Vitis vinifera ssp. sativa) is a perennial crop especially important for wine and fruit production. The species is highly polymorphic with thousands of different varieties selected by farmers and clonally propagated. However, it is still debated whether grapevine domestication from its wild ancestor (V. vinifera ssp. sylvestris) has been a single event or rather it occurred on multiple occasions during the diffusion of its cultivation across the Mediterranean. Located in the center of the Basin, Sicily is its largest island and has served as a hotspot for all civilizations that have crossed the Mediterranean throughout history. Hundreds of unique grapevine cultivars are still cultivated in Sicily and its surrounding minor islands, though most of them are menaced by extinction. Wild grapevine is also present with isolated populations thriving along riverbanks. With the aim to evaluate the phylogenetic relationships among Sicilian varieties, and to assess the possible contribution of indigenous wild populations to the genetic makeup of cultivated grapevine, we analyzed 170 domestic cultivars and 125 wild plants, collected from 10 different populations, with 23 SSR markers. We also compared our data with published dataset from Eurasia. Results show that Sicilian wild populations are related to the cultivated Sicilian and Italian germplasm, suggesting events of introgression and/or domestication of local varieties.Grapevine ( Vitis vinifera L.) is one of the most widespread and economically important perennial crops on the planet. It was estimated that in 2016 the world vineyard area was 7.4 million hectares, with a production of 76 million tons of fresh grapes and 269 million hectoliters (mhl) of wine (http://www.oiv.int/). The cultivation of the domesticated grape (V. vinifera L. subsp. sativa (DC.) Hegi) is believed to have started at least 7,000–8,000 years ago from its wild progenitor (V. vinifera L. subsp. sylvestris (Gmel.) Hegi  Hegi . Archaeol.) Hegi  and the l.) Hegi , then thl.) Hegi . During l.) Hegi . Differel.) Hegi . Nowadayl.) Hegi . The genl.) Hegi . Severalultivars .The domestic and the wild vine can be distinguished by morphological differences concerning leaves, flowers, and fruits, although in most cases the distinction of wild grape is hampered by the gene flow between the two subspecies . The wilV. vinifera subsp. sylvestris is also present in the region with isolated populations mainly thriving along riverbanks , from the main Island and surrounding archipelagos, and 125 wild plants (V. vinifera spp. sylvestris) collected from 10 different Sicilian populations.In order to provide meaningful insights into grapevine evolution and domestication in the Mediterranean Basin, in this work, we compared the unique plant material constituted by the relict populations of Sicilian wild grapevine to the cultivated local germplasm, as well as to grapevine accessions from Western Europe and Central Asia. To evaluate the phylogenetic relationships among Sicilian varieties, and to assess the possible contribution of indigenous wild populations to the genetic makeup of cultivated grapevines, we analyzed with 23 nuclear SSR markers 170 local cultivars  located in Collesano district , Italy .Vitis germplasm was collected during several surveys between 2007 and 2016 in the main mountainous and protected areas of Sicily , i.e., VVS2 , VVMD5, sylvestris and cultivated accessions using Bayes' theorem with the R/Solomon package  phylogenetic tree was designed by using R/ggtree package  with NeiGenetic relationships among the studied genotypes were investigated by Discriminant Analysis of Principal Components (DAPC). DAPC, implemented in the R/adegenet , was pervia Covariance matrix with data standardization.Principal coordinates analysis (PCoA) was performed by GenAlEx 6.5 ad hoc statistic, DELTA K. For each K, 20 independent runs  were carried out. All runs were performed with the admixture model. The 20 runs were averaged using the software CLUMPP  was similar between the cultivated pool (11.8) and the wild pool (10.8 as average), with wild populations ranging from 2.8  to 7.0 (P9) , whereas the rightmost vertex included the Eastern sylvestris populations . Similarly, the PCoA graph differentiated the Eastern from the Western wild samples. However, all the cultivated samples, including the Sicilian and the Italian, and the wild Sicilian grouped together in this analysis  and the residual Italian, Croatian, and Georgian sylvestris that were grouped in cluster B in the first round. Subcluster BB included most of the Sicilian germplasm, both sylvestris (98%) and sativa (94%), and more than half of the Italian sativa accessions (58%). Subcluster BC included almost all the Spanish and French cultivated accessions .Finally, we run a second round of STRUCTURE on the samples closely associated to cluster B, which included most of the Sicilian germplasm and the For millennia, grapevine cultivation has been central in the culture and economy of many regions across the Mediterranean Basin and Middle East, with thousands of different varieties selected. Yet, the exact dynamics of grapevine domestication remains elusive, and subject of a passionate debate among scholars of different fields. In the last decades, molecular techniques have expanded our ability to dig into the genetic signatures left along the history of crossing and migrations. Recent studies have generated genetic profiles for hundreds of cultivated and wild grapevine accessions, though the geographical origin of the sampled material is generally unequal, with Eastern and Southern populations highly underrepresented. In this study, we compared the genetic structure of wild and cultivated grapevine germplasm from Sicily, a region that has been so far overlooked. Due to its size and its central position in the Mediterranean, bridging between Europe and Africa, Sicily has always played a key role in the migration routes crossing the Mediterranean basin, both for natural dispersion of species and during human migrations. The recent discovery of a jar containing wine residues dates back the winemaking culture in Sicily to the 4th–3rd millennium BCE, i.e., at approximately the same time of the oldest wine traces found in the Armenia, and about 2,000 years later