Cherax tenuimanus in the upper reaches of the Margaret River in southwestern Australia is under threat following the introduction and spread of its congener Cherax cainii. Here, we examine the extent of hybridization and introgression between the two species using twelve polymorphic microsatellite loci. Our study reveals there are three times more C. cainii than C. tenuimanus at our study site in the upper reaches of the Margaret River. There is also evidence of hybridization and introgression between C. tenuimanus and C. cainii at this site, with F1, F2 and backcrossed individuals identified. While interbreeding was confirmed in this study, our simulations suggest that the levels of introgression are much lower than would be expected under random mating, indicating partial reproductive barriers exist. Nevertheless, it is apparent that hybridization and introgression with C. cainii pose a serious threat to C. tenuimanus and their survival in the wild will require active adaptive management and continued genetic monitoring to evaluate management effectiveness.Hybridization and genetic introgression following the introduction of exotic species can pose a significant threat to the survival of geographically restricted species. A remnant population of the critically endangered freshwater crayfish  The survival of a species can be detrimentally affected by invasive species, especially if accompanied by hybridization and introgression –3. The tCherax tenuimanus (Decapoda: Parastacidae) , when the last genetic assessment of the population was made. The mutation rate was set at 0.001 [To determine whether the observed levels of hybridization and backcrossing in the admixed Margaret River population are consistent with random mating and whether there has been sufficient time for random mating to breakdown GD and genetic differentiation between subpopulations, simulated data sets were created using a modified version of EASYPOP 2.0.1 . The simat 0.001 ,28. BothFST ~ 0.5) allele frequency differences between subpopulations. In models with maximum genetic divergence, subpopulations were fixed for different alleles at each locus. Subpopulations with intermediate genetic divergences were set up by randomly selecting alleles from all possible allelic states for one subpopulation and randomly selecting alleles from a subset of possible allelic states for the other subpopulation. This also resulted in a difference in allelic richness between subpopulations, which is what we observed in the C. tenuimanus samples.The simulations ran for three generations (the estimated number of generations since 2002 assuming a generation time of three years ) and wert-tests.At each generation 40 individuals were randomly selected from each subpopulation  and used to test for population structure and ancestry using STRUCTURE and NEWHYBRIDS, as undertaken with the original data. Ten simulated data sets were generated and tested for genetic differentiation for each model. Simulated data sets from each generation were also analyzed with FSTAT to assess levels of GD between pairs of loci. The significance of differences between the observed and simulated results was tested using one sample Cher08) was identified as having null alleles in the control Shannon River population using MICROCHECKER, and was therefore removed from further analyses for all populations.One locus . Further pairwise testing using Wilcoxon’s signed-rank tests revealed significantly higher allelic richness and gene diversity within the population sample from Margaret River compared to all other population samples . There was also a significant difference in gene diversity between the C. tenuimanus control and C. cainii Warren River population samples (P = 0.045), with higher gene diversity in the C. cainii sample. All other pairwise tests were non-significant.Genetic diversity statistics for the remaining 12 loci were typically higher in the Margaret River population sample than the ulations . FriedmaC. tenuimanus captive breeding population had significantly positive multilocus FIS values indicating a higher than expected proportion of homozygotes  and ΔK across different values of K. These clusters were representative of C. cainii and C. tenuimanus, as determined by the assignment of all individuals from the C. tenuimanus control population sample to one cluster and all individuals from the C. cainii Warren and Shannon River populations to the other genetic cluster , though the differences between observed and simulated results were greater  the percentage of individuals that were assigned to the parental subpopulations (i.e. not admixed) after three generations of random mating was much lower than observed, while the percentage of individuals that were Fobserved . Similar greater .K = 2 as the most likely number of clusters, irrespective of whether the simulations were started with maximum and intermediate genetic divergence between subpopulations. The simulations also indicated that high levels of genotypic disequilibrium (GD) are not unexpected after three generations of random mating under some scenarios. However, the observed levels of GD were significantly higher than expected under random mating , and had remained stable until their study in 2009. Microsatellite analysis indicates that the proportion of C. tenuimanus in the site sampled is less than 20% of the total marron population. It is unclear whether this difference reflects a temporal change in the composition of the population in the upper reaches of the Margaret River or is due to the microsatellites being better able to detect hybrid individuals, thereby reducing the reported proportion of pure C. tenuimanus. This study also used a different sampling site to previous studies (precise sampling locations in previous studies were not given), which may also explain the different estimates.The Margaret River population of marron is admixed, containing individuals that are pure f et al.  reportedtenuimanus and C. cainii. Previous estimates of the level of hybridization in the upper reaches of the Margaret River range from 7.5% in 1981 to 12.5% in 1992 [C. tenuimanus, it appears that hybridization and introgression still pose a serious threat to the viability of the current C. tenuimanus population.The identification of an admixed Margaret River population validates the results of previous allozyme , allozym in 1992 . The NEWOrconectes propinquus and O. virilis in North America have been completely replaced by their congener O. rusticus [O. virilis by O. rusticus, hybridization and introgression appear to play an important role in the extirpation of O. propinquus [O. rusticus genes by 36% above ecological replacement mechanisms alone [C. tenuimanus populations, with their complete replacement by C. cainii in the lower and middle reaches of Margaret River within 13 years of its introduction to this river system [C. tenuimanus populations.The introduction of non-indigenous crayfish species is regarded one of the greatest threats to freshwater crayfish biodiversity . For exarusticus ,30. Whilopinquus . Indeed,r system ,11. ThisC. cainii are displacing C. tenuimanus from their home range. The rate of displacement of C. tenuimanus in the lower and middle reaches suggests competitive mechanisms are in place [C. cainii may give rise to larger C. cainii males, which may prevent C. tenuimanus males getting access to mates, contributing to the decline of the pure C. tenuimanus population. Another hypothesis is that earlier breeding times give C. cainii offspring a competitive size advantage over C. tenuimanus offspring, making them less susceptible to predation or better able to secure limited resources (e.g. suitable habitats). Sympatric juvenile coho salmon (Oncorhynchus kisutch) and steelhead trout (O. mykiss) experience strong asymmetric competition, whereby coho salmon are naturally larger than steelhead trout resulting in poorer habitat selection by the smaller trout [C. tenuimanus population, resulting in fewer conspecific encounters and matings between C. tenuimanus individuals. Investigating these processes in an experimental setting would provide valuable insights into the factors causing the decline of C. tenuimanus populations and increasing proportion of C. cainii and hybrids in Margaret River.It is not known why in place . There iin place , howeverer trout . PseudocC. tenuimanus and C. cainii. Such patterns are not unexpected in recently admixed populations [C. cainii from the lower reaches of the Margaret River is preventing the population in the upper reaches reaching equilibrium, though the relatively stable proportions of C. cainii in the upper reaches over the last 11 years suggests that migration rates are low.The Margaret River admixed population exhibits significantly higher levels of gene diversity and allelic richness when compared to populations of pure ulations ,33. The FST values revealed there were significant differences in allelic frequencies between the Margaret River population sample and all others. Further analyses using only individuals identified as pure C. cainii based on the STRUCTURE results may shed light on the origins of C. cainii introduced to the Margaret River. However, it would first be necessary to sample widely throughout the C. cainii range to confidently address this question. The significant differences in allelic frequencies between the C. cainii Shannon River and Warren River population samples suggest that the population divergences required for such a study are present. This result is also in agreement with the only previous microsatellite study [The pairwise te study , which iC. tenuimanus and C. cainii as independent species has been questioned due to reports of widespread hybridization in natural and artificial environments [C. tenuimanus and C. cainii. Further, the analyses clearly identified the presence of two distinct genetic populations, corresponding to each of the current species. Therefore, regardless of interbreeding, C. tenuimanus and C. cainii should continue to be considered as separate evolutionary significant units [The taxonomic status of ronments . While int units  and distnt units .C. cainii should be regarded as serious threats to C. tenuimanus. Without intervention, demographic and genomic extinction of C. tenuimanus in the wild appears inevitable. More accurate methods to rapidly identify C. cainii and hybrid individuals are being developed to assist with projects aimed at removing them from the upper reaches of the Margaret River. In addition, intensive sampling of the marron in the upper reaches is being used to determine the levels of effort required for physical removal to achieve significant benefits for the C. tenuimanus population. There is also an effective captive breeding program underway which, in light of these results, remains a high priority. Although the C. tenuimanus control population had lower gene diversity than the C. cainii Warren River population, allelic richness and gene diversity in the C. tenuimanus captive breeding population were comparable to levels in both the Warren and Shannon River populations of C. cainii, which will be important for the long-term viability of the species. Regular monitoring is needed to ensure that genetic diversity is maintained in the captive breeding population. Furthermore, to maintain the genetic integrity of the captive population, all wild-caught individuals added to the captive breeding population should be subjected to microsatellite genotyping to ensure that hybrid or backcrossed individuals are not mistakenly introduced.Although lower than expected under random mating, hybridization and introgression with


