Dendrolimus punctatus, is one of the most destructive forest pest insects and is an endemic condition in China, we still do not fully understand the patterns of how its distribution range varies in response to Quaternary climatic oscillations. Here, we sequenced one maternally inherited mitochondrial gene (COI) and biparentally inherited nuclear data (ITS1 and ITS2) among 23 natural populations across the entire range of the species in China. A total of 51 mitotypes and 38 ribotypes were separately obtained using mtDNA and ITS1 data. Furthermore, significant phylogeographical structure  were detected. The spatial distribution of mitotypes implied that two distinct groups existed in the species: one in the southwest distribution, including 10 locations, and the other located in the northeast region of China. It is suggested, therefore, that each group was derived from ancestors that occupied different isolated refugia during previous periods, possibly last glacial maximum. Mismatch distribution and Bayesian population dynamics analysis suggested the population size underwent sudden expansion, which is consistent with the results of ecological niche modeling. As a typical phytophagous insect, the history of population expansion was in accordance with the host plants, providing abundant food resources and habitat. Intraspecific success rate of barcoding identification was lower than interspecific ones, indicating a level of difficulty in barcoding individuals from different populations. However, it still provides an early insight into the pattern of genetic diversity within a species.Although the Masson pine moth, https://doi.org/10.5061/dryad.2df87g2. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.This article has been awarded an Open Data and Open Materials. All materials and data are publicly accessible via the Open Science Framework at  The oscDendrolimus punctatus  distributed in subtropical China was adopted in our study to identify whether the genetic differentiation of host plants, caused by climate changes during the ice age, affects genetic variation of their predators, the phytophagous insects. Dendrolimus species (Lepidoptera: Lasiocampidae) are one of the most serious phytophagous pests worldwide and caused extensive forest damage  and second spacer (rDNA ITS2). Two different inherited molecular markers were included in this study: mtDNA marker (COI) represented maternal inheritance; the ITS fragments represented the biparental inheritance. In addition, these three molecular markers shared barcoding sequences well and proved to be excellent methods to help explain the interspecific relationship among the sibling species of D. punctatus . The same PCR primers and amplification reaction of Dai et al.  to check whether the amplification reactions were successful. Then, the amplification products were used for sequencing, which was performed with an ABI3130 sequencer at Biomed Biotechnology Co., Ltd. All sequences have been deposited in GenBank , and the COI barcodes were also submitted to the BOLD system (accession numbers: GBGL14669–GBGL14904).DNA samples were isolated from the legs of adult individuals using DNeasy Blood and Tissue kit (Biomed) following the manufacturer's protocol. Sequences of mitochondrial gene i et al.  were use2.3COI sequences were translated into amino acid sequences using the MEGA program  inference and Bayesian inference (BI). Two specimens from species Dendrolimus superans  were used as outgroups. The best‐fit model of nucleotide substitution was selected from 88 models using the Akaike information criterion (AIC) with jModelTest 2  haplotype networks were constructed to draw an unrooted network with Network 5 (Fluxus Technology Ltd.) based on COI gene, as well as ITS1 and ITS2.The raw DNA sequences were all checked manually and aligned with software MUSCLE Edgar, . Aligned2.4COI gene and the ITS fragments. We used DnaSP 5.10 , number of haplotypes (Nh), haplotype diversity (h), and nucleotide diversity (π). The program PERMUT (available at www.pierroton.inra.fr/genetics/labo/Software/) was applied to estimate the average gene diversity within a population (HS), total gene diversity (HT), and between‐population divergence  with 1,000 permutations tests.All analyses were executed separately for the FST), and Mantel test were all calculated to detect the population structure with Arlequin V3.5 software , the average values of population differentiation  was applied to estimate time since expansion (in generations) using the equation t = τ/2u , k represents the average DNA sequence length, and g represents the generation time in years.To evaluate the population history of jima's D  and Fu's's D , and we discarded the burn‐in of the first 20% steps. The evolutionary models using AIC were selected for COI (GTR) and ITS1 (TVMef+I) using jModeltest2  implemented in BEAST v2.0 were also used to evaluate the timing of the population expansion . We compared the suitable habitats and potential distributions of D. punctatus in two periods: the present day and the LGM. The environmental layer of the present and LGM were built at a resolution of 2.5 min, and the LGM dataset was based on the Community Climate System Model (CCSM). A distribution was generated using 15 climatic variables from the WorldClim database  for the current climate, with an estimate based on other distribution localities that were collected  computed using equation (20) from Zhang et al. (The distance between intraspecific variation (the DNA barcoding gap) is considered an important criterion in DNA barcoding. To explore the feasibility of populations that identify within g et al. . The BCMg et al. .33.1COI dataset, which contained 43 parsimony informative sites. These polymorphic sites defined 51 unique haplotypes (H1–H51) from the 236 sampled individuals of D. punctatus, 18 of which were found in single individuals. A quarter of haplotypes (15 of 51) were unique to single populations, while others were shared (Table π = 0.0075 and varied across populations (ranging from 0.0011 to 0.0082). Within all populations, GZXY  at the species level, and ZJLX  was much lower than total gene diversity (HT)  were recovered (R1–R38), two of which occurred in single populations separately. Meanwhile, the other haplotypes were shared by more than two populations. The most widespread ribotypes1 was R26 occupied by 19 populations. Ribotype1 diversity was estimated to be h = 0.773 at the species level, ranging from 0.000 to 1.000 in different populations. At the species level, nucleotide diversity was 0.008 (p = 0.022), but it varied across populations, ranging from 0.000 to 0.0189, with the GDXN  were defined (N1–N29). Overall nucleotide diversity was 0.0028, and haplotype diversity was 0.818 (ranging from 0.167 to 1.000). N15 was the most popular ribotypes2, which was found in 16 populations of the southern region. Similar results were detected comparing with the mitochondrial dataset. Within‐population gene diversity (HS) was lower than total gene diversity (HT)  and two monotypic clades (Clades IV and V)  Figure a. The ha) Figure b display) Figure .ITS1 dataset, the topological structure of the phylogenetic trees, reconstructed by two methods, were similar , indicating a strong phylogeographic structure in D. punctatus. The Spatial AMOVA (SAMOVA) method represents a useful method to define partitions of sampling sites that are maximally differentiated from each other without any priori assumption of population structure. However, the analysis did not clearly reveal one single group of maximally differentiated populations, as FCT values increased progressively with increasing values of K ; however, the first level of divergence (K = 2) revealed a well‐defined group including those populations from the east region of China with a high frequency of mitochondrial haplotype Clade I, which were differentiated from the remaining populations. The composition of the groups detected by SAMOVA that had increasingly larger values of K mainly corresponded to the geographical distribution of haplotypes  in 1,000 permutation tests between two geographic regions: 0.393 for COI, 0.363 for ITS1, and 0.523 for ITS2, respectively. This suggested high genetic differentiation between these two geographic groups.Significant genetic differentiation was identified in es Table . Further3.4COI, ITS1, ITS2, or combined ITS datasets, the null hypothesis that showed sudden population expansion historically existed could not be rejected  and 0.17 Mya (ITS2)  compared with genetic variation within population . The former was roughly 1.1 (1.1038) and 1.6 (1.6111) times larger than the latter. Similarly, there were no positive barcoding gaps or a blurred discriminations among subpopulations with ITS1 and ITS2  of 46.83%–55.55% (MD), 35.40% (95% CI: 31.33%–39.68%) (BCM), while corresponding interspecific success rates achieved values of 97.40% (95% CI: 95.60%–98.47%) and 96.60% (95% CI: 94.62%–97.87%), respectively  in D. punctatus, which might reflect a longer evolutionary history and the wide distribution of this species. However, the AMOVA analysis based on COI dataset revealed genetic differentiation among all 23 populations and most variation was from within populations (47.75%), followed by variation among groups (28.74%) .The mtDNA diversity was relatively high HT = 0.97 in D. pITS1 were corroborated with our COI findings, and distinct phylogeographic signals  were found in entire distribution regions, including western and eastern regions. A relatively low value of total diversity was recorded for ITS dataset in D. punctatus , which was lower than the mitochondrial dataset, largely due to reduced genetic divergence , as within‐population diversity was also decreased compared with mtDNA (HS = 0.595 for ITS1 and HS = 0.587 for ITS2). The primary cause of this discrepancy is due to the different modes of inheritance, maternal for mtDNA and biparental for ITS. The latter method leads to higher levels of gene flow. Compared with the protein‐coding COI region, the noncoding ITS marker was more sensitive at detecting gene flow among interbreeding populations. Similar inconsistencies were shared in many other Lepidopteran insets, for example, Biston suppressaria   Table . This phy) Table . Althougy) Table . Continuy) Table , which my) Table . RestricD. punctatus , which experienced drastic reduction in population size due to the coverage of ice sheet during the Quaternary  . During the interglacial periods of Penultimate Glaciation, populations could be effectively preserved in the mountainous areas of southern China. In addition, D. punctatus is one of the most serious and economically damaging insect pests, which have the genetic characteristics of population outbreak initiated by high temperature and drought  and Eurycorymbus cavaleriei (Sapindaceae), and several postulating separate refugia  in subtropical China , a closely related species of the host plant P. massoniana, were demonstrated among group FST differentiation, suggesting that Yungui Plateau and the Mts Nanling were a major refugium  and postglacial‐Holocene warm climate (0.018–0 Mya). The warm climate and high sea level influenced interglacial continental vegetation distribution.Temperate evergreen forest, such as D. punctatus suggests that several isolated refugia were existed where mtDNA divergence occurred. In addition, population dynamic analysis indicated that the D. punctatus species experienced population expansion, and an obvious northward recolonization was detected. The haplotype distribution of the present investigation seems further to indicate that the common fixture of the same haplotype among the disjunct populations was mainly caused by the founder effect during the population recolonization in this region. The highest latitude population  formed a monophyletic clade (Figure COI gene, less population structure was also found for ITS genes on the phylogenetic trees (Figure D. punctatus populations, which also impaired the success rate of population assignments and resulted in no positive barcoding gaps being detected (Figure To assign unknown individuals to certain populations within a species or to identify population from DNA sequences is not a new approach (Lou & Golding, s Figure . For thes Figure , 5. The d Figure .D. punctatus. It would be interesting to investigate further the applicability of barcoding information in the study of genetic diversity of other species.Despite the fact that the sequence information collected from DNA barcoding was not sufficient to solve population‐level questions, it reflected genetic diversity among different population (Bazin, Glémin, & Galtier, 5D. punctatus based on the genetic variation of mtDNA as well as nuclear markers. A history of postglacial recolonization of the populations and multiple refugia during the ice age of the Quaternary was inferred. Many previous phylogeographic studies have paid close attention to the effects of very large mountainous systems such as the Mts Hengduan and Mts Qinling, but less attention was paid to the effect of the smaller mountain regions in the subtropical regions of China. The findings of this study proved that several potential refugia and geographic barriers existed in these hilly areas. These results provide a phylogeographical hypothesis for further testing; however, given the small sample size and limited molecular markers, many low‐copy nuclear genes and mitochondrial genomes are required to further examine the genomic divergence within D. punctatus.We investigated the population structure and dynamics of None declared.Jing Li wrote the MS and Qian Jin performed the experiment. Jing Li and Qian Jin analyzed all the data together. Geng‐ping Zhu and Chong Jiang provided great help in ecological niche modeling analysis. Ai‐bing Zhang defined the project as the main supervisor and considered the whole analysis.


