Mytilus spp. is based on the exploitation of natural populations and aquaculture. Aquaculture activities include transfers of spat and live adult mussels between various geographic locations, which may result in large-scale changes in the world distribution of Mytilus taxa. Mytilus taxa are morphologically similar and difficult to distinguish. In spite of much research on taxonomy, evolution and geographic distribution, the native Mytilus taxa of the Southern Hemisphere are poorly understood. Recently, single nucleotide polymorphisms (SNPs) have been used to clarify the taxonomic status of populations of smooth shelled mussels from the Pacific coast of South America. In this paper, we used a set of SNPs to characterize, for the first time, populations of smooth shelled mussels Mytilus from the Atlantic coast of South America.Throughout the world, harvesting of mussels Mytilus spp. samples were collected from eastern South America. Six reference samples from the Northern Hemisphere were used: Mytilus edulis from USA and Northern Ireland, Mytilus trossulus from Canada, and Mytilus galloprovincialis from Spain and Italy. Two other reference samples from the Southern Hemisphere were included: M. galloprovincialis from New Zealand and Mytilus chilensis from Chile. Fifty-five SNPs were successfully genotyped, of which 51 were polymorphic. Population genetic analyses using the STRUCTURE program revealed the clustering of eight populations from Argentina (Mytilus platensis) and the clustering of the sample from Ushuaia with M. chilensis from Chile. All individuals in the Puerto Madryn (Argentina) sample were identified as M. platensis × M. galloprovincialis F2 (88.89%) hybrids, except one that was classified as Mediterranean M. galloprovincialis. No F1 hybrids were observed.M. platensis (or Mytilus edulis platensis) and M. chilensis are distinct native taxa in South America, which indicates that the evolutionary histories of Mytilus taxa along the Atlantic and Pacific coasts differ. M. platensis is endangered by hybridization with M. galloprovincialis that was introduced from Europe into the Puerto Madryn area in Argentina, presumably by accidental introduction via ship traffic. We confirm the occurrence of a native M. chilensis population in southern Argentina on the coast of Patagonia.We demonstrate that The online version of this article (10.1186/s12711-018-0376-z) contains supplementary material, which is available to authorized users. Mytilus spp., including exploitation of natural populations and aquaculture, is approximately 1.2 million tons per year [Mytilus taxa [Mytilus taxa are morphologically similar and difficult to distinguish. Replacement of native species by an invasive taxon can go unnoticed [Mytilus taxa can hybridize in areas where their populations merge and coexist. Hybridization zones have been described in the Danish Straits at the entrance to the Baltic Sea [Mytilus taxa [Correct recognition and identification of species are important to understand the phylogeography of living resources and to ensure the conservation of their biodiversity, their management, sustainable exploitation and traceability. The world production of mussels, lus taxa –5. Mytilnnoticed . Individltic Sea –10, on tltic Sea , Great Bltic Sea , 13, Greltic Sea , and theltic Sea . In addilus taxa –19. Nucllus taxa , 20.Mytilus galloprovincialis, one of the most invasive mussel species, threatens native populations of Mytilus on a global scale [M. galloprovincialis with native Mytilus taxa, i.e. Mytilus trossulus on the eastern and western North Pacific coasts [M. galloprovincialis in Australia and New Zealand [Mytilus in Europe. The introduced M. galloprovincialis interbreeds with native Mytilus edulis are cultivated in Scotland. Northern Hemisphere M. galloprovincialis is also a threat for local farms that are located south of the Gulf of Arauco in Chile and use native populations of Mytilus chilensis from South America [Introduction of al scale , 22. Irrc coasts –26 and n Zealand –29. Mich Zealand  reviewed America .Mytilus are in South America. South American smooth shelled blue mussels of the genus Mytilus are present on the Atlantic coast of the south of Brazil [Mytilus edulis [M. platensis [M. chilensis [Mytilus on the Atlantic coast of South America, i.e. M. platensis was described by d’Orbigny in 1846 [M. chilensis was described by Hupé in 1856 [M. platensis, were later synonymised to M. edulis or assigned a subspecies rank [M. platensis [M. edulis platensis [M. platensis [M. chilensis [M. edulischilensis [M. platensis to differentiate it from the Chilean mussel, M. chilensis. Later, these mussels were considered as subspecies (M. edulis platensis and M. edulischilensis) since the number of diagnostic characters to assign them to species rank was not sufficient [M. e. platensis and M. chilensis have been used in Argentina and Chile, respectively.The longest Pacific and Atlantic coastlines that are continuously inhabited by native populations of f Brazil , throughf Brazil , 34 and f Brazil , where tlatensis , 36–38 ohilensis . The nat in 1846  using sp in 1856  based onies rank . Ecologilatensis , 44 and latensis , while tlatensis , M. chilhilensis , 48. Bashilensis  named thfficient , 49. ForM. e. platensis was developed in Argentina [Mytilus and Aulacomya) in Argentina fell from over 250 tons in 2011 to 11.20 tons in 2016 [M. e. platensis is cultivated only in the San Matías Gulf [M. chilensis in the Canal Beagle, Ushuaia [Aquaculture of Atlantic rgentina . Currentrgentina . Product in 2016 , 53. Dueías Gulf  and M. c Ushuaia .Mytilus edulis [M. galloprovincialis [M. chilensis [M. chilensis was a subspecies, M. edulischilensis, whereas Ouagajjou et al. [M. chilensis to be a valid species. Analysis of mtDNA and nuclear DNA Me15/16 restriction fragment length polymorphisms (RFLP) revealed the occurrence of native M. chilensis and the alleged presence of the native Atlantic blue mussel M. edulis, Northern Hemisphere M. galloprovincialis, M. trossulus genes  and hybrids (M. chilensis × M. edulis) in the region of the Strait of Magellan [M. chilensis should be named M. edulisplatensis [M. platensis [Mytilus populations from Argentina and Uruguay using 30 enzyme loci and five DNA markers  showed that native Atlantic South American populations are closely related to North Atlantic M. edulis [M. edulis at 10 loci, a taxon name Mytilus edulisplatensis was proposed for native mussels from the Atlantic coasts of South America. Consequently, these names have been used in ecology and aquaculture-related publications e.g. [M. chilensis and M. platensis were placed in the same clade, representing conspecific variants rather than distinct species [M. planulatus or M. e. platensis was proposed for all native Mytilus populations in South America [The natural distribution of Chilean mussels ranges from Bahia Concepcion (36°45′S) to Punta Arenas (53°S), on the edge of the Strait of Magellan , 57. Thes edulis , 59 and incialis , 61 was incialis , or evenhilensis . Based ohilensis  suggesteu et al.  in a stuMagellan . Recent latensis  or M. pllatensis . A genet. edulis . Becauseons e.g. , 71. Bas species . In addi America .Mytilus taxa, including a set of reference samples from North America, Europe and New Zealand. According to their results, the Pacific coast South American native mussel is genetically distinct from the reference species M. edulis, M. galloprovincialis and M. trossulus, and should be recognised as M. chilensis Hupé 1854 [Mytilus from the coast of South-Western Atlantic and adjacent waters. The aim of our research was to extend the knowledge of the taxonomic status and distribution along the Atlantic coast of South America and Patagonia of native Mytilus taxa and to identify areas where they are potentially endangered by hybridization with the invasive M. galloprovincialis.To clarify the taxonomic status of native populations of smooth shelled mussels from the Pacific coast of South America, Larrain et al.  used SNPupé 1854 . In our Mytilus spp. samples that consisted of 359 individuals of mixed ages and sizes (5 to 50 mm) were collected from ten localities in Argentina and one from Chiloe in Chile between 2012 and 2014  was tested by 10,000 permutations of alleles between individuals. Departures from Hardy–Weinberg equilibrium (HWE) were tested by exact test, and significance was determined by Markov chain Monte Carlo simulations. The most informative loci were detected by identifying FST outlier loci. The upper limits of the 95% confidence interval were identified with 20,000 iterations and characterized by estimating the 0.05 and the 0.95 and 0.99 quantiles of the distribution. The false discovery rate (FDR-BY) was applied to correct significance (P) values after multiple testing [FST values in Arlequin. The FST distance measures in the Newick format, which were obtained from SNP allele frequencies data in POPTREEW [K) was estimated by computing likelihood over 10 runs for values of K ranging from 1 to the number of populations studied plus 1. At the plateau of the graph curve, the value of K captures the main structure of the populations. The best-fit number of genetic clusters was determined by calculating the logarithmic probability LnP(K) using the ΔK method [Arlequin v.3.5.1.2  was used testing , 77. GenPOPTREEW  were usePOPTREEW . RobustnPOPTREEW , 81. STRK method . ThreshoK method . IndividK method . A MarkoCorrespondence analysis (CA)  implemenM. platensis, M. galloprovincialis, F1 hybrids, F2 hybrids and two types of backcrosses.Following preliminary analyses that indicated that the Argentinian population of Puerto Madryn (PMD) contained possible hybrids, the software NewHybrids v1  was usedOf the 79 SNPs assayed, 55 were successfully genotyped with an acceptable quality score, and among these, 51 were polymorphic for 562 mussels from 19 samples [see Additional file FST values at individual SNPs ranged from 0.014 to 1 [see Additional file FST values significantly different from zero. Several groups of samples were tested to detect highly informative SNPs that are effective for differentiation between groups. Populations and individuals (hybrids) with an admixture of different groups (taxa) were excluded from the analysis. An outlier test was carried out for the group of eight populations from Argentina (without PMD and UBC) and the group of reference samples from Chile, USA, Canada, Northern Ireland, Spain, Italy and New Zealand. The test indicated 14 outlier loci, of which seven were characteristic only for M. trossulus and seven  that differentiated the Argentinian samples. These latter same seven SNPs were identified as outliers in a test between M. platensis and M. galloprovincialis samples.M. edulis samples from America and Europe. This test highlighted six outlier SNPs , of which three had not been previously reported. These findings show that 19 SNPs are sufficient to differentiate Argentinian populations from those of all other regions and highlighted their distinct taxon status. Five SNPs were identified as highly informative (P < 0.01): BM106B, BM12A, BM151A, BM21B, and BM6C.Four SNPs were effective at differentiating between the Argentinian and Chilean populations, and among these, two were significant also in the outlier analysis (BM151A and BM21B) while BM203C and BM57A were new. Subsequently, an outlier test was carried out to detect SNPs that could differentiate populations from Argentina and FIS measures , an excess of homozygotes was found only in one population from Argentina (COM) and two reference samples .The proportion of polymorphic SNPs  that exhibited admixture in the Mytilus taxa. The population from Ushuaia (UBC) clustered with M. chilensis. Internal branches were short between M. chilensis and M. platensis populations, whereas they were long between M. edulis and M. galloprovincialis populations. Pairwise FST values were significantly different from zero