FROH) and signatures of selection were determined by calculating runs of homozygosity (ROH). The results build the basis for a better understanding of the genetic development of Swiss dairy cattle populations and highlight differences between the original populations  and those that have become more popular in Switzerland as currently reflected by their larger populations .Domestication, breed formation and intensive selection have resulted in divergent cattle breeds that likely exhibit their own genomic signatures. In this study, we used genotypes from 27,612 autosomal single nucleotide polymorphisms to characterize population structure based on 9214 sires representing nine Swiss dairy cattle populations: Brown Swiss (BS), Braunvieh (BV), Original Braunvieh (OB), Holstein (HO), Red Holstein (RH), Swiss Fleckvieh (SF), Simmental (SI), Eringer (ER) and Evolèner (EV). Genomic inbreeding  differ more than the other pairs of populations. The original Swiss cattle populations OB, SI, ER, and EV are clearly genetically separated from the Swiss cattle populations that are now more common and represented by larger numbers of cows. Mean levels of FROH ranged from 0.027 (ER) to 0.091 (BS). Three of the original Swiss cattle populations, ER (FROH: 0.027), OB (FROH: 0.029), and SI (FROH: 0.039), showed low levels of genomic inbreeding, whereas it was much higher in EV (FROH: 0.074). Private signatures of selection for the original Swiss cattle populations are reported for BTA4, 5, 11 and 26.The levels of genetic diversity were highest and lowest in the SF and BS breeds, respectively. Based on The low levels of genomic inbreeding observed in the original Swiss cattle populations ER, OB and SI compared to the other breeds are explained by a lesser use of artificial insemination and greater use of natural service. Natural service results in more sires having progeny at each generation and thus this breeding practice is likely the major reason for the remarkable levels of genetic diversity retained within these populations. The fact that the EV population is regionally restricted and its small census size of herd-book cows explain its high level of genomic inbreeding.The online version of this article (10.1186/s12711-017-0358-6) contains supplementary material, which is available to authorized users. FST was highest for Holstein /[number of non-missing loci − number of expected homozygous loci]) and by using the option –homozyg (FROH) with the following non-default settings: at least 50 SNPs to define a ROH  for each breed were calculated according to McQuillan et al. [LAUTO is the length of the autosomal genome that spans SNP positions [2,497,129 kb in the current study;  were derived for the 9214 bulls based on pedigree data with the Software CFC [FPED and FROH were compared using linear regression and Pearson’s correlation coefficients, across all animals or only for animals with at least 95% known ancestors across the last five generations .Inbreeding coefficients . If the average di value of a window exceeded the 99th percentile of the empirical distribution of di, it was considered significant, resulting in 25 windows as putative signatures of selection for each breed.The http://www.ncbi.nlm.nih.gov/projects/mapview/; NCBI annotation release 104). Knowledge of population-specific characteristics  to 0.155 (BS), whereas the mean observed heterozygosity ranged from 0.357 (BS) to 0.399 (SF)  of pairwise genetic distances was used to visualize relationships between the 9214 bulls. Plotting the first dimension versus the second dimension revealed five distinct clusters  to 0.156 (BS/HO) in the investigated populations  to 47.5% (SI)  estimates (Table FPED was highest for BS (7.1%), followed by BV (5.9%) and HO (5.7%) and average genomic inbreeding  and 0.67 (p < 2.2e−16), respectively , at least 25% of the investigated animals within a population shared a ROH  are in Table di (25.7) was on BTA18 (14.2 Mb) in HO (Table di (24.3) in RH and was also significant in SF, SI and OB. The window with the third highest di (18.6) was on BTA14 (24.4 Mb) in RH. This latter window was significant in all populations and was previously reported to harbor signatures of selection , 47di hdi was on BTA11 (68.6 Mb) in OB and was also significant in BV, SI, SF and ER/EV. It was previously reported in OB [di of 12.9, the preceding window (i.e. on BTA11 between 65.5 and 67.5 Mb) was also significant in OB. A window with a significant di higher than 14 was found on BTA16 (between 24.7 and 26.7 Mb) in BV and BS and was also significant in OB, RH, SF and SI. In the same region, another window with a di higher than 10 was significant in BS (BTA16 between 26.5 and 28.5 Mb) and was also significant in BV and OB.The window with the fourth highest ed in OB . With a di higher than 10) were observed for HO on BTA20, i.e. in windows spanning 29.2–31.2, 43.5–45.6 and 46.6–48.6 Mb. All three windows were also significant in RH whereas the third window was significant in SF and SI. In the top two significant windows that spanned 43.5–45.6 and 46.6–48.6 Mb on BTA20, we did not identify any candidate genes (Table GHR gene was not significant in HO but moderately significant in RH (di = 7.4) and in SF (di = 5.9)  and on BTA26 (between 21.5 and 23.5 Mb). The BTA26 window was also significant in OB, another local Swiss cattle population. Among the nine loci localized in the region of the significant window on BTA4, no obvious candidate gene was identified. A second signature of selection on BTA4 (between 78.6 and 80.6 Mb) was private to ER/EV.For ER/EV, two windows with di of 13.1 was observed on BTA6 (between 69.3 and 71.3 Mb) and was also significant in BS, BV, HO and SF. The KIT gene (BTA6: 71.8–71.9 Mb) is located near this region. Based on the filtered dataset, the available SNP density in this region was very limited. Similarly, the window on BTA5 (between 16.3 and 18.3 Mb) was significant in five populations . On BTA5, two additional windows (between 55.6 and 57.6 Mb and between 60.5 and 62.5 Mb) were highly significant in the sampled SI individuals, with the first window (55.6–57.6 Mb) being also significant in the RH and SF populations. Two other windows on BTA5 were significant: i.e. the window between 75.1 and 77.1 Mb in SI, BS and BV and the window between 76.4 and 78.4 Mb in BS, BV and OB.In SI, a window with a di higher than 10 were detected on BTA5 (between 46.2 and 48.2 Mb and between 60.5 and 62.5 Mb) and BTA6 (between 32.4 and 34.4 Mb and between 77.4 and 79.4 Mb) but no candidate genes were identified in any of these windows.Additional windows with a FST, BS and HO were more differentiated (0.156) than all other pairs of populations  than from RH (0.024). This is also apparent in the MDS plot, where SF is more distant from SI than from RH. The comparison of within-population diversity, which was quantified either from genomic relationships or from observed heterozygosity, showed that the two extreme populations were SF and BS with the highest diversity observed in SF and the lowest diversity in BS. This is not surprising for two reasons: SF is well known as a composite population of SI and RH [We assessed the population structure of nine Swiss cattle populations by using 27,612 autosomal SNPs and showed that genetic diversity was highest in SF and lowest in BS. Based on and 1910 . Accordiand 1910 , the BS and 1910 , 50. SF I and RH  and, as I and RH .FROH and FPED using both 50k and HD SNP data and thus, they concluded that 50k SNP data are sufficient to identify ROH and to estimate genomic inbreeding. Because the parameters used to detect ROH vary among analyses, it is not easy to compare results from different ROH studies. The setting of the parameters used to derive ROH is crucial to account for the effects of SNP density correctly. Using the PLINK 1.9 [FROH. In our study, we defined ROH as tracts of homozygous SNPs that spanned a minimum of 50 consecutive loci, in regions with a minimum density of one SNP every 100 kb, a maximum gap length of 1800 kb, while allowing up to two missing genotypes per window but no heterozygous loci. Most ROH were observed in the length class of 5 to 10 Mb  to 0.091 (BS). The results for BS (0.091) and BV (0.074) are consistent with their low levels of genetic diversity. In contrast, the three original Swiss cattle populations ER (FROH: 0.027), OB (FROH: 0.029), and SI (FROH: 0.039) had lower levels of inbreeding. Natural service is still commonly used in these three populations .Various studies on signatures of selection in cattle using genome-wide SNPs have been published and for dairy cattle populations such as Holstein, Red Holstein and Brown Swiss, major signatures of selection have been described (e.g. ). In theKIT, MC1R, ABCG2, LCORL/NCAPG and PLAG1 [PLAG1 being significant in all eight population groups. The pleiotropic nature of this region [Genes known to be linked to strong signatures of selection in cattle such as nd PLAG1  are fulls region  and a pos region  are undePOLL locus, the MSTN and DGAT1 genes, and the genes from the casein cluster have not left any recognizable signatures of selection among the investigated populations. Based on phenotypic evidence, it is presumed that the region that includes the casein cluster is under selection but that it has not yet reached fixation in any of the Swiss populations. We did not detect any signature of selection in the DGAT1 gene, possibly because of the low SNP density in this region of BTA14. Since the Swiss dairy cattle populations are historically horned and not influenced by any double-muscled breed, the lack of signatures of selection around the POLL locus and the MSTN gene was not surprising.Selection at the INHBA gene represents a relevant candidate gene since it is involved in feed intake in Angus cattle [FGF8 and SCD. These two candidate genes are known to influence carcass quality, fatty acid composition of meat and milk and fertility traits [CAPN14, CAPN13, LBH and LCLAT1 genes that are located in this region and influence meat quality [PMEL and GDF11 genes is characterized by a di higher than 20 in SI (results not shown) but it was omitted from the final derivation of signatures of selection, because it did not fulfill the minimal SNP density of four loci per window. Based on these results, it is suggested that the extended BTA5 region between 55.6 and 62.5 Mb played an important role in the differentiation of SI. Besides many other loci, we propose STAT5, GDF11 and PMEL as potential drivers of the differentiation of SI from other cattle populations.The common ancestries between BS, BV and OB, between HO, RH and SF and between SF and SI are well known and were previously described , 38. Thus cattle . We detey traits –74. Due y traits  assigned quality , 76 shouFor several of the significant regions representing signatures of selection, we did not identify any candidate genes either due to the lack of genes with functional evidence in these regions  tree based on FST distances.Additional file 8: Figure S6. Relationship between the number of ROH and the total length of genome in ROH.Additional file 9: Figure S7. Distribution of the number of ROH in different length classes and for each population.Additional file 10: Table S4. Number of ROH per chromosome and population.Additional file 11: Figure S8. Regression of FPED on FHOM for all 9214 individuals. Multiple R-squared: 0.45.Additional file 12: Table S5. Correlations between FPED and FROH and between FPED and FHOM including all animals. Table S6. Correlations between FPED and FROH and between FPED and FHOM including only animals with PCI > 0.95.Additional file 13: Figure S9. Proportion of animals with this SNP within a ROH.Additional file 14: Figure S10.. Genomic distribution of the di statistic for all 1-Mb windows across all autosomes and the eight groups of populations. The dashed red line denotes the 99th percentile for each population group .Additional file 15: Table S7. Genomic coordinates (chromosome and start– and end–position) of all significant windows, the population for which di was calculated, and genes within this region.


