Rhodiola crenulata, a perennial herb almost exclusively restricted to rock crevices on mountain peaks, and to test whether the nunatak or massif de refuge hypotheses could explain its distribution pattern.Quaternary climatic oscillations had tremendous effects on the current distribution of species. Here, we aim to elucidate the glacial history of R. crenulata and its closest relatives was dated have occurred ca. 0.65 Mya, during the Naynayxungla glaciation on the QTP. Mismatch distribution analysis suggested that the species experienced a range expansion around 0.31 Mya. Populations with high genetic and haplotype diversity were found on the QTP platform as well in the Hengduan Mountains. The ecological niche modeling results showed that there were suitable habitats on both the QTP platform and in the Hengduan Mountains during the LGM.Six haplotypes and six ribotypes were detected in the cpDNA data set and the ITS data set, respectively. The divergence of R. crenulata. We also confirmed that Quaternary climatic oscillations could promote plant speciation in some circumstances. This study adds to a growing body of evidence suggesting that the QTP plant lineages exhibited diverse reactions to the Quaternary climatic oscillations.Our results support a scenario that both nunataks and the massif de refuge hypotheses could explain the distribution of The online version of this article (10.1186/s12862-018-1270-6) contains supplementary material, which is available to authorized users. Mountains have long been recognized as island-like systems –3. TheseThe current distribution of genetic lineages of species in the Northern Hemisphere was greatly influenced by the Quaternary climatic oscillation , 6. DuriRhodiola crenulata (HK. f. et Thoms.) H. Ohba, our study species, is almost exclusively restricted to rock crevices on mountain peaks  to conduct a phylogeographic study. The ENMs that are widely used in phylogeographic studies, can provide important evolutionary and biological insights by allowing the evaluation of biogeographic hypotheses or to interpret genetic diversity patterns [aks Fig. , from 43of years . The accpatterns , 22.Fig.R. crenulata during the summers of 2016 to 2017. For each population, we generally sampled 10–20 individuals. Each sampled individual in every population was at least 20 m away from any other to avoid sampling clonal individuals. In total, we collected 253 individuals and dried their leaves with silica gel. Based on a previous phylogenetic study, we chose one individual of R. wallichiana (Hk.) S. H. Fu to act as an outgroup [Our sampling did not require specific permits for the locations involved as we held a permission letter from College of Life Sciences, Shaanxi Normal University, Xi’an relating to colleting samples. We sampled 16 populations across the distribution range of outgroup .trnL-F, c and f [trnS-G, trnS and trnG [We used a Plant Genomic DNA Kit  to extract DNA from silica-gel dried leaves. PCR amplification was conducted in a 20 μl system , using primers for ITS, ITS-1 and ITS-4 , for trn c and f  and for and trnG . We usedand trnG . For chland trnG  was usedtrnL-F and trnS-G) were concatenated into a matrix because the chloroplast genome can be considered to have evolved as a single entity in plants. Segregating sites and haplotypes were extracted using DnaSP v5 [Sequences from two cpDNA regions  in Modeltest v. 3.7 , 33 . We then used both the ITS and plastid data sets to conduct a dating analysis using the BEAST software [R. crenulata and its relatives available; we therefore used substitution rates to estimate divergence times. It has been reported that the substitution rate of nrITS in shrubs and herbs varies from 3.46 × 10− 9 to 8.69 × 10− 9 s/s/y [− 9 1.590 × 10− 9). The evolutionary rate of the plastid markers was assumed to be 2 × 10− 9 s/s/y [To infer the divergence time between lineages, we first examined the molecular clock hypothesis with a likelihood ratio test in PAUP* v. 4.0b10. The results showed that both the ITS and cpDNA data sets fit the molecular clock hypothesis  and nucleotide diversity (π) for each population  and at the species level . We also calculated average gene diversity within the population (HS), total gene diversity (HT) and between population divergence  using the program PERMUT (available at https://www6.bordeaux-aquitaine.inra.fr/biogeco/Production-scientifique/Logiciels/Contrib-Permut/Permut) with 1000 permutation tests. If the NST value is larger than GST, it means that closely related haplotypes will appear in the same area with higher probability than remotely related ones, thus indicating the presence of phylogeographic structure [We used DnaSP v5  to estimtructure . The Mantructure .R. crenulata, we used SAMOVA v. 1.0 [FCT value, the program can search different K values that the user defines as groups of geographically adjacent populations. We ran the program repeatedly with K values from 2 to 10. After defining geographic groups, we calculated the amount of variation among populations within a geographic group and within a population using the hierarchical analysis of molecular variance (AMOVA) framework, which was run using ARLEQUIN v. 3.5 [To see if there is any spatial structure in populations of A v. 1.0  to assesN v. 3.5 . We usedD and Fu’s Fs statistics [D and Fs statistics are significantly negative if there is population expansion, as an excess of rare new mutations will originate during the expansion process. A previous study has shown that mismatch distribution is not affected by population structure [SSD) and the raggedness index (HRag) [τ was used to estimate the expansion time (in generations) with the equation t = τ/2u [u = μ × k × g, in which μ represents substitution rate (s/s/y), k represents the length of aligned matrix, and g is the generation time in years. For the present study, k was 1531 bp, and the substitution rate for the cpDNA genome was assumed to be 2 × 10− 9 s/s/y [Then we inferred signatures of demographic expansion by calculating Tajima’s atistics , 43 and atistics , 45. D atructure , thus wetructure , 47 was x (HRag)  with 100t = τ/2u , 46, whe 9 s/s/y , 49. GenR. crenulata in response to glacial climatic oscillations. Locations of R. crunulata were obtained from field collections of the authors and from herbarium records online . Data from online databases were checked to exclude misidentification. A total of 88 spatially unique localities were used for analysis. MAXENT v. 3.3.3e [R. crenulata. Twenty replicates were employed, from which 80% of the distribution coordinates were used for training and 20% for testing. Environmental layers of 19 bioclimatic variables  was much higher than within-population gene diversity (HS)  was also much lower than total gene diversity (HT)  in all 253 individuals, which were determined by four substitutions. Among the six ribotypes, three occurred in only one population. R1 was the most widespread ribotype, occurring in 15 of the 16 populations we sampled , i.e. in the Pleistocene , still in the Pleistocene    Fig. . We founJuniperus przewalskii [Picea crassifolia [Potentilla glabra [Aconitum gymnandrum [Juniperus tibetica complex [Hippophae tibetana [Many authors have investigated how the Quaternary climate oscillations have shaped the distributions patterns of plants on the QTP and its adjacent areas , 56–58. ewalskii  and Picea glabra  and Aconmnandrum . In addi complex  and Hipptibetana  provide R. crenulata, which is also the case for Potentilla glabra [Aconitum gymnandrum [R. crenulata, both cpDNA and ITS data sets showed high genetic diversity and private haplotypes in population ML  and four out of the six ribotypes (67%) are endemic to one population. Furthermore, only one haplotype and one ribotype were widespread across the distribution range  from WorldClim [Rhodiola crenulata. (DOCX 67 kb)orldClim . VariablAdditional file 2:Table S2. Haplotype composition of 16 sampled populations of Rhodiola crenulata based on the cpDNA data set. (DOCX 16 kb)Additional file 3:Table S3. Haplotype composition of 16 sampled populations of Rhodiola crenulata based on the ITS data set. (DOCX 15 kb)Additional file 4:Figure S1. Divergence time of R. crenulata and its closest relative based on the plastid DNA haplotypes estimated with BEAST. Gray bars indicates 95% highest posterior density intervals. (TIF 93 kb)Additional file 5:Figure S2. Correlation between the F statistics and grouping number (K = 2–10) from the SAMOVA results. (a) results based on cpDNA haplotypes; (b) results based on ITS ribotypes. (TIF 887 kb)Additional file 6:Figure S3. Historical demography for overall populations and in each clade based on the plastid DNA dataset. Clade I and clade II correspond to the groups in the Bayesian phylogenetic tree in Fig.