than the oldest evidence of wine production in Georgia, considered the primary domestication center for grapevine . For allsativa, matching the segregation observed along the secondary axis of DAPC and PCoA. These results, together with the Fst, and Nei values, that were highest in the pairwise comparisons of P4 with the other wild groups, suggest that P4 is very different from the other Sicilian wild populations, as confirmed by the Fis value, and that is related to the cultivated accessions. We had already noticed the peculiar genetic makeup of P4 before, though using only six SSR markers  can favor the expression of a more sylvestris-like morphology, while more sativa-type traits are masked. Finally, during our collection surveys, we deliberately sought plants possessing all the typical sylvestris features, such as a dioecious flower, ignoring those with hermaphroditic flowers, that could randomly appear at each generation in some individuals if a population maintains a significant sativa contribution. Yet, P4 forms a compact cluster in all the analyses we have performed, therefore the putative introgression events must have occurred in a common ancestor of all current individuals. Alternatively, P4 might represent the residues of an ancient sylvestris population that contributed to the genetic structure of many modern Sicilian and Italian cultivars, as discussed below.We first focused on the comparison between cultivated and wild populations within the Sicilian germplasm. The DAPC and the PCoA distinguished the cultivated from the wild germplasm along the main axis, reflecting major genetic distances between the two groups. Wild samples were especially scattered, indicating a high degree of diversity, probably due to the genetic isolation of most of the populations. The exception was the wild population P4, which separated from the cultivated pool only along the secondary axis, and was not connected with the other wild populations. STRUCTURE confirmed the major divergence between cultivated and wild samples. However, P4 initially grouped together with the cultivated pool, validating the affinity suggested by DAPC and PCoA. Only a second round of STRUCTURE separated P4 from  markers . It was sativa pool characterizes P8, since this population largely mixed with the cultivated pool in all our analyses. Moreover, the STRUCTURE ancestry values of most accessions from P8 were weak, indicating mixed profiles between the sativa and sylvestris clusters. Confirming this hypothesis, the pairwise genetic distance between P8 and the cultivated pool was very low, and individuals from P8 were dispersed among sativa accessions in the phylogenetic tree. Population P8 is located in the Anapo Valley at Pantalica, a site with a large Neolithic necropolis that is known to be actively inhabited since ancient times, and that is currently surrounded by cultivated fields. To a minor extent, we also observed evidence of introgression in single individuals of P1 and P6, since a few samples clustered together with the sativa group in the DAPC, PCoA, STRUCTURE, and the phylogenetic tree. The genetic isolation of these populations was also confirmed by the positive inbreeding coefficient value (Fis). We suspect introgression with cultivated germplasm also for P3, P5, P7, P10 given that the observed heterozygosity (Ho) values are higher than the expected (He). Nevertheless, the negative Fis value showed by the latter populations, indicating an excess of heterozygosity, could be also due to the following different factors: the small population size, overdominant selection favoring heterozygote survival (heterosis) self-incompatibility system effect, proportion of asexual reproduction and effect of clonal reproduction on the number of heterozygotes   . All thesylvestris (289 accessions). The exact geographical origin of each accession is not specified in the paper, yet we ascertained that Riaz et al.'s dataset does not comprise any Sicilian sample , as opposed to the larger collection from which the wild Italian samples from Riaz et al. derived  a Western sylvestris pool, ii) an Eastern sylvestris pool, and iii) the cultivated germplasm, regardless of their origin. The latter cluster also contained the wild Croatian samples. The samples falling in the transition zones among these clusters might suggest events of gene flow between wild populations and the cultivated germplasm in these regions, as previously reported from several investigations  into the cultivated Italian pool. The latter hypothesis is consistent with the assumption that grapevine cultivation in Italy spread from the Southern regions northward since the second part of the 1st millennium BCE  and the wide scale analysis, might depend on the number of SSR markers used  and on the larger genetic diversity present in the world dataset, that may hinder the smaller differences within the Sicilian accessions. However, the second round of STRUCTURE clearly distinguished a cluster including nearly all the Sicilian germplasm, both sylvestris and sativa, and more than half of the Italian cultivated accessions, thus confirming the affinity among these groups, already observed in the DAPC analysis. The hierarchical STRUCTURE also separated the cultivated accessions from other regions of the world in two additional clusters. One included almost all samples from Western Europe (Spain and France); a third cluster included the Eastern sativa accessions  plus sylvestris form Georgia, Croatia and Italy. Interestingly, about half of the Italian (42%) cultivated accessions also showed affinity for this cluster. Therefore, the Italian cultivars are very different from the rest of Western Europe, and appear as mix between the Eastern group and the Sicilian pool. Accordingly, the list of Italian sativa accessions grouping together with the Sicilian germplasm consists predominantly (15 out of 18) of cultivars from Southern regions  with the exception of three cultivars that are from Northern Italian regions , pointing to a close relationship of this group with the Sicilian germplasm. Conversely, the remaining Italian cultivars, which show affinity with the Eastern pool, are varieties mostly cultivated in the Northern regions .The two-step analysis through STRUCTURE provided some additional