A widely accepted two-wave scenario of human settlement of Oceania involves the first out-of-Africa migration circa 50,000 years ago (ya), and the more recent Austronesian expansion, which reached the Bismarck Archipelago by 3,450 ya. Whereas earlier genetic studies provided evidence for extensive sex-biased admixture between the incoming and the indigenous populations, some archaeological, linguistic, and genetic evidence indicates a more complicated picture of settlement. To study regional variation in Oceania in more detail, we have compiled a genome-wide data set of 823 individuals from 72 populations (including 50 populations from Oceania) and over 620,000 autosomal single nucleotide polymorphisms (SNPs). We show that the initial dispersal of people from the Bismarck Archipelago into Remote Oceania occurred in a “leapfrog” fashion, completely by-passing the main chain of the Solomon Islands, and that the colonization of the Solomon Islands proceeded in a bidirectional manner. Our results also support a divergence between western and eastern Solomons, in agreement with the sharp linguistic divide known as the Tryon–Hackman line. We also report substantial post-Austronesian gene flow across the Solomons. In particular, Santa Cruz (in Remote Oceania) exhibits extraordinarily high levels of Papuan ancestry that cannot be explained by a simple bottleneck/founder event scenario. Finally, we use simulations to show that discrepancies between different methods for dating admixture likely reflect different sensitivities of the methods to multiple admixture events from the same (or similar) sources. Overall, this study points to the importance of fine-scale sampling to understand the complexities of human population history. The Pacific is a vast region, encompassing an entire hemisphere of our planet, and the human settlement of the far-flung Pacific Islands has long been of intense interest. A convenient division of the Pacific consists of Near and Remote Oceania, with the border located between Makira, the most easterly island of the main Solomon Island Archipelago, and the islands of Santa Cruz , Vanuatu, and New Caledonia  online. The languages spoken today in Near Oceania are classified as either Papuan or Austronesian. The indigenous Papuan languages are so deeply rooted and varied that the relationships between them are still poorly understood ; they doWhile there is growing consensus for the above “dual-wave” model for the colonization of Near and Remote Oceania, many questions remain. For example, there is an ∼40,000–45,000 year hiatus and regional isolation between the initial colonization of Near Oceania and the Austronesian expansion; are there any detectable signals in the genomes of modern Oceanian populations of any other migrations during this time period? Whereas some studies of mtDNA variation have claimed to find evidence for pre-Austronesian contact between Southeast Asia and Near Oceania , the mosKayser et al. 2000Another question concerns the timing of the contact that occurred between the Austronesian-speaking groups and the resident populations of Near Oceania , prior to the colonization of Remote Oceania. The contemporary populations of Remote Oceania all carry both Asian-related and Papuan-related ancestry , and varA further question is whether the movement of people from Near to Remote Oceania followed a simple wave-of-advance model , or whether movement was more complicated. There are already some indications that migration was more complicated than a simple wave-of-advance model; for example, obsidian from the Bismarck Islands is found in archaeological sites on Santa Cruz but not elsewhere in the Solomon Islands , suggestTo address these and other issues concerning the relationships among and between populations of Near and Remote Oceania, we have assembled a data set of 823 individuals from 72 populations, including 50 populations from Near and Remote Oceania  online, To obtain an overview of the data structure and potential associations between the samples in the data set, we applied two different approaches: principal components analysis (PCA), based on individual SNPs as well as on segments shared identical-by-descent (IBD), and the clustering algorithm ADMIXTURE . PCA basPCA based on segments shared IBD  indicates substantial haplotype sharing . Since tAsupplementary figs. S5, AS6, and supplementary fig. S5B, K = 5 , nor was it inferred for the aboriginal Australians.We first applied the 3-population test  in the fA, supplementary fig. S10B, supplementary fig. S11A, supplementary fig. S11B, Next, we used TreeMix  to buildsupplementary fig. S10In summary, both the f3 statistics and TreeMix clearly identify the genetic impact of the Austronesian expansion on Oceanic populations, and TreeMix confirms many of the more local-scale admixture signals . However, neither test infers NGH and Bougainville ancestry in aboriginal Australia, nor have any other studies of genome-wide data in aboriginal Australians e.g., . The likTo infer the date when Asia-related (presumably Austronesian-related) ancestry entered Near Oceania, we first used PCAdmix  to inferTo understand the source of this discrepancy we compared the WT method to ALDER using data simulated under a variety of different admixture scenarios. For this comparison, we used coalescent simulations implemented in the software MaCS . The basTo more clearly elucidate the patterns of recent relatedness and local migrations postdating the Austronesian expansion, we analyzed IBD blocks, that is, segments of DNA that were inherited without recombination by each pair of individuals from their most recent common ancestor . DetectiA, supplementary fig. S13B, supplementary fig. S13C, supplementary fig. S13D, In terms of between population IBD sharing, we observe that most populations of New Britain (the largest island in the BA) show substantial sharing of IBD segments with their neighbors, namely populations from New Ireland (BA), Bougainville, and the eastern part of the SI chain, and also with the PO and Tongans. The exceptions are: 1) the Baining groups, who share only with the neighboring Sulka and Tolai, and with Santa Cruz ; and 2) the Nakanai Loso, Ata, and the Mamusi groups, who share only with their neighbors, and in agreement with the ADMIXTURE result  reveal nsupplementary fig. S13E, Isabel and Santa Cruz depart from these general patterns of ancestry sharing for the SI. Isabel shows extensive recent genetic sharing with most populations from the BA and the SI (Santa Cruz being the notable exception), and with all sampled populations of Polynesian ancestry. Santa Cruz shares more segments IBD with New Britain than with New Ireland, Bougainville, or eastern or western SI (including Isabel). Peculiarly, Santa Cruz exhibits recent genetic ties with the Tolai, as do some other islands in the Solomons. Santa Cruz also shows equally low levels of sharing with all populations of Polynesian ancestry; this nonspecific sharing may indicate that Santa Cruz did not experience any additional post-Austronesian gene flow from Polynesia; by contrast, other populations exhibit ubiquitous sharing of long IBD segments with either Tongans , or Tongans and the PO , suggestive of contact postdating the Austronesian expansion  a considerable recent post-Austronesian expansion gene flow, which is diverse both in terms of its sources and direction, across many of the islands in Near Oceania; 2) different patterns of sharing in western versus eastern Solomons , charactNext, to better understand the chronology of gene flow across Near Oceania, we applied the Admixture History Graph (AHG) approach , which eA, supplementary fig. S13B, supplementary fig. S13D, supplementary fig. S13D, supplementary fig. S13D, supplementary fig. S13E, As already discussed, most of the components inferred by the ADMIXTURE analysis in Oceania do not reflect distinct ancestries, yet an Austronesian-related signal is clearly present, as seen in the f3 results that indicate admixture involving Taiwan Aboriginals  online. supplementary fig. S13The AHG approach is based on differences in the covariance between the various ancestry components inferred by ADMIXTURE . Since tWe assessed recent genetic drift in the Oceanian populations by two approaches. Firstly, we analyzed patterns of sharing of IBD blocks within populations. Because the genomes of individuals in a drifted population would exhibit a congruence of genealogies, we expect to see more IBD segments shared by individuals within such populations  as opposThis pattern is further corroborated by the genome-wide analysis of runs of homozygosity (ROH), which are uninterrupted chromosome regions in which all loci in an individual are homozygous, due to identical chromosomal segments being inherited from both parents. Thus, as with IBD segments, ROHs provide a useful measure of parental relatedness and degree of endogamy, historical population size and geographical isolation e.g., . Again, As previously mentioned, the placement of Santa Cruz in the PC analysis is rather unexpected; given the geographic location, we would expect Santa Cruz to fall closest to the other populations of Remote Oceania. Instead they are positioned closest to populations from New Britain in the BA, and remain so positioned at higher PCs  online. The sharing of IBD segments within Santa Cruz is low, especially in comparison to the Polynesian Outliers  online, A, supplementary fig. S17B, supplementary fig. S17C, Z = 3.96 and Z = 6.12 standard errors from zero for the difference between SC and the BA versus SC and Makira, and between SC and the BA versus SC and Choiseul, respectively.Next, we examined the decay in correlation of genome-wide LD between Santa Cruz and other populations. The basic logic behind this analysis is that following divergence, the correlation in genome-wide LD between the two daughter populations will decay exponentially over time, with the rate of decay dependent only on the recombination distance between the markers . For thisupplementary fig. S17To summarize, in all analyses Santa Cruz falls consistently closer to populations from the Bismarcks than to those from the Solomon Islands, and appears to be quite different from the other populations of Polynesian origin. The high frequency of Papuan-like ancestry in Santa Cruz, and the gradient of this ancestry diminishing across the eastern SI in an east–west direction, is in accordance with previous suggestions  that theIn conclusion, our analyses support the view that the initial dispersal of people from Bismarck Archipelago into Remote Oceania occurred in a “leapfrog” manner, completely by-passing the main chain of the Solomon Islands, and that the colonization of SI proceeded then in a bidirectional manner, underlying the divergence between western and eastern Solomons, in agreement with the Tryon–Hackman line. Santa Cruz remains a puzzle, exhibiting strong ties to the Bismarcks and high levels of Papuan ancestry not found elsewhere in the SI, yet also not exhibiting any signal associated with a founder event. It appears there was substantial Papuan-related gene flow to Santa Cruz that bypassed the rest of the SI. We also report ubiquitous recent post-Austronesian gene flow across the Solomons, both from the west and from the east, which could have been precipitated by natural calamities, such as volcanic eruptions (known to have occasioned the movement of the Tolai people) and droughts, wars, trade, or changes in cultural practices, like the introduction of sweet potato over the last four hundred years , as wellThe data set includes samples from 50 populations from Near and Remote Oceania  online: PCA was performed using the StepPCO software  on the er2 threshold: –indep-pairwise 200 25 0.4  were inferred using the fastIBD method implemented in BEAGLE , 2011. W phasing . All res phasing . To run  phasing ; the inv phasing .ROH (consecutive stretches of homozygous SNPs) provide information about relatedness among individuals and population isolation. ROH were identified using PLINK  with def2) in allele frequencies by randomly selecting seven individuals from each population  and further adjusting the measurement for each pair of SNPs to account for missing data (Genome-wide LD was estimated by binning the genome-wide data into 50 evenly spaced recombination distance categories (0.005–1.000 cM). For each population and for every pair of SNPs within each distance category, we calculated the squared correlation  The data will only be used for studies of population history; 2) the data will not be used for medical or disease-related studies, or for studies of natural selection; 3) the data will not be distributed to anyone else; 4) the data will not be used for any commercial purposes; and 5) no attempt will be made to identify any of the sample donors.Molecular Biology and Evolution online.Supplementary DataClick here for additional data file.


