Tabanus nigrovittatus Macquart) as a bioindicator of marsh health. This species is bound to coastal marshes, since its larvae develop as top invertebrate predators in the marsh soil. Immediately after the oil spill (2010–2011), populations of this horse fly declined in oiled areas of Louisiana marshes with significant impacts on genetic structure. In this follow-up study five years after the catastrophic event (2015–2016), we now report signs of recovery of populations in formerly oiled areas. Fly numbers increased compared to previous counts. Previously detected genetic bottlenecks in oiled populations have disappeared. Migration into oiled areas began to replenish formerly depleted horse fly populations in impacted regions with populations from non-oiled areas as an important source of migrants. Parameters of family structure that had been impacted by the oil spill  rebounded to levels similar to or exceeding those in non-oiled control areas.The Deepwater Horizon oil spill in April 2010 had unprecedented impact on the Gulf of Mexico. We established the greenhead horse fly ( Coastal Louisiana received the largest share of contamination by oil/dispersant mixture of all the Gulf States impacting its valuable and vulnerable salt marshes6.In April 2010, the Deepwater Horizon (DWH) oil spill caused the largest man-made accidental marine oil spill to date with a total release of approximately 5 million barrels of oil into the Gulf of Mexico8. Microbial and meiofaunal communities decreased in diversity and community function shifted towards oil degrading taxa12. The large majority of infauna that serve as prey for higher trophic levels  experienced high mortality in both heavily and moderately oiled sites in Louisiana salt marshes with their lowest diversity and density occurring 18 months after the oil spill16. Furthermore, McCall and Pennings17 reported that arthropod predators, herbivores, parasitoids and detrivores were suppressed by 50% at oiled sites in 2010.When the oil made landfall, wide-spread destruction of marsh vegetation was observedTabanus nigrovittatus Macquart (Diptera: Tabanidae), in oiled areas18. We established this horse fly species as our prime bioindicator model of marsh health for several reasons: 1. this species is native and linked to Spartina marshes from Texas to Nova Scotia; 2. the carnivorous larvae of this species develop for 3–9 months as the top predators within the invertebrate food web in the marsh soil, and their development is dependent on and a sign for the presence of a healthy food web in the sediment; 3. adult populations are highly visible, easy to catch, and 4. have shown immediate responses to the presence of oil in the marsh19. Population census and genetic studies of T. nigrovittatus showed population decline in both adult and larval numbers in oiled locations compared to unoiled locations in 2010 and 201118. Genetic bottlenecks, loss of breeders, shrinking family sizes, and reduced migration rates provided combined proof for a loss of effective population size and a change in population structure in oiled areas. The immediate decline in the adult population was likely due to attraction to oil sheen on the water since oiled lakes have been shown to be insect traps20.One of the most extensive studies on the impact of the oil spill on an arthropod species with close association to the salt marsh was our study of population changes of the greenhead horse fly, Spartina marsh and the food web it contains. Therefore, we followed up on the fate of the greenhead horse fly in Louisiana Spartina marshes 5 to 6 years after the oil spill by pursuing the following scientific questions.Have census numbers of adult horse flies in oiled areas increased in 2015 and 2016 compared to the years immediately after the oil spill (2010–2011)?Have successful migrants replenished formerly depleted populations in oiled areas and has migration changed the population genetic structure over time?Do the previously detected genetic bottlenecks in oiled populations still exist?Have parameters of family structure that were impacted by the oil spill changed to indicate increased effective population size and population recovery when reexamined in 2015 and 2016?We presumed that the recovery of the greenhead horse fly would be closely linked to the decline of oil toxicity and subsequent recovery of infaunal communities reported in the literature and could be used as a measurable indicator for the underlying recovery of the Immediately after the oil spill 2010, 2011), adult horse fly trap catches were magnitudes lower in oiled areas; yet in 2016, numbers of adult horse flies caught near oiled areas in Plaquemines Parish were not significantly different from those caught in the non-oiled areas of Cameron Parish. In 2016, adult horse fly trap catches in formerly oiled areas, i.e., Elmer’s Isle, Grand Isle (Jefferson Parish) and Grand Bayou  determined by microsatellite genotyping. However, not all of the 18 trap samples  collected in 2016 were significantly genetically differentiated based on their allele frequencies at the 5% level . We combined samples that were not genetically differentiated and collected within the same location. This process resulted in eight genetically independent populations for 2016. For comparison, these genotyping data were analyzed together with data collected immediately after the oil spillT. nigrovittatus in oiled and non-oiled areas over a period of six years following the DWH oil spill.Population genetic analysis via TESS assigned the individual tabanids from the 26 sample populations spanning the years from immediately after the oil spill (2010 and 2011) to six years after (2015 and 2016) to eight major genetic clusters  among non-oiled populations showed no significant difference among the years  being significantly lower compared to 2010  and 2011 . Distances were also lower (P = 0.037) in 2015  than in 2010 and marginally lower (P = 0.097) in 2011  being significantly lower than those from previous years  exceeded the minimum value required for informative levels obtained from simulations in BAYESASS21. None of the populations from oiled areas showed meaningful levels of migration rates in 2010. Gene flow among geographically close oiled areas connecting Grand Isle and Grand Bayou populations with migration rates ranging from 0.026 to 0.261, was first detected in 2011, one year after the spill18, but continued in 2015 and 2016  among populations collected in 2015 and 2016 ranged from 0.1 to 25% with 68–99% being derived from the source populations in each generation . In 2016, migration was observed from most of the non-oiled regions into all formerly oiled regions with reciprocity in one instance  that contributed small proportions of migrants (m) to oiled areas in the east (GIP: m = 0.017 and GB3: 0.073) in 2011The average emigration rate from non-oiled regions almost doubled in 2015 and 2016 and was significantly higher than in the two years after the spill Table . EmigratIn 2016, immigration was significantly higher than in both 2010 (P = 0.001) and 2011  and EI (m = 0.119). Finally, in 2016, migrant proportions ranging from 0.03 to 0.14 originating from non-oiled areas  contributed to all formerly oiled populations   and GB2 (m = 0.073)18, none of the populations collected in 2015 and 2016 showed signs of recent genetic bottlenecks. No heterozygote excess resulting from a population crash induced rapid loss of allele numbers was detected under the three mutation models  with significant interactions of “year” and “condition” on the number of parents  and percent of half-sibs  and the years were, thus, treated separately. When 2015 and 2016 data sets were added to the 2010/2011 data, “year” had significant effects on the number of parents , the number of family clusters , the percentage of half-sibs , and a marginal effect on the effective population size . “Condition” had a significant effect over all years on effective population size , and the number of parents .Year had no significant effect in the previous data sets from 2010/2011 (GLM)18). These differences between non-oiled and oiled regions persisted in the year 2015 . However, in 2016 both effective population size and number of parents had increased significantly in formerly oiled areas compared to 2010/2011  and 2015  and reached a level that is not significantly different from those of non-oiled areas across the years (P > 0.20).Effective population size and number of parents contributing offspring to population samples were equally high in non-oiled areas across all years Fig. . In the The number of family clusters increased significantly in oiled regions in 2015  and 2016  compared to the number in oiled areas in 2010/2011. In 2015, the number of family clusters in oiled areas was not significantly different from the numbers in non-oiled areas anymore. Moreover, in 2016 the number of family clusters in formerly oiled areas became significantly higher than in control areas .T. nigrovittatus can be polygamous18. The number of partners each individual mated with and the number of offspring per partner did not differ across the years or between oiled and non-oiled regions. The percentage of full-sib pairs found in the sample populations was only different between non-oiled areas immediately after the oil spill and oiled areas in 2016. The percentage of half-sibs was significantly lower in oiled areas in 2016 compared to most other samples (except non-oiled 2015). In particular, the percentage of half-sibs was reduced in oiled areas sampled in 2016 compared to oiled areas in 2010/2011  and 2015 .Both sexes of A holistic understanding of the progression of long-term effects of oil spills as well as the timelines for recovery of salt marshes is essential for assessing the resilience of the marshes’ vital ecosystem services and fundamental ecological patterns and processes. Native infauna, i.e., the meio- and macrofauna developing in the salt marsh soil, are postulated to be the most informative indicators of ecosystem recovery after oil spills due to their important interactions as part of the food web. Here we discuss the observed recovery of the greenhead horse fly 5–6 years after the Deepwater Horizon oil spill in the framework of the multi-layered recovery of infauna with different life history, diversity and function.T. nigrovittatus, with carnivorous sediment dwelling developmental stages placed at the top of the invertebrate food chain, should be most sensitive22 and, thus, most valuable bioindicators of marsh health. The DWH oil spill caused a severe drop in the numbers of horse fly larvae and adults in oiled areas, accompanied by underlying changes in population genetic and family structure18. This led to the question if, when, and how the process of recovery might mitigate or even restore the genetic make-up of tabanid populations colonizing or residing in oiled areas.We postulated that species native to the marsh, like 23. Trap catches vary due to seasonal fluctuations18, weather conditions and the occasional presence of horse fly predators such as dragonflies and bembix sand wasps, which cause horse flies to avoid traps24. Although increases in adult numbers in formerly oiled areas were small, the incidence rate of detecting larvae in the marsh soil clearly increased. The larva numbers (0.9 larvae per sample) in the formerly oiled Grand Bayou location reached the range of larva recovered from control areas. Since the larvae are cannibalistic, finding approximately one per sample would be expected in a productive habitat. The other formerly oiled areas (Elmer’s Isle and Grand Isle) were heavily impacted by clean-up and reconstruction efforts, which may explain why the ecosystem has not yet rebounded to sustain peak larval development. Although there were no pre-event data to know the “normal” baseline number of horse flies in specific areas before the oiling, the rising census numbers in formerly oiled areas could be interpreted as the first sign of population recovery, especially since recovery subsequently was confirmed six years after the spill by genetic measures.Six years after the oil spill, adult census numbers and the frequency of larvae being found in the soil were increased in formerly oiled areas compared to our findings immediately after the oil spill. Adult numbers as stand-alone, however, should be interpreted with caution. Census data often overestimate effective population size26 that are typically predicted and observed in slow recovering populations28. Most of the populations of 2015 and 2016 showed significant homozygote excess in at least one of the mutation models. While inbreeding is part of the life history of T. nigrovittatus19, the novel rise of heterozygote deficiency in populations of the formerly oiled areas likely indicates a reverse bottleneck effect, i.e., a recent population expansion. Population expansion in formerly oiled areas could be caused