after FDR-BY correction between most pairs of samples . Pairwise FST estimates between the seven populations from Argentina (M. platensis) were not significantly different from zero (0 to 0.025), which indicated that these seven populations are mostly homogenous.We constructed a neighbour-joining tree based on les Fig.  and [seeMytilus, correspondence analyses (CA) were carried out on 18 samples of M. edulis, M. galloprovincialis, M. chilensis and M. platensis, by excluding M. trossulus for higher resolution .To characterize the population structure of ion Fig. . Figure K = 3, and then K = 5. This result indicated that the best-fit number of genetic clusters was found for K = 3, for which differentiation between clusters corresponding to M. trossulus, M. galloprovincialis and all other Mytilus taxa occurred. The high value of ΔK for K = 5 suggested further subdivision, with five clusters corresponding to five taxa: M. trossulus, M. galloprovincialis, M. edulis, M. chilensis and M. platensis  increase was largest (i.e. ΔK was highest) for sis Fig. . These rK = 5, most of the 562 individuals were properly assigned to their original samples with a genome admixture value q higher than 0.8 [see Additional file M. platensis), whereas the sample from Ushuaia (UBC) clustered with the two M. chilensis samples from Chile. A few exceptions were observed: one individual from UBC was assigned to M. platensis, single individuals from ARG73 and IRD were considered potentially admixed . Furthermore, the entire PMD population from Argentina showed very high levels of admixture: one individual was assigned to M. galloprovincialis with q higher than 0.8, whereas all other individuals were ambiguously assigned to two clusters, mainly to M. galloprovincialis and M. platensis, with q ranging from 0.2 to 0.8. In addition, two individuals were assigned to two other clusters M. edulis and M. galloprovincialis, and M. chilensis and M. galloprovincialis. In general, most individuals had the highest q for the M. galloprovincialis cluster followed by the M. platensis cluster.Using the panel of 51 SNPs, two methods of analysis were used to test the assignment of individuals from Argentina to the most likely population and regions, based on reference taxa. In the STRUCTURE analysis at M. galloprovincialis from the Mediterranean and Atlantic populations and more than 30% to the Argentinian population from Isla de los Pajáros (IPL). Four individuals were placed in the M. edulis population from North America and, for one individual the highest probability occurred for M. chilensis populations from Chile (PZC).Individuals were assigned to baseline populations based on region of origin with a success rate of 97.8% using GeneClass2 [see Additional file M. platensis and Northern Hemisphere M. galloprovincialis, we carried out additional analyses to identify the type of hybrid (F1 or F2). Using the program NewHybrids, 19 SNPs that are effective at differentiating between populations from Argentina were used to analyze all the Argentinian populations and three groups of reference samples: M. platensis, Northern Hemisphere M. galloprovincialis, and M. chilensis. All PMD individuals were identified as hybrids except one that was classified as M. galloprovincialis and F2 hybrids that carry M. platensis and M. galloprovincialis alleles were detected in 88.89% of the PMD individuals with a probability higher than 90%. We did not identify any F1 hybrids and detected only one backcross (probability of ~ 60%) to M. platensis. In addition, using M. chilensis as a reference sample, two F2 hybrids that carry M. chilensis and M. galloprovincialis (PMD) alleles and M. chilensis and M. platensis (ARG73) alleles were identified.Because most of the individuals from PMD were assigned mainly to two clusters using STRUCTURE analysis, M. trossulus, M. galloprovincialis, M. edulis, M. chilensis and M. platensis  and M. edulis samples  [see Additional file M. platensis samples differed from Mediterranean M. galloprovincialis (FST = 0.65). The level of differentiation observed between M. platensis and M. edulis was comparable to that between M. edulis and Atlantic M. galloprovincialis  and was highest between M. platensis and M. trossulus and Southern Hemisphere M. galloprovincialis taxa .To determine the degree of similarity between taxa, we compared the different groups of populations without hybrids . We constructed a NJ tree by using only non-admixed individuals as identified by STRUCTURE and GeneClass2 analysis, which revealed five well-supported clades that coincide with five separate taxa: sis Fig. . Based oMytilus in Argentina were composed of three groups: (1) eight samples of native M. platensis, (2) a southern group represented by the UBC population of native Chilean blue mussel (M. chilensis), and (3) the population from Puerto Madryn (PMD) composed of hybrid individuals . Argentinian populations showed a low level of hybridization, with the exception of the PMD population, which is composed mainly of hybrid individuals.Overall, these results indicate that the samples of Mytilus populations e.g. [Mytilus populations from the Atlantic coast of South America and Isla Grande de Tierra del Fuego are strongly differentiated and belong to at least three taxa. Native M. platensis is a separate taxon from other Mytilus species, including native M. chilensis. M. platensis predominates on the Argentinian coast south of Rio de La Plata, whereas M. chilensis inhabits the southernmost part of the South American coasts including the area of Isla Grande de Tierra del Fuego. Invasive European M. galloprovincialis and introgressed specimens were found in one location (PMD). These findings shed new light on the genetic relationships between South American Mytilus taxa. M. platensis was first described by d’Orbigny in 1846 [M. platensis (M. e. platensis) was reported on the Atlantic coast, from Uruguay including Maldonaldo in the north [M. chilensis (M. e. chilensis) on the Argentinian coast of the Isla Grande de Tierra del Fuego including the Ushuaia Bay was tentatively assumed from studies on similar topics [In most previous studies of South American smoothed-shelled blue mussel ons e.g. , 68, onlons e.g. . In our  in 1846  based onhe north , 91–93 ahe north  to Golfohe north , 95, 96.r topics –99.M. chilensis × M. platensis hybrid and one M. platensis × M. edulis hybrid in more northern sampling sites. Oyarzún et al. [M. chilensis and native Atlantic Mytilus in the Magellan Region (southern Chile). However, since they used molecular markers with lower diagnostic power for Southern Hemisphere Mytilus taxa, their results cannot be directly compared with those reported here. We observed no hybridization in populations of M. chilensis from neighbouring sampling sites (Punta Arenas and Ushuaia) and detected only pure individuals of M. chilensis and one M. platensis individual in the area of the Isla Grande de Tierra del Fuego.In our study, we detected only one native individual n et al.  reportedM. galloprovincialis with Southern Hemisphere native mussels in Chile, New Zealand and Australia was reported [Based on the use of single molecular markers, hybridization of invasive Northern Hemisphere reported , 99, 100reported , 31. We reported , 20.M. galloprovincialis from the Northern Hemisphere might be related to past aquaculture activities in the Nuevo Gulf, although Dellatorre et al. [M. e. platensis. In addition, the National authorities in Argentina did not issue any permission for any experiment related to the introduction of M. galloprovincialis . It was further confirmed that no related aquaculture experiments were performed in Argentina . Another more likely scenario is the accidental introduction of M. galloprovincialis by means of ship transportation. Fishing vessels as well as big cruise ships enter Puerto Madryn from Europe, North America and Chile [Mytilus sp. in the Puerto Madryn local ecosystem [Semimytilus algosus was transported to Puerto Madryn (Nuevo Gulf) by a fishing vessel and released during in-water hull cleaning [M. galloprovincialis.The introduction of e et al.  clearly nd Chile  with thend Chile , which rcosystem . For exacleaning , which cM. galloprovincialis was detected in Punta Arenas and named M. galloprovincialis planulatus Lmk 1819 [M. planulatus from Port du Roi George, Nouvelle-Hollande  in 1819. To confirm or not its occurrence in South America, new genetic markers such as SNPs could be used. However, our results did not confirm its presence in the studied area and a study of Mytilus populations from Chile, including Punta Arenas did not detect it [M. planulatus in South America [The presence of Southern Hemisphere Lmk 1819 . HoweverLmk 1819  originaletect it . Therefo America .M. platensis (or M. e. platensis) and M. chilensis are differentiated and native taxa in South America, which indicates a distinct evolutionary history of Mytilus taxa from the Atlantic and Pacific coasts. Our analysis identified 19 SNPs (five of which are highly informative) that are effective in differentiating populations from Argentina. M. platensis is endangered by hybridization with the introduced European M. galloprovincialis in one area in Argentina (Puerto Madryn), presumably due to accidental introduction from ship traffic. We also confirmed the occurrence of M. chilensis on the Isla Grande de Tierra del Fuego, southern coast of Argentina. Knowledge of the origin of the mussels is very important for the conservation of native populations in the context of aquaculture activities in this area. The occurrence of the M. platensis × M. galloprovincialis hybrid population in Puerto Madryn that demonstrates one of the most invasive species, Northern Hemisphere M. galloprovincialis, is a threat to native populations. Continued monitoring is needed to check for the spread between these two taxa on the Atlantic coast of South America.We have demonstrated that Additional file 1: Table S1. SNP polymorphisms in populations of Mytilus spp. studied. Description: Information is presented on SNP properties, genome location, substitution type, FSTP value associated with test for outlier status, minor allele frequency, GenBank annotation and references.Additional file 2: Table S2. Allele frequencies of 51 SNPs for 19 Mytilus spp. sample. Description: The data show frequencies of all alleles at the studied SNP loci in all samples.Additional file 3: Figure S1. Neighbour-joining tree of native South American and the reference Mytilus taxa. Description: Neighbour-joining tree shows genetic relationship between 19 Mytilus spp. samples from Argentina and reference populations of M. edulis, M. trossulus, M. galloprovincialis and M. chilensis from America, Europe and New Zealand based on the FST distance measures obtained with POPTREEW and visualised with MEGA version 6.Additional file 4: Table S3.FST distance matrix for 19 Mytilus spp. samples for 51 SNPs. Description: Values of FST with P < 0.05 after Benjamini–Yekutieli (FDR-BY) correction are marked in bold. Site names and locations are in Table Additional file 5: Table S4. Result of population assignment algorithms STRUCTURE and GeneClass for 19 populations of mussels. Description: Two methods of analysis were used to test the assignment of individuals from Argentina to the most likely population and regions, based on reference taxa. In the STRUCTURE analysis at K = 5, most individuals were properly assigned to their original samples. Individuals were assigned with GeneClass2 to baseline populations based on region of origin with a success rate of 97.8%.Additional file 6: Table S5.FST distance matrix for Mytilus spp. samples. Description: FST distance matrix is presented for Mytilus spp. samples from Argentina and reference populations of M. edulis, M. trossulus, M. galloprovincialis and M. chilensis, obtained with POPTREEW for individuals without admixture (q > 0.8 or q < 0.2) identified by STRUCTURE analysis.