Allium species, A. neriniflorum and A. tubiflorum. We sequenced three cp DNA (cpDNA) fragments  of two closely related Allium species, with samples from 367 individuals in 47 populations distributed across the total range of these species. The interspecific and intraspecific divergence times of the two species were in the Quaternary glaciation. The population divergence was high for the cpDNA variation, suggesting a significant phylogeographic structure . Remarkable ecological differentiation was also revealed by Niche models and statistical analyses. Our results suggest the speciation event of the two species was triggered by violent climatic changes during the Quaternary glaciation.This study investigated the effects of climate oscillations on the evolution of two closely related  Most species of Allium have an onion flavor and adapt to arid climatic conditions with developed underground storage organs. However, it is very difficult to resolve Allium taxonomy due to the great diversity in the morphology, biological learning, and ecological adaptation of Allium  G. Don (A. neriniflorum), Allium tubiflorum Rendle (A. tubiflorum), and Allium inutile Makino (A. inutile)  and Japan  Are A. neriniflorum and A. tubiflorum significantly different from molecular levels? Is the genetic diversity of A. neriniflorum and A. tubiflorum analogous? (b) Where were the refugia of both species during the climatic oscillations? Did they survive in situ or retreat to somewhere below to 30°N during climatic oscillations? (c) How did the climatic oscillations or other geological factors effect on the population evolutionary history of these two species?In this study, we sampled populations across the distribution range of 22.1A. inutile in Anhui, China. As for A. neriniflorum and A. tubiflorum, we found that their morphological characters are very similar. According to our statistical analysis, the main difference in the shape of these two species based on quantitative characters is that the plant height and pedicel length, A. neriniflorum are usually taller in plant height and longer in pedicel length than A. tubiflorum , A. tubiflorum , and three probable hybrid populations  in China  and ‐rps‐R2 (5′‐TCG GGA TCGAAC ATC AAT TGC AAC‐3′)  and ‐trnL (5′‐CTGCTT CCT AAG AGC AGCGT‐3′)  and ‐trnTGUC (5′‐CTA CCA CTG AGT TAA AAG GG‐3′) ; annealing ; extension ; and a final extension for 10 min at 72°C.The total genomic DNA was extracted from 100 to 150 mg dry leaves using a plant genomic DNA kit .Three cpDNA fragments, The PCR products were separated in 1.5% (w/v) agarose TAE gel and purified by the Wizard PCR Preps DNA purification system  following the manufacturer's instructions. The cycle sequencing reactions were carried out with the purified PCR product, AmpliTaq DNA polymerase, and fluorescent Big Dye terminators. The sequencing products were generated using an ABI Prism 310 DNA sequencer  that sequenced in both directions using the PCR primers.2.32.3.1rps16, rpl32‐trnL and trnD‐trnT were joined into one cpDNA dataset. Sequences were aligned using ClustalX ver. 1.81  haplotype network, which was constructed using NETWORK ver. 5.0  analyses were carried out using RAxML‐HPC BlackBox 7.7.1  and nucleotide diversity (π), were estimated with DNASP version 6.0 , total gene diversity (HT), and two‐population differentiation parameters (GST and NST) at the species level were statistically compared using 1,000 permutations in the program Permut CpSSR 2.0   of A. tubiflorum and its congener A. neriniflorum experienced a recent population expansion, a mismatch distribution analysis was carried out using the DNASP program , Specimen National Specimen Information Infrastructure . Based on the 57 records, the potential habitats of A. tubiflorum and A. neriniflorum were modeled with the widely used program Maxent 3.4.1   spatial resolutions were retained: the mean diurnal range, isothermality, temperature seasonality, mean temperature of the driest quarter, mean temperature of the warmest quarter, annual precipitation, precipitation of the wettest month, and precipitation seasonality.Nineteen climate variables based on mean values from 1970 to 2000 were downloaded from the WorldClim database , only the linear + quadratic + hinge functional forms were used . We used a binary threshold from DIVA‐GIS of zero, which excluded the zero‐suitable habitats, and the minimum training presence cumulative threshold was from Maxent  was performed to plot the occurrences of A. tubiflorum and A. neriniflorum in relation to the eight climate variables using Canoco 5.0  using SPSS. The significance of Wilks’ k was tested by chi‐square tests. If the p‐value of chi‐square is <0.05, a significant ecological difference between the two species can be concluded.Three statistical approaches were used to investigate the ecological divergence between 33.1rps16, rpl32‐trnL and trnD‐trnT sequences of Allium were 819, 888 and 777 bp in length, respectively. A 2,500‐bp data set with 36 nucleotide substitutions and 11 indels was obtained when all the three fragments were combined (Supporting Information Table rpl32—trnL), MG709290–MG709317 (rps16) and MG709318–MG709345 (trnD—trnT).The aligned A. neriniflorum, 14 populations of A. tubiflorum and three probable hybrid populations in central and northern China  were only found in A. tubiflorum. Haplotypes H1–H3, H5, H7, H8, H15, H17, and H18 occurred in two or more populations, and the other haplotypes just existed in a single population , followed by haplotype H5 (frequency 10.1%), which was found in nine A. neriniflorum populations ; for A. tubiflorum, the most common haplotype was H18 (frequency 10.1%), which was identified in five A. tubiflorum populations . The total cpDNA diversity of all populations (Hd = 0.887) was very high. The total cpDNA diversity was significantly higher in A. tubiflorum (Hd = 0.826) than in A. neriniflorum (Hd = 0.734).Twenty‐five haplotypes H1–H25) were detected from 30 populations of –H25 wereA single tree was obtained by maximum likelihood analysis, and two clades were identified: clade I and clade II. Clade I consists of H1–H13, which is distributed in the northern region. Clade II include H15–H25, all occurring in the southern region  is 0.265 ± 0.0427, and the total gene diversity (HT), 0.883 ± 0.0304. The coefficients of differentiation of the two species NST (0.844) were high and higher than GST (0.798) (permutation test: p = 0.01), indicating significant population differentiation and suggesting the existence of a phylogeographical structure . Approximately 76% of the variation was attributed to the species differentiation, and among‐population variation accounted for just one‐quarter (16%) of the total variation . This result showed that the gene flow among populations of A. tubiflorum was lower than that among populations of A. neriniflorum. Overall, these results strongly indicate that the haplotypes are geographically structured across the species distribution range.AMOVA analysis revealed that, in all the sampled populations, the variation attributed to differences between species was 76.22%, which is very significant, while that variation among and within population differences were 15.85% and 7.93%, respectively (Table A. tubiflorum had higher levels of genetic diversity based on cpDNA (Hd = 0.826) than A. neriniflorum (Hd = 0.734) (Table A. tubiflorum populations (FST = 0.87) was also far higher than that among A. neriniflorum populations (FST = 0.55). Accordingly, the calculated gene flow among A. tubiflorum populations (Nm = 0.07) was lower than that among A. neriniflorum populations (Nm = 0.40).Cp DNA data shows that 4) Table . Moreove3.3A. neriniflorum and A. tubiflorum underwent a recent demographic population expansion, we plotted the mismatch distribution of each group based on the observed number of differences between pairs of haplotypes. The mismatch distributions of the two species were clearly multimodal curves  were significantly positive values, which confirmed that the A. neriniflorum and A. tubiflorum populations had not undergone a recent expansion.To test whether population groups of s Figure . The tes3.4A. neriniflorum and A. tubiflorum was inferred using BEAST 2.5 , demonstrating a reliable predictive model performance. Model exhibited distinctive ecological niches between A. neriniflorum and A. tubiflorum   Table .44.1A. neriniflorum and A. tubiflorum, although both species show close morphological similarities  in the edge of the Taihang Mountains and Qinling Mountains   and central region  of the range  Tian, . For A. e Figure . Additio4.3A. neriniflorum and A. tubiflorum, the divergence time was estimated based on genetic distances . In this period, Taihang Mountains nearly formed a S‐N‐striking uplifted mountain range within the central North China block. However, the divergence time is different from that in Hauenschild et al.  at approximately 1.3446 Ma. Almost simultaneously, the intraspecific divergence of A. tubiflorum occurred at approximately 1.168 Ma. These data show that the simultaneous intraspecific differentiation of A. tubiflorum and A. neriniflorum resulted from common climate events in the Quaternary , in the late stage of the Early Pleistocene (Cui et al., A. tubiflorum and A. neriniflorum can be attributed to the climate oscillation of the largest glaciation in the Quaternary (Zhang, Fengquan, & Jianmin, A. neriniflorum and A. tubiflorum mainly resulted from the violent climate changes that occurred in the Quaternary glaciation.We inferred that the populations of ancestral taxon about None declared.JTY and XJH designed the experiment. JTY collected the field data. JTY, SDZ, and DQH analyzed the data. JTY drafted the text. All authors revised it critically and significantly improved the text.https://doi.org/10.6084/m9.figshare.6224939.DNA sequences: Genbank accessions MG709262–MG709345. Climate data and MaxEnt input files, morphological data, Sampling locations: Figshare.  Click here for additional data file.