Schistosoma japonicum, has long been a major public health problem. An understanding of fundamental evolutionary and genetic processes in this species has major implications for its control and elimination. Intensive control efforts have greatly reduced the incidence of schistosomiasis in China, but little is known about the genetic consequences of these efforts.Schistosomiasis is one of the neglected tropical diseases. The causative agent of schistosomiasis in China, S. japonicum had persisted despite control efforts and genotyped these samples using ten microsatellite markers. Our main hypothesis was that parasite genetic diversity would be greatly reduced across this time period.To investigate this, we sampled twice (years 2003 and 2011) from two endemic regions where populations of S. japonicum populations has not been reported before.There was no apparent reduction in allelic diversity, and a non-significant reduction in clonal diversity in these parasite populations between 2003 and 2011. We did, however, detect temporal genetic differentiation among the samples. Such a significant temporal genetic variation of  Schistosoma japonicum has long been a major public health problem in China [S. japonicum from cattle and humans to snails. Effective interventions were implemented from 2005 to 2007, including keeping cattle away from snail-infested grasslands, providing farmers with mechanised farming tools and improving public environmental sanitation by supplying tap water and building lavatories. Annual synchronous rounds of chemotherapy are also routinely used [Schistosomiasis is a serious parasitic disease, infecting over 200 million people and threatening the health of about 779 million people around the world . Schistoin China . Specifiely used . It has S. japonicum populations among different geographical regions [S. japonicum populations. There have also been reports on spatio-temporal modeling for the prevalence of S. japonicum infection [S. japonicum populations.Genetic diversity is of great importance for any organism to adapt in a changing environment and, in the case of parasites, to respond to the pressure of intervention programs . Differe regions , as well regions  or host  regions . Some re regions , 12, 13 nfection , 15. HowS. japonicum populations were present in 2003. In 2011, the parasite was only detected in two of the seven locations, suggesting a high degree of intervention success at several locations. For the two locations at which parasites had persisted, we hypothesised that a considerable reduction in genetic diversity  would have occurred between these time points as a consequence of the interventions. In addition, we applied a range of population-genetic analyses under the hypothesis that there should be no temporal changes between the time points.In this study, we sampled again in 2011 from seven endemic regions where All procedures involving animals were carried out according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. Our protocol followed institutional ethical guidelines that were approved by the ethics committee at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention .S. japonicum [In 2011, we re-sampled seven endemic sites in China that had been investigated in 2003 for our previous project . In the aponicum  to such S. japonicum cercariae isolated from infected snails (Oncomelania hupensis) collected from two locations (i.e. Yueyang and Shashi) in 2011. For each location, we screened 5,000 snails in 2011 and detected three infected snails from Shashi and 25 infected snails from Yueyang. After 45 days, adult worms were obtained by perfusing the hepatic portal system and mesenteric veins of each infected mouse. All worms were washed at least three times with normal saline (0.9 % NaCl) to remove the host tissues before being stored in 95 % ethanol at 4 °C. The gender of each worm was noted.Ten laboratory-raised Kunming mice were each infected with about 1,000 In this study, we randomly selected 44 worms per location from the year 2011 samples for the genetic analyses. Genomic DNA was extracted individually from adult schistosomes using a DNeasy Blood & Tissue Kit , following the manufacturer’s instructions, and then stored at -20 °C until use.S. japonicum was genotyped at ten microsatellite loci  from our previous work [The DNA of each individual ous work . The PCRous work . Then thS. japonicum populations between 2003 and 2011, we included microsatellite data from 51 worms from Yueyang and 23 worms from Shashi, which were collected in 2003 and published previously [To compare the genetic variation of eviously . This yiR = (G-1)/(N-1) [Ar; sample size was corrected) were estimated using FSTAT V2.9.3.2 [Clonal diversity (R) was calculated manually as 1)/(N-1) , where G1)/(N-1) . Somatic1)/(N-1) . Inclusi1)/(N-1) . PCoAs wV2.9.3.2 . As the FST in Arlequin 3.11 [FST value was tested using 104 permutations. Hierarchical analysis of molecular variance (AMOVA) was applied to quantify genetic variances into spatial, temporal and within-sample components in Arlequin 3.11.Genetic differentiation between pairs of samples was calculated as Wright’s uin 3.11 , and theS. japonicum individuals based on ten microsatellite loci. No shared MLG was found among the eight subpopulations . The PCoA revealed that the 138 unique MLGs fell into a main cluster (Cluster 1) with small outlying clusters . The value of 1.00 , but not significantly (P = 0.06).Importantly, allelic richness among the 82 retained MLGs and corrected for sample size did not change dramatically at either site between 2003 and 2011  and the AMOVA. Spatial structure (between Yueyang and Shashi) was also indicated according to FST values, but not according to the AMOVA results.Using microsatellite markers, we noted a non-significant decrease in clonal diversity between the two time periods, but no obvious change in allelic richness. Measures of gene flow and population structure indicated substantial temporal genetic change (between 2003 and 2011) according to population differentiation tests (R = 0.94) was detected in S. japonicum populations from the year 2003. However, by 2011, this had decreased but not significantly. The clonal diversity in subpopulation YY11M was very low (0.35), perhaps indicating that the sample contained many sibling cercariae from a single male miracidium. Such a temporal change in clonal diversity of S. japonicum populations has not been observed previously.Clonal diversities were in agreement with previous studies using microsatellites , 20. A hS. japonicum in the region, but apparently not to an extent sufficient to impact markedly on allelic richness. Similarly, the extent of temporal population-genetic structure was unexpected. Such temporal changes are not previously known for S. japonicum, although structure at small spatial scales has been reported [S. japonicum over the vast region of the mid-Yangtze basin, from which our samples came. Local extinction followed by repopulation from elsewhere in the region might partly explain the extent of genetic differentiation observed. Given the known fluxes of water in the region [S. japonicum subject to substantial dispersal and gene flow.The maintenance of high allelic richness across time was not expected. Infected snails were more difficult to find in 2011 than in 2003 . This suggests a reduction in overall population size of reported , 11, 16.e region , especiaS. japonicum, few strong conclusions can be drawn.At our study locations, intensive control measures  had been implemented during the investigation period (spanning 9 years), which might have acted as a strong selective pressure and been responsible for the temporal changes observed. There were also marked ecological changes during this period, in particular the completion of the Three Gorges Dam (TGD), which significantly reduced the amount of water reaching our sampling locations (Dongting and Poyang lakes) and has moderated the annual floods. Given the lack of baseline data from prior to the construction of the TGD, and the lack of understanding of metapopulation dynamics in et al. [S. mansoni. However, we used mammal hosts only for a single passage (field-collected snails were used to infect laboratory hosts from which adult worms were harvested) and not serial passages such as used by LoVerde et al. [0 generation of laboratory hosts, in which very little deviation from the parental population was observed [S. japonicum can be long-lived in the mammal host, 47 years being reported in one human case [There are several limitations of our study. One is the use of different mammal hosts between 2003 (rabbits) and 2011 (mice). LoVerde et al.  noted the et al. . In effeobserved . Anotherman case . Long-liS. japonicum populations would be reduced following control programs between the years of 2003 and 2011. We did, however, find significant genetic differentiation across time in these populations. Overall, our findings provide a deeper understanding of molecular epidemiology and population genetics of S. japonicum, which may be of value for effective control and elimination of schistosomiasis.In conclusion, using ten microsatellite loci, we rejected the hypothesis that genetic diversity in two


DNA should evolve fixed differences more rapidly than nuclear DNA due to its smaller effective population size and faster mutation rate. As a consequence, molecular systematists have focused on the criteria of reciprocal monophyly in mitochondrial DNA for delimiting species boundaries. However, mitochondrial gene trees do not necessarily reflect the evolutionary history of the taxa in question, and even mitochondrial loci are not expected to be reciprocally monophyletic when the speciation event happened very recently. The goal of this study was to examine mitochondrial paraphyly within the Orchard Oriole complex, which is composed of Icterus spurius (Orchard Oriole) and Icterus fuertesi (Fuertes' Oriole). We increased the geographic sampling, added four nuclear loci, and used a range of population genetic and coalescent methods to examine the divergence between the taxa. With increased taxon sampling, we found evidence of clear structure between the taxa for mitochondrial DNA. However, nuclear loci showed little evidence of population structure, indicating a very recent divergence between I. spurius and I. fuertesi. Another goal was to examine the genetic variation within each taxon to look for evidence of a past founder event within the I. fuertesi lineage. Based on the high amounts of genetic variation for all nuclear loci, we found no evidence of such an event – thus, we found no support for the possible founding of I. fuertesi through a change in migratory behavior, followed by peripheral isolates speciation. Our results demonstrate that these two taxa are in the earliest stages of speciation, at a point when they have fixed differences in plumage color that are not reflected in monophyly of the mitochondrial or nuclear DNA markers in this study. This very recent divergence makes them ideal for continued studies of species boundaries and the earliest stages of speciation.As two lineages diverge from one another, mitochondrial  For the last 25 years, neutral molecular markers, especially mitochondrial DNA (mtDNA), have been used to examine closely related species – elucidating their evolutionary histories and looking for evidence of gene flow. Studies of closely related taxa often address species delimitation by looking for reciprocal monophyly in mitochondrial genes Moritz . MitochoHowever, there are many examples where species delimitation based solely on mtDNA would disagree with boundaries delimited using other characters , which breeds from southeastern Canada, across the eastern United States and into north‐central Mexico, and Icterus fuertesi (Fuertes' Oriole), which breeds in a geographically restricted region along the coastal lowlands of southern Tamaulipas and Veracruz, Mexico . They found that the two species were paraphyletic in their mtDNA with intermixed haplotypes , for which I. fuertesi showed lower haplotype diversity than I. spurius – which is the predicted pattern if the lineage leading to I. fuertesi was founded from a few individuals whose genetic diversity only represented a portion of the available diversity within the ancestral population  and through an increase in sample size, adding nine I. fuertesi samples. We sequenced multiple nuclear loci to obtain a broader picture of the genomic divergence between these two taxa. Using multiple loci, we attempted to avoided either stochastic or deterministic processes that might have only affected a part of the genome or one type of marker , C.R. (KU903080–KU903118), SLC9 (KU903119–KU903152), RDP2 (KU903153–KU903221), and TGFB2 (KU903222–KU903282). For additional information about the five loci, see Table In this study, we had 32 samples already available to us and we collected nine additional We extracted DNA from muscle tissue of all samples using a DNeasy Blood and Tissue Kit . PCRs were then performed on each locus using a Gene Amp PCR System 9700 , and we verified our product using 1% agarose gels with ethidium bromide. We cleaned PCR product using QIAquick PCR Purification Kits (QIAGEN). The amplified loci were sequenced using ABI's BigDye v. 3 Terminator Cycle Sequencing Kit on the Gene Amp PCR System 2400 (Applied Biosystems). We used EDTA‐ethanol precipitation to remove excess dye terminators. Chromatograms for each sequence were produced using the University of Maryland, Baltimore County's ABI3100 DNA Sequencer (Applied Biosystems). We edited and aligned the chromatograms using Sequencher 4.1 .Individuals within our dataset that contained more than one polymorphic