The aim of this study is to reveal the genetic variability and population structure of maternal lines of the Polish Konik—a native Polish horse breed of the primitive type included in the Polish conservation programme. The analysis was carried out on the basis of 17 microsatellite markers routinely used for parentage testing. The structural analysis indicated the likelihood of three genetic clusters (using the Structure and Structure Harvester software). F-statistics indicated a low level of inbreeding. All mean population coefficients were close to those obtained for representatives of male founder lines. The population seemed to be stable. However, due to a previous bottleneck event, breeding strategies should focus on avoiding inbreeding depression, preventing the decrease of genetic variability, and sustaining the active female lines.IT was 0.0188, coefficient of differentiation FST was 0.0304, and mean inbreeding index value FIS was −0.0119). Variability monitoring should be carried out in order to avoid inbreeding depression, while breeding strategies should be designed to prevent the decrease of genetic variability in the Polish horse breed and to sustain the active female lines.The aim of the conservation programme is to maintain the population size of endangered livestock breeds of less economic importance at a level that ensures the survival of the breed, the preservation of genetic diversity, and the preservation of as many pedigree lines as possible. The Polish Konik, a native Polish primitive-type horse breed and is one of the breeds included in such a programme in Poland. Presently, there are only 16 , some of which are endangered. We examined the genetic variability and structure of the Polish Konik maternal lines  on the basis of the pedigree data and 17 microsatellite markers (STRs) from parentage testing. The overall mean number of alleles was 7.647 (±0.411), the effective number of alleles was 3.935 (±0.271), the mean number of alleles for which the frequency was equal to or lower than 5% was 4.471 (±0.286), and the mean information index was 1.506 (±0.087). The structure of the population and admixture patterns were calculated with the Structure and Structure Harvester software. The structural analysis indicated three likely genetic clusters; as the most optimal K value was estimated as 3, with ∆K of 15.4188. The F-statistics results indicated a low level of inbreeding (average inbreeding coefficient F Equus caballus)—sometimes called Polish primitive [The Polish Konik horse is one of the horse breeds covered by the conservation programme in Poland, which considers six horse breeds: Polish Konik, Hucul horse, Wielkopolska, Malopolska, Silesian, and Polish Coldblood (sokolski and sztumski types). The Hucul horse and Polish Konik studbooks were closed in the 1980s; therefore, these breeds have been kept pure, and the admixture of other breeds has been forbidden . The Polrimitive  or Polisrimitive —is a natrimitive . The lasrimitive . Polish rimitive . Polish rimitive ,8. Polisrimitive ,9. They World War II had a ruinous impact on the breeding of Polish horses  includinPresently, Polish Konik are broadly used for hippotherapy, agritourism, and recreational riding , as wellThe first studbook of the breed, which was published in 1962, included 35 maternal and 6 paternal lines. In the 1980s, one more female line—Geneza—was implemented into the conservation programme. Unfortunately, 19 of maternal lines became extinct . MoreoveThe breed was included in the Global Strategy for the Management of Farm Animal Genetic Resources . Genetic diversity monitoring is crucial for livestock management, especially for small, endangered livestock breeds. A recent study of the genetic diversity of the Polish Konik showed that the inbreeding coefficient increased from 4.8% in 1980 to 8.6% in 2011 .Polish Konik horses are unique. They must be preserved for future breeding works as a specific reservoir of genetic resources . This stWe examined the genetic variability, and population structure of the Polish Konik breed within all 16 active maternal lines.We verified maternal lines based on pedigree data and identified all 16 maternal lines. We randomly selected representative samples of maternal lines from our database. Samples with doubtful or excluded pedigree were eliminated. As a result of a random selection among the selected samples, representatives for fifteen of the sixteen lines were found; therefore, the representatives of the missing line were selected manually. Finally, we tested 176 horse samples representing the sixteen maternal lines, where the distribution of the individuals among the lines was as follows: Białka , Dzina I (14), Geneza (3), Karolka (15), Liliputka I (15), Misia II (3), Ponętna (8), Popielica (15), Tarpanka I (14), Traszka (15), Tunguska (15), Tygryska (12), Urszulka (15), Wola (15), Zaza (14), and Bona (2). We used peripheral blood and hair follicle samples collected for routine horse parentage testing (a collection of DNA samples from different regions of Poland) and the panel of 17 microsatellite markers, which was currently recommended by the International Society for Animal Genetics (ISAG).Isolation, using a Wizard Genomic DNA Purification Kit  or Sherlock AX  and from hair follicles using Sherlock AX (A&A Biotechnology), were conducted as in our previous study. Amplification of multiplex reactions, electrophoresis conditions, and fragment analysis were carried out with the same methods as described in our previous report . The 17 We used the Structure 2.3.4 software  to analy1) and both parental genotypes (PE2), and the probability of identity (PID).We estimated the genetic diversity using GenAlEx6.5 ,23, FSTAThe results showed that the seventeen microsatellite loci analyses for 176 Polish Konik samples represented all sixteen maternal lines. We identified the overall mean number of alleles as 7.647 (±0.411), where the effective number of alleles was 3.935 (±0.271) for all samples. The mean number of alleles with a frequency equal to or lower than 5% was 4.471 (±0.286). The mean information index was 1.506 (±0.087).We tested the population structure and admixture pattern within maternal lines using the Structure 2.3.4 software. We used the same running length as in our previous research and set the number of clusters (K) from 1 to 20, with ten independent simulations for every K. Next, we identified the most likely K through ∆K using the Structure Harvester v0.6.94 software to investigate the genetic groups in the data set. The structural analysis results are presented in In the structural analysis, three and fourteen inferred clusters best suited the data set, considering the highest ∆K values. The results were not unambiguous and did not answer the question of how many genetic clusters there were. To additionally visualise the relation among dam lines, we calculated the Fst values between pairs of populations based on Nei’s estimator using the adegenet package for R , which aThe average allelic richness estimated for each locus for all samples was 7.635. Allelic richness per locus (<5.000) was the lowest at loci HTG6 (4.956) and HTG7 (4.978), and the highest (>8.000) were at ASB2, HTG10, VHL20, ASB17, and ASB23 (from 8.983 to 10.989). The allele’s mean number for the whole group studied was 7.647 (±0.411), while the effective allele’s mean number was 4.471 (±0.286).ST) was low, the mean inbreeding coefficient (FIT) was close to zero (0.0188), while the mean value of inbreeding index was negative (−0.0119). The coefficients describing the population were then calculated  or both (CPE2) parents were known. The precise values of those coefficients were 99.9321% and 99.9996%, respectively.The mean value of expected heterozygosity (0.7111) was higher than the observed heterozygosity (0.7019). The power of discrimination and exclusion were nearly 1 . The polymorphism information content (PIC) was close to or higher than 0.7 for a majority of microsatellite (STR) markers in the studied horse population . The cumulative values of the remaining coefficients were high for both probability of exclusion and power of discrimination as well the cumulative parentage exclusion probability, when one  and FIT (0.0188) were close to zero. The mean FIS was also negative (−0.0119), indicating a low level of inbreeding. The mean Ho (0.7019) in this study was a little higher than that calculated for paternal representatives (0.6519), but the general value of heterozygosity in both research works was the same. Ho for X-linked LEX3 was higher in this study for obvious reasons. The PIC values were generally high . All remaining mean population coefficients were overall the same as for representatives of male founder lines [The F-statistic coefficients were similar to those estimated in our previous study for the paternal line representatives: Five −0.01, indicato and He , as well as the PIC value (0.6512) [We also obtained similar coefficients to the results presented by Gralak et al.  and Mack(0.6512) . We also(0.6512)  for Poli1 and CPE2 coefficients of the Hucul horse are also high [o and He reported by Mackowski et al. [o and He were reported for Romanian Hucul horses [Another Polish breed covered by the conservation programme is the Hucul horse. Polish Konik and Hucul horses are sometimes compared. Both breeds are regional and native with historical similarities; both breeds are at risk of extinction  and, as endangered horse breeds, have been maintained for many years ,33. Furtlso high , similari et al. . The saml horses  or Hucull horses .Our study, together with our previous report , allowedOur study was carried out on the STR set currently recommended by the ISAG, where fourteen of them have been recommended by the Food and Agriculture Organization (FAO) for molecular generic characterisation. We analysed all maternal line representatives in order to illustrate that their genetic diversity is still not numerous as an endangered breed. The Polish Konik population experienced a bottleneck event, in which the number of dam lines was reduced from 35 to 16. Genetic variability and polymorphism, assessed on the basis of maternal line representatives, confirmed no significant inbreeding in the analysed Polish Konik population. However, monitoring of variability should be carried out in order to avoid inbreeding depression. Breeding strategies should be designed to prevent a lack of sustainable continuation, to continually develop active female lines, and to prevent the decrease of the genetic variability in the Polish Konik horse breed. Using molecular markers to assess genetic diversity can be a reasonable tool for use in conservation programmes. The results obtained should be supplemented in the future through research concerning mtDNA, as well as variability on the Y chromosome, to obtain a more complete view of the founding lines.


