a major component of the northern boreal and temperate forests, especially dominating the boreal forest which comprises one of the largest biomes in the world ulations -32. Thisulations ,32, all n of two ,28, all Thuja occidentalis L.) provides an ideal model species to study SGS in core versus peripheral populations of declining and long-lived plant species with mixed mating system. Eastern white cedar is a wind-pollinated, monoecious, evergreen tree species belonging to the Cypress family (Cupressaceae). It reproduces both sexually and vegetatively. Vegetative reproduction occurs mainly in swampy sites by layering [Eastern (or northern) white cedar , easentalis, -41. It ientalis, . Althoug, [Thuja -41,43, tjaponica , which reN, and higher anthropogenic fragmentation as compared to the core populations [The aim of the present study was to determine within-population fine-scale genetic structure of eastern white cedar growing in core and peripheral populations. Our hypotheses were: (i) since eastern white cedar is a mixed-mating long-lived tree with high levels of selfing rate, and has capability to reproduce vegetatively, this species is expected to have some levels of SGS within population; (ii) the peripheral populations of eastern white cedar are expected to have higher levels of SGS than the core populations due to their lower population density; potentially restricted gene dispersal, smaller ulations -13.Picea mariana) and balsam fir .Four natural populations of eastern white cedar were sampled from two Maritime Provinces of Canada. Two of these were core (Edmundston and Perth Andover) populations from New Brunswick and other two were peripheral (Cedar Lake and Rockland) populations from Nova Scotia Figure ; Table 1A plot consisting of ~ 100 contiguous eastern white cedar trees was established in each of the four sampled populations irrespective of the plot area Figure . All of Small amounts of foliage samples were collected from each of the 394 sampled trees in plastic Ziplock bags containing silica gel. Upon arrival to the lab, the foliage samples were stored at -20°C until processed for DNA extraction.Thuja plicata [Chameacyparis obtusa [2, 0.20 mM of each dNTP, 0.50 pmol of each primer and 0.25 U Taq DNA polymerase . The epGradients Master Cycles  were used for PCR amplification of the sample DNA, with the following thermocycling profile: initial denaturation step at 95°C for 5 min, followed by 33 cycles each of 95°C for 45 sec, annealing temperature at 55 to 65°C for 45 sec, 72°C for 45 sec and final extension step of 72°C for 5 min. PCR products were separated on LI-COR4300 genetic analyzers . The genotypes of individual trees were first determined by scoring the LI-COR gels with the SAGA GT program  and later verified by manual scoring.Total genomic DNA was extracted from the needle samples of individual trees using the DNeasy Plant Mini Kit . Microsatellite DNA markers of the nuclear genome were used for genotyping the sampled trees. A total of 40 microsatellite markers developed for the Cupressaceae species were tested in eastern white cedar. Of these, 37 were developed for  plicata ,45, [L.Ms obtusa . Only siF) were calculated using the program GENALEX [R) was estimated using the program FSTAT [O and GAE) as the sum of genotypes observed and expected under Hardy-Weinberg equilibrium over the loci [The standard population genetic diversity parameters  and fixation index ( GENALEX . Allelicam FSTAT . Genotypthe loci . Deviatithe loci .eN) for each of the four populations was estimated using the maximum likelihood method of coalescent-theory and MIGRATE program [-3 per generation. The e Nwas calculated as θ/(4×10.-3).The effective population size . The weighted edge correction [Pattern of spatial distribution of individual trees in the four eastern white cedar populations were estimated using univariate second-order Ripley's K-functiorrection  method wrrection .K at this distance class. Similar approach was used by Oddou-Muratorio et al. [Sorbus torminalis.We used Smouse and Peakall's  spatial o et al.  to selec2t) and multi-class (ω) test criteria were used to test the heterogeneity of spatial autocorrelation (r) values between single and subsets of populations. Heterogeneity in SGS for single distance classes between any two populations or a subset of populations was determined by performing the squared paired- sample t test (t2) [In addition to the estimation of multilocus SGS parameters for each of the four populations separately, SGS analyses were also performed for combined subset each of core , and peripheral (CL and RL) populations, as well by combining all four populations to infer degrees of heterogeneity of SGS within and between subsets based on the method of Smouse et al.  using GEest (t2) . SGS hetest (t2)  under thr) with e N, genetic diversity parameters and density of eastern white cedar trees within the sampled populations were determined by calculating the Pearson correlation coefficients (R), using the MINITAB 15 package.The correlations of the SGS parameters , where -^bF is the regression slope of the autocorrelogram and ^F1 is the mean kinship coefficient (Fij) [Sp statistics, Wright's neighborhood size Nb (Nb = 1/Sp) was also estimated. Nb is defined as Nb = 4πeDσg2, where σg2 is the axial variance of gene dispersal and e Dthe effective population density. Estimates of gene dispersal