site within a locus were phased using PHASE 2.1.1 , the haplotype diversity (Hd), the nucleotide diversity (π), the average number of nucleotide differences per site (p), and the number of nucleotide polymorphisms (θ) were all calculated using DnaSP  and the allelic frequency parameter (k) to be inferred from the dataset, rather than being fixed. All runs had a burn‐in of 50,000, followed by 250,000 iterations to collect data to estimate posterior probabilities. We tested K values from 1 to 10, and replicated each run 20 times for all K values.The number of haplotypes  and daughter populations (θ1 and θ2), migration rates between the two daughter populations (m1 and m2), and the time the two daughter populations diverged from each other (t), and the contribution of the ancestral population to each daughter population  (Hey and Nielsen m1 and m2), the maximum time of population splitting (t), the duration of the burn (b) and the duration of the run (l), the heating parameters (g1 and g2), the number of chains (n) and the number of chain swap attempts per step (k), and the estimates of maximum ancestral and daughter population sizes .To determine whether or not we could estimate how much of the shared DNA variation between these two taxa is due to retained ancestral polymorphisms versus gene flow (either historic or ongoing), we analyzed mtDNA, nuclear DNA, and the combined mtDNA and nuclear DNA datasets using both the programs IM and IMa  to 0.889 (TGFB2 for I. fuertesi) , three of the five loci  have higher nucleotide diversity for I. spurius and two (GADPH11 and SLC9) have higher diversity for I. fuertesi.Both taxa show high levels of haplotype diversity (Hd) for all of the nuclear loci, ranging from 0.724  was detected for K = 1 .The results from STRUCTURE analysis of the four nuclear loci provide no evidence of population structure. The highest log‐likelihood probability for Ln = 1 Fig. , and str= 1 Fig. . The AMO= 1 Fig. . Thus, tFor the IM analyses of our nuclear loci, the IM runs consistently failed to converge even after more than twenty runs with the autosomal loci – the last five of which included the Z‐linked intron SLC9 – and using multiple random number seeds. , indicating there is evidence of a great deal of structure between the two taxa for this locus. With the addition of more I. fuertesi, the mtDNA haplotype frequencies are clearly statistically significantly different.The results of the AMOVA for mtDNA CR are a clear contrast to the nuclear DNA Table . These rST value . The amount of variation explained at the among‐species level in the AMOVA for TGFB2 is 5.1%, indicating that a small portion of the genetic variation can be explained by species boundaries. If the mtDNA paraphyly first described by Baker et al. . Even with the inclusion of eight more I. fuertesi samples, only one haplotype is shared between the two taxa for this locus, and only two haplotypes are intermixed and showing mtDNA paraphyly. Two haplotypes that we found only in I. spurius are only one base pair different from I. fuertesi haplotypes . Thus, one of the nuclear loci shows some evidence of segregation, indicating that the nuclear genomes of the two taxa are beginning to diverge.Although the majority of variation within the nuclear loci exists at the level of variation within populations, one autosomal locus showed a small amount of variation that could be explained by species boundaries. TGFB2 had roughly 5% of its variation being explained by variation within each taxon (Corvus (corone) corone and C. (corone) cornix, showed significant divergence in the genes that controlled for plumage coloration of the two taxa, yet a lack of divergence in neutral genetic markers due to hybridization and widespread introgression, showing that assortative mating and sexual selection can lead to genetic differentiation in regions under selection . Based on these results, we found no evidence of a founder event in I. fuertesi's recent past. This result is surprising because the distribution of I. fuertesi suggested that it might have evolved through this mechanism. Instead, both taxa show a great deal of allelic diversity, indicating relatively large historic population sizes in spite of the very restricted range of I. fuertesi. Nevertheless, it is conceivable that I. fuertesi was established through a large number of founders, and/or that subsequent gene flow from I. spurius has added a lot of genetic diversity through introgression. As the IM analyses did not converge, we are unable to completely eliminate this possibility without including many additional loci. However, a classic splitting of a single more widespread population clearly fits our data. This taxon pair would be ideally suited to next generation sequencing approaches to study this and other aspects of their divergence . Whole genome comparisons would most likely provide more insight into both the divergence between these two taxa, and any potential gene flow that has occurred since this divergence. The statistical results of the Ramos‐Onsins and Rozas R2 for both taxa , but also within the nuclear locus TGFB2, indicates that I. spurius and I. fuertesi are in the process of diverging genetically from one another. Based on this and other evidence such as coloration and migratory patterns, it is likely that these two taxa have become evolutionarily distinct groups, regardless of any potential gene flow.Based on the lack of convergence in IM, due largely to the lack of structure within the nuclear loci, we are unable to determine how much of the shared variation is the result of retained ancestral polymorphisms or is the result of gene flow. The control region shows far more evidence of structure, with only one shared haplotype and two intermixed haplotypes within all individuals included in this study. There are two possible explanations: (1) significantly different haplotype frequencies in mtDNA between the taxa indicates gene flow is likely greatly reduced or nonexistent and is not being captured at the nuDNA level due to retained ancestral polymorphisms, or (2) haplotype frequency differences for only the mtDNA could also be explained by Haldane's rule in the face of ongoing gene flow  shows a small, but significant ΦST divergence between the two taxa, confirming the formation of two distinct genome pools. Based on these molecular findings, as well as the fixed differences in plumage, migratory behavior, and bioclimatic niches, there is strong evidence that these two taxa are just beginning to diverge from one another, making them ideal study organisms for the early stages of speciation and the formation of species boundaries (Hofmann et al. None declared.Table S1. Specimen numbers and sampling locations for tissue samples.Click here for additional data file.


Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (Ne) are much smaller than those expected in a fishes with large population census sizes (Nc). Estimates of Ne indicate low effective population sizes, even before the population collapse. The ratio Ne/Ne ranged between 10−6 and 10−8, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy ( Population dynamics are driven by a complex set of ecological and evolutionary variables that mold population demography on several spatial and temporal scales . UnfortuNc)  , but this drop was not statistically significant. However, values of NA increased significantly after 1989. These results clearly show the influence of demographic fluctuations on genetic variability of Adriatic anchovies, which has also been observed in other marine populations [Temporal trends in genetic variables are concordant with abundance trends in the Chioggia and Vieste time series. However, the Chioggia time series showed greater changes in genetic variables than the Vieste time series. In the Chioggia time series, a significant reduction in declines . After 2atic Sea . Values ulations , 62. Theulations .He in northern Adriatic anchovies is consistent with a bottleneck in Ne and with a recent 10 to 20-fold drop in fishery biomass [He in response to large declines in population abundance [Ne [Ne among samples from Chioggia from 1978 to 2000, as well as that among samples from Vieste from 1985 to 1989.Even though a significant reduction in  biomass , 20, 22,bundance . Second,bundance  reviewedbundance . The lowance [Ne , and thiHe values between 1978 and 2000 in Chioggia and between 1985 and 1989 in Vieste, may be consistent with the loss of genetic variability as a consequence of a bottleneck. Observed NA values between 1978 and 1987 in Chioggia and between 1985 and 1987 in Vieste were close to simulated values. These simulations also indicated a decrease in genetic diversity in the late 1980s that is consistent with bottlenecks in population size.The use of demographic simulations with BOTTLESIM showed that most of the observed Reduced genetic variability as a consequence of a bottleneck, especially in the northern Adriatic Sea, is also indicated by significant deviations from Hardy-Weinberg expectations for Chioggia in 1978 and 1987, as a consequence of homozygote excess. These genetic signals support the hypothesis that a reduction in genetic variability began earlier than the decline in effective population size. Although unlikely in small pelagic fishes, other marine species characterized by high fecundities and a high variance in reproductive success (sweepstakes recruitment) often show signs of inbreeding , 67.Ne in the Chioggia and the Vieste time series were characterized by good converges for this parameter, which gives some confidence in the results. We observed low levels of effective population sizes (Ne) of only 100s of fish in the pre-collapse and early post-collapse periods in both historical time series. These results indicate that the reductions in genetic diversity were largely driven by genetic drift [Ne from pre-collapse years were as low as those for 1987. Our global estimates of Ne for Adriatic anchovy indicate values of Ne between one hundred and a few thousand fish, values that have previously been reported in other small pelagics, such as European sardines [The MCMC methods used to evaluate temporal variability in ic drift  and thatsardines , 12, 27,sardines , 66. TheNe/Nc in Adriatic anchovies was between 10−6 and 10−8 orders of magnitude and are smaller than those typical of many marine species (Ne/Nc ≥ 10−5) [Ne with conservation guidelines provided by the revised 50/500 rule [Ne that come close to those recommended by 50/500 rules, suggesting that severe population declines place Adriatic anchovies at risk of losing evolutionary potential in the absence of stable connectivity among anchovy subpopulations in the Adriatic basin.Estimates of  ≥ 10−5) . These s500 rule . The 50/He, NA and Ne over time indicates that both localities have recovered from a population bottleneck, but that the recovery appears to have started earlier in Vieste (from 1989) than in Chioggia (from 1994). The relative geographic genetic homogeneity of anchovy populations in the Adriatic  may indicate that migration between subpopulations is important for the recovery of genetic diversity in local populations. In this way, migration between the Chioggia and Vieste populations likely led to the recovery of most of the genetic diversity lost in the Chioggia population over about seven generations.Despite fluctuations in genetic variables in response to severe population declines, anchovies in the Adriatic Sea generally showed little genetic heterogeneity both between northern and middle-southern samples and among temporal samples in these areas. We detected a lack of genetic differentiation and a single population comprising all the Chioggia and Vieste samples. The occurrence a single genetic stock is also confirmed by a microsatellite study of samples from throughout the Adriatic basin . The lack of temporal variability confirms that this genetic composition has been maintained over at least the past four decades. Similar evidence was found in other temporal studies of small pelagics , 69 and This study confirms the utility of archived DNA to address evolutionary and fishery-related problems. Our results indicate that, contrary to the common assumption that abundant species, such as anchovies, are not immune to the loss of genetic variability, because sweepstakes recruitment can lead to small effective population sizes. The effective population sizes of Adriatic anchovies are several orders of magnitude smaller than census population sizes. This large discrepancy indicates that anchovy populations are more vulnerable to fishery pressures and environmental change than previously thought, and these small effective sizes must be taken into account in the management of fishery harvests. Our results also indicate that temporal genetic data provides an assessment of the impact of demographic disturbances on the persistence of local populations.S1 Fig(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableMicrosatellite loci primer sequences, reference genome and authors.