Plasmodium falciparum populations can be used to describe the resilience and spatial distribution of the parasite in the midst of intensified intervention efforts. This study used microsatellite analysis to evaluate the genetic diversity and population dynamics of P. falciparum parasites circulating in three ecological zones of Ghana.Genetic diversity in P. falciparum parasites.A total of 1168 afebrile children aged between 3 to 13 years were recruited from five (5) Primary schools in 3 different ecological zones , Forest (Konongo) and Coastal (Ada and Dodowa)) of Ghana. Asymptomatic malaria parasite carriage was determined using microscopy and PCR, whilst fragment analysis of 6 microsatellite loci was used to determine the diversity and population structure of P. falciparum by microscopy and nested PCR respectively. The genetic diversity of parasites in the 3 ecological zones was generally high, with an average heterozygosity (He) of 0.804, 0.787 and 0.608 the rainy (peak) season for the Sahel, Forest and Coastal zones respectively. The mean He for the dry (off-peak) season were 0.562, 0.693 and 0.610 for the Sahel, Forest and Coastal zones respectively. Parasites from the Forest zone were more closely related to those from the Sahel than from the Coastal zone, despite the Coastal zone being closer in physical distance to the Forest zone. The fixation indexes among study sites ranged from 0.049 to 0.112 during the rainy season and 0.112 to 0.348 during the dry season.Out of the 1168 samples examined, 16.1 and 39.5% tested positive for A large asymptomatic parasite reservoir was found in the school children during both rainy and dry seasons, especially those in the Forest and Sahel savannah zones where parasites were also found to be related compared to those from the Coastal zone. Further studies are recommended to understand why despite the roll out of several malaria interventions in Ghana, high transmission still persist.The online version contains supplementary material available at 10.1186/s12879-021-06120-9. Ghana comprises of three distinct ecological zones; Sahel zone (Northern Ghana), the Forest zone (Middle belt) and the Coastal zone (Southern Ghana). The prevalence of malaria varies across the three ecological zones, with the Forest zone having relatively higher parasite prevalence 22.8%)  compared2.8%  comP. falciparum parasites primarily results from recombination between different clones in addition to within clone polymorphisms including chromosomal deletions, gene duplication, number of repeat sequences and point mutations at various genetic loci  [1-Σpi2]), where n is the number of isolates and pi is the frequency of the ith allele.Results obtained after the capillary electrophoresis was analyzed using the genetic analysis software GeneAlEx  and was IAS), which quantitatively measures the strength of LD ranges from 0 (lack of LD in the loci) as seen in most high parasites locations to 1 (strong LD in loci) as in low transmission settings [IAs values [Multi locus linkage disequilibrium (LD) was assessed from each of the sites using LIAN 3.5 . The stasettings , 34 and s values  at 10,00s values . at 10,0Among all 119 samples, 107 were polyclonal infections and 83 of these samples showed > 1 allele in two or more loci  or polyclonal if the infection contained multiple alleles at any of the 6 loci [Multiplicity of infection (MOI) refers to the number of distinct parasites clones in a single infection and was determined using the ratio of the total number of distinct parasites clones for a gene to the number of samples positive for the same gene. e 6 loci .FST analysis was conducted using θ, an FST-estimator in SPAGeDi v1.2e [FST values were tested for significance using 10,000 permutations. The pair Wright’s Fixation index (FST) was used to determine the population structure with FST value ranged from 0 to 0.05 indicates low genetic variability, 0.05–0.15 indicates moderate genetic variability, 0.15–0.25 indicated great genetic differentiation and > 0.25 indicates substantial genetic differentiation [An Di v1.2e . The FSTntiation .K) was determined by simulating a range of K values from 1  to 5 . The posterior probability of each value was then used to detect the modal value of ΔK, a quantity related to the second order rate of change with respect to K of the likelihood function [6 iterations of each chain was performed to ensure convergence of the MCMC. Each MCMC chain for each value of K was run ten times with the independent allele frequency option that allows individuals with ancestries in more than one group to be assigned into one cluster. Individuals were assigned into K clusters according to membership coefficient values (Q) ranged from 0 (lowest affinity to a cluster) to 1 (highest affinity to a cluster). The partitioning of clusters was visualized with DISTRUCT [P. falciparum clones at two different levels: (i) within polyclonal individuals where only individuals with multiple alleles detected in a single locus were included and differentiated into separate clonal genotypes; (ii) among monoclonal samples from all study sites. The squared Euclidean distance, which is based on the number of times a certain allele found in two clones [A model-based Bayesian method implemented in STRUCTURE v2.3.4 was performed to examine partitioning of individuals to genetic clusters . The numfunction . PosteriDISTRUCT . NeighboDISTRUCT  to show o clones  was calco clones .p = 0.718)  were males and 634 (54.3%) females. The mean ages of the children from all the five sites were similar . Malaria parasite carriage in the dry season was highest in Tamale (13.6%) and lowest in Kumbungu (7.1%). In the rainy season, malaria parasite carriage was highest in Konongo (24.1%) and lowest in Ada (12.8%)  in the rainy season than the dry season. From a zonal perspective, parasite carriage estimated by microscopy in the children from the Coastal zone (Ada and Dodowa) were similar across the dry and rainy seasons, but parasite carriage were significantly higher  in children in the Forest (Konongo) and the Sahel  zones during the rainy season than the dry season . All microscopy positive samples were confirmed to be positive for P. falciparum by PCR. Children living in Konongo had the highest prevalence of PCR detectable parasites during both the dry (59.8%) and the rainy season (58.8%)  in the rainy season than in the dry season  of parasite prevalence identified 39.5% 461/1168) of the children to be positive for /1168 of on Table .A total 119 parasites were genotyped at six polymorphic loci Fig. b Table. P = 0.9630).Allelic variation patterns in each of the six MS loci genotyped in the rainy season were not significant, allelic size variation were between 1 and 10 bp except TA40 locus which recorded allelic variation of 25 bp between 2 alleles in the marker Fig.  Table 2.He (0.886) and ARA2 the lowest He (0.735), average He = 0.804 in the Sahel zone . In the Coastal zone (Ada), Poly_α similarly had the highest He (0.829) and ARA2 the lowest He (0.227), average He = 0.608. However, parasites from the Forest zone (Konongo) showed the highest diversity at the TAA81, He = (0.877) and TA40 the lowest He (0.602), Average He = 0.787 (He (0.705) and ARA2 the least He (0.444) at Sahel zone  with average He of 0.562. In the Coastal zone (Ada), TA87 had the highest He (0.812) and Pfpk2 had the least He (0.338) with average He of 0.610. In the forest zone (Konongo) Poly_α locus had the highest He (0.805) while TA87 had the least He (0.600) with an average He of 0.693  were higher than those from the Coastal zone (Ada).Samples from the Forest zone (Konongo) had the highest number of different parasite clones, with MOI range of 2.6 (TAA81) to 1.3 (TA40) and a mean MOI of 2.0. Multiplicity of infection in the Coastal zone (Ada) ranged between 1.8 (TA87) and 1.1 (ARA2) with a mean MOI of 1.4. The average MOI in samples from the Sahel zone  was similar to that reported from samples collected from the Forest zone (Konongo), MOI range from 2.3 (Poly_α) to 1.5 (ARA2), mean MOI was 1.7 in the rainy season. In the dry season, MOI in the Coastal zone (Ada) ranged from 1.6 (Poly_α) to 1.0 ARA2, mean MOI was 1.4 compared to MOI of 1.3 (Poly_α) to 1.0 (TA40) with mean MOI of 1.1 for the Sahel zone  and 2.4 (Poly_α) to 1.0 (TAA81) with mean MOI of 1.4 for the Forest zone (Konongo). The average MOI of asymptomatic infections of P. falciparum infections 12 (10.1%) of the infections were monoclonal and 107 (89.9%) were biclonal and polyclonal across the three ecological zones  in the 6 MS loci genotyped the Monte Carlo simulation model was used [IAS values ranged from 0.036 in the Konongo (Forest zone) to 0.047 in Tamale  to 0.140 in Ada  values were used to estimate the population structure of the six loci across the three ecological zones. The greatest differentiation was observed between Tamale (Sahel zone) and Ada  (Two most probable genetic clusters (pink and blue) were identified by Bayesian inference for the 119 samples collected from the three zones  Table 3.P. falciparum infections are a hindrance to malaria control as they can serve as reservoirs for gametocyte carriage [P. falciparum carriers in the Coastal zone compared to the Forest and Sahel zones might mean that ongoing malaria interventions might have worked better in the Coastal zone than the others. The higher prevalence of asymptomatic P. falciparum carriers in the Sahel and Forest zones is despite additional malaria control interventions such as indoor residual spraying (IRS), which is not carried out in the Coastal zone.Asymptomatic carriage  and at tcarriage . Investicarriage . In thisThis high prevalence of asymptomatic malaria parasite carriage by the children in both the peak and dry malaria season may mean that additional malaria control interventions that target asymptomatic parasite carriage such as mass test and treat exercises could be implemented to reduce parasite burden .P. falciparum identified in the Coastal zone (Ada) relative to the Forest (Konongo) and Sahel zones  could be due to relatively lower transmission intensity and consequently lower densities of mosquitoes in the Coastal zone relative to the Sahel and Forest zones . The low parasite diversity in the Coastal zone could also imply that the malaria control interventions implemented in the Coastal zone have been more effective and/or successful than in the Forest and Sahel zones as discussed already [The genetic diversity of the parasites identified across the three ecological zones of Ghana was generally moderate to high. In the rainy season, genetic diversity in the Sahel and Forest zones were higher than diversity in the Coastal zone (Ada), consistent with earlier findings that areas with the highest prevalence of asymptomatic parasite carriers would have the greatest genetic diversity and multiplicity of infection . Parasit already . The higThe level of recombination, genetic variation and genetic structure from LD and clustering analyses reflect the patterns of gene flow and transmission intensity among the parasites circulating within the three ecological zones. In areas with high transmission, the frequent recombination among parasite strains, low genetic differentiation and large parasite gene pools result in high levels of genetic variation as seen in most malaria endemic areas in sub-Saharan Africa , 50, 51.The mean MOI in samples from the Sahel zone  was slightly lower than that identified in the Forest zone (Konongo) in the rainy season most likely due to the residents being exposed to a higher incidence of infectious mosquito bites , with eaHigh parasites diversity and MOI may imply high parasite survival and successful transmission in the midst of malaria control interventions . AsymptoP. falciparum parasites identified in this study had high genetic diversity and polyclonalty. Parasites from the Forest zone were more related to parasites from the Sahel zone than parasites from the Coastal zone despite the sites from the Coastal zone being closer to the Forest zone than the Sahel zone.A large asymptomatic parasite reservoir was found in the school children during both rainy and dry seasons, especially those in the Forest and Sahel savannah zones. Further studies are recommended to understand why despite the roll out of malaria interventions in Ghana, high transmission still persist. The asymptomatic Additional file 1: Table S1. Primers used in the study. Table S2. Microsatellite analysis. 2a. Genetic diversity. 2b. Complete Microsatellite data set. Table S3. Results of the Bayesian inference for the 119 samples collected from the three zones.


