Recent studies have detailed a remarkable degree of genetic and linguistic diversity in Northern Island Melanesia. Here we utilize that diversity to examine two models of genetic and linguistic coevolution. The first model predicts that genetic and linguistic correspondences formed following population splits and isolation at the time of early range expansions into the region. The second is analogous to the genetic model of isolation by distance, and it predicts that genetic and linguistic correspondences formed through continuing genetic and linguistic exchange between neighboring populations. We tested the predictions of the two models by comparing observed and simulated patterns of genetic variation, genetic and linguistic trees, and matrices of genetic, linguistic, and geographic distances. The data consist of 751 autosomal microsatellites and 108 structural linguistic features collected from 33 Northern Island Melanesian populations. The results of the tests indicate that linguistic and genetic exchange have erased any evidence of a splitting and isolation process that might have occurred early in the settlement history of the region. The correlation patterns are also inconsistent with the predictions of the isolation by distance coevolutionary process in the larger Northern Island Melanesian region, but there is strong evidence for the process in the rugged interior of the largest island in the region (New Britain). There we found some of the strongest recorded correlations between genetic, linguistic, and geographic distances. We also found that, throughout the region, linguistic features have generally been less likely to diffuse across population boundaries than genes. The results from our study, based on exceptionally fine-grained data, show that local genetic and linguistic exchange are likely to obscure evidence of the early history of a region, and that language barriers do not particularly hinder genetic exchange. In contrast, global patterns may emphasize more ancient demographic events, including population splits associated with the early colonization of major world regions. The coevolution of genes and languages has been a subject of enduring interest among geneticists and linguists. Progress has been limited by the available data and by the methods employed to compare patterns of genetic and linguistic variation. Here, we use high-quality data and novel methods to test two models of genetic and linguistic coevolution in Northern Island Melanesia, a region known for its complex history and remarkable biological and linguistic diversity. The first model predicts that congruent genetic and linguistic trees formed following serial population splits and isolation that occurred early in the settlement history of the region. The second model emphasizes the role of post-settlement exchange among neighboring groups in determining genetic and linguistic affinities. We rejected both models for the larger region, but found strong evidence for the post-settlement exchange model in the rugged interior of its largest island, where people have maintained close ties to their ancestral lands. The exchange (particularly genetic exchange) has obscured but not completely erased signals of early migrations into Island Melanesia, and such exchange has probably obscured early prehistory within other regions. In contrast, local exchange is less likely to have obscured evidence of population history at larger geographic scales. On the Origin of SpeciesThe Descent of ManIn Anthropologists Genetic and linguistic correspondence may also form through a process that is analogous to the genetic model of isolation by distance Earlier studies have not provided convincing support for either the branching or isolation by distance processes for gene-language coevolution. Cavalli-Sforza and colleagues Several factors may account for the lack of evidence for gene-language coevolution. First, genes and languages may disperse in very different ways simply because biological transmission is solely vertical but linguistic transmission is both vertical and horizontal The lack of strong support for coevolution may also reflect deficiencies in the methods used to examine linguistic variation. Many studies employ controversial language classifications estimated from cognate data In this study, we compared detailed genetic and linguistic patterns from data collected across a set of particularly diverse populations in the Southwest Pacific. To construct a linguistic classification and estimate linguistic distances, we used data from over 100 structural linguistic features  that may avoid some of the limitations associated with cognate data The datasets come from Northern Island Melanesia, a region well-known for its complex history and remarkable biological and linguistic diversity The languages of Northern Island Melanesia (NIM) belong to two major groups: Oceanic and Papuan. Oceanic is a major branch of the widespread Austronesian language family that appeared in the region about 3,300 years ago The standard method of constructing the historical relationships between languages, called the Comparative Method, is a tree-building technique that relies on recognizing sets of words in different languages that are related in meaning and form (cognates) and which show regular sound changes  demonstrating that they derive from a single ancestral language. Because cognates change relatively rapidly, reconstructions using the Comparative Method cannot generally be made beyond 8,000 years Recently, Dunn and colleagues The branching model predicts that the patterns of linguistic and genetic variation will be treelike, so that for our datasets, the Oceanic- and Papuan-speaking populations will cluster on separate branches of the language and genetic trees, and it also predicts that the topologies within the separate Oceanic and Papuan clusters will be similar in both trees. We tested these predictions by comparing simulated and observed patterns of genetic variation and the topologies of gene and language trees.The isolation by distance model predicts that genetic and linguistic distances will be correlated with one another not because of congruent tree-like evolution but because of ongoing genetic and linguistic exchange between neighboring populations. If genetic and linguistic exchange have occurred independently of one another, the genetic-linguistic distance correlation will lose statistical significance when geographic distance is held constant. If they have moved largely in concert with one another, the genetic-linguistic distance correlation will remain significant when geographic distance is held constant. These predictions were tested using computer simulations, matrix correlation and partial correlation tests, and by examining plots of genetic, linguistic and geographic distances.The detailed genetic and linguistic datasets were recently collected from 33 populations located on the major islands of the Bismarck Archipelago and Bougainville in NIM The population names are linguistically based. Where genetic data were collected from more than one group in a language area, we added a distinguishing letter . Our basic unit of genetic similarity is the allelic identity between individuals, defined as the probability that two alleles of the same locus drawn from two random individuals, either within the same population or from two different populations, are identical We used coalescent-based computer simulations to construct the predicted pattern of allelic identity variation for the branching model. The simulations are detailed in ST genetic distances Trees were constructed with different techniques. The unrooted language tree was constructed from the 108 structural linguistic items across the 23 languages using the Bayesian approach described by Huelsenbeck and Ronquist 2 random variable, with degrees of freedom equal to s(s+1)/2 minus the number of nodes in the language tree, where s is the number of populations. The expected value of Λ is equal to the degrees of freedom if the language tree “fits” perfectly. Further details of the method are provided in To compare the trees formally, a modified version of the Cavalli-Sforza and Piazza a is the likelihood ratio statistic of the baseline tree with adf degrees of freedom, and Λb is the likelihood ratio statistic for the language tree. Lewis and Long To further compare the linguistic and genetic structure, we also fitted the simplest possible tree in which all populations diverged from a common ancestor at the same time in the past. In this tree, there is only one internal node connecting all of the populations. Because the tree contains no internal structure other than this single internal node, it can serve as a baseline against which the fit of the language tree can be compared. The fit of the language tree relative to this baseline tree was estimated with an F-test, distances between populations from the observed allelic identity matrix, then estimated genetic distances from the language tree-expected allelic identity matrix, and finally plotted these two sets of genetic distances against one another. Besides allowing a simple visual comparison of the correspondence between the observed genetic pattern and the predicted pattern for the language tree, the residuals of the plot may be examined to assess the specific causes of any observed lack of correspondence. Genetic distances were estimated from the allelic identities using the formula of Nei kJ and lJ are the allelic identities in populations k and l, and klJ is the allelic identity between populations k and l.As a third way to evaluate the fit of the language tree to the genetic structure of NIM populations, we estimated the genetic We used the coalescent-based computer simulations to estimate an allelic identity matrix for the isolation by distance model see  and thenThe last column of However, the allelic identities show an even more pronounced relationship to the coastal/inland residential distinction. Without exception, the coastally-located populations have lower allelic identities than the inland populations. Two of the coastally-located Papuan-speaking groups (Sulka and Kuot) fall in this lower allelic identity coastal grouping, and two of the inland Oceanic-speaking groups (Mamusi and Nakanai-S) fall in the higher allelic identity interior