information. In the first round of STRUCTURE, the cultivated Sicilian germplasm clustered together with most of the other sylvestris and the Sicilian and Italian sativa pools, observed in the wide DAPC, PCoA, and STRUCTURE analyses, is intriguing and can be explained by two different hypotheses. First, it is possible that many Sicilian wild populations suffer from introgression of sativa germplasm. This scenario is plausible, considering many different factors, such as the relatively small extension of the island, its millennial history of exploitation, the ancient reduction of its original forest cover, the importance of viticulture in the local economy with extensive fields, and the diffusion of recent diseases threatening the natural populations , it is plausible that the genetic affinity among current Sicilian sylvestris and sativa germplasms derives from early domestication events occurred in this region. The data set and the results presented here, in a region of primary interest for understanding domestication, migration, and expansion of grape around the Mediterranean basin, may contribute to facilitate future investigations to further unravel the phylogenetic history and population dynamics of grapevine.The comparison of the genetic structure of Sicilian All datasets for this study are included in the article/ RM and FC conceived and supervised the project. RM, AG, LA, AM, GG and FC contributed to collect plant materials. RM, DC and FB performed the genetic characterization of the plant material. RM, AG and IF analysed the data. RM, AG, IF, and FC analysed and interpreted data. RM, AG, DP and FC wrote the first draft. All authors made a substantial, direct and intellectual contribution to this work. All of the author approve the final version of the manuscript.This research was supported by Regione Siciliana .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Xyela concava and the design of primer pairs flanking those motifs. Sixteen of those primer pairs were selected and implemented successfully to answer questions on the phylogeography and population genetics of X. concava. The markers were characterized in three geographically distinct populations of X. concava and tested for cross-species amplification in two additional Xyela and one Pleroneura species (Xyelidae). All markers showed substantial polymorphism as well as revealing subtle genetic structures among the three genotyped populations. We also analyzed a fragment of the nuclear gene region of sodium/potassium-transporting ATPase subunit alpha (NaK) and a partial mitochondrial gene region coding for cytochrome oxidase subunit I (COI) to demonstrate different genetic resolutions and sex-biased patterns of these markers, and their potential for combined use in future studies on the phylogeography and population genetics of X. concava. Although a limited number of populations was analyzed, we nevertheless obtained new insights on the latter two topics. The microsatellites revealed a generally high gene flow between the populations, but also suggested a deep historical segregation into two genetic lineages. This deep genetic segregation was confirmed by NaK. While the high gene flow was unexpected, because of assumed restricted dispersal ability of X. concava and the discontinuous distribution of the host trees between the populations, the segregation of two lineages is comprehensible and could be explained by different refuge areas of the hosts during glacial times. The COI results showed a discordant strong genetic structure between all populations, which might be explained by the smaller effective population size of the mitochondrial genome. However, given the frequent evidence of a similar nature in recent studies on sawflies, we also consider and discuss mitochondrial introgression on population level as an alternative explanation.Microsatellites are widely used as powerful markers in population genetics because of their ability to access recent genetic variation and to resolve subtle population genetic structures. However, their development, especially for non-model organisms with no available genome-wide sequence data has been difficult and time-consuming. Here, a commercial high-throughput sequencing approach (HTS) was used for the very first identification of microsatellite motifs in the genome of  Xyelidae have always attracted the attention of taxonomists and systematists. They represent the sister group of the rest of the megadiverse insect order Hymenoptera , which iThe rich fossil record of Xyelidae includes the earliest fossil forms of Hymenoptera dating from the Middle–Upper Triassic . Proper Xyela concava  and cliff-rose (Purshia spp.), from which they gather pollen for nutrition with their adapted mouthparts . Finally, 98 larvae of X. concava were selected for the analysis  according to the manufacturer’s protocol, but with an extended 2 h incubation time at 55 °C  for cell lysis. The extracted DNA was stored at −20  °C until later use. The integuments were retained and stored in 70% ethanol.X. concava  were used for tests of polymorphism. Furthermore, 12 specimens of X. deserti  and 5.0 µl of 2×Multiplex PCR Plus Master mix (QIAGEN). PCR reaction conditions were as described above with the respective optimal annealing temperature for each primer pair mix. Reactions were diluted 1:2 and sent to Macrogen Europe  for fragment analysis.Total genomic DNA of a single female of  deserti , 12 specna group , and six1910 see   were teAllele sizes were scored using GeneMapper 5.0 (Applied Biosystems). No marker showed strong stutter peaks or intensive background signal. Two primer pairs appeared to be monomorphic and were excluded from further analyses. Sixteen primer pairs showed apparent polymorphism for the four tested samples and were finally selected .COI). The first 658 bp of this fragment (from the 5′ end) correspond to the standard barcode region of the animal kingdom (NaK) was amplified.Primers used for amplification and sequencing are listed in  kingdom . AdditioMK265017 –MK265114 and MK264919 –MK265016, for detailed data see PCR reactions were carried out in a total volume of 20–25 µl containing 1.5–3.0 µl DNA, 1.2–2.5 µl of primers (5 pmol each) and 10.0–12.5 µl of 2×Multiplex PCR Plus Master mix (QIAGEN). The PCR protocol consisted of an initial DNA polymerase (HotStar Taq) activation step at 95 °C for 5 min, followed by 38–40 cycles of 30 s at 95 °C, 90 s at 49–59 °C depending on the primer set used, and 50–120 s (depending on the amplicon size) at 72  °C; the last cycle was followed by a final 30 min extension step at 68 °C. 3 µl of the PCR product was visualized on a 1.4 % agarose gel. Primers and dNTPs were inactivated with FastAP and Exonuclease I (Thermo Fisher Scientific). 