Martes americana), a species rediscovered on Isle Royale 76 years after presumed extirpation. We characterized the genetic composition of martens throughout the Great Lakes region using nuclear and mitochondrial markers, identified the source of Isle Royale martens using genetic structure analyses, and used demographic bottleneck tests to evaluate (eliminate redundancy of test). 3 competing colonization scenarios. Martens exhibited significant structure regionally, including a distinct Isle Royale cluster, but mitochondrial sequences revealed no monophyletic clades or evolutionarily significant units. Rather, martens were historically extirpated and recolonized Isle Royale from neighbouring Ontario, Canada in the late 20th century. These findings illustrate the underappreciated dynamics of island communities, underscore the importance of historical biogeography for establishing restoration baselines, and provide optimism for extirpated and declining Isle Royale vertebrates whose reintroductions have been widely debated.Island ecosystems are globally threatened, and efforts to restore historical communities are widespread. Such conservation efforts should be informed by accurate assessments of historical community composition to establish appropriate restoration targets. Isle Royale National Park is one of the most researched island ecosystems in the world, yet little is actually known about the biogeographic history of most Isle Royale taxa. To address this uncertainty and inform restoration targets, we determined the phylogeographic history of American martens ( Due to their seeming simplicity, islands are often useful models to understand ecological interactions5 and the evolutionary histories of regional taxa6. Moreover, islands have served as refugia in the face of global change throughout history8, often resulting in endemic species or unique genetic lineages due to long-term isolation9. Yet, contemporarily, islands are some of the most altered ecosystems10 and are regularly subject to introduced species11, novel diseases12, and overexploitation13. Consequently, extensive conservation programs have been established to protect island biodiversity15 and to restore departed island communities17. Historical community composition, though, is not always clear, leading to ambiguous restoration baselines and the potential mismanagement of native and invasive species19.Island ecosystems, and the unique taxa they feature, have fascinated biologists since the inception of ecology and evolution21. Moreover, Isle Royale is home to the longest running predator-prey study on record, resulting in a comprehensive understanding of wolf (Canis lupus)-moose  interactions, and a deeper appreciation for paired population and trophic dynamics23. Recent declines in wolf abundance have also sparked widespread discussions on conservation ethics, wilderness management, and the restoration of island communities25. Despite this attention, little is actually known about the 17 other mammals that inhabit Isle Royale, and widespread species turnover throughout the 20th century has obscured historical community composition26. Indeed, historical assemblages of vertebrates have been constructed entirely from museum surveys and anecdotal accounts28. Furthermore, lake ice formation, a primary mode of colonization to Isle Royale, has become increasingly stochastic due to regional climatic warming, potentially disrupting historical connectivity to the mainland29. Thus, the restoration of Isle Royale fauna is confounded by uncertainties in both historical community composition and future colonization potential26. Nevertheless, the reintroduction of Isle Royale carnivores has garnered widespread consideration31 and illuminated the need for a priori restoration baselines derived from phylogenetic histories of past and present community members26.Isle Royale National Park is an isolated archipelago in the western reaches of Lake Superior, USA. A national park since 1931 and a designated wilderness area since 1976, Isle Royale is widely regarded as one of the most pristine island ecosystems in the worldth century that included the extirpation of Canada lynx (Lynx canadensis), woodland caribou (Rangifer tarandus caribou), and, presumably, American martens 32. Though historically abundant on the island27, martens were valuable furbearers and trapped heavily until 1917 when the last recorded specimen was collected33. Following four decades of presumed absence, the National Park Service initiated a program to reintroduce martens from Ontario, Canada to Isle Royale in 1966; however, the translocation of martens to the island was never documented, though such a release cannot be completely discounted34. A quarter century later marten tracks were observed, and in 1993 martens were once again confirmed on Isle Royale34. Martens have since remained rare following this apparent 76-year absence, and the origins of this extant population are unknown.Prior to establishment as a National Park, Isle Royale was subject to significant anthropogenic disturbances at the turn of the 20c. 11,000 years, resulting in unique lineages of several taxa36. Like other historical community members , martens could constitute an evolutionarily unique population. Throughout the Lake Superior Basin, however, martens have a dynamic history of extirpation and reintroduction that has resulted in a complex configuration of local populations with unique genetic structures38. Indeed, following widespread local extirpations, martens have since been reintroduced to Wisconsin and Michigan from source populations in Minnesota and Ontario, respectively, while Wisconsin also received translocated martens from Colorado now known to be non-native Pacific martens (Martes caurina39)  Fig. . Consequ38, we then assessed the potential for Isle Royale martens as an evolutionarily significant unit (ESU41). Finally, we used demographic bottleneck tests to assess three putative colonization scenarios: (1) an historic, pre-settlement colonization; (2) a successful 1966 reintroduction; and (3) a modern colonization consistent with the timing of rediscovery. Due to their historic prevalence, and the ability of martens to maintain cryptic populations for millennia19, we hypothesized that the extant marten population on Isle Royale stemmed from a historical colonization and presents an endemic evolutionary lineage. We predicted significant structure among nuclear markers, with martens on Isle Royale representing a distinct genetic cluster, and we expected reciprocal monophyly across mtDNA sequences for martens on Isle Royale.Herein, we identified the source and colonization history of the recently rediscovered marten population on Isle Royale using population genetic and demographic analyses. We explored the phylogenetic history of martens on Isle Royale using nuclear and mitochondrial DNA from biological samples collected across the Lake Superior basin. Given the potential for endemism on Isle Royale and the complex genetic structure of marten populations regionallyM. caurina) exhibited the highest proportion of unique alleles, while martens in Wisconsin revealed high degrees of both allelic richness and private alleles despite being a reintroduced, state endangered species42. All populations exhibited deviations from HWE, and Isle Royale and Wisconsin each exhibited linkage disequilibrium  and G′ST (0.01–0.45) ranged widely, with martens from Isle Royale and Colorado displaying the largest differences (>0.1) from other populations . Structure analyses were consistent with and without the use of M. caurina as an outgroup  and 129 individuals for the cytochrome b gene (CytB). All sequenced scat samples were confirmed as martens via BLAST. All COI sequences included a section of 12 undetermined sites and were therefore concatenated to 174 bp fragments for all subsequent analyses. Haplotype and nucleotide diversity were low to non-existent for COI, as multiple locations exhibited a single haplotype (Table M. caurina) exhibited reciprocal monophyly  that did not overlap 1. Further, MIGRAINE estimated an historic effective population size (N0) of 2947 (1208–14248) and a current effective population (N1) of 2.05 (2.00 − ∞), resulting in an estimated bottleneck time of 0.50 (0.0041–1.22) generations. Similarly, the loss of heterozygosity test from the source population in Ontario estimated that Ne = 1.94 for Isle Royale martens, while tests in the program LDNe44 estimated effective population sizes of 3.2 (2.2–7.6) and 257 (53 − ∞) for Isle Royale and Ontario, respectively.Program MIGRAINENe ≅ 2) that derived from a much larger population of several hundred to thousands of individuals. This is consistent with our structure analyses showing that martens on Isle Royale were most closely related to individuals in Ontario, which is home to a large, panmictic marten population45. Thus, we conclude that the rediscovered marten population on Isle Royale stemmed from a recent but cryptic colonization via mainland Ontario. Any apparent genetic differentiation in nuclear markers is likely the result of founder effects, and the low allelic richness, limited number of unique alleles, and reduced heterozygosity of Isle Royale relative to Ontario support this conclusion. Given the estimated effective population size of approximately 2 individuals, contemporary Isle Royale martens were likely founded by a pair of colonizing individuals or a fertilized female.Martens on Isle Royale exhibited considerable differentiation in nuclear markers from other regional marten populations, but mitochondrial sequences revealed no reciprocally monophyletic marten lineages within the Lake Superior Basin. Rather, martens from Isle Royale overwhelmingly shared mtDNA haplotypes with other populations. Thus, contrary to our hypothesis and despite significant population structure in nuclear markers across the region, we found no evidence of ESUs in martens of the Lake Superior Basin. This incongruity between nuclear and mitochondrial analyses indicates that while extant martens on Isle Royale are a distinct and isolated population, their colonization of the island was recent. Demographic analyses confirmed that martens on Isle Royale were subject to a recent population change and detected a significant bottleneck. Moreover, all demographic analyses showed that martens on Isle Royale are a small cohort , possibly using an ice bridge across Lake Superior, as has occurred for other Isle Royale carnivores48. Our MIGRAINE estimates indicate martens on Isle Royale experienced a significant bottleneck within the last generation (i.e. 5 years). While we know that extant martens have occupied Isle Royale for approximately 25 years34, samples used in this analysis were primarily collected from 2006–2009, putting the timing of colonization within a decade of marten rediscovery. This estimate is consistent with our other analyses that suggest martens colonized Isle Royale recently and are still recovering from a significant population bottleneck. Moreover, given the prevalence of ecological research on Isle Royale in the 20th century49 it seems unlikely that martens would have gone undetected for decades following an earlier colonization event like the potential 1966 reintroduction. Thus, we postulate that martens were historically extirpated from Isle Royale but recolonized the island around the time of their rediscovery, c. 1991. Historical ice data shows numerous ice bridges connected Isle Royale to mainland Ontario in the 10 years preceding marten rediscovery29, thereby making a natural colonization possible. It is worth noting, however, that martens are the most widely translocated carnivore in North America50 and have a history of human-assisted island colonization – both sanctioned and unsanctioned19. Thus, human-assisted dispersal of martens to Isle Royale around the time of rediscovery cannot be precluded.Assuming the extant marten population on Isle Royale was founded contemporarily by way of Ontario, we tested three plausible colonization scenarios. Given the low mutation rates of CytB and COI in vertebrates30. For instance, wolves, coyotes (Canis latrans), and martens have all colonized the island, while Canada lynx, coyotes, and martens were also extirpated at one point or another28. The dominant herbivore, woodland caribou, was replaced by moose, and white-tailed deer (Odocoileus virginianus) were introduced but quickly extirpated. In addition, beavers (Castor canadensis) recolonized Isle Royale after apparent extirpation in the 19th century51, and foxes were likely introduced for fur farming52. This dynamic history of colonization and extinction in an ecosystem that has been protected for most of the last century complicates the baseline for restoring historical Isle Royale communities and interactions. Moreover, such dynamics, coupled with the lack of historical and prehistorical information regarding past Isle Royale communities, has precipitated questions about the necessity of reintroducing or augmenting mammal populations on the island26. Our data suggests that martens, one of the smaller mammals on Isle Royale, were historically extirpated but recolonized the