by 1. successful migration into oiled areas, 2. sufficient rate of reproduction in oiled areas to overcome residual larval mortality, if any, and 3. increased survival of larvae developing in formerly oiled areas due to decreased oil residue toxicity to larvae and/or increased availability of their food web.The observed increase in effective population size, number of breeders and number of family clusters in formerly oiled areas, which reached the level of control populations in 2016, supported the rise in census numbers and explains the mitigation of genetic bottlenecks recorded after the oil spill. The quick (≤5 years) recovery from bottlenecks of tabanid populations from oiled areas is expected to reduce negative consequences on evolutionary potential and resilience of the impacted populations30. In the present study we recorded shrinking genetic distance (FST) over the years between populations from non-oiled and oiled areas and among populations from oiled areas, which suggests increasing migration rates compared to the low gene flow among populations after the oil spill. Therefore, we tested the hypothesis that populations from non-oiled areas were an important source for repopulation of formerly depleted oiled areas.1. In terms of migration capacity, tabanids rank as strong fliers and can disperse across considerable distances32; and/or mortality of migrants into oiled areas, because flies were attracted by polarized reflections from oil sheens while searching for fresh water34. However, in 2011 (one year after the spill), some degree of migration was detected among geographically close oiled areas (Grand Isle and Grand Bayou) suggesting sufficient reduction of oil in the water and/or in the sediment to sustain survival of migrants. Still, larvae were almost entirely absent from formerly oiled areas in 201118. The most important parameter in terms of recovery was the significant increase in migration from non-oiled areas into oiled areas over the years with 2016 showing migration from most of the non-oiled regions into all formerly oiled regions and even occasional contributions of migrants from oiled areas into non-oiled areas. This flow of migrants from populations of non-oiled regions (or closely related unsampled populations with the same genetic signature) into oiled areas was reflected in the increasing homogenization of population structure in the later years, which stands in contrast to the distinct genetic signatures of populations immediately after the oil spill in 2010.The lack of migration of a typically strong flyer immediately after the oil spill in 2010 could be explained by reduced dispersal capacity of tabanids from oiled areas  was shown for taxa that lack larval dispersal and those whose later stages are also poor dispersers due to their association with the marsh sediment35.These results highlight dispersal ability as an important driver of recovery. As a general rule, populations of organisms with rapidly dispersing life stages were the first to recover as soon as oil contamination and soil conditions were sufficiently improved to sustain survival. Similar patterns were also found in long-term colonization and succession studies of invertebrates in newly constructed salt marshesT. nigrovittatus, the dispersal and recovery of the latter was quicker than expected despite the fact that this tabanid has sediment-dwelling carnivorous (top of the food chain) larvae. The larvae are accomplished swimmers and can be observed hunting in the tidal zone, which likely facilitates dispersal at the larval stage to some degree . In addition, adults are excellent fliers, which explains the observed gene flow from unoiled into oiled areas as soon as one year after the spill.In comparison to these macro- and meiofauna invertebrates that are similarly native and tightly bound to the marshes as T. nigrovittatus, successful recolonization of formerly oiled areas also requires reproductive success. Invertebrates with slow recovery include the tube-dwelling oligochaetes, polychaetes and tanaids, whose relatively small offspring numbers develop confined within the maternal tubes37. In contrast, quick recovering invertebrates  produce and release high numbers of progeny into the water column16. Tabanus nigrovittatus is known to lay >200 eggs per batch and is an autogenous species, i.e., females can produce the first batch without the need for a blood meal and produce subsequent batches after blood meals have been obtained. The pursuit of a blood meal and subsequent oviposition would ensure dispersal of individuals and recovery of depleted areas. Genetic data suggest that males and females might be able to mate multiple times (polygamy) and have multiple offspring with each partner18. The high reproductive and dispersal capacity of tabanids should translate into rapid population growth and re-colonization of formerly oiled areas, but only if larvae can develop in the sediment. The observed rebound in effective population size of adults, number of successful breeders and families in formerly oiled areas required not only a certain degree of decontamination of formerly oiled sediments, but also sufficient secondary (heterotrophic) production and food web support for larval development.2. While dispersal capacity was certainly a strong factor to facilitate population recovery of 38 paved the way for vegetation recovery. Even in heavily oiled areas with near complete plant mortality initially, some recovery of was reported six years after the spill albeit total live aboveground biomass was <50% of reference marshes. In moderately oiled areas above ground vegetation recovered within 2.5 years39. Most microalgae and 90% of meiofauna , which form the base of the food web in the marsh sediments, recovered within three years after the DWH oil spill following closely the re-growth of Spartina marshes except for certain polychaetes, amphipods, kinorhynchs, ostracods and gastropods, that had not reached levels of reference marshes after 4 years16. According to McCall and Pennings’ study17, the terrestrial arthropod community, including mostly herbivorous insects, and intertidal crabs had largely recovered one year after the spill despite suppression by oil exposure a year earlier. The guts of tabanid larvae contain mostly insect DNA as revealed by a recent 18S metagenome sequencing study (unpublished data). The quick return of insects to oiled marshes was, therefore, vital for the tabanid recovery.3. The process of recovery of the food web had started bottom-up almost immediately after the oil spill setting the stage for the return of horse flies to oiled marsh regions. Microbial bioremediation of oiled marsh sitesTabanus nigrovittatus populations’ sensitivity to oil contamination evidenced by the immediate population crash following landfall of DWH oil18 and their recovery following in succession with regrowth of Spartina and recovery of 90% of the meiofauna16 makes this species a valuable bioindicator to assess the process of marsh reconstitution . The sites were selected relative to available access, personal observation (LF) of high horse fly abundance prior to the oil spill, absence of confined livestock, and vicinity to Spartina marshes. Cameron Parish and Cypremort Point in St Mary’s Parish in West Louisiana were not impacted by the oil spill (non-oiled controls). However, oiling was reported at Elmer’s and Grand Isle on May 24, 2010 and at Grand Bayou on June 18, 2010 . The exact daily trapping period (ranging from 2–8 hours) was recorded for each site at each location, and the highest three trap counts were used for analysis. The captured flies were transferred on dry ice and stored at −80 °C. Specimens were identified as T. nigrovittatus and counted using a dissecting microscope and cold plate, and returned to −80 °C for storage. Voucher specimens were deposited in the Louisiana State Arthropod Museum at the Department of Entomology of the Louisiana State University Agricultural Center.For population census data, adult female flies were collected using canopy traps baited with dry ice18 for longitudinal comparison. For the data to be comparable to our microsatellite data set from 2010 and 2011, we genotyped horse flies that were collected during the same month (June) in 2015 and 2016. Samples were taken from single traps representing three different trap sites in the non-oiled marshland along the coast of Western Louisiana  and three trap sites from regions in Eastern Louisiana 18. Since a formerly monomorphic locus (7FA)19 showed multiple alleles in several samples of 2016, the number of polymorphic loci was increased from 10  to 11 (2016). Locus characteristics, GenBank accession numbers, and other summary statistics have been published previously19.Total DNA was extracted from thoraces of up to 30 adult female horse flies per sample (trap) using the DNeasy Tissue Kit  and genotyped using the Li-Cor 4300 automated DNA analyzer  as described in Husseneder et al.18, but included for comparison to adult tabanid counts obtained six years after the oil spill.Exploratory analyses indicated that data were not normally distributed. The average numbers in each trap collection were transformed to y = log(x + 1) to normalize prior to statistical analysis. A two-way ANOVA  was used to compare log transformed tabanid catch data across years and locations. Model main effects were Year  and Region . All possible interactions among the main effects in the model, and a Tukey-Kramer adjustment (α = 0.05) were used to separate the means. The catch data for 2010 and 2011 were published in Husseneder G-Statistics with standard Bonferroni corrections at a 0.001 nominal level for multiple comparisons (FSTAT)42 and genetically separated samples (populations) were then used as population genetic units of analysis. Genetic distances among populations (FST) were calculated using the methods of Weir and Cockerham (1984) in FSTAT. For comparison of pairwise distances two-tailed difference of means tests were performed with resampling techniques (300 permutations) using a modified Excel worksheet available at woodm.myweb.port.ac.uk/nms/diffofmeansconfidence.xls.Samples from 5 traps in June 2015 and 18 traps in June 2016 were tested for significant genotypic differentiation using pairwise log-likelihood 18 was combined with the genotypes obtained for 2015 and 2016 to allow for longitudinal comparisons to detect changes in population genetic structure over time. For ad hoc approximations of the number of genetic clusters in the total population (2010–2016) and the genetic relationships among the 26 populations, individual flies were probabilistically assigned to genetic clusters based on their multilocus genotypes using TESS 3.143. For exploratory analysis, the simulations were run 10 times with and without admixture for each Kmax ranging from 2–26 for 1,200 sweeps with 200 sweeps burn-in. The maximum number of genetic clusters (Kmax) was determined from changes in the deviance information criterion  were averaged using CLUMPP 1.1.245. Finally, the estimated membership coefficients of each individual multilocus genotype to each genetic cluster were plotted with STRUCTURE PLOT Version 246.The data set from 2010 and 201121. Preliminary runs were used to assure that delta values for allele frequency, migration rate and inbreeding stayed between 20–60% of the total chain length21. The final settings were 3,000,000 iterations, a sample frequency of 2000, a burn-in period of 999,999 and delta values of 0.25.Bayesian statistics were employed (BayesASS v. 1.3) to estimate the proportion of migrants Table  and immi47. In general, SMM and TPM are recommended when using microsatellite data since they are taking evolutionary relationships among alleles into account47. However, in our study over a limited geographical and time scale migration likely exceeded mutation as source of genetic variation and, therefore, we also presented results for the IAM, an implicit model that makes no assumptions on ancestry of alleles and origin of diversity.Immediately after a genetic bottleneck, allele numbers are reduced faster than heterozygosity. To detect signs of bottlenecks, genotypes of individuals in each population were tested for heterozygote excess across loci using a Wilcoxon sign-rank test under different mutation models , as implemented in BOTTLENECK v. 1.2.0248. Program choice and parameter settings are described in detail in Husseneder et al.18. To infer the best configuration of relationship structure, populations were subjected to full maximum likelihood calculations49. Based on the Best Maximum Likelihood Configuration output of COLONY, we determined the number of inferred parents contributing offspring to each population  and the number of family clusters in each population. Values were corrected for uneven sample sizes by calculating each for a sample size of 30 individuals genotyped per population. We also estimated the