Barbitistes vicetinus Galvagni & Fontana, 1993 is a recently described species that was considered rare until ten years ago, when unexpected population outbreaks causing severe defoliations across forests and crops were observed in north-eastern Italy. A genetic approach was used to analyse the origin of outbreak populations. The analysis of two mitochondrial regions (Cytochrome Oxidase I and II and 12S rRNA-Control Region) of 130 samples from the two disjunct ranges (Euganean and Berici Hills) showed high values of haplotype diversity and revealed a high geographical structure among populations of the two ranges. The high genetic variability observed supports the native origin of this species. In addition, results suggest that unexpected outbreaks are not a consequence of a single or few pestiferous haplotypes but rather the source of outbreaks are local populations which have experienced an increase in each area. The recent outbreaks have probably appeared independently of the genetic haplotypes whereas environmental conditions could have affected the outbreak populations. These findings contribute to a growing understanding of the status and evolutionary history of the pest that would be useful for developing and implementing biological control strategies for example by maximizing efforts to locate native natural enemies.Insect outbreaks usually involve important ecological and economic consequences for agriculture and forestry. The short-winged bush-cricket  Herbivorous insect outbreaks usually involve important ecological and economic consequences for agriculture and forestry. In most cases, it concerns the accidental introduction of an exotic species into a new geographical area due to international trade and human movement –3. In thBarbitistes, , occasional outbreaks were reported only for B. constrictus Brunner von Wattenwyl, 1878 in conifer forests of central and eastern Europe, for B. ocskayi  in broadleaf forests of north-eastern Italy [B. serricauda  in vineyards of north Italy. In spring 2008, the first outbreak of the species B. vicetinus Galvagni & Fontana, 1993 was recorded in a restricted area of north-eastern Italy (Euganean Hills) [Until a decade ago, among bush-crickets belonging to the genus n Hills)  S1 Fig)BarbitistB. vicetinus have been reported only in this area. Only since 2005 this species started to be more common in some localities and easy to be found also without bioacoustics technics. The recent discovery of the species  and increase in these north-eastern areas affected by outbreaks during the past two decades has sparked a debate about the possible exotic origin of B. vicetinus. Recently, as a consequence of the outbreaks, some authors have thoroughly studied important aspects of the biology and ecology of B. vicetinus [Interestingly, the bush-cricket was first described just in 1993 as a rare endemic species of north-eastern Italy, being found only in small confined hilly areas . From thicetinus , 13 but icetinus . This inicetinus –17. Seveicetinus –23. Howeicetinus , 24.B. vicetinus populations from the two disjunct ranges where it currently outbreaks . The COI and COII are considered slowly evolving genes of the mitochondrial protein-coding genes [Among the genetic tools to be exploited, maternally-inherited mtDNA is widely used since it is relatively conserved compared to some nuclear genes, and is thus suitable when searching for historical processes . In thisng genes . The nonng genes , 28 and ng genes . Both mang genes –35.By studying the genetic structure of the bush-cricket this study aims to answer three main questions:is there any evidence for an alien origin of the bush-cricket?B. vicetinus populations genetically distinct?are are either of the recent outbreaks caused by a single outbreak population followed by a spread pattern or have they derived from multiple local populations?B. vicetinus and could provide insight to assist management of this outbreak pests.This information can help understand the factors related to the outbreak events of The study area was located in north-eastern Italy (Veneto Region) and included the two disjunct ranges where outbreaks have occurred (Euganean Hills and the Berici Hills) and a third range where no outbreaks have been reported to date and the species is rare (Lessini Mountains). The three ranges are separated from each other by cultivated and inhabited alluvial plains devoid of woody vegetation .2 and comprise approximately 100 hills of volcanic origin emerging from the alluvial sedimentary plain, with the highest elevation of approximately 600 m above sea level. They are characterised by numerous narrow and deep valleys, steep hills with different sun exposures and many microclimatic conditions that influence the vegetation. The annual mean temperature is approximately 12°C and precipitation ranges from 700 to 900 mm [The Euganean Hills cover an elliptical area of approximately 180 kmo 900 mm  even if 2 with a maximum elevation of approximately 450 m above sea level [The Berici Hills are an isolated plateau situated on the southern Vicenza plain, they cover almost 160 kmea level . The cliea level .2, at the transition between the Fore-Alps and the River Po Plain [The Lessini Mountains are a triangular-shaped tableland, which occupies some 800 kmPo Plain . The mouB. vicetinus were collected, using a sweep net, throughout the spring of three successive years . Specimens from the north, centre and south part of both outbreak areas (the Euganean and Berici Hills) as well as from two sites on the Lessini Mountains were collected from bush and tree canopies on marginal side of the forests  and Vicetinus_P4R (5’-TCCACAGATTTCTGAACATTG-3’). The universal primers SR-J14610 (5’-ATAATAGGGTATCTAATCCTAGT-3’) and T1-N18 (5’-CTCTATCAARRTAAYCCTTT-3’) [The genomic DNA was extracted from tissue samples taken from the hind femora of each specimen separately, according to a previously described salting-out protocol . Two mitCTTT-3’)  were useCTTT-3’)  consistiPCR products were checked through electrophoresis on 1.0% agarose gels stained with SYBR® (Invitrogen), purified using Exonuclease and Antarctic Phosphatase  and sequenced at the BMR Genomics Service . Primers used for amplification were also used for sequencing.DNA sequence chromatograms were quality checked, manually corrected when necessary, and aligned using MEGA X . Low-quaHaplotype and nucleotide diversity, as well as the pairwise genetic distances between populations, were calculated with Arlequin 3.5  using a A haplotype parsimony network with a probability cut-off of 95%, was reconstructed using the TCS 1.21 software  and PopAThe study of the past demographic history of the species was inferred using Arlequin 3.5 through the Tajima’s D and Fu’s Fs tests , 50 and For those populations that did not deviate from sudden expansion (p > 0.05) the time since expansion was calculated considering the relationship τ = 2*u*t . The exB. vicetinus . Unfortunately, only 12 specimens were collected from the Lessini Mountains due to the rarity of the species in this range  to exclude the presence of any nuclear mitochondrial pseudogenes. This bioinformatics tool translates nucleic acid sequences to their corresponding peptide sequences and identifies stop codons. Since these pseudogenes (NUMTs) are characterized by the accumulation of in-frame stop codons and indels [The two fragments of the mitochondrial genes, COI-tRNALeu-COII and 12S-CR were successfully amplified and sequenced in 130 samples, with an average of 19.6 specimens for each of the three areas within the two main ranges . After quality assessment and trimming the sequences, high-quality sequences 799 bp long for the COI-tRNALeu-COII and 898 bp long for the 12S-CR fragment were obtained. Sequences of the COI-tRNALeu-COII fragment were translated with Transeq . Sequences of each haplotype of COI-tRNALeu-COII and 12S-CR obtained in this study are available through GenBank accession numbers MW405351- MW405381.Partition homogeneity test confirmed that COI-tRNALeu-COII and 12S-CR fragments bear a homogeneous signal (P = 0.28), allowing data to be pooled for further analyses. A final dataset including 130 concatenated sequences of 1697 bp was obtained. Further analyses were conducted considering the combined dataset.The diversity indexes for the concatenated dataset ranged between 0.28 and 0.90 for the haplotype diversity (H) and between 0.02 and 0.24 for the nucleotide diversity (π). Among populations from both the main outbreak areas, those from the north and the centre part of the Berici Hills showed the highest H and π values  while populations from the south part of the Berici Hills showed the lowest variability. The distribution of haplotypes among all populations analysed and other summary statistics are shown in B. vicetinus genotypes were grouped based on the six main collection sites , the locus-by-locus AMOVA revealed a significant geographic structure between populations of the two main disjunct ranges (P = 0.0014) (Euganean vs Berici Hills). To avoid bias as a result of the small sampling size of Lessini Mountains, populations from this area were not included in this analysis. The analysis showed that 40% of the variation was explained by differences among groups (Euganean and Berici Hills), whereas about 48% of genetic variation was explained within populations (The presence of population differentiation was confirmed by the tests of population differentiation (P>0.001). When ulations .TCS Network of the combined dataset revealed the presence of 32 haplotypes of which 15 were exclusive to samples from the Euganean Hills, 13 were exclusive to the Berici Hills and 2 regarded samples only from the Lessini Mountains . Only 2 Going deeper into the network’s characteristics, the most common haplotype, A6, included 34 samples coming from all three parts of the Euganean Hills . Ten rare haplotypes including samples exclusively from the Euganean Hills were connected, separated