island. Dispersal ability in mammals is strongly correlated with body mass53, indicating that if martens did indeed immigrate to Isle Royale naturally, they were less likely to do so than other, larger-bodied carnivores. Moreover, ice bridges generally do not form until January55, months after the natal dispersal period in martens, and dispersal success is largely a function of available cover56, none of which is present over ice. Thus, if martens did naturally recolonize Isle Royale despite such barriers, the natural repatriation of larger carnivores may also be possible.National Park Service policy aims to restore historical communities and ecosystem processes where appropriate, and the mammal community on Isle Royale appears to be an ideal candidate for reintroductions due to significant turnover in the last century29. This loss of gene flow will ultimately reduce genetic diversity in Isle Royale mammal populations, the effects of which are already evident in wolves57. Consequently, population augmentation will likely be necessary to maintain genetic diversity in most extant Isle Royale community members 48 and for any potentially reintroduced species29. It is therefore critical to understand the current genetic composition of other Isle Royale mammal populations in order to preserve potential endemic lineages or local adaptations59, and to identify sources for potential translocations. We conducted the most thorough evolutionary assessment of an historical Isle Royale mammal to date and found that extant martens do not constitute a unique genetic lineage and could ultimately be augmented from a number of marten populations in the Lake Superior Basin with which they share haplotypes, though Ontario was identified as the most closely related population. Regardless, martens on Isle Royale possess reasonably high heterozygosity and maintain low inbreeding coefficients despite a significant bottleneck, indicating that direct genetic management is currently unnecessary. Previous studies, however, found that other Isle Royale mammals like red squirrels and deer mice (Peromyscus maniculatus) also appear unique, but these divergences have only been described morphologically36 and via random amplifications of polymorphic DNA (RAPD)60, respectively. Our results show that population bottlenecks and founder effects can generate such putatively unique lineages, despite only recent divergence; thus, without a more complete assessment of evolutionary history, future management of these potentially endemic populations is uncertain. Furthermore, both red squirrels and deer mice, along with other Isle Royale mammals like snowshoe hares (Lepus americanus), have limited dispersal capabilities61, and more thorough genetic assessments of these populations are needed to assess colonization, historical community baselines, and the potential for human-assisted dispersal.The frequency of ice bridges connecting Isle Royale to the mainland is decreasing due to climate change, thereby reducing the probability of natural immigration to the island for many species62 and effectively managing both native and non-native species19. Our work also illustrates the importance of continued regional monitoring, the relevancy of historical surveys, and the need for genetic techniques to establish robust and defensible conservation targets. While often difficult to obtain, the combination of these approaches has proved particularly useful19, so we emphasize the importance of multiple, interdisciplinary stakeholders when establishing conservation and restoration programs. Finally, our findings show that martens, one of the least likely island colonizers, repatriated Isle Royale following extirpation, providing optimism for the natural recolonization of other extirpated or declining carnivore populations. Moreover, given that restoration initiatives often center around charismatic megafauna – species that generally possess the greatest dispersal power – conservationists are likely underrepresenting the natural colonization potential for most other taxa. Thus, future efforts aiming to re-establish island communities should first assess the colonization histories of smaller, more dispersal limited species, to inform and evaluate restoration efforts a proiri.Isle Royale National Park is a notable wilderness area with a pristine reputation and a storied history of ecological research. Yet, like many systems, Isle Royale has experienced significant anthropogenic change, and has a dynamic history of species colonization and extinction. Consequently, very little is known about the history of the island’s vertebrate community as a whole. Our study revealed an unexpected and dynamic pattern of extinction and recolonization for a small-bodied carnivore and illustrates that even federally protected or historically pristine ecosystems have experienced more community turnover than previously appreciated. Given that anthropogenic disturbances have driven the turnover of fauna globally, identifying the biogeographic origins of extant species and documenting historical community composition are critical guideposts to establishing restoration baselinesa priori using a cotton-tipped applicator to collect epithelial cells. We extracted DNA from all samples using commercially available kits . Samples from 2006–2008 were identified as martens by Wildlife Genetics International  via sequencing of the 16S rRNA gene63, while samples from 2012–2013 were identified using fragment analysis of control region segments64. Second, we sampled populations of martens on the mainland surrounding Lake Superior to capture regional genetic diversity and potential sources to Isle Royale. Specifically, we used existing genotypes from hair, scat, and tissue samples to characterize the reintroduced marten population in the Chequamegon National Forest of Wisconsin and existing genotypes from tissue samples to characterize its source population in the Superior National Forest of Minnesota  muscle tissue from Colorado37 as an outgroup population to characterize the relative genetic diversity of martens in the Lake Superior basin and to assess the potential introduction of non-native alleles to the region. All sampling was approved by the University of Wisconsin Animal Care and Use Committee (A005239-A01) and conducted ethically under the guidelines established by the American Society of mammalogists65.To identify the source and colonization history of martens to Isle Royale, and to assess ESUs for the martens across the Lake Superior Basin, we analyzed biological samples from three distinct biogeographical regions Fig. . First, ota Fig. 42. We thrio Fig. . Lastly,66, as well as Mer022, Mer041, and Mvis07267. Polymerase chain reactions were conducted following Manlick et al.42, analyzed on an ABI 3730xl DNA analyzer , and scored using GeneMapper® . All samples were independently genotyped in duplicate to minimize potential genotyping errors68, and genotypes in disagreement were successively re-scored via independent PCRs until genotypes could be confirmed or the sample was consumed. All unresolved scores (i.e. mismatching) were censored at the locus in question, and genotypes were screened for potential allelic dropout and null alleles in program CERVUS69 throughout the scoring process. We calculated an overall genotyping error rate of 0.066. Genotypes were combined across studies42, therefore the number of loci analyzed varied by site 42, we identified unique individuals prior to population genetic analyses. We used all available samples to first generate a genotype accumulation curve in the R package poppr71 and, consistent with our genotyping procedure, determined that ≥7 markers were needed to identify unique individuals sib( in program CERVUS72. All samples with PID)sib in poppr using permutation tests with 1000 iterations71, and we assessed linkage disequilibrium in Genepop73 using a sequential Bonferroni correction. Population metrics including observed and expected heterozygosity, allelic richness, private alleles, and inbreeding coefficients were calculated using the R packages diveRsity74 and PopGenKit75.Because all Isle Royale and some Wisconsin samples were collected noninvasively  in GENETIX v. 4.0577 to capture variation among individuals and we visually identified population clusters. Finally, we implemented a series of hierarchical AMOVAs78 in poppr to first test for significant population structure when considering each sampled population separately, and then to explore the amount of variance explained by combining clustered populations identified via FCA.We quantified genetic structure between sampled populations by first calculating pairwise K) within the Lake Superior basin we employed Bayesian clustering models in STRUCTURAMA 2.079. Unlike most clustering algorithms, STRUCTURAMA does not assume a fixed number of populations and instead makes K a random variable to estimate the number of populations under a given Dirichlet process prior, the mean expected number of populations E(K). We ran seven variations of this model with a prior E(K) ranging from 2 to 8 populations to test the sensitivity of model results to prior distributions. All models used a single MCMC chain with 106 generations sampled every 1,000 steps and an additional 10% burn-in. Using the estimated K that maximized likelihood, we then employed the classic Pritchard et al.80 model with admixture and correlated allele frequencies for 105 iterations with a 10% burn-in to assign individuals to genetic clusters.To quantitatively assign individuals to genetic clusters and estimate the number of unique marten populations  in the r package adegenet82 to assign individuals to populations. DAPC is a multivariate statistical approach that does not assume HWE, but instead transforms genotypic data to principle components and assigns individuals to populations by maximizing variation between genetic clusters 82. We calculated the number of principle components (N = 12) via alpha-score optimization, identified the number of populations using K-means clustering and Bayesian Information Criterion, and then used DAPC to generate assignment probabilities for all individuals83. Genetic structure diagrams were constructed for both the DAPC and STRUCTURAMA results and plotted using the R package strataG84.Bayesian clustering models assume populations are in HWE and assign individuals to clusters that minimize disequilibrium; however, not all populations we analysed were in HWE (Table b (CytB) and cytochrome c oxidase subunit I (COI) mtDNA genes. We sequenced all unique Isle Royale individuals and 20 randomly chosen individuals from each mainland population. All mainland samples were restricted to high-quality tissue samples; however, individuals from Isle Royale were identified from scat samples that also included potential prey DNA, thereby precluding the use of generalized mtDNA primers previously used to characterize martens85. Consequently, we developed marten-specific primers and amplified a 370 bp fragment of CytB and a 186 bp fragment of COI  were then entered into a GenBank nucleotide BLAST search to confirm species identity. All sequences were deposited in GenBank (accession nos. MH684021- MH684285).To identify potential evolutionarily significant units and further quantify genetic variation among marten populations in the Lake Superior Basin, we amplified and sequenced fragments of the cytochrome 88. We then combined data for all samples successfully sequenced across both genes and assessed relatedness and monophyly among marten populations using a median-joining network developed in POPART.We calculated haplotype and nucleotide diversity for CytB and COI in all sampled populations using the program POPART43. Migraine employs a class of importance sampling algorithms and a generalized stepwise-mutation model (GSM) for microsatellite loci to generate point estimates and 95% coverage confidence intervals for the scaled parameters ancestral population size (2θ = 2N0μ), current population size (2θ = 2N1μ), and time of the demographic change in generations (T = T/2N1). Using these parameters, ancestral (N0) and current (N1) effective population sizes were estimated assuming a marten microsatellite mutation rate (μ = 3 × 10−4)19 and then used to solve for T. We estimated an additional parameter, Nratio (N1/N0), to quantify historical population expansion (>1) or contraction (<1), with significant demographic changes identified by estimates with 95% confidence intervals that did not overlap 143. Because MIGRAINE is sensitive to the number of loci used43, we combined the aforementioned Isle Royale marten genotypes with additional loci independently genotyped by WGI for individual assignment for a previous study. Specifically, we used 27 individuals sampled on Isle Royale from 2006–2008 and combined 9 polymorphic loci used for structure analyses with 9 unique WGI loci for a total of 18 markers  model in program MIGRAINE v. 0.5.2Ne) based on the loss of heterozygosity89 and linkage disequilibrium44. Given that the timing of colonization is not known, we employed a simplified loss of heterozygosity model and estimated Ne following89H1 was the observed heterozygosity of the identified source population and H0 was the observed heterozygosity of the Isle Royale population. Secondly, we used the linkage disequilibrium-based estimator of Ne in the program LDNe44 to estimate effective population sizes for the source and Isle Royale populations. This approach assumes discrete generations and therefore cannot estimate Ne directly; thus we interpreted LDNe results as an estimate of the breeding population size90. We applied a random mating model, used the conservative, unbiased threshold of 0.05 for lowest allele frequency (Pcrit)91, and calculated 95% CIs via permutation tests.To further assess demographic variation of martens on Isle Royale we estimated the current effective population size (Supporting Information