number of partners that individuals mated with and the number of offspring produced per parent. The percentages of full-sib and half-sib pairs in each population were inferred from sibship assignment plots48. Effective population size (Ne) was inferred with the sibship assignment method in COLONY48. The sibship assignment method showed superior accuracy for “real-world” data sets compared to other methods since it does not assume isolated populations or random mating and performs comparatively well even with small sample sizes and numbers of loci50.Pedigree structure in each population was inferred using the full pedigree likelihood method implemented in COLONY v.2.0.3.1P-values ≤ 0.05 were considered significant and P-values up to 0.10 were considered marginal.General Linear Model (GLM) analyses were used to test for effects of “year” and “condition” (oiled vs non-oiled) on the variables of mating structure and population size. For each variable pairwise comparisons were performed between populations from oiled and non-oiled regions and between each year using two-tailed t-tests . https://data.gulfresearchinitiative.org/in the data sets by L.F. Adult tabanid population data and available voucher specimens (10.7266/N75718ZM) and Tabanid adult and larval collection data, coastal Louisiana, 2016 (10.7266/N7MS3R4Z). Raw data of microsatellite genotypes are available at https://data.gulfresearchinitiative.org/ in the data sets by C.H. Tabanus nigrovittatus microsatellite data for the assessment of population genetics of 13 populations sampled from coastal Louisiana, 2010-2011 (10.7266/N71J97N2) and Tabanus nigrovittatus microsatellite data for the assessment of population genetics of populations sampled from coastal Louisiana, 2012–2017 (10.7266/N7MK6B8V).Metadata are archived and available through the GRIIDC site of the Gulf of Mexico Research Initiative Information and Data Cooperative. Detailed sample information including GPS location, dates, trap times and horse fly counts and can be found at Supplementary Information


Acropora hyacinthus along the Kuroshio Current, including recently expanded (<50 years) populations. Among the three cryptic lineages found, only one was distributed in temperate regions, which could indicate the presence of Kuroshio-associated larval dispersal barriers between temperate and subtropical regions, as shown by oceanographic simulations as well as differences in environmental factors. The level of genetic diversity gradually decreased towards the edge of the species distribution. This study provides an example of the reduced genetic diversity in recently expanded marginal populations, thus indicating the possible vulnerability of these populations to environmental changes. This finding underpins the importance of assessing the genetic diversity of newly colonized populations associated with climate change for conservation purposes. In addition, this study highlights the importance of pre-existing temperate regions as coral refugia, which has been rather underappreciated in local coastal management.As corals in tropical regions are threatened by increasing water temperatures, poleward range expansion of reef-building corals has been observed, and temperate regions are expected to serve as refugia in the face of climate change. To elucidate the important indicators of the sustainability of coral populations, we examined the genetic diversity and connectivity of the common reef-building coral  However, reef-building corals have a relatively narrow range of temperature tolerance and are considered especially vulnerable to climate change. The most well-known impact of warming oceans on corals is the increase in mass coral bleaching2. In addition, climate change exacerbates local stresses by reducing water quality, which further threatens coral reefs2. In contrast to tropical and subtropical regions where coral abundance is declining due to increasing water temperatures, rapid poleward expansions of the ranges of hermatypic corals have been reported in temperate Japanese regions3 and the Mediterranean Sea4. Given that all of the range-expanding coral species are classified as Near Threatened or Vulnerable on the IUCN Red List, temperate regions may serve as refugia for hermatypic coral species in the near future3 if the coral species can overcome the seasonal fluctuations in temperature and reduced light intensities5. Among the multiple definitions of refugia, we use the term as defined by Keppel and Wardell-Johnson6. In this paper, authors noted that “Refugia are habitats that components of biodiversity retreat to, persist in and can potentially expand from under changing environmental conditions and facilitates the persistence of species under anthropogenic climate change.” Population connectivity and relatively high genetic diversity are crucial for a population to expand and persist under climate change7; thus, these characteristics are important for the populations in potential refugia.Coral reef ecosystems harbour high biodiversity, supporting almost 30% of marine coastal specieshttp://www.data.jma.go.jp/gmd/kaiyou/data/shindan/a_1/japan_warm/japan_warm.html), causing coral expansion and producing potential coral refugia at the edges of their distribution ranges3. While such temperate region can be potential coral refugia, temperate peripheral populations may suffer from reduced genetic diversity due to small population sizes and isolation8. However, no studies have assessed the genetic diversity or structures of such coral populations, including the recently colonized temperate populations that expanded within the last 50 years as a result of climate change along the Kuroshio Current. In addition, there is a lack of knowledge of the oceanographic larval dispersal processes that shape the genetic variations and population structures of corals in the high-latitude regions of Japan.Winter water temperature has risen by 1.45 °C over the last 100 years in the temperate Kuroshio Current region in Japan , is widely distributed in the tropical, subtropical, and some part of temperate regions of the Indo-Pacific9. Northward range expansion of A. hyacinthus has been observed in the temperate regions of Japan for the last 50 years3. Previous studies that included some temperate populations found cryptic lineages (species) of A. hyacinthus11. The A. hyacinthus species complex is a hermaphroditic, broadcast-spawning species whose fertilization takes place in the water column. More than 70% of the larvae settle within one week12 but can survive in the water column for up to 5–6 weeks13. A. hyacinthus is categorized as Near Threatened on the IUCN red list. A. hyacinthus is one of the key reef-building species on tropical reefs14 and can represent as much as 36% of the coral coverage in some high-latitude areas15. These ecological features of this species are suitable for comparing the genetic diversity and connectivity of populations among temperate and subtropical regions. We defined the region south of the Watase Line16 as subtropical and the region north of the Watase Line as temperate  indicated that the most likely number of clusters was K = 2, followed by 3  in P < 0.001 were more frequently found in the temperate region than in the subtropical region , which indicated that asexual reproduction (such as fragmentation) likely occurred more frequently in the temperate region than in the subtropical region  . Notably, all temperate populations, except for one north Yakushima population (KAHyn), had more than one fixed (monomorphic) locus. All the recently expanded populations had 2 to 4 fixed loci out of 8 loci   only when the recently expanded populations were excluded, which possibly occurred because the recently expanded populations are not in genetic equilibrium.Significant IBD patterns within the green lineage were detected only when regressed onto oceanographic distance (05) Fig. . A weak A. hyacinthus lineage that was examined in this study  or excluding  the recently expanded populations. The AMOVA results revealed non-significant genetic differentiation between the recently expanded populations and the pre-existing temperate populations  and between the populations along the main stream of the Kuroshio Current vs the Tsushima Current  in the temperate region  results suggested overall significant genetic structuring within the K = 1 best explained the data , which implies that very small genetic differences exist between the two regions.STRUCTURE analysis was again performed within the green lineage with and without prior information . The results indicated that a Suppl. , which s= 2 Fig.  with a sThe estimation of recent gene flow based on the assignment method using GeneClass2 showed that 95 out of 240 individuals were successfully assigned to one of the populations. Most of the successfully assigned individuals (15 out of 16) in the recently expanded populations suggested self-seeding. Only one out of 76 (1.3%) temperate individuals were possible recent immigrants from subtropical populations 23, reduced aragonite saturation24, light intensity25, and high turbidity26 around northern populations  was observed in the recently expanded ns Table . Alternas Suppl. , could a27. In the Atlantic, another coral species, Montastraea cavernosa, showed lower genetic diversity in peripheral populations28. These studies suggested that a small founding population, small effective population size of genetic drift, or inbreeding depression caused reduced genetic diversity in peripheral coral populations. However, expanding coral populations do not always follow this trend of reduced genetic diversity, as was demonstrated in the Mediterranean coral Oculina patagonica29.Some previous studies have also provided evidence for reduced genetic diversity in peripheral coral populations. For example, both brooder and broadcaster coral species in the southernmost reef in the Pacific showed lowered genetic diversityA. hyacinthus. In Acropora palmata, different rates of asexual reproduction are known to be related to different habitat characteristics30.The clonal rates were higher in temperate regions than in subtropical regions. Although the mechanism is unclear, different habitat characteristics  potentially caused different rates of sexual reproduction success in F-statistics; Tables 31. Recently expanded populations were therefore possibly seeded from other temperate populations by occasional long dispersal and then sustained themselves by self-seeding.While the historical gene flow analysis  had low genetic diversity and a small number of samples was analysed (MGL = 9), one individual had a private allele. One possible explanation is that harsh environments around GAH Suppl.  caused sFST) significantly increased with geographic distance. This result indicates that overall, the effect of genetic drift is stronger than the effect of gene flow between distant populations.Significant IBD patterns were detected only when oceanographic distance was used Fig. , which iFST calculations, which assume equilibrium. Similarly, the genetic distance of sea stars across the Indo-Pacific region was best explained by Euclidean distance and not modelled larval distance in a previous study34 in which the authors considered that the estimated FST reflected historical gene flow rather than contemporary gene flow. Another explanation for the weak correlation with IBD patterns is the lack of biological characteristics  in the larval dispersal models used in this study. A more sophisticated model that includes the biological aspects of A. hyacinthus would improve the correlation with genetic distance, as in a previous study35.We expected that the larval dispersal model rather than oceanographic distance would have significant IBD patterns because genetic structure is considered to be most associated with larval dispersal. However, the IBD patterns that were derived using the number of larvae between sites based on numerical simulations did not improve in comparison to those using Euclidean distances and oceanographic distances Fig. . This re3. Many climate change predictions of the species distributions of corals show a general loss of reef habitats in many areas except for the relatively robust eastern marginal Pacific region, including temperate Japan36. Given the moderate levels of neutral genetic diversity and the persistence and increase in coral cover before and after the rise in sea water temperatures in pre-existing temperate coral populations, we propose that some of the pre-existing temperate regions in Japan would be good refugia for at least one of the A. hyacinthus lineages. Pre-existing temperate regions are indeed the potential habitats that “part of biodiversity retreat to, persist in and can potentially expand” under climate change conditions. In temperate regions, seaweed beds have been replaced by temperate corals, including A. hyacinthus, and the coral cover is currently expanding