Notechis scutatus occidentalis) is a predator of wetlands in the Swan Coastal Plain, a unique bioregion that has suffered substantial degradation through the development of the city of Perth, Western Australia. Within the urban matrix, tiger snakes now only persist in a handful of wetlands where they are known to bioaccumulate a suite of contaminants, and have recently been suggested as a relevant bioindicator of ecosystem health. Here, we used genome-wide single nucleotide polymorphism (SNP) data to explore the contemporary population genomics of seven tiger snake populations across the urban matrix. Specifically, we used population genomic structure and diversity, effective population sizes (Ne), and heterozygosity-fitness correlations to assess fitness of each population with respect to urbanisation. We found that population genomic structure was strongest across the northern and southern sides of a major river system, with the northern cluster of populations exhibiting lower heterozygosities than the southern cluster, likely due to a lack of historical gene flow. We also observed an increasing signal of inbreeding and genetic drift with increasing geographic isolation due to urbanisation. Effective population sizes (Ne) at most sites were small (< 100), with Ne appearing to reflect the area of available habitat rather than the degree of adjacent urbanisation. This suggests that ecosystem management and restoration may be the best method to buffer the further loss of genetic diversity in urban wetlands. If tiger snake populations continue to decline in urban areas, our results provide a baseline measure of genomic diversity, as well as highlighting which ‘islands’ of habitat are most in need of management and protection.Urbanisation alters landscapes, introduces wildlife to novel stressors, and fragments habitats into remnant ‘islands’. Within these islands, isolated wildlife populations can experience genetic drift and subsequently suffer from inbreeding depression and reduced adaptive potential. The Western tiger snake ( Urbanisation, the anthropogenic transformation of natural ecosystems via the growth of cities , introduIsolated populations are expected to experience increased levels of genetic drift–stochastic loss of alleles through time–and differentiation, in conjunction with reduced genetic diversity within populations . In instNotechis scutatus) are a ~ 1 m elapid snake restricted to the cooler, wetter climates of Australia Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. ORIGINAL SUBMISSION1.1. Recommendation Accepted with minor revision2. COMMENTS TO AUTHORAuthors: Damian C. Lettoof, Vicki A. Thomson, Jari Cornelis, Philip W. Bateman, Fabien Aubret, Marthe M. Gagnon, Brenton von TakachTitle: Top predator snake shows genomic signatures of natural and anthropogenic barriers to gene flow2.1. Overview and general recommendation:This study describes the effects of urbanization on tiger snakes using population genomics around the Perth region in Australia to access and inform conservation strategies. For this study, the authors used RAD-associated SNPs to evaluate the genetic structure among populations of Notechis scutatus occidentalis of different localities along with its distribution. This article is well-written, with a few issues related to interpretation that need to be considered. Therefore, I accepted this manuscript for publication, but with minor revision. Please, find below my comments and consideration of this manuscript.I think that the results about the genetic distance among populations are over-interpreted. Although Carnac has the greatest genetic distance among other populations (0.40-0.29), the genetic distance between Carnac and Black Swan/Kogolup is moderate (0.29). Also, it is moderate when the genetic distance is compared among distant populations, for example, Yanchep and Black Swan (0.26). So, I conclude that the genetic distance among populations is not high but moderate to low, and they are not entirely isolated.About the investigation of the cross-entropy scree plot produced using the tess3r package, in Figure 3, I do not see the plot of K=4 in the figure. I just saw the plot of K=2 to K=3. Where is this plot (K=4)? In Figure S3, I just saw the plot of K=5 to K=7. Also, in Figure 3 and Figure S3, I suggest that the south and north lakes should be indicated in these figures because it is tough to understand what the authors present in results and what they have shown in these figures.Also, following the cross-validation interpretation, to choose the best K, in Figure S2, the author said in Materials and Methods: “We considered ancestry coefficients for K values of 2, 3 and 4 as these values showed the largest improvements in cross-entropy, with K = 4 showing a substantial change in slope, and plotted these for each individual”.In the manual of tess3r they mentioned: “The best choice for the K value is when the cross-validation curve exhibits a plateau or starts increasing… Be cautious about over-interpreting the value of K and the folkore around the choice of this value. Population structure is often hierarchical, and the estimation of K strongly depends on sampling and genotyping efforts. The number of genetic groups detected by ancestry estimation programs does not necessarily correspond to the number of biologically meaningful populations in the sample (Francois and Durand 2010). If I am correct, following the Figure S2 the best K in your analysis is K=4 to K=6, so why you choose K=2 to K=4?Also, the author mentioned, “At K=3, Herdsman Lake is recovered as a distinct cluster, potentially due to isolation from a sea of urban infrastructure”. I do not see this in figure 3, I see a strong differentiation, but I see that there is gene flow with other populations. I suggest that the authors use additional analysis to see gene flow, as Ima2 and FastSimCoal. Also, the author can use Estimated Effective Migration Surface analysis (EEMS); in this analysis, you can see the landscape and the genetic structure among populations. Also, you can evaluate some geographic barriers that can explain the population structure.Reviewer #2: A comprehensive study that makes important theoretical contributions as well as using its data to guide management action for conservation.I have only minor comments to make.Line 2: "Top predator" probably not justifiable, nor necessary in title, or elsewhere in the MS. No evidence that tiger snakes are top or apex predators is presented in the MS or cited references. Seems to just be words added to colour the MS.Line 19: Abstract. Discovery of the much greater than predicted heterozygosity on Carnac was not an aim of the study but becomes a significant finding. It should be mentioned in the Abstract.Line 30: why the quotes around ‘fitness’?Line 95: Study Sites and Sample Collection. In this section could the authors comment on the unevenness of the sampling at the different sites? Low samples at some sites could reflect low population numbers, but other factors such as snake detectability, habitat complexity or access could also influence sampling. This unevenness, leading to potential population underestimates, could then be part of the potential set of explanations for differences between sites.This topic could be part of the Discussion around population size estimates in lines 429-450Line 202: again, why the quotes?Line 383: isolated due to urbanisationLine 390: and reduced survival probabilityLine 425-26: incomplete sentence: While Fraser, Ironside….habitat.Line 439: typo: KogolupLines 466-67: Awkward sentence structure – needs rewrite.**********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,  22 Sep 2021Reviewer 1I think that the results about the genetic distance among populations are over-interpreted. Although Carnac has the greatest genetic distance among other populations (0.40-0.29), the genetic distance between Carnac and Black Swan/Kogolup is moderate (0.29). Also, it is moderate when the genetic distance is compared among distant populations, for example, Yanchep and Black Swan (0.26). So, I conclude that the genetic distance among populations is not high but moderate to low, and they are not entirely isolated.These values are not genetic distance, they are genetic differentiation . As the number of migrants strongly influences the level of differentiation, we can conclude that the population with the highest level of differentiation from other populations is likely to be the most isolated. We suggest that a differentiation value of 0.4 indicates quite strong isolation from other populations. However, we agree that we can express the results in a more communicable fashion, and have now revised the relevant paragraph in the Results section to state:“Carnac Island was the most differentiated from other populations (pairwise G″ST = 0.29–0.40). The most geographically distant pair of sites, Yanchep and Black Swan, exhibited a moderate level of differentiation (G″ST = 0.26).”About the investigation of the cross-entropy scree plot produced using the tess3r package, in Figure 3, I do not see the plot of K=4 in the figure. I just saw the plot of K=2 to K=3. Where is this plot (K=4)? In Figure S3, I just saw the plot of K=5 to K=7. Also, in Figure 3 and Figure S3, I suggest that the south and north lakes should be indicated in these figures because it is tough to understand what the authors present in results and what they have shown in these figures.We accidently submitted the old figure with incorrect labelling. Fig 3 shows K2, 3 & 4. We have attached the correct version, and added additional labels clarifying the populations north/south of the rivers. We also state in the figure caption that the dashed line is the divide between north/south populations. Also, following the cross-validation interpretation, to choose the best K, in Figure S2, the author said in Materials and Methods: “We considered ancestry coefficients for K values of 2, 3 and 4 as these values showed the largest improvements in cross-entropy, with K = 4 showing a substantial change in slope, and plotted these for each individual”.In the manual of tess3r they mentioned: “The best choice for the K value is when the cross-validation curve exhibits a plateau or starts increasing… Be cautious about over-interpreting the value of K and the folkore around the choice of this value. Population structure is often hierarchical, and the estimation of K strongly depends on sampling and genotyping efforts. The number of genetic groups detected by ancestry estimation programs does not necessarily correspond to the number of biologically meaningful populations in the sample (Francois and Durand 2010). If I am correct, following the Figure S2 the best K in your analysis is K=4 to K=6, so why you choose K=2 to K=4?With large genomic datasets utilising thousands of SNPs, the K value with the lowest cross-entropy is typically the same value as the overall number of sampling localities, as we have the power to identify even fine-scale differentiation among geographically close sites. This is not very informative because it doesn’t group populations into ancestral clusters or allow for inference of meaningful population structure across the landscape. A large drop in cross-entropy implies a big improvement in the explanation of genetic structure and so is useful for inference about higher levels of the hierarchy. We have now reworded the relevant paragraph of the Materials