Sphyrna tiburo, is a small elasmobranch distributed in the Eastern Pacific from southern California to Ecuador, and along the Western Atlantic, with preferences for continental margins of North, Central and South America, the Gulf of Mexico, and the Caribbean. Recent studies have suggested that it could be under a process of cryptic speciation, with the possibility to find different species in similar geographic locations. Here we assessed the population structure and genetic diversity of this highly philopatric and non-dispersive species in the Bocas del Toro Archipelago, Panama. Fragments of the mitochondrial genes cytochrome oxidase I and control region, were used to test the genetic structure of adult and juvenile S. tiburo in this area, and were compared with other locations of the Western Atlantic and Belize. We found significant genetic differentiation between Caribbean bonnethead sharks from Bocas del Toro and Belize, when compared to bonnetheads from other locations of the Western Atlantic. These results also suggest that Bocas del Toro could constitute a different genetic population unit for this species, whereby bonnethead sharks in this area could belong to a unique stock. The information obtained in this study could improve our understanding of the population dynamics of the bonnethead shark throughout its distribution range, and may be used as a baseline for future conservation initiatives for coastal sharks in Central America, a poorly studied an often overlooked region for shark conservation and research.The bonnethead shark,  Molecular approaches can provide powerful tools for augmenting our understanding of the population features, connectivity, and conservation needs of highly dispersed, mobile marine species, such as sharks –4. The uSphyrna and one to the genus Eusphyra [Sphyrna genus, the bonnethead shark Sphyrna tiburo (L.1758) is one of the five small-bodied species , whose biological processes are different than those of the larger species (e.g. Sphyrna lewini) [Sphyrna tiburo is distributed in the Eastern Pacific from California to Ecuador, and throughout the Western Atlantic from North Carolina (USA) to southern Brazil, including the Gulf of Mexico and the Caribbean [Hammerhead sharks are members of the family Sphyrnidae, which is composed of 11 species, of which 10 belong to the genus Eusphyra . From th lewini) . Sphyrnaaribbean ,15. Thisaribbean ,16–18.S. tiburo is considered highly productive due to its rapid growth rate, short gestation period and high biological productivity [S. lewini, S. mokarran, S. zygaena) [Hammerhead sharks are known to migrate long distances where mating and parturition areas are often separated ,19, theructivity ,20,23, rzygaena) ,18,25. Hzygaena) . Howeverzygaena) .S. tiburo is considered to be a widely-distributed species, composed of a single panmictic population in the Western Atlantic [Taxonomically, Atlantic ,27. HoweAtlantic ,13,20,27ND2 gene, from twelve bonnethead sharks from the Gulf of Mexico, and two individuals from Trinidad. It was suggested that the animals from the two locations belong to two different species (the Trinidad specimens were designated as S. cf. tiburo). Additionally, Escatel-Luna et al. [ITS-2 genes, and maintained the two species (S. tiburo and S. cf. tiburo) proposed by Naylor et al. [Previously, Naylor et al.  found laa et al.  found sia et al.  providedr et al.  remarkinS. tiburo, may add important geographical resolution, since S. tiburo is commercially important in the U.S., Mexico, Brazil, Belize, Panama, and the Caribbean [An improved understanding of genetic population structure and clarification of taxonomic differences between populations of aribbean ,29,30. Taribbean ,13,21,26S. tiburo in Bocas del Toro (BDT), Panama, by analyzing fragments of the mitochondrial genes cytochrome oxidase I (COI) and control region (CR). We confirmed species identity by barcoding and subsequently compared the obtained sequences with similar data available from Belize (BZ), the Western Atlantic (WA) and the Gulf of Mexico [S.cf. tiburo in BDT, and subsequently, for management implications [The primary aim of this study was to investigate the genetic diversity and population structure of f Mexico ,31–33. Fications ,21.The BDT archipelago is located in the northwest of the Republic of Panama, between 8°30' and 9°40'N and between 82°56' and 81°8'W. This area in the Caribbean Sea, comprises a continental shelf, seven large islands, and other smaller islands fringed by mangrove cays –36. Thishttp://www.bioline.com/us/isolate-ii-genomic-dna-kit.html).Samples were collected between October 2016 and January 2017. Fifteen bonnethead sharks were captured at Hospital Point  during n5´TCWAC-CAACCACAAAGAYATYGGCAC) and FishCoxI R1 (TAR-ACTTCWGGGTGRCCRAAGAATCA), modified from Ward et al. [A 639 base pair (bp) fragment of the mitochondrial COI region was amplified for all samples (n = 15), using the primers FishCoxI F1  and 12SrRNA (5´AAGGCTAGGACCAAACCT3´), modified from Quintanilla et al. [A 1064 bp fragment of the mitochondrial CR was amplified for all samples (n = 15), using the primers Pro-L  and aligned using MacClade 4.08 software, which was also used to identify haplotypes [S. tiburo were obtained from GenBank from the WA: North Carolina (NC = 23), and from the following locations in Florida: Tampa Bay (TB = 27), Florida Bay (FB = 25) and Panama City (PC = 25), using the data from Portnoy et al. [All sequences were edited and checked manually using Geneious v.3.6.1  was used to build a COI maximum likelihood tree, including an outgroup Carcharhinus leucas (GenBank: FJ519612.1), and sequences from sister species Sphyrna lewini (GenBank: FJ519636.1) and Sphyrna tudes (GenBank: FJ519524.1). For the CR, the best model selected was HKY+i, that was used to create the maximum likelihood tree in PAUP [Sphyrna media (GenBank: GU385317.1) [S. lewini (KY315830.1).A statistical parsimony network for each gene fragment was constructed using the software TCS v. 1.21 , providi in PAUP . For the85317.1) , and S. h) and nucleotide diversity (π) for each gene fragment. Population differentiation was tested via pairwise comparisons of both FST and ΦST using ARLEQUIN v.3.5.1.2 [P-value was also performed to take into account the number of pairwise comparisons for the localities.Genetic diversity was calculated as haplotype diversity . Another 44 COI sequences reported in GenBank from other localities of the WA and BZ  was low and similar for all the sites, with values ranging from 0.00% , 0.053% for TX, and 0.1369% for BZ , were i% for BZ .FST and ΦST values (FST = 0.72961 P = 0.000+-0.000), which is also consistent with the phylogenetic tree. AL was not included in this analysis due to small sample size. In general, ΦST values showed higher values when compared to FST estimates, ranging from ΦST = 0.000 to 1.000. However, these values exhibited high variation between sites, showing significant differences for FST, revealing that BDT and BZ samples were distinct from all other sites. This was also confirmed by the maximum parsimony network, and the maximum likelihood tree, which segregated these two sites apart from other locations.Pairwise estimates of T values  revealedST: 0.9568. P< 0.000). Pairwise FST was also significant, showing high population structure between sites (FST: 0.72961 P: 0.000).The AMOVA indicated, that the genetic differentiation was significant between the combined BDT and BZ, and the other WA sites , defined by 12 variable sites, were identified for BDT ; anotherh = 0.898) with the lowest value for NC (h = 0.7194), which contained six haplotypes dominated by H43, whereas other localities contained haplotypes in lower frequencies. For BDT, haplotype diversity was also high (h = 0.9429). Mean nucleotide diversity (π) was 0.23%, with differences between haplotypes being based on two variable sites, with values ranging from 0.10% for NC to 0.32% for BDT . There was also no significant differentiation between several locations in the WA , while NC showed significant differentiation from all other sites . FST values showed a similar pattern, with BDT and BZ as significantly differentiated from all WA locations , and NC also significantly differentiated from all other sites (FST= 0.0551 to FST= 0.1754). Population structure was also detected between BDT and BZ, suggesting two differentiated populations in this lineage . A global AMOVA , suggesting genetic heterogeneity within localities. The distance tree (ML) for the CR clearly showed a reciprocally monophyly between BDT and BZ, and the WA, which is consistent with the COI results showing high bootstrap values  and the WA. Specifically, the significant FST and ΦST values, as well as the result of the ML trees (for both COI and CR), support the conclusion that there is significant population structure between BDT and BZ, and the WA. We found two reciprocally monophyletic clades (A and B) both in the COI and the CR genes. Based on these results, we suggest that BDT is a genetically distinct population, and add new support to the notion that there are two different lineages of bonnethead sharks (Caribbean and WA). The haplotype networks (COI and CR) also showed a clear segregation of haplotypes between BDT and BZ, and the WA, suggesting geographical isolation and limited or no gene flow between the populations analyzed.This study represents the first genetic analysis of the population structure of In a previous study, Fields et al.  found siThe presence of a common haplotype in COI (ST02) and one in CR (H55) between BZ and the WA, suggests that the ABH and CBH could be occurring in sympatry in BZ, indicating that the genetic connectivity between sites or hybridization could still be possible, as it has been observed in other shark species . AlternaS. tiburo female philopatry can limit the genetic connectivity between sites, as restricted home ranges and natal nursery areas have been found, even in connected geographical regions [Interestingly,  regions ,19,20,23Nevertheless, in this study we only included information on mitochondrial markers, which are maternally inherited and, therefore, we have no information regarding possible connectivity due to male migration. Additional analyses, including nuclear bi-parentally inherited markers should be used in order to further investigate population connectivity and potential male-mediated gene flow.C. limbatus [C. plumbeus [The COI haplotypes showed that all the BDT samples belonged to a single haplotype (ST01), only shared with BZ; the CR haplotype network also showed that BDT and BZ (CBH) constitute a different lineage than the populations from the WA (ABH). While the populations of the WA (ABH) appear to be in expansion corroborated by the large number of CR haplotypes (44) and the star-like topology , the BDTlimbatus ,46 and tplumbeus . This hiplumbeus ,20,24. HBDT is composed of many islands, which are characterized by coral reefs, linked seagrass- mangroves ecotones, and estuaries, all of which constitute an ideal environment for bonnethead sharks, given the benefits of high prey abundance and refuge from larger elasmobranch predators. Evidence of a pattern of latitudinal variation in life history traits and reproduction of bonnethead sharks  has been reported for this species in closely related populations of the WA (e.g. Florida Bay and Tampa Bay) ,24. WhilS. tiburo. Lastly, sampling of other nearby Caribbean localities may resolve questions regarding genetic connectivity, or to find potential contact zones. Nevertheless, genetically differentiated populations should be a priority for conservation and management, since divergences in mtDNA can reflect long-term restriction of gene flow between different populations [Our study also supports the conclusion that BDT and BZ constitute two differentiated populations, and as a primary result we provide preliminary mitochondrial evidence for BDT, to be assessed as a unique stock for management and conservation purposes. The lack of nuclear data precludes our ability to consider this area as an evolutionarily significant unit ,49. Compulations .S1 Table(DOCX)Click here for additional data file.S2 TableSamples from the Caribbean: Bocas del Toro (BDT) and Belize (BZ). Samples from the Western Atlantic (WA): North Carolina (NC) and three locations along the Gulf Coast of Florida: Florida Bay (FB), Tampa Bay (TB) and Panama City (PC).(DOCX)Click here for additional data file.