Betula nana) has a widespread boreal distribution but has declined significantly in Britain where populations are now highly fragmented. We analyzed the genetic diversity of these fragmented populations using markers that differ in mutation rate: conventional microsatellites markers (PCR-SSRs), RADseq generated transition and transversion SNPs (RAD-SNPs), and microsatellite markers mined from RADseq reads (RAD-SSRs). We estimated the current population sizes by census and indirectly, from the linkage-disequilibrium found in the genetic surveys. The two types of estimate were highly correlated. Overall, we found genetic diversity to be only slightly lower in Britain than across a comparable area in Scandinavia where populations are large and continuous. While the ensemble of British fragments maintain diversity levels close to Scandinavian populations, individually they have drifted apart and lost diversity; particularly the smaller populations. An ABC analysis, based on coalescent models, favors demographic scenarios in which Britain maintained high levels of genetic diversity through post-glacial re-colonization. This diversity has subsequently been partitioned into population fragments that have recently lost diversity at a rate corresponding to the current population-size estimates. We conclude that the British population fragments retain sufficient genetic resources to be the basis of conservation and re-planting programmes. Use of markers with different mutation rates gives us greater confidence and insight than one marker set could have alone, and we suggest that RAD-SSRs are particularly useful as high mutation-rate marker set with a well-specified ascertainment bias, which are widely available yet often neglected in existing RAD datasets.Dwarf birch ( N much greater than u and s) there is nearly complete fixation, little variation, little effect of selection and thus a static condition modified occasionally by chance fixation of new mutations leading inevitably to degeneration and extinction” , a monecious wind-pollinated, subarctic dwarf tree. Betula are generally considered self-incompatible, however, low levels of selfing has been reported in many species (Clausen Our study species is dwarf birch ( Clausen . Dwarf b Clausen . Threats Clausen . The spe Clausen  since thti) and transversions (RAD-SNPtv), and microsatellites data-mined from RAD-sequencing reads (RAD-SSRs). Typically, indel mutations generating variation at SSR loci occur more frequently than substitution mutations that cause variation of SNPs , single-nucleotide polymorphisms from RAD-sequencing reads divided into transitions  protocol published in   and linkage-disequilibrium (LD) using the software GENEPOP v4 Rousset , with te Nichols  implemen Nichols  over 50, Nichols .PstI, as previous analysis of the draft B. nana genome identified an appropriate number  of cut sites , identified as an optimum threshold in Ilut et al.  and other parameters set to default. Subsequently, cstacks and sstacks were manually rerun to rebuild and match to the catalog.Raw libraries were demultiplexed and barcode sequences checked for errors using the t et al.  and a mipopulations module of the software Stacks  and transversion  datasets based on the type of polymorphism recorded and analyzed these independently. Finally, SNPs were exported to other analysis software using custom scripts and PGDSpider 2.1.0.1  of <0.01 were excluded. Similarly, to reduce LD these data were filtered to retain only a single locus per scaffold or contig in the To generate RAD-SSR markers we filtered raw Illumina RADseq reads for SSR motifs by searching the consensus catalog for loci with di-nucleotide repeats (see Ar) estimates were rarefied to allow correction for uneven sample size and estimated in ADZE  was calculated across all populations and markers by region in Hierfstat . In order to investigate the relative importance of recent genetic drift in generating these patterns, we required an estimate of the current effective population size, (Ne), at each location. An estimate was obtained using the LD-based method of Do et al. (B. nana census population size (see above).Some statistics characterize genetic patterns that may have accumulated over long time periods, such as the local genetic diversity (heterozygosity) or differentiation among populations /FST, and then plotted against log transformed geographic distance.Two different approaches were employed to assess population differentiation. In both cases we filtered for <25% missing data, genotypes in every population, and a minor allele frequency >0.05. First, pairwise an Dixon . To lineFST was defined; here termed as Maximum Likelihood FST (ML-FST). Although FST is often thought of as a statistic describing the degree of genetic differentiation among a group of sub-populations, it can also be expressed in this way, since it can be formulated as a genetic correlation within a particular subpopulation  relative to the regional mean, using the multinomial Dirichlet likelihood function proposed by Balding and Nichols . Finally, to evaluate the influence of sample size, we estimated ML-FST across all loci from a single individual drawn from each population.Second, to quantify the genetic divergence of each population from the regional mean, an alternative estimate of  Balding . We estiTo infer the evolutionary and demographic history of dwarf birch in Britain since colonization, we used coalescent-based approximate Bayesian computation (ABC) implemented in DIYABC v2.0 , to test for evidence of a historic bottleneck immediately on colonization (Scenario 2) or multiple bottlenecks after population differentiation (Scenario 1), potentially as a result of recent anthropogenic impacts. Subsequently, we incorporate census population information into the best-supported scenario (Scenario 1b), permitting independent British population Ne values to refine our posterior estimates. We test this against the null meta-population scenario from the previous analysis (Scenario 1). To further reduce the complexity of the analysis, we subsampled our full dataset . This is perhaps due to reference-based mapping performing better at distinguishing paralogs. A threshold of 70% presence resulted in 6092 loci containing 23,882 SNPs. Quality filtering retained 17,694 SNPs with an overall Transition/Transversion ratio of (11:10). With minor alleles occurring at a frequency of <0.01 removed, 4208 loci and 8081 SNPs were retained, of which 4775 were transition and 3306 transversion polymorphisms (144:100); thus proportionately more of the low frequency polymorphisms were transitions.A total of 339,291,129 reads were generated from two single-end Illumina HiSeq lanes, of which 81.66% passed strict quality controls. Three individuals were removed due to low sequencing coverage. The number of reads retained per individual ranged from 373,727 to 2,852,627, with a mean of 1,397,192 Table . Across Simple sequence repeat motifs were initially identified in 3507 RADseq loci, with 196 loci meeting initial filtering criteria. We excluded three further loci with >40 unique genotypes due to the risk of a high number of sequencing errors in these reads. The number of alleles per locus ranged from 2 to 31 with a total of 1758 alleles across all populations. No evidence of clones was found in any of the RADseq samples.ti. Data are summarized by marker and region in Table Ar) and expected heterozygosity (He) displayed a similar pattern (Table FIS) were lowest in SNPs datasets, and higher among SSR data, with RAD-SSR data reporting the highest values. As expected, SSR marker FIS values were lower when co-estimated with null allele frequency.The number of alleles across all samples ranged from 414 in PCR-SSRs to 9428 in RAD-SNPt-test, all p ≤ 0.01), with the exception of PCR-SSR He, which displayed the opposite trend. Fixation indices were higher in Scandinavia than in Britain for both RAD-SNP datasets . For SSR markers, fixation indices were not significantly different in Scandinavia and Britain, and null alleles affected fixation index estimates more in Scandinavia than in Britain, likely because our markers were first developed using British populations. Global FST was significantly higher in Britain across all RAD markers, with 95% confidence intervals only weakly overlapping in PCR-SSR markers. The rather small difference in overall levels of genetic diversity in Scandinavia and Britain is reflected in the diversity data for individual populations , but much less so in Scandinavia (s = 0.02 [0–0.17])  see .Fig. 3Plr = –0.003, p = 0.46; Scandinavia: r = 0.36, p = 0.055), RAD-SNPti  and RAD-SNPtv , except in PCR-SSRs  .Overall we found little evidence for isolation by distance (IBD) across markers, RAD-SSRs . This is further supported by STRUCTURE analysis, which shows a stronger pattern of population structure in Britain compared to Scandinavia , but showed no significant correlation in Scandinavia . Model checking, bias and precision estimates in these data are reported in Supplementary Table We estimated combined posterior parameter distributions based on scenario 1b. These supported differentiation of British and Scandinavian lineages t2 = 789 (CI = 521–1274) generations ago, presumably shortly after colonization of Britain; an event that would have substantially limited gene flow. The estimates suggest that scandinavia maintained a somewhat larger historic Ne  than Britain , although their confidence intervals overlap substantially. Subsequently, British populations appear to have become differentiated very recently at t1 = 23 generations ago (CI 10–415). The ti biallelic loci and 3306 RAD-SNPtv biallelic loci. We successfully genotyped 1066 individuals with the PCR-SSRs and 187 individuals with the RAD-based markers. The number of loci, alleles and samples for these marker sets reflect their abundance in the genome, the dynamics of their mutation and the ease of assaying them.We generated population genomic datasets for dwarf birch populations in Britain and Scandinavia using four types of markers: 18 PCR-SSR loci with a total of 414 alleles, 193 RAD-SSR loci with 1758 alleles, 4775 RAD-SNPti > RAD-SNPtv. Figures FST is higher for the SNP markers than in the PCR-SSR markers, with the RAD-SSRs being intermediate. We expected all markers to give the same FIS estimates, as nonrandom mating should affect all markers equally. However SSR based markers reported slightly higher FIS values, which can be explained by our higher estimates of null allele frequencies, particularly in Scandinavia , show that differentiation is clearly related to current population sizes with a strong effect in populations currently around ten individuals. This effect is also seen in loss of allelic richness values in smaller populations , setting a lower bound on generation time. A more central estimate can be obtained if we assume divergence occurred between the ancestral British and Scandinavian dwarf birch populations (t2 = ~789 generations) just after the last ice age 10–12,000 years ago. Dividing by the number of generations gives a generation time is 10–15 years, which is biologically plausible. This calibration would place the divergence among British populations at 230–345 years ago (t1 = ~23). That would date the ABC estimate for the time of current population subdivision to a the period when large scale anthropogenic activity including lowland agriculture had begun to shape the distribution of woody plants in Scotland . Taken at face value, the estimate of 11 generations (tb1 = ~11) is sufficient for the very smallest populations to have lost a substantial part of their genetic variation, and corresponds to 110–165 years. Accelerated forest clearance is known to have occurred in the sixteenth to nineteenth century, combined with a shift to livestock farming during the highland clearances (Dodgshon and Olsson B. nana populations are preserved in different fragments, so that overall levels of diversity in British B. nana populations are only moderately lower than the large continuous populations of Scandinavia. The