Drosophila subobscura. However, there is a lack of knowledge on the genetic variation in genes within inversions, possibly targets of climatic selection, across a geographic latitudinal gradient. In the present study we analysed four candidate loci for thermal adaptation, located close to the breakpoints, in two chromosomal arrangements of the sex (A) chromosome of Drosophila subobscura with different thermal preferences. Individual chromosomes with A2 (the inverted arrangement considered warm adapted) or AST  were sequenced across four European localities at varying latitudes, up to ~ 2500 Kms apart.The role of chromosomal arrangements in adaptation is supported by the repeatable clinal variation in inversion frequencies across continents in colonizing species such as ST orientation but not in the distal ones, independently of their location inside or outside the inversion. This can be possibly due to variation in the levels of gene flux and/or selection acting in these regions.Importantly, we found very low differentiation for each specific arrangement across populations as well as no clinal patterns of genomic variation. This suggests wide gene exchange along the cline. Differentiation between the sex chromosome arrangements was significant in the two more proximal regions relative to the AGene flow appears to have homogenized the genetic content within-arrangement at a wide geographical scale, despite the expected diverse selective pressures in the specific natural environments of the different populations sampled. It is thus likely that the inversion frequency clines in this species are being maintained by local adaptation in face of gene flow. The differences between arrangements at non-coding regions might be associated with the previously observed differential gene expression in different thermal regimes. Higher resolution genomic scans for individual chromosomal arrangements performed over a large environmental gradient are needed to find the targets of selection and further elucidate the adaptive mechanisms maintaining chromosomal inversion polymorphisms.The online version of this article (10.1186/s12862-018-1178-1) contains supplementary material, which is available to authorized users. There is increasing evidence of the role of chromosomal inversion polymorphisms in adaptation and speciation , 2. SeveSeveral hypotheses have been advanced to explain the adaptive character of inversions (see ), with tD. subobscura found an extensive genetic differentiation between arrangements occurring regardless of the distance to the nearest breakpoint, with selection likely implicated [In(3R)Payne of Drosophila melanogaster, both in North American and Australian localities, implicating a role of this inversion in the response to climatic selection [Empirical studies on the molecular variation associated with inversions have frequently detected high levels of genetic differentiation between chromosomal arrangements ; with an enrichment for candidate genes located inside arrangements [D. pseudoobscura with multiple genes presenting expression differences among arrangements [D. subobscura inversions as well as address their variation across the species geographic gradient to search for targets of climatic selection.One of the most emblematic examples of the adaptive role of chromosomal polymorphisms comes from  America . Thermalde scale . Also, sat waves  as well at waves –38 and bat waves , 39. Impngements . This sungements . ConsequD. subobscura and found no differentiation for the same arrangement between Barcelona and Greece indicating high levels of gene flow and the absence of geographic varying selection at least in those two southern regions. In a study in D. subobscura involving several localities across western Europe, Simões et al. [2), suggesting adaptation at neighbouring loci.Pegueroles et al.  analyseds et al.  using mis et al. , 43). NeDrosophila subobscura across a geographic gradient. Specifically, we intend to (1) characterize genetic variation and differentiation between arrangements, addressing the impact of inverted regions at the genetic level; (2) analyse patterns of within-arrangement differentiation, searching for evidence of different epistatic combinations in different populations; (3) identify latitudinal patterns of genetic variation, which might indicate targets of climatic selection at a geographical scale. In this study we targeted AST and A2, the two most frequent inversions of the A chromosome in our sampled localities. These two arrangements present clinal variation of frequencies in Europe, as well as in the two recently colonized American hemispheres, with A2 increasing frequency towards warmer locations and AST showing the corresponding opposite pattern [PhKgamma, Ubc-E2H, Hfw and Nipsnap). These genes are good candidates to search for evidences of climatic latitudinal selection associated with the Drosophila subobscura inversion clines as they (1) have been previously implicated in thermal adaptation , and the only two arrangements present in all analysed locations  with an estimated age of 160.000 years [2   chromosome . This p2 or AST) had been previously scored as described above. By amplifying and directly sequencing males we avoided the problems associated with sequencing of diploid data. The karyotype of these males was inferred from their descendants (see above) making it possible to obtain both genetic and karyotypic data for the same individual. Genomic DNA extraction was performed following the protocol described in [Nipsnap that was localized nearby but outside the inversion  and Nipsnap  are located outside the A2 inversion, and Ubc-E2H  and Hfw  within that inversion  is involved in lateral inhibition and protein polyubiquitination, PhKgamma (Phosphorylase kynase enzyme) is involved in embryonic morphogenesis and glycogen metabolism and Hfw  is involved in intracellular signal transduction and pupariation and no involvement in biological processes is yet ascribed to the Nipsnap gene although a putative role in vesicular transport has been proposed (the function of each gene is according to Flybase). Primers used for amplification and sequencing reactions are listed in Table PhKGamma and around 0.574 Mb for the other 3 genes  whose chromosomal arrangements , number of polymorphic sites (S), the nucleotide diversity (π), nucleotide diversity in synonymous sites and non-coding positions (πsil), heterozygosity in silent sites (ϴsil) and divergence per silent site (Ksil) using D. pseudoobscura as outgroup [ST [xy [PhKgamma and Ubc-E2H gene regions, pairwise FST values were used to compute a Principal Coordinate Analysis (pcoa in R package ape version 3.5 (http://ape-package.ird.fr/). A locus-by-locus analysis of Molecular variance (AMOVA) was performed on Arlequin v3.5.2 [CT) between arrangements at each specific polymorphic site. A hierarchical structure was considered with two groups corresponding to the two arrangements (A2 vs AST), with the different localities and individuals sampled within these groups.Several standard parameters for each locality and arrangement were estimated using DnaSP v5 : the numoutgroup , with throup [ST  and Dxy  [ST [xy  as well  [ST [xy  were obt [ST [xy  was appln v3.5.2  to test ZnS was estimated for informative sites. LD was also estimated between polymorphic informative sites through r2 estimates. Deviations from neutrality were tested using Tajima’s D [2 [D. subobscura genes whether located inside or outside chromosomal arrangements (see [Gene conversion tracts (GCTs) were detected using the algorithm defined by . Linkagejima’s D  for eacha’s D [2 . Statistnts (see ). All nents (see  was usedST and A2), we analysed the four genomic regions in the two most geographically distant localities (Málaga and Groningen), and two gene regions for the two central localities (Rasquera and Dijon) . No differences in the divergence per silent site values (Ksil) relative to D. pseudoobscura were found between the two arrangements . Higher nucleotide diversity was found for Hfw and Nipsnap  and Ubc-E2H .As low levels of within-population variation – as is the case in stimates , 64 we hST individuals from different locations in non-coding regions, but not for A2 individuals. Considering each gene region, we found high differentiation between arrangements in upstream regions for Ubc-E2H and both upstream and intronic regions for PhKgamma while no relevant differentiation was observed for the other two genes in any region analysed  between arrangements at each specific polymorphic site using a hierarchical structure (see Methods). Again, differentiation between arrangements was observed exclusively in non-coding regions  and non-coding (non-exon) regions for the four genes concatenated data set for Málaga and Groningen , showed that the significant differentiation between groups of individuals carrying different arrangements was due solely to variation in non-coding regions , or AST . Similar results are also obtained for the two genes separately . For Ubc-E2H, one fixed difference was found between the two arrangements (position 60 in the 5’UTR). No fixed differences were found in the other two genes.Gene conversion tracts (GCT) were detected only in 2) showed only 35 significant comparisons out of a total of 2415 . Importantly, the majority of the significant LD comparisons were observed between sites from the same gene region, indicating short-range LD . In the analysis including only the individuals carrying the A2 inversion just 3 out of a total of 496 comparisons indicated significant LD after Bonferroni correction . However, in these three cases, the significant LD comparisons were always between sites within the same gene region  were found between arrangements and sites in PhKgamma  and also in Ubc-E2H . Interestingly, all positions in PhKgamma are also in complete LD among themselves . All these sites are located in non-coding/intronic regions. The fixed position in the Ubc-E2H gene is located 547 bp upstream from the first exon. The fixed positions at 358–360 bp in the PhKgamma gene are located in an intronic region just 26 bp downstream of the first exon. Comparison against D. pseudoobscura orthologous gene indicates more similarity to AST sequences. The fixed position between arrangements at 205 bp is within the 5’UTR region 36 bp upstream of the start coding site of PhKgamma, and possibly within the core promoter region, despite the absence of characteristic sequence motifs such as TATA box and the TFIIB recognition element [In general, levels of linkage disequilibrium (LD) were low in all gene regions and arrangements analysed  presented significant differentiation between arrangements particularly in non-coding regions, regardless of the arrangement’s