To determine newest the most accurate allele frequencies for 15 short tandem repeat (STR) loci in the Bosnian and Herzegovinian population, calculate statistical parameters, and compare them with the relevant data for seven neighboring populations.® 16 System to obtain allele frequencies for 15 polymorphic STR loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA. The calculated allele frequencies were also compared with the data from neighboring populations.Genomic DNA was obtained from buccal swabs of 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using PowerPlexThe highest detected value of polymorphism information content (PIC) was detected at the PentaE locus, whereas the lowest value was detected at the TPOX locus. The power of discrimination (PD) values had similar distribution, with Penta E showing the highest PD of 0.9788. While D18S51 had the highest value of power of exclusion (PE), the lowest PE value was detected at the TPOX locus.Upon comparison of Bosnian and Herzegovinian population data with those of seven neighboring populations, the highest allele frequency differentiation was noticed between Bosnian and Herzegovinian and Turkish population at 5 loci, the most informative of which was Penta E. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population was between the mentioned cluster and Serbian population. To determine more accurate distribution of allelic frequencies and forensic parameters, our study included 1000 unrelated individuals from all regions of Bosnia and Herzegovina, and our findings demonstrated the applicability of these markers in both forensics and future population genetic studies. Short Tandem Repeats (STRs) are common markers in population biodiversity research, paternity testing, and forensic analysis of biological evidence. Reliability of STR amplification provides a high level of individualization that is crucial for population genetic studies. To obtain precise and reliable results of analysis, it is necessary to use population data obtained from a sufficient number of the samples . CurrentThe latest recommendations regarding the official publication and forensic usage of STR population data highlighted the need for increasing the size of population sample for its calculation. Therefore, the main aim of this study was to determine latest and more accurate allele frequencies and forensic statistical parameters for 15 most used STR loci in the Bosnian and Herzegovinian population and compare them with the relevant data for other neighboring populations.Biological buccal swab samples were collected from 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Samples were randomly collected from routine casework performed during the period of 2006-2016 at the Institute for genetic engineering and biotechnology, University of Sarajevo. Only unrelated adults over 18 years of age were included in this study. Informed consent for the use of collected biological material and data was obtained from all subjects.DNA extraction was performed using the Qiagen DNeasyTM Tissue Kit . DNA conP < 0.01 and P < 0.001 for the population differentiation test. The number of effective alleles (AE) was estimated as 1/ ∑ pi2, where p denotes the allele frequency for a particular locus. Ratio of effective and detected numbers of alleles and its statistical significance was calculated as suggested by Pojskic et Kalamujic , power of discrimination (PD), power of exclusion (PE), and typical paternity index (TPI) were calculated within Microsoft Excel workbook template – PowerStats . Powermaalamujic  with Allalamujic . A Z-scoer et al . The neier et al . These cer et al .P > 0.05 for all), except at the D8S1179 locus. However, after applying the Bonferroni’s correction, no statistical significance was revealed either (P = 0.015). Excess of heterozygosity was detected for D3S1358, D21S11, D18S51, D16S539, vWA, and TPOX loci (E/AN) was detected for the TH01 (0.652) and lowest for the TPOX (0.319) locus (P = 0.001), D18S51 , Penta E , PentaD , D8S1179 , TPOX  and FGA  loci showed statistically significant ratio effective and detected number of alleles (P < 0.01) indicating a sharp-cut departure of effective number of alleles from the detected number of alleles.Allele frequencies and statistical parameters including heterozygosity (observed and expected), results of exact test, PD, and PE for the 15 STR markers were calculated . No statPOX loci . A totalPOX loci . The hig9) locus . HoweverThe highest value of polymorphism information content (PIC) was found for the Penta E, and the lowest for the TPOX locus. The same results were obtained for PD, while the highest PE value was detected for D18S51 . The lowThe neighbor-joining dendrogram based on result of genetic distance analysis showed the relationship between the Bosnian and Herzegovinian population and neighboring populations -23. It sOur results were concordant with the findings of the previous study conducted in Bosnian and Herzegovinian population . This waAllele frequencies for 15 STR loci in the studied Bosnian and Herzegovinian population did not differ significantly from those found in populations of Slovenia , Serbia The neighbor-joining dendrogram showed the relationship among 8 populations on the basis of result of genetic distance analysis. Bosnian and Herzegovinian population has the highest genetic distance from the Turkish population and the lowest from the Serbian population. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population is placed between the mentioned cluster and the Serbian population. The results of genetic distance analysis are in concordance with conclusions about similarity among neighboring populations based on of STR profiles .Although a study that was based on 100-150 respondents was sufficient at the time  and cons


Parmelina carporrhizans in the Mediterranean region. Using eight microsatellite and mating-type markers we showed that fungal populations are highly diverse but lack spatial structure. This is likely due to high connectivity and long distance dispersal of fungal spores. Consistent with low levels of linkage disequilibrium and lack of clonality, we detected both mating-type idiomorphs in all populations. Furthermore we showed that the Macaronesian Islands are the result of colonization from the Mediterranean Basin. The unidirectional gene flow, though, seemed not to be sufficient to counterbalance the effects of drift, resulting in comparatively allelic poor peripheral populations. Our study is the first to shed light on the high connectivity and lack of population structure in natural populations of a strictly sexual lichen fungus. Our data further support the view of the Macaronesian Islands as the end of the colonization road for this symbiotic ascomycete.The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, represents a center of diversification for many organisms. The genetic structure and connectivity of mainland and island microbial populations has been poorly explored, in particular in the case of symbiotic fungi. Here we investigated genetic diversity and spatial structure of the obligate outcrossing lichen-forming fungus  Cryptic biodiversity is an essential component of biodiversity that will be considerably affected by global climate change in the next decades5Lichen-forming fungi are a successful group of nutritionally specialized fungi that form obligate symbioses with photosynthetic partners. Molecular studies of lichen-forming fungi have repeatedly indicated that numerous distinct lineages, i.e. cryptic species, can be hidden under a single taxon. This shows that species diversity of lichen-forming fungi might be vastly underestimated, mainly because of the plasticity of morphological and chemical characters and the frequency of homoplasy and convergence of characters78Lichen-forming fungi are interesting subjects for population genetics studies. One of the reasons for this is the vast range of reproductive strategies, i.e., from purely asexual to exclusively sexual, that could differentially shape their population structures111314Buellia frigida, a crustose lichen of continental Antarctica, an SSR-based study revealed high intra-population diversity, local population differentiation and limited dispersal, probably resulting from harsh habitat conditions and scarce spatial and temporal habitat availabilityLobaria pulmonaria, SSR-based studies have shown highly clonal and structured populations, resulting from low level of recombination and limited dispersal capacity of vegetative propagules20Parmotrema tinctorumParmelina tiliaceaLobaria pulmonaria is also the only lichen species for which population-level data on the distribution of mating-type idiomorphs is available so farFor investigating intra-population variability and inferring the role of reproductive strategies in shaping population dynamics, highly variable molecular markers such as microsatellites (SSRs) are one of the most appropriate tools, especially for highly clonal organisms such as lichens17Parmelina carporrhizans24. Parmelina carporrhizans is a foliose, epiphytic macrolichen with the center of distribution in the Mediterranean region. The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, is an important centre of diversification for many plant and animal species2526In this study we analyzed intra-species genetic diversity, population structure, and mating-type distribution in natural populations of the obligate outcrossing lichen-forming fungal species P. carporrhizans is a result of migration from the Macaronesian Islands to the continent or from the continent to the Macaronesian Islands.To test these hypotheses, we used highly variable, species-specific microsatellite and mating-type specific markers. First, we analyzed the species population genetic diversity and evaluated how it is hierarchically and spatially partitioned. We assessed the extent to which alleles are geographically restricted or shared across regions. Second, we calculated the relative abundance of the two mating-type idiomorphs in the populations. Self-incompatible species require the presence of both mating types in the population for sexual reproduction. Third, we estimated migration rates among mainland and island groups. Combining genetic diversity and gene flow estimates we inferred whether the distribution of P. carporrhizans.We successfully amplified the MAT-genes fragments in 93% of the samples. In each sample we found a single band corresponding to one idiomorph . These rThe overall difference in frequency of the mating-type idiomorphs was not significantly different, with MAT1-1 found in 55% of the samples, and MAT1-2 in 45% of the samples. Only two