by only one mutational step, to A6 in a star-shape. Five of them  were represented by only one sequence, six  by two sequences, and one (E6) by 4 sequences. In addition, haplotype A1, which included samples from both the Euganean and Berici Hills, was also connected to A6 separated from it by only one mutational step.The second most common haplotype, I1, was composed of 20 sequences:15 belonged to samples from the three geographical populations of Berici Hills  while 5 were represented by samples from site 23 (Lessini Mountains). This haplotype (I1) was separated from the most dominant haplotype (A6) by two mutational steps. A star-like pattern could also be observed in this part of the network, with haplotype I1 surrounded by nine haplotypes, eight of which  exclusively represented by samples from the Berici Hills. Among them, haplotype I5 was shared by 19 samples from the southern and central populations of the Berici Hills while haplotypes I2, I3, I15, K2 and L9 were rare (3 sequences each at most) and included only samples for the north part of the Berici Hills. Two haplotypes, O1 and N17, were exclusive to samples from site 22 (Lessini Mountains) the furthest site from the outbreak areas .The parsimony networks of the single markers (COI-tRNALeu-COII and 12S-CR) showed a similar pattern, with most haplotypes including exclusively samples from only one disjunct distribution range (Euganean Hills or Berici Hills or Lessini Mountains)  Figs.B. vicetinus populations of this hilly area did not conform to the theory of neutral evolution , suggesting a past population expansion after a period of low effective sample size . Berici volution .The mismatch distribution plots of both ranges were smooth and unimodal, revealing that these populations were undergoing population expansion . They weEstimation of the expansion time showed that populations from the Euganean Hills lineage started to expand about 10,700 years ago (with a 2.6% substitution rate) and 12,400 years ago (with a 2.3% substitution rate) . PopulatB. vicetinus in its outbreak areas and reveals the presence of a high geographical structuring among populations of the two outbreak ranges analysed (Euganean and Berici Hills).This study presents the first population genetic analysis of the species B. vicetinus is a native species. Conversely, populations of invasive alien species are traditionally thought to have reduced genetic variation relative to their source populations because of genetic founder effects linked to small population size during the introduction and establishment phases of an invasion [B. vicetinus may be an invasive species accidentally introduced.Populations from both ranges showed high values of haplotype diversity, a typical characteristic of ancestral populations –65, suppinvasion . FurtherEven though outbreaks have been reported on both the Euganean and Berici Hills, different haplotypes have been found in these two distribution ranges. Ninety-four percent of haplotypes were exclusive to a single distribution range (i.e. either Euganean or Berici Hills) and only one haplotype, scarcely represented, was found in samples from both hilly areas. These results suggest that outbreaks are not a consequence of a single or few pestiferous haplotypes but rather that the source of outbreaks is due to local populations which experienced a demographic increase in each area. Thus, it seems that outbreaks have appeared independently from the genetic origin, as also found in some studies which indicate that outbreak events are often more affected by environmental conditions than by genetic characteristics of the local population , 67–69.B. vicetinus has been reported to show a low dispersal ability [Pholidoptera griseoaptera, [B. vicetinus outbreak areas, it has been observed that habitat loss and the presence of patchy areas of the non-host alien tree Robinia pseudoacacia play an important role in reducing population density and dispersion [The geographical separation between populations of the Euganean and Berici Hills, observed in the network and confirmed by population differentiation test and AMOVA, indicates a limited gene flow among populations. In phytophagous insects, dispersal capacity, geographical or reproductive barriers, host plant, and habitat fragmentation are reported as the main drivers of genetic structure , 70, 71. ability , as welloaptera, ). In addoaptera, . In B. vspersion . These fB. vicetinus from the Euganean Hills and to a lesser extent from the Berici Hills  and recent changes (e.g. habitat loss) have contributed to determining the genetic structure and diversity of the bush-cricket in its outbreak range, resulting in genetically structured populations.High haplotypic diversity, low levels of sequence divergence (nucleotide diversity) and a star-like phylogeny were observed in populations of ci Hills ; Table 1ci Hills . The busB. vicetinus population structure.Further studies increasing the sampling size, mostly in areas where the species is rare (e.g. Lessini Mountains), combined with analysis of other markers could help obtain a better picture of B. vicetinus, we have achieved an important step in understanding this native species. Shedding light on the origin of a species is also important for its biological control. Given that natural enemies and their host tend to coevolve, biological control programmes often rely on the use of parasitoids present in the native areas. Failure to identify the correct origin of a pest may lead to the use of unsuitable species as biocontrol agents with negative effects on control programmes [B. vicetinus is currently in outbreak status, control programme strategies should consider the intrinsic vulnerability of an endemic species. Its low dispersal ability and small and fragmented distribution range could lead this native species to quickly shift from outbreaking to endangered.The current findings contribute to a growing knowledge of the status and evolutionary history of the pest. Here, by removing doubt about the origin of the ogrammes , 77. Maxogrammes  will be S1 Fig(TIF)Click here for additional data file.S2 FigNetwork obtained with the COI-tRNALeu-COII data set.(PDF)Click here for additional data file.S3 FigNetwork obtained with the COI-tRNALeu-COII data set. B) Network obtained with the 12S-CR data set. Network obtained with the 12S-CR data set.(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file. 1 Mar 2021PONE-D-20-40749Studying genetic population structure to shed light on the demographic explosion of the rare species Barbitistes vicetinus PLOS ONEDear Dr. Martinez-Sañudo,Thank you for submitting your manuscript to PLOS ONE. 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Reviewer #1: he authors present analyses of mtDNA variation intended to shed light on the origin of a recent pest insect outbreak. The analyses are sound as are the conclusions. I have two main comments and a few minor issues outlined below.line 169 I think this paragraph should be deleted since the tree is not mentioned (maybe on line 379?) or presented anywhere. Nor is it needed given that assumptions of bifurcating relationships is likely violated (which is presumably the justification for the TCS network).lines 309-313 One major issue seems to be neglected: how do the estimates of the timing of demographic change compare to the ideas presented in the introduction that the change has occurred very recently? The inferences from molecular data indicate a post-Pleistocene time frame for demographic expansion, but the introduction (line 54) indicates the outbreak has occurred over the last decade. As presented, the attempt to time the demographic events seems to indicate that the authors are thinking of the very recent outbreak, though I suspect, given the nature of the data and the analyses, that only historical events can be detected. I suggest the authors re-arrange the questions and methods to separate these issues and to be more clear on why they are interested in timing the demographic events (which don't have anything to do with the last decade). Temporal sampling with 100's to 1000's of bi-parentally inherited markers might be required to detect changes in Ne over the very near term.minor commentsline 41 this is not clear - what is meant by "genetic origin"?line 44 how does mtDNA help with biocontrol strategies? This idea could be expanded by including some of the logic that appears in the discussion.line 52 I think that some more precise language is needed rather than "ecological balance", a phrase that has had a controversial and tortuous history in ecology.line 97 DO the authors have the requiste geographical sampling to answer this question of alien origin?? It seems that some basis for comparisons is required for this.line 114 Change "expositions" to "exposures"lines 106-121 refer to map fig 1line 169 I think this paragraph should be deleted since the tree is not presented and not mentioned (maybe on line 379?) or presented anywhere. Nor is it needed given that assumptions of bifurcating relationships is likely violated (which is presumably the justification for the TCS network)?line 181 delete "a" before coalescentline 192 delete "occurred"line 216 it might be useful to describe what you are looking for, i.e. what distinguishes numts from mtDNA genes?line 229 delete the percent signline 379 what aspects are unresolved? The data presented seem to me to provide a fairly clear picture. Further, why "phylogenetic" resolution? The network clearly demonstrates shared haplotypes indicative of gene flow or, more likely, incomplete lineage sorting for which phylogenetic methods are inappropriate - see comment above .FIg 1 top: what do the lines within haplotype circles signify? I think the authors might be indicating proportions shared between regions (haplotypes A1 and I1) or among subregions  but this is not clear from the colouration.Reviewer #2: A great application of these techniques. It would be nice to see similar application to other pest species.I have only minor questions:(1) How significant is the limited geographical mobility to your interpretation? Would you not come to similar conclusions on species that can disperse but usually show site fidelity?