Astragalus edulis (Fabaceae) is an endangered annual species from the western Mediterranean region that colonized the SE Iberian Peninsula, NE and SW Morocco, and the easternmost Macaronesian islands (Lanzarote and Fuerteventura). Although in Spain some conservation measures have been adopted, it is still necessary to develop an appropriate management plan to preserve genetic diversity across the entire distribution area of the species. Our main objective was to use population genetics as well as ecological and phylogeographic data to select Relevant Genetic Units for Conservation (RGUCs) as the first step in designing conservation plans for A. edulis. We identified six RGUCs for in situ conservation, based on estimations of population genetic structure and probabilities of loss of rare alleles. Additionally, further population parameters, i.e. occupation area, population size, vulnerability, legal status of the population areas, and the historical haplotype distribution, were considered in order to establish which populations deserve conservation priority. Three populations from the Iberian Peninsula, two from Morocco, and one from the Canary Islands represent the total genetic diversity of the species and the rarest allelic variation. Ex situ conservation is recommended to complement the preservation of A. edulis, given that effective in situ population protection is not feasible in all cases. The consideration of complementary phylogeographic and ecological data is useful for management efforts to preserve the evolutionary potential of the species. Although one of the central concepts in biodiversity conservation is that genetic diversity is crucial to ensure the survival of species, until now the conservation of plant genetic resources has received less attention than it deserves. Plant-conservation strategies have been commonly based on general premises, leading to more or less standardized systems for evaluating the extinction risks of the species . HoweverBoleum asperum Desv. , is an annual plant that inhabits semidesertic areas of south-eastern Spain, western North Africa, and the Canary Islands (Fuerteventura and Lanzarote) (The species selected for this study nzarote) . It is anzarote) . Specifinzarote) . This spA. edulis; (3) to identify which populations should be prioritized to better represent the genetic singularity and geographic variability for both ex situ and in situ conservation.Our specific aims are: (1) to evaluate the distribution of the genetic diversity among the different populations, and/or geographical areas; (2) to assess the number of populations that should be sampled or preserved in order to establish a representative percentage of the total genetic variation of Astragalus edulis Bunge (Fabaceae) is a short-lived therophytic, hermaphroditic plant. Until now, no information has been available on population sizes, except for the rough estimates by Astragalus edulis is rare , 8 from Morocco (AE7 to AE14) and 3 from the Canary Islands (AE15 to AE17), spanning the entire distribution range of the species ; Fig. 1.Total DNA was isolated following the 2x CTAB protocol  with mintrnG-trnS, trnC-rpoB, and tabF-tabC (A. edulis : 38 from Iberian Peninsula (IP), 17 from Morocco (M) and, 6 from Canary Islands (CI). PCR products were purified using PCR Clean-Up with ExoSAP-IT Kit  following the manufacturer’s instructions. The cleaned amplification products were analysed with a 3,730 DNA Genetic Analyzer capillary sequencer . All sequences were deposited in GenBank  was performed with the software ARLEQUIN 3.5.1.2 . The anadat for R (r),  obtained with ARLEQUIN was used to estimate the total number of populations that should be targeted, according to the n is the number of populations to be sampled to represent a given proportion (P) of the among-population genetic diversity. For A. edulis, a P value of 99.9% of the total genetic diversity was established, to cope properly with high conservation standards.First, the population-differentiation coefficient N2  and expected (Le) probabilities of loss were calculated. The negative natural logarithms (−Log Lo and −Log Le) of those values were plotted (y-axis) against the mean frequency of each rare allele (x-axis) and used to calculate the respective linear regressions. The representative R value  was calculated as the quotient between the slope of the expected regression line and the slope of the observed regression line, i.e. R = m(−Log Le)/m(−Log Lo)  and their associated probabilities of loss, the probability that a sample size on lculated . For thi1 − p)2N  was used present . For eac−Log Lo) .2 vs. large >1 km2, ii) population size: high >1,000 individuals vs. low <1,000 individuals), iii) vulnerability: stable = with no disturbances or with minor disturbances/declining = with clear disturbance of both individuals and habitat/critically declining = major disturbances, with major disturbance of individuals and habitat; and iv) conservation status of the area: protected vs. unprotected.Several qualitative features of the populations and habitat disturbances were recorded during the field work in order to combine them with the measures of genetic diversity. For this, we selected population variables that were accounted as follows  show associations with qualitative population and conservation features. Beforehand, to enhance the robustness of the models, we resampled the cases 10,000 times by bootstrapping using the R boot package  to a maximum of 0.155  and the diversity values were similar across all other populations studied. The total species diversity was 0.108. Regarding rarity, the genetically most distinctive population (DW = 5.713) appeared to be AE16 in Fuerteventura, while the lowest DW values were found in the easternmost part of the Iberian core .A total of 1134 reliable polymorphic bands  were found from the three primer pairs selected for the 360 individuals studied. The final error rate was insignificant (1.67%). The number of rare alleles, DW values and Nei’s genetic diversity values corresponding to each population are given in Both the unrooted NJ tree and the PCoA based on the entire data set  revealedST = 0.289, p < 0.001). The results of a hierarchical AMOVA confirm that a population division into the three geographic groups defined by NJ and PCoA analyses reveals 24.44% of the variance attributed to differences among these geographical areas , while only 10.14% of the variance is attributed to differences among populations within these three geographic groups.AMOVA analysis of the entire data set as a single group  revealedA. edulis was remarkably low , and all the mutations together defined a total of 7 haplotypes. The completeness of haplotype sampling estimated using The length of the three cpDNA regions for 61 individuals was 712 to 926 bp, and resulted in an alignment of 2545 bp (2549 characters with indels coded). The genetic variability within A. edulis would be represented by just 6 populations (N = 5.69). This should be the minimum number of populations to be targeted for suitable conservation. Of the total 1134 alleles detected by the AFLP analysis, 273 complied with the established rarity criteria . Based on the mean frequencies of the rare alleles, as well as on their distribution among populations, the areas where each of these alleles had the highest probability of being found by randomly sampling one population were: IP (124), M (92), and CI (57). Thus, the optimal proportion of populations to be sampled for conservation purposes from each geographical group can be expressed as 0.45 (IP): 0.34 (M): 0.21 (CI).According to our results, 99.9% of the overall genetic diversity through the entire distribution range of criteria . Of thesA. edulis populations (9/17) occupy large areas (>1 km2), but only 7 populations exceed 1000 individuals , and within Morocco the AE8 and AE9 populations , and their lowest levels in AE15 (Lanzarote). Considering that both islands emerged as a single proto-island and remained together as recently as the late Pleistocene , the curJ. thurifera, which shows a wider distribution area, and surprisingly they also parallel those shown by the perennial shrubs B. asperum and V. pseudocytisus ssp. paui, which are very narrow endemics from NE Spain. It is well known that long-lived and outcrossing species retain most of their genetic variability within populations and, by contrast, annual and/or selfing taxa allocate most of the genetic variability among populations  genetic variability are significantly lower  variability, while a smaller percentage of the total variation appeared among populations . Compariulations . Neverthly lower . These dAstragalus edulis has a relatively high number of populations and number of individuals (at least in the large Spanish core), hampering the protection in situ of the entire distribution range of the species, and thus populations need to be identified to apply conservation measures. To select the populations deserving protection, by means of RGUCs, we propose the consideration of factors that could have influenced the evolutionary history of the species lineages :0.34(M):0.21(CI). Considering the diversity and rarity values found for each population based on AFLP data and also this optimal proportion of populations to be sampled for conservation purposes from each geographical group, we would initially recommend the priority selection of populations AE1, AE4 and AE5 (IP), AE8 and AE9 (M) and AE16 (CI). Nevertheless, linking genetic diversity and rarity with qualitative population and conservation features, we have found that Astragalus edulis exhibit a significantly higher genetic diversity and rarity in populations occupying larger areas, with higher numbers of individuals, stable populations, and locations in protected areas. That is the case of populations AE4, AE5 but not of populations AE1, AE9 and AE16.lineages . The selA. edulis genetic diversity and divergence. Haplotypes endemic to restricted areas represent singular genetic variants that may have evolved separately from each other and, therefore, they deserve particular conservation efforts. Within the Iberian distribution range of the species, populations AE4 and AE5 show maximum diversity and rarity values and their sampling may warrant conservation of the Iberian endemic haplotypes III and V, apart from the widely distributed haplotypes I and IV , our final decision on which particular populations from N Africa deserve priority for conservation would probably be more accurate if based on the consideration of these rare or restricted haplotypes. From this perspective, AE10 and AE11 could be prioritized over AE8 or AE9, although this decision should be taken with care given that our sampling may be low despite the results obtained from Dixon’s test. The protection of large populations and smaller dispersed patches usually help preserve genetic integrity and diversity  in shaping I and IV ; Fig. 3.iversity , but somAstragalus edulis at long-term, including the evolutionary potential of its populations, are needed ex situ collections (e.g. botanical gardens and seed banks; Several conservation measures could be implemented for the populations selected, e.g. studies to gather data on spatial distribution, population-size fluctuations, habitat quality, and fitness trends , reinforThe identification of highly representative populations based on genetic data is essential to design appropriate conservation guidelines, especially because this species is listed in a threat UICN category. In biological conservation it is useful to combine molecular data with additional environmental, ecological, and biological data sets in multidisciplinary approaches . The met10.7717/peerj.1474/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.1474/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.1474/supp-3Supplemental Information 3Appendix 1. Probabilities of loss of rare alleles when all populations of Astragalus edulis are considered as one single management unit and preferred sampling area.Click here for additional data file.