distance (σg) were determined assuming ratios of effective to census density, De/D, of 0.1 and 0.5 [^bF and ^F1 were carried out using SPAGEDI program [P ≤ 0.05) of the analyses was performed by permutation tests with 10,000 replications.In order to quantify SGS and to compute neighborhood sizes and gene dispersal distances in the four eastern white cedar populations, nt (Fij)  between nt (Fij) . Using t and 0.5 ,21. Esti program . Statiste Nwas 432 and 910, with an average of 671 in the core populations, and 110 and 310, with an average of 210 in the peripheral populations  are presented in Figure Results of the multilocus spatial genetic structure (SGS) analysis based on Smouse & Peakall's  spatial r) were observed for the first (0-15 m) distance class for individual or combined populations  to 0.114 (RL population)  was considerably higher than in the intact populations (r = 0.058).The maximum values of spatial autocorrelation coefficient  Table . SignifiSp statistics and dispersal estimates in the four eastern white cedar populations are presented in Table Sp values were 0.009 (PA) and 0.018 (ED) in the core populations, and 0.016 (CL) and 0.031 (RL) in the peripheral populations. Average Sp value was higher in the peripheral (0.023) than in the core (0.014) populations Average value for Wright's neighborhood size (Nb) was higher in the core populations (81.57) as compared to the peripheral populations (48.04). Gene flow distances (σg) were 44 and 120 m in the core populations and 46 and 167 m in the peripheral populations (Table Results of tions 81.7 as compr) with the population genetic diversity measures. The correlations (Pearson correlation coefficient R) of r with genetic diversity measures were as follows: mean number of alleles per locus (A) = -0.85 P = 0.034), observed heterozygosity (HO) = - 0.89 (P = 0.022), expected heterozygosity (HE) = -0.82 (P = 0.044), observed genotypic richness (GAO) = -0.88 (P = 0.024) and expected genotypic richness (GAE) = 08 P = 0.040). The correlation of eastern white cedar stand density with r a -0.69 P = 0.096). The significant SGS r values showed negative high correlation (Pearson R = -0.65)wt Ne, although the correlation was not statistically significant (P = 0.139).There was negative correlation of the significant SGS . These results are discussed later in this section.Larix larcinia (up to 20 m) [Larix larcinia was explained primarily due to the effect of founding population. In contrast, predominantly-outcrossing species showed either weak or no within-population SGS e.g. [The extent of SGS for combined four eastern white cedar populations (up to 45 m) is more than two times of that observed in one 20-years old population of another mixed-mating (mean selfing rate of 27% ) speciesto 20 m) . The exiSGS e.g. ,22,28. TBesides higher levels of selfing or inbreeding, limited gene flow and founding gene pool may be responsible for the observed short-distance SGS in eastern white cedar. Restricted gene flow due to limited pollen and seed dispersal contributes to local grouping of individuals with similar alleles or genotypes . In eastEurya emarginata. They explained that the restriction in gene flow due to limited dispersal of seed and pollen could be the main reason for the observed SGS in the species.Vegetative propagation through natural layering has been reported in eastern white cedar ,64. SincK function indicated significant aggregation of trees in all four populations of eastern white cedar. Within population spatial patterns of individual plants can be influenced by several factors such as variation in microsites, level of disturbance, seed dispersal patterns [Pinus clausa and Pinus halepensis populations, respectively, and they explained that the observed aggregation could be due to either limited seed dispersal or recruitment of aggregated seedlings after stand disturbance due to fire. These factors may have contributed to the observed spatial pattern of individual trees in eastern white cedar populations. The effect of limited seed dispersal on the significant aggregation of individual trees may be evident from the significant SGS (15-75 m) observed in the four populations. Although we are not aware of any records of fire in the studied populations, past occurrence of fire on the studied populations cannot be ruled out because fire is one of the main agents of natural disturbance in boreal forest [The analysis of spatial distribution pattern using Ripley's patterns ,66. Parkpatterns  reportedl forest .P = 0.001). Both Nova Scotia populations (CL and RL) showed the highest within-population SGS, and the lowest genetic diversity, stand density and eN. These populations are highly fragmented and affected by human disturbance for over 400 years ever since the arrival of European settlers in the Province. The several-folds higher SGS levels observed in the two marginal and highly fragmented populations from Nova Scotia is likely due to reduced stand density, gene dispersal, genetic diversity and e Nobserved in these populations  was up to six times of that was observed in the core 15 m) populations. The difference in SGS between the core and peripheral populations was statistically significant  than the predominantly outcrossing species with relatively high genetic diversity, such as Sitka spruce. However, this aspect remains to be further tested in more details