populations (Morocco and Herbés) had a significantly skewed higher frequency of MAT1-1 idiomorphs .All eight microsatellite loci were highly polymorphic. The analysis of 220 samples with eight microsatellite markers resulted in a total of 220 microsatellite alleles, ranging from 16 alleles (Pcar1) to 46 alleles (Pcar7) . No idenOverall we found a high unbiased haploid diversity (mean uh = 0.846), ranging from 0.744 (Tenerife) to 0.898 (Sicily). Populations had high population allelic richness (mean = 5.244), ranging from 3.973 (Cádiz) to 6.047 (Gredos). Private allelic richness was moderately low in all populations, ranging from 0.447 (Tenerife) to 1.072 (Gredos). At the regional level, the Macaronesian Islands had lower allelic (11.486) and private (3.095) allelic richness than the mainland populations  .P = 0.014), Cádiz , Famalicão  and Herbés  (P. carporrhizans populations occurs within populations (92.1%), with lesser amounts among populations within regions (2.3%) and among regions (5.6%) (ST = 0.079) and regions (FCT = 0.056) are not significant .We detected low levels of linkage disequilibrium (LD) across populations (mean = 0.069). Low but significant LD values were found in four populations, namely Gran Canaria 1  . The AMOs (5.6%) . PermutaST values between populations were low, ranging from 0 to 0.023 (ST = 0.016) were statistically significant.As expected, pairwise Fto 0.023  and onlyBased on the discriminant analysis of principal components (DAPC), three genetic clusters were considered optimal to describe the data . The scatter plot of local densities of distances  that were significantly geographically restricted (P < 0.001). The overall observed mean distance between geographically restricted alleles was also significantly smaller than the expected mean , but higher than the overall observed mean geographic distance between all shared alleles thus indicating over-dispersion  of the Macaronesian Islands group (1.16) was considerably smaller than that of the mainland group (16.6). The migration analysis detected high, unidirectional gene flow from the mainland group to Macaronesia .P. carporrhizans in the Mediterranean region. Sexually reproducing fungal species are expected to show higher genetic diversity and weaker population structure than vegetatively reproducing species11P. carporrhizans exhibit high levels of genetic diversity and a complete absence of clonality. This is strikingly different from the trend reported for predominantly asexual lichen species, e.g. L. pulmonaria, whose populations harbor, almost always, a high number of clonal multilocus genotypes  is different from P. carporrhizans, which might also have contributed to the differences in population structures of the two species. As compared to vegetatively reproducing species however, as expected, P. carporrhizans displayed lower genetic structure. For example, a recent study based on sequencing data, showed high population differentiation in the closely-related vegetatively reproducing (isidiate) species, Parmelina tiliaceaP. carporrhizans, the Macaronesian populations of P. tiliacea were the most genetically diverse.Another species, for which the genetic variability and population structure have been analyzed using SSR markers, is P. carporrhizans indicates high frequency of sexual recombination resulting in novel genotypic combinations in each generation. We obtained evidence for this prediction by analyzing the distribution of mating-type idiomorphs in P. carporrhizans populations.The high genetic diversity of P. carporrhizans is a heterothallic, i.e. self-incompatible, species, as previously found using RAPD fingerprinting on cultured spore isolateset al.P. carporrhizans population seems to be sufficient to ensure panmictic populations and thus low levels of linkage disequilibrium.Our results based on mating-type ratios corroborate that P. carporrhizans suggested that core populations of the Mediterranean Basin and edge populations in the Macaronesian Islands are still highly connected via gene flow. To corroborate this hypothesis, we estimated migration rates between these two population groups. Our results indicate that populations across the southern range of the species are still highly connected. However, we found that gene flow is unidirectional, i.e., from core towards edge populations. This may well explain the smaller population size and lower allelic richness of the island populations. Moreover, on the Canary Islands, P. carporrhizans does not grow at low altitudes, where it is replaced mainly by Parmotrema species, but grows in the Erica–Myrica heath  communities on well-lit borders of the laurel forest (‘monteverde’), on sweet chestnut, elms, poplars, pines, etc. from 800 to 1500 m a.s.l. The composition of the forests (at least in Tenerife) has undergone major changes during the Late Holocene, in particular, suitable habitats such as arboreal species of the vegetation alongside laurel forest have considerably declined as a consequence of human activity and climatic variationGenetic diversity and population structure analyses of Based on these observations, we propose that migratory fluxes from core populations can only partially compensate for the detrimental effects of drift, likely caused by founder effects, by maintaining relatively high genetic diversity in peripheral populations. Several studies on different organisms  towards the Macaronesian Islands.In conclusion, we showed that the strictly sexual, heterothallic lichen-forming fungus Parmelina carporrhizans represents an ideal system to test these predictions for strictly outcrossing lichen-forming fungi. Additional studies in this direction can further shed light on the effect of dispersal mode on the co-phylogeographic dynamics of the lichen symbiosis.The apparent absence of dispersal barriers found in our study implies that the establishment of newly dispersed fungal spores is not limited by the availability of compatible photobionts17474849P. carporrhizans from 11 localities to extensively cover the south-western distribution range of the species, from the Macaronesian Islands and Northern Africa, to Southwestern Europe where the species is most abundant  of terminal lobes of freshly collected thalli or recent herbarium specimens were used for DNA isolation with the DNeasy Plant Minikit .Small pieces (0.5 cmWe genotyped each specimen at eight fungus-specific microsatellite markers (Pcar1–8). Details of the markers, PCR conditions and multiplexing are given in ref. P. carporrhizans-specific MAT-genes primers we obtained partial MAT1-1 and MAT1-2 sequences from the scaffolds of the P. carporrhizans genomeXanthoria elegans, Xanthoria polycarpaLobaria pulmonariaFor designing 2 tests.To identify the mating-type idiomorph of each sample, we performed multiplexed PCR reactions in a total volume of 25 μL with 0.65 μL of each MAT1-1 primer (10 μM), 0.6 μL of each MAT1-2 primer (10 μM), 0.4 μL dNTP’s  and 0.5 μL DNA polymerase  and 2.5 μL of 10–50 ng DNA. PCR reactions were performed with an initial denaturalization at 94 °C for 4 min, followed by 10 cycles of denaturalization step at 95 °C for 10 sec, annealing at 55 to 50 °C for 20 sec and extension at 72 °C for 30 sec, followed by 30 additional cycles with annealing temperature of 50 °C for 20 sec and an additional extension at 72 °C for 5 min. We tested if both idiomorphs were evenly distributed in the entire area and in each individual sampling locality with χWithin-population genetic diversity was calculated by estimating allelic richness and private allelic richness using a rarefaction approach, implemented in ADZEpoppr of the R package poppr v.2.1.159To analyze the level of linkage disequilibrium we estimated rBarD (unbiased index of associationST) were calculated using Arlequin v3.5.To quantify genetic differentiation among and within populations we carried out a global analysis of molecular variance (AMOVA) as a weighted average over loci using Arlequin v3.5adegenet 2.0.15363To detect population structure we used the multivariate, model-free method DAPC  analysis with the package et al.To further analyze the overall spatial pattern of alleles and test for areas with geographically restricted alleles  we calculated the observed distribution of geographic distances between allelic occurrences in the overall data set and per marker and compared it with the panmictic distribution. To test for significant deviations of the observed mean distances from the expected under panmixia, we performed 1,000 nonparametric permutations of the allele-by-location data sets.6 generations, recording every 100 Kth step after a burn-in period of 5 × 104 generations. Convergence was monitored with Tracer (http://beast.bio.ed.ac.uk). All effective sample sizes of the MCMC chain were larger than 104.To test for the presence and infer the directionality of gene flow between the Macaronesian Islands and the mainland populations, we used the coalescent-based method MIGRATE-N 3.2.6How to cite this article: Alors, D. et al. Panmixia and dispersal from the Mediterranean Basin to Macaronesian Islands of a macrolichen species. Sci. Rep.7, 40879; doi: 10.1038/srep40879 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Meretrix petechialis is an important economic shellfish species in Northwestern Pacific, but little is known about its phylogeographical pattern. Here, we analyzed 311 samples from 22 locations along the northwestern Pacific using combined profiling of one mitochondrial gene  and one nuclear DNA marker  to investigate contemporary genetic structure and reconstruct phylogenetic history of this species. The results revealed that two distinct phylogeographic lineages dominated marginal seas—the East China Sea (ECS) and the South China Sea (SCS) respectively. The estimation of divergence time between two lineages was 2.1–3.8 Ma, corresponding to a period of the early Pleistocene to late Pliocene. The vicariance of the two lineages was connected to the historical isolation of marginal seas and sea surface temperature (SST) gradient, pointing that SST might play an important role in maintaining phylogeographical patterns of M. petachialis. Significant overlaps between two lineages were observed in 23° to 29° N, located at the adjacent area of the ECS and SCS, which might be promoted by the connectivity of China Coast Current. However, the influence of ocean currents on mixings between two lineages was limited. In comparison, significant relationships were found between genetic distances and geographic distances if the North and South populations were analyzed separately, result of which might be due to some small reciprocal, rotating flows along coastal areas