Corallium rubrum. Our analyses reveal partially contrasting PEIs between shallow and mesophotic populations separated by approximately one hundred meters, suggesting that red coral populations may potentially be locally adapted to their environment. Based on the effective population size and connectivity analyses, we posit that genetic drift may be more important than gene flow in the adaptation of the red coral. We further investigate how adaptive divergence could impact population viability in the context of warming and demonstrate differential phenotypic buffering capacities against thermal stress. Our study questions the relevance of the deep refugia hypothesis and highlights the conservation value of marginal populations as a putative reservoir of adaptive genetic polymorphism.Studying population-by-environment interactions (PEIs) at species range margins offers the opportunity to characterize the responses of populations facing an extreme regime of selection, as expected due to global change. Nevertheless, the importance of these marginal populations as putative reservoirs of adaptive genetic variation has scarcely been considered in conservation biology. This is particularly true in marine ecosystems for which the deep refugia hypothesis proposes that disturbed shallow and marginal populations of a given species can be replenished by mesophotic ones. This hypothesis therefore assumes that identical PEIs exist between populations, neglecting the potential for adaptation at species range margins. Here, we combine reciprocal transplant and common garden experiments with population genetics analyses to decipher the PEIs in the red coral,  In the human-dominated Earth ecosystem, global change raises the question of the capacities of populations to cope with the increase of selective pressures  are stronger in the shallow (0–30 m depth) compared to the mesophotic (30–100 m depth) zone, resulting in an intense decline of shallow compared to mesophotic populations within species  within the upper edge of this species’ bathymetric range. We combine reciprocal transplants and We conducted the study at two localities: Riou Island (RI)  and Palazzu Island (PZ)  Fig.. These lThe following experiments were designed to account for the low abundance and low population dynamics of the red coral and the limitations induced by fieldwork conducted via SCUBA diving in the mesophotic habitat (40 m).We performed two independent RTEs Fig. at the tA CGE Fig. was condBased on the biological characteristics of the red coral  and the depths of the experiments, which limit the fieldwork, we selected three phenotypic traits to assess the impact of environmental conditions on the colonies. We analyzed the mean maximum growth in diameter in the RTEs and the level of tissue necrosis and the survivorship of the colonies in the CGE. These traits are a good proxy for fitness because the reproductive output in gorgonians is function of their size  were genotyped for 12 microsatellites loci. Due to extensive partial or total mortality of the colonies in the CGE, genotyping was not performed. PCR amplifications were conducted following Ledoux et al. . To avoin = 9999) avoiding the normality assumptions required for parametric tests. Analyses were carried out using PRIMER v.6 .To represent the genetic distances between the samples, we produced a phenogram using POPULATIONS 1.2.30  set to 4 (number of populations) using a locprior model . The computations were performed with LDNe under the random-mating model, excluding rare alleles with frequencies of less than 0.02 and using the jackknife option to estimate confidence intervals  allows the estimation of the relative impacts of neutral and selective processes on populations divergence. Divergent selection is suggested when QST exceeds FST whereas when FST is greater than QST, stabilizing selection drives the evolution of the considered trait. If the trait evolves neutrally, then QST should be equal to FST ; and c represents the proportion of the total variance presumed to occur because of additive genetic effects across populations  and PST were compared based on their 95% confidence intervals (95% CI). The 95% CI of θ was computed using bootstraps over loci in FREENA. We estimated the 95% CI of the PST by resampling over the maximum growth of the individuals within each sample for the RTEs and over individual necrosis rates within each sample for the CGE.To complement the field experiments, we therefore computed  Brommer . We firsPST-FST comparisons are questioned due to intrinsic characteristics of the two measures . Considering that the PST-FST comparisons were conducted based on the results obtained for populations in the same habitat, the c value is likely to be low. We implemented this sensitivity analysis for the PST-FST comparisons that were significant under the null hypothesis (see Results). We evaluated the robustness of each comparison by estimating the c/h2 value beyond which PST is significantly higher than FST; Brommer θ and FSTall. Considering the intrinsic characteristics of the PST and the FST previously exposed, caution is necessary when interpreting PST-FST comparisons. In this context, Brommer ( Hedrick . Neverth Hedrick  or D (Jok D Jost  in PST-F Brommer . Accordi Brommer  and Brom Brommer  recommen Brommer  showed thttp://cran.r-project.org/), and an example of the scripts used for the PST-FST comparisons and the sensitivity analyses is presented in The computations were performed in R (P < 0.01), but not the origin of the sample (P = 0.09). A significant interaction between these two factors (P < 0.01) was highlighted. In the two habitats, the local sample exhibited a significantly higher value than the foreign one , the transplantation depth (P = 0.55) or their interaction (P = 0.62). Therefore, we could not rejected the null hypothesis of no “local vs. foreign” differences   Table. MoreoveP > 0.05 after FDR correction). Significant linkage disequilibrium was observed in PZ-40 for one pair of loci (Mic22-Mic26) after FDR correction. The mean (± standard deviation) observed heterozygosity over the loci ) ranged from 9.4 ± 7.6 for PZ-20 to 12.4 ± 9.2 for RI-40, showing a mean value of 10.7 ± 1.3 over the four samples . The pairwise values and 95% CI for θ ranged from 0.02 (95% CI: 0.01–0.03) for RI-20 vs. RI-40 to 0.14 (95% CI: 0.09–0.19) for RI-20 vs. PZ-20. The exact tests for genotypic differentiation were significant at the global level and for the six pairwise comparisons . This PST was significantly higher than θ and FSTall with PST for c/h2 = 1 was 0.06 (95% CI: 0.01–0.24), which was not significantly different from θ and FSTall. Regarding the RTE in Palazzu and considering the shallow habitat and c/h2 = 1, the PST was 0.29 (95% CI: 0.01–0.68), which was not significantly different from θ and FSTall. Regarding the RTE in Palazzu, the PST values computed in the shallow and mesophotic habitat were 0. For the CGE, the PST under the null hypothesis was 0.44 (95% CI: 0.42–0.49), which was significantly higher than θ and FSTall. The θ and shifted to 0.15 when considering FSTall  and vely Fig.. In the Tall Fig..Patterns and processes linked to ecology and evolution in marginal habitats are central in our understanding of the responses of natural populations facing global change  was significantly higher than the FST in the shallow habitat in Riou. The sensitivity analyses showed that this difference between the two measures of differentiation might be considered as relatively robust. Indeed, the θ or FSTall was lower than or slightly superior to 0.2, which is a threshold that has been suggested for realistic approximations of QST by PST  was significantly less affected by a thermal stress than the population dwelling at mesophotic depth (40 m). Our study substantially refines these results because the differential response was observed in situ and between individuals originating from shallow and mesophotic populations separated by approximately one hundred meters. This is concordant with the independent functioning of populations suggested by the significant genetic structure reported at this spatial scale (Ledoux et al. FST and the PST, was significant and robust (The PST-FST comparisons, genomic approaches should also be considered to confirm the role played by divergent selection in the observed patterns. Nevertheless, we demonstrated the occurrence of contrasting PEIs with potential for local adaptation at fine spatial scales in a survey involving only four populations. This implies the existence of putatively important adaptive diversity in marginal red coral populations.Complementary studies involving more populations and phenotypic traits are needed to generalize our conclusions. Considering the limitations of the From a conservation perspective, genetic drift is an important evolutionary force due to its impact on genetic diversity and inbreeding and, ultimately, on the evolutionary potential of a population (Frankham et al.