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Acer mono, populations in region II and the influence of physiographic patterns on intraspecific genetic diversity. Analyses of chloroplast sequences and nuclear microsatellites indicated high levels of genetic diversity. The diversity distribution was spatially heterogeneous and a latitudinal cline existed in both markers. The spatial distribution pattern between genetic diversity within A. mono and the diversity at species level was generally consistent. Western subtropical evergreen broad-leaved forest subregion (IVb) had a unique ancient chloroplast clade (CP3) and a nuclear gene pool (GP5) with dominance indicating the critical role of this area in species diversification. Genetic data and ecological niche model results both suggested that populations in region II disappeared during the last glacial maximum (LGM) and recovered from south of Changbai Mt. and the Korean Peninsula. Two distribution centers were likely during the LGM, one in the north edge of warm temperate deciduous broad-leaved forest region (III) and another in the south edge of region III. This was reflected by the genetic pattern with two spatially independent genetic groups. This study highlights the key role of region III in sustaining genetic diversity in the northern range and connecting diversity between southern and northern range. We elucidated the diversity relationship between vegetation regions which could facilitate the understanding of biodiversity origin and maintenance in East Asia.East Asia has the most diverse temperate flora in the world primarily due to the lack of Pleistocene glaciation and the geographic heterogeneity. Although increasing phylogeography studies in this region provided more proofs in this issue, discrepancies and uncertainty still exist, especially in northern temperate deciduous broad-leaved and coniferous mixed forest region (II). And a widespread plant species could reduce the complexity to infer the relationship between diversity and physiographical pattern. Hence, we studied the evolution history of a widespread temperate tree,  Juglans mandshurica . Discrepancies between pollen record and molecular data in region II require additional analysis to accurately locate the northern extent of glacial refugia.The Sino-Japanese Floristic Region (SJFR) in East Asia is one of the most diverse temperate floras in the world, with approximately twice as many plant species as eastern North America, where holds similar size and environment Qian . SeveralHigh physiographical heterogeneity is considered to be another major influence on the extreme high floral diversity within the SJFR . PCR assays were performed in 40 μL containing 10–20 ng of template DNA, 1× buffer, 10 mmol/L Tris-HCl (pH = 8.3), 50 mmol/L KCl, 200 mmol/L each dNTP, 2.0 mmol/L MgCl2, 0.1 mmol/L each primer, and two units of Taq . PCR amplifications were performed as follows: an initial denaturation step at 94°C for 5 min, followed by 24 cycles of 50 sec at 94°C, 50 sec at the annealing temperature (Tables S2) and 50 sec at 72°C, with a final extension step at 72°C for 10 min. PCR products were sequenced at the Beijing branch of the Beijing Genomics Institute. All the cpDNA sequences were uploaded to the GenBank. The GenBank accession numbers of these sequences were KC767550–KC767586.Two intergenic spacers within the chloroplast genome, of rpl16  were ampA. mono and the annealing temperature  with the Clustal module. The genetic diversity of populations and vegetation regions were calculated in Arlequin 3.5 , the program simulated population groups with the highest variance among groups (FCT). We calculated the FCT value for each group number from 2 to 13 and set the number of simulated annealing processes to 100.The genetic structure and the potential genetic barriers for populations were analyzed using a spatial analysis of molecular variance  belonging to four sections of the genus  of A. mono clade as a normal distribution with mean (±1 SD) = 23.42 (±5) Mya , total genetic diversity over all populations (Ht), expected heterozygosity (He) and observed heterozygosity (Ho) were calculated using FSTAT 2.9.3 , private allele frequency (PAR), expected heterozygosity (HE), and observed heterozygosity (HO) using GenAIex v6.1 ) and ΔK, introduced by Evanno et al.  as well as the 63 records from this study. In total, 261 presence records of A. mono were obtained from throughout the species range. Six bioclimatic variables . We utilized seafloor topography data (ETOPO1) produced by the National Geophysical Data Center of the National Oceanic and Atmospheric Administration  to estimate the paleocoast lines (−130 m compared with the present) and the paleoclimate surfaces of the exposed seafloor area during the LGM. The LGM paleoclimate layers were at a 2.5-arcmin resolution.The established model was projected onto the climatic conditions reconstructed during the LGM, as simulated by MIROC3.2, provided by the Palaeoclimate Modelling Intercomparison Project (π) was 3.49 × 10−3 , while CP3 had the lowest genetic diversity . The diversity of CP1 was intermediate  , suggesting a Miocene split between the two clades. In contrast, the CP2 and CP1 subclades likely diverged in the late Pliocene . The time of the most recent common ancestor for CP1 was estimated to be 4.127 Mya (95% HPD: 2.222–6.349), while that for CP2 was 4.127 Mya (95% HPD: 2.222–6.032), and that for CP3 was 0.952 Mya (95% HPD: 0.000–2.857). Based on the cpDNA chronogram, BEAST analysis provided an average substitution rate of 3.15 × 10−10 s/s/y, ranging from 2.89 × 10−10s/s/y (rpl16) to 3.50 × 10−10s/s/y (psbA-trnH).The estimated crown age of Ht, was 0.886 (0.746–0.926), and the genetic differentiation was significant for all loci . The total genetic diversity over all populations, 6 to 38. ΔK was highest for K = 2 (387.4), but the log-likelihood value of the data (lnP(D)) was the highest for K = 6  was detected predominantly at the central area of Taihang Mt. , GP2 (Yanshan Mt.), and GP3 (Korean Peninsula and south of Changbai Mt.), while the southern group was divided into GP4 , GP5 (Hengduan Mt.), and GP6 (north of Qinling Mt.).In the STRUCTURE analysis, or K = 6 . The twoio Figs. , 6. BothMt. Fig. . For K =Two genetic barriers with high bootstrap support were detected. One was located between north of Taihang Mt. and Yanshan Mt., extending south to the Jiaodong Peninsula. The other barrier was on border of the south of Taihang Mts. and Qinling Mt., extending north to the valley between Taihang and Lvliang Mt. Fig. A.A. mono distribution were predicted with a high probability: (1) middle and high elevation areas of Hengduan Mt.(region IVb); (2) Qinling Mt., at the boundary between region III and IV; (3) Taihang and Lvliang Mts. in the southwest region III; (4) Bo Sea Coastal region in northeast region III, including Yanshan and Qianshan Mts. and the Jiaodong Peninsula; and (5) Changbai Mt. in region II and northeast North Korea. The predicted distribution based on the model was similar to the actual species distribution ]. Under the present climate, five areas of on IVb);  Qinling on Figs. A.Under the LGM climate, the species distribution in south China appeared little changed. In contrast, extremely low distribution probabilities were predicted for north of Changbai Mt. (region II) during the LGM. Additionally, the distribution probabilities in the Taihang-Lvliang Mts. Range decreased, while the distribution area in the Bo Sea Coastal region increased Fig. B.A. mono populations. Genetic diversity and privacy decreased from the south to the north; populations in region II had the lowest diversity and privacy  to Jiaodong Peninsula and the Korean Peninsula. This northern region III area might represent an independent distribution center during the glaciation, which was different from another center in Qinling Mt.. This hypothesis is supported by the high genetic diversity and privacy in the northern region III. The genetic composition of region II was nested within northern region III, suggesting that region II populations probably originated from expansion of this northern distribution center after the LGM.The genetic data and the ENM simulation indicated that during the LGM, A. mono can be suggested. The northern nuclear genetic group can be further divided into three gene pools in the STRUCTURE analysis  had greater diversity and endemism than region II and III (northern); while within region IV, region IVb (western) was greater than region IVa (eastern). Hengduan Mt. in region IVb had the highest total number of seed plants species (7954) and endemic species (2988), while region III (north China) and region II (northeast China) had the lowest species diversity  and endemism  and privacy (Private sites = 37). Diversity decreased with increasing latitude, and region II had the lowest diversity in both abundance and endemism . Microsatellite data revealed a pattern similar to that of the chloroplasts , but low privacy . This genetic diversity pattern may have originated from the transition status of region III between region II (northern) and region IV (southern). Region III held both chloroplast haplotypes from CP1 and CP2, and nuclear gene pool of both southern and northern ancestral origin . There is a clear boundary between southern and northern genetic types located in the north of Taihang Mt. . Similarly, the shrub Ostryopsi davidiana exhibited high genetic diversity on Taihang Mt., particularly in the northern region. Northern populations of O. davidiana shared haplotypes with southern populations  and another in southern region III (Qinling Mt.)  Fig. B. These The Korean Peninsula, although surround by ocean, is similar to the environment of region III. During the glacial periods, the peninsula was connected with the mainland due to lower sea level Fig. B. The chA. mono, however, the genetic diversity in subregion IVb was much lower than subregion IVa or region III in both chloroplast and microsatellite data, and approximately equal to region II , which harbored the most diverse and endemic species in East Asia and is one of the 25 worldwide biodiversity hotpots  due to movement of the Himalayas Li . The moue 2000 m . AlthougA. mono. The results showed high genetic diversity throughout its range and a distinctive genetic structure. A continental ice cap did not cover the temperate deciduous broad-leaved and coniferous mixed forest region (II) in northeast China during the last glaciation maximum, but A. mono populations retreated to the south due to decreased temperature and humidity and formed a distribution center during LGM at the northern edge of the warm temperate deciduous broad-leaved forest region (III). Present day populations in region II probably recolonized from south of Changbai Mt. or from the Korean Peninsula.We used chloroplast sequences and nuclear microsatellite markers to study a Tertiary relict temperate forest tree species A. mono, was consistent with the species level diversity pattern. Therefore, abundance and privacy were higher in the south than in the north. There were two exceptions. One was in region III where the populations with the highest genetic diversity probably originated from the transition between northern and southern genetic types. Another one was in the western subtropical evergreen broad-leaved forest subregion(IVb) with very low genetic diversity, probably due to the limited sample size in this region. Whereas the ancient lineage or haplotypes dominated in IVb and adjacent area (population 7) indicated the critical role of this area in species origin and diversification. By comparing the diversity pattern at two levels, we highlighted the significance of region III, including the Korean Peninsula, in reserving temperate floristic biodiversity during climatic oscillations and connecting the diversity between region IV (southern) and region II (northern). Physiographic heterogeneity represented by diverse terrain and landforms in region III may contribute to this vital function.In general, the intraspecific genetic diversity of the widespread species, The Japanese Archipelago appeared to have closer chloroplast relationships with central China. However, there were insufficient samples from Japan to make further inference regarding the relationship between the Japanese Archipelago and the mainland.