grouping. These linguistic “outlier” populations probably reflect recent population movements between the New Britain coast and interior.As mentioned, In sum, the observed pattern of allelic identity variation is not consistent with the branching model. It shows that significant genetic exchange has occurred between local populations within islands whether they belong to the same major language group or not, but that genetic exchange between islands may have been relatively restricted for some time.The language and genetic trees in The results of the model-fitting procedure are shown in Λ is much lower for the fitted language tree than it is for the fitted baseline tree . The F-tThe lower plot in The contributions of other populations to the lack of correspondence between the observed and expected genetic distances are shown in The revised 18-population language tree is shown in In the simulations, the populations are arrayed next to one another in a linear stepping stone pattern, but the 33 sampled NIM populations are not located next to one another in a simple linear fashion. However, the lack of congruence between the heat plots is not because of this difference. Isolation by distance predicts decreasing allelic identity with increasing geographic distance regardless of the actual sampling locations, and this pattern does not occur for the observed allelic identities. This conclusion is supported by additional simulations reported in the last section of Plots 7C and 7D show the linguistic-geographic distance correlations, with the different sets highlighted as before. As one might expect for the linguistic correlations, the coastal and interior strata are less clearly distinguished than the Oceanic and Papuan strata. This is again consistent with the argument that there has been little linguistic exchange between Oceanic and Papuan languages where they occur in neighboring groups . The poorer distinction for the interior and coastal strata is caused by these outliers. Plots 7E and 7F show the genetic-linguistic distance correlations with similar highlighting. They suggest that any linguistic-genetic correlation is driven solely by the Papuan-speaking populations, but as The plots also show that for any given geographic distance, the interior/Papuan-speaking populations have higher genetic and linguistic distances among them than do the coastal/Oceanic-speaking populations. The correlation coefficients are also generally larger between interior/Papuan populations than they are between coastal/Oceanic populations. This distinction is the result of the comparatively restricted movement in the rugged highland interiors The correlations are particularly high in the New Britain interior . The genThe results on the New Britain coast suggest a separate isolation by distance pattern there as well. All of the correlation coefficients there are high, but only the genetic-linguistic distance correlation is statistically significant . The p-vThe tests of the branching model in Northern Island Melanesia show that genetic and linguistic exchange between local populations has erased evidence that may have once existed for a branching process there. Genes have tended to move freely between nearby populations, regardless of the languages they speak. On the other hand, structural linguistic exchange has been particularly limited between neighboring Oceanic and Papuan languages. In these instances, the Oceanic-speaking populations have become very similar genetically to their Papuan-speaking neighbors , and we then examined the correlation between these distances and the genetic and geographic distances. None of the correlations were statistically significant. If not for the structural linguistic data, we would have failed to identify any linguistic relationship to genetic or geographic patterns at all.Another relevant point is that the linguistic data and methods typically used in studies of coevolution have usually been of comparatively poor quality. To illustrate the higher quality of our structural linguistic dataset, we employed the commonly used method of node counting to estimate linguistic distances between NIM languages in a classification constructed using the Ethnologue  also undoubtedly led to our finding of comparatively high correlations in our various analyses. Many recent studies have used mitochondrial d-loop data and Y-chromosome data to investigate genetic and linguistic correspondence in various world regions The implications of our results for broader issues in Pacific prehistory are important but must be interpreted carefully. While our results provide little support for the branching model in Northern Island Melanesia, this is different from arguing that branching did not occur in very early periods there, or elsewhere in the Pacific, and it does not mean that our microsatellite data lack important information about the deeper prehistory of the entire region.For example, two contrasting scenarios for the origins of the Polynesians have persisted in recent Pacific prehistory debates, and they bear a very close relationship to the two models examined in this paper. The first has been called the phylogenetic model The genetic data have been interpreted to support several of these Polynesian origin scenarios. Some have indicated that a clear phylogenetic signal exists between Taiwan Aborigines and Polynesians, with little intermixture taking place in Near Oceania, while other datasets have been interpreted to suggest heavy intermixture with, or major contributions from, Near Oceanic and Wallacean populations Text S1Supplemental (0.09 MB DOC)Click here for additional data file.


Most studies of European genetic diversity have focused on large-scale variation and interpretations based on events in prehistory, but migrations and invasions in historical times could also have had profound effects on the genetic landscape. The Iberian Peninsula provides a suitable region for examination of the demographic impact of such recent events, because its complex recent history has involved the long-term residence of two very different populations with distinct geographical origins and their own particular cultural and religious characteristics—North African Muslims and Sephardic Jews. To address this issue, we analyzed Y chromosome haplotypes, which provide the necessary phylogeographic resolution, in 1140 males from the Iberian Peninsula and Balearic Islands. Admixture analysis based on binary and Y-STR haplotypes indicates a high mean proportion of ancestry from North African (10.6%) and Sephardic Jewish (19.8%) sources. Despite alternative possible sources for lineages ascribed a Sephardic Jewish origin, these proportions attest to a high level of religious conversion (whether voluntary or enforced), driven by historical episodes of social and religious intolerance, that ultimately led to the integration of descendants. In agreement with the historical record, analysis of haplotype sharing and diversity within specific haplogroups suggests that the Sephardic Jewish component is the more ancient. The geographical distribution of North African ancestry in the peninsula does not reflect the initial colonization and subsequent withdrawal and is likely to result from later enforced population movement—more marked in some regions than in others—plus the effects of genetic drift. The genetic diversity of human populations in Europe has been the subject of intense scrutiny since the first “classical” markers became available.Although evidence of the cultural impact of historical events can be gleaned from sources such as archaeology, place names, and linguistic elements, there is often debate about the weight of their corresponding demographic impact. Genetic analysis of modern populations can offer a more direct approach to recognizing the impact of migrations and invasions in historical times, especially when source populations for migrations are clearly differentiated from recipient populations. The Iberian Peninsula is of particular interest in this context, because it has a complex recent history over the last two millennia, involving the long-term residence of two very different populations with very distinct geographical origins and their own particular cultural and religious characteristics—North African Muslims and Sephardic Jews.North Africa and the Iberian Peninsula are separated by a mere 15 km of water at the Gibraltar Strait, making the region a potential migration route between Africa and Europe. Historically documented contact began dramatically in 711 CE, when a Berber army under Arab leadership crossed from Morocco, winning a key battle the following year.convivencia) ensued in the twelfth and thirteenth centuries, but after 1492 , religious intolerance forced Spanish Muslims to either convert to Christianity  or leave.moriscos were relocated across Spain on occasion, and, finally, during 1609–1616, over 200,000 were expelled, mostly from Valencia.The established population of the Iberian Peninsula prior to 711 CE has been estimated at 7–8 million people, ruled by about 200,000 Germanic Visigoths,Sepharad, the Hebrew word for Spain. The Jewish presence was very long-established, with some evidence that it predated the Christian era; many Jews, however, are thought to have arrived during the Roman period, either voluntarily or as slaves brought from the Middle East after the defeat of Judea in 70 CE.convivencia, their population size in Spain was around 100,000.conversos (in Spain) or cristãos novos .The people encountered by the Islamic invaders in the eighth century were not a religiously and culturally uniform group; they included among the Catholic Christian majority a substantial minority of Jewish people. They and their descendants are known as Sephardic Jews, from Alu insertion polymorphisms,Previous genetic studies of the Iberian Peninsula included analyses of classical marker frequencies,A total of 1140 DNA samples isolated from males from the Iberian Peninsula and the Balearic Islands were analyzed; below, we refer to these samples as “Iberian,” for brevity. All samples were collected with appropriate ethical approval and informed consent. Individuals were assigned to geographical locations on the basis of paternal grandfather's place of birth, and they were then grouped on the basis of traditional