(2) I would like to see some discussion of application to other species.(3) What light can such techniques shed on the efficacy of geographically-based pest control methods (such as quarantines)?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  25 Mar 2021Reviewer #1 The authors present analyses of mtDNA variation intended to shed light on the origin of a recent pest insect outbreak. The analyses are sound as are the conclusions. I have two main comments and a few minor issues outlined below.line 169 I think this paragraph should be deleted since the tree is not mentioned (maybe on line 379?) or presented anywhere. Nor is it needed given that assumptions of bifurcating relationships is likely violated (which is presumably the justification for the TCS network).Ok. Thank you. We deleted this paragraph.lines 309-313 One major issue seems to be neglected: how do the estimates of the timing of demographic change compare to the ideas presented in the introduction that the change has occurred very recently? The inferences from molecular data indicate a post-Pleistocene time frame for demographic expansion, but the introduction (line 54) indicates the outbreak has occurred over the last decade. As presented, the attempt to time the demographic events seems to indicate that the authors are thinking of the very recent outbreak, though I suspect, given the nature of the data and the analyses, that only historical events can be detected. I suggest the authors re-arrange the questions and methods to separate these issues and to be more clear on why they are interested in timing the demographic events (which don't have anything to do with the last decade). Temporal sampling with 100's to 1000's of bi-parentally inherited markers might be required to detect changes in Ne over the very near term.The analyses of past demographic events were conducted to understand how past events could have shaped the genetic structure of B. vicetinus populations. The time since the expansion events revealed by the analysis was calculated to infer the historical period in which the expansion could have taken place, being aware that this expansion is different from the recent outbreak events. However, this analysis is secondary in importance compared to the genetic diversity analysis. To be clearer and avoiding misunderstandings we delete one sentence in the abstract, modified the third question in the introduction (line 101), as well as some sentences in discussion (lines 366-368), paying more attention to keep separated these issues.line 41 this is not clear - what is meant by "genetic origin"? We changed this unclear word. Please see line 40line 44 how does mtDNA help with biocontrol strategies? This idea could be expanded by including some of the logic that appears in the discussion. Thank you for your suggestion. We briefly expanded it. Please see lines 43-44.line 52 I think that some more precise language is needed rather than "ecological balance", a phrase that has had a controversial and tortuous history in ecology. Done. Please see lines 52.line 97 DO the authors have the requiste geographical sampling to answer this question of alien origin?? It seems that some basis for comparisons is required for this. We agree with the reviewer. However, the distribution range of B. vicetinus is limited to the small area of north-east Italy where the sampling was performed. The species has not been reported elsewhere, so far.In addition, we have conducted a preliminary phylogenetic study of the species belonging to the Western-Palearctic genus Barbitistes (through barcoding). Sequences of B. vicetinus were different from sequences of other Barbitistes sp. and all the B. vicetinus specimens grouped in a monophyletic clade (unpublished data). We added more details in the text. Please see line 67-68.line 114 Change "expositions" to "exposures” Done. Please line 115.lines 106-121 refer to map fig 1 Done. Please line 111.line 169 I think this paragraph should be deleted since the tree is not presented and not mentioned (maybe on line 379?) or presented anywhere. Nor is it needed given that assumptions of bifurcating relationships is likely violated (which is presumably the justification for the TCS network)? Done.line 181 delete "a" before coalescent Done. Please see line 179.line 192 delete "occurred" Thank you. Done. See line 190.line 216 it might be useful to describe what you are looking for, i.e. what distinguishes numts from mtDNA genes? Ok. Thank you. We added more information. See line 214-218.line 229 delete the percent sign Done. Please see line 232.line 379 what aspects are unresolved? The data presented seem to me to provide a fairly clear picture. Further, why "phylogenetic" resolution? The network clearly demonstrates shared haplotypes indicative of gene flow or, more likely, incomplete lineage sorting for which phylogenetic methods are inappropriate - see comment above . We agree with the reviewer. We modified it. Please see line 384-385.FIg 1 top: what do the lines within haplotype circles signify? I think the authors might be indicating proportions shared between regions (haplotypes A1 and I1) or among subregions  but this is not clear from the colouration. Thank you for your suggestionWe added this information in the figure legend trying to make it clearer:Lines within haplotypes circles indicate the proportions shared between collection areas(Please see lines 263-264) Reviewer #2 A great application of these techniques. It would be nice to see similar application to other pest species.I have only minor questions:(1) How significant is the limited geographical mobility to your interpretation? Barbitistes vicetinus tends to stay close to its hatching site during the whole life cycle, likely due to its low dispersal ability. This characteristic, coupled to the low density of the species until 2008, probably helped to maintain the strong genetic structure of the species. However, in correspondence with the outbreaks, a tendency of the insect to disperse more than before was reported . During the outbreaks, B. vicetinus have colonized new habitats and moved far from the hatching areas, perhaps to avoid intraspecific competition. Besides the low mobility of the bush-cricket other factors such as geographical barriers and spatial configuration could have favoured the absence of gene flow between B. vicetinus populations. The lowland areas between the Euganean and Berici Hills, with several agricultural fields and without woody vegetation could have acted as a geographical barrier, notwithstanding the short geographical distance. We have also observed that patchy areas of the non-host alien tree Robinia pseudoacacia have played an important role in reducing population density and dispersion of the pest . (1) b) Would you not come to similar conclusions on species that can disperse but usually show site fidelity? The low dispersal ability is undoubtedly a contributing factor to maintaining a high levels of genetic differentiation. Other species that have the ability to move but show fidelity to their life site could also show a strong genetic structure, although it may be less marked as with low-mobility species. Species showing breeding site fidelity could move if necessary  and it could modify the genetic structure.(2) I would like to see some discussion of application to other species. Similar studies can be applied to a wide range of insect species. Population genetic strategies have been widely used for examining patterns and magnitude of dispersal of several insects over geographic and temporal scales. Regarding invasive species, for example, population genetics studies can be conducted for highlighting and discovering the sources and colonization routes of these alien insects. The information obtained with genetic analyses can be also useful for an effective conservation of endangered species. Indeed, analysing the genetic diversity patterns of endangered species has become an integral component of many management strategies. Some examples of the application of these techniques to other species can be found in lines 74-83 and 93-95.(3) What light can such techniques shed on the efficacy of geographically-based pest control methods (such as quarantines)? The genetic techniques used in this study have allowed us to realize that population outbreaks are not always a consequence of a single or few pestiferous haplotypes. As reported here, any individual of this species, regardless of its genetic haplotype, had an equal chance of producing outbreaks. It follows that control strategies must always be applied (e.g.: strategies based on geographic control). For species characterized by a reduced movement behaviour throughout their lifespan (such as the bush-cricket here studied), gene flow appears low enough to maintain a high genetic structure among populations. In this case, control programs based on isolated treatments  could be also effective. Thus, these results based on genetic techniques strengthen the need to apply and enhance control methods such as quarantines or other strategies based on geographic control.