Centotheca lappacea is one of the most abundant alien grass species in plantations and in jungle rubber (an extensively used agroforest), but largely missing in natural rainforests. Here, we investigated putative genetic differentiation and signatures for adaptation in the introduced area. We studied reproductive mode and ploidy level as putative factors for invasiveness of the species. We sampled 19 populations in oil palm and rubber monocultures and in jungle rubber in two regions (Bukit Duabelas and Harapan). Amplified fragment length polymorphisms (AFLP) revealed a high diversity of individual genotypes and only a weak differentiation among populations (FST = 0.173) and between the two regions (FST = 0.065). There was no significant genetic differentiation between the three land-use systems. The metapopulation of C. lappacea consists of five genetic partitions with high levels of admixture; all partitions appeared in both regions, but with different proportions. Within the Bukit Duabelas region we observed significant isolation-by-distance. Nine AFLP loci  were under natural diversifying selection. All studied populations of C. lappacea were diploid, outcrossing and self-incompatible, without any hints of apomixis. The estimated residence time of c. 100 years coincides with the onset of rubber and oil palm planting in Sumatra. In the colonization process, the species is already in a phase of establishment, which may be enhanced by efficient selection acting on a highly diverse gene pool. In the land-use systems, seed dispersal might be enhanced by adhesive spikelets. At present, the abundance of established populations in intensively managed land-use systems might provide opportunities for rapid dispersal of C. lappacea across rural landscapes in Sumatra, while the invasion potential in rainforest ecosystems appears to be moderate as long as they remain undisturbed.Intensive transformation of lowland rainforest into oil palm and rubber monocultures is the most common land-use practice in Sumatra (Indonesia), accompanied by invasion of weeds. In the Jambi province,  Elaeis guineensis), and rubber (Hevea brasiliensis), an extensive management system of rubber trees planted into rainforest, also called “jungle rubber”, was established in the early 20th century 38] were C. lappacea inflorescences of genotypes from different collection sites. Selected inflorescences were trapped in sulphite paper bags 2–5 days before flowering and harvested between 45 and 60 days after anthesis. Presence of full caryopses was checked with a Leica S6E stereoscope .Pollen-pistil self-compatibility was tested by bagging Centotheca lappacea from 19 populations were scored and analyzed . However, rubber land-use systems differed significantly from both jungle rubber and oil palm by lower means of panmictic heterozygosity  between geographic distances and genetic divergence among populations , all of them with a diversifying effect. Six out of these nine loci were concordantly depicted by an alternative search algorithm implemented in Mcheza. A principal component analysis (PCA) based on all nine loci from BayeScan shows a tendency of a separation between Bukit Duabelas and Harapan individuals along the first ordination axis  megaspore . No apoCentotheca lappacea. Here, we focused on population genetic diversity, genetic structure, mode of reproduction, and ploidy level in three different agroforest systems in Sumatra.We provide the first study on putative factors linked with invasiveness of the tropical grass C. lappacea in Sumatra mostly distributed within populations . The FST value (0.173), estimating genetic differentiation among populations, is lower than grand means of plant populations in early successional stages (0.37) and more similar to late successional stages  was also reported for sexual Solidago canadensis populations in the invasive range in China, where the species was introduced c. 85 years ago [Chromolaena odorata [C. lappacea, we cannot reconstruct colonization history with our data alone. However, the distribution of diversity within populations would fit better to a scenario of established outcrossing populations rather than to recently founded populations [nd half of the 19th century and was regularly sampled from the 1910s and 1920s onwards [Our population genetic study suggests that genetic diversity is in the invasive grass  markers ). A low ears ago . A scena odorata , cannot ulations . Herbari onwards , which cC. lappacea is in the whole area partitioned into five gene pools, which all occur in both landscapes (Harapan and Bukit Duabelas), but with unequal proportions within the areas. The populations in Harapan were more homogeneous and showed no isolation-by-distance, while in Bukit Duabelas the populations showed a weak but significant geographical substructure, which may relate to a higher landscape heterogeneity in the hillside region of Bukit Duabelas. Altogether, the populations in the Jambi region appear still like a big “metapopulation” with only a weak geographical substructuring and continuous gene flow over the whole area. Interestingly, populations from rubber plantations in both landscapes exhibited a significantly lower gene diversity / heterozygosity compared to jungle rubber and oil palm . Acs , needs Flaveria bidentis [Gene flow among populations of the species in land-use systems may have been enhanced by efficient seed dispersal mechanisms via spikelets with adherent bristles . Diasporbidentis . Also a bidentis .C. lappacea in land-use systems in Sumatra. These basic data can be useful for estimating the potential of the species to invade natural rainforests. The species does occur in jungle rubber, it is also elsewhere shade-tolerant  and thut  and (b) panmictic heterozygosity (Hs). Sample size (N) indicates number of plots which were analysed within each land-use system. Kruskal-Wallis test: (a) H = 4.263, P = 0.119; (b) H = 9.491, P = 0.009, Mann-Whitney comparisons: rubber x jungle rubber (P = 0.010/Bonferroni corrected P = 0.031), rubber x oil palm (P = 0.017/Bonferroni corrected P = 0.051).Box plot summary of the genetic variation within the three land-use systems expressed as mean gene diversities per plot inferred from (a) Nei’s gene diversity (H(EPS)Click here for additional data file.S2 Figr = -0.327, P = 0.172).An estimate of the allelic richness (expressed as band richness after rarefaction to 5), was plotted against sample size. The both variables are not significantly correlated ((EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Osmanthus fragrans Lour., an evergreen small tree, has the rare sexual system of androdioecy , once with wide-spread natural distribution in the areas of the South Yangzi river basin. However, due to excessive human utilization, natural distribution became fragmented and the number and size of natural populations reduced sharply. With four different types of natural populations from the same region as research object, we aim to provide a comparative analysis on the relationships among genetic diversity, sexual system, population structure and size, and geographic isolation by ISSR. In genetic parameters of Ne, He, and I, the LQGC population had the highest value and the LQZGQ population had the lowest value. These indicated that LQGC population showed the highest genetic diversity, followed by QDH and JN population, and LQZGQ population exhibited the lowest genetic diversity. Genetic diversity in populations is closely related to population structure, reproduction mode, and sex ratio. However, there seems to be no obvious correlation between genetic diversity and population size. The results of AMOVA showed that genetic variations mostly occurred within populations. It indicates that no significant genetic differentiation among populations occurs, and geographic isolation has no significant effect on genetic diversity. Osmanthus fragrans Lour. (Oleaceae), an evergreen small tree with the sexual system of androdioecy , is one of the most important ornamental plants and is also a famous aromatic plant in China. The species has been utilized for many years and has a long history of cultivation (more than 2500 years) [ Osmanthus fragrans consists of four cultivar groups, namely, Aurantiacus, Albus, Lutes, and Asiaticus, with approximately 120 cultivars [0 years) . Osmanthultivars . However O. fragrans. According to Chang et al. [ O. fragrans is mainly distributed in the southwest area of China. However, with gradual in-depth field investigation, natural populations were found to be distributed in the south area of Yangzi River basin, especially in Zhejiang, Fujian, Hunan, Jiangxi, Guizhou, Guangxi, and Guangdong provinces [China is a center of distribution and origin ofg et al. , O. fragrovinces –10. Howe O. fragrans, sex ratios of natural populations are mostly 1 : 1  [ O. fragrans is distributed in evergreen broad-leaf forests in limestone mountain areas [The genetic diversity in plants is not only related to internal genetic background and reproductive system but also affected by population structure, spatial distribution pattern, and reproduction mode. Inhrodite) . The genhrodite) –14. Habiin areas –10. Natuin areas –18. O. fragrans have been reported [ O. fragrans and provide the theoretical basis for the protection of natural populations.Intersimple sequence repeat (ISSR) has been widely used in researches on genetic diversity of natural populations of woody plants –21. Relareported –24. ThisA total of 188 samples from four natural populations in Zhejiang Province were collected from Jinning (JN), Longquan , and Jiande  . Approxi O. fragrans, with a large distribution density. Individuals were distributed closely and continuously, with most intervals being less than 0.5 m. Since regeneration seedlings were observed in the forest, the primary reproduction style focused on sexual reproduction. The proportion of male and hermaphroditic individuals meets the ratio of 1 : 1. The LGQC population was distributed in evergreen broad-leaves forest in active limestone mountains and individuals of O. fragrans in a patchy shape were distributed around the forest gaps. The intervals between individuals were more than 1.5 m. A certain quantity of clonal seedlings was found in the forest. Population reproduction included both clonal propagation and seed reproduction. The proportion of male to hermaphroditic individuals was 1 : 1. The LQZGQ population was distributed in evergreen broad-leaves forest also in active scree mountains. The individuals were all hermaphrodite, and the intervals