in these regions37. Although a direct association between neutral genetic diversity and resilience of coral populations against environmental change is partly under debate8, some coral genotypes are known to be associated with stress tolerances such as disease and higher water temperature38. Thus, moderate genetic diversity in pre-existing temperate populations may indicate stability and higher tolerance against unexpected extreme environmental changes such as local anthropogenic stress. Nonetheless, much less attention has been paid to coral assemblages and their conservation in temperate regions than in subtropical regions. Unlike in subtropical regions, where tourism, recreation, and fisheries are directly associated with the abundance of corals, temperate corals are sometimes regarded as a nuisance by local fishermen in temperate regions because subtropical fishes are of low economic value in these areas. In addition, expensive fishing nets become entangled on and broken by hard corals, causing pecuniary damage to fishermen . Because reef-building corals help to increase overall biodiversity39 by providing food and habitats with complex structures in coastal ecosystems, there is a need to review the value of temperate corals for conservation.Although corals in subtropical regions are threatened due to climate change, they are expanding and propagating into temperate regionsA. hyacinthus lineages were found in only the subtropical region, and the highest genetic diversity was found in the subtropical region, indicating that these areas are nonetheless important.The importance of conserving the pre-existing temperate corals discussed in this study does not dismiss or discourage the conservation of subtropical corals. Rather, two out of the three This study indicated that recently expanded marginal populations could have low genetic diversity and are possibly more vulnerable to environmental changes than pre-existing populations. This finding underpins the importance of assessing the genetic diversity of populations that have recently expanded due to climate change for conservation purposes, in particular when anticipating the roles of these areas as refugia under climate change.A. hyacinthus samples were collected using SCUBA from 22 populations along the Kuroshio and Tsushima Currents . For Amil2-2h and Amil2-23h, we used modified primers to increase PCR efficiency for A. hyacinthus  size standard  for genotyping on an ABI 3730xl automated sequencer , which was used to determine the fragment sizes  of all samples. All genotyping was manually checked. Whenever we found ambiguous patterns or no peak, we re-extracted the genomic DNA and re-amplified it or removed the sample from the analysis. We tested for the presence of null alleles using MICRO-CHECKER ver. 2.2.3FIS), was evaluated with GenAlEx ver. 6.50244. Genotypic linkage disequilibrium across 8 loci was examined using GENEPOP on the web (ver. 4.0.10)45 by estimating exact P values using the Markov chain method with 10,000 dememorization steps, 1,000 batches, and 10,000 iterations per batch.The clonal structure of each coral population was examined by identifying the same MLGs based on 8 microsatellite loci; these were identified as possible clones , and all except one were excluded from subsequent analyses. The inbreeding coefficient of a population (O) and expected (HE) heterozygosity using GenAlEx ver. 6.50244, and allelic richness (A) using FSTAT ver. 2.93246.Different measures of genetic diversity were estimated for each population, including the number of alleles per locus (Na), the observed  chains used a burn-in of 500,000 chains followed by 500,000 MCMC replications. Ten independent chains were run for each K = 1 to 10 to confirm convergence. The coefficient of ancestry was calculated and visualized for each individual across 10 runs for the most likely values of K (Delta K47), in CLUMPAK48 Among the lineages identified in the STRUCTURE analysis above, we used only the lineage distributed in both temperate and subtropical regions in subsequent analyses  using the LOCPRIOR model53. After calculating global FST, we evaluated the genetic structure using the following prior groupings: (1) all temperate populations vs subtropical populations, (2) recently expanded populations vs pre-existing temperate populations, and (3) populations along the temperate main stream of the Kuroshio Current  vs populations along the Tsushima Current . Pairwise FST and pairwise DEST (Jost’s direct measure of differentiation54) values across populations were calculated to estimate relatively isolated populations using GenAlEx 6.50344. The significance of population differentiation was also determined based on Fisher’s exact test using Genepop on the web ver. 4.0.1045 with Markov chain parameters with 10,000 dememorization steps, 1,000 batches, and 10,000 iterations per batch. Sequential Bonferroni correction was applied to determine statistical significance for all pairs of populations (α < 0.05).To examine the degree of genetic connectivity between temperate and subtropical populations, population genetic structure was examined by AMOVA using Arlequin ver. 3.555 was used to estimate recent gene flow between temperate and subtropical populations using the criteria and the computational algorithm of Rannala and Mountain56 with default settings. Individuals were assigned to a source population when an individual had the highest assigned probability >70%, and others were regarded as unassigned. Because the two geographically close Wakayama populations (WAHs and WAHk) and the two Goto populations (GAHo and GAHk) were not significantly differentiated  Euclidean distances between sites, (2) oceanographic distances avoiding physically impossible routes  that were manually estimated using Google Earth and (3) simulated number of accumulated larvae based on a particle tracking model with iterations after 1000 generations  ocean-current analysis/reanalysis products (https://hycom.org). CMS can evaluate a full range of transport and fate variabilities at high computational efficiency. In the particle tracking model, larvae are treated as passive particles transported on the surface by horizontal current velocities. The particles regarded as larvae were totally released from 111 habitat zones of 1/12° × 1/12° rectangles (~10 km2)  were determined by trial-and-error adjustment using previous records of Acropora spawning timing in Japan. The estimated spawning dates were in general agreement with the records of previous spawning timing . The time-series dataset of sea surface temperature used to estimate spawning timing at each site was obtained by extraction from the Global HYCOM analysis/reanalysis products. Spawning events were assumed to take place 3 days before the spawning full moon and to continue for 10 days. From each site, 50 particles were released each day, and a total of 500 particles were released in one year. The maximum larval dispersal period was set for 42 days, which is the maximum length of pelagic larval duration of A. hyacinthus. It was assumed that larvae could begin settling on coral habitats three days after particle release. When the particles passed by the coral habitats, the particles were counted as successful recruitments (the number of larvae dispersed from one place). Finally, a connectivity matrix was obtained by conducting the particle tracking simulation for a total of 24 years from 1993 to 2016. Using the average of the 24-year connectivity matrix, we iterated particle dispersal to simulate the long-term accumulation of dispersed larvae and consider the stepping-stone-like connectivity. This calculation was repeated until the number of the accumulated particle stabilized over time (1000 generations).To estimate the probability of larval dispersal between different sites based on ocean current systems, we conducted Lagrangian particle tracking simulation using the Connectivity Modelling System (CMS)m2) Fig. . The timSuppl. 9Suppl. material


Bioko is one of the few islands that exist around Africa, the most genetically diverse continent on the planet. The native Bantu-speaking inhabitants of Bioko, the Bubi, are believed to have colonized the island about 2000 years ago. Here, we sequenced the genome of thirteen Bubi individuals at high coverage and analysed their sequences in comparison to mainland populations from the Gulf of Guinea.We found that, genetically, the closest mainland population to the Bubi are Bantu-speaking groups from Angola instead the geographically closer groups from Cameroon. The Bubi possess a lower proportion of rainforest hunter-gatherer (RHG) ancestry than most other Bantu-speaking groups. However, their RHG component most likely came from the same source and could have reached them by gene flow from the mainland after island settlement. By studying identity by descent (IBD) genomic blocks and runs of homozygosity (ROHs), we found evidence for a significant level of genetic isolation among the Bubi, isolation that can be attributed to the island effect. Additionally, as this population is known to have one of the highest malaria incidence rates in the world we analysed their genome for malaria-resistant alleles. However, we were unable to detect any specific selective sweeps related to this disease.By describing their dispersal to the Atlantic islands, the genomic characterization of the Bubi contributes to the understanding of the margins of the massive Bantu migration that shaped all Sub-Saharan African populations.The online version of this article (10.1186/s12864-019-5529-0) contains supplementary material, which is available to authorized users. This moDuring their extensive geographical migration, the Bantus encountered and admixed with local rainforest hunter-gatherer (RHG) tribes. Several Bantu-speaking groups have been studied from a genetic point of view over the years, especially using mitochondrial DNA (mtDNA) and Y chromosome markers , 17–20. Despite these efforts, the genetic patterns of variation within the Bantus remained largely unexplored on a genomic scale until fairly recently , 15, 22.There are hypothesis that could potentially be explored with genome-wide data from the Bubi. First, their relatively long period of isolation on the island is a potential way to test the age and extent of the Bantu admixture with RHG tribes, an event that supposedly took place among the coastal Bantu groups after isolation of Bubi ancestors on the island. Second, studying a tribe from one of the few islands around Africa could provide information about potential effects of endogamy and isolation that are less likely to occur in mainland tribes. Finally, genome-wide data from the Bubi can offer clues regarding the adaptation of this group to local conditions. For example, it is known that the population of Equatorial Guinea has one of the highest levels of malaria infection in the world  and rankWe show in this work that the Bantu population of Bioko Island mirrors the genetic makeup of the extant, coastal Bantu-speaking groups, also clarifying the dynamics of this human expansion into the Atlantic islands of Africa.We sequenced 13 Bubi genomes obtaining a depth of coverage up to 21x-32x  to human coordinates in 546,558 single nucleotide polymorphisms (SNPs) of our dataset. We have subsequently computed f4 statistics in the form , to examine the homogeneity of the Bubi population  separate the RHG from the Bantu speakers and the Western Africa non-Bantu populations Fig. c. When PMoreover, ADMIXTURE analysis (K = 4) . Our results indicate that non-Bantu Western African populations such as Yoruba, Bariba, Fon and Azhizi, as well as Bantu-speaking groups from Angola such as Kongo, Ovimbundu and Kimbundu show more genetic affinities with the Bubi, apparently because they all show lower hunter-gatherer ancestry as compared to other groups , , ,  . The Bubi are placed within the range of Western African and other Bantu-speaking populations , and also with a Bantu-speaking group from Cameroon . On the other hand, we found that the Bubi displayed the highest Fst values when compared with RHG populations ; however, no admixing events could be identified. We also used fineSTRUCTURE to plot a PCA based on haplotype differentiation. This method clusters the populations into the same groups as those used when considering genotypes. In this analysis, the Bubi present a clear intermediate position between Western African and Bantu-speaking populations  tracks to help elucidate the recent co-ancestry links between the Bubi and mainland populations. We found the Bubi to share the highest number of IBD blocks longer than 2 cM  with poACKR1 [CD36 genes [G6PD [ATP2B4 [GRK5 [, and IL-10 [ABO, HBB [TIRAP [st scan between the Bubi and Yoruba, using all the variable positions with MAF > 0.05 and missing genotypes < 0.05, plotting the mean Fst values in 0.5 Mb windows. We set a threshold of significance in 0.25 [We found