1.7–2.2 U of both enzymes were added to 17–22 µl of PCR solution and incubated for 15 min at 37 °C, followed by 15 min at 85 °C. Purified PCR products were sent to Macrogen Europe  for sequencing. To obtain unequivocal sequences, both sense and antisense strands were sequenced. Sequences were aligned manually with Geneious 11.0.5. Ambiguous positions  due to heterozygosity or heteroplasmy were coded using IUPAC symbols. Sequences have been deposited in the GenBank (NCBI) database (accession numbers NA), observed (HO) and expected heterozygosity (HE) as well as deviations from Hardy-Weinberg equilibrium (HWE) for each locus for all X. concava populations using ARLEQUIN 3.5.2.2 . The program GENEPOP 4.7.0 . GENEPOP was also used in combination with the ENA correction implemented in the program FreeNA . Number of genotypes (NG) in the dataset was counted with Excel. To test for isolation by distance, a Mantel test for the microsatellite data was performed  in ALLELES IN SPACE  was assumed, since correlated frequency models, though more powerful in detecting subtle differentiations, are more sensitive to departure from model assumptions  but either had a considerably narrow confidence interval or were significant or approaching the level of significance (P = 0.055). The FST values for NaK and COI were, in comparison, higher (0.215–0.740). While the values for NaK showed the same pattern as the microsatellite markers in respect of genetic relationship of the populations, the FST values for COI indicated relatively high differences between all populations . While spatial assignment tests for NaK and the mircosatellites came up with the same pattern as the FST values—as indicated by the assignment of two populations with high posterior probabilities to one genetic group or lineage  (The ountains . In geneulations . The Man lineage  and 3—th (K = 3) –3D.Xyela and Pleroneura, four markers showed polymorphic products and five were apparently monomorphic for X. deserti. Eight markers showed polymorphic products for the new Xyela species of the alpigena group, while no or unspecific fragments were amplified for Pleroneura koebelei . Furthermore, homozygote genotypes equally distributed across all populations indicated haploidy for altogether 26 specimens and may have had an impact on the discrepancy between the observed and expected heterozygosity. Thelytokous parthenogenesis—producing solely female offspring—which is known in xyelids  the segregation could still be detected, but also a recent state of admixture was indicated. To test this hypothesis and a possibly ongoing admixture of the segregated lineages, populations of X. concava in hybrid zones and overlapping distribution areas of the host species should be included in future studies.Second, the proposed geographically remote and restricted refugia of the host species during glacial times , and theNaK, FST values and Bayesian statistics for the mitochondrial COI region showed a clear non-congruent pattern with a strong genetic structure among all three populations. One explanation could be the different effective population sizes (Ne) of the mitochondrial and nuclear genome, which will cause differences in the diversity of the genetic structure of populations over time , unique specimen identifier (DEI-GISHym), GenBank accession number, and collection data of the analyzed imagines (♀♂) and larvae (L) of Click here for additional data file.10.7717/peerj.8010/supp-2Table S2Unique specimen identifier (DEI-GISHym). Allele sizes in base pairs (bp). Empty cells indicate no amplificationClick here for additional data file.


In the present study, we investigated the genetic polymorphisms of 20 short tandem repeat (STR) loci in a Qinghai Han population, as well as its genetic relationships with other populations. A total of 273 alleles were identified in 2 000 individuals at 20 loci, and the allelic frequency ranged from 0.000 2 to 0.532 7. The 20 STR loci showed a relatively high polymorphic rate in the studied group. Observed and expected heterozygosities ranged 0.613 0–0.907 5 and 0.614 8–0.920 0, respectively. The combined power of discrimination, and the probability of exclusion in duo and trio cases were 0.999 999 999 999 999 999 999 999 34, 0.999 996 0 and 0.999 999 996 5, respectively. Analyses of interpopulation differentiation revealed that the most significant differences were found between the Qinghai Han and Malaysian, while no significant differences were found between the Qinghai Han and Han people from Shaanxi and Jiangsu. The results of principal component analysis, multidimensional scaling analysis and phylogenetic reconstructions also suggested the close relationships between the Qinghai Han and other two Han populations. The present results, therefore, indicated that these 20 STR loci could be used for paternity testing and individual identification in forensic applications, and may also provide information for the studies of genetic relationships between Qinghai Han and other groups.China is a multinational country composed of 56 ethnic groups of which the Han Chinese accounts for 91.60%. Qinghai Province is located in the northeastern part of the Qinghai–Tibet Plateau, has an area of 72.12 km The Han population is the largest of the 56 officially recognized ethnic groups in China. Findings from the 6th National Population Census of 2010 suggest that they make up 91.60% of the overall Chinese population with a population of 1 220 844 520, and they are also distributed worldwide. The Chinese language, used as the spoken and written language of Han people, belongs to the Sino–Tibetan language family. The appellation ‘Han’ can be traced back to the Han dynasty of the second and third centuries and represents the majority of the Chinese population to date.Qinghai Province has the