between two individuals were 1 to 5 m. The JN population also with 1 : 1 sex ratio was distributed in evergreen broad-leaves forest with a fragmented distribution, in which severe man-made damage had occurred.The QDH population is basically pure stand of2+), Taq DNA polymerase, dNTPs, and 2000 bp DNA marker for amplification . The following amplification profile was used: predenaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 50°C to 57°C for 45 s, and extension at 72°C for 90 s, with 32 cycles; extension at 72°C for 8 min and holding at 4°C. The PCR amplification reaction was performed on a PTC-100TM PCR instrument (German Biometra Company). The amplified product was subjected to electrophoresis on 1.5% agarose gel. After electrophoresis, the gel was photographed using gel imaging analyzer (US Bio-rad Company).Modified CTAB method  was usedNa) and number of effective alleles (Ne); (3) expected heterozygosity (He); (4) Shannon's Information Index (I). GenAlEx 6.41 [Фst) among populations, Nei's genetic distance (D), and genetic identity (IN) were also calculated. The number of population migrants per generation, which reflects the gene flow level, was also calculated based on the following formula: Nm = (1 − Fst)/4Fst [According to the bands in the electrophoretogram, the positions with the same migration rate on the gel and with DNA bands were recorded as “1,” and those without DNA bands were recorded as “0.” POPGENE 1.32 software  was usedlEx 6.41  softwaret)/4Fst .An average of 4.27 bands with molecular weights ranging from 200 bp to 2000 bp was amplified for each primer. The number of total bands from QDH population was the highest (63), and that from LQZGQ population was the lowest (39) and did not show any specific bands. The proportions of polymorphic loci (PPL) in populations were different, among which QDH population was the highest (96.92%) and LQZGQ population was the lowest (55.38%) . The PPLI) as well as the expected heterozygosity (He) of all populations was ranked in the following descending order of LQGC > QDH > JN > LQZGQ, with an average of 0.332 and 0.217, respectively (Na) of QDH population was the highest (1.938) and that of LQZGQ population was the lowest (1.154), and the estimated allele frequency with number of effective alleles (Ne) of LQGC population was the highest (1.406) and that of LQZGQ population was the lowest (1.331). According to the results of molecular variance analysis (AMOVA), genetic variance mostly occurred within populations and accounted for 85% of the total genetic variance  [Gst = 0.33) [The population genetic structure in plants depends not only on its genetic background and mating system but also on genetic drift, gene flow, natural selection, and so forth –31. The  on RAPD . The tot = 0.21)  and also = 0.33) . The resHe of the four populations ranged from 0.189 to 0.241, and the I ranged from 0.282 to 0.366. Among four populations, LQGC showed the highest genetic diversity, followed by the QDH and JN populations. The LQZGQ population exhibited the lowest genetic diversity.A certain degree of genetic difference existed among populations. In terms of PPL, the QDH population was the highest, and LQZGQ was the lowest. This value is lower than PPL of 19 cultivars by ISSR , but hig Geum reptans, clonal reproduction did not cause severe consequences for population genetic variability and neither did older age or higher elevation of the populations; gene flow and repeated seedling recruitment during succession could have been more frequent than commonly suggested [ O. fragrans; however, population size and geographic isolation due to habitat fragmentation seemed to have no obvious influence on genetic diversity.Field investigations showed significant differences in population structure , regeneration mode, and sex ratio among these four populations. Sex ratios  in LQGC, QDH, and JN population were all 1 : 1 . All induggested . The QDH O. fragrans, genetic variances of natural populations mostly existed within populations and genetic differentiation among populations was small. Among populations, LQGC showed the highest genetic diversity and LQZGQ population exhibited the lowest genetic diversity. Therefore, as a protective strategy, high genetic diversity populations and populations carrying special genetic information should be protected primarily. The LQGC population had the highest genetic diversity, complicated population structure, and rich individual trait variations. The QDH population was distributed in limestone areas with relatively stable habitats, whose genetic diversity was also high and contained the most specific bands. The PCA results indicated that LQGC and QDH populations contained almost all genetic information of all populations carried. Consequently, the LQGC and QDH populations should be protected primarily. Simultaneously, the PCA results and amplified specific bands showed that LQZGQ carried the specific genetic information being absent in the other three populations. Thus, protecting LQZGQ population will help in the preservation and analysis of genetic diversity in this species.In


FST genetic distances did not reveal statistically significant differences in haplogroup frequency distributions among populations from the different regions. The genetic differences observed among regions were, however, consistent with the colonization history of the country. The sample from the Northern region presented the highest Native American ancestry (8.4%), whereas the more pronounced African contribution could be observed in the Northeastern population (15.1%). The Central-Western and Southern samples showed the higher European contributions . The Southeastern region presented significant European (86.1%) and African (12.0%) contributions. The subtyping of the most frequent European lineage in Brazil (R1b1a-M269) allowed differences in the genetic European background of the five Brazilian regions to be investigated for the first time.The non-recombining nature of the Y chromosome and the well-established phylogeny of Y-specific Single Nucleotide Polymorphisms (Y-SNPs) make them useful for defining haplogroups with high geographical specificity; therefore, they are more apt than the Y-STRs to detect population stratification in admixed populations from diverse continental origins. Different Y-SNP typing strategies have been described to address issues of population history and movements within geographic territories of interest. In this study, we investigated a set of 41 Y-SNPs in 1217 unrelated males from the five Brazilian geopolitical regions, aiming to disclose the genetic structure of male lineages in the country. A population comparison based on pairwise  Due to the way it was formed, the Brazilian population exhibits some peculiar characteristics. In the early sixteenth century, Brazil came under the influence of three main groups: the Native Americans who already inhabited the region at the time; the Portuguese, who arrived in the territory in the mid-1500s, and the Africans that were brought by the Portuguese during the slave trade period. By this time, French and Dutch settlers also arrived in an attempt to colonize the region, but they were soon expelled by the Portuguese. From 1808 on, other people migrated to the country, including Spaniards, Italians, Germans, Syrians, Lebanese and Japanese, also contributing to the formation of the current population [Brazil is a country of continental extension, and it is currently divided into five main geopolitical regions  with diverse histories of colonization and settlement, a fact that is reflected in the genetic structure of the current Brazilian population –8.The existing heterogeneity among Brazilian populations was mainly shaped by differential maternal heritage in a biased mating environment. Indeed, the early European colonization involved mainly sailors, soldiers, deportees and wood traders, and the migration of European women was insignificant, which favored mating between European men and indigenous or African women ,4.When using genetic markers with low mutation rates and different inheritance patterns, a clear heterogeneity can be observed in the genetic contribution of each parental population to the different geopolitical regions of the country as a result of diverse colonization histories –8. HowevDue to the high mutation rate of the Y-STRs and the predominant European male contribution to the Brazilian population, most studies based on the analysis of Y-STRs have failed to detect statistically significant differences between admixed populations in the country –11. In tDespite having a lower intra-population discrimination power than Y-STRs, Y-SNPs are more powerful in revealing the differences among populations characterized by diverse levels of admixture. The non-recombining nature of the Y chromosome associated with a high geographical specificity of the haplogroups formed by sets of SNPs distributed along the chromosome make the Y-SNPs the most appropriate genetic markers to detect population stratification of male lineages in admixed populations .Until now, a limited number of studies using Y-SNPs have been performed to characterize admixed populations in Brazil, and they have focused on small population groups, reporting only estimates of the portions of Native American, European, and African contributions to the current populations –19. ThesNevertheless, no attempts have been made to detect the differences concerning the different European sources of the male lineages currently existing in Brazil, by increasing the resolution of the main European haplogroups.Therefore, to better characterize the male lineage background of Brazil, in the present work a large set of highly informative Y-SNPs was for the first time investigated in representative samples from all Brazilian geopolitical regions. Moreover, to discriminate contributions from different countries in Europe, a group of Y-SNPs was selected to increase the resolution inside haplogroup R1b1a-M269, the main representative of the European male lineages present in Brazil. In this way, we were able to not only determine the different continental source of the Y chromosomes present in Brazilian admixed populations belonging to the five Brazilian regions but we could also predict the origin of the samples inside R1b1a-M269 to investigate the differences in the European background of these regions.Samples involved in this study are long-lasting anonymized DNA extracts previously obtained with informed written consent from healthy individuals for research purposes.This