that all Bubi individuals possessed the malaria-resistant allele of the ACKR1  and CD3636 genes , in addies [G6PD , ATP2B4  [ATP2B4 , GRK5 [3B4 [GRK5 , and IL-nd IL-10 . MoreoveB [TIRAP  . Based on the evidence that Bantu expansion likely moved from Angola northwards [Within the Bantu-speakers, the Bubi are more closely related to Angolan than to the geographically closer Cameroon groups  that is known to protect against Plasmodium vivax and P. knowlesi, seems to be fixed, or at least is present at extremely high frequencies, in the Bubi population. In fact, this is a common trait in all Western Africa. At the beginning of the twenty-first century, malaria was responsible for a child mortality rate of 152 per every 1000 births in Bioko island, a figure that is only beginning to decrease thanks to recent malaria control projects [The Bubi, like other groups from the Gulf of Guinea, display a high frequency of some mutations associated with protection against malaria. Other mutations, however, are absent or segregating. The underlying mutation for the Duffy-negative phenotype , additional sampling with a broader geographical distribution should be undertaken in the future.In addition to the general population affinities of the Bubi, we have unveiled genetic evidences of a certain degree of isolation, which can be related to the insular conditions; this trait is quite unique in most of African mainland populations. Our study of the genomic composition of the Bubi not only adds further information to the current genetic diversity within Africa and its Atlantic islands, but also points to the importance of the genome-wide analyses in unravelling population affinities, selective pressures and past migrations that can be correlated with linguistic and archaeological information. We conclude that the origins of the Bantu expansion still needs further research and that future retrieval of ancient genomes from Central and Western Africa could shed need light on the cradle of the Bantu migrations.All thirteen individuals analysed in this study are members of the Cultural Bubi Association of Fuenlabrada, Madrid (Spain). We obtained informed consent from all subjects. We discarded 25 of the interviewed individuals because of admixed ancestry; many of them had a recent Fang ancestor from the mainland. Even though most of the individuals were not born in Bioko, we verified that the selected individuals had all grandparents born in the island; many of the volunteers’ direct ancestors come from Malabo, Bariobé and Baney, which are located in the Northeast region of Bioko . The procedure produced a PCR-free library with 350 bp average insert size that requires 20 ng/ul (in 50 ul samples). DNA samples were randomly fragmented by Covaris system and sequenced in HiSeqX10 (Illumina) with hiseq2x150bp settings plus 65 bp paired-end adapters at Macrogen (South Korea).We evaluated the paired-end sequenced reads with FASTQc to check their quality. The sequencing adapters were then removed using Adapter removal , reads sp-value below 1e-6.Unique aligned reads were processed with Base Quality Score Recalibration (BQSR) implemented in the GATK version 3.7 software . Even ifst between both samples were also removed. Based on the colonial history of Bioko, we have assessed the presence of potential genetic admixture of the Bubi with Spanish individuals, adding Iberian samples from 1000 Genomes [p-values below 1e-10.We merged our filtered variants with 690,739 SNPs from 1235 genotyped individuals belonging to 35 Western Africa populations. This dataset includes: Bantu-speaking populations, hunter-gatherers and Western African groups ,using Pl Genomes  to the SFor most of the analyses, we have extracted a sub-dataset with representative populations from Western and Central Africa. This reduced dataset includes 14 populations and 169 individuals  of the human mtDNA . After cTo situate the Bubi within the present diversity of the Gulf of Guinea and Western Africa, a principal components analysis (PCA) with the reduced dataset was generated using EIGENSOFT software , Results2 = 0.5 in windows of 50 SNPs. ADMIXTURE analyses were performed with K from 2 to 15 and were repeated five times. The ADMIXTURE iterations were consolidated using CLUMPP with the large K greedy algorithm [pophelper [ADMIXTURE plots were generated to estimate the proportions of K ancestral components on each individual genome  of the rlgorithm  and the ophelper .3 statistic is a useful test to determine the closest population to a target one using one outgroup population and measuring the amount of shared genetic drift with a test population. San were selected as outgroup, as they represent the most distant African population with genome-wide data, Bubi population was compared to all other populations in the dataset. The f3  statistic was calculated with popstats [f statistics can also be implemented in order to determine which populations exhibited the highest genetic drift with the Bubi people, to do so, we used the popstats software to compute the f4 statistic , , , . These combinations allow us to dissect the genetic admixture of the tested populations with the Bubi in relation to all the representative source of genetic ancestry in Western Africa: Eastern RHG, Western RHG, Western-African populations and Bantu-speaking populations.Outgroup fpopstats  and the st) is a measure of population differentiation. We calculated the mean pairwise Fst values between all the populations present in the global dataset. All autosomal SNPs were included in this analysis using the approach of Cockerham and Weir integrated in Plink 1.9 [The fixation index  iterations and output printed every 10,000 iterations. The best tree was calculated with 10,000 state attempts. We also generated a haplotype-based PCA with fineSTRUCTURE.To identify any admixture events between Bubi ancestors and other populations during the last 4500 years, we used the GLOBETROTTER  softwareIdentity by descent (IBD) blocks are defined as identical chromosome fragments present in multiple individuals that have been inherited from the same ancestral chromosome . We haveROHs (> 1000 kb) were estimated with Plink software. First, we calculated the average (in kilobases) of the genome that it is in homozygosis for each population. Second, we calculated the average of the number of genomic fragments that are in homozygosis for each population.p < 0.001).Relevant mutations associated with malaria resistance in 10 different genes Additional file 2:Supplementary figures. (PDF 1206 kb)


Calliphora vicina is a common species distributed throughout Europe that can play an important role as forensic evidence in crime investigations. Developmental rates of C. vicina from distinct populations from Germany and England were compared under different temperature regimes to explore the use of growth data from different geographical regions for local case work. Wing morphometrics and molecular analysis between these populations were also studied as indicators for biological differences. One colony each of German and English C. vicina were cultured at the Institute of Legal Medicine in Frankfurt, Germany. Three different temperature regimes were applied, two constant (16°C & 25°C) and one variable . At seven time points , larval lengths were measured; additionally, the durations of the post feeding stage and intrapuparial metamorphosis were recorded. For the morphometric and molecular study, 184 females and 133 males from each C. vicina population  were sampled. Right wings were measured based on 19 landmarks and analyzed using canonical variates analysis and discriminant function analysis. DNA was isolated from three legs per specimen (n = 61) using 5% chelex. A 784 bp long fragment of the mitochondrial cytochrome b gene was sequenced; sequences were aligned and phylogenetically analyzed. Similar larval growth rates of C. vicina were found from different geographic populations at different temperatures during the major part of development. Nevertheless, because minor differences were found a wider range of temperatures and sampling more time points should be analyzed to obtain more information relevant for forensic case work. Wing shape variation showed a difference between the German and English populations (P<0.0001). However, separation between the seven German and English populations at the smaller geographic scale remained ambiguous. Molecular phylogenetic analysis by maximum likelihood method could not unambiguously separate the different geographic populations at a national (Germany vs England) or local level.The bluebottle blow fly  Samplomegalen , isomorpomegalen , 7, curvomegalen  or accumomegalen . Blow flomegalen . Publishomegalen . Such poCochliomyia hominivorax and C. macellaria [Chrysomya albiceps and C. megacephala [Anopheles (Nyssorhynchus) [C. bezziana, from Africa  and Asia .Morphometrics are defined as the quantitative studies of biological size and shape, shape variation, and its covariation with other biotic or abiotic factors  and can cellaria  or Chrysacephala . Wing mohynchus) . Hall ethynchus)  have repBeside morphometric tools, species identification based on molecular analysis is common practice in forensic entomological case work . Such DNCalliphora vicina, commonly called the bluebottle fly, is a very common blow fly species distributed throughout the Palaearctic region that plays an important role as forensic evidence in crime investigations [C. vicina as the consequence of the warming of an urban area in London (UK), therefore some might argue that using reference data derived from, e.g., Russia cannot be used to estimate the age of, e.g., an Austrian population, because of adaptations due to local climate conditions. The main aim of this study was, therefore, an intermediate approach to compare two relatively close geographically but clearly separated populations. We analysed for the first time developmental rates of two geographically distinct C. vicina populations from Germany and England under the same temperature regimes in the same laboratory. In addition, we evaluated the wing venation and cytochrome b sequence of flies from different locations of England and Germany to check the potential for these tools to discriminate specimens from both regions, thereby introducing a marker(s) that could indicate possible biological differences.igations , 26. Sevigations , 9, 27. igations  showed tC. vicina were first classified into two populations; German flies and English flies. The German population was further divided into 3 groups: Frankfurt am Main (coded as CVG1), Dortmund (CVG2) and Frankfurt laboratory colony (CVG3); the latter had been maintained for about 1.5 years without refreshment, i.e., no new specimens from the field. The English population was divided into 4 groups: Exeter (coded as CVE1), Haywards Heath (CVE2), Liverpool (CVE3) and London laboratory colony (CVE4), which had been in colony for less than six months. The geographic location of these samples is shown in For wing morphometrics and molecular analysis, adult flies of C. vicina, one from Frankfurt (Germany) and one from London (England), were processed as follows. Adult flies were held in rearing cages at room temperature  and a 12:12 L:D cycle. They were provided with water and sugar ad libitum. A piece of fresh pork liver was regularly placed into the cage as a protein source. At the beginning of each experimental run, the liver was checked for eggs 3 hours after placement. Resulting eggs were transferred into an incubator (LinTek MKKL 600/2), set at 25±1°C. Twenty-four hours after transferring the eggs into the incubator, hatched larvae were used for further experiments.Two established colonies of Specimens of each geographic population were exposed to 3 different temperature regimes, two constant (16°C & 25°C) and one fluctuating . For each temperature, five groups of 20 freshly hatched larvae were transferred from the oviposition medium to approximately 20 g mixed minced meat (pork/beef) in a plastic cup, with each group in a separate cup. These cups were placed in 12 cm × 12 cm × 8 cm plastic containers which were filled with 2 cm of sawdust, serving later in development as the medium for pupariation. This protocol was repeated three times, leading to the analysis of 300 larvae (3 x 100) per temperature and geographic region.The plastic containers with cups were moved daily within the incubators to avoid possible incubator-specific effects. At seven time points , 10 larvae (2 per container) were randomly selected, killed with hot water, and their length measured by using a geometrical micrometer . AdditioFine forceps were used to remove the right wing of each fly. Each wing was placed on a drop of Euparal  on a glass slide, then a thin layer of Euparal was added to the wing and a cover slip was placed on top. After drying at room temperature for 24 h, each wing was photographed using a digital camera (AxioCam ICc1) attached to a stereomicroscope (Olympus SZ61) at 1×magnification. Tps files of each wing were created using TpsUtil V.1.70 software  to minimThe established tps files contained digitized coordinates of 19 homologous landmarks from all wings and were subjected to the MorphoJ software  for alighttp://folk.uio.no/ohammer/past/) to observe phenetic relationships among 7 locations.For wing size variation analysis, the overall measure of wing size was estimated by the centroid size. As a normality test of centroid size showed non-normal distribution, wing size difference among species was evaluated by Kruskal-Wallis test (significance level 0.05). Furthermore, MorphoJ software was used to determine wing shape variation. The variation in the shape space was assessed using a principal component analysis (PCA) to display the major features in a dataset and discover patterns in the relations among specimens. Canonical variates analysis (CVA) was used to determine the most important shape characteristics for discriminating between groups (Germany and England) and among multiple groups of specimens . The statistical significance of pairwise differences in mean shapes was tested using permutation tests  with Mahalanobis distances and Procrustes distances. Additionally, discriminant function analysis (DFA) and cross-validation test was used to confirm the individuals to the correct geographic regions. An UPGMA (unweighted pair-group method with arithmetic average) dendrogram was constructed based on Mahalanobis distances from CVA using PAST V.3.09 software (C. vicina were collected using chicken liver baited fly traps (Red Top) and hand-held sweep nets in Germany and England. Geographic locations and number of specimens are shown in Adult Genomic DNA was isolated from three legs per specimen using 5% chelex suspension. DNA isolation was performed in a 1.5 ml reaction tube. 200 μl of 5% chelex-suspension was added, the sample homogenized, and mixed thoroughly by vortex. Homogenized samples were incubated at 50±5°C for 15 min, vortexed, heated at >100°C for 8 min in a heat block, and centrifuged at 13,000 rpm/15,000 ×g for 5 min. Isolated DNA was stored at -20°C until processing.5’-ATTTCACgCTCATTAACT) and CB_CAL-F07 (5’-gTWATAggAACAgCTTTTATRgg) previously published by Ready et al. [Only those specimens that generated a reasonable amount of DNA (n = 61) were further analysed. Here, a 784 bp long fragment of the mitochondrial cytochrome b gene was amplified using PCR Primers CB_PDR-WR04  following a slightly modified manufacturer’s protocol. The sequencing reaction was performed using only 1 μl BigDye Ready Reaction Mix. Thermal cycling conditions were 28 cycles of the following profile: 96°C for 10 s, 50°C for 5 s, and 55°C for 4 min. Sequencing reactions were purified using gel-filtrated columns  following the manufacturers protocol. Purified Sequencing reactions were run on an ABI3130 genetic analyzer (Life Technologies).P<0.05.Statistical analysis was conducted in SPSS V.22.0 software . Length differences of specimens for each temperature at seven time points was analysed by Kruskal-Wallis test, the times for reaching the various stages of development  were compared by Chi-Square test. Differences were accepted as statistically significant if The tps files containing digitized coordinates of 19 homologous landmarks from all wings were subjected to the MorphoJ software  and thenhttp://folk.uio.no/ohammer/past/).For wing size variation analysis, the overall measure of wing size was estimated by the centroid size. The normality test of centroid size showed non-normal distribution, so wing size difference among species was evaluated by Kruskal-Wallis test. Furthermore, MorphoJ software was used to determine wing shape variation. The variation in the shape space was assessed using a principal component analysis (PCA). Canonical variates analysis (CVA) was used to determine the important features discriminating between groups. The statistical significance of pairwise differences in mean shapes was tested using permutation tests  with Mahalanobis distances and Procrustes distances. Additionally, discriminant function analysis (DFA) and cross-validation test was used to confirm the individuals to the correct geographic regions. An UPGMA (unweighted pair-group method with arithmetic average) dendrogram was constructed based on Mahalanobis distances using PAST V.3.09 software  and Stomoxys uruma (EU851371.1) as outgroups.Quality control and assembly of sequences were performed using CodonCode Aligner . Alignments and phylogenetic analysis were conducted with MEGA 6 (Center for Evolutionary Medicine and Informatics) . Tree coC. vicina at a constant 16°C showed differences in average larval length between both populations at 850, 1200, 1450 and 1800 ADH, with English specimens significantly longer than their German conspecifics  to reach the post-feeding (Pf), puparial (Pu), and adult (A) stages at almost all experimental temperatures: only the durations to the post-feeding stage at 16°C were similar  between male and female. The mean of centroid size ±SD in German flies (CVG1-3) was 1157.04±80.62  and 1285.65±82.94 , whereas for English flies (CVE1-4) it was 1217.95±77.21  and 1300.45±99.52 . Furthermore, wing size measured by centroid size ±SD in each location showed significant differences between CVG1-CVG2, CVG2-CVG3, CVG1-CVE4 and CVG3-CVE4 (Tables The different populations (CVG1-3 and CVE1-4) showed significant wing size variations. The mean ±SD of centroid size was 1227.04±103.88 (CVG1-3) and 1268.80±99.82 (CVE1-4). For the effect of gender on size, both populations showed a significant difference  and 0.0122 (P<0.001). However, Procrustes distances between individual populations were also significantly different , except between CVG1-CVE3, CVG2-CVE1, CVE1-CVE2, CVE1-CVE3, and CVE2-CVE3 , except for CVE1-CVE2, CVE1-CVE3 and CVE2-CVE3. Cross-validation tests showed 73–94% (CVG1), 67–89% (CVG2), 96–99% (CVG3), 55–90% (CVE1), 35–95% (CVE2), 47–89% (CVE3) and 95%-100% (CVE4), which indicated a high percentage of correct separation of CVG3 and CVE4 from other groups.The results of the DFA revealed significant difference of shape between the two countries and showed correct classification at 81% . DFA from wing shape between single groups was also significantly different  model . The anaMelipoua beecheii (Hymenoptera: Meliponini), in Central America. The PCA results clearly indicated the formation of two separated clusters, one comprising the samples from Mexico and the other with the samples from Guatemala, El Salvador, Nicaragua, and Costa Rica. The dendrogram of morphological proximity also supports the existence of genetic lineages, with the population of Mexico in an isolated branch and the other populations sub-divided in another branch. However, using geometric morphometrics of wing shape of dwarf honey bees, Apis florea (Hymenoptera: Apidae), in four states of Iran demonstrated a high level of variation in this species between four states in Iran, with significant differences demonstrated by DFA [C. bezziana were separated according to collection area, Africa  and Asia  [Phlebotomus papatasi (Diptera: Psychodidae) in Morocco [Cochliomyia spp. [Amenia spp. [Drosophila spp. [Eubazus spp. [In this study, geometric morphometric analysis of wing variation from CVA showed overlapping areas for almost every location. Procrustes distances revealed no variation of wing shape between Frankfurt am Main–Liverpool, Dortmund–Exeter, Exeter–Haywards Heath, Exeter–Liverpool, and Haywards Heath–Liverpool, whereas the DFA indicated the separation between two groups of observations between Exeter–Haywards Heath, Exeter–Liverpool, and Haywards Heath–Liverpool. The UPGMA dendrogram divided the populations into two groups, one from Germany and the other from England plus Frankfurt am Main (CVG1). In the latter group, the population from Frankfurt am Main was included within the same branch as the London laboratory colony. This might indicate that the German and English laboratory populations are converging towards a similar form and maybe mirror a potential effect of colonization in the laboratory—in a laboratory environment flight is not so important as in the field. Francoy et al.  comparedd by DFA . Observadonesia)  and anot Morocco . Furtheryia spp.  and Amennia spp. , sand flnia spp. , fruit fila spp. , 45, andzus spp. . Hence, zus spp. , 48. Winzus spp. , 50.Calliphora vicina populations all appeared in a single branch of the tree  using COI. Neighbour joining tree based on COI sequence showed two main groups, one branch comprised Penang and Selangor populations while another consisted of Johor, Pahang, and Sabah populations [Melipona beecheii from Mexico, Guatemala, El Salvador, Nicaragua, and Costa Rica, using the internal transcribed spacer 1 of the ribosomal gene (ITS1). Furthermore, a strong differentiation between North American and West European populations of the blow fly Phormia regina was proven by analyzing the mitochondrial COI, COII, and cyt b genes [C. bezziana by analysis of nuclear and mitochondrial genes [We could not unequivocally discriminate ulations . Includiulations  determin b genes  and cleaal genes , 58C. vicina population by use of the English data. The higher the temperatures are, the less the differences and discrepancies would be, as the impact of applying the lower developmental threshold becomes smaller. A mean time from egg hatch to the point of pupariation for both populations at 16°C, 25°C, and RT (average 21.5°C) was 11 days (3960 ADH), 7 days (4032 ADH), and 9 days (4428 ADH), respectively. According to Donovan et al. [C. vicina growth rate was examined at temperatures between 4°C and 30°C, the minimum developmental temperature was estimated to be 1°C and 4700 ADH were required for development from egg to puparial stage. Furthermore, calculating the number of hours to develop to the puparial stage rearing at 5–29°C (average 13°C) from Wilson and Barnett’s formula showed 391.66 h (16.3 days) [C. vicina at 13°C [C. vicina showed, for example, a 1.2 cm third instar larva which has grown at 25°C would be classified as 30–50 h old with a median age of 35 h [Developmental study of both populations showed significant differences in larval length at certain time points, given as ADH, but is this difference of forensic relevance? Using an example of 1.2 cm larval length growing at 16°C reveals an ADH difference between German and English flies of about 130 see . Applyin, 7 days 032 ADH, .3 days) , 60 Simi at 13°C  and 15.8 at 13°C  demonstr11 days 360 ADH, 7C. vicina show different rates of growth related to their geographical origin.Despite the fact that we recorded different developmental rates at some ADH landmarks, a calculation using the equation of Wilson and Barnett (59) gave for both populations a PMImin estimation on the same day with a natural variation up to ±12 h. Nevertheless, this should not be the end of the story. Our findings highlight an important fact, the need to estimate the temperature threshold of a species, because using no or a wrong value could have dramatic consequences for the age estimation of a forensically important insect when applying the ADD/ADH method. Moreover, it also shows that we need more data on development at temperatures at the margins of development, as the impact of applying a lower threshold is greater at lower ambient temperatures. In times of climate change and extreme weather scenarios, not only lower thresholds of a species should be taken into account. Calliphora spp. could adapt to lower temperatures much better than warm-adapted taxa like L. sericata [C. vicina were observed to survive at high temperatures (35°C) but didn’t make it to pupation [C. vicina’s population died at this temperature [C. vicina is not able to complete its development at a constant temperature of 29°C [C. vomitoria, Lucillia illustris, Sarcophaga argyrostoma) have been demonstrated to develop more rapidly under fluctuating temperatures than at a constant temperature, if the temperatures do not exceed the optimal range for the organism [This study focused on whether or not populations of sericata , but it  Barnett 9 gave foperature . Similar of 29°C . Analyziorganism .C. vicina by the maximum likelihood method did not provide evidence for a consistent difference between the German and English country scale populations like the wing morphometric data did, although even the latter could not separate the different geographic populations from each other at a local level. Future studies using more genes, specimens and locations would be very worthwhile, producing molecular profiles which could serve as an indication of possible population differences useful in forensic case work.Molecular phylogenetic analysis of S1 Data(XLSX)Click here for additional data file.S2 Data(TPS)Click here for additional data file.S3 Data(TXT)Click here for additional data file.