Bathyacmaea nipponica that colonizes both vents and seeps across ∼2,400 km in the Northwest Pacific is a feasible model to answer these questions. We analyzed 123 individuals from four vents and three seeps using a comprehensive method incorporating population genomics and physical ocean modeling. Genome survey sequencing and genotyping-by-sequencing resulted in 9,838 single-nucleotide polymorphisms for population genomic analyses. Genetic divergence and demographic analyses revealed four habitat-linked  genetic groups, with the vent genetic group established via the opportunistic invasion of a few limpet larvae from a nearby seep genetic group. TreeMix analysis uncovered three historical seep-to-vent migration events. ADMIXTURE and divMigrate analyses elucidated weak contemporary gene flow from a seep genetic group to the vent genetic group. Physical ocean modeling underlined the potential roles of seafloor topography and ocean currents in shaping the genetic connectivity, contemporary migration, and local hybridization of these deep-sea limpets. Our study highlighted the power of integrating genomic and oceanographic approaches in deciphering the demography and diversification of deep-sea organisms. Given the increasing anthropogenic activities  affecting the deep ocean, our results have implications for the conservation of deep-sea biodiversity and establishment of marine protected areas.Hydrothermal vents and hydrocarbon seeps in the deep ocean are rare oases fueled by chemosynthesis. Biological communities inhabiting these ecosystems are often distributed in widely separated habitats, raising intriguing questions on how these organisms achieve connectivity and whether habitat types facilitate intraspecific divergence. The deep-sea patellogastropod limpet  The discoveries of deep-sea hydrothermal vents in the late 1970s and hydrocarbon seeps in the early 1980s have significantly changed our understanding of how life has evolved on Earth. Unlike shallow-water ecosystems that are mainly driven by photosynthesis, deep-sea vent and seep ecosystems distributed in tectonically active areas and along continental margins are primarily supported by chemosynthesis, with many species strictly endemic to either vent fields or seep areas . NeverthGigantidas platifrons and the galatheoid squat lobster Shinkaia crosnieri. Bathymodioline mussels, at least the species for which the dispersal mode has been studied, produce planktotrophic larvae that can disperse a long distance in the upper ocean currents   understand the habitat colonization and evolutionary divergence of its vent and seep populations; 2) test whether these populations exhibit a habitat-linked genetic differentiation; and 3) investigate the migration dynamics and gene flow of B. nipponica under the intricate interactions amongst local habitats, seafloor topography, and ocean currents.The patellogastropod limpet  limpets . To dategami Bay A, with d5, 2021) . Here, wThe Illumina whole-genome sequencing generated 111.2 Gb of raw reads. After removing low-quality reads, a total of 102.9 Gb reads were retained for genome assembly using Platanus, which produced 2,733,936 scaffolds with a total size of 950.7 Mb and an N50 of ∼2.3 kb . BUSCO aFST between all the local population pairs ranged between 0.0054 and 0.6137 , intermediate between the Kuroshima Knoll seep population and the Okinawa Trough vent populations (range: 0.2777–0.3431), and very low between the vent local population pairs in the Okinawa Trough (range: 0.0054–0.0475).Genetic differentiation measured by pairwise d 0.6137 . In partThe phylogenetic network showed four habitat-linked genetic groups, including three seeps and one vent genetic groups C. SpecifK = 2, all individuals from the Jiaolong Ridge and the Off Hatsushima seeps, together with two individuals from the Hatoma Knoll vent and three individuals from the Izena Hole Field vent in the Okinawa Trough, formed one genetic group. Most of the other individuals from the Okinawa Trough vents along with all individuals from the Kuroshima Knoll seep formed the other genetic group. In addition, two individuals from the Izena Hole Field vent were detected to exhibit genetic architecture of both genetic groups. At K = 3, all individuals from the Kuroshima Knoll seep were segregated from those inhabiting the Okinawa Trough vents to form the third genetic group. At K = 4, all individuals from the Jiaolong Ridge seep and the Off Hatsushima seep were further separated to form two distinct genetic groups. Moreover, two individuals from the Hatoma Knoll vent and three individuals from the Izena Hole Field vent in the Okinawa Trough were found to be migrants from the Off Hatsushima seep, and two individuals from the Izena Hole Field vent were detected to be hybrid descendants between the Okinawa Trough vent and the Off Hatsushima seep genetic groups.ADMIXTURE analysis revealed a habitat-linked genetic structure D and E tK = 3  provided further evidence to support a habitat-linked population divergence F. In parK = 3 D. AdditiK = 3 C and the3 K = 4; D.Hobs), nucleotide diversity (π), and inbreeding coefficient (FIS), compared with the other three seep genetic groups  and private SNPs , as well as the highest values of the observed heterozygosity  and to the Izena Hole Field vent population in the middle Okinawa Trough (P < 0.00001), as well as one from the Kuroshima Knoll seep population to the phylogenetical ancestry of the southern Okinawa Trough vent populations (P < 0.005).TreeMix analysis uncovered four migration events m = 4; A. AmongsdivMigrate analysis unveiled intense gene flow between the Jiaolong Ridge seep genetic group and the Off Hatsushima seep genetic group, weak gene flow from the Off Hatsushima seep genetic group to the Okinawa Trough vent genetic group, and very limited gene flow between the Kuroshima Knoll seep genetic group and the other two seep genetic groups in the Jiaolong Ridge and the Off Hatsushima B. ExtensB. nipponica  genetic group and the shallower seep  genetic group occurred ∼24,400 generations ago; opportunistic invasion by a small number of limpet larvae (∼650) from the Kuroshima Knoll seep genetic group to vent fields in the Okinawa Trough occurred ∼11,800 generations ago; establishment of the Okinawa Trough vent genetic group occurred ∼6,410 generations ago, and further genetic separation of the deeper seep genetic group into the Jiaolong Ridge and the Off Hatsushima seep genetic groups occurred ∼1,220 generations ago .Amongst the four genetic groups, the Jiaolong Ridge seep genetic group had the smallest effective population size of ∼10,700, whereas the Okinawa Trough vent genetic group had the largest effective population size of ∼98,200 .Seasonal-mean lateral ocean currents in the study region at selected depths during the five model years (2011–2015) are illustrated in J) and the Kerama Gap  and one vent  genetic groups. These results were contrasted with those of a previous study based only on the nuclear histone H3 and the mitochondrial cytochrome c oxidase subunit I (cox1) genes, in which B. nipponica in the Northwest Pacific was inferred to form one panmictic metapopulation  and the other in the shallower seep . Water depths have been noted as a key factor driving the diversification of animal groups in deep-sea chemosynthesis-based ecosystems, such as some vesicomyid clams in the Northwest Pacific   , genetical film) . Such dial film) , which mng there . In addiit e.g., .FST estimation, phylogenetic network, ADMIXTURE results (K = 2), and PCA plots consistently implied that the Kuroshima Knoll seep genetic group had high genetic similarities to the vent genetic group established in the Okinawa Trough , and PCA concordantly revealed that all the vent local populations in the Okinawa Trough belonged to one metapopulation , a high degree of inbreeding , and extensive contemporary gene flow with a direction from the shallower to the deeper vents for individuals inhabiting the Okinawa Trough. Such a larval dispersal potential of B. nipponica would also help to explain its different population segregation patterns in comparison with those of the bathymodioline mussel G. platifrons. This mussel has been considered to produce planktotrophic larvae that may mainly disperse in the upper currents, with the local populations in vent fields in the Okinawa Trough and the Off Hatsushima seep in the Sagami Bay sharing high genetic similarities , and PCA plots; that is, five Okinawa Trough vent individuals inferred to be immigrants from the Off Hatsushima seep, and two Okinawa Trough vent individuals inferred to be hybrid descendants between individuals from the Okinawa Trough vent and the Off Hatsushima seep genetic groups  . These dipponica : Althougipponica . Detectaas e.g., . Due to as e.g., .B. nipponica in the Northwest Pacific, including three seep genetic groups inhabiting hydrocarbon seeps in the Jiaolong Ridge, the Kuroshima Knoll, and the Off Hatsushima, as well as one vent genetic group dwelling in four hydrothermal vents in the Okinawa Trough. The assumed lecithotrophic larvae of B. nipponica were considered to mainly disperse along the intermediate to the deeper ocean currents. The most likely demographic history of B. nipponica was deciphered to have occurred in the following sequences: 1) an initial split into two seep genetic groups, one deeper  and one shallower ; 2) a small number of limpet larvae from the shallower seep genetic group invaded vent fields in the Okinawa Trough, adapted to the local environments, and thereafter established a distinct vent genetic group; and 3) two deeper seep populations diverged very recently into two distinct genetic groups with detectable genetic differentiation. Assuming a generation time of one to two years for B. nipponica, formation of its vent genetic group may have been linked to the historical shifting of the Kuroshio Current. Additionally, diversification of the two deeper seep genetic groups might be associated with the recent reduction of seepage intensity in the Jiaolong Ridge seep along with the barrier effect of the Luzon Strait. Physical ocean modeling results elucidated the seafloor topography of the Okinawa Trough may have contributed to the genetic homogeneity of B. nipponica among different vent local populations in this region, and the North Pacific Intermediate Water might have facilitated the opportunistic migration of a few limpet larvae from the Off Hatsushima seep in the Sagami Bay into the Okinawa Trough, and thus led to their hybridization in the Okinawa Trough vents. Altogether, this study has enhanced our knowledge on the historical population divergence and contemporary gene flow of deep-sea organisms inhabiting hydrothermal vents and hydrocarbon seeps under the intricate interactions amongst local habitats, seafloor topography, and ocean currents. More importantly, these results could serve as the scientific basis for a better conservation of chemosynthesis-based ecosystems and an effective establishment of marine protected areas.Using an integrated approach combining population genomics and physical ocean modeling, four distinct genetic groups with a habitat-linked pattern were disclosed for the deep-sea patellogastropod limpet B. nipponica were collected from four hydrothermal vents and three hydrocarbon seeps in the Northwest Pacific between 2002 and 2016  in Novogene . A DNA sequencing library with an insert size of 350 bp was constructed using the NEBNext DNA Library Prep Kit and paired-end sequenced on an Illumina NovaSeq 6000 sequencer with a read length of 150 bp. Raw reads were filtered using Trimmomatic v.0.38  < 0.5 calculated using popStats . In brief, genomic DNA from each individual was digested using the restriction enzymes v.0.20.0  under thv.0.7.17 . The resv.0.7.17 . Duplicav.0.7.17 . Callingv.0.7.17 . Filtratv.0.7.17  to retaipopStats  to avoidpopStats . Based opopStats  and