long-term viability of B. nana in Britain may be enhanced by artificially restoring gene flow between populations, perhaps by culturing seedlings within, or transplanting among populations (Aitken and Whitlock Our results suggest that the long-term prospects for many dwarf birch populations in Scotland may be good, as there are levels of genetic diversity comparable to Scandinavia, despite fragmentation and reduced population size. Our ABC models suggest only moderate loss of diversity in the British populations upon post-glacial colonization, and that losses of diversity since then occurred in localized populations during their divergence from one another. Thus, different sub-sets of the ancestral variation of British sensu lato Hampe and Petit The very smallest populations have diverged in allele frequency and lost genetic diversity at the genome-wide marker loci we surveyed. We can explain these effects as a consequence of recent severe isolation and reductions in effective population size. We would, therefore, expect loss of alleles conferring adaptive variation and inefficient selection to maintain traits at the local adaptive optimum. Consequently these populations are unlikely to harbor adaptive rear-edge diversity that needs to be conserved (While, the potential for orders of magnitude more data from SNP analyses based on modern NGS technology is widely appreciated, comparative studies combining different categories of population genetic markers remain sparse (examples include Bradbury et al. Raw PCR-SSR data available from the Dryad Digital Repository: 10.5061/dryad.v75rj24.Illumina read data from RADseq libraries has been uploaded to the European Nucleotide Archive project PRJEB26807, sample accessions ERS2598190- ERS2598376Newly developed PCR-SSR primers are reported in the Supplementary Materialshttps://github.com/JamesBorrell/RAD-SSR_extraction_scripts.gitRAD-SSR marker data mining script available on Github: FST function available on Github: https://github.com/qmwugbt112/FstCalcMaximum Likelihood Supplemental Material


Climate oscillation may have a profound effect on species distributions, gene flow patterns and population demography. In response to environmental change, those species restricted to montane habitats experienced expansions and contractions along elevation gradients, which can drive differentiation among sky islands.Pachyhynobius shangchengensis) is a cool stream amphibian restricted to high-elevation areas in the Dabie Mountains, East China. In the present study, we used mtDNA genes (Cyt b and ND2) of 193 individuals and 12 nuclear microsatellite loci genotyped on 370 individuals, representing 6 populations  across the taxon’s distribution area, to investigate their genetic variation and evolutionary history of P. shangchengensis. Most populations showed unusually high levels of genetic diversity. Phylogenetic analyses revealed five monophyletic clades with divergence times ranging from 3.96 to 1.4 Mya. Accordingly, significant genetic differentiation was present between these populations. Bayesian skyline plot analyses provided that all populations underwent long-term population expansions since the last inter-glacial (0.13 Mya ~ 0.12 Mya). Msvar analyses found recent signals of population decline for two northern populations (JTX and KHJ) reflecting a strong bottleneck (approximately 15-fold decrease) during the mid-Holocene (about 6000 years ago). Ecological niche modelling has shown a discontinuity in suitable habitats for P. shangchengensis under different historical climatic conditions.The Shangcheng stout salamander  contains supplementary material, which is available to authorized users. Geological, climatic, and hydrological histories are the major drivers that have determined present-day patterns of biodiversity . These fSky islands are montane regions isolated from one another by intervening valleys with drastically different environmental conditions, in a similar way to how the sea separates oceanic islands . In sky Pachyhynobius shangchengensis) is a stream salamander narrowly distributed in the Dabie Mountains and is endemic to the cool and oxygen-rich streams above 500 m [P. shangchengensis is found distributed as six isolated areas/populations   Fig.. In prevTTZ, BYM ). In thelethodon , 13, 14.P. shangchengensis throughout its range and used multiple molecular markers combined with GIS–based environmental niche analyses to better understand population genetic relationships and evaluate diversification processes. Firstly, we used statistical phylogenetic methods to test whether each geographically isolated sky island population is reciprocally monophyletic and whether the patterns of phylogenetic diversification identified are consistent with past climatic oscillation. Secondly, we examined whether the isolated sky island populations maintained genetic diversity effectively. Lastly, we used ecological niche modelling (ENM), combined with paleoclimate data to infer historical causes for the isolated distribution pattern of P. shangchengensis.Here we sampled P. shangchengensis between 2012 and 2015 , throughout the distribution range of b (Cyt b) and NADH dehydrogenase 2 (ND2) were amplified using primers designed from Ranodon sibiricus (NC004021) and P. shangchengensis (NC008080) mitogenome using Primer v5 [Taq polymerase , 0.6 mM of each primer and sufficient pure molecular biology grade water. The amplification program included an initial denaturation step of 95 °C for 5 min followed by 32 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s and an extension at 72 °C for 80 s, with a final extension at 72 °C for 10 min. PCR products were purified using EasyPure PCR Purification Kit (TransGene), and sequenced on an ABI Prism 3730 automated sequencer using the BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems).Total DNA was extracted from samples using a standard proteinase K/phenol-chloroform protocol , and purrimer v5   and sufficient water. The amplification program was conducted with following conditions: 5 min at 95 °C; followed by 35 cycles of 30 s at 95 °C, 20 s at the annealing temperature  and haplotype diversity (Hd) in DnaSP v.5.10. Micro-Checker [Na), observed heterozygosities (HO), expected heterozygosities (HE), inbreeding coefficients (F) and pairwise FST.Sequences were assembled using SeqMan in DNAStar . All DNA-Checker  was used-Checker . Several-Checker  and GENE-Checker , includib and ND2) using BEAST v.1.8 [R. sibiricus (NC004021) as outgroup. The best partition strategy and nucleotide substitution model for each data partition were determined with Partitionfinder v.2.1.1 [P. shangchengensis and the outgroup, based on the time calibrated molecular phylogenetic tree of Hynobiidae [We reconstructed the phylogenetic relationships and divergence time of combined sequences  between populations was estimated in Arlequin v.3.5.1.3 [A minimum-spanning network based on mitochondrial genes was created with Network v.4.6.1 , using the full median-joining algorithm  to visua.3.5.1.3  based onA Bayesian analysis of population structure using STRUCTURE  was carrA spatial cluster analysis was conducted with the Geneland package  in R 3.3D [Fs [P. shangchengensis using the Bayesian skyline plots (BSP) with the model selected by Partitionfinder. BSP were performed in BEAST to reconstruct historical population size dynamics by using the same parameter sets as previous divergence time estimation. Each dataset was run four times for 20 million MCMC iterations, sampling every 2000 iterations and discarding the first 25% of the iterations as burn-in. Tracer was used to check the convergence of the MCMC analyses .Mismatch distributions were estimated to test the null hypothesis of recent population growth using Arlequin. A goodness-of-fit test was used to determine the smoothness of the observed mismatch distribution  and the degree of fit between the observed and simulated data  . Due to D  and Fu’sD [Fs  using 10P. shangchengensis using the microsatellite data. The method assumes that a current population (of size N0) passed through a demographic change (a bottleneck or an expansion) at time T in the past, which changed its size from N1 to N0 following an exponential model. The coalescent simulations were conditioned with various prior distributions to assess the lack of dependency of the posterior estimates of the three inferred parameters . The prior and hyperpriors used in Msvar were showed in Additional file N0 and N1 had the same prior upper bound, (ii) where N0 had a wider prior than N1  and (iii) where N1 had a wider prior than N0 . In addition, various upper bound values for the prior of the time of change were also tested. As to the microsatellite mutation rate, we used an average vertebrate microsatellite mutation rate of 10− 4 [− 2 and 10− 6  at 30 arc-seconds resolution (~ 1 km2) and clipped these coverages to a region that encompassed the entire Dabie Mountains’s range  [We use ENM to simulate the distribution pattern of  3.3.3 k . For the~ 117°E) . We perf~ 117°E) , we proj~ 117°E) , to obta~ 117°E)  were useWe performed 15 replicates for the model with subsamples, 10,000 background points and 25% of points set for random testing. Analyses were run using default program parameter cumulative outputs, a 0.00001 convergence threshold, and a maximum of 5000 iterations. The area under the curve (AUC) of the receiver operating characteristic (ROC) plot were used for model evaluation . All modND2 gene and 1127 bp of the Cyt b gene from 193 individuals. Total Hd and π across all populations were 0.990 and 0.0345, respectively (Table Hd = 0.983) while the KJY population possessed the lowest value (Hd = 0.746). The BYM population exhibited the highest nucleotide diversity (π = 0.0030) while KHJ presented the lowest one (π = 0.0015).We sequenced 984 bp of the Na ranging from 8.83 (JTX) to 17.42 (BYM), HO ranging from 0.596 (KHJ) to 0.756 (BYM), while HE varied from 0.699 (KHJ) to 0.837 (BYM). Overall, among the six populations, BYM showed higher genetic diversity than the other five populations  and southern four populations  respectively Fig. . In the FST values among populations were all significant and ranged from 0.494 (BYM and KJY) to 0.962 (JTX and MW) for mtDNA data and from 0.087 (MW and TTZ) to 0.590 (JTX and KJY) for the microsatellite data  shows that 61.02% of the genetic variation occurs among groups, 33.83% among populations within groups and 5.15% within populations , mismatch distribution analysis and BSPs were conducted on the mtDNA sequence data and excluding the two KJY haplotypes clustered within the BYM population. Fu’s Fs and Tajima’s D were negative in all six populations, however, while Fs was significant for all populations except KJY, only MW and BYM presented a significant D  have an N0  almost 15 times smaller than their N1  indicating that these populations passed through a drastic bottleneck approximately 6000 years ago   Fig. b. Based P. shangchengensis populations that have been isolated from each other for more than one million years, however, each populations still harbored relatively high levels of genetic diversity when compared to other narrowly distributed salamanders, such as Batrachuperus tibetanus  [P. ouachitae, P. fourchensis and P. caddoensis [Pseudopaludicola falcipes) in South America’s Pampas or Southwest China’s Leishan spiny toad (Leptobrachium leishanense) [P. shangchengensis under different climate condition during the LIG, LGM and mid-Holocene periods, probably through the presence of favorable environments in long-term and stable montane stream habitats that prevented the collapse of most of its populations and thus favored the maintenance of their genetic variation.Small and isolated populations for prolonged periods often encounter a number of genetic risks, such as the loss of genetic variation due to genetic drift, inbreeding the potential accumulation of weakly deleterious mutations –59. In tCaudata)  and variddoensis , 13, 14,hanense) –63. Sevehanense) . In genehanense) , 12. BasP. shangchengensis. However, this observation is