globally.The spatial distribution of genotypes was found to be random in the core populations of Sitka spruce, whereas significant positive SGS up to 500 m was observed in the peripheral populations of this species . In eastSp statistic was also higher in peripheral than in core populations of eastern white cedar. Vekemans and Hardy [Sp value in populations with low density as compared to the populations with high density. Since the density of trees in the peripheral populations was much lower than in the core ones, the effect of density on the results of Sp cannot be ruled out. Except in the population PA, Sp values observed in the other three populations of eastern white cedar were higher than the average value observed in five wind-dispersed temperate trees (Sp = 0.012) and 18 outcrossing plant species [Sp = 0.037) of seven mixed mating species [Sp values in fragmented than in continuous populations of another conifer species, Pinus pinaster, and they explained that the lower effective population size due to few reproductive individuals in fragmented populations may have resulted in restricted gene flow via seed and increased the level of SGS in the populations. This explanation may also be applicable to eastern white cedar because the peripheral populations were highly affected by anthropogenic fragmentation. The average gene flow distance in eastern white cedar was higher in peripheral populations than in the core ones. Higher gene flow distances would be expected in fragmented and peripheral populations than in core and intact populations because of lower tree density in peripheral populations resulting in lower obstruction for pollen movement [In agreement with the SGS results estimated using Smouse and Peakall's  autocorrnd Hardy  reported species . However species . De-Lucamovement ,69, whicmovement  also repNe and SGS comparisons in core versus peripheral populations suggest that the evolutionary potential of the studied peripheral eastern white cedar populations may have been adversely affected. The results of our study have significance and implications for conservation and management of genetic resources of eastern white cedar - a declining to endangered species in Atlantic Canada. For ex-situ conservation via seed and vegetative propagule collection, a minimum distance of 75 meters in peripheral and 20 m in core populations is advisable to capture maximum genetic diversity and avoid sampling genetically-related trees. The core populations in New Brunswick should be conserved in-situ. Both of the peripheral Nova Scotia populations have suffered genetic degradation as a result of extensive human disturbance and edge effect. Here, both ex-situ and in-situ genetic resource conservation efforts supplemented with genetic restoration will be required given that eastern white cedar is a red-listed species in Nova Scotia. The results of our study are relevant for within-population sampling of individuals for population genetic studies in this species. The results may also be applicable for conservation and management of genetic resources of other long-lived, declining species with similar mixed-mating characteristics.The results of genetic diversity, In conclusion, our results clearly demonstrate that the studied eastern white cedar populations have significant fine-scale genetic structure at short distances. Peripheral populations have several-folds higher within-population fine-scale genetic structure than core populations. Vegetative propagation does not apparently contribute to SGS in eastern white cedar. Anthropogenic disturbances and fragmentation presumably have significant effects on fine-scale genetic structure in eastern white cedar. The magnitude of evolutionary forces such as founder effect, genetic drift, gene flow and selection, operating in core versus peripheral populations is likely to be different. This may be the first report comparing SGS in intact core versus fragmented peripheral populations of a long-lived, mixed-mating declining plant. The results of our study contribute to the knowledge of poorly-understood spatial genetic structure of core versus peripheral populations in plants.T: Total number of alleles; A: Mean number of alleles per locus; AE: Effective number of alleles; AR: Allelic richness; AP: Number of private alleles; b-log (^bF): Slope of the regression of kinship with ln(dist); CL: Cedar Lake; D: Census density; De: Effective density; ED: Emundston; F: Fixation index; Fij: Mean kinship coefficient; GPS: Geographical positioning system; GAO: Observed genotype additivity (richness); GAE: Expected genotype additivity; HO: Observed heterozyogosity; HE: Expected heterozygosity; Nb: Wright's neighbourhood size; Ne: Effective population size; PA: Perth Andover; r: Spatial autocorrelation coefficients; RL: Rockland; Sp: Sp statistics; σg: Estimate of gene flow distance from Sp; SGS: Spatial genetic structureABoth authors have contributed significantly to the work reported in the manuscript. MP was a Postdoctoral Fellow, who generated 50% of the eastern white cedar genotype data, performed data analysis, and drafted the manuscript. OPR is the principal investigator of the project and contributed to the conception of the study and its experimental design, provided overall guidance and direction and funding, and revised the manuscript. Both authors have read and approved the final manuscript.