geographical origin. Several molecular studies in Drosophila have documented significant genetic differentiation between chromosomes carrying different arrangements, particularly within inverted regions [2 - around 160.000 years [PhKgamma and Ubc-E2H did not show similar patterns [Our study shows that these arrangements shape the patterns of genetic variation likely by reducing recombination between certain regions within chromosomes. In fact, the two proximally located regions in A regions , 33, 66  regions , 21, 23) regions , 18. Fur regions ). In add regions , particu regions , 69. Thi regions . In our  regions  the age patterns  - namelyST/A2 complex, Nóbrega et al. [2. In our study, we show that the reduction of recombination is not homogeneous in regions around the vicinity of the breakpoints of the AST/A2 complex, with significant genetic differentiation and linkage disequilibrium between inversions occurring only in the proximal region of AST. Interestingly, linkage disequilibrium occurred in one region (PhKgamma) located outside the inversion, although near the breakpoint. Other studies have suggested that suppressed recombination can extent to 2.5 Mb or more outside inversion breakpoints [PhKgamma and Ubc-E2H is in fact adaptive.In the specific context of the Aa et al.  also fouakpoints , 71. FunPhKgamma and Nipsnap have shown to be positively associated to latitude in semi natural populations of D. subobscura from a similar geographic gradient, but not when the same populations were maintained in a common garden [We did not find evidence for geographical (i.e. climatic) selection within arrangements, as very low within-arrangement genetic differentiation was detected across localities. It is possible that the differences in gene expression described previously for these genomic regions between thermal regimes  were conn garden . Thus, aD. subobscura found very limited evidence for geographical molecular variation within inversions. Pegueroles et al. [D. subobscura and found no significant differentiation within each arrangement for individuals sampled in Barcelona (Spain) and Mount Parnes (Greece). Evidence for similar patterns of generally low within-arrangement differentiation was also described in other D. subobscura studies [D. melanogaster Payne [Other studies in s et al.  also ana studies , 74 as wogaster Payne , 34, perAtpα gene, another candidate gene for thermal adaptation, Pegueroles et al. [Within-arrangement differentiation across localities would be expected if different epistatic interactions evolved  in different localities due to adaptation to the specific local conditions as proposed by the Dobzhansky’s coadaptation model . The lacs et al.  found stD. subobscura localities of the European cline found very low within-arrangement differentiation across localities as well as no clinal patterns of genetic variation. This indicates that gene flow across the European continent has homogenized the genetic content of these potentially selected regions at a wide geographical scale. Given the strong effect of gene flow in this species, it is possible that considerable within-arrangement differentiation at a geographical scale is only maintained at relatively few loci subjected to stronger selective pressures in the different environments. In this scenario, one can argue that restricted gene flow cannot explain the observed inversion frequency clines and that local adaptation is maintaining the clinal variation in this species, counteracting the homogenizing effects of the high gene flow. We also found an important effect of the AST/A2 complex in shaping genetic variation and preventing relevant gene exchange in different genomic regions, although its impact was not uniform across the different regions analysed. More generally, our study adds to a body of evidence showing that inversions act as barriers to recombination even outside inversions and can potentially have a role in the spread of favourable genic combinations and consequent adaptation to local habitats.Our genetic analysis of candidate loci for thermal adaptation  across gAdditional file 1:Nipsnap gene. (PDF 108 kb)New hybridization location for Additional file 2:PhKgamma and Ubc-E2H. A) Genetic differentiation (FST) between all localities and arrangements in PhKgamma  and Ubc-E2H . B) Genetic differentiation (Dxy) between all localities and arrangements in PhKgamma  and Ubc-E2H . (XLSX 12 kb)Measures of genetic differentiation within and between arrangements for Additional file 3:Nucleotide diversity (π) for all genes, arrangements and localities. (PDF 178 kb)Additional file 4:ST) between Malaga and Groningen in Hfw, Nipsnap and the concatenated dataset (four loci). (XLSX 10 kb)Genetic differentiation  for coding and non-coding regions in the concatenated dataset for all 4 genes (A) and for coding, intronic and 5’ UTR regions for PhKgamma (B). (XLSX 13 kb)Genetic differentiation  for all genes. 5’UTR, intronic and exonic regions are discriminated  and Ubc-E2H (B). (PDF 344 kb)Genetic differentiation between arrangements for each locality in Additional file 8:Linkage Disequilibrium parameters for all regions and the concatenated dataset. (XLSX 10 kb)Additional file 9:2) against the nucleotide distance between compared sites. Significant comparisons after Bonferroni correction are presented in red . A) LD between arrangements; B) LD within AST; C) LD within A2. (PDF 248 kb)Linkage disequilibrium (LD) between pairs of polymorphic sites


Sturnira is one of the most species-rich genera in the Neotropics, and it is found from Mexico and the Lesser Antilles to Argentina. This genus forms a well-supported monophyletic clade with at least twenty-one recognized species, as well as several others under taxonomic review. Sturnira parvidens is a widespread frugivorous bat of the deciduous forests of the Neotropics, is highly abundant, and is a major component in fruit dispersal to regenerate ecosystems.S. parvidens. We analyzed millions of resulting reads with specialized software to extract those reads that contained di-, tri-, tetra-, penta-, and hexanucleotide microsatellites.We used a technique based on Illumina paired-end sequencing of a library highly enriched for microsatellite repeats to develop loci for S. parvidens. We observed medium—high genetic variation across most loci, but only 12 were functionally polymorphic. Levels of expected heterozygosity across all markers were high to medium . We examined ascertainment bias in twelve bats of the genus, obtaining null/monomorphic/polymorphic amplifications.We selected and tested 14 polymorphic  microsatellites. All markers were genotyped on 26 different individuals from distinct locations of the distributional area of S. parvidens in particular, and for the genus in general, could be suitable for further genetic analysis, including taxonomic inconsistencies, parentage/relatedness analysis, and population genetics assessments.The Illumina paired-end sequencing system is capable of identifying massive numbers of microsatellite loci, while expending little time, reducing costs, and providing a large amount of data. The described polymorphic loci for  Sturnira parvidens) is primarily associated with lower elevations , and is found mainly in tropical/subtropical habitats and ecotones . S. parvidens has been caught in the understory and subcanopy of tropical and subtropical forests, in xeric scrubs, and in secondary and temperate forests. They are commonly found roosting in the foliage of forests of advanced successional stages, but their home ranges include mature and secondary forest (Cecropia peltata), pioneer herbs , or pioneer shrubs . adrianae . Lastly,arvidens .Sturnira parvidens; our goal was to isolate and characterize polymorphic microsatellite loci for the species by using Next-Generation Sequencing. The development of these markers can be useful for understanding the genetic structure of subpopulations in its distributional range. They can be used to identify the impact of humans on the fragmentation of the populations and assess the divergent lineages formed by genetic drift. They can also be used to evaluate movements of individuals in the mosaic-fragmented landscapes, and discern the genetic component in the social structure of the population by assessing relatedness and paternity. We showed cross-species amplification in twelve species of the Sturnira genus, under the hypothesis of having a positive ascertainment bias due to the phylogenetic relatedness among species  for shotgun sequences, and we used the Universal Salt Protocol to extract DNA from the remaining specimens . An Illu2 (25 mM), and 1.0 U of FlexiTaq polymerase. PCR cycling conditions were as follows: initial denaturation at 95 °C for 3 min; followed by 30 cycles of 95 °C for 3 min, gradient temperature (ranging from 56 to 60 °C) for 30 s, and 72 °C for 2 min; extension of 68 °C for 8 min; and final ending of 4 °C. Exact annealing temperatures for each primer are given in S. parvidens.Once positive reads were identified in PAL_FINDER, they were batched to a local installation of the program MSATCOMMANDER v 0.8.2 for primer design . We reco® v. 2.4.2  and peaks were scored by hand. We obtained the number of homozygotes and heterozygotes by scoring data. We estimated the proportion of polymorphic loci and the average number of alleles per locus by using the GDA software (HO) and the expected heterozygosity (HE), linkage disequilibrium, and Hardy–Weinberg proportions by using Genepop 4.2 , containing perfect, imperfect, and compound microsatellites . DinucleSturnira parvidens. Polymorphic information content (PIC) presented values above 0.5 showing a significant content of alleles per locus. Allele frequencies showed a remarkable diversity of alleles per locus, driving a superior number of valuable loci to be used in different genetic analyses (HE) ranged from medium to high for all markers . In the majority, there was no evidence of null alleles, but three loci  showed significant frequencies of null alleles , followed by S. bakeri (7). S. mordax had the lowest number of amplified loci (4).Cross-species amplification showed differences for the twelve related species . S. new Sturnira parvidens. This method has been probed for several bat species, and it is becoming a standard method for acquiring specific molecular markers . Cross-species amplification was effective for the 12 related species, but with no positive amplifications in several cases.We used Illumina Paired-Sequences to efficiently develop microsatellite loci for 10.7717/peerj.3367/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.3367/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.3367/supp-3Supplemental Information 3Click here for additional data file.