Mosquito colony populations often show significant changes in their population genetic make-up compared to the field populations that were used as founding source. Most of the changes that have been reported are indicators of depletion in the overall genetic diversity of the colony populations. The Sterile Insect Techniques programme of mosquito control that is underway in Northern Sudan uses sterilized males produced from a laboratory-maintained colony population. The genetic diversity of an advanced generation of this colony population was quantitatively assessed and compared to the field population from which the colony was derived.Anopheles arabiensis mosquito samples from the 13th generation of the colony, and from the locality that was the source of the first generation of the colony, were genotyped at 11 microsatellite loci distributed throughout the species’ genome. Standard population genetic analyses were carried out to quantify and compare their population genetic make-up and diversities.The colony samples showed significant reduction in the total number of alleles, the numbers of rare and private alleles, and the fractions of heterozygote individuals at all the loci. The pattern of change is consistent with the expected effect of the use of a small number of mosquitoes when the colony was established. Departure from Hardy–Weinberg equilibrium in the direction of homozygote excess was observed at some loci and attributed to the presence of null-alleles.This study highlights the need for broad sampling when initiating colony populations and for ongoing assessment of the population genetic make-up of colony populations. Previous assessments of survivorship, dispersive behaviour and swarm formation indicate that the inbreeding and reduced genetic variability reported in this study may not have had direct fitness consequences yet. However, noting the lessons learned in other SIT programmes about the impact of colonization on male sexual behaviour and longevity, as well as other inbreeding related adverse effects, a systematic investigation of these potential effects is recommended because they have direct impact on the ultimate success of the programme. Insect species are reared in laboratories to produce colony populations from which a steady supply of the desired life-stage can be obtained for research as well as for biological control purposes. The laboratory colonies are either representative of the field populations from which they originate or serve as a stable standard to which other populations may be compared. Many studies have examined the genetic consequences of artificial rearing when compared to wild populations; some with focus on the implications on using the laboratory animals in pathogen transmission studies. These studies have revealed that the genetic make-up of colony populations often shows significant departures from the originally sampled field populations. Common features of the departures include: reduced number of alleles, divergence in allele frequency, presence of new alleles, reduced heterozygosity, fewer multi-locus genotypes, fewer rare alleles, and, reduced number of private alleles. Most of the changes that have been reported are indicators of depletion in the overall genetic diversity –4. The cAedes triseriatus, was reported and it was concluded that lethality of homozygous recessives at these loci accounts for the elevated heterozygosity 20] were Microsatellite genotyping was carried out according to . The forThe software package Tandem  was usedAn. arabiensis mosquitoes collected from Dongola area, henceforth referred to as field population, were compared to 51 samples from generation 13 of the An. arabiensis colony maintained at the TMRI insectary, referred to as colony population. DNA from each sample was PCR amplified using the species specific primers to confirm species identification and subsequently genotyped at 11 microsatellite loci, nine of which have been mapped to linkage groups (chromosomes) 2 and 3 while two are on the X chromosome [A total of 58 romosome . Binned Figure A private allele is an allele present in one, but not the other of the two populations that were compared. The total number of private alleles combined across all loci was 40, of which 36 were private to the field population. Averaged across all 11 loci, the number of private alleles in the field population was almost ten times as many as that in the colony population, 3.27 vs 0.36 private alleles per locus (Table E) is a measure of allele diversity, which is used when comparing populations in which the number of alleles and their frequency distributions differ drastically. It is defined as the number of equally frequent alleles it would take to achieve a given gene diversity, represented by the expected heterozygosity (He) of a population. It is computed as AE = (1 − (1/He). Again, a consistent trend of reduction in the effective number of alleles at all but one locus is evident in the colony population compared to the field population  based mosquito control programme underway in Northern Sudan that uses the TMRI population as source of sterile male mosquitoes.This study confirms that microsatellite allelic and genotypic diversities have declined considerably in the TMRI colony of mosquitoes compared to the field population from which it was established. Comparable loss of diversity has been reported in Mali by Norris et al.  at nine n et al.  also repThe contrasting features of the genetic diversities of the wild and colony populations, taken individually or combined, could be due to the founder effect. When a new population is founded from a small, random sub-sample of a genetically diverse population the common alleles in the source population are much more likely to be sampled than the rarer alleles, and, there is bound to be a reduction in diversity. This is borne by the data in that the common alleles retained in the colony population are also common in the field population Fig. . The redAn. arabiensis under semi-field conditions to laboratory-rearing in small cages have reported that the former retained a higher level of genetic diversity, showed less inbreeding and were phenotypically more similar to the founding wild population [A number of recent studies comparing colonization of pulation , 15, 26.pulation –29. Howepulation –32.An. arabiensis males to survive, disperse and participate in swarms occurring at varying distances from the release site, showed that sterile An. arabiensis males released into the field were able to find and participate in existing swarms. Helinski et al. [An. arabiensis males in the current SIT setting and laboratory-reared and irradiated An. arabiensis males from sixty generations were able to inseminate wild females at rates comparable to wild males. Mating competitiveness experiments showed that irradiated male mosquitoes are fertile as wild counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions [All novel insect control strategies involving mass release must address the issue of the evolutionary trajectory of genetic diversity in the released control agents relative to the source. This study has not confirmed or excluded the possibility that the elevated inbreeding in the colony population compared to the source population has led to loss of mating vigour in the sterile males, or any other change in their reproductive characteristics, or the evolution of premating isolation mechanisms. However, relevant insight has been gained from other studies focusing on the impact and consequences of colonization, mass rearing and loss of diversity within the SIT trial programme in northern Sudan. Excoffier and Lischer , who stui et al.  and Hassi et al.  reportednditions .This study highlights the need for broad sampling when initiating mosquito colony populations. Ongoing assessment of the population genetic make-up of colony populations at regular intervals is also recommended. The SIT programme in Northern Sudan has an active research programme that has looked into survivorship, dispersive behaviour and swarm formation in the colony population we studied. These studies have indicated that the inbreeding and reduced genetic variability we report may not have had direct impact on the traits that were investigated. However, we note the lessons learned in other SIT programmes about the impact of the population genetic makeup of release populations on male sexual behaviour , on the


Triatoma patagonica is a secondary vector of Chagas disease endemic of Argentina, and it has been found colonizing domiciles and most commonly peridomiciliary structures in several Argentine provinces and morphological variation along its distribution range have been described. To asses if population differentiation represents geographic variants or true biological species, multiple genetic and phenotypic approaches and laboratory cross-breeding were performed in T. patagonica peridomestic populations. Analyses of chromatic variation of forewings, their size and the content of C-heterochromatin on chromosomes revealed that populations are structured following a North-South latitudinal variation. Cytochrome c oxidase I mitochondrial gene (COI) nucleotide analysis showed a mean genetic distance of 5.2% between the most distant populations. The cross-breeding experiments suggest a partial reproductive isolation between some populations with 40% of couples not laying eggs and low hatching efficiency. Our findings reveal phenotypic and genetic variations that suggest an incipient differentiation processes among T. patagonica populations with a pronounced phenotypic and genetic divergence between the most distant populations. The population differentiation here reported is probably related to differential environmental conditions and it could reflect the occurrence of an incipient speciation process in T. patagonica.The epidemiological importance of Chagas disease vectors largely depends on their spreading ability and adaptation to domestic habitats.  Trypanosoma cruzi, etiologic agent of Chagas disease. This subfamily includes 150 species of obligatory hematophagous insects, classified in 5 tribes and 18 genera mainly distributed in the New World [Chagas disease is a neglected tropical disease considered a serious threat to human health in Latin America that also represents an emerging health problem in the United States and Europe . In the ew World .Triatoma infestans is the most successfully triatomine species adapted to domestic environments and the most important vector in the Southern cone of Latin America. Since 1991, large-scale insecticide spraying campaigns were successfully implemented in several countries for the elimination of domestic T. infestans. In some regions, when the primary vector is eliminated, other triatomine species become epidemiologically important. Included in these secondary vectors is Triatoma patagonica, an endemic species of Argentina with a wide geographical range from the north to the south of this country [Trypanosoma cruzi vector [The epidemiologic importance of Chagas disease vectors mainly depends on the spreading ability and adaptation to domestic habitats . Triatom country  Fig 1)..Triatoma country ,6. Diffei vector ,8.Phenotypic and genetic studies are essential to known taxonomic status, population structure as well as the differentiation and dispersal processes. The interplay among gene flow, selection and genetic drift across biogeographic regions often results in a mosaic of phenotypically differentiated populations –11. FactTriatoma genus [Triatominae species are considered plastic insects that respond rapidly to environmental changes, which might represent a common evolutionary route to new species . In Triama genus , 20. DNAma genus –24.T. patagonica are very scarce. Different patterns on the corium and connexivum related to their geographical distribution and wet conditions have been described [T. patagonica is restricted to two mitochondrial gene fragments (16S and 12S rDNA) [Population studies on escribed . Also, vescribed . Chromosescribed . Sequenc2S rDNA) ,26.T. patagonica populations to provide an assessment of their phenotypic and genetic variation, with the aim of understanding if they represent geographic variants or true biological species. We tested the hypotheses that both phenotypic and genetic variation will be structured in a latitudinal variation, associated with environmental conditions