Plasmodium vivax apical membrane antigen 1 (Pvama1) gene in a parasite population from the China-Myanmar border and compared it with global P. vivax populations.With the premise of diminishing parasite genetic diversity following the reduction of malaria incidence, the analysis of polymorphic antigenic markers may provide important information about the impact of malaria control on local parasite populations. Here we evaluated the genetic diversity of Pvama1 sequences acquired from the China-Myanmar border as well as 615 publically available Pvama1 sequences from seven global P. vivax populations.We performed evolutionary analysis to examine the genetic diversity, natural selection, and population differentiation of 73 Pvama1 haplotypes were identified among the global P. vivax isolates. The overall nucleotide diversity of Pvama1 gene among the 73 China-Myanmar border parasite isolates was 0.008 with 41 haplotypes being identified (Hd = 0.958). Domain I (DI) harbored the majority (26/33) of the polymorphic sites. The McDonald Kreitman test showed a significant positive selection across the ectodomain and the DI of Pvama1. The fixation index (FST) estimation between the China-Myanmar border, Thailand (0.01) and Myanmar (0.10) showed only slight geographical genetic differentiation. Notably, the Sal-I haplotype was not detected in any of the analyzed global isolates, whereas the Belem strain was restricted to the Thai population. The detected mutations are mapped outside the overlapped region of the predicted B-cell epitopes and intrinsically unstructured/disordered regions.A total of 308 Pvama1 in the P. vivax parasite population from the China-Myanmar border with DI displaying stronger diversifying selection than other domains. There were low levels of population subdivision among parasite populations from the Greater Mekong Subregion.This study revealed high levels of genetic diversity of The online version of this article (doi:10.1186/s13071-016-1899-1) contains supplementary material, which is available to authorized users. Plasmodium vivax is the major malaria species; around 2.5 billion people are living in areas of P. vivax transmission [www.apmen.org), P. vivax is often sympatric with P. falciparum, P. ovale and P. malariae [P. vivax has become the predominant species in recent years [P. vivax presents a major challenge for malaria elimination. Within the GMS, malaria is distributed very unevenly; malaria transmission is concentrated along international borders, whereas central plains are mostly free from malaria [In South and Southeast Asia, Latin America and Oceania, smission . In the nt years . With th malaria , 4. The  malaria . In thes malaria , 8. Sinc malaria , 10, traP. vivax proteins, including Duffy-binding protein (DBP), apical membrane antigen 1 (AMA1), and merozoite surface proteins (MSPs), have been selected as vaccine candidates for their essential functions during erythrocytes invasion and their antigenicity in natural host immune response [ama1 gene has been extensively studied in a number of Plasmodium species [ama1 gene, it has been used as a molecular marker for population genetic studies [The genetic diversity of antigens in malaria parasites has been extensively studied not only because of their importance as malaria vaccine candidates , but alsresponse –15. Amonresponse . The ama species . As a ty species –24. Anti species –27. The  species . DI harb species . Within  species . Evidenc species , 32. In  species . Due to  studies , 35.P. vivax ama1 (Pvama1) has been extensively studied with samples from Asia, Oceania and South America [Pvama1 gene from the China-Myanmar border area. We wanted to determine whether this potentially isolated parasite population was genetically different from those of other endemic regions. Furthermore, we wanted to learn whether the intensified malaria control efforts resulted in evident reduction of genetic diversity of the parasite population as observed elsewhere in Southeast Asia [Pvama1 gene in the China-Myanmar border parasite population.Although the genetic diversity of  America –39, therP. vivax has become the predominant parasite species [P. vivax infections were based on microscopy of Giemsa-stained thick smears. The study protocol was approved by the IRB of Pennsylvania State University and by the Health Department of Kachin.Parasite sample collection was carried out from April 2011 to October 2012 around the township of Laiza, which is located in the northeast Kachin State, along the China-Myanmar border . This re species , 41. Lik species , 42. FilPvama1 gene encoding the extracellular region  148–1,437 of the Sal-I sequence; GenBank accession no. AF063138) was amplified and sequenced with six oligonucleotide primers listed in Additional file 4, 250 nM of each primer, 0.4 units of KOD Plus neo polymerase , and 1 μl genomic DNA as template. The reaction was run at 94 °C for 5 min, followed by 45 cycles of 94 °C for 15 s, 56 °C for 15 s, and 68 °C for 90 s, and extension at 68 °C for 5 min. PCR fragments were analyzed by electrophoresis on a 1.2% agarose gels. Direct sequencing of purified PCR fragments was carried out on both strands with primers listed in Additional file Genomic DNA from dried filter paper blood spots of slide-positive samples was extracted by using a QIAamp DNA Blood Mini kit . A DNA fragment of the P. vivax infections amplified by PCR, five samples showing dual peaks suggestive of a mixed infection were excluded from further analysis. Pvama1 was successfully sequenced from 73 samples (sequences submitted to GenBank under accession nos. KX495505–KX495577). A single contiguous 1,290 bp of Pvama1  was derived for each of the 73 Pvama1 sequences, which include DI (codons 94–247), DII (codons 265–363) and DIII (codons 388–451). The consensus of the sequences were aligned to the P. vivax Sal-I strain by using the CLUSTAL W program in MEGA6.0 [P. vivax isolates  were also analyzed as additional reference sequences [S), the total number of mutations (η), the average number of pairwise nucleotide differences (k), nucleotide diversity (π), the number of haplotypes (H) and haplotype diversity (Hd), and their corresponding standard deviation were computed using the options available in DnaSP v5.10.01 software [Pvama1 gene was analyzed using the sliding window approach.Of the 78 equences . AlignedPvama1 sequence was constructed by using the NETWORK software Version 4.6.1.3 with the Median-Joining method [Pvama1 sequences representing seven P. vivax populations were retrieved from the GenBank: South Korea [RM) [C [D’ [R2 [FST among populations were calculated by using DnaSP v5.10.01 [A haplotype network based on the g method . A totalth Korea , India [th Korea , Sri Lanth Korea , Thailanth Korea , Iran [4th Korea , Venezueth Korea , and Papth Korea . LD was ea [RM)  and to g[RM) [C . The rel) [C [D’  and R2 [D’ [R2 . To assev5.10.01 .D, Fu and Li’s D* and F*, and McDonald-Kreitman indices were calculated using DnaSP v5.10.01 and via a sliding window method [D test, departure from neutrality is determined by comparing the values of θ (estimated nucleotide diversity) derived from π (observed average pairwise nucleotide diversity) and the total number of segregating sites (S) [D* and F* tests determine departure from neutrality by differences between estimation of θ derived from the number of singletons and that based on either the total number of mutations (D*) or the average pairwise diversity (F*) [P. cynomolgi ama1 sequence . A phylogenetic analysis of Pvama1 sequences  with Myanmar samples [To determine departure from neutrality, Tajima’s w method . In Tajiites (S) . The Fu ity (F*) . The McDity (F*)  was appl X86099; ) as the  samples  was perf samples . The booThe potential B cell epitopes of DI-III in PvAMA1 were predicted by using the ABCpred server . In ordePvama1 was amplified from 73 P. vivax samples acquired from the China-Myanmar border. Of the 73 Pvama1 sequences, there were 41 haplotypes, giving overall haplotype diversity (Hd) of 0.958 (Table k) for the entire 1,290 bp sequenced region, DI, DII, and DIII were 10.895, 7.433, 1.385, and 0.419, respectively  of 023 Fig. . The ect023 Fig. . Of thes023 Fig. . Within Pvama1 sequences obtained from the China-Myanmar border (n = 73) were then compared to previously published sequences from seven other geographic regions and primate-adapted P. vivax isolates . A total of 117 mutations were identified among the 615 global and reference Pvama1 sequences, including 38 synonymous SNPs (SP SNPs) and 79 non-synonymous SNPs (NS SNPs) (Table NS SNPs (53.2%) were clustered within DI, resulting in a peak nucleotide diversity (π = 0.020) for this region . Nucleotide diversity was the highest in the Iranian and Thai populations and lowest in the South Korean population (Table Pvama1 sequences from the China-Myanmar border population did not form distinct clades with the Myanmar isolates [The s) Table . The majder n = 7 were the SNPs 53.% were clisolates  by phyloPvama1 from the China-Myanmar border samples provided estimates of the minimum number of recombination events of six, while values of the recombination parameter C between adjacent sites and per gene were 0.043 and 25.5, respectively  with increasing nucleotide sites distance within the Pvama1 gene of the China-Myanmar border isolates, indicating a high meiotic recombination rate  was detected between the South Korean population and the China-Myanmar border population, whereas a low to moderate level of genetic differentiation was detected among other worldwide populations (ranging from 0.03 to 0.24). A moderate range of FST values (0.13–0.16) were detected when comparing the China-Myanmar border populations with the India, Iran, Venezuela, and PNG populations, but a much lower genetic difference was identified when comparing the China-Myanmar border population with the Thai population (FST = 0.03)  were also observed when comparing part of Pvama1 DI (nt 322–737) among Myanmar, Thai and China-Myanmar border populations . This result suggests that polymorphisms found for Pvama1, especially in the DI, are maintained by diversifying selection, presumably due to host immune pressure.To examine whether natural selection contributed to the generation of the diversity in Pvama1 DI global isolates, 162 unique haplotypes were identified in 615 sequences, of which 54.9% was singleton. Individual haplotype prevalence ranged from 0.6 to 45.1%. Of the reference strain Pvama1 sequences, only the Belem haplotype was detected, but was restricted to the Thai population. No haplotype matched that of the Sal-I strain. Twelve of the 27 China-Myanmar border haplotypes (44.4%) were shared with other populations, of which 91.7% (11/12) were identical to some of the Pvama1 haplotypes observed in the Thai population. Haplotype 21 is the predominant haplotype both in the China-Myanmar border and Thai populations, with a frequency of 24.7% and 23.8%, respectively. Intriguingly, an independent haplotype analysis performed among the China-Myanmar border, Myanmar, and Thai populations indicates that Haplotype 21 is also the only haplotype shared among these three populations  and Sou = 0.007 ) populat = 0.009 ) and Wes = 0.010 ) populatntensity –64, it in border .C (C = 4Nr), these results indicate that recombination is very frequent in the Pvama1 gene of diverse geographic populations [C  was detected for Pvama1 (0.043 and 25.5) in the China-Myanmar border population. This C value observed for Pvama1 is much higher than that of Pfama1 (0.003 and 3.8) from a sympatric population of P. falciparum [Ne) is only 1.3 × 103 when calculated by using the average recombination rate of P. falciparum (~6 × 10-7 Morgans/base) [Pvama1 of the China-Myanmar border population are common, and that this area appears to have a considerably larger effective population of P. vivax than P. falciparum. In P. falciparum, strong LD and low genetic diversity often are observed in low-transmission areas [P. vivax populations, as we observed LD and high-level haplotype diversity (0.958) of Pvama1 in the China-Myanmar border, where malaria transmission is low [Pvama1 in the border regions. The ability to relapse in P. vivax increases the prevalence of mixed infections and chances for genetic recombination in the mosquito vectors. In addition, heavy human population flow across the porous international borders further favors mixing of parasite populations and increasing parasite genetic diversity.The LD indices decline rapidly with the increasing distance between sites. Considering the high values of recombination parameter, ulations , 47–50. lciparum , for whins/base) . We concon areas . HoweverFST comparisons only detected a moderate level of genetic differentiation (FST = 0.23) among global populations. This may reflect the functional constraints of this protein and similar balancing selection from human immunity on this protein. Despite this, major geographical genetic differentiation does exist. For instance, the Korean parasite population was drastically different from other parasite populations from the world. The Thai P. vivax populations collected from three regions  from 1996 and 2007 also showed significant population subdivision [P. vivax populations with only slight genetic differentiation in different regions of the GMS, suggesting the lack of major gene flow barriers in this region. Together with the fact that similar allelic forms of Pvama1 are circulating in the Myanmar, China-Myanmar border, and Thai P. vivax populations [Pairwise division , which mdivision . Yet, veulations , 59, thePvama1 showed that both domains are under diversifying selection, suggesting a significant departure from neutrality as confirmed by the McDonald and Kreitman test. These results are consistent with previous reports of Pvama1 DI from various global isolates [Pvama1 has been reported as highly immunogenic [P. vivax populations [Pvama1 as confirmed by neutrality tests, which is in agreement with several other reports [The pattern of polymorphisms observed in the ectodomain as well as DI of the China-Myanmar border isolates , 47–50. unogenic , 69, andulations , no evid reports , 50. ThePvama1. This extreme polymorphism indicates that each haplotype might be recognized as immunologically distinct by the immune system. Further observation of Pvama1 DI showed that only haplotype 30 in the China-Myanmar border is shared with populations outside of Asia, and haplotype 10 is the only one shared among the majority of analyzed Asian populations. Together with the complex network analysis results, covering diversity of P. vivax will be a great challenge for a PvAMA1-based vaccine design. Although highly diverse, the observation that the majority of the China-Myanmar border Pvama1 haplotypes (44.4%) are shared with Thai populations and that the dominant haplotype (H21) is identical in both populations suggested little differentiation of parasite populations within GMS, and a PvAMA1-based malaria vaccine may be effective in this entire region. Notably, the vaccine strain Sal-I haplotype was not identified in any of the analyzed population in the current study. Thus, the dominant Pvama1 alleles from field parasite populations should be considered in the future studies.A total of 308 haplotypes, with a haplotype diversity of 0.988, was identified in global isolates when analyzing the ectodomain of Pvama1. The majority of the diversity clustered within Pvama1 DI, as has been reported previously for P. falciparum and P. vivax [Pvama1 are comparable to those previously reported [P. falciparum, the c1 (positions 187 to 231) and c1L (positions 196–207) clusters contain the most polymorphic residues [Pvama1 polymorphic sites of the China-Myanmar border isolates were located in the Pfama1 c1 cluster. These mutation sites are comparable with those observed in the PNG isolates [Compared to the Sal-I sequence, 46 SNPs resulting in 43 amino acid substitutions were identified in the China-Myanmar border P. vivax , 37, 50.reported , 47–50, reported , 71. In residues , 71. Howisolates . Previouisolates . ComparaPvama1 in 73 P. vivax isolates from the China-Myanmar border area, as well as global isolates, was exceptionally high, and that Pvama1 DI is the dominant target of positive diversifying selection. Furthermore, a low level of genetic differentiation among the Myanmar, China-Myanmar border and Thai populations suggests high levels of gene flow within the GMS.Our study demonstrated that the genetic diversity of the


Tubastraea coccinea and T. tagusensis are spreading quickly and outcompeting native species in the Atlantic Ocean, there is little information regarding the genetic structure and path of introduction for these species. Here we present the first data on genetic diversity and clonal structure from these two species using a new set of microsatellite markers. High proportions of clones were observed, indicating that asexual reproduction has a major role in the local population dynamics and, therefore, represents one of the main reasons for the invasion success. Although no significant population structure was found, results suggest the occurrence of multiple invasions for T. coccinea and also that both species are being transported along the coast by vectors such as oil platforms and monobouys, spreading these invasive species. In addition to the description of novel microsatellite markers, this study sheds new light into the invasive process of Tubastraea.Although the invasive azooxanthellate corals  Mnemiopsis leidyi, which caused the collapse of the fishing industry  . Among tindustry , the algoduction  and the Atlantic .Tubastraea were introduced and are spreading rapidly throughout the Western Atlantic Ocean    to South80°02′W) ; (2) T. an coast ; and (3)f Mexico . All thrf Mexico .Tubastraea is largely restricted to shaded or marginal habitats, studies on oil rigs in the Gulf of Mexico have shown that both T. coccinea and T. micranthus are excellent competitors and can overgrow other species  and also from a monobuoy (IMODCO 4) at the São Sebastião channel , Brazil. Additional samples of T. coccinea and T. tagusensis, collected from Todos-os-Santos Bay (TSB), northeastern Brazil , and Ilha Grande Bay (IGB) , southeastern Brazil , were used to test the markers and evaluate their genetic diversity (Microsatellite development was performed using samples of iversity . Samplesiversity  prior tode novo assembly. The programs MSATCOMMANDER 0.8 (http://bioinfo.ut.ee/primer3-0.4.0/) and primer characteristics were checked using OligoAnalyzer 3.1 . Forward primers were designed with a M13 tail at their 5′ end (TGT AAA ACG ACG GCC AGT) for dye labeled  primers annealing to the replicated strand during PCR reactions  using the 454 Genome Sequencer FLX platform . Reads wNDER 0.8  and SSRfeactions .T. coccinea and 48 T. tagusensis were amplified by Polymerase Chain Reactions (PCRs). PCRs were performed in 10 µl reactions including 0.2 µM of forward primer with M13 tail, 0.4 µM of labeled primer , 0.8 µM of reverse primer, 1U GoTaq , 1× PCR Buffer (Promega), 0.20 mM dNTPs , between 1.5 and 2.5 mM MgCl2 , was calculated following Psex < 0.001, samples are considered ramets belonging to the same genet. In order to avoid the overestimation of genotype numbers due to scoring errors or somatic mutations , where G represents distinct multilocus lineages (MLL) and N is the total number of individuals sampled. The index ranges from zero  to one ; and (2) the genotypic evenness, to evaluate the equitability in the distribution of the MLL, calculated by the Simpson’s complement evenness index (V = (D − Dmin)∕(Dmax − Dmin)), where D represents the observed diversity, Dmax the value assumed if all genets have the same number of ramets, and Dmin the diversity value when all but one genet has one individual  tested varied from one to three with 15 iterations for each K-value. A higher range in the number of clusters (K ranging from one to five) was also tested to verify possible substructure within the populations. The most likely K-value was estimated by estimating the “log probability of data” for each value of K (mean LnP(K)) )  using ST LnP(K)) . The ΔK ent work .Tubastraea coccinea and T. tagusensis respectively (Accession numbers: KY198738 –KY198749). While two loci failed to amplify for T. tagusensis (Tco36 and Tco38), this species also exhibited two loci at a single locus with no evidence of linkage disequilibrium between them (Tco32a and Tco32b), so both were included in these analyses.The two 454 runs resulted in a total of 329,832 reads with an average size of ±708.5 bp. A total of 1,077 regions with 2–6 bp microsatellite repeats with at least four units were found. Among these regions, 39 were selected for primer design, based on the size and position of the repeat within the sequence, and the primer characteristics . Within these, 11 and 10 were successfully amplified and genotyped for T. coccinea TSB population was observed in the same two loci (Tco36 and Tco38) that failed to amplify for T. tagusensis. Since both loci had only homozygote genotypes at the two analyzed localities, these loci were removed from the genetic diversity analyses. The loci Tco1 and Tco9 showed evidence of linkage disequilibrium with other loci and were also removed from the remaining analyses. The number of alleles per locus ranged from one to five in T. coccinea and one to four in T. tagusensis. Between localities, Ho ranged from 0.38 to 1 (TSB) and 0.17 to 1 (IGB) for T. coccinea and from 0.2 to 1 (TSB) and 0 to 1 (IGB) for T. tagusensis. He ranged from 0.31 to 0.76 (TSB) and 0.15 to 0.58 (IGB) for T. coccinea and from 0.18 to 0.62 (TSB) and 0 to 0.61 (IGB) for T. tagusensis  for all but two and seven individuals of T. coccinea and T. tagusensis respectively. Thus, these data do not support the hypothesis of several individuals with the same MLG having originated by chance from distinct sexual reproduction events. A high proportion of clones were observed at both localities for both species  and three (at IGB) unique MLLs among the 24 sampled colonies at each locality , with only six MLLs out of 24 individuals, while TSB has nearly half of the individuals comprised of clones  (V = 1.13−16). Conversely, the TSB population of T. coccinea had more equally distributed MLLs, with the most common one being shared among only 4 individuals (V = 0.85). For T. tagusensis, both populations were composed mainly of clones, with very low clonal richness . Similarly to what was observed for T. coccinea, MLLs were more equally distributed at TSB, with 14 individuals belonging to the same MLL (V = 0.54), while in IGB the most common one was shared among 22 individuals (V =  − 1.04p−16).Clonal richness observed for  = 0.55) . In addiT. coccinea presenting the more accentuated differences  and T. tagusensis . The inbreeding coefficient (FIS), although not significant, was negative for both localities and in both species, indicating an excess of heterozygotes  and 0.13 (p = 0.07) for T. coccinea and indistinguishable from zero (p = 0.69 and p = 0.69) for T. tagusensis. The lack of significant population structure among the sampled localities indicates similar allele frequencies for both species across these sites. Although Bayesian analysis recovered two genetic clusters for T. coccinea for both ranges of K tested, these groups are not a function of population structure between localities  have high proportions of clones at both localities on the Brazilian coast with identical multilocus lineages (MLLs) found in sites separated by more than 1,500 km. The results indicate that asexual reproduction dominates in the invasive range of Tubastraea spp. in the Southwestern Atlantic and despite the large distance between localities, no significant population structure could be found. In contrast, there are clear signs of population structure across this same region in an endemic spawning coral species  that typically display gregarious settlement  both ttlement  and (ii) regions , and areAtlantic . Thus, oAtlantic .Tubastraea spp. are spreading quickly throughout the Atlantic, in some areas covering up to 100% of the available surface (Tubastraea species globally. This study provides the first survey of genetic diversity and likely reproductive strategies along the southwestern Atlantic coast, demonstrating that asexual reproduction has an important role in the population dynamics of both T. coccinea and T. tagusensis and is probably a relevant feature leading to their invasive success. Results also indicate that there were likely at least two different populations of T. coccinea introduced into the southwestern Atlantic. A molecular systematic examination of the genus is highly recommended in order to check for the occurrence of cryptic species. Future studies should focus on the identification of potential source populations and the global phylogeograpy of Tubastraea with the goal of tracking and limiting future invasions, as well as the establishment of effective management and prevention strategies.Invasive  surface  and outc surface . Despite10.7717/peerj.3873/supp-1File S1Click here for additional data file.10.7717/peerj.3873/supp-2File S2Raw data used in the present work, including allele sizes for each genotyped individual.Click here for additional data file.