Oncorhynchus keta) populations demonstrate weak genetic structure, but invoking contemporary gene flow as the basis for the low divergence is problematic because salmon home to their natal streams and some of the populations are thousands of kilometers apart. We used genotypes from microsatellite and single nucleotide polymorphism loci to investigate alternative explanations for the current genetic structure of chum salmon populations from western Alaska. We also estimated current levels of gene flow among Kuskokwim River populations. Our results suggest that weak genetic structure is best explained by physical connections that occurred after the Holocene Thermal Maximum among the Yukon, Kuskokwim, and Nushagak drainages that allowed gene flow to occur among now distant populations.Low genetic divergence at neutral loci among populations is often the result of high levels of contemporary gene flow. Western Alaskan summer-run chum salmon ( The genetic structure of populations results from contemporary and historical processes. Typically, allele frequencies at many loci explain the divergence among sub-populations and a vicariant or a dispersal event associated with their geography is used to explain the reason for that divergence see Avis . The genOncorhynchus keta, Figure Oncorhynchus tshawytscha), sockeye (Oncorhynchus nerka), coho (Oncorhynchus kisutch), and steelhead (Oncorhynchus mykiss); Quinn 2  and yet they display genetic divergence that is 5–10 times lower than the rest of western Alaska  and markers . The second set included the largest number of populations within CSW (n = 21) genotyped at 50 SNP loci 21P50L. Populations were divided into eight regional groups: A – Kotzebue Sound, B – Norton Sound, C – Lower Yukon, D – Middle Yukon, E – Lower Kuskokwim, F – Middle Kuskokwim, G – Upper Kuskokwim, and H – Bristol Bay . G-tests can be summed across loci and different regional groups to identify significance at hierarchical levels. Simulations have shown that G-tests can have high power but high type I error . FSC and FCT were estimated with Arlequin 3.5  was performed on the allele frequency data from the 25P70L data; all allele frequencies were arcsine-square root transformed prior to the analysis in SYSTAT  is estimated from:The relationship between the genetic and water distances (De) and the mean-squared parent-offspring distance  of the Kuskokwim River. Although this is likely an underestimate, it provides an upwardly biased estimate of dispersal distance and is therefore a conservative over-estimate of the potential gene flow among populations. The annual Nc estimate also includes fall-run fish, but those populations are small relative to the summer-run populations and the estimate of Bue et al.  can be considered to be the efficiency of passing genes from one generation to the next. For salmon, the determination of Ne can be challenging because of fluctuating population sizes and overlapping generations  except for the populations from the Middle Kuskokwim River , which may reflect the weak genetic structure of the CSW populations. The tests with the 21P50L data set were similar  from the null distribution and indicated that those or linked loci did not conform to neutral expectations . In contrast, the test for IBD was significant (P < 0.03) when the geographic distances were calculated assuming an historic connection between those rivers and the slope of the line was approximately fivefold higher  when the connection between the Stony and Mulchatna rivers was assumed , but the slope for the test for IBD within the Kuskokwim was significant (P < 0.04). The IBD analysis within the Kuskokwim was similar for both data sets (results not shown). There were insufficient samples to test for IBD within the Nushagak River for either data set.The med Fig. . We alsoIf long distance straying among CSW populations is responsible for the weak genetic structure, then estimates of dispersal should be large. We used the slope and the mean density from the IBD analysis with data from the Kuskokwim River populations in the 25P70L data set  to estimate the straying distance for The IBD analyses support our hypothesis that the reduced genetic divergence among CSW chum salmon populations is a result of past physical connections among now divided drainages, not from contemporary migration among populations. Erosion of genetic divergence from straying could result either from high levels of long-distance migration or serial migration among neighboring populations, but the dispersal distances do not approach those necessary for long distance migrations; and they are likely over-estimates because we used underestimates of the total census size. Multiple serial migrations among neighboring populations are unlikely given that there is a signal for IBD within the Kuskokwim River, but not between the Yukon and Kuskokwim or between the Kuskokwim and Nushagak drainages and few populations exist between the Yukon and Kuskokwim river mouths to act as vectors for dispersal. A more detailed historical exploration of when and how these connections may have occurred could provide useful information for predicting the response of the present day populations to climate change or reveal subtle genetic structure that may not be evident if groupings are based on present-day geographical and fluvial information.Chum salmon populations between Japan and Kamchatka and between the Gulf of Alaska and the Pacific Northwest have genetic diversity estimates that are several-fold higher than for populations in the geographic area between Kotzebue Sound and Bristol Bay ; the populations were probably small and ephemeral. However, as sea levels rose, the shorelines retreated rapidly  there should have been time to establish equilibrium  and recent colonizations by populations of other species of salmon display much greater divergence. For example, the observed divergence estimates among Chinook salmon that were introduced into New Zealand rivers at the end of the 19th century (Kinnison et al. A more recent timing for extirpation, recolonization and/or geneflow is suggested by the fact that the divergence estimates of CSW populations are an order of magnitude lower than for chum salmon in western Alaska overall (θRegardless, the difficulties associated with applied genetic work to chum salmon populations from these dynamic coastal regions of western Alaska may be the result of very recent events coupled with a long-term history of environmental disruptions and drainage interconnections. This conclusion combined with the fact that large numbers of neutral microsatellite, allozyme, and SNP markers have been unable to demonstrate more divergence than we have shown here suggests that the addition of more neutral markers may be inadequate to demonstrate significant divergence among CSW populations, which are necessary for applied genetics work. As more data sets become available, they can be merged to further test the hypotheses here. Markers responsible for local adaptation may demonstrate larger divergence estimates than our current analysis.