Gossypium hirsutum are classified under seven races. Five Mesoamerican races would have been derived from the wild race ‘yucatanense’ from northern Yucatán. ‘Marie-Galante’, the main race in the Caribbean, would have developed from introgression with G. barbadense. The racial status of coastal populations from the Caribbean has not been clearly defined. We combined Ecological Niche Modeling with an analysis of SSR marker diversity, to elucidate the relationships among cultivated, feral and wild populations of perennial cottons. Out of 954 records of occurrence in Mesoamerica and the Caribbean, 630 were classified into four categories cultivated, feral (disturbed and secondary habitats), wild/feral (protected habitats), and truly wild cotton (TWC) populations. The widely distributed three first categories cannot be differentiated on ecological grounds, indicating they mostly belong to the domesticated pool. In contrast, TWC are restricted to the driest and hottest littoral habitats, in northern Yucatán and in the Caribbean (from Venezuela to Florida), as confirmed by their climatic envelope in the factorial analysis. Extrapolating this TWC climatic model to South America and the Pacific Ocean points towards places where other wild representatives of tetraploid Gossypium species have been encountered. The genetic analysis sample comprised 42 TWC accessions from 12 sites and 68 feral accessions from 18 sites; at nine sites, wild and feral accessions were collected in close vicinity. Principal coordinate analysis, neighbor joining, and STRUCTURE consistently showed a primary divergence between TWC and feral cottons, and a secondary divergence separating ‘Marie-Galante’ from all other feral accessions. This strong genetic structure contrasts strikingly with the absence of geographic differentiation. Our results show that TWC populations of Mesoamerica and the Caribbean constitute a homogenous gene pool. Furthermore, the relatively low genetic divergence between the Mesoamerican and Caribbean domesticated pools supports the hypothesis of domestication of G. hirsutum in northern Yucatán.Perennial forms of  Gossypium spp.) is unique among crop plants in that four species have been independently domesticated in four different regions of the world: two tetraploids, G. hirsutum L. in Mesoamerica, G. barbadense L. in South America, and two diploids, G. herbaceum L. in Arabia and Syria and G. arboreum L. in the Indus Valley of India and Pakistan Cotton  range encompasses most of Mesoamerica and the Caribbean, with two centers of morphological and genetic diversity, one in Southern Mexico-Guatemala, considered a primary center of diversity, and one in the Caribbean, where some introgression took place with G. barbadensePhylogenetic investigations in G. hirsutum exhibits a diverse array of perennial forms, which Hutchinson G. barbadense into Central America and the Antilles and its introgression with G. hirsutum in these areas G. hirsutum. The remaining four races present a more restricted geographic distribution, with race ‘palmeri’ in the Mexican states of Oaxaca and Guerrero, race ‘morrilli’ in the central Mexican plateau, race ‘richmondi’ along the Pacific side of the Isthmus of Tehuantepec, and race ‘yucatanense’ limited to the northern coast of Yucatán. The latter is known only as a small, highly branched, sprawling shrub forming a dominant constituent of undisturbed beach strand vegetation. Hutchinson In these two regions, G. hirsutum has been the subject of considerable debate. On one hand, most germplasm collections came from man-made habitats, such as field plots and house yards, or highly disturbed habitats, such as roadsides and secondary vegetation, indicating that spontaneous cotton plants were escapes from cultivation. Furthermore, morphological differentiation appears similar and parallel for both landraces and feral plants The persistence of wild populations of G. hirsutum, Stephens In a study of the effects of domestication in G. hirsutum in the Pacific Ocean. Fryxell and Moran G. hirsutum. He presented a hypothesis relating this coastal adaptation and capacity for diffusion via ocean currents to the significant mobility of shorelines during the Pleistocene. In 1979, Fryxell further developed his views in his monograph on the Malvaceae G. hirsutum has been further complicated by the recent description of wild populations of G. hirsutum in Paraguay Long-range seed dispersal also explains the presence of G. hirsutum and its wild progenitor.” Thus, the initial stages of cotton (G. hirsutum) domestication would have taken place in northern Yucatán and the human-mediated transfer of the first ‘punctatum’ cottons out of the species' natural range would have triggered the process of concomitant differentiation into new and improved races, agronomic developments, and long range germplasm diffusion. This process would explain the current distribution of G. hirsutum diversity. The SSR study of Lacape et al. Despite its importance for cotton genetics and breeding, the question of truly wild cottons has spawned relatively few genetic studies. In their RFLP study, Brubaker and Wendel G. hirsutum diversity by successive waves of diffusion of genetic and agronomic developments, from northern Yucatán to inland Yucatán (race ‘punctatum’), then to southern Mexico and Guatemala (race ‘latifolium’), and finally to all Mesoamerica and the Caribbean, imply an early cotton domestication. This is consistent with the contributions of historical linguistics and archaeology. Thus, words for cotton can be reconstructed in Proto-Otomangue, a language that was spoken in Central Mexico at least 6500 BP The views of Brubaker and Wendel G. hirsutum has been generally accepted and it is found practically unchanged in the most recent syntheses G. hirsutum is a perennial whose regressive forms thrive in disturbed human habitats and xerophytic secondary vegetation, domestication was not necessarily a linear process, moving from littoral strands to the agricultural field through the dooryard. Wendel et al. G. hirsutum achieved widespread distribution and regional differentiation as a wild plant prior to domestication, or if it was widely distributed as a perennial semi-domestic by the pre-Columbian people from a much smaller native range”. Casas et al. in situ domestication process is a mosaic of habitats and useful plant populations with particular morphological, genetic, and even reproductive characteristics, according to management intensity. Similar management practices may have been used for cotton. Stephens The domestication and diffusion scenario proposed by Brubaker and Wendel If we recognize that Caribbean wild cotton populations may have been involved in the domestication process, we must also question the distinction between a primary centre of diversity in Mesoamerica and a secondary centre in the Caribbean. The strong dominance of race ‘Marie-Galante’ in the latter region, as well as in southern Central America and northern South America, poses the question of its origin and even of its possible separate domestication G. hirsutum is not only crucial for understanding the biogeography of tetraploid cottons, and their evolution and diffusion under domestication, but also for the continuation of the domestication process. Further improvement of the crop requires both a better exploitation of the available germplasm and better genetic tools to manipulate important economic traits The question of the natural distribution of G. hirsutum was recently published by Wegier et al. G. hirsutum populations, feral cotton (escaped from cultivation), and presumably or truly wild populations of races ‘yucatanense’ and ‘punctatum’. Potential distribution of G. hirsutum was predicted for modern climatic conditions as well as for climatic parameters modeled for the Last Glacial Maximum (LGM). The underlying idea is that the original distribution of wild cotton in the early Holocene was necessarily related to its distribution during the Pleistocene, following an approach validated by several studies We present here a double approach to investigate this question, combining Ecoclimatic Niche Modeling (ENM) and neutral genetic markers to assess whether coastal cotton populations are “truly wild,” and investigate their relationship with inland perennial cottons. ENM methods derive an envelope for the environmental requirements of a taxon from a set of its occurrence localities. They have provided a powerful tool for investigating the ecology and distribution of both plant and animal species. An ENM study on As the ENM study confirmed the particular ecology and “truly wild” status of a number of coastal cotton populations, SSR neutral genetic markers were then used to characterize them and investigate their relationship with neighboring feral cottons.G. hirsutum, i.e., Mexico, Central America, and the Eastern Caribbean (from the coasts of Venezuela to Florida through the Antilles). From now on, we will collectively refer to this region as Mesoamerica and the Caribbean. Geographical and ecological information was extracted from the CIRAD cotton germplasm database and records Our ENM study focused on the centers of diversity of The information associated with the collections/observations was also used to classify cotton occurrences according to their status on a wild to cultivated scale, using four categories: ‘cultivated’ (fields and dooryards), ‘feral’ , ‘wild/feral’ (plants found in preserved habitats and/or forming persistent populations), and ‘truly wild’ . This categorization is partially analogous to that used by Stephens For each occurrence record, 19 bioclimatic variables were extracted from WorldClim, a package consisting of global surfaces of climate, with a 2′30″ grid resolution (corresponding roughly to 4.4×4.6 km) G. hirsutum observations, discarding those variables whose contribution appeared marginal. The factors with an eigenvalue above 1 were retained and a normalized varimax rotation was applied to maximize the sum of the variances of the squared loadings, simplifying the interpretation of the results. The different categories of populations were then plotted on the principal components plane to visualize and compare their ecoclimatic range.For ENM, we chose the widely used Maxent machine learning method. It estimates the probability distribution of maximum entropy (i.e. closest to uniform) subject to the constraint that the expected value of each environmental variable (or its transform and/or interactions) under this estimated distribution matches its empirical average G. hirsutum at LGM, the MIROC climatic model http://www.worldclim.org/) and used on a dataset restricted to the ‘truly wild’ category.To predict the potential distribution of G. hirsutum cotton populations used for SSR genotyping comprised 