Zostera marina (common name “eelgrass”) is one of the seagrass bed-forming species distributed widely in the northern hemisphere, including the Korean Peninsula. Recently, however, there has been a drastic decline in the population size of Z. marina worldwide, including Korea. We examined the current population genetic status of this species on the southern coast of Korea by estimating the levels of genetic diversity and genetic structure of 10 geographic populations using eight nuclear microsatellite markers. The level of genetic diversity was found to be significantly lower for populations on Jeju Island , which is located approximately 155 km off the southernmost region of the Korean Peninsula, than for those in the South Sea , which is on the southern coast of the mainland. South Korean eelgrass populations were substantially genetically divergent from one another (FST = 0.061–0.573), suggesting that limited contemporary gene flow has been taking place among populations. We also found weak but detectable temporal variation in genetic structure within a site over 10 years. In additional depth comparisons, statistically significant genetic differentiation was observed between shallow (or middle) and deep zones in two of three sites tested. Depleted genetic diversity, small effective population sizes (Ne) and limited connectivity for populations on Jeju Island indicate that these populations may be vulnerable to local extinction under changing environmental conditions, especially given that Jeju Island is one of the fastest warming regions around the world. Overall, our work will inform conservation and restoration efforts, including transplantation for eelgrass populations at the southern tip of the Korean Peninsula, for this ecologically important species.Seagrasses provide numerous ecosystem services for coastal and estuarine environments, such as nursery functions, erosion protection, pollution filtration, and carbon sequestration.  Seagrasses, marine angiosperms, play a pivotal role in ecosystem functioning and services in coastal zones. For example, they are primary producers and seagrass beds also provide important habitats, serving as both nursery and grazing areas for other marine organisms –3. They 2 of seagrass meadows have been lost during the past 127 years  within the 16 populations of Z. marina on the Korean coasts are summarized in PID and PIDsib were 0.0017 with a range of 0–0.0175 and 0.0436 with a range of 0.0074–0.2215, respectively, indicating a reasonable power to identify unique clones using our eight microsatellite markers and sample sizes  (HO) and expected (HE) heterozygosity in Jeju Island  were much lower than those in the South Sea  (n = 44) than for Jeju Island (n = 11). The KJ samples collected in 2015 showed a slightly higher level of AR than those collected in 2005  , suggest= 0.001) , Fig 2. ± 0.142] , Fig 2. = 0.483] . MoreoveFIS values within the Jeju Island and South Sea regions ranged from −0.198 (HD) to 0.251 (SH) and from −0.129 (AG-D) to 0.243 (KJ15-D), respectively. As a consequence, two populations  on Jeju Island and three populations  in the South Sea might be experiencing non-random mating  at the eight loci analyzed, based on our multilocus tests for Hardy-Weinberg equilibrium (HWE) expectations  to 0.243 (KJ15-D). The estimated frequencies of null alleles at the eight loci were close to zero, ranging from 0.020 (GA1) to 0.1521 (CT20), suggesting a low probability of null alleles. Tests of LD between the eight loci were not significant after a sequential Bonferroni correction, suggesting that all of the loci analyzed can be considered independent markers.The ctations . In JejuBOTTELNECK analysis revealed that only two populations (HD and OJ) within Jeju Island and one population (KJ05-S) within the South Sea had allelic distribution shifts (mode-shift), which are typically regarded as evidence of population bottlenecks . HoweverNe) of only 0.9 [95% confidence interval (CI): 0.6–1.5] and infinity (95% CI: 1.6 – ∞) for the WJ population and the OJ population on Jeju Island, respectively  for KJ05-D to an infinite Ne (95% CI: 50.1 – ∞) for JD-S , and FST values ranged from 0.009 (AG-S vs AG-M in the South Sea) to 0.573 (HD vs OJ in Jeju Island) (FST = 0.054 − 0.573) than for the South Sea region (FST = 0.005 − 0.346), suggesting that more restricted population connectivity was evident among populations in Jeju Island  , indicatu Island .FST statistics between shallow and deep populations for the KJ05 samples showed no significant difference . However, these populations after 10 years became significantly genetically divergent . Among samples from three depths  within the AG population, one population-pair was significantly genetically differentiated . There was no significant genetic differentiation detected between shallow and deep populations for JD   . The pai in 2015 .FST) for all 14 populations  , Jeju Island populations showed a lack of a significant correlation between them .The Mantel test showed a significant positive correlation between geographic (km) and genetic distances ( < 0.01) . Especia < 0.01) . While tZ. marina are most likely to form two genetically unique clusters (K = 2), which corresponds well to their geographical proximities   with an admixed population (SH), and two clusters , respectively  are shown in STRUCTURE analysis found that the 14 populations of uth Sea) . Two disuth Sea) . The numectively . GeneticK = 3) that appeared to be distributed broadly across the depths and sampling years, perhaps due to low levels of genetic divergence between the samples , STRUCTURE analysis suggested three genetic clusters  than those from the South Sea. The observed lower levels of genetic diversity in five populations from Jeju Island (mean AR = 1.92), which is located approximately 155 km off the mainland, relative to those from the South Sea (AR = 2.69), which is situated on the southern coast of the mainland, suggest that effective population sizes (Ne) have been smaller for the former than for the latter. Multifaceted lines of evidence support the hypothesis that Jeju Island populations have a smaller current Ne. Our microsatellite-based LD estimates of Ne indicate that a median of Ne at Jeju Island [mean Ne = 17.55 when OJ (Ne = infinity) was excluded] is approximately eight times smaller than that at the South Sea [mean Ne = 143.50 when NP and JD-S (Ne = infinity) was excluded], providing direct support for our hypothesis  of the temperate seagrass species, in Korea , harbor pothesis . Two of 2, WJ = 310 m2, OJ = 841 m2, TK = 4438 m2, and SH = 275736 m2; S.R. Park, unpublished data) estimated from Jeju Island appears to be commensurate with those previous reports. The SH population, which has the largest meadow area, shows the highest level of genetic diversity (AR = 2.26) whereas the HD population, which has the smallest area, shows the lowest level of genetic diversity (AR = 1.52). Although the WJ population’s coverage is approximately half that of OJ, the level of genetic diversity in WJ (AR = 2.12) is even higher than in OJ (AR = 1.69). This unexpected, relatively high genetic diversity in WJ is possibly due to a human-mediated transplantation project undertaken using other populations as a source material around this region in 2009 [Ne has been augmented in this population, which is supported by our estimates of Ne  but also small Ne.Geographically disconnected and genetically isolated populations with a relatively smaller  alleles . This di alleles . This is alleles . Therefo alleles , a high  alleles . In thisZ. marina on the southern coast of the Korean Peninsula comprises genetically divergent populations on a 0.7–250 km geographic scale at a given time. Our multifaceted analyses of a microsatellite dataset clearly reveal a noticeable level of genetic structure between populations in Jeju Island and those in the South Sea, as suggested by spatial AMOVA as well as an individual based Bayesian population assignment test and also FCA analyses. The observed significant genetic structuring between the Jeju Island and South Sea regions suggests a very low level of ongoing gene flow occurring between populations across the South Sea of Korea. Even within each region, microsatellite genetic differentiation was fairly high , suggesting that dispersal via rafting seems highly unlikely in this region, perhaps due to local currents. The restricted population connectivity, particularly among Jeju Island populations might, at least in part, explain why levels of within-population genetic diversity on Jeju Island are generally lower than in the South Sea, and this may ultimately lead to a reduction in population fitness, thereby elevating the risk of local extinction. On the other hand, directional selection can cause a reduction in genetic diversity rapidly, although it increases average population fitness. Yet, this scenario seems unlikely, given presumed selective neutrality of microsatellite markers used in this study.Most, but not all, other population genetics studies on spatial genetic variation in the eelgrass lometers ,71. This species ,19. Ther. marina : (1) sho. marina  and (2) . marina  and even. marina ,75. In tZ. marina population was maintained through only asexual reproduction  before 2006; however, seedling shoots via sexual reproduction have been observed since 2006, and their density has gradually increased in both shallow and deep zones . The observed weak temporal variation in the genetic structure can also be explained by the temporally varying clonal dynamics and genetic mosaic of eelgrasses [We also observed a statistically significant change in population structure between temporal samples (2005 vs 2015) in a shallow population of KJ in the South Sea . Twelve lgrasses . Our datZ. marina is known to be affected by various environmental parameters such as different light regimes [The life history or reproductive strategy of  regimes ,76, sali regimes . Accordi regimes , a deep Z. marina at two (AG and KJ) of the three localities in the South Sea region analyzed  and deep populations of analyzed . The resNe, and limited levels of genetic exchange among populations on Jeju Island–suggest an urgent need to conserve these vulnerable populations. Other populations with higher levels of genetic diversity could be used as source materials for restoration of the eelgrass populations through transplantation [Posidonia oceanica, a seagrass species endemic to the Mediterranean Sea, enriched genetic diversity of source populations was significantly positively correlated with individual survival, increased rhizome length , and number of ramets in the transplanted shoots [Knowledge about the population genetics of seagrass is an important issue for its effective conservation and restoration management ,25,28. Mantation . Howeverd shoots . Howeverd shoots .Zostera marina) populations, which will contribute to the establishment of appropriate management, conservation, and restoration plans for future persistence of this ecologically valuable species. We genotyped eight microsatellite loci for 454 individuals sampled from 16 populations  along the southern coastal regions of the Korean Peninsula . We found significantly lower levels of genetic diversity, smaller Ne and more restricted population connectivity  for Jeju Island compared with the mainland populations, suggesting that the southernmost populations off the Korean Peninsula are more vulnerable to local extinction under future changing environments. We suggest that the Jeju Island eelgrass populations should be conserved with high priority, given that this region is known to harbor the highest level of biodiversity in spite of being one of the fastest warming regions around the world. Further studies of Z. marina along the western and eastern coasts of the Korean Peninsula would help us to better understand the broader pattern of population genetic structure of Korean eelgrass and also to develop effective restoration strategies for this species.This study first provides the information on the genetic diversity and genetic structure of South Korean eelgrass Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