regions. Andalusia was divided into western and eastern (including Murcia) parts; Castilla y Leon was divided into northeast and northwest Castile; and a set of individuals from the Pyrenees, including some from north of the Spain-France border, were pooled as “Gascony.” Samples from Portugal were divided into two sets, those north and those south of the Mondego river.10831). Two individuals derived for M45 but ancestral for M207 (hgP∗[xR]) were analyzed with the markers MEH2 and M3 and could thus be assigned to hgQ∗(xQ3). Samples derived for M173 but ancestral for SRY10831.2 and M17 (hgR1∗[xR1a]) were further analyzed with multiplex IV—which, to our knowledge, is previously unpublished—containing the markers M65, M153, M222, M269, and SRY-2627. Ten individuals carry reversions of the marker P25 through gene conversion,Binary markers  on the n∗ refers to E3b∗, also known as E-M35∗; and R1b3∗ refers to R1b3∗, also known as R-M269∗.Nomenclature of haplogroups is in accordance with the Y Chromosome Consortium,Nineteen Y-chromosomal STRs  were typed in three multiplexes, as described previously,∗. Haplogroup G was also undetermined in the published Algerian and Tunisian data,∗ cases was predicted with high confidence to belong to hgG (100% [Bayesian] and 96% [SVM]); this low level of the haplogroup in North Africa is consistent with the Moroccan and Saharawi samples and with an independently published set of Algerian data.Comparative data for North African populations were obtained from the literature.Comparative data for Sephardic Jewish populations were extracted from a large collection of Y haplotypes assembled by D.M.B. and K.S. The term “Sephardic Jews” is used here in its narrow sense,In many cases, sample sizes for haplogroup-based analyses are larger than those used for the same populations for Y-STR-based analyses. For the Moroccan sample , this is because only a subset (n = 104) was typed for Y-STRs. For the remainder, small reductions in sample sizes are due to the removal of chromosomes carrying STR-allele duplications, or “partial” alleles, which cannot be readily accommodated in STR-based analyses.FST [for haplogroups] and RST [for Y-STR haplotypes]) were calculated with Arlequin.FST and RST matrices was carried out with PROXSCAL in SPSS 14.0.Summary statistics  was calculated as described previously.∗, is by far the most common (55%). When all chromosomes derived for the marker M269 are considered , this figure approaches 66%.A total of 30 binary markers were typed in a set of 1140 Y chromosomes belonging to 18 populations from the Iberian Peninsula and the Balearic Islands . Of the ∗(xJ2), and G.To provide a context in which to consider the issue of a North African genetic contribution, we compiled haplogroup frequency data for four North African populations: Moroccans and Saharawi,Haplogroup frequencies in these Iberian, North African, and Sephardic Jewish populations are displayed graphically in STF matrix  plot based on a pairwise T matrix A displaySTR estimates based on Y-STRs. Most of the Iberian samples had been previously typed with a set of 19 Y-STRs;Ascertainment bias of the SNPs used to define haplogroups is a potential problem in Y-chromosomal diversity analysis , this is indeed true for the mainland.North Africans entered the Iberian Peninsula from the south, and after a rapid northward expansion soon retreated southwards, being finally expelled from Andalusia over 700 years after their arrival. Thus, they apparently spent the least amount of time in the north, and we might therefore expect a south-north gradient of North African ancestry proportions. However (and in agreement with studies of independent samplesIt is important to consider factors that might act to elevate the apparent proportions of Sephardic Jewish ancestry that we estimate, because these values are surprisingly high. Choice of parental populations in admixture analysis can have a major effect on the outcome, and among the parental populations in our analysis, the Sephardic Jewish population has a different status compared to the two others: whereas Basque and Moroccan samples are drawn from sizeable populations that have maintained their existence in situ, with a probable low level of admixture with the other parentals, the Sephardic Jewish sample is taken from a comparatively small group of self-defined individuals whose ancestors have lived in various parts of the Iberian Peninsula and were themselves probably subject to some degree of admixture with Iberians. This potential past admixture would have the effect of increasing the perceived level of Sephardic Jewish ancestry compared to the actual proportion. The presence of the typically western European lineage hgR1b3 at a frequency of 11% in the Sephardic Jewish sample might be a signal of such introgression. To examine this, we constructed a network of hgR1b3 Y-STR haplotypes in Iberian, Sephardic Jewish, and Moroccan samples . Twelve ∗, and J. These influences fall into two different time periods: the Neolithic era, beginning in 10 KYA, the demographic effects of which are a matter for heated debate;An additional factor that could lead to overestimation of Sephardic Jewish ancestry proportions is the effect of other influences on the Iberian Peninsula from eastern Mediterranean populations that might have imported lineages such as G, KOur study has focused on the Y chromosome, but can we say anything about whether admixture has been predominantly male-mediated? Some mtDNA studiesIn this study, we have demonstrated the dramatic impact of recent events on the genetic landscape of an important part of the European continent. Immigration events from the Middle East and North Africa over the last two millennia, followed by introgression driven by religious conversion and intermarriage, seem likely to have contributed a substantial proportion of the patrilineal ancestry of modern populations of Spain, Portugal, and the Balearic Islands. In studies that seek to trace the imprint of key events in the earlier prehistory of Europe, the impacts of such recent episodes of gene flow and integration must be taken into account.


Acropora digitifera at 19 sites in seven regions along the 1,000 km long island chain of Nansei Islands, Japan. This area includes both subtropical and temperate habitats. Thus, the coral populations around the Nansei Islands in Japan are northern peripheral populations that would be subjected to environmental stresses different from those in tropical areas. The existence of high genetic connectivity across this large geographic area was suggested for all sites (FST≤0.033) although small but significant genetic differentiation was detected among populations in geographically close sites and regions. In addition, A. digitifera appears to be distributed throughout the Nansei Islands without losing genetic diversity. Therefore, A. digitifera populations in the Nansei Islands may be able to recover relatively rapidly even when high disturbances of coral communities occur locally if populations on other reefs are properly maintained.Recently, reef-building coral populations have been decreasing worldwide due to various disturbances. Population genetic studies are helpful for estimating the genetic connectivity among populations of marine sessile organisms with metapopulation structures such as corals. Moreover, the relationship between latitude and genetic diversity is informative when evaluating the fragility of populations. In this study, using highly variable markers, we examined the population genetics of the broadcast-spawning coral  Coral reefs support the highest biological diversity of all marine ecosystems. Reef-building corals play an important role in structuring and maintaining coral reef ecosystems and in forming the framework of coral reefs. However, coral populations worldwide have been decreasing recently due to anthropogenic disturbances such as overfishing, sediment pollution, and nutrient influx and are also under threat from global warming and ocean acidification FST-based method). For more detailed examinations on the genetic connectivity of corals, analyses over ecological timescales using highly variable markers  are required. Recently, microsatellite markers have been developed for some corals  different from those in tropical areas.Our target species A. digitiferaA. digitiferaA. digitifera maintains populations at the northern limit of its distribution, we used a population genetic approach using microsatellite markers based on a large sample size  from across a wide geographic area  covering most of the Nansei Islands  to examine genetic connectivity and the relationship between genetic diversity and latitude  for latitude .PVA) were found at some sites: in particular, two private alleles were detected at the SNS site in the Kerama region and three were at the KRS site in the Sekisei Reef region  to the number of individuals sampled (N) ranged from 0.91 to 1.00 . The mean heterozygosity value of all loci was 0.508–0.687 at every site, and the total mean value ± standard error (SE) was 0.592±0.012 for all sites . No signf region . Departull sites . FIS vaCKER see . The rat to 1.00 . The valA. digitifera populations in this area, which is also supported by the small values of pairwise FST (≤0.033). Significant differences were detected between some sites . Significant differentiations were also detected between SNS site in the Kerama region and some sites in the Ishigaki, Sekisei Reef regions. Also, pairwise FST values between some sites in the southern part of the Nansei Islands  and KSN site in the Amami region tended to be significantly different from zero. Despite the short geographic distance between them, significant genetic differentiations were observed between IRB site in the Miyako region and UGN site in the Ishigaki region, between sites in the Miyako region and those in the Sekisei Reef region. However, the sites in the Miyako region were genetically closer to some sites in the Amami, Okinawa, and Kerama regions. A similar pattern showing no correlation between geographic and genetic distances was found between the Sekisei Reef region and other regions. For example, no significant differences were found between some sites in the Sekisei Reef region and those in the Tanega-shima, Okinawa, Kerama regions. These results did not change after