probably exacerbated by the demographic history of these two populations that passed through a relatively recent and strong bottleneck during the mid-Holocene further leading to a decrease in their genetic variation in comparison to the southern populations  resulting in the classic phylogeographic structure associated with stream drainages and mountains [B. tibetanus  is a typical stream salamander in the Qinling Mountains showing a split into two deeply divergent lineages that formed in the past 3 to 4 Mya as a response to the orogenesis of Qinling Mountains during the late Cenozoic [Species restricted to montane sky island habitats often show high levels of inter-population genetic divergence and unique patterns of genetic structure , 68–70, ountains , 13, 14.Cenozoic .P. shangchengensis has changed over different historical periods and climatic conditions, there has always been extensive suitable habitat for different populations   [Leucodioptron canorum) [B. thoracica) [Podarcis siculus and P. filfolensis, respectively) [Generally, mid-latitude regions, and especially mountainous areas, experienced the most significant climatic changes due to the fluvial and glacial-dominated conditions during the Quaternary , 12. In ulengeri ) these ealophus) , the Chicanorum)  and the oracica) . In mid-ctively) , 86. Obvctively) , 85, 86.P. shangchengensis populations, except the two northern populations (JTX and KHJ), remained stable during the Holocene Additional file 2:Table S2. Prior and hyperpriors used in Msvar based on microsatellite data. (DOCX 17 kb)Additional file 3:Figure S1. STRUCTURE clustering results for K = 4, 5 and 6. (TIF 2521 kb)Additional file 4:Table S4. Multiple runs for inferring the number of populations of P. shangchengensis with Geneland. Bold indicates the highest average posterior probability. (DOCX 16 kb)Additional file 5:Table S3. Results of hierarchical AMOVA based on mtDNA and microsatellites. Groups are set as JTX and KHJ, MW and TTZ, BYM and KJY. (DOCX 17 kb)Additional file 6:Figure S2. Mismatch distributions analyses for the six populations of P. shangchengensis based on the mtDNA sequence data (a–f). The two KJY individuals clustering with BYM were excluded from the demographic history analyses. The x coordinate represents the number of differences between pairs of sequences and the y coordinate represents the frequencies of pairwise differences. The blue histograms are the observed frequencies of pairwise divergences among sequences and the green line refers to the expected shape of the distribution under the model of population expansion. (TIF 2468 kb)Additional file 7:Table S5. Results of demographic statistics of P. shangchengensis based on mtDNA data. (DOCX 18 kb)Additional file 8:Figure S3. Bayesian skyline plot of effective population size in P. shangchengensis based on the mtDNA data (a–f). The BSP analyses of the KJY population was calculated excluding the individuals with two haplotypes clustered more closely to the BYM population. The x-axis indicates time in Mya BP, and the y-axis indicates the effective population size in units of Neτ (the product of effective population size and generation time in Mya). The blue areas represent 95% highest posterior density. Time is expressed in million years. (TIF 2011 kb)Additional file 9:Table S6. Results from Principal Components Analysis on environmental variables used in comparison of environments among occurrence localities for P. shangchengensis and the relative contribution of each variable to the niche model. (DOCX 16 kb)


Gobiobotia filifer, is an endemic benthic fish in the Yangtze River and is a good model for ecological and evolutionary studies. This study aimed to decode the population structure of G. filifer in the Yangtze River and reveal whether divergence occurred before or after population radiation. A total of 292 specimens from eight locations in the upper and middle reaches of the Yangtze River were collected from 2014 to 2016 and analyzed via mitochondrial DNA Cyt b gene sequencing. A moderately high level of genetic diversity was found without structures among the population. However, phylogenetic and network topology showed two distinct haplotype groups, and each group contained a similar proportion of individuals from all sampled sites. This suggested the existence of two genetically divergent source populations in G. filifer. We deduced that a secondary contact of distinct glacial refugia was the main factor creating sympatric populations of G. filifer, and climate improvement promoted population expansion and colonization.The ecosystem and Pleistocene glaciations play important roles in population demography. The freshwater gudgeon,  The results revealed two sympatric populations coexisting in all geographical locations, and similar proportions of sympatric populations among the geographical populations. It appeared that ecosystem and historical event played important role to shape the population patterns. According to the river characteristics, the river section above Yichang City, Hubei Province, is considered the upper reaches, and the section below Yichang City the middle and lower reaches. There are approximately 361 fish species inhabiting the river basin, 177 of which are endemic  is a group of small freshwater gudgeon distributed in the rivers of East Asia in Korea and China  to examine population genetic diversity of G. filifer. We aimed to (a) reveal whether genetic divergence occurred within the population and (b) determine whether divergence occurred before or after population radiation.Own to the specialized position in phylogeny and relatively broad distribution, 22.1G. filifer were collected from eight locations from 2014 to 2016; four locations were in the upper reaches of the Yangtze River and four in the middle reaches  following the manufacturer's instructions.2.2b (Cyt b) gene was amplified using polymerase chain reaction (PCR) with primers L14742 and H15915  and aligned with the Clustal X 1.81 program  and nucleotide diversity (π) in the DnaSP 6.0 software . Phylogenetic analysis of haplotypes was conducted using the neighbor‐joining (NJ) method and Bayesian inference (BI). The NJ analysis with the Kimura 2‐parameter distance method was carried out in MEGA 7.0 .A median‐joining haplotype network was constructed in Network v4.6  were used to assess the population configuration in Arlequin v3.5  and four populations in middle reaches (MPG). Estimation of parameters in MIGRATE was done using Bayesian approach Beerli, .D  and the Harpending's raggedness index .Historical demographic expansions were investigated with neutral tests, such as Tajima's  Tajima,  and Fu's, Fs Fu, , and pai, Fs Fu, . Departu3b dataset of 1,009 bp in size was obtained, and a total of 57 polymorphic sites were detected including 14 singleton variable and 43 parsimony informative sites. Among 292 sequences, 66 unique haplotypes were determined. The mean haplotype diversity and nucleotide diversity were 0.893 (range from 0.692 to 0.989) and 0.0072  and p = .93 for the chi‐square test , whereas HapGroup 2 had 32 haplotypes and 89 individuals (30.5% of samples; Table st Table . HaplotyFST = 4.55% (Table FST values further revealed that genetic differences between YHJ and other populations were moderately large and significant. The divergence between XXJ and the four populations in the upper reaches was also significant (Table UPG→MPG = 869.7; MMPG→UPG = 86.8; Table The global AMOVA showed that no significant differentiation occurred among geographical populations with 5% Table . Pairwisnt Table . The migD, and three populations, including XXJ, HapGroup 1, and HapGroup 2, and the total sample had highly significant negative values for Fu's Fs (Table τ values for the HapGroup populations. Based on μ = 0.76% for cyprinid fish Cyt b gene (Zardoya & Doadrio, The local and global neutral test showed that only HapGroup 1 had a significantly negative value for Tajima's b (Figure Eighty‐five COI sequences with 570 bp in size were also obtained and identified to 11 unique haplotypes. Genetic distance among haplotypes ranges from 0.002 to 0.022 Table . The NJ b Figure .44.1b gene sequences have been used to study population genetics of some fishes in the Yangtze River, for example, Zacco platypus (Perdices, Cunha, & Coelho, Leiocassis longirostris (Xiao, Xia, & Ma, Leptobotia elongata (Tian, Duan, Wang, Liu, & Chen, Siniperca chuatsi (Tian et al., Saurogobio dabryi (Li, Tang, Yu, & Liu, G. filifer is currently high. Although the FST test did not detect genetic differentiation among the sampled populations, the pairwise analysis (Table MtDNA Cyt is Table  revealedis Table . Howeveris Table  and had G. filifer to migrate from downstream to upstream, and dam construction has further hindered the upward migration. Therefore, ecological barriers and dam should promote the existence of higher genetic diversity in downstream reaches than in the upper reaches.Two dams were constructed in Yichang City Figure  in 1981 4.2G. filifer coexisting in all eight sampled sites, which reflects a so‐called sympatric population. Genetically different populations of fishes are commonly detected between geographical stocks because of isolation or distant barriers (Yang, Tang, et al., Astraptes fulgerator is the best example (Hebert et al., G. filifer, the greatest genetic distance for K2P was 2.2% between haplotypes 8 and 10 of COI (Table In this study, we found two distinct mtDNA haplotypes of OI Table , which wOI Table . TherefoG. filifer has been examined, and most of its food sources are benthic organisms, such as mosquito larvae, Limnoperna fortunei, and aquatic insects (Wu et al., G. filifer population.Second, sympatric intraspecific divergences in mtDNA could be shaped by long‐term isolation coupled to food niche and/or reproductive separation (Wimberger, Cottus spp. (Nolte, Freyhof, Stemshorn, & Tautz, Poecilia reticulata (Lindholm et al., Sander lucioperca (Eschbach et al., G. filifer. Lineages were usually detected in invaded habitats, and the proportions of population size in each lineage were varied with the scales and plasticity of the introduced population (Eschbach et al., G. filifer.Third, distinct lineages in the same site can also be mixed populations from geographical subpopulations through invasion or introduction. Correlations between admixture genetic lineages and invasion events have been observed in sculpins Pagellus erythrinus in the central Mediterranean Sea (Angiulli, Sola, Ardizzone, Fassatoui, & Rossi, Rhinichthys cataractae in the rivers of southeastern British Columbia, Canada (Ruskey & Taylor, Salmo trutta in two tiny subarctic Swedish lakes (Andersson et al., G. filifer. Global homogeneity implied that the two distinct populations came in contact postglacially at one position and then colonized novel environments synchronously. Population expansions (Table Leiocassis longirostris (Yang, Xiao, et al., Squalidus argentatus (Yang, Tang, et al., Gymnocypris dobulai (Chan, Li, Hu, Liu, & Xu, Parabramis pekinensis (Chen et al., Fourth, divergent populations could have originated from secondary contacts of two distinct glacial refugia. Geographical isolation with secondary contacts would predict a high level of divergence between the mtDNA haplotypes (Grant & Bowen, ns Table  occurredns Table . Many fi4.3G. filifer is likely not the only example of a sympatric population in the Yangtze River.To our knowledge, this is the first report about sympatric genetically populations of fish in the Yangtze River. These fishes are not a cryptic species, but instead represent a secondary contact of distinct glacial refugia. This study highlights that historical and ecological factors play an important role in population patterns for fishes in the Yangtze River. None declared.Wang DQ, Gao L, Tian HW, Dong WW, and Duan XB collected samples. Wang DQ, Dong WW, and Duan XB performed the experiments and analyzed the data. Wang DQ, Liu SP, and Chen DQ prepared the data and wrote the manuscript. All authors read and approved the final manuscript. Click here for additional data file.