SLC30A6 with a weighted-average STF (WA-STF = 0.015), while other ZTGs exhibit considerably large population differences, such as SLC30A9 (WA-STF = 0.284). Overall, ZTGs harbor many more highly population-differentiated variants compared with random genes. Intriguingly, we found that SLC30A9 was underlying natural selection in both East Asians (EAS) and Africans (AFR) but in different directions. Notably, a non-synonymous variant (rs1047626) in SLC30A9 is almost fixed with 96.4% A in EAS and 92% G in AFR, respectively. Consequently, there are two different functional haplotypes exhibiting dominant abundance in AFR and EAS, respectively. Furthermore, a strong correlation was observed between the haplotype frequencies of SLC30A9 and distributions of zinc contents in soils or crops. We speculate that the genetic differentiation of ZTGs could directly contribute to population heterogeneity in zinc transporting capabilities and local adaptations of human populations in regard to the local zinc state or diets, which have both evolutionary and medical implications.Zinc transporters play important roles in all eukaryotes by maintaining the rational zinc concentration in cells. However, the diversity of zinc transporter genes (ZTGs) remains poorly studied. Here, we investigated the genetic diversity of 24 human ZTGs based on the 1000 Genomes data. Some ZTGs show small population differences, such as  SLC30A family lower intracellular cytoplasmic zinc by mediating zinc efflux from cells or influx into intracellular vesiclesSLC39A family transporters mobilize zinc in the opposite directionZinc (Zn), an essential trace mineral, is required for the structures and functions of many proteins, including enzymes and transcription factors, and is critical for their biological activities, such as cellular metabolism and gene expression14SLC39A4 alleles, leading to a diminished uptake of dietary zinc, increased sensitivity to zinc deficiency, and severe growth retardation121314et al. found that rs13266634, a non-synonymous SNP (R325W) in SLC30A8 is related to type 2 diabetesSLC30A8 encodes a zinc transporter which is expressed solely in secretory vesicles of β-cells and the overexpression of SLC30A8 in insulinoma cells increases glucose-simulated insulin secretionSLC39A8 was observed to have relationship with body mass indexBecause of the fundamental roles in biological processes, mutations in ZTGs are likely to break the balance of zinc in cell compartments, resulting in improper biological functions of zinc-dependent proteins and subsequent severe diseases or impaired development. For instance, studies have shown that acrodermatitis enteropathica OMIM 201100) was caused by the loss of function of one of or both 0 was cauet al. reported that the human intestinal zinc uptake transporter, SLC39A4, has undergone positive selection in Sub-Saharan African populationsSLC39A4 which was shown to lead to reduced zinc uptake in the African isoform. According to previous studies, zinc contents in soils or crops are extremely diverse across continents or countries and it is estimated that African populations have undergone severe zinc deficiency, according to various kinds of zinc deficiency indicatorsOn the other hand, functionally important ZTGs may also be subjected to positive selection for acquired changes that increase fitness. Recently, Engelken and Carnero SLC30A9 was underlying natural selection in both East Asians (EAS) and Africans (AFR) but in different directions. By performing a correlation, we found that the evolutionary force underlying the selective sweep of SLC30A9 may be the uneven worldwide zinc distribution in soils or corps. Moreover, we predicted 17 potentially functional SNPs, which may guide the study of molecular mechanism of ZTGs. Our results may subsequently increase our understanding of the evolutionary forces that affect ZTGs, as well as augmenting our knowledge of gene function on zinc homeostasis in different populations and the mechanisms of zinc-related diseases.We, therefore, systematically analyzed the patterns of genetic diversity and signals of natural selection for 24 14ZTGs in 14 worldwide populations. In this study, we showed that ZTGs harbor many more highly population-differentiated variants compared with random genes and discussed the potential underlying forces shaping the genetic diversity of ZTGs. Further, we reported that SLC30A9 and SLC30A3 had a greater proportion of variance among continental groups than within group , indicating that the two genes were genetically differentiated among continental populations. On the contrary, SLC30A6 showed a much lower proportion of variance between groups compared with most of the other genes genome-wide , suggesting that SLC30A6 may be functionally conserved.We first performed an analysis of molecular variance (AMOVA) to examine whether genetic variance among four continental regions is significantly different from populations within each region . Our anaSTF (referred to as WA-STF) for each gene. WA-STF employs a weighted average of the STF values calculated from variants in a gene or a genomic region. Among the 24 14ZTGs, SLC30A9 and SLC30A3 showed high WA-STF values , indicating that the variation composition is substantially different between populations. Therefore, the functional variants associated with specific haplotypes are also distributed heterogeneously across populations. On the contrary, SLC30A6 with a low WA-STF  may have limited genetic heterogeneity across populations  as the cutoff for a significant signal of population differentiation. As a result, compared to the STF distributions of 24 random genes, ZTGs showed significantly higher percentages of variants with high STF and average STF values . In total, there were 361 (of 19888) (1.82%) SNPs in ZTGs with STF > = 0.183, while the percentage was only 0.56%  in random genes  was also significant (see next section for more details). All these results suggested that the differentiations of ZTGs among populations were unlikely to be caused by any stochastic factors.We then calculated the unbiased locus-specific om genes . In parter genes . Finallyer genes . When weSTF distribution of all loci in ZTGs. However, the genetic variants that directly affect protein functions and gene expressions are functional SNPs, which are more likely to have true associations with zinc transporting or diseases. In our study, SNPs that (i) change the amino acid sequence of protein , (ii) correlate with gene expression (eQTL (expression quantitative trait loci) SNPs), (iii) alter splicing (splicing SNPs) and (iv) are associated with diseases  were investigated .The above analyses focused on general patterns of STF distributions of random genes, the ZTGs showed significantly higher percentages of variants with high STF; i.e. there were 932 out of a total 19,888 14SNPs with STF > = 0.092 (the top 5% STF cutoff) . We first predicted the functional types of all 19,888 14SNPs in ZTGs and 5,728 14SNPs located within the upstream and downstream 10 14Kb of ZTGs by consulting the variance effect prediction tools from the Ensembl website  (Compared to the  website . As show in GKB) .STF values across populations and therefore were assumed to have a higher potential functional impact on zinc transporting or were associated with diseases. Non-synonymous SNPs can cause a structural change in the protein product, which potentially leads to a minor or major phenotypic change. We applied Polyphen-2SLC39A4, predicted by HMMTOP  but was 0.080 in YRI , and high DAF in YRI but low DAF (<0.05) in CEU and CHB were significantly enriched in ZTGs (P = 0.019)  . TherefoSTF analyses revealed overall patterns of ZTGs genetic differentiations among worldwide populations. We then applied two commonly used statistics, the integrated haplotype score (iHS) and the composite likelihood ratio (CLR) , to detect the signals of selection in the ZTGs. We found ZTGs exhibiting evidence of recent positive selection. There was a certain consensus between the results of the iHS and CLR tests, which supported the reliability of the analysis  (SLC30A8 appeared to have been selected in primarily American populations (MXL and CLM)  and two African populations (YRI and LWK)  exhibiting significant signals in the CLR test. For instance, SLC39A9 shows strong selection signals in YRI and LWK. Four genes  showed overlapping signals in both methods in some populations. For example, SLC30A7 harbored both iHS and CLR signals in LWK and ASW with SLC30A8 in MXL and CLM. SLC39A11 had overlapping signals for both methods in YRI and ASW. Specifically, a previous study reported that SLC30A9 was strongly selected in East Asian populations, and it is among the top 10 selection signals in the genome-wide detection of positive selection in human populationsSLC30A9 underwent selection in different continental groups, including Africans and East Asians using both iHS and CLR tests   and S6. and CLM)  and S7. and LWK)  and S8.  and CLM  and S9. LR tests  and S8, SLC30A9 in each population and found that the haplotype favored in the African population was different from that found in East Asians and Europeans  with large negative values (13 of 26)  and rs28620429 (C) (blue color), it had long-range haplotype homozygosity, which was unusual given their frequency in CHB. However, if the haplotype included ancestral alleles (red color), it extended less compared with the comprised derived ones in CHB. However, the trend in YRI was completely the opposite. That is, the extended haplotype in YRI comprised the ancestral allele of rs4362859 (C) and rs28620429 (T), while the haplotype that included derived alleles had short-range homozygosity  , while i3 of 26) . That is3 of 26) . For the3 of 26) . As showzygosity  and F. TSLC30A9 in each population. Non-synonymous SNPs that may cause a structural change in protein can potentially lead to a minor or major phenotypic change. We observed that 9 different kinds of haplotypes of SLC30A9 were composed of 7 14nSNPs    , changine (M50V) . PreviouSLC30A9, we performed a correlation between the haplotype frequency of H2 in each population and the corresponding zinc deficiency status. Previous studies have estimated the global prevalence of zinc deficiencies based on zinc availability in national food supplies and the prevalence of stuntingSLC30A9. For instance, in YRI the frequency of H1 was 84.1%, but the zinc deficiency state was 21%, which is the highest among all populations. However, H2 was dominant in the populations with a low prevalence of zinc deficiency. For example, the frequencies of H2 in CEU (76.5%) and CHB (96.4%) were much higher than in YRI (8%), but the zinc deficiency states in CEU (9.6%) and CHB (11%) were lower than in YRI (21%). We then demonstrated a strong correlation between the haplotype frequency of H2 and the zinc deficiency state  in CEU and CHB are significantly enriched in ZTGs (P = 0.019)  , which hSTF, which revealed patterns of genetic differentiations that are incompatible with neutral expectations in ZTGs among worldwide populations. We then applied two commonly used approaches, iHS and CLR, to detect natural selection in ZTGs; several genes exhibited significant signals  that are shared among different continental populations. In principle, haplotypes inherited from ancestral populations might also lead to sharing selective signals between populations. However, this is likely a small effect because recombination for >1000 generations will break down such unusually long haplotypesThe analysis above was based on locus-specific  signals . In geneSLC30A9 was reported to be strongly selected in East Asian populations; it was among the top 10 selective signals in the genome-wide detection of positive selection in human populations26SLC30A9 in corresponding populations   . We