StacpopStats .FST was estimated using Arlequin v.3.5.2.2  was assessed using the cross-validation (CV) procedure implemented in ADMIXTURE with the --cv flag. The barplot function in R was used for result visualization with the selected K. The PCA implemented in the R package SNPRelate (Hexp), observed heterozygosity (Hobs), nucleotide diversity (π), and inbreeding coefficient (FIS), were calculated using the POPULATIONS module implemented in Stacks v.2.5 , three demographic scenarios were hypothesized to infer how the four identified genetic groups of B. nipponica had formed and diversified in the Northwest Pacific (T1 < T2 < T3 < T4 < TA) with details summarized in Demographic history was reconstructed using the approximate Bayesian computation (ABC) analysis implemented in DIYABC v.2.1.0 . Accordi Pacific A. Each sB. nipponica initially diversified into two seep genetic groups at TA. The first seep genetic group inhabited the shallower seep area in the Kuroshima Knoll and formed the Kuroshima Knoll seep genetic group. A few limpet larvae from the Kuroshima Knoll seep genetic group  invaded and settled in the Okinawa Trough vents by chance at T3, which later diverged to form the Okinawa Trough vent genetic group at T2. The second seep genetic group inhabited the deeper seep areas, which diverged into two distinct seep genetic groups in the Jiaolong Ridge and the Off Hatsushima seeps at T1. In scenario 2, B. nipponica inhabited the deeper seep areas was hypothesized to be ancestral. A few limpet larvae from this ancestral genetic group  incidentally invaded and settled in the shallower seep area in the Kuroshima Knoll at TA, which gradually formed the Kuroshima Knoll seep genetic group at T4. Afterward, a few limpet larvae from the Kuroshima Knoll seep genetic group  occasionally dispersed and settled in the Okinawa Trough vents at T3, which thereafter formed the Okinawa Trough vent genetic group at T2. More recently, the ancestral deeper seep genetic group further diversified into two distinct seep genetic groups in the Jiaolong Ridge and the Off Hatsushima seeps at T1. In scenario 3, B. nipponica inhabited the shallower seep area in the Kuroshima Knoll was speculated to be ancestral. A few limpet larvae from the Kuroshima Knoll seep genetic group  fortuitously invaded the deeper seep areas at TA, which then established the deeper seep genetic group at T4. Furthermore, a few other limpet larvae from the Kuroshima Knoll seep genetic group  incidentally entered and settled in the Okinawa Trough vents at T3, which afterward formed the Okinawa Trough vent genetic group at T2. Lately, the deeper seep genetic group further differentiated to establish two distinct seep genetic groups in the Jiaolong Ridge and the Off Hatsushima seeps at T1.In scenario 1, FST, proportion of zero values and mean of complete distribution Nei’s distances, were calculated between pairs of genetic groups. Posterior probabilities and 95% confidence intervals for each scenario were estimated based on the 500 simulated data closest to the observed data using the emphdirect method and 1% of the simulated data closest to the observed data using the logistic regression method. Posterior distribution of each demographic parameter of the best scenario was calculated according to 1% simulated data closest to the observed data with the logistic regression method. Fitness of the parameter priors to the best scenario was illustrated using PCA with the function of Perform Model Checking.To obtain robust results, a total of 3,000,000 simulations were performed as recommended by DIYABC for all three demographic scenarios. Summary statistics, including proportion of zero values, mean and variance of nonzero values, and mean of complete distribution, were calculated for each genetic group. In addition, summary statistics, including mean and variance of nonzero values of B. nipponica between its vent local populations in the Okinawa Trough, and weak contemporary gene flow from its Off Hatsushima seep to the Okinawa Trough vent genetic groups (details refer to the Results section). To establish a deeper understanding of these gene flow patterns from an oceanographic aspect, physical ocean modeling data were further analysed to uncover the 1) seasonal-mean lateral ocean current patterns and 2) topographic constraints and fluid dynamics in the study region via two sets of numerical particle release experiments.Population genomic analyses conducted in the present study uncovered extensive contemporary gene flow of https://www.hycom.org, last accessed April 10, 2021) in the study region. This set of ocean modeling data has integrated multiple sources of ocean observational records, including satellite measurements of the sea surface height/temperature and in-situ hydrographic information documented by the XBTs/Argo floats/moored buoys. Seasonal-mean lateral ocean current patterns at 0, 500, 800, 1,000, and 1,250 m depths were elucidated by averaging the daily velocity fields within each season  across an ensemble of five model years (2011–2015). This calculation effectively filtered out high-frequency processes, such as transient mesoscale eddies, which allowed investigation of the present-day large-scale ocean circulations.Velocity fields of lateral ocean currents were extracted from the HYCOM + NCODA Global 1/12° Reanalyses (experiment sequence: 53.X) modeling output (B. nipponica within the Okinawa Trough (details refer to the Results section), the first set of numerical particle release experiments were performed by uniformly releasing numerical particles within a 25-km radius from the Futagoyama Field vent coordinates , which then flew passively with ocean currents for around three years since 2011. Based on the seasonal-mean lateral ocean current patterns in the study region and the migration dynamics of B. nipponica (details refer to the Results and Discussion sections), B. nipponica larvae were considered to mainly disperse in the intermediate to the deeper water similar to those of S. crosnieri . To examine this hypothesis, the second set of numerical particle release experiments were performed by uniformly releasing numerical particles within a 25-km radius from the Off Hatsushima seep coordinates at 500 and 800 m depths. These two depths were selected based on the seasonal-mean lateral ocean current patterns in the study region (details refer to the Results section), topographic constraints of the Okinawa Trough inferred from the results of the first set of numerical particle release experiments (details refer to the Results section), and depth range of the North Pacific Intermediate Water , aiming to reflect the topographic constraints and fluid dynamics of the study region. No data are available on the swimming capability of B. nipponica larvae. Therefore, the present study is not applicable for determining the exact travel time of B. nipponica larvae along ocean currents, such as from the Off Hatsushima seep in the Sagami Bay to vent fields in the Okinawa Trough.The two sets of numerical particle release experiments were analyzed based on the horizontal and time-varying ocean current velocity. To attain statistically robust results, a broader domain (15°N–40°N × 115°E–180°E) than the study region was selected and over 10,000 numerical particles were released for each set of experiments. This approach accommodated long-range trajectories of particles following the Pacific Ocean currents . Trajectories of these particles were calculated using a step-adapting fourth/fifth-order Runge–Kutta method with interpolations done using a cubit method . SeasonaMolecular Biology and Evolution online.msab278_Supplementary_DataClick here for additional data file.


Ruditapes philippinarum. Cells were exposed to three concentrations of C6O4  and the effects on haemocyte viability, haemocyte morphology, differential haemocyte count, lysosomal membrane stability, superoxide anion production, acid phosphatase, and β-glucuronidase activities, as well as on the percentage of micronuclei and chromosomal aberrations were evaluated. The results demonstrated that C6O4 significantly affected haemocyte morphology, lysosomal membrane stability, hydrolytic enzyme activity, and superoxide anion production, and promoted chromosomal aberrations. To the best of our knowledge, this is the first study revealing the in vitro effects of C6O4, a substitute for PFOA, on haemocytes from a bivalve species.Alternative chemicals to per- and poly-fluoroalkyl substances have recently been introduced in various industrial processes. C6O4 -1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a replacement for perfluorooctanoic acid (PFOA). From an ecotoxicological point of view, in vitro assays are useful tools for assessing the negative effects and understanding the mechanisms of action of chemicals at the cellular level. Here, we present the results of an in vitro study in which the effects of C6O4 were evaluated—for the first time—on haemocytes of the clam  Per- and polyfluoroalkyl substances (PFAS) are classified as persistent organic pollutants (POPs) due to their high resistance to degradation . HoweverOne of the mainly used PFAS is perfluorooctanoic acid (PFOA), which has been recently included, along with its salts, into Annex A of the Stockholm Convention . PFOA haPerfluoroalkyl ether carboxylic acids (PFECAs) are a relatively new class of short-chain PFAS used to replace PFOA . Some ofIt is classified as perfluoro ether non-polymer with saturated bonds, and dozens of tons are produced every year ,15,16. IRuditapes philippinarum haemocytes. It is well-known that haemocytes are involved in immune responses in bivalves pyrene  [Planorbarius corneus, exposed for 14 days to pentachlorophenol (10 to 450 µg/L) [P. viridis to various concentrations  of PFOS, PFOA, PFNA, and PFDA caused a significant increase in MN frequency of haemocytes, at least at the two highest concentrations tested [Oryzias latipes, time- and concentration-dependent increases in both MN frequency and cell abnormalities  were observed in erythrocytes after the exposure of fish for 28 days to 0.1, 0.5, and 2.5 mg/L of PFDA [Micronuclei induction is a biomarker commonly used to detect the genotoxicity of pollutants to aquatic organisms ,47,48. I000 ppb) , while c50 µg/L) . As for  of PFDA . As for  of PFDA . In the  of PFDA  demonstrR. philippinarum. The ability of the compound to increase intracellular ROS production, which, in turn, could cause damage to macromolecules, such as lipids, proteins, and DNA, is noteworthy. In this regard, increases in the frequency of cell/chromosomal abnormalities are suspected to be a consequence of the cytotoxic potential of C6O4. In any case, further studies are necessary to investigate the mechanisms of action of C6O4 in R. philippinarum in depth, as well as those in other bivalve species.The results of this study demonstrate that C6O4 can affect the morpho-functional features of haemocytes from