Tursiops truncatus) under the IUCN is ‘least concern’. However, in the Caribbean, small and localized populations of the ‘inshore form’ may be at higher risk of extinction than the ‘worldwide distributed form’ due to a combination of factors including small population size, high site fidelity, genetic isolation, and range overlap with human activities. Here, we study the population genetic structure of bottlenose dolphins from the Archipelago of Bocas del Toro in Panama. This is a small population characterized by high site fidelity and is currently heavily-impacted by the local dolphin-watching industry. We collected skin tissue samples from 25 dolphins to study the genetic diversity and structure of this population. We amplified a portion of the mitochondrial Control Region (mtDNA-CR) and nine microsatellite loci. The mtDNA-CR analyses revealed that dolphins in Bocas del Toro belong to the ‘inshore form’, grouped with the Bahamas-Colombia-Cuba-Mexico population unit. They also possess a unique haplotype new for the Caribbean. The microsatellite data indicated that the Bocas del Toro dolphin population is highly structured, likely due to restricted movement patterns. Previous abundance estimates obtained with mark-recapture methods reported a small population of 80 dolphins (95% CI = 72–87), which is similar to the contemporary effective population size estimated in this study . The combination of small population size, high degree of genetic isolation, and intense daily interactions with dolphin-watching boats puts the Bocas del Toro dolphin to at high risk of extinction. Despite national guidelines to regulate the dolphin-watching industry in Bocas del Toro and ongoing educational programs for tour operators, only in 2012 seven animals have died due to boat collisions. Our results suggest that the conservation status of bottlenose dolphins in Bocas del Toro should be elevated to ‘endangered’ at the national level, as a precautionary measure while population and viability estimates are conducted.The current conservation status of the bottlenose dolphin ( Tursiops truncatus) is a cosmopolitan species [The bottlenose dolphin . As  species , 6, 7, 8 species , 9, 10.  species , 13, 14) species , 15. It  species , 9.Some coastal populations of bottlenose dolphins live in small populations characterized by low genetic diversity and low gene flow between neighboring populations, suggesting local founder events at least in some areas or recent isolation e.g., , 18, 19), 1918, 1T. truncatus spp. ponticus from the Black Sea (classified as ‘endangered’) [Conservation priorities and ultimately, funding to monitor marine mammal populations is largely dependent on the IUCN conservation status of the species. Given their worldwide distribution, the bottlenose dolphin is classified by the IUCN as ‘least concern’ , with thngered’) . Howeverngered’)  and presngered’) . Howeverngered’) .Like other coastal bottlenose dolphins, the dolphin population found in the Archipelago of Bocas del Toro in Panama is small. Preliminary estimates suggest a population of 80 dolphins (95% CI = 72–87)  and bothHere, we evaluate the conservation status of the bottlenose dolphins of Bocas del Toro by estimating the genetic diversity and population structure using nine microsatellite markers and around 750 base pairs (bp) fragment of the mitochondrial DNA Control Region (mtDNA-CR). We test the hypothesis that there is a high degree of genetic distinctiveness in dolphins from Bocas del Toro in comparison to other bottlenose dolphin populations in the Caribbean, largely due to the small population size and high individual philopatry.The Archipelago of Bocas del Toro (BDT) is located on the Caribbean coast of Panama . This arThe methodology for sample collection was approved by the Smithsonian Tropical Research Institute IACUC . Samples were collected with permission from the Autoridad Nacional del Ambiente–Panamá .Following Krützen et al. , tissue 5′-TGTAAAACGACAGCCAGTTCACCCAAAGCTGRARTTCTA-3′) and Dlp8G (5′- GGAGTACTATG TCCTGTAACCA-3′). Amplification followed the protocol from Baker et al. [2, 200 μM dNTP, 2 units BSA, 0.5 μM of each primer, and 1 U Biolase DNA polymerase (Bioline USA). PCR products were purified following a Polietilenglicol protocol (PEG 20%), and DNA was sequenced using the Sanger sequencing method [SRY gene and ZFY/ZFX genes of males and females as positive controls. The haplotypes sequences identified in this study were submitted to GenBank as accession number KX833116 for Bocas del Toro population, and KY817220 and KY817221 for individuals from Costa Rica. These two haplotypes from Costa Rica were successfully amplified in only two samples.DNA was extracted from skin samples using the DNeasy kit . The portion of the mtDNA-CR (approximately 750 bp) was amplified by the polymerase chain reaction (PCR), using the primers pairs, t-Pro-whale M13Dlp1.5 . These microsatellite data of the individual dolphins from Bocas del Toro and Costa Rica are available in Dryad , and samples from Jamaica and the US Virgin Islands were excluded (n = 1)   performeFST  and φST ce model  was used (n = 1) .n = 25) with nuclear data of samples from Costa Rica (n = 5) and previously published nuclear data from six different Caribbean geographic locations, including The Bahamas (n = 11), Colombia (n = 3), Cuba (n = 53), Honduras (n = 6), Mexico (n = 30), and Puerto Rico (n = 20) [To determine the genetic structure of bottlenose dolphins from Bocas del Toro in the Caribbean, we compared our microsatellite results  [K set from 1 to 10 for each value of K with the admixture model and correlated frequencies in the software STRUCTURE. After, using the program STRUCTURE HARVESTER [LnP(D) of different values for K using an ad hoc statistic ΔK, which calculates the second order rate of change of Ln P(D). The corresponding values for each K were plotted to determine the uppermost level of population structure for our dataset.To examine the genetic structure in Bocas del Toro and the Wider Caribbean, we used the software STRUCTURE version 2.3.4 , with a a LnP(D) , we firsARVESTER , we compHo), the expected heterozygosity (HE), levels of polymorphism , and the linkage disequilibrium (LD) (10000 permutations). We estimated the number of alleles (NA) and allelic richness (AR) per locus using the rarefaction test implemented in the software HP-RARE [HWE) using the Microsoft Excel add-in GENALEX software version 6.5 [FST and RST values for each population’s pair to estimate gene flow using the software GENALEX version 6.5. (1000 permutations) [FST and global corrected FST using the HIERFSTAT package in R version 3.3.3 [We assessed genetic differentiation and diversity among population units defined previously by STRUCTURE using the software ARLEQUIN version 3.5 . We calc HP-RARE . We testsion 6.5 . We assesion 6.5  as implesion 6.5 . We calctations) . We alsoon 3.3.3 . To deteon 3.3.3  in the pon 3.3.3 .Nm) among pairs of population units of bottlenose dolphins in the Caribbean, we ran the software MIGRATE version 3.0.3 with microsatellite data. This software uses the coalescent approach of Markov chain Monte Carlo (MCMC) to run the maximum likelihood with all population units simultaneously [To estimate the number of migrants per generation  for the Bocas del Toro population, using the bias-corrected version of the method of linkage disequilibrium [Ne in small populations [Finally, we assessed the contemporary effective population size , Dolphin Bay (n = 10), Loma Partida (n = 3), Pastores Islands (n = 2), Popa (n = 2), Shark Hole (n = 2), and Tierra Oscura (n = 2)  . AdditioFrom the 25 samples collected in the Archipelago of Bocas del Toro, only one haplotype was identified (760 bp). We compared this haplotype to two sequences (760 bp and 540 bp) obtained from bottlenose dolphins of Gandoca-Manzanillo in Costa Rica, located only 35 km north of Bocas del Toro, and with 40 published bottlenose dolphins haplotypes found in multiple locations in the Wider Caribbean. Our results revealed that the haplotype found in Bocas del Toro is unique, as it is not found in any other bottlenose dolphin population in the Caribbean . The difTtruCAR-E), Cuba , Mexico , and Puerto Rico (TtruCAR-JJ)  ; Table 1TtruCAR-MM), Cuba , Honduras (TtruCAR-G), Mexico (TtruCAR-QR1), and Puerto Rico (TtruCAR-GG). In addition, the Costa Rican haplotypes nested with the haplotype TtCAR-C, an ancestral WDF haplotype related to WDF haplotypes from Madeira [The haplotype from Bocas del Toro was not shared with any other population. In contrast, the two haplotypes from Gandoca-Manzanillo (Costa Rica) were grouped with ‘worldwide distributed form’ (WDF) haplotypes from the Caribbean see ; Table 1 Madeira , which iP = 0.000) was found among both population units at the FST and ΦST levels (P < 0.1) since we found a value of ФCT = 0.205 (P = 0.052), ФST = 0.427 (P = 0.000), and the ФSC = 0.279 (P = 0.000).In the analysis of population structure, we defined two population units, where Bocas del Toro (BDT) was grouped with other subpopulations: BDT-Bahamas-Colombia-Cuba-Mexico, meanwhile Costa Rica was grouped with Honduras and Puerto Rico . SignifiT levels . In geneK) present in the Caribbean. The evaluation of the K values using the ΔK method produced a clear peak at K = 2 (K = 2 as the most likely number of groups present in the data (ΔK = 267.317) (K = 4 as the number of populations units present in the Caribbean according to the same microsatellite data (without including Bocas del Toro and Costa Rican samples). Based on four population units identified in this previous work , and in order to identify population structure between the Bocas del Toro population and individuals from Costa Rica, we assessed the population structure analyses in K = 6, in which Bocas del Toro (Panama) and Gandoca-Manzanillo (Costa Rica) belong to two distinct population units (K = 6), where the X axis corresponds to each individual, it is notable that the Bocas del Toro cluster conforms a discrete population unit. This plot also shows that the Bocas del Toro population unit is highly differentiated from other population units and that individuals do not appear to maintain present gene flow with other dolphin population in the Caribbean, including Gandoca-Manzanillo, and if they do, it is minimal.Based on the assumption that Caribbean bottlenose dolphins have a common ancestor, we performed the STRUCTURE analysis under the admixture model with correlated frequencies, in order to assess the number of units  . Howeveron units . In Fig Nm = 1.070) (Nm = 0.132), suggesting asymmetrical migration. Individuals from Gandoca-Manzanillo also showed low rates of migration toward other locations in the Caribbean. While both Bocas del Toro and Gandoca-Manzanillo dolphins show low migration rates, other dolphin populations in the Caribbean seem to maintain connectivity with at least one migrant per generation . Similarly, Colombia-Honduras-PuertoRico, The Bahamas, and Mexico population units contribute one migrant per generation towards Cuba , and the Mexico population unit receives migrants from Cuba (Nm = 1.598). Hence, all populations units in the Caribbean show a certain level of connectivity, except for Bocas del Toro and Gandoca-Manzanillo.The results from the MIGRATE analysis indicated that there was only one migrant per generation from Bocas del Toro to the neighbor population in Gandoca-Manzanillo (= 1.070) , and ratneration . For insFST  and corrected FST  values showed a population structure among