Sus scrofa, is an extant wild ancestor of the domestic pig as an agro-economically important mammal. Wild boar has a worldwide distribution with its geographic origin in Southeast Asia, but genetic diversity and genetic structure of wild boar in East Asia are poorly understood. To characterize the pattern and amount of genetic variation and population structure of wild boar in East Asia, we genotyped and analyzed microsatellite loci for a total of 238 wild boar specimens from ten locations across six countries in East and Southeast Asia.Wild boar, Our data indicated that wild boar populations in East Asia are genetically diverse and structured, showing a significant correlation of genetic distance with geographic distance and implying a low level of gene flow at a regional scale. Bayesian-based clustering analysis was indicative of seven inferred genetic clusters in which wild boars in East Asia are geographically structured. The level of genetic diversity was relatively high in wild boars from Southeast Asia, compared with those from Northeast Asia. This gradient pattern of genetic diversity is consistent with an assumed ancestral population of wild boar in Southeast Asia. Genetic evidences from a relationship tree and structure analysis suggest that wild boar in Jeju Island, South Korea have a distinct genetic background from those in mainland Korea.Our results reveal a diverse pattern of genetic diversity and the existence of genetic differentiation among wild boar populations inhabiting East Asia. This study highlights the potential contribution of genetic variation of wild boar to the high genetic diversity of local domestic pigs during domestication in East Asia. Sus scrofa, is one of the most widely distributed mammalian species, native throughout Europe, North Africa, and much of Asia as far south as Indonesia. Wild boar populations have also been artificially introduced in some areas of the world including the Americas and Australasia, principally for hunting, or through escapes from captivity. Sus scrofa is the most common wild ancestor of the domestic pig, with which it freely hybridizes[Wild boar, bridizes. The Fambridizes.th and 19th centuries after independent domestication[Since wild boar is a co-existing wild ancestor of domesticated pig, the patterns and origins of pig domestication worldwide are of increasing interest, not only in economic contexts, but also academically. Previous phylogenetic studies based on the mtDNA D-loop sequence revealed that continental wild boars and domestic pigs are clearly divided into eastern and western clades-5. Thesetication. A recentication.et al.[It has been well known that the cosmopolitan wild boar originated and spread from Islands of Southeast Asia. Knowledet al. ascertaiIn this study, we aimed to characterize genetic relationships and genetic structure of wild boars from East Asia by examining genetic variation at microsatellite loci for a total of 238 wild boar individuals from six countries. Our results shed light on the genetic relationships among populations and help define population boundaries of wild boar in East Asia.FIS, ranged from 0.017 to 0.279 with a mean of 0.091. Most of the populations except for two sample locations, Japan and Vietnam, showed non-significant FIS values, implying no signature of significant inbreeding , Yunnan, China , Vietnam , and Indonesia . The highest number of alleles  was found in wild boars from Indonesia, of which 33 were private alleles. Inbreeding coefficients, HE and ≥ 7.3 in allelic richness), followed by the Russian Far East (Primorsky Krai) and mainland Korea (except Jeolla-do). The lowest level of genetic diversity was found in the samples from Jeolla-do, Korea , and Jeju Island  , are presented in Table FST values ranged from 0.020  to 0.314 . Gene flow estimates (Nm) derived from FST ranged from 0.546 to 12.250. All wild boar population pairs were significantly differentiated from one another except population pairs from the north-central region of Korea. The wild boar population on Jeju Island showed the highest degree of genetic differentiation from other populations (mean FST = 0.253). The lowest mean FST value was found in southeastern Korea (Gyeongsang-do) vs. other populations (mean FST = 0.123).Between population genetic differences, as indicated by pairwise A genetic distance showed wild boars from Vietnam and Indonesia grouped together, forming a basal cluster to all other populations  they represent. Model-based clustering analysis revealed that wild boars in Eastern Asia had the highest ΔK when K was set to 3, assuming three inferred populations: 1. mainland Korea ; 2. Jeju Island (KJIW); and 3. Southeast Asia   and southern region (KGSW and KJLW), although they displayed a genetically admixed pattern  and 11.4% among populations within region (FSR = 0.114), to explain the proportion of genetic variance among populations to the total (FST = 0.198)  Table .2 = 0.078; P = 0.140). However, when the Jeju Island population was excluded, a significant regression was detected   exhibited the lowest genetic diversity among all populations sampled from East Asia. Negligible gene flow from the Korean mainland  were reintroduced to the mainland a decade ago[Archaeological evidence suggests that wild boars appeared on the Korean peninsula in the mid-Pleistocene, ca. 780,000 to 130,000 years before present. Howevercade ago. Archaeocade ago,24. ModeK = 7) showed that wild boars from mainland Korea are represented by two genetic clusters  support a gradual cline in genetic structure in mainland Korea , Japan (JPNW), China , Vietnam (VIEW), Indonesia (INDW) and South Korea. This experimental work was conducted with permission by the Conservation Genome Resource Bank for Korean Wildlife (CGRB) that provided wild boar samples for this study. All samples were legally collected and deposited into CGRB. The procedures involving animal samples followed the guidelines by Seoul National University Institutional Animal Care and Use Committee (SNUIACUC). Wild boars in South Korea were divided into five regional groups according to the province of collection and other geographic considerations: Gyeonggi-do (KGGW), Gangwon-do (KGWW), Gyeongsang-do (KGSW), Jeolla-do (KJLW) and Jeju Island (KJIW)  according the manufacturer’s instructions. The 16 microsatellite loci were amplified using the Multiplex PCR Kit (QIAGEN). Touchdown PCR was carried out under the following conditions: initial denaturation for 15 min at 95°C, followed by seven touchdown cycles starting at 94°C for 30s, 67°C for 90s, and 72°C for 60s, with annealing temperature decreasing by 2°C per cycle to 53°C. The touchdown cycles were followed by an additional 25 cycles at 94°C for 30s, 53°C for 90s, 72°C for 60s, and a final extension at 60°C for 30 min. Individuals were genotyped using a DNA Sequencer .Ad) per locus, observed heterozygosity (HO), and expected heterozygosity (HE) under Hardy-Weinberg assumptions, were estimated using the Microsatellite Tool Kit[Ar)[FIS, and the level of genetic differentiation between each pair of populations, pairwise FST estimates, and their significance values were calculated using a permutation approach with FSTAT v. 2.9.3[Nm, effective number of migrants per generation) were calculated from FST using the equation of Wright[FST/(1-FST), and geographic distance among locations using Mantel’s test in GenAlEx v.6.0, and significance was determined based on 999 permutations. The DISPAN computer program[A genetic distance by the neighbor-joining (NJ) method[Measures of genetic diversity, including mean number of alleles (Tool Kit. AllelicKit[Ar) is a funKit[Ar). Inbreedv. 2.9.3. Signifiv. 2.9.3. Indirecf Wright. The soff Wright was usedf Wright by testi program was used) method,38.K) was estimated according to the method of Evanno et al.[K is based on the rate of change in the log probability of data between successive K values. Ten runs were carried out for each K, from 1 to 12, to quantify the amount of variation of the likelihood value. Initially, we obtained the highest ΔK value when K was set to 3 (see Results). Three main clusters, therefore, were further analyzed according to the hierarchical island model to probe for possible hidden substructure for each predefined cluster[To assess population structure, STRUCTURE 2.3.3 software was usedo et al., where a cluster.The authors declare that they have no competing interests.SKC carried out the molecular genetic studies, participated in the experiments, data analyses, and drafted the manuscript. KSK and HL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. JEL participated in the experiment. YJK, MSM, IV, AM, JGO, THK, IS, NI, LY, YPZ and NM provided genetic materials and helped to draft the manuscript. All authors read and approved the final manuscript.K), based on 10 iterations per K, generated by the STRUCTURE program [K analysis of LnP(D), according to Evanno et al.[Figure S2: Regression of genetic distance on geographic distance between pairs of East Asian wild boar populations. A. Analysis using all populations included (P = 0.140); B. Analysis after excluding wild boars from Jeju Island (P = 0.002). Mantel’s test for correlations was carried out with 999 permutations. Table S1: Genetic characteristics of 16 microsatellite DNA loci for ten sampling locations in East Asia. See Table Plot of mean posterior probability (LnP(D)) values per clusters ( program , and delno et al.. Figure Click here for file