work follows the ethical principles stated in the Helsinki Declaration (2000) of the World Medical Association, and it was approved by the institutional review board of the Laboratório de Genética Humana e Médica, Instituto de Ciências Biológicas, Universidade Federeal do Pará.A total of 1217 non-related male samples were collected to represent the five geopolitical regions of Brazil  analysis using the SNaPshot kit (Thermo Fisher Scientific Inc.). To avoid genotyping all SNPs in each sample, a hierarchical approach based on the phylogeny reported by Van Oven et al.  was usedWe first performed an initial screening of the major clades present in our population sample by typing all samples using the Multiplex Major South American previously described by Geppert et al. .Subsequently we performed four specific multiplexes according to the previous results: (a) samples with the derived allele at M213 were further genotyped for the markers included in the Multiplex GIJ ; (b) samHaplogroup frequencies were determined by direct counting. Population comparisons and genetic diversities estimated according to Nei  were perFST and the significance was tested with 10,000 permutations. Pairwise genetic distances were visualized in two dimensional space using the multi-dimensional scaling (MDS) analysis included in the StatSoft, Inc. (2007) program, STATISTICA , ver.8.0 (www.statsoft.com). This software was also used in Principal Component Anaysis (PCA).Population pairwise genetic distances were conducted based on −10 [Migration rates for the Brazilian populations were estimated using ADMIX 2 . This so−10 . All theA total of 382 out of the 630 samples belonging to haplogroup R-M207 were genotyped for the downstream SNPs M269, L23, U106 (S21), S116 (P312), U152 (S28), M529 (S145), M153 and M167 (SRY2627).The frequency of each sub-haplogroup detected inside haplogroup R1b1a-M269 were compared with those found in Portugal, Spain, France, Italy, Germany, the Netherland and Turkey. The sub-haplogroup frequencies for the European populations were calculated using published data by Myres et al.  except fA total of 22 different haplogroups were detected in the whole sample, revealing the three major continental origins of the current Brazilian population, namely from America, Europe and Africa .More than 50% of the Y chromosomes belong to the R1 branch, namely to the sub-lineage R1b1a-M269, which presents high frequencies throughout Western Europe. Previous studies have shown that Y-STR haplotypes are rather uninformative towards detecting R1b1a-M269 sub-haplogroups (and geographic origins), due to a recent expansion of this haplogroup in Europe, in a relatively short time period ,33. HoweHaplogroup R1b1a-S116*, which has its greatest frequency in Iberia was, by far, the most frequent haplogroup observed in our sample, representing 32.5% of the Y chromosomes investigated ,35. OtheOther typical European haplogroups were also observed, namely J-P209, I-M170, G-M201, KLT-M9 and R-M207*. When adjusted to the total number of European lineages, these haplogroup frequencies are in the same range of those usually observed in Iberian populations .Haplogroup E-P170 can be observed in Africa, Europe and the Middle East. Inside haplogroup E, some lineages originating in sub-Saharan Africa were detected in Brazil, namely E1a-M33, E1b1a-M2 and its M191 sub-lineage, E1b1a-M154 and E1b1b-M35, accounting for 8.2% of all Y chromosomes investigated here. E1b1b-M78 can be found at similar frequencies in Europe and Africa; hence, samples from this haplogroup were not included when estimating ancestry proportions. E1b1b-M81 exhibits a high prevalence in North Africa and Iberia, the latter being the probable origin of most Y chromosomes in Brazil belonging to this haplogroup. A low frequency of E1b1b-M123 was found in our sample, a haplogroup that is spread all over Europe and also frequent in some West Asian countries such as Turkey, Syria and Lebanon .The haplogroup Q1a2-M346 and its sub-lineages, mainly Q1a2-M3, are almost completely restricted to Native American populations . These lOnly very few and isolated populations in Brazil did not receive European admixture, either through early contact with the Portuguese or through more recent contact with other Europeans or European descendants in Brazil –19. As eHaplogroup diversity remained constant among the five geopolitical regions of the country , with a ST genetic distance did not reveal statistically significant differences in haplogroup frequency distributions among populations from different regions in Brazil . The estimates for the European male contributions are 95.2% and 93.6% for the Central-West and the South, respectively. A clear difference emerges when examining the distribution of European haplogroups in these two samples, with haplogroup E1b1b-M123 ten times more frequent in the Central West than in the South; and haplogroup J-P209 is also two times more frequent in the Central-West sample. Moreover, haplogroups E1b1b-M123 and J-P209 present higher frequencies in Central-Western Brazil  than in Portugal .R1b1a-M269 is the most frequent haplogroup in Europe, presenting a cline distribution with high frequencies in the West, decreasing towards the East. The highest frequencies of this haplogroup were reported for populations in Ireland . Its preR1b1a-M269 is also present in significant proportions in South American populations as the most frequent haplogroup in the great majority of admixed, non-Native populations from Brazil, Colombia and Argentina ,53,54.In a survey of 75 Y chromosomes from African Americans and European Americans belonging to the haplogroup R1b1a-M269, Sims et al.  were ablFurther studies using SNPs to increase the discrimination between lineages inside haplogroup R-M207 were performed in large samples from West Asian and European populations, revealing different gradients for R1b1a-M269 sub-clades inside Europe ,31. The Concerning the European lineages, the haplogroup R1b1a-S116* was the most frequent in the five geopolitical regions of the country emphasizing the strong influence of the early Portuguese colonization ,31.ST values calculated among Brazilian and some European populations representing potential sources of male immigrants to Brazil including Portugal, Spain, Italy, Germany and Netherlands. The sample from Turkey was also included to represent the immigration from the Middle East because no data are available for other countries in this region with reported historically affinities with Brazil (like Syria and Lebanon) [To investigate possible signs of differential European colonization in the five regions of Brazil, the frequency distributions of the R1b1a-L23*, U106, S116*, U152, M529, M153 and M167 sub-haplogroups with respect to total R1b1a-M269 were compared by means of pairwise FST values were found between Brazil and the remaining populations, associated with very low values of non-differentiation probabilities. In the MDS plot of the pairwise genetic distances  were found in the Eastern populations. Historically, the Eastern regions were important destinations of African slaves, which entered Brazil through the port cities of Salvador (in the northeast Atlantic coast), Rio de Janeiro and Santos (in the southeast Atlantic coast) .A detailed analysis of the European Y chromosome gene pool also allowed for the detection of differences among Brazilian populations and the evaluation of the genetic impact of the early Portuguese colonization and the more recent migrations from other European countries. The Northeast region showed the largest genetic distance to Portugal and a slight increase in the frequency of haplogroup R1b1a-L23*, which can be explained by the Eastern European influx .Most of the Germans and Italians that arrived in Brazil during the nineteenth and early twentieth centuries settled in the South . The freThe Central-Western region was the last to be settled in the country by migrants from other Brazilian regions, mainly the Northeast and Southeast. This situation attracted many Arab traders who arrived and settled. The high frequencies of haplogroups R1b1a-L23*, E1b1b-M123 and J-P209 found in this region can be explained by the influx from Near East, since these lineages are frequent there –36.ST genetic distances among populations from the different regions did not reveal statistically significant differences in haplogroup frequency distributions, which can be explained by the high predominance of Western European Y chromosomes in all populations. However, the close agreement between the genetic differences observed among the geopolitical region and the history of colonization and settlement of the country supports a possible population stratification of the paternal lineages in Brazil that needs to be further investigated in larger sample sets of each region.Population comparison based on pairwise FS1 Fig(PDF)Click here for additional data file.S2 FigThe haplogroups are named in accordance with Van Oven et al. .