fourth largest land area in China. It is located in the northeastern part of the Qinghai–Tibet Plateau, which has an altitude over 3 000 m above sea level. The history of Qinghai Province began during the Han dynasty when General Huo Qubing built the military fortress known as Xipingting. This was the former site of Xining, which appeared during the Ming dynasty between 1368 and 1644. In 2010, Qinghai had a population of 5 626 723, of which 39% was taken up by minorities including Tibetans, Mongolians, Kazaks and the Hui, Tu and Salar.D8S1179, D21S11, D18S51, vWA, D3S1358, FGA, TH01, D5S818, D13S317, D7S820, CSF1PO, D16S539 and TPOX loci.In the present study, the genetic distributions of 20 short tandem repeat (STR) loci and the sex-determining locus amelogenin were studied in the Qinghai Han population. Additionally, the phylogenetic relationships between the Qinghai Han and other reference populations  were stuBlood samples were collected from 2  000 unrelated healthy Han individuals living in Qinghai Province, China, whose ancestors over the past three generations were Han individuals who had not migrated or interbred with other ethnic groups. All of the participants signed an informed consent form and completed a questionnaire providing information about their direct blood relatives over three generations. The experimental procedures conformed to the human and ethical research principles of Xi’an Jiaotong University Health Science Center, China. Genomic DNA extraction was performed using the Chelex-100 procedure as described by Walsh et al. .® 21 System . The total volume of PCR reactions was 25 µL, containing 5 µL PowerPlex® 21 5× Master Mix, 5 µL PowerPlex® 21 5× Primer Pair Mix, 1 ng template DNA and amplification grade water. Amplification was carried out using a GeneAmp PCR System 9700 Thermal Cycler  under the manufacturer’s specifications. The AB PRISM 3130 Genetic Analyzer (Applied Biosystems) was used to obtain sample genotypes. Raw data were analysed using GeneMapper ID 3.2 software (Applied Biosystems). 9947A DNA was used as a positive control.PCR amplification was performed using the PowerPlexP-values for exact tests of Hardy–Weinberg equilibrium were calculated using the modified Powerstat (version 1.2) spreadsheet. Linkage disequilibrium (LD) analysis of pairwise STR loci was calculated using Genepop version 4.0.10 [P-values) between the Qinghai Han population and other referenced populations were conducted using Arlequin software version 3.1 with the method of analysis of molecular variance (AMOVA) [AD) and fixation index (FST) of the studied Han group and other populations were calculated using the DISPAN program [AD and FST between these populations were plotted by R software (https://www.r-project.org/). Principal components analysis (PCA) of these populations was drawn using MVSP software version 3.1 [Allelic frequencies of 20 STRs, their forensic relevant parameters, and n 4.0.10 . Based o (AMOVA) . Populat (AMOVA) . The pai program  and Arlesion 3.1  based onDP), and the probability of exclusion (PE) in duo and trio cases were 0.792 6, 0.203 5 and 0.306 8, respectively, at the TPOX locus, while the highest values were 0.987 5, 0.720 5 and 0.810 8, respectively, at the Penta E locus. With the exception of locus TPOX, the polymorphism information content (PIC) of all remaining loci reached above 0.6. Highest observed heterozygosity (Ho) and expected heterozygosity (He) values were observed at the Penta E locus, while the lowest Ho and He values were at the TPOX locus. The combined DP, and PE in duo and trio cases were 0.999 999 999 999 999 999 999 999 34, 0.999 996 0 and 0.999 999 996 5, respectively. These results revealed that these 20 STR loci are highly polymorphic, and have the potential to be used in both forensic human identification and paternity testing in the Qinghai Han population.The allelic frequencies of 20 autosomal STR loci and their corresponding forensic relevant parameters are shown in P-values of two  out of the 190 pairwise comparisons were below the significant level (0.000 263). LD can be influenced by many factors, such as selection, the rate of recombination, the mutation rate, genetic drift, the system of mating, population structure and genetic linkage. As the loci are located on different chromosomes, genetic linkage cannot explain the observed LD. However, additional studies are required to determine the role of other factors in LD.The results of LD tests are shown in P-values of genetic differentiation comparisons are shown in P < 0.05) were observed between the Qinghai Han and the following groups: the Malaysian at 13 loci, the Tibetan and She at 11 loci, the Uygur and Shui at 10 loci, the Zhuang at five loci, the Dong and Yi at three loci and the Hui, Guangdong Han and Russian at two loci. No significant differences were observed among the Qinghai Han, Jiangsu Han, and Shaanxi Han groups. The highest ethnic diversity was observed at the D18S51 locus, where significant differences were found in eight out of 13 compared groups. The lowest ethnic diversity was observed at CSF1PO and TPOX loci, where significant differences were found in only three out of 13 compared groups.The K settings (K = 2–7) for the 13 overlapping loci dataset are shown in Supplementary Figure S1. Population components of the 14 populations at K = 3 are shown in We explored the population structures of the Qinghai Han and other published populations by the Structure program. Different Supplementary Tables S1 and S2. As shown in Pairwise genetic distances of the Qinghai Han population and other reference populations are shown in PCA of the Qinghai Han population and 13 other reference groups was performed according to the normalized allelic frequencies of the shared STR loci. As shown in Phylogenetic analysis was used to explore genetic relationships between the Qinghai Han group and other populations, as shown in The present study indicated that the loci examined were highly polymorphic in the studied Qinghai Han population. We also found that the Qinghai Han population has the close genetic relationships with Shaanxi Han and Jiangsu Han populations. These results suggest that the loci can be used for paternity testing and forensic human identification, and could also provide information about the genetic relationships between Qinghai Han and other groups.The experimental protocol in the present research involving human blood samples conformed to the human and ethical research principles of Xi’an Jiaotong University Health Science Center.