110 feral and wild accessions supplemented by a modern cultivar, ‘FM966’  of young leaves using the MATAB protocol He = 1−Σpi2 where pi is the frequency of the ith allele.Twenty-six SSRs showing strict and unambiguous bi-allelic patterns  were selected. The 26 SSRs were mapped on 18 of the 26 chromosomes . ExpecteThe data matrix of bi-allelic codings for the 26 SSRs and 111 genotypes was imported into the DARWin5 software A total of 954 datapoints were gathered, of which 630 could be ascribed to our four categories . Figure Among the 544 datapoints from Central and Mesoamerica, few have been assigned to a geographical race: 2 for race ‘morrilli’ (state of Guerrero), 8 for ‘palmeri’ (Guerrero), 5 for ‘richmondi’ (Oaxaca), 41 for ‘punctatum’ (Yucatán peninsula and Socorro Island), and 32 for ‘yucatanense’ (state of Yucatán). Albeit poor, this information is consistent with their original description by Hutchinson For the Eastern Caribbean (410 accessions from Venezuela to Florida), most observations were from breeders, so the racial composition is much better documented. It shows a strong dominance of race ‘Marie-Galante’ (278 acc.). The only other identified race is ‘punctatum’, ascribed to the TWC category (64 datapoints) or, exceptionally, to the ‘wild/feral’ category (one datapoint). In our dataset, these TWC are classified as ‘punctatum’, following the early views of Hutchinson G. hirsutum for the last three millennia at least, i.e. a period of very active agricultural development, during which modern climatic conditions were already established G. hirsutum appears related to the diffusion of race ‘Marie-Galante’, as the favorable areas close to the Guatemalan-Salvadoran border and in western Nicaragua show good correspondence with the distribution of this race, presented by Stephens Given the relative over-representation of wild and feral materials in our sample, The great majority of ‘wild/feral’ populations , maximal temperature of the warmest period (Bio5), precipitation of the wettest and driest periods (Bio13 and Bio14), precipitation seasonality (Bio15), and precipitation of the driest and warmest quarters (Bio17 and Bio 18).G. hirsutum was not observed in regions that are both cooler and wetter  the wild populations of G. barbadense (North and South of the Guayas estuary) and (ii) the wild tetraploid species G. darwinii, a close relative of G. barbadense, endemic to the Galapagos islands Both the ENM and factorial analyses clearly show that TWC populations of G. tomentosum, while an unusual wild form of G. hirsutum is locally common in Wake Island G. tomentosumGossypium species (probably G. hirsutum) G. hirsutum, only Fiji and Samoa do not appear climatically fit for this species according to our extrapolation; however, this can be related to the rarity of G. hirsutum var. taitense Roberty in both archipelagos In the fourth favorable area (not shown), further west into the Pacific, Worldclim coverage is incomplete, particularly for small atolls, so all climatically suitable sites could not be detected. Among those cases where the extrapolation results can be compared to data from the literature, worth mentioning are the Hawaiian Islands , Wake Island, the Republic of Kiribati, Fiji, Samoa, and French Polynesia. Indeed, Hawaiian Islands are home of the endemic wild tetraploid G. hirsutum and the actual distributions of wild tetraploid species  provides a very interesting example of ecological niche conservatism in evolution The excellent correspondence between areas potentially favorable to wild forms of G. hirsutum for LGM climates, i.e. about 21,000BP. Sea level was ca. 125 m lower at that time, and rose markedly from 17,000 to 7,000 BP G. hirsutum. In contrast, three areas show a considerable extension at LGM, with many more favorable emerged lands: (i) northern Yucatán, (ii) southern Florida, the Bahamas and Virgin Islands, and (iii) the western shore of Venezuela and a small area on the northeastern Colombian shores. On the whole, G. hirsutum distribution was probably much more extended in the Caribbean and in the Gulf of Mexico during late Pleistocene and early Holocene. The main picture is consistent with the hypothesis of Fryxell Further south, in equatorial and southern America, LGM climatically favorable areas appear essentially similar to modern ones, except for the Brazilian Northeast, which was less favorable  than in G. hirsutum populations as described above; although several similar populations could not be sampled. It appeared very early in the analysis that these TWC populations, including the ‘yucatanense’ population of Yucatán as well as those from diverse places in the Caribbean, showed no racial or geographic differentiation, so we have pooled them in the following presentation. In our sample the ‘feral’ group was represented by 53 ‘Marie-Galante’ accessions of northern South America and the Caribbean and by 15 other accessions  countries  ensure aHe values varied among markers, confirming previous results He shows only limited differences between the different races/categories (He is slightly higher in wild accessions (28.2%) as compared to feral ones (22.0%). The genetic dissimilarity was also higher within the TWC group (D = 0.51) than within the feral group (D = 0.38)  .Both distance-based methods implemented with DARwin, NJ classification  and prinThe STRUCTURE analysis and ΔK method of Evanno in situ hybridization cannot be dismissed, as they show an important level of admixture (<50% membership to TWC): W105 (acc. INC035) from Socorro Island, W58 (acc. AS0653) from Yucatán, W86 (acc. BPS1157) from Bonaire, W148 (acc. BPS1239) from Puerto Rico, and W95 (acc. BPS1225) from Dominican Republic. For the latter, the collector mentioned a “different” phenotype with orange pollen and yellow petals The 42 accessions from TWC populations form a fairly homogenous group  and S3 wG. hirsutum or G. barbadense.”The 53 accessions (22 countries) of race ‘Marie-Galante’ have an average membership of over 81%. This group encompasses the same geographical distribution as the TWC group except for Mexico . Four ‘MLastly, the mostly ‘punctatum’ branch of feral cottons presents the lowest level of admixture (>97% membership). With 16 accessions, this group includes 12 ‘punctatum’ accessions , one representative of Mexican races, ‘morrilli’, ‘richmondi’ and ‘palmeri’, and the modern cultivar (FM966) from Australia. The homogeneity of this group indicates that the genetic differentiation among Mexican races G. hirsutum still exist, but they are ecologically and genetically distinct, occupying a narrow and well defined habitat. Their genetic distinctiveness and homogeneity invalidate any racial or specific distinction among TWC populations of G. hirsutum, such as their classification into race ‘yucatanense’ in northern Yucatán and into race ‘punctatum’ in the Caribbean. A fortiori, our results do not support any particular status for the wild perennial cottons from the Dominican Republic, which were the most ‘inland’ collections among our TWC samples (see G hirsutum race ‘ekmanianum’) or specific status, as G. ekmanianum Wittmack 6 (other 5 tetraploid species being denoted as AD1 to AD5). Our results do not support such proposals as these specimens fall within the overall range of TWC accessions  demonstrates that the major driver organizing this collection is the status, feral or wild, of the cotton population, rather than any geographical factor. Thus, ENM and genetic analyses converge in discriminating TWC populations from feral populations, as assessed in our categorization exercise. We can conclude definitively that, not only do ‘truly wild’ populations of ples see . These wcessions . Insteadsensu Schwendiman and feral populations is clearly stronger than the splitting of the latter into two clusters corresponding to (i) races of pure G. hirsutum from Mesoamerica, and (ii) the Caribbean and Central American representatives of race ‘Marie-Galante’ resulting from an introgression with G. barbadense. This comparison indicates that domestication resulted in a major infraspecific division in G. hirsutum. In any case, the low level of admixture between neighboring TWC and feral populations shows the effects of surprisingly strong reproductive barriers and/or very strict ecological adaptation, resulting in very limited gene flow, despite their geographical proximity.The genetic and ecological divergence between race ‘yucatanense’ G. hirsutum. For example, the wild population of Portland Point in Jamaica was mentioned by Britton in 1908 G. hirsutum is a pioneer plant colonizing disturbed coastal habitats, but that “this (habitat) instability is in itself highly stable” and very ancient, so “that the pioneers are simultaneously old residents”, as Fryxell Gossypium are intolerant to competition, particularly at the seedling stage. Contrary to feral cotton, the realized niche of TWC populations is narrower than their fundamental niche.The much stronger differentiation of TWC populations is reminiscent of the study of Stephens Spondias purpurea L.), a small fruit tree native from the dry forests of southern Mexico and Central America, and cultivated for its fruit and/or as a fence. They found that the climatic envelope of the wild populations is nested in that of the cultivated forms. In other words, domestication and cultivation mostly expanded the range of the species. Miller and Knouft Thus, the present study provides an opportunity to analyze the effect of domestication on the distribution of cultivated perennials, a rarely studied aspect of domestication. Miller and Knouft G. hirsutum differs in its relatively high level of autogamy and endogamy G. hirsutum through selection and genetic adaptation? As this envelope is common to feral and cultivated populations, and the feral populations depend on the permanent contribution of cultivated cotton, the most likely answer is that the much wider distribution of these two categories is essentially related to the reduction of competition in cultivated and neighboring disturbed habitats, not to a genetic effect. Furthermore, as the domestication syndrome involves seed characteristics  that are essential for the survival of wild populations, most feral cottons are unable to re-colonize, and persist in, the original habitat of the species. Thus, the apparent paradox is that, although the geographical distribution of perennial G. hirsutum has been considerably widened by domestication and cultivation, its niche has been reduced by the loss of reproductive capacity in its natural habitat. In fact, there would have been a true paradox if cultivation, while reducing exposure to both extreme aridity and competition, had increased the competitive potential of G. hirsutum in secondary habitats.As compared to purple mombin and