of the different ecological regions (eco-regions). Moreover, experimental reciprocal crosses between most distant T. patagonica populations were carried out in order to evaluate their reproductive compatibility and genetic isolation.In this paper multiple approaches were performed on some Specimens were originally collected from peridomestic structures in five Argentinean provinces, representing North , Center (San Luis—SL) and South  regions .T. patagonica is not an endangered or protected species. Insects analyzed came from natural populations (SF and SL) and 3 laboratory colonies  with no more than 3 laboratory generations. The geographic distance between the most distant collection sites (SE and RN) is around 1,300 kms. The geographic localization and number of specimens studied from each collection site, as well as the techniques applied, are described in SF individuals were collected from goat corrals, usually made of piled thorny shrubs and vertical posts. SL, LP, RN and SE insects were collected from chicken coops but with differences in their building materials. In SL, chicken coops were built with wire mesh and wood sticks and thatched. In LP and RN, chicken coops were made of cairns of stones, while in SE they were built with walls made out of wood sticks or mud bricks. Insects were supplied by the “Centro de Referencia de Vectores (CeReVe)” from the National Health Ministry from Argentina. All specimen collections were carried out by technical personnel from the National Health Ministry from Argentina. In all cases, owners of the domiciles gave permission to collect specimens. T. patagonica belong to 3 different ecological regions from Argentina , and become more reduced in the semiarid western region of Argentina [It has been proposed that rgentina . We compMost individuals were concurrently analyzed with all applied techniques . Wings athe geometric coordinates of each landmark were digitalized using tpsDIG 2.17 [Forewings were mounted between microscope slides and cover slips . PhotogrDIG 2.17  Fig 2A)the geomeDIG 2.17 . The CS http://www.flywings.org.uk/morphoj_page.htm). The relationship between CS and shape discrimination among populations  was estimated by the regression on size variation of the canonical axes derived from shape. We did not found allometric effects for any of the studied populations (results not shown).A generalized least squares Procrustes superimposition was used  for wingAir-dried chromosome preparations were made by squashing gonads in 50% acetic acid, freezing them in liquid nitrogen and removing the cover slip with a razor blade. Chromosome preparations were treated for C-banding to observe C-heterochromatin chromosomal distribution during mitosis and meiosis as previously described . Microschttp://www.blast.ncbi.nlm.nih.gov/Blast.cgi) to find the homologous sequences in GenBank. Sequences were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov), under accession numbers KR139998 to KR140009 and KU842348 to KU842352. For phylogenetic analyses, we used as a triatomine species included in the same subcomplex (T. guasayana) [http://treegraph.bioinfweb.info).For DNA extraction, one or two legs fixed in 70% ethanol were used from each specimen and total DNA was isolated by standard phenol-chloroform techniques. For each specimen, a 569-bp fragment of COI gene was amplified by PCR . The PCRasayana)  using COasayana)  with a sasayana)  using 103) covered with nylon mesh and containing vertically folded paper to allow insects’ access to food and to avoid humidity excess. During all experiments, insects were kept in the laboratory at 26 ± 2°C, 60–70% RH and a 12:12 h (L:D) photoperiod. Bugs were fed regularly every 15 days on pigeons (Columba livia). Once bugs had reached the adult stage, 28 crosses were made and each couple was placed in separate glass bottles. Once a week the number of eggs laid and eggs hatched were recorded. Crosses within each population were not performed since the number of fifth instar nymphs that CeReVe could supply was very limited. Nevertheless, intra-population crosses were performed with the same environmental conditions and feeding frequency for RN population and previously published [Laboratory crosses were conducted in the Laboratory of insect vectors from the FCEFyN  from fifth instar nymphs from SE and LP. Insects were kept in cylindrical glass bottles (500 cmublished .Three patterns of chromatic variation of forewings were observed across populations: dark pattern in North group (SF and SE populations), intermediate darkness pattern in South group (RN and LP) and a light pattern in Center group  . ConnexiComparisons of wing size between populations were shown in For wing shape analyses, the first two axes of the CVA performed with PCA scores, explained 37.11% and 27.15% of the variation, respectively. The factorial map distribution of each individual in the space of the first CVA axes showed that the SL and LP populations were highly differentiated in this space from the rest. RN, SE and SF individuals were overlapped .Male and female specimens from all populations analyzed showed the same diploid number (2n = 22) constituted by 20 autosomes plus two sex chromosomes . All individuals presented normal meiosis without meiotic irregularities. All specimens analyzed exhibit a C-heterochromatic Y chromosome and the autosomes may possess or lack C-heterochromatic regions. C-banding technique also revealed the existence of three C-heterochromatin distribution patterns. The North pattern includes all individuals of the North region (SE and SF populations). All autosomes showed large C-heterochromatic blocks in one or both chromosomal ends , represeThe 569 bp COI sequences analyses of 17 individuals showed 8 haplotypes, 3 of them represented by only one individual. Overall, there were 75 polymorphic sites, 17 of which were singletons. Maximum likelihood phylogenetic tree retrieved, using General Time Reverse (GTR) model, was well-supported containing two clades (100% bootstrap support): North clade including SF and SE individuals (3 haplotypes) and Center-South clade formed by SL (3 haplotypes) plus LP and RN individuals (2 haplotypes) .T. patagonica populations have genetic distances greater than 8.9% compared to T. guasayana used as out-group (Mean genetic distance (K2-p) between North and Center-South clades is 5.2 ± 0.9% (from 5.0 to 5.3 ± 0.9%). Genetic distance within Center-South clade  is from 0.5 to 1.3 ± 0.4%. All ut-group .Of the 28 experimental couples between North (SE) and South (LP) populations, 60% of them produced eggs, of which an average of 53% (37% and 67%) have hatched eggs .The number of laid and hatched eggs per couple varied greatly between the crosses SE x LP and LP x SE with RN x RN . These rTriatoma patagonica presents the southernmost distribution within all the Triatominae subfamily with broad North-South and East-West ranges that include a wide variety of environmental conditions, especially with regards to temperature and humidity [Speciation in Triatominae has been related to rapid morphological changes associated with ecological or environmental factors ,35. Durihumidity . Meteorohumidity  does notWing size as well as the C-heterochromatin content showed latitudinal variation. It has been proposed that wing size variation may be strongly influenced by ecological attributes of populations, while wing shape is thought to be affected by genetic and historical attributes . Wing siTriatoma species from North [T. infestans, marked C-heterochromatin population differentiation could be reflecting adaptive genomic changes that contribute to the ability to survive and reproduce in different environments [T. patagonica populations could also be related to adaptive changes associated with different environmental conditions.C-heterochromatin content differentiates three groups with a North-South decreasing C-content . Althougom North  and Soutom North ,20,31. Eom North . Changesom North . In T. ironments ,42. HeteT. patagonica clades (T. sordida sensu stricto and a new putative species T. sordida Argentina (5.3%) [For mitochondrial COI gene fragments, the biggest mean pairwise nucleotide distance (5.2%) was observed between North and Center-South a clades . A simila (5.3%) , but lowa (5.3%) . ConsideT. patagonica populations, it would be necessary to analyze hybrids fertility, including chromosomal behavior in F1 meiotic products.Hybridization studies are powerful tools for determining the existence of isolating mechanisms that lead to population divergence and speciation . ReproduT. patagonica populations are undergoing an incipient speciation process. All these results suggest that T. patagonica should be considered as a highly polymorphic species with a pronounced phenotypic and genetic divergence between the most distant populations. The genetic and phenetic variations here reported are probably associated with broad eco-regional differentiation . Analyses on other populations not sampled in this study, such as humid Chaco, humid Pampa, and the Patagonian steppe, could help to clarify the mechanisms involved in the T. patagonica population differentiation.Regardless of which processes are acting, the extreme chromosomal differentiation (C-heterochromatin amount), forewings corium and connexivum chromatic variation, wing size differences, COI gene sequences nucleotide differentiation and partial reproductive isolation determined by experimental crosses, suggest S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Sebastiscus marmoratus in 59 individuals from three populations in Chinese coastal waters. Based on all SNPs, there was little evidence of genetic differentiation among populations. However, outlier tests revealed 329 SNPs putatively under divergent selection across populations. Structural and phylogenetic topology analyses based on the outliers showed clear genetic differentiation among populations. Gene Ontology (GO) annotation results revealed that most of these outliers are known or hypothesized to be involved in metabolic process. Together with previous work using mitochondrial cytochrome b sequences, the present results further suggest that the population structure is strongly influenced by locally adaptive pressure. Overall, adaptive evolution in a heterogeneous environment plays an important role in inducing genetic differentiation among local populations. This study increases understanding of the factors  promoting and constraining population genetic differentiation in marine organisms.Advances in next-generation sequencing techniques have allowed for the generation of genome-wide sequence data, to gain insight into the dynamics influencing genetic structure and the local adaptation of marine fish. Here, using genotyping-by-sequencing (GBS) technique, we identified 31,119 single nucleotide polymorphisms (SNPs) for  Many marine fish species are distributed widely across heterogeneous landscapes, and across these ranges, natural selection can promote genetic differentiation and local adaptation. However, the evolution of ecological divergence may be impeded if high rates of migration homogenize the gene pool among populations5.Inferring the degree of genetic differentiation among populations of marine fish species is key to successfully managing fishery resources, allowing the identification of management units, assignment of individuals to geographic regions, and detection of product mislabeling and fraud6, its wide latitudinal range and its complex geological history7, the Northwestern Pacific (NWP) is a good region to investigate the patterns of genetic population structuring in marine fish species. Numerous studies on the phylogeographic patterns of marine fish based on neutral markers have been unable to detect genetic structures over such large geographic distances8 , even for species with weak migration abilities . This failure is understandable for fish species with strong migration ability, because the gene flow is unimpeded by ecological homogeneity and a lack of clear dispersal barriers in marine habitats15. However, owing to local adaptation and a lack of genetic exchange17, this observation makes less intuitive sense for settled or short-distance-migration fish species. Local adaptation plays an important role in promoting genetic divergence between populations, whereby natural selection increases the frequency of traits that enhance the survival or reproductive success