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 8 Apr 2021Studying genetic population structure to shed light on the demographic explosion of the rare species Barbitistes vicetinus PONE-D-20-40749R1Dear Dr. Martinez-Sañudo,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Bi-Song Yue, Ph.DAcademic EditorPLOS ONE 22 Apr 2021PONE-D-20-40749R1 Barbitistes vicetinus  Studying genetic population structure to shed light on the demographic explosion of the rare species Dear Dr. Martinez-Sañudo:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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The domestication and spreading of grapevine as well as the gene flow history had been described in many studies. We used a high-quality 7k SNP dataset of 1,038 Eurasian grape varieties with unique profiles to assess the population genetic diversity, structure, and relatedness, and to infer the most likely migration events. Comparisons of putative scenarios of gene flow throughout Europe from Caucasus helped to fit the more reliable migration routes around the Mediterranean Basin. Approximate Bayesian computation (ABC) approach made possible to provide a response to several questions so far remaining unsolved. Firstly, the assessment of genetic diversity and population structure within a well-covered dataset of ancient Italian varieties suggested the different histories between the Northern and Southern Italian grapevines. Moreover, Italian genotypes were shown to be distinguishable from all the other Eurasian populations for the first time. The entire Eurasian panel confirmed the east-to-west gene flow, highlighting the Greek role as a “bridge” between the Western and Eastern Eurasia. Portuguese germplasm showed a greater proximity to French varieties than the Spanish ones, thus being the main route for gene flow from Iberian Peninsula to Central Europe. Our findings reconciled genetic and archaeological data for one of the most cultivated and fascinating crops in the world. Vitis vinifera L. subsp. sativa) is one of the earliest domesticated and most cultivated crops worldwide, having a great impact on the agri-food economy and prized for its fruits and wines. Europe is the largest producer , followed by Asia and America. Nowadays, worldwide grape production reaches nearly 78 M tons (http://faostat.fao.org/).Grapevine , when human populations began to collect and propagate The Italian Peninsula seems to have had a key role in the spreading of grapevine and viticulture practices from Greece to Central and Western Europe, due to its strategic position on the Mediterranean Sea. In the eighth century BC, the establishment of Greek colonies in the Southern Italy was the main driver of viticulture expansion and development in these areas. In the beginning, the settlers conceivably introduced vines from their places of origin, nonetheless they plausibly also cultivated local populations crossed with Greek varieties  and the Italian Register  list more than 13,000 and nearly 800 Eurasian grapevine varieties, respectively.In Europe, a long period of significant successes for viticulture has been witnessed due to the human selection of new and valuable cultivars, up to the current age. To date, the Vitis International Variety Catalog  loci were developed   decay, and ancestry coefficients, as well as, inferring migration events through the evaluation of putative scenarios.https://www.crea.gov.it—and AGER consortium—https://www.progettoager.it) showing unique SNP profiles and covering most of the Italian germplasm were selected from 615 genotypes firstly investigated for parentage analysis  on both the Italian (384 genotypes) and whole  grapevine dataset were performed to estimate the overall relationship among the varieties. The distance-based dendrogram with Nei's genetic distance Nei, , UPGMA aK) was computed for both datasets using an admixture model performed through fastSTRUCTURE  was chosen applying the algorithm for multiple choices, chooseK.py. To extract the optimum K, the prediction error for each K was computed by the cross validation (CV) function. Bayesian analyses for both datasets were performed also by discriminant analysis of principal components (DAPC) implemented in the R/adegenet. The Bayesian information criterion (BIC) method , a population pairwise index  segments were detected in the whole dataset. Relatedness analysis using IBD estimation was conducted through R/SNPrelate  was calculated between each pair of molecular markers . Migration edges were tested 10 times from 1 to 5 with different random seeds each time and using blocks (k) of 20 SNPs, to check for convergence in terms of the likelihood value of each model, and the variance explained in each migration event was added. Standard errors (SE) and bootstrap replicates (bootstrap) were used to assess the confidence in the inferred tree topology and the weight of migration events. To automate the choice of the best migration event, an ad hoc statistic based on the second-order rate of change in the likelihood weighted by SD was adopted through R/OptM  explained 99.8% of the variance in ancestry between groups, only the model showing this cutoff was believed to be the best one -tree of data collected and a gene flow model among the geographic groups  were devM Fitak, . HoweverK was chosen after a cross-entropy criterion evaluation for each K  approach implemented with DIY-ABC 2.0 software was adopted to establish the most likely grapevine gene flow scenarios  Scenario 10 assumed a gene flow from EMCA-MFEAS-RUUK to Northern Africa (MAGH) and then to IBER (ii) Scenario 11 assumed that an admixture event between MAGH and EMCA-MFEAS-RUUK germplasm flowed into the IBER genotypes; (iii) Scenario 12 assumed a contrary hypothesis to Scenario 10.5 simulated datasets, considering all scenarios. Mean gene diversity across all loci, mean across loci of Fst distances between pairs of geographic groups, and mean across loci of Nei's genetic distances between pairs of geographic groups were calculated. The most appropriate scenario for each hypothesis was selected comparing the summary statistics of simulated and observed datasets. The posterior probabilities [with 95% confidence interval (CI)] of each competing scenario were also estimated following a logistic regression approach on the 1% of simulated datasets closest to the observed dataset. The ability of ABC approach to discriminate between scenarios (ABC performance) was evaluated. Per each scenario, Type I and II error probabilities were also estimated  varieties, except for 14% of ITAP-south that clustered in the Northern Italian group and  samples . The Cen samples . A PCoA  samples , splittiK was firstly evaluated with fastSTRUCTURE, providing an optimal value between 2 and 6 . Based on the prediction error, the lowest model complexity within the range was explained at K = 5  were attributable to a specific pool with a high percentage (ancestry membership >70%) by fastSTRUCTURE. About sixty-two (87%) out of 71 ITAP-north samples formed Pool 1, the ITAP-center genotypes were mainly split between Pools 1 (28) and 3 (19), while Pool 2 is formed almost only by the ITAP-south genotypes (88%), and four from ITAP-center . About 5K = 3  Italian varieties to one of the three major pools, setting a cut-off of 70% posterior membership probabilities . Almost K = 3 , the samfunction .The DAPC results matched with that of PCoA and the unweighted pair group method with arithmetic mean (UPGMA). According to these results, the Northern and Central Italian genotypes were joined and named ITAP-north-center .Cluster analysis distinguished genotypes from the IBER, ITAP-north-center, and WCEUR in three clusters . Nearly A similar distribution was obtained through PCoA analysis , with thK = 9, using both prediction error and BIC method . The lowest Fst pairwise-value (0.009) was recorded between WCEUR and ITAP-north-center groups, followed by EMCA-MFEAS-RUUK and BALK groups (0.011) and ITAP-south and BALK(0.011)  . The NJ t-values  split upt-values .Based on genome-wide IBD pairwise distances, the MDS analysis showed a distinctness of IBER (red), as well as EMCA-MFEAS-RUUK (cyan), and BALK (blue) populations, while the other populations were not clearly separated . The aveThe average LD declined with the increase of physical distance between markers and appeared fast in all the populations. The fastest decay was observed in EMCA-MFEAS-RUUK population, followed by BALK and in the pair from ITAP . In contm) were added in sequence, showing the saturation of the model likelihood at four additional migration edges  for all migration edges recorded was chosen with the best fits at four inferred migration events (p < <0.001), from EMCA-MFEAS-RUUK to IBER , from IBER to WCEUR , and from ITAP-north-center to EMCA-MFEAS-RUUK  groups  groups .m was also verified through the Evanno and linear methods, inferring two admixture events from ITAP-south to ITAP-north-center  and from EMCA-MFEAS-RUUK to IBER  populations (m = 4 with the highest f (0.998 vs. 0.992) and the lowest residual values (SE ± 2.7 vs. SE ± 6.8)   were obtThe ancestry estimation was performed adding 21 Northern African (MAGH) genotypes  to the Mediterranean Basin . MoreoveFour groups of scenarios were tested using ABC approach to define the possible East-to-West grapevine migration routes . The ABCScenario 1 showed the best fitting values, assuming a gene flow from BALK to ITAP-south, then to ITAP-north-center . For SceThe second question related to the gene flow between ITAP and WCEUR populations  establisAmong the scenarios hypothesized to evaluate the possibility of a gene flow from ITAP-south to WCEUR population through IBER population , ScenariThe last ABC test confirmed Scenario 10 as the most likely model  taking ivia incipient domesticated populations  and Greeks (7th-6th BC). The former domesticated wild grapevines in ITAP-north-center, while the Greeks introduced their varieties in ITAP-south. Differences between these areas occurred in winegrowing, the choice of varieties, and in the way to manage the vines  including South-Italian varieties and two others overlapped, the first mainly counting North-Italian varieties (Pool 1), and the second (Pool 3) including varieties from all Italian areas  on the whole dataset, where ITAP-south germplasm resulted as the main contributor to xed pool .Fst index among all pairwise comparisons  of which seven pools showed the highest ancestry membership values in agreement with the geographic origin. Similarly, Pools from 1 to 5 were assigned to IBER, EMCA-MFEAS-RUUK, Georgian, ITAP-north-center, and