(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Tablep-values are indicated in red; for a significance level of 0.00042, obtained by applying the Bonferroni correction for multiple tests.Significant non-differentiation (PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableFST values were calculated based of the frequencies of R1b1a-L23*, R1b1a-U106, R1b1a-S116*, R1b1a-U152 and R1b1a-M529 haplogroups that are indicated in (PDF)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Dikerogammarus villosus has colonized most of the European main inland water bodies in less than 20 years, having deteriorating effect on the local benthic communities. Our aim was to reveal the species phylogeography in the native Black Sea area, to define the source populations for the colonization routes in continental Europe and for the newly established UK populations. We tested for the loss of genetic diversity between source and invasive populations as well as along invasion route. We tested also for isolation by distance. Thirty three native and invasive populations were genotyped for mtDNA  and seven polymorphic nuclear microsatellites to assess cryptic diversity (presence of deeply divergent lineages), historical demography, level of diversity within lineage , and population structure. A wide range of methods was used, including minimum spanning network, molecular clock, Bayesian clustering and Mantel test. Our results identified that sea level and salinity changes during Pleistocene impacted the species phylogeography in the Black Sea native region with four differentiated populations inhabiting, respectively, the Dnieper, Dniester, Danube deltas and Durungol liman. The invasion of continental Europe is associated with two sources, i.e., the Danube and Dnieper deltas, which gave origin to two independent invasion routes (Western and Eastern) for which no loss of diversity and no isolation by distance were observed. The UK population has originated in the Western Route and, despite very recent colonization, no drastic loss of diversity was observed. The results show that the invasion of the killer shrimp is not associated with the costs of loosing genetic diversity, which may contribute to the success of this invader in the newly colonized areas. Additionally, while it has not yet occurred, it might be expected that future interbreeding between the genetically diversified populations from two independent invasion routes will potentially even enhance this success.The amphipod  Biological invasions are the inherent symptom of global changes and a major threat to biodiversity –3. AlienMolecular markers proved to be powerful tools in tracking invasion patterns and dynamics , 10. TheDikerogammarus villosus , also known as the ‘killer shrimp’, which colonized most of the European main inland waters in less than 20 years [Such information is still scarce in case of some prominent invaders. One example is the amphipod 20 years –33. This20 years –37, char20 years –41 as we20 years –45.D. villosus have been proposed [Occurrence of the species in its native area is associated predominantly with brackish lagoons (limans) and lower reaches of large rivers draining to the Black Sea. Its phylogeographic history in the native area is unknown, although it could bring key information for understanding current invasion dynamic, as in the case of other Ponto-Caspian intruders ,47. The proposed . The easD. villosus has not been found in the waterways joining the Vistula and the Oder and the dates of first records of D. villosus from both river systems suggest that it was present in the Oder prior to colonization of the Vistula and Bug rivers [D. villosus’s presence in northern Germany and western Poland. If correct, there would be two fronts of invasion in Poland (eastern and western), presently 150 km apart and likely to get in contact in the near future [First, the origin and genetic diversity of populations found in northern and central Europe  is not clear. Colonization of these sites could be a westward expansion of the eastern route, from the Bug and Vistula rivers, based on the fact that other Ponto-Caspian invasive species followed such direction . Howeverg rivers . Therefor future .These two fronts might be genetically distinct. First, they originated from different parts of the native area . Second,D. villosus. So far, only three studies dealt with its genetic diversity. They focused either on molecular identification of D. villosus versus two congeneric species present in the Danube [D. villosus invasion. In particular, genetic variation within the eastern route has not yet been explored. Additionally, the genetic variation of the populations recently established in UK [A second question is the global impact of the invasion process on the genetic variation of e Danube ,52 or one Danube . The lated in UK , as wellD. villosus populations from the western part of the Black Sea basin (native area), as well as invasive populations from the presumed western and eastern colonization routes and from UK were genotyped for mtDNA  and seven polymorphic nuclear microsatellites, in order to answer the following questions: (1) What is the species phylogeography in the native Black Sea area, including the assessment of cryptic diversity (presence of deeply divergent lineages), historical demography, level of diversity and genetic differentiation between populations being potential sources for the two presumed colonization routes? (2) Is it possible to associate a distinctive genetic signature to the two presumed colonization routes in continental Europe? (3) Is there a loss of genetic diversity between source populations and colonized areas, and is there a loss of genetic diversity and an isolation by distance along the colonization routes? (4) What was the source(s) for the newly-established UK populations and was this colonization associated with a genetic bottleneck?In this study, Dikerogammarus villosus was collected from 33 sites, both in the native area (hereafter N) and the invaded part of Europe  and COI (654 bp) which were concatenated to perform analyses. Haplotypes were retrieved using DnaSp v5 both for individual markers and concatenated data [DNA from 876 samples was extracted with a standard phenol-chloroform method after Hillis et al. . Air-drilgorithm  using Bited data . HaplotySeven microsatellite loci (msat) were used as nuclear co-dominant molecular markers: DikS, DikF , Dv11, DArlequin 3.5.1.2 [Mega 6.2 [Gammarus was applied for the analyses [jModelTest2 [To visualize molecular divergence of mtDNA haplotypes, a Minimum Spanning Network was generated using  3.5.1.2 . PairwisMega 6.2 . For anaMega 6.2  was usedMega 6.2 . The Hasanalyses . Two runanalyses , with reanalyses . To add analyses  with 100delTest2 . ML analdelTest2 .To reveal historical demography in the Ponto-Caspian region we used 133 individuals from nine localities . In ordeAr) and private allelic richness (PAr) corrected for a common sampling size using rarefaction approach [Hp-Rare 1.1 [Ar was tested using the non-parametric Mann-Whitney U-test in Statistica 10 [HO), expected heterozygosity (HE) and fixation index (FIS) were calculated, when appropriate, for microsatellite markers using Fstat [FST estimators: ΘST with Tamura-Nei distance for mtDNA [Arlequin, statistical significance being measured using 10000 permutations. Genetic diversity and FST were assessed either pooling sampling sites, or not, according the hypothesis tested, e.g. between fronts in Poland.Diversity was assessed by calculating: (1) allelic-haplotypic (msat/mtDNA) diversity (k), (2) allelic richness  were repeated 20 times. Each run used a burn-in of 500000 iterations, a run length of 750000 iterations. All simulations were performed using the admixture and correlated allele frequencies models with no prior information. Selection of most probable value of K relied on the ΔK method developed by Evanno et al. [Population structure was also analyzed using individual-based Bayesian clustering method implemented in re 2.3.4 . SimulatGoogleEarth v.7.1.2. IBD was tested using Mantel test between FST / (1- FST) and geographic distance as recommended by Rousset [GenePop on the Web 4.2 [Based on 20 sites along the WR we tested if microsatellite differentiation increased positively with distance between sites  but also if diversity  was associated with geographical distance from the source area (Danube delta). The distances were estimated using  Rousset  for test Web 4.2  and ISOLD. villosus in the Durungol liman experienced steady growth for the last 20 ky, while populations of the remaining two lineages in the native area remained stable for most of the last 30k years, with accelerated growth starting less than 10 ky ago  16S and COI mtDNA sequences . First, 0 ky ago .Ar = 5.3 and 5.73 respectively); the locality harboring also the highest private allelic richness (PAr = 5.3 and 1.08) (a priori) source areas of invasion, i.e. Dnieper and Danube deltas, did not exhibit the same level of mtDNA diversity (Ar = 2 and 3) but harbored some specific haplotypes (Ar = 3.94 and 3.57) and low private allelic richness (PAr = 0.05 and 0).The highest diversity for mtDNA and msat was observed in Durungol (nd 1.08) . The potplotypes , Fig. 2.FST = 0.180 and ΘST = 0.693) and the lowest between Danube and Dniester (FST = 0.048 and ΘST = 0.103). The results of Bayesian clustering suggest that the four selected areas may represent four genetic clusters, although the division is not strict. The Durungol and Dnieper populations are the most homogeneous ones, while the Dniester and Danube populations show symptoms of migration or very recent common ancestry  compared to differentiation among sites belonging to different routes (0.157 < FST < 0.215)  and Danube (6+7+8+9), respectively , while d< 0.215) . Bayesia< 0.215) .2 = 0.0047, P = 0.42).Both fronts in Poland had the same level of diversity for msat compared to their putative source in the native region i.e. in Dnieper and Danube respectively . This isST < 0.298) and msat (0.138 < FST < 0.172), for pairwise comparisons between ER in Poland and two pooled sites in the Netherlands (23+24) or any UK site. On the opposite, pairwise comparisons between two sites in the Netherlands and each UK site showed no significant differentiation for mtDNA (FST < 0.035) than between the UK site and ER in Poland (0.138 < FST < 0.172). The UK site 33 showed a less conclusive picture for FST , the most common in continental Europe, occurred in UK. Haplotype 4, while common in ER, was absent from the UK. In Grafham Water site  we observed no loss of diversity for msat compared to the Netherlands with rsus 3.8 . Diversir values .D. villosus, is complex, with multiple routes, and that the loss of genetic diversity during the course of colonization is weak. We evidenced that colonization of the UK was originating from one (here Western) out of the two genetic units associated with continental invasion.The Ponto-Caspian region has been recognized as the most prominent donor of non-indigenous hydrobionts to Europe and to the North American Great Lakes system. Their taxonomic spectrum is wide including amphipods, mysids, cladocerans, gastropods and fishes –82. PhylD. villosus in its native region. The 17 mtDNA haplotypes showed shallow divergence with an overall K2p genetic distance of 0.0009, far below the threshold of 0.03–0.055 identified between crustacean species [Level of cryptic diversity for invertebrates in the Ponto-Caspian region is highly variable. Deep level of divergence, but below the species threshold, was observed for e.g. cladocerans  and mysi species . Such loST) between populations is high in the Black Sea area. The turbulent Pleistocene hydro-geological history of the region with recurrent changes of sea level and salinity may be among the most powerful driving forces explaining this pattern [Although shallow, the divergence between mtDNA haplotypes is geographically structured with lineage A including a set of 8 haplotypes (out of 17) and being restricted to the Durungol liman. This pool of haplotypes separated from others ca 200 kyr BP. In addition, the