Glyphis glyphis from the last three river drainages they inhabit in northern Australia.Mitochondrial DNA markers have long been used to identify population boundaries and are now a standard tool in conservation biology. In elasmobranchs, evolutionary rates of mitochondrial genes are low and variation between distinct populations can be hard to detect with commonly used control region sequencing or other single gene approaches. In this study we sequenced the whole mitogenome of 93 Critically Endangered Speartooth Shark ΦST =0.28283, P <0.00001). Surprisingly, the comparison with single gene sub-datasets revealed that ND5 and 12S were the only ones carrying enough information to detect similar levels of genetic structure. The control region exhibited only one mutation, which was not sufficient to detect any structure among river drainages.Genetic diversity was extremely low (π =0.00019) but sufficient to demonstrate the existence of barriers to gene flow among river drainages  contains supplementary material, which is available to authorized users. Genetic markers have long been used to discriminate between populations and define evolutionary significant units  or managProgeny generally inherit their mtDNA from the female parent. As such, the analysis of mtDNA reflects matriarchal lineage processes only, providing information unobtainable from any nuclear marker . This isHaploblepharus pictus using the COXI gene [Paratrygon aiereba using the ND6 gene [Mitochondrial DNA markers have been widely used in studies of teleost and elasmobranch fish populations over the past decade with much attention focused on the D-loop or control region (CR). This region has been shown to be extremely variable in many vertebrates including humans , teleostOXI gene  followedND6 gene . Also, NND6 gene  collecteND6 gene ,22,23. OND6 gene . These rChelonia mydas [Martes pennanti (a weasel) [Orcinus orca [Recent advances in next generation sequencing have significantly reduced costs in a way that could easily facilitate routine investigation of intraspecific variation of whole elasmobranch mtDNA genomes at typical sampling levels used for standard population analyses. In the past, whole mitogenome sequencing has only been used in phylogenetic studies, where the number of individuals sequenced was generally low. Compared to single gene approaches, whole mitogenome sequencing can sometimes provide higher resolution of phylogenies and increased precision of divergence time estimates . More reia mydas  and the  weasel) . It has  weasel)  and the nus orca . There aGlyphis glyphis is a rare, habitat- and range-restricted, river shark of northern Australia and southern Papua New Guinea [Environment Protection and Biodiversity Conservation Act 1999. Little is known of its biology: juveniles and sub-adults have been recorded across a large salinity range in rivers, however, adults of this species have never been recorded and their range and habitat requirements are unknown [G. glyphis and the sympatric Northern River Shark G. garricki and Bull Shark Carcharhinus leucas, but no intraspecific variation was found across 12 and 17 G. glyphis individuals from the Northern Territory and Queensland, respectively, thus preventing any assessment of potential population structure.The Speartooth Shark w Guinea . It is cw Guinea . Its rar unknown . An unde unknown  developeG. glyphis as a case study. We anticipated that this approach would provide useful information for the conservation of this Critically Endangered species, as well as giving insights into the intraspecific genetic variability of the different mitogenomic regions in elasmobranchs.The aim of this study was to explore the potential of whole mitogenome sequencing for phylogeographic studies in elasmobranchs, using G. glyphis were successfully sequenced and assembled . Individual mean coverage ranged from 152 to over 6,000 fold ensuring a high quality of mitogenome sequencing. The duplicated sequencing of the G. glyphis reference mitogenome [The complete mitogenomes of 93 togenome  providedG. glyphis samples were successfully mapped on the Largetooth Sawfish Pristis pristis mitogenome, the other elasmobranch species present in the Miseq run. Those reads were mostly mapped on the highly conserved 16S region, thus suggesting cross contamination did not occur during library preparation.Less than 0.5% of the reads from G. glyphis mitogenome was calculated to be 16,701 bp in length. Interestingly, no transversions were observed but there were 19 transitions spread across 12 haplotypes. Nucleotide diversity (π) was 0.00019 and haplotype diversity (Hd) 0.76. Only one amino acid change was found in the complete dataset, at position 1348 of the ND5 alignment; this was not river drainage specific. The highest genetic diversity was found in the Alligator Rivers (π = 0.00024 and Hd = 8), whereas the Wenlock River exhibited the lowest genetic diversity (π = 0.00003 and Hd = 2). River drainages each contained from one to four drainage-specific haplotypes . The pairwise differentiation ΦST values ranged from 0.17755 to 0.53088 and all were significant  having lower values than the Alligators-Wenlock and Adelaide-Wenlock .Significant genetic structure was detected among the three river drainages  pooling more libraries on a given sequencing run [The whole mitogenomic survey using next generation sequencing  proved to be an efficient and accurate method for routine surveys of cing run ; or (ii)cing run . Whole mG. glyphis revealed particularly low levels of genetic diversity. Previous to this study, the species that exhibited the lowest recorded genetic diversity inferred from a whole mitogenome was the Giant Squid, Architeuthis sp., in a survey of 43 individuals from 10 sampling locations [G. glyphis. The low diversity in the Giant Squid is believed to be due to a recent bottleneck followed by expansion, possibly coupled with a low mutation rate [G. glyphis, suggesting that a bottleneck followed by population expansion is unlikely to be the best explanation for this species.Mitogenomic sequencing in ocations . This spion rate . With thG. glyphis. All species included in this review had populations with higher nucleotide diversity indices; 42 out of 50 species exhibited nucleotide diversity indices at least one order of magnitude higher than G. glyphis  and especially low levels of molecular evolution were proposed to explain current levels of genetic diversity [Glyphis glyphis has a naturally very limited range, being recorded in only a small number of tidal rivers and their estuaries in northern Australia and southern Papua New Guinea resulting in a low expected total number of individuals for this species [G. glyphis is probably due to the low evolutionary rate of mitochondrial genes  combinespecies, . These fn et al. . Low lev glyphis .G. glyphis. Haplotype diversity appears to be partitioned in a way that suggests strong female philopatry as each of the three river drainages is home to a distinct genetic population. Prior to our study, no evidence of population structure had been observed, however this was inferred from single gene approaches [G. glyphis used in this study were obtained from juvenile and sub-adult animals captured in the Adelaide, Alligators and Wenlock River drainages. In Australia, this represents the entire known extant distribution of G. glyphis with the population in the Bizant River, Eastern Cape York Peninsula presumed extinct [G. glyphis is not fully understood, although given the apparent absence of adults from river systems, adults presumably occur in marine habitats [C. leucas, population structuring occurred only at very large scales and not between adjacent rivers [Glyphis glyphis either has more discrete reproductive habitat preferences than C. leucas  or the two gene approach used by Tillett et al. [G. glyphis using a similar approach  , the combined  Alligator Rivers (n = 47) (referred to as the Alligator Rivers) in the Northern Territory, and the Wenlock River (n = 23) in Queensland, Australia . Whole mitogenomes were amplified in two overlapping fragments using Takara LA Taq, a proofreading Taq polymerase mixture . The PCR reaction mix consisted of 1 unit of Takara LA Taq, 2 μl of 10x LA PCR Buffer II (Mg2+ free), 2 μl of 25 mM MgCl2, 3.2 μl dNTP (2.5 mM each), 0.5 μl of each primer at 10 mM, and 10.6 μl sterilized distilled water. The primer pairs GL965F – 5′ TATTTCTCCAACAAGAGGAGGCAAGTCGTAAC 3′/GL10307R - 5′ GTAGTTGGTCAGAACCGTGT 3′ and GL10014F - 5′ GTCCAAATCAAGACCGCTAA 3′/GL1403R – 5′ CTTTTGCCACAGAGACGG 3′ were designed using previously acquired conserved areas of whole mitogenomic sequences of  glyphis  and G. ggarricki . PCR conP. pristis, another elasmobranch species (data not shown).The two purified fragments obtained for each individual were pooled at equimolar concentrations and subjected to library preparation using Nextera XT DNA Sample Preparation kits . Individual Nextera XT libraries were simultaneously fragmented and barcoded using the 96 sample Nextera Index kit . All libraries were quantified by a fluorometric method, using the Qubit dsDNA HS assay kit , and concentrations were normalized to give equal mitogenome coverage from each individual fish. Libraries were then pooled and sequenced on a Miseq desktop sequencer using 2 × 250 bp paired-end reads Miseq reagent kit v2 . The sequencing was done in two different runs, approximately half of the samples for each run being from G. glyphis sample were mapped on the reference mitogenome of the second elasmobranch species included in the Miseq sequencing runs.Fastq files were imported into GENEIOUS PRO (v. 7.0.6) . Before mitogenome assembly, reads were paired; 5′ and 3′ ends as well as regions with more than 5% chance of an error per base were trimmed. Reads with more than 15 low quality bases and/or shorter than 150 bp after trimming were discarded from subsequent analyses. Reads for each individual were then mapped onto a previously published reference sequence  using ‘MPreliminary reconstruction of a maximum likelihood haplotype tree indicated that the level of nucleotide variation among haplotypes was too low to provide a well-supported phylogenetic tree  . This ind . Tajima’d  and Fu’sd [s  statistiST) to estimate the degree of genetic differentiation among the three sampling locations. This was done for the whole mitogenome dataset and for each sub-dataset containing variable sites. The model of nucleotide evolution used for the AMOVA and to calculate ΦST values was Tamura-Nei [Analyses of molecular variance (AMOVA) were conducted with ARLEQUIN (v 3.5.1.2), to test for the existence of genetic structure among animals from different river drainages, and we calculated the fixation index  in elasmobranchs (Additional file Mitogenomic sequences: Genbank accessions KM100613 - KM100704.


