Amphilophus spp. has been proven to be a powerful model system for the study of ecological specialization, sexual selection and the mechanisms of sympatric speciation. Here we present a comprehensive and integrative phylogeographic analysis of the complete Midas Cichlid species complex in Nicaragua  covering the entire distributional range, using two types of molecular markers . We investigated the majority of known lake populations of this species complex and reconstructed their colonization history in order to distinguish between alternative speciation scenarios.Elucidation of the mechanisms driving speciation requires detailed knowledge about the phylogenetic relationships and phylogeography of the incipient species within their entire ranges as well as their colonization history. The Midas cichlid species complex We found that the large lakes contain older and more diverse Midas Cichlid populations, while all crater lakes hold younger and genetically less variable species assemblages. The large lakes appear to have repeatedly acted as source populations for all crater lakes, and our data indicate that faunal exchange among crater lakes is extremely unlikely. Despite their very recent (often only a few thousand years old) and common origin from the two large Nicaraguan lakes, all crater lake Midas Cichlid radiations underwent independent, but parallel, evolution, and comprise distinct genetic units. Indeed several of these crater lakes contain multiple genetically distinct incipient species that most likely arose through sympatric speciation. Several crater lake radiations can be traced back to a single ancestral line, but some appear to have more than one founding lineage. The timing of the colonization(s) of each crater lake differs, although most of them occurred more (probably much more) recently than 20,000 years ago.The genetic differentiation of the crater lake populations is directly related to the number of founding lineages, but independent of the timing of colonization. Interestingly, levels of phenotypic differentiation, and speciation events, appeared independent of both factors. Despie.g., ), specia., [e.g. ). Albeit., [e.g. ,11,13-23., [e.g. ,24.e.g., [The study of biogeography and phylogeography can provide baseline information on the question as to whether speciation requires complete geographic isolation or not, by revealing contemporary and historical gene flow among populations and incipient species . One proe.g., ). One ape.g., . In ordee.g., ,28,29. He.g., [Cichlids (Family Cichlidae) are tropical freshwater fishes occurring in southern and Central America, Africa, Madagascar and India. They are one of the best models for the study of biological diversification, since this family of fish comprises both the fastest rates of evolution e.g., ,30-36) a-36 ae.g.e.g., ,37-43. Te.g., ,41,44,45e.g., ,41,46-48e.g., ,49.Cichlids have also radiated albeit to a lesser extent in smaller-scale lake environments. Two well-known examples of such radiations are the Cameroonian  and the Amphilophus radiation exist in an group of crater lakes that are located on the western part of Nicaragua in Central America right on the Pacific Ring of Fire, as well as in the Large Nicaraguan Lakes, Managua and Nicaragua [Cichlasoma citrinellum - now Amphilophus citrinellus), and a specialist thick-lipped form restricted to the large lakes Managua and Nicaragua  he goes on: "It is possible that more than one species should be recognized here, and no doubt such will some day be the case, especially if some enthusiastic student of fishes has at his command far less amount of material than I have had the opportunity to examine."Species of the icaragua -57; Figuicaragua . Initialabiatus, ,60). A tzaliosus ). Severazaliosus ,56,61,62zaliosus  about C.e.g., whether particular body plans or trophic strategies evolved only once or multiple times, or whether given lineages are the consequence of single or repeated colonization events within confined water bodies - it is compulsory to have detailed knowledge of the phylogeography, population history and colonization history of the species complex throughout its range . To . To e.g.et al. [ca. 100 fish from 9 water bodies, covering the majority of the area of distribution. Here, using over 2000 fish samples and two types of molecular markers  we reconstruct the phylogeography of the entire Midas cichlid complex. We first describe the phenotypic diversity encountered in each lake. We assign individuals to populations based on nuclear genetic data to test for the genetic cohesiveness of each lake/crater lake population. We then focus on the Large Nicaraguan lakes, which represent the source populations, because they contain the largest, oldest and most stable populations, and investigate the genetic diversity and structure of their species and populations. Following this, we investigate the age and colonization history of each crater lake, and estimate the timing, source and size of the colonization events. We discuss the relative importance of the colonizing population size versus the timing of colonization, and relate it to present levels of intralacustrine phenotypic differentiation. We also discuss the apparent disconnection found between current genetic and phenotypic diversity within lakes. Later we will discuss how our results compare with those of the study by Geiger et al. [Previous studies have partially reconstructed the phylogeography of the Midas Cichlid species complex in some of the lakes mostly based on mitochondrial DNA sequences ,66,67 GeA. citrinellus with cryptic dark normal and conspicuous gold coloration  and Momotombo. In Lake Nicaragua A. labiatus was observed at all sampled localities (but >15 samples were only collected in Isletas in Granada where this species was highly abundant). In Tisma we found both normal and gold A. citrinellus. In crater lakes Asososca Managua and Asososca Leon we found mostly high-bodied A. citrinellus forms, but also a few elongated individuals . In crater Lake Xiloá we found two types of high-bodied individuals, one with yellowish coloration breeding at smaller sizes and living in shallower habitats, and another type with both normal coloration (most abundant) and gold coloration individuals, which breed at bigger sizes and inhabit deeper habitats. We also found an elongated form living in the open water with both normal and gold coloration. These three forms have been described as new species . In crater Lake Apoyo, similarly to Xiloá, we found two types of high-bodied fish, one breeding at smaller sizes and living in shallower habitats, and another breeding at bigger sizes and inhabiting deeper habitats. These two morphs have been described as new species  similar to the small high-bodied form but with yellowish coloration. We collected a few individuals that responded to this description (Lorenzo J. Lopez Pérez pers. com.) (< 15). A detailed list of all specimens included in this study, voucher identifications and their sampling localities is provided in Additional file The number of samples obtained in each of the studied water bodies differed which was by differences in accessibility and abundance. Localities from which we were able to collect less that 15 samples where not used for population genetic approaches. We found diverse body plans and color types in all studied lakes. In the large lakes we found high-bodied ion (see ) in all ctively; ). A thicctively; , but altectively ); we alsA. citrinellus, Lake Nicaragua). Details on descriptive statistics and genetic diversity indexes averaged across loci are shown in Table A. citrinellus and A. labiatus) followed by the populations in Lake Managua and Tisma Pond. Genetic diversity was significantly higher in the large lakes and Tisma Pond than in the crater lakes . There was no evidence for systematic scoring errors according to MICRO-CHECKER. Linkage disequilibrium between pairs of loci was non-significant for every comparison. Genotype distributions were generally in accordance with expected Hardy-Weinberg proportions, and only 19 out of 180 population-locus combinations showed significant deviations, but did not consistently involve the same loci.The 15 microsatellite loci analyzed  plus Tisma Pond, and Lake Nicaragua (A. citrinellus and A. labiatus). We did a further clustering analysis restricted to the populations within the large lakes, and found no differentiation between the two species, A. citrinellus and A. labiatus within any of the two lakes. STRUCTURE revealed k = 2  clearly differentiated the species in crater Lake Apoyo from all other populations, as did the previous clustering method. PCoA2  separated, although not significantly, the species within crater Lake Apoyo, and populations in crater lakes Apoyeque and Asososca León from the rest . PCoA2  separated these two populations. Hence, the third analysis  distinguished most clearly the species in crater Lake Xiloá  and crater Lake Masaya (which showed some overlap with Lake Nicaragua) from the rest, and less clearly the samples from crater Lake Asososca Managua. PCoA2  separated crater lakes Xiloá and Masaya from crater Lake Asososca Managua. The last analysis included only the samples from the two large lakes in Nicaragua (both A. citrinellus and A. labiatus) and Tisma Pond. PCoA1  discriminated between the two lakes but revealed some degree of overlap, and clumped the samples from Tisma Pond together with those of Lake Managua  to investigate the relative position of each lake population within a multidimensional space Figure . The rest Figure . The sect Figure  was perfs Figure  was perfST Φ= 0.23; microsatellites ST F= 0.16). The few exceptions from this general finding involved the fish from the large lakes, fish from Tisma Pond and fish from the River San Juan  and Tisma Pond, contained the largest amount of genetic diversity with the largest maximum distance between haplotypes  were detected in the two large lakes. We found signatures of extremely recent expansions in crater Lake Apoyeque . Significant Tajima's D with very negative values were found for all populations but those from Tisma Pond, Asososca León and Asososca Managua  are rivA. citrinellus from the upper Rio San Juan are genetically indistinguishable from those in the lake , probably due to habitat similarity and local adaptations to similar environmental conditions. Some hidden diversity (both genetic and phenotypic) could still remain undiscovered due to the relative turbidity of the large lakes, although this possibility seems unlikely because of the extensive work in the area over the years.The sheer size of the large Nicaraguan lakes, they are the largest in Central America, might favor the divergence of geographically distant populations within lakes. Cichlid species widespread in the large African lakes often show allopatric diversification, even forming allopatric local species in some instances (see review in ). In botA. citrinellus, and the seemingly more specialized and only locally abundant thick-lipped species A. labiatus. These two species are morphologically quite distinct, which suggests that they also are ecologically segregated, although ecological studies are still lacking. Amphilophus labiatus have very characteristic fleshy lips [A. labiatus, as other fleshy-lipped cichlids, linked to the presence of rocky habitats-which is also reflected in our sample collection, while A. citrinellus was mostly found on sandy substrates