bottlenose dolphins in the Wider Caribbean. The calculated pairwise population differentiation indices FST and RST for all six proposed dolphin populations are described in RST values are higher than FST values. This suggests an older differentiation among dolphin populations with some recent genetic connectivity [FST and RST indicate that the Bocas del Toro population is differentiated from the other Caribbean dolphin populations, suggesting a certain degree of isolation. The other dolphin populations in the Caribbean show relatively low but significant FST and RST values. However, all these populations units did not show strong differences in FST and RST values, probably because some populations still present some migration  was estimated for the Bocas del Toro population using minimum allele frequencies of 0.05 and 0.02. The Ne varied from a low of 73  to a high of 122 . Despite Ne showing a wide confidence interval (CI), it is a good indicator of number of adults reproductively contributing to the Bocas del Toro dolphin population [The contemporary effective population size , number of alleles (NA), and expected heterozygosity (HE). We obtained genetic diversity values for nine microsatellites loci from the six populations units analyzed along with deviations from HWE , dolphin populations in Colombia-Honduras-PuertoRico and Cuba showed three loci out of HWE, meanwhile Bocas del Toro showed one locus , decreaectively , therefoectively . In contely [HWE .HE) values were similar among the six populations, with Bocas del Toro and The Bahamas showing slightly higher values. Particularly for the Bocas del Toro dolphin population, heterozygosity values were moderate and similar for most of the loci  or selection against heterozygotes [FIS) for the Bocas del Toro population, and although it was positive, it was not significant . No significant differences between HE and Ho were found (P > 0.05), meaning that there appear to be no significant reduction of diversity. Nevertheless, it is possible that we cannot detect significance because there is high variation within each locus. MICRO-CHECKER did not detect evidence for large allele dropout in all loci, but the software detected null alleles in some loci; however, no one locus presented null alleles in more than two populations. Thus, it is possible that HE was higher than Ho due to presence of null alleles in some loci [Microsatellite expected heterozygosity . The loci Tur48 and Tur117 showed the lowest NA  and the lowest mean AR . The NA observed in each dolphin population ranged from two to nine, and the AR varied between 1.222 and 3.115 (NA ranged from two to nine) and allelic richness (AR varied between 1.330 and 3.115) compared with the Bocas del Toro dolphin population . In contrast, individuals from Costa Rica showed the highest allelic richness values in TexVet5, Tur91, MK9, and Tur117 loci (see The nd 3.115 . In geneloci see .This study provides the first description of the population structure and genetic diversity of bottlenose dolphins from the Archipelago of Bocas del Toro (Panama) using both mtDNA-CR and microsatellite data. Our results also provide further evidence that ‘inshore’ bottlenose dolphin populations face higher risks of extinction than worldwide distributed dolphin populations. The Bocas del Toro population has a small effective population size, show high levels of genetic isolation, and are aggressively targeted on daily basis by the local dolphin-watching industry. Combined these factors can potentially reduce population viability and recovery time.pers.com. 2017). Furthermore, dolphins in Bocas del Toro show higher residency [The multiple analyses conducted here identify the Bocas del Toro dolphin population as another ‘inshore form’ in the Wider Caribbean. However, unlike other Wider Caribbean dolphin populations, it possess a unique mtDNA-CR haplotype that is grouped with other ‘inshore’ bottlenose dolphin haplotypes found in The Bahamas, Cuba, Mexico, and Puerto Rico , 13. Intesidency  than thoesidency . While sesidency , 15,17),MK6, TexVet5, and Tur91 loci) [The strong population structure of Bocas del Toro is however, not unique. Many coastal populations of bottlenose dolphins show high levels of philopatry and genetic isolation. Among these are the bottlenose dolphins from Gulf of California , 17, New91 loci) .HE mean = 0.53), when compared with other coastal dolphin populations such as the one found in San Antonio Bay, Argentina (HE mean = 0.19) [HE values [Microsatellite expected heterozygosity was high  = 0.19) , but ver = 0.19) , the Wes = 0.19) , and the = 0.19) , 100. Ho = 0.19) ) and it  = 0.19) . Similar , but veE values . In lighTursiops have gone through a recent diversification in the Holocene [pers.com. 1997, In: [Based on mtDNA-CR haplotype clustering, we identified two bottlenose dolphin population units in the Caribbean: BDT-Bahamas-Colombia-Cuba-Mexico and CostaRica-Honduras-PuertoRico. The population unit containing haplotypes from Bocas del Toro, The Bahamas, Colombia, Cuba, and Mexico contained a considerable number of individuals that were assigned to the ‘inshore form’ . In contHolocene , coincidHolocene , 105, coHolocene , 10. GeoHolocene , and tha997, In: ). TherefNe estimated falls within this range (73 to 122) is of concern and should be considered as a measure for conservation [Ne less than 100 individuals have an increased extinction risk [Ne of 50 reproductive individuals can maintain genetic health in a population in the short term, and 500 mature individuals to the long term [Preliminary estimates of population size suggest that a small population of 80 bottlenose dolphins are found in Bocas del Toro (95% CI = 72–87) . Our conervation , since pion risk . Accordiong term , 108. Thong term , 107.Ne), genetic isolation, and intense commercial dolphin-watching activities can potentially impact the survival and fitness of the Bocas del Toro dolphin population. These dolphins sustain the largest dolphin-watching industry in Panama, interacting daily with a high number of boats [Therefore, the combination of small dolphin population size . Duringof boats . Local oof boats , 110. Inof boats . Dolphinof boats . Many ofof boats . Furtherof boats , 113, esof boats . These pof boats , 117.Despite the intense interactions with boats, there is no evidence that the dolphins of Bocas del Toro are moving out of this area. The cost of leaving appears to be greater than staying, even when boat traffic continues to grow. The Archipelago of Bocas del Toro provides a protected area from potential predators and rough weather, and mangroves, sea grasses, and coral reefs provide a rich source of food supply. In addition, males in this population do not appear to disperse to other areas, as is commonly described for other bottlenose dolphin populations , 20, 118Similar to the situation in Bocas del Toro, there are at least 50 other dolphin populations that are small, isolated, and vulnerable to human activities . Even soS1 Data(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Steindachneridion parahybae (Family Pimelodidae) is endemic to the Paraíba do Sul River basin in southeastern Brazil. This species was heavily exploited by artisanal fisheries and faces challenges posed by dams, introduced species, and deterioration of critical habitat. The remaining populations are small and extirpated from some locales, and the species is listed as critically endangered in Brazil. Screening variation at a partial mitochondrial control region sequence (mtCR) and 20 microsatellite loci, we: (i) describe the patterns of genetic diversity along its current distributional range; (ii) test the null hypothesis of panmixia; (iii) investigate the main factors driving its current population structure, and (iv) propose management of broodstock for fostering recovery of wild populations through genetically cognizant restocking. Our microsatellite data for 70 individuals from five collections indicate moderate levels of heterozygosity (HO = 0.45) and low levels of inbreeding (FIS = 0.016). Individual-based cluster analyses showed clear genetic structure, with three clusters of individuals over the collection area with no mis-assigned individuals, suggesting no recent migration among the three clusters. Pairwise DEST values showed moderate and significant genetic differentiation among all populations so identified. The MUR population may have suffered a recent demographic reduction. mtCRs for 70 individuals exhibited 36 haplotypes resulting from 38 polymorphic sites. Overall, mitochondrial haplotype diversity was 0.930 (±0.023) and nucleotide diversity was 0.011 (±0.002). Significant population structure was observed, with ϕST = 0.226. Genetic markers could be used in a hatchery-based restoration program emphasizing breeding of pairs with low kinship values in order to promote retention of genetic diversity and avoid inbreeding. Individual average kinship relationships showed 87.3% advised matings, 11.0% marginal matings, and 1.7% advised against. While these results comprise a contribution toward planning better breeding management and monitoring, parallel actions to be undertaken include surveying healthy riverine habits for reintroduction and continued searching for wild individuals to introduce new variation into the captive broodstock to avoid adaptation to captivity and to minimize inbreeding.The long-whiskered catfish  Steindachneridion (Order Siluriformes: Family Pimelodidae), which encompasses six recognized species of migratory long-whiskered catfishes that inhabit deep water in mid-sized streams with rocky bottoms  is a medium-sized, dorsoventrally flattened, whiskered catfish endemic to the Paraíba do Sul River basin in southeast Brazil  describe the patterns of genetic diversity of the S. parahybae along its current distributional range; (ii) test the null hypothesis of panmixia in this region; (iii) investigate the main factors driving its current population structure, and (iv) propose ex situ genetic breeding management for recovery of wild populations through genetically cognizant restocking programs.Once abundant in artisanal fisheries, ulations . ManagemAll field work for fish sampling complied with the legal regulations of Brazil .S. parahybae were captured through an extensive occurrence survey along the tributaries of the Paraíba do Sul River basin in Brazil. Sites of occurrence were located with the help of local artisanal fishers and the Companhia Energética de São Paulo . Wild S. parahybae were collected at five different locations  within the Paraíba do Sul River basin in 2004 and between 2010 and 2015, consisting of: 1 individual from the Paraíba do Sul River (PSP) in São Paulo State , 24 individuals from the Paraíba do Sul River (PRJ) in Rio de Janeiro State , 5 individuals from the Preto River (PRE) in Rio de Janeiro State , 3 individuals from the Pomba River (POM) in Rio de Janeiro State , and 37 individuals from the Muriaé River (MUR) in Rio de Janeiro State . All individuals were transferred live to aquaculture facilities belonging to CESP and kept as an ex situ germplasm bank  described by KU821557 to KU821572 and KX578067 to KX578070. Microsatellite alleles were genotyped using a 6.5% denatured polyacrylamide gel matrix  and a Li-Cor 4300 DNA Analyzer  using the IRDye® 700 marker and universal M13 tail primer described by ® 700) using the SagaGT Client program . All recommendations for minimizing genotyping errors originating from DNA quality and handling procedures , allelic richness (Ar), observed heterozygosity (HO), expected heterozygosity (HE), and fixation index (FIS) using the Adegenet package  using means and variances of kinship coefficients. Three relationship categories  were used, with 5,000 pairs of simulated individuals for each category. In this evaluation, seven rxy (KE) kinship estimators were used as follows: trioml , 1x buffer , 1.5 mM MgCl2, 2.5 mM dNTPs, 10 mM of each primer, and ultrapure water. The PCR thermal-cycling profile consisted of initial denaturation at 94°C for 3 min; followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min, and extension at 72°C for 1:30 min; and a final extension step at 72°C for 10 min.A partial mitochondrial DNA control region (mtCR) was amplified by polymerase chain reaction (PCR) using two external primers, SPf (5′TCTAACTCCCAAAGCTAGAATC3′) and SPr (5′GGAACTTTCTAGGGTTCATCTTAAC3′), developed in this study. Amplification was performed in a 20-μl reaction containing: 20 ng/μl of genomic DNA, 0.5 IU The amplicons were purified using ExoSAP enzymes  following the manufacturer’s instructions, except that the shortest time for deactivation of the enzymes was 15 min. The amplicons were sequenced on an Applied Biosystems 3130 Genetic Analyzer, and bases were called using Applied Biosystems Sequencing Analysis Software version 5.2. The sequences were edited manually when necessary using CodonCode .Hd) and nucleotide (π) diversities, number of haplotypes (h) and polymorphic sites (S) – were calculated in DnaSP v. 5.10 , PRE (n = 5), and POM (n = 3) sites, data from those collection areas were included only to provide preliminary insights into how these collections may be related to the others.The estimated probabilities were corrected using Holm–Bonferroni sequential adjustments for multiple tests . Due to S. parahybae, R2  for seven pairs of loci in a single population (MUR), which may be the consequence of many factors, including limited numbers of parents in the preceding generation, as well as natural selection, gene flow, assortative mating, and linkage. There was no evidence of genotyping errors, such as stuttering, null alleles, or large allele drop-out. The highest number of alleles (13) was amplified at the Spa18 locus, while Spa3, Spa7, Spa16, Spa23, and Spa28 all exhibited only two alleles. The number of alleles per locus within particular populations ranged between 2 and 13, with a mean of 4.7 ± 3.2. The allelic richness values ranged between 6.1 and 10.4, with average of 2.65 ± 2.07. The observed heterozygosity values ranged between 0.0 and 0.89, with average of 0.45 ± 0.24. Private alleles were observed in the MUR (14), PRE (1), and PRJ (2) collections. The FIS inbreeding coefficient values ranged between -0.002 and 0.356, with average of 0.016. The summary statistics for all loci are shown in Supplementary Table All samples of Figure 2). Overall, individuals from the MUR and PRJ sites showed a 100% likelihood of assignment to their original collection area (hereafter termed populations). Individuals from POM, PSP, and PRE collections present a group of individuals of mixed ancestry . Even though they do not form a same genetic cluster, we decided to assume a single cluster due to the low sample number. Neighbor-joining analysis  also corroborated the three clusters of individuals: MUR, PRJ, and the POM/PSP/PRE sites. No mis-assigned individuals were observed, suggesting that there may have been no recent migration among these well-defined clusters.Results of the individual-based DAPC and NJ cluster analyses showed clear genetic structure, with three clusters of individuals over the entire collection . The pairwise DEST values for populations identified previously by NJ and DAPC analysis were congruent with the results of individual-based analysis, showing moderate and significant genetic differentiation among all populations (Table 1).The genetic differentiation indices were highly significant  for the MUR population using the SMM mutation model (Table 2). However, there were no significant values for TPM model, which is more powerful for fewer than 20 loci  and 0.011 (±0.002), respectively. The haplotype diversity values ranged from 0.868 ± 0.049 (MUR) to 0.99 ± 0.272 (POM), and nucleotide diversity values ranged from 0.005 ± 0.004 (POM) to 0.013 ± 0.007 (PRJ) (Table 3). Haplotype H2 (n = 13) was the most common, occurring mainly in MUR, and shared by all populations except for PSP  showed mainly singleton haplotypes in populations MUR and PRJ. Haplotype H2 was most frequent haplotype, shared by all S. parahybae populations except for PSP. Significant population structure was observed for S. parahybae along the Paraíba do Sul River basin based on mtCR data .A total of 791 bp of the mtCR were resolved from 70 SF metric presented high values, but there were no significant values (Table 3). In contrast, the R2 values were high and significant (p < 0.05), indicating a significant departure from neutrality. According to R2 test is more powerful in detecting demographic events in small sample sizes, whereas Fu’s FS is better for large sample sizes.Fu’s ex situ germplasm bank, sampling variances for the kinship estimators (KE) ranged from 0.0198 to 0.1046. The lowest variance was found in the triadic likelihood estimator (TrioML). Considering that the KEs had high correlations and little influence on the inference of relatedness, paired kinship coefficients are reported only for the TrioML. Values estimated by the TrioML of kinship were low, indicating a low inbreeding index for 67 individuals, and thus, a range of permissible pairings among the broodstock candidates. Only three individuals with the highest values of inbreeding were classified as moderately endogamous. Means and variances for kinship likelihood estimators are showcased in Supplementary Table Among prospective broodstock in the Table 4. Supplementary Tables When we matched the 44 females with the 21 males among the broodstock candidates, we obtained 924 interactions that were added to the possible interactions with the five individuals of indeterminate sex , obtaining 1,269 possible matings. The index obtained for kinship values was split into three categories according to expected average theoretical values . That isS. parahybae populations reflected the endangered status of this Neotropical catfish in its area of occurrence. Attempts to locate wild populations proved the first indicator of the imperiled status of S. parahybae in the Paraíba do Sul watershed. Many efforts have been made during recent years to find and collect S. parahybae individuals to be kept in an ex situ gene bank so that a genetically cognizant restocking program  and number of alleles per locus (A = 4.7) for wild S. parahybae populations were low across the 20 loci screened in this study.Assessing the genetic differentiation of wild populations and individuals kept in tinction . SummaryFIS = 0.016) was below the average value (FIS = 0.05) for 18 endangered animal species kept in ex situ conservation programs  and microsatellites (DEST = 0.082) can be interpreted as reflecting historical and recent structuring, especially for the female component of the population, with some degree of homogenizing gene flow for males, with or without the effects of recent anthropogenic isolation. Cluster analyses  showed considerable population structure, indicating the division of the S. parahybae gene pool into three distinct groups, MUR, PRJ, and a mixed group formed by POM, PSP, and PRE. The strongly supported assigned clusters for MUR and PRJ (q > 0.9) denoted virtual absence of migration between the two groups. The identification and characterization of an Evolutionary Significant Unit (ESU), based on population genetic structure and adaptive differentiation, would provide a useful basis for conservation, as well as a more complete understanding of the role of adaptive evolution in the natural history of the species  does not have the star-like shape typical of populations that show a signature of a species that has expanded its numbers from a modest number of founders  at 100 so that allelic diversity can be retained at above 90% of current allelic diversity for at least four generations. In the present study of endangered S. parahybae populations, we used this same approach, also based on multilocus microsatellite markers, to genetically characterize the few remaining wild populations and broodstock currently maintained in an ex situ germplasm bank. These genetic data comprise a contribution toward planning better breeding management and monitoring any changes in the genetic makeup of the broodstock, as well as in the reintroduced fingerlings in their new environments. Parallel actions to be taken would include surveying healthy riverine habits for reintroduction and continued searching for wild individuals to introduce new variation into the captive broodstock to avoid adaptation to captivity and to prevent inbreeding. Successes in other genetic marker-assisted restoration programs suggest that application of this approach might also prove effective for S. parahybae. Oncorhynchus gorbuscha in a supportive breeding program on the Dungeness River in Washington State, United States, providing the basis for hatchery-based supplementation. Following cessation of stocking from outside sources, microsatellite marker-assisted hatchery-based supplementation of walleye Sander vitreus (Use of estimators for pairwise relatedness and individual inbreeding coefficients based on multilocus microsatellite markers has proven an efficient strategy to achieve better responses in reintroduction programs.  vitreus  contribuAH conceived and supervised the project. FF performed the experiments, analyzed data, and drafted the manuscript, RD performed statistical analyses and contributed to manuscript writing, AH and EH wrote and critically edited the final manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eupallasella percnurus) is critically endangered. In this paper we characterize the genetic properties of this fish over its range of occurrence in Poland and propose the use of this knowledge in its active protection. Twelve populations of lake minnow from across its range in Poland were investigated. 