Plectranthus edulis (Vatke) Agnew  is one of the most economically important edible tuber crops indigenous to Ethiopia. Evaluating the extent of genetic diversity within and among populations is one of the first and most important steps in breeding and conservation measures. Hence, this study was aimed at evaluating the genetic diversity and population structure of this crop using collections from diverse agro-ecologies in Ethiopia.P. edulis based on EST sequences of P. barbatus deposited in the GenBank. These markers were used for genetic diversity analyses of 287 individual plants representing 12 populations, and a total of 128 alleles were identified across the entire loci and populations. Different parameters were used to estimate the genetic diversity within populations; and gene diversity index (GD) ranged from 0.31 to 0.39 with overall mean of 0.35. Hierarchical analysis of molecular variance (AMOVA) showed significant but low population differentiation with only 3% of the total variation accounted for variation among populations. Likewise, cluster and STRUCTURE analyses did not group the populations into sharply distinct clusters, which could be attributed to historical and contemporary gene flow and the reproductive biology of the crop.Twenty polymorphic expressed sequence tag based simple sequence repeat (EST-SSRs) markers were developed for P. edulis and hence are valuable genetic tools that can be used to evaluate the extent of genetic diversity and population structure of not only P. edulis but also various other species within the Lamiaceae family. Among the 12 populations studied, populations collected from Wenbera, Awi and Wolaita showed a higher genetic diversity as compared to other populations, and hence these areas can be considered as hot spots for in-situ conservation as well as for identification of genotypes that can be used in breeding programs.These newly developed EST-SSR markers are highly polymorphic within The online version of this article (10.1186/s12863-018-0682-z) contains supplementary material, which is available to authorized users. Ethiopia is one of the countries in the world that has the greatest crop genetic diversity and considered as a primary gene center for several crops –3 includPlectranthus edulis (Vatke) Agnew (Lamiaceae), also locally known as Ethiopian potato syno. Ethiopian dinich, is one of the most economically important indigenous tuber crops . A mixture of all the components except genomic DNA was included as a negative control. Amplification was performed using GeneAMP PCR 9700 thermocycler (Applied Biosystems Inc. USA) according to the following five-stage PCR protocol: (1) initial 15 min denaturation at 95 °C, (2) 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at optimized annealing temperature for each primer-pair  and pairwise FST were computed using GenAlEx ver. 6.501 [Peak identification and fragment sizing were done using GeneMarker V2.6.0 (SoftGenetics®) with default settings and 200 threshold intensities. Then, the allele size (bp) data at each locus were exported to excel for statistical analyses. Locus based diversity indices: major allele frequency (MAF), the number of alleles (NA), gene diversity (GD), and polymorphic information content (PIC) were determined using PowerMarker ver. 3.25  Table 3. To deter. 6.501 , and sigST, corrected) [Simple matching dissimilarity coefficient-based Neighbor-Joining tree and Nei’s standard genetic distance (Drrected)  based Unrrected)  tree wasrrected)  and POPTrrected) , respectrrected) . The resrrected)  and FigTrrected) . Gene flrrected) .A Bayesian model-based clustering algorithm in STRUCTURE ver. 2.3.4 , 44 was The 20 newly developed EST-SSRs markers Table , out of The maximum number of alleles detected per locus was 12  including richness in private alleles (Arp) with Ar of 3.33 and Arp of 0.28  and index of clonality (G/N) over the entire samples (populations) was 13.08 and 0.55, respectively , observed heterozygosity (Ho) and expected heterozygosity (He), gene diversity (GD) and Shannon diversity index (I). However, 28 Table . Jim andly Table . The analy Table .ST value was very small (0.03)  and geographical regions (GR). In all cases, variation within individuals accounted for at least 97% of the total variation. The variation among populations, AR and GR accounted for only 3, 2 and 2% of the total variation, respectively. Hence, most of the within population variation is due to the heterozygosity of the individuals within each population. The overall FAw vs WS, Wen vs HKT, WS and YeL). The mean genetic distance of each population from the other populations ranged from 0.02 to 0.04 and, in terms of this parameter, Wen is the most distantly related population with a mean genetic distance of 0.04 (Table ST) [Wen and YeL populations.Nei’s standard genetic distance between populations ranged from 0.02 to 0.05. The highest pairwise genetic distance (0.05) was observed between four pairs of populations (04 Table . Similarable ST) , 48 rangThe neighbor-joining based cluster analysis of 60 individual samples randomly selected across the 12 populations (five samples per population) resulted in four major clusters C1, C2, C3, and C4) with the first three clusters further divided into two sub-clusters. Each of the four clusters comprised individual plants from different collection zones (geographic regions). However, samples are more or less grouped according to their geographic region of origin at sub cluster levels (designated as i and ii on each major cluster) although there is considerable intermixes  containing SSRs. This proportion suggest that EST-SSRs are less abundant in P. barbatus than in Salvia miltiorrhiza (14.7%) [Lavandula species (18.8%) [P. barbatus than in Mentha piperita (8.4%) [P. barbatus is higher than their abundance in cereal crops such as rice (4.7%), sorghum (3.6%), barley (3.4%) and maize (1.4%) [s-genera  and cross-genera  transfer (14.7%)  and Lava (18.8%) , and slia (8.4%) , which ae (1.4%) . Howevere (1.4%) .Plectranthus [P. edulis has not been reported and, hence, further in-depth cytogenetic analysis is important to confirm the finding of this study.A maximum of two alleles are expected per individual plant at single copy microsatellite loci in diploid species. In the present study, a maximum of two alleles per plant were detected at each of the 20 loci, indicating that they are all single copy and have disomic inheritance regardless of the thus far reported basic chromosome number and ploidy level for several members of the genus tranthus . HoweverThe EST-SSR markers developed in the present study contained di-, tri- penta- and hexanucleotide repeats. Studies have shown that di, tri and tetra-nucleotide repeat SSRs are the most commonly used motifs in molecular genetic studies . Tri-nucP. edulis genetic materials for the association of particular alleles with traits of interest and for conservation. Such alleles are useful in comparing diversity between species or populations [The use of molecular markers for efficient selection of genotypes with desirable traits and enhancing the efficiency of breeding by allowing effective simultaneous selection of various desirable traits is a well-established approach , 65. Henulations  and alsoulations .M. piperita (61%), which shares the same family with P. edulis. However, it was similar with that reported for 28 alfalfa accessions (97%) [Opium poppy accessions (96%) [P. edulis genetic resources for efficient conservation and breeding.The average percent polymorphism per population revealed in the present study across the 20 loci (94%) is by far greater than the level reported by Kumar et al.  for 13 ans (97%)  and 37 Ons (96%) . Such hins (96%) , and theThe loci studied displayed differences between Ho and He in which half of them showed excess heterozygosity that led to a significant departure from HWE across populations. Such excess heterozygosity is expected in historically outcrossing species that maintain their heterozygosity through vegetative propagation, or if other factors such as natural and artificial selection pressure favor heterozygosity. Similar results have been reported in sweet potato , 72.P. edulis by other crops. In addition, farmers’ preferences for selected traits of economic importance such as tuber size, tuber skin color, tuber texture, maturity etc. and asexual mode of reproduction of the crop , which was evidenced from the considerably higher global clonality index (G/N), could have contributed to limited genetic variation in P. edulis populations. There are similar reports on potato cultivars from Yunnan province, China [Higher genetic diversity is expected in larger and older populations when compared with small and newly established ones because of higher levels of accumulated and maintained genetic variation  which ise, China .Wen, WS, and Aw populations are genetically more diverse than the other populations as estimated by parameters such as gene diversity, heterozygosity and Shannon diversity index and hence the areas representing these populations could be considered as genetic diversity hot spots and a potential in-situ conservation sites for P. edulis. Among the populations, EW has the least genetic diversity, which might suggest current rapid genetic erosion from the area (population bottleneck) or intensive artificial selection pressure to maximize tuber yield. In terms of allelic richness, Gur, Jim, WS and WSh populations are the top four in that order, and hence are more interesting in terms of genetic and evolutionary studies on this crop because allelic richness is more informative in this regard as it is sensitive to the presence of rare alleles [WS bear a relatively high proportion of private alleles which may indicate certain level of independent evolution of their gene pools that allowed maintenance of private alleles at a population level [ alleles   [ST averaged across all loci (FST = 0.03) and pairwise FST for all pairs of populations  was generally low to moderate on the bases of Wright [ST is greater than 0.25. This could be partly attained if gene flow, which is a powerful force to decrease differentiation among populations, is low (Nm < 1) [P. edulis has very little population sub-structuring. The low population differentiation is supported by high gene flow (mean Nm = 18.29) owing to step-wise pollen movement across populations, germplasm exchange in the form of tubers and seeds through sharing common markets among several of the adjacent areas where different populations were collected. This study also showed the minimal effects of regions or geographic origins of populations on genetic variation in P. edulis. This could be partly explained by the extensive exchange of tubers as planting materials among farmers (gene flow), common origin of the populations, the clonally propagating nature of the crop in which only a limited number of individuals contribute tubers to the next generation, which gradually leads to recent or old population bottlenecks and hence facilitate genetic drift.AMOVA revealed that ricosum) , 79, as f Wright  and Hartf Wright  suggestif Wright  indicate(Nm < 1) . Hence, ST value among pairs of populations (ranging from 0.01 to 0.07). The highest population differentiation was observed between Wen and HKT, Wen and WS, Wen and YeL as well as between Aw and WS populations. Wen population showed the highest (0.05) pairwise Nei’s standard genetic distance with WS and HKT populations and is the most genetically distinct population with a mean Nei’s standard genetic distance 0.04 Additional file 2:Allele frequency distribution and overall percentage of rare alleles (f ≤ 0.01) across populations. (XLSX 9 kb)Additional file 3:Estimates of the overall Nei’s heterozygosity, population differentiation measures and proportion of progenies produced by selfing. (DOCX 18 kb)