haveSLC30A9, which exhibited a great proportion of high STF  play pivotal roles in decreasing or increasing zinc concentrations in cells and thus keep homeostasis of zinc in human body. Genetic variations in ZTGs could directly contribute to population heterogeneity in zinc transporting capabilities. In this study, we outline for the first time the genetic differentiation of worldwide populations and footprints of natural selection upon a comprehensive list of ZTGs. We demonstrated that high differentiations exist in ZTGs among populations. Besides, we identified 17 potentially functional SNPs with allele frequency highly differentiated among populations, which may affect either the protein structures or the expression levels of ZTGs. These results may enhance our understanding of the importance of zinc levels in human evolutionary history and facilitate further functional studies of ZTGs and medical studies on worldwide nutrient problem as well as zinc-related diseases.http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase1/analysis_results/supporting/ancestral_alignments/).We analyzed the latest release of the data  from the 1000 Genomes Project with autosomal SNPs of 1,092 individuals representing 14 populations worldwideSLC30 (ZnT) family and the SLC39 (Zip) familySLC30A1-10 and SLC39A1-14, respectively. Gene coordinate information was obtained from the UCSC Table Browser (http://genome.ucsc.edu/cgi-bin/hgTables) to infer the start and end position (hg19) for each gene  of multiple sites from haplotypes of each gene across all 14 populations. The genetic differentiation between populations of each locus was measured using the unbiased estimates of STF, following Weir and HillSTF value greater than the cutoffs were considered as highly differential SNPs (selected SNPs) or genes. As a result, the highest 1% and 5% of the genome-wide locus-specific STF is 0.183 and 0.092 with the average being 0.017.To evaluate whether the genetic variance between the four continental regions is significantly different from the genetic variance among populations within each region, AMOVA was carried out using ArlequinThe functional effects of each SNP from each ZTGs gene were obtained based on the variance effect prediction tools from the Ensembl database47STF distribution of ZTGs was different from random genes of the whole genome. We randomly sampled 24 unrepeated genes from the whole genome gene list 10,000 times. In every instance, we calculated the proportion of SNPs with STF values higher than 0.183. Finally, we obtained 10,000 percentage values representing the whole genome and 1 value (0.0182) for ZTGs; we then sorted these values in descending order. Thus, the index  of value for ZTGs represents the P-value of this permutation, which is 0.0478. The distribution of percentage values shows that the ratio (0.0182) of ZTGs is larger than the highest 5% cutoff value (0.0180) of the whole genome  in Africans are enriched in ZTGs, and to exclude the possibility that drift leads to this pattern. We randomly sampled 24 genes from the UCSC database without repetition 10,000 times. Every time, for random genes we calculated the proportion that the number of nSNPs with high DAF in YRI but low DAF (<0.05) in CEU and CHB accounted for the number of SNPs with high STF.The other permutation attempts to examine whether nSNPs with high Two approaches, the integrated haplotype score (iHS) and the composite likelihood ratio (CLR) test, were used to detect the signals of recent positive selection. Because of the hitchhiking effect, positive selection might bring a selected allele into high frequency rapidly enough that recombination does not have time to break down this haplotype, resulting in a long haplotype in high frequencyThe CLR test, a model-based method, is a statistic to compute the likelihood ratio of selective sweeps by comparing the spatial distribution of allele frequencies in a given window, compared to the frequency spectrum of null distribution, such as all the autosomal regionsSLC30A9, EHH plot and bifurcation diagrams were drawn using an R package rehhSLC30A9 spans about 97 14Kb in the chromosome region 4p13. We defined that SLC30A9 haplotypes were composed of 7 non-synonymous SNPs .Three indicators of zinc status at the population level has been recommended: (1) the percentage of the population with plasma (serum) zinc concentrations below an appropriate cut-off, (2) the prevalence of usual dietary zinc intakes below the Estimated Average Requirement (EAR), and (3) stunting prevalence535455The transmembrane helices and topology of ZTGs were predicted using HMMTOPSupplementary Information


Pleistocene climatic oscillations have greatly influenced the evolutionary history and distribution pattern of most extant species. However, their effects on species on the Qinghai-Tibet Plateau (QTP) are not well understood. To investigate the effects of past climatic shifts, particularly the Last Glacial Maximum (LGM), on plateau fish, we analysed the phylogeographic structure and demographic history of five closely related taxa of the subfamily Schizothoracinae, a representative endemic taxon of the QTP, from nine endorheic lakes on the central QTP and three peripheral exorheic rivers using the mitochondrial control region (D-loop) sequence and 12 microsatellite (SSR) markers. Phylogram from D-loop haplotypes revealed two well-supported lineages (North and South) separated by the Tanggula Mountains. The results from the D-loop and SSR revealed that endorheic populations possess high genetic diversity and a unique genetic structure. The most recent demographic expansion occurred post-LGM for most endorheic populations and in the last interglacial period for Siling Co and all exorheic populations. Phylogeographic structure, together with species distribution modelling, supports the scenario of multiple glacial refugia on the QTP during the LGM and suggests that Siling Co (4540 m asl) is a cryptic glacial microrefugia for plateau fish, which would be the highest glacial microrefugia known. During these glaciations, many species were excluded from large parts of their ranges and forced into refugia by the expanding ice sheets, followed by recolonization and population expansion as the glaciers retreated1. For example, in Europe, palaeontological records and genetic analyses have revealed that many species were retreated southward to the three Mediterranean peninsulas  during the LGM and expanded northward into their current distribution areas in the subsequent warming period3. Recently, biogeographic studies have led to the widespread acceptance of northern extra-Mediterranean refugia from the Atlantic coast of France in the west to the Black Sea region in the east, for example, the Carpathian Basin and even northern Central Europe4. Using phylogeographic studies, increasing numbers of previously unknown, or cryptic, refugia are identified5.The advances and retreats of glaciations through the Pleistocene, especially most recently the Last Glacial Maximum , have significantly affected the demographic history, abundance and distribution pattern of modern species2 and has an average altitude of 4500 m above sea level (asl). The QTP and adjacent mountains are the largest glaciated tracks outside the polar region6. Both plateau uplifting and Pleistocene climatic oscillations have greatly influenced the biotic pattern on the QTP8. Although the uplifting progress of the QTP remains controversial, most studies agree that the QTP attained its current state at least 150 kya before the last interglacial period (130–25 kya)9. The present biotic pattern on the QTP was mainly shaped by Pleistocene climatic oscillations6. Unlike Europe and North America, where continental glaciations occurred, only mountain and valley glaciers formed on the QTP during the LGM11. Therefore, in the common “edge refugia” model, the low-altitude southern and eastern edge of the QTP served as the glacial refugia for the plateau species; this model has proved insufficient for a comprehensive understanding of the effects of glaciations on the biota of the QTP14.The Qinghai-Tibetan Plateau (QTP) is the world’s highest and largest plateau, covering an area of 2.5 million km15. Some studies of endemic birds and plants have suggested that the south-eastern edge of the QTP acts as the glacial refugia18, while others have failed to detect “edge refugia” but suggest multiple refugia on the platform of the QTP20. Correspondingly, two main scenarios for the biota on the QTP have been proposed, i.e. postglacial recolonization from peripheral refugia or the survival of the LGM in situ, with several refugia on the platform. For both scenarios, evidence for the location of refugia and postglacial colonizing routes is scant15. Previous studies have mainly focused on plants, birds and amphibians in the periphery of the QTP. Therefore, more studies about additional endemic species on the central QTP are necessary for a thorough understanding of the influence of Pleistocene glaciations on the QTP biota.The influence of Pleistocene glaciations on the QTP have been studied in various species, but several basic questions, such as the locations of glacial refugia and the process of postglacial colonization, remain poorly understood21. Schizothoracine fishes (Teleostei: Cyprinidae) account for the largest component of the QTP ichthyofauna and are widely distributed in local lakes and rivers23. They have evolved a number of adaptations to cold-water environments and are regarded as the most diverse vertebrates on the central QTP25. Many lakes are located on the central QTP, and most are endorheic lakes26. As the migratory range of fish was limited by watercourses, it is likely that fishes in central QTP lakes recolonized postglacially from local refugia rather than “edge refugia”. Therefore, these lakes are ideal sites for investigating the potential of the central QTP to harbour glacial refugia.Freshwater fishes are regarded as the ideal subjects to explore the effects of glaciations because of their restriction in glacial refugia that are peripheral to ice sheets and the restrictive aquatic requirements for postglacial dispersalIn this study, we analysed the phylogeographic structure and demographic history of schizothoracine fishes on the central QTP and their closely related species in peripheral exorheic rivers using mitochondrial DNA (mtDNA) control region (D-loop) sequence and nuclear microsatellite markers (SSR). Species distribution modelling (SDM) was also used to predict the suitable habitat ranges for these schizothoracine fishes during the LGM. We sought to (i) assess the genetic diversity, phylogeographic structure and demographic history of those populations; (ii) infer whether the present schizothoracine fishes in the central QTP endorheic waters were recolonized from low-altitude peripheral rivers or persisted on the plateau platform through the LGM; and (iii) determine whether cryptic glacial refugia on the central QTP exist.Gymnocypris namensis and Schizopygopsis spp.) in nine endorheic lakes on the east Changtang Plateau, which is the central part and the earliest uplifted block of the QTP27, as well as their closely related species , in peripheral exorheic rivers . A total of 839 specimens belonging to five closely related taxa were collected from 19 sites in 2010 and 2015, ranging from 23–89 individuals per site  shared by 455 individuals were identified. The most common haplotype Hap10  was private to endorheic populations. In total, 20 haplotypes were shared across drainages  was high for most populations (0.854 ± 0.028), while the observed heterozygosity (HO) was lower (0.6134 ± 0.026). For all endorheic populations, Siling Co showed the highest h, HO and HE  and maximum likelihood (ML). The BI and ML methods obtained similar topologies; therefore, we only present the BI tree Fig. .In accordance with the phylogenetic analyses, spatial genetic analyses of mtDNA haplotypes revealed high values of uncorrected pairwise