13 microsatellite loci were investigated to evaluate genetic variation and distance among populations. The magnitude of the genetic bottleneck or founder effects was investigated. In the studied populations, the allelic diversity and heterozygosity showed that genetic variation in this species is low. At most loci, only 2–3 alleles per population were detected. The average number of alleles detected across all loci was 35, and ranged from 24 to 53. The average observed heterozygosity (Ho) across all investigated loci was 0.38 (range 0.21–0.59); the average expected heterozygosity (He) was 0.36 (range 0.18–0.55). The populations remained in Hardy-Weinberg equilibrium. The average Garza-Williamson M index value for all populations was low (0.47), suggesting a reduction in genetic variation due to a founder effect or a genetic bottleneck. Genetic distance among populations was high or very high ; this was likely a consequence of low gene flow among isolated populations, a founder effect or other genetic bottleneck, and strong genetic drift. The large genetic differences among the investigated lake minnow populations are likely to also exist among other populations of this species, and knowledge of these differences should inform active protection programs based on translocation of wild or cultivated fish of this species. The method presented here can potentially be applied to any population of lake minnows or closely related species.The lake minnow ( Eupallasella percnurus , is a tiny cyprinid fish species that is widely distributed in the Northern Hemisphere, from Poland in the west, throughout Northern Asia, and to the Pacific coast in the east , , . Thus, tAdult fish from 12 lake minnow populations from throughout the species' entire range  were cau3. Material for genetic analyses was then taken by clipping approximately 20 mm2 from the ends of the fishes’ left pelvic fins; these fin fragments were collected from 48 fish from each population. Immediately after collection of these fragments, the fish were released into the same water bodies where they had been caught. All of these procedures were approved by the Ethics Commission in Olsztyn, Poland (permit number 22/2010). The wet samples were first placed on a 15 x 12 cm plate that was wrapped in foil, and then the samples were taken to an air-dryer for preservation. After drying each fin fragment was numbered, wrapped in new fragment of aluminum foil, and placed in separate 1.5 ml Eppendorf tube.To anesthetize the fish, they were placed in water that contained 0.45 g 2-phenoxyethanol /dmGenomic DNA was extracted and purified from fin tissues using a Sherlock AX DNA Extraction and Purification Kit, or a DNA and Genomic Mini AX Tissue SPIN DNA Extraction and Purification Kit. The extraction procedure was performed following the manufacturer’s recommendations (A&A Biotechnology). DNA samples were stored at a temperature of –20°C. The integrity of the DNA samples was visually inspected following electrophoresis in a 1.5% agarose gel stained with etidium bromide. All agarose gels were photographed using a gel imaging system and the images were digitally recorded. Samples of the DNA yields were quantified by spectrophotometric analysis; only samples containing more than 30 pg/μl of double-stranded DNA qualified for the PCR stage.Ca3 Ca4, and Ca12, were taken from ST) , , . This meIn Poland, the most common lake minnow habitats are old (50–100 years) peat excavations, which have shallowed to less than 1 m at their deepest point because of intensive plant succession . HoweverM) for all the investigated lake minnow populations were very low, which indicates that all of them experienced severe genetic bottlenecks and/or founder effects. Knowledge of these values makes it possible to compare the probable reductions in population size in lake minnow populations to those in other species of finfish, reptiles, and mammals reported by M) for alHo and He were low or very low in all populations, both on the level of a single locus and on the population level. It is noteworthy that, for example, Ho and He values for Ca3 or Ca4 were lower than those recorded in other fish species, such as Campostoma anomalum  an an32] ananomalum . When thtocholas  or Anaecispanica . The aveorted by . The simorted by  confirm In the present study, population BE was found to have the highest number of alleles and one of the highest levels of heterozygosity of all the investigated populations. The differences in the genotypes of individuals in this population indicate that admixtures with other populations took place. These admixtures could have occurred because this population is located in the upper part of the Tyśmienica River valley with several other lake minnow populations only 1–2 km away. Thus, the relatively high heterozygosity and allelic diversity of population BE suggests that local gene flow takes place among all the nearby lake minnow populations during the flood events that occur there from time to time. Such a situation is not experienced by any of the other 11 populations in the present study.Ho and He values differed significantly at some markers but not at others. Although average Ho is close to He it is not clear if all populations remain at H-W equilibrium. Departures from H-W equilibrium at locus Z9878 are known to occur in the genomes of cyprinid species [ species , althougAlthough there was little variation within the populations of lake minnow investigated in the present study, genetic variation between these populations was large, and it seems likely that the situation is similar in the majority of all lake minnow populations in Poland.Although this conclusion differs from the results obtained by , those aST method was comparable to or even larger than those of geographically distanced or isolated populations of sturgeons [2 method were comparable to the values of bears inhabiting various regions of North America [2 method between lake minnow populations were higher than those of other endangered animal species, such as the natterjack toad Bufo calamita, which inhabits various regions of Great Britain [The analysis of molecular variance shows that the genetic differences between lake minnow populations in the present study is the major component of genetic variation in all investigated samples. In general, the genetic differences between pairs of populations are very large. In many cases, the magnitude of the genetic distance calculated using the Fturgeons . The mag America . In many Britain , or Mong Britain .ST and δμ2 methods were similar. The Mantel test showed that between all pairs of lake minnow populations, the results obtained for genetic distance using the two methods were in close agreement. Moreover, the sizes of pairwise within-population and interpopulation genetic differences were found to confirm the results of the genetic assessment test. We did not detect a clear correlation between geographic distance and the size of genetic differences existing between pairs of populations.Generally, the distances estimated by the FThe results of the Mantel test suggest that the geographical distance between lake minnow populations may have some effect on genetic distance, but it is definitely not the key factor determining the extent of genetic differences.As was indicated above, the Polish lake minnow likely still inhabits its natural range of occurrence in the country. This commonly accepted view is also supported by the comparison of currently extinct lake minnow sites  was begun in 2002 . The proThe method developed and applied in this study has the potential to help maintain the genetic variation of lake minnow populations. The set of molecular markers used in this study enable identification a group of males and females within given population that are most genetically different from each other. These individuals can be assembled in spawning pairs using Genassemblage software  to produ


Clintonia udensis (Clintonia) is a primary perennial herb widely distributed in China with two karyotypic characteristics—diploid and tetraploid and thereby used to understand the ploidy and distribution. This study unraveled the patterns of genetic variation and spatiotemporal history among the cytotypes of C. udensis using simple sequence repeat or microsatellites. The results showed that the diploids and tetraploids showed the medium level of genetic differentiation; tetraploid was slightly lower than diploid in genetic diversity; recurrent polyploidization seems to have opened new possibilities for the local genotype; the spatiotemporal history of C. udensis allows tracing the interplay of polyploidy evolution; isolated and different ecological surroundings could act as evolutionary capacitors, preserve distinct karyological, and genetic diversity. The approaches of integrating genetic differentiation and spatiotemporal history of diploidy and tetraploidy of Clintonia udens would possibly provide a powerful way to understand the ploidy and plant distribution and undertaken in similar studies in other plant species simultaneously contained the diploid and tetraploid.Polyploidy is an important factor shaping the geographic range of a species.  The approaches of integrating genetic differentiation and spatiotemporal history of diploidy and tetraploidy of Clintonia udens provide a powerful way to understand the ploidy and plant distribution and could be undertaken in similar studies in other plant species simultaneously contained the diploid and tetraploid.l Kanai, . The dipn Figure . Those a22.1C. udensis. Among the four populations, the karyotypic characteristics of HLN and JHL populations are diploid cytotype while HLB and MLZ are tetraploid cytotype. The HLN and HLB populations located at Hualongshan Mountains in Shaanxi Province while JHL and MLZ located at Shennongjia Mountains in Hunan province. This study was included total of 152 individuals  of 2.2The leaves were used to extracted genomic DNA followed the method of CTAB  of individuals were assumed in Hardy–Weinberg and linkage equilibria were given number from K = 1 to K = 4 to investigate under the correlated allele frequencies model by running 100,000 iterations. A Neighbor‐joining (NJ) tree was performed the genetic relationships among populations  and heterozygosity within each population (Hs) . The average number of effective alleles per locus and high values of heterozygosity was similar in all populations. Significantly higher values were also found in all populations for the Shannon's information index (I) (Table p = .035). Two cytotyped were significant differences found in these diversity estimators    or tetraploid  populations, the genetic variability was also both mainly found among populations.PhiPT distances were ranged between 0.673 (HLN/HLB) and 0.813 (JHL/MLZ) and were statistically significant between all studied populations Table . It indiR2 = 0.024, p = .001). Bayesian analysis showed populations of diploid JHL, and HLN were grouped into one cluster while populations of tetraploid MLZ and HLB were grouped into the other cluster that with the value of K = 2, which was dominant in each of the two cytotype; the population of diploid HLN and population of tetraploid HLB solely gathered a cluster, respectively, with the value of K = 3; four populations were, respectively, gathered different cluster and were easily distinguished with the value of K = 4  were arised from the isolated and different ecological surroundings, which could be evolutionary capacitors to preserved distinct karyological, and diploid–tetraploid complex that exhibits an intriguing pattern of eco‐geographic differentiation. However, polyploidy is a significant and complex mode of species formation in plants, therefore, firstly, solely depend on the genome DNA of SSR markers of plant distribution and polyploidy was limited and Reduced‐Representation Genome Sequencing should be used to the polyploidy genome analysis of phylogeography to confirm the relationship of the ploidy and plant distribution in plant species simultaneously contained the diploid and tetraploid in the future; secondly, only four populations simultaneously contained the diploid and tetraploid are difficult to explain the factors involved in the origin and establishment of polyploids in nature, and the rest samples of other regions of YLXS  and DGK, HSK, LW, MLG, GBS, LTDZ, HHL, TBS, FP, KZQ, EMS, JYH  should be collected and analyzed in the future study.In summary, polyploidy is contributed to shape the geographic range of a species. Two regions (Hunan and Shaanxi provinces) distributed the diploid  and tetraploid (HLB and MLZ) of None declared.JH, SNW, and YLW designed the study; JL and ZLF performed data management; JH and SNW analyzed the data and interpreted the results; JH, SNW, and YLW wrote the manuscript.