Loxosceles rufescens is native to the Mediterranean but considered cosmopolitan because it has been dispersed worldwide. A previous study revealed 11 evolutionary lineages across the Mediterranean, grouped into two main clades, without any clear phylogeographic pattern. The high genetic diversity within this species (p-distances of up to 7.8% in some Mediterranean lineages), together with the results obtained with different species delimitation methods  could indicate the existence of cryptic species. Here we compare the mitochondrial and microsatellite diversity to elucidate if the lineages of L. rufescens in the Mediterranean should be considered different species (cryptic species) or populations of the same species. To do so, we analyzed the cox1 diversity of 196 individuals, of which, we genotyped 148, sampled from 19 localities across the Mediterranean. STRUCTURE analyses of microsatellite data identified two genetic clusters of L. rufescens. One cluster included individuals from Western Mediterranean localities  and Israel, while the second one grouped individuals from Italian and Greek localities, including Sardinia, Sicily and Tunisia. These patterns suggest that geographic proximity is the more significant factor in the clustering with microsatellite data and shows the existence of gene flow between the nearest geographic areas, even if the individuals belong to different mitochondrial lineages or clades. The lack of correspondence between both genetic markers suggests that the evolutionary lineages found within L. rufescens should not be considered different species. We conclude that these phylogenetic linages and their distribution may be the result of the maternal evolutionary history of the species and human-mediated dispersion.The species  Loxosceles Heineken and Lowe, 1832 (Araneae: Sicariidae) are widely known for their medical importance . Th. ThLoxos but see ,15–18. D but see  suggestens group . Given t species .L. rufescens has been introduced, it can be found inhabiting infrastructures and areas related to human activities [Loxosceles species, it has a low dispersal capacity because, unlike other spiders, it does not disperse by ballooning [L. rufescens individuals tend to be sedentary and show a strong preference to stay in the same location for long periods of time or their entire life [Its ability to expand into new territories is enhanced by its high synanthropism ,6,21 andtivities ,22. Simillooning ,14. L. rire life .L. rufescens in the Mediterranean Basin Planas et al. [cox1). They also point out that these 11 lineages could be considered as putative species based on the results obtained with different methods of species delimitation . The lineages were grouped into two clades: A and B. The first clade is a combination of six lineages (A1 to A6), and the second clade is composed of five (B1 to B5). However, the lineages were adjusted to two completely different phylogeographic patterns: while some lineages from Morocco and the Iberian Peninsula were well structured, the remaining lineages were distributed all over the Mediterranean without a definite geographical pattern. The authors [L. rufescens within the Mediterranean Basin and mixed the geographic structuration of the lineages. The same authors indicated that the uncovered phylogeographic patterns should be strengthened with additional nuclear data.Studying the genetic diversity of s et al.  proposeds A1 to A, and theL. rufescens to elucidate if the mitochondrial lineages of L. rufescens that exist in the Mediterranean should be considered different species (cryptic species) or if these are populations of the same species. To do so, we sampled numerous populations of L. rufescens throughout the Mediterranean by considering the geographical distribution and the representation of the mitochondrial lineages.In this contribution, we compare the mtDNA and the microsatellite diversity within If the mitochondrial lineages must be considered different species, we would expect to find a correspondence between the mitochondrial and microsatellite clusters, which would suggest that alleles of individuals from different lineages or clades would remain distinctive after secondary contact. In contrast, if the microsatellite information does not match with the outlook provided by mitochondrial data, the microsatellite loci would delimitate geographically structured genetic clusters whose individuals could belong to different mitochondrial lineages. Since alleles would be able to mix, we expect that the allelic frequencies would show more similarity between nearer populations than distant ones and favoring the hypothesis that the current genetic variability would have originated in several glacial refugia during the Pleistocene and that their posterior mixture around the Mediterranean Basin would have been facilitated by human-mediated dispersion.cox1 sequences obtained in Planas et al. [cox1 partial sequences were aligned in GENEIOUS v6.1.6 [Our dataset includes a total of 196 terminals , 128 coxS v6.1.6  using ClS v6.1.6  with defS v6.1.6  under a To infer the mitochondrial lineage of each individual, a maximum likelihood (ML) analysis was performed using RAx-ML 7.0.3  with theL. rufescens [ox1 linage in R [We genotyped 148 individuals sampled from 19 localities across the Mediterranean Basin  for seveufescens . We condufescens . We calcage in R  using cuTo identify the genetic clusters, we used microsatellite data with a method based on Bayesian clustering, which is implemented in the program STRUCTURE . With thTo evaluate the effect of the missing data in the previous analysis, we tested five additional subsets of the most complete dataset. First (subset 1), we removed individuals lacking information for two out of seven microsatellites loci. In subset 2, we removed all the individuals with missing data. In subset 3, we removed the microsatellite locus with the highest value of missing data. In subset 4, we removed the locus with the highest value of missing data and individuals