and special geographical conditions.The marine clam  Historical climatic changes in sea level, water temperature and ocean currents can have a powerful impact on the distribution and genetic structure of the marine biota ,2. DurinMugil cephalus in northwestern Pacific [Ligia occidentalis in North America [Lasaea australis in Australia [Sea surface temperature (SST) is always an important factor governing dispersal range of marine organisms . Studies Pacific , the sup America , and theustralia . Furtherustralia .Cyclina sinensis [Ruditapes philippinarum [Tegillarca granosa [Thais clavigera [Cellana toreuma [Ocean currents have an important influence on secondary contract by promoting larval dispersal and enriching population connectivity –23. In Csinensis , Ruditapppinarum  and Tegi granosa , rock shlavigera , the lim toreuma . HoweverMeretrix petechialis is an important economic shellfish species, widely distributed along the coastal areas of the northwestern Pacific, as far north as the Bohai Sea, and as far south as Beibu Gulf. The water temperature in reproductive period for M. petechialis was 20–30°C [M. petechialis was able to live in a salinity ranging from 17.3 to 28.4‰ in incubation period, 9.2 to 29.3‰ in pelagic larval stage and 9.2 to 36.0‰ in larval period, and was more successful in lower salinity environments [M. petechialis has been developed rapidly to meet the huge demand of market, and consequently this clam had become one of the most commercially maricultured bivalves in China. In order to ensure sustainable exploitation, management and conservation must rely on population genetic structure and gene flow among populations of the species [M. petechialis was often misidentified as M. meretrix. A previous study reported that M. meretrix was only distributed in the South China Sea, while M. petechialis was more widely distributed throughout the coasts of China [M. petechialis populations have been performed in China Seas [ 20–30°C . This eu 20–30°C  with a rronments . Culture species . M. peteof China . Geneticina Seas ,42, howeRuditapes philippinarum [Cellana toreuma [Thais clavigera [M. petechialis populations in the northwestern Pacific.Mitochondrial DNA is well established to study population genetics and phylogeography due to its comparatively fast rate of evolution and lack of recombination . Becauseppinarum , Cellana toreuma , the scy toreuma  and the lavigera . MtDNA mlavigera . Combinilavigera ,47. ITS1lavigera ,31,33. IM. petechialis; (2) calculate approximate divergence time between major lineages; (3) elucidate the factors that may affect the genetic population structure.This work aims to (1) understand the contemporary genetic structure of M. petechialis samples were collected from 22 locations along the coastal areas of China, Korea and Vietnam  under the following conditions: an initial denaturation for 3 min at 94°C, followed by 35 cycles of 40 s at 94°C, 40 s at 48°C in COI or 40°C in ITS, 40s at 72°C, and completed with a final extension for 5 min at 72°C.Genomic DNA was extracted from the ethanol-preserved adductor muscle using an improved phenol-chloroform procedure, which was described by Li et al . A fragmGTTAC-3’ . For a fFor COI, PCR products were directly sequenced using respective primers on an ABI 3730 automated sequencer. For ITS, PCR products were purified by DNA gel extraction kit UNIQ-10 following the recommended protocol, then were cloned into cloning vector pEASY-T1 (TaKaRa). For each individual, we selected two to five clones to sequence by M13 primers.Sequences of COI and ITS were assembled and checked by SeqMan in DNASTAR software. The consensus sequences were then aligned with BioEdit  using ClM. lamarckii (KY318175) was used as outgroup.The neighbor-joining (NJ) tree with bootstrap analysis (1000 pseudoreplicates) was constructed using MEGA 6.0, and parameters setting was same as distance analyses. Bayesian inference (BI) was performed in MrBayes 3.2.0 , in whicHierarchical analyses of molecular variance (AMOVA)  were perST values among populations within lineages were evaluated using COI gene by Arlequin 3.5, and the statistical significance was tested by 10000 random permutations. Mantel tests to test the relationship between genetic distances and geographic distances were performed on the Isolation by Distance Web Service (IBDWS at http://ibdws.sdsu.edu/~ibdws/) [Pairwise Φ~ibdws/)  with 10,M. petechialis, Fu’s Fs [M. lusoria as outgroup species. Because ITS-1 was not a protein-coding gene, only COI sequences were analyzed for the McDonald-Kreitman test. In cases where expansion was evident based on neutrality statistics, statistics in mismatch distributions were performed in DnaSPv5 to test whether a sudden population expansion occurred in the history. Sum of squared deviation (SSD) [To infer if there had been a past population expansion or selective sweep of  Fu’s Fs  and Taji Fu’s Fs  statisti Fu’s Fs  was condon (SSD)  and Harpon (SSD)  were calM. petechialis precludes an estimate of a species-specific molecular clock, which will influence any estimates of divergence time. Molecular clocks have been estimated for the COI gene in several bivalves such as Cyclina sinensis (0.7–2.4%/Myr) [Ruditapes philippinarum (0.9–3.35%/Myr) [Tegillarca granosa (2–2.4%/Myr) [Atrina pectinata (2.4%/Myr) [M. petechialis, the rate was used to estimate a rough time for elucidating phylogeographic hypotheses. The mean substitution rates defined in our analyses were obtained by dividing the mean sequence divergence rates by two [Divergence time between the major distinct lineages was estimated in Beast v1.7.5 software  only on .4%/Myr) , Ruditap35%/Myr) , Tegilla.4%/Myr)  and Atri.4%/Myr) , but the.4%/Myr) , 69 and .4%/Myr) , 71 to es by two . Convergs by two  and the The aligned COI and ITS-1 gene sequences were obtained with lengths of 747 bp and 860 bp, respectively. The obtained 305 COI gene sequences without indels contained 112 variable sites and 68 parsimony informative sites, yielding 97 haplotypes, of which 72 (74.2%) haplotypes were singletons, being represented by a single sequence in the sample. Wenzhou population showed the highest values of nucleotide diversity (0.01339 ± 0.00701) and mean number of pairwise differences (9.99900 ± 4.70302) because lineage overlap was observed in this population. Overall values of COI gene for haplotype and nucleotide diversities were 0.9483 ± 0.0054 and 0.03364 ± 0.01638, respectively. The obtained 345 ITS-1 gene sequences contained 194 variable sites and 65 parsimony informative sites yielding 286 haplotypes, in which 260 (90.9%) haplotypes were singletons. Overall values of ITS-1 gene for haplotype and nucleotide diversities were 0.9968 ± 0.0009 and 0.00734 ± 0.00386. No genetic recombination was detected within ITS-1 sequences. Overall, values of haplotype diversity, nucleotide diversity and mean number of pairwise differences for ITS-1 gene were higher than those of COI gene .M. petechialis into two lineages with high nodal support . A smaller  amount of genetic diversity occurred within populations, and the smallest (0.80%), yet significant (P < 0.01), of the total variation was present among populations within lineages. For ITS-1 gene, the result showed that the most of the total genetic variation  was apportioned within populations, followed by a smaller  amount occurred between groups, and only a small part  among populations within groups.When populations were grouped by sea basins, for COI gene, hierarchical analyses of AMOVA  indicateBased on the COI, two haplogroups were formed in the network , which wST values based on COI sequences among populations within lineages were shown in Tables ST) and geographic distances , ruling out the possible of the selective sweep. Estimates of neutral tests for two lineages conducted on both COI and ITS data indicated population expansion by significant negative values of Fu’ Fs and Tajima's D statistics . The obsM. petechialis are shown in Results of the COI divergence time estimations for M. petechialis was connected to the historical isolation of marginal seas, which is consistent with the observations in mitten crab Eriocheir sensu stricto [Chelon haematocheilus [Bostrychus sinensis [Cyclina sinensis [Tegillarca granosa [M. petechialis, giving rise to divergent lineages. The divergence time in our study is similar with two subspecies of oyster (2.0–3.6 Ma) [M. petechialis was also detected through star-like type of network topology, mismatch distribution analysis and neutrality tests.Based on the network relationships and phylogenetic reconstruction for COI markers, significant population differentiation were observed between northern (ECS) and southern groups (SCS), and overlapped at the adjacent area of the ECS and SCS . In addi stricto , marine ocheilus  and Bostsinensis ) and bivsinensis  and Tegi granosa ). The di granosa ,77. Beca–3.6 Ma)  and two –3.6 Ma) . Above a–3.6 Ma) . In post–3.6 Ma) ,79,80. TM. petechialis. Firstly, the boundary of two M. petechialis groups is approximately near to Taiwan Strait. In the northwestern Pacific, the Tropic of Cancer is the boundary between the warm-temperate and tropical regions, and 20°C isotherm of annual mean temperature running through the Taiwan Strait, which is a major biogeographic boundary for many species [Mugil cephalus [M. petechialis. Moreover, from 1982, SST have continued to rise [One major cooling period was occurred in the 2.2–1.0 Ma, which are reflected in the increasing SST gradient in the subtropical area, where SST cooled significantly (decreased by 3.3–5.4°C and 1.0–2.1°C in winter and summer respectively), while SST showed little or no cooling (decreased by 0.9°C and 0.6°C in winter and summer respectively) in the tropical western Pacific . SST gra species . Secondlcephalus , the dis to rise ,84, whic to rise .Tegillarca granosa and Cyclina sinensis, plankton larval duration of M. petechialis is about 5–8 days, however, M. petechialis migrates from mid-tidal region to low-tidal region along with tidal currents extending from late May to late June and mid-September to late-September. This specific feature may increase opportunity for gene flow among different populations. During the breeding season of M. petechialis (spring and summer), the China Coast Current with a common velocity of 20 cm/s [ΦST) and geographic distances when the North and South populations were analyzed respectively, after fitting IBD model. In addition, some pairwise ΦST values were significant between populations within each lineage. For North lineage, the genetic difference between some Bohai Sea populations (Changyi and Weihai) and East Sea populations were statistically significant. For