GLC) is a complex structure with astonishing properties: isotropic 2D/3D amorphous films. The many models all have compromises: neither all bad nor entirely satisfactory. Properties are understood in a single framework given by topological and geometrical properties. To do this, we present the basic tools of topology and geometry at a ground level for 2D surface, graphene being the best candidate to do this. With this in mind, special attention is paid to the hyperbolic geometry giving birth to triply periodic minimal surfaces. Such surfaces are the basic tools to understand the GLC network architecture. Using two theorems (the classification and the uniformisation), most of the GLC properties can be tackled at least at a heuristic level. All the properties presented can be extended to 2D materials. It is hoped that some researchers may find it useful for their experiments.Glass-like carbon ( GLC) )A compact surface is classified in terms of its boundary number β, its orientability number ω and its Euler characteristic χ. These numbers are topological invariants and are preserved under homeomorphism. Then, we can speculate that, under laser annealing, these numbers remain constant during a “continuous deformation” . This is of prime importance for a comprehensive study of the non-graphitisable versus graphitisable process.For the following, we need other definitions:A and B, the connected sum of A and B is formed by cutting a disk from A and a disk from B and gluing the surfaces together along the boundary.For two surfaces One obtains a surface with a boundary by taking any surface without a boundary and punching some holes in it by removing open discs. Using connecting sum, two surfaces can be connected by “gluing” the edges of the open disks together.2D crystal is a periodic arrangement of units, e.g., squares). The torus is homeomorphic to the unit square A planar model of a surface is a polygon whose vertices and edges are identified, or glued together,“ in some specified way” (cut and paste methods are used in the proof of the classification of surfaces). This abstract concept is the basis of the common crystallography where polygons are duplicated with some rules. We can create a cylinder by using a piece of paper and gluing the ends together. Stretching the cylinder and gluing them, the two opposite sides gives a flat torus (a donut from a cooking point of view). Native people of the 2-torus look forward and see them from behind. They have the illusion of seeing a copy of them. The flat torus mimics the periodic BVK (Born–Von Karman) conditions in solid state physics commonly observed in standard crystallography  (Other conditions than ompounds ), electrompounds ,65. Thernd torus . Figure orphism) .If we consider the periodic arrangement of atoms, the single way is the torus, but, if we consider the properties of the electronic wave functions , other ways can be considered. M point in the Brillouin zone. First, check that the inhabitant is the same. To do this, he can move in momentum space with the periodic conditions. Starting from one selected M point, the inhabitant visits all four corners labelled M before coming back home. The inhabitant position in the torus is given arbitrarily in K point visits three sites before coming back home. Likewise, an inhabitant living at K’ visits the three other points in the hexagon. From a topological point of view, one needs two inhabitants for visiting the six corners. These two inhabitants are entangled but different (twin state). This is the keypoint for the Dirac points at K and K’ . T. Tsp3 anr carbon , boron [r carbon  or phospr carbon . Such a e family  and prestructure ; thus, Tthan 0.2 . For exathan 0.2 , but elathan 0.2 . There it (0.17)  but is at (0.17) . Manohart (0.17) . Once agt (0.17)  predicteGLC ranging 1–Diffusion (or permeability) in carbon compounds depends to the geometry of pores. Kowalczyk et al.  reported0−9 m2/s . Such vaGLC. GLC are totally impermeable to gas with a large open structure (see the low density and the TEM observation). The first naive answer is that GLC are impermeable to gas because of closed pores. This is in agreement with the apparent porosity of 0.2–0.4% in GLC as compared to 8–15% in impregnated carbon [K is related to both parameters in the Tomadakis–Sotirchos model [GLC, it would be the diameter of the spaghetti) and the porosity 3D random networks GLC, the low permeability suggests a porosity close to Porosity is probably the most amazing issue in d carbon . The peros model : the fibrosity ϵ .(20)K=ϵk) and pore fluid diffusivity (D) in the global permeability [tortuosity as the parameter which describes the sinuosity and the interconnectedness of the pore space as it affected transport processes. From a topological point of view, tortuosity can be defined by the simple formula :i is the number of elemental steps. A 2D example is given in 2D fractal space filling curves (Hausdorff dimension 2) [n is the iteration step in the fractal sequence: nThen, tortuosity tends to infinity (if eability . Unfortu Clennel  defined nsion 2)   fullerenes with -TPMS) with TPMS are metastable structures. The excess in “strain” energy is evidenced after relaxation of a P-TPMS unit cell with boundaries [GLC cannot accommodate large holes that characterise giant Schwarzites (with a high number of hexagons) because of a complete relaxation. This is not the case for the smallest Schwarzites which are not smooth surfaces. Willmore energy states for smooth surfaces. One can define the discrete Willmore energy after tiling [The nanotubes with undaries  ≥2π2 . In carbP6m. The symmetry operators in graphene are, respectively, a reflection line , we can introduce the cost of a orbifold . To do that, we use the defect formula  Equatio):29)χo=orbifold ,147. Theχo=orbifo2D plane groups have a zero cost orbifold regardless of the type of tiling. Likewise, let us consider the fullerene Note that, for the Coxeter class, 2D spherical orbifolds, *235 has the low cost energy (m3m) has a larger cost ν=1+ectively . BM and −1.52 eV . The surTPMS [This model can be applied to nanotubes , fullereTPMS .(35)ESWeVR the radius in Å, energies in TPMS.GLC is determined by the local values of both parameters, which are highly sensitive to the t parameter in Equation  ,151, thes (POAV) . First, al shape ,(40)sinTPMS in the standard form is far away the GLC structure. The first step is to modify the density. The problem is achieved by studying gyroids of Constant Mean Curvature (CMC). To do this, one considers the deformation of a minimal surface by embedding it into a continuous family of constant mean curvature surfaces [TPMS keeping the same symmetry. Deformations of minimal surfaces are obtained by minimising the Willmore or bending energy under a volume constraint. The density is a key parameter. t factor  is of prime importance to do calculations  Moreover, these structures (including randomisation) do not have any edges. This may explain the chemical robustness: the physi-chemisorption is on the dangling bonds of the edge atoms. Nevertheless, all the modified TPMS check the two congruent labyrinths and then are “unknot structures”. As mentioned previously, “unknot structure” is the best way for graphitisation (let us keep at mind that GLC are non-graphitisable structures). To summarise, random CMC are a good ground to model GLC. The low difference in cohesive energy between structures with positive curvature  and negative curvature such as TPMS favours a mixing between them in GLC carbon. Random CMC trie to capture the essence of all the GLC properties but requires improvementThe simulation of the GLC can be understood at first glance. The presence of n-gons with GLC and the entire 2D compounds) are good candidates to do this. The possibility to synthesise GLC with well controlled conditions should allow in the future a better possible confrontation of problems thanks to topology. It remains a fascinating subject to study with many secrets yet to be discovered.Thanks to the uniformisation theorem (geometry) and the classification one (topology), some amazing properties of


Monodonta sp. within a narrow range of peripheral oceanic islands, the Ogasawara Islands. Genetic analyses were performed not only with a mitochondrial DNA marker but also with a high-throughput SNPs dataset obtained by ddRAD-seq. The results of the mtDNA analyses did not show genetic divergence among populations, while the SNPs dataset detected population genetic differentiation. Population demographic analyses and gene flow estimation suggested that the genetic structure was formed by sea level fluctuation associated with the past climatic change and regulated by temporal oceanographic conditions. These findings provide important insights into population genetic patterns in open and connectable environments.Molecular studies based on the high resolution genetic markers help us to grasp the factor shaping the genetic structure of marine organisms. Ecological factors linking to life history traits have often explained the process of genetic structuring in open and connectable oceanic environments. Besides, population genetic divergence can be affected by fragmented habitat, oceanic current, and past geographical events. In the present study, we demonstrated the genetic differentiation of marine gastropod  In the open and connectable oceanic environment, several marine organisms tend to exhibit a high level of gene flow among populations4. For marine benthic organisms with low mobility in the adult phase, life history traits such as development types and the length of the planktonic larval phase are related to the level of dispersal ability and connectivity among populations. It often explains the process of genetic structuring in marine situations8. However, the length of the larval phase does not necessarily completely explain dispersal distance and genetic structure9. Accumulated molecular studies have demonstrated that other ecological characteristics such as habitat range and usage patterns are effective factors that cause population genetic differentiation12. Besides, the process of genetic population structuring is influenced not only by the above ecological factors but also by past geographical events, fragmented habitats and oceanic currents15. Climate fluctuation influences the population demographic history of various taxa, and genetic drift is a major factor in genetic differentiation17. After these events, it is predicted that temporal oceanographic conditions regulate dispersal via seawater and influence population genetic structures.Understanding the level of genetic differentiation and what factors caused it is a central issue in marine molecular studies21.Delineating the genetic dynamics of marine species depends on the resolution of genetic markers. To detect the level of gene flow with a high degree of precision, molecular analyses using high-throughput data have been performed. For wild and non-model species, recent progress in genotyping methods through genome-wide variation of single-nucleotide polymorphisms (SNPs) have provided powerful tools, including restriction site-associated DNA sequencing (RAD-seq), double-digest RAD sequencing (ddRAD-seq), multiplexed ISSR genotyping by sequencing (MIG-seq), and genotyping by random amplicon sequencing (GRAS-Di). Our understanding of the process of genetic differentiation of various marine taxa is growing rapidly due to these remarkable genomic technologies22. Since the voyage of the HMS Beagle, many of Darwin’s followers have regarded oceanic islands as a kind of ‘evolutionary laboratory’ due to their outstanding species diversification, mainly in terrestrial taxa26. In addition, oceanic islands harbour distinct marine fauna composed of species that could reach and settle into these distant environments29. Some species isolated from their continental relatives have evolved to become endemic to the islands. Oceanic islands provide a simple model for studying the process of genetic differentiation following divergence from ancestral species.Oceanic islands, which are remote and isolated from continental landmasses, have been considered a suitable model for evolutionary studies32. Naturally, several marine and freshwater species have also successfully colonised the islands, some of which were investigated using molecular methods and identified as endemic, including fish, crabs, and molluscs38. These endemic species provide a suitable study system for investigating the factors shaping genetic structure in the Ogasawara Islands after divergence from their continental relatives. However, even though on the famous oceanic islands such as Hawaii and the Galapagos Islands, population genetic studies for endemic species were performed within the archipelago40, little is known about the Ogasawara Islands.The Ogasawara