Bathymodiolus azoricus and B. puteoserpentis, living along the Mid-Atlantic Ridge.The analysis of hybrid zones is crucial for gaining a mechanistic understanding of the process of speciation and the maintenance of species boundaries. Hybrid zones have been studied intensively in terrestrial and shallow-water ecosystems, but very little is known about their occurrence in deep-sea environments. Here we used diagnostic, single nucleotide polymorphisms in combination with one mitochondrial gene to re-examine prior hypotheses about a contact zone involving deep-sea hydrothermal vent mussels, Admixture was found to be asymmetric with respect to the parental species, while introgression was more widespread geographically than previously recognized. Admixed individuals with a majority of alleles from one of the parental species were most frequent in habitats corresponding to that species. Mussels found at a geographically intermediate vent field constituted a genetically mixed population that showed no evidence for hybrid incompatibilities, a finding that does not support a previously inferred tension zone model.B. azoricus and B. puteoserpentis hybridize introgressively across a large geographic area without evidence for general hybrid incompatibilities. While these findings shed new light onto the genetic structure of this hybrid zone, many aspects about its nature still remain obscure. Our study sets a baseline for further research that should primarily focus on the acquisition of additional mussel samples and environmental data, a detailed exploration of vent areas and hidden populations as well as genomic analyses in both mussel hosts and their bacterial symbionts.Our analyses indicate that The online version of this article (doi:10.1186/s12862-016-0862-2) contains supplementary material, which is available to authorized users. Hybrid zones, regions where genetically distinct species interbreed to form genotypes of mixed origin, have achieved considerable interest in evolutionary research. Such zones of interbreeding can provide significant insights into the mechanisms that underlie speciation and the integrity of species , 2. InteBathymodiolus azoricus inhabits shallower vents (813–2251 m) clustered near the Azorean Triple Junction [Bathymodiolus puteoserpentis inhabits deeper vents (2432–3480 m) distributed along the central MAR [B. azoricus [B. azoricus takes place between January and March [B. puteoserpentis is unknown.Although most hybrid zone research has focused on terrestrial, freshwater and coastal ecosystems, three examples of hybridization and intergradation were described from deep-sea hydrothermal vent environments –13. UnliJunction , whereastral MAR , 18. Thetral MAR , 11. Elstral MAR . They arazoricus –24. SpawND4) haplotypes and leucine-aminopeptidase (Lap) allozymes. Parental genotypes greatly surpassed the frequency of recombinants, indicating limited reproduction and self-recruitment at the environmentally stressful BS locality. Concordant step-clines across a number of allozyme loci suggested that introgression into the parental ranges was limited. Together these patterns were interpreted as evidence for a tension zone maintained primarily by immigration of parental species and possibly hybrid unfitness. Faure et al. [B. azoricus and B. puteoserpentis split as recently as 0.76 million years ago. The coalescent history of intragenic recombination between semi-diagnostic GF1B alleles led these authors to surmise that secondary intergradation probably followed a brief period of allopatric divergence. Although biogeography can be useful in the interpretation of genetic patterns [Won et al.  found the et al.  developepatterns , a lack patterns .B. azoricus and B. puteoserpentis. For the present study, we used a diagnostic subset of these SNP markers and the mitochondrial ND4 gene (i) to identify the geographical range of hybridization along the MAR axis, (ii) to thoroughly determine different classes of hybrids, and (iii) to find evidence for or against the tension zone model.To remedy these limitations, the present study builds upon a recent development. Breusing et al.  designedB. azoricus and B. puteoserpentis, we used a subset of 18 species-diagnostic markers from a starting panel that included 90 SNPs [B. azoricus) and SM (B. puteoserpentis)  and B. puteoserpentis (0.92) to set these as thresholds for hybrid identification. As STRUCTURE q-values do not consider interspecific heterozygosities and are therefore not suitable for distinguishing between different hybrid genotypes, we did not attempt to further classify potential hybrid individuals with this program. For INTROGRESS, hybrid categorizations essentially followed the classification scheme by Milne & Abott [h) and interspecific heterozygosities (IH). Reference values of h and IH were determined for artificial hybrid and parental types and subsequently adjusted as given in Table For STRUCTURE, we identified the highest and lowest average  & Abott , consideB. azoricus and B. puteoserpentis along the MAR. Simulations based on the SNP data were run according to the admixture model with correlated allele frequencies [K with 10 replicates. Model runs involved 107 iterations after a de-memorization period of 106. For identifying the most likely value of K we applied the ΔK correction as described in Evanno et al. [http://evolution.unibas.ch/salzburger/software.htm). Haplotype networks for population structure analyses of the mitochondrial ND4 locus were created with NETWORK v4.6.1.2  were performed with the Benjamini-Yekutieli False Discovery Rate (BY FDR) procedure [STRUCTURE was used to determine population genetic structuring and patterns of hybridization between quencies , testingo et al.  and inspo et al. . STRUCTUing.com; –42) as iing.com; . To asseing.com;  using 10rocedure , which hrocedure .h and interspecific heterozygosities IH based on genotypic information at the 18 diagnostic SNP markers, we used the maximum likelihood approach implemented in the R package INTROGRESS, defining MG and SM as parental populations of B. azoricus and B. puteoserpentis, respectively. To support the findings of the INTROGRESS program, we performed additional simulations in NEWHYBRIDS. Runs were replicated five times with different seed values, using 100,000 iterations of the MCMC chain and a burnin of 10,000.To estimate individual hybrid indices x generation hybrids and parental genotypes were easily distinguishable from each other using the 18 diagnostic SNP markers, as all programs showed a very high assignment certainty for individuals from these classes .Simulations of hybrid categories confirmed that different F2–4 hybrids  or pure species . STRUCTURE categorized up to 87.5% incorrectly as parental genotypes given that q-values largely overlapped between these classes  or comprised a mixture of mostly pure B. azoricus and a low amount of hybrids . By contrast, at the southern vents SP, IR, QS and SM populations contained only B. puteoserpentis individuals  or were a mixture of pure B. puteoserpentis and a few hybrid mussels . At the intermediate BS locality 60% of mussels seemed to be hybrids, whereas the rest of the individuals were classified as pure B. puteoserpentis.The STRUCTURE analyses confirmed the presence of two genetic groups corresponding with the recognized species, tis Fig. . At the B. azoricus were detected at MG (4.0 − 12.0%) and LS (10.0 − 26.7%), backcrosses to B. puteoserpentis were detected at SP (16.7 − 76.7%), IR (8.3 − 39.6%), QS (6.7 − 33.3%) and SM (0.0 − 32.5%). At the central vent BS 40 − 50% of the individuals were categorized as multi-generation hybrids (F2 to F4) with a similar allelic contribution from both parental species  and backcrosses thereof (20.0 − 40.0%), whereas no B. azoricus were found at this locality. In contrast to the INTROGRESS program, NEWHYBRIDS identified a few backcrosses to B. azoricus (6.7%) at BS, although these might be misclassified F-generation hybrids, as indicated by results from the artificial population data set , IR 8. − 39.6%,ND4 haplotypes that formed lineage-specific clades differing by at least 12 mutations , no significant deviations from HWE were found in any of the sampled demes. By contrast, tests for LD (BY FDR α = 0.0072) revealed 48 significant associations of non-homologous alleles, mostly (85.4%) involving the BS sample  detected admixed individuals outside the BS hybrid zone. As many backcross genotypes were often identified as pure species and as the use of diagnostic markers might underestimate the level of introgression [Studies of interspecific hybridization in animals have a long-standing history in terrestrial, coastal and shallow-water research, but virtually nothing is known about contact zones in deep-sea environments. We used 18 diagnostic SNP markers and the mitochondrial erpentis , 11. Witgression , these oBathymodiolus azoricus and B. puteoserpentis appear to segregate by depth along the MAR [B. azoricus and B. puteoserpentis ND4 haplogroups in the northern and southern regions due to geographic premating barriers resulting in genetic drift or due to co-evolution of nuclear and cytoplasmic genomes [B. azoricus genotypes at the intermediate BS locality could simply be a matter of sampling bias. Firstly, mussels are very difficult to distinguish from surrounding rock at this vent field as they are coated with sulphide-enriched sediments [B. azoricus genotypes, if they are relatively infrequent or adapted to environmental patches at BS that we did not explore during our cruises. Likewise, this could be the case for F1 hybrids, which were surprisingly not observed in our sample. Given that we were able to detect B. azoricus at other localities and that the assignment certainties for F1 individuals were very high, it is improbable that our marker panel did not have enough power to detect these genotypes at BS. Therefore, it is justified to conclude that our results mirror a true pattern rather than a statistical artefact.Different, mutually non-exclusive explanations could account for the asymmetry in admixture patterns and the differential abundance of genotype classes among vent locations.  