in the large lakes [Two species of the Midas Cichlid complex co-occur in sympatry in the large Nicaraguan lakes, the widespread and common shy lips , a charashy lips , and morshy lips , traits shy lips ,75. The shy lips . In agrege lakes .Amphilophus labiatus was collected from several sites in Lake Nicaragua, and populations across the lake showed some allopatric differentiation. Sympatric populations of the two species showed genetic differentiation, but the Bayesian assignment tests did not discriminate between them , and A. labiatus from this site was consistently genetically significantly different from all A. citrinellus populations. However, since no sympatric sample of A. citrinellus from this site could be collected, it is not possible to distinguish between species segregation due to ecological adaptation or allopatric differentiation.A. citrinellus and A. labiatus are morphologically and ecologically clearly differentiated, they remain genetically surprisingly indistinguishable . This genetic distinction has also been found using AFLP data on a smaller sample of Midas cichlids, corroborating the robustness of this result [An interesting result of this study is that despite the recent origin of most crater lakes , the Midas Cichlid populations in each of them had their own characteristic genetic signature, clearly differentiated from any other population in the area , but also the number of founding lineages . Other factors likely to influence genetic composition are demographic events after colonization.The unique genetic signature of each crater lake population appears to be influenced by the combination of two main factors, the age of colonization  our estimations would correspond to establishment of the recent population rather than colonization per se. Older populations in the Midas Cichlid system are characterized by higher numbers of private alleles and a distinct allelic composition, but not necessarily by pronounced phenotypic differentiation. Younger populations are typically genetically undifferentiated from the source population, although in some cases contain novel morphotypes, that have not been described in the large source lakes. Remarkably, we find a pronounced disconnection between genetic and phenotypic diversity. According to the demographic analyses based on mtDNA sequences, the oldest colonization was most likely in crater Lake Asososca León, which expanded within the lake about 3-4 mutations ago . Population expansions occurring less than one mutation ago were consistently found in Asososca Managua  and Apoyeque , linhttp://www.ineter.gob.ni/), and young estimated demographic history. This is the case for crater lakes Xiloá and Masaya  .http://www.ineter.gob.ni/; [Both crater lakes Asososca Managua and Apoyeque have a signature typical of small and genetically homogeneous colonizations, since all mtDNA haplotypes are connected to each other by single mutations, and the amount of genetic diversity is muted. Alternatively, those populations might have experienced a bottleneck due to volcanic activity after colonization. The time since their colonization is estimated to be particularly short  which igob.ni/; ). Periodgob.ni/; ,79, The gob.ni/; . It shoui.e., all mtDNA haplotypes are private to the lake; Figure A. zaliosus, an endemic species adapted to open water, originated in this lake and evolved sympatrically with the ancestral Midas cichlid population within the lake [in situ in the lake, and make alternative evolutionary scenarios , far lenization ).e.g., a river, thus explaining the lack of sharing of mtDNA haplotypes with their populations. Samples of other fish species from this lake would allow exploring this, albeit unlikely, hypothesis. Although this lake has not been thoroughly studied, no major morphological or genetic diversification among the Midas cichlids from there has been detected, despite the presence of a few more elongated individuals.Making inferences about the evolutionary processes that have occurred in crater Lake Asososca León is rendered difficult since the colonization of this lake occurred a relatively long time ago compared to the other crater lakes. We show here, as we did previously also for crater Lake Apoyo , that alin situ, we find the strongest genetic differentiation among species in lakes where founder populations were small and genetically homogeneous, regardless of their age. Bottleneck effects most strongly shape the genetic structure of crater lake populations at neutral loci. Interestingly, the evolution of phenotypic differentiation does not seem to be related to genetic diversity . Most likely, differences in ecological opportunity  among crater lakes determine the degree of morphological, behavioral and ecological diversity contained in each system. The validation of this hypothesis would require a more formal description of all the phenotypic diversity as well as the characterization of the ecological niches contained in each lake, an effort that is still incomplete.The combined effect of both timing and size of the colonizing populations will influence the genetic composition of newly formed crater lake populations, together with possible demographic events after colonization linked to volcanic activity. Although the longer ago a colonization event occurred, the more likely it is for populations to have diversified The large lakes are the most likely source populations of all crater lake Midas cichlids. The origin of the Midas Cichlid in crater Lake Asososca León is difficult to reconstruct since it shows no obvious genetic affinities to any other studied population. Crater Lake Apoyeque has a genetic composition that would be compatible with several population sources, since the most common haplotype within the lake is also the overall central one (haplotype C); also the most common haplotype in the majority of the other lakes, including the large lakes. Its geographical position suggests that its fauna could have its origin in Lake Managua. Crater Lake Xiloá could be a potential source as well, and nuclear markers cluster these populations together , suggest that evolution Fish of the Midas Cichlid species complex were collected during the dry season (February-March) of 2001 and the wet seasons (November-December) of 2003 and 2005 with gill nets, harpooning while scuba diving, and with the help of local fishermen, from twelve different locations in Nicaragua, covering the main range in which these species are found . The collections included the two Large Nicaraguan Lakes - L. Managua and L. Nicaragua ; BurtKit, Salzburger et al. unpubl.; see Additional file Total DNA was isolated using a proteinase pitation . Individ, Acit6, ; Unh011,: TmoM7, ; Unh002,: TmoM7, ; and fiv: TmoM7, ,  method. Posterior probabilities were calculated in multiple runs, where each run consisted of 500,000 iterations with a burn-in period of 50,000. We set K between 1-15, and performed 10 replicates for each value of K. Pritchard et al. [STRUCTURAMA v.1.0 [Two different approaches were used in an effort to examine the genetic clustering of the populations in the different lakes. First, the Bayesian clustering method as implemented in E v. 2.2  was usedd et al.  suggest d et al. . TherefoMA v.1.0 , where bMA v.1.0 . This meMA v.1.0 . The resMA v.1.0 .ST differentiation per axes were calculated with 1000 permutations with PCAGEN v.1.0 [Second, a Principal Coordinate Analysis (PCoA) of individual diploid genotypes was performed as implemented in GENALEX v.6  to visuaEN v.1.0 .In the last two analyses (PCoA and Bayesian clustering) in order to have a compensated number of samples due to software limitations, we used only a subset of the samples representing all localities and morphotypes of the fish with a maximum of 100 individuals per lake and species.The complete mitochondrial control region (836 bp) was PCR amplified using published primers and reaction conditions  and verified by eye. The alignment was collapsed into the representative haplotypes with COLLAPSE v. 1.2 iversity  per lakeiversity . A modeliversity . jMODELT0 (2 μN0) and θ1 (2 μN1) using a parametric bootstrap approach (1000 simulations), where μ is the mutation rate and N is the female effective population size. Tajima's D [A mismatch analysis was performed to estimate the demographic history of the species complex from each of the lakes Figure , Table 3jima's D  analysisF-statistics [Genetic differences among and within lakes were estimated with pairwise atistics  as impleatistics .MB and AM designed the study and obtained the material. MB did the molecular work and the genetic analyses, and wrote a first draft of the manuscript. Both authors completed and approved the final manuscript.Marta Barluenga is an evolutionary ecologist interested in speciation and the origin of adaptive radiations. Axel Meyer is an evolutionary geneticist interested in the ecological, developmental and genomic aspects of speciation and organismal diversification.Supplementary Table 1. Table with all the individuals included in the study with species identification, individual ID, lake of origin, GenBank accession numbers of mitochondrial control region sequences, and haplotype ID (see Figure Click here for fileSupplementary materials. This additional file contains information about the microsatellite primers used in the study, and additional details on the results of the clustering analyses with STRUCTURE and STRUCTURAMA, and pairwise statistics.Click here for file


Rhinopithecus roxellana) by comparing genetic variation found in MHC and microsatellite loci. There are few studies of this kind conducted on highly endangered primate species.Small, isolated populations often experience loss of genetic variation due to random genetic drift. Unlike neutral or nearly neutral markers , major histocompatibility complex (MHC) genes in these populations may retain high levels of polymorphism due to balancing selection. The relative roles of balancing selection and genetic drift in either small isolated or bottlenecked populations remain controversial. In this study, we examined the mechanisms maintaining polymorphisms of MHC genes in small isolated populations of the endangered golden snub-nosed monkey  population. Historical balancing selection was revealed at both the DQA1 and DQB1 loci, as revealed by excess non-synonymous substitutions at antigen binding sites (ABS) and maximum-likelihood-based random-site models. Patterns of microsatellite variation revealed population structure. FST outlier analysis showed that population differentiation at the two MHC loci was similar to the microsatellite loci.Two MHC genes were sequenced and sixteen microsatellite loci were genotyped from samples representing three isolated populations. We isolated nine MHC genes and microsatellite loci showed the same allelic richness pattern with the lowest genetic variation occurring in SNJ, suggesting that genetic drift played a prominent role in these isolated populations. As MHC genes are subject to selective pressures, the maintenance of genetic variation is of particular interest in small, long-isolated populations. The results of this study may contribute to captive breeding and translocation programs for endangered species. Understanding how levels of genetic variation influence the survival of threatened species is of fundamental interest to evolutionary and conservation biologists because many natural populations are threatened by