ITAP-south populations, respectively (K2 (including EMCA-MFEAS-RUUK germplasm) and K6 pools. Therefore, Greece appears to be the main bridge between Near Eastern germplasm and the Mediterranean Basin (ITAP), supporting the theory that grapevines were spread from the Greek shores to the Southern and then Northern Italian ones , appearing to be the most plausible candidate group for the genetic exchange between IBER and WCEUR, highlighted by TreeMix computations and DAPC analysis. This hypothesis is also supported by ancestry coefficient analysis, where Portugal is painted with two colors  can be found in the article/supplementary material.FM, GDL, MC, and FS: conceptualization. FM and GDL: methodology, formal analysis, and writing—original draft. AM, MZ, CM, PR, LB, CD'O, MB, CB, LP, and VN: investigation. CM, PR, LB, CD'O, MB, CB, LP, and VN: resources. FM, MC, and FS: supervision. CM, PR, LB, CD'O, MB, CB, LP, FS, and VN: funding acquisition. All authors contributed to the writing—review and editing, article, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ne) was estimated to be 199 individuals, and total combined population size (NC) was 499, with each cluster showing a relatively high level of genetic variability, suggesting that migration has been sufficient to counteract genetic erosion. The mtNDA results were congruent with the microsatellite data, and the three genetic clusters were matched predominantly with an equal number of mtDNA haplotypes that belong to the brown bear Western mitochondrial lineage (Clade 1), with two haplotypes being globally new and endemic. The detection of a fourth haplotype that belongs to the Eastern lineage (Clade 3a1) in three bears from the western distribution places the southernmost secondary contact zone between the Eastern and Western lineages in Greece and generates new hypotheses about postglacial maxima migration routes. This work indicates that the genetic composition and diversity of Europe's low‐latitude fringe population are the outcome of ancient and historical events and highlight its importance for the connectivity and long‐term persistence of the species in the Balkans.In human‐dominated landscapes, connectivity is crucial for maintaining demographically stable mammalian populations. Here, we provide a comprehensive noninvasive genetic study for the brown bear population in the Hellenic Peninsula. We analyze its population structuring and connectivity, estimate its population size throughout its distribution, and describe its phylogeography in detail for the first time. Our results, based on 150 multilocus genotypes and on 244‐bp sequences of the mtDNA control region, show the population is comprised by three highly differentiated genetic clusters, consistent with geographical populations of Pindos, Peristeri, and Rhodope. By detecting two male bears with Rhodopean ancestry in the western demes, we provide strong evidence for the ongoing genetic connectivity of the geographically fragmented eastern and western distributions, which suggests connectivity of the larger East Balkan and Pindos‐Dinara populations. Total effective population size ( Using microsatellites and mtDNA, we reveal the genetic structure, diversity, population size of the brown bear in Greece, and its detailed phylogeography for the first time, revealing a new matrilinear contact zone Events, such as population size depletion, range shifts, and bottlenecks, have undoubtedly shaped the distribution of genetic variation of European wildlife Hewitt, . One groUrsus arctos L., 1758) was primarily shaped by the Late Pleistocene glaciations when bears primarily took refuge in the Mediterranean Peninsulas and most likely also in the Carpathian–Caucasus and some unknown refugia in the east, from where the species recolonized the continent, reaching as far north as Scandinavia , stool (n = 90), and blood (n = 10) samples with the latter used to create reference genotypes for allelic acceptance. For the stools, we followed the modified protocol of Skrbinšek et al.  and 1μL of GS500LIZ size standard (Applied Biosystems) for fragment analysis in an ABI 3,730 (Applied Biosystems) sequencer.DNA was amplified for 11 commonly used microsatellite loci G1D, G10C, G10H, G10L, G10P, G10X, Mu09, Mu10, Mu15, Mu23, Mu50, Mu59, and the SRY marker  simulations after a 2 x 105 burn‐in period. K was allowed to vary from 1 to 10, and a total of 10 independent simulations were run for each K. We selected the admixture model with correlated allele frequencies without prior identification of subpopulations and allowed alpha to vary. Log‐likelihood [Ln(K)] values were plotted in Structure Harvester v0.563 . In GENELAND, a Poisson–Voronoi tessellation was run in a two‐step mode, performing a number of tests to determine the best fit of the parameters. For the first step, 105 MCMC iterations were performed in five independent runs where K was set to vary 1–10 with thinning of 100. We used the uncorrelated model which performs better . The modal population was selected after a burn‐in period of 10 x 100 iterations. In the second step, we selected the run with the highest posterior probability and run the analysis an additional 30 times to assign individuals into clusters, setting K fixed to the optimal value suggested by the first run, with all other parameters kept the same as in step one. As Bayesian inference can cause artificial clustering , which is independent of sample size, was calculated in FSTAT  in GenAlEx  was estimated in GENEPOP and corrected for sample size , allele frequencies (dA), and inbreeding coefficient (dF), we adjusted the values to 0.1, 0.25, and 0.35, respectively, to achieve acceptance rates for changes to the parameters between 40% and 60%  was based on two capture histories generated for each cluster (Appendix n = 7) were excluded from the analysis. Effective population size (Ne) was computed using the linkage disequilibrium method in LDNe, which is recommended for both unlinked and linked loci and has greater power in small sample sizes , Peristeri (n = 10), Rhodope (n = 9), and for the sample from Olympus (n = 1). Using the primer pair 1 from Keis et al.  and the remaining bears were sampled from Rhodope (n = 22). Amplification success was higher for stool (76%) than for hair (66%) samples. No locus had an estimated frequency of null alleles above 0.1. PIDSIB analysis. Waits et al.  whereas 74% of the hair samples collected from rub surfaces belonged to males, 23% to females, and 3% were undetermined. Most individuals were sampled in the western distributions  approaches the highest value but still has a low variance, increasing only slightly or starting to plateau for higher K. In our analyses, this occurred at K = 3, suggesting three subpopulations and no bias due to the presence of family groups  value had larger variance than K = 3; however, a clear‐cut distinction of the Q values was observed between the eastern and the western population, with further substructuring resulting in genetic clusters matching the geographical distribution  for the western distribution as a whole and within Pindos  and Peristeri . The result after correction for clustering was r = −0.08 (p < .001).GENELAND confirmed that the most likely number of demes was 3.3HE) decreases from north to south by almost equal intervals . Three times as many private alleles were found in Rhodope than in the Pindos subpopulation with the relative percentage of these values being 6.9% in Pindos, 9.8% in Peristeri, and 23.9% in Rhodope. Mean FIS across all clusters was 0.036 (n = 3) suggesting low inbreeding and connectivity within demes.All loci were found to be polymorphic and the number of alleles ranged from 5 to 11 across all populations with Rhodope population being the most polymorphic in all measures  and lowest between Pindos and Rhodope (Nm = 0.1), whereas between Peristeri and Rhodope Nm = 0.6.Pairwise ns Table . Bayesians Table . All tenns Table  and the 3.4NE was highest for Pindos with 97.4 (95% CI: 64.3–163.8), followed by Peristeri with 59.1 (95% CI: 32.8–181.1), and 42.2 (95% CI: 25.3–97.7) for Rhodope. All three populations exhibited signatures of recent bottlenecks under IAM and Pindos under TPM . Singletons may inflate population estimates , Peristeri (n = 9), Rhodope (n = 9), and Olympus (n = 1). Trimming and alignment with homologous sequences from GenBank yielded a dataset of 51 haplotypes with a final sequence length of 244 bp, spanning pos. 15452–15716 according to reference sequence HQ685901 (pos. 15526–15542 containing the pyrimidine tract were excluded due to high level of homoplasy). The analyzed samples yielded four haplotypes in total  was found in bears exclusively from Rhodope; haplotype 31 (n = 38) was carried by 36 bears from Pindos and three from Peristeri, whereas haplotype 32 (n = 7) was confined to Peristeri and the single bear from Olympus , which belongs to the Eastern mtDNA lineage (clade 3a1)  while nucleotide diversity was 0.0133 ± 0.0028 (π ± SD) for the whole Greek population  and deltaK agreeing on the optimum number of clusters (K = 3).In the first comprehensive multi‐source noninvasive genetic study at range scale of the brown bear population in Greece, utilizing both microsatellite and mtDNA markers we report the presence of three highly differentiated genetic groups Figure . Using tK = 3 illustrating in more detail the spatial pattern of the structuring and the contact zones of the populations inferred by the microsatellites  where the habitat creates corridors for dispersing bears to use. GENELAND supported the case for 0.85° whes Figure  especials Figure . This arPylidis, .4.2The detection of two male bears in the western distribution with Rhodopean ancestry illustrates for the first time to our knowledge, recent demographic and genetic connectivity between the Pindos and Rhodope distributions in Greece and, by extension, connectivity of the wider Dinaric‐Pindos and East Balkan