the MAR , 11. Suc genomes , 48. Altediments . Secondlediments . ConsequB. puteoserpentis-specific bacterial strain or double infections by symbionts from both species. By contrast, no single infections by the B. azoricus-specific type were found. Mixed infections would only be possible, when both pure species co-occur at BS (sampling gap hypothesis) or when the first contact happened at another locality (hybrid zone movement hypothesis). Both hypotheses seem to be plausible for several reasons. On the one hand, secondary contact zones between recognized species often emerge under environmental conditions to which parental genotypes are not well adapted [B. azoricus and B. puteoserpentis and lead to hybrid advantage in environmentally intermediate vent areas. Addressing this aspect will be helpful to identify the relative importance of selective forces and neutral processes, which can cause the same introgression and admixture patterns as natural selection [Another explanation for the observed patterns could be a southward movement of the contact zone from an unknown, possibly now extinct vent locality north of BS. Under this scenario, the early generation hybrids would be immigrants that reproduced locally. Both hybrid zone movements and potential sampling gaps would be consistent with the composition of thiotrophic symbionts in BS individuals . Based o adapted , 51–53. election .On the other hand, a recent study by Breusing et al.  indicateND4 and Lap), Won et al. [Based on information from two loci (n et al.  reportedn et al. , 57.B. azoricus and B. puteoserpentis is introgressive and that gene flow extends across more vent localities than reported in former work. While our results give new insights into the genetic structure of this hybrid zone, several additional analyses need to be performed to test further hypotheses on its nature and evolutionary implications. Most importantly, it will be necessary to obtain new samples from the BS vent field and to thoroughly search surrounding areas for the existence of other mussel populations. Metagenomic studies of the composition and functional variation of both thiotrophic and methanotrophic symbionts might provide critical informative data about the extent and type of ecological differentiation between parental species and hybrids. In addition, genome-wide scans could be used to design additional diagnostic markers, which—in combination with a standardized sampling design, measurements of environmental conditions and determination of phenotypic traits—would help to resolve the true degree of introgression and admixture along the MAR and the role of differential adaptation in this system. Such insights will be crucial for our general understanding of the role of hybridization in the evolution of deep-sea taxa.In contrast to previous research our study shows that hybridization between


Canarina eminii to explore causal explanations for this pattern. Phylogeographic analyses were undertaken using plastid regions and AFLP fragments. Population genetic analyses, Statistical Parsimony, and Bayesian methods were used to infer genetic diversity, genealogical relationships, structure, gene flow barriers, and the spatiotemporal evolution of populations. A strong phylogeographic structure was found, with two reciprocally monophyletic lineages on each side of the Great Rift System, high genetic exclusivity, and restricted gene flow among mountain ranges. We explain this pattern by topographic and ecological changes driven by geological rifting in Eastern Africa. Subsequent genetic structure is attributed to Pleistocene climatic changes, in which sky-islands acted as long-term refuges and cradles of genetic diversity. Our study highlights the importance of climate change and geographic barriers associated with the African Rift System in shaping population genetic patterns, as well as the need to preserve the high levels of exclusive and critically endangered biodiversity harboured by current patches of the Afromontane forest.The Eastern African Afromontane forest is getting increased attention in conservation studies because of its high endemicity levels and shrinking geographic distribution. Phylogeographic studies have found evidence of high levels of genetic variation structured across the Great Rift System. Here, we use the epiphytic plant species  The Afromontane Floristic Region179Geographically, the Eastern Afromontane region is divided longitudinally by the Great Rift System, a 4830 km north-south fissure whose terrestrial section extends from Djibouti to Mozambique. This orogenic fault marks the line along which the African Plate is splitting into two tectonic plates, the Somalian Plate and the Nubian Plate . The forThe Afromontane vegetation is divided into three altitudinal belts, each with unique floristic composition14151171819Interest on geographic patterns of genetic variance in Eastern African species has increased in recent years. Many of these phylogeographic studies  the81723241819Canarina eminii Asch. & Schweinf, a strictly Afromontane species with a geographic distribution extending from the Ethiopian massifs to the SMSI in the northern end of Lake MalawiHagenia J.F.Gmel., Conopharyngia G. Don, and Afrocarpus (J. Buchholz & E.G. Gray) C.N. PageC. eminii, except that the species is pollinated by sunbirds (unpublished field observations) and exhibits floral traits that have been associated with this type of ornitophilous pollinationet al.Canarina L., a small genus of three species within tribe Platycodoneae (family Campanulaceae). This genus exhibits a wide disjunct distribution across North Africa and probably originated from a Central Asian ancestor, which arrived to Eastern Africa in the Mid Miocene (13.7 Ma) through the Arabian PlateC. abyssinica diverged first, followed by the split between C. eminii and the endemic Canarian species C. canariensisC. eminii was traced back to 1.76 Maet al.C. eminii.Here, we examine support for these two hypotheses, and the role of the Rift Valley as a long-term dispersal barrier, by reconstructing patterns of genetic variation in C. eminii among and within populations. Our aims were to: i) describe the geographic distribution of genetic variation within this species; ii) examine the role of the Rift Valley as a long-term dispersal barrier; iii) understand how Pleistocene climatic fluctuations might have affected population ranges, especially in relation to the “mountain-forest bridge” hypothesis and the role of isolated “sky islands”; and iv) provide insights into the phylogeography of an Afromontane epiphytic species. Since the epiphytic growth of this species is tied to dominant tree species of the Afromontane forests, it makes C. eminii a good case study for tracing the history of fragmentation and expansion of forest coverage in this region.In this study, we reconstruct the phylogeographic history of C. eminii, we sequenced three highly variable plastid (pDNA) regions covering the entire distributional range of this species . We generated 237 new sequences from 79 individuals: rpl32-trnLUAG (79 sequences), trnSGCU–trnGUCC (79 sequences), and petB1365–petD738 (79 sequences). The number of nucleotide sites ranged from 648 in trnSGCU–trnGUCC, 834 in pet B1365–petD738, to 963 in rpl32-trnLUAG. The concatenated matrix consisted of 79 sequences of C. eminii with 2445 nucleotide sites. The haplotype network recovered by TCS identified nine haplotypes  (In order to examine patterns of genetic variation within see M&M) ; these ssee M&M) .K = 4, ST and geographic distance , we carried out an analysis of fragment length polymorphism (AFLP) in the nuclear compartment. The final data set after scoring comprised 773 loci. The Bayesian software STRUCTURE assigned individuals to four geographic clusters based on patterns of genetic variance K = 4, . These cT values . All intT values  gave a sT values . HierarcT values . The relT values  might beet al.Canarina (8.98 My.) and the stem-age of C. eminii (7.14 My.)  . The firC. eminii populations. This agrees well with phylogeographic studies in other Afromontane groups  for gradual SSD migration through forest connections. Results from the plastid DNA suggest seed dispersal on each side of the Rift Valley, with more recent connectivity among the eastern side populations . Genealogical relationships among haplotypes were inferred via the statistical parsimony algorithmet al.Canarina and nine outgroup taxa from Platycodoneae and Campanulaceae, was used to inform the clock rate of a linked population-level dataset of C. eminii, under a mixed Yule-coalescent modelHaplotype divergence times were estimated using Bayesian relaxed clocks implemented in the software package BEAST v.1.7.5et al.C. eminii. It is based on a continuous-time Markov Chain process where the discrete states correspond to the geographic locations and the state transition rates to the migration rates between areas . Bayesian Stochastic Search Variable Selection  of Lemey EcoRI and MseI and linked to the adaptors EcoRI (5’-CTCGTAGACTGCGTACC-3’/5’-AATTGGTACGCAGTCTAC-3’) and MseI (5’- GACGATGAGTCCTGAC-3’/5’-ATCTCAGGACTCAT-3’). We tested 32 combinations of selective primers and chose the four pairs that produced the most polymorphic and clear profiles: 1-EcoRI6-FAM-ACT/MseI-CAA; 2-EcoRI6-FAM-ACT/MseI-CAT; 3-EcoRIVIC-AGG/MseI-CTA, and 4-EcoRIVIC-AGG/MseI-CTT.Laboratory molecular protocols for the AFLP analysisAmplified fragments were analysed using GeneMapper 3.7 software (Applied Biosystems), and peaks ranging between 100 and 500 bp recorded. AFLP Scorer softwareST), the percentage of polymorphic fragments per population (P), and the bootstrapped Nei’s genetic distance matrix between individuals and populations70IS) was set to 0.1 as suggested by HardyST values. Genetic distances between individuals, populations and geographic groups were also calculated. We used AFLPdat75The resulting AFLP presence/absence matrix was analysed using AFLPSURV v.1.0K values of 1–10, with ten repetitions each. For each K value, only the run with the highest maximum likelihood value was considered. The LnP (D) for the successive decomposition of groups was used in all STRUCTURE analysesC. eminii populations, correlations between genetic (measured as FST) and spatial distances between pairs of populations were determined using the Mantel permutation procedure in Ntsys. The genetic distance matrix used was based on the presence/absence matrix; the geographic distance matrix was based on absolute distances between the geographic coordinates for each collected population. In addition, the kinship multilocus coefficient (FIJ) was estimated using SPAGeDiC. eminii populations, based on genetic distances. The significance of these was examined with 1000 bootstrapped distance matrices obtained using AFLPsurv.To distinguish genetic groups of individuals in the AFLP dataset, a comparison was made by constructing a pairwise similarity matrix for all individuals: Dice’s coefficient was calculated and the resulting matrix was transformed using principal coordinates analysis (PCO) with Ntsys v.2.180DNA sequences: Genbank Accession nos KF028817–KM189329. GenBank accessions, sampling locations and/or online-only appendices uploaded as online How to cite this article: Mairal, M. et al. Geographic barriers and Pleistocene climate change shaped patterns of genetic variation in the Eastern Afromontane biodiversity hotspot. Sci. Rep.7, 45749; doi: 10.1038/srep45749 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Fusarium isolates was collected from blighted wheat spikes as well as from rice stubble with perithecia originating from nine sampling sites in five provinces in Southern China. Fusarium asiaticum dominated both wheat and rice populations, although rice populations showed a higher species diversity. Chemotype analysis showed that rice is the preferred niche for NIV mycotoxin producers that were shown to be less virulent on wheat. In contrast, 3ADON producers are more prevalent on wheat and in wheat producing areas. The 3ADON producers were shown to be more virulent on wheat, revealing the selection pressure of wheat on 3ADON producers. For the first time, members of the Incarnatum-clade of FusariumIncarnatum-Equiseti Species Complex (FIESC) were found to reproduce sexually on rice stubble. The pathogenicity of FIESC isolates on wheat proved very low and this may cause the apparent absence of this species in the main wheat producing provinces. This is the first report of the Fusarium population structure including rice stubble as well as a direct comparison with the population on wheat heads in the same fields. Our results confirm that the perithecia on rice stubble are the primary inoculum of FHB on wheat and that cropping systems affect the local Fusarium population.In recent years, Fusarium head blight (FHB) outbreaks have occurred much more frequently in China. The reduction of burning of the preceding crop residues is suggested to contribute to more severe epidemics as it may increase the initial inoculum. In this study, a large number of  Triticum aestivum L.), barley (Hordeum vulgare L.) and other small grains worldwide [Fusarium head blight (FHB) is a devastating disease of wheat  consists of at least 16 phylogenetically distinct species [F. graminearum is the most widely distributed species and occurs in most FHB areas around the world [F. asiaticum is the main FHB pathogen present in Asia [ species : F. gramhe world , while F in Asia ,6,7. Mem in Asia : NIV and in Asia . A numbe in Asia ,10,11. M in Asia .Fusarium. In agreement with this hypothesis, several studies have found a strong association between the distribution of FGSC species and cropping systems (host preference). F. asiaticum dominated in regions where rice is grown in rotation with wheat, as can be found in China and Korea, whereas F. graminearum was more common in wheat-maize rotation systems [F. graminearum was found to be dominant in wheat [F. asiaticum in rice [F. meridionale was determined as the predominant FGSC species on maize ears and stalks [F. boothii was shown to be the best colonizer and mycotoxin producer on maize whereas F. graminearum was predominant on wheat [F. boothii was prominent when maize is cultivated without rotation with wheat or rice [Fusarium and there are few reports on the saprophytic stage on crops residues, which is crucial to the lifecycle and epidemiology of the pathogen. So far, there is no direct evidence for the overlap between Fusarium populations from wheat heads and the previous crop debris, which is important to understand the epidemiology of this pathogen.FHB pathogens usually overwinter on infested crop residues . In spring, perithecia form on the surface of these residues, and ascospores released from perithecia on this crop debris serve as the primary inoculum of FHB. Hence, the presence of crop debris is essential for the overwintering of  systems ,12. A si systems , while iin wheat , F. asia in rice , and F. d stalks . In Souton wheat ,18. Simi or rice . HoweverFusarium isolates from both wheat heads and rice stubble that were sampled from the same fields in five provinces along the Yangtze River. These populations were characterized at the species and chemotype level and their composition was compared. Subsequently, the pathogenicity of Fusarium species from wheat and from rice towards wheat was determined to identify possible differences in this host. Finally, these findings are discussed in the context of rice populations as the main primary inocula of FHB and the selection of wheat vs. rice on the different chemotypes of FGSC with respect to the different cropping systems.In China, wheat-rice rotation within one year is the predominant cropping system in the regions along the Yangtze River, where FHB outbreaks are very frequent ,20. AbunF. asiaticum (n = 1110), F. graminearum (n = 67), and F. meridionale (n = 9). Based on partial TEF-1α gene sequences, the remaining 60 strains were identified as 10 species including F. incarnatum-F. equiseti Species Complexes (FIESC) (n = 30), F. acuminatum (n = 1), F. tricinctum (n = 1), F. armeniacum (n = 6), F. kyushuense (n = 2), F. fujikuroi (n = 7), F. proliferatum (n = 6), F. concentricum (n = 3), F. nygamai (n = 3), and F. verticillioides (n = 1). The distribution of these isolates is summarized in F. asiaticum. In the rice population, we found more species (12 species) than on wheat, but again F. asiaticum was predominant, although its frequency was lower (85.6%). The fact that F. asiaticum dominated both populations indicates that it can circulate in the wheat-rice rotation system.A total of 1246 single spore strains were isolated from nine sampling sites in five provinces A, 702 frF. graminearum isolates 86.1% (55/67) were isolated from Sichuan Province; F. graminearum is rarely found in the provinces located in the middle and lower reaches of the Yangtze River. All nine F. meridionale strains collected originated from Sichuan Province and exhibited a significantly  uneven distribution among rice and wheat, as only one isolate originated from wheat and eight from rice. Most non-FGSC species are at very low frequency except FIESC, which accounted for 50% of them (30/60). FIESC isolates were identified in all five provinces but were most commonly found on rice stubble (27/30). Phylogenetic analysis of FIESC showed that almost all isolates (29/30) belong to the Incarnatum clade with the exception of one isolate (IPP 14080) that belonged to the Equiseti clade  dominated in Sichuan Province, especially on rice, where no other chemotypes were found. 