Africa is the origin of modern humans within the past 300 thousand years. To infer the complex demographic history of African populations and adaptation to diverse environments, we sequenced the genomes of 92 individuals from 44 indigenous African populations.Genetic structure analyses indicate that among Africans, genetic ancestry is largely partitioned by geography and language, though we observe mixed ancestry in many individuals, consistent with both short- and long-range migration events followed by admixture. Phylogenetic analysis indicates that the San genetic lineage is basal to all modern human lineages. The San and Niger-Congo, Afroasiatic, and Nilo-Saharan lineages were substantially diverged by 160 kya (thousand years ago). In contrast, the San and Central African rainforest hunter-gatherer (CRHG), Hadza hunter-gatherer, and Sandawe hunter-gatherer lineages were diverged by ~ 120–100 kya. Niger-Congo, Nilo-Saharan, and Afroasiatic lineages diverged more recently by ~ 54–16 kya. Eastern and western CRHG lineages diverged by ~ 50–31 kya, and the western CRHG lineages diverged by ~ 18–12 kya. The San and CRHG populations maintained the largest effective population size compared to other populations prior to 60 kya. Further, we observed signatures of positive selection at genes involved in muscle development, bone synthesis, reproduction, immune function, energy metabolism, and cell signaling, which may contribute to local adaptation of African populations.We observe high levels of genomic variation between ethnically diverse Africans which is largely correlated with geography and language. Our study indicates ancient population substructure and local adaptation of Africans.The online version of this article (10.1186/s13059-019-1679-2) contains supplementary material, which is available to authorized users. Paleontological and genetic evidence indicates that modern humans originated in Africa within the past 300 thousand years (ky)  and spreAfrican populations practice a wide variety of subsistence patterns including hunting-gathering, pastoralism, fishing, agriculture, and agro-pastoralism , 9, 10. Because all modern humans originated in Africa, a better understanding of the pattern of genetic variation in African genomes is important not just for understanding African demographic history but also, more generally, for deepening our understanding of the origin of modern humans, the genetic basis of adaptation to different environments, and genetic factors influencing disease susceptibility , 10, 19.To extend our knowledge of patterns of genomic diversity in Africa, we generated high coverage (> 30×) genome sequencing data from 43 geographically diverse Africans originating from 22 ethnic groups, representing a broad array of ethnic, linguistic, cultural, and geographic diversity  method, which assumes no admixture events. Thus, individuals who cluster near each other in the tree could either share a recent common ancestry and/or experienced gene flow. The resolution of the reconstruction is excellent; bootstrap values of most nodes are greater than 90. The San lineage (including Khomani San and Juǀ’hoan) is the basal lineage of all modern human lineages. The other African populations mainly cluster in the tree based on their current geographic location, with the exception of the CRHG and some pastoralist/agro-pastoralist populations such as the Mada and Luo, the latter of which have migrated over long distances and admixed with neighboring populations. We found that the CRHG populations from central Africa, including the Mbuti from the Demographic Republic of Congo (DRC), Biaka from the CAR, and Baka, Bakola, and Bedzan from Cameroon, also form a basal lineage in the phylogeny. The other two hunter-gatherer populations, Hadza and Sandawe, living in Tanzania, group with populations from eastern Africa   is the mean and sd[FST] is the standard deviation of the FST between populations i and j. Outlier variants were defined as d values within the top 0.1% of the empirical distribution. To identify independent outliers , percentiles were calculated for all variants  and the “—clump” command from plink v1.9 was used to cluster independent groups of outliers (with parameters --clump-p1 0.001 --clump-p2 0.01 --clump-kb 1000). This returned a set of independent “tag” variants for each independent cluster. All variants in strong LD (r2 > 0.8) with these tags were considered as potential locally adaptive. Low sample size per population limits use of methods to detect signatures of natural selection based on the allele frequency spectrum or extended haplotype homozygosity [We first merged the populations into six meta-ancestry groups and then calculated a per site le sizes  between zygosity , 80.P values were calculated as the number of permuted scores higher than the observed score, with the P values less than the 0.05 family-wise error rate (P < 5.56 × 10−3) considered significant (Bonferroni-corrected for the number of annotations tested). To identify biological functions of genes near outlier windows, regions spanning all variants in strong LD with tag variants were identified and merged. These merged windows were used as test regions in GREAT with default parameters [To test for functional enrichment of outlier variants, functional genomic regions including DNase I hypersensitive sites (DHS) annotated as promoters, enhancers, and dyadic regions ; enhancerameters . All terAdditional file 1:Table S1. Sample information in this study. (XLSX 12 kb)Additional file 2:Figure S1. A phylogeny of African lineages used the Altai Neandertal as outgroup constructed using Treemix allowing for six migration events. Figure S2. Principal component analysis of 44 African and 32 west Eurasian populations using principal component analysis. Figure S3. ADMIXTURE analysis of 92 African and 62 West Eurasian individuals from K = 2 to 10. Figure S4. Effective population size of African Khoesan-speaking populations. (PDF 414 kb)Additional file 3:Table S2. Enrichment test results of positively selected loci in different populations using GREAT. (XLSX 58 kb)


Limnoperna fortunei a highly invasive species in Brazil, has generated productive, economical, and biological impacts. To evaluate genetic structure and variability of L. fortunei populations present in fish farms in the reservoirs of Canoas I (CANFF), Rosana (ROSFF), and Capivara (CAPFF) , eight microsatellite loci were amplified. Five of those eight loci resulted in 38 alleles. The observed heterozygosity (Ho) was lower than the expected heterozygosity (He) in all populations, with a deviation from the Hardy–Weinberg equilibrium (HWE). The average value for the inbreeding coefficient (Fis) was positive and significative for all populations. There was higher genetic variability within populations than among them. The fixation index (Fst) showed a small genetic variability among these populations. The occurrence of gene flow was identified in all populations, along with the lack of a recent bottleneck effect. The clustering analysis yielded K = 2, with genetic similarity between the three populations. The results demonstrate low genetic structure and suggest a founding population with greater genetic variability (ROSFF). Our data point to the possible dispersal of L. fortunei aided by anthropic factors in the upstream direction. It was concluded that the three populations presented a unique genetic pool for Paranapanema River, with occurrence of gene flow.The golden mussel,  Another invasive mollusk, Zebra mussel (Dreissena polymorpha), has shown low genetic differentiation among populations of the Great Lakes, North America  populations in three reservoirs of the Paranapanema River, Paraná, collaborating to the understanding of invasion patterns in the assessed regions.Genetic structuring analyses performed in previous studies by Zhan et al.  and Ghab22.1The samples were collected in December 2014 from fish farms located in three reservoirs of the Paranapanema River, State of Paraná . The mollusks were mechanically removed from the colonies, which remained fixated to the nets of the tanks. After removal, the mussels were placed in buckets and taken to nearby locations, where they were cleaned to remove any type of superficial dirt . The animals were subsequently kept for 2 hr in recipients containing ice (approximately 5°C) to induce numbness before they were killed. At this stage, they were cleaned again to remove dirt that remained between the mussels and were placed into labeled plastic bags. These bags were sealed and placed in thermos boxes filled with ice, before transporting them to the laboratory for analyses.Samples were collected from fish farms that use the open‐net tank production system to grow the Nile tilapia  was discarded, and the samples were dried at room temperature for 20 min. The samples were added with 35 μl TE (Tris/EDTA) and 5 μl RNAse (30 μg/ml) and kept in a water bath at 37°C for 40 min before storage at −20°C. DNA was quantified using a PICODROP® spectrophotometer . The samples were diluted to a final concentration of 30 ng/μl. In order to assess DNA quality, an electrophoresis run in 1% agarose gel was conducted at 70 V for 1 hr.After centrifugation, 800 μl of the supernatant was removed from each sample and placed into a new sterile microtube, before the addition of 700 μl cold absolute ethanol for DNA precipitation. In order to increase the efficiency of the process, the microtubes were stored at −20°C for 2 hr. The samples were then centrifuged at 11,270 2.3L. fortunei , 0.08 μl 5 p.m. forward primer, 0.3 μl 5 p.m. reverse primer, 0.3 μl 5 p.m. M13 primer labeled with a fluorophore , 1 μl DNA (30 ng/μl), and 3.82 μl nuclease‐free water in a final volume of 10 μl.The amplification was performed at the Laboratory of Molecular Markers and Plant Cytogenetics at the Department of Biology, State University of Londrina (UEL). Eight primer pairs  previously developed for ® Size Standard .The PCR reactions were performed in a PTC200 thermocycler  using a gradient program. All eight primer pairs were tested, but only five of them were used  as they presented the expected results: they were polymorphic and amplified the microsatellite alleles consistently and reproducibly. The respective PCR reactions using these five primer pairs were performed using the following program: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, respective AT (°C) for each primer for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. The final amplification product was subjected to an electrophoresis in the automatic sequencing machine 3500xL  with the Formamide reactions Hi‐Di (8.8 μl), GeneScan LIZ600 Size Standard (0.2 μl), and DNA . The software Gene Marker v. 2.6 was used to calculate alleles size. It enables the determination of the allele size using the