of individuals expressing them19. Although the marine environment is relatively homogeneous compared with freshwater, numerous studies have revealed that differential thermal tolerance may result in ecological adaptation in marine species20. Moreover, with the development of sequencing techniques, a growing number of studies using a high number of genome-wide polymorphic makers suggest that marine populations are not as connected as might be presumed22. Hence, these techniques allow for inference of neutral population structure and also provide information on local adaptation or speciation events23. Consequently, local adaptation in marine fish species with little intraspecific population structure may commonly arise through ecologically different environments. The low intraspecific genetic structure reported in previous studies might be due to the limitation of markers and/or the short divergence time.Owing to its distinct geographical features with a series of marginal seas24, has facilitated the identification of such local adaptation through genome-wide scans. For instance, the GBS technique and related approaches have been used to investigate the genetic structure and local adaptation in marine fish species, such as sailfin molly Poecilia latipinna25, ide Leuciscus idus26, threespine stickleback Gasterosteus aculeatus28, small yellow croaker Larimichthys polyactis22, and Atlantic mackerel Scomber scombrus L21. Using such methods to investigate populations undergoing ecological divergence is particularly useful because these techniques provide good opportunities to detect genomic loci under divergence selection5, and such loci are expected to stand out with high Fst estimates against a background of low genetic divergence29. Such genome-wide scans can serve as a useful first pass  to identify the candidate loci for ecological divergence, which can then be subjected to further functional tests5. Given that the factors that promote or hinder local adaptation in marine organisms are still relatively unexplored, genomic studies that examine genetic structure and local adaptation are of great help.The advent and increasingly widespread use of high-throughput next-generation sequencing, such as the genotyping by sequencing (GBS) techniqueSebastiscus marmoratus, is an excellent marine organism to lend insight into the balance between local adaptation and genetic exchange because it is influenced by several factors that alternately promote and hinder local adaptation. S. marmoratus has a wide latitudinal range: it inhabits littoral rocky bottoms along the northwest Pacific coast from Japan to the Philippines30. Across this wide distribution, individuals encounter highly heterogeneous environments, thus indicating that natural selection might favour local adaptation. In fact, previous genetic work investigating mitochondrial cytochrome b (Cyt b) sequences has also shown that the northern and southern populations of this species are locally adapted to thermal stress, with population-specific amino acid substitution . In addition, S. marmoratus has strong site fidelity and usually appears within narrow home ranges in the daytime and hides in holes or crevices during the night30. Therefore, the latitudinal variation and ecological differentiation between the northern and southern rockfish populations used in this study may potentially drive local adaptation. However, population genetic studies of S. marmoratus using the mitochondrial control region sequences and the amplified fragment length polymorphism (AFLP) technique31 have detected no apparent genetic structure across its distribution area. Compared with the results based on the mitochondrial Cyt b sequences, the observed pattern of genetic structure may be derived from either a high level of gene flow in the present or a recent divergence with high levels of gene flow in the past.The marbled rockfish, S. marmoratus populations. We were particularly interested in characterizing the genomic variation across study sites with a background of genetic similarity and identifying genomic regions showing footprints of adaptive selection. Therefore, in this study, we sampled a total of 59 S. marmoratus individuals from three populations from the northernmost to the southernmost populations across its distribution range in China and then performed genotyping by sequencing (GBS) to examine genome-wide population structure and test for genetic signatures of local adaptation. In particular, we sought to elucidate the extent of the effect of local adaptation on the genetic differentiation of this settled rocky fish with a background of population homogeneity.This study’s objective was to gain insight into the balance between local adaptation and genetic exchange in geographically distinct http://www.ncbi.nlm.nih.gov/Traces/sra) under accession number SRP095927. After quality filtering, a total of 68.37 G of clean data was retained, which presented a nearly 100% effective rate. Overall, our GBS results showed a high phred quality , a high enzyme catch ratio ranging from 97.1% to 99.5% and a stable GC content ranging from 40.14% to 41.89%  for 59 individuals, with an average of 1.16 G (~515 million base pairs) per individual, which ranged from 0.63 G (~279 million base pairs) to 1.67 G (~743 million base pairs) (see Supplementary Table π) for all SNPs was 0.374 (standard error (SE) = 0.118) and ranged from 0.364 to 0.384 within each population (Table Ho) and expected heterozygosity (He) across all populations were 0.391 (SE = 0.173) and 0.374 (SE = 0.132) respectively , of which 1005, 833 and 773 SNPs were identified in the Fangchenggang-Rushan comparison, Fangchenggang-Zhoushan comparison and Zhoushan-Rushan comparison, respectively  for 16 fish species Fig. . The BLATo test the possible effects of local adaptation on inferring population genetic structure, the population genetic structure was analyzed by using two different SNP data sets: all putative neutral SNPs  and candidate outlier SNPs (329 SNPs). When all SNPs were used to construct the NeighborNet topology, three main clusters generally defined by geographic localities were recovered . However, only 0.74% of the variation arose among groups and among populations within groups, and the remaining 99.3% arose within populations , indicating a high level of genetic structure. Additionally, a relatively large proportion of variation (13.81%) occurred among groups and among populations within groups compared with only 0.74% for the all SNPs 35. The present data also supported this stepping stone hypothesis with robust evidence of patterns of IBD , under the most severe environmental conditions in glacial periods, S. marmoratus might have become extinct over large parts of its distribution and have survived in glacial refugium. When favorable conditions returned, population expansions from glacial refugium would be expected in S. marmoratus, and genetic similarity would be expected in the recolonized regions37 given the relatively young postglacial ecosystems 38. In the present study, the Zhoushan population showed higher nucleotide diversity and expected heterozygosity than other populations 41 and the small yellow croaker22. In this sense, the high degree of regional differentiation between northern and southern Chinese populations might be common in marine fish species, owing to local adaptation to heterogeneous environment. For instance, with the average annual sea surface temperature ranging from 10.9 °C in the Bohai Sea to 26.5 °C in the South China Sea , these differences in the thermal environment may result in divergent selection on specific genes42. More work is needed to verify the hypothesis of regional differentiation between the northern and southern Chinese populations.Recent studies have shown that high-throughput sequencing technology provides a novel approach for investigating local adaptation in natural populations of marine fishesNPs Figs . Given t43. For further analyses performed well with few false positives, three pairwise datasets were analyzed, and the results were contrasted with a parallel model. Among the three datasets, the number of candidate outliers was the highest in the Fangchenggang-Rushan comparison (1005/2274) whereas it was the lowest in the Rushan-Zhoushan comparison (773/2274). The results were consistent with the pattern of PCA plotting  of the SNPs represented variation that might be linked to adaptive selection, showing nearly 19 times the variation than when all SNPs were used. Similar results were shown by Wang et al.41, in which 7.3%  of the SNPs demonstrated substantial population differentiation in the spotted seabass. Likewise, 6%  of the SNPs in the study of the small yellow croaker were found to show significant divergence22. Much like S. marmoratus, earlier studies of small yellow croaker using mitochondrial fragments also failed to find similar levels of differentiation10. Hence, the higher number of genetic markers  in our study could be a possible explanation for the lack of correspondence with earlier studies on S. marmoratus, in which only a handful of genetic markers were used. A higher number of SNPs may be used as a window to gain relatively comprehensive insights into the dynamics influencing genetic differentiation among S. marmoratus populations. In summary, the estimated genetic variation should not be interpreted too literally as a direct measure of gene flow15, because the degree of divergence resulting from local adaptation might be underestimated among S. marmoratus populations.The analytical approach used herein provided a very conservative estimate of S. marmoratus populations in Chinese coastal waters should be considered as one fishery management unit31. Given the high level of genetic divergence revealed through selective markers within the same management unit, there is a clear mismatch between fishery management units and genetic population structure, which may lead to unsound management of local populations45. Similar suggestions were made for the fishery managements of marine fish with genetic backgrounds of population homogeneity  across Chinese coastal waters. Much like S. marmoratus, a recent study of small yellow croaker using genome-wide SNPs has also found a high level of population divergence within one management unit22. Large numbers of genome-wide markers is useful not only for determining traditional genetic structure resulting from gene flow, but also for identifying the hidden genetic structure in populations of marine fish species caused by local adaptation. As a result, given the robust patterns of genetic differentiation, sound management can be implemented for scientific and sustainable development.From the viewpoint of fishery management, the results in our study revealed a need for further studies and possible refinements in fishery management. Owing to the genetic homogeneity revealed by a handful of genetic markers, previous population studies suggested that S. marmoratus specimens was conducted in strict accordance with Animal Care Quality Assurance in China.Ethical approval was not required for this study because no endangered animals were involved. All handling of 46. The samples were collected by trawl net or hook fishing in offshore waters at the spawning season, thus ensuring that the samples collected were representative of the local populations. Muscle tissues were preserved in 95% ethanol for DNA isolation by using the standard phenol-chloroform extraction protocol.The samples were collected in the winter of 2015 from three separate sites: one from the coast of Rushan , Yellow Sea; one from Zhoushan , East China Sea; and one from Fangchenggang , South China Sea  to enable reads mapping and facilitate SNP calling in stage 2, using the PEAR 0.9.849 and USEARCH 9.050 software packages with default parameters. The “MockRef_Genome.fasta” file was used for read alignment and the “MockRef_Clusters.fasta” file was used for the downstream gene annotation. After a reliable reference was generated, all quality filtered reads were then sorted according to their unique index sequences and aligned to the reference assembly assembled from paired-end reads by using the bwa-mem algorithm in BWA 0.7.1251 with default parameters. After the alignment, SNP calling was performed using a conservative Bayesian approach, as implemented in the package SAMtools 1.3.152. SNP filtering was produced using VCFtools53 with