populations. Bears are capable of dispersing over long distances with a proven capacity to overcome barriers such as water bodies and highways  is not far from the migration fixation index which predicts that when FST = 0.2 then Nm is expected to be ≤ 1). A possible explanation for the observed differentiation is that the mountainous terrain in Northern Greece may act as a physiographic barrier limiting dispersal between Peristeri and Pindos and in conjunction with the female philopatry; further enhance differentiation. Mountain ranges act as biogeographical barriers preventing gene flow and inducing differentiation even in highly mobile species with large home ranges and long‐distance dispersal capacity , but much higher than the other populations of Mediterranean Peninsulas. Perez et al. . As unique alleles arise faster through mutation under conditions of limited gene flow and genetic isolation ; Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Resources (lead); Validation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Peeter Anijalg: Formal analysis ; Investigation ; Validation ; Writing‐original draft ; Writing‐review & editing . Urmas Saarma: Formal analysis ; Investigation ; Validation ; Writing‐original draft ; Writing‐review & editing . Deborah A. Dawson: Methodology ; Supervision ; Validation . Nikoleta Karaiskou: Methodology (supporting). Roger Butlin: Methodology (supporting); Supervision (supporting). Yorgos Mertzanis: Project administration (supporting); Resources (supporting). Alexios Giannakopoulos: Investigation (supporting); Resources (supporting). Yorgos Iliopoulos: Investigation (supporting); Resources (supporting). Andrew Krupa: Data curation (supporting); Methodology (supporting); Validation (supporting). Terence A. Burke: Resources (supporting); Supervision (supporting).Appendix S1–S7Click here for additional data file.


Culter alburnus. We sought to discover the differentiated intestinal structure and functional divergence in the two geographically isolated populations. Our study indicated that significantly different intestinal structures, digestive enzymes activities, and intestinal microbiomes were presented in the two populations, which may have been due to adaptative evolution caused by the long-term geographical isolation.Population differentiation usually forms between geographically isolated populations. We compared the intestinal structures, digestive enzyme activities, and intestinal microbiomes in the Xingkai (XK) Lake and Dianshan (DS) Lake populations of Culter alburnus inhabiting different regions of China present substantial differences in their reproduction and development characters. This study compared the intestinal structures, digestive enzyme activities, and intestinal microbiomes in the Xingkai (XK) Lake and the Dianshan (DS) Lake populations of C. alburnus collected in two isolated and contrasting river systems. We wanted to discover whether the intestinal structure and functional divergence were formed in the two populations due to adaptive evolution caused by geographical isolation. Our study indicated that higher intestinal villi, thicker intestinal mucosa layer and intestinal muscle layer, and significantly higher activity of α-amylase were identified in the XK population. Moreover, quite different intestinal microbiomes were presented in the two populations, with the higher abundance of Bacteroidetes and Firmicutes in the XK population. The significantly different intestinal microbiome in the XK population was functionally enriched in carbohydrate, lipid, and amino acid metabolism by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Our findings indicated that substantial adaptative divergence in the intestinal structures and intestine microbiomes was formed in the two populations due to long-term geographical isolation, which may have strongly affected the digestion and absorption ability of the XK population compared with the DS population.Geographical populations of  Significant population differentiation or environmental adaptation may lead to the formation of new, distinct species, which is a process called speciation . It is wCulter alburnus) is a fish species with high economic value distributed throughout China [C. alburnus are formed and distributed in Xingkai Lake, Yellow River, Huai River, Yangtze River, and other river systems, which present differentiated population structures and substantial divergent characters [C. alburnus is Xingkai Lake, located in Heilongjiang Province, in the northern part of China . Due to its unique geographic position and water environment, the C. alburnus population of Xingkai Lake forms an independent population structure, and presents divergent characters such as the characteristics of eggs, body shape, growth, and development compared to other populations of C. alburnus in the southern part of China, such as in the Yangtze River region [C. alburnus.Topmouth culter  Lake   and the Dianshan (DS) Lake   in 2014 (p > 0.05) were collected from two respective ponds. The collected C. alburnus individuals (n = 10) from the XK and DS populations were immediately dissected. The midgut tissue was sampled, snap-frozen in liquid nitrogen, and stored at −80 °C in a freezer. This study was approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University . The sampling procedures complied with the guidelines of the Institutional Animal Care and Use Committee on the care and use of animals for scientific purposes.In this study, two  in 2014 . Both coThe collected midgut tissues from the XK and the DS populations were stored in 4% paraformaldehyde overnight. Then, tissue slices were prepared and stained according to the hematoxylin-eosin (HE) staining method. The slice thickness was set to be 5 um, and after HE staining, the intestinal structure was observed under a microscope system . The height of the intestinal villi, the thickness of the intestinal mucosal layer, and the thickness of the intestinal muscle layer were measured and compared between the two populations .n = 6). The enzyme activities of LPS (U/gprot), the α-AMS (U/gprot), and the Trys (U/gprot) were measured using a lipase assay kit, an α-amylase assay kit, and a trypsin assay kit, respectively  and normalized using the total protein content. The data was presented with mean ± SD, and statistical analysis was performed using the Student’s t-test.Digestive enzyme activities such as lipase (LPS), α-amylase (α-AMS), and trypsin (Trys) of the midgut tissue were measured in the XK and the DS populations . Then, paired-end sequencing libraries (PE250) were constructed using a TruSeqTM DNA Sample Prep Kit . The constructed sequencing libraries were sequenced on an Illumina NoveSeq 6000 platform . The raw reads were deposited into the NCBI SRA database (NCBI accession number: PRJNA792693).DNA was extracted from the midguts of the 10 individuals in each population (XK and DS) using the HiPure Stool DNA Kits  according to the manufacturer’s protocols. The concentration and purity of extracted DNA were evaluated with a NanoDrop 2000 platform . The 16S rDNA target region (V3-V4) was amplified by using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers . The ampt-test.Raw sequencing reads were first quality-filtered to remove the adaptors and the low-quality reads before data processing using FASTP v0.19.6 software . Noisy st-test using the Vegan package v2.5.3 implemented in the R project. The unweighted_unifrac distance matrixes generated by Vegan package v2.5.3 were used to calculate the beta diversity, and were visualized via principal coordinates analysis (PCoA) using the ggplot2 package implemented in the R project. The analysis of similarities (ANOSIM) test was conducted to detect differences between the XK and DS populations using QIIME software with unweighted_unifrac distance [t-test [The alpha diversity of Sobs, Chao, ACE, Shannon, Simpson, and Pielou’s evenness indices were calculated using QIIME v1.9.1 . Alpha ddistance . The KEG [t-test .C. alburnus (p < 0.05), and the thickness of the intestinal mucosa layer (IMT) and intestinal muscle layer (MLT) also were higher in the XK than in the DS population (p < 0.05)  D.A total of 2,504,505 paired-end sequences were obtained after sequencing and raw read filtering. After sequence clustering and annotation, a total of 2261 different OTUs, representing 30 bacterial phyla and 482 bacterial genera, were identified . The rarp < 0.05) (p > 0.05) (p = 0.001)  . No sign > 0.05) . Beta di > 0.05) A. The AN= 0.001) B.The XK and the DS populations presented substantially different intestinal microbiome compositions and abundances. At the phylum level, the top five dominant bacterial phyla were Proteobacteria (60.68%), Firmicutes (14.98%), Bacteroides (14.14%), Actinobacteria (4.39%), and Cyanobacteria (1.18%) in the XK population. In contrast, those in the DS population were Proteobacteria (61.98%), Firmicutes (10.88%), Bacteroides (7.89%), Planctomycetes (4.62%), and Verrucomicrobia (4.08%) A. At thet-test, p < 0.05; t-test, p < 0.05). The proportion of Cellvibrio, Pseudoxanthomonas, Luteimonas, Devosia, Lysobacter, and other genera were significantly higher in the XK than in the DS population, and only the proportion of Polynucleobacter was significant higher in the DS group (p < 0.01)  B.Putative microbiome functions were predicted using PICRUSt2 software. The functional categories, which represented a set of genes influencing the same functional profile, varied significantly between the two populations . In partC. alburnus has been reported to develop adaptative divergence such as spawning floating eggs, while other geographical populations in the southern part of China, such as the DS population, spawn adhesive eggs [Reproductive isolation between or among populations usually causes them to be genetically differentiated, and if maintained for a sufficient period, leads to adaptative evolution and ultimately speciation. The most documented and probably most widespread cause of reproductive isolation is geographic isolation . The XK ive eggs . Moreoveive eggs ,25. In tCyprinus carpio, and cold exposure or cold transplantation increased the gut size and absorptive capacity in mice [The intestinal tract is an essential organ for fish to digest and absorb food resources. The digestion and absorption capacity of organisms can be assessed by the structure of the intestine and the activities of related digestive enzymes. The height of intestinal villi; the thickness of intestinal mucosa and intestinal muscle layers; and the activities of LPS, Trys, and α-AMS directly affect the organisms’ digestion and absorption capacity ,12. Our  in mice ,27. The The intestinal microbiome of fish species plays essential roles in digestion, metabolism, and other biological processes ,28,29. IIn summary, different intestinal structures, digestive enzyme activities, and diverged intestinal microbiomes in the Xingkai Lake population compared with the Dianshan Lake population indicated functionally diverged digestion and absorption abilities, which may have been due to adaptative evolution caused by the long-term geographically isolation in the Xingkai Lake population compared with the Dianshan Lake population.