intense reduction and fragmentation of habitat, leading to isolation, declining populations, and decreasing genetic diversity ,2. Loss Cervus dama) [Mirounga angustirostris) [Triturus cristatus) [Petrogale lateralis lateralis) [FST outlier analysis [FST) [FST values are computed for all genes to distinguish the genes under selection from those non-selective genes [However, some functionally important genes that are maintained by balancing selection, such as major histocompatibility complex (MHC) genes, may have a different evolutionary pattern compared with neutral markers. The multi-gene MHC family is found in vertebrates, codes for cell surface glycoproteins, and is important in animal conservation due to its role in resisting pathogens . Compareus dama) , northerrostris) , great cistatus) , and theteralis) . These rteralis) . To elucanalysis  is widelis [FST) . The FSTve genes .Rhinopithecus roxellana), an endangered primate endemic to China where it inhabits three isolated areas: Sichuan and Gansu provinces (SG); the Qinling Mountains, Shaanxi province (QL) and the Shennongjia Forestry District, Hubei province (SNJ)  in SNJ is approximately 80 individuals [We studied MHC and neutral genetic variation in the golden snub-nosed monkey  Figure . Currentividuals . The smaividuals ,31. In tHE) and observed heterozygosity (HO) was in the SG population, while the lowest level was in the SNJ population . Linkage disequilibrium occurred in the SG population (p = 0.01). Population recombination analysis using LDhat revealed that the DQB1 locus had a higher recombination rate (ρ = 7) when all alleles were included between the MHC loci .No more than two alleles were present per individual, suggesting that for each MHC locus one gene copy was sequenced with these primer sets. No stop codon or insertion/deletion was detected in K (ΔK = 95.500 at K = 2). The pairwise FST was the largest between SNJ and the other 2 populations among all of the FST values , and results showed that in addition to the divergence between SG and QL, there was also divergence within the SG and QL populations. These results agree with earlier studies, where the differences within the SG and QL populations resulted from their different origins and habitat fragmentation [DQA1 locus or the DQB1 locus . No clear clade could be identified from the phylogenetic tree at these two loci. Populations had more private alleles at DQB1 than DQA1  among 16 DQB1 exon 2 alleles were shared among all of the populations . Comparisons of pairwise FST values of MHC and microsatellite sites are shown in Figure FST values showed lower divergence among populations compared with microsatellite loci (except SG-SNJ and QL-SNJ at the DQB1 locus). Half of the FST values at the MHC loci were higher than 0.05, indicating that differences existed among populations, especially between SG and SNJ. The population difference was more distinct at the DQB1 locus: the differentiations between the SG and SNJ populations and between the QL and SNJ populations were even larger than the differentiation at the microsatellite.Microsatellite-based analyses detected significant genetic structures among the populations Figure . Two gens Figure , which sentation ,34. The dN) mutations was greater than the number of synonymous (dS) mutations using the Z-test   matched MHC better than the other models based on the Akaike information criterion (AIC) values  Table . AlthougFST outlier analysis did not reveal a high or low level of differentiation among populations at the DQA1 and DQB1 loci compared with 16 microsatellite loci  [Ursus arctos) [Canis simensis) [Bison bonasus) [Panthera tigris tigris) [Panthera pardus pardus) [DQA1 and 16 DQB1 alleles found in 64 individuals. However, we found lower MHC variation in the SNJ population, which also showed lower genetic diversity in microsatellites and mitochondrial genes [Habitat isolation generates barriers to gene flow among populations that often result in loss of genetic diversity through genetic drift and inbreeding . For vergorilla) , brown b arctos) , Ethiopiimensis) , Europeabonasus) , Bengal  tigris) , and Nam pardus) . In thisal genes ,34. Smalal genes .For each MHC locus, regardless of how many clones were sequenced from an individual, no more than 2 alleles were observed in an individual, a strong indicator that we amplified single loci in all cases. We assumed that all alleles were from a single functional gene. The assumption that our sequences were from functional genes was supported by three findings: 1) sites inferred to have been exposed to significant selection, most of which were ABS sites, indicating historical selection at functional genes; 2) no reading frame or stop codon disruptions found in any alleles; and 3) the sequences identified from cDNA were parts of or included sequences obtained from DNA.DQA1 ABS may due to a weaker recombination within DQA1 as a lower recombination rate has been shown before. Without higher recombination and stronger selection, some DQA1 alleles might be lost when population size decreased. The sharing of MHC alleles among populations also indicates that MHC alleles may have been conserved by selection [Golden snub-nosed monkey MHC genes reflect historical balancing selection in that an excess of non-synonymous substitutions was mainly concentrated in the ABS  showed a FST value that was lower than the neutral level, indicating its linkage with other genes under selection [Although selection historically maintained MHC diversity, recent population isolation and fragmentation has increased the role of genetic drift in shaping patterns of MHC variation in snub-nosed monkeys. First, compared with neutral forces, balancing selection is supposed to diminish population differentiation as measured by conventional pairwise FST,52. ThusFST. Howevers Figure . Second,election . Lastly,election . Maintenelection . In all,Ne . Therefore, the smaller Ne becomes, the greater the likelihood of genetic drift [Triturus cristatus) [Petrogale lateralis lateralis) [Sphenodon spp.) [Neophron percnopterus) [The positive correlation in the allelic richness of MHC and microsatellites, together with other evidence, indicates that genetic drift has a great influence on the maintenance of MHC variations in small, isolated populations of snub-nosed monkeys ,25,55. Nic drift . The SNJic drift . Other aistatus) , black-fteralis) , tuataraon spp.) , and theopterus) . These rVariation at MHC loci is widely accepted as being maintained by balancing selection (reviewed in 2), even with a low level of neutral variability in some species ,59. ThisR. roxellana samples  from the current population  , and 10–100 ng DNA template. The amplification profile consisted of 5 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C (DQA1) or 58°C (DQB1), and 30 s at 72°C, with a final extension of 10 min at 72°C. The following primers were used for amplifying exon 2: 5'DQA1-AAGCCCA TAATATT TGAAAGTCAGT and 3'DQA1-TATGTGATTTTAGAGATGGGAGATG, or 5'DQB1-TGTAAAC GACGGCCAGTTCCCCGCAGAGGATTTCGTG and 3'DQB1-TGCTCTAGAGGGCGACGACGACGCCTCACCTC [DQA1 primers were designed based on BLAST sequences from relative species. According to the manufacturer’s protocols, a Wizard PCR Preps DNA Purification Kit (Promega) was used to purify the PCR products. Purified PCR products were cloned using the pMD-18T vector (Takara) following manufacturer’s instructions. Ten to twenty clones containing inserts from each individual were sequenced on an ABI 377 or ABI-PRISMTM 3100 Genetic Analyzer (Applied Biosystems Inc.) with the Prism BigDyeTM Terminator Ready Reaction Kit (Applied Biosystems Inc.).The PCR was carried out in a 50 μL solution including 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 2.5 mM MgClCTCACCTC . The DQAMacaca mulatta exons. All amplified cDNA products were analysed by cloning and sequencing as described above.Expression analyses were conducted to validate the expression of the obtained MHC II-DQA and DQB alleles. The RNA was isolated from 2.5 mL whole blood using Trizol (Invitrogen) following the manufacturer’s instructions. To ensure that the genomic DNA was removed from the isolated RNA, a second DNA digestion was performed using the DNase I RNase-free Set (Promega). The cDNA synthesis was obtained using 200 U of M-MuLV Reverse Transcriptase (Promega) in a 25 μL reaction tube containing 1μL Oligo(dT)12–18 primer , 2 μg total RNA as a template, and 25 U ribonuclease inhibitor, RNase-free water, 5 μL dNTP mix (10 mM), and 5 μL 5× reaction buffer. The reactions were incubated at 42°C for 60 min. The cDNA was acquired from 2 samples and was amplified by PCR using the primer sets  and expected heterozygosities (HE) [FIS) and allelic richness (AR) analyses were performed in Fstat version 2.9.3 [R calculations were set to the smallest population size correction. Pairwise FST among populations and Hardy–Weinberg equilibrium were analysed across all loci for each population in an exact probability test in Genepop 4.0 [enepop 4.0 [Genetic diversity was measured as observed (ies (HE) . The aboies (HE) . Inbreedon 2.9.3 . The AR epop 4.0 . Using tepop 4.0  in Genepepop 4.0 . To deteepop 4.0 .ega 4 [http://blast.ncbi.nlm.nih.gov/Blast.cgi) from NCBI. Because some obtained sequences are false alleles potentially corresponded to PCR amplification artifacts [ega 4 to detect the variable and parsimony-informative sites (sites with at least 2 different nucleotides or amino acids), to compute the mean number of nucleotide differences, and to derive the overall mean genetic distances of nucleotide sequences. Standard estimate errors were detected using 1,000 bootstrap replicates. Hardy–Weinberg equilibrium tests, pairwise FST values, and LD were calculated in Genepop. The AR and FIS of DQA1 and DQB1 were compared among populations using the comparison among groups of samples option in Fstat version 2.9.3 [AR estimates were adjusted to the smallest sample size.Sequences were aligned and translated into the amino acid sequences in Mega 4 . The genrtifacts , a new srtifacts . We usedon 2.9.3 . The AR tructure 2.1 [K) tested range was set from 1 to 10, and 10 independent runs were executed for each analysis. The Markov Chain Monte Carlo (MCMC) iterations lengths and burn-ins were set at 5,000,000 and 200,000, respectively. For most situations, ΔK had a mode at true K; thus, true K was selected using the ΔK statistic, which was calculated according to the rate of the log probability change of the data between successive K values [To detect the genetic structures of microsatellite loci, Bayesian clustering was used in Sture 2.1 . Becauseture 2.1 . The cluK values .odeltest 3.7 [etwork 4.600 [er), the composite-likelihood method [e) and rate per generation (r), and is estimated without prior information [The relationship of the MHC allele phylogenesis was constructed using a maximum likelihood method in PhyML 3.0 . Before test 3.7 . The K80d method  in LDhatd method  was usedormation . Even foormation .dN/dS) [dN/dS > 1. Six models  allowing for different selection intensity among sites were tested [Two methods were used to detect historical selection. First, we calculated the rates of non-synonymous and synonymous substitutions at all amino acid sites, ABS sites, and non-ABS in Mega 4 by the Nei-Gojobori method with the Jukes-Cantor correction  and 1,00dN/dS)  among coe tested ,92. The FST outlier analysis [FST values were likely under selection. The FST was estimated for