Tapinoma melanocephalum is listed as one of the most important invasive pest species in China. Information regarding the patterns of invasion and effects of geographic isolation on the population genetics of this species is largely lacking.South China.DNA sequencing to compare colony genetic structure of T. melanocephalum on the mainland and islands of South China.To address this problem, we genotyped 39 colonies (two colonies were collapsed due to genetic similarity) using microsatellite markers and mitochondrial SCD) was significantly lower than that of the other colonies. We also found six haplotypes in 111 mitochondrial DNA COI sequences. The relatedness (r) value between colonies of SCD was 0.410, higher than that of the other populations. The genetic clusters among colonies were not related to geographic locations and exhibited admixture likely due to frequent human‐mediated dispersal associated with trade between the mainland population and the islands. Pairwise FSTs between populations showed differentiation among mainland populations, while SCD displayed high levels of divergence (FST > 0.15) from most mainland populations. There was no significant isolation by distance among colonies. Most populations showed signs of a bottleneck effect.An analysis of the colony genotypes showed that the genetic diversity of the mainland population was slightly higher than that of the island populations but not significantly so. However, the observed heterozygosity on Shangchuan Island (SCD population stand out from all the others.Our study suggests that there was no significant difference in the genetic diversity among the islands and the mainland; however, the lower genetic diversity, the higher degree of genetic divergence from other colonies, and the higher relatedness among nestmates made the  T. melanocephalum samples were collected at 23 and 16 points from the mainland and islands, respectively . The alleles were scored using the software PROSize 2.0 .DNA from at least 18 workers from each collection point was extracted using the TIANamp Micro DNA Kit . In total, 778 workers were genotyped at six microsatellite loci: locus 911, locus 1315, locus 364, locus 434, locus 592, and locus 1178 (GenBank accession nos.: KX641151–KX641156). The polymerase chain reaction (PCR) amplification was performed in 25 μl containing 1.5 μl of DNA extract (25 ng/μl), 1 μl of each primer  Co., Ltd., Shanghai, China), 12.5 μl Premix rTaq , and 9 μl of ddHThe partial cytochrome oxidase subunit I (COI) gene of the mitochondrial DNA was amplified and sequenced from 111 individuals from all colonies using the primers LCO1490 (5′‐GGT CAACAAATCATAAAGATATTGG‐3′) and HCO2198 (5′‐TAAACTTCAGGGTGACCAAAAAATCA‐3′)  for each pair of loci were tested by GENEPOP version 4.5.1  to determine the most likely number of genetic clusters (K) using the method of Evanno et al. ; a medium degree of genetic differentiation (0.05 ≤ FST < 0.15); a high degree of genetic differentiation (0.15 ≤ FST ≤ 0.25); and a very high degree of genetic differentiation  and the natural log of the geographic distance (km) were run using GENEPOP version 4.5.1 with Mantel tests . For the SCD population, the SCD2 and SCD3 points, which were 80 m apart, were grouped into one colony . All collection points from the HBD (three colonies), DAD (four colonies), and WZD (two colonies) populations were shown to be unique colonies . For the mainland populations, each pair of collection points was significantly differentiated  and therefore considered separate colonies . Therefore, we considered all loci as independent. Null alleles were estimated by FreeNA software, and their frequency varied from 0 to 0.45. Locus 364 had the highest null allele frequency (0.45), and it exceeded 0.3 in most of the populations. Locus 364 was dropped from further analysis.Four of the six microsatellite loci exhibited significant deviations from Hardy–Weinberg equilibrium . In addition, we also analyzed the genetic diversity of each colony using microsatellite and mitochondrial DNA data. The number of alleles for each colony ranged from 4.4 to 9.0, and the allelic richness varied from 4.3 to 8.8 . Therefore, with the exception of the SJ and BH populations, the genetic diversity of the SCD population was significantly lower than that of the mainland populations (p < .05). Regarding the mitochondrial DNA data  and allelic richness (AR) than the island populations Table , but the3.4T. melanocephalum populations by Bayesian clustering analysis was grouped into two branches  (see Table The relatedness FST < 0). However, comparing the SCD population with most of the mainland populations showed high levels of genetic differentiation (range of FST = 0.1129 to 0.325), except for the nearby SJ population. Two colonies from BH and WZD were grouped into one cluster and showed no differentiation (FST < 0), but ZJ paired with NZD, with a similar geographic distance, exhibiting a high degree of differentiation (FST = 0.1721), which grouped into different clusters. The colonies from ZH, DAD, and HBD were mixed, suggesting a shared ancestry or migration between the islands and mainland due to human activity or species dispersal. When evaluating the correlation between the pairwise FST/(1 − FST) and the geographic distance with all colonies of the island or mainland populations, no significant correlation between genetic differentiation and geographic distance was observed  than within the populations  and within region  in 11 geographic populations, except for the BH, NZD, and SCD populations under the Wilcoxon signed‐rank test of the infinite allele model . The stepwise mutation model (SMM) showed no significant heterozygosity excess in any population. However, the sign test with the stepwise mutation model exhibited significant heterozygosity excess in all populations except GZ, ZH, and HBD populations (p < .05).There was no significant heterozygosity excess in any of the populations determined by the sign test performed with the infinite allele model   and the lowest allelic richness; these colonies all clustered together. Similar to a previous report, invasive species in introduced regions tend to form supercolonies or colonies (Holway et al., Our results indicated low relatedness within all individuals of colonies. But, in the SCD population, colonies exhibited the highest Monomorium pharaonis (Wilson, Lasius sakagamii ant species (Yamauchi, Kinomura, & Miyake, T. melanocephalum, the Argentine ant, and Anoplolepis gracilipes (Bustos, Cherix, Lachaud, & Fourcassié, Anoplolepis gracilipes, which suggested kinship as one factor causing the formation of a supercolony. Kinship also facilitates colonization (Drescher, Blüthgen, & Feldhaar, A. gracilipes undergo random mating; intranidal mating system in this ant can be observed due to genetic cohesion in these two supercolonies (Thomas et al., Many reports of invasive species in different geographic regions suggest that social behavior is related to colony breeding structure. In its native range, the Argentine ant forms small supercolonies that are effectively unicolonial by intranidal mating and kin recognition (Pedersen, Krieger, Vogel, Giraud, & Keller,  Wilson,  and LasiT. melanocephalum should also be a focus of future studies as well the relationship between colonies in China and those in other parts of the world.Closely related colony members in polygynous nests may arise from inbreeding, reproductive dominance of one queen, and/or high relatedness among queens (Giraud, Blatrix, Poteaux, Solignac, & Jaisson, None declared. Click here for additional data file.