the following parameters: (1) the SNP was called in at least 50% of individuals (--max-missing 0.5), (2) the minor allele frequency (MAF) was > 10% (–--maf 0.1), (3) the average read depth was < 150 (–--max-meanDP 150), (4) only two alleles were present , (5) loci with quality score < 98 were eliminated (–--minGQ 98), (6) only sites with quality value > 30 were included (–--minQ 30), (7) sites that contained an indel were excluded (–--remove-indels), (8) sites that failed the Hardy-Weinberg equilibrium (HWE) test at P < 0.05 were excluded (–--hwe 0.05). Subsequently, contigs consisting of putative SNPs were aligned with the S. marmoratus mitochondrial genome (NC_013812) by using Geneious 10.0.654 software, and SNPs contained in aligned contigs were excluded. All datasets were reformatted using PGDSpider 2.0.5.255.The generated GBS-tags were analyzed using the GBS-SNP-CROP pipeline for SNP calling56 was used to estimate nucleotide diversity (π), observed heterozygosity (Ho) and expected heterozygosity (He) of each population. The fixation index (Fst) for each pairwise comparison was also estimated using Arlequin. In addition, to characterize the genome-wide pattern of genetic variation, the TASSEL v5.2.3157 program was used to estimate nucleotide diversity (π) and Tajima’s D value at each SNP for all individuals, and the Arlequin 3.5 program was used to estimate expected heterozygosity (He) at each SNP site. The patterns of genomic variation of each population based on all SNPs as reflected in He, π and Tajima’s D were visualized using the R package RCircos58.To characterize the pattern of population differentiation, the program Arlequin 3.5http://map.baidu.com). These measures of genetic and geographic distance between each of the three sites were used to test for isolation by distance as implemented in IBDWS (http://ibdws.sdsu.edu/~ibdws/distances.html)60. According to Slatkin’s recommendation61, the log of both genetic and geographic distance was used as inputs for the Mantel test for matrix correlation between the genetic and geographic distance. Reduced major axis regression using 1000 randomizations was also performed to calculate the intercept and slope of the regression line of genetic distance vs. geographic distance.Geographic (shortest straight line) distances between each site were calculated using Baidu Map . Any locus with Fst higher or lower than the neutral distribution was considered a candidate that might be under selective pressure63. To gain insight into the dynamics influencing adaptive divergence, outlier analyses were implemented on the basis of three pairwise comparisons: (i) individuals in population Rushan and Fangchenggang, (ii) individuals in population Rushan and Zhoushan, and (iii) individuals in population Zhoushan and Fangchenggang. Lositan was first run using all loci under attempted neutral mean Fst, 50,000 simulations, 99.5% confidence interval, infinite alleles mutation model, and false discovery rate of 0.1%, following the procedure described in Antao et al.62, to decrease the bias in the estimation of the mean neutral Fst by eliminating extreme loci from the estimation. After the first run, all loci that were outside the confidence interval were removed, and the mean neutral Fst was recalculated. Only the supposed neutral loci were used in this run under the same parameters as above. The third run used all loci and the newly calculated neutral Fst, with all other parameters maintained. Loci recovered as outliers in the last run were inferred to be under selection. To perform analyses with few false positives, outliers detected on the basis of at least two datasets were chosen for further analyses. The results were shown through a Venn diagram constructed in Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/index.html).The program Lositan64 and equal angle splits transformation65 using the SplitsTree v 4.14.4 program66 on the basis of (i) all loci and (ii) loci identified as being under selection with the program Lositan. This approach was applied to compare the power of discrimination of individuals within populations of likely non-neutral (or adaptive) markers and putatively neutral markers68. To assess the robustness of the topology of the tree, 1,000 bootstrap replicates were performed using the SplitsTree program.We constructed a splits tree with NeighborNet distance transformationadegenet69 to determine whether sampled individuals reflected a history of differentiated populations by outputting individual coordinates along axes of genetic variation within a statistical framework that corresponded to the first two principal components.Principal component analyses (PCA) based on the two datasets were implemented using the R package 70 and analyzing the two datasets, with a maximum-likelihood method. The best value of the coancestry cluster (K) was estimated using a corss-validation procedure in the admixture software. The best value of the coancestry cluster exhibited the lowest cross-validation error (CVE) compared with the other cluster values. The CVE values of K 1–3 were estimated, and the lowest CVE value was represented by the K = 1  and K = 3 (for outlier SNPs). We increased the coancestry clusters spanning from 1 to 3 and ran the analysis with 10,000 iterations.To estimate the individual admixture assuming different numbers of clusters, the population structure was investigated by utilizing the program admixture 1.3.0We also conducted an analysis of molecular variation (AMOVA) in Arlequin 3.5 to examine the variation within and among groups of genetically similar populations by using two datasets. The hierarchy for this analysis was chosen on the basis of the clustering from the PCA analysis: (i) populations Rushan and Zhoushan, and (ii) population Fangchenggang. The significance of the covariance components associated with the different possible levels of genetic structure was tested using 1000 permutations.71. This software conducts BLAST similarity searches and maps Gene Ontology (GO) for homologous sequences. Blast2GO produces GO annotations as well as corresponding enzyme commission numbers (EC) for sequences with E-value < 1E-6, annotation cut-off > 55 and a GO weight > 5. Subsequently, the annotated sequences with corresponding ECs obtained from Blast2GO were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database.The contigs containing outlier SNPs identified using the program Lositan were used as queries in nucleotide searches with BLASTX  against the nr database at the National Center for Biotechnology Information (NCBI) website. In case of multiple hits, the best match was chosen. Then, the functional annotation of these genes were obtained using the Blast2GO softwareSupplementary Information except Table S2Supplementary Information Table S2


Protonectarina sylveirae (Saussure) presents a broad distribution from Brazil, Argentina and Paraguay, occurring widespread in the Atlantic rainforest and arboreal Caatinga, being absent in the Amazon region. Given the peculiar distribution among swarm-founding wasps, an integrative approach to reconstruct the evolutionary history of P. sylveirae in a spatial-temporal framework was performed to investigate: the presence of genetic structure and its relationship with the geography, the evolution of distinct morphologic lineages and the possible historical event(s) in Neotropical region, which could explain the observed phylogeographic pattern. Individuals of P. sylveirae were obtained from populations of 16 areas throughout its distribution for DNA extraction and amplification of mitochondrial genes 12S, 16S and COI. Analysis of genetic diversity, construction of haplotype net, analysis of population structure and dating analysis of divergence time were performed. A morphometric analysis was also performed using 8 measures of the body of the adult (workers) to test if there are morphological distinction among populations. Thirty-five haplotypes were identified, most of them exclusively of a group and a high population structure was found. The possibility of genetic divergence because of isolation by distance was rejected. Morphological analysis pointed to a great uniformity in phenotypes, with only a small degree of differentiation between populations of south and the remaining. Divergence time analysis showed a Middle/Late Miocene origin, a period where an extensive marine ingression occurred in South America. Divergence of haplogroups began from the Plio/Pleistocene boundary and the last glacial maximum most likely modeled the current distribution of species, even though it was not the cause of genetic breaks.Swarm-founding wasps are endemic and common representatives of neotropical fauna and compose an interesting social tribe of vespids, presenting both complex social characteristics and uncommon traits for a eusocial group, such as the absence of castes with distinct morphology. The paper wasp  Comprehension of the evolutionary processes that generate and maintain the biological diversity of tropical fauna has been challenging biologists for centuries and should be the underpinning of conservation strategies . CurrentPhylogeography focuses on the processes governing the distribution of genealogical lineages within species across the geographical landscape , 7. PhylA single model of vicariance or recent climate changes cannot explainthe origin of Neotropical biota . This reP. sylveirae is very low, once there is only one or a few reproductives in the colony. The swarm strategy plus the polygyny (presence of more than one queen) greatly reduced the risk of loss of the reproductives, increasing the survive of the colony [Swarm-founding wasps of the Epiponini compose an interesting social tribe of vespids, presenting both complex social characteristics  and uncommon traits for a eusocial group, such as castes with the same or with a very similar morphology , 17. As e colony , 20, 21.e colony , 23.Protonectarina sylveirae (Saussure) is the only species of the genus Protonectarina Ducke, and these aggressive wasps form colonies of large populations that build very large and perennial arboreal nests, usually suspended from a twig [Protonectarina is the only one that is not found in the Amazon region, occurring widespread in the Atlantic rainforest and arboreal Caatinga  forests and brejo forests [The Brazilian Atlantic Forest is the second largest tropical forest in South America, covering an area of > 1,000,000 kmrgentina –30. Comprgentina , 31, whi forests . For thi forests , amphibi forests , 35, rep forests , 37, bir forests ,39, bats forests  and plan forests . Moreove forests , 42.P. sylveirae, may reveal useful information regarding how such species responded to past climatic changes and how these events may have driven the evolutionary history of populations [The most well-known hypothesis on the origin of diversity in the Brazilian Atlantic rainforest is based on the classical Pleistocene Refugia Model , 44, whiulations .P. sylveirae in a spatial-temporal framework. The aim of this study was to answer the following questions to investigate the phylogeographic and demographic history of this species: (1) Do populations of P. sylveirae present genetic structure? (2) If yes, is this structure related to geography? (3) Does the genetic structure also lead to the evolution of distinct morphologic lineages? (4) Is the genetic diversity of this species a result of past habitat fragmentation or recent range expansion? (5) Is there some historical event(s) in the Neotropical region that can be related to the observed phylogeographic pattern?Given the widespread  and the peculiar distribution among Epiponini wasps (absent in the Amazon region), in the present study, we used an integrative approach to reconstruct the evolutionary history of Wasps were actively collected along the distributional area of the species with entomological nets in the vegetation or directly from nests and preserved in ethanol (96–100%). Only worker wasps were collected to avoid bias in morphometric analysis due to the dimorphism among castes . One samcytochrome oxidase subunit I (COI) and 16S and 12S ribosomal RNA. PCR was performed using a PCR Ready-To-Go Beads (Amersham Biosciences) kit. Primers used to amplify sequences of COI and 16S were developed by Schulmeister et al. [http://mafft.cbrc.jp/alignment/server/).DNA was extracted from legs, antennae and mesosomal muscles of wasps using DNeasy Blood & Tissue (Qiagen) and Illustra tissue & cells genomic Prep mini spin (GE) kits. The extracted DNA was used to amplify part of the mitochondrial genes r et al. , whereasr et al.  and aligr et al.   version 1.7 [P. sylveirae. An Analysis of Molecular Variance (AMOVA) was performed to test the presence of populational genetic structure. This analysis is based on F-statistics and assumes the hypothesis that the genetic diversity within two populations is not significantly different from that resulting from the joining of the two populations [a priori population group; grouping populations according to the geography; and grouping populations in groups that resulted from the arrangement obtained in the haplotype network. AMOVA was also performed in PopART.To determine the relationships among the haplotypes, a network was constructed in PopART  was performed considering the premise of panmictic populations using the program DnaSP v. 5.101 [The distribution of genetic differences between pairs of haplotypes  as out-groups under the GTR nucleotide substitution model, which was identified as the best model for the data in MEGA 7.0.7 [Agelaia electra (20.43 to 13.65 mya) [The time since the most recent common ancestor (TMRCA) was estimated under a Bayesian approach using BEAST 1.8.3 . AnalyseGA 7.0.7 . An unco.65 mya)  was used.65 mya) .P. sylveirae from the 13 populations sampled  was used to derive the measures in the images. Comparisons between populations within the species were performed. To determine the degree of discrimination and identify which measures best explained the data, a multivariate discriminant analysis with a posterior canonical discriminant analysis was performed [Digital images of the bodies of 128 individual  sampled  were mea , which was the most widespread haplotype geographically. Additionally, samples of SGRA and VIC shared haplotype 1 . All theThe Mantel test showed performed for all the samples, no correlation between genetic distance and geographical distance, indicating that the genetic divergence among populations could not be attributed to isolation by distance .P. sylveirae, with the peculiar haplotype composition for each sample and the presence of “hypothetical haplotypes” (black circles), indicating some haplotypes were already lost, clearly demonstrated. Nevertheless, a lack of samples cannot be discarded.From the topology of the haplotype network, three groups were delimited according to geography, composed by haplotypes from Northeast (NE), South (SO) and Central (CE) regions of Brazil. Notably, although geographically close to the cities of BON and BOD, haplotypes from DOU were closely related to those from the South (haplogroup C). Some haplotypes were separated by many mutations, including within these geographical groups . At least six distinct haplogroups, A to G, were delimited ; Fig 4A.ST (0.948). The source of variation was 94.81% among populations and only 5.19% within populations (CT (0.90299) compared with that in the analysis with groups defined by geography (0.76017), andninety percent of the variation was found among the groups  considering all populations as a single group resulted in a high value of Fulations . This reulations . Analysie groups .Mismatch distribution for overall samples resulted in a multimodal distribution, which suggested populational stability. The same pattern was found for the three groups according to geography. A multimodal pattern was also found for haplogroups C and E, in contrast to groups A, B and F that exhibited a unimodal pattern, suggesting a recent demographic expansion [The xpansion , 78 or axpansion , 80  and the others (at 1.83 mya), followed by the separation between lineages from the South  and the others (at approximately 1.35 mya). More recently, at 0.36 mya, the Central lineages became separated in two primary groups: SACA, SJRP and LAV and BON, BOD, IND, SGRA and VIC  in functions generated by the value of Wilks’ λ were determined to identify those that were important to discrimination among groups (populations), and only the measures MeL and MeH were not significant . AnalysiThe ordination plots  show theProtonectarina sylveirae were clarified in this study. Across the distribution range, populations were genetically highly structured, as confirmed by AMOVA, presumably reflecting long lasting isolation in the past. Populations within the same geographical region were genetically more similar, such as populations of BON and BOD, or presented a low degree of genetic divergence, suggesting restricted gene flow between geographically separated groups of populations. However, the haplotype composition for most populations inside the same geographical region implied a condition of restricted gene flow not related to isolation by distance but most likely caused by a barrier. A unique haplotype composition was found for populations of P. sylveirae, in which exclusive alleles were found across most of the populations. A similar pattern was found for Eugenia uniflora from the south of the Atlantic Forest, which became geographicaly isolated during glacial cycles in fragments with appropriate environmental conditions, resulting in lineage divergence [P. sylveirae suggests a long-term persistence of these populations, rather than recent migrations. Moreover, the founding-nest behavior of reproductive females most likely reinforced the highly structured pattern of P. sylveirae. Indeed, individuals sampled in geographically close nests shared very similar haplotypes with low nucleotide diversity, which is also found within some species of ants with restricted female dispersion [The evolutionary history and the pattern of genetic variability for the species vergence . In the spersion –84.A swarm has a limited dispersion capacity once the queen is constrained by the dispersing ability of nestmates . The lowP. sylveirae showed low haplotype sharing and long-term divergence. This divergence is not surprising when the distribution and the molecular markers of this species are considered. The use of only mitochondrial markers has some limitations, mainly because it represents the study of a single locus, showing only a small part of the history of populations and reflecting the matrilineal history, which may differ from the population's history when analyzing, for example, nuclear genes [Lineages of ar genes , 89. Morar genes .Although reciprocal monophyly of the alleles at a given locus in allopatrically or parapatrically distributed sets of populations is commonly interpreted as an indication of distinct species , 91, othP. sylveirae, reduces queen mortality risk and increases homeostatic control of the colony, but the dispersal process could be restricted with populations prone to local extinction because the swarm is vulnerable to harsh climatic conditions during the migration [P. sylveirae are not common and are not easily located in the field, with nests usually built in high trees and found only in areas with forested vegetation. Even samples collected in dry environments, such as those from CANO, CAT and MOS in the Caatinga biome, were found in high trees. These biological requirements likely shaped the distribution of populations and contributed to the low rates of gene flow among populations. Areas with open vegetation and/or dry conditions most likely were not a barrier for the expansion of populations but were for the establishment of colonies.The type of social organization in vespids has generated profound effects in several aspects of the group . Non-swaigration , 94. NesP. sylveirae. The divergence among haplotypes and the genetic structuration suggested a deep divergence instead of ancestry based on coalescence, indicating low rates of gene flow and resulting in the high index of FST. Considering the predictions of the theory of coalescence, a direct relationship occurs between haplotype frequency and the age of haplotypes in which the most common haplotypes are the oldest [Sufficient gene flow between populations can slow down or prevent the process of geographic differentiation and leave a signature of little population structure over large areas. This signature is commonly observed in flying insect species, specifically in those species that migrate or are good dispersers . Neverthe oldest . Hence, e oldest . In thise oldest . NeverthST [P. sylveirae, only a few individuals of the colonies are reproductive. After a swarm, colonies start with several queens and workers, and as the colony develops, some queens disappear or adopt a worker role, and the number of queens reduces until one or a few queens remain [Angiopolybia pallens and Synoeca surinama, two species of paper wasps in the same tribe as Protonectarina. The author found a lower genetic diversity for S. surinama than that for A. pallens, which was attributed to the differences in biology between the two species . In the same way, both genera diverge earlier than Protonectarina [Rapid cycles of extinction and colonization can also affect the genetic structure of populations in which frequent founding events during colonization may increase the value of FST . NeverthST . The comST . In eusos remain . Carvalhs remain  also fouectarina  and showThe multimodal Mismatch distribution suggested stability in populations , 79. HowMorphometric analysis indicated differences among populations, which were statistically significant, although morphologically imperceptible. Furthermore, according to the results, the selected measures were adequate for discrimination of groups. From the graphic of discriminant analysis , a diffeConsidering other morphological traits, such as cuticular punctuation and color, no different patterns were observed among populations. In fact, color showed intra-population differences. Several studies reveal deep intraspecific phylogeographical structure with multiple lineages (phylogroups) , which cDifferences concerning patterns obtained from molecular and morphological data rely on the way in which selection acts to drive evolution . FunctioP. sylveirae in the eastern part of Brazil, explaining the absence in the Amazon region.The dating analysis showed a time of origin of the species in the Middle Miocene (approximately 13 mya). During the Miocene, one of the largest and most extensive humid environments in geological history was formed in the Amazon region. The evolution of this system was influenced primarily by the rise of the Andes in association with other factors such as global sea level change, subsidence and a seasonally humid climate . In the P. sylveirae the oldest ones. Analysis of palynological records for areas of the Caatinga in the states of Bahia and Pernambuco showed hot and wet weather [P. sylveirae populations.Divergence times estimated for the primary genetic breaks were placed within the Plio–Pleistocene periods (approximately 2 mya), a period in which the average temperature of the earth began to decrease rapidly and for which extensive records document changes in forest distribution associated with climatic cycles , 118–126 weather –129 unti weather . For Cat weather , 129. ThFrom the current vegetation, the Caatinga is clearly inhospitable to most rainforest species and even more stressful to arboreal ones . Living P. sylveirae. The Atlantic Forest may have been able to maintain a reasonable degree of continuity during such climatic events because of the complex topography of the region and the potential for vertical migration [P. sylveirae included altitudes from 400 to more than 1000 m, which can be explained by vertical migration. Based on the mountain refuge hypothesis [P. sylveirae.Climatic fluctuations and the effect of Pleistocene arid phases were greater in the southern portion of the biome than in the northern region , 133, whpothesis , 135, dupothesis . MoreoveThe DOU population was in the SO group, and these populations share the Paraná watershed and a common climate region. However, the DOU population was morphologically different from the other populations, including those of the same group. A possible cause is the Paraná River between DOU and the other SW populations, which could have hindered gene flow, reinforcing the differences among populations. Another notable result was that, despite the spatial proximity, the population of DOU was not in the same haplogroup of populations of BON and BOD. However, BON and BOD are in a different watershed (basin of Paraguay). Moreover, DOU is in the