Macaca, which shows frequent inter-specific hybridization and predominantly female philopatry. We found that hybridization and admixture were prevalent within these species. For three cases of suggested hybrid origin of species/subspecies, Macaca arctoides, Macaca fascicularis ssp. aurea, and Chinese Macaca mulatta, we examined the level of admixture of X chromosomes, which is less affected by male-biased migration than that of autosomes. In one case, we found that Macaca cyclopis and Macaca fuscata was genetically closer to Chinese M. mulatta than to the Indian M. mulatta, and the admixture level of Chinese M. mulatta and M. fuscata/cyclopis was more pronounced on the X chromosome than on autosomes. Since the mitochondrial genomes of Chinese M. mulatta, M. cyclopis, and M. fuscata were found to cluster together, and the mitochondrial genome of Indian M. mulatta is more distantly related, the observed pattern of genetic differentiation on X-chromosomal loci is consistent with the nuclear swamping hypothesis, in which strong, continuous male-biased introgression from the ancestral Chinese M. mulatta population to a population related to M. fuscata and M. cyclopis generated incongruencies between the genealogies of the mitochondrial and nuclear genomes.The role of sex-specific demography in hybridization and admixture of genetically diverged species and populations is essential to understand the origins of the genomic diversity of sexually reproducing organisms. In order to infer how sex-linked loci have been differentiated undergoing frequent hybridization and admixture, we examined 17 whole-genome sequences of seven species representing the genus  The role of sex-biased hybridization for the origin of species is essential to understand the mechanisms of generating ecological diversity. Most of the previous studies dealing with this issue analyzed mitochondrial and Y-chromosomal data to study sex-biased migration rate, but here we focus on the genetic diversity of X chromosomes.Here we investigated 17 macaque genomes. Contrasting the pattern of autosomal and X-chromosomal genetic diversity provides strong evidence of a particularly interesting demographic model, which is called the nuclear swamping model.Our analysis showed strong sex-biased migration explained the observed pattern of unusual genetic differentiation among macaque species, highlighting the importance of sex-biased demographic parameters plays an important role for shaping the genomic diversity of organisms.A key issue in evolutionary genetics is understanding the mechanisms by which populations are genetically differentiated and continue diverging to form separate species. Genetic differentiation is often attributed to geographical isolation, and genetically differentiated populations evolve into new, reproductively isolated, species. However, hybridization and admixture among genetically divergent populations occasionally occur, impeding further genetic differentiation. Hybridization and admixture have frequently been observed in populations of wild animals and between different species .Individuals of different sexes have shown different dispersal patterns in a wide range of sexually reproducing organisms. Sex-biased migration affects the pattern of genetic differentiation across genomes. In animals with an X-Y sex chromosome system, the X and Y chromosomes, as well as the mitochondrial genomes, can be used as markers to track sex-biased migration. The effect of migration on mitochondrial genomes depends solely upon female migration, whereas that of Y chromosomes depends solely upon male migration. Similarly, the migration of males is expected to have less impact on X chromosomes than on autosomes, because a female carries two X chromosomes but a male has only one. Genetic differentiation at these sex-linked markers, therefore, provides insights into the way in which sex-biased migration has shaped animal genomes through hybridization and admixture among populations and species .Macaca consists of 24 species distributed in the part of the Eurasian and African continents, mostly in the South, Southeast, and East Asia ; the mulatta group including Macaca mulatta (the rhesus macaque), Macaca cyclopis (the Taiwanese macaque), and Macaca fuscata (the Japanese macaque); the sinica group, including Macaca sinica (the toque macaque), Macaca thibetana (the Tibetan macaque), Macaca assamensis (the Assamese macaque), and Macaca radiata (the bonnet macaque); and the arctoides group, including Macaca arctoides (the stump-tailed macaque). A phylogenetic analysis using nuclear genome data showed that the fascicularis and mulatta groups and the sinica and arctoides groups are sister pairs  Macaicularis . A genetal locus . 3) The  species .Incongruent phylogenies constructed using sex-linked loci have been often considered to be the result of male-biased migration in macaques. Because current macaque populations show predominantly female philopatry, the lineage of mitochondria is assumed to reflect the genetic lineage of the recipient population of migrants, whereas the other autosomal and Y-chromosomal genomes in the recipient population may have been “swamped” by the genomes of donor (introgressing) populations , 2013. TM. arctoides and the mulatta-group species; 2) between M. fascicularis ssp. aurea and the sinica-group species; and 3) among the mulatta-group species. We clarified the phylogeny of seven macaque species using 17 whole-genome sequences, by combining newly and previously determined whole-genome sequences of macaques, and compared the level of genetic admixture at the mitochondrial genome, autosomes, and X chromosomes.In order to investigate how sex-biased migration affects the inter-specific hybridization of genomes, we used the above-mentioned three cases that showed incongruent species trees between mitochondrial and nuclear loci: 1) between M. fuscata and M. cyclopis were obtained from the Primate Research Institute (PRI), Kyoto University. The monkeys were cared for and handled according to the guidelines established by the Institutional Animal Welfare and Animal Care Committee of PRI. They were anesthetized, and peripheral blood was obtained. The DNA samples were used for library preparation, and paired-end sequences of 101 bp were determined using HiSeq 2500 . All experimental procedures were approved by the Institutional Animal Welfare and Animal Care Committee of PRI (No. 2015-138).DNA samples of M. fascicularis samples were obtained from temporally caught wild animals in Thailand. The animals were anesthetized, and blood samples were withdrawn from the femoral vein. The protocol was approved by the Institutional Animal Care and Use Committee of the Faculty of Science, in accordance with the guidelines for the care and use of laboratory animals prepared by Chulalongkorn University, Protocol Review No. 1423010. DNA was extracted from the buffy coat using a standard phenol-chloroform method as described in a previous study  using the BWA-MEM algorithm  were estimated using the PLINK1.90 software using “--homogyz-kb 500” option . Per-samM. fascicularis (CMV1) were assembled using the same method with the data obtained by Macaca nigra, Bornean Macaca nemestrina, and Macaca tonkeana were assembled using the same method, with the data obtained by P. anubis were downloaded from the public database. The DDBJ/EMBL/GenBank accession numbers of sequences are given in We assembled mitochondrial genomes from short-read sequences using the NOVOPlasty software . Of the To construct a neighbor-joining tree and neighbor-net network, all pairwise identity-by-decent (IBD) distances were calculated using the PLINK1.90 with “–distance 1-ibs” option. All filtered sites on autosomes including monomorphic sites were considered. The neighbor-joining tree and neighbor-net network were constructed using the phangorn package in R  using thf3 and f4 statistics , where the sample C is the outgroup species (OLB). The larger f3 indicates that the sample A and B have a larger amount of shared genetic drift, which means A and B are genetically close. We represent the configuration of f4 statistics as f4, where A–D represent the names of the populations to which the samples belong. We fixed the species A to OLB, since we assumed that OLB has not experienced any recent gene flow with our macaque populations. As f4 statistics are equivalent to D statistics, the statistics do not deviate from 0 when the genetic relationship between the four species/populations is represented as a tree structure, indicating no gene flow between species/populations , one male M. fascicularis ssp. aurea , one male M. fascicularis ssp. fascicularis from Southern Thailand , one male M. fuscata , and one male M. cyclopis  , two Indian M. mulatta , three Mauritian M. fascicularis ssp. fascicularis , one Vietnamese M. fascicularis ssp. fascicularis , one Chinese M. assamensis ssp. assamensis , one M. thibetana , and one M. arctoides from China . The abbreviations of the sample names are summarized in This study has performed whole-genome sequencing of five macaque samples: one female 1, TWM1)  online. 