Islands, located in the northwestern region of the Pacific Ocean and approximately 1000 km from the Japanese mainland, are typical oceanic islands as they have never been connected to the Eurasian continent. The Ogasawara Islands comprise 30 islands that are categorized into four regions: the Mukojima Islands, Chichijima Islands, Hahajima Islands, and Volcano Islands. The terrestrial ecosystem of the Ogasawara Islands is unique and commonly referred to as an ‘Oriental Galapagos’, since it is characterized by a large number of endemic taxaMonodonta sp. ; it does not extend into the Volcano Islands, which constitute the southernmost region of the Ogasawara Islands41. The life cycle of Monodonta sp. on the Ogasawara Islands is not well understood, but knowledge of East Asian Monodonta species including M. confusa, which is a continental relative of Monodonta sp., is available45. According to these studies, Monodonta species undergo a planktonic larval duration of 3 days (M. confusa and M. perplexa). After larval settlement and metamorphosis, M. confusa matures to a shell height of approximately 10 mm, which takes 1 to 2 years. Around the Japanese archipelago, a previous molecular study on M. confusa detected a significant genetic population structure, but the level of differentiation was smaller than that of closely related species46. For Monodonta sp. distributed in the Ogasawara Islands, previous phylogenetic analyses based on standard molecular markers showed no differentiation within the Ogasawara Islands35. However, given the relatively short planktonic larval duration of Monodonta, it is possible that Monodonta sp. is genetically differentiated across the archipelagos. To clarify the genetic structure of marine species within the Ogasawara Islands, it is necessary to carry out a high resolutive molecular approach.In this study, we focused on an endemic marine snail species with a planktonic dispersal ability distributed in the intertidal zone of the Ogasawara Islands: sp. Fig. . This spMonodonta sp. as our model. In addition, we tested the hypothesis that genetic differentiation occurred due to historical climatic events and regulated temporal oceanographic conditions. Genetic analyses were performed using a mitochondrial DNA (mtDNA) marker, and a high-throughput SNPs dataset obtained by ddRAD-seq. Based on the dataset obtained by these two types of genetic markers, we discuss the genetic structure, temporal population connectivity, and timing of genetic divergence in open and connectable marine environments of peripheral oceanic islands.Here, we aimed to determine the level of genetic differentiation and patterns of genetic structuring within a narrow range of peripheral oceanic islands using marine organisms . Monodonta sp. had nine haplotypes from 59 individuals of four sampling islands. The haplotype diversity and nucleotide diversity of each population are shown in Table Monodonta sp.  and Yomejima Island ] and the southernmost Hahajima Island . On Chichijima Island , which is located centrally in the Ogasawara Islands, the two genetic clusters were mainly mixed. A scatterplot of the PCA also showed that individuals fell into two genetic clusters  and a total alignment length of 158,328 bp (9.20% of missing sites) were obtained  and the southern (Chichijima Island and Hahajima Island) islands are genetically differentiated at 8971 generations ago (t2) Fig. . The recMonodonta sp. endemic in the Ogasawara Islands. In the case of our study, the mitochondrial DNA (mtDNA) did not provide sufficient information on genetic polymorphisms compared to the above high resolution genetic marker. For molecular ecological studies, mtDNA has been commonly used to demonstrate the population genetic structure and evolutionary history of various taxa. However, mitochondrial-specific traits such as frequent introgression may bias the investigation of population genetic structuring and dynamics47. Incomplete lineage sorting also makes the interpretation of the genetic results difficult. The discordance often occurs between mtDNA and nuclear DNA (nDNA) due to their different inheritance patterns49 and mtDNA seems to lose the ancestral polymorphisms faster than nDNA51. In contrast, genetic traces left in mtDNA are important for understanding the complex evolutionary history between organelle and nuclear genomes52. By using both organellar DNA and genome-wide datasets, we can understand the population genetic dynamics in detail. From the above context, the present mtDNA results provided useful information on the differences in genetic diversity between Monodonta sp. on the Ogasawara Islands and its closely related continental species, M. confusa. The mtDNA of M. confusa displays a higher level of genetic diversity around the Japanese archipelago and the continental side than Monodonta sp.53. This reduced genetic diversity of Monodonta sp. observed in mtDNA is attributed to the effects of bottlenecks, and similar patterns have been detected in other organisms of the Ogasawara Islands54. These results indicate that endemic marine species underwent a strong bottleneck when colonising the Ogasawara Islands, which is considered a fundamental pattern in peripheral oceanic islands40.The present study tested the level of population genetic differentiation of an intertidal gastropod species distributed within a narrow range of peripheral oceanic islands using two types of genetic markers. A genome-wide single nucleotide polymorphisms (SNPs) dataset obtained by double-digest RAD sequencing (ddRAD-seq) was able to detect genetic differentiation among populations of Monodonta sp. accumulated genetic differentiation within a relatively narrow range from Kitanoshima Island to Hahajima Island (< 120 km). The genetic components of Monodonta sp. are divided between the Mukojima Islands (Kitanoshima Island and Yomejima Island) and the southern islands (Chichijima Island and Hahajima Island). The genus Monodonta is known to undergo a planktonic larval phase during its life cycle. In the case of M. confusa, which is the closest continental relative to Monodonta sp. endemic to the Ogasawara Islands, the planktonic duration is 3 days45. Besides, trochid snail species related to Monodonta have relatively short PLD 55. Dispersal ability during the planktonic larval phase is thought to be one of the most important factors for genetic differentiation among populations in the marine environment. However, a theoretical study indicated that population genetic connectivity is not necessarily determined by planktonic larval dispersal duration56. Isolated island populations have experienced significant founder effects and bottlenecks when colonising and could not receive migrants from the source continental populations. The genetic drift is thus important for the genetic differentiation of island endemic species58.Populations of 59. Although the Kuroshio Current and its countercurrent are effective factors in the dispersal of marine organisms distributed in the North Pacific ocean60, the detailed system of oceanic currents around the Ogasawara Islands has not been sufficiently studied62. In seed dispersal plants Pandanus boninensis (Pandanaceae) distributed in the Ogasawara Islands, molecular studies with gene flow estimations demonstrated that migration mainly occurs from the south islands (Hahajima Island) to the north islands (Mukojima Island) via Chichijima Island and also from the Mukojima to the Chichijima Islands62. The result of gene flow estimation of Monodonta sp. also showed that the opportunities for migration to Chichijima Island from Yomejima and Hahajima Islands tended to occur relatively frequently, while reverse migration occurred less frequently. Although migration via oceanic currents vary among taxa around the Ogasawara Islands, there may be currents heading toward Chichijima Island from the south and north islands. In addition, frequent migration to Chichijima Island may also be due to the habitat preference of Monodonta sp. While Monodonta sp. is common in the rocky intertidal zone of the Ogasawara Islands, their inhabitation is less common in environments that are exposed to strong waves63. Chichijima Island may hold many suitable habitats for Monodonta sp. since it is the largest of the Ogasawara Islands and has a long coastline and large bay. Furthermore, the Chichijima Islands are composed of three large islands  and Kita-iwoto Island (approximately 150 km from Hahajima Island)65. This suggests that planktonic larvae of Monodonta sp. are unable to settle in an environment attacked by strong waves or it may be difficult to disperse over distances of 100 km.The geographical distance at which genetic differentiation occurs depends on the oceanographic conditions and specific characteristics of speciesMonodonta sp. on the Ogasawara Islands is not well understood, but the knowledge of M. confusa can be applied to estimate the results of population demographic estimation44. According to these studies, M. confusa matures in 1 to 2 years. If we apply the above generation time to interpreting the population demographic estimation of Monodonta sp. on the Ogasawara Islands 66. At 8971–17,942 years ago (t2), northern  and southern populations (Chichijima and Hahajima Islands) started to become genetically divergent. In the late Pleistocene period, the shoreline was longer and the range of the intertidal zone (suitable habitat for Monodonta sp.) was wider, because the last glacial maximum  caused a drop of approximately 100 m in sea level67. During the LGM period on the Ogasawara Islands, the islands constituting the Mukojima Islands  were connected. Similarly, the islands constituting the Chichijima Islands , and the islands constituting the Hahajima Islands  were connected, respectively68. Following LGM, because the expansion of the intertidal zone was completed and the sea level began to rise, genetic differentiation of Monodonta sp. may have been initiated. Although the present pattern of genetic structure, which includes two different genetic components in the two most external islands and a mixture of the two in the central islands, seem to fit the stepping stone model, it is not confirmed by gene flow estimation. Therefore, this pattern was established by genetic differentiation between the north and south islands due to the above discussed past climate event and is thought to be regulated by the oceanic current moving to Chichijima Island.The life cycle of nds Fig. , the bot71. Larval dispersal is considered to have several advantages, including colonising new areas and decreasing the likelihood of inbreeding and extinction due to genetic exchanges between populations73. Dispersal also fundamentally has various unavoidable risks. Even if Monodonta sp. maintains similar dispersal ability of its continental relatives, the dispersed larvae may not be able to settle in suitable habitats on isolated oceanic islands. If few suitable habitats are available, dispersed larvae may fail to settle successfully, thereby increasing mortality74. For the intertidal snail species genus Littorina, direct developing species are present on isolated and remote islands, while there are no closely related species with a pelagic larval stage 75. The possibility that isolated situations may promote genetic differentiation due to the disadvantages of larval dispersal is an interesting issue to be solved by future ecological studies on larval behaviours.In addition, population genetic differentiation of endemic species may be promoted by the disadvantages of dispersals in remote environments like oceanic islands isolated from the continental landmass27. Around the Ogasawara Islands, several marine species were confirmed to be endemic38. However, the level of genetic connectivity and differentiation within the Ogasawara Islands is poorly understood in other marine species. In the case of coral species with far-reaching