without information for two of the remaining six loci. Finally, in subset 5, we again removed the locus with the highest value of missing data and all the individuals with missing data.cox1 alignment included a total of 196 sequences, 148 of which came from individuals that were also used in the microsatellite’s analyses. The phylogenetic ML tree , 4.2% for subset 4 and 0% for subsets 2 and 5. The number of alleles in subset 1 was reduced to 54, that in subsets 2, 3 and 4 was reduced to 51, and in subset 5, the number was reduced to 48. The number of alleles per locus ranged between five in ME067 and ME031 and 11 in ME012 in subset 1, between five in ME067 and 11 in ME067 in subsets 2 and 5, and between five in ME067 and 12 in ME012 in the subsets 3 and 4.We obtained K = 2 as the optimal number of clusters in the STRUCTURE analysis following Evanno’s method . The majThese results show moderate geographical structuration of the genetic clusters obtained based on the microsatellite information Figs  and 3. CThe results obtained with subsets 1 to subset 5 did not greatly differ from the results obtained with the complete dataset. The assignment value of individuals closest to 50% only changed in some datasets , which sIn this study, we observed a general incongruity between the microsatellite clusters and the mitochondrial lineage, that is, not all the individuals that belong to one mitochondrial lineage correspond to the same microsatellite clusters as inferred by STRUCTURE . In bothWe observed a general geographic pattern by considering the microsatellite clusters. As noted above, one genetic cluster is compounded by the individuals from the western Mediterranean localities (Morocco and Iberian Peninsula) and others by individuals from Italy and Greece. This pattern suggests that geographic proximity is a more significant factor in the clustering with microsatellite data. This is despite the fact that in some cases, the assignation of the individuals is not markedly homogeneous for the individuals of the same locality, for example, TN1 (n = 6), where half of the individuals were assigned to cluster 1 and the other half to cluster 2. However, the west-east geographic pattern obtained by microsatellites is broken by the assignation of the easternmost individuals (LE1) to the western cluster. This fact is probably a consequence of scarce sampling in North Africa and the Eastern Mediterranean, or a consequence of human-mediated dispersal, since 7 of the 8 individuals analyzed belong to clade B2, where most of them are from Iberian Peninsula. If we exclude this exception, the results show a clear west-east geographic pattern.Overall, our results suggest that neither the mitochondrial lineages nor the clades should be considered different species. Our dataset included several localities  with individuals belonging to different mitochondrial clades or lineages. Since the individuals were clustered together with the microsatellite data and thus were not structured according to the mitochondrial lineages or clades, the hypothesis of the existence of cryptic species is not supported. Several localities  included individuals of a single mitochondrial lineage,  and were assigned to different microsatellite clusters, again not supporting the hypothesis of the existence of different cryptic species.L. rufescens as a consequence of external factors and mostly the influence of human-mediated dispersal. These external factors would have altered  the phylogeographic structuration of this species, which allows populations from different Mediterranean regions to resume interaction. This hypothesis is supported by the results obtained by microsatellite data, which show the existence of gene flow between the nearest geographic areas even if the individuals belong to different mitochondrial clades. Therefore, the different mitochondrial linages could have been mixed up in the Mediterranean Basin.Planas et al.  proposedL. rufescens around the Mediterranean Basin. Using STRUCTURE, we detected the presence of two different genetic clusters. These two clusters are approximately geographically structured in a way such that the individuals from Morocco and the Iberian Peninsula would be differentiated from representatives of Italy and Greece.In the present study, we analyzed multiple microsatellite loci from different localities of cox1 do not correspond to those obtained from microsatellites clusters. Our interpretation of the lack of correspondence between the two genetic markers is that the different evolutionary lineages found within L. rufescens should not be considered different species. The different lineages originated in distinct glacial refugia during the Pleistocene and were distributed through human-mediated dispersion. However, the employment of a higher number of microsatellites and more localities with larger sample sizes would be necessary to be able to conclusively determine the existence of these genetic clusters and resolve how the populations of L. rufescens are structured in the Mediterranean Basin. Additionally, the analysis of other nuclear genetic markers would be beneficial to increase our knowledge regarding the evolutionary history of this species.The mitochondrial lineages and clades inferred from S1 Fig2: STRUCTURE results based on the microsatellite loci for K = 2 without individuals with more than one loci missing. 3: STRUCTURE results based on the microsatellite loci for K = 2 without individuals with missing loci. A. Including all the microsatellite loci. B. Excluding microsatellite locus ME031. Abbreviations: GR (Greece), IB , IP (Iberian Peninsula), IT , LE (Israel), MA (Morocco), SA (Sardinia), SC (Sicily), TN (Tunisia).(PDF)Click here for additional data file.S1 TableAbbreviations: GR (Greece), IB , IP (Iberian Peninsula), IT , LE (Israel), MA (Morocco), SA (Sardinia), SC (Sicily), TN (Tunisia).(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableAbbreviations: GR (Greece), IB , IP (Iberian Peninsula), IT , LE (Israel), MA (Morocco), SA (Sardinia), SC (Sicily), TN (Tunisia).