South lineage, some populations located in Beibu Gulf  were significantly divergent from Wenzhou, Xiamen, Shantou and Zhanjiang populations. This is most likely attributed to the low-dispersal ability of the clam and the existence of small but persistent reciprocal flow and rotating flow along coastal areas, which impede the exposure of larvae to currents and their ability to be transported effectively [Similarly,  20 cm/s  flowing  20 cm/s . Signifiectively .Periophthalmus modestus because of an older coalescence time, a putative ancestral haplotype, higher genetic diversity, more proportion of private haplotypes and higher species diversity of the genus Periophthalmus. Similarly, we proposed that South China Sea is the earlier refuge for M. petechialis. First of all, north lineage originated from refugium in the Okinawa Trough about 0.7 Ma, whereas South lineage derived from refugium in South China Sea about 0.8 Ma. Although they are approximate times, the relative coalescence times of two populations are reasonable. Moreover, the portion of private haplotypes from South lineage (75.9%) was higher than that from North lineage (72.1%), which supported the South China Sea was the colonization origin [Meretrix Lamarck, 1799 are broadly distributed in the West Pacific and the Indian Ocean, M. petechialis is the only Meretrix species in the ECS regions.He et al.’s study indn origin . In addin origin . FinallyM. petechialis into two lineages, while nrITS data showed a slightly different topology. The net average genetic distance (± SD) between lineages was 5.85 ± 0.95% for COI, but only 0.08 ± 0.03% for ITS. Result of AMOVA also displayed that nrITS were not significantly differentiated between groups. This inconsistent pattern has also been found in black-throated tits Aegithalos concinnus [Atrina pectinata [M. petechialis into two lineages, but no obvious difference and discernible geographic pattern was found between the two lineages using the ITS gene. We also found samples from different mtDNA lineages shared the same haplotype for ITS gene, for example, samples from Busan, Beihai and Caotan shared the same haplotype. Therefore, incomplete lineage sorting is more likely to be the reason of the discepancy between mitochondrial and nuclear topologies.MtCOI gene analysis separated oncinnus  and the ectinata . A largeectinata . Howeverectinata . COI genectinata , becauseectinata  (a fourfectinata ). In ourectinata . In our M. petechialis, we sequenced the COI and ITS1 genes from 311 individuals sampled from 22 locations along the coastal areas of China, Korea and Vietnam. The results provided evidence for strong genetic divergence between two lineages, which might be attributed to variance of sea level changes and SST gradients. Here, the combinatory use of nuclear ITS gene and the mitochondrial COI gene helped elucidate the phylogeographical pattern of M. petechialis, such as population structure and population expansion analyses. With the objectives of getting more detailed information about phylogeography of M. petechialis with higher genetic resolution, analyses with multiple nuclear genes would be conducted.To better elucidate phylogeographic pattern of


HLA class I  and class II  genes. A total of 56 Arab populations comprising 10,283 individuals were selected from several databases, and were compared with 44 Mediterranean, Asian, and sub-Saharan populations. The most frequent alleles in Arabs are A*01, A*02, B*35, B*51, DRB1*03:01, DRB1*07:01, DQB1*02:01, and DQB1*03:01, while DRB1*03:01-DQB1*02:01 and DRB1*07:01-DQB1*02:02 are the most frequent class II haplotypes. Dendrograms, correspondence analyses, genetic distances, and haplotype analysis indicate that Arabs could be stratified into four groups. The first consists of North Africans , and the first Arabian Peninsula cluster , who appear to be related to Western Mediterraneans, including Iberians; this might be explained for a massive migration into these areas when Sahara underwent a relatively rapid desiccation, starting about 10,000 years BC. The second includes Levantine Arabs , along with Iraqi and Egyptians, who are related to Eastern Mediterraneans. The third comprises Sudanese and Comorians, who tend to cluster with Sub-Saharans. The fourth comprises the second Arabian Peninsula cluster, made up of Omanis, Emiratis, and Bahrainis. It is noteworthy that the two large minorities (Berbers and Kurds) are indigenous (autochthonous), and are not genetically different from “host” and neighboring populations. In conclusion, this study confirmed high genetic heterogeneity among present-day Arabs, and especially those of the Arabian Peninsula.This is the first genetic anthropology study on Arabs in MENA (Middle East and North Africa) region. The present meta-analysis included 100 populations from 36 Arab and non-Arab communities, comprising 16,006 individuals, and evaluates the genetic profile of Arabs using  HLA) system plays a key role in self-nonself recognition, and is divided into class I  and class II  loci, and comprises 220 genes in a 3.6 Mb region found on the short arm of chromosome 6. HLA system is highly polymorphic, and in excess of 17,000 alleles were detected. For example, there are 4,828 B, 3,968 A, and 3,579 C class I alleles, compared with 2,103 DRB1, and 1,142 DQB1 class II alleles. Several HLA alleles were associated with various auto-immune and infectious diseases  17]. P. PHLA alHLA allele frequencies must be obtained by molecular typing, and that subjects should be typed for at least one of the following: HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1. Publications were excluded in case of serological data; sample size less than 35 individuals, typed individuals (or controls) were either related and not randomly selected, presentation of duplicate data sets. Studies were also excluded if they presented incomplete/partial allele frequencies, or there were significant ambiguities in the typing.All included studies met the following criteria. HLA-A, -B, -C, -DRB1, and -DQB1 allele frequencies, haplotype frequencies, region, country, and typed loci.Studies were independently selected by two authors (H.A and H.L). An external referee was invited in case of disagreements not resolved by both reviewers. Data extracted from selected papers included publication year, study type , sample size, A three-dimensional correspondence analysis and bi-dimensional representation were performed using VISTA V5.02 software . PhylogeHLA datasets, of which 315 were deemed relevant to the study. Of these, 42 articles and 11 HLA datasets containing information on 56 Arab populations, and meeting the study criteria, were included. The study flow is illustrated in HLA datasets which meet the criteria of this study, containing complete information on 44 other populations were selected, but without going through systematic review. The populations used in the comparison were chosen mainly from neighboring Arab countries. This study relied on a database consisting of 100 populations (of which data of 11 populations were extracted from association studies) from 36 countries Arab and worldwide countries, and belonging to Asia, Europe, and Africa. The distribution of populations by region is illustrated in The use of more than fifty key words allowed identification of 5,456 papers and HLA datasets  selected provided information on 56 populations   . The stuForty-four worldwide populations , 68–85 cHLA-A, -B, -DRB1, or DQB1.Data of only three populations , 75, 84 HLA-A and -B alleles in Arab populations. A*02 was the most prevalent allele, and its frequency exceeded 25% in some populations, such as Saudis (30.4%) , and Saudis (0.0231).HLA A-B haplotypic data are extremely rare in Arabs. The most frequent A-B haplotypes in Arabs are shown in A*02:01-B*50:01 (9.0%) and A*02:01-B*44:02/03 (7.5%) were the haplotypes with the highest frequencies in Berbers of Zrawa. Diversity in A-B haplotype frequencies are found among Arabs, hence demonstrating comparable frequencies of A-B haplotype in Arab populations, which did not exceed 5.3% in Gabesians (Tunisia). For example, while A*34:02-B*08:01 and A*29:01-B*45:01 characterize Tunisians, A*01-B*57(02.9%), A*30-B*18 (01.50%), and A*33:01-B*14:01 (02.50%) characterize Algerians. Several haplotypes identified in Arabs were also seen in other Mediterraneans. For example, A*32:01-B*40:02 was seen in Greeks (2%) [A*02:01-B*50:01 was seen in Italians (2%) [A*24:02-B*08:01 (4.75%) and A*30:02-B*53:01 (3.48%) were only identified in Saudis.eks (2%)  and Spaneks (2%) , while Aans (2%) , Portuguans (2%) , and Morans (2%) . A*24:02DRB1-DQB1 haplotypes with significant LD in Arabs are listed in DRB1*03:01-DQB1*02:01 haplotype was the most frequent DRB1-DQB1 haplotype in Arabs  [DRB1*07:01-DQB1*02:02 is also frequent in Arabs, such as Moroccans (16.70%), and is reportedly common in Spaniards (17.3%) [DRB1*07:01-DQB1*02:01 is also a common DRB1-DQB1 haplotype, and its frequency exceeds 4% in several Arab populations.The most frequent in Arabs , and its (17.5%) , Morocca (17.5%) , Algeria (17.5%) , and Cre (17.5%) . In addi (17.3%) , and Mor (17.3%) , but rarDRB1*16:01-DQB1*05:01 and DRB1*04:01-DQB1*03:02, rare in neighboring populations and Mediterraneans, were identified only in Lebanese and Bahraini Arabs. The high frequency of DRB1*11:01-DQB1*03:01 haplotype (31.42%) among Lebanese is noteworthy, since it is the highest in all populations studied, but rare in Saudi (2.2%). Furthermore, DRB1*11:01/04-DQB1*03:01, identified in Arabs, is also frequent in Cretans (18.5%) [DRB1*01:02-DQB1*05:01 was seen in Spaniards (6.30%) [DRB1*13:01-DQB1*06:03 was also reported for Spaniards (13.23%) [DRB1*15:01-DQB1*06:02 was observed in Cretans (2.6%) [In addition,  (18.5%)  and Basq (18.5%) , while D (6.30%) . Varied 13.23%) , Cretans(13.23%) , and Ger(13.23%) . Likewiss (2.6%) , German s (2.6%) , and Sou% , CretaHLA alleles and haplotypes between North Africans and Arabian Peninsula populations. On the contrary, key differences were noted between Levant Arabs , and other Arab populations, highlighted by high frequencies of A*24, B*35, DRB1*11:01, DQB1*03:01, and DRB1*11:01-DQB1*03:01 haplotype in Levantine Arabs compared to other Arab populations. Class I haplotype frequencies are lower than Class II haplotypes, because of weak LD between A and B loci, due to long physical distance between them, compared to DRB1 and DQB1 loci. The identification of shared haplotypes between Arabs and other Mediterranean and Asian populations is attributed to the higher admixture of Mediterraneans and Asians in Arab populations.This meta-analysis is the first genetic anthropology study in MENA