and Methods section to improve our explanation of the method, which now reads:“The ‘tess3’ function incorporates the latitudes and longitudes of each sampled individual to account for the influence of isolation-by-distance on ancestry coefficients, with large drops or plateaus in the scree plot identifying useful values of K for inference of population genetic structure . We considered K values of 2, 3 and 4 most useful for describing the high-level genetic structure across the region, as these values showed the largest reductions in cross-entropy, with a plateau starting to form at K > 4. For finer-scale investigation of population structuring we also plotted ancestry coefficients of K = 5, 6 and 7 .”Also, the author mentioned, “At K=3, Herdsman Lake is recovered as a distinct cluster, potentially due to isolation from a sea of urban infrastructure”. I do not see this in figure 3, I see a strong differentiation, but I see that there is gene flow with other populations. I suggest that the authors use additional analysis to see gene flow, as Ima2 and FastSimCoal. Also, the author can use Estimated Effective Migration Surface analysis (EEMS); in this analysis, you can see the landscape and the genetic structure among populations. Also, you can evaluate some geographic barriers that can explain the population structure.Our apologies for inserting an old figure. The reviewer was thus interpreting these findings on K=2, not K=3. The new figure should improve understanding in this regard. To address the issue of visualising the pattern of population structure across the landscape, we have now included maps of the ancestry coefficients output from tess3r, for K values of 2 to 6, in the Supplementary Material. We believe this adequately allows readers to visually identify barriers to gene flow.Reviewer 2Line 2: "Top predator" probably not justifiable, nor necessary in title, or elsewhere in the MS. No evidence that tiger snakes are top or apex predators is presented in the MS or cited references. Seems to just be words added to colour the MS.Although there is no formal study quantifying the trophic tier for tiger snakes in Australia, they are well recognised by herpetolologists - including very experienced authors of this MS – as not, or rarely, suffering predation once they attain adult size. This is supported by the lack of adult predation records in the literature. As an alternative, we propose changing the phrase in the title to “Bioindicator snake” – as there are currently six publications justifying the use of tiger snakes as bioindicators .Line 19: Abstract. Discovery of the much greater than predicted heterozygosity on Carnac was not an aim of the study but becomes a significant finding. It should be mentioned in the Abstract.Our abstract is currently at the word limit and we believe it adequately summarises the key findings. Although the Carnac discovery was interesting, it was not a primary focus of the MS and we cannot describe the findings – with appropriate context – in an already filled abstract.Line 30: why the quotes around ‘fitness’?We have removed these quotes.Line 95: Study Sites and Sample Collection. In this section could the authors comment on the unevenness of the sampling at the different sites? Low samples at some sites could reflect low population numbers, but other factors such as snake detectability, habitat complexity or access could also influence sampling. This unevenness, leading to potential population underestimates, could then be part of the potential set of explanations for differences between sites.This topic could be part of the Discussion around population size estimates in lines 429-450Kogolup, Black Swan and Carnac Island were opportunistically sampled to add sites to this study, and received less survey effort than Herdsman, Joondalup, Bibra and Yanchep. We are confident that our detection ability in catchers was the same at every site, but the lower sample size was related to survey effort. We have added a sentence in the method to clarify “Kogolup Lake, Black Swan Lake and Carnac Island were surveyed less than the other sites , which resulted in lower sample sizes for these sites.”Line 202: again, why the quotes?We have removed these quotes.Line 383: isolated due to urbanisationWe have amended this sentence.Line 390: and reduced survival probabilityWe have added ‘reduced’.Line 425-26: incomplete sentence: While Fraser, Ironside….habitat.We have restructured the sentence to “While Fraser, Ironside (88) suspect that large available habitat is responsible for maintaining large population sizes in deer populations isolated by urbanisation.”Line 439: typo: KogolupAmended.Lines 466-67: Awkward sentence structure – needs rewrite.We have rewritten to “It is possible that the founding population was sourced from many genetically diverse populations (e.g. including the east coast subspecies), if that was the case however, we would expect the Carnac Island snakes to separate from our sampled populations at lower K values, and the geographically closest sampled populations to show little-to-no shared ancestry with Carnac Island in our admixture plots.”AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 13 Oct 2021Bioindicator snake shows genomic signatures of natural and anthropogenic barriers to gene flowPONE-D-21-24399R1Dear Damian ,,In the manuscript authors have fairly done the good work and have scientific merit. My suggestions is to accept the manuscript after incorporating the suggestions.Kind regards,Randeep SinghAcademic EditorPLOS ONEAdditional Editor Comments :In the manuscript authors have fairly done the good work and have merit. My suggestions is to accept the manuscript after incorporating the suggestions.Reviewers' comments: 21 Oct 2021PONE-D-21-24399R1 Bioindicator snake shows genomic signatures of natural and anthropogenic barriers to gene flow Dear Dr. Lettoof: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. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Randeep Singh Academic EditorPLOS ONE


Didymella rabiei, is an important disease affecting chickpea (Cicer arietinum L.) in many countries. We studied the genetic diversity and population structure of 96 D. rabiei isolates collected from three geographic populations in Ethiopia using simple sequence repeat (SSR) markers. We confirmed the genetic identity of 89 of the D. rabiei isolates by sequencing their rRNA internal transcribed spacer region genes. The chickpea blight pathogen isolates were genetically diverse, with a total of 51 alleles identified across 6 polymorphic SSR loci, which varied from 3 to 18 (average 8.5) alleles per SSR marker. The observed heterozygosity and expected heterozygosity ranged from 0.01 to 0.92 and 0.19 to 0.86, respectively. The mean polymorphic information content value of the D. rabiei populations was 0.58, with a mean gene diversity of 0.61 among loci. Gene flow (Nm = number of migrants) for the three populations of D. rabiei isolates ranged from 1.51 to 24.10 (average 6.2) migrants/cluster. However, the genetic variation between the D. rabiei populations was small (8%), with most of the variation occurring within populations (92%). Principal component analysis to visualize genetic variation showed that the D. rabiei isolates obtained from most of the chickpea samples formed roughly three groups on a two-dimensional coordinate plane. Similarly, the clustering of individuals into populations based on multi-locus genotypes (using Clumpak) grouped isolates into three clusters but with individual isolate admixtures. Hence, no clear geographic origin-based structuring of populations could be identified. To our knowledge, this is the first report of D. rabiei diversity in Ethiopia. Virulence studies should be conducted to develop chickpea varieties that are resistant to more aggressive pathogen populations.Ascochyta blight, also known as chickpea blight, which is caused by the fungal pathogen,  Cicer arietinum L.) is the most important grain legume in the world after the common bean (Phaseolus vulgaris) and pea (Pisum sativum) [Chickpea (sativum) . Ethiopisativum) . Ethiopisativum) , and thesativum) . The crosativum) .Ascochyta rabiei (Pass) Labr. (Teleomorph: Didymella rabiei (Kovachevski) var. Arx.), also known as chickpea blight, is the most important yield- and quality-limiting factor worldwide [Ascochyta blight of chickpea  in the major chickpea growing area  medium . After 5–6 days on a rotary shaker at 150 rpm and 23 °C, mycelia from the flasks were lyophilized in 9 cm Petri dishes. The mycelia of each isolate were finely ground using liquid nitrogen, and total genomic DNA was extracted following the manufacturer’s protocols for the Plant Kit® .Isolates of chickpea plant parted infected by ing area . The colD. rabiei isolates was amplified using two primers, ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′), as described by White et al. [® , and 0.5 µL of each primer. Cycling conditions consisted of an initial denaturation at 95 °C for 15 min followed by 35 cycles at 94 °C for 30 s, 56 °C for 1 min, at 72 °C for 1 min, and a final extension at 72 °C for 7 min. Amplified PCR products were visualized on a 1% agarose gel stained with ethidium bromide (4 ng/mL) and were purified with a QuikClean Gel Extraction Kit  according to the manufacturer’s protocols. A 100 base-pair (bp) DNA ladder  was run in the outer lanes of the gel as a size standard. Representative isolates (n = 89) of the amplified ITS region of rRNAs were sequenced.The rRNA ITS1-5.8S-ITS4 gene region of the e et al. . The priD. rabiei  sequence (5′-TGT AAA ACG ACG GCC AGT-3′) along with a complementary FAM-labeled M13(-21) universal primer (FAM-5′-TGT AAA ACG ACG GCC AGT-3′). PCRs were conducted in 10-µL volumes containing 1 µL of genomic DNA, 5 µL of PCR master mix HotStarTaq® (QIAGEN Group), 0.5 µL each of forward and reverse primer as well as the FAM-labelled M13(-21) universal primer. Amplifications were performed in a GeneAmp PCR System 9700 thermocycler  with the following cycling conditions: 95°C for 15 min followed by 35 cycles at 94 °C for 30 s, 56 °C for 1 min, 72°C for 1 min, and a final extension at 72 °C for 7 min. GeneScan 500 LIZ dye size standard  was used according to the manufacturer’s recommendations, and PCR without template DNA was used as control. Fragment analysis was outsourced from the University of Illinois, USA .Six SSR primers developed by Geistlinger et al.  were use. rabiei . The D. D. rabiei infection, homologous ITS sequences identified using the Basic Local Alignment Search Tool (BLAST) in GenBank  were aligned to those obtained from 89 isolates using Geneious Prime 2020.2.3 software.For confirmation of Peak identification and fragment sizing of SSR loci were performed using Geneious software with the default settings. Then, the allele size data at each locus were exported to Excel for statistical analyses. Polymorphisms were calculated for basic parameters of locus-based diversity indices: major allele frequency (MAF), number of alleles (NA), gene diversity (GD), and polymorphic information content (PIC) using PowerMarker ver. 3.25 . The numTo estimate the variance components of the populations and the distribution of gene diversity, analysis of molecular variance (AMOVA) was estimated using GenAlEx molecular software. Population differentiation (PhiPT) of the whole population and pairwise PhiPT among all pairs of populations were determined, and