Hippocampus erectus) can be commonly found in three western Atlantic zoogeographic provinces, though inhabitants of the temperate northern Virginia Province are often considered tropical vagrants that only arrive during warm seasons from the southern provinces and perish as temperatures decline. Although genetics can locate regions of historical population persistence and isolation, previous evidence of Virginia Province persistence is only provisional due to limited genetic sampling . To test alternative hypotheses of historical persistence versus the ephemerality of a northern Virginia Province population we used a RADseq generated dataset consisting of 11,708 single nucleotide polymorphisms (SNP) sampled from individuals collected from the eastern Gulf of Mexico to Long Island, NY. Concordant results from genomic analyses all infer three genetically divergent subpopulations, and strongly support Virginia Province inhabitants as a genetically diverged and a historically persistent ancestral gene pool. These results suggest that individuals that emerge in coastal areas during the warm season can be considered “local” and supports offshore migration during the colder months. This research demonstrates how a large number of genes sampled across a geographical range can capture the diversity of coalescent histories (across loci) while inferring population history. Moreover, these results clearly demonstrate the utility of population genomic data to infer peripheral subpopulation persistence in difficult-to-observe species.Understanding population structure and areas of demographic persistence and transients is critical for effective species management. However, direct observational evidence to address the geographic scale and delineation of ephemeral or persistent populations for many marine fishes is limited. The Lined seahorse ( In warmer seasons, the waters lining the concrete bulkheads, wooden piers, estuaries, and sandy beaches of the temperate Northeastern United State’s mid-Atlantic coast become home to numerous tropical fish species ,2. Over Hippocampus erectus [H. erectus is commonly found in coastal zones in three zoogeographic provinces: Caribbean , Carolina (warm-temperate) and Virginia (temperate)  . Some red fishes , and eard fishes ,12.Hippocampus species [Ecologists and evolutionary biologists often focus on questions at different temporal scales, but both fields are increasingly making use of genetic data to test hypotheses about population history, estimate the movement of individuals between local populations, and characterize the spatial distribution of genetic variation for effective species management ,13,14. O species . Most of species , to wide species , and dif species ,21.H. erectus individuals north of Cape Hatteras are the result of an ephemeral deme that is seasonally replenished from demographically persistent southern populations , or in contrast, there are persistent and isolated populations on either side of Cape Hatteras . A previous study of the H. erectus complex utilized mitochondrial DNA and five more slowly evolving nuclear loci across many individuals (n = 115), yet rejected H2 in favor of H1, with little divergence and evidence of isolation across Cape Hatteras [Here we test whether the presence of Hatteras . Now, wiHatteras .Clupea herangus) [H. erectus from the eastern Gulf of Mexico to Long Island, NY  datasets generated by restriction site associated DNA sequencing  have been utilized to study several fish species. Examples utilizing RADseq datasets include the support of cryptic differentiation between populations of the Baltic Sea herring (erangus) , the deterangus) , geneticerangus) –28, and erangus) . To testland, NY .n = 23), data from large numbers of unlinked loci allow highly resolved inference even with few individuals [Although we base our inference from only 4–9 individuals per each of the three zoogeographic provinces . Samples were collected from 2009–2013 from the following locations: Apalachicola, FL, Tampa Bay, FL, Charlotte Harbor, FL, the Florida Keys, Jacksonville, FL, and Indian River Lagoon, FL, Chesapeake Bay, New Jersey, the Hudson River and Long Island, NY. The specimens collected in this study were carried out in accordance and approval of the Queens College Institutional Animal Care and Use Committee (IACUC) (Permit # 137), which approved all aspects of specimen use in this study. Domestic fishing of Hippocampus is neither under direct regulation within the United States nor under species protection and no specific permissions were required for these locations; however we collaborated with the following authorities for samples, and if standard collection permits were required they were issued for each collection location. The Florida specimens used in our study were collected under the authority of the Florida Fish and Wildlife (FFW) as part of the FFW: Southeast Area Monitoring and Assessment Program. Samples from the Chesapeake Bay were collected in collaboration with the Virginia Institute of Marine Science (VIMS), which is authorized to collect any fishes necessary for research under the Code of Virginia. Lastly, samples collected in New Jersey and New York were collected in collaboration with Rutgers University under the New Jersey Department of Environmental Protection and the New York State Department of Environmental Conservation (DEC) Special Licensing Unit, License No. 1638 with additional samples collected under DEC License No. 1405.Samples of n >100) over multiple collection years to ensure genomic similarity was not the result of non-independent relatedness. Total Genomic DNA was extracted using Puregene extraction (Qiagen) from tail muscle tissue and treated with RNAase A following standard protocols. Genomic DNA quality was checked on an agarose gel to ensure that the majority of DNA was >10,000bp and equalized to 30 ng/uL using Qubit Fluormetric Quantitation (Invitrogen). Library construction and restriction site associated DNA sequencing (RADseq) protocol followed [Sequenced samples were randomly chosen from a large number of individuals  was implemented to determine if sampled individuals reflect a history of differentiated populations by outputting individual coordinates along axes of genetic variation within a statistical framework  that corTreemix [Treemix incorporates a model to allow for population divergence in the presence of post-divergence admixture/migration (m) given that incorporation of this parameter can improve the likelihood fit of a bifurcating phylogeny. More specifically, the m parameter represents the proportion of admixture from one population to another [The program Treemix  was util another . The res another , with brst utilized between subpopulations [st estimates [Population genetic statistics  were genulations  specificstimates .st (Fst/(1-Fst)) and shoreline distance (km) [st, calculated in vcftools, and distance of coastlines between sampling locations in kilometers was determined using Google Earth Tools. For this Mantel test, the centroid distance between sampling locations for the Gulf-Keys subpopulation was utilized and results indicated a non-significant correlation between geographic and genetic distance (p = 0.4925). See To investigate the visual similarity between genetic and geographic distance from the PCA analysis , we condnce (km) . PairwisH. erectus coming from a persistently breeding and isolated ancestral gene pool, rejecting the categorization of it being composed of seasonal migrants. The sNMF-based estimates of ancestry coefficients support three distinct subpopulations with limited admixture (K = 3)  : 1) the  (K = 3) . Here, tThe elevated heterozygosity found in Gulf-Keys individuals  could beH. erectus representing a persistently isolated independent subpopulation from other regional ancestral gene pools, there are several conceivable non-mutually exclusive causes of this divergence. First, seagrass is a preferred breeding habitat of H. erectus and a long gap without coastal seagrasses exists along the Georgia and South Carolina coastlines (roughly 600km) [Syngnathus floridae also shares a similar pattern of genetic divergence across this region of unsuitability, though the area of absence extends from the southern end of the Florida Peninsula to near Cape Hatteras, with the northern population extending from North Carolina to Chesapeake Bay [H. erectus is observed to occur via Sargassum rafting driven by ocean currents [H. erectus may help decouple the potential drivers of temperate subpopulation genetic isolation. Although our observed patterns of genetic isolation could have emerged via a continuous isolation-by-distance regime without clear breaks driving the isolation, a Mantel test resulted in a non-significant relationship between genomic and geographic distance (p = 0.4925).Given the strong evidence we report for Virginia Province inhabitants of y 600km) . This bay 600km) ,59. The currents . Under tcurrents , and futH. erectus from Virginia Province during winter months. While extreme temperature changes influence latitudinal movement of many species [H. erectus is unlikely due to its relatively weak swimming ability [Syngnathus fuscus), which has similar life history traits to H. erectus [Hippocampus [S. fuscus  and found that they closely resemble that of H. erectus, further supporting intercontinental shelf overwintering .Our results also support local offshore migration to account for the coastal absence of  species , substan ability . To avoi erectus , and haspocampus . As a quH. erectus as “vulnerable” based on it being commonly collected as by-catch and sold by trawl fishermen to supply the aquarium trade [Overall, our results demonstrate the utility of supplementing life history information with population genomic data when a small number of unlinked genetic loci may be insufficient to discern the range of persistence in difficult-to-observe fishes. Currently, the IUCN (World Conservation Union) Red List categorizes um trade . Our resS1 Table(PDF)Click here for additional data file.S1 Methods(PDF)Click here for additional data file.S2 Methods(PDF)Click here for additional data file.