adjusting for the genotypes using MICROCHECKER FST values were changed slightly  me sites , but FSslightly .A. digitifera at 19 sites  from A. digitifera are remote from other plots. The results of PCA did not change after adjusting for the genotypes using MICROCHECKER (data not shown). The result of STRUCTURE K, i.e., K = 3 to 7. Also, the estimated log probability of the data, LnP(D) gradually decreased with increases in K value, K = 2: LnP(2) = −11396.3, K = 3: LnP(3) = −12003.0, K = 4: LnP(4) = −12668.2, K = 5: LnP(5) = −12313.1, K = 6: LnP(6) = −12141.3, K = 7: LnP(7) = −12223.5.Principal coordinate analysis PCA;  revealed19 sites , the firA. digitifera populations over the large geographic area  of the Nansei Islands was confirmed. FST values for A. digitifera were 0.033 and smaller for all site combinations  suggest that A. digitifera colonies in the Nansei Islands propagate mainly through sexual reproduction. Thus, the wide range of genetic connectivity among A. digitifera populations in the Nansei Islands is believed to be maintained by the dispersal of sexually produced planula larvae, and not by asexual reproduction.In our study, the existence of genetic connectivity among inations . These vA. digitifera was high in the Nansei Islands, significant genetic differentiation between close sites in some regions were confirmed in some cases . In addition, PCA indicated that the FST values of this species varied considerably among sites within a region. Although one must note that low FST values may be easily detected when using highly variable markers such as microsatellites A. digitifera population genetic study using allozymes, Nishikawa et al. A. digitifera in the Ryukyu archipelago. Therefore, our result appears to have been caused by ecological or biological factors rather than by the characteristics of the markers. Physical and topological factors may affect the movement and habitat selection of this species, causing local genetic differentiation. Miller and Ayre Pocillopora damicornis on Lord Howe Island, and they found that FST values throughout the North Bay and other sites in a protected lagoon  were lower than at sites in the Old Gulch (only 1 km away) located in the open sea. They suggested that the existence of a shallow reef interfered with genetic exchanges between the close sites. Such topographic effects may also be present around Okinawa Island because the coastal topography of the Nansei Islands is complex. Many gulfs exist along the coast of Okinawa Island, and the shapes of reefs around the Nansei Islands vary greatly, which would influence larval recruitment. For example, in contrast to the geographic distances between them, a population of Goniastrea aspera at Sesoko on the west coast of Okinawa Island was genetically closer to a population in the Kerama Islands located west of the Okinawa Islands than to a population at OHD on the southeast coast of Okinawa Island A. digitifera observed in our results. In addition, differences in the environmental factors  affecting larval recruitment and the survival of adult corals may also be related to the local genetic differentiation observed in this study because environmental conditions around the Nansei Islands vary greatly among sites While the degree of the genetic connectivity of A. digitifera. To date, some studies of marine animals have shown that genetic diversity decreases at the boundaries of geographic distribution Batillaria zonalis has low mitochondrial genetic diversity at the northern and southern edges of its geographic distribution range in Japan Zostera marina, the numbers of leaf shoots, dry biomass, and faunal abundance were found to increase according to the increase in microsatellite genotypes, despite near-lethal sea temperatures, and declines in genotypic diversity were related to decreases of leaf shoots, dry biomass and faunal abundance Acropora tenuis in the northwest of Australia, significant differences in genetic diversity were detected between inshore and offshore regions, with lower diversity observed on higher-latitude reefs A. digitifera populations to decrease with latitude. Decreased heterozygosity was not detected in the Tanega-shima region, which is almost the northern limit of A. digitifera's geographic distribution, even though the sample size was small (N = 11). The exchange of larvae creates and maintains high levels of genetic diversity, which is crucial in terms of resilience against disturbance A. digitifera populations across a large geographic area, even if mass bleaching occurs, A. digitifera populations might be able to show rapid numerical recovery, even in temperate regions. A possibility exists that A. digitifera populations in the Nanei Islands remain relatively tolerant to environmental changes such as sudden elevations in sea temperature by sharing a variety of alleles. This potential would have been maintained by strong gene flow caused by the Kuroshio Current We found no significant relationship between genetic diversity and latitude in A. digitifera populations in the Nansei Islands seem to have high recovery potential even after high levels of disturbance, provided that populations on unaffected reefs are maintained. However, note that we examined only one species and that these results should not be simply applied to other coral species, including other Acropora spp. Therefore, comparative analyses of genetic connectivity in several coral species, including both spawners and brooders, should be performed using highly variable markers such as microsatellites to enhance the management of coral communities in our study area based on a more generalized view of coral populations at the periphery of their geographical distribution. Also, if connections and recruitment from outside decrease, peripheral populations are expected to exhibit reduced levels of genetic and genotypic diversity due to the combined effects of bottlenecks, inbreeding, and site-specific selection In conclusion, our results show that regardless of the latitude of their habitat, A. digitifera were taken at each sampling site from haphazardly selected colonies that were at least 3 m apart. Coral fragments were preserved in 100% ethanol in 1.5-ml Eppendorf tubes and were then transported to the laboratory.We established seven geographic sampling regions  in the Nansei Islands . All samA. digitifera fragments by removing the coral skeleton from sample tubes containing coral tissues. We usually extracted genomic DNA from the surface tissues as above, but in the case of samples from Sekisei Reef, genomic DNA was extracted not only from the surface tissues but also from skeletons. The skeletons were washed in 100% ethanol, dried, crushed with a pestle, and suspended in 400 µl SE buffer  in a 1.5-ml Eppendorf tube. We then added 3 µl Proteinase K (20 mg ml−1) and incubated samples at 37°C to 50°C for 24 to 72 hours. After that, we added 160 µl 5 M NaCl and 530 µl chloroform and vigorously mixed the samples using a vortex mixer before centrifuging. We then took the water layer and carried out standard ethanol precipitation.Using an AquaPure Genomic DNA kit , genomic DNA was extracted from the surface tissues of A. digitifera by Nakajima et al. Acropora palmata by Baums et al. Acropora millepora by van Oppen et al. A. digitifera (−1 (multiC: <1 ng µl−1) template DNA, and MilliQ water  for a total reaction volume of 5 µl. Amplifications were carried out in a PC-818 touchdown thermocycler  operated under the following conditions: 95°C for 5 minutes, followed by 35 cycles at 95°C for 30 seconds, 50°C (gradient: −0.1°C cycle−1) for 30 seconds, 72°C for 1 minute, and a final 72°C extension for 30 minutes. Allelic variations were analyzed using a DNA capillary sequencer . When alleles were unclear or not detected, normal PCR  was conducted under the 100 nM primers for 1 locus . We did not find shifts in allele size between multiplex and normal PCR. In Sekisei Reef samples, when we could not detect clear genotypes using DNA from tissues of A. digitifera, we also used DNA extracted from the skeleton (∼50–300 ng µl−1) as a template. For regular PCR with the Sekisei Reef samples, we diluted skeletal DNA (up to 10 times) to obtain the appropriate DNA concentration, if required.We used microsatellite marker developed for gitifera . We amplgitifera , 0.125 UNg and Ng/N values, pairwise AMOVA FST values, principal coordinate analysis  and expected heterozygosity (He) under HWE are in proportion to the values of the inbreeding coefficient (FIS), that is, positive and negative FIS values suggest deficits and excesses of heterozygotes, respectively. The exact test for departure from HWE was also performed using FSTAT. Significance levels were adjusted using a false discovery rate (FDR) correction following The numbers of alleles, allele frequencies, and the number of private alleles were calculated using the GenAlEx program (Ver. 6.2) sis PCA; , estimatNg represents an estimate of the minimum number of clones present in a population. When N indicates the number of collected and genotyped individual colonies, Ng/N provides an index of the effects of asexual reproduction and suggests genotypic richness Ng/N = 1, all of the collected colonies in a population are unique (no clones); Ng/N approaches zero when a population has only a single genotype .The extent of asexual reproduction was estimated from the genotypic diversity of each population. If several unique multilocus genotypes were detected, F-statistics via AMOVA. This analysis was carried out using GenAlEx (Ver. 6.2) FST indicates a high extent of gene flow and vice versa. Significance levels were adjusted using FDR corrections. Adjusted allele data by MICROCHECKER  Table S1Acropora digitifera at 19 sites. N is the number of analyzed colonies. ADJ and % suggest the number and rate of individuals adjusted by MICROCHECKER, respectively. *We excluded MS166 from subsequent analyses because alleles of many individuals were adjusted due to null alleles (over 20%).The number and rates of individuals changed the allele pattern to heterozygosis from homozygosis after the adjustment by MICROCHECKER Click here for additional data file.Table S2Acropora digitifera pairwise population FST via AMOVA values estimated among sites in the Nansei Islands and adjusted by MICROCHECKER Click here for additional data file.