(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Melanoplus sanguinipes, appears to be a sink population derived from low elevation populations. While dispersal ability has a clear connection to the genetic structure in different species, genetic distance does not predict growth, reproductive, or physiological trait variation in any species, requiring further investigation to clearly link phenotypic divergence to local adaptation.Species responses to environmental change are likely to depend on existing genetic and phenotypic variation, as well as evolutionary potential. A key challenge is to determine whether gene flow might facilitate or impede genomic divergence among populations responding to environmental change, and if emergent phenotypic variation is dependent on gene flow rates. A general expectation is that patterns of genetic differentiation in a set of codistributed species reflect differences in dispersal ability. In less dispersive species, we predict greater genetic divergence and reduced gene flow. This could lead to covariation in life‐history traits due to local adaptation, although plasticity or drift could mirror these patterns. We compare genome‐wide patterns of genetic structure in four phenotypically variable grasshopper species along a steep elevation gradient near Boulder, Colorado, and test the hypothesis that genomic differentiation is greater in short‐winged grasshopper species, and statistically associated with variation in growth, reproductive, and physiological traits along this gradient. In addition, we estimate rates of gene flow under competing demographic models, as well as potential gene flow through surveys of phenological overlap among populations within a species. All species exhibit genetic structure along the elevation gradient and limited gene flow. The most pronounced genetic divergence appears in short‐winged (less dispersive) species, which also exhibit less phenological overlap among populations. A high‐elevation population of the most widespread species,  Melanoplus sanguinipes, follows a stepping stone pattern, with the highest elevation population appearing to be a sink population derived from low elevation.Four grasshoppers species exhibit genetic structure and limited gene flow along elevation gradients in the Rocky Mountains. The strength of genetic divergence is associated with dispersal phenotypes and phenological patterns. The most widespread species,  Reproductive traits, including ovariole number and resource allocation to eggs, vary among populations along the gradient, and in different directions for long‐winged and short‐winged species. Physiological traits related to temperature tolerance and performance also vary among populations. Clinal differences in thermal tolerances  and preferences (preferred body temperature) are greater for the long‐winged species (C. pellucida and M. sanguinipes) than the short‐winged species, while these species also exhibit higher thermal limits , but do not vary with elevation for the other species  at the high‐elevation site in response to warming . We followed the manufacturer's protocol with one modification: 4 μl RNase A (100 mg/ml) was added after the initial lysis step. DNA was eluted in 2 × 100 μl of the supplied elution buffer. DNA concentrations were quantified with a Qubit® 2.0 Fluorometer using a dsDNA High Sensitivity Assay Kit (Life Technologies). At least 300 ng of DNA per individual was sent to the University of Wisconsin‐Madison Biotechnology Center for library preparation and sequencing using a double‐digest genotype‐by‐sequencing (GBS) protocol , two mismatches allowed between loci when building the SNP catalog (n parameter), and the presence of the SNP in at least two of the focal populations (p parameter).Raw reads were split by individual barcode using the astphase v1.4  and expected (HE) heterozygosity, within‐population gene diversity (HS), and Wright's inbreeding coefficient (FIS). Each statistic was averaged across loci within populations, and also calculated overall for each species. Additionally, for each species, we calculated overall gene diversity  and Dest  in a pairwise manner using Nei's D . Pairwise FST between populations within a species was computed in hierfstat using the “pairwise.neifst” function. Additionally, we tested for association of genetic divergence with geographical distance (isolation by distance) using the Mantel test function in the vegan package in R , observed  for which the model has best predictive accuracy, testing K = 1 to K = N where N is the total number of populations sampled. We conducted 10 independent runs at each value of K and compared the average cross‐validation error. Data conversions were done using custom R scripts and plink 1.9  in R, on centered and scaled SNP datasets for each species. Second, we examined genetic admixture using Francis,  in R. CL2.5FST). We used the vegan package in R to compute dissimilarity matrices using Euclidean distance, and we conducted matrix correlation tests for each species. Mantel tests were used to test the Pearson product‐moment correlation of phenotypic variation to each predictor variable independently, using 999 permutations to test for significance. These tests assume a linear relationship between the two variables and a constant scalar relationship among populations.Previously published phenotypic data include multiple variables representing growth, reproductive and physiological traits. Growth traits include body mass and femur length, which is correlated with body size. Reproductive traits include clutch size, ovariole number, egg mass, and egg clutch weight. Physiological traits include preferred body temperature, critical thermal minima, and critical thermal maxima. For each trait type, variables were combined to compute the phenotypic distance among populations based on mean values. We then tested for phenotypic correlations with the following predictor variables: geographical distance, elevation, environmental distance , and genetic distance  at elevations above ~3,000 m a.s.l, with adults sourced from lowland populations. Therefore, for this species, we additionally tested a source‐sink model where all populations formed an island model and could contribute unidirectional gene flow to the upper elevation population D1. The ABC method estimates the posterior distribution of model parameters without calculating the explicit likelihood of these parameters. Instead, data are simulated under each model by randomly