the majority of perennial fruit crops, G. hirsutum in Mesoamerica and the Caribbean are the identification of potential areas for the early domestication processes and the comparative characterization of domesticated versus wild cottons. Our distribution maps do not contradict the hypothesis of Brubaker and Wendel G. hirsutum in northern Yucatán, as this region, home of the most extensive wild populations, indeed corresponds to the largest favorable area . Even though these wild cottons are excellent sources for widening the genetic base for breeding because of their complete interfertility with modern cultivars of G hirsutum, for example at the transcriptome level ad hoc categorization as attempted in the present study.Lastly, a further understanding of the domestication process, through the comparison of the domesticated and wild pools of G. hirsutum restricted to littoral or related habitats, on the shores of the Caribbean Sea and the Gulf of Mexico from Venezuela to Florida, and even as far as Polynesian islands in the Pacific Ocean. The obvious niche conservatism expressed in the strong similarity of the natural habitats of all five allotetraploid species shows that their speciation was essentially driven by the geographic, rather than ecological, isolation of their highly scattered populations.Ocean diffusion and ecological constraints, related to extreme aridity and low levels of competition, best explain the past and current distribution of truly wild populations of G. hirsutum and G. barbadense, would have allowed genetic introgression in southern Central America and/or northern South America, resulting in the development of race ‘Marie-Galante’. The close genetic relatedness between ‘Marie-Galante’ and the Mesoamerican domesticated races shows that the introgression process was anterior to the diffusion of domesticated G. hirsutum to the Antilles.Our ecological and genetic data consistently support the hypothesis of Brubaker and Wendel Even where domesticated and TWC forms grow in close proximity, they hybridize only sporadically. As a result, the level of genetic divergence between them overwhelms differentiation among domesticated races and/or geographic regions.Our understanding of plant evolution under domestication is more limited for perennial plants than for seed-propagated annual crops G. hirsutum, with seeds only sparsely covered with short fibers but with adaptation to extreme environmental conditions, contrast with cultivated cotton, with its highly valued long-fibered seeds but adaptation to less demanding ecologies. Owing to the advances of genomics and genome sequencing and the ability to scan the genomes of wild species for new and useful genes, we may now be in a position to unlock the genetic potential of the wild germplasm resources of crop plants G. raimondii and the genome A of G. arboreum, have already been published G. hirsutum and G. barbadense is underway. It should be a relatively easy step now to systematically scan wild germplasm for useful genetic variants. However, this presupposes that ex situ collections are adequate, accessible and safe, and that in situ preservation efforts are effective in safeguarding material not yet in gene banks, and still evolving in the field. This work should facilitate the development not only of efficient strategies for exploiting cotton diversity for crop improvement, but also of strategies for its long-term conservation.Wild forms of Figure S1Maps of the 11 locations/islands where truly wild cottons (red dots) were sampled for the SSR-based genetic analysis (Socorro Islands not shown). Purple dots represent sampling locations for feral populations in the vicinity. The comments attached to each map are extracted (and translated from French) from collecting reports as indicated.(DOC)Click here for additional data file.Figure S2Potential distribution of G. hirsutum during the Last Glacial Maximum  in South America, extrapolated according to the MIROC climatic model.(DOC)Click here for additional data file.Figure S3Unrooted neighbor-joining tree based on dissimilarities between 110 perennial, and one cultivated, accessions of Gossypium hirsutum based for 26 SSR markers.(DOC)Click here for additional data file.Figure S4Prediction of the best value of K , from K = 2 to 5 clusters, from the STRUCTURE analysis of 111 perennial cottons of G. hirsutum.(DOC)Click here for additional data file.Table S1Detailed records of the 111 accessions used in the SSR-based genetic analysis.(DOC)Click here for additional data file.Table S2List of 26 SSR markers, their chromosome localization on the consensus map , and summary statistics : total number of alleles, He value as expected heterozygosity, numbers of alleles and unique alleles within groups .(DOC)Click here for additional data file.Table S3Mean observed heterozygosity (in %) of 26 SSR markers as observed among 110 perennial accessions of G. hirsutumth accession was 100% homozygote). Click here for additional data file.Table S4Mean dissimilarities within and between groups of 110 perennial accessions of G. hirsutum. MG, PU and TWC refer to ‘Marie-Galante’, ‘punctatum’ and truly wild cottons.(DOC)Click here for additional data file.


Empoasca (Matsumurasca) onukii Matsuda. Previous morphological study that revealed variation in the structure of the male genitalia within and among populations of this species suggested that there may be significant population-level genetic variation. To provide powerful molecular markers to explore the population genetic diversity and population genetic structure of this pest in China, microsatellite markers were obtained by AFLP of sequences containing repeats (FIASCO).Tea green leafhopper is one of the most dominant pests in Chinese tea plantations. Recent evidence, including morphological and molecular data, revealed that tea green leafhopper in China is the same species as in Japan, E. (M.) onukii, Two related empoascine leafhopper species were selected to test the transferability of the markers. Population genetic structure of E. (M.) onukii was detected using Structure analysis, principal coordinate analysis (PCoA) and variance analysis. The identified markers were polymorphic with total number of alleles ranging from 6 to 24 per locus, observed and expected heterozygosity ranged from 0.133 to 0.9 and 0.183 to 0.926, respectively, and the polymorphic information content value over all populations varied from 0.429 to 0.911.Eighteen polymorphic markers were evaluated for five populations of E. (M.) onukii in Chinese tea plantations. There is obvious genetic differentiation between the two populations in the Southwest tea area. These microsatellite markers will be the powerful tools for genetic studies of E. (M.) onukii and improve understanding of the microevolution of this species.This is the first study to demonstrate that microsatellite markers provide valuable information for genetic structure of The online version of this article (doi:10.1186/s12863-016-0420-3) contains supplementary material, which is available to authorized users. Empoasca vitis (Goëthe), was applied to this species for nearly 30 years in China and caused confusion in academic and applied research on the pest [Empoasca (Matsumurasca) onukii Matsuda [Tea green leafhopper, one of the most dominant pests in Chinese tea plantations recognized since the 1950s, causes considerable yield loss each year by piercing and sucking young leaves of tea . An incothe pest , but rec Matsuda , 8.COI and 16S rRNA gene regions from different populations [Empoasca it is important to examine the male genitalia of specimens used in such studies to ensure accurate identification. Also in these previous studies only a few genetic markers were used for a single individual, and their variation was too low for studying population structure. As a result, the population structure of E. (M.) onukii is still poorly understood.In China, tea-growing regions are fairly widespread across different climatic zones and tea green leafhopper has adapted to different habitats, conditions that may have given rise to genetic differentiation among populations. Previous studies have attempted to explore the genetic diversity of tea green leafhopper using either RAPD (Random Amplified Polymorphic DNA) techniques to assess the genetic polymorphism and relationships among seven populations of this species  or sequeulations –12. HoweScaphoideus titanus Ball and Empoasca vitis (Goëthe), and subsequently showed that European S. titanus populations originated from northeastern North America [Hishimous phycitis using the mitochondrial cytochrome oxidase I (COI) gene and nine microsatellite DNA markers isolated by FIASCO [Empoasca, belonging to a separate cicadellid subfamily , and we were unable to consistently amplify the microsatellite markers developed for these species in Chinese E. (M.) onukii populations.Based on their high polymorphism, codominance, abundance, and stability, microsatellite markers have been widely adopted to obtain multilocus genotypes using minute quantities of DNA , 14. The America –18. Shaby FIASCO , 20. UnfE. (M.) onukii [E. (M.) onukii populations both within and between the recognized areas.Tea production originated in Southeast China more than 3000 years ago and is now widespread in tropical and subtropical regions of the world . Our pre) onukii , 22. Thi) onukii . Given tE. (M.) onukii populations in the Jiangbei and Southwest tea areas. These microsatellite markers will provide the tools needed for genetic studies of E. (M.) onukii aimed at elucidating the microevolution and population dynamics of this species in China. This information will help pinpoint the origin of this pest and its routes of dispersal, which will be used to develop environmental friendly control strategies against this species in different tea areas.The aim of this study was to develop microsatellite markers and to use them to analyze genetic structure of Tea green leafhopper specimens used in this study were collected from tea plantations in five provinces representing five geographic populations see Fig. . To redu12, (AC)12 and (AAC)8-enriched partial genomic libraries were constructed by FIASCO. 