other sampled areas are also characterized by high private haplotypic richness. Overall, the level of differentiation  between the four native sampled areas, that were divided in four genetic clusters in Bayesian analyses. If the Durungol liman is clearly isolated from the other sampled areas, the latter show connectivity as pointed out by unclear Bayesian assignment of some individuals. One of the explanations can be a shorter geographic distance between these populations. The north-western Black Sea is also the shallowest and least saline, due to massive sedimentation and inflow of riverine waters from e.g. Dnieper and Danube rivers [The msat analyses pointed out high level of differentiation  not even encountered in the native area, probably due to its very low frequency. For microsatellites, we found no loss of diversity along the route and no isolation by distance. Globally, it suggests that no bottlenecks occurred along WR. Similar conclusions were made by Müller et al.  and WattD. villosus first colonization [Only one site (site 16) in the middle section of River Vah, was characterized by both low diversity and high level of differentiation from other sites. Strong founder effect is likely to explain this pattern as, probably, the site was sampled very recently after nization , 100.D. villosus into UK in 2010 was noticed in popular media [FST and Bayesian clustering of msat allowed us to exclude the populations from ER as donors for the UK sites. In addition, the haplotype 4, both frequent and private to ER, was not detected in UK, although our sampling size was limited in this case.The overseas introduction of ar media . It provar media , 102. BoD. haemobaphes, has already been recorded in UK [D. villosus and D. haemobaphes it has been estimated that more than 60% of the UK waterbodies is suitable and vulnerable to colonisation by these two invaders [Diversity in any UK population was not different from the continent and no bottleneck effect was observed. Apparently, the propagule pressure was high enough to alleviate diversity loss. We are not able to conclude whether the introduction to UK was a single event followed by secondary colonization or multiple introductions. Anyway, the killer shrimp is spreading very efficiently throughout the UK. Furthermore, another congeneric invader, the “demon shrimp”, ed in UK . Based oinvaders , 104–107invaders , due to invaders .Dikerogammarus villosus in the Black Sea native region and presence of two differentiated source populations, i.e. the Danube and Dnieper deltas. These sources are associated with two independent invasion routes (Western and Eastern) in continental Europe for which no loss of diversity is observed. We can expect further spread of the killer shrimp in continental Europe, even in smaller tributaries. MacNeil & Platvoet [D. villosus as mainstay in smaller tributaries. This may pose a threat to native gammarids occupying such refuges [Our results identified impact of the Pleistocene sea level and salinity fluctuations on the phylogeographic structure of Platvoet  pointed  refuges .The UK population has probably originated in the Western Route and despite very recent colonization, no drastic loss of diversity was observed. This recent overseas conquest provides rather non-optimistic message, accounting that the UK authorities implemented preventive biosecurity protocols and risk assessments. The Great Lakes of North America are likely to be the next step, since other Ponto-Caspian invertebrates already managed to reach them , 111.Finally, the Dniester native area is characterized by high msat allelic diversity . Thus, even if at the moment it is not a source population for the colonization of Europe, it may act as a donor for other source areas if the anthropogenic transport increases, enhancing the local genetic diversity.S1 Table(DOCX)Click here for additional data file.


Bufo bufo) inhabiting a naturally strongly fragmented habitat at the Northern fringe of the species’ range: islands offshore the Norwegian coast. A total of 475 individuals from 19 populations (three mainland populations and 16 populations on seven adjacent islands) were genetically characterized using nine microsatellite markers. As expected for a highly fragmented habitat, genetic distances between populations were high , with however little differences between populations separated by ocean and populations separated by terrestrial habitat (mainland and on islands). Despite a distinct cline in genetic variation from mainland populations to peripheral islands, the study populations were characterized by overall high genetic variation, in line with effective population sizes derived from single‐sample estimators which were on average about 20 individuals. Taken together, our results reinforce the notion that spatial and temporal scales of fragmentation need to be considered when studying the interplay between landscape fragmentation and genetic erosion.The last decades have shown a surge in studies focusing on the interplay between fragmented habitats, genetic variation, and conservation. In the present study, we consider the case of a temperate pond‐breeding anuran (the common toad  In ecological and evolutionary research, populations with pronounced spatial structure are often the focus of genetic investigations. Studies can be conducted across an entire species’ range, for example to provide a better understanding of the evolutionary history underlying an observed distribution , and all islands are inhabited by humans. Six of these seven islands are connected with other islands or the mainland through 16–100 year old road bridges, and pairwise geographic (line‐in‐sight) distances between populations ranged between 1.20 km and 73.78 km. Although we lack historical records about the studied B. bufo populations we assume that they are of natural origin.Field work was conducted in an area of approximately 30 × 35 km south of Bergen Fig. , and forca. 4 m above present sea level. A transgression took place between 8500 and 7200 years B.P. when the shore level rose to ca. 11 m above the present level. Between 7200 and 6000 years B.P. the shore level was almost constant before a slow regression. Since all islands have peaks between 50 and 750 m above the present sea level, they exist since the last ice age without land bridges between them, at however varying size dynamics (detailed data not shown).The postglacial sea level changes in the coastal area of western Norway are characterized by uplifting of land masses after the recession of glaciers Kaland , and a sDNA samples were collected through dip‐netting for larvae or through collecting eggs before raising them in water‐filled containers until late egg stages, and stored in absolute ethanol. When performing the sampling, care was taken that the whole pond shore was evenly sampled and that the number of eggs per egg string was minimized; whenever possible, sites were visited several times. Long‐term monitoring of a subset of populations revealed that population size estimates based on single clutch counts are not representative for true population size (data not shown). We therefore lack good approximations of population census sizes.Bbuf11, Bbuf13, Bbuf15, Bbuf24, Bbuf46, Bbuf49, Bbuf54, Bbuf62, Bbuf65). Each 10 mL PCR contained 10–50 ng DNA, 5 pmol (5 mmol/L) of each primer, 0.15 mmol/L of each dNTP, 1.5 mmol/L MgCl2, and 0.5–1.0 U Taq polymerase  in the manufacturer's buffer. The PCR profiles were 94°C for 2 min, followed by 39 cycles of 94°C for 30 sec, the primer‐specific annealing temperatures as in Brede et al. .DNA extractions were performed using standard phenol–chloroform procedures  and expected (He) heterozygosities, and departures from Hardy–Weinberg equilibrium at each locus and population were computed with genepop 4.0, using the implemented Markov Chain method (106 runs) to obtain unbiased estimates of Fisher's exact tests . Isolation‐by‐distance scenarios were tested using Mantel tests to correlate Fst and log‐transformed geographic distances as implemented in the software IBDWS version 3.23  allowed to discern between within‐ and between‐island differentiation using analysis of molecular variance (AMOVA) as implemented in Ne) were obtained. We used the linkage disequilibrium approach , 2/19 populations for locus Bbuf15, and 1/19 populations for locus Bbuf54; all other loci and populations revealed no evidence for null alleles or large allele dropouts (detailed data not shown). Given the geographic location close to the northern border of the species’ geographic range as well as the assumed ecological isolation, the studied populations are characterized by moderate to high overall genetic variation ; the overall PCR success rates across genotyped loci was 78%. The analysis conducted in Fst values were significant for all but three comparisons which involved population T3 for which only five samples were available . The analyses of molecular variance (AMOVA) revealed similar patterns for the four scenarios of population grouping  on rocky outcrop islands also along the Scandinavian coast  estimates obtained previously (Scribner et al. Ne values for amphibian populations are usually accompanied by high standing amounts of genetic variation (Beebee Ne values in comparison to population census sizes when census sizes are low (genetic compensation, Jehle et al. Ne were rather similar across populations, supporting that genetic compensation can also act for B. bufo; we however lack detailed population census size data to further investigate this assumption. The genetic mating system of B. bufo is expected to depend on sex ratios and population densities (Sztatecsny et al. Ne values for distal populations such as on offshore islands can be higher relative to census sizes than for core populations. Despite islands having a long history as natural laboratories for evolutionary studies, we still miss a comparative investigation which compares Ne/N values between island and mainland populations.The obtained estimates of n Beebee , which wWhat do our inferences tell us about conservation concerns for populations in landscapes recently fragmented by humans? We demonstrate that amphibian populations are able to maintain significant levels of genetic variation in naturally strongly fragmented landscapes, despite clear genetic effects of fragmentation though high differentiation. While genetic erosion in naturally small populations is expected to accumulate over time, it can be compensated for when the environment is stable (e.g. Kaeuffer et al. None declared.