Venturia inaequalis Cooke (Wint.) is one the important diseases of trade and industrial significance in apple. In present study variability studies in pathogen isolates were studied, which is one of the most important factors for devising management studies of scab disease in apple. Genetic diversity of 30 Venturia inaequalis isolates from 12 districts of two geographical distinct regions of Jammu and Kashmir was calculated based on the allele frequencies of 28 SSR markers and the internal transcribed spacer (ITS) region of the ribosomal DNA. The ITS based characterized sequences were submitted to NCBI GenBank and accession numbers were sanctioned. Dendrogram showed that all the accessions formed 2 main clusters with various degree of sub clustering within the clusters. Analysis based on SSR study reveals that the heterozygosity ranged from 0.0 and 0.5, with an average value of 0.39. The expected heterozygosis or gene diversity (He) ranged from 0.0 to 0.50 with an average of 0.40. The Fst value ranges from 0 to 0.6 with an average of 0.194. Diversity within each population (HS) values ranging from 0.26 to 0.33. Average differentiation among populations (GST) was 0.11 and populations were isolated by significant distance . From the AMOVA analysis, 25% of variation was observed among population, 9% among individuals and 66% within individuals observed in the population. Structure analysis grouped isolates into two populations. Principle coordinate analysis explained variation of 36.6% in population 1, 14.30% in population 2 and 13.10% in population 3(Admixture) with 64.07% as overall cumulative percentage of variation. This indicates that extensive short-distance gene flow occurs in Kashmir region that dispersal over longer distances also appears to occur frequently enough to prevent differentiation due to genetic drift. Also it is evident that Jammu and Kashmir most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the Jammu region compared to the mountainous inland Kashmir region. The results of present study would help to understand the genetic diversity of V. inaequalis from Jammu and Kashmir that would lead in the development of more effective management strategies and development of new resistant cultivars through marker-assisted selection.Apple scab caused by  Malus × domestica Borkh.) is most widely and commercially cultivated species in the genus Malus throughout temperate regions of world[Venturia inaequalis Cooke (Wint.) whichis an ascomycetous heterothallic and hemibiotrophicfungus. Earlier, this genus was included in the family Venturiaceae, order Pleosporales, according to its “Pleospora-type centrum and bitunicateasci” [Venturia inaequalis (Cke) and parasitic [Apple . It overapomi Fr). In earlapomi Fr).Venturia inaequalis population[Venturia inaequalis in numerousapple growing districts of Jammu and Kashmir was carried out.One sexual and multiple asexual cycles, of this pathogen annually causes noteworthy variations in opulation. Recombiopulation. In deviopulation. Detaileopulation and alsoopulation. In wideopulation. As rRNAopulation. Besidesopulation. As per opulation. KeepingSamples were collected from 12 apple growing districts of Jammu and Kashmir India during the year 2017–18 as shown in The fungus from the infected samples was isolated and purified using monoconidial method by streaking out spores on plates containing 2% water agar, pure fungal cultures were obtained by transferring single germinated conidium on potato dextrose agarcontaining antibacterial chloromphenicol (50 μg/ ml) to avoid bacterial contaminations. Total 3Cultured fungal isolates in 100ml of potato dextrose broth was kept in incubator-shaker at 19°C for about 25–30 days under continuous dark. The mycelia harvested was blotted dry between the tissue layers and immediately frozen in liquid nitrogen. After freeze-drying, DNA was extracted using Fungal DNA isolation kit (GCC Biotech India Pvt. Ltd). The DNA was quantitatively and qualitatively checked using a Nanodropspectrophotometer (Themoscientific) and was further diluted to a workingconcentration of 30ng/μl and stored at -20°C for further use.2. The PCR was normalized after repetitive cycles till optimal amplification was achieved and consists of 35 cycles involving initial denaturation step at 94°C for 5 min, followed by 94°C for 30 s, annealing at 53°C for 45 s, extension at 72°C for 1min and final extension at 72°C for 15min[All 30 isolates were amplified using polymerase chain reaction (PCR) in a thermal cycler (Takara Japan) using 30ng of genomic DNA in a final volume of 25 μl per reaction. The universal ITS primers with ITS 1 as forward and ITS4 as reverse primerwere used for PCR amplification . ThePCR was performed in a 0.2-ml tube containing 0.5μM forward and reverse primer, 200 μM eachdNTP, 1 unit kappa Taqpolymerase and 1ul of genomic DNA in a 10xkappa buffer and 5 mM MgClfor 15min. The PCRfor 15min. The fraTaq DNA polymerase (kappa), 1μM of each primer and 1μl DNA template [For diversity and structure analysis of selected fungal samples, 28 published SSR primer pairs were used template . The amphttp://ncbi.nlm.nih.gov/BLAST). The sequence generated from the present study and reference strain sequences retrieved from GenBank were used to construct phylogeny by neighbour joining method with 1000 replications for each bootstrap value using MEGA 7.0 software version [Venturia pirina and nashicola were also included in phylogeny to separate Venturia inaequalis from these species. For validation of results, an out group non-fungal pathogen Pseudomonas syringae was selected.Amplified PCR products were sequenced at Agri Genome Labs . Primers for the sequencing PCR product were the same as for the PCR amplification. The sequences of PCR products were assembled using DNA baser V.4 program to produce complete contig. These were further aligned using CLUSTAL W method of Bio-Edit software and aligned sequences were deposited in NCBI GenBank. A database search of homologous sequences was performed by BLAST analysis at NCBI , and PCoA. , 27. TheVenturia inaequalis.Identification based on morphological characters from fungal culture  revealedVenturia inaequalis GenBank submitted sequences. Sequences were submitted to NCBI GenBank and accession numbers were received  in phylogeny , cluster IIcontained 18 isolates (M1to M18) and Cluster IIIincluded only 1 isolate (M19). Both The Cluster I and II were further subclustered into two subclusters  were ascertained by choosing higher ΔK value, with respect to the number of clusters inferred by Structure [Structure analysis revealed that isolates of tructure . As per is for all the 28 marker loci was 0.126, while Fit was 0.343 across the clusters. Pair wise Fst values showed significant differentiationamong all the pairs of sub-populations ranging from 0.248 to 0.881 suggesting that all the threegroups were significantly different from each other. The Fst values and their distribution pattern show clear differentiation of sub populations from each other. This result was also validated by the principal coordinate analysis (PCoA), ,  where coV. inaequalis. The molecular characterization elucidates the genetic diversity among the isolates and for better resistance against any pathogen, the diversity must be known and accordingly resistant varieties can be developed. Hence in present study the molecular characterization of V. inaequalis was undertaken in order to provide better management strategies for this disease in the form of resistance and cisgenic breeding approaches.Apples are grown in high altitude areas of India particularly in J&K. It is tVenturia sequences , two clades emerged: one with all the V. inaequalis sequences and another with the V. pirina and V. nashicola sequences that further separated into two subclades. Bilal et al[V. inaequalis isolates, which can help to identify characteristics such as resistance or susceptibility toward a particular anti-fungal agent.We used ITS ribotyping of 30 isolates collected from two distinct regions. The noncoding ribosomal DNA ITS sequences doesn’t change more rapidly than the coding sequences and may diverge between species and populations . Analyzilal et al had simiV. inaequalis.Microsatellites or SSRs are very useful markers for population genetics analysis because of their high specificity, polymorphism, and reproducibility. These are major advantages of using SSR markers over Random Amplification of Polymorphic DNA. The SSR markers used in this study were highly variable and results generally corresponded with previous population genetics studies conducted in Europe. However, in this study the markers1tc1a, 1tc1b, 1tc1g, 1aac3b, 1aac4b, 1aac4f, and 1aac4h showed only one allele compared to eight to ten alleles reported previously, 42. TheV. inaequalis isolates from geographically and topologically distinct regions. We observed a high level of diversity, which could be expected due to the climate differences. Xu et al[V. inaequalis isolates obtained from dissimilar apple cultivars in a solo apple orchard. This was credited to assortment pressure applied by diverse cultivars. Another possible explanation of variation in diversity is sexual recombination.The distance between the Jammu region and the Kashmir region where we collected samples is approximately 400 km and allowed us to collect  Xu et alalso obseV. inaequalis populations in