significance was tested based on 1000 bootstraps. An unweighted neighbor-joining dendrogram was constructed. The AMOVA based on codominant SSR loci was estimated using GenAlEx version 6.503 .2)); I = Shannon’s information index (−1 × Sum (pi × Ln (pi))); No. of private alleles = number of alleles unique to a single population; He = expected heterozygosity = (1 − Sum pi2); and uHe = unbiased expected heterozygosity = ((2N/(2N − 1)) × He).The equations used are as follows: Na = number of different alleles with a frequency ≥ 5%; Ne = number of effective alleles  was completed using GenAlEx software to show the genetic differentiation pattern of the populations. Population structure was inferred using a Bayesian model-based clustering algorithm designed in STRUCTURE version 2.3.4. To determine the most appropriate number of populations (K), a burn-in period of 25,000 was used in each run, and data were collected over 100,000 Markov chain Monte Carlo replications from K = 1 to K = 10. This procedure groups individuals into populations and estimates the proportion of membership in each population of individuals [K value was determined by the log probability of data (Ln P(D)) based on the rate of change in LnP(D) between successive K [K value was predicted following the simulation method of Evanno et al. [D. rabiei isolates.The phylogenetic relationships of 96 isolates were constructed using the unweighted neighbor-joining approach with 1000 bootstrap replicates using DARwin molecular software version 6.0.010 . A single consensus nucleotide sequence was obtained and was deposited in GenBank (Accession number OK161019).High quality ITS nucleotide sequences (502 bp) were obtained from 89 isolates, all of which shared 100% identity with the D. rabiei isolates that were studied, and the number of alleles per locus ranged from 3  to 18 (ArH02T), with an average of 8.5 per locus  polymorphic and informative SSR markers were used for genetic diversity analyses. The overall allele size ranged from 164 to 435 bp . Across er locus .The observed heterozygosity and expected heterozygosity ranged from 0.01(ArR01D) to 0.92 (ArH02T) and 0.19 (ArA08T ArR01D) to 0.86 (ArH02), respectively. The maximum observed heterozygosity was detected at the ArH02T locus with a value of 0.92, and the minimum was at the ArR01D locus with a value of 0.01; the expected heterozygosity (He) ranged from 0.19 to 0.86, with an average of 0.56. The effective number of alleles (Ne) ranged from 1.24 to 7.23.FST value (0.039) was obtained according to Nei’s genetic distance analysis. With the exception of ArA08T, all of the evaluated markers significantly deviated from the Hardy-Weinberg equilibrium.Many loci were polymorphic, and the mean PIC was 0.58, with a range between 0.29 (ArR01D) and 0.86 (ArH02T). The highest (0.82) major allele frequency was observed at the ArR01D locus, whereas the lowest was observed at the ArH02T locus. The mean gene diversity was 0.61, whereas the highest (0.86) was observed at the ArH05T locus, and the lowest (0.31) was observed at the ArR01D locus. The ArH02T and ArH05T loci provided the most information in this diversity study, with a higher  allele number, and the fewest alleles were found at the ArR01D and ArA08T loci . The number of genotypes was higher at the ArH05T locus (18), but this number was lower at ArH08T and ArR01D, which had a value of four. Pairwise cluster estimates of gene flow (Nm) for the three populations ranged from 1.51 to 24.10 (average 6.2) migrants per cluster, and a low I = 1.179, He = 0.564, and uHe = 0.568) showed higher diversity than Pop B and Pop-C. The percentage of polymorphic loci per population ranged from 66.67% (Pop-C) to 100% (Pop-A), with an average of 83.33%. The number of private alleles was higher in Pop-C (0.67) than it was in the other populations.The mean values for the number of different alleles (Na) and the effective number of alleles (Ne) across all six loci of the overall population were 4.22 and 2.68, respectively , and theD. rabiei populations. Hence, most of the within-population variation is due to the heterozygosity of the individuals within each population. In addition, population differentiation, the PhiPT of the total populations, and the pairwise PhiPT among all of the pairs of populations were determined and were found to be significant . Most of the observed variation occurred within populations (92%) . In addinificant .D. rabiei populations revealed that the effective number of alleles (Ne) and expected heterozygosity (He) across all six loci varied from 2.28 to 3.36 and 0.40 to 0.56, respectively (p < 0.001) and 3.73, respectively  with some isolates being present in different clusters . The oveD. rabiei isolates, showed that the isolates were roughly grouped into three major groups, and the first axis explained 34.41% of the total variation, the second axis explained 48.35% of the variation, explaining 82.76% of the total variation , which was used to identify genetic variation pattern among ariation . Howeverclusters . Clumpakclusters .D. rabiei was the causative agent of chickpea blight in Ethiopia and that there are no sequence differences in the ITS1-5.8S-ITS2 regions of the isolates. This result could be due to the origin of the source pathogen and the involvement of humans in pathogen movement via seeds, contributing to geographic spread [This study confirms that c spread . Ethiopic spread ; this geD. rabiei isolates were polymorphic, with a mean PIC of 0.58, ranging between 0.86 and 0.29. Three loci were highly informative (0.5 < PIC > 0.25), and three were reasonably informative (0.5 < PIC > 0.25) [The six SSR loci of  > 0.25) . The PIC > 0.25) .D. rabiei have been reported from other countries, we detected relatively high (H = 0.61) genetic variability in Ethiopian populations, which were comparable to the highest levels of diversity that were observed in Iran (0.79) and Turkey (0.69) [Though different levels of genetic diversity of y (0.69) ,42. Tuniy (0.69) ,44,45. Ly (0.69) ,47. GeneST value (0.063) between the D. rabiei populations could be due to gene flow among the populations. Gene flow could have resulted from anthropogenic activities such as the exchange of infected seeds for planting among framers, the introduction of infected s breeding material seeds, genetic drift, and migration [ST across all of the loci (FST = 0.03) and the pairwise FST for all of the population pairs  were generally low to moderate based on Wright (1943), which indicates that genetic differentiation among populations can be considered high, as the value of FST was greater than 0.25 [The average estimated gene flow (Nm) (the movement of genes into or out of a population) among the populations was 3.73, and the relatively low Figration . Informaigration , when thhan 0.25 . A separWright 193, which han 0.25 .D. rabiei populations as the low temperature required for sexual reproduction does not exist in the country [D. rabiei, which is controlled by a specific gene, was identified from different chickpea-producing countries with different levels of disease severity [Host resistance to chickpea blight in chickpea has not been durable in some countries because of the high pathogen virulence variability in popular chickpea cultivars . Sexual  country . The genseverity ,46,55,56D. rabiei isolates from the different geographic locations in Ethiopia did not show clear genetic differences (admixture). A higher mean number of different alleles per locus (Na) was observed in the central population (Pop-A), but this could be due to the broadest geographical range of this population. The relative genetic distances among populations did not completely correlate with the geographical distances of their sampling locations. Genetic diversity may not indicate pathogenic aggressiveness, but it is important to test the aggressiveness of pathogens on a set of hosts with different levels of resistance [The sistance ,56,57,58Based on this preliminary study, we suggest additional studies with multiple isolates of both local and global strains to evaluate the long-term stability of this characteristic in haplotype classification. The Ethiopian isolates clearly showed genetic differences within and between collection areas, and the isolates collected from research stations and farmer fields did not show significant differences; the isolates from geographically distinct populations indicate that admixture has occurred. Most of the genetic variability was attributable to diversity within locations rather than between locations.Ascochyta teleomorph D. rabiei, is the most important chickpea disease in most chickpea growing areas around the world. We found genetically diverse Ascochyta blight pathogens in Ethiopia, where chickpea production has increased every growing season, even in areas where chickpea has not been previously grown, as producers earn reasonable prices for this cash crop. Unless the pest burden can be managed, there will be great economic losses for producers and the country in case of epidemics. Understanding of the genetic diversity of this pathogen will help in the development of improved disease management strategies for the sustainable production of chickpea as a source of food and income. The genetic variability of Ethiopian isolates may result from the genetic recombination of the pathogen or may be acquired through seeds imported for variety trials. Genetic diversity still does not necessarily indicate pathogenic aggressiveness. Hence, the virulence of this pathogen should be studied on appropriate hosts or on chickpea blight differential lines, and phytosanitary rules should be applied when moving seeds within the country or from overseas.Chickpea blight, caused by the fungal