100 ng of DNA was digested with MseI  and ligated to prepared MseI AFLP adaptors (5′-TACTCAGGACTC AT-3′/5′-GACGATGAGTCCTGAG-3′) using T4 DNA ligase  The linker-adaptor-PCR was performed in a final volume of 50 μL containing: 2 mM MgCl2, 250 uM dNTP, 0.8 uM MseI AFLP adaptors and 0.32 U of Taq DNA polymerase . The reaction procedure included denaturing at 94 °C for 3 min, followed by 20 cycles of 30 s at 94 °C, 1 min at 53 °C, 1 min at 72 °C, and a final extension of 10 min at 72 °C. 300–750 bp DNA fragments were purified and separated using a QIAquick PCR Purification Kit . The product was then denatured for 5 min at 95 °C and hybridized with biotinylated probes ((AC)12, (AG)12 and (AAC)8) for 2–3 h, respectively.Microsatellites were enriched and isolated by FIASCO , 25. GenMseI-N (5′-GATGAGTCCTGAGTAAN-3′). The PCR products were ligated to pMD19-T vectors  and transformed into Escheri-chia. coli strain Trans1-T1 as follows: cells were cultured at 37 °C for about 16 h on LB agar plates containing ampicillin, X-gal, and IPTG for blue/white selection. Insert-positive bacterial clones were transfered into liquid medium with ampicillin in 96-well plates and cultivated at 37 °C for about 4 h. PCR amplification was performed in a volume of 25 μL containing: 1 μL bacterium solution, 2 mM MgCl2, 250 uM dNTP, 0.8 uM each of M13 forward and M13 reverse primer and 0.32 U of Taq DNA polymerase . The reaction procedure included denaturing at 94 °C for 3 min, followed by 35 cycles of 30 s at 94 °C, 40 s at 57 °C, 50 s at 72 °C, and a final extension of 10 min at 72 °C. PCR products were considered as positive clones when two or more bands appeared in the 100–500 bp size range and were sequenced by Sangon  after purification.DNA fragments were selectively captured by streptavidin-coated magnetic breads . Nonspecific binding and redundant DNA was eluted by several non-stringent and stringent washes. The microsatellite-enriched DNA fragments were amplified with E. (M.) onukii individuals was amplified in 10 μL total volume with 20 ng DNA and containing 2.5 mM MgCl2, 250 uM dNTP, 1uM each of forward and reverse primer and 0.2 U of Taq DNA polymerase .The results were screened for SSR motifs by SSR Hunter 1.01 . PrimersE. (M.) onukii populations. From the results of electrophoresis, polymorphism was defined as the presence of more than two alleles (bands of different size). Polymorphic markers were selected based on their performance in PCR and number of alleles.All of the primers were screened by “touchdown PCR”, which included 4 min denaturation at 94 °C: 30 s at 94 °C, 30 s at 50 °C to 60 °C (dropping 0.3 °C/cycle), 30 s at 72 °C, 30 cycles, then 35 cycles of 30 s at 94 °C, 30 s at 57 °C, 30 s at 72 °C, and one cycle of 7 min at 72 °C. Thereafter, the optimum annealing temperature of primers was determined by gradient temperature PCR, including a 4 min denaturation at 94 °C, 30 s at 94 °C, 30 s at 50 to 65 °C (dropping 1 °C/sample), 30s at 72 °C, 30 cycles, then 7 min at 72 °C. Finally, fragment length polymorphism of products amplified with the optimum annealing temperature were further analyzed by 6 % polyacrylamide gel electrophoresis for samples representing eight individuals from four Empoasca (s. str.) sp. (n = 8) and Alebrasca actinidiae Hayashi & Okada (n = 8). Genomic DNA was extracted from each single individual by CTAB, yielding a concentration of > 20 ng/μl and polymorphism was assessed by using labeled primers and the PCR protocol noted above. PCR products were then run by automated capillary electrophoresis using a genetic analyzer . Data were analyzed using GeneMapper v4.0 .Thirty-one selected primers were synthesized and labeled as forward primers (FAM). To obtain accurate allele frequencies, 30 individuals per population  collected from the five different populations were used to evaluate polymorphism (Table A), observed heterozygosities (HO), expected heterozygosities (HE) and polymorphism information content (PIC) were calculated by Cervus 2.0 [AR) was estimated using a minimum sample size of 25 diploid individuals in HP-Rare v1.0 [t-test in Spss Statistics 20 (IBM) was used to examine if the genetic diversity significantly differed between the Jiangbei and Southwest populations. Differences in allelic frequencies were tested with Fisher’s method (exact G test) using Genepop v3.4.The frequency of null alleles was evaluated by Micro-Checker . The numrvus 2.0 . The allare v1.0 . Deviatiare v1.0 . Linkageare v1.0 . IndepenPopulation structure was defined on the basis of Structure analysis and principal coordinate analysis. Structure 2.3.4 was used to generate clusters of individual genotypes by Bayesian assignment . An admiFST indices.Analyses of molecular variance (AMOVA) and fixation indices were performed by Arlequin 3.11 . AMOVA aE. (M.) onukii populations  sp.  and three in the more distantly related Alebrasca actinidiae Hayashi & Okada  (Table E. (s. str.) sp. However, for A. actinidiae, all three amplified markers yielded single amplified fragments.One hundred and seventy-three clones were selected to be sequenced from three microsatellite-enriched libraries, (AC)n, (AG)n and (AAC)n. After searching for repeats, 74 sequences were deemed adequate for designing primers. 56 pairs of primers were obtained, 31 of which successfully yielded clear single target bands of predicted size, with the others showing multi-banding patterns or no amplification when products were visualized on 2 % agarose gels. 21 markers showed fragment length polymorphism, accounting for 37.5 % of total markers. The sequences of the 21 markers have been uploaded to GenBank and accession numbers are shown in Additional file Eo-4-5, Eo-1-77 and Eo-F-8 exhibited a significant excess of homozygosity and the amplication rates were lower than 50 %. So rejecting Eo-4-5, Eo-1-77 and Eo-F-8, the remaining 18 markers were selected for population genetic studies. After Bonferroni correction, nine markers deviated from Hardy-Weinberg equilibrium (pHWE < 0.01) in different populations, but linkage disequilibrium was not detected for any pair of markers. Eo-54, Eo-1-61, Eo-1-52, Eo-83, Eo-E-12, Eo-70, Eo-36, Eo-1-65 and Eo-1-5 deviated from HWE because of null alleles . Similarly, the mean expected heterozygosity were significantly different between the Jiangbei and Southwest populations . Analysis of allelic frequencies across all markers showed significant differences between each population pair . Allelic frequencies for each marker in each population were shown in Additional file The number of alleles for the 18 markers varied from 4 to 18 among different populations. The mean number of alleles and allelic richness per population ranged from 8.7 to 9.4 and from 8.4 to 9.0, respectively. The mean expected and observed heterozygosity per population ranged from 0.665 to 0.758 and from 0.516 to 0.632, respectively . The proportion of genetic variance was larger between groups (4.56 %) than between populations (2.66 %). The fixation indices between groups  and between populations  were significant  revealed that majority of the genetic variance originated from variation among individuals within populations (92.78 %) and was highly significant  onukii populations failed. This is not surprising, given that two of the three species in these previous studies belong to the distantly related leafhopper subfamily Deltocephalinae and that leafhoppers, in general, are a phyletically diverse and widespread group of insects. The results of cross-species amplification indicate that, in this group of leafhoppers, microsatellite markers are highly species-specific, and amplification rate of markers developed for one species decrease proportionally according to the genetic distance in other species [Although previous studies reported 27 microsatellite markers for three different leafhopper species , 17, 20, species .Eo-4-5, Eo-F-8,Eo-1-77 due to lack of amplification, monomorphic fragments or unstable polymorphism, may have various causes including mutations within primer regions or presence of secondary DNA structures that prevented amplification. Among the 18 remaining markers, Eo-54, Eo-1-61, Eo-1-52, Eo-83, Eo-E-12, Eo-70, Eo-36, Eo-1-65 and Eo-1-5 deviated from HWE because of null alleles and a deficit of heterozygotes. But null allele frequency of 18 markers, ranged from −0.048 to 0.152, below 0.200 and induced little effect on the result of genetic diversity and genetic structure [E. (M.) onukii populations.Sufficient numbers of specimens from each isolated population and tests of polymorphism for markers are the key factors for successfully developing microsatellite markers. Accordingly, tea leafhopper specimens in this study were collected from sites at least 500 km distant from each other. Following the recommendations of Hale et al. , we usedtructure , 44. As E. (M.) onukii populations revealed by 18 microsatellite markers is probably due to some combination of geographic isolation and climatic variation among the tea-growing regions in which these populations occur. Genetic diversity and genetic structure based on 18 microsatellite markers highlighted the existence of significant genetic difference between the Sichuan and Yunnan populations in the topographically complex Southwest tea area. Furthermore, different mean allelic richness and mean expected heterozygosity were found between the Jiangbei and Southwest populations  onukii in the Jiangbei and Southwest tea areas. However, the data available so far are not sufficient to reconstruct invasion routes. The markers developed here will be used in future, more detailed, analyses of genetic structure of E. (M.) onukii populations in Chinese tea plantations and to study the evolutionary mechanisms yielding the observed variation.The microsatellite markers developed here revealed genetic differentiation of E. (M.) onukii microsatellite sequences were obtained and analyzed. 18 polymorphic markers were selected to analyze five populations of E. (M.) onukii. Study of the genetic structure of five Chinese populations demonstrate that the newly developed markers provide valuable information on the genetic structure of E. (M.) onukii in Chinese tea plantations. The Structure analysis and PCoA for populations reveals that there is significant genetic differentiation between the Sichuan and Yunnan populations and that these have similar genetic diversity to that present among the Henan, Shandong and Shaanxi populations. These microsatellite markers will be powerful tools for genetic study of E. (M.) onukii and yield an improved understanding of the microevolution of this species.Seventy-four