Crocodylus suchus, across its distribution in West and Central Africa. We genotyped 109 individuals at nine microsatellite loci from 16 sampling localities and used three Bayesian clustering techniques and an analysis of contemporary gene flow to identify population structure across the landscape. We identified up to eight genetic clusters that largely correspond to populations isolated in coastal wetland systems and across large distances. Crocodile population clusters from the interior were readily distinguished from coastal areas, which were further subdivided by distance and drainage basin. Migration analyses indicated contemporary migration only between closely positioned coastal populations. These findings indicate high levels of population structure throughout the range of C. suchus and we use our results to suggest a role for molecular tools in identifying crocodile conservation units for this species. Further research, including additional sampling throughout the Congo and Niger drainages, would clarify both the landscape connectivity and management of this species.Landscape heterogeneity, phylogenetic history, and stochasticity all influence patterns of geneflow and connectivity in wild vertebrates. Fine-scale patterns of genetic partitioning may be particularly important for the sustainable management of widespread species in trade, such as crocodiles. We examined genetic variation within the rediscovered African crocodile,  Repeated phylogeographic patterns across diverse taxa are slowly revealing how both deep and recent phylogeographic events have shaped inter- and intraspecific biodiversity across Africa . EnvironProper understanding of the often-complex natural histories of species inhabiting large geographic regions can help elucidate current patterns of biodiversity. Conservation and management of widespread species can be improved with proper understanding of patterns of intraspecific genetic diversity and the identification of appropriate management units. In many cases, distinct population segments and genetic units are at risk of extinction due to ongoing and predicted rates of habitat loss, climate change, and unsustainable utilization . A betteThough crocodiles have generally been considered an ancient, wide-ranging, and relatively homogenous group, numerous recent studies have shown patterns of both considerable intraspecific  and specCrocodylus suchus  on the IUCN Red List  and is ms suchus . A redesCrocodylus niloticus is one of the most well known crocodilians globally, very little is known about C. suchus. This species is distributed across Central and West Africa, spanning known regions of complex biogeographic history like the Dahomey Gap and Cameroonian Highlands, for example  for C. suchus to be managed under the conservation of evolutionary processes paradigm .We screened eleven crocodile specific microsatellite loci developed by . suchus . Of the He), observed heterozygosity (Ho), and number of alleles (A), and HP-Rare 1.1 (AR) and private allelic richness (PAR).We examined microsatellite data for scoring errors and null alleles using MICRO-CHECKER . We asseRare 1.1  to calcuWe employed three different Bayesian clustering methods that identify clusters of individuals based on different underlying assumptions of inheritance to assess genetic population structure: STRUCTURE 2.0 , BAPS 6.K clusters in a way that minimizes deviations from Hardy–Weinberg Equilibrium and linkage equilibrium. We implemented a correlated allele frequency model with admixture and no sample locality information. For each analysis we conducted 20 independent replicate runs for each a priori assumed number of clusters (K) where K-values varied from 1 to 16, with 16 representing the number of sampling localities  by examining the Ln P(X∣ K) and ΔK in the program STRUCTURE HARVESTER , to assess optimal partitions of the data and allows for the inclusion of geographic coordinates for each sample locality as biologically relevant non-uniform priors to help the algorithm identify meaningful genetic clusters . Spatialdividual . We visudividual .K = 2 to K = 16 in the admixture analysis with spatial locations for all individuals. TESS requires unique coordinates for each individual sampled, so coordinates were randomly created within TESS for populations that lacked specific coordinate data for each individual (K), we used the deviance information criterion (DIC) to evaluate runs for convergence . We preferred these results over the other two methods because it does not a priori incorporate spatial data, which we felt could introduce a potential source of bias given the unequal distribution in sampling across Central and West Africa. In addition, we excluded individuals sampled at the Accra Zoo (n = 2) and Kinshasa Reptile Park (n = 2) from the following two analyses (FST and BAYESASS) due to unreliable original locality information.We used the results of the STRUCTURE analysis to determine clusters to be analyzed in the following two analyses (FST) amongst clusters in ARLEQUIN 3.5.1.3  ranged from 0.237 to 0.674 and estimates of allelic richness ranged from 1.77 to 3.47 with estimates of genetic diversity across all measures appearing higher in the west of this species range than in the east (One hundred and nine (of 125) samples, including samples from all 16 localities , successthe east . The isoK (K = 8) for the dataset  = − 3,192)  . The cluKThe TESS analysis only identified FST values ranged from 0.110 to 0.535 and were statistically significant (p < 0.05) in all pairwise comparisons (FST < 0.20) and, thus, we found significant levels of population genetic structuring throughout the range of C. suchus. The PCoA more clearly exhibits the genetic relationships among sampled populations  have origins in the Sahelian woodland savannahs. We identified population clusters corresponding to the upper (savannah woodland) and middle  reaches of some of these river systems as distinct from their coastal (lowland humid forest) clusters. Congruent patterns of genetic partitioning have been observed in Nile monitors (V. niloticus) (Habitat for loticus)  and two loticus) . These, loticus) . The fluloticus) .In coastal areas, the pattern was more complex. Population clusters from adjacent coastal regions exhibited lower levels of differentiation, but were still partitioned by associated drainage. However, we found extensive evidence for contemporary gene flow between the San Pedro dam site and the western Ivorian coastal cluster. Additionally, the low levels of variation and limited admixture with individuals from the nearby Volta River drainage suggest that the unique cluster at the isolated site near Digya National Park  may be an artifact of inbreeding and a single founder event.C. acutus, in nine delineated ecoregions explaining “planning for threatened species conservation ideally requires maintenance of viable populations across the full range of ecosystems in which they exist.” Justification for this perspective was largely based on maximizing the conservation of ecological variability. However, a more comprehensive approach to identifying CCU’s would be to incorporate information about underlying evolutionary processes in the form of identifying evolutionarily significant lineages or populations (C. suchus CCU’s.ulations . OverlayFST value. This result could reflect the historic isolation or it could be that the growth of Abidjan, one of West Africa’s largest cities, in the middle of this lagoon system continues to keep these populations in isolation.The C. suchus may additionally prove informative for monitoring trade and trafficking in crocodiles and crocodile products. Crocodilian leather is widely used for the fabrication of artisanal leather products throughout West Africa (C. suchus products. For example, our analyses allowed us to “source” two individuals sampled from the Accra Zoo. These two individuals were reportedly collected from the wild just outside of Accra; however, our analyses show strong evidence that they were collected from northern Ghana and transported either to the zoo for display or were later housed in the zoo after being confiscated by wildlife officials. Two individuals from Kinshasa were sampled at a private reptile park. Our analyses showed them to be highly admixed and most likely represent as yet fully sampled genetic diversity in this species. The owners were unsure of their specific origin, but felt strongly that they originated from DRC, coming from either the Congo River upstream of Kinshasa or from the far east of the country. Crocodiles from far eastern DRC were recently shown to most likely be C. niloticus (C. suchus (In addition to identifying CCU’s, the observed patterns of intraspecific variation within iloticus , while c. suchus . Our abi. suchus .It is now widely understood that the failure to recognize unique lineages and distinct population segments can result in steep declines in global biodiversity. In particular, West Africa’s vertebrate fauna has suffered from the lack of recognition of patterns of local and regional endemism. Until recently, widespread taxa including such high profile species as giraffe, lion, cheetah, and even elephant were managed primarily as panmictic populations throughout Africa. Over the past decade, however, molecular tools have uncovered deep divergence between West African and other lineages in these  and manyCrocodylus suchus (Our study presents the first broad scale analysis of molecular variation within the newly resurrected species s suchus  and supps suchus . This prCrocodylus suchus. Prior to its recent rediscovery, surveys for the “Nile crocodile” throughout western Africa were noted as highest priority by the IUCN/SSC Crocodile Specialist Group due to habitat destruction and decreasing availability of nesting sites (Following ng sites . Such sung sites .10.7717/peerj.1901/supp-1Data S1Click here for additional data file.10.7717/peerj.1901/supp-2Figure S1Click here for additional data file.


P. unifilis, popularly known as tracajá, leaves long-lived animals with late sexual maturity, characterized by a small mortality of adult animals, but high mortality rate of embryos and hatchlings, with natural predation as one of the most important factors of low hatching success of these species. This species is one of the most important representatives of the fauna of the Amazon turtle, but their eggs, meat and offal provide food for local communities, and have their hooves used as adornment and household items. The implementation of large hydropower enterprise cause many environmental damage. The scientific emphasis in EIA/RIMA should be held primarily in the study of special targets, and should take into account the genetic structure of populations. Mitochondrial molecular markers have particular maternal characteristics and high rates variation, which allow us to analyze the behavior of populations. The deployment of hydropower modifies the natural habitat of the turtles, which have difficulty in adapting. This work aimed to study the genetic structure of populations of P. unifilis analyzing the behavior of females in conditions of ambient modified by Tucuruí, and Antonina-PA and Xambioá-TO, where will be built the dam of Santa Isabel.The turtles are usually associated with low substitution of individuals in the population because they have longer life than any other vertebrates. The order Testudines or Chelonia, the oldest among all vertebrates, comprising the turtles, terrestrial, marine and freshwater. These have as main characteristics, low growth rates and long periods required for mature, causing many turtles are vulnerable to extinction. Like other turtles, We performed the sequencing of fragments of the control region of mitochondrial DNA, generating data of gene diversity and nucleotide, numbers of haplotypes and segregating sites, AMOVA, levels of structuring by pairwise comparison of ΦST and Mantel test in ARLEQUIN program. Clades were grouped checked by software Treefinder and the haplotype tree was held at hapview software.The study populations were similar to each other and have high diversity within them, but the region of rocks and waterfall in Xambioá associated with hydroelectric Tucuruí, act as barriers to gene flow. Tucuruí has the largest number of groups haplotypes, upriver this number decreases by Antonina and especially Xambioá. The Mantel test showed no correlation between genetic variation and spatial distribution, noting that the species does not present a long-distance migratory pattern. The region Xambioá and Tucuruí should be considered distinct management units due to the influence of anthropogenic activities differently in each place.