standard molecular weight marker, GeneScan™ 600‐LIZ2.4p < 0.05; calculated by chi‐square test for Hardy–Weinberg equilibrium) for each locus, and genetic distance  was Lf06 , followed by Lf07 and Lf23 , and Lf21 and Lf22 . A low frequency of null alleles  was identified in the L. fortunei populations. Two loci showed null alleles in CANFF (Lf06 and Lf07), two in ROSFF (Lf06 and Lf22), and one (Lf06) in CAPFF.Three of the eight loci tested  did not amplify or presented unspecific products, and were therefore removed from the analyses. The five loci used  were polymorphic and amplified consistent and reproducible microsatellite alleles, with the expected sizes between 57 bp (Lf23) and 291 bp (Lf21). In total, 38 alleles were detected for 75 individuals from the three natural populations of p < 0.05) for deviation from the HWE. The average Ho in each population ranged between 0.457 (ROSFF) and 0.275 (CANFF). The p values of Fis were compared to alpha of 0.05 after indicative adjusted nominal level (0.003), and the results presented a significant positive average value (p < 0.05) for all populations. Analysis using the Bottleneck program for the three mutation models indicated that in the populations, there has been no significant reduction in size in a short period of time. In other words, no recent bottleneck effect was observed. It was observed low genetic distance between the three populations .Considering the results presented to verify the estimates of genetic diversity parameters of the five loci Table , the avep < 0.05) in CANFF × ROSFF and CAPFF × ROSFF (0.046). Significant linkage unbalance associations (p = 0.05) were observed in the pairwise analyses performed for the evaluated loci in all populations. Four associations were observed in CANFF, five in ROSFF, and four in CAPFF.Using the analysis of molecular variance (AMOVA), a greater genetic variability was identified within than between the studied populations Table . LikewisIn the Bayesian analyses for the number of migrants, the average percentage of nonmigrants was 0.788 for the CANFF population, 0.785 for ROSFF, and 0.766 for CAPFF. All the values are significant with confidence interval of 95%. Gene flow occurred in all populations, with an average rate of 0.110 in CANFF for migrants originating from ROSFF and 0.101 for those originating from CAPFF; 0.205 in ROSFF for migrants coming from CANFF and 0.010 for those coming from CAPFF, the latter being the lowest rate observed, and 0.020 in CAPFF for migrants originating from CANFF and 0.213 for those from ROSFF, the latter being the highest rate observed.K = 2), with genetic similarity among all populations and greater proximity between CANFF and CAPFF  demonstrated overlapping individuals from the three populations Figure a. This rF Figure .4L. fortunei. It is probable that the genetic differentiation of this species in new colonization sites has promoted changes in the flanking region of the primer, making it impossible the binding of these in the specific region. The low locus pairs in linkage disequilibrium suggest that they are not closely linked on the chromosomes  across the three populations. This difference was characterized by a heterozygote deficit compared to HWE. Zhan et al.  and the effective alleles (Ae) were higher in ROSFF than in CANFF and CAPFF. The low Ae values compared to Ra indicate high frequency alleles. With the exception of ROSFF, the mean Ra values of CANFF and CAPFF were lower than those found by Zhan et al.  and Ghabn et al.  and Ghabn et al.  observed Holland  observedL. fortunei population are composed by females, that is, the lower proportion of males increases the chance of recurrent inbreeding  that indicated two distinct genetic groups but with mix of individuals from all three populations  identified a higher genetic variability within than among the three populations, and the fixation index (Fst) indicated a low degree of differentiation among them. Likewise, the occurrence of gene flow was detected in all populations, along with a small genetic distance among them. Finally, corroborating our results, the FCA demonstrated an overlap in the distribution of genetic variability among populations, which was corroborated by clustering analysis K =  that inds Figure .L. fortunei, Pessotto & Nogueira  and artificial (hydroelectric dam) barriers.In relation to the dispersion potential of Nogueira  found inNogueira  revealedNogueira . Based oL. fortunei in South America started in Rio de la Plata (Argentina), following an upstream direction along the Uruguay, Paraguay, and Paraná basins . The three populations presented a unique genetic pool for the entire stretch of the sampled river.The authors reported no potential conflict of interest.The study was coordinated by NMLB; samples collection was performed by PJFM, EM, and ARPP; FPS and PJFM extracted and quantified the DNA; LMB, CFR, and AMUR performed DNA amplification and capillary electrophoresis; statistical analyses were performed by EAR, CFR, and FPS; PJFM, NMLB, and ECSL performed the writing of the manuscript, and all authors contributed to editing the manuscript. Click here for additional data file.


Gasterophilus species are widely distributed around the world. The larvae of these flies parasitize the digestive tract of equids and cause damage, hindering horse breeding and protection of endangered species. However, study of the genetic structure of geographically distinct Gasterophilus populations is lacking. Here, we analyzed the genetic diversity of Gasterophilus pecorum, G. intestinalis, G. nasalis and G. nigricornis from three typical grasslands  in China as compared to published sequences from Italy, Poland and China (Daqing and Yili), based on the mitochondrial cytochrome c oxidase cox1 and cox2 gene sequences.Gasterophilus populations. Due to the unique natural climatic conditions of the alpine steppe, there were high levels of genetic differentiation among different geographical populations of G. pecorum and G. nasalis, indicating that environmental variations influenced population genetic structure. Frequent exchanges between meadow and desert steppe Gasterophilus species resulted in low genetic differentiation. The highest exchange rates were found among G. intestinalis populations. Genetic differentiation was only observed on a large geographical scale, which was confirmed by analyzing population genetic structure. Three species, G. pecorum, G. intestinalis and G. nasalis, from meadow steppe showed a high emigration rate, indicating that the direction of Gasterophilus dispersal in China was from east to west.Haplotype diversity and nucleotide diversity of mitochondrial genes was generally high in all Gasterophilus species have a high level of genetic diversity and different degrees of genetic differentiation and gene flow among different populations of the same species, reflecting their potential to adapt to the environment and the environmental impact on genetic structure. Knowledge of the genetic structure, population history, and migration will help understand the occurrence and prevalence of gasterophilosis and provide a basis for controlling the local spread of Gasterophilus spp.Our results show that the four The online version of this article (10.1186/s13071-018-3042-y) contains supplementary material, which is available to authorized users. Gasterophilus species are common obligate parasites in equines and are widely distributed worldwide. The genus Gasterophilus (Diptera: Oestridae) includes nine species [G. pecorum, G. intestinalis, G. nasalis, G. nigricornis, G. haemorrhoidalis and G. inermis [Gasterophilus larvae parasitize the gastrointestinal tract of equids for 10–11 months and cause mucosal lesions, gastrointestinal ulcers, peritonitis, anemia and gastric rupture, which can be severely debilitating [G. pecorum, which lays its eggs on grass [G. intestinalis and G. nasalis, with the other Gasterophilus species rarely observed [Gasterophilus larvae is more complicated than in other regions. For instance, Xilin Gol Grassland of Inner Mongolia Autonomous Region is an important pastoral area in China with an abundance of grass and water. Six species of Gasterophilus were found in Inner Mongolia, the predominant species being G. intestinalis followed by G. nasalis, which is consistent with the prevalence of Gasterophilus in other countries and regions [Gasterophilus in this region was found to be 1.31 [Gasterophilus species have been found in Xinjiang and Inner Mongolia, but G. pecorum was the predominant species (96.17%) in Xinjiang, where the Gasterophilus species diversity index was very low (0.21) [G. pecorum and G. nasalis have been documented, with the former being the predominant species (93.71%) and contributing to a low diversity index (0.23) [ species ; of thes inermis –4. Gastelitating , 6. The on grass . In Euroobserved . In trad regions . The div be 1.31 . The samw (0.21) . In Madux (0.23) .c oxidase cox1 and cox2 genes have been used to examine inter- and intraspecific relationships in Diptera, Lepidoptera, Coleoptera, Hymenoptera and Hemiptera [cox1 gene sequences revealed a high degree of genetic diversity that enabled differentiation of the Italian and Polish populations by PCR-restriction fragment length polymorphism [G. intestinalis from Daqing (DQ) and Yili (YL), China, genetic information is available for three mitochondrial genes, including cox1, NADH dehydrogenase subunit 5 gene (nad5) and 23S ribosomal RNA gene [Gasterophilus populations. In the present study, we explored the population genetic structure and haplotype distribution patterns in several populations of G. pecorum, G. intestinalis, G. nasalis and G. nigricornis in China based on analyses of mitochondrial cox1 and cox2 gene sequences.Mitochondrial DNA markers have been widely used in studies of insect taxonomy, population genetics and evolution. More specifically, mitochondrial cytochrome emiptera –19. Prevmorphism . In G. iRNA gene . HoweverGasterophilus larvae were collected in Kalamaili Nature Reserve (KNR) of Xinjiang Uigur Autonomous Region, Duolun County (DL) of Inner Mongolia Autonomous Region and MD of Qinghai Province  and Gco1an (5'-AGG AAG TTC AGA ATA GCA GTG TTC-3') [cox2 gene was amplified with primers TL2-J-3037 (5'-ATG GCA GAT TAG TGC AAT GG-3') and TK-N-3785 (5'-GTT TAA GAG ACC AGT ACT TG-3') [cox1 was performed according to a previously described protocol [cox2 were as follows: 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, and 25 °C for 2 min. PCR products were visualized by using 1% agarose gel electrophoresis, purified with the BigDye XTerminator Purification kit  and sequenced on an ABI 3730XL Genetic Analyzer . Sequences obtained were deposited in the GenBank database under the accession numbers MG815141-MG815634.Total genomic DNA was extracted using a standard