Alu markers were studied in 574 unrelated individuals from 10 endogamous groups and 2 hill tribes of Tamil Nadu and Kerala states. DNA was isolated, amplified by PCR-SSP, and subjected to agarose gel electrophoresis, and genotypes were assigned for various Alu loci. Average heterozygosity among caste populations was in the range of 0.292–0.468. Among tribes, the average heterozygosity was higher for Paliyan (0.3759) than for Kani (0.2915). Frequency differences were prominent in all loci studied except Alu CD4. For Alu CD4, the frequency was 0.0363 in Yadavas, a traditional pastoral and herd maintaining population, and 0.2439 in Narikuravars, a nomadic gypsy population. The overall genetic difference (Gst) of 12 populations (castes and tribes) studied was 3.6%, which corresponds to the Gst values of 3.6% recorded earlier for Western Asian populations. Thus, our study confirms the genetic similarities between West Asian populations and South Indian castes and tribes and supported the large scale coastal migrations from Africa into India through West Asia. However, the average genetic difference (Gst) of Kani and Paliyan tribes with other South Indian tribes studied earlier was 8.3%. The average Gst of combined South and North Indian Tribes (CSNIT) was 9.5%. Neighbor joining tree constructed showed close proximity of Kani and Paliyan tribal groups to the other two South Indian tribes, Toda and Irula of Nilgiri hills studied earlier. Further, the analysis revealed the affinities among populations and confirmed the presence of North and South India specific lineages. Our findings have documented the highly diverse (micro differentiated) nature of South Indian tribes, predominantly due to isolation, than the endogamous population groups of South India. Thus, our study firmly established the genetic relationship of South Indian castes and tribes and supported the proposed large scale ancestral migrations from Africa, particularly into South India through West Asian corridor.Seven human-specific  India is served as the important corridors for human migration and evolution. A small group of modern humans ventured ‘out-of-Africa’ through the southern coastal route to colonize the Middle East, India, Southeast Asia, Australia and subsequently the other parts of the globe ,2. The IAlu sequences are thought to be ancestrally derived from 7SL RNA gene was mobilized through a RNA polymerase III derived transcript by a process called ‘retro position’ process [Alu insertion polymorphisms identify the patterns of human genetic diversity and history, race determination, gender identification, personal identification, paternity testing. Alu insertional elements are a family of SINEs and presence of an AluI recognition site in the sequence [Alu repeats, which represents ~11% of nuclear DNA [Alu insertions are 300 bp length, dimeric in structure, composed of 3’ oligo (dA)-rich tail and short flanking repeats [Alu polymorphism has an important application in phylogenetic analyses of human populations [Alu’ polymorphic loci were previously been studied for many Indian populations [Alu ACE, Alu TPA25, Alu FXIIIB, Alu Apo, Alu D1, Alu Pv92 and Alu CD4 among castes and tribes of the state of Tamil Nadu, South India.The genomic variations among individuals may help to understand the evolutionary and migrational course of populations. The genetic variations and/or polymorphism at loci that code for expressed profess are commonly deleterious and therefore, are often negatively selected and hence eliminated. On the other hand, allelic polymorphisms, especially in the non-coding regions of the human genome are expected to be evolutionarily neutral. Of late, several insertion/deletion polymorphism have been discovered in the human genome.  process . Alu inssequence . The humlear DNA . It is olear DNA . Alu ins repeats –15. The ulations –18, 19. ulations , 22, 23.ulations , 27, 28.5 ml of blood samples were collected from 574 unrelated volunteers from twelve different population groups from South India. The populations selected for the present study includes Pallan, Nair  Namboothiri , Kani, Vanniyar, Paliyar, Narikuravar, Sourashtra, Iyer, Vettuva Gounder, Kallar and Yadava. They belong to different geographical locations in the states of Tamil Nadu and Kerala. The sample size, location of sampling and anthropological information was given in Alu elements . The protocols for these markers have been described elsewhere [The DNA samples were extracted from peripheral blood lymphocytes using a standard salting-out procedure . Each DNlsewhere –22, 30  method [Allele frequencies were calculated by direct counting for each population. Heterozygosities at individual loci and the overall average heterozygosity were calculated by using the allele frequencies for each population. Hardy-Weinberg equilibrium was tested using a χ) method . PrincipThe allele frequencies and heterozygosities for the insertion (+) and/or deletion (-) alleles for two tribal populations, Kani and Paliyar  are presented in Alu locus was determined. The gene diversity analysis was performed based on the polymorphism data for eleven tribal populations, two from the present study , five South Indian Tribes (SITs) such as Badga, Irula, Kota, Kurumba and Toda [The genetic differentiation or genetic differences among tribal populations, Gst values (a measure of the interpopulation variability) for each and Toda  and fourand Toda  from preAlu ACE, TPA25, FXIIIB, APO, D1, Pv92 and CD4 of 12 caste populations were presented in Alu CD4 exhibits low level of polymorphism in many of the populations studied. All the populations showed very high levels of polymorphism for all Alu loci except CD4. Among 12 population groups studied, the heterozygosity of seven loci ranged between 0.291 (Kani)—0.468 (Yadava).The allele frequencies for the insertion (+) and deletion (-) for loci Gst values for each polymorphic locus were determined among populations and the results were presented separately for each locus and also for all loci taken together . The totThe genetic affinities among eleven tribal groups, 2 tribal populations of the present study namely Kani and Paliyar and the 9 tribal populations studied previously , 25 wereThe phylogenetic relationship of 10 caste and 2 tribal population groups studied in the present work was presented in Alu insertion marker [Alu insertion loci . The allele frequencies and heterozygosity vAlues observed in the present study are comparable, however with minor deviations with the frequencies of other South Indian endogamous populations studied previously. The overall average heterozygosity of all the loci analyzed ranges between 0.292–0.468. The lowest level of heterozygosity in the present study was observed in Kani (0.292), a primitive tribe inhabiting Western Ghats  Tamil Nadu. A classical study was undertaken by Watkins et al [Alu and HLA loci were attributed to isolation from other populations for considerably long periods of time and entertaining a tribal life in hill regions. The heterozygosity vAlue of Kani (0.292) was very close to Vysyas (0.299) reported previously by Watkins et al., [Alues (0.351–0.449) observed for different population groups of India [Of late, the ns et al  to elucins et al . Previouns et al . High le et al., . Nonetheof India –26, 33.Alu loci such as mtNUC, Alu ACE, Alu APO, Alu FXIIIB, Alu D1, Alu CD4, Alu PLAT, Alu TPA25, Alu PV92 [Alu markers was 9.5%. Thus, the present and previous studies have confirmed the higher Gst values for north and South Indian tribes confirmed the presence of different genetic elements in Indian caste and tribal populations.The observed average genetic differences (Gst) within the each caste populations of the present study was 3.6%. Stoneking et al.  have repAlu PV92  and WestAlu PV92 . Thus, oAlu PV92  have repAlu PV92 . PreviouAlu PV92 . HoweverAlu PV92  have docAlu PV92 . HoweverAlu PV92  and KaniR1a and R1a1 Haplogroup. The R1a1 Haplogroup have spread in people from regions of Central Europe, East Europe, Scandinavia and Punjab. These and other findings have reiterated the fact that the Badaga tribe of Nilgiri hills of South India might have originated from the Eurasia. The tribal groups presently studied such as Kani and Paliyar are linguistically similar but are living in distant geographical locations in Western Ghats, of South India. The phylogenetic analyses have revealed that these two tribes overlap genetically with two other South Indian tribes, Toda and Irula. In the present study, Paliyar tribe forms a separate cluster, and overlap with North Indian Bagdi which belongs to Indo-Aryan ancestry. Kani tribe also overlaps with North Indian middle class Agharia and South Indian Narikuravars , two populations that possess Indo-Aryan ancestry. The Narikuravars speak an Indo-Aryan language called Vagriboli which is a western Indian language  belongs to the Indo-Aryan linguistic family. Alu marker based studied have documented that the North Indians are genetically highly diverse populations with variations scattered between individuals. With a glut of human migratory episodes and admixtures, the paternal genealogy of North Indians have revealed the genetic foot-prints and legacy of the Indo-Aryan speaking populations.Phylogenetic analysis as depicted in The Principal Components Analysis provides alternative methods for examining the inter-population relationships. The present study of endogamous caste populations revealed close relationships and/or proximity in PCA as well as in NJ phylogenetic tree. The results suggested that these populations might have a common ancestry. It is highly interesting to note that an upper class population, Iyers of Madurai, overlaps genetically with UP-Brahmin. Balakrishnan et al.  have repAlu polymorphism based affinities of the South Indian populations (castes and tribes) forms a potential genetic data for mapping population migrations, histories, genetic similarities and gene-disease linkage analysis in a country known for the practice of strict endogamy and higher level of prevalence of infectious diseases. Our study thus provides (i) an evidence of presence of North/South differences in the frequencies of Alu alleles and (ii) affinities of South Indian endogamous caste and tribes with middle East and West Asian populations. These observations thus confirmed the well established notion of peopling of South India by coastal migrations of man from Africa.Indian sub-continent has witnessed a massive gene flow from varied ethnic sources over the historical periods. The gene flow could be occurred prior to the subdivision of this population into largely endogamous caste groups. Thus, it was suggested that, after the migration of modern humans from Asia, there were many rapid population explosion (s) following an initial period of isolation. To conclude, the studied Indian populations have revealed higher heterozygosities as compared to African populations. The present study thus concluded that the endogamous populations of South India have showed the amalgamation of various populations coming–in from different directions and geographical locations by the process of admixture and miscegenation. Thus, the S1 File(DOCX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Pacifastacus leniusculus) is one such example, where rapid spread across Japan in recent decades is believed to be the result of only three founding populations. To infer the history and explore the success of this remarkable crayfish invasion, we combined detailed phylogeographical and morphological analyses conducted in both the introduced and native ranges. We