DQA1, DQB1, and 16 microsatellite loci using Lositan [HE and FST were printed for each of the 100,000 simulations. The infinite allele model was used in simulations that were performed using Fdist2. The aforementioned FST outlier analysis was also carried out on each population respectively. For each population, simulation parameters were similar using the same parameter set.The analysis  was used Lositan ,93. SimuLuo MF carried out the molecular genetic studies, performed the statistical analysis, and drafted the manuscript. Pan HJ participated in the sequence alignment. Liu ZJ participated in the design of the study. Li M conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Table S1. Microsatellite data of 64 samples.Click here for fileTable S2. MHC haplotype distributions in populations.Click here for fileFigure S1. Median-joining networks for MHC alleles of the snub-nosed monkeys. A) Network for DQA1 alleles; B) Network for DQB1 alleles. The circles represent alleles , with the area proportional to the frequency of the alleles in the 3 populations.Click here for fileTable S3. Alignment of the deduced amino acid sequences of Rhro-DQA1 exon 2 sequences. Identical amino acids are shown by points, * represent ABS site, and sites revealed to be under significant selection in PAML are shown by dash.Click here for fileTable S4. Alignment of the deduced amino acid sequences of Rhro-DQB1 exon 2 sequences. Identical amino acids are shown by points, * represent ABS site, and sites revealed to be under significant selection in PAML are shown by dash.Click here for fileTable S5. cDNA primer sets for DQA and DQB (primer sets with * were those that amplified successfully in cDNA). Their location within the gene is shown in Additional file Click here for fileFigure S2. Schematic representation of the position of cDNA primer sets used in the study of MHC II variation.Click here for file


Telestes genus is of special interest: the Telestes species have a wide distribution range across Europe (from the Danubian district to Mediterranean districts) and have not been prone to translocation. Molecular data  were gathered from 34 populations of the Telestes genus, almost encompassing the entire geographic range. Using several phylogenetic and molecular dating methods interpreted in conjunction with paleoclimatic and geomorphologic evidence, we investigated the processes and timing of differentiation of the Telestes lineages. The observed genetic structure and diversity were largely congruent between mtDNA and microsatellites. The Messinian Salinity Crisis (Late Miocene) seems to have played a major role in the speciation processes of the genus. Focusing on T. souffia, a species occurring in the Danube and Rhone drainages, we were able to point out several specific events from the Pleistocene to the Holocene that have likely driven the differentiation and the historical demography of this taxon. This study provides support for an evolutionary history of dispersal and vicariance with unprecedented resolution for any freshwater fish in this region.Investigating processes and timing of differentiation of organisms is critical in the understanding of the evolutionary mechanisms involved in microevolution, speciation, and macroevolution that generated the extant biodiversity. From this perspective, the  Three analytical levels can be defined when dealing with the processes of evolutionary differentiation For European freshwater fishes, these geomorphologic events are usually related to (i) the Messinian Salinity Crisis MSC , which sThe processes of colonisation of freshwater European ichthyofauna have received particular attention during the last decade, especially in Cyprinids. Many studies based on mitochondrial DNA (mtDNA) explored the major diversifications of this family in Europe and proposed an array of evolutionary scenarios for species or groups of species . These species are thus not valuable for fishing and are therefore less prone to be translocated, an activity that is well known in economically valuable freshwater fish species From this perspective, the Telestes, we gathered molecular data from a 1232 bp long mitochondrial sequence (including the complete cytochrome b gene and 92 bp of flanking tRNA genes) and 11 nuclear microsatellite loci to investigate the major mechanisms and events responsible for the genetic structure and diversity pattern in Telestes. We explored whether (i) the MSC/Lago Mare event, (ii) the Pleistocene glaciation cycles, and/or (iii) the post-LGM/Holocene period could account for the current species distribution pattern and their genetic structure. Furthermore, we stressed the importance of watersheds crossing by upstream river capture periglacial lakes and downstream river connections in the Telestes genus evolution.Using 970 specimens, almost encompassing the entire geographic range of www.iucnredlist.org). The fishes collected for this study were killed rapidly (using clove oil as anesthetic) or were returned to the wild. The DDTs  from the Alpes-de-Haute-Provence, Hautes-Alpes, Vaucluse, Ardèche, Haute-Savoie, Doubs, Ain and Saone districts issued the permits for France, where fishing was done in collaboration with Officers of the ONEMA . Permits for the localities in Romania, Italy, Greece and Montenegro were supplied respectively by the Institute of Biology of Bucarest (representative Petru Bănărescu), the Universita di Napoli ‘Federico II’ (representative Pier Giorgio Bianco), the Aristotle University, Thessaloniki (representative Panos Stavros Economidis) and the University of Montenegro, Podgorica (representative Drago Marić). The permit for the Slovenian sampling was supplied by the Angler Society of Idrija, the Angler Society of Sevnica and the Fisheries Research Institute of Ljubljana as part of the research project 9E0073 funded entirely by the Tour du Valat Foundation (representative Alain Crivelli) and the WWF International.This study was conducted according to relevant national and international guidelines regarding the care and welfare of fishes. Field studies did not involve fish that were endangered or protected  following the manufacturer's instructions. A 1275 bp mitochondrial DNA (mtDNA) coding region was amplified by PCR using the Taq CORE Kit  with primers L14350C (5′-ACCACCGTTGTAGTTCAACTAC-3′) and H15620U (5′-AGGGGTGGGAGTTAAAATCTC-3′). PCRs were conducted following standard conditions in a total volume of 40 µL with 1 U of Taq polymerase and 2 µL of 1∶10 diluted DNA extract . The amplicons were sequenced by GATC Biotech  using internal primers Cb-L500 (5′-CAATGAATCTGAGGCGGTTT-3′) and Cb-H650 (5′-GAGAAGTATGGGTGGAAAGA-3′), resulting in a 1232 bp long fragment including the entire cytochrome b (Cyt b) gene and 92 bp of flanking tRNA genes. Chromatograms were checked for quality and sequences were aligned using SeqScape 2.5 (Applied Biosystems). Sequences were deposited in Genbank (accession numbers JQ651395 to JQ652369).Whole DNA was extracted from 25 mg of muscle or 0.25 cm3; Genbank ID JQ652370) using primers BL1-36F (6FAM-5′-GATGACTGTGCGATGAATGC-3′) and BL1-36R (5′-TGTGTGTGCAGTTTGTGTGG-3′). The eleven loci were amplified through four multiplex PCRs. Amplicons were analyzed and allele sizes were scored using protocols and conditions described by Eleven microsatellite loci were successfully genotyped in 889 individuals. Ten out of the 11 primer pairs used in this work  were described previously rlequin 3.11 k), the gene diversity (H) θ = 2Nμ: πθkθSθenepop 4.0 icro-Checker 2.2.3 Ho) and expected (He) heterozygosities were estimated for each population sample using Arlequin 3.11. For all population samples and groups of populations, the mean number of alleles per locus (nA), the average Ho and average He over loci were estimated with Arlequin 3.11. As the observed number of alleles in a sample is highly dependent on sample size, we used the rarefaction procedure dze 1.0 rA) pA) pA was therefore estimated for groups of populations within the Rhone and Danube drainages.In order to explore the diversity of the mitochondrial gene pool, ATelestes genus and from five other Leuciscinae species that were used as outgroups (odeltest 3.7 b fragment and the tRNA collated flanking regions using the Akaike Information Criterion rBayes 3.1 www.ucsc.canterbury.ac.nz). Bayesian analysis consisted of two independent runs of 2.0×107 generations with random starting trees and 10 Markov chains (one cold) sampled every 1000 generations. Adequate burn-in was determined by examining convergence onto stationarity on bivariate plots of the split frequencies, cumulative split frequency for all splits for the two runs of the simulation and symmetric tree-difference score wtyWe collated 1232 bp long mtDNA sequences obtained from 970 individuals of the utgroups . We seleTelestes lineages, as well as a time frame for the diversification, we used a relaxed Bayesian molecular clock with uncorrelated lognormal rates  was used to compute a Median-Joining (MJ) network T. souffia complex (T. muticellus haplotype from BEV sample was used to root the network). A statistical parsimony (SP) network of haplotypes cs 1.21 cs failed to resolve the topology of some parts of the phylogeny that were resolved by the MJ network. Furthermore, several TMRCA were estimated from the Median-Joining network through Network 4.05 using the ρ statistic methodology Nhylip 3.69 Genetic relationships between populations based on microsatellite allele frequencies were also investigated using the Cavalli-Sforza chord distance (Dc) Telestes microsatellite dataset  K test Fst test tructure does not export Fst values between clusters when the I-model is used. Only the first two approaches were thus used for the analyses dealing with the different Telestes species. Finally, T. alfiensis individuals were discarded from the Structure analyses since assignment tests are biased for populations of limited sample size A Bayesian-based approach was used to search for the occurrence of independent genetic groups (K) in the ated see . Actuallenetix 4.05 In order to graphically summarize the allelic frequencies variation for the different populations, Factorial Correspondence Analyses (FCA), which are well suited for qualitative variables  Fst) rlequin 3.11. In order to test isolation by distance (IBD) within the Rhone basin we used Mantel test rlequin 3.11. As recommended for analyzing differentiation in elongated habitats Fst/(1−Fst) and (unmodified) geographical distance. A Mantel test was also conducted to explore the correlation between the microsatellite Fst/(1−Fst) and the mtDNA Fst/(1−Fst). Since null alleles were detected at two loci for a few populations (see below), FreeNA Fst including a correction for null alleles. Samples VAR, BES, HER, VID and SEI were discarded from the IBD analyses, since these are populations that are currently disconnected from the Rhone river system.Population differentiation was estimated by pairwise F-statistics  TD) Fs neutrality tests rlequin 3.11. Using microsatellites, we tested for potential recent reduction in Ne through the Wilcoxon test implemented in Bottleneck 1.2 ottleneck analyses, as well as a two-phase mutation model (TPM) Using mtDNA sequences, the demographic history of populations and major lineages was also investigated by computing Tajima's D  and INN (T. souffia) populations. Lsou10 was polymorphic in T. muticellus , T. montenigrinus (POD) and T. s. agassii 1 (BRN and SLO) only.All the microsatellite