distance (Fst) between the upper Yangtze River (UY) and other populations (0.849 ± 0.09), suggesting that two defined groups were significantly differentiated. Fst values among populations south of the Tanggula Mountains were low (0.185 ± 0.117). Most of the Fst values among drainages (97%) were significantly higher than zero, suggesting a distinct genetic structure among drainages Table .Table 3AThe SSR marker analyses also revealed clear genetic differentiation among drainages. When conducting the STRUCTURE analysis, LnP (D) showed no peak and increased gradually up to K = 14, but the increase slowed after K = 6. In addition, ∆K declined severely from K = 2 to K = 4 and then showed a clear peak at K = 6 (data not shown). Therefore, K = 6 was considered as the most likely number of clusters. At K = 6, the drainages were separated from each other, except for Pongok Co (PC) and Nam Co (NC), which comprised a complex cluster with populations of Siling Co drainage Fig. . The SSRD were obtained for most populations, and significantly negative values of Fu’s Fs were obtained for more than half populations  and the raggedness index (Ri) calculated by goodness-of-fit tests were not significant (P > 0.05) for most populations  and the model for interdisciplinary research on climate (MIROC) predicted similar suitable habitat ranges during the LGM; therefore, we only show the result from the CCSM  eastern edge of the QTP acted as refugia for fish on the central QTP, the endorheic populations would not have a distinct genetic lineage, and the genetic diversity would have gradually decreased from peripheral populations to the interior. This was not the case in our study.The results from the phylogeny and genetic structure analyses of schizothoracine fishes on the central QTP were inconsistent with the hypothesis of “edge refugia”. Commonly, glacial refugia tend to harbour more ancestral haplotypes and higher genetic diversity than recolonized regions, and populations derived from separated refugia show clear genetic differentiation due to driftsin situ. The above results suggest that schizothoracine fishes on the Qiangtang Plateau survived the LGM in local refugia and that Siling Co, the largest lake in Tibet (2 200 km2), which is lower than most surrounding lakes32 and is likely a cryptic glacial refugia for local fishes.In our study, phylogenetic analyses and network analyses showed that the sublineage S1 of D-loop haplotypes was generally private to endorheic populations Figs . Secondl3. The demographic history reconstruction revealed that the most recent population growth of three exorheic rivers and Siling Co began approximately 100 kya, corresponding to the last interglacial period (130–25 kya). For most other endorheic populations, fast growth began approximately 20 kya, corresponding to post-LGM  was approximately 800 m lower than the equilibrium line during the LGM. In our sampling sites, Yueqia Co  was approximately 800 m lower than the equilibrium line. Now that schizothoracine fishes can currently complete their life history in Yueqia Co, they were likely able to complete their life history in Siling Co during the LGM. Using phylogeographic analyses, Wang et al.35 and Wang et al.36 discovered the highest microrefugia (just above 4000 m asl) ever reported. With an elevation of 4540 m asl, Siling Co is the highest glacial microrefugia known.The results from demographic history reconstruction, SDM and previous geological studies support our speculation. Glacial refugia provide protection from glaciations; therefore, populations in refugia would have a longer demographic history than would postglacially expanded populationsLGM Fig. . The Nagiod 130–2 kya. For37. In total, four or five glaciations have occurred on the QTP since the Pleistocene, the last being the LGM38. During the warm and wet interglacial periods, increased rainfall and melting water expanded the lake’s area and generated several temporary channels among drainages39. For example, during the last interglacial period, the QTP experienced a “high lake level period” (40–25 kya), in which the temperature was 2–4 °C higher and precipitation was 40–100% higher than today40. During that period, many presently isolated lakes, such as Nam Co, Siling Co and Pangkog Co, were expanded several times and connected with each other. In addition, the endorheic and exorheic drainages were also connected at certain times by temporary watercourses42. These temporary connections propelled gene flow among drainages, reducing their phylogenetic and genetic differentiation. They also served as migration routes for glacial retreat and postglacial dispersion.Phylogram from D-loop haplotypes revealed two well-supported lineages (lineages N and S) separated by the high intervening Tanggula Mountains. Lineage S was further divided into four main sublineages. Freshwater fish have restrictive aquatic requirements for dispersal. However, we noticed that the bootstrap values of the four sublineages are low Fig.  and 2. I43. Compared with mtDNA sequences, SSR markers presented higher variability and faster mutation rate and provided more information to detect recent divergence44. In this study, most results from mtDNA data are consistent with those from nuclear microsatellites, except for the Bam Co and Gomang Co populations. In mtDNA analyses, haplotypes from Bam Co and Gomang Co group with those from other endorheic populations  to be a cryptic microrefugia for schizothoracine fish on the central QTP that survived the LGM. Our study also illustrates how postglacial lake expansion has influenced the genetic structure among the drainages of those species, especially for the endorheic drainages.The methods involving animals in this study were conducted in accordance with the Laboratory Animal Management Principles of China. All experimental protocols were approved by the Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.22. Measurements were taken immediately after collection. The fin tissue of each specimen was collected and preserved in 95% ethanol. Total genomic DNA was extracted using a standard phenol–chloroform method46. DNA concentration was estimated using a NanoDrop 2000 supermicro spectrophotometer  and adjusted to 50 ng /μL for polymerase chain reaction (PCR).Specimens were sampled with gill nets and electrofishing. Fish identification was according to Fauna Sinica2), dNTPs at 150 μM, forward and reverse primers at 10 pM, 1.0 unit Taq polymerase (TaKaRa) and 50 ng genomic DNA. PCR was performed with a preliminary denaturation at 94 °C for 3 min; followed by 30 cycles of strand denaturation at 94 °C for 40 s, annealing at 55 °C for 50 s and primer extension at 72 °C for 45 s; and a final extension at 72 °C for 10 min. The PCR products were sequenced on an ABI 3730 capillary sequencer from the Sangon Biotech Company . Sequences were checked with Bioedit v. 7.2.547 and corrected manually.The mtDNA D-loop sequence was amplified using the pair of primers DL (5′-ACCCCTGGCTCCCAAAGC-3′) and DH (5′-ATCTTAGCATCTTCAGTG-3′) in a 60 μL mixture containing 6 μL 10 × PCR buffer , mean number of pairwise differences (p), haplotype (h) and nucleotide (π) diversity) were calculated for each population with Arlequin v 3.5.150.The D-loop sequences were initially aligned with the program Clustal × 2.151 and PhyML 3.052 respectively. Two D-loop sequences of Gymnocypris przewalskii and Gymnocypris eckloni were designated as the outgroup. The best nucleotide substitution model (GTR + G + I) was selected among 88 alternative models by the Bayesian Information Criteria (BIC) using jModelTest v2.1.453. For Bayesian analyses, two independent runs with four  Markov Chain Monte Carlo (MCMC) were conducted with the GTR + G + I substitution model, run for 20 000 000 generations and 1/1000 sample frequency. Based on convergence diagnostics, the first 4 000 trees (standard deviation of split frequencies <0.01) were discarded as burn-in. Nodal support was calculated using the mean posterior probability (BBP) values of each node of the resulting consensus tree after burn-in. For ML analysis, the node support was assessed using the nonparametric bootstrap method with 1000 replicates. The tree was visualized with FigTree v1.4.254.Phylogenetic analyses of D-loop haplotypes were conducted using BI and ML, by MrBayes v3.2.355 with a 95% criterion for a parsimonious connection. Excessive connections in the network were removed based on coalescent theory56. Genetic differentiation among populations was assessed by pairwise genetic distance (Fst) values. In this section, we treated every river as a single site. The genetic structure within and among drainages was further estimated by AMOVA. Populations within one drainage were pooled into one group. Both analyses were implemented in Arlequin v3.5.150.A statistical parsimony network of haplotypes was constructed using TCS1.2.1D57 and Fu’s Fs58 tests to detect population expansions. The significance of both values was calculated from 10 000 simulated samples using a coalescent algorithm. Second, we performed pairwise mismatch distribution analysis (MDA) to compare theoretical distributions under sudden expansion with observed data. Goodness-of-fit was evaluated by the SSD and Raggedness index (Ri) between observed and expected mismatch distributions. The above two analyses were conducted using Arlequin 3.5.150. Finally, we generated the BSP for each population using a Bayesian MCMC algorithm in BEAST 1.8.2 and BEAUti 1.8.2 to convert the molecular sequences of each lake into the input file59. The best nucleotide substitution model was selected by jModelTest 2.1.4., using the Akaike Information Criterion (AIC).To assess the historical changes in effective population size, we used multiple approaches. First, we calculated Tajima’s b sequence of the genus Gymnocypris has been calibrated by the isolation between the upper reaches of the Yellow River and Qinghai Lake, which is a reliable geological event25. We calculated the divergence rate of cyt b and D-loop sequences between H. microcephalus in Tuotuo He and Schizopygopsis pylzovi in the Yellow River using MEGA 6.060. Based on the observed divergence rate of 1.98% for cyt b and 2.32% for the D-loop, we estimated an evolutionary rate of 1.07% per million years for the D-loop sequence. We used a strict clock model and four independent runs of 200 million generations, sampling every 2 000 runs. Tracer 1.6 was used to confirm stationary and convergence, determine the burn-in value (10%) and create the BSP61. All runs were combined using LogCombiner 1.8.2 and created a consensus tree using TreeAnnotator 1.8.2, from the BEAST package.An evolutionary rate of 0.91% per million years for the cyt Schizopygopsis younghusbandi63 were used to screen polymorphisms in 40 individuals from four populations . PCR was performed as previously described, except for some variations in annealing temperature. The amplified fragments were separated on a 12% non-denaturing polyacrylamide gel. We chose 12 pairs of highly polymorphic SSR primers that amplified reliably across four populations for further genetic analyses  and expected heterozygosity (HE) were calculated using the program AUTOTET65. Fst values among populations were estimated by POLYSAT66. The population genetic structure was assessed by a Bayesian model-based method in STRUCTURE 2.3.467. We chose the admixture model for the ancestry of individuals and the correlated model for allele frequencies. The length of burn-in was set to 10 000, with 100 000 MCMC replication runs after burn-in. To avoid bias, we performed the program from K = 1 to 14, with 30 iterations at each value of K. The optimum value of clusters (K) was determined by comparison of mean log probability Ln P (D) and by calculating the ∆K value for each K68.The number of alleles per locus (Na), allelic richness (Ai) and observed , with a spatial resolution of 2.5 arc-minutes. The altitude data for these sites were recorded by GPS. Highly correlated environmental variables were removed using multicollinearity test by ENMtools v1.370, and six bioclim-variables  and altitude were ultimately used.We used SDM to predict the potential range of these five taxa in the present and during the LGM. Models were generated with MaxEnt v3.3.315 Table . We down67. Model evaluation was based on the AUC71.Both the CCSM and MIROC were used to generate the environmental variables for the LGM. Those data layers were also downloaded from WorldClim. We chose the climatic-topographic models (climate model) for both time periods and set the convergence threshold and number of maximum iterations at default values of 0.00001and 500 respectively. We assigned 75% of the occurrence point as training data and the remaining 25% as testing data. The number of maximum iterations and convergence threshold were set at default values of 500 and 0.00001, respectively. We ran five fold cross-validation for each model and produced a single map per time period using the logistic output format, with suitability values ranging from 0 to 1Supplementary information