Mizuhopecten yessoensis) is a cold-tolerant bivalve that was introduced to China for aquaculture in 1982. In this study, amplified fragment length polymorphism (AFLP) markers were used to investigate levels of genetic diversity within M. yessoensis cultured stocks and compare them with wild populations. Six pairs of primer combinations generated 368 loci among 332 individuals, in four cultured and three wild populations. High polymorphism at AFLP markers was found within both cultured and wild M. yessoensis populations. The percentage of polymorphic loci ranged from 61.04% to 72.08%, while the mean heterozygosity ranged from 0.2116 to 0.2596. Compared with wild populations, the four hatchery populations showed significant genetic changes, such as lower expected heterozygosity and percentage of polymorphic loci, and smaller frequency of private alleles, all indicative of a reduction in genetic diversity. Some genetic structures were associated with the geographical distribution of samples; with all samples from Dalian and Japan being closely related, while the population from Russia fell into a distinct clade in the phylogenetic analysis. The genetic information derived from this study indicated that intentional or accidental release of selected Japanese scallops into natural sea areas might result in disturbance of local gene pools and loss of genetic variability. We recommend monitoring the genetic variability of selected hatchery populations to enhance conservation of natural Japanese scallop resources.Japanese scallop ( Moreoet al. .Mizuhopecten yessoensis) has a wide distribution on the cold coasts of northern Japanese islands, in the northern part of the Korean Peninsula, Russian Primorye, Sakhalin, and Kuril Islands, [M. yessoensis  were first introduced into China from Japan by Liaoning Ocean and Fisheries Science Research Institute [Chlamys farreri) and the introduced bay scallop (Argopecten irradians). By 2005, the annual production of Japanese scallop had reached about 150,000 tons in China [M. yessoensis farming is mainly distributed in the region of Liaoning and Shandong provinces, and Dalian (Liaoning) scallop aquaculture accounts for about 90% of the total national production [The Japanese scallop  [EcoRI, M = Mse I): E-AAC/M-CCA, E-ACA/M-CTC, E-ATC/M-CCT, E-ATC/M-CTT, E-ATG/M-CCT, and E-ATG/M-CTG , percent polymorphic loci (P), and FST values were estimated using the TFPGA program [Fragment data were transferred to a binary (1/0) data matrix. Average heterozygosities ( program . The con program . Average program . Confide program  to produvs. 66%) and mean heterozygosity values (0.2285–0.2442 vs. 0.2380) similar to the natural seed population LSK from Dalian. But natural seed population JA was observed to have significantly higher percent polymorphic loci  and mean heterozygosity values  than all the populations from Dalian, indicating that higher genetic diversity existed in JA population than in those in Dalian. This may be due to a very large natural scallop population existing in Japan, while all populations around Dalian originated from a limited number of stocks introduced in the 1980s from Japan [Six AFLP primer combinations were used to genotype 332 scallops from 7 populations. Of the 350 original individuals, 18 scallops were omitted from the analysis because of a high incidence of questionable bands, presumably because of low quality DNA. A total of 368 bands were identified across the remaining 332 scallops, 337 of which were polymorphic bands, with an overall mean heterozygosity of 0.2601 . Some vaOne of the objectives of this research was to provide an initial determination of the genetic structure of the scallop population. The genetic similarity analysis, using Nei’s genetic distance with UPGMA method, revealed significant differentiations among the scallop populations . All popOstrea edulis [Haliotis discus [This research uses AFLP markers for the first time for the analysis of genetic resources of Japanese scallop. Because of the large numbers of bands and high levels of polymorphism, AFLP analysis allows large genome coverage with limited resources . Before a edulis  and Pacis discus , but ourThe results of the UPGMA dendrogram analyses indicated that there was population differentiation in the scallop populations studied. Population RU was separated from other populations and formed a single clade. All other populations, regardless of their seed breeding methods, were clustered into the other single clade, indicating their geographic differentiation. One interesting finding was that the natural seed population LSK clustered closely together with the artificial population ZI with a high bootstrapping confidence value, despite the geographic distance (about 100 km) between the two farming locations. It is possible that the natural seed population LSK was spawned or reproduced by the scallop population in Zhangzi Island and then drifted with the ocean currents to the Lüshun Kou area. Because no Japanese scallop population previously inhabited the Lüshun Kou coast, the natural spats or juveniles collected must come from somewhere else nearby. Our recent driftage test (unpublished data) has shown that there is an ocean current flowing from Zhangzi Island to the Lüshunkou area that lasts about 20 days in spring (from February to March). Further research is needed to test this hypothesis. Population XM is comprised of the hybrid scallops produced by female scallops of the population originally introduced in the 1980s crossed with male scallops imported in 1998 from Aomori, Japan. Population LSB was also made up of scallops cultured by suspended cage culture, and it clustered in the same clade. The population LSB and GI clustered together, and there may have been broodstock exchanges between the hatcheries in LSB and GI in the past.M. yessoensis cultured stocks and compared them with three wild populations. The results indicate that some differentiation existed between wild and cultured populations, even though the ancestors of the currently cultured scallops were introduced from Aomory, Japan, and artificial breeding operations have had no substantial effect on the genetic structure of the scallop populations examined in Dalian. The data can be used as baseline information for further study of the interactions between hatchery based artificial breeding and natural seed collection of Japanese scallop in China.In the present study, we used AFLP markers to investigate levels of genetic diversity within four


Thymallus thymallus) is a salmonid fish native to Europe, with a distribution ranging from England and France to the Ural Mountains of north-western Russia. The majority of grayling populations inhabit freshwater rivers and lakes but some populations also occupy brackish water in northern parts of the Baltic Sea. Previous population genetic studies have demonstrated that grayling populations in Finland, Estonia and Russia belong to a single mitochondrial lineage and exhibit high levels of differentiation even at a small geographic scale. As a result, we predicted that grayling populations should not cluster regionally. Despite the extensive amount of genetic research that has been carried out on grayling, comprehensive national-level information on population structure of grayling in Northern Europe is still lacking. Yet this is the level at which populations are currently managed.The European grayling (We found unanticipated population structure of grayling clustering into three groups largely corresponding to the northern, Baltic and south-eastern geographic areas of Finland using 13 microsatellite loci. We also found a high level of genetic differentiation among the groups and moderate to high differentiation within the groups. This combined with low variability strongly indicates that genetic drift and limited migration have a major impact on grayling population structure. An allele size permutation test indicated that mutations at microsatellite loci have not significantly contributed to genetic differentiation among the three Finnish groups. However, at the European scale, mutations had significantly contributed to population differentiation.This research provides novel genetic information on European grayling in its northern distribution range and has clear implications for supporting country-scale conservation efforts. Specifically, the strong between population divergence observed indicates that single populations should generally be recognized as separate management units. We also introduced an alternative prioritization strategy for population conservation based on the evaluation of the relative roles of different evolutionary forces shaping the gene pools. We envision that the proposed approach to categorize populations for conservation will be a useful tool for wildlife researchers and conservationists working on a diverse range of organisms. Thymallus thymallus, is a salmonid fish highly appreciated by recreational anglers. It is native to Europe with a distribution from England and France to the Ural Mountains of north-western Russia. It can inhabit stream, riverine or lacustrine habitats and show large phenotypic differences in gill raker number, body size, weight at first spawning and fecundity . Later, "pooled" sum(X2) and sum(Y2) were calculated accordingly. One-thousand pooled RXY were subsequently obtained as XY were ranked and the corresponding P-value was calculated.An association between the geographic distance and genetic divergence matrices was examined using the Mantel test implemented in the GenAlEx 6.1 package  supportedistance . For detdistance  to yieldRST is analogous to FST, where RST is a stepwise mutation based measurement of genetic differentiation taking into account the variance of microsatellite allele size [FST and RST are expected to have similar values. If stepwise-like mutations have significantly contributed to the differentiation RST is expected to be larger than FST. In the allele size permutation test, the distribution of RST values from 10 000 permutations (pRST) was compared to observed RST. The test of allele size permutation was performed at three different hierarchical levels; among population groups within Finland as identified by PCA and Structure analyses, among countries and among 25 grayling populations.To test whether stepwise-like mutations have significantly contributed to genetic differentiation between populations, the allele size randomization test  was emplele size . When thDST and GST) components of each population. These parameters are relative