phenol/chloroform protocol , with mi TTC-3') , and a 6T TG-3') . PCR ampprotocol . PCR conG. intestinalis  and G. nasalis (GenBank: GU265749-GU265758 and GU299284-GU299286) were used for data analysis. Raw sequences were proofread and edited using BioEdit v.7.0.9.0 [Hd), and nucleotide diversity (π) were estimated using DnaSP v.5.0 [Fst) statistics and gene flow (Nm) values. Phylogenetic trees were constructed by the neighbor-joining (NJ) method using MEGA and were assessed with 1000 bootstrap replicates [θ), exponential growth rate (g) and migration rate based on the maximum-likelihood approach. The search strategy included three replications of 10 short initial chains and two long final chains. The initial chains were run with 500 samples and the sampling interval was set to 20. The final chains were performed with 10,000 samples with the same sampling interval. A burn-in of 1000 samples was used for each chain.Sequences generated in this study as well as published sequences of SP v.5.0 . The Kimplicates . Haplotyplicates  with a 9plicates  was usedcox1 sequences were obtained for G. pecorum, G. intestinalis, G. nasalis and G. nigricornis, co-analyzed with 41 published sequences of G. intestinalis from China (DQ and YL), Italy and Poland, and of G. nasalis from Italy and Poland. An analysis of all cox1 sequences identified a total of 154 haplotypes. The haplotype distribution, Hd and π of each population based on the mitochondrial cox1 gene are shown in Additional file Hd and π were as follows: G. pecorum ; G. intestinalis ; G. nasalis ; and G. nigricornis . For each species, only two to five common haplotypes were observed within localities in China and there were no shared haplotypes across G. intestinalis and G. nasalis populations in Italy and Poland.A total of 247 cox2 sequences of the four Gasterophilus species defined 127 haplotypes. The haplotype distribution, Hd and π of each regional population based on the mitochondrial cox2 gene are shown in Additional file Hd and π were as follows: G. pecorum ; G. intestinalis ; G. nasalis ; and G. nigricornis . Similar to cox1, only two to six common cox2 haplotypes were observed within populations of each species.The 247 cox1 haplotypes of G. pecorum had three sub-networks  were shared between KNR and DL and three  were shared between KNR and MD, while no common haplotypes were detected between DL and MD. The sub-networks formed a star-like structure that was derived from haplotype GpH4. Most unique haplotypes in the DL population were distributed around GpH4, whereas those unique to the MD population mostly surrounded GPH11. Haplotypes in the KNR population were more evenly distributed throughout the network. Individuals of GpH37 and GpH48 constituting a single sub-network were all from MD. In general, the cox2 haplotypes formed two sub-networks with GpH17 and GpH43 as the central haplotypes  were shared between KNR and DL, four  were shared between KNR and MD, and GpH2 was shared by all three populations.The minimum spanning network calculated with TCS software using rks Fig. . Of the pes Fig. . The hapcox1 haplotypes of G. intestinalis had a single network and no sub-networks. Of the 48 cox1 haplotypes, three  were shared by KNR and DL, and one each was shared by KNR and DQ (GiH13), DL and DQ (GiH26), and DL and YL (GiH20). The network structure was dominated by a single haplotype (GiH4)  in the center and distributed across all geographical regions (KNR and DL)  Fig. . HaplotyDL) Fig. . The seccox1 haplotypes of G. nasalis had two sub-networks. Four  of the 33 haplotypes were shared between KNR and DL, and two (GnH6 and GnH17) were shared among KNR, DL and MD , 0.023 (KNR and MD) and 0.024 (DL and MD), whereas distances based on cox2 gene sequences were 0.006 (KNR and DL), 0.015 (KNR and MD) and 0.017 (DL and MD). These results demonstrate that the genetic distance was smallest between KNR and DL populations and larger than for other populations. Fst values for cox1 sequences were 0.03245 (KNR and DL), 0.19067 (KNR and MD) and 0.22841 (DL and MD), while the corresponding Nm values were 7.45416, 1.06116 and 0.84452, respectively. Fst values for cox2 sequences were 0.04216 (KNR and DL), 0.17652 (KNR and MD) and 0.17893 (DL and MD), and Nm values were 5.67979, 1.16627 and 1.14719, respectively. These results indicate that there was a low degree of genetic differentiation  between KNR and DL populations, and moderate genetic differentiation (0.15 < Fst < 0.25) and low level of gene flow (Nm < 4) between MD and the other two populations.Mean genetic distances between different populations were calculated based on cox1 sequences are shown in Table cox2 sequences was 0.006. Fst and Nm values for cox1 sequences among the six populations are shown in Table Fst > 0.25) and low level of gene flow (Nm < 4) occur among Italian, Polish and Chinese populations, but restricted by the very small sample size of Italian population, this result remains to be verified. Genetic differentiation was low among KNR, DL and DQ populations  and moderate among the remaining populations in China (0.05 < Fst < 0.15), which also exhibited a low degree of gene flow (Nm < 4). Fst and Nm values for cox2 sequences between KNR and DL were 0.01847 and 13.28546, respectively, indicating a low level of genetic differentiation (Fst < 0.05) and frequent gene flow (Nm > 4).Genetic distances among different populations based on cox1 sequences are shown in Table cox2 sequences were 0.009 (KNR and DL), 0.015 (KNR and MD) and 0.013 (DL and MD), and those based on cox1 sequences ranged between 0.009–0.023, with the closest genetic distance observed between KNR and DL populations. The sequence dataset for cox2 also revealed a close genetic distance between KNR and DL and showed that MD was distant from the other populations. Fst and Nm values for the cox1 gene among the four populations are shown in Table Fst < 0.05) and frequent gene flow (Nm > 4) between KNR and DL, the other populations showed high genetic differentiation (Fst > 0.25) and low gene flow (Nm < 4).Mean genetic distances among populations based on cox1 and cox2 sequences were 0.019 and 0.013, respectively. Fst and Nm values for the cox1 gene between KNR and DL were 0.07371 and 3.14167, respectively, while those for the cox2 gene were 0.08215 and 2.79321, respectively. These results showed that there was little genetic differentiation (Fst > 0.05) and low level of gene flow (Nm < 4) between KNR and DL.The mean genetic distances between KNR and DL populations calculated based on D and Fu’s Fs  of G. intestinalis, and in the KNR population of G. nasalis, whereas the other populations had remained relatively stable.Populations are often far from equilibrium, and not all deviate in the same direction. The neutrality of all populations was estimated using Tajima’s θ) and exponential growth rate (g) were relatively high for the DL population followed by the KNR population of G. pecorum, while the low θ value and negative g value of the MD population indicated a declining trend. Migration was high from DL to KNR and from KNR to MD, whereas the emigration and immigration rates of MD and DL, respectively, were low. Among G. intestinalis populations, DQ had the highest θ and g values followed by KNR. High migration was observed among Chinese populations, including from DL to KNR, DL to YL, DL to DQ and YL to DQ. In general, KNR populations had low emigration and high immigration rates, while the opposite was true for DL. In European populations, migration was more frequent from Poland to Italy than in the other direction. The relationship between the Italian and Chinese populations was more complex than that between the Polish and Chinese populations, but the results may be biased due to the limited number of samples from Italy. Among G. nasalis, the highest g and θ values were observed in the MD and Italy populations, respectively. In China, the highest rates of migration were from DL to KNR and MD. The migration rates from Italy to Poland were even higher, while those between the Chinese and European populations were generally low. The θ values of G. nigricornis in KNR and Dowered were similar, whereas g values were higher in DL than in KNR. Migration rates between the two populations were similarly high, with migration from KNR to DL being slightly higher.The LAMARC analysis  [Gasterophilus populations in KNR of Xinjiang and DL of Inner Mongolia share similar a genetic structure and species type.Host migration affects the genetic structure of parasite populations , 37. The region) , 25; conGasterophilus pecorum, G. intestinalis and G. nasalis from DL showed a consistently high migration rate, suggesting that DL is the origin of Gasterophilus flies in Mongolia-Xinjiang and Qinghai-Tibet regions of China. The Xinjiang Uygur Autonomous Region includes KNR at the heart of the Eurasian continent. Historically, the Silk Road of China passed through this area, which saw frequent trade of animals such as horses that were important means of transportation [Gasterophilus flies in KNR indicate that Gasterophilus spp. in the area spread from multiple locations, likely reflecting the frequent movement of their hosts. Although we sampled a limited number of sites in this study, our results nonetheless demonstrate that Gasterophilus spp. migrated westward from the grasslands of eastern China. The neutrality test results showed that G. pecorum and G. intestinalis underwent significant population expansion and were the predominant species in KNR and DL, respectively.ortation . The genGasterophilus species of different geographical populations based on analyses of mitochondrial cox1 and cox2 genes. Based on these results, we propose dispersal patterns of these species in China’s three major pastoral areas.We investigated the genetic structure and inter-population relationships of four common Additional file 1:Figure S1. Map of sampling sites in China. (PDF 5098 kb)Additional file 2:Table S1. Number of individuals (n), haplotypes, number of haplotypes (denoted in parentheses), haplotype diversity (Hd), and nucleotide diversity (π) based on the mitochondrial cytochrome c oxidase subunit 1 gene in G. pecorum, G. intestinalis, G. nasalis and G. nigricornis. Table S2. Number of individuals (n), haplotypes, number of haplotypes (denoted in parentheses), haplotype diversity (Hd), and nucleotide diversity (π) based on the mitochondrial cytochrome c oxidase subunit 2 gene in G. pecorum, G. intestinalis, G. nasalis and G. nigricornis. Table S3. Neutrality test for G. pecorum, G. intestinalis, G. nasalis and G. nigricornis in different geographical locations. Table S4. Maximum likelihood estimates of population size (θ), exponential growth rate (g), and migration rate for different populations of G. pecorum, G. intestinalis, G. nasalis and G. nigricornis. (DOCX 27 kb)