Cycas dolichophylla-an endemic but widely distributed Cycas in Southwest China and North Vietnam along the RRF were carried out based on four chloroplast DNA intergenic spacers (cpDNA), three nuclear DNA sequences (nDNA) and 16 simple sequence repeat variations (SSR). Two different phylogeographical patterns were detected: a Southwest-Northeast break across the RRF disclosed by chlorotypes and a China-Vietnam separation revealed by SSR. A Bayesian skyline plot from cpDNA data demonstrated a historical increasing, but a recent declining, dynamic in population size during the Pleistocene. Consequently, we infer it is the local environmental variation during Cenozoic that contributed to the complex landscape and microclimate mosaics, facilitating speciation and divergence of C. dolichophylla. Subsequently, the Quaternary climatic fluctuations coupled with human activities profoundly influenced the genetic structure and demographic history of this species.Dramatic crustal deformation and river incision in Southwest China induced by the Indo-Asian collision have long been argued to contribute to the complicated landscapes, heterogeneous environment and abundant biodiversity in this region. However, biological impacts in promoting intraspecific phylogeographical subdivision and divergence along the Red River Fault zone (RRF) remain poorly understood. To investigate the possible biological effects of tectonic movements and environment variations within the RRF, the phylogeography of  Cycas worldwide is supposed to be not much older than ~8 Ma161718In recent years, accumulating evidence from tectonics, paleontology and climate simulations increasingly supported a view that the main body of the Tibetan Plateau (TP) reached its current height 4–5 km) since the mid-Eocene123–5 km sin1234111Cycas dolichophylla K. D. Hill, T. H. Nguyen & K. L. Phan is an ideal species for inferring the potential biological/biogeographical model within the RRF. On one hand, the distribution of this organism is restricted to the Red River drainage region , three nuclear sequences (nDNA) and 16 simple sequence repeats (SSR) in 13 populations across its entire range with an attempt to test the following hypotheses: (i) local environmental variation in its habitats influenced the lineage divergence of C. dolichophylla; (ii) climate variations, especially the glacial-interglacial cycles during the Quaternary re-sculptured this species’ genetic structure and accelerated its diversification.Here, a phylogeographical study of C. dolichophylla to be 1.94 Ma (95% HPD: 1.30–2.62 Ma) and retrieved the ‘northeastern’ and ‘southwestern’ chlorotypes as reciprocally monophyletic lineages, with coalescence time dated to about 1.84 Ma (95% HPD: 1.22–2.51 Ma) (Fst (Fst > 0.25) suggested significant genetic differentiation among populations based on Wright’sNm < 1)  . AMOVA r2.51 Ma) . The higsNm < 1) .GTP, PHYP and PPRC, were 573–574 bp, 940–946 bp as well as 720 bp, respectively. For GTP, 14 polymorphism loci were identified with 12 haplotypes confirmed  and BN was restricted to P1 although this haplotype was shared by CP, HS, MG and XL    . However0.87 Ma) . Apart fRC: 86%) .C. dolichophylla populations when the ΔK statistics were applied. When considering the second fittest number of grouping, which suggested as K = 3, it was almost similar to the case in K = 2 with only minor subdivision. Additionally, all the structure analysis derived from UPGMA and PCo , GTP (55.08% vs 44.92%) and PPRC (59.24% vs 40.76%). However, 74.3% of genetic variations within populations and 25.7% among populations were detected from SSR data. The fixation indexes calculated by these three nuclear genes as well as SSRs were all greater than threshold 0.25, indicating significant genetic variation between populations. The gene flows inferred from nuclear genes were lower than the critical value 1  was much lower than the total diversity (HT = 0.994), and both NST (0.834) and GST (0.678) were high , which was different from estimating of population subdivision , that was, significant phylogeographical structure was detected in southwest (P = 0.018) but not in northeast (P = 0.051). In accordance with results of phylogeographical structure inference, Mantel test uncovered a significant pattern of IBD for cpDNA at the range-wide scale    and in sPHYP  than in GTP  or PPRC . Consequently, both the NST and GST estimated by PHYP were lower than the other two genes. However, no significant difference between NST and GST was detected on these three gene fragments. In other words, no significant effect of IBD was detected, as the correlation between genetic distance and geographic distance was not significant (P > 0.05).The genetic diversities  were mucC. dolichophylla populations as well as northeastern and southwestern groups displayed a multimodal distribution pattern with non-significant SSD statistic and HRag value under a sudden demographic expansion model, except for the significant SSD value detected at the whole chlorotypes level. Similarly, positive values of Fu and Li’s D*, Fu and Li’s F*, Fu’s Fs rejected the sudden expansion model   .C. dolichophylla experienced population expansion  and a northeastern clade occupying the northeast region . On the other hand, most of the tectonic process in Southwest China happened during or even before the Miocene, such as uplift, crustal thickening, strike-slip and river incision123Cycas species in this region. Subsequently, the local environmental variation contributed to the formation of microclimate pocketsC. dolichophylla, as well as the subsequent differentiation of the Southeastern and Northwestern subcladesThe discovery of two distinct groups with cpDNA data was not unexpected, given the complex topography and heterogeneous environmental conditions in Southwest China, especially along the RRF. However, for a newly radiated species, the local habitat differences in the immense region with varying altitudes, soils, slopes as well as ecological selection from pollinators and seed transporters, seemed more promising factors to explain the diversification of ocene 1.9 Ma. On tC. dolichophylla to undergo spatial expansion and population growth. Nonetheless, with the persistence of cold conditions until the late Ionian Stage (0.3–0.13 Ma) together with the onset of Riss glacial cycle, the enlarged C. dolichophylla populations began to shrink. Moreover, during the last stage of the last glacial period (32–12 ka), cold and arid climatic conditions established, accompanied by a strengthened Asian winter monsoon dominating over continental Southeast AsiaC. dolichophylla to scattered habitats and thus hindered gene flow between populations. Although the Bottleneck analysis based on SSR data failed to register any recent bottleneck event on this species, the small G-W indices implied a historical bottleneck, which coincided with the demography history (C. dolichophylla estimated by LDNe (During the largest Naynayxungla Glaciation (0.72–0.50 Ma) when valley glaciers dominated history . Concurr by LDNe .C. dolichophylla. That is, tectonic activities, river incision as well as the reinforcement of Asian monsoon systems didn’t work directly on speciation, but local habitat differences coupled with ecological selection from pollinators and seed transporters gave birth to the speciation of C. dolichophylla. Whereas the alternation of glacial and interglacial periods from the mid-Pleistocene onwards played critical roles in shaping this species’ population structures and demographic history.In conclusion, our results together with evidence from previous researches jointly support the perspective that it is the climate changes and environmental heterogeneity (pockets of microclimate) that gradually cast the evolutionary history of C. dolichophylla in RRF were collected, which covered almost the entire range of this species PHYP) and hypothetical protein (PPRC) , were selected for subsequent PCR amplifications analysis. Microsatellite markers were selected from recently developed 65 nuclear microsatellites in Cycas394041424344Genomic DNA was extracted from the silica-dried leaves by modified CTAB methodHs), total genetic diversity for species (HT) and coefficient of population differentiation C. edentata and C. rumphii served as outgroups. For the MP trees, multi-trees heuristic search and tree bisection-reconnection branch swapping were adopted with 1000 repeats bootstrap analysis performing on PAUP. For BI tree search, the Markov chain Monte Carlo (MCMC) runs started from independent random trees and extended for 1.0 × 107 generations, were repeated twice with trees sampled every 1000 generations implemented in MrBayes 3.1.2Prior to conducting all analyses, congruence test was generated by PAUP* 4.0 b10Cycas, previously estimated rates for seed plants, 1.01 × 10−9 and 5.1–7.1 × 10−9 substitutions per site per year for synonymous sites (s/s/y) for cpDNA and nDNA, respectively, were adopted in this analysis7 generations, sampling once every 1000 generations. All the above parameters were performed by BEAUti 1.6.1 in the BEAST package. TreeAnnotator 1.6.1 was used to create a maximum clade credibility tree with the first 10% trees discarded as burn-in5758C. dolichophylla was inferred from Bayesian skyline plots, which were generated by BEAST. Maps were drawn using the software ArcGIS version 10.2 (http://desktop.arcgis.com).Divergence times among haplotypes were estimated under a Bayesian framework in BEAST 1.6.1Nm) between populations were calculated with formula Nm = (1-FST)/4FSTFST acquired from Arlequin. AMOVA analysis was performed on Arlequin. Isolation by distance (IBD) was tested by performing Mantel tests in GenAlEx using a correlation of geographic distance with genetic distance for all pairs of populations. To infer genetic structures of sampled populations and individuals, unweighted pair group mean analysis (UPGMA) were carried out on TEPGA, version 1.3O) was visualized by the program MVSP, version 3.125 iterations and 1 × 105 subsequent MCMC steps were performed. In addition, the second-order rate of change of the natural logarithm probability of the data with respect to the number of clusters (ΔK) was evaluated to deduce the best fit number of grouping with the online tool STRUCTURE HARVESTER V.0.6.93The indices of genetic diversity and genetic differentiation were calculated using GenAlEx version 6.4.1NE) with the linkage disequilibrium (LD) method implemented in LDNeTo detect recent bottlenecks caused by reduction in effective population size, the observed gene diversity was compared with the equilibrium gene diversity given the observed number of alleles implementing in BOTTLENECK version 1.2.02How to cite this article: Zheng, Y. et al. Tectonic and climatic impacts on the biota within the Red River Fault, evidence from phylogeography of Cycas dolichophylla (Cycadaceae). Sci. Rep.6, 33540; doi: 10.1038/srep33540 (2016).