plateau zone of the state of Mato Grosso do Sul, whereas BON and BOD are in Serra da Bodoquena, which are two different geological formations.Angiopolybia pallens and Synoeca surinama [et al. [Synoeca, with subsequent diversification of extant species occurring in the Plio-Pleistocene. The calibration used was different for both works and also differed in this study. The inference of lineage divergence time from molecular data has been criticized, primarily because dating is prone to many types of error  [Previous studies with other genera of Epiponini also found a recent time of divergence. A phylogeographic analysis of surinama  found ea [et al.  found a bration) , one of bration) . Hence, Protonectarina sylveirae presented a strong structuration, suggesting the presence of barriers, which restricted or even interrupted the genetic flow among populations. Moreover, deep phylogeographical divisions within taxa also suggested extensive periods of isolation among populations of the species. The possibility of genetic divergence because of isolation by distance was rejected. From the haplotype network, three primary groups were delimited based on geography: Northeast, South and Central. However, at least six haplogroups were delimited genetically. Demographic analysis showed stability for most populations, indicating that the populations had remained constant throughout their evolutionary history. However, signs of recent expansion were found for populations of the south region and Catimbaú. Importantly, note that the eusociality of P. sylveirae also strongly influenced genetic and geographic patterns. Despite the uniformity in phenotypes, a small degree of morphological differentiation was also found for populations in morphometric analysis, revealing a distinction between populations of SO and the others and a particular morphology for the sample of DOU. No differences in other morphological features such as color or punctuation were observed for different patterns among populations.The genetic diversity of Protonectarina in the limit of Middle/Late Miocene, approximately 11 mya. In this period, an extensive marine ingression acted as a barrier, which could explain the absence of the species in the Amazon region. Divergence of haplogroups began from the Plio/Pleistocene boundary. No clear overall pattern indicated the origin of most of the South American species during the Pleistocene, as predicted by the refuge theory. However, the last glacial maximum (LGM) most likely modeled the current distribution of species, as observed for P. sylveirae. Nevertheless, the climatic changes that occurred in the LGM could not be the cause of genetic breaks among lineages because that event occurred more recently than the divergence found in our analysis. The divergence times obtained might provide a crude estimate of the relative order of events isolating the major haplotype lineages of P. sylveirae in the Brazilian Atlantic Forest.The dating analysis indicated the origin of Protonectarina sylveirae and increase understanding of the complex pattern of diversification for the enormous biodiversity in the Brazilian Atlantic Forest. Further studies using a multiple loci approach and increasing the range of geographical sampling of the species will provide a more complete picture of the evolution and diversification of paper wasps and contribute more pieces to solve the jigsaw of historical diversification of biota in South America.Our results provide insights on the phylogeography of S1 FigLines represents RMA regression. The values of p were not significant for any of the analyzes. Z, Pearson's correlation coefficient between matrix; r, Pearson’s statistics.(TIF)Click here for additional data file.S1 Tableforward; R, reverse; bp, base pair; *approximate size of the amplicon. COI, cytochrome oxidase subunit I; 12S ribosomal RNA, 16S ribosomal RNA.F, (DOCX)Click here for additional data file.S2 TableAccession numbers provided after submission of obtained gene sequences.(DOCX)Click here for additional data file.S3 TableThe matrix shows, for each population, the number of cases classified correctly and incorrectly according the discriminant model. Rows: observed classification; columns: predicted classification.(DOCX)Click here for additional data file.S4 TableNumbers in bold show the contribution of the two functions that best explained the discrimination found among populations. HL, head length; HW, head width; MW, mesoscutum width; MeL, mesosoma; MeH, mesossoma height; HFL, hind femur lenght; T2L, 2° metasomal terga length and T2W, 2° metasomal terga width.(DOCX)Click here for additional data file.


Cheiracanthium punctorium, a medically important species in Central Europe. Using microsatellite markers and DNA sequences, morphological and climate niche analyses, we identify factors associated with the spider's expansion success. Our results indicate that the species' initial expansion has been triggered by environmental change and preadaptation in the source populations. However, despite extensive gene flow, expanding populations maintain genetic and morphological differentiation from native ones, which is correlated with climatic niche differences. Moreover, expanding spiders might have temporarily escaped an eggsac parasite that causes high mortality in the native range. Hence, our results paint a complex picture of diverse factors associated with expansion success. We speculate that expanding populations might be capable of adapting to novel ecological conditions in northern Europe. This could allow a substantial range expansion, much farther than by environmental change alone. Our distribution model predicts that the spider will soon massively spread over most of northern Europe, bringing along considerable health concerns.Although poleward range expansions are commonly attributed to global change, a complex interaction of ecological and evolutionary factors might contribute to expansion success. Here, we study the expansion of the yellow sac spider  Global ecosystems are currently exposed to unprecedented environmental change. Although global change is often associated with range contractions or even extinctions , offers an interesting example of an expanding spider species. It is a comparably large, free ranging, and nocturnal hunter, living on fallows and meadows. Their historical European distribution was largely limited to warm climate regions in southern Europe with the southern Upper Rhine Plain marking the northern limit of its distributional range . A total of nine Iberian and nine Baltic DNA samples were chosen each from three populations, and their concentration measured on a NanoDrop Fluorospectrometer . Equal DNA amounts were pooled for the two geographic regions, and each pool sequenced in 300 bp paired reads on one flow cell of an Illumina MiSeq. Library preparation and sequencing were conducted according to the manufacturer's protocols . The resulting DNA reads were quality trimmed with a minimum quality of 20 using Popoolation  and the mitochondrial COI gene (for 399 specimens) were PCR amplified using the Qiagen Multiplex PCR kit according to the manufacturer's protocol and using the primers 28Srd4.8a and 28Srd7b1 . Russian, Iberian, and populations from northeastern Germany did not show significant differences in diversity.We did not find a pronounced difference of genetic diversity between native and expanding spider populations. This held true for allelic richness (AR) and expected heterozygosity (EH). The highest diversity was found in Russia , followed by Iberia  and Italy, then northeastern Germany , and finally, the lowest in the Baltic States . The only significant difference in diversity was found for the Baltic populations, which showed a reduction of allelic richness and expected heterozygosity compared to all other populations , while haplotype diversity was more comparable . Similar to the Russian populations, about 30% of the specimens from the Baltic States also carried haplogroup B. Their haplotype was distinct from the Russian one by a single substitution. Their nucleotide diversity was high, while their haplotype diversity was reduced compared to native populations . An even more pronounced reduction of diversity was found for expanding populations in northeastern Germany, which carried only two slightly divergent haplotypes from group A . In contrast to the mitochondrial data, the 28S rDNA was completely monomorphic across all of Europe, with all 105 sequenced specimens sharing the same 28S alleles.The mitochondrial haplotype network Fig.  showed aanova, Games–Howell post hoc test, P < 0.05). In addition, we found a significant size difference between the two expanding populations, with specimens from northeastern Germany being larger than those from the Baltic. Overall, the body size data were highly congruent with our microsatellite analysis.Our body size measurements indicated a homogenous native population from Russia (average body size ± standard deviation = 4.72 ± 0.59 mm), Italy (4.82 ± 0.51 mm) and the Iberian Peninsula (4.62 ± 0.41 mm), as well as two isolated expanding ones in the Baltic states (4.06 ± 0.47 mm) and northeastern Germany (4.42 ± 0.50 mm) Fig. A. Body sanova, Games–Howell post hoc test, P < 0.05). Russian spiders and those from the two recently colonized locations shared a very similar relative leg length . This analysis thus corroborated a closer relationship of Russian and expanding populations.The relative leg length showed slightly different results Fig. B. Here, Sarcophaga sexpunctata , a widely distributed flesh fly known to parasitize eggsacs of different spider species. We screened 10 expanding and nine native populations for S. sexpunctata infections. Of nine screened native Italian populations, all showed infections. We estimated the abundance of S. sexpunctata infections in four of these populations. Between 30% and 90% of all eggsacs were parasitized, on average 50%. At the same time, none of the 10 analyzed expanding populations from the Baltic States and northeastern Germany were affected by brood parasitism. Inquiries with fellow arachnologists did not reveal any incidence of parasitism in additional expanding populations in Sweden and northeastern Germany (Jonson and Friman pers. comm.).DNA barcoding identified the eggsac parasite as Principal component analysis revealed four PCs with eigenvalues >1. PC1 was predominantly composed of temperature and precipitation‐related variables . PC2 was most strongly correlated with Bio14: Precipitation of the Driest Month and Bio18: Precipitation of the Warmest Quarter, while PC3 was driven by Bio2: Mean Diurnal Temperature Range, Bio4: Temperature Seasonality, and Bio7: Temperature Annual Range. PC4 represented mainly ‘Precipitation Seasonality’ (Bio15). For all factor loadings, see Supplementary Material S4.BWD Native = 3172.6; Volume BWD Expanding = 19.7; Volume BWD Unknown = 5.3; Volume MMCP Native = 722.3; Volume MMCP Expanding = 236.2; Volume MMCP Unknown = 108.2). For visualization, see Fig. BWD Native/ Expanding = 57.0; Intersection MMCP Native / Expanding = 6.4) leading to low–very low Soerensen indices . This indicates a strong niche shift between native and expanding populations, wherein the combination of PC1 and PC3 had the highest contribution to the differentiation . For the analyzed populations, the genetic differentiation decreases with increasing summer temperatures . In contrast, neither geographic distance nor winter cold explain the observed genetic differentiation.An association of genetic structure with bioclimatic variables and longitude and latitude was tested using GESTE. The highest probability model of our GESTE analysis suggests an association of only Bio10 (Mean Temperature of Warmest Quarter) with genetic structure over Europe  will be required to rule out phenotypic plasticity as the driving force of the observed expansion. In the coming decades, an unprecedented expansion of the venomous yellow sac spider into northern Europe is expected, highlighting one of the health‐related perils of the ongoing climatic changes.http://dx.doi.org/10.5061/dryad.2q8p6): COI sequences and 28S rDNA sequences Dryad  computed only for genotyped native and expansive European populations. C. Estimated hypervolumes based on multivariate minimum convex polygons (MMCP) computed only for genotyped native and expansive European population.Click here for additional data file.