measurements and are evaluated as the contribution of each population to total allelic richness  pooled from all populations. Thus, negative values indicate that the diversity or the differentiation of a population is lower than the mean of the whole dataset. For prioritizing grayling populations within Finland, foreign populations were excluded from the analysis in order to avoid the underestimation of the differentiation component within Finnish populations. Two Finnish populations (Iso and Rau) were also excluded because of their mixed origin, from hatchery stocking of non-native fish [In order to rank populations in terms of their conservation priority, Contrib 1.20 ,34 was uive fish , which wo = 0.21; He = 0.20) and highest in SweHol . No deviation from Hardy-Weinberg equilibrium was observed for any locus after the sequential Bonferroni correction (k = 13). Genotypic linkage equilibrium tests suggested linkage disequilibrium (P < 0.0001) between five population-locus pairs: BFRO7-BFRO13 in Esa; BFRO11-BFRO13 and BFRO13-BFRO18 in Kit; BFRO12-Cocl3 and BFRO15-Cocl23 in Juu. However, as each of these five linkage disequilibrium occurred only in a single population and none involved the same two loci, these loci are likely not in physical linkage. Beside these five locus pairs, no deviation from genotypic linkage equilibrium was observed after the sequential Bonferroni correction . Therefore, for subsequent analyses, all loci were assumed to have independent segregation of alleles.The average number of alleles per locus varied from 1.6 (Kai) to 5.4 (Tor) while the average allelic richness ranged from 1.6 (Kai) to 4.6  were very distinct compared to the rest along the first principal component axis (data not shown). This deep divergence corresponds to two mtDNA lineages described by Koskinen et al. . When onSimilar to the PCA results, population clustering analysis using the program Structure without prior information about population of origin of individuals initially distinguished three main groups of Finnish grayling corresponding to 'northern', 'Baltic' and 'south-eastern' groups figure . After tFST across all 25 populations was 0.342 indicating a very high level of differentiation among individual grayling populations. Within Finland, the global FST was 0.294 and the FST within the three population clusters was 0.145, 0.146 and 0.256 for the northern, Baltic and south-eastern group, respectively. Pairwise FST among populations is detailed in the supplementary material [see Additional file The global FCT = 0.191). The two alternative hierarchical groupings explained smaller proportions of variation .The hierarchical analysis of molecular variance revealed that the proportion of total genetic variance due to differences between groups was highest for grouping 1 . In addition, pooled within-group comparisons did not result in significant IBD signal . The Mantel test using ln distance provided the same conclusion as employing the raw geographic distance.A significant isolation-by-distance (IBD) signal was observed when all Finnish populations were included in the analysis . In the among five countries comparison, the observed multilocus RST also fell well above the upper limit of the 95% confidence interval (CI) of the null distribution of pRST . However, RST was not significantly larger than pRST when comparing Finland with Sweden . When the SweHol sample from the Baltic sea was excluded from the analyses because it essentially belongs to the same Baltic group as Kru, Pera, Kit and Iso RST was still very similar to pRST indicating the predominant role of genetic drift and migration over mutation when comparing Finnish and Swedish populations.The allele size permutation test indicated that mutations at microsatellite loci did not significantly contribute to genetic differentiation among the three Finnish groups compared to genetic drift and migration. At the European scale, the permutation test indicated that mutations had significantly contributed to population differentiation  high diversity-high differentiation group, 2) high diversity-low differentiation group; 3) low diversity-high differentiation group and 4) low diversity-low differentiation group figure . Arguablsensu Moritz [In accordance with other population genetic studies in European grayling ,13,36,37u Moritz . One of u Moritz ,13 of gru Moritz ,39,40.Rather surprisingly, several different types of analyses strongly indicated that the Kitkajärvi population (Kit) groups together with the Baltic populations even though this population inhabits an inland lake that is relatively distant from the Baltic Sea (174 km) and, moreover, currently flows into the White Sea basin. However, it is known that Kitkajärvi (lake Kitka) had postglacially bifurcated outflow; the eastward channel drained into the Baltic sea via the system of Livojärvi (lake Livo) until around 8 400 years ago, and the westward channel flowed into the White Sea via Kitkajoki (river Kitka) as it still does . The posSalmo salar), brown trout , whitefish (Coregonus hoyi) and rainbow trout (Oncorhynchus mykiss), the mean expected heterozygosity measured at microsatellite loci commonly varies from 0.63 to 0.76 (e.g. [r = 1.81) and low heterozygosity (He = 0.24), which is in accordance with the demographic history of the population as it is known that NorLes was most likely established by a small number of individuals in 1880 [r = 1.56, He = 0.20). As most of the study populations are from the area that was completely covered by an ice sheet during the last glacial period, it is possible that the low variability of grayling in northern Europe reflects postglacial colonization and subsequent founder effects [r = 3.23, He = 0.41) and other studies concentrating on the southern distribution range of European grayling have shown low diversity levels [Among salmonid fishes, European grayling is one of the least genetically variable species, exhibiting low diversity even across highly variable markers such as microsatellites. In Atlantic salmon (76 e.g. -45) whil whilSalm in 1880 . However effects ,47. Howey He = 0.4, which y levels ,48. ThisDespite the clear separation of the three population groups in Finland there was no evidence that mutations have significantly contributed to the genetic differentiation among these groups. Thus, even though we observed relatively high differentiation between the three population groups, the allele permutation test indicated that patterns of differentiation are mostly driven by genetic drift and low migration rather than accumulation of new mutations. However, the allele size permutation test strongly suggested that mutations significantly contributed to population differentiation on a broader geographic scale.sensu Petit et al. [The "central dogma of conservation genetics" is that genetic variability is beneficial and therefore it is often assumed that increasing genetic variability enhances population survival . Anothert et al. , it is pWhen examining diversity and differentiation components in grayling populations, we identified four categories where the relative importance of different evolutionary forces (namely drift and migration) varies. These categories are high diversity-high differentiation (category 1), high diversity-low differentiation (category 2), low diversity-high differentiation category 3) and low diversity-low differentiation category 4). From the conservation perspective, the populations falling into the high diversity-high differentiation category  may be less affected by genetic drift and migration as they represent large isolated populations. This high diversity-high differentiation category has the highest likelihood of containing unique genetic material. Notably, all three Baltic members fall into this category and given that grayling populations in the Baltic Sea are likely adapted to the brackish environment, they are hence highly relevant for conservation . Populat and low . From thThis research provides genetic information on European grayling in its northern distribution range in order to assist country-scale conservation. We found unanticipated population structure of grayling, clustering into three groups largely corresponding to the northern, Baltic and south-eastern geographic areas of Finland and we recommend that these three groups should be used as the starting point for developing a national grayling conservation strategy. However, as the observed clusters extend beyond the borders of Finland, international co-operation for broader scale conservation management is warranted. We also found high levels of genetic differentiation among the groups and moderate to high differentiation within the groups. Such strong divergence indicates that single grayling populations should generally be recognized as separate management units. We also developed an alternative prioritization strategy in the conservation perspective by categorizing populations based on the evaluation of the relative role of various evolutionary forces affecting the indigenous gene pool. We envision that the proposed population categorization approach could be useful for a diverse range of organisms.The authors declare that they have no competing interests.AS carried out the molecular genetic work, statistical analyses and wrote the first draft of the manuscript. MTK performed genotyping in some populations. JP conducted the majority of the fieldwork. MTK, JP, AV and CRP co-designed the research theme. AV and CRP were involved in developing later drafts of the manuscript. All authors read and approved the final manuscript.Characteristics of microsatellite loci used in the study. Primer concentration and summary statistics of microsatellite loci.Click here for fileElectropherograms of four individuals, each genotyped at 15 microsatellite loci in a single multiplex polymerase chain reaction. Electropherograms generated by GeneMapper software.Click here for fileRST  and FST .Pairwise genetic distance as measured with  Pairwise genetic distance of the grayling populations as measured with RST and FST.Click here for file