Camellia flavida is an endangered species of yellow camellia growing in limestone mountains in southwest China. The current classification of C. flavida into two varieties, var. flavida and var. patens, is controversial. We conducted a genetic analysis of C. flavida to determine its taxonomic structure. A total of 188 individual plants from 20 populations across the entire distribution range in southwest China were analyzed using two DNA fragments: a chloroplast DNA fragment from the small single copy region and a single-copy nuclear gene called phenylalanine ammonia-lyase (PAL). Sequences from both chloroplast and nuclear DNA were highly diverse; with high levels of genetic differentiation and restricted gene flow. This result can be attributed to the high habitat heterogeneity in limestone karst, which isolates C. flavida populations from each other. Our nuclear DNA results demonstrate that there are three differentiated groups within C. flavida: var. flavida 1, var. flavida 2, and var. patens. These genetic groupings are consistent with the morphological characteristics of the plants. We suggest that the samples included in this study constitute three taxa and the var. flavida 2 group is the genuine C. flavida. The three groups should be recognized as three management units for conservation concerns. Camellia (Theaceae) species with yellow flowers, known as yellow camellia, grow in parts of south China and north Vietnam. Studies have reported 10–16 species in China .Yellow camellia are valuable ornamental plants and genetic resources for breeding. Moreover, yellow camellia leaves and flowers are used in traditional Chinese medicine and commercially available teas  according to the classification by Ming and Bartholomew  C. ptilosperma and C. longruiensis should be considered synonyms of C. longgangensis, (2) C. longgangensis var. patens and C. multipetala should be classified as a synonym of C. quinqueloculosa, and (3) C. wumingensis is an independent species . Effective conservation of endangered species requires accurate taxonomic classification, and incorrect taxonomy can lead to erroneous conservation decisions  and a single-copy nuclear gene. Plastid and nuclear markers are frequently used in phylogeographic and population genetic studies, as they present different features  clarify the species classification and boundaries, and (3) propose recommendations for guiding future preservation actions.Molecular genetics analyses can help resolve the taxonomic uncertainties and define management units within species  was amplified and sequenced using a primer design based on the sequences from Camellia taliensis  region containing several genes was amplified using the following three primer pairs: SSC1, CP30 , 5 μL of dNTP mix (10 mM), 0.5 μL of each primer (50 μM), and 2.5 U of LA Taq DNA polymerase . DNA was amplified in a thermal cycler , programmed for an initial denaturation at 94°C for 3 min; followed by 30 cycles of 30 s denaturation at 94°C, 30 s annealing at various annealing temperatures  . Cloned PCR products were purified and sequenced by Sangon Biotech . At least four clones were sequenced per PCR product (maximum eight clones). All haplotype sequences in this study were deposited in GenBank: KX751947–KX751960, KU669063, KU669073, KU669071, and KX751961–KX752048.Previously reported karyotype analysis demonstrated that by Clark . For seqPAL gene was tested using DAMBE software . A post-processing MP calculation was used to search for the shortest tree. Several values of the parameter ε were explored, without significant changes in network topology. The topology presented was obtained at the default settings (ε = 5). The geographical distribution of populations was mapped using ArcMap GIS , the gene diversity (h) Nei, , and theIS ESRI, .Camellia oleifera (JQ975031) and Camellia pitardii (KF156837) and nuclear sequences of C. taliensis H16 (JX161631) and H17 (JX161632) were used as outgroups for analysis of C. flavida.Phylogenetic analyses of the identified haplotypes for each marker were performed. Maximum-likelihood (ML) tree generation and bootstrap analyses were performed with the program RAxML-HPC-SSE3  calculated using DNAsp v5 was used to assess the degree of genetic differentiation between populations.The presence of phylogeographic structures was inferred by testing for significant differences between FCT value after the 100 independent simulated annealing processes is retained as the best grouping of populations. In addition, for nuclear DNA data, analysis of the Monmonier's algorithm was implemented with the BARRIER 2.2 program, which identifies possible barriers to gene flow among the most differentiated groups of populations, creating a Delaunay triangulation network to connect adjacent populations and, consequently, a Voronoi tessellation set  was conducted with SAMOVA 2.0. This approach defines groups of populations that are geographically homogeneous and maximally differentiated from each other  . Haplotype C_15 was separated from C_16 and C_17 by a minimum of eight mutational steps   Table .GST = 0.976 and NST = 0.974, NST < GST; P > 0.05). Low gene flow (Nm = 0.01) among the 20 C. flavida populations was detected by the cpDNA sequences.The contribution of phylogenetic relationships between haplotypes to among-population differentiation was nonsignificant  and found that they were significantly smaller than the corresponding ISS.C values , indicating that substitution in the PAL gene region is not saturated. The alignment of 372 PAL gene sequences from 186 samples was 653 base pairs long and contained 52 parsimony informative sites, 17 single-nucleotide polymorphisms, and no indels, resulting in 87 distinct haplotypes  or shared only very common haplotypes . Total haplotype and nucleotide diversities were 0.97993 and 0.00880, respectively (Table h) ranged from 0.27895 to 0.92105, and nucleotide diversity (π) ranged from 0.00044 to 0.00968 , and the lowest were in population WM  , group II , and group III .In the SAMOVA analysis, ps Table . These tPAL haplotype network is presented in Figure patens (H_67 through H_87) were clustered together and situated in the middle of the network, separating haplotypes of var. flavida 1  from those of var. flavida 2 (H_58–H_66). In the ML tree, although the bootstrap value was low, all the haplotypes of var. flavida 2 and most haplotypes of var. patens clustered together respectively . Nm value (0.35) detected by nuclear DNA sequences indicated that gene flow among populations was limited.Our analyses of C. flavida populations were grouped into the three PAL SAMOVA groups (Table PAL SAMOVA groups (Table flavida and var. patens in the PAL data was low (17.81%) and accounts for 53.28% of the genetic variation within populations  was observed within populations, but the variation among groups was much higher (31.19%). Thus, three genetic groups might be optimal for these populations.Over 60% of the genetic variation in cpDNA was attributable to variation among varieties; 57.95% of the variation was explained if the ps Table . The witps Table . In contns Table . When thA), the haplotype diversity (h), and the nucleotide diversity (π) of each PAL SAMOVA group are shown in Table flavida 1 group had the highest level of genetic diversity . The var. flavida 2 group  and var. patens  have a relatively lower level of genetic diversity.The number of haplotypes . The Monmonier algorithm (barrier program) suggested four main barriers to gene flow in the distribution range of all populations . Three barriers  separate group var. flavida 2 populations  from a group of var. flavida 1 populations  separates WM from SG , barriers were found in geographically close populations in addition to the barriers between distant populations  in C. flavida were higher than those in the congener, C. taliensis, which is believed to possess abundant variation  , similar to plant species with the highest cpDNA differentiation .This study of 20 populations of PAL gene data was also high (GST = 0.253 and NST = 0.414) compared with that in other angiosperm species . AMOVA analysis indicated that most of the genetic diversity existed within populations (51.58%), but the among-groups variance (31.19%) is also very large. In our analysis of this nuclear marker, the gene flow (Nm) among populations was 0.35, and was primarily achieved by pollen transfer, given the lack of gene flow via seeds; however, gene flow between populations appears to be rather restricted. The mode of pollination and dispersal of C. flavida are poorly understood; however, bees are effective pollen vectors for its congeners Camellia oleifera  and Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection , Ministry of Education, China (grant number ERESEP2015Z01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amphiprion akallopsisos) across the Indian Ocean, and on a smaller geographic scale in the Western Indian Ocean (WIO). Highly restricted gene flow was discovered between populations on either side of the Indian Ocean using the control region as a mitochondrial marker (mtDNA). We verify this conclusion using 13 microsatellite markers and infer fine scale genetic structuring within the WIO. In total 387 samples from 21 sites were analysed using mtDNA and 13 microsatellite loci. Analysis included estimation of genetic diversity and population differentiation. A haplotype network was inferred using mtDNA. Nuclear markers were used in Bayesian clustering and a principal component analysis. Both markers confirmed strong genetic differentiation between WIO and Eastern Indian Ocean (EIO) populations, and a shallower population structure among Malagasy and East African mainland populations. Limited gene flow across the Mozambique Channel may be explained by its complex oceanography, which could cause local retention of larvae, limiting dispersal between Madagascar and the East African coast. Two other potential current-mediated barriers to larval dispersal suggested in the WIO, the split of the SEC at approximately 10° S and the convergence of the Somali Current with the East African Coast Current at approximately 3° S, were not found to form a barrier to gene flow in this species.In this contribution, we determine the genetic population structure in the Skunk Clownfish ( Coral reefs are threatened worldwide by several human induced factors, such as overfishing, tourism, or pollution. They are at risk to convert to an alternative, macroalgae-dominated state that would alter their ecosystem characteristics. This process is further accelerated by human-induced environmental stresses, such as global warming and ocean acidification, making urgent conservation measures a necessity . A very Since gene flow is almost exclusively realised through larval dispersal in coral reef associated organisms, patterns of larval dispersal have been studied to evaluate connectivity in these organisms using various methods, such as population genetics and phylogeography, direct tagging of larvae, parentage analysis, otolith chemistry, and biophysical models , 5. An oResults of population genetic studies of reef associated organisms spanning the whole Indian Ocean basin display a wide variety of population structure, with some revealing the existence of a barrier to gene flow between the Western Indian Ocean (WIO) and the Eastern Indian Ocean (EIO) –10, whilPleuronectes platessa, a mtDNA marker enabled to define population structure more precisely than nuclear markers, i.e. microsatellite loci [st-values and allele drop out [Another factor capable of influencing the results of a phylogeographic study is the choice