dispersal ability, a low level of genetic differentiation among the three populations on the Ogasawara Islands was detected by microsatellite markers76. In contrast, Monodonta sp., with a relatively short larval phase, exhibited a genetic structure within this narrow range of archipelagos. Similarly, other endemic marine species with short dispersal ability may also show genetic differentiation within a similar range. To understand the connectivity among populations of the Ogasawara Islands, comparative genetic studies using multiple taxa are needed. The present study indicated that it is necessary for marine conservation on the Ogasawara Islands to focus on both the north and south island populations which differ genetically. Besides, the genetic structure of marine species may be more differentiated than previously thought within the Ogasawara islands. High resolution genetic analyses of various marine taxa will provide invaluable insights into marine conservation strategies in highly endemic water regions such as oceanic islands78. By clarifying the genetic structure and population demographic history of various species, we can set up a marine reserve system at an appropriate geographic scale80.The marine species inhabiting the Ogasawara Islands are mainly characterised by taxa that once successfully colonised these islands after long-distance travel. Endemic phenomena in oceanic islands are affected by the distance of the island and time elapsed since colonisationIn conclusion, we detected the population genetic divergence of intertidal gastropod with a planktonic larval phase, even within a narrow range of the Ogasawara Islands. This was caused by historical climatic events and maintained by temporal oceanographic conditions. To accurately grasp the degree of genetic differentiation and population history, high-throughput genetic data is useful. In the genomic era in which we live, unexpected genetic differentiation is bound to be detected. These findings provide important insights for understanding the population genetic patterns of open and connectable environmental situations observed in remote oceanic islands.Monodonta sp. were collected from the four localities of the Ogasawara Islands  were performed under the following conditions: 94 °C for 3 min followed by 5 cycles at 94 °C for 30 s, 45 °C for 30 s, 72 °C for 1 min, followed by 35 cycles at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. The products were then purified using ExoSAP-IT . Cycle sequencing was performed using the PCR primers with the BigDye Terminator Cycle Sequencing Ready Reaction Kit  and the products were directly sequenced from both directions using an ABI 3130xl automated sequencer (Applied Biosystems). The validity of the sequences was verified with the software package 4Peaks82, and the forward and reverse sequences were assembled using CLUSTALW83. The sequences were aligned using MUSCLE v. 3.884. Sequence data are available on Genbank .Total DNA was obtained from tissue samples using the NucleoSpin Tissue  according to the manufacturer’s instructions. Fragments of the COI gene were amplified using the primers CoxAF (5′-CWAATCAYAAAGATATTGGAAC-3′) and CoxAR (5′-ATATAWACTTCWGGGTGACC-3′)85. The population genetic structures were estimated using analysis of molecular variance (AMOVA)86. Pairwise FST between populations was calculated by Arlequin v. 3.5 with 1000 permutations. To visualize the geographical distribution pattern of haplotypes, haplotype networks were reconstructed using a median-joining network87 implemented in PopART88.Two genetic diversity indices  were calculated for each population using Arlequin v. 3.5Monodonta sp. were used  was used to collect approximately 450 base pair (bp) DNA fragments. The DNA fragments were amplified in eight single PCR reactions, and the products were cleaned using the NucleoSpin gDNA Clean-up kit. The constructed DNA library was sent to Oregon State University’s Center for Genome Research and Biocomputing and sequenced using Illumina HiSeq 4000 single-end sequencing, yielding maximum read lengths of 100 bp. Raw sequence data were deposited in the DDBJ Sequence Read Archive (DRA013313). The raw Illumina sequence reads were demultiplexed and processed with the ipyrad pipeline90. If the obtained reads were low in quality or contained adapter sequences, it was trimmed and set to a minimum length of 35 bp using ipyrad’s parameter setting. Reads were clustered at 85% sequence similarity. The minimum number of samples was set to 26/29 (0.896%). Other parameters followed the default setting of the ipyrad pipeline and output datasets for subsequent genetic analysis.To prepare a library of double digest restriction site-associated DNA sequencing (ddRAD-seq), 29 genetic samples of ed Table . RNase whttps://ipyrad.readthedocs.io/en/latest/index.html). The number of preassigned genetic clusters (K) was assumed to range from 1 to 10, and seven independent runs were performed for each K value. Each run included 50,000 burn-in iterations and 100,000 iterations. We also performed a principal component analysis (PCA) with GenoDive v. 3.0391 and PCAs were plotted by PAST 4.0492. Temporal gene flow rates and inbreeding coefficient among four populations were estimated by BA3‐SNPs93. The delta values  selected were 0.3250, 0.5500, and 0.0750, respectively. The number of runs were 30,000,000 MCMC iterations including 6,000,000 burn-ins (20%) and 1000 sampling intervals. Tracer v. 1.6.0 was used to verify the Bayesian convergence and parameter values throughout generations94. The AMOVA and pairwise FST between populations were calculated with Arlequin v. 3.5.To grasp genetic population structure, we estimated individual genotypes using STRUCTURE v. 2.3.4 according to the ipyrad analysis toolkit  and the timing of genetic differentiation (t2) between the northern (Kitanoshima Island and Yomejima Island) and the southern (Chichijima Island and Hahajima Island) islands. The pilot run with a broad range of parameters was conducted and the parameter settings were determined according to the results of pilot run (Table https://github.com/isaacovercast/easySFS). We carried out 100 independent runs of 100,000 coalescent simulations in each model. Then, we used a simulation with the highest likelihood values for parameter estimation.The above results of genetic analyses showed that the populations of the northern (Kitanoshima Island and Yomejima Island) and the southern (Chichijima Island and Hahajima Island) islands are genetically differentiated see “” sectionun Table . We calcSupplementary Information.


Trichiurus japonicus accounts for a major amount in the northwest Pacific fishery. Previous studies based on mitochondrial DNA markers reported incongruences in its population structure, hence prompting the need for high-resolution markers and avoiding possible shortcomings in its management. Here we genotyped ten novel de novo-assembled transcriptome-derived microsatellite markers on a total of 150 samples across five major fishing grounds (encompassing latitudes 22–39°N). These markers presented a high number of alleles and heterozygosity compared to other marine fishes, corresponding to the large effective population size of ~20,000 per location and cohort differentiation. Population structuring analyses suggested T. japonicus to be a homogenous well-mixed population. This configuration is likely attributed to the majority of its effective population migrates across locations, and the absence of oceanographic barriers at the continental shelves. Qingdao with reportedly high ocean productivity could be a genetic pseudosink based on the high heterozygosity and migratory preference. Moreover, the results of sign tests suggest that T. japonicus experienced a recent bottleneck likely concurrent with historical glaciation events. Further, we demonstrated satisfactory cross-amplifications of our markers on several congeners, indicating a great promise to use these markers to study the population genetics of trichiurids. Together, our findings will serve as an essential groundwork for enhancing resource conservation and management of cutlassfishes.Cutlassfishes, also known as hairtails, include multiple predatory fishes of the family Trichiuridae. They constitute a top marine fish commodity globally, yet the knowledge about their composition and intraspecific genetic structures is still limited.  Trichiurus japonicus [Cutlassfishes or hairtails are predatory fish species of the family Trichiuridae. They are characterized by extremely elongated and compressed bodies and inhabit the tropical and temperate continental shelves , 2. Tricaponicus –7.T. japonicus. Life-history traits such as growth rate, maturation time, and maximum body length, as well as an inverse correlation to temperature, were found variable [T. japonicus for multiple cohort productions per year [Intra-specific variations were observed in the NW Pacific-endemic variable . Besidesper year . In addiper year . These vT. japonicus populations, which was caused by the exposure of Taiwan Strait during the glacial periods [Environmental factors such as oceanographic features and geological barriers are known to differentiate populations in various marine organisms . In the  periods .T. japonicus have only been studied using mitochondrial DNA markers, including cytochrome b (Cyt-b) [b and control region revealed two genetically differentiated populations at the East and South China Seas, with Taiwan Strait as their transient zone [T. japonicus. Other genetic markers, which are non-mitochondrial in origin and have a higher capacity to yield better resolutions, would help elucidate this unresolved intra-specific genetic pattern.Genetic tools have proven to be efficient in identifying and characterizing the presence of different populations or the so-called stocks in fisheries –23. Thes (Cyt-b) , 25, con (Cyt-b) , and 16Sent zone . These iT. haumela [T. lepturus [T. nanhaiensis [Eupleurogrammus muticus [Lepturacanthus savala [L. savala) along the coast of China [T. japonicus from the NW Pacific are still unavailable.Microsatellites or simple sequence repeats (SSRs) are nuclear DNA with short and tandemly repeated simple sequences. SSRs have been widely used as markers for population genetics due to their high polymorphicity . Their h haumela , T. leptlepturus , T. nanhhaiensis , Eupleur muticus , and Leps savala . Only onof China . PopulatT. japonicus. We hypothesized the presence of an isolated population in the Yellow Sea based on its semi-enclosed topology, unique oceanographic dynamics, and the freshwater barrier from the Yangtze River , Qingdao (QD), Zhoushan (ZH), Badouzi (GE), and Tsukuan (T), from August 2018 to November 2019 .One , Taiwan , Table 1de novo assembled in Trinity v2.2.0 [The sequence read quality was checked and filtered with FastQC v0.11.5  and subjy v2.2.0 , 54. Cory v2.2.0  was thenMicrosatellite identification tool (MISA) v1.0 was used for microsatellite loci detection in the unigenes , 57. TheT. japonicus samples were genotyped for the ten microsatellite loci: TJ-2, TJ-7, TJ-8, TJ-9, TJ-10, TJ-14, TJ-17, TJ-18, TJ-20, TJ-21 (2) , 1 μl of template DNA, 0.5 μl of each primer, and ddH2O with a final volume of 20 μl; (2) For TJ-2, TJ-17, and TJ-20, the solution contained 10 μl SuperRed PCR Master Mix (2x) (4 mM MgCl2) , 1 μl of template DNA, 0.8 μl of each primer, and ddH2O with a final volume of 20 μl. Reactions had an initial step at 95°C for 3 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 54.5–66.5°C depending on the primers  algorithm [p-values of locus-specific F-statistics conditioned on observed levels of heterozygosities [The departure of each microsatellite locus from Hardy-Weinberg equilibrium was assessed following procedures described by Louis and Dempster (1987)  and perflgorithm . The deflgorithm . Coalescgosities , 70. Unugosities .E) and observed