Astyanax mexicanus in order to better understand the evolutionary origins of the cave forms, the basic genetic structuring of both cave and surface populations, and the degree to which present day migration among them affects their genetic divergence.Cave animals converge evolutionarily on a suite of troglomorphic traits, the best known of which are eyelessness and depigmentation. We studied 11 cave and 10 surface populations of To assess the genetic structure within populations and the relationships among them we genotyped individuals at 26 microsatellite loci. We found that surface populations are similar to one another, despite their relatively large geographic separation, whereas the cave populations are better differentiated. The cave populations we studied span the full range of the cave forms in three separate geographic regions and have at least five separate evolutionary origins. Cave populations had lower genetic diversity than surface populations, correlated with their smaller effective population sizes, probably the result of food and space limitations. Some of the cave populations receive migrants from the surface and exchange migrants with one another, especially when geographically close. This admixture results in significant heterozygote deficiencies at numerous loci due to Wahlund effects. Cave populations receiving migrants from the surface contain small numbers of individuals that are intermediate in both phenotype and genotype, affirming at least limited gene flow from the surface.Cave populations of this species are derived from two different surface stocks denoted "old" and "new." The old stock colonized caves at least three times independently while the new stock colonized caves at least twice independently. Thus, the similar cave phenotypes found in these caves are the result of repeated convergences. These phenotypic convergences have occurred in spite of gene flow from surface populations suggesting either strong natural or sexual selection for alleles responsible for the cave phenotype in the cave environment. The mechanisms underlying the evolution of convergent phenotypes in independent natural populations pose long-standing questions in evolutionary biology. The extent to which convergent or parallel changes draw on preexisting genetic variation in ancestral populations versus new mutations is still debated ,2. The mConvergence is also of interest to evolutionists because it provides an element of replication to evolutionary studies that is often otherwise absent. Replication allows for the powerful testing of evolutionary hypotheses. Cave-dwelling organisms provide the best known examples of convergences, sharing similar phenotypes such as loss of eyes and pigmentation across diverse taxonomic groups -5.Astyanax mexicanus) is nearly unique among cave animals because the cave forms have closely related surface conspecifics and the two forms are fully interfertile [The Mexican blind cavefish : Pachón, Yerbaniz, Japones, Arroyo, Tinaja, Curva, Toro, and Chica, respectively. In the Sierra de Guatemala we sampled two caves, Molino (N1) and Caballo Moro (N2) and in the Micos area we sampled one of three closely clustered caves . An overview of the geographical distribution of the sampling area of cave and surface locations is presented in Figure et al. (2006). All samples were profiled at 26 microsatellite markers with primers previously developed for QTL studies [DNA extraction and genotyping were done according to Protas  studies . The for studies . In addi studies ,10,12.The database was optimized for analysis using the coalescent-based program Migrate-N 3.2.6. -20. The o) and unbiased expected (He) heterozygosities [IS statistic estimations, using Markov chain Monte Carlo (MCMC) runs for 1000 batches, each of 2000 iterations, with the first 500 iterations discarded before sampling [We calculated observed  for each value of K were evaluated by calculating ΔK, the rate of change in the log probability of data between successive K values [The program STRUCTURE 2.3.3  was usedK values . We alsoK values  in orderWhile these clustering methods can be quite powerful, particularly when there is a high divergence between populations , they ofPrivate allele estimates and allele richness were calculated, grouping the independent geographical regions obtained by clustering methods. In order to estimate rarified allelic richness and private rarified allelic richness, the rarified method in HP-RARE  was usedST pairwise estimates [In order to estimate the variance between the groups of populations, pooled sample structuring was estimated using analysis of molecular variance (AMOVA)  and 20,0stimates  as implestimates .The coalescence-based program MIGRATE-N 3.2.6. -20 was ue is the effective population size and μ is the mutation rate per generation per locus, as well as mutation-scaled migration rates M = m/μ, where m is the immigration rate per generation among populations. The model comparison was done using Bayes factors that need the accurate calculation of marginal likelihoods. These likelihoods were calculated using thermodynamic integration in MIGRATE-N 3.2.6 [MIGRATE-N 3.2.6 -20 also st/(1-Fst)) versus straight line geographic distance by application of the Mantel test as implemented in GENALEX [We tested for isolation by distance among populations and sample locations comparing genetic distance , N3 (n = 72) and O8 (n = 119). Photos were taken in the lab using a digital camera with the fish placed on a Cartesian coordinate grid. Measurements were made using ImageJ (NIH). In order to correct for individual size differences, relative eye size was standardized as a proportion of standard body length .16 = 11.6, t10 = 8.7, respectively, P < 10-6 for both). The Micos cave population (N3) had an intermediate average number of alleles per locus (2.98); previous studies have shown this population to contain both cave and surface-dwelling phenotypes [expected heterozygosities (He) were also higher and significantly different (0.82 ± 0.04) in surface populations than in the El Abra  or Guatemala  populations, while the Micos population (N3) exhibited intermediate heterozygosities of 0.66  as described in MICRO-CHECKER v2.23 . This method is mostly based on significant heterozygote deficits relative to HWE . However biological factors such as Wahlund effect or inbreeding might be easily misconstrued as evidence for null alleles . Null alleles seem to be present mostly in the Micos and Chica populations (data not shown). If, however, null alleles were the cause of the lack of heterozygotes, we would expect them to be equally represented in all the other populations, as well as locus-specific; this we did not observe. Thus, loci out of HWE owing to heterozygous deficit seem to be caused not by a technical artifact such as null alleles, but rather by population genetic phenomena. Only one locus (213B) was out of HWE in many populations (9 out of 21)   and detected 71 significant departures from HWE (based on 0.05 level of significance and standard Bonferroni corrections). Most of the significant loci showed heterozygote deficiency characterized by a positive FAs a starting point to infer the relationships among populations we used the clustering algorithm implemented in the program STRUCTURE . We explThese five independent population groups are: 1) El Abra caves (O1-O7), 2) El Abra cave mixed population (O8), 3) the new cave populations to the north in the Guatemala region , 4) the new cave mixed population in the southwest Micos region (N3) and 5) the surface populations Figure  and 2C. We further tested the genetic distances among populations using the metric of shared alleles. Figure Private allele estimates were calculated based on groupings of populations united by geographical proximity, which also corresponded well to the groupings revealed both by STRUCTURE and the shared allele distance analysis. The private allele content is significantly higher in surface populations than in cave populations Figure  Kruskal and 3B svs."new" [vs. the Guatemala and Micos caves (N1 - N3) pooled with surface populations (S1 - S4) was significant (P < 0.0001) and explained 4.52% of the variance among groups. This supports the hypothesis emerging from the STRUCTURE analysis that two different stocks of surface fish were ancestral to the present day cave populations. We tested by AMOVA all inferences of structuring that emerged from the shared allele distance and STRUCTURE analyses. The largest proportion of variance in all of the groups was within individuals  and Micos cave (N3) (FST range from 0.23 to 0.36) than was seen in comparisons among caves of the El Abra cluster (FST range from 0.20 to 0.51). This low genetic divergence is notable because the Sierra de Guatemala and Micos caves are more than 100 kilometers (km) apart. Consistent with the AMOVA analyses, FST values among surface populations were generally low (the highest FST = 0.09), suggesting that many of these populations from multiple and distant geographical regions essentially have high levels of allelic exchange. On the basis of FST values, general divergence between cave and surface pairs seems to be related to the level of the physical isolation of the particular caves from the surface water. Four cave populations  show the highest FST values against the surface populations  was 0.230 ± 0.021 (SEM), which was significantly higher than the average FST values between O8 and the ten surface populations .As is the case with O1, all seven Fed Table . The avee) and migration rates among populations were performed with MIGRATE-N 3.2.6, using Bayesian inference and the Brownian motion mutation model. The model allows for mutation rates differing among loci by using the number of alleles per locus to estimate locus specific relative mutation rate modifiers. All the estimates of the mutation-scaled effective population size Θ were scaled using a microsatellite mutation rate of 5.56 × 10-4 per locus per generation [e). Effective population sizes varied among different surface clusters (Ne from ~1011 to ~5058) but were generally greater than in cave populations  to 1326 (O2)  were the highest we observed and were mostly symmetrical . The phenotype we used was relative eye size and the genotypic designations for each of the 26 loci were obtained from the STRUCTURE analyses  and surface populations also confirm that these populations have recently diverged  than between Chica and the other El Abra populations (O1 through O7), but the opposite is the case   has been a long standing question in the terature . Our datterature ,44. If Cis Table , all of Astyanax and other surface species, including Tilapia, in certain caves, such as Yerbaniz (O2), Chica (O8) and Micos (N3). The significance of Tilapia's presence is that it was introduced into Mexican waters and only became common in the late 1980's [ST values between Pachón and the other El Abra populations are significant  cave and the closest of the Guatemala caves, Caballo Moro (N2), is over 90 km and there is one ridge and two open valleys between them. No documented underground route currently exists between the two regions. Thus, the Micos and Guatemala cave clusters likely represent separate invasions.Astyanax in NE Mexico. We envision that the area was originally colonized by surface Astyanax of the old stock which independently