P. vivax (PvMSP-7), a gene member encoding PvMSP-7E was analyzed among 92 Thai isolates collected from 3 major endemic areas of Thailand . In total, 52 distinct haplotypes were found to circulate in these areas. Although population structure based on this locus was observed between each endemic area, no genetic differentiation occurred between populations collected from different periods in the same endemic area, suggesting spatial but not temporal genetic variation. Sequence microheterogeneity in both N- and C- terminal regions was predicted to display 4 and 6 α-helical domains, respectively. Signals of purifying selection were observed in α-helices II-X, suggesting structural or functional constraint in these domains. By contrast, α-helix-I spanning the putative signal peptide was under positive selection, in which amino acid substitutions could alter predicted CD4+ T helper cell epitopes. The central region of PvMSP-7E comprised the 5’-trimorphic and the 3’-dimorphic subregions. Positive selection was identified in the 3’ dimorphic subregion of the central domain. A consensus of intrinsically unstructured or disordered protein was predicted to encompass the entire central domain that contained a number of putative B cell epitopes and putative protein binding regions. Evidences of intragenic recombination were more common in the central region than the remainders of the gene. These results suggest that the extent of sequence variation, recombination events and selective pressures in the PvMSP-7E locus seem to be differentially affected by protein secondary structure.Merozoite surface protein 7 (MSP-7) is a multigene family expressed during malaria blood-stage infection. MSP-7 forms complex with MSP-1 prior to merozoite egress from erythrocytes, and could affect merozoite invasion of erythrocytes. To characterize sequence variation in the orthologue in  Plasmodium vivax mainly coexists with Plasmodium falciparum, both of which affect global health burden and contribute remarkably to economic loss . Th. ThPvMSPPvMSP-7E could support functional constraint on the N-terminal part of this protein.The central region of PvMSP-7E was highly enriched in polar and charge amino acids with a number of glycine and proline residues whilst non-polar and non-charge residues were sparse in this region . ImportadS and dN has shown evidence for positive selection in the central region of PvMSP-7E. However, signals of positive selection occurred exclusively to the 3’ portion of this region corresponding to the dimorphic central domain of the protein. Besides being a potential binding region to the primary processing fragments of MSP-1, the dimorphic domain could be responsible for immune evasion. Consistently, mice immunized with P. yoelii MSP-7 could elicit antibody response, but failed to protect mice against lethal infection with the virulent strain [The occurrence of long stretch of unstructured or disordered region in the central part of PvMSP-7E could lead to increased in intrinsic plasticity that has been considered to be an important characteristic for molecular recognition of or interaction with its protein targets. The protein-protein binding regions were also predicted to be located in the central part of this protein . Importat strain . AlthougPvMSP-7E locus of P. vivax populations from 3 major endemic areas of Thailand has shown a remarkably lower level of haplotype diversity in Yala and Narathiwat than other endemic areas, in which only 3 haplotypes circulated in the study population whereas 19 and 34 haplotypes were sampled from Ubon Ratchathani and Tak, respectively. These findings are in agreement with previous reports on analysis of other genetic loci that encode merozoite surface protein-5, apical membrane antigen-1 and thrombospondin-related adhesive protein of P. vivax in this country [P. vivax and P. falciparum populations from Yala and Narathiwat occurred as a result of population bottlenecks in the parasites caused by control measures [PvMSP-7E of P. vivax populations surveyed ranged from 1 in Yala-Narathiwat population to 11 in Tak population collected during 2008–2009. The correlation between the number of recombination breakpoints and the levels of mean haplotype diversity was approaching a significant value , suggesting that intragenic recombination could have partly shaped the extent of haplotype diversity. The non-zero recombination breakpoints in PvMSP-7E of P. vivax isolates from Yala and Narathiwat provinces further support that bottleneck effects rather than strict clonal expansion occurred in the population.The implementation of integrated malaria control program in Thailand has markedly reduced the annual parasite incidences of the country during the past 3 decades. However, foci of malaria transmission remain in several provinces bordering Myanmar, Cambodia and Malaysia albeit being considered hypoendemic areas . Analysi country , 52, 53.measures . AlthougFst estimates among P. vivax populations in this study have revealed significant genetic differentiation of P. vivax population between endemic areas. However, the fixation index between populations from Tak province collected in 1996 and during 2008–2009 exhibited low and non-significant value. This is in accord with our previous analysis on genetic diversity in the PvTRAP locus of parasites in Thailand that displayed spatial but not temporal variation [FST value, suggesting gene flow or genetic admixture between these parasite populations. Recent malaria epidemic in Ubon Ratchathani province during sample collection period was mainly due to forest encroachment for illegal logging by both native people and migrants from other provinces, whereas pre-epidemic malaria cases in this province were mostly indigenous cases who acquired infections locally. Undoubtedly, population migration could shape genetic diversity of P. vivax populations in Thailand.Phylogenetic analysis neither shows specific clusters of sequences for each endemic province nor unique clades for Thai or Colombian isolates. Pairwise ariation . SurprisPvMSP-7E locus among P. vivax populations in Thailand that could have been influenced by selective pressure and intragenic recombination. The levels of haplotype diversity displayed geographic variation in which a few haplotypes were circulating in southern parasite population as a result from bottleneck effects as previously noted [In conclusion, our analysis revealed extensive polymorphism in the ly noted . DiffereS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


This first pan-Mediterranean analysis of genetic diversity in wild narrow-leafed lupin revealed strong East–West genetic differentiation of populations, an historic eastward migration, and signatures of genetic adaptation to climatic variables.Lupinus angustifolius L.) as a model to understand adaptation in a wild crop ancestor. A set of 142 wild accessions of narrow-leafed lupin from across the Mediterranean basin were subjected to genotyping-by-sequencing using Diversity Arrays Technology. Phylogenetic, linkage disequilibrium and demographic analyses were employed to explore the history of narrow-leafed lupin within the Mediterranean region. We found strong genetic differentiation between accessions from the western and eastern Mediterranean, evidence of an historic West to East migration, and that eastern Mediterranean narrow-leafed lupin experienced a severe and recent genetic bottleneck. We showed that these two populations differ for flowering time as a result of local adaptation, with the West flowering late while the East flowers early. A genome-wide association study identified single nucleotide polymorphism markers associated with climatic adaptation. Resolving the origin of wild narrow-leafed lupin and how its migration has induced adaptation to specific regions of the Mediterranean serves as a useful resource not only for developing narrow-leafed lupin cultivars with greater resilience to a changing climate, but also as a model which can be applied to other legumes.Most grain crops suffer from a narrow genetic base, which limits their potential for adapting to new challenges such as increased stresses associated with climate change. Plant breeders are returning to the wild ancestors of crops and their close relatives to broaden the genetic base of their crops. Understanding the genetic adaptation of these wild relatives will help plant breeders most effectively use available wild diversity. Here, we took narrow-leafed lupin (The online version of this article (10.1007/s00122-017-3045-7) contains supplementary material, which is available to authorized users. Agriculture is facing the ‘perfect storm’ of factors that threaten global food security including continued population growth, a rapidly changing climate, reduced water available to agriculture, soil erosion and increasing costs of fertilisers —as a model to study migration and climatic adaptation in the ancestral wild populations of this species. Narrow-leafed lupin (Lupinus angustifolius L.) is an important minor grain legume crop, being grown over 750,000 hectares mainly in Australia, Poland, Russia and Germany  gene . While narrow-leafed lupin domestication was only completed in the twentieth century, there is extensive evidence of its use by Mediterranean cultures for centuries  . This subset of 142 accessions were selected from 1248 wild accessions characterised by Berger et al.  using 13http://www.Invitrogen.com/qubit).DNA was extracted from leaves of single plants from each accession using a standard CTAB method  genotyping at Diversity Arrays Technology Pty Ltd, Canberra, Australia  were effectively amplified in 30 rounds of PCR using the following reaction conditions: 94 °C for 1 min, then 30 cycles of 94 °C for 20 s, 58 °C for 30 s and 72 °C for 45 s, finishing with 72 °C for 7 min. After, PCR equimolar amounts of amplification products from each sample of the 96-well microtiter plate were bulked and applied to c-Bot (Illumina) bridge PCR followed by sequencing on Illumina Hiseq2500. The sequencing (single read) was run for 77 cycles.Only “mixed fragments”  sequences per barcode/sample were used in marker calling. Finally, identical sequences were collapsed into “fastqcall” files. These files were used in the secondary pipeline for DArT PL’s proprietary SNP and SilicoDArT (presence/absence of restriction fragments in representation) calling algorithms (DArTsoft14). Co-dominant SNP markers were the focus of the study, as they are able to distinguish heterozygous and homozygous loci, which was necessary for downstream analyses, and were filtered using different thresholds for various analyses. For demography analyses, SNP markers which had positions mapped to pseudo-chromosomes were used. For phylogenetic, population structure, linkage disequilibrium and association studies, SNPs with more than ≥ 25% missing data or ≥ 12.8% heterozygosity were eliminated. DArTseq reads were aligned to the K = 2 to K = 12, using default parameters  was performed using EIGENSTRAT to assess genetic diversity and to correct for population stratification  using GAPIT  for every SNP within a window of 1 Mb using Plink  as measured by L. angustifolius, we used the models implemented in the software dadi ; the central population containing samples from France and Italy (n = 21); and the eastern population containing the samples from Greece, Cyprus and Turkey (n = 45). As the ancestral