loci were polymorphic in at least one population, and only two loci (BL1-36 and Lsou10) were found to be monomorphic in several populations. BL1-36 presented only two alleles (namely 195 and 197). Allele 195 was found in ottleneck analyses). MicroChecker analyses were conducted on populations with significant heterozygote deficiency. Neither allele dropout nor scoring errors were detected. Null alleles were detected in three loci: BL1-T2, BL1-98 and BL1-153. BL1-T2, which was originally developed from a T. souffia genomic library T. muticellus populations. BL1-98 and BL1-153 present null alleles in all eight populations with heterozygote deficiency. Null alleles are believed to have a negligible impact on assignment tests FstFst were estimated for T. s. souffia populations , for which only three populations  present null alleles at one locus each. We therefore assumed a limited impact on Fst and genetic diversity summary statistics estimations. With regard to Fst, this limited impact was confirmed by the FreeNA estimation of Fst including a correction for null alleles: the corrected Fst were highly correlated to uncorrected Fst , and one haplotype within T. s. souffia (shared with six populations of the Rhone drainage and very frequent in the Var River)  were polymorphic for mtDNA data . From θπr River) . This coV sample .kθ is known to be very sensitive to Ne fluctuations, more than H, πθ or Sθπθ and Sθ, this sample exhibited a very low kθ (0.28) when compared to most of the other Telestes population samples (up to 8.48 in T. muticellus and 12.46 in T. souffia). This may indicate a hybridization event associated with a sharp bottleneck event, which is also suggested by the very low rA value.rA were similar to those encountered in the Rhone drainage (except for INN), the values of pA were about two-fold higher (even for INN). In the Rhone basin, kθ values tended to be higher in populations from the Durance basin (Southern Rhone drainage) than in most of the other more northern Rhodanian populations, whether considering individual populations or groups of populations. This pattern was similar when rA (estimated from microsatellites) was considered, except for the Ardeche basin, Sorgue River and Ain River that were very similar to the Durance basin. On the contrary, pA tended to be lower in the Durance basin (0.60) than in the northern areas of the Rhone drainage (except the Upper Rhone), with a peak of pA in the Ardeche basin (0.95). Contrasted diversity patterns were observed within the French Mediterranean coastal rivers . Their genetic diversity indices ranged from comparable to or slightly higher than some Rhodanian populations (in BES) to very low genetic diversity (in VID).Within the Danube drainage, it is noteworthy that although mtDNA diversity and Telestes dataset, with 96 in the T. souffia complex and 71 in T. s. souffia alone. Bayesian phylogenetic tree reconstruction (Telestes genus being >0.95. Three major clades were recovered: (i) T. souffia complex comprising four highly divergent lineages, (ii) an Italian/Balkan clade with T. muticellus+T. montenigrinus, and (iii) a Greek clade with T. pleurobipunctatus+T. alfiensis. Nevertheless, a few ambiguities remain: the basal split among these three Telestes clades and the relationships among the four lineages within the T. souffia complex remain poorly supported. Moreover, T. pleurobipunctatus, T. muticellus and T. alfiensis are also each subdivided into highly divergent lineages that do not spatially overlap (except for T. muticellus). The same topology  was recovered in the Bayesian relaxed molecular clock time calibration (T. souffia complex and T. muticellus+T. montenigrinus (pp = 0.72), between T. muticellus and T. montenigrinus (pp = 0.76), between T. s. agassii 3 and T. s. agassii 1+T. s. agassii 2 (pp = 0.75) and between T. s. agassii 1 and T. s. agassii 2 (pp = 0.78).A total of 140 distinct mitochondrial haplotypes were found in the entire truction  providedibration . Most noT. muticellus+T. montenigrinus (BP = 96), T. pleurobipunctatus+T. alfiensis (BP = 78) and the Telestes souffia complex  with INN (T. s. agassii 3) displaying basal position. Within T. pleurobipunctatus, the GLB and GLO populations formed differentiated clusters (BP = 63). Moreover, the 21 populations of T. s. souffia form a clade . As for the FCA conducted on populations  T. souffia populations together on the other hand.The topology of the ML tree based on microsatellites  was congulations , it clusK analyses of Structure runs conducted on 14 populations representative of the Telestes genus (T. s. agassii 3 and T. montenigrinus grouped together for K = 4) and no more than half of the runs were convergent. On the contrary, for K = 13, 9/10 runs converged and all population samples were discriminated, except CAR+VIC (T. muticellus) and GLO+GLB (T. pleurobipunctatus).Four values of K were found to fit the data from Δes genus . HoweverT. souffia complex. From the Bayesian phylogenetic tree reconstructions . The three other clusters are distributed in distinct patches in the headwaters of the Danube River catchment with INN population harboring an exclusive lineage assigned to T. s. agassii 3; VIZ and TIZ in Romania assigned to T. s. agassii 2 and BRN and SLO from the Lim and Soca rivers assigned to T. s. agassii 1. From the MJ network, these Danubian clades are separated by 12 to 16 mutational steps, while they are separated from T. s. souffia haplotypes by 19 to 21 mutational steps .Our dataset allowed us to focus further on the genetic pattern and structure of the ructions , the T. al steps . The samal steps  and usinT. s. souffia with 23 populations and 681 individuals. All these populations display some degree of haplotype sharing and the T. s. souffia haplotype network  values. For K = 3, the Mediterranean coastal river Var on the one hand and the rivers Herault and Vidourle on the other hand are discriminated from the rest of the Rhone basin. These three coastal rivers are characterized by very low or null mitochondrial diversity and low microsatellite diversity  and populations from the North of the Rhone basin  and from the South Rhodanian populations (SOR and Durance basin). Interestingly, both AIN (Northern Rhone basin) and BES  are hardly differentiable from the South Rhodanian populations, and SEI (from the Seine basin) is similar to the North Rhodanian populations. For K = 7, VID was discriminated from HER and a new cluster characterized the Upper Rhone (USS and FIL). Nevertheless, the genetic structure was less clear and gene flow between the different parts of the Rhone drainage could be inferred from the results: (i) AIN sample, from the Northern part of the Rhone drainage, was hardly differentiable from the South Rhodanian populations for all K values, and (ii) some traces of gene flow from the southern and northern parts of the Rhone drainage were detectable in USS and (to a lesser extent) in ARD.Bayesian clustering using microsatellites revealed distinct subpopulations. Three values of K  were retained from the analysis of the Sure runs : K = 3 asity see . Moreovesity see  showed tFst/(1−Fst) values estimated from microsatellites  , a Mantel test found the correlation between microsatellites Fst/(1−Fst) and mtDNA Fst/(1−Fst) to be significant . This minor discrepancy is probably due to the four-fold lower effective size of the mtDNA that makes this marker more prone to local genetic drift, which may have distorted the imprint of IBD. When using Fst corrected for null alleles (microsatellites data), Mantel tests gave similar results: R = 0.561 and P = 0.000 for linear distances vs. Fst/(1−Fst); R = 0.508 and P = 0.005 for microsatellites Fst/(1−Fst) vs. mtDNA Fst/(1−Fst).IBD was observed in the Rhone drainage using linear distances vs. = 0.000) . AlthougTelestes species is 1.055E10−2 subs/site/Myr (95%CI 0.738–1.402E10−2). Our evolutionary rate is in agreement with previous estimates for Leuciscins Cyt b gene The estimated calibrated rate of evolution of the Telestes that corresponds with the separation between the four Telestes species groups  ranged from 6.7 [8.9-4.6] Myr to 4.9 [6.9-3.0] Myr ago, which corresponds to Late Miocene and matches the age of the MSC (6.0-5.3 Myr) (T. pleurobipunctatus) to ∼0.1 Myr ago (within T. souffia)  ; Fig. 2Bsouffia) ; Fig. 2BT. souffia complex, we obtained an estimation of the divergence between T. s. souffia and the other T. souffia lineages of ∼1 Myr ago, while the sampled Danubian lineages split between 0.7 and 0.6 Myr ago. These differentiation events fall within the Early-Middle Pleistocene transition (∼0.5–1.2 Myr), the period of some of the most severe glacial stages of the Pleistocene T. souffia lineages were younger with means comprised between 160 Kyr (T. s. souffia) and 90 Kyr (T. s. agassii 2), which correspond to the Late Pleistocene  in T. s. souffia and T. s. agassii 2 populations only (TD and Fs tests).Tajima and Fu's neutrality tests suggested population expansion  evolutionary history of the species and populations.No signal of recent population contraction was detected from BTelestes species have been reported . This asymmetric pattern suggests a past hybridization event with the subsequent sorting of the nuclear genome from T. s. souffia while both mitochondrial genomes subsisted. Our sampling scheme aimed at better characterizing the introgression between T. muticellus and T. s. souffia previously detected in the Var basin T. muticellus mtDNA haplotypes in VAR, nor any evidence from microsatellites. Furthermore, any sign of hybridization between T. s. agassii 1 and T. muticellus was found in the Soca River (SLO), which represents a contact zone between these two species according to Telestes. This statement is not trivial considering the importance of this process in Cyprinid speciation Several studies demonstrated extensive introgressions in Leuciscinae e.g.  and caseTelestes genome evolution well.The patterns of genetic structure and diversity obtained from mtDNA and microsatellites are largely congruent. We are therefore confident that our mtDNA-based time estimates represent the Telestes species groups  ranged from 6.7 [8.9-4.6] Myr to 4.9 [6.9-3.0] Myr. These dates are consistent with those found from 16S rDNA sequences (split between T. souffia and T. muticellus) b sequences (split between T. souffia and its sister species T. beoticus and T. pleurobipunctatus) T. muticellus, T. souffia and T. turskyi) Telestes genus is also suggested by the distribution of the different species in independent ichthyological districts of the Mediterranean area: Rhone and Danube Rivers representing the SF and DB districts, the Po River for the PV district, the Moraca River for the AB district and the Louros and Alfios Rivers for the WG district. The Lago Mare phase that occurred at the end of the MSC may have favored the dispersal of Telestes across these Mediterranean districts. The Lago Mare phase then ended abruptly during the Zanclean ∼5.33 Myr Telestes populations that had scattered across the Mediterranean rivers.The mtDNA-based estimations of divergence time between the four T. souffia (Danubian) and T. montenigrinus+T. muticellus (both Adriatic) ∼5.6 Myr suggests that Telestes colonized the Danube drainage as early as the Lago Mare stage.Furthermore, during the Lago Mare, both the Mediterranean and the Parathetys were reduced to a network of lakes, among which was the Pannonian Lake (covering the current