Medaka is a model organism in medicine, genetics, developmental biology and population genetics. Lab stocks composed of more than 100 local wild populations are available for research in these fields. Thus, medaka represents a potentially excellent bioresource for screening disease-risk- and adaptation-related genes in genome-wide association studies. Although the genetic population structure should be known before performing such an analysis, a comprehensive study on the genome-wide diversity of wild medaka populations has not been performed. Here, we performed genotyping-by-sequencing (GBS) for 81 and 12 medakas captured from a bioresource and the wild, respectively. Based on the GBS data, we evaluated the genetic population structure and estimated the demographic parameters using an approximate Bayesian computation (ABC) framework. The genome-wide data confirmed that there were substantial differences between local populations and supported our previously proposed hypothesis on medaka dispersal based on mitochondrial genome (mtDNA) data. A new finding was that a local group that was thought to be a hybrid between the northern and the southern Japanese groups was actually an origin of the northern Japanese group. Thus, this paper presents the first population-genomic study of medaka and reveals its population structure and history based on chromosomal genetic diversity. Oryzias latipes) is a small fresh-water fish native to East Asia that has attracted attention as a vertebrate model for population genetics . Medaka is divided into four mitochondrial groups: the northern Japanese (N.JPN), southern Japanese (S.JPN), eastern Korean (E.KOR) and western Korean/Chinese groups (W.KOR)  and S1, b gene sequences, N.JPN and S.JPN are composed of 3 and 11 subgroups, respectively  and north end (Ichinoseki in Iwate) inhabited by S.JPN were 11.6° and 988 mm in 2017, respectively (http://www.jma.go.jp/jma/indexe.html). The phylogenetic data from these studies also suggested that N.JPN and S.JPN have been spreading in the Japanese archipelago at different times. Particularly, the origin of S.JPN, which has the largest habitat, has been suggested to be the northern part of Kyushu Island based on mtDNA (“Out of Northern Kyushu” hypothesis)  using all 81 local populations maintained as wild lab stocks at the University of Tokyo. We examined an individual sampled from each population stock and 12 wild individuals captured from the northern part of Kyushu Island, where  at the University of Tokyo in 2014. The lab stocks originated from geographically distinct populations in East Asia and have been maintained since 1985 . These let al.  for the individual sample. The data have been submitted to the DDBJ Sequence Read Archive (DRA) database under project accession ID: DRA006353.One-third of the medaka body was dissolved in a 600 µl lysis buffer containing 1.24% SDS, 0.124 M EDTA and 0.062 mg/ml proteinase K . The total genomic DNA was extracted and purified using phenol-chloroform and isopropanol precipitation. After a 70% EtOH wash, an isolated DNA pellet was resuspended in 100 μl TE buffer and then treated with RNase A  for 1 hr at room temperature. Then, the DNA was purified again using phenol-chloroform and isopropanol precipitation. For ninety-three samples, the GBS process was outsourced to Macrogen Japan in Kyoto. The procedures for constructing libraries and performing Illumina HiSeq 2000 single-end sequencing were the same as those described by Poland FASTQ Quality Filter in FASTX-Toolkit version 0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/download.html) using the following options: -Q 33 -v -z -q 30 -p 90. The draft genome of the medaka sequenced by the PacBio sequencer  was used to align the reads using BWA backtrack 0.7.12-r1039 (Samtools v1.2 (Our single-end reads were filtered using 12-r1039  using thStacks pipeline (version 1.35) and the Stacks workflow (https://github.com/enormandeau/stacks_workflow) were used to generate SNPs and sequences for each individual separately (Stacks parameters were as follows: minimum stack depth (-m), 3; and number of mismatches when building the catalog (-n), 1. In the ‘rx’ step of Stacks, we used the bounded SNP model set to 0.1, the ε upper bound set to 0.1 and the log likelihood set to 10. Following this pipeline, five datasets were constructed using the population program in Stacks.The parately . The selThe first was the “PopStat” dataset, which was generated using the “-p 5 -r 0.66” options and mitochondrial grouping , respectively, including the 15 boundary populations  with or without the Kyushu populations . These datasets were generated using the “-p 3 -r 0.70” (without Kyushu) and the “-p 4 -r 0.70” (with Kyushu) option, respectively, to assess the genetic population structure and the history of the boundary population for the Tajima-Tango group.The fifth was the “Local” dataset, including two Kyushu deme samples: one was Umejiro (US), which was sampled in the northern part of the Saga prefecture, and the other was Ogi (SO), which was sampled in the southern part of the Saga prefecture. These samples were used to estimate the time of Honshu’s population divergence from Kyushu to infer the timing of the “Out of Northern Kyushu” event , the SNPs with strong linkage disequilibrium were randomly removed, one from each pair of SNPs with rSNPRelate program  of the “Global” dataset as implemented in the RE v1.23 .population program with the “-p 58 -r 1.00–phylip_all” option in the Stacks pipeline. The dataset included 4,638 partitions and 217,986 bp of nucleotide sequences to compensate for the loci that could not be sequenced by invariable sites across all samples and “N” at the variable site. The dataset including 4,638 partitions and 217,986 bp nucleotide sequences was analyzed via the IQ-TREE program  to visualize the phylogenetic tree.We considered an individual as representative of a population and generated the individual sequences using the DIYABC ver2.1.0 . The same prior parameters were defined for four scenarios based on previous studies, and the prior distribution of each parameter is presented in Using the HZ-2 dataset, which added five Kyushu populations to 15 boundary populations of the HZ-1 data set, the population history, including the possibility that the Tajima-Tango group occurred by the admixture of N.JPN and S.JPN, was inferred from 1,014 SNPs using ver2.1.0 ; (III) NDIYABC  in DIYABC and accepted the parameters that were non-scaled and scaled by the mean effective population size for the HZ-2 and Local datasets, respectively. Using the infinite-sites model in the Wright-Fisher populations of a constant size, a rough value of Ne was estimated using the relationship π = 4Neµ  and deposited into the international DNA database DDBJ/EMBL/GenBank (accession number: LC335803).To examine the mitochondrial introgression in the tested samples, we generated and analyzed the mitochondrial DNA (mtDNA) partial sequences from the GBS-read data. We mapped the reads to the complete mtDNA sequence of “Clade C” . GBS data are available at DDBJ Sequence Read Archive (DRA) database with the accession number: DRA006353. The complete mtDNA sequence is available at the international DNA database DDBJ/EMBL/GenBank with the accession number: LC33580. Supplemental material available at Figshare: https://doi.org/10.25387/g3.7358042.Wild lab-stocks are available upon request , southern Japanese (S.JPN), eastern Korean (E.KOR) and western Korean/Chinese (W.KOR) groups— and hybridization group which is Tajima-Tango group, their population-genetic summary statistics were calculated using the “PopStat” dataset that included the alignment regions across all groups and contained approximately 45 kb of sequences and 2,453 SNPs . All groTo reveal the medaka population structure based on the chromosomal SNPs, we performed genetic clustering analyses using the “Global” dataset. First, we investigated the genetic relationship by a principle component analysis (PCA) using 8,361 SNPs on 24 chromosomes. The PCA showed that the wild lab-stocks were divided into two clusters, N.JPN/Tajima-Tango and S.JPN and others from E/W.KOR, which is similar to the grouping based on the mtDNA data  and S6. Although Tajima-Tango was considered to be a hybridization group between N.JPN and S.JPN , all indIQ-TREE. The maximum likelihood tree showed relatively high bootstrap values on each branch and almost the same topology as previous trees based on mtDNA , S.JPN medakas were assigned to two distinct ancestries  tree based on 4,638 short fragment sequences  using cestries  and S2. i.e., specific alleles in Tajima-Tango. These proportions are near those of a previous study based on 96 genomic regions (4 loci for each chromosome) . We classified the fixed alleles between the groups into three states: shared by N.JPN and Tajima-Tango, shared by S.JPN and Tajima-Tango, and shared by N.JPN and S.JPN. Then, we summarized each state’s total numbers, as shown in table in To investigate the mitochondrial introgression in boundary populations, we reconstructed the phylogenetic tree based on partial mtDNA sequences generated by mapping short reads from the GBS of the complete mitochondrial genome . The mtDNA sequences from Toyooka and Kinosaki in Tajima-Tango were clustered and closely related to those from Kaga, which was a root population in N.JPN. Considering that Tajima-Tango was a subgroup of N.JPN, which we inferred from the ML tree based on chromosomal sequences, these phylogenetic positions on the mtDNA tree would have also reflected an evolutionary history in which N.JPN diverged from the Tajima-Tango group. We confirmed that Amino, Kumihama and Hamasaka in Tajima-Tango had the mitochondrial genomes of phylogenetically distant populations classified into S.JPN . Additionally, the mitochondrial genome of Ayabe in S.JPN was classified into N.JPN but diverged from Tajima-Tango. These results suggest that mitochondrial genome introgressions occurred independently, meaning that they occurred not only from S.JPN to Tajima-Tango but also from N.JPN to S.JPN.Ne), time (T), and proportion of the admix (r), we analyzed the “HZ-2” dataset based on the coalescent theory using an approximate Bayesian computation (ABC) framework. We performed the model selection to identify the best explanation scenario for the observed data from the four scenarios; (I) Tajima-Tango originated in N.JPN, (II) Admixture of N.JPN and Honshu, (III) N.JPN originated in Tajima-Tango, and (IV) Admixture of Tajima-Tango and Honshu  for model selection. Based on each criterion, scenario III (Honshu diverged from Kyushu and then N.JPN diverged from the Tajima-Tango group without admixture with Honshu) and scenario IV (Honshu diverged from Kyushu and then Tajima-Tango occurred by an admixture with Honshu at r rate) were supported by the directional  and logistic  approaches, respectively . Our data could not distinguish between scenarios III and IV (see the Discussion section).To infer the demographic parameters, effective population size (d Honshu . DIYABC DIYABC output) indicated that non-scaled parameters fit the observed data better than scaled parameters. These estimates showed that the time to the most common ancestor (TMRCA) between S.JPN and N.JPN in scenarios III and IV were dated to 4,560,000  and 5,160,000  years ago, respectively. However, although we inferred the TMRCA at three divergence events , which were split at first on the S.JPN lineage  by mtDNA sequences and allozymic electrophoresis patterns  the nucleotide diversity in aN.JPN is higher than that in dN.JPN , (ii) S.ADMIXTURE analysis shows that the two ancestry components ; however, it contains the lowest watershed (sea level 95.45 m) in the Japanese archipelago. The medaka has possibly moved in both directions across the watershed. From the above, the most plausible scenario is as follows . AncestrOur genome-wide SNP analysis reconstructed the detailed population structure and reliable history of medaka that evolved in the Japanese archipelago. Since the distribution of the subgroups was highly consistent with the geographical features, several adaptive traits could have evolved in each subgroup. Furthermore, the boundary populations were not caused by a hybridization event but instead were the origin of the populations dispersed to a northeastern part of the Japanese archipelago on the Sea of Japan side. A better understanding of the population structure and history of medaka will support association studies for phenotypes and genotypes related to environmental adaptation.