Within this specialised fauna, the tube-dwelling polychaete Alvinella pompejana showed reciprocal monophyly at mtCOI on each side of the Equator (9°50'N/7°25'S), suggesting potential, ongoing allopatric speciation. However, the development of a barrier to gene flow is a long and complex process. Secondary contact between previously isolated populations can occur when physical isolation has not persisted long enough to result in reproductive isolation between genetically divergent lineages, potentially leading to hybridisation and subsequent allelic introgression. The present study evaluates the strength of the equatorial barrier to gene flow and tests for potential secondary contact zones between A. pompejana populations by comparing the mtCOI gene with nuclear genes.Comparative phylogeography recently performed on the mitochondrial Alvinella pompejana populations along the East Pacific Rise. Migration was oriented north-to-south with a moderate allelic introgression between the two geographic groups over a narrow geographic range just south of the barrier. Multilocus analysis also indicated that southern populations have undergone demographic expansion as previously suggested by a multispecies approach. A strong shift in allozyme frequencies together with a high level of divergence between alleles and a low number of 'hybrid' individuals were observed between the northern and southern groups using the phosphoglucomutase gene. In contrast, the S-adenosylhomocysteine hydrolase gene exhibited reduced diversity and a lack of population differentiation possibly due to a selective sweep or hitch-hiking.Allozyme frequencies and the analysis of nucleotide polymorphisms at three nuclear loci confirmed the north/south genetic differentiation of A. pompejana. This separation also coincides with demographic expansion in the southern East Pacific Rise. Our results suggest that allopatry resulting from ridge offsetting is a common mechanism of speciation for deep-sea hydrothermal vent organisms.The equatorial barrier leading to the separation of East Pacific Rise vent fauna into two distinct geographic groups is still permeable to migration, with a probable north-to-south migration route for  Many population genetic models have been proposed to describe how individuals disperse between geographically separated localities including n-island models ,2, steppDeep-sea hydrothermal vents represent a patchily distributed habitat (sometimes separated by thousands of kilometres) suggestive of a one-dimensional stepping-stone model over 60 000 kilometres of globe-encircling ridge crests . BecauseAlvinella pompejana, which only lives at the top of hydrothermal-vent chimneys of the East Pacific Rise (EPR) between 38°S and 27°N (Guaymas Basin), has been described as a pioneer species because it is the first species to colonise new hot-sulphide edifices [et al. [A. pompejana's larval biology - cannot be maintained across vent fields if larvae travel more than 15-30 days in the water column [et al. [A. pompejana embryos can arrest their development in cold water, potentially delaying their metamorphosis during dispersal from vents. To better explain the absence of isolation-by-distance in the northern EPR, three hypotheses have been proposed: (1) allozymes lack the power to detect genetic differentiation between populations; (2) balancing selection - due to the homogeneity of vent conditions along the whole ridge system - maintains homogeneous allozyme frequencies; or (3) displacement of habitat patches over geological times promote metapopulation dynamics, thereby maintaining gene flow [et al. [A. pompejana larvae [Amongst vent species, the deep-sea hydrothermal vent polychaete edifices . The laredifices . This woedifices . During edifices . This kiedifices . Given t [et al. ,21 have  [et al. ). A hydrr column . Neverthr column . These rr column . Accordi [et al.  reportedene flow . The firene flow  who suggcytochrome c oxidase subunit I (mtCOI) gene between the northern and southern EPR populations of A. pompejana [et al. [mtCOI haplotype distributions over the geographic range of the species, including the southern EPR, was performed to better address the ability of allozymes to detect genetic differentiation and to discriminate between the three hypotheses described above. Three additional nuclear genes, including the PGM locus, were used to evaluate the strength of this equatorial barrier to gene flow and to test for potential secondary contact. We also attempted to quantify migration and/or introgression rates between the previously isolated northern and southern EPR populations to disentangle the effects of selective and demographic processes contributing to the genetic break.Recently, genetic divergence of nearly 1% was observed at the mitochondrial ompejana ,29, suggAlvinella pompejana polychaetes were collected with the manned submersible Nautile during two oceanographic cruises in 1999 (HOPE) and 2002 (PHARE) at northern EPR sites , samples were measured at the fourth setigerous segment, sexed, and cut in two: the cephalic portion was frozen immediately at - 80°C for allozyme analyses and the body was preserved in 95% ethanol for DNA analyses.et al. [et al. [Allozymes, encoded by nine enzyme systems, were examined from seven hydrothermal vent field samples Table , followiet al. . Protein [et al. . Allozymet al. [A. pompejana mtCOI sequences from this previous study and the sequences of the outgroup A. caudata were obtained with the 'universal' LCOI and HCOI primers [S-adenosylhomocysteine hydrolase (SAHH) and globin X (GlobX) genes [phosphoglucomutase gene sequence in A. pompejana previously obtained using degenerate primers . A non-synonymous change was discovered in exon 4 of PGM and specifically targeted in subsequent analyses. Sizes of the DNA fragments and their exon-intron structures (boundaries) are given in additional file Genomic DNA was extracted using a CTAB extraction procedure following the protocol described in Plouviez et al. . A. pomp primers  using thX) genes ), or froet al. [2, 0.05 mM (0.25 mM for PGM) of each dNTP, 0.48 μM (0.4 μM for PGM) of each primer . PCR products were isostochiometrically mixed, purified using QIAQuick™ columns, and ligated into a pGEM-T vector . For each cloning experiment, 32 positive clones were sequenced on both strands at Genoscope (Evry) with universal plasmid primers (SP6 and T7), leading to a total number of approximately 3500 nuclear sequences. Sequences from all genes were proofread using CodonCode Aligner 2.0.6 http://www.codoncode.com/aligner/. Sequence alignments were initially performed with ClustalW [in vitro recombinants between individuals from the same cloning set were detected by their abnormal combination of 5'-tails and removed from the dataset. Multiple recaptures also allowed us to discard intra-individual in vitro recombinants  and artefactual/somatic mutations.Polymerase chain reactions (PCR) and cloning were performed using the mark-recapture (MR) method developed by Bierne et al.  with 4-nClustalW  in BioEdf and tested with 1000 permutations. To detect putative barriers to gene flow, genetic differentiation was estimated along the EPR from a multilocus Fst as calculated using Weir and Cockerham's θ [Linkage disequilibrium between allozyme loci was examined for each locality and for the complete dataset using Genetix version 4.05.2  to detecrham's θ , adding rham's θ  for all mtCOI marker, this test was also performed without the 13°N population. To better understand population structure, the number of genetically-differentiated groups (K) was evaluated using individual assignments to these a priori groups based on their multilocus allozyme genotypes from an admixture model with a 150 000 burn-in length period followed by 50 000 MCMC repetitions. The most appropriate number was obtained by testing K values from 1 to 15 using the Structure program (version 2.2) [Isolation-by-distance was tested along the whole EPR using a Mantel Spearman test  with 500ion 2.2) . ConvergGlobX, SAHH and PGM genes, respectively. For all statistical analyses based on recaptured sequences, one consensus allele was randomly selected per individual to avoid any putative bias due to the cloning method. Allele diversity Hd, nucleotide diversity Nπ, and Watterson's theta W θwere estimated for northern and southern EPR sites separately with DNAsp 4.10.3 [st) were computed using Arlequin 3.1 [Average recapture rates were 0.60, 0.74 and 0.63 for p 4.10.3 . For eacp 4.10.3  to searcquin 3.1  from nucBecause most coalescent-based approaches impose strong assumptions on the absence of recombination, recombinants were detected by the Hudson and Kaplan 's four-gmtCOI to adjust for their expected population sizes. The Hasegawa-Kishino-Yano model [Population sizes, migration rates, and the time of population splitting were estimated using the isolation-with-migration model  by grouno model  was chosIMa) were converted into years. Because mutation rates have not yet been estimated for alvinellid polychaetes, with the exception of the mtCOI gene [mtCOI, as well as PGM and SAHH for which a mutation rate has been obtained from the hydrothermal vent gastropod Lepetodrilus fucensis across the Blanco transform fault [Bathymodiolus thermophilus across the Easter microplate [GlobX gene was not used because there is no adequate calibration for its mutation rate. Before calculating the geometric mean, an average mutation rate was assessed per locus per generation accounting for different lengths of the three genes , assuming one generation per year. The geometric mean U = 0.64 × 10-6, close to the value previously found using eight genes  on B. thermophilus [IMa /U).To better estimate the date since the splitting event, IMa parameters of divergence between populations . A restriction fragment length polymorphism (RFLP) analysis combined with genotyping insertion-deletions (indels) was performed to create a composite PGM genotype for each individual. Among the four clades observed, clade 3 displayed two restriction sites for the RsaI enzyme whereas the three remaining clades only had one. Clade 4 displayed two restrictions sites for SspI whereas the three remaining clades have three sites. Clades 1 and 2 differed by several fixed substitutions for which no restriction site was observed and the presence of a 9 bp indel. RsaI and SspI digestions were performed on PGM PCR products following manufacturer's protocols and the presence/absence of the indel was genotyped using new nested primers defined from each side of the indel. Individuals were amplified in 15 μl solutions that included 1X buffer (supplied by manufacturer), 1.25 mM MgCl2, 0.2 mM each dNTP, 0.1 μM of fluorescent-labelled (IRD700™) forward primer, 0.27 μM of unlabelled forward primer, 0.33 μM of unlabelled reverse primer . This test compares divergence and polymorphism at several loci to determine if any loci display a departure from neutral evolution. McDonald Kreitman tests  in northern and southern EPR populations  using Sweep_bott [Because gene genealogies contain