of genetic markers. Because of its matrilineal inheritance, mtDNA is likely to be more severely influenced by bottleneck events in populations compared to nuclear DNA due to lower effective population size. Consequently, mtDNA diversity is often very low in populations having suffered bottlenecks and may not have returned to equilibrium state yet after such an event. This will mask more recent genetic differentiation and means that mtDNA will often only provide evidence for historical gene flow . The useite loci . Microsadrop out , 28. Micdrop out . Considedrop out .Myripristis berndtii, while mtDNA did not [Lutjanus kasmira both markers were congruent [A. ocellaris in the Indo-Malay Archipelago, results from both markers were congruent, but population structure inferred from mtDNA was more pronounced [In studies where both marker types are simultaneously used, mito-nuclear discordance, or differences between results from mtDNA and nuclear markers, is common. Several explanations, such as introgression, sex-biased dispersal, selection, and different effective population sizes have been suggested . More sp did not , but in ongruent . A few sonounced . In threonounced –36.The population structure of the skunk clownfish has been studied using mtDNA, showing highly restricted gene flow between populations in the WIO and the EIO. However, only a weak genetic population structure was detected in the WIO and none in the EIO . In thisA. akallopisos individuals were sampled between one and 25 m depth at 18 sites in the WIO and three in the EIO  Ali Hassan Mwinyi Road, Kijitonyama Area. P.O. Box 4302, Dar es Salaam, Tanzania; Zanzibar Research Committee, PO Box 239, Stone Town, Zanzibar, Tanzania; Kenya: National Council for Science and Technology, Nairobi, Research permit No: NCST/RRI/12/1/BS/250; Mozambique: Universidade Eduardo Mondlane, Escola de Ciências Marinhas e Costeiras (ECMC), Quelimane; Indonesia: Universitas Hasanuddin, Makassar, Research permit No: 55/PSTK/UH/XI/04; Madagascar: Ministère de l'Environnement, de l'Ecologie et des Forêts; Autorisation de recherche: N°287/15/MEEMF/SG/DGF/DAPT/SCBT. DNA from the samples from Kenya, Tanzania, Indonesia, and Madagascar (partly) was extracted with the QIAGEN  extraction kit, and DNA from samples from Madagascar (Sainte Marie) and Mozambique was extracted with the E.Z.N.A. Tissue DNA Kit , following the manufacturer’s protocol.CR-A (5’-TTCCACCTCTAACTCCCAAAGCTAG-3’) and CR-E (5’-CCTGAAGTAGGAACCAGATG-3’) [2, 0.4 μM of each primer, 0.2 mM dNTPs, and 1 U Taq polymerase. The following temperature profile was used: 94 °C for 5 minutes, followed by 35–40 cycles of 1 minute at 94 °C, 1.5 minutes at 45 °C and 1 min at 72 °C. Final extension was conducted at 72 °C for 5 minutes. Sequencing was done with an ABI 3770XL automated sequencer . The sequences were edited using the software ChromasPro . A multiple alignment was done using Clustal W [s-test [A fragment of the CR was amplified by polymerase chain reaction (PCR) using the primers GATG-3’) . The PCRlustal W  as implelustal W . Haplotylustal W . The prolustal W  was usedlustal W  and Fu’s [s-test , as well [s-test  were als [s-test .Amphiprion , and 2.5 μL H2O. The following temperature profile was used: 95 °C for 5 minutes once, then 95 °C for 30 seconds, 1.5 minutes at 57 °C for annealing and 30 seconds at 72 °C for extension, repeated during 35 cycles. Finally, a last extension was done at 68 °C for 30 minutes. The PCR product was diluted 20 times and mixed with buffer (HiDi) and GeneScan™-500 LIZ® size standard  and then analysed on an ABI 3100 Automated Sequencer (Applied Biosystems). The resulting electropherograms were scored manually with GeneMarker V2.6.3 . The dataset was tested for the presence of null alleles with Microchecker v1.0 [Multiplex PCR was conducted using the four different fluorescent labels 6-FAM, VIC, PET and NED  with these two sets of primer pairs. The PCR was conducted in an Eppendorf Ep S Mastercycler with a volume of 12.5 μL for each PCR that contained 2.5 μL DNA template and 10 μL of a master mix containing 1.25 μL primer mix (containing 2 μM of each primer), 6.25 μL Multiplex PCR Master Mix , containing optimised concentrations of HotStarTaqker v1.0 . To testker v1.0 . Arlequiker v1.0  was usedker v1.0 .Finally, a Principal Component Analysis (PCA) and Discriminant Analysis of Principal Components (DAPC), which uses an algorithm that clusters individuals in groups optimising between group variance and minimising within-group variance, was done using the program AdeGenet in R .st-values  and therefore false positive errors in population structure inference, although a higher number of loci seems to temper this effect [https://www.dcode.fr/tirage-au-sort-nombre-aleatoire, last accessed 26/12/2017) and discarded the other individuals. We then compared the ϕst, ϕct, Fst, and Fct-values obtained in these reduced datasets with the values obtained using the complete dataset. We calculated the difference between the value of the complete dataset and the reduced datasets and then tested whether the set of differences was statistically different from 0, using the z-test function in Excel.When analysing population genetic structure, the accuracy of the analysis critically depends on the capacity of the sampled individuals to represent the gene pool of the population they originated from . Especias effect , 54. Ourst-values (< 0.1), even though significantly different from 0, can be misleading as estimates for gene flow [st-values and Fct-values of AMOVA significantly different from 0 in Arlequin using 20.000 permutations and only accepted the ϕ/Fst-values where the 95% confidence interval did not overlap with 0. Only when the 95% confidence interval does not overlap with 0, it can be accepted as an indication of limited gene flow among populations [Furthermore, it has been pointed out that low ϕ/Fene flow . To compulations .Sequences from 74 individuals from three different sites in Mozambique as well as from 23 additional individuals from two different sites in Madagascar were successfully edited and aligned with the 263 sequences from Huyghe & Kochzius (2016) , resultiOne of the 16 initially selected primer pairs AC137;  did not In the dataset of 360 individuals, 98 haplotypes were identified, of which none occurred simultaneously in the WIO and the EIO . Just liAs for the microsatellite data, mean observed heterozygosity over all loci was moderate in both the EIO populations (0.769–0.646) and the WIO populations (0.743–0.520). Allelic richness was higher in the EIO (11.15–8.85) than in the WIO (9.62–4.69).st-values as significantly different from 0 applying the False Discovery Rate approach was set at p<0.0241. All values significantly different from 0 were tested for overlap of the 95% confidence interval with 0  and pairwise ϕst-values . Population structure was clearly caused by the differentiation of the population of Sainte Marie at the East coast of Madagascar from the other WIO populations . Hierarand Tofo . Within and Tofo .The results from the Migrate analysis confirmed the very limited gene flow between the WIO and the EIO. The 95% posterior distribution interval for migration in both directions between WIO and EIO overlapped with 0. The distribution interval gives the probability that the actual value of the assessed parameter falls within the interval. Within the WIO, 95% posterior distribution also overlapped with 0, except for estimates of migration from West-Madagascar to East-Madagascar. In the EIO, gene flow was equal and relatively high in both directions .st—values significantly different from 0 were equally checked for 95% confidence interval overlap with 0  as for mtDNA analysis was applied, and Fp with 0 . Differeto 0.151 . This wato 0.151 . Low levto 0.151 .The highest ΔK in Evanno’s test was reached for K = 2 (ΔK = 1312.862). In a STRUCTURE analysis conducted under this scenario the WIO populations were almost exclusively assigned to cluster one and EIO populations almost exclusively assigned to cluster two . When a st = 0.012; p < 0.001). Three distinct groups could be derived from the pairwise Fst-values. One population from the North of Kenya, Watamu, had a high number of significant pairwise Fst-values (12/17 pairwise comparisons) with the other populations of the WIO, as well as the three populations from Madagascar: Nosy Bé, Tuléar and Sainte Marie  followed by K = 3 (89.823). When a STRUCTURE analysis was conducted with both scenarios, however, clustering of individuals did not correspond to two or three geographically distinct areas, but rather failed to assign individuals to either of the two or three clusters .In PCA analysis, individuals belonging to the Northern Kenyan populations (Watamu and Lamu), Madagascar, and the rest of the WIO populations, were not differentiated . Howeverst-values that were significantly different from 0 dropped from 81 to 56 (69%) and 55 (68%) in Control Region analysis  and 64 (59%) in the reduced datasets  based on microsatellites. This could be a consequence of the low sample size at this site. As illustrated with tests using a reduced number of samples for all populations , a lowerAnother possible explanation can be derived from the fact that mtDNA is maternally inherited. The skunk clown fish, like all anemone fishes, lives in size-based hierarchical groups with only one female per group of four to eight individuals , 63. Effst-values based on microsatellite markers suggested a moderate genetic break between the North Kenyan population of Watamu and other populations in the WIO. Geographically, genetic differentiation between the population at Watamu and populations further South corresponds to a suggested barrier to larval dispersal mediated by the confluence of the Somali Current and the EACC (barrier 3), which can prevent larval dispersal across this zone [Contrary to the mtDNA analysis, pairwise Fhis zone , 16, 17.his zone . GeneticThis study confirms earlier findings on a barrier to gene flow and larval dispersal separating the WIO and EIO populations of the skunk clownfish. It also identifies fine scale population structure within the WIO. Two of the three suggested current-mediated barriers to larval dispersal, the split of the SEC at approximately 10° S, and the convergence of the Somali Current with the EACC at approximately 3° S, were not found to form a barrier to gene flow in this species. The third, caused by the complex oceanographic nature of the Mozambique Channel, could promote genetic isolation among Malagasy and African mainland populations. The exact geographic location of this differentiation, however, shifted depending on the genetic markers used. Estimated gene flow within the WIO was found to be stronger from East to West than in the other direction, which corresponds to the direction of the dominant sea surface current, but was also different between marker types. The latter could be an indication of variability of gene flow over time. Furthermore, significant population structure has been detected among populations of the skunk clownfish within the Mozambique Channel, confirming the possible restricting influence of eddies on gene flow in the Mozambique Channel, at least in some reef fish species .S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(A) Pairwise ϕst-values among populations using reduced datasets. (B) Pairwise Fst-values among populations using reduced datasets. (C) Composition of reduced populations with eight randomly chosen individuals. (D) ϕct–values of hierarchical AMOVAs. (E) Fct–values of hierarchical AMOVAs.(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.