established cave populations in the south of the El Abra (O8), in central El Abra (O2 - O7), and in its north (O1). Subsequent to this, the surface stock went extinct locally and was eventually replace by surface fish of the new stock. These gave rise to cave populations in the two geographically distinct regions of the Gautemala (N1 and N2) and Micos (N3)  also contained both cave and surface forms and, as in Chica (O8), we observed departures from HWE due to Wahlund effects. In contrast to the situation in Chica, food is not abundant in this cave, thus the surface fish are prone to starvation, leading in most cases to reduced fitness and inefficient mating . NeverthA. mexicanus cave populations is correlated with the influx of alleles from surface populations, as well as by their effective population sizes [Poecilia sulphuraria is relatively low, even though there are few physical barriers to movement [Poecilia, the barrier seems to be the extreme environment of the sulphidic caves, which requires physiological adaptation to high levels of H2S, a condition to which cave Astyanax are not exposed.The MIGRATE-N analysis also detected relatively high rates of gene flow from the Pachón cave population (O1) to their nearby surface populations, supporting an earlier suggestion of a route for alleles from cave to surface  Figure . Estimaton sizes . The relmovement . In the 3 individuals and broad 95% confidence intervals ranging from about 1.5 × 103 to 17.0 × 103 [e in cave population varied from 2.8 to 7.3 × 103, with the exception of Curva (O6) and the admixed populations Micos (N3) and Chica (O8). While consistent with the estimates from mark-recapture studies [The migration rate analysis revealed that surface fish in the region form a metapopulation, with extensive exchange of genetic material among its component populations. Thus, there is high genetic diversity within and little genetic differentiation among surface populations. In strong contrast, cave populations live under dramatically different ecological conditions and often have lower population densities. MIGRATE-N results also show that the effective sizes of surface populations are generally larger than those of cave populations, consistent with earlier studies based on estimates of nucleotide diversity  Figure . Mark an studies .e (θ*M/4) can exceed 1.0, implying that migration from surface to cave populations could significantly affect allelic frequencies at neutral loci [We note that the mutation-scaled immigration rate (M) from surface populations into cave populations often exceeds 1.0 ; Ap is the mean number of alleles per locus, expected (He) and observed (Ho) heterozygosity, FIS. Bold FISvalues are estimates significantly different from zero after Bonferroni correction.Click here for fileMIGRATE-N 3.2.6, runtime conditions and methods. Summary of methods and conditions.Click here for fileAllelic frequencies of 26 microsatllite loci in the studied populations. Summary of allelic frequencies for all loci and all populations.Click here for fileA detailed hydrological map of the El Abra region. El Abra region map with the indication of surface and subsurface water divide. Points at, or near, base level (orange line) are indicated by solid circles; fish-inhabited pools by solid circles closer to the high water profile (blue dotted line) .Click here for fileGenetic isolation by distance. Results of the Mantel test for correlation between genetic distance (Fst/(1-Fst)) and geographic distance for populations of the El Abra region (O1 - O8) .Click here for fileEstimates of gene flow based on Bayesian inferences of migration rates and population sizes. Results of MIGRATE-N 3.2.6 on Astyanax mexicanus population clusters within each geographical region. Mutation scaled immigration rate, M, between different population groups. M is the ratio of the immigration rate over the mutation rate. The central box of the plots represents the values from the lower to upper quartile (25 to 75 percentile). The middle dot represents the median posterior values over all loci. The horizontal line extends from the 2.5% percentile to the 97.5% percentile. Populations compared are designated to the left of the boxes.Click here for fileSummary of the proposed models and conclusions of the paper. Proposed model with five independent origins of cave adapted Astyanax in NE Mexico as estimated by the data. The first wave of surface fish led to three independent subterranean invasion events establishing the "old" cave populations. The second wave gave rise to two independent invasions establishing "new" cave populations. The arrows signify that the ancestral stock moved into the area from the south but are not meant as specific routes.Click here for file


Palinurus elephas in the northwest Mediterranean Sea by means of ten polymorphic microsatellite markers. Non-metric Multidimensional Scaling coupled with assignment tests were used as a new approach to further delimit a reference population inside a genetically homogeneous pool of individuals and test for the presence of long distance immigrants. From this approach, we found that most early benthic juveniles collected while settling in the northwest Mediterranean Sea originated from a common larval pool. However, 4.2% of the individuals were classified as immigrants from other genetically differentiated populations, with more immigrants in the south than in the north of the sampled basin. Given currents in the northwest Mediterranean Sea and the long pelagic larval phase of P. elephas that lasts several months, this result suggest a restricted homogenized zone in the studied basin with some individuals probably coming from more