information on demographic and/or selective events, neighbour-joining trees (distances obtained by the maximum composite likelihood) were performed using Mega 4.0.2 , separatjima , Fu and and Fay[F*, [H, ) indexes[F*, [H,  was compsts  were aleep_bott  with 100Linkage disequilibria were not significant for any pair of loci in any population (p > 0.05 using the Garnier-Géré and Dillman  test), iθ [θ = 0.097, p > 0.05, 1000 permutations) but Fst significantly differed from zero with the Robertson and Hill's correction . The exact G test for genic differentiation was significant for the overall populations (p < 0.05). Tests for isolation-by-distance showed a significant correlation between the genetic distance of allozymes θ/(1-θ) and geographic distance across the entire EPR region (Mantel Spearman test: p = 0.014) but was not significant when the 13°N site was removed from the dataset (p = 0.115). Partitioning genetic structure using the Bayesian clustering method of Pritchard et al. [The multilocus estimate of Weir and Cockerham's θ  over allrection  and mtCOI were analysed in the northern and southern populations of A. pompejana across the Equator  in the northern and southern EPR, respectively : clades 1 and 2 were mainly found in the northern EPR, whereas clades 3 and 4 were distributed in the southern EPR . Although populations appeared to be genetically differentiated across the Equator at the GlobX locus , these populations were genetically homogeneous across regions at the SAHH locus .DNA sequence variation among the three nuclear genes  and from the south to the north (ms) shows that gene flow occurred predominantly from the north to the south with no migration being detected in the opposite direction  model of Hey and Nielsen  was usedn Figure .PGM, SAHH, and GlobX were not significantly different from zero on the basis of their 90% highest posterior density (HPD) interval. This was clearly suggested by the allele networks for PGM and GlobX  at this locus.Single-locus estimates of migration also exhibited asymmetry from the north to the south, although the parameters for X Figure  for whicX Figure , indicatPGM network  between the northern and southern groups were detected at 7°25'S, two (16.7%) at 14°S and only one (1.7%) at 13°N. No 'hybrids' were found at the other latitudes.Based on the k Figure , we deteD) and Fu and Li (F*) indexes were significantly negative for all loci in the south, but only SAHH displayed significant values in the north. All genes exhibited departures from neutral equilibrium between drift and mutation in the southern EPR, but not in the northern EPR. Similarly, for each nuclear gene, Fay and Wu tests were not significantly different from the neutral expectation in the northern populations, but were significantly negative in the southern populations.Tajima , whereas trees from the two other nuclear genes (SAHH and GlobX) had more star-like topologies typical of a selective sweep , indicating that at least one gene has experienced positive selection (or gene hitchhiking). After excluding mtCOI, the HKA test became nonsignificant (p χ2 > 0.05), suggesting this gene could be experiencing strong purifying selection or a sweep. However, McDonald-Kreitman tests failed to reject neutral expectations at any locus (p > 0.05) when comparing A. pompejana polymorphism to the divergence of the A. caudata outgroup.The p Figure . The HKASAHH gene, indicating that in addition to a bottleneck this gene may have undergone a selective sweep, at least in the northern populations. In effect, the M2 and M3 models are not mutually exclusive if a gene has experienced a moderate sweep in a bottlenecked population.The Sweep_bott analyses performed on northern and southern groups of populations using the four loci are summarised in Table mtCOI gene between the northern and southern EPR populations has been identified in previous studies by using a large number of hydrothermal vent species [Lepetodrilus ovalis and the bivalve Bathymodiolus thermophilus, a clinal distribution of the northern and southern mtCOI haplotypes was observed and attributed to the occurrence of a secondary contact zone between the northern and southern EPR [A. pompejana exhibited a clear equatorial break at this locus, raising the question of the permeability of this barrier to gene flow. The strength of the break between the northern and southern populations of A. pompejana appears to have significantly affected both allozyme and nuclear loci despite the recent formation of the barrier. Although the SAHH gene exhibited very little differentiation across this barrier, both the GlobX and PGM loci displayed a clear geographic separation of alleles. Divergence between northern and southern clades was estimated at ~1-2 Mya using the IMa multilocus estimation with very low migration rates. This estimate is in agreement with the shared vicariant event estimated at ~1.3 Mya from a series of vent species using approximate Bayesian computation on the mtCOI gene [A genetic break at the  species ,29. For COI gene -61) reinCOI gene .PGM locus  is there emergence of a secondary contact between previously isolated populations due to the relaxation (i.e. attenuation of the transverse circulation) of the barrier?Questions addressing potential migration during an ongoing allopatric isolation process may be explored in two ways, depending on the primary cause for separation of populations (transform faults or bottom water circulation): (1) is there a progressive isolation of the north and south populations of n (north to south) was shifted in comparison to ms  raises the question of whether the barrier is becoming increasingly impermeable to dispersal with time, or whether it represents a semi-permeable genetic barrier following recent secondary contact. In the first case, one can expect that most "migrating" alleles  would coalesce deeply in the evolutionary tree as they will represent incomplete lineage sorting. In the other case (emergence of a tension zone), all "migrating" alleles should coalesce shortly in the coalescence tree, leading to very low mtimes (times at which gene flow was maximum).The fact that gene flow is virtually absent for at least two loci  revealed the occurrence of a concomitant demographic expansion in the southern EPR within the last 0.5 My [et al. [SAHH). The two former explanations seem unlikely. In the first case, the expectation would be an ancestral population size nearly similar to that of the northern one. In the second case, the Sweep_bott analysis (based on the same sequence dataset) would have validated the bottleneck scenario in the northern populations. The detection of a bottleneck in the southern populations does not preclude the possible additional effect of a selective sweep at any specific locus. The SAHH locus was the only locus that conformed to a selective sweep model on both sides of the EPR with virtually no genetic differentiation across the expected barrier, if an initial isolation step in allopatry is assumed. An advantageous allele  is more likely to cross a genetic barrier and to spread within populations of the recipient lineage if the allele is advantageous for both lineages . This suggests that the lack of lineage sorting found in A. pompejana at the SAHH locus is probably not the result of a slow evolutionary rate at this locus but rather a case of high migration rate across the barrier. The spread of an allele across a permeable barrier is expected to be related to the magnitude of the selective advantage [et al. [SAHH gene may be indeed very sensitive to selection or linked to a potential positively selected gene.A comparative phylogeographic approach involving seven vent species using the t 0.5 My . In the t 0.5 My ,56), the [et al. . Multipl [et al. ,76, woul [et al. ,78. This [et al. ,80. Howees e.g. ,82). Ana. AnamtCOdvantage , suggest [et al.  proposedA. pompejana populations confirmed the presence of an equatorial barrier, which plays an important role in structuring populations of the species A. pompejana, as proposed by Plouviez et al. [mtCOI and nuclear genes strongly supports the southern demographic expansion hypothesis previously suggested by a multispecies analysis using mtCOI only. Combining multilocus and multispecies analyses therefore allows us to propose that allopatry is probably one of the major forces promoting speciation and biodiversity within the vent fauna along the one-dimensional framework of oceanic ridges.In summary, our multilocus study of z et al. . This geA. pompejana. FHL organised two of the three oceanographic cruises, helped sample specimens and provided helpful elements of discussion. DJ designed the study, carried out sampling, performed analyses with SP and edited the manuscript. All authors have read and approved the final manuscript.SP carried out sequence acquisition and RFLP/indel genotyping as well as the molecular genetic analyses and drafted the manuscript. DLG genotyped individuals using allozymes. OL annotated the cDNA library of This study is part of SP's PhD thesis on the comparative phylogeography of deep-sea hydrothermal vent species along the East Pacific Rise (EPR). Her research involves multiple species and markers to examine speciation and demographic processes along the EPR. DLG is lab technician in charge of genotyping and biochemical analyses. OL works on comparative genomics and bioinformatics. FHL has broad knowledge of vent ecology and has spent most of his career working on the physiology of deep-sea hydrothermal vent species. DJ is a population geneticist working on the evolution and dispersal of deep-sea hydrothermal vent species. He has recently developed a genomic approach to better understand the role of gene adaptation in the evolution of vent species.Alvinella pompejanaPrimers designed for DNA amplification on . Table of sequences for forward and reverse primers used in the study.Click here for fileSize and exon-intron structure of the four sequenced genes. Schematic drawings of the gene portion used in which exonic regions are represented by boxes.Click here for fileAllele frequencies, expected and observed heterozygosities and Fis estimated from enzyme loci across vent fields. Table of allozyme frequencies and heterozygosities for each sampled population.Click here for fileEstimates and 90% Highest Posterior Density (HPD) intervals of demographic parameters from IMa multilocus analysis. Table of 90% HPD intervals for population sizes, migration rates and time since population splitting estimated from the IMa Bayesian computations.Click here for file