The utilization of biological data to infer the geographic origins of human populations has been a long standing quest for biologists and anthropologists. Several biogeographical analysis tools have been developed to infer the geographical origins of human populations utilizing genetic data. However due to the inherent complexity of genetic information these approaches are prone to misinterpretations. The Geographic Population Structure (GPS) algorithm is an admixture based tool for biogeographical analyses and has been employed for the geo-localization of various populations worldwide. Here we sought to dissect its sensitivity and accuracy for localizing highly admixed groups. Given the complex history of population dispersal and gene flow in the Indian subcontinent, we have employed the GPS tool to localize five South Asian populations, Punjabi, Gujarati, Tamil, Telugu and Bengali from the 1000 Genomes project, some of whom were recent migrants to USA and UK, using populations from the Indian subcontinent available in Human Genome Diversity Panel (HGDP) and those previously described as reference.Our findings demonstrate reasonably high accuracy with regards to GPS assignment even for recent migrant populations sampled elsewhere, namely the Tamil, Telugu and Gujarati individuals, where 96%, 87% and 79% of the individuals, respectively, were positioned within 600 km of their native locations. While the absence of appropriate reference populations resulted in moderate-to-low levels of precision in positioning of Punjabi and Bengali genomes.Our findings reflect that the GPS approach is useful but likely overtly dependent on the relative proportions of admixture in the reference populations for determination of the biogeographical origins of test individuals. We conclude that further modifications are desired to make this approach more suitable for highly admixed individuals.The online version of this article (doi: 10.1186/s12863-017-0579-2) contains supplementary material, which is available to authorized users. The formulation of appropriate methods to decipher the geographic origins of human populations has been a long-standing quest with biologists and anthropologists. Given that a significant correspondence between geography and genetics has been reflected previously , 2, variIndia and its neighbouring areas in South Asia are a rich melting-pot of genetic and ethno-linguistic diversity interwoven with unique social practices. Several lines of evidence allude to the presence of prominent signatures of the Late Pleistocene era in Indian population history –15. The Given the complex history of population dispersal and gene flow in the Indian subcontinent, here we have employed the GPS tool to interrogate publically available whole genome sequence (WGS) data from the Indian sub-continent to ascertain its utility in geo-localization of the corresponding populations. Our dataset included five populations of South Asian ancestry (SAS) available in the 1000 Genomes Project (Phase 3 release), namely two populations originating from the North-Western region of the Indian subcontinent, Gujarati (GIH) and Punjabi (PJL), two populations from the Southern Indian subcontinent,Tamil (STU) and Telugu (ITU), and one population from Eastern region of the Indian subcontinent, Bengalis from Bangladesh (BEB) . NotablyN = 197) [N = 378) [http://www.internationalgenome.org/data/). The single nucleotide polymorphisms (SNPs) that passed the default quality control check and were marked as ‘PASS’ in the VCF files were used for further analysis. No additional quality control measures were employed for the HGDP and previously described datasets [N = 1583) for performing a global admixture analysis. A total of 89,727 SNPs were assessed for the global dataset.The datasets used in the present study comprised of the five South Asian populations available in 1000 Genomes Project, namely Gujarati Indians from Houston, USA (GIH), Punjabis from Lahore, Pakistan (PJL), Indian Telugu from UK (ITU), Sri Lankan Tamil from UK (STU), and Bengalis from Bangladesh (BEB), assessing a total of 489  samples ; eight PN = 197) , 35 and N = 378) . File coN = 378) , VCF tooN = 378) . The VCFdatasets . All thrdatasets , 35, 99 datasets  to the 1K) was discerned by minimizing the cross-validation error (CVE) [N = 1583), the lowest CVE was estimated for K = 13  the lowest CVE was estimated for K = 8   implemenBiogeographical analysis was performed using the Geographic Population Structure (GPS) algorithm . Given aPrior to applying GPS to elucidate the biogeographical affinity of South Asian populations, we sought to test its accuracy on selected Indian populations corresponding to known latitudes and longitudes. To this end we analyzed 57 individuals from four Indian populations  described previously  and estiN = 489) [Subsequently we mapped five South Asian populations (N = 489) , using pN = 489)  and PakiN = 489) , 35 as thttps://CRAN.R-project.org/package=geosphere). For GIH, ITU, and STU genomes that were pertaining to the South Asian diaspora, the capital cities of their ancestral region or country, Ahmedabad (India), Hyderabad, (India) and Colombo (Sri Lanka) respectively were used as their native regional location. Similarly, for PJL and BEB samples the geographic coordinates of Lahore (Pakistan) and Dhaka (Bangladesh) respectively were used for the geographic distance estimation.Geographic distances  between the physical location of the query samples from 1000 Genomes project and their corresponding GPS assigned locations was calculated using the R package geosphere (K) was achieved for both datasets by minimizing the cross-validation error (CVE). For the global dataset (N = 1583), the lowest CVE was estimated for K = 13  the lowest CVE was estimated for K = 8 , Chinese (CHB), African (YRI), Papuan, Melanesian, Surui and Karitiana samples were assigned homogeneously to unique populations putatively representing k1, k5, k8, k9, k10, k11 and k12 ancestral components  and Andamanese (Onge) populations. Finally, the ancestral component k13 was exclusively found in the Kalash population that is a genomic isolate [k3) and South Indian (k4) ancestral components with variable fractions of k1, k5 and k6 in their genomes.In the global admixture analysis, at  isolate  from PakK = 8, a discernible degree of genetic admixture between the North and South Indian populations was evident from the admixture analysis      Fig. , while t01) Fig. . The Wes01) Fig. . As repo01) Fig. , in addileave-one-out procedure [Prior to applying GPS to elucidate the biogeographical affinity of South Asian populations, we sought to trial its accuracy on 57 individuals belonging to four Indian populations  corresponding to known latitudes and longitudes, as described previously . We applrocedure , 10 at tNext, we applied the GPS algorithm to infer the biogeographical affinity of the five SAS populations from 1000 Genomes Project  using SoGPS assignment accuracy was ascertained for each SAS individual to investigate the proximity of their predicted location to that of their likely regional location. Overall more than 56% and 88% of all queried genomes were positioned within 600 km and 1000 km, respectively from their regional location   in Brahmins and Kashmiri Pandits is in concordance with a previous study that demonstrated the presence of 11.4% and 10.6% of Northern Eurasian and Mediterranean components, respectively, in the Brahmin genome [k1 in Brahmins and North-West Indians indicates a potential shared ancestry between the Brahmins and Europeans. Finally the GPS assignment accuracy was found to be poor for the Bengali (BEB) individuals, wherein ~ 98% BEB genomes were assigned to locations more than 1000 km of their native region, Dhaka (Bangladesh)  among Bengalis and Northeast Indians supports the presence of at least three [Our trial analysis of four Indian groups, Brahmin, Kshatriya, Madiga, and Kurumba  using thion Fig. . Among o k4 Fig. . Overall k4 Fig. . We obta k2 Fig. ; >90% of k2 Fig. . GPS pla k2 Fig. . While t k1 Fig. , it alsoly Figs. . We note01) Fig.  which li01) Fig. . Furtherons Fig. , while ioup Fig. . We surmyas Fig. . The pren genome . As specn genome  a highersh) Fig. . We notest three  to four st three  major adst three .Inference of the geographic origin of individuals on the basis of their genetic information poses a formidable challenge and is prone to misinterpretations. In this study the GPS algorithm employed the unsupervised ADMIXTURE analysis where an appropriate number of admixture components for the model in question is deduced, evaluating both the query and reference populations against the same putative ancestral populations. GPS correlates the relative proportions of admixture in the query and reference populations to extrapolate the geographic location of the former on the basis of the geographic coordinates (latitude and longitude) of the latter. Therefore, the reference populations can be conceived as drawing the query groups on the basis of the corresponding genetic proximity till an agreement of geographical locations is achieved . Our resWhile in theory the GPS approach can have putative applications in various fields of science, including genealogical research, where GPS can aid adopted individuals to localize their home regions; it may be employed in forensics, where it can improve the assignment of geographic ancestry to DNA evidence . FurtherDespite its success in tracing ancestry of several modern-day populations and several other likely applications, our findings exemplify that the GPS approach is heavily dependent on the relative proportions of admixture in the reference populations to articulate the population history and biogeographical origins of test individuals. Given the perils of bias in the GPS predictions, interpretation of its results must be performed with adequate caution. Finally, we conclude that further modifications are desired to make this approach more suitable for highly admixed individuals.