3ADON is the main chemotype (80.5%) in the Hubei, Anhui, and Jiangsu Provinces, which are located in the middle and lower reaches of the Yangtze River. However, we found an uneven distribution of chemotypes in rice and wheat populations in these provinces. Significantly higher ratios  of NIV producers were observed on rice stubble (20–35%) than on wheat kernels (7–10%) in these provinces .Trichothecene chemotypes of all 1186 FGSC isolates were identified by MLGT. The results are shown in rovinces B. In theF. graminearum, F. meridionale, and FIESC as well as NIV, 3ADON, or 15ADON producers of F. asiaticum for pathogenicity analyses. Ten strains were selected randomly from each population. The three populations of F. graminearum (15ADON), F. asiaticum (3ADON), and F. asiaticum (15ADON) showed the highest incidence of infected spikelets (IIS), at 41.5 ± 5.2, 43.3 ± 6.3, and 45.1 ± 4.2, respectively. NIV-type F. asiaticum strains were clearly less aggressive (27.5 ± 3.1), but their pathogenicity was still significantly higher than that of F. meridionale isolates (22.6 ± 3.0), while FIESC strains showed the lowest IIS (6.3 ± 1.0), as most isolates in this population only caused symptoms in the inoculated spikelet which never extended to adjacent spikelets (In order to understand how the hosts (wheat or rice) could influence their distribution of Fusarium species chemotype combinations, we selected 10 strains of each population including pikelets .Fusarium composition on wheat kernels and on rice stubble in the same locations in five provinces of Southern China. The rice populations revealed higher species diversity than the wheat populations. F. asiaticum dominated both pathogenic and saprophytic populations, and there is no significant difference  between the ratio of F. asiaticum in the two populations. This indicated that rice stubble may be selective for F. asiaticum rather than for other FHB species. It also confirmed that the perithecia on rice stubble are likely to act as the primary inoculum for FHB of wheat in Southern China. Del Ponte et al. (2015) compared the Fusarium composition on wheat heads and on corn residue in Brazil and observed a significant difference in two places; while F. meridionale dominated the saprophytic stage, F. graminearum was predominantly found in the pathogenic stage [F. meridionale on wheat—although they may show better fitness on corn residue (saprophytic stage), they failed in the competition with F. graminearum on wheat (pathogenic stage). In this study, we found similar pathogenicity levels in F. asiaticum with the 3ADON genotype and F. graminearum with the 15ADON genotype. We thus hypothesize that the preference of rice stubble in the saprophytic stage is crucial. Most F. graminearum strains (55/67) were isolated from Sichuan Province, which is consistent with our previous report [F. graminearum as F. graminearum is prevalent on maize.FHB is one of the most important problems in wheat production worldwide. In the last decade, FHB outbreaks became much more frequent in China, where the epidemic area covered over 4 million ha in seven of 10 years. The popularization of straw-returning instead of burning of the preceding crops was suggested as the main reason, as it leads to increased numbers of pathogen propagules in the field. In this study, we surveyed the ic stage . This maF. asiaticum with the 3ADON genotype to dominate the Middle-Lower Yangtze River Plain, which is the area most frequently affected by FHB epidemics. However, NIV-producing F. asiaticum isolates were reported to be associated with almost all rice agroecosystems outside China. This difference may be caused by the selection on wheat. A strict wheat-rice rotation within one year is unique to this large area in China. Consequently, the Fusarium population is forced to go through two selection cycles, one on rice and one on wheat every year. In agreement with previous studies [F. asiaticum with 3ADON and F. graminearum with 15ADON was significant higher than that of F. asiaticum with the NIV genotype on wheat  than that on rice in Hubei, Anhui, and Jiangsu, while in Sichuan and Fujian, 3ADON producers were only isolated on wheat but not on rice  in Cerro Largo, which is a new wheat production zone in traditionally rice growing areas. This can be explained by assuming that NIV-producing F. asiaticum isolates represent an old FHB population that existed before wheat was implemented, while the replacement by 15ADON-producing F. graminearum is still in progress in the new wheat production zone. Monitoring the species and chemotype shift of FGSC in this region could further validate our hypothesis and may be important for food safety because of the differences in toxicity of the trichothecenes.In agreement with previous studies in China ,21,22,23 studies , we founon wheat . The bet on rice . In the th Korea  and Brazd States  and Brazd States , and theF. asiaticum isolates on wheat are NIV-type, much higher than 3ADON producers (29%) [F. asiaticum NIV producers. In Fujian, the situation is distinct from other provinces in this study because farmers abandoned wheat cultivation due to severe FHB epidemics more than 10 years ago. In the present study, we could only collect samples in a small nursery dedicated to evaluating FHB resistance of wheat varieties from other provinces. NIV-producing F. asiaticum dominated both wheat and rice populations and only a few 3ADON producers were found on wheat, which may be introduced by wheat seeds from the Middle-Lower Yangtze River Plain. The presence of abundant NIV-type F. asiaticum in non- (Fujian) and less-wheat (Sichuan) provinces revealed that not only wheat selects 3ADON producers, but also that rice agroecosystems favor NIV producers. This strong association between NIV producers and rice is in agreement with previous reports from Korea [F. asiaticum on both rice and on wheat in Fujian, which is rare in wheat growing areas. This population showed a similar level of pathogenicity to 3ADON-producing F. asiaticum and 15ADON-producing F. graminearum on wheat, but higher aggressiveness than NIV producers of F. asiaticum and F. meridionale . This may be due to the decrease of the wheat production in Sichuan. The mountain region in this province restricts the use of machines and, along with the increase of the labor costs, more and more farmers have abandoned wheat cultivation. Consequently, the selection pressure for more virulent species on wheat also decreased. In addition, the mountains and the small holder farming in this region also limits the spread of successful populations on wheat. In Japan, where wheat cultivation is rare and rice production occurs across the entire country, 70% of the rs (29%) . Also, iom Korea , Japan [om Korea , the Uniom Korea , Uruguayom Korea , and Braom Korea , furtherom Korea ,24, we aidionale .F. meridionale isolates collected originated from Sichuan. Previous studies reported the presence of F. meridionale in Sichuan and Yunnan on wheat [F. meridionale has been reported as the main species on maize in Nepal and in Northern Argentina [F. meridionale were also found in Korea [F. meridionale population was identified on wheat in Brazil, which was thought to be related to the two harvests of maize in one year [F. meridionale was also shown to be less competitive on wheat than F. graminearum isolates [F. meridionale to be less virulent than both F. graminearum and F. asiaticum on wheat. This may be the main reason for the uneven distribution of F. meridionale on wheat (1/9) vs. rice stubble (8/9).All nine on wheat  and Sichon wheat  in Chinargentina ,26. Smalin Korea  and in Sin Korea . Recentlone year . F. meriisolates . SimilarFusarium isolates where obtained and half of these strains were identified as FIESC. Among them, three isolates were found on wheat while the other 27 were isolated from rice stubble. In the pathogenicity assays, most FIESC isolates caused only symptoms on the inoculated spikelets but no spreading was observed, which may explain the absence of this species in the main wheat producing regions. Phylogenetic analysis showed that 29 strains were clustered in the Incarnatum clade and the remaining one in the Equiseti clade. Lineages in the Incarnatum clade are not known to have a sexual stage [In addition to FGSC, 60 non-FGSC F. graminearum, while a wheat/rice rotation selects for F. asiaticum. F. boothii is selected when maize is cultivated without rotation [Fusarium population on rice stubble on a large scale and made comparisons with populations from wheat kernels. We confirmed that perithecia on rice stubble were the primary inoculum for FHB on wheat. Based on our findings and previous reports across China, we conclude that wheat is selective for DON-producing F. graminearum and F. asiaticum due to higher pathogenic fitness. In contrast, rice favors F. asiaticum with the NIV chemotype, possibly due to increased saprophytic fitness. Depending on the cropping system, this divergent selection can shape Fusarium populations and this can help us to predict possible prevalent species and subsequently predict which mycotoxin contamination can be expected.Many previous studies on the occurrence and prevalence of FGSC exist and the composition of populations is reported to change over time . Howeverrotation . In thisRice stubble with black perithecia was collected from nine sampling sites in five provinces A in SoutA small mycelial plug was transferred to 6-cm Petri plates containing potato dextrose agar (PDA) and incubated at 26 °C for 5 days. Mycelium was harvested with scalpel blades and vacuum freeze-dried at −20 °C overnight. The mycelium was lyophilized using liquid nitrogen and grinding by vigorous shaking in a MiniBeadbeater-96 . Total genomic DNA was extracted using the SP Fungal DNA Kit  according to the manufacturer’s instructions. DNA concentration was determined by NanoVue Plus . Finally, DNA samples were diluted to 5 ng/mL for each sample in 96-well microtiter plates, which were stored at −20 °C.TEF-1α, 700 bp) gene sequences were generated with the previously reported primers [TEF-1α sequence, a maximum likelihood tree of FIESC isolates together with several reference strains  was constructed in Geneious 9.1 using PhyML, and F. concolor (NRRL 13459) was set as an outgroup.Multilocus genotyping (MLGT) assay  was used primers . These s primers  and/or iF. meridionale population) for pathogenicity analyses. Conidia were prepared by mung bean broth cultures as mentioned above. At anthesis, 10 heads in each block were inoculated by injecting 20 mL conidial suspension (106 conidia/mL) of one isolate into a central floret of a spike (injection inoculation). A total of 30 heads was infected by a single strain. Ten heads in each block were also selected to be inoculated with 20 μL sterilized water as a control group. Pathogenicity was assessed as the incidence of infected spikelets, which was determined visually by counting the number of infected spikelets per head at 14 days after inoculation and expressed as a percentage of the total number of spikelets.A medium resistant winter wheat cultivar, Yangmai 158, was used for the pathogenicity tests. Wheat was planted in three different blocks (2 m × 4 m) according to normal agronomic practices at the Langfang Farm (Experiment Farm of the Institute of Plant Protection) in 2015. We selected 10 strains from each population randomly (nine isolates from