J&K. Asmost of the apple cultivation area in Jammu and Kashmir is dominated by single cultivar Red Delicious and no resistant variety is under cultivation yet, so there is no selection pressure on pathogen to bring some change, hence this could be the reason having high percentage of identical allelesamong V. inaequalis populations. The movement of the V. inaequalis from one place to other can be through planting material, high speed winds, but the planting material was one of the most important factors of introduction of V. inaequalis to India. This was also observed in the magnitude of migration between the regions. The Kashmir region seems to have the highest migration rate towards it and the lowest away from it. Migration towards the Jammu region was the lowest, indicating that this region is isolated, probably due to warmer winter temperatures. Overall the migration results indicated the possible free movement of the pathogen between the regions. The present study undoubtedly shows that there is high diversity of V. inaequalis in the Kashmir valley and reveals that the larger part of variability existed within the individuals.The isolates in this study shared a high percentage of identical alleles, indicating considerable gene flow among all isolates of V. inaequalis isolates. Pair-wise Fst values, ranging from 0.248 to 0.881 showed noteworthy demarcation between the subpopulations. This signifies that the two groups are notably dissimilar from one another. This outcome is also validated by the principal coordinate analysis, whichdistributed the isolates into two major populations with one admixture (two isolates). The admixture is of smallsize, hence can’t be considered as a population.Structure analyses divided the isolates into two populations (K = 2) with a clear differentiation between the two apple-growing regions. Pathogen distance seems to be the most significant factor in steep gene flow because it explains 50% of the variation among the V. inaequalis from different apple growing regions in Jammu and Kashmir shows significant levels of genetic variation within the populations in the similar fashion as observed in other V. inaequalis population’s studies conducted in Europe and elsewhere. Results indicated that gene flow between regions is occurring and has significant implications for the apple industry if fungicide resistant strains move between regions. Based on ITS sequencing, a database can be maintained to list out the sequence-based isolation of various fungal strains or species. In order to control such menace caused by scab, proper prediction and forecasting systems are need of the hour to prevent apple scab disease well in advance and also understanding the host pathogen interaction, which can provide new insights for effective management of this disease. Cisgenesis can be one of the approaches for introgression of resistance gene through biotechnological intervention under control of its own regulatory sequences from same species or related species which can also maintain the original cultivar characteristics.The genetic variation and population structure of scab causing S1 Table(DOCX)Click here for additional data file.


Papilio polytes, that exhibits mimetic polymorphism on multiple islands of the Ryukyus, Japan, and performed field surveys and genetic analysis. We found that the mimic ratio of P. polytes was strongly correlated with the model abundance observed on each of the five islands, suggesting negative frequency‐dependent selection is driving the evolution of polymorphism in P. polytes populations. Molecular phylogenetic analysis indicated that the southern island populations are the major source of genetic diversity, and the middle and northern island populations arose by relatively recent migration. This view was also supported by mismatch distribution and Tajima's D analyses, suggesting a recent population expansion on the middle and northern islands, and stable population persistence on the southern islands. The frequency of the mimetic forms within P. polytes populations is thus explained by variations in the model abundance rather than by population structure. Thus, we propose that predation pressure, rather than neutral forces, have shaped the Batesian mimicry polymorphism in P. polytes observed in the Ryukyus.Batesian mimicry is a well‐studied adaptation for predation avoidance, in which a mimetic species resembles an unpalatable model species. Batesian mimicry can be under positive selection because of the protection gained against predators, due to resemblance to unpalatable model species. However, in some mimetic species, nonmimetic individuals are present in populations, despite the benefits of mimicry. The mechanism for evolution of such mimetic polymorphism remains an open question. Here, we address the hypothesis that the abundance of mimics is limited by that of the models, leading to mimetic polymorphism. In addition, other forces such as the effects of common ancestry and/or isolation by distance may explain this phenomenon. To investigate this question, we focused on the butterfly,  In other words, the mimic and nonmimic types have equal fitness independent of their frequency, and therefore, their gene frequencies in local populations change through genetic drift and migration , southern Japan: Kikai (abbreviated as KIK), Okinawa (OKI), Miyako (MYK), Ishigaki (ISG), Taketomi (TKT), Amami (AMM), Aguni (AGN), and Tarama (TRM) , sex, and mimic type were recorded, live all specimens were stored at −30°C in a freezer until DNA extraction  Figure . The buton Table , enablin7 Tables  and S2. ed Table .2.2Whole middle and hind legs of the butterflies were used for DNA extraction with a DNeasy Blood and Tissue Kit . Samples were gently cut with hand shears and manually homogenized using disposable pestles in 180 µl of buffer ATL. The tissue homogenates were digested overnight at 55°C with 20 µl of Qiagen Proteinase K solution (600 mAU/ml) and subjected to DNA extraction. Total DNA was eluted from the spin column in 200 µl of buffer AE and stored at −30°C after confirmation of the concentration and quality using a Nanodrop 2000C (Thermo Scientific).2.3P. machaon (GenBank accession number: HM243594), P. helenus (KM244656), P. maackii (KC433408), P. syfanius (KJ396621), and Troides aeacus (EU625344) were used to amplify P. polytes mtDNA (GenBank accession number: NC_024742) . This approach prevented amplification of nonpreferred sequences and contamination by short nuclear pseudogenes of mitochondrial origin, which often produce misleading results in evolutionary analyses  Table . PrimeSTThe PCR products were used to prepare sequencing libraries with a Nextera XT DNA Library Prep kit, which uses a transposome‐based approach, and an Index kit v2 (Illumina). To improve the fidelity of library amplification, the Nextera PCR master mix (NPM) was replaced with the same volume of KAPA HiFi HotStart ReadyMix (Kapa Biosystems). The amplified libraries, containing sequences ranging in length from 300 to 800 bp, were dissected from 1.0% L03 agarose gels (Takara) using a MinElute gel extraction kit (Qiagen), quantified by a Qubit fluorometer with a dsDNA high‐sensitivity assay kit (Thermo Scientific), and sequenced using a MiSeq Reagent Kit v2 (Illumina) to generate 2 × 250‐bp paired‐end reads (run numbers 1 to 3) and 2 × 150‐bp paired‐end reads (run numbers 4 to 8) (see Table 2.4P. polytes mtDNA amplicons (DRA accession numbers: DRA006999 and DRA008115) were subjected to primary data processing. The overall data quality was evaluated by FastQC  using a custom Perl script.Quality‐filtered sequences were analyzed to estimate the haplotype sequence of the subject mtDNAs using the software described below for short read mapping and variant calling. Sequence reads were aligned to the reference mtDNA sequence of 2.5P. polytes in the Ryukyu Islands, we performed molecular phylogenetic and population genetic analyses. The mtDNA sequences described above were aligned with MAFFT  among islands was calculated based on AD divided by the average number of pairwise differences among islands.To address the demographic history and evolution of Batesian mimicry of D‐tests , 7.5 × 10−9 (intermediate), and 9.5 × 10−9 (fast) substitutions per site and year.Population genetic analyses, including Tajima's  Tajima, , 1989b a Tajima, , were pe Tajima,  based on Tajima, , 1989b.  Tajima, , and a s Tajima, . The val2.6P. polytes were defined as follows:The MR and the advantage index (AI) of Batesian mimicry of P. polytes females” from the denominator because the nonmimic females  of P. polytes do not possess warning signals for avian predators and thus do not affect the mimicry advantage.The definition of MR above is identical to relative abundance (RA) as defined by Sekimura, Fujihashi, and Takechi . The def2.7AD, geographic distance, and difference in MR among islands were assessed by Mantel test . We obtained mtDNA sequence for the Cytochrome c oxidase subunit 1 (COI) gene and the neighboring region  for 137 individuals. In addition, for 70 of the 137 individuals, mtDNA sequence was also obtained for the Cytochrome c oxidase subunit 3 (COIII) gene and the neighboring region (442 bp) and the Cytochrome b (Cyt b) gene and the neighboring regions (568 bp). For a summary of the regions used, see Tables We obtained a total of 2,823,806 reads , an average of 20,612 ± 35,667 3.2P. polytes consistently indicated two major clades strongly associated with the geographic distribution  implied similar relationships with those depicted by the heatmap. The populations were separated into two distinct clusters concordant with their geographic distribution: southern island populations (ISG and TKT) and northern island populations   was detected among the populations of these five islands, except for two pairs (ISG–TKT and MYK–KIK) based on 1,273 bp of the COI gene and the neighboring region (Table FST) based on 1,273 bp of the COI gene and the neighboring region was not detected among these islands .Heatmap visualization of pairwise genetic distances calculated from 1,273 bp of the n Figure a,b showen Figure a,b, whern Figure a,b. A UP) Figure c. Modest3.3P. polytes populations on the five islands of the Ryukyus . The analyses were conducted using the haplotype data for the COI gene and the neighboring region  from 130 individuals from all five islands . Mismatch distribution analysis showed that the northern and middle island populations  had unimodal distributions, which imply a recent population expansion or rapid turnover , geographic distance, and MR differences among P. polytes populations, no