region, and included 100 populations from 36 Arab and neighbouring countries, and comprising in excess of 16,000 individuals. A main outcome of the study is the lack of striking differences in the distribution of The relatedness between North Africans and Iberians was previously discussed , 86, 88.HLA markers prompted the conclusion that all Berbers of North Africa constitute a homogeneous genetic unit, except for small isolates, such as the Berbers of Djerba, who display a Berber genetic profile.The analyses performed showed that current North Africans are closely related to Tunisian (Zrawa and Matmata) and Moroccan (Sousse-Agadir and Eljadida) Berbers, suggesting that North Africans have a genetic Berber profile. On the contrary, North Africans displayed a greater distance from the Arabs of Levant , indicating low genetic contribution of Phoenician and Levant Arab invasion of North Africa. These observations based on HLA data on Eastern Saudi Arabia that shed light on pre-Islamic history; some ancient people may have originated from old Persians, but quantification is difficult and undetermined [HLA-A,-B, -DRB1, and DQB1 markers support the notion that Saudis along with the Kuwaitis and Yemenis are closely related to North Africans.Saudi populations used in this study originated from Eastern Saudi Arabia, especially from Riyadh province. There is no reliable termined . The genThe most plausible explanation for West Arabia and Yemen clustering with Iberian/North Africans is a possible important massive migration that occurred when Sahara underwent desiccation in all directions , 93. CulHLA Kuwait genetic similarity to this group seems more difficult to achieve but interaction between Arabian Peninsula and Mesopotamia through this strategic Kuwait area is documented since 6,500 years BC (Ubard Period) [Explanation to  Period) .HLA class I  and class II markers (DRB1 and DQB1) analysis. However, Levant Arabs are distant from North African Arabs  and Iberians . The strong relatedness between Levant Arab populations is explained by their common ancestry, the ancient Canaanites, who came either from Africa or Arabian Peninsula via Egypt in 3300 BC [Using genetic distances, correspondence analysis and NJ trees, we showed earlier , 62 and  3300 BC , and set 3300 BC . The relHLA markers, may be due to three reasons. First, Egypt is a neighbor to Levant Arab countries, and historically part of the Levant. Second, the Egyptians invaded the Levant several times throughout history; the most significant was 1468 BC invasion, where they settled for 12 centuries [The close relatedness of Levant Arabs to Egyptians, as confirmed genetic distances using enturies . Third, Historically, Levant is a wider region that included countries along the Eastern Mediterranean with its islands, and extended from Greece to Cyrenaica . BroadlySudanese are close to sub-Saharan Africans , and North Africans, in particular Egyptians, suggesting that the genetic profile of Sudanese is the admixture between North Africans  and sub-Saharan Africans throughout history. The close relatedness of Sudanese to sub-Saharan Africans suggests a reduced genetic effect of Arabs on Sudanese. Also, the Comorians  are close to sub-Saharan Africans  , EgyptiaA*30-B*18-Cw*5 basic HLA haplotype is very high in Sardinia, Iberia, and North Africa) [Bahrainis, Emiratis, and Omanis are geographically similar populations, which explains their genetic relationship as demonstrated in this study. These three populations tend to form a heterogeneous group with Pakistanis, Indians, Iranian Arabs (Famoori), Sardinians , Egyptians, and some sub-Saharan Africans, such as Congolese. These populations appear close to certain Eastern Mediterranean populations including Greeks, Macedonians, and those further, in particular North Africans, hence explaining their intermediate grouping, and distinction from two main clusters. Collectively, this suggests high admixture in these populations brought about by their commercially important position. Sardinia is a relative genetic isolate “founded” by Iberian Norax/Nora  and Iberians/North Africans may be genetically related to Sardinians ( Africa) .The Kurds and Berbers are the two major ethnic minorities in Arab world. Berbers are indigenous North African ethnic group found over a vast area stretching from Atlantic Ocean to Siwa Oasis in Egypt, and from Mediterranean Sea to Niger River. Berbers number about 20 million people, and constitute 40–45% of Moroccans, 20–25% of Algerians, and 2–7% in both Libya and Tunisia. The Kurds live in the northern regions of Iraq (15–20%) and Syria (10%). They constitute an Indo-European ethnic group, and speak Kurdish. Less important minorities include Armenians, Nubians, Assyrians, and Turkmen .Berbers populations used in this work are closely linked to each other, as well as to present-day North Africans, and to Western Mediterranean populations, especially Iberians. Indeed, the Moroccan Berbers are not genetically different from the current Moroccans, nor those of neighboring populations, like Algerians and Tunisians. This also applies to Tunisian Berbers, except those of the island of Djerba, who appear to be related to Eastern Mediterranean populations, including Levant Arabs. This suggests that North African Berbers are in perfect harmony with their environments, and that differences between them are cultural rather than genetic due to 7th century Arabization of the region.Clustering and genetic distances analyses demonstrated that Iraqi and Iranian Kurds are not genetically different from Iranians or neighboring populations, including Levant Arab, and are close to Turks and other Eastern Mediterranean populations. This suggests that Kurds originate from the region, and are in genetic harmony with neighboring populations, despite the clear cultural differences. This suggests that Kurds, Syrians, Jordanians, Palestinians, Iraqis, Lebanese, and Iranians probably share the same genetic profile, with few differences. Accordingly, our findings confirm the results of an earlier study of Arnaiz-Villena on Iraqi Kurds .Sunni Muslims constitute the majority (80%) of Arab populations, followed by Shi'a Muslims (10%) who are present in parts of Iraq, Lebanon, Saudi Arabia, Kuwait, Yemen, and Bahrain. Non-Muslims make up about 10% of all Arabs, and Christianity (6%) is the second largest religion among Arabs, with about 20 million Christians living in Lebanon, Egypt, Iraq, Syria, and Jordan. Other minor religions (4%) such as Judaism, Druze and others are practiced on a much smaller scale .HLA data on Sunni and Shiite Arabs are not available, same as comparison of Muslims to Christians. The only available data are those concerning Arab Jews. In this study, data are available for three Jewish populations, including two from North Africa (Moroccan and Libyan Jews) and one from the Arabian Peninsula (Yemenite Jews). While genetic distances separating these three groups of Jews are small , Saudis, Kuwaitis, and Yemenis, with relatedness to Western Mediterraneans, including Iberians. The second includes Levantine Arabs , Iraqi, and Egyptians, who appear to be related to the Eastern Mediterranean and Iranians, who in turn belonged to 'Great Levant' historically described. The third consists of Sudanese and Comorians who associate with Sub-Saharan Africans. Finally, the fourth group of Arabs comprises Omanis, Emiratis, and Bahrainis. This group associates with heterogeneous populations . Lastly, the two main indigenous minorities, Berbers and Kurds, are not genetically different from the ‘host’ and neighboring populations.S1 Checklist(DOC)Click here for additional data file.S1 FigPopulations’ data were taken from references detailed in Tables (TIF)Click here for additional data file.S2 FigPopulations’ data were taken from references detailed in Tables (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


We sampled additional 11 dog populations  at continuous altitudes in China from 300 to 4,000 meters to validate and test the association between the haplotype frequency of AMOT gene and altitude adaptation. The results suggest that AMOT gene may be a notable candidate gene for the adaptation of TM to high-altitude hypoxic conditions. Our study shows that X chromosome deserves consideration in future studies of adaptive evolution.Genome-wide studies on high-altitude adaptation have received increased attention as a classical case of organismal evolution under extreme environment. However, the current genetic understanding of high-altitude adaptation emanated mainly from autosomal analyses. Only a few earlier genomic studies paid attention to the allosome. In this study, we performed an intensive scan of the X chromosome of public genomic data generated from Tibetan Mastiff (TM) and five other dog populations for indications of high-altitude adaptation. We identified five genes showing signatures of selection on the X chromosome. Notable among these genes was angiomotin  EPAS1, EGLN1, PPARA and HBB identified from human populations), energy metabolism  and DNA repair , have been identified from studies of native highlanders with different time depths of exposure to high-altitude. These included human populations 1356789211121314154171819Adaptation to high-altitude environments, characterised by extreme conditions like hypoxia, low temperature, and high ultraviolet radiation, has been of great interest in evolutionary biology research12345Drosophila melanogaster populations revealed that some X-linked genes had undergone positive selection and were responsible for hypoxia toleranceNotably, most of the current understanding of genetic basis of high-altitude adaptation is based on selective scans of autosomes, despite the chromosomes of diploid organisms usually harbouring both autosomes and allosomes. Relative to autosomes, X chromosome shows reduced population size and hemizygote state in male individuals. These characters can make it be more sensitive to demographic history and selective effects21ALG13, AMOT, DCX, LHFPL1 and TRPC5) on the X chromosome of TM population. Notable among these genes is angiomotin (AMOT) gene, which was indicated to regulate the process of angiogenesis252627AMOT gene and altitude adaptation. The results corroborated the initial findings that AMOT gene may be an important target for TM’s adaptation to hypoxic conditions at their high-altitude habitat.Recently, the high-altitude adaptation of Tibetan Mastiff (TM), an ancient dog breed that migrated to the Tibetan Plateau  with humans, has received wide attention41718In the present study, the publicly available genome-wide re-sequencing data sets from six populations of 60 dogs, including a Tibetan Mastiff (TM) population  and five other dog populations , were usedvs. 