Hungary). And yet, the freshwater Parathetys drained into the freshwater Mediterranean, connecting the Adriatic/Ionian area with the forthcoming Danube drainage T. souffia sub-species range between 0.6 and 1.0 Myr. The estimation of the divergence time between the Rhodanian T. s. souffia and its Danubian sister sub-species is 1.0 [0.6–1.6] Myr from time calibrated phylogeny and 1.0 [0.6–1.5] Myr from the ρ statistic method. These dates are consistent with the mtDNA Control Region based estimate of Telestes souffia might have benefited a series of similar upstream river capture events to colonize the Rhone drainage from the Danube, possibly via the Rhine drainage. Moreover, the formation of periglacial lakes at the edge of glaciers are known to have transiently connected the Rhine and Rhone drainages during the LGM or the Younger Dryas promoting the dispersal of Cottus gobio lineages from the Rhine drainage to the Danube drainage T. souffia in the Rhone drainage during the Middle Pleistocene.Divergences among T. souffia lineages in the Danube are comprised between 0.7 and 0.6 Myr. T. souffia does not currently occur in the main course of the Danube T. s. agassii 1, T. s. agassii 2 and T. s. agassii 3 are reciprocally monophyletic). The estimated TMRCAs fall within the transition between Early and Middle Pleistocene (0.5–1.2 Myr c. 870-880 Kyr) and MIS16 (c. 650-620 Kyr)  and Tisza river systems. Subsequent milder climate conditions, notably the interglacial MIS15  than in T. souffia, a similar pattern was also found in the cold-adapted and rheophilic Barbatula barbatula, for which populations were maintained in the upstream portion of the rivers Rhodeus amarus, a species common in lotic and lentic habitats The crown ages of the different Kyr) see . During T. s. souffia, T. s. agassii 1 and T. s. agassii 2 populations are all very similar  is ∼120-160 Kyr old, but Danubian and Adriatic populations share a population expansion ∼60 Kyr ago. Despite lack of precision, molecular dating suggests that the upstream river capture responsible for the occurrence of T. s. agassii 1 in an Adriatic river likely dates back to the Late Pleistocene. Upstream river catchment has been previously suggested for T. muticellusThe T. s. agassii 1 and T. s. souffia ∼10 Kyr (T. souffia lineages therefore suggest that the end of the Younger Dryas (rather than the end of the LGM) was critical (as suggested for Cottus gobioDemographic expansion was detected in  ∼10 Kyr . The LGM ∼10 Kyr . The ageT. s. souffia (Rhone drainage), the processes involved in this expansion could be investigated in more detail given the completeness of the sampling. When all populations are considered, a clear signal of demographic expansion is detected using mtDNA . However, only samples from the Durance River (in the southern Rhone basin) exhibit signs of population expansion when considered individually. This result suggests a glacial refugium during the LGM/Younger Dryas in the South of the Rhone drainage. This southern Rhone refugium hypothesis is further supported by higher mtDNA diversity in Durance (with respect to kθ) than in the other parts of the range. Considering microsatellites, the pattern of pA also supports a range expansion from the Southern Rhone: higher pA values in populations from the Northern Durance River  fit the so called allele surfing model T. s. souffia, we can hypothesize a demographic and range expansion that would have started in the Southern Rhone drainage at the beginning of the Holocene.For T. s. souffia, several elements suggest that two potential microrefugia also co-occurred during the LGM/Younger Dryas. As a matter of fact, the Ardeche basin and the Upper Rhone are currently connected to the rest of the Rhone basin but hold the genetic signature of past isolation: high microsatellite rA values and multiple divergent and private mtDNA haplotypes. As previously recognized for some other taxa Along with this major glacial refugium in the Southern Rhone basin for T. s. souffia populations that are isolated today. In fact, before the Rhone River was channelized and fragmented during the 19th and the 20th centuries, T. s. souffia occurred in the main course of the Rhone River Interestingly, the Bayesian clustering analysis  does notThe differentiation pattern and lack of genetic diversity observed in the isolated populations of the French Mediterranean coastal rivers  could be explained by recent downstream connections during phases of sea level drops linked to the glacial periods. A subsequent rise in sea level at the beginning of the Holocene would have led to isolation of the populations and genetic differentiation. MtDNA haplotype sharing, the Bayesian clustering analysis (K = 3 and K = 5) and (to a lesser extent) the FCA, suggest that HER and VID stemmed from the same source population. However, this scenario is unlikely for the Var River population. In fact, the topology of the seafloor in this region does not support the possibility of a past connection between the Var and the Rhone rivers, even when the sea was at its lowest level. Alternatively, the topography of the Var drainage and of the Verdon drainage (Durance basin) supports possible episodic upstream connections either linked to the Alpine orogenesis or to periglacial lakes. Moreover, these three coastal river populations clearly experienced sharp genetic drift, suggesting that the connection was very limited in time and that a founder effect characterized their settlement processes.th century by a channel devoted to irrigation and human water consumption, the Canal de ProvenceT. s. souffia. A similar scenario involving human influence could also be invoked for the pattern observed in SEI, which is indistinguishable from the other Northern Rhone drainage populations, DOU and SAO (based on Bayesian clustering approach applied to microsatellites). In the mid-19th century, the Seine, Rhone, Rhine and Loire basins were connected through the construction of a network of navigation channels. These channels have been previously proven to have allowed some cyprinid species to cross basins. They were notably instrumental in the invasion of French hydrographic systems by Chondrostoma nasusHowever, these scenarios were not supported for the Argens coastal river (BES). The fact that BES is both indistinguishable from the Durance basin (based on Bayesian clustering approach) and has experienced a recent bottleneck  supportsT. pleurobipunctatus from the Louros River exhibit an important genetic differentiation based on microsatellites and harbor very distinct mitochondrial lineages. These results confirmed previous work that used allozymic data T. pleurobipunctatus mtDNA lineages  seems to have been the major process, parapatric differentiation may have played a non-negligible role in the Telestes genus evolution.Although they are found in the same river system, the two populations of lineages , Table 2Telestes. These events range from geomorphologic, climatic to anthropogenic factors and date back from Late Miocene to Holocene. Most of them constituted a series of alternations between dispersal and vicariance that critically impacted the evolution of the Telestes genus. Additionally, the dynamics of recolonisation of a species from glacial refugial zones in a hydrographic system are still poorly understood . These results could constitute important foundation information for improving the conservation strategies for European freshwater fishes.We were able to establish several clear links between major historical events and the genetic differentiation in  but see . The detFurthermore, the existence of general patterns of colonisation for the European freshwater fishes Click here for additional data file.Supporting Information S2Results from HW equilibrium tests conducted on microsatellites.(XLS)Click here for additional data file.Supporting Information S3Fst and Fst corrected for null alleles (Fstc).Pairwise relationship between uncorrected (PDF)Click here for additional data file.Supporting Information S4Results from LD tests conducted on microsatellites.(XLS)Click here for additional data file.Supporting Information S5Genetic diversity and demographic parameters obtained from mtDNA and microsatellites data.(XLS)Click here for additional data file.Supporting Information S6T. s. souffia, T. s. agassii 1 and T. s. agassii 2.Bayesian Skyline Plots for (PDF)Click here for additional data file.


FCT = 0.054; mtDNA ΦCT = 0.222): (i) Okhotsk Sea–Norton Sound–Aleutian Islands, (ii) southeastern Bering Sea–western Gulf of Alaska, and (iii) Southeast Alaska. Populations in the Bering Sea and in Southeast Alaska are genetically heterogeneous, but populations in the center of the range are homogeneous. Mitochondrial DNA diversity drops from h = 0.91 in the northwestern Pacific to h = 0.24 in the Southeast Alaska. Bayesian skyline plots (BSPs) indicate postglacial population expansions, presumably from ice-age refugia. BSPs of sequences simulated under a demographic model defined by late Pleistocene temperatures failed to detect demographic variability before the last glacial maximum. These results sound a note of caution for the interpretation of BSPs. Population fragmentation in the Bering Sea and in Southeast Alaskan waters requires population management on a small geographic scale, and deep evolutionary partitions between the three geographic groups mandate regional conservation measures.Observed patterns of genetic variability among marine populations are shaped not only by contemporary levels of gene flow, but also by divergences during historical isolations. We examined variability at 15 SNP loci and in mtDNA sequences  in red king crab from 17 localities in the North Pacific. These markers define three geographically distinct evolutionary lineages (SNPs,  Evolutionary biologists and marine fishery managers are challenged with two questions. First, how do genetic differences arise among marine populations? A prevailing paradigm postulates that populations diverge, in part, because of contemporary processes, including random drift and selection. Despite large potentials for dispersal of planktonic larvae in many marine species, shoreline topography and ocean currents, in some cases, isolate populations and promote divergence . IdeallyParalithodes camtschaticus  in the North Pacific and Bering Sea. This species is the largest and most abundant of the king crabs in the North Pacific and extends from the Sea of Japan  and mitochondrial (mt) DNA markers to define population structure for harvest management of red king crabs and to lay a foundation for possible stock enhancements. We use the analysis of allele frequencies to detect the effects of drift between populations, and the analysis of mtDNA variation to resolve historical events. Maternal inheritance of mtDNA produces lineages that trace the dispersals and colonizations that have shaped a deep phylogeographic structure in red king crabs . We usedIn total, 1501 red king crabs were sampled at 17 locations extending from the Okhotsk Sea, through the Bering Sea and western Gulf of Alaska to Southeast Alaska , Table 1m of both polymerase chain reaction (PCR) primers, and 200 nm of each probe , 1 μm of forward and reverse primers, and 5 U GoTaq Flexi DNA polymerase . PCR amplifications were conducted in ABI 9700 thermocyclers with an initial denaturation of 1 min at 95°C, 37 cycles of 40 s at 95°C, 40 s at primer annealing temperature 