differentiated populations through the Almeria-Oran Front or the Strait of Sicily.We investigated the genetic structure of early benthic juveniles of the spiny lobster  Connectivity between populations is a key process in understanding marine life cycles and has become a central issue in the fields of conservation and management of marine wildlife. In the marine world, connectivity is mainly determined by dispersal processes throughout oceanic currents and water masses movements. For most benthic fish and invertebrates, whereas the adult stages are generally sedentary or exhibit limited mobility, the larval phase is planktonic, potentially highly dispersive and therefore playing a key role in population connectivity  is an economically important species inhabiting a wide geographic range that extends from the northeast Atlantic to the eastern Mediterranean. The overfished status of its populations is attributed to changes in fishing practices together with growing fishing effort in recent decades . In such perspective, we focus, for the first time, on collections of recent post-puerulus individuals to investigate, for the first time in that species, patchiness in the recruitment and connectivity pattern. Investigating post-puerulus reduces the variance in genetic differentiation due to cumulating adults of multiple years of recruitment in collections and therefore various network of connectivity from year to year. Using ten microsatellite markers previously developed by Palero and Pascual  by the Centro Oceanográfico de Baleares of the InstitutoEspañol de Oceanografía (COB-IEO). Three regions within the northwest Mediterranean Sea, separated by more than 200 km  were selected and within each region, four locations separated by approximately 10 km were sampled . In totaDNA was extracted from a total of 238 sampled recruits following the QIAGEN® DX Universal Tissue Sample DNA Extraction protocol. Fourteen polymorphic microsatellites of 15 previously developed for this species  performed in Arlequin version 3.5.1.2  implemented in the same software. Piry et al.  and the mean observed heterozygosity (0.702 ± 0.037) were high and not statistically different. No evidence of linkage disequilibrium was found between any pair of loci. Four loci  showed deficit of heterozygotes. This deficit is likely to be linked to the presence of null alleles as shown by the results of Micro-checker and therefore we excluded them from further analysis. All 10 remaining loci were considered statistically independent, in Hardy–Weinberg equilibrium and highly polymorphic. Values of observed and expected heterozygosities and number of alleles for the 14 loci are summarized in FST equal to 0.0005 . The results of both Bayesian methods (STRUCTURE and nMDS) also confirmed the absence of genetic clustering even while not considering the sample structure. Results from the graphic representation of Evanno's ΔK revealed the presence of a peak at K = 3.0, but when plotting the relative membership of each individual to each of the three clusters, all individuals presented equal probabilities to belong to each one of the three clusters. Finally, results of the nMDS representation (stress = 0.26) also supported the presence of one single homogeneous population with a single cloud of points representing each individuals , as they are not assigned to the reference population. Three of these immigrants are located within the 10% most peripheral points (to the barycenter) in the nMDS, whereas two immigrants belong to the 75–90% area and five individuals belong to the 50–75% area  among the most divergent are distinguished as immigrants  using Bayesian methods, whereas classic frequency analysis approaches were showing homogeneity. However, the main limitation of these approaches is that they are poor predictors of genetic structure and fail to correctly assign individuals to populations when gene flow among populations is moderate or, in our case, high (FST < 0.05) . The use of this combination was implemented to overtake different problems given by Bayesian approaches. It has been proven that Bayesian methods are powerful approaches that overtake problems encountered with P. elephas following oceanic currents. Two main currents enter in the northwest Mediterranean Sea. The first one, coming from the East, enters through the Corsica Channel (Salat P. elephas and seems to play a role in the differentiation of eastern most populations (Palero et al. P. elephas is differentiated from the Mediterranean population, but with populations of Azores and Portugal coast closely related to the northwest Mediterranean populations. The little connectivity found in these studies and the distribution of immigrants documented here both lend support to a limited gene flow of P. elephas through the AOF. However, given the lack of outgroups in our study, we cannot determine whether one hypothesis is better than the other. Thanks to the combination of assignment methods and nMDS representation and the geographic pattern found in the repartition of immigrants, we can speculate that individuals coming from more differentiated Atlantic populations disperse into the Mediterranean Sea through the Almeria-Oran Front. Obviously, further analyses, comparing immigrants' genetic signature with populations' genetic signatures in both Atlantic and Mediterranean populations need to be performed to precisely define their origins.Given the differentiation of the northwest Mediterranean population compared with other regions, the long larval phase and the adapted morphology of the larvae to be dispersed by currents, immigrants can come from the entire distribution of el Salat  and can el Salat  has rece