significant association was detected among the five islands of the Ryukyu Islands  in the Ryukyu Islands for several decades.Regarding genetic distance (4P. polytes populations the frequency of the Batesian mimicry morph is limited by the abundance of the model species (P. aristolochiae) and investigated populations of these two butterflies on eight of the Ryukyu Islands, Japan  in the wild or in field surveys; however, our preliminary data for beak marks on P. polytes indicate predation by birds. The strength of the predation pressure can be estimated from beak marks . Furthermore, Katoh et al.  population of  D Table  estimate D Table . On MYK,ts Table . This viP. polytes populations examined in this study. Restriction site‐associated DNA sequencing (RAD‐seq), multiplexed intersimple sequence repeat genotyping by sequencing (MIG‐seq)  gene could provide a useful perspective, because the dsx gene is thought to be responsible for mimicry polymorphism in P. polytes (Kunte et al., P. polytes mimicry in the Ryukyu Islands. Molecular analysis to detect the signatures of selection in the dsx gene would also be worthy of future study.In future works, massive and comprehensive SNP analysis of the nuclear genome could be used to confirm the evolutionary history of the None declared.KT and KT‐S designed the study. KT‐S, YS, MK, and EK performed the research. YS, KT‐S, EK, and HT analyzed data. All authors contributed to writing the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Caretta caretta) sampled from the southeastern coast of the United States, including 42 individuals experimentally confirmed for gonadal sex. By performing reference-based SNP calling in independent runs of Stacks, 3,901–6,998 SNPs and up to 30 potentially sex-specific genotypes were identified. Up to 68 pairs of loci were found to be in complete linkage disequilibrium, potentially indicating regions of natural selection and adaptive evolution. This study provides a valuable SNP diagnostic workflow and a large body of new biomarkers for guiding targeted studies of sea turtle genome evolution and for managing legally protected nonmodel iconic species that have high economic and ecological importance but limited genomic resources.In the era of genomics, single-nucleotide polymorphisms (SNPs) have become a preferred molecular marker to study signatures of selection and population structure and to enable improved population monitoring and conservation of vulnerable populations. We apply a SNP calling pipeline to assess population differentiation, visualize linkage disequilibrium, and identify loci with sex-specific genotypes of 45 loggerhead sea turtles ( Caretta caretta) are found in the Atlantic, Pacific, and Indian Oceans, with major loggerhead rookeries spanning from southwest Florida to North Carolina . Recent research indicates that sea turtle populations may be at further risk due to increased proportion of females attributed to temperature-dependent sex determination (TSD) and rising global temperatures  technologies have revolutionized the detection of genetic variation and signatures of selection within populations . Single-C.caretta) sampled from the southeastern United States in compliance with authorized federal and state agency permitting and established tissue collection operations. To describe global and loci-specific signatures of natural selection, we use GBS to call thousands of high-quality SNPs, estimate population structure and linkage disequilibrium, identify potential sex-specific loci, and functionally annotate identified SNPs. Other endangered populations for which there are limited genomic resources can benefit from the outlined procedure that facilitates understanding of the adaptive potential of populations and can aid future conservation efforts . Florida Bay turtles were captured from the Everglades National Park in June 2009 for the National Park Service marine turtle inventory . Sex was determined by testosterone radioimmunoassay  and immediately frozen at −20 °C . To extrwww.clemson.edu/centers-institutes/itg; last accessed September 12, 2019) according to the procedure developed for high diversity maize genomes by ApeKI. We designed barcodes of variable length for multiplex sequencing .GBS libraries were prepared in cooperation with the Clemson University Genome Institute (quencing  online. Stacks (version 2.2)  and Chrysemys picta bellii  genome assemblies using BWA-MEM and performed SNP calling via the gstacks program while requiring base call accuracy to be >95%   and Florida Bay C. caretta (FB). SNP calls were filtered to require that a locus be present in a minimum of 75% of individuals within a group (-r 0.75), have a minor allele frequency of at least 0.01 (–min_maf 0.01), and have a heterozygosity <0.70 (–max_obs_het 0.70) to reduce paralogs.To demultiplex and clean raw reads, we used the script “process_radtags.pl” from the program ion 2.2) , 2013. Fha 0.05) . The thrP values <0.001 (-hwcutoff 0.001), excluded individuals with more than 0.80 missing data (-missingCutoff 0.80), and noted the distance (in basepairs) between loci and the number of pairs of loci that display an r-squared value equal to 1, D′ > 0, or significantly associated loci pairs with log odds ratio (LOD) > 2. Due to the possibility of strong linkage disequilibrium existing simply due to short distance between loci pairs, we consider loci pairs at a minimum distance of 250 basepairs to better infer the potential effects of selection. The populations parameter option –write_single_snp was enabled for STRUCTURE (version 2.3.4) analysis to select only the first SNP in a locus to ensure SNPs are unlinked. The program STRUCTURE was used to describe population structure with estimated number of clusters (K) varied from 2 to 5 with burnin length of 10,000 and 20,000 iterations    and estierations , and admophelper . Optimal6, 2018)  with Chrin files .process_radtags” filtering  and SNPs  called compared with Chr. picta reference-based method with consideration of sea turtles as either a single group or two separate groups for population genetics analyses  among AC and FB groups reveal that population differentiation is limited and ranges from 0.0137 (Ch. mydas as reference) to 0.0141 (Chr. picta as reference), differing significantly according to Wilcoxon signed rank tests. Observed homozygosity at variant sites is relatively similar in FB and AC loggerheads, and nucleotide diversity (π) , nonzero inbreeding coefficients (FIS) at variant positions and “all” (variant and nonvariant) positions, indicating a greater excess of homozygotic calls found when considering AC and FB loggerheads as separate groups rather than a single group. Nucleotide diversity is decreased and observed homozygosity is increased for calls produced without distinguishing AC and FB individuals into two groups.le group . Fixatiosity (π)  tends tosity (π)  online. r2 = 1), display D′ > 0, or were significantly associated with a log odds score (LOD) of at least 2  and 30 (Ch. mydas as a reference) loci with sex-specific genotypes according to Fisher’s exact test (q-value <0.05, Hundreds of pairs of loci were found to be in complete linkage disequilibrium ( least 2  online. K from 2 to 5 with 10 replicates per K and observed the admixture proportion of each individual (K determined via the Evanno method indicated that the optimal K using either Ch. mydas or Chr. picta as a reference with consideration of AC and FB individuals as separate or a single group was K = 2. Using Chr. picta or Ch. mydas as a reference, at K = 2, most individuals show more than 80% admixture from a single cluster. Most AC and FB individuals show similar patterns of admixture, again indicating detection of relatively little population differentiation. As K increases, admixture plots using SNP calls that had separately grouped AC and FB individuals indicate that almost all individuals can be assigned to one of K clusters. Additionally, multidimensional scaling reveals clustering and decreased genetic distance among FB individuals relative to AC individuals (To describe population structure in loggerheads, we varied the number of clusters or populations for dividual . With coividuals  online.Cheloniamydas, the green sea turtle, is more closely related to loggerhead sea turtles than is Chr. picta, the western painted turtle, and the use of Ch. mydas as a reference identifies 1,117–1,764 more SNPs than using Chr. picta as a reference (FST estimates are independent of coverage. Using Ch. mydas as a reference and with consideration of AC and FB loggerhead sea turtles as either a single group or two groups during population genetics analyses, 20,666–119,653 loci and 3,901–6,998 SNPs were identified, in comparison to previous studies which have identified <100 haplotypes and have relied on fewer than 50 microsatellite markers to study loggerhead populations (We describe the application of a SNP calling pipeline to Atlantic loggerhead sea turtles that identifies loci in linkage disequilibrium and potentially sex-specific loci, calculates population genetics statistics, and assesses population structure. Reference-based SNP discovery has the potential to identify more SNPs than de novo methods; greater relatedness of the reference to the focal species tends to result in a greater number of SNPs called . Chelonieference . The useulations .Stacks populations analyses indicate slightly lower genetic diversity and nucleotide diversity in FB compared with AC loggerheads, but it is likely that the sampled individuals are members of several breeding populations in an overlapping geographic region (Average population genetics statistics are comparable to previous studies of sea turtle population genetics that used nuclear and mitochondrial DNA haplotype-based analyses  online , 2014. Gc region . The admc region , there ic region , 2013. RLevels of linkage disequilibrium are affected by natural selection, genetic drift, gene flow, and changes in population size . LinkageCh. mydas as a reference for SNP calling over Chr. picta argues for the practical value of establishing genome assembly resources for target species of conservation priority over less informative de novo approaches to biodiversity studies.The development of new molecular markers is necessary for future population genomics analysis of sea turtles . In thisManagement strategies for sea turtle populations should maintain large population sizes by preserving natural habitats to facilitate outbreeding and prevent inbreeding depression . AttentiGenome Biology and Evolution online.evz190_Supplementary_DataClick here for additional data file.