sequenced 16S mitochondrial DNA of signal crayfish from across the introduced range in Japan  and the native range in western North America . Because chela size is often related to aggressive behavior in crayfish, and hence, their invasion success, we also measured chela size of a subset of specimens in both introduced and native ranges. Genetic diversity of introduced signal crayfish populations was as high as that of the dominant phylogeographic group in the native range, suggesting high propagule pressure during invasion. More recently established crayfish populations in Japan that originated through secondary spread from one of the founding populations exhibit reduced genetic diversity relative to older populations, probably as a result of founder effects. However, these newer populations also show larger chela size, consistent with expectations of rapid adaptations or phenotypic responses during the invasion process. Introduced signal crayfish populations in Japan originate from multiple source populations from a wide geographic range in the native range of western North America. A combination of high genetic diversity, especially for older populations in the invasive range, and rapid adaptation to colonization, manifested as larger chela in recent invasions, likely contribute to invasion success of signal crayfish in Japan.Successful invasion by nonindigenous species is often attributed to high propagule pressure, yet some foreign species become widespread despite showing reduced genetic variation due to founder effects. The signal crayfish ( Mounting evidence suggests that nonindigenous species may become successful invaders despite showing low genetic variation  is among the world's most notorious freshwater invaders and has impacted native biodiversity throughout its introduced ranges via predation, competition, ecosystem engineering, or transmission of diseases ) determined that the introduced signal crayfish in Japan consisted of three founders, that is, Lake Mashu (Hokkaido Prefecture), Tankai (Shiga Prefecture), and Akashina (Nagano Prefecture), because these populations  are characterized by different composition of branchiobdellidan species  the three founding populations of the introduced signal crayfish in Japan originate from multiple sources in North America; (2) the introduced signal crayfish populations have undergone a loss of genetic diversity relative to native populations, or following successive invasions and secondary spread within Japan; and (3) recently established signal crayfish in Japan demonstrate patterns of morphological change  consistent with expectations of increased aggression or boldness in invasive populations. Results from this study provide the first intercontinental phylogeographic comparison between the native and an introduced range for this major invasive crayfish, thereby testing whether this species has experienced reduced genetic diversity where introduced. Our results inform current management of introduction pathways and secondary spread of the signal crayfish in Japan and provide fundamental scientific insight into the genetic and morphological correlates of invasion success at biogeographical scales.The signal crayfish is native to the northwestern United States and southwestern Canada, including the Columbia River and its tributaries and adjacent coastal rivers. The species has also been widely introduced within the western United States, where it is a notable invasive species in the states of California and Nevada . Neither cryptic group, nor other species of the crayfish genus Pacifastacus, have been observed in Japan by our or previous studies. Therefore, in this study, new genetic data from the introduced range in Japan were used together with a portion of previously published sequence data from the native range in North America . Using the 16Sar‐L and 16Sbr‐H primers .All sequences found in the native range have been previously deposited in GenBank  and 128 crayfish from 23 sites in the Pacific Northwest native range . As for the genetic data, morphological data from the introduced range in Japan are newly reported in this study, while those from the native range in North America use previously published data from Larson et al. .Morphological measurements were made using Vernier callipers to 0.01 mm. Only male crayfish with ≥20 mm carapace length were used in our morphological analysis because crayfish chela tends to be larger in males relative to females Stein  and thisWe used the program TCS 1.21  for use in subsequent spatial analysis of molecular variance (SAMOVA). To identify best genetic groups that are maximally differentiated from each other, we performed SAMOVA in SAMOVA 2.0 for all sites in the introduced and native ranges  ranging from 2 to 10 without geographic constraints with 100 annealing processes as starting conditions. We determined the most likely number of groups when φCT reached a plateau , we subsequently performed analysis of molecular variance (AMOVA) in Arlequin ver. 3.5.2.2 , we performed jModeltest 2.1.8 to select the best model for DNA sequence evolution of among‐site variation (Guindon and Gascuel Nh), haplotype diversity (h), and nucleotide diversity (π) in each site in introduced and native ranges. We calculated the genetic diversity indices in Arlequin for the sites comprising three or more individual crayfish samples or specimens. To test for differences in Nh, h, and π between signal crayfish populations in introduced and native ranges, we conducted Wilcoxon rank‐sum tests using R version 3.2.0 , haplotype diversity (h = 0.81–0.83), and nucleotide diversity (π = 0.0039–0.0041) relative to the initial or founding population . Nine recently invaded sites  in Hokkaido and Fukushima Prefectures contained haplotypes A, G, and/or K, which were also found in Touro or Iwabokki but not in Lake Mashu. Therefore, the Kushiro River probably acted as a source for tertiary invasions to these sites.Among the three founding populations in Japan, the number of haplotypes was greatest in Lake Mashu, whereas haplotype diversity and nucleotide diversity were greatest in Tankai Table . Only onThe haplotype network showed that the native signal crayfish populations consisted of five lineages which were connected by one to seven missing haplotypes  were selected. In both cases, all introduced populations were clustered into the same group as the Main group in the Pacific Northwest. Subsequent pairwise AMOVAs or pairwise population differentiation tests (when only one population consisted of each group) showed high φCT or φST between the six genetic groups . Thus, all introduced populations in Japan likely originate from the Main group in the native range.In SAMOVA, ps Table . When AMNh) in introduced signal crayfish populations in Japan  was as high as that of all sites in the native range   or the Main group in the native range   . Haplotype diversity (h) was not significantly different between introduced signal crayfish populations in Japan  and all native range populations  or native range populations belonging to the Main group . Likewise, there was no statistical difference in nucleotide diversity (π) between the introduced Japanese populations  and all groups in the native range  or the Main group populations in the native range . These results were not generally sensitive to inclusion of all Pacific Northwest signal crayfish genetic groups or the use of a more restrictive native range definition . The only exception was that π of signal crayfish was higher in the Main group in the restrictive native range relative to that in Japan .The number of haplotypes . However, there was a significant negative relationship between the number of haplotypes and the year of establishment or discovery when only specimens of the Hokkaido (or Lake Mashu originating) group were considered in the analysis . However, we found a significant positive relationship between ChA.CL and the year of establishment or discovery when only specimens of the introduced Hokkaido group were considered in the analysis  or that in the Hokkaido group  or genetic admixture (Lake Mashu and Tankai), as suggested from the distributions of haplotypes in the native range and SAMOVA grouping. Some of these putative source populations from the Pacific Northwest to Japan may themselves be introductions  of the introduced signal crayfish were as high as those of the Main group in the native range regardless of whether or not we consider a more restrictive or expansive native range classification.In theory, invasive populations are expected to suffer from loss of genetic variation due to founder effects, genetic bottlenecks, and genetic drift. In contrast to these expectations, studies investigating genetic diversity of aquatic species often show little sign of reduced genetic variation following biological invasions, perhaps because biological invasions in aquatic ecosystems are often associated with high propagule pressure  relative to their putative source populations in the native range, even if some of these native range populations  may also represent human introductions of the species within the Pacific Northwest. Furthermore, there was a positive relationship between chela size and the year of establishment or discovery in the Hokkaido introduced group. An increase in the size of crayfish chelae in recently introduced populations may be a response to interactions with conspecific predators/competitors, avian, mammal, or fish predators or other biotic or abiotic factors. In particular, crayfish with large chela are expected to have advantages in acquiring limited resources, such as food, shelter, and mates, because dominance hierarchy or survivorship in crayfish is largely determined by chela size  may have contributed to the subsequent success of this species and its widespread distribution in Japan. However, younger populations produced by secondary spread or subsequent introductions within Japan generally have lower genetic diversity, and high genetic variation or admixture from multiple source populations does not seem to be a prerequisite for invasion success in this species  and number of clusters (K) in the signal crayfish in Japan and North America based on spatial analysis of molecular variance (SAMOVA) using the Kimura 2‐parameter evolution model with gamma correction (gamma = 0.03).Figure S2. Comparisons of genetic diversity indices  of signal crayfish between North America and Japan when restrictive native range definitions and genetic groups are considered (C and D) or not (A and B).Click here for additional data file.Table S1. Correspondence between each 16S mitochondrial DNA haplotype and the GenBank accession number of signal crayfish (Pacifastacus leniusculus) in North America and Japan.Click here for additional data file.