state of each SNP is uncertain, we folded the SFS in dadi before fitting the Isolation with Migration (IM) model. Preliminary analysis suggested that singletons, i.e. alleles found only once in the population, were under-represented in all populations, suggesting a bias in the SNP calling protocol. To avoid biasing the demographic inferences due to this, we excluded all singletons from the calculations of the SFS.For SFS analysis, we included all 38,948 SNPs with known position in the pseudo-chromosomes of the We performed two types of analyses. First, we investigated the demographic history of each population separately, by fitting the default single population models available in dadi Table . We thenDArTseq analysis generated 45,230 co-dominant SNPs markers in 142 wild narrow-leafed lupin accessions. For the demography analyses, we used a subset of SNPs that mapped to pseudo-chromosomes which included 38,948 SNPs. For other analyses, loci with excessive missing and heterozygote values were removed . Due to the evidence of whole-genome triplication in the genome of narrow-leafed lupin  depicted a vivid division between East and West Mediterranean accessions Fig. . The firK2–12) (Online Resource 5). The internal algorithm in fastSTRUCTURE for multiple choices of K determined that population numbers K = 7 or K = 8 best explained the variation in the dataset. To determine which one of these two models (K = 7 or K = 8) better fitted the data, we subjected both models to phylogenetic analysis. Again, as both models appeared plausible, the simpler model (K = 7) was selected , and seven accessions from Italy and one accession from Greece that fell within the western clusters .To explore further how genomic diversity differs between the distinct eastern and western Mediterranean population groups and to infer the historic levels of genetic diversity in each population group, we investigated linkage disequilibrium (LD) in these two population groups and conducted demographic analyses. The western population group showed far lower average maximum LD (0.2) than the eastern population group 0.4), and the LD decayed more rapidly in the western populations  model with each pair of populations. The IM model had six free parameters: the relative size of the two populations after they split, time of the split, modern population sizes of each population and effective migration rates in two directions. We found that population split sizes were highly unequal, with each pairwise comparison suggesting that the eastern population emerged from a very small proportion of the ancestral population (p < 0.01 and degree of freedom of 140) associated with some climatic/eco-geographic variables, including precipitation in different months/quarters , latitude and annual mean temperature (BIO1). Positive correlations were observed between flowering time and latitude, and between flowering time and precipitation in the driest month and driest quarter, while the correlation between flowering time and annual mean temperature was negative.We investigated adaptation parameters distinguishing West and East populations. Climatic variables from the sites of origin of each accession (extracted from the WorldClim database) were correlated with phenotypic traits . As separate correlation analyses for Western and Eastern populations showed similar trends, we only present one correlation matrix for both West and East together Fig. . Variatip < 0.05) variation in phenotypic traits (such as flowering time) and climatic variables  between samples from West/East Mediterranean. Details of significant variation traits are provided in Table p < 0.05 Table We next sought to determine how geographical location  could explain observed phenotypic variation. In total, 53 and 89 accessions were labelled as eastern and western groups, respectively (Online Resource 1). A one-way analysis of variance (ANOVA) indicated there was significant  with precipitation of driest month/quarter and LaDArT_SNP09086 locus on chromosome NLL-05  was significantly associated (p = 1.089e−08) with annual mean temperature , as well as association between markers and climatic variables. Although GWAS analyses did not highlight any significant association between markers and phenotypic traits, it pointed out a strong association with two of the climatic variables. A SNP on position 4,582,844 bp of chromosome NLL-07 (LaDArT_SNP18940) was found to be strongly associated Supplementary material 2 (XLSX 24600 kb)Supplementary material 3 (XLSX 6624 kb)Principal Component Analysis (PCA) for 142 accessions of wild narrow-leafed lupin colour-coded by phylogeny results (east/west Mediterranean) (PPTX 214 kb)Supplementary material 5 (PPTX 220 kb)Supplementary material 6 (XLSX 11 kb)Below is the link to the electronic supplementary material.


Bos taurus and Bos indicus populations were clearly distinguished, whereas Japanese Shorthorn and Japanese Polled clustered with European populations. Furthermore, STRUCTURE analysis demonstrated the distinct separation between Bos taurus and Bos indicus (K=2), and between European and Asian populations (K=3). In addition, Japanese Holstein exhibited an admixture pattern with Asian and European cattle (K=3‐5). Mongolian (K=13‐16) and Japanese Black (K=14‐16) populations exhibited admixture patterns with different ancestries. Bos indicus populations exhibited a uniform genetic structure at K=2‐11, thereby suggesting that there are close genetic relationships among Bos indicus populations. However, the Bhutan and Bangladesh populations formed a cluster distinct from the other Bos indicus populations at K=12‐16. In conclusion, our study could sufficiently explain the genetic construction of Asian cattle populations, including: (i) the close genetic relationships among Bos indicus populations; (ii) the genetic influences of European breeds on Japanese breeds; (iii) the genetic admixture in Japanese Holstein, Mongolian and Japanese Black cattle; and (iv) the genetic subpopulations in Southeast Asia.In this study, we genotyped 117 autosomal single nucleotide polymorphisms using a DigiTag2 assay to assess the genetic diversity, structure and relationships of 16 Eurasian cattle populations, including nine cattle breeds and seven native cattle. Phylogenetic and principal component analyses showed that  In Asia, 258 cattle breeds have been reported, and some of these breeds (11%) are classified as being at the risk of extinction Scherf . In addiet al. et al. et al. et al. et al. et al. The bovine whole genome sequence has been determined and over 2.2 million putative single nucleotide polymorphisms (SNPs) were identified  and Bos indicus . We collected samples of Black Angus and Hereford from Australia, Hanwoo from Korea and Japanese Holstein from nine prefectures in Japan: Tosa Japanese Brown from Kochi Prefecture, Higo Japanese Brown from Kumamoto Prefecture, Japanese Shorthorn from Iwate Prefecture, Japanese Polled from Yamaguchi Prefecture and Japanese Black from 17 prefectures in Japan. We selected 30 unrelated samples from each breed and population, and DNA was extracted from the blood, sperm or muscle.We collected 480 DNA samples from 16 Eurasian cattle breeds and populations of et al.  and observed (Ho) heterozygosities were calculated as indices of genetic diversity. Analyses of pairwise population differentiation (Fst) and within population differentiation (Fis) were performed with ARLEQUIN ver 3.5.1.2  for a given sample. To obtain a representative value of K for modeling the data, we performed 16 independent runs of the Gibbs sampler for each K (1 ≤ K ≤ 17) with a 20 000 initial burn‐in used to minimize the effect of the starting configurations, followed by 100 000 Markov Chain Monte Carlo iterations, as recommended by Falush et al.  was higher than that in Bos indicus (0.195). We also calculated Ho and He within populations  to 0.394 (Mongol) and from 0.308 (Higo Japanese Brown) to 0.389 (Mongol), respectively. In contrast, those in Bos indicus populations were relatively low ranging from 0.229 (Laos) to 0.287 (Bhutan) for Ho and from 0.250 (Bangladesh) to 0.294 (Bhutan) for He. No deviations from the Hardy‐Weinberg equilibrium between Ho and He were observed in all populations (P > 0.05).We estimated the allele frequencies of 117 SNP markers in 16 populations. The mean MAF in each population ranged from 0.077 (Mongol) to 0.419 (Bangladesh) , whereas those among Mongol cattle and Bos indicus populations were relatively low (0.140‐0.186). The highest value was observed between Japanese Polled and Bangladesh (0.326) and the lowest value was observed between Cambodia and Vietnam or Laos (0.008). In Bos taurus, the distance between Tosa Japanese Brown and Japanese Shorthorn was highest (0.203), and that between Mongol and Hanwoo was lowest (0.042). The distances among Bos indicus populations were relatively low (0.008‐0.041).We estimated Nei's genetic distance and ns Table . The genBos taurus and Bos indicus clades. The Bos indicus populations were clustered close together. The Japanese Shorthorn and Japanese Polled were clustered with the European populations.Two types of phylogenetic constructions, NJ tree Fig. A and UPGBos taurus and Bos indicus were distinguished by PC1 (17.6%). European and Asian breeds were separated by PC2 (4.5%), whereas Japanese Polled and Japanese Shorthorn were clustered with European breeds. Japanese Holstein were located in an intermediate position between the European and Asian populations. Bos indicus populations were more closely clustered than Bos taurus populations as well as results of phylogenetic constructions.According to the PCA Fig. , Bos tauK values. We inferred the optimum number of genetic ancestral populations (K) by computing the log likelihood of the data (LnP(D)) and the second order rate of change of the likelihood function with respect to K (ΔK)  and on the Indian subcontinent (Bos indicus).In this study, we evaluated the genetic diversity, relationships and structure of 16 cattle populations using 117 autosomal SNPs. The genetic indices Table , phylogeet al. Japanese Shorthorn and Japanese Polled clustered with European groups in the phylogenetic trees, PCA and STRUCTURE analyses Figs , 2, 3. Jet al. K=5 in STRUCTURE, Tosa Japanese Brown formed a distinct group from the Asian cattle group, including Korean cattle, Japanese Black and Mongolian cattle. At present, the Tosa Japanese Brown is maintained with a very small population size (<3000), which has likely resulted in genetic drift, thereby leading to a different genetic structure compared with the other Asian cattle populations.Although Tosa and Higo Japanese Browns were categorized as the same breed, they have different breeding improvement histories  the genetic influences of European breeds on Japanese breeds; (iii) the genetic admixtures in Japanese Holstein, Mongolian, Korean and Japanese Black cattle; and (iv) the presence of the genetic subpopulations in Southeast Asia. The genetic information obtained in this study would contribute to illuminating the genetic construction of Asian native cattle and facilitate future breeding improvements.In conclusion, we successfully determined the genetic diversity, structure and relationships of 16 cattle populations by using 117 autosomal SNPs genotyped in a DigiTag2 assay. Our results suggested: (i) the close genetic relationships among Supporting info itemClick here for additional data file.