41°C, and 1 min at 72°C; the final cycle was at 4°C for 5 min. Cycles were made at a ramp speed of 1°C/s. The PCR amplifications were sequenced in the forward and reverse directions by the High-Throughput Genomics Unit . Sequences were aligned with a published sequence for red king crab (GenBank accession no. AB211303) with the muscle algorithm  universal primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198  in arlequin 3.11  or haplotype (mtDNA) frequencies. Additional amovas of mtDNA haplotypes were based on an FST analog statistic (ΦST) that accounts for both haplotype-frequency differences between samples and sequence divergences between haplotypes. We used the amovas. The probability an F statistic was larger than 0.0 was estimated with 50 000 permutations of individuals among samples.Single nucleotide polymorphism genotypes for the 15 loci were tested for fit of genotypic frequencies to Hardy–Weinberg expectations (HWE) in each sample with exact probability tests of 20 batches of 10 000 permutations and a burn-in of 10 000 permutations  computed with NTSYS . Haplotype richness was estimated with HP-rare (π) and haplotype diversity (h) and their standard deviations were estimated with arlequin. A 95% plausible parsimony network of mtDNA haplotypes in 1278 crabs was made with TCS 1.21  can be estimated from the results with Neμg, where μ is the mutation rate and g is generation time (about 5 years for red king crab). However, estimates of μ are problematic, as no temporally anchored phylogenetic estimates are available for Paralithodes. Even so, phylogenetic estimates appear to greatly underestimate contemporary mutation rates and do not appear to be appropriate for BSP calibrations (n = 200) of sequences (bp = 665) with Mesquite 2.74  with st 1.6.1  with MCMbrations . To helpite 2.74 . The scaite 2.74 . We reasodelTest . This emHO = 0.148 (western Gulf of Alaska) to HO = 0.220 , so these samples were combined. SNP allele frequencies differed significantly among the 17 samples . An amova indicated that the greatest heterogeneity occurred among four groups, [1]  [4–11] [12–17] , or among three groups [1–3] [4–11] [12–17] . Samples 1 and 3 were significantly different , but not 1 and 2 . Eight samples from the southeastern Bering Sea and western Gulf of Alaska [4–11] were genetically homogeneous . However, a comparison between samples from the southeastern Bering Sea and western Gulf of Alaska was significant . A significant amount of heterogeneity appeared among the six samples from southeast Alaska [12–17] . A core group of samples  showed no heterogeneity . However, samples 15  and 16  each introduced heterogeneity into this core group.No significant temporal shift in SNP frequencies overall loci was detected between two samples from Deadman Reach  and were pooled. A PCoA of ΦST values revealed the same three groups detected with SNPs, with an addition of the outlier sample from Seymour Canal  and haplotype divergence  corresponds to large differences among groups. Most of this divergence was because of differences among the three regional groups [1–3] [4–11] [12–17]  . Unlike SNPs, samples 1 and 2 differed significantly , but not 1 and 3 . As with SNPs, samples 4 and 5 from the southeastern Bering Sea were not significantly different from each other . The six samples from the western Gulf of Alaska [6–11] were homogeneous . As with SNPs, the addition of  to this group did not produce significant heterogeneity . However, a comparison of haplotype frequencies between samples  and [6–11] was marginally significant , but not for frequencies plus haplotype divergences . In Southeast Alaska, a core group  was genetically homogeneous , but the inclusion of sample 15 from Seymour Canal to the group produced a significant amount of heterogeneity .A significant amount of overall haplotype-frequency heterogeneity ( df = 2) . The comn = 551) had a central haplotype (JF738160) that occurred in 57.2% of the crabs. In the haplotype network for the combined Southeast Alaska samples [12–17], JF738160 occurred in 80.7% of the crabs. Several low-frequency haplotypes, unique to the region, were one substitution from the common haplotype. A unique small lineage appeared in Seymour Canal [15], in which the central haplotype was one mutation removed from the most common haplotype.The haplotype networks  varied cDT was not significant in samples from the western Pacific, Bering Sea, and western Gulf of Alaska, but was significant and negative for each of the samples from Southeast Alaska, indicating excesses of low-frequency mutations  with statistical power of 95%, or greater. However, these markers provide only a small amount of power (10–50%) to detect finer differences among populations (FST = 0.001). Such small differences likely reflect short-term frequency shifts from unequal recruitment among populations, or temporary isolations stemming from North Pacific ocean–climate regime shifts. Hence, our discussion is focused on larger-scale geographic patterns resulting from glacial isolations, postglacial dispersals, and population expansions.Previous genetic studies of red king crabs in the Alaskan waters did not resolve some details of population structure reported here. The SNP and mtDNA markers and samples sizes used in this study can detect low levels of differentiation among populations  that are typical of many marine species , and populations in Southeast Alaska had even lower diversities .A striking feature of red king crab phylogeographic structure is a strong gradient in both mtDNA haplotype and nucleotide diversity across the North Pacific. While some SNPs show allele-frequency clines across the North Pacific, a comparable drop in diversity within populations was absent, most likely because SNPs are assays of specific nucleotides and, hence, are not suitable for surveying geographic patterns of within-population diversity. Western populations have high levels of mtDNA diversity  for the last 800 000 years . Each cyDT, by a unimodal mismatch distribution with a mean number of mismatches <1.0, and by a large number of private haplotypes.The high levels of genetic diversity, ragged mismatch distributions, and long coalescence times in the northwestern Pacific indicate that these populations have remained large over the Pleistocene and likely supplied colonists for extirpated eastern North Pacific areas on long time scales  red king crabs, on the other hand, are at the extremes of a large group of genetically homogeneous populations in the northern Gulf of Alaska. Southeastern Bering Sea populations are tied to northern Gulf of Alaska populations by recent common ancestry and by dispersals from the western Gulf of Alaska. Other species, including flatfishes  and WallThe genetically homogeneous populations in this region may have spread from the Kodiak refuge. Parts of the haplotype network for this group consist of a star-shaped mtDNA genealogy, indicating a recent population expansion. It is uncertain, however, whether the genetic similarity among populations is a legacy of the expansion or whether these populations are connected by high levels of larval dispersal in the fast-moving Alaska Coastal Current ; Fig. 2aOther information from stock assessments may provide insights into genetic population structure. Synchronous responses among red king crab populations to climate variables may reflect genomic similarities. For example, populations in the southeastern Bering Sea and western Gulf of Alaska showed similar recruitment patterns over several decades, but these patterns were significantly different from those in Norton Sound and Southeast Alaska . In receThe results of this study clarify issues in the management of red king crab harvests and possible stock enhancements. The genetically defined groups of red king crabs largely coincide with the State of Alaska’s registration areas. Populations in the northeastern Bering Sea (Q), Bristol Bay (T), and the Aleutian Islands (O) are genetically divergent from one another, because of different biogeographical histories. However, oceanic current patterns or biotic interactions prevent large-scale mixing between these populations. The genetic discontinuity between western Aleutian and southeastern Bering Sea populations coincides with a biogeographical boundary at Samalga Pass in the Aleutians that has been attributed to oceanic features . AdditioFST, which is based on frequencies, but not with ΦST, which additionally incorporates sequence divergences between haplotypes  and western Gulf of Alaska , and possibly the northern Gulf of Alaska . Genetic similarity among these populations likely reflects a postglacial radiation from a common glacial refuge and may not indicate close ties by gene flow. While no overall significance was detected among populations in this group, significant SNP and mtDNA divergence was detected with plotypes  and 3. Lplotypes . These gplotypes , indicatIn contrast, Southeast Alaskan populations show much greater levels of subdivision. These populations may have expanded into Southeast Alaskan waters from a glacial refuge population, but are presently isolated in semi-enclosed fjords that limit larval dispersal. Private mtDNA haplotypes in all of these populations indicate isolation and population self-recruitment. These populations may also have diverged from one another because of adaptive shifts to local environmental conditions. While all of these populations fall into a single registration area, they are best managed on a finer geographic scale, because of their demographic independence.Research is underway to develop hatchery techniques to raise crabs for restoring depressed populations, or for stock enhancement. If stock restoration is warranted because of reproductive limitations, several genetic factors must be considered in devising a stock restoration plan. A close genetic match between hatchery broodstock and the depressed population is desirable. While the results of population genetic studies provide important background information, the molecular markers used in these studies are generally neutral to selection and may not detect important adaptive differences among populations. Patterns of gene flow between local populations and the spatial scale of the adaptive landscape for red king crab are unknown. In addition to broodstock choice, artificial culture may alter the genetic profiles of hatchery-reared crabs, because more larvae survive in a hatchery setting than in the wild, setting the stage for selective or random shifts in genetic profiles. Hybridizations between hatchery-reared crabs and wild crabs may compromise the genetic integrity of the wild populations, and persistent hatchery releases may displace wild populations with crabs of hatchery ancestry .In conclusion, the distributions of SNP and mtDNA population markers portray the effects of ancient isolations and dispersals, contemporary gene flow, and random drift on population structure. These findings provide managers with a sound basis for managing harvests and restorations on a population level. However, the failure to find significant differences among populations in some areas with neutral molecular markers does not necessarily indicate that populations are connected with large amounts of gene flow. Historical expansions from a common ice-age refuge can produce the appearance of panmixia, even though populations may be demographically independent from one another.