Psalidodon scabripinnis populations of Serra da Mantiqueira, previously known as Astyanax scabripinnis have been deeply studied in the last years. These populations are small and isolated and occur very close to the watershed between Paraíba do Sul River basin and Upper Paraná River basin, in Serra da Mantiqueira region in the Atlantic Rainforest. These conditions arouse the interest in knowing theor genetic conservation status and how they responded to the separation between the two rivers basins. Therefore, we accessed the genetic diversity of five P. scabripinnis populations of this region with microsatellites and mitochondrial data. The results showed a complex structure pattern that doesn’t match the simple basin separation and a reasonably conservation status when compared with other populations of the same family or with similar natural history.Recently renamed,  Serra da Mantiqueira (SM) is a mountain range formed by crystalline rocks, located at Atlantic Rainflorest in southeastern Brazil. In general, its origin is associated with tectonic movements of the lower Cretaceous plate some 70 million years before the present time , identified is these work as Astyanax sp.1, a species now renamed Psalidodon scabripinnis by The ichthyofauna of the Paraíba do Sul River has also been characterized and studied by other authors who identified endemic regions, species introduction, and some possible headwater capture points in the basin . Astyanax scabripinnis complex  using the cytochrome c oxidase I (COI) gene. In addition, we sent another sample from each point for confirmation to the Museum of Zoology at the State University of Londrina. The locations, codes assigned to the populations, sample number, municipalities, and geographic coordinates of the collection points are presented in Psalidodon (~Astyanax) altiparanae (Characiformes), whose transferability has already been tested to P. scabripinnis   describeripinnis  marked wGenotyping was performed in an ABI 3500 XL sequencer (Applied Biosystems), using the GeneScan 600 LIZ™ (ThermoFisher Scientific) as the size standard and the electropherograms were interpreted using the program GELQUEST , with the alleles being named for their size. Subsequently, the software TANDEM  was usedST values. Subsequently, to investigate the presence of linked loci, we tested the Linkage disequilibrium in two programs that showed similar results: ARLEQUIN 3.5.2 , the effective number of alleles (NE), the number of private alleles (NAP), the levels of observed and expected heterozygosity , and the gene fixation indices for each population were calculated using GENALEX 6.501 (R) was calculated using the HP-Rare program , using the on line tool The patterns of population structure were investigated using STRUCTURE 2.3.4  was performed in ARLEQUIN 3.5.2 , with 10m, is the migration rate, which is the fraction of individuals in a population made up of immigrants or the probability that a randomly chosen individual is an immigrant and the second is, of course, the mutation rate (µ). Thus, Mi→j= mi→j /µ represents the rate of immigrants, within the effective population size, in the population j coming from the population i. The value of the mutation rate used here was 5.56 x 10-4  for each population using MIGRATE 3.6.11 . This prTo try to identify the gene flow patterns, a chain with 500,000 steps without replicates was used, being sampled every 100 steps, the first 10,000 steps being discarded (burn-in period), and single-step mutations model as parameters for the Bayesian inference. For these analyses, we used three strategies: (a) investigation of the gene flow only among the populations in the Sapucaí basin; (b) investigation of gene flow only among populations in the Paraíba do Sul basin; and (c) investigation of gene flow between the Sapucaí basin and between the Paraíba do Sul basin. In all analyses, the geographical distances between the collection points were used.2, 0.2 mM dNTPs, 0.2 µM of each primer (Forward and Reverse), and 0.2 unit of Taq DNA Polymerase and 25 ng of DNA from each individual, supplemented with 2.5 µl of enzyme buffer (10X) and ultrapure water for a final volume of 25 µl. The amplification cycles occurred with an initial denaturation of 95 oC for two minutes, followed by 35 cycles of 30 s at 94 oC for denaturation, 30 s at 54 oC for the annealing of primers and one minute at 72 oC for extension. After the cycles, a final extension was performed of 72 oC for 5 min. For ATPase 6/8), the primers L8331 (5’-AAGCRTYRGCCTTTTAAGC-3’) and H9236 (5’-GTTAGTGGTCAKGGGCTTGGRTC-3’)  and FishR1 (5’-TAGACTTCTGGG TGGCCAAAGAATCA-3’)  according to the manufacturer’s instructions. After the purifications, the COI amplified fragments were sent for sequencing in ACTGene, linked to Ludwig Biotecnologia Ltda, located in Alvorada-RS-Brazil. ATPase fragments were sent to Macrogen, Seoul-KOR.After obtaining the sequences, we compared each individual one with deposited sequences in GenBank, to confirm similarity. Subsequently, we checked for the presence of gaps or misaligned bases in the MEGA 7.0 program (h), and nucleotide diversity (π) and analysis of population structure by AMOVA . Also in ARLEQUIN, we carried out selective neutrality tests D of Tajima  and the numerical comparison between the expected and observed heterozygosities suggest that there is no deficit of heterozygotes only in the RP population and frequent inbreeding between populations. Only five loci among the populations (about 14%) were found outside the EHW (The highest heterozygosity was observed in the RP population (0.698), while the lowest was observed in the LV population (0.563), while the largest heterozygote deficiency was in the RG population (0.207). Gene fixation indices  and their probabilities.The hypothesis of the formation of three groups or clusters, tested by AMOVA for both microsatellites and sequences, seems to best explain the population structure, as our data show . This hyWT), the most robust when there are less than 20 microsatellite loci, revealed a significant heterozygosity excess, suggesting a recent bottleneck only for the PE and RG populations (significance level of P<0.05). The probability values can be seen in The investigation of bottlenecks, assuming the two-phased mutation model (TPM), according to the Wilcoxon test (PThe amplification of the cytochrome c oxidase I (COI) gene resulted in fragments of, on average, 652 bp, whereas the amplifications for the ATPase gene, resulted in fragments of, on average, 884 bp, after the alignment and editing of the sequences.The values of haplotypic and nucleotide diversity for the ATPase gene ranged from 0 to 0.8333 and from 0 to 0.0073, respectively. As for the COI values, they ranged from 0 to 0.4 and from 0 to 0.37, respectively. No significant values were obtained for the majority of neutrality tests (D for Tajima and Fs for Fu). The results of the neutrality tests, as well as the diversity values, are shown in The results of the AMOVA for the mitochondrial markers , showed P. scabripinnis populations on the Campos do Jordão plateau does not follow the division of the basins. Thus, the simple hypothesis of the hierarchy of the populations of P. scabripinnis in the Serra da Mantiqueira in two groups equivalent to the basins of Sapucaí-Grande-Alto Paraná and Paraíba do Sul, does not seem to explain the genetic variation between them. This is indicated by the results of the Bayesian inference for the investigation of population structure, carried out by the STRUCTURE program. The methodology suggested by Although the Serra da Mantiqueira has proven to be an efficient barrier to ichthyofauna , our datSimilarly, the hypothesis of hierarchization in two groups formed by the two basins, tested by AMOVA, both for microsatellite markers and for mitochondrial DNA, was not able to satisfactorily explain the variation. The variation can be better explained by hierarchizing the data into groups , as suggested by the Bayesian analysis of Structure. The analysis of the AMOVA results of both markers  also sugST point in the same direction as the AMOVA and Bayesian inference of STRUTURE. Values above the global mean (FST=0.098) are found between RP and any other of the four populations and between CP and LV, populations from the same basin  populations with microsatellite data are extremely rare, despite of the large number of studies with with chromosomal markers  altiparanae in the Upper Paraná River basin high levels of heterozygosity ranging from 0.5 to 0.852, and high levels of gene fixation in almost all loci.The populations of P. scabripinnis studied here, ranging from 0.563 to 0.698, with the studies mentioned above, it is noted that they are relatively high and less variable than the heterozygosities of populations of A. mexicanus. It is interesting to note here that the majority of the populations in the studies cited and our results showed a deficit of heterozygotes  the numbers varied from 2.23 to 15, as in A. mexicanus , makes comparisons difficult, and restricts the power of broader inferences. However, there is a greater variation in several parameters of A. mexicanus, such as levels of heterozygosity and allelic richness. This may be due to the large number of existing and studied populations of this species, which enabled description of the colonization of caves from various origins and in several different times . The subdivision of populations can lead to an increase in inbreeding, which would, consequently, increase homozygosity and reduce heterozygosity. Reductions in effective population size are often linked to population genetic bottlenecks. In this scenario, genetic diversity tends to decrease, leading to an increase in inbreeding and decreasing the viability of populations  and indications of inbreeding . Similarly, mitochondrial DNA data indicate that these two populations have low levels of both haplotypic and nucleotide diversity for the COI gene and moderate for ATPase. The set of these factors, together with the small effective population size (Ne), are indications that both populations went through bottlenecks. According to .Comparing PE and RG with the other populations of Salmo truta caspius - Salmoniforms: Salmonidae) and detected a population bottleneck for one of them. This population also presented low genetic diversity, which, according to the authors, may have been caused by overexploitation of fishing, pollution, or destruction of spawning sites. Geophagus braziliensis studied, and the only population that did not present a bottleneck is the one in the least anthropized environment.In fish, the decrease in population sizes can be better detected in species with high commercial interest, as well as which the genetic effects of this decrease. P. scabripinnis populations in the Serra da Mantiqueira is complex and must have been influenced by different geological events in a non-simultaneous manner. Although they are small and isolated, the populations living there present good levels of genetic variation, when compared to similar populations or those with similar biology.Our data showed that the genetic structure of


Megalobrama is the economically most important freshwater fish genus in China. In recent years, germplasm resources of Megalobrama have been depleting as a result of environmental degradation and artificial factors. In this study, we established the whole genome database of Megalobrama populations using the whole genome re-sequencing technology, explored population genetic structure, and inferred comprehensive evolutionary relationships using principal component analysis and population structure analysis. Furthermore, employing selective sweep analysis, we identified candidate genes related to variations in feeding habits, revealing the molecular mechanisms of environmental adaptability in Megalobrama populations. Taken together, this study describes the population history and genetic diversity of Megalobrama populations and also the molecular mechanisms likely involved their environmental adaptability. These findings will make a substantial contribution to conservation and utilization of Megalobrama germplasm resources. Megalobrama, a genus of cyprinid fish, is an economically important freshwater fish widely distributed in major waters of China. Here, we report the genome resequencing of 180 Megalobrama fish including M. amblycephala, M. skolkovii, M. hoffmanni, and M. pellegrini. Population structure indicated that geographically divergent Megalobrama populations were separated into six subgroups. A phylogenetic tree showed that M. skolkovii was more closely related to M. pellegrini than other species and M. hoffmanni was clustered apart from other Megalobrama species, showing a high nucleotide diversity in geographic groups. Treemix validated gene flow from M. amblycephala to M. skolkovii, suggesting that introgression may provide an important source of genetic variation in the M. skolkovii populations. According to the demographic history analysis, it is speculated that Megalobrama might have been originally distributed in the Pearl River with some spread to Hainan