Aquila chrysaetos canadensis) in three geographic regions of western North America to conduct a preliminary study of the genetics of the North American subspecies, and to provide data for United States Fish and Wildlife Service (USFWS) decision-making for golden eagle management. We used a combination of mitochondrial DNA (mtDNA) D-loop sequences and 16 nuclear DNA (nDNA) microsatellite loci to investigate the extent of gene flow among our sampling areas in Idaho, California and Alaska and to determine if we could distinguish birds from the different geographic regions based on their genetic profiles. Our results indicate high genetic diversity, low genetic structure and high connectivity. Nuclear DNA Fst values between Idaho and California were low but significantly different from zero (0.026). Bayesian clustering methods indicated a single population, and we were unable to distinguish summer breeding residents from different regions. Results of the mtDNA AMOVA showed that most of the haplotype variation (97%) was within the geographic populations while 3% variation was partitioned among them. One haplotype was common to all three areas. One region-specific haplotype was detected in California and one in Idaho, but additional sampling is required to determine if these haplotypes are unique to those geographic areas or a sampling artifact. We discuss potential sources of the high gene flow for this species including natal and breeding dispersal, floaters, and changes in migratory behavior as a result of environmental factors such as climate change and habitat alteration. Our preliminary findings can help inform the USFWS in development of golden eagle management strategies and provide a basis for additional research into the complex dynamics of the North American subspecies.Understanding the genetics of a population is a critical component of developing conservation strategies. We used archived tissue samples from golden eagles ( An understanding of genetic diversity and structure is vital for developing conservation strategies for species of concern . GeneralAquila chrysaetos) are Holarctic in distribution and some studies have evaluated genetic diversity and structure for the species [A. c. canadensis) occurs, Katzner et al. [Golden eagles , has primary authority for the management of golden eagles in the US. Some golden eagles are harvested by Native Americans and falconers ; current management also allows for additional “take” of golden eagles under certain circumstances. The Eagle Act defines “take” as “…pursue, shoot, shoot at, poison, wound, kill, capture, trap, collect, or molest or disturb…”. Recently, the USFWS began to permit a limited unintentional “take” of eagles at energy projects , but witGenetic data can help identify subpopulations and are useful for designing conservation and management plans ,15,34,35We investigated population genetic variation and structure among sampled golden eagles from three geographic areas in western NA using a combination of mitochondrial DNA (mtDNA) D-loop sequences and microsatellite loci. We used archived blood samples from birds that spent the summer (breeding) season in Idaho/eastern Oregon, in California, and in Alaska and samples of eagles wintering in Idaho that represented both migrants and year-round residents. Our research goals were to: 1) investigate the extent of gene flow among golden eagles from these three geographic areas, 2) determine if summer resident eagles from different geographic locations in western NA can be distinguished based on their genetic profiles and 3) determine if the natal origin of winter residents in Idaho can be determined from their genetic profiles. We discuss our results as initial data for understanding the genetics of the subspecies, as a consideration for USFWS in evaluating the effects of permitting actions, and in development of conservation and management plans that must account for complex lifetime movements of individuals.n = 45), tissue (n = 3), feathers (n = 9) and fecal material (n = 1). In addition, we used blood samples from eagles that wintered in Idaho (n = 9) to determine if we could assign them to geographic summer locations based on our results from known origin birds. DNA was extracted from blood, tissue and feather samples using a modified DNeasy Blood and Tissue Kit protocol  [The samples from golden eagles were collected by the authors or were contributed  ,37, and n = 6) and California , were tested for amplification success and variability in birds from Idaho .® Terminator v3.1 Cycle Sequencing Kit. Sequencing products were cleaned using the BigDye® Xterminator™ Purification Kit according to the manufacturer’s protocol . Sequences were run on a 3130xl Genetic Analyzer  and analyzed using Sequencher 4.7 . Sequences were aligned by eye in Sequencher 4.7 (Gene Codes Corporation), and the number of haplotypes was determined using the filter redundant taxa option in MacClade 4.08 [www.ncbi.nlm.nih.gov/genbank/).The sample of western golden eagles was also assessed using mitochondrial DNA (mtDNA) sequencing of the D-loop region . A ~415bp fragment of the D-loop region was amplified using the primers GOEA_CR1L and GOEA_CR595H . PCR proade 4.08 . Golden ade 4.08  were dows [The number of alleles, observed and expected heterozygosity and the probability of identity (PID and PIDsibs) were calculated for each microsatellite locus using Gimlet 1.3 . The eigs ) were pes .A median joining network was drawn using Network 5 . Included in the network were sequences from an additional 6 eagles that summered in Alaska .ST in Arlequin 3.5 for both the microsatellite loci and the D-loop haplotype data [The samples from Alaska were excluded from population differentiation analyses due to small sample size. The degree of genetic differentiation between California and Idaho summer resident golden eagles was assessed by calculating pairwise Fype data ,45.To evaluate whether individuals can be classified into genetic groups based on their genetic signature, we used an assignment test approach as implemented in the software Geneclass 2 ,47, plusTwenty-five of the 36 microsatellite loci amplified successfully and produced bands in the correct size ranges  and all 6 samples from Alaska provided by Domenech and Sherrington. Eight mtDNA D-loop haplotypes were detected  and Idaho populations .Haplotype (h) and nucleotide (π) diversity was similar between California (h = 0.74 ± 0.064 SD), (π = 0.0025 ± 0.0019 SD) and Idaho (h = 0.68 ± 0.085 SD), (π = 0.0023 ± 0.0018 SD) and lower for Alaska (h = 0.49 ± 0.18 SD), (π = 0.0013 ± 0.0013 SD). Tajima’s D and Fu’s FST value calculated from the microsatellite data between Idaho and California was low but significantly greater than zero  indicating that the two populations are not in panmixia, but genetic differentiation is low between the Idaho and California golden eagle populations. The pairwise FST value calculated from the D-loop haplotype data between Idaho and California was also low and not significantly greater than zero .The pairwise FST values less than 0.03 [The assignment test implemented in Geneclass 2 correctly assigned an individual eagle to its sampling region of origin 60% of the time. This suggests there is not enough of a difference between allele frequencies in the California and Idaho populations to confidently assign individuals to their sampling population with the current number of loci. The results from program STRUCTURE indicate that the value of K with the greatest log likelihood is one when analyzing the Idaho and California samples only, and we obtained the same result when the 6 samples from Alaska were included . Programhan 0.03 . Three oResults of the AMOVA showed that 97% of the mtDNA haplotype variation was within the geographic populations while 3% of the haplotype variation was among populations . Using gSimilar to other work on raptors in NA ,12,13, wS using mtDNA sequence data from eagles in Idaho and California provide no evidence for recent population expansion or bottlenecks.Our findings of low genetic structure and high connectivity are consistent with earlier preliminary comparisons of mtDNA and nDNA structure of 44 eastern and 25 western NA golden eagles that found no evidence for population substructure and shared mtDNA haplotypes across these two regions . HoweverST levels (0.03) also indicated high gene flow. Our study adds to the growing understanding of golden eagle genetics in NA and similar to Doyle et al.’s recent work [In contrast to our findings, Sonsthagen et al.  were ablent work , furtherGenetic research can contribute to understanding the extent to which animals rely on resources and face threats beyond their natal area . Some diNatal dispersal is integral to genetic structure, species demography, and conservation . In IdahBreeding dispersal by adults also could contribute to gene flow among our sample populations. Unsuccessful golden eagle breeders can disperse  and reprUnderstanding the role that floaters and other immigrants play in the population dynamics at a locale is difficult to do without knowledge of the genetics of that portion of the population. Undetected replacement of breeders can mask mortality in breeding locales . For exaGolden eagles can encounter multiple risks  across S1 File(DOCX)Click here for additional data file.