heterozygosity (HO) were calculated for each sampling location following Weir and Cockerham (1984) [O) was directly calculated from the proportion of heterozygotes in the sample, whereas expected heterozygosity (HE) was calculated following Nei (1978) [ST) and inbreeding coefficients (FIS) between and within each location based on the F-statistics method [Expected heterozygosity (Hm (1984)  using GEm (1984) . Observei (1978) . GENETIXs method  with 100T. japonicus was assessed through four methods. First, an unrooted neighbor-joining (NJ) phylogenetic tree was constructed for the five locations using the DA distance method [T. japonicus individual to its most probable cluster by Structure Harvester [ad hoc statistic of ΔK values was also calculated based on the rate of change of log probability of the data [The potential genetic differentiation of e method  with 100e method . Second,e method . A burn-arvester . An ad hthe data . Third, the data . The intthe data  on the me) and the number of migrants per generation were calculated based on the Maximum likelihood (ML) method in MIGRATE v1.2.32 [e was inferred from the frequency of allele exchange among the locations and was calculated by the estimated population size parameter (mutation rate = 10−3) [The effective population size (N v1.2.32 . Ne was  = 10−3) . The ass = 10−3) , 84. Com = 10−3) , 86. We  = 10−3)  and Wilc = 10−3) , which w = 10−3) . The sim = 10−3)  and Two- = 10−3) . The pro = 10−3) .T. brevis, T. lepturus, and T. nanhaiensis, which also occur in the NW Pacific waters. Two DNA samples of each species were collected, extracted, and subjected to the PCR with conditions elaborated earlier.The application of the ten microsatellite markers developed in this study was also tested on three congeneric species, T. japonicus transcriptome and found 40,027 (85.84%) that contained microsatellites. On all the motif repeat sizes, mononucleotide was the most abundant (41.29%)  . A total(41.29%) .P < 0.05)  . Detecti < 0.05) . As the  < 0.05) .a), expected heterozygosity (HE), and observed heterozygosity (HO), per locus and location, were listed in The number of alleles  to 0.01476 . For the FST values of the ten loci across all locations, the highest value was recorded from ZH (0.008019), while the lowest value was from DL (0.002937). All FST values were below 0.05, which indicated that there is no differentiation in each location. The FIS values ranged from 0.00819  to 0.13604 . For the FIS values of ten loci in all five locations, the highest value was observed at DL (0.072342) and lowest at ZH (0.065282). All the FIS values and the 95% confidence interval (0.04785–0.08527) were very low, indicating a low level of inbreeding in each location.The F-statistics results of FT. japonicus. The NJ tree showed poor resolution on the genetic relationships of the five populations of T. japonicus, represented with low node supporting values  to 0.00766 (between DL and QD). They were significantly different for DL/QD/ZH vs T; QD vs DL; ZH vs T; and ZH vs the rest of the locations  of DL, QD, ZH, GE, and T were 21,343.79; 24,014.94; 19,529.13; 20,971.69; and 22,655.89, respectively. The number of migrants per generation ranged from 15,739.67 (DL to T) to 19,656.04 (GE to QD) (The effective population size (NE to QD) —more thaE to QD) .T. japonicus were highly successful in cross-amplifying with the three congeneric species  were required for amplifying the pure target products in some cases.The ten microsatellite markers developed from  species . For allT. japonicus in the NW Pacific using one of the NGS platforms—Illumina, which will lay the foundation for future related studies.Genome-wide genetic information can help us better understand the biological characteristics and molecular mechanisms of organisms. In recent years, Next Generation Sequencing (NGS) has become an efficient and cost-effective method to generate genome-scale sequence data, which advanced the formulation of molecular markers for both model and non-model organisms . CutlassMolecular markers are an important tool to study population genetic diversity. Microsatellite DNA markers are particularly useful in monitoring genetic variability due to their inherent high mutation rates, thus have also been successfully applied to the management of fisheries species . In thisT. japonicus being the dominant cutlassfish species in the NW Pacific and the estimated effective population size per sampling locality also ranged between 19,529 to 24,014. Inbreeding is neither a feasible reason because the inbreeding coefficients are positive yet very low [The null hypothesis on genetic studies states that populations are selectively neutral, meaning they are not influenced by natural selection but by random fixation , 97. Thevery low . We did very low . In thisvery low .T. japonicus.Another potential explanation for the departures from Hardy-Weinberg equilibrium is the Wahlund effect . In popuFinally, we tried to remove loci with departures from the Hardy-Weinberg equilibrium from multiple locations, repeated all analyses, and still obtained the same results. Therefore, we consider that these non-neutral loci might have a weak influence on our subsequent analyses. Also, the inclusion of multiple loci would help average the effects of selection ; therefoT. japonicus than other reports on commercial marine fishes. The average number of alleles per locus of T. japonicus (23.14) was relatively higher than that of striped bass Morone saxatilis (14.125) [Trachinotus ovatus (5.875) [Larimichthys polyactis (5.82) [T. japonicus was relatively higher than 12 other marine fish species (20.6) based on 66 loci [Morone saxatilis (0.766) [Trachinotus ovatus (0.318) [Nibea albiflora (0.8033) [T. japonicus were higher than other cutlassfish species such as T. haumela  [T. lepturus  [T. nanhaiensis  [L. savala  [Genetic diversity indices, including the number of alleles and observed heterozygosities of the ten microsatellite markers showed higher values in (14.125) , pompano (5.875) , and smas (5.82) . Whether 66 loci . The ave (0.766) , pompano (0.318) , and yel(0.8033) ; and als(0.8033) . Among t = 0.71) , 109, T.= 0.804) , T. nanh= 0.807) , and L.  = 0.81) , 50.T. japonicus. In the NW Pacific, T. japonicus has the widest distribution range compared to its congeners [The high genetic diversity indices typically indicate a larger population size and/or higher mutation rate . Since mongeners  and has ongeners , 112, wiongeners . This diongeners . This heongeners .T. japonicus in the NW Pacific. The NJ tree yielded poor phylogenetic resolution to separate populations, which was indicative of a homogenous structuring. AMOVA showed that there was no significant differentiation at all geographic hierarchical levels. PCoA displayed that all samples were mixed in the quadrant and no obvious grouping was evident. The results of STRUCTURE showed that both K = 1 and K = 2 population schemes were the most probable; however, only a few individuals from Qingdao were assigned to the second cluster, and the reason behind that is still unknown. Although differentiation among some locations was statistically significant based on F-statistics, the FST values were all much lower than 0.05 suggesting minimal differentiation [T. japonicus was also previously reported to be seasonal in the China Seas and potentially related to spawning seasons [T. japonicus is congruent with previous genetic results using mitochondrial DNA markers [The results of the NJ tree, AMOVA, PCoA, and STRUCTURE all suggested a single well-mixed population of ntiation . In addintiation , 117. Wentiation , 119–whentiation . This ov markers , 26. Thi markers , 123.A recent bottleneck event was proposed for the majority, if not all locations, based on the sign tests. Although the timing of this event was not estimated, we suspected that it corresponded to the sea-level change in the Pleistocene. It had been well recognized that the cyclical fluctuations of the global climate in the Pleistocene caused drastic changes in sea level, which can affect the distribution range and biomass of marine organisms , 124. ThT. japonicus in the late Pleistocene based on mitochondrial DNA markers [The Bohai Bay, the Yellow Sea, and the Taiwan Strait all disappeared completely. After the glacial period, rising sea levels brought the fauna back to a broader habitat and open opportunities to expand the population size. He, et al. (2014) and Xiao, et al. (2014) proposed a population expansion of  markers , 26. The markers .T. japonicus has high genetic diversity and large population size, suggesting that this highly exploited species is resilient to previous impacts. Moreover, a single population with high gene flow across locations had been identified throughout the NW Pacific. Recent fisheries data have shown that the top exploited Trichiurid relative, T. lepturus, has already been experiencing a decreasing catch rate since the last decade [T. japonicus management schemes as this species could potentially be the next sought target.A downward trend of the total catch of cutlassfish in the NW Pacific area has been reported –134. Somt decade . This seBased on the above genetic characteristics, we suggest recognizing the entire sampling localities as a single fishery unit and developing international cooperative management. More stringent measures can also be applied to fishing grounds with relatively higher recorded heterozygosity and migration preference  to establish more substantial protection for the existing genetic diversity. The successful amplification of the EST-microsatellite markers developed in this study towards congeneric species implies their usefulness for characterizing genetic diversity on other highly targeted Trichiurids and confirms the advantage of transferability across related species which economized time and resources . With thT. japonicus. The ten microsatellite loci developed from transcriptome displayed high polymorphism and genetic diversity thus proved as ideal markers to study intra-specific variations. We found that T. japonicus has higher genetic diversity than other marine fishes including the cutlassfish relatives, which might be attributed to its large population size and/or cohort genetic differentiation. Besides, a well-mixed single population was identified for T. japonicus in the NW Pacific, which has experienced a bottleneck and subsequent expansion likely facilitated by the strong migratory ability and the lack of dispersal barriers. High heterozygosity and migration preference were also reflected for Qingdao, and hence considered as a genetic pseudosink. Based on the above genetic characteristics, we suggest treating the entire NW Pacific population as one fishery unit and developing international cooperative management for the species. The developed microsatellite markers have proven effectiveness for transferability in congeneric species investigations.This is the first study applying microsatellite markers to depict the population genetics of S1 FigST assuming a stepwise mutation mode.Dashed lines are one-sided confidence interval limits obtained from simulation-based expected distributions of F(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableNa: number of alleles; He: expected heterozygosity; Ho: observed heterozygosity. Dark gray means number of alleles > 30 and heterozygosity > 0.9; medium gray means number of alleles between 20 to 30 and heterozygosity between 0.85 to 0.9; light grey means number of alleles < 20 and heterozygosity < 0.85.(DOCX)Click here for additional data file.S5 TableST: The fixation index; FIS: The Wright’s fixation index.F(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Tabled.f., degree of freedom.(DOCX)Click here for additional data file.S8 Tablem = Number of migrants. Location 1 = DL, 2 = QD, 3 = ZH, 4 = GE, and 5 = T.N(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.