Entomacrodus vomerinus  is a demersal fish with planktonic larvae endemic to this marine province, inhabiting shallow tidal pools in continental and oceanic reef environments. We evaluated the population structure, genetic diversity and gene flow of E. vomerinus using mitochondrial data (CYTB and COI) and nuclear  DNA sequences. We sampled a total of 85 individuals, comprising 46 from three oceanic archipelagos with varying distance from the coast  and 39 from two localities in northeastern Brazilian coast (Rio Grande do Norte—RN and Bahia—BA). Multilocus analysis revealed the presence of three Evolutionarily Significant Units—ESUs , which are in accordance with distinct marine ecoregions. Coalescent analyses showed that the central ESU has a larger effective population size than the other two, suggesting strong asymmetries in the genetic diversity across the species range. Moreover, they showed that gene flow is highly asymmetric, suggesting a source-sink dynamics from the central ESU into the remaining ones, in agreement with oceanic currents. Together, these results provide insights in the evolutionary mechanisms facilitating diversification in this marine province.The Tropical Southwestern Atlantic is characterized by prominent ecosystems with large-scale oceanographic complexity. Yet, the evolutionary processes underlying genetic differentiation and connectivity in this region remain largely unknown.  Phylogeographic studies seek to reveal biogeographical history of species and the habitats they occupy via the spatial association between clusters of alleles with geographic/ecological barriers, and via estimates of historical population size ,2. A morMany studies of marine species assume that the presence of planktonic larval period gives a greater dispersal ability, increasing gene flow between geographically isolated populations and hence reducing the population structure. However, a meta-analysis involving molecular data of marine organisms from 300 studies suggests that a long larval period is not a good predictor of gene flow between populations . Thus, iEntomacrodus vomerinus  is endemic to the Tropical Southwestern Atlantic province  and sedentary behavior, almost exclusively inhabiting shallow tidal pools  and Cytochrome B [CYTB]) and a nuclear gene (the exon Rhodopsin [RHO]) were amplified by polymerase chain reactions (PCR). The COI fragment was amplified using the primers FISH-BCL  and FISHd HMVZ16 . A 25 μlablished . All PCRAfter purification, the amplicons were sequenced using forward primers in the Sequencing facility from Centro de Biociências, Universidade Federal de Pernambuco. RHO sequences that showed heterozygous sites were additionally sequenced with the reverse primer to confirm polymorphisms.All DNA sequences were edited and aligned using Bioedit 7.2 . SubsequMitochondrial genes (COI and CYTB) were concatenated and incorporated as a single non-recombining marker in all analyses (mtDNA hereafter). Due to the presence of polymorphic sites in the diploid RHO sequences, all alleles were reconstructed using PHASE 2.1  implemenh), nucleotide diversity (π)] was assessed by measuring haplotype and nucleotide diversity. Changes in population size were tested using Fu’s Fs [Molecular diversity indices [number of polymorphic sites (S), haplotype number (H), percentage of private haplotypes (Hp), haplotype diversity . Three competing hypotheses of population structure were tested using the analysis of molecular variance (AMOVA): 1) an hypothesis contrasting insular against coastal habitats that is congruent with morphological differentiation (SS+FE+RA / RN+BA); 2) an hypothesis based on the four ecoregions sampled (SS / FE+RA / RN / BA); and 3) an hypothesis based on the three ecoregions (SS / FE+RA / RN+BA). This last hypothesis considers the coastal sampled area as the same ecoregion because localities in BA are situated in the border of two ecoregions. Those tests were performed in Arlequin 3.5 [The genetic structure among the sampling sites of quin 3.5 .6 iterations in each) of K from 1 to 10 was used to determine the most probable number of populations (or clusters).Haplotype networks were obtained for mtDNA and nuDNA data with PopART 1.7 , in ordeA splits into two extant populations that may differ in population size (Ne1 and Ne2), and where migration in both directions (m1 and m2) may occur after divergence. Comparing this 6-parameter model to a simpler 4-parameter model without migration, a likelihood ratio test can be used to test for ‘migration’ versus ‘isolation’ between descendent taxa [Demographic parameters were estimated using the multilocus coalescent method implemented in IMa2 . In thisent taxa . This apBecause this method assumes no intragenic recombination, this assumption was tested on phased data using the PhiPack software , with a Entomacrodus vomerinus. A total of 15 sites were polymorphic (seven parsimony informative), resulting in 12 haplotypes. CYTB yielded 580 bp sequences from 71 individuals, with 17 polymorphic sites (ten parsimony informative) in 15 haplotypes. Concatenated mitochondrial data included 1,124 bp from 65 individuals with 29 polymorphic sites in 18 haplotypes.Sequences of COI of 544 bp were obtained from 72 individuals of RHO resulted in 441bp sequences from 83 individuals, and eight polymorphic sites. PHASE analysis recovered the alleles of 81 diploid sequences . Two individuals from BA were removed from the analysis because their likelihood probabilities of the estimated allele sequences were lower than 70%. Nine alleles were identified with six polymorphic sites (five parsimony informative) .The number of haplotypes was higher in the coastal area  relative to the oceanic islands (9 and 5). The majority of haplotypes (72%) recovered from mtDNA were exclusive of a certain locality, although they were mainly singletons. Coastal habitats presented more private haplotypes  than the insular habitats (66% and 60%).In mtDNA, haplotype diversity showed very similar values at both continental and island habitats, while the diversity of nuDNA was higher in the insular habitats .Statistical significance of both neutrality tests suggested a population expansion event in BA (indicated by nuDNA). Bayesian Skyline Plot indicated no change in demography history .ST) in the data from the three genetic markers .The Mantel test showed no correlation between geographic and genetic distances , except between the closest oceanic islands (FE and RA) and between the coastal sites (RN and BA). Genetic differentiation was strongest between SS and all other localities.The pairwise For nuDNA . Both mtCT , and P values marginally significant (0.07 and 0.06).The three scenarios tested with the AMOVA were not significant  for eithThe haplotype network of mtDNA data shows a high number of haplotypes shared between FE and RA individuals as well as between RN and BA, and a few shared with the other localities. For mtDNA, only one haplotype (hap1) was shared among all localities. Moreover, two haplotypes (hap2 and hap4) were found in all localities except in SS, also evidencing low genetic diversity in SS, with only two haplotypes, including one exclusive that is several mutational steps distant from the others The nuDNA showed a very similar scenario, but the allele diversity was lower. There were two alleles (hap1 and hap2) shared among all localities, three alleles shared by coastal specimens  and four alleles exclusive for a certain locality  .ST results, suggesting three genetically different groups. K = 3 has the highest posterior probability in all the nine runs (33% to 38.5%), followed by K = 2 and K = 4  and Central populations (FE+RA), and to a second comparison involving the Central (FE+RA) and Coastal (RN+BA) populations. There was no significant signal of intragenic recombination. The estimation of demographic parameters reached very high values of ESS and consistent estimates across all runs, irrespective of the priors, indicating a good sampling of the parameter space and convergence in the same result.For both comparisons, there were marked differences in effective population size (Ne) and population migration rates (2Nm) . In the Entomacrodus vomerinus [Previous morphological study pointed out differentiation between the oceanic  and coastal individuals of omerinus , suggestomerinus ,17.E. vomerinus. However, small sampling sizes could also result in low statistical power of the AMOVA analysis to detect population differences [When testing the morphological hypothesis using an AMOVA, we did not find support for insular and coastal groups, suggesting either low statistical power based on single locus analysis or that a different population structure approach could better explain the patterns of genetic differentiation in ferences .ST) based on mtDNA and nuDNA data was more consistent with evolutionary divergence within E. vomerinus in three distinct populations (SS / FE+RA / RN+BA). In fact, when using the multilocus assignment test implemented in Geneland .Notably, the North population (SS) was always the most differentiated locality both in mtDNA and nuDNA, suggesting that it might be genetically isolated from the remaining populations. This ecoregion (SS) seems to be a result of a young active tectonism and are composed by very rugged scarps with very few rockpools . It is lE. vomerinus, particularly the break separating the North group, were also detected in other reef fish species, such as Chromis multilineata (Pomacentridae) and Caranx lugrubis (Carangidae) [E. vomerinus. The three genetic clusters inhabit geographically and ecological distinct regions. Therefore, oceanographic and ecologic features have resulted in three Evolutionary Significant Units (ESUs) that need to be taken into consideration when accessing current population dynamics and conservation efforts.Similar phylogeographic breaks to those reported here in angidae) ,57, suggEntomacrodus vomerinus.Genetic diversity might not be equally partitioned among populations due to important demographic processes that occur after divergence, such as differences