Kala bhat (Black soybean) is an important legume crop in Uttarakhand state, India, due to its nutritional and medicinal properties. In the current study, the genetic variabilities present in Kala bhat were estimated using SSR markers and its variability was compared with other improved soybean varieties cultivated in Uttarakhand state, India.Seventy-five genotypes cultivated in different districts of Uttarakhand were collected, and molecular analysis was done using 21 SSR markers. A total of 60 alleles were amplified with an average of 2.85 alleles per locus. The mean value of gene diversity and PIC was estimated to be 0.43 and 0.36, respectively. The unrooted phylogenetic tree grouped soybean genotypes into three major clusters, where, yellow seed coat (improved varieties) genotypes were grouped in one cluster, while reddish brown (improved varieties) and Kala bhat showed intermixing. Population structure divided the soybean genotypes into six different populations. AMOVA analysis showed 12% variance among the population, 66% variance among individual and 22% variance was observed within individuals. Principal Coordinate Analysis (PCoA) also showed that yellow seed coat genotypes were grouped in one cluster, whereas, the Kala bhat showed scattered distribution and few genotypes of Kala bhat showed grouping with red and yellow genotypes.The different genetic diversity parameters used in the present study indicate that Kala bhat genotypes were more diverse than the yellow seed coat and brown seed coat colour genotypes. Therefore, Kala bhat genotypes can be a good source for the soybean breeding programme due to its better genetic diversity as well as its medicinal properties. Soybean (Glycine max (L.) Merr) is an important legume crop which contains 37–42% protein, 17–24% oil and 35% carbohydrates [In soybean, evaluation of genetic diversity is enhanced by the use of DNA markers. Researchers have studied the genetic divergence among soybean genotypes for various agronomic traits –8 with mBlack seed coat soybean, locally known by different names such as Bhat, Bhatmash and Kala bhat is grown in Kumaon and Garhwal region and in frontiers of Uttarakhand state . In UttaAnalyses of the genetic variation and population structure of Kala bhat genotypes are important for their effective conservation and utilization of the valuable genetic resource. The present study was done to estimate the genetic variability and population structure present in Kala bhat cultivated in Uttarakhand state using SSR markers, as the information on the level of diversity present in local landraces  and population structure had not been studied systematically. The genetic diversity of Kala bhat was also compared with other improved soybean varieties cultivated in Uttarakhand.Seeds of 75 soybean genotypes were procured from NBPGR regional station located at Bhowali, Uttarakhand, India. The Seeds were sown in pots under controlled conditions inside the Green house of NBPGR, New Delhi. Black seed coat genotypes were the landraces  whereas, reddish-brown and yellowish-white genotypes were improved varieties, which were introduced earlier and naturalized as the population in that agro-ecological region. The leaf samples were collected at 3–4 leaves stage for DNA isolation. The details of each genotype along with passport data, National ID, i.e. Indigenous Collection (IC) number, cultivar name, seed colour, district, region and state are given in Table Five grams of young fresh leaves were crushed in liquid nitrogen using a motor pestle and DNA was isolated using CTAB method . The DNATotal 51 SSR markers were selected for initial screening. Gradient PCR was done for each primer with selected soybean samples to standardize the temperature for amplification (Ta). 21 SSR primers , 0.2 μl of Taq DNA polymerase  and 5.6 μl distilled water. Amplification was performed in a thermocycler  using following program; Initial denaturation at 94 °C for 4 min followed by 36 cycles of 94 °C for 30 s, Ta for 45 s, 72 °C for 1 min and a final extension at 72 °C for 10 min. The amplified products were analyzed on 4% metaphor agarose gel for 4 h at a constant supply of 120 V. Gel pictures were recorded using gel documentation System .PCR reaction was set in a total volume of 10 μl containing 2 μl genomic DNA (10 ng/μl), 1 μl of 10X buffer, 0.8 μl of 25 mM MgClSSR bands generated near expected product size were scored visually for all 75 genotypes of Soybean. The band size of amplified products was determined by comparing with 100 bp DNA ladder . The SSR bands scored in soybean genotypes was subjected to statistical analysis. Major allele frequency, gene diversity, heterozygosity and polymorphic information content (PIC) for each locus for SSR markers were calculated using Power Marker 3.25 . In addiTotal 21 SSR primers were used for genetic diversity study of 75 soybean genotypes. A total of 60 alleles were amplified with an average of 2.85 alleles per locus. The number of alleles amplified per SSR primer varied from 2 to 4  of soybean genotypes based on seed coat color was performed to analyze the distribution of genetic diversity between and within the populations. AMOVA analysis showed 12% diversity among populations, 22% diversity within individuals and a maximum of 66% diversity among individuals Table .Table 4SPrincipal coordinate analyses (PCoA) showed two distinct groups represented by Kala bhat and yellow seed coat colour soybean respectively. The brown seed coat colour soybean got distributed in both the groups. The yellow seed coat colour soybean was confined to one group, a similar pattern was also observed during the cluster analysis. The first three axes of PCoA have explained a cumulative percent variation of 33.15% Fig. . This shSince the similar pattern of a grouping of genotypes was observed in the hierarchical cluster as well as in population structure, therefore, the Co-linearity between a grouping of genotypes in hierarchical cluster and model based population structure was confirmed using Venn diagram Fig. . The VenThe assessment of genetic diversity is not only important for crop improvement but also important for the efficient management and protection of the available genetic resource. The reliable and authentic results of molecular profiling have made it preferred in genetic diversity study. The molecular study is less influenced by environmental fluctuations, stands another reason for its preference in breeding . Also, imax and G. soja in China. Ghosh et al. [The SSR primers used in the present study amplified an average number of 2.61 alleles per locus with a gene diversity value of 0.43. Li et al.  reportedh et al.  reportedh et al.  studied h et al.  while sth et al.  reportedh et al.  in Koreah et al.  reportedOur study showed that Kala bhat, which has medicinal properties possess large diversity in comparison to yellow and brown seed coat soybean genotypes cultivated in Uttarakhand, India. This study confirms the hypothesis that the landraces are thought to possess rare alleles and therefore, good genetic diversity. This study also provides useful insights about the Kala bhat (black coloured soybean) among different districts of Uttarakhand and simultaneous isolation of yellow coloured soybean. Improving the genetic base requires an introduction of new alleles into the breeding program, and this can only be done by exploiting the genetic variability found in Kala bhat.