207 in autosomes per 50 kb) and transition/transversion (Ts/Tv) ratio (1.76 in X chromosome vs. 2.15 in autosomes)  for each dog were obtained . After Stosomes)  and S5. tosomes) . In subsvs. 1.3E-3 in autosomes) , we found that the introduction of GS or KM both resulted in a significantly higher Fst level as compared to TM vs. YJ pair (P < 2.2E-16) (The genetic diversity or pairwise nucleotide diversitytosomes) . Among ttosomes) . Third, tosomes) . Interes2.2E-16) . These rALG13, AMOT, DCX, LHFPL1, TRPC5 and ZCCHC16) showed highest densities  has been reported to code for angiomotin protein, which is located on the surface of vascular endothelial cells (VECs), and has been thought to be crucial for angiogenesis252627The X-chromosome-wide selection scans were conducted on TM with the highest altitude of 4,380 m and on YJ population with the lowest altitude of 800 m. Based on a total of 306,678 SNPs from the two populations, using the threshold of 1% top level , we identified 3869  and 4049  SNPs as outliers . Accordiensities . FurtherZCCHC16, the other five genes ALG13, AMOT, DCX, LHFPL1, and TRPC5 with the highest outlier SNPs densities in the previous analyses were also observed among the 21 shared genes. Therefore, we considered the five genes as candidate positively selected genes (PSGs).Since both TM and DQ are high-altitude populations, 4,380 m and 3,300 m respectively, we combined the two populations and compared them against YJ, a low-altitude group, and rescanned the X chromosome following the process and criteria stated above. Interestingly, we identified 64 genes with outlier SNPs . MoreoveAMOT in our analyses and its cited importance in angiogenesis, we sought to attentively assess AMOT with respect to high-altitude adaptation. From annotations of outlier SNPs in AMOT, we observed two nonsynonymous mutations (p.Ser971Ala and p.Leu1025Pro) and two synonymous mutations. Interestingly, for the two nonsynonymous mutations, the reference alleles showed significant divergent distributions between TM and YJ .It has been reported that angiogenesis could be activated by hypoxia-inducible factor (HIF) pathway in hypoxic conditions1113AMOT gene for four dog populations , were calculated. TM and YJ had the lowest genetic diversity compared to DQ and LJ populations  scans on about 1 million bp region, covering the ulations . This paYJ to TM , which iTM . Then we used these data to test whether the observed TM-YJ genetic divergence could be due to genetic drift instead of selection. For each run, we calculated Fst values of the 108 SNPs from the simulated data sets, and compared the result to the Fst values from the real data. Our analyses showed that the probability of the real Fst values attributable to genetic drift was only 2.32E-3, implying that the genetic pattern observed on AMOT was likely not caused by genetic drift, and AMOT may be a target of positive selection during adaptation to hypoxia.Given that genetic drift can also generate similar genetic patterns as positive selection did, we performed simulations for 1,000,000 times to generate allele frequency data sets of TM and YJ populations with AMOT . Interestingly, one kind of haplotype (‘CGTTCGCTTTG’) occupied a very high ratio (about 82% on average) in Tibetan Mastiff populations  to further validate the selective signatures of AMOT . In thisulations . Moreoveulations .AMOT could be a key target gene of positive selections, and could be associated with high-altitude hypoxia tolerance among Tibetan Mastiff.Taken together, the scans of X chromosome of TM and other dog populations, and the subsequent analyses of the genetic diversity, haplotype distributions, as well as the correlation between haplotype frequency and altitude, all provide evidence suggesting that Several selective scans on X chromosome have been reported in previous animal domestications and breeding studies. Specific regions or genes have shown likely involvement in the process of domestications of pig and sheep3839EPAS1 and HBB, which are associated with HIF pathway and oxygen transportation, and one PSG, i.e., PLXNA4, which functions in angiogenesis by interacting with VEGFAMOT gene has been reported to act in the process of angiogenesis252627AMOT in TM and YJ populations based on a large genetic region were similar low. As high and low elevations represent two different environments, divergent selective pressures may have acted on AMOT in TM and YJ resulting in the similar genetic diversity patterns, although a narrowed scan showed a marked haplotype divergence between the two populations. Interestingly, studies on Atlantic Salmons and dog populations have observed signals of divergent selections in some regions, in which the selective effects caused increased genetic differentiation between populations and reduced genetic diversity at linked loci42Here, we chose Tibetan Mastiff, which is a typical representative of domesticated animal that migrated to the high-altitude plateau with humans, as our study model. TM has been investigated in several studies for its high-altitude adaptation via autosome genome analyses417VEGF, VAV3, NOS3, SRF, TXNRD2, ADAM17 and RNASE4, have been proposed as candidate genes in the adaptation of highland human and wild animals to high-altitude conditions810111316AMOT, which is also an angiogenesis related gene, may have played a key role in the high-altitude adaptation of TM. Hence, it is likely that, besides the HIF pathway, angiogenesis also played a similarly critical role in the adaptation of TM populations to their high-altitude hypoxic habitat.Although several angiogenesis-related genes, including et al.This study presents important insights for understanding the contribution of sex chromosomes to hypoxia adaptation, as most previous genome-wide studies have often excluded sex chromosome because it represents a different effective population size. Relative to autosomes, our genetic diversity assessments demonstrated that X chromosome had lower genetic diversity across all six dog populations , consistAMOT as a target candidate gene provides a foundation for future functional studies to further elucidate its role in adaptation to hypoxia.In summary, our study suggests that X chromosome, like autosomes, may have a contribution in the adaptation of Tibetan Mastiff to hypoxic conditions at high-altitudes, indicating that X chromosome warrants a more attention in future studies of adaptive evolution. In addition, the identification of All methods were performed in accordance with the guidelines approved by the Kunming Institute of Zoology, Chinese Academy of Sciences. All experimental protocols were approved by the Kunming Institute of Zoology, Chinese Academy of Sciences.http://picard.sourceforge.net) was used to remove duplicate sequences in each individual, then all the Bam files were processed with a standard pipeline for local re-alignment and base-recalibration in the Genome Analysis Tool Kits (GATK) (version: 2.5-2-gf57256b)Raw sequence reads were mapped to the dog reference genome (version: Canfam3.1) using BWA-MEM (version: 0.7.8-r455)Genome-wide SNPs calling were handled by UnifiedGenotypeCaller module in GATK. Raw SNPs were recalibrated with the Variant Quality Score Recalibration (VQSR) module, where a list of known or verified SNPs/INDELs was downloaded from Ensembl Database and used as the training data set. Then the recalibrated SNPs were filtered with rules as described below: 1) SNPs that were within 5 base pairs proximity to inserts and deletions (INDELs) were filtered out, and the INDELs also abandoned; 2) SNP locus at which the ratio of individuals missing SNP detection was greater than 20% of all the samples was excluded; 3) Only bi-allelic SNPs were retained; 4) The remaining SNPs were sorted by their depths, with depths ranging from 2.5% to 97.5% retained; and 5) SNPs with Quality value greater than 40 were retained. Finally, all high-quality SNPs were annotated by ANNOVARhttp://pngu.mgh.harvard.edu/purcell/plink/; Parameter: –indep-pairwise 50 10 0.1), to make sure that each SNP evolved independently. Based on the whole autosomal SNPs from our downloaded genome-wide data setsvs. YJ, KM vs. TM, GS vs. YJ, GS vs. TM, and YJ vs. TM) were also conducted.With the availability of complete gender information for the six dog populations, we made corrections to the allele frequencies of the study populations before assessing population genetic diversity and structure. Here, we first computed pairwise nucleotide diversity (π)P-values (after FDR correction) in Fisher’s exact test, were considered as candidate loci of selection.Two approaches were applied for positive selection scans: Fst test, which reflects the genetic differences among populationset al.The genetic diversity (π) scans of regions around candidate genes were performed with our custom Perl scripts, applying 10 k-bp non-steps windows. Prior to our haplotype analyses, haplotypes of the four dog populations  were inferred by SHAPEIT softwareAMOT gene were caused by genetic drift instead of selection. First, using G-PhoCS tools−9 per year−9 per year as the mutation rate in the X chromosome. Our results showed that TM diverged from YJ about 3,400 years ago, and the effective population size of their common ancestor was approximately 37,800. On the other hand, the effective population size of extant YJ and TM was 8,681 and 4,058 respectively. With parameters drawn from previous analyses, we used ms toolsAMOT region (about 47 k bps) for both TM and YJ populations in a neutral model. In each simulation, we calculated the Fst values of simulated data sets, and compared them to those computed from the real data sets, with the significance of the divergence measured by t-test. After 1,000,000 simulations, the distribution of all P-values was evaluated to determine whether selective footprints that were produced by the real data sets were generated by genetic drift.We applied dense computational simulations to test whether the selective signals of AMOT identified from X-chromosome-wide scans, we sampled additional 175 dogs from 11 different places in China  and added them into our present study  for each individual, and used PHASE (version: 2.1)59To verify the selective signals of nt study . Total gAccession codes: All newly generated sequences of the 11 dog populations have been deposited in GenBank (KX245540-KX245889).How to cite this article: Wu, H. et al. Identifying molecular signatures of hypoxia adaptation from sex chromosomes: A case for Tibetan Mastiff based on analyses of X chromosome. Sci. Rep. 6, 35004; doi: 10.1038/srep35004 (2016).