Genetic variations of the TCF7L2 gene are associated with the development of Type 2 diabetes (T2D). The associated mutations have demonstrated an adaptive role in some human populations, but no studies have determined the impact of evolutionary forces on genetic diversity in indigenous populations from Mexico. Here, we sequenced and analyzed the variation of the TCF7L2 gene in three Amerindian populations and compared the results with whole-exon-sequencing of Mestizo populations from Sigma and the 1000 Genomes Project to assess the roles of selection and recombination in diversity.2 values of linkage disequilibrium were significantly higher in the Mexican population than in Africans from the 1000 Genomes Project. We observed reticulations in both the haplotypes network analysis from seven marker associates and the neighborNet tree based on 6061 markers in the TCF7L2 gene identified from all samples of the 1000 Genomes Project. Finally, we identified two recombination hotspots in the upstream region and 3’ end of the TCF7L2 gene.The diversity in the indigenous populations was biased to intronic regions. Most of the variation was low frequency. Only mutations rs77961654 and rs61724286 were located on exon 15. We did not observe variation in intronic region 4–6 in any of the three indigenous populations. In addition, we identified peaks of selective sweeps in the mestizo samples from the Sigma Project within this region. By replicating the analysis of association with T2D between case-controls from the Sigma Project, we determined that T2D was most highly associated with the rs7903146 risk allele and to a lesser extent with the other six variants. All associated markers were located in intronic region 4–6, and their rThe lack of diversity in intronic region 4–6 in Indigenous populations could be an effect of selective sweeps generated by the selection of neighboring rare variants at T2D-associated mutations. The survivors’ variants make the intronic region 4–6 the area of the greatest population differentiation within the TCF7L2 gene. The abundance of selective peak sweeps in the downstream region of the TCF7L2 gene suggests that the TCF7L2 gene is part of a region that is in constant recombination between populations.The online version of this article (doi:10.1186/s12863-016-0372-7) contains supplementary material, which is available to authorized users. Type 2 diabetes (T2D) is a leading global health problem. The diabetic population is estimated to increase from 366 million to 552 million by the year 2030. T2D-related genetic variants explain only a small portion of the heritability of T2D. Ethnic differences in the prevalence of T2D may be attributable to distinct genetic backgrounds of these populations. High-throughput sequencing and genotype data for ethnic groups other than those in which most of the known variants have been discovered will enable the discovery of new variants and an exploration of their role in T2D . GeneticTCF7L2 is a transcription factor in the Wnt signaling pathway and is expressed in many tissues, including fat, liver and pancreatic islets of Langerhans . GeneticAlthough the primers were designed primarily for exons of the TCF7L2 gene See , most ofThe majority of mutations identified in the Indigenous population were new and were located in noncoding regions Table , particu2 and XP-EHH  in Latino population, flanked to regions with a strong positive selection . It is worth mentioning that most of selected regions only in the Latino population  were localized in regions adjacent to TCF7L2 gene. The upstream region included the genes MIR4295 and VTI1A, while the downstream region harbored the genes DCLRE1A and NHLRC2. This downstream region was located within of a block hard selection sweeps, which covers several genes involved in metabolism, for example, genes PLEKHS1 and MIR4483. However, the values significant of r2 were found in a region of soft selection sweeps in TCF7L2 gene  of the TCF7L2 gene were less than 0.4 when we used only the African population. By contrast, we observed an increase in the values of r2 , the correlation between the loci associated with T2D in the intronic regions could be under selective constraints in the Amerindian populations. The TCF7L2 gene is located between two recombination hotspots  had an effect on enhancer activity, resulting in higher TCF7L2 gene expression [http://neandertal.ensemblgenomes.org/Homo_sapiens/Location/Genome)  of the Sigma Project exomes (data not shown). We observed a decrease in the variability of the TCF7L2 gene (80 variants) in the Indigenous population. The localization of SNPs along TCF7L2 gene was not constant, i.e., regions with low and high density of mutations were observed. The last exons (6–14) of the gene harbored the higher density of SNPs Fig. . We obsepression . Comparime) Fig.  revealedme) Fig. . This diAllelic frequencies are subject to selective and recombination processes. For example, the diversity pattern of the rs7903146 risk allele suggests a positive selective sweep that may have rapidly driven fixation in East Asians and decreased the frequencies in Europeans and Africans . We obseThe reticulations on the internal nodes are produced by recombination effects. The rapid growth of the human population also facilitated the spread of rare variants among different races. The presence of recombination hotspots inside and outside the TCF7L2 gene suggests constant genetic flow, and the phylogenetic interpretation is consequently unclear. We therefore constructed a NeighborNet tree see  based on2= 0.73) [2= 0.5 to 1) than the African population (r2= 0.2 to 0.5), and the SNPs in LD were not associated with T2D, suggesting a vertical inheritance of genotypes.TCF7L2 harbors variants with the strongest effect on T2D. The phenotypic changes associated with the risk genotype suggest that T2D arises as a consequence of impairment of several vital functions in the pancreatic islet . In addi2= 0.73) . By contPhylogenetic analysis of exon 4 suggested that the rs7903146 risk allele is an ancestral character and tends to recombine with a variety of haplotype backgrounds, particularly in the relatively diverse West African population ; howeverSelective sweeps can increase the genetic differentiation among populations, cause allele frequency spectra to depart from the expectation under neutrality, and also alter the allele frequencies of single nucleotide polymorphisms (SNPs) in the vicinity of the selected allele . We obseThe impact of the TCF7L2 gene on metabolic diseases is fundamental because this gene has multiple targets in the pathways for insulin synthesis . The hepThe TCF7L2 gene is the locus most strongly associated with T2D risk in different populations . In addiThe Amerindian population, based on self-reporting, comprised 60 Mazatecan from San Lorenzo Cuaunecuiltitla, Oaxaca (Mexico), 34 Nahuas from Xoteapa, Veracruz (Mexico) and 30 Zapotecan from the Mexican Diversity Project. Henceforth, these populations are referred to as the Indigenous population. Another group of 30 mixed-race Mexicans (Mestizo population) from the Mexican Diversity Project  participDNA was extracted using the QIAamp DNA Blood Maxi Kit, QIAGEN. We designed primers for each exon that also covered part of the introns surrounding the exon . This project contains five super-populations: African (AFR), Ad Mixed American (AMR), East Asian (ASN), European (EUR), and South Asian (SAN). The numbers of sampled individuals were 661, 347, 504, 503 and 489, respectively. The family relationship is included in the *.bed files along with the gender and geographic origin of the individuals.The variability (SNPs) of chromosome 10 was obtained from the 1000 Genomes Project ((v4.20130502) To assess variability inside the Latino population, particularly on chromosome 10, we used the Sigma Project database [The quality of Sanger sequencing  of the Indigenous populations was assessed by the fastqc program , with prReplication of the association test of case–control for T2D in Latinos with Amerindian ancestry greater than 70 % (described above) was performed by the plink program  with theThe area of study for selective sweep analysis and recombination focused only on chromosome 10 because this region harbors the TCF7L2 gene. The region was defined by referring to the human genome (hg19 version) and delimited with the coordinates: 113,925,369 – 115,925,369. This region encompasses the following genes: GPAM, TECTB, MIR6715A, MIR6715B, GUCY2GP, ACSL5, ZDHHC6, VTI1A, MIR4295, LOC103344931, TCF7L2, HABP2, NRAP, CASP7, PLEKHS1, MIR4483, DCLRE1A, NHLRC2, ADRB1 and CCDC186.Based on the variation identified in chromosome 10 from the whole-exome-sequencing of the Sigma Project (described above), we constructed a nucleotide sequence composed mainly of variation (SNPs) for each individual. If a mutation was not observed at a particular position for an individual in the alignment of all samples, we used the existing nucleotide in the hg19 reference genome (The Genome Sequencing Consortium). This process comprised two parts. First, we extracted the nucleotide variation for each individual using the pseq program  with flaThe traditional tests used: iHS , XPEHH  and nSL The nSL scans were performed and normalized using Selscan program  followinThe XP-EHH (Cross Population Extended Haplotype Homozygosity) statistic compares the integrated EHH profiles between two populations at the same SNP  and is eThe whole-genome tools: The ω statistic proposed by Kim and Nielsen  was ableLD was calculated for chromosome 10, particularly for the regions of the TCF7L2 gene. We extended this region 1000 kb downstream and upstream of the TCF7L2 gene (described above). The plink program  was usedThe mutations from the Sigma Project localized to the extended region (described above) and were searched against a GWAS database to identify the diseases associated with this genomic region. Additionally, the peaks of selective sweeps (determined above) and mutations of the Indigenous population previously identified were integrated by the LocusZoom program .To determine the relationships among the different genotypes associated with T2D (describe above), we performed Network median joining using the Network program . We usedThe mutations were analyzed based on their genomic coordinates in the ANNOVAR  and SNPShttp://genome.ucsc.edu/cgi-bin/hgGateway).The SNPs could create, destroy, or modify the efficiency of miRNA binding to the 3’UTR of a gene, resulting in gene dysregulation. Consequently, we explored each mutation identified in the Indigenous population in the following databases: miRdSNP , mrSNP  and RNAsThis study was approved by the Research Committee, Research Ethics Commitee and Institutional Biosafety Commitee from the National Institute of Genomic Medicine. All individuals gave informed consent before they were included in the study. Mestizo and Zapotecan participants gave written consent; Mestizos and Zapotecan samples were taken with anonymized consent; Verbal consent from Nahuatl population was taken and written consent from Mazatecan population was taken. The consent from Nahuatl population was verbal, because the samplings were made with the consent of the leaders of the ethnic group and each participant gave his approval to participate in the study. The oral approval was obtained with a translator and answers were given to all questions about the investigation.