Due to the unparalleled genetic diversity of its peoples, Africa is attracting growing research attention. Several African populations have been assessed in global initiatives such as the International HapMap and 1000 Genomes Projects. Notably excluded, however, is the southern Africa region, which is inhabited predominantly by southeastern Bantu-speakers, currently suffering under the dual burden of infectious and non-communicable diseases. Limited reference data for these individuals hampers medical research and prevents thorough understanding of the underlying population substructure. Here, we present the most detailed exploration, to date, of genetic diversity in 94 unrelated southeastern Bantu-speaking South Africans, resident in urban Soweto (Johannesburg).Participants were typed for ~4.3 million SNPs using the Illumina Omni5 beadchip. PCA and ADMIXTURE plots were used to compare the observed variation with that seen in selected populations worldwide. Results indicated that Sowetans, and other southeastern Bantu-speakers, are a clearly distinct group from other African populations previously investigated, reflecting a unique genetic history with small, but significant contributions from diverse sources. To assess the suitability of our sample as representative of Sowetans, we compared our results to participants in a larger rheumatoid arthritis case–control study. The control group showed good clustering with our sample, but among the cases were individuals who demonstrated notable admixture.Sowetan population structure appears unique compared to other black Africans, and may have clinical implications. Our data represent a suitable reference set for southeastern Bantu-speakers, on par with a HapMap type reference population, and constitute a prelude to the Southern African Human Genome Programme. The Human Genetic Diversity Project (HGDP) provides genotyping information for populations residing in the Central African Republic, the Democratic Republic of Congo and Senegal [The African continent continues to attract a growing proportion of research attention due to the unprecedented level of genetic diversity of its peoples ,2. In pa Senegal , whilst  Senegal ,5.Less well represented in current research, however, are inhabitants of the southern Africa region. Defined here as the collection of Botswana, Lesotho, Swaziland, Namibia and South Africa , southeThe lack of local genetic information with robust allele frequency distributions currently serves as a significant hurdle to designing biomedical research and may have important medical implications. With the highest worldwide prevalence of HIV/AIDS  and risiAs a prelude to the SAHGP, we sought to investigate the genetic diversity amongst urban black South Africans residing in the Soweto-Johannesburg metropolitan area of the Gauteng province - one of the urban centres in South Africa most densely populated by southeastern Bantu-speakers. Soweto is a major contributor to South Africa’s leading rates of urbanization , retainiIt is commonly accepted that the inadequacy of SNP chips is exposed when used to assess most African populations . Thus, tTo contextualise Sowetan genetic variation, PCA plots (based on 460 568 SNPs) were generated from the combined dataset Figures  and 3 whPrincipal components 3 and 4 more clearly distinguished African populations from one another. Component 3 highlights the separation between Europeans, Oriental populations and Gujarati Indians, the latter of which appears as an extended cluster. Component 4 disaggregates African populations along a north–south gradient, with a correspondingly clear distinction between Sowetans and the more northern African groups. Southeastern Bantu-speakers (SEB) typed by Schlebusch et al.  remainedTo investigate the distinctions between African populations further, we generated an intracontinental plot that included only African populations, namely the BSO, SEB, YRI, MKK, LWK, KAR (Karretjie), KHO (Khomani) and NAM (Nama) Figure . In agreK=2 to K=5 and K=2 to K=6, for intracontinental and intercontinental datasets respectively, are shown in Figure K=2, YRI individuals were already distinguished from their African counterparts. By K=3, clear separation between BSO, YRI, MKK and LWK populations was evident, along with a clear link between southeastern Bantu-speakers and the southern Khoe-San, in confirmation of previous reports [K=5, BSO and SEB presented with greater diversity in admixture than northern Africans. Intercontinentally  before separating as a distinct population at K=4. With increasing K clusters, African populations are increasingly distinguished. In particular, Bantu-speakers appeared to be significantly different with relatively small contributions from all 6 ancestral populations; a result that was not typical for members of the other populations investigated.ADMIXTURE results for ancestral populations  reports ,30. At Ky Figure b, K=2 seTo examine how well our reference Soweto sample represented another larger and independently selected sample of unrelated black Sowetans, we performed a comparison with data from a case–control study for rheumatoid arthritis. PCA results are displayed in Figure The rapid urbanisation of Soweto and its subsequent epidemiological transition are largely representative of the transformations occurring across the developing southern Africa region ,31-33. CUsing principal component analysis, we noted two important observations. Firstly, we confirmed that southeastern Bantu-speakers (BSO and SEB) occupy a distinct space from northern Africans. Secondly, we observed a relatively loose clustering of BSO individuals, consistent with the demographic “melting pot” of the urban Soweto community. In confirmation, ADMIXTURE results suggested Sowetans comprise of small contributions from a diverse assortment of ancestral populations, more so than was evident for other African populations investigated, with the exception of the Khoe-San. Such varied contributions, however, were not significant enough to detract from the general homogeneity of the group , suggesting that most migration and admixture into Soweto is likely from areas where individuals have a similar genetic heritage. Consequently, the average level of admixture is unlikely to significantly interfere with the analysis of disease association studies. However, individuals with significant admixture also form part of the Sowetan population [as witnessed in Figure Amongst the numerous and diverse sources of genetic variation, Bantu-speakers are specifically known to display levels of Khoe-San admixture ,34,35. OIn confirmation that our sample was a good representation of the larger Soweto population, we investigated its similarity to a sample of over 600 individuals from a recent rheumatoid arthritis case–control study . The cases and controls were separately identified and in the comparisons, the controls clustered tightly with the BSO group, reflecting their common origin, and thus strengthening the applicability of our data as a suitable reference for Soweto and South Africa [according to Statistics South Africa , the 201CYP3A4 gene in a sample comprised of Khoe-San, Xhosa and Mixed Ancestry individuals from South Africa identified 24 SNPs, two of which were novel, non-synonymous variants [The performance of the Omni5 in assessing African genetic variation merits comment. Based on our comparisons, the platform performs well in typing common variation in Africans, and will have use in genome-wide association studies. Beneficially, the superior marker density improves the chances for positive associations, which are more likely to progress to the identification of causal variants due to the limited linkage disequilibrium (LD) of African populations . Conversvariants . Only onvariants , especiavariants . DespiteSeveral limitations to the present study are acknowledged. Ideally, a larger sample size and complete genome sequences would more accurately reflect the full spectrum of genetic diversity across southeastern Bantu-speakers. Our sample of 94 individuals does, however, compare in size to those of the HapMap and 1000 Genomes Projects, which have more than demonstrated their value as reference panels for specific populations. The comparison to Sowetans in the rheumatoid arthritis study was done primarily with markers related to loci relevant to autoimmune disease, which may have introduced some bias, since they may have been involved in significant selection pressures as highlighted by Schlebusch et al. . Lastly,inter alia, present interesting genetic diversity in their own rights, distinct from that seen amongst the more populous southeastern Bantu-speakers. Several studies have already commenced with the documentation of this variation [Our data have begun to address the paucity of southern Africa genetic information, although considerable work remains in sampling more broadly across the region. Numerous other ethnicities, including the Cape Mixed Ancestry, southwestern Bantu-speakers (Herero) and Afrikaner populations ariation ,30,44, bhttp://sbimb.core.wits.ac.za/data/SNPgenotyping_01.html.To conclude, our investigative search into Sowetan genetic variation is, to date, the most detailed of its kind. We have observed a distinct genetic profile for these individuals, different from other more widely studied African populations, supported by principal component analysis as well as ADMIXTURE. Combined, these results aligned well with demographic and historical knowledge on the inhabitants of the Soweto region, clearly highlighting the significant, but relatively small genetic contributions from far and wide, that have been made to the local gene pool. We have demonstrated that this dataset is a good reference sample for future research on black South Africans who speak southeastern Bantu languages. Most importantly, some of the implications for future medical policy and research are highlighted. Lastly, the dataset may be considered a first step toward the SAHGP, and is available at Study participants included 94 unrelated southeastern Bantu-speaking South African individuals , residing in the Soweto-Johannesburg metropolitan area whose ethnicity was captured from municipal birth notification forms. These individuals are existing participants in a longitudinal birth cohort and were all born in 1990 . Followihttp://www.illumina.com/support/documentation.ilmn). Beadchips were scanned on the Illumina iScan . Raw data were inspected using Genomestudio (version 2011.1) and genotype calls were made based on a clustering manifest supplied by Illumina.Participants were genotyped using Infinium Omni5 beadchips . DNA samples were prepared in accordance with the Infinium LCG assay  (2012/01/31 release). We also obtained genotyping data for southeastern Bantu-speakers and the southern Khoe-San groups from Schlebusch and colleagues (2012) . Briefly, 304 affected individuals and 318 healthy controls  were typed on the Illumina Infinium Immunochip  , consistsmartpca.perl script, part of the EIGENSTRAT suite , was used to calculate Eigen vectors that determined the relative principal components. These components were then plotted using Gnuplot (version 4.6) [K=2 to K=6 ancestral populations where applicable, calculated from 100 permutations. To ensure no bias was introduced into the PCA analysis due to variations in sample size, we conducted 50 random samplings of 50 individuals from each population studied (the Khoe-San were treated as a single group). Inter- and intracontinental PCAs using these subsamples demonstrated negligible variation in general patterning and clustering when compared to PC analysis of the full sample sizes (data not shown).PLINK was used to generate the necessary minor allele frequency statistics that allowed the assessment of the performance of BSO samples on the Omni5 chip. To compare variation between populations, the ion 4.6) . ADMIXTUion 4.6) , CLUMPP ion 4.6)  and Distion 4.6)  were use1kGP: 1000 Genomes project; BSO: Black southeastern Bantu-speakers in Soweto, Johannesburg; CEU: Utah residents with ancestry from northern and western Europe; CHB: Han Chinese in Beijing, China; GIH: Gujarati Indians from Houston, Texas; GWAS: Genome-wide association study; JPT: Japanese in Tokyo, Japan; KAR: Karretjie in South Africa; KHO: Khomani in South Africa; LD: Linkage disequilibrium; LWK: Luhya in Webuye, Kenya; MKK: Maasai in Kinyawa, Kenya; NAM: Nama in Namibia; NK: Niger-Kordofanian; PC: Principal component; PCA: Principal component analysis; SCA: Black Sowetan case individuals with rheumatoid arthritis; SCO: Black Sowetan control individuals; SEB: Southeastern Bantu-speakers; SNP: Single nucleotide polymorphism; YRI: Yoruba in Ibadan, Nigeria.The authors declare that they have no competing interests.AM assisted with the genotyping and quality control, performed the data analysis and drafted the manuscript. SH contributed in a major way to the quality control of the data and public datasets, as well as the data analysis with regard to PCA and ADMIXTURE. YL and CH oversaw and implemented early quality control procedures on the raw Omni5 data. NG and MT provided the rheumatoid arthritis data. SAN contributed the BSO samples and KJJ, NH, FS and SAN, provided expertise on the experimental platform and critical comment on the manuscript. MR conceived the study design and was involved in guiding the analysis and critical revisions of the drafted manuscript. All authors read and approved the final manuscript.