1, TWM1)  online: Because we focused on admixture between different species groups, the reads were mapped to the reference genome sequence of OLB, which should be equally distant from all the analyzed samples, in order to avoid mapping bias due to short reads. The mapping rate to the OLB genome was greater than 99% except for one sample, RMC2  online. The coverage of the genomes and the per-sample nucleotide diversity on autosomes  are summarized in M. arctoides clustered with the mulatta group, whereas M. fascicularis ssp. aurea clustered with the sinica group. The tree also indicated that M. mulatta samples were paraphyletic; the Indian M. mulatta samples were considered an outgroup for the Chinese M. mulatta, M. fuscata, and M. cyclopis samples. The result of our study is largely consistent with the result of the previous one  was closer to the mulatta-group than the other M. fascicularis.Next, we constructed a neighbor-joining tree using the genetic distances between autosomes with the draft genome sequence of OLB as an outgroup . The tre studies , 2018. W studies  and a gr studies . These r studies  online b studies  online. fascicularis–mulatta clades, which is contradicting to the conventional taxonomic classification of M. fascicularis ssp. aurea. In order to statistically evaluate the genetic relationship between CMA1/CMA2 and other fascicularis and mulatta group species, we evaluated f3 statistics (or outgroup f3 statistics) using the OLB genome as the outgroup. In the following analyses based on population genetics theory, we use three-letter symbols to represent the names of the populations to which samples belong. For example, RMC represents the population of which RMC1, RMC2, and RMC3 are members. Although we have only one individual for each of the CMT, CMV, TWM, JPM, STM, TIM, and ASM populations, we can assume that the genotype of the sampled individual reflects the allele frequency of SNVs in each population. However, we treated samples CMA1 and CMA2 as if they were in different populations, since we do not have any priori assumptions about their population structure. The f3 statistics showed that CMA1 and CMA2 were generally closer to CMT and CMV than to CMM or the populations in the mulatta group , supporting that CMA1 and CMA2 were fascicularis-like, rather than mulatta-like, populations.As shown in ta group A. For exmulatta group—M. mulatta, M. fuscata, and M. cyclopis—has not previously been examined at the whole-genome level. In the neighbor-joining tree shown in M. mulatta samples clustered with the Indian M. mulatta samples, and M. mulatta was monophyletic and sister to M. cyclopis. However, the branching pattern of the tree may be distorted by gene flow between species/populations. The f3 statistics agreed with the pattern in the neighbor-joining tree and indicated that M. mulatta and M. cyclopis were the sister pair . Hereafter, we use the symbol f4A to represent f4 statistics computed using an autosomal genome. We computed f4A by choosing B, C, and D for all 220 combinations of sampled populations. Of these, 160 combinations were found to be significant, according to the f4 statistics (P < 0.0001 after Bonferroni correction) in the best-fitted tree topologies  was strongly negative . Similarly, the populations in the sinica group were significantly closer to CMA1 and CMA2 than to the populations in the other fascicularis–mulatta group. Besides, CMV and CMT were more closely related to RMC than to RMI.The deviation from the tree structure was found to be particularly strong when the configuration included STM, CMA1, CMA2, CMT, and CMV. The all populations in f4 statistics for each SNV across the non-PAR of the X chromosome. We found that an approximately 10-Mb length region proximal to PAR1 boundary showed an unusual pattern of differentiation. An example of the f4 across the non-PAR of the X chromosome is shown in f4 values near the PAR1 boundary were observed to be strongly negative and showed unusually high variance. This pattern was consistently observed in comparisons involving few samples. Although we could not identify the reason for this unusual pattern of differentiation, we excluded the region from further analysis. Hereafter, we designate the f4 statistics on the X chromosome excluding the PARs and the 10-Mb region proximal to PAR1 boundary as f4X.Before estimating the level of admixture on the X chromosome, we investigated the regional heterogeneity of genetic differentiation on the X chromosome. We computed f4 values between autosomes and X chromosomes under the situation presented in f4 statistics becomes more strongly negative on X chromosomes than on the autosomes.We compared the pattern of genetic differentiation between autosomes and X chromosomes. If incongruent genealogies between mitochondrial and autosomal loci, as observed above, are due to a nuclear swamping process, we expect that X chromosomes are less affected by introgression than autosomes. To test the bias, we statistically compared fascicularis–mulatta-group populations. The values of f4A, where B represents a population in the fascicularis or mulatta group, were significantly biased in the negative direction  would be lower than f4A. The results showed that the f4X had very large variance across the chromosome and were not significantly different from f4A. In addition, there was no consistent trend in the differences between the f4X and f4A, as shown in We first examined the level of admixture between STM and the irection A. The f4f4A and f4X including CMA1 and CMA2. We computed the f4, where D represents a population in the fascicularis or mulatta group other than CMA1 and CMA2. The f4A values were observed to be strongly negative, indicating that ASM was significantly more closely related to CMA1 and CMA2 than to the other fascicularis- and mulatta-group populations  and f4. In both cases, the f4A and f4X values were significantly negative, indicating that JPM and TWM were more closely related to RMC than to RMI , which shows that RMC were more similar to JPM and TWM on the X chromosomes than on autosomes, a pattern expected under the nuclear swamping hypothesis.We finally looked at the n to RMI C. Since ies pair , this remulatta group  compared with the other samples . The reason for the large number of deviant comparisons might be that the f4 test is too sensitive. We tested the deviation of f4A, where C and D represent individuals from the same populations, and estimated the degree of deviation from treeness (STM was selected with arbitrary decision). Among seven possible comparisons, only one pairs f4A showed slight deviation from the null model . We cannot confirm whether these deviations are due to technical artifacts or to hidden population structure. However, most of the observed degrees of deviation in our main analysis were much higher than these values; 62 out of the 220 comparisons had a Z score greater than 20 or smaller than −20, indicating that most of the inferred admixture events accurately reflect past demographic events.In this study, we observed significant levels of gene flow among many pairs of species. Of the 220 possible configurations of M. arctoides and M. fascicularis ssp. aurea, we did not find evidence supporting the nuclear swamping hypothesis for their origination. Given the significant gene flow among species, the evolutionary history of the fascicularis and sinica groups would be extremely complicated and may have blurred the signature of the past sex-biased migration. Alternatively, the incongruence between autosomal and mitochondrial genealogies could be explained by the introgression of mitochondrial genomes. Studies with larger sample sizes may be conducted to answer the question in the near future.For M. mulatta, if we assume that the mitochondrial locus is evolutionarily neutral, the observed pattern provides evidence supporting the nuclear swamping hypothesis. However, several studies have suggested that the introgression of mitochondrial genomes might be deleterious because they may disrupt the interactions between nuclear and mitochondrial alleles, a phenomenon which is referred to as mitonuclear incompatibility (We should also note that the different patterns of genetic differentiation between autosomal and X-chromosomal loci expected under nuclear swamping are also explained by strong natural selection against the introgression of X chromosomes. These factors are not mutually exclusive, and their relative importance should be examined using multiple sources of evidence. In the case of Chinese tibility . In suchIn this study, we investigated the complex evolutionary history of macaques with rampant gene flow among species, using a whole-genome sequence data set. As shown in previous studies, incongruencies in genealogical relationships between nuclear and mitochondrial genomes were observed among species. We showed that comparing the pattern of admixture between autosomal and X-chromosomal loci is a potentially valuable approach to statistically evaluate the power of sex-biased migration in shaping the pattern of genome evolution. We detected a statistically significant difference in admixture levels between these loci, which could be explained by strong male-biased migration in one of the three cases tested. Comparisons between an autosomal and an X-chromosomal evolutionary pattern using a larger data set in other species would reveal a more detailed evolutionary history in future studies.Genome Biology and Evolution online.evaa209_Supplementary_DataClick here for additional data file.