in effective population size or differences in the amount and direction of gene flow. Using coalescent methods , Fig 3, E. vomerinus has a relatively low abundance, 0.07 fishes/20 m2 [2 in FE island [E. vomerinus, which can be explained by the small suitable intertidal area for the species.Effective population size (Ne) was notably different between ESUs. Both in North vs Central and in the Central vs Coastal comparisons, the Central ESU always showed to be much larger than the other ESUs. This suggests that, irrespective of the census size of each population (N), the number of reproducing individuals contributing to the gene pool (Ne) is larger in the Central ESU. This can be explained by differences in habitat suitability and species abundance, which is known to strongly vary along the species distribution. In the coast, es/20 m2 , which mes/20 m2 . In the E island . In factE island  and it cE. vomerinus. Since adults are sedentary and criptobentic, the connectivity between populations probably occurs mainly by larval transport [Population migration has also shown to be highly asymmetric. In both pairwise comparisons, the Central ESU does not receive gene flow either from the North or from the Coast ESU. Nevertheless, there is significant gene flow from the Central ESU into the others , suggestransport , which aransport  and wherransport ,63 movesEntomacrodus is unknown, this period in other species of the Blenniidae family varies between 50 days  and 29 s pholis ). Thus, s pholis , and fros pholis ,66.E. vomerinus. Most importantly, our results show that it acts as an important source of gene flow, while the remaining populations act as sinks, highlighting the importance of this ESU for the evolutionary dynamics in this species.Together, these results suggest that the Central ESU, occupying the Fernando de Noronha and Rocas Atoll, has a key role in the genetic connectivity among populations of Entomacrodus vomerinus showed three ESUs based on mtDNA and nuDNA data, North (SS), Central (FE+RA) and Coastal (RN+BA). Of those, three oceanic archipelagos  are in marine protected areas (MPAs): the Rocas Atoll Biological Reserve (RA), Fernando de Noronha Marine National Park (FE) and Fernando de Noronha Environmental Protected Area . The Central ESU is the main source of gene flow to the Coastal and North ESUs.Evolutionarily Significant Unit (ESU) is a qualitative criteria based on the distribution of population genetic diversity and is used as a basis for species conservation efforts . EntomacE. vomerinus genetic diversity along the Brazilian coast. Even though this species is not currently threatened, it is essential to conserve their shallow reefs habitats that are under different human-mediated pressures, ensuring the preservation of genetic variability of the tidepool communities, including some endemic species, mainly in the oceanic islands [E. vomerinus may reflect more general biogeographic patterns, reinforcing its importance as an indicator species for these ecoregions.This result corroborates the importance of these Central oceanic islands MPAs for the maintenance of  islands . FurtherGreater efforts should be targeted to population bioecological assessments on the Brazilian coast in order to understanding the low density in this region, which also reflects is lower genetic variability and effective population size. Future efforts may be able to test if the observed low genetic diversity is due to naturally small populations limited by competition with other species, or if the species respond to human-mediated coastal impacts.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Ramayana, one of the Great epics of India. Though there have been studies about their affinity based on classical and haploid genetic markers, the molecular insights of their relationship with other tribal and caste populations of extant India is expected to give more clarity about the the question of continuity vs. discontinuity. In this study, we scanned >97,000 of single nucleotide polymorphisms among three major ancient tribes mentioned in Ramayana, namely Bhil, Kol and Gond. The results obtained were then compared at inter and intra population levels with neighboring and other world populations. Using various statistical methods, our analysis suggested that the genetic architecture of these tribes (Kol and Gond) was largely similar to their surrounding tribal and caste populations, while Bhil showed closer affinity with Dravidian and Austroasiatic (Munda) speaking tribes. The haplotype based analysis revealed a massive amount of genome sharing among Bhil, Kol, Gond and with other ethnic groups of South Asian descent. On the basis of genetic component sharing among different populations, we anticipate their primary founding over the indigenous Ancestral South Indian (ASI) component has prevailed in the genepool over the last several thousand years.Kol, Bhil and Gond are some of the ancient tribal populations known from the  Vedas, which are four in number, namely Rigveda, Yajurveda, Samaveda and Atharvaveda. Then, there are Puranas, Upanishads, Brahmanas and Aranyakas, of which Vedas are said to be the precursors [Vedas as well as the historical dates for the various Puranas, Upanishads and epics [Knowledge about the past comes through different disciplines where researchers look at history through different lenses. And in many cases, these interdisciplinary studies land on the same conclusions ,2. Howevecursors . There ind epics –10. A cond epics ,11–15.Ramayana era, Indian society was well-stratified [Ramayana, particularly in the portions known as the Ayodhyakanda, Aranyakanda and Kishkindhakanda [Our survey on mythological sources has revealed detailed information about the ancient Indian society structure as well as relations of different tribal and caste groups and their rituals ,11,16–18ratified ,21,23–26dhakanda ,20,22–27dhakanda .The Bhils are primarily from Central India and speak the Bhil language . They haMore than 25 years of genetic research on Indian tribal and caste populations involving classical markers to mtDNA/Y chromosome and more recently autosomes, have indicated complex demographic history of the subcontinent ,30–39. Avs. discontinuity of the genetic thread connecting the different populations of India. Second, keeping in mind the pivotal information extracted from Ramayana, we look specifically into the question: whether and to what extent the three major tribes  share their genetic ancestry among them as well as with other contemporary caste and tribal populations?Therefore, in the present study, we make an attempt to evaluate two schools of thought emerging from the current scenario. The first school suggests that the tribal people are the aboriginal inhabitants, while the later migrants, i.e., the Dravidians followed by the Aryans have pushed them back in to small pockets in South India –52. Accoidentity by state (IBS) scores for all pairs of individuals [This study was performed using control samples collected, genotyped and published for various population studies conducted in the last few years  37–39,4,61–63. Aividuals . We usedividuals  in orderividuals  and ADMIividuals , we thinsmartpca programme (with default settings) of the EIGENSOFT package [We performed PC analysis using  package  in orderFst distance measure by following the methods as described by Cockerham and Weir [f3 statistics [Mean pairwise differences between different population groups were computed using and Weir , Phylip and Weir  and MEGAand Weir  were useand Weir  was usedatistics , taking atistics , we madeatistics . A coancatistics , fineSTRFst (population differentiation) algorithm [We combined hundreds of thousands of autosomal markers generated from different studies  37–39,4,61–63 anlgorithm  and drewlgorithm ,71, rootlgorithm ,74,75. TFst analysis. The PCA on Eurasians placed Indian populations between East and West Eurasia  and ADMI Eurasia . The cli Eurasia . FurtherConsistent with previous observations ,38, the Ramayana, we have considered these tribal populations to be ancient inhabitants of India, surviving from the times of the Stone Age [vs. discontinuity of prehistoric heritage. In case of continuity, we should see largely similar genome composition among contemporary caste and tribal populations of modern India. On the other hand, in case of discontinuity, these tribal populations should show a unique genome composition or they should emerge as an outliers in our cluster based analysis. Our extended analysis on Fst, PCA and ADMIXTURE showed similar genome composition of these tribal populations, carrying both the ANE and ASI components  suggested that most of the derived alleles of Bhil, Kol and Gond are overwhelmingly shared with Indian caste and tribal populations  migration either of the present Dravidian or the Indo-European speaking populations to the subcontinent. We also concluded that the Indian populations preserve strong genetic signatures in support of a common ancestry. The studied tribal populations do share large number of genome among theselves as well as from o caste and tribal poulations. Notebly, the placement of various populations along the Indian cline is not solely governed by the geography, but also by the caste-tribe interaction and various other selectional forces. These patterns point to a complex demographic history of the subcontinent which has been shaped in-situ by admixture events at different time scale, as well as by intricate geographical heterogeneity and long term effect of several evolutionary forces.S1 FigIn the inset, the heatmap showing the inter and intra regional genetic affinity of the three tribal populations under investigation.(TIF)Click here for additional data file.S2 Figf3 values are plotted on Y axis against the X- targeted populations on X axis. C_Asia- Central Asia, IN_IE_Caste- Indian Indo-European Caste, IN_IE_Tribe—Indian Indo-European Tribe, IN_DRA_Caste- Indian Dravidian Caste, IN_DRA_Tribe—Indian Dravidian Tribe, IN_AA- Indian Austroasiatic (Munda).The (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.