Island and northern China due to lower sea levels during the glacial period. Whole-genome selective sweeps analysis demonstrated that M. amblycephala likely developed an enhanced energy metabolism mostly through fatty acid degradation pathways whereas M. hoffmanni possibly regulate lipid absorption via the cholesterol metabolism pathway. Taken together, this study provides a valuable genomic resource for future genetic investigations aiming to improve genome-assisted breeding of Megalobrama species. Megalobrama is a cyprinid fish genus that belongs to the subfamily Cultrinae . It is one of the economically most important fish species, as well as the main aquaculture species in China. The genus Megalobrama contains four recognized species, M. amblycephala, M. skolkovii, M. hoffmanni, and M. pellegrini [M. amblycephala is distinguished by the morphology of the upper orbital bone and oral fissure, and also the caudal peduncle depth/length ratio [M. skolkovii differs from the other three species in the longer length of the first swim bladder [M. hoffmanni has pigment deposits on the scale base that form dark spots on its body surface and exists mostly in the Pearl River and Hainan Island [M. pellegrini dwells in the middle and upper reaches of the Yangtze River and has a unique upper jaw and upper orbital bone shape [Megalobrama species, the phylogenetic relationships among M. amblycephala, M. skolkovii, M. hoffmanni, and M. pellegrini remain unknown [llegrini . Previouth ratio . It is mth ratio . M. skol bladder . It inha bladder . M. hoffn Island . M. pellne shape . Althoug unknown ,8.Megalobrama species have distinct feeding behaviors. For instance, M. hoffmanni is an omnivorous fish that feeds mostly on benthic creatures such as freshwater shellfish, river clams, organic detritus, and some aquatic plants [M. skolkovii and M. pellegrini are omnivorous as well, however, they primarily feed on aquatic plants [M. pellegrini, the development of the upper and lower jaws as well as the thickening of the stratum corneum have strengthened the scraping function, making it more conducive to feeding on stationary organisms [M. amblycephala lives in aquatic plant-rich lakes and its diet consists primarily of aquatic plants, emergent plants, floating-leaf plants, and submerged plants. It is classified as herbivorous fish that mainly feeds on aquatic vascular plants [Megalobrama species must have evolved metabolic mechanisms that allow them to adapt to varying dietary compositions. However, it is not clear how Megalobrama species regulate carbohydrate, lipid, and protein metabolism to maintain energy balance in the body.Four c plants . M. skolc plants . Importarganisms . M. amblr plants . ObviousMegalobrama germplasm resources have been rapidly depleting in recent years as a result of many factors, including environmental degradation factors. There are a few reports on Megalobrama genetic resource conservation and population genetics [M. amblycephala as reference genome, this study was designed with the following objectives: (1) establish the whole genome database of Megalobrama population using the whole genome re-sequencing technology; (2) explore the genetic structure of populations and infer detailed evolutionary relationships by phylogenetic tree, principal component analysis (PCA), and admixture analysis; (3) illustrate the demographic history of Megalobrama species by pairwise sequentially Markovian coalescent (PSMC) method; (4) determine candidate genes related to variations in feeding habits utilizing selective sweeps analysis that would indicate the molecular mechanism (s) of environmental adaptability of Megalobrama populations. This study will ascertain the genetic diversity, population history, and improve the understanding of the evolution of Megalobrama populations and their molecular mechanisms of environmental adaptability. These findings will represent a significant contribution to conservation and utilization of Megalobrama’s germplasm resources.genetics ,2,4. By genetics ,13. For genetics ,15. UsinM. amblycephala from Liangzi Lake , Poyang Lake , Yuni Lake , Tiane Island , Dongting Lake , Jinsha River Reservoir of Hongan County , Shaoguan , Yingde ; six populations of M. skolkovii from Fuyuan , JS (n = 12), Qiantang River , SG (n = 10) and YD (n = 5), DTL (n = 1); four populations of M. hoffmanni from Qingyuan , Zhaoqing , Fengkai , and Hainan , and one population of M. pellegrini from Longxi River  were collected. The fin tissues from all samples were preserved in 95% ethanol and stored at −80 °C.A total of 180 samples, eight populations of http://github.com/BGI-flexlab/SOAPnuke (accessed on 9 September 2020)) software. The specific filtering conditions were as follows: removed reads containing adapter, low-quality reads , and reads with N bases greater than 10%. All cleaned reads were mapped to the M. amblycephala reference genome (mixed assembly of second and third generations) using BWA-MEM (0.7.13-r1126) with default parameters [Genomic DNA was extracted from the fin of each fish using a DNA extraction kit . Paired-end sequencing libraries with an insert size of approximately 300–400 bp were constructed for each individual using the BGISEQ 500 platform and sequenced on an MGISEQ-2000 sequencer. After removing joint pollution and low-quality reads, the filtered high-quality clean data were for subsequent comparative analysis. The filtering was performed using SOAPnuke (v1.5.6)  with the UnifiedGenotyper method . Single K, and CLUMPP (v1.1.2) software was used to merge 50 analytical results [K = 2 to 8) with 200 bootstrap replicates. No admixed individuals were used to construct the phylogenetic tree by FastTree v2.1 software [The principal component analysis (PCA) was performed using EIGENSOFT (version 4.2) and the significance level of eigenvectors was determined with the Tracy-Widom test . Admixtu results . DISTRUC results . Populatsoftware .r2 can be used to calculate the degree of linkage disequilibrium between two markers. In this study, the software PopLDdecay (v3.40) (https://github.com/BGI-shenzhen/PopLDdecay (accessed on 4 December 2020)) was used to set the parameters (-MaxDist 1000 -MAF 0.05 -Miss 0.1) to perform linkage disequilibrium analysis on SNPs [2 value is used as the decay distance of the population. Based on all credible SNP data, vcftools (v0.1.13) software was used to calculate the nucleotides polymorphisms (π) of the four Megalobrama species and the differentiation index (Fst) between species [Linkage disequilibrium (LD) is the non-random association between alleles of different loci in the population and  on SNPs . LD is m species .n migration events, and each migration event is calculated separately. Two analyses were performed. In one, the different Megalobrama populations were divided into four subgroups: M. hoffmanni, M. skolkovii, M. amblycephala, and M. pellegrini, with M. hoffmanni as the out group. In the other, M. hoffmanni was recognized as an outgroup with nine subgroups including M. pellegrini from Longxi River; M. skolkovii from Fuyuan, Shaoguan, Qiantang River, and Jinsha River Reservoir; and M. amblycephala from Dongting Lake, Poyang Lake, and Liangzi Lake added for gene flow analysis. Treemix software was used to investigate the gene flow between populations . The anaMegalobrama ancestors [Based on the phylogenetic tree generated by the credible SNPs, we used the statistical dispersal–vicariance analysis (S-DIVA), dispersal-extinction-cladogenesis (DEC), and Bayesian binary MCMC (BBM) models of reconstruct ancestral state in phylogenies (RASP) 4.0 software to reconstruct the geographical distribution of ncestors ,30. In DMegalobrama species (at least 20 ×) and infer the historical changes in their effective population sizes and population separations. The PSMC model was utilized to estimate changes in the effective population size using heterozygous sites across the genome [M. amblycephala genome, an average mutation rate (μ) of 0.1 × 10−8 per base per generation and a generation time (g) of 2.5 years were used for analysis.The pairwise sequentially Markovian coalescent (PSMC) method was used to model the history of four e genome . The nume genome . Based oM. amblycephala and M. hoffmanni dietary adaptation, genomic regions were scanned based on genome-wide calculations for selective sweeps by differentiation index (Fst), nucleotide polymorphism ratio (PiR), and extended haplotype homozygosity between populations (XP-EHH). vcftools software [M. amblycephala populations, 132 regions with a total length of 6.68 M contained 166 genes were identified from the comparison between M. amblycephala and M. skolkovii; 133 regions with a total length of 6.67 M containing 179 genes identified from the comparisons between M. amblycephala and M. pellegrini; 97 regions with a total length of 5.32 M containing 191 genes identified from the comparisons between M. amblycephala and M. hoffmanni. For the M. hoffmanni populations, 75 regions with a total length of 4.14 M containing 126 genes were identified from the comparisons between M. amblycephala and M. hoffmanni. Genes from these selective regions were subjected to a functional enrichment analysis of selected genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)  [p-value was based on Bonferroni Correction and the functional terms or pathways with corrected p-value < 0.05 defined as significantly enriched functional terms.To investigate the potential selective signals during software  was usedsoftware  was applM. amblycephala in LZL and M. hoffmanni in QY populations were selected. The total RNA of these samples was extracted by Trizol reagent . The quality and quantity of RNA were detected by NanoDrop 2000 , agarose gel electrophoresis and a UV spectrophotometer . Then 1 µg total RNA was reverse-transcribed into cDNA using cDNA Synthesis Kit . qPCR primers are listed in β-actin, Gapdh, and 18S rRNA), and found that β-actin was more stable than other reference genes . Calculations for relative expression were conducted by the 2−ΔΔCt method. Statistical analysis was performed with SPSS 16.0 software and results are reported as mean ± standard error (SE). The data were analyzed by independent sample t-test, and a p-value < 0.05 was considered to indicate a statistical significance.In this study, the liver and spleen tissues from ce genes . Thus, βMegalobrama fish representing different geographical populations in China were selected for genome resequencing  = 6, six geographically distributed ancestral components (K) were labeled as: HN and Pearl River M. hoffmanni populations; M. amblycephala populations; FY and other M. skolkovii populations; and M. pellegrini populations  and was consistent with the phylogeny and biogeographic distribution  provided supporting evidence for these groupings  and M. pellegrini (1.888 × 10−3). The low genetic differentiation (Fst) between M. skolkovii and M. pellegrini (0.148), and the high Fst between M. hoffmanni and M. amblycephala (0.4091), M. skolkovii (0.3516), and M. pellegrini (0.4259)  and correlation coefficient (r2) values were calculated. The decay of LD reached half the maximum average r2 at a distance of 24 Kb, 4 Kb, 1.8 Kb, and 0.2 Kb for M. amblycephala, M. pellegrini, M. skolkovii, and M. hoffmanni, respectively  of (0.4259) B are conectively C. TherefMegalobrama species reached 0.5 suggesting a split between M. hoffmanni and other species 3–5 Mya. The cross-coalescence analysis suggested a decline to 0.5 between M. amblycephala and M. skolkovii or M. pellegrini at ~1.3 Mya, and a decline to 0.5 between M. skolkovii and M. pellegrini around 0.3–0.4 Mya  peak of M. amblycephala and M. pellegrini was 2.5 Mya and 4 Mya, respectively, compared to 3–4 Mya of the Ne peak for the M. skolkovii subgroups. After that, the FY M. skolkovii subgroup continued to shrink, whilst the Ne curves of other M. skolkovii subgroup split at 0.2 Mya expanded  among the four –0.4 Mya A. The PSexpanded B. Moreovl period C. Interei Rivers .M. amblycephala dietary adaptation, we scanned the genomic regions based on genome-wide calculations for selective sweeps by estimating Fst, PiR, and XP-EHH values. Candidate genes were discovered in the common region of top 5% Fst, High/Low top 5% PiR, and High/Low top 5% XP-EHH, inferred from the comparisons between M. amblycephala and the other three species  involved in cholesterol metabolism , and carnivorous (Gadus morhua and Cynoglossus semilaevis) fish [M. hoffmanni may have developed unique metabolic pathways for animal food.The diet of ntestine . Bile acntestine . Cyp46a1ntestine . Furtherntestine . A previntestine . Cholestntestine . We founis) fish ,51. TakeMegalobrama. The screening of selective genomic regions related to the Megalobrama diet elucidated a significant role in the environmental adaptation mechanisms of the four Megalobrama species. Importantly, this study has provided a valuable genomic resource for future genetic studies that can contribute to improvement of the Megalobrama species using genome-assisted breeding.Population genomic research provided new insights into phylogenetics, population structure, demographic history reconstruction, and feeding habits of the genus


Musa balbisiana, an important CWR of plantains, dessert and cooking bananas. Musa balbisiana has its origin in subtropical and tropical broadleaf forests of northern Indo-Burma. This includes a large part of northern Vietnam where until now, no populations have been sampled. We screened the genetic variation and structure present within and between 17 Vietnamese populations and six from China using 18 polymorphic SSR markers. Relatively high variation was found in populations from China and central Vietnam. Populations from northern Vietnam showed varying levels of genetic variation, with low variation in populations near the Red River. Low genetic variation was found in populations of southern Vietnam. Analyses of population structure revealed that populations of northern Vietnam formed a distinct genetic cluster from populations sampled in China. Together with populations of central Vietnam, populations from northern Vietnam could be subdivided into five clusters, likely caused by mountain ranges and connected river systems. We propose that populations sampled in central Vietnam and on the western side of the Hoang Lien Son mountain range in northern Vietnam belong to the native distribution area and should be prioritised for conservation. Southern range edge populations in central Vietnam had especially high genetic diversity, with a high number of unique alleles and might be connected with core populations in northern Laos and southwest China. Southern Vietnamese populations are considered imported and not native.Crop wild relatives (CWR) are an indispensable source of alleles to improve desired traits in related crops. While knowledge on the genetic diversity of CWR can facilitate breeding and conservation strategies, it has poorly been assessed. Cultivated bananas are a major part of the diet and income of hundreds of millions of people and can be considered as one of the most important fruits worldwide. Here, we assessed the genetic diversity and structure of  Crop wild relatives (CWR) are wild species closely related to cultivated plants, including all direct progenitors of crops. Because of their relatedness, CWR provide useful trait characteristics for crop improvement such as stress tolerance and yield increase . CWR areMusa acuminata Colla  and Musa balbisiana Colla (“B” genome). Only a small number of cultivars contains genetic information of other Musa species such as Musa schizocarpa Simmonds and members of the former Australimusa section [With an estimated production of 158.4 million tonnes in 2019, bananas (74%) and plantains (26%) can be considered as one of the most important fruits and staple foods of the world . Over on section .Musa balbisiana is a diploid (2n = 22) wild perennial herb of the “Musa” (former Eumusa) section of the genus Musa [M. balbisiana on the Ryukyu islands, the Philippines, and Papua New Guinea is assumed to be feral after the introduction of this species by humans for cultivation purposes [nus Musa . Its natnus Musa . The prepurposes . Asexualpurposes . Inflorepurposes . The flopurposes ,30. Seedpurposes .M. balbisiana (BB) is one of the ancestors of diploid (AB), triploid , and tetraploid  plantains, cooking, and dessert bananas [Musa acuminata , the presence of the B genome has been associated with increased drought and cold tolerance [Xanthomonas wilt [M. balbisiana populations [ bananas ,31,32. N bananas –36. In colerance –40, resinas wilt –43, and nas wilt . The avanas wilt  and receulations ,46–49. Sulations ,50–53.M. balbisiana. Using Amplified Fragment Length Polymorphism (AFLP) markers, Ude et al. [M. balbisiana accessions obtained from gene banks and field collections. Their study was of the first to detect a high level of genetic diversity among M. balbisiana accessions. Ge et al. [M. balbisiana populations using AFLP and Simple Sequence Repeat (SSR) markers. The highest heterozygosity levels were observed in the Yunnan province, close to the Vietnamese border. Recently, Bawin et al. [ex situ seed collections in Arusha (Tanzania) and Kampala (Uganda).To date, only a few studies have investigated the intraspecific genetic diversity within e et al.  assessede et al.  and Wange et al.  found hin et al.  used 18 Musa balbisiana populations at the southern edge of their native distribution range  might be exceptionally interesting. Populations may hold specific alleles adapted to better cope with drought stress, interesting for cultivated material containing the B genome, especially in a changing climate.Species are often assumed to have larger population sizes, high genetic diversity, high gene flow and low differentiation in the centre of their distribution range . In contMusa balbisiana. More specifically, we assessed the genetic structure and diversity in 17 Vietnamese M. balbisiana populations using 18 SSR markers. As large parts of northern Vietnam belong to the putative region of origin of the species, populations are expected to hold levels of genetic variation similar to those found in populations sampled in China. In addition, genetic variation in 6 wild populations of neighbouring Chinese provinces are compared with the Vietnamese populations to allow us to better understand the native character of the Vietnamese populations. Four questions are addressed: (1) How does genetic variation in Vietnamese M. balbisiana populations relate to that in Chinese populations? (2) How does genetic diversity vary among Vietnamese M. balbisiana populations? (3) Are M. balbisiana populations in Vietnam genetically structured along a geographic gradient? (4) Can southern populations of Vietnam be considered as wild?In this study, we for the first time quantify the genetic variation in Vietnamese populations of the banana crop wild relative Musa balbisiana were sampled in Vietnam  were newly acquired and used in the present study. Finally, data from Bawin et al. [Field research was carried out as a collaboration between Meise Botanic Garden (Belgium) and the Plant Resources Center . The Plant Resources Center had permission for working with Meise Botanic Garden under the Bilateral cooperation project: the Vietnamese National Foundation for Science and Technology Development (NAFOSTED). Field permits for Yen Bai (564/UBND-NV), Ha Giang (255/SNN-CCLN), and Lao Cai (1060/UBND-NC) were obtained. During multiple collection missions carried out between 2017 and 2020, 274 individuals belonging to 17 populations of wild  Vietnam , Fig 1. n et al.  for threMusa balbisiana “Pisang Klutuk Wulung” in the Banana genome hub [Banana leaf material was dried for preservation in the field using silica gel . Seed emnome hub . An expenome hub .PCR of microsatellite fragments was carried out with Qiagens Type-it Microsatellite PCR Kit . Subsequently, 1 μl of diluted PCR sample was added to 12 μl HiDi Formamide mixed with 0.4 μl of the MapMarker 500 labelled with DY-632 , after which 1.5 μl of this product was genotyped on an ABI 3730 system  at the Université Libre de Bruxelles (ULB), Belgium. For more recent samples, 20 μl of PCR product was sent to Macrogen  for genotyping on an ABI 3730 system. A minimum of ten samples per genotyping run were ran in duplicate to deal with potential differences in fragment sizing. Raw data were scored and checked for errors in Geneious Prime 2021.0.3  with a third order least squares method. To ensure a uniform scoring of alleles, raw data obtained from the two populations of Yunnan and one from Hainan from the study of Bawin et al.  were re-The average number of alleles per locus with a frequency larger than 5% (Na), the average number of private alleles unique to a single population (Np), Shannon’s Diversity Index (H), proportion of polymorphic loci (P), Unbiased expected Heterozygosity (uHe), and Observed Heterozygosity (Ho) were calculated with the GenAlEx 6.51 Excel Package . InbreedST. Within and between population variation including all samples was additionally visualised by a PCoA constructed using a Euclidean distance matrix with the “dudi.pco” function in R package “ade4” [To explore our genetic data and to visualize the genetic structure of populations, we assessed the pairwise genetic differentiation between populations through codominant genotypic distances and used a Principal Coordinate Analysis (PCoA) as visualisation. With GenAlEx, an analysis of molecular variance (AMOVA) was run using 999 permutation steps to assess the genetic variation present within and between sampled populations while taking the sampled region into account. Here, genetic differentiation was measured as Fe “ade4” .Population genetic structure was assessed using Bayesian clustering in STRUCTURE 2.3.4 . All runFirst, the optimal number of genetic clusters to which individuals could be assigned to was determined by testing 10 independent runs for a K value ranging from one to 20 using a model with correlated allele frequencies and a default initial α of 1.00 for all clusters. For each independent run, 100,000 Markov Chain Monte Carlo (MCMC) iterations were sampled after a burn-in of 100,000 iterations. The optimal number of genetic clusters was then determined based on ΔK and the log posterior probability of the replicates over each K . This vaTwo datasets were used to run the clustering analyses in STRUCTURE. The first dataset contained all samples from all populations after removing any outlying samples, allowing us to directly compare populations of Chinese with those of Vietnamese origin. The second dataset consisted of only Vietnamese populations in their native distribution range (northern Indochina). This allowed us to evaluate the population genetic (sub)structure of these populations. Structure plots with the optimal K were made with the CLUMPAK software as implemented in StructureSelector .To estimate whether genetic distance also increases when populations are geographically more distant (Isolation by distance), a geographic distance matrix was calculated based on the geographic coordinates of each population. This was done for the two different datasets, one including all sampled populations and one only including populations sampled in northern and central Vietnam. For both datasets, a paired Mantel test analysis was performed between the geographic and genetic distance matrices (Edward’s distance) with R package “ade4” to assess whether genetic isolation by distance was significant . All R pBlasting SSR marker sequences against the reference genome showed that they are spread across 8 out of 11 chromosomes . No markM. balbisiana populations had an excess in heterozygotes  .The average number of alleles per locus with a frequency larger than 5% was low in all sampled populations, ranging from 1.11 in populations VTN-N8 and VTN-S1 to 1.94 in VTN-C1 . FourteeIn the Principal Coordinate Analysis of the full dataset, the first two axes explained together 26.6% and 17.8% of the genetic variation present between the populations respectively . The ChiIn both sets of genetic clustering analyses using STRUCTURE with correlated allele frequencies, the optimal number of clusters was different for the three assessment criteria, making it hard to determine the optimal number of clusters . BecauseM. balbisiana populations (p < 0.01) when assessing all populations. However, the regression curve only accounted for 15% of the observed response   . When asf 0.153) .Musa balbisiana and complemented them with populations sampled in the Yunnan and Guangdong province of China, as well as a population of Hainan island. For this, we used a set of 18 polymorphic microsatellite markers that were combined and in four multiplex PCRs in a previous study [M. balbisiana [M. balbisiana populations was rather low, with observed heterozygosity levels ranging from 0.05 to 0.28 with an average of 0.19. Low genetic variation in populations can be expected in small, fragmented populations or with low gene flow between populations and high levels of inbreeding [M. balbisiana and other crop species with clonal reproduction capacities [M. balbisiana due to the way the flowers develop. The flowers at the base of the inflorescence are typically female, followed by hermaphrodite flowers with aborted female and male organs at the tip of the inflorescence. Due to different flowering times, self-pollination is largely prohibited. Second, the species can propagate clonally. Clonal reproduction might result in a heterozygosity excess because of a low number of genotypes present in a population (low expected heterozygosity) while maintaining heterozygosity for a large proportion of the assessed loci [In this study, we assessed for the first time the genetic variation of Vietnamese populations of us study . This allbisiana ,49. Blaslbisiana  found tolbisiana –82), thebreeding . Comparapacities ,85, we fsed loci . Lastly,sed loci ,84,85,87M. balbisiana populations, high relative levels of genetic diversity were also found in populations sampled in Yunnan, Guanxi and southern Guangdong, especially for the population of Yunnan in the northern Indochina subtropical region [M. balbisiana populations is potentially caused by the presence of long-tongued fruit bats (Macroglossus sobrinus) that act as a long-distance pollinator in western China.Despite the relatively low observed levels of genetic variation, clear differences were found between populations. Excluding the population from Hainan, Chinese populations on average had moderate to high percentages of polymorphic loci, expected and observed levels of heterozygosity as well as low levels of inbreeding. In previous studies on genetic diversity in Chinese l region ,49. Theyo ranging from 0.175 to 0.243), the genetic diversity in Vietnamese Musa balbisiana showed larger differences between populations (Ho ranging from 0.051–0.278). Populations sampled in south Vietnam, near the Red River Basin  as well as VTN-N3 in Lai Chau had low genetic diversity, with a low number of different alleles, a limited number of private alleles, a low number of polymorphic loci, and low heterozygosity levels. Three populations had high levels of these indices and low levels of inbreeding , while the other populations had moderate levels. Most notable is that the populations of central Vietnam  had the highest levels of different alleles and number of polymorphic loci and in particular VTN-C2, which had the highest proportion of private alleles compared to the other populations. Compared to the Chinese populations, similar levels of genetic diversity were found in northern and central Vietnam, excluding VTN-N1, VTN-N3, VTN-N7, and VTN-N8. Noteworthy, according to the bats checklist of Vietnam [Macroglossus sobrinus has only been recorded in the close vicinity of those central Vietnamese M. balbisiana populations . However, other sources also report M. sobrinus bats further North (including where VTN-N9 and VTN-N10 occurred) [In contrast to the relatively high genetic variation observed in Chinese populations  .2 value of the IBD pattern for all assessed populations. This can be caused by either limited gene flow due to geographic barriers or a lack of suitable long-distance pollinators on the one hand, or excessive gene flow homogenising genetic variation among populations on the other hand. The former has been linked with a potential decline in fruit bats due to habitat loss, bushmeat hunting, and climate change [M. balbisiana populations can be considered to be a part of the native distribution area of the species based on their levels of observed heterozygosity. However, the origin of the two remote M. balbisiana populations located in southern Vietnam (Kon Tum province) remains more uncertain. Populations in southern Vietnam might be more connected to M. balbisiana populations in Laos and Cambodia than to other populations in Vietnam, which may explain the genetic differentiation of the southern populations from other Vietnamese populations included in this study. Nevertheless, the distribution of M. balbisiana in these neighboring countries is obscure, impeding the inference of any potential link with the populations in southern Vietnam. Alternatively, the southern M. balbisiana might be feral, originating after the recent human introduction of the species in the region as a source of food, medicine, fibre, and animal fodder. M. balbisiana was known by locals in northern Vietnam because of its higher tolerance to cold, drought and potential plant diseases, encouraging its cultivation in other parts of the country [M. balbisiana, especially VTN-S1, were also found close to cultivated varieties. Moreover, these populations were located far outside the range of the northern Indo-Burma subtropical forests, the native habitat of the other Vietnamese M. balbisiana populations. Taken together and in absence of information on M. balbisiana in neighboring countries, southern Vietnamese populations of M. balbisiana are supposedly no part of the native distribution area of the species.In the PCoA considering all sampled populations, Vietnamese populations could clearly be distinguished from Chinese populations . This wae change ,90. Beca country ,39,42. TM. balbisiana populations could be subdivided into five different groups. This result was supported by both the PCoA and the STRUCTURE analyses. Interestingly, the genetic diversity indices followed to a large extent this subdivision. In this section, we discuss the observed patterns of genetic structure among the northern and central Vietnamese M. balbisiana populations.By inferring genetic clusters solely among the Vietnamese populations that occur in the northern Indochina subtropical forests, the northern and central Vietnamese Cycas [M. balbisiana is known to be introduced from China to the Ryuku isles in Japan already in the 16th century for fiber use, making it hard to consider all sampled populations as wild, especially when they have low genetic variation and are located close to cultivated areas [In northern Vietnam, four populations  should be studied as well, as they are likely suitable for Musa balbisiana were genetically assessed and considerable differences between sampled populations were identified. Populations in central Vietnam, as well as a subset of populations in northern Vietnam, exhibited relatively high levels of genetic diversity comparable to native populations in China. Interestingly, Vietnamese populations showed no pattern of isolation linked to distance, indicating either excessive gene flow homogenising variation among populations or low gene flow between populations due to geographical barriers, limited long-distance pollination, and the ability to easily establish a new population after colonisation. We propose that combined clonal and sexual propagation are drivers of the observed patterns of genetic variation and structure, together with the mountain and river systems in northern Vietnam. Two populations sampled in more southern regions of Vietnam were considerably less diverse and were most likely introduced. We suggest a broader sampling of Musa balbisiana, as many regions with a similar suitable climate have not been investigated yet as well as neighbouring countries also in northern Indo-Burma . Moreover, a large scale phylogeographic study including populations from the multiple countries of the natural distribution range of M. balbisiana and in regions where M. balbisiana was introduced is necessary to further understand how this species has evolved and to what extent this was caused by human interference.For the first time, Vietnamese populations of S1 FigThe marked population was clearly an outlier and was removed from the study.(TIF)Click here for additional data file.S2 FigMarker locations are labeled in blue by the marker ID.(TIF)Click here for additional data file.S3 FigWithin population variance (Within Pops), among population variance (Among Pops), and variance among regions are presented. Regions include Northern Vietnam, Southern Vietnam, Central Vietnam, Western China, Southern China, and Hainan.(TIF)Click here for additional data file.S4 FigEach individual was coloured by population.(TIF)Click here for additional data file.S5 FigThe optimal number of K was determined for the full dataset (panels at the left hand side) and for native Vietnamese populations (panels at the right hand side): (A) MedMed K and MaxMed K through the Puechmaille method; (B), ΔK/K; (C), LnP(K)/K with K the number of clusters assumed.(TIF)Click here for additional data file.S1 TableTGTAAAACGACGGCCAGT; Q2 = TAGGAGTGCAGCAAGCAT; Q3 = CACTGCTTAGAGCGATGC .The underlined part of the reverse sequences indicates the sequence of the primer tail: Q1 = (DOCX)Click here for additional data file.S2 Table16 nuclear markers with the corresponding GenBank accession number of each sequence obtained from Wang et al. (2011)  and Rotc(DOCX)Click here for additional data file.S3 TableThe optimal number of clusters K was determined using MedMedK and MaxMedK according to the Puechmaille method, ΔK/K, and Mean LnP(K)/K. The number of clusters that were chosen for consecutive analyses are indicated in bold.(DOCX)Click here for additional data file.


During the Last Glacial Maximum, a small band of Siberians entered the Beringian corridor, where they persisted, isolated from gene flow, for several thousand years before expansion into the Americas. The ecological features of the Beringian environment, coupled with an extended period of isolation at small population size, would have provided evolutionary opportunity for novel genetic variation to arise as both rare standing variants and new mutations were driven to high frequency through both neutral and directed processes. Here we perform a full genome investigation of Native American populations in the Thousand Genomes Project Phase 3 to identify unique high frequency alleles that can be dated to an origin in Beringia. Our analyses demonstrate that descendant populations of Native Americans harbor 20,424 such variants, which is on a scale comparable only to Africa and the Out of Africa bottleneck. This is consistent with simulations of a serial founder effects model. Tests for selection reveal that some of these Beringian variants were likely driven to high frequency by adaptive processes, and bioinformatic analyses suggest possible phenotypic pathways that were under selection during the Beringian Isolation period. Specifically, pathways related to cardiac processes and melanocyte function appear to be enriched for selected Beringian variants. The Beringian ecology provided a refuge for this migrant population as the LGM intensified6. Plant macro-fossils and fossil pollen from Beringia suggest that it was a productive dry grassland ecosystem7 inhabited by a variety of large mammals8. However, North American glacial coverage and inhospitable Siberian environments during the LGM effectively sealed off the migrant population in the Beringian refugium, preventing either forward or backward movement until approximately 15kya when the surrounding glaciers receded, opening up both coastal and interior corridors of entry into the North American continent10 , approximately 30 thousand years ago (kya)18. However, the genomic impacts of the Beringian experience are still being discovered. Several factors suggest that there was a great deal of opportunity for genetic evolution in the Beringian population. Importantly, the population originated from a small group of founders and maintained a small size for millennia21. The combination of a founder effect and prolonged bottleneck would have greatly enhanced genetic drift22. It is well-known that genetic drift will reduce variation and provide a descendant population with only a subset of the variation that was present in their ancestors23. The current literature documents such a reduction in variation, and the subset pattern, in Native Americans in comparison to Eurasians and Africans24. However, in addition to the loss of alleles, genetic drift can elevate the frequencies of rare alleles and new mutations27. This will occur to a much lesser extent than the loss of standing variation. Nonetheless, full genome analyses make it possible to observe instances of such ‘allele gains’. The gain of novel variation tracing back to the Beringian occupation has been less studied than the loss of variation. Allele gains are the major focus of this paper.The demographic and paleo-ecological features of the Beringian experience have been well characterizedFADS) gene cluster, potentially modulating a unique lipid profile in response to a protein-rich diet28. Similarly, Hlusko and colleagues29 have argued that an amino acid substitution in the ectodysplasin A receptor (EDAR) may have evolved in response to vitamin D deficiency created by the low UV at high latitude. A survey of the genome may reveal more locations of adaptive changes.We expect that most alleles gained through founder effects and bottlenecks will be outside of gene coding and regulatory sequences, and therefore selectively neutral. However, it is also possible that some of the alleles gained through enhanced genetic drift will affect the expression of phenotypes. A portion of the alleles gained may have health consequences. In addition to genetic drift, positive natural selection by Beringian environmental conditions may have produced some allele gains. Indeed, a strong signature of positive selection has been found in several variants contained in the fatty acid desaturase . A GSP is a common allele in one group of people that is absent or nearly absent in all other groups. After a founder effect, GSPs will be present in both the ancestral and descendant populations. Ancestral and descendant populations can be distinguished after a founder effect by the mix of ancestral and derived alleles. GSPs in the ancestral population will be composed of a mix of ancestral and derived alleles. By contrast, GSPs in the descendant population will be almost entirely derived alleles that were gained from the founder effect. We analyzed whole autosome DNA sequences from the Thousand Genomes Project Phase 3 (TGPP3) sample to identify Group Specific Polymorphisms (GSP).Figure 32.We used coalescent simulations to verify that the observed pattern of derived GSP alleles is consistent with the reduction in heterozygosity that was seen in short tandem repeat and single nucleotide polymorphism data sets that support the serial founder effects model for genetic diversity in contemporary human populationsFigure Panels CNTNP2) gene on chromosome seven, 69 variants in the region of the Makorin Ring Finger Protein (MKRN9P) gene on chromosome twelve, and 62 GSPs in the region of the Melanoma-Associated Transcript-6 (MEAT6) gene on chromosome six. Figure Figure The Beringian GSPs located in genic versus inter-genic regions of the genome are presented on the first line of Table 33 analysis  for the 20,424 SNPs in the GSP set to test the hypothesis that some Beringian GSPs are environmental adaptations. These tests yielded 2,820 candidate loci with ihs scores exceeding the generally accepted threshold |2.5|. Typically, in tests for selection where many loci pass a minimum significance threshold, only the outliers in the top 1-5% are considered. Here, the top 5% of significant iHS scores across the entire genome includes 141 Beringian GSPs . A GSP is a common allele in one group of people and absent or nearly absent in all other groups. As such, a GSP will be diagnostic of ancestry from that group, and conversely, group membership will be a reasonable indicator that an individual will carry the allele. After a founder effect, GSPs will be present in both the ancestral and descendant populations. However, the ancestral and descendant populations can be distinguished by the mix of ancestral and derived alleles. GSPs in the ancestral population will be composed of a mix of ancestral and derived alleles, whereas GSPs in the descendant population will be almost entirely derived alleles. Our analyses reveal GSPs in only three groups: Sub-Saharan Africans, Eurasians, and admixed Americans. Sub-Saharan African GSPs have the greatest number of GSPs . These alleles clearly identify Sub-Saharan Africans as the ancestral population as 71% percent are the ancestral state and 29% are the derived allele state. By contrast, the set of Eurasian GSPs is composed almost exclusively of derived variants (99%), and further document that despite some archaic admixture, the Eurasian gene pool was primarily established by the Out of Africa (OOA) migration. The American GSPs are composed of 99.9% derived alleles and show the Beringian founder effect. It was surprising at first glance that GSPs specific to European, South Asian, and East Asian populations are absent or rare. However, the results are consistent with our simulations of the serial founder effects model and are easily explained by the fact that Eurasia was settled in a narrow time window after the out of Africa migration.One of the most striking features of Beringian GSP architecture is the distribution of GSPs throughout the 22 autosomes. Over 90% of the GSPs are distributed evenly with the spacing pattern approximating that seen in the set of 20,424 random SNPs. The evenness in the GSP distribution is punctuated by distinct clusters in 16 chromosomal regions  significantly exceeds the percentage of Random SNPs in coding sequence (38%). Second, in Beringian GSPs that do occur in coding sequence, the ratio of non-synonymous to synonymous nucleotide substitutions . The relevant phenotypes that favored survival and reproduction of individuals cannot be directly inferred from the nucleotide sequence data alone. Therefore, we have used bioinformatic analyses to gain provisional insights into potential phenotypes influenced by these alleles. Our analyses were performed by parsing the candidate loci according to three criteria, individual SNPs with outlying iHS scores, specific genes that harbor a disproportionate number of putatively selected GSPs, and gene ontology classes enriched for putatively selected GSPs. In combination, these three lines of evidence suggest adaptations in the Beringian Isolation Period are related to cardiac function and melanogenesis.EPHA3 which includes the GSP with the most extreme iHS score  determined from next-generation sequencing of 2,504 individuals. Each individual was sequenced for the whole genome using mean depth We analyze whole autosomes from the Thousand Genomes Project Phase 3 (TGPP3) sample to identify SNPs originating in the ancient Beringians Table 36. Brief37. Taken together, the Indigenous American proportions are equivalent to approximately 119 unadmixed genomes. The approach outlined below extracts information about genetic variants contributed by Indigenous American ancestors shared by all three populations.These are (We define a group specific polymorphism (GSP) as an allele that is at high frequency within a group of populations, private to that group of populations, and shared by all populations within the group Fig. . As such38. To search for African, European, South Asian, East Asians, and non-African GSPs we made the comparisons illustrated in Fig. The occurrence of GSPs depends on how groups of populations are defined. In this study, we consider the following groups determined by geographic locations, and descent groups formed by the well-documented out-of Africa migration that took place approximately 60,000 years before presentTo identify Indigenous American GSPs it was necessary to control for non-indigenous American admixture. Thus, in the Mexican American sample, the 30% GSP threshold was transformed according to a 47% Native American ancestry component (32. Demographic parameters for these simulations were estimated by fitting a tree to data from the CEPH-HGDP short tandem repeat (STR) data set39.We used coalescent simulations to verify that the observed pattern of derived GSP alleles is consistent with the loss of variation from which the serial founder effects model was inferred40. The non-South African populations from Europe and Asia were chosen to match the populations in the thousand genomes project data as closely as possible. Native American populations were included to estimate demographic parameters for the Beringian Isolation period.We began by choosing a subset of 27 populations from the CEPH-HGDP dataset: San and Kxoe from South Africa; Mandenka, Brong, Igala, Yoruba, and Luhya from Central Africa; Russian, Tuscan, Orcadian, Basque, and French from Western Europe; Punjabi, Tamil, Bengali, Gujarati, and Telugu from South Asia; Cambodian, Dai, Han, North Chinese Han, and Japanese from East and South-East Asia; and Pima, Maya, Mixtec, Embera, and Cabecar who are Native Americans. The San and Kxoe served to root the tree for the remaining 25 populations39 to compute Nei’s minimum genetic distances between all pairs of populations41. We re-scaled these distances by multiplying by 2. Using the re-scaled genetic distances, we built a neighbor-joining tree for the 27 populations. We rooted the neighbor-joining topology on the branch between the San-Kxoe and the remaining African and non-African populations, and then fitted branch lengths using the maximum-likelihood method proposed by Cavall-Sforza and Piazza44. The branch-lengths on the tree constructed in this manner measure the increase in gene identity (homozygosity) accrued between each pair of nodes moving from the root to the extant populations. The nodes on this tree were assigned chronological dates using estimates of the times at which modern humans inhabited the various regions of the globe. These dates were inferred from archaeological sources and independent genetic data46. We estimated effective population sizes for each branch of the tree by iteratively solving for the population size that would reproduce the genetic distance on that branch while allowing step-wise mutations to occur at a rate of Next we used 619 microsatellite loci47. The times of each mutation in the simulated coalescent histories were recorded, as were the frequencies of the mutant (derived) allele in the contemporary population. From this simulated data, we constructed the probability density for the age of a high frequency derived allele found in a population that inhabited a specific geographic region of the world. Thus, we were able to assess the probability that a derived allele arose, on a branch, at a time, that would render it exclusive to a particular set of populations or geographic region.With the chronological dates and effective population size estimates obtained as outlined above, we simulated single-copy DNA sequences using an infinite sites mutation model and mutation rate of For comparative purposes, we constructed a random sample of 20,424 SNPs selected from across the genome. The allele frequency distribution and proportion of SNPs per chromosome in the random sample were matched to the set of Beringian GSPs that we discovered (See Results).We searched for spatial clustering within the Beringian GSPs and random SNPs by tabulating the median distance from each SNP to its nearest neighbor within a window size of +/−10 SNPs.48 (https://rdocumentation.org/packages/annovarR/versions/1.0.0)bto categorize each variant from the Beringian GSPs and Random SNP Sets according to a variety of genomic properties. All SNPs were annotated as either intergenic, non-coding RNA (ncRNA), or genic . Exonic variants were further annotated to reflect synonymous and non-synonymous substitutions. We further annotated variants from the Beringian and Random SNP sets with known gene associations as reported by NCBI’s gene database. The gene associations included intergenic SNPs that fell within known regulatory regions of specific genes. Genes impacted by Beringian GSPs and Random SNPs were then grouped according to similar functional properties defined by both the Kyoto Encyclopedia of Genes and Genomes(KEGG) categories49 and gene ontology categories (GO) using the Enrichr https://cran.r-project.org/web/packages/enrichR/vignettes/enrichR.html and Panther version 16.0 (http://www.pantherdb.org/pathway/) software packages, respectively50.We used the ANNOVAR annotation suite51 and compared it to the same measure on a random set of SNPs. Genic SNPs in both the Beringian and random sets were annotated as synonymous or non-synonymous substitutions using the ANNOVAR suite. To calculate 51. To determine whether the GSPs displayed an atypical pattern of natural selection in comparison to SNPs chosen from the genome at random, we applied the above steps to the set of 20,424 random SNPs. seqinr package version 4.2-552 (http://seqinr.r-forge.r-project.org/) on gene sequences downloaded from the NCBI database.To measure purifying selection, we calculated the iHS)53 using the rehh package in R54. We calculated an To identify signals of positive selection, we calculated integrated haplotype homozygosity scores 55. Results from this analysis were rank ordered using the Enrichr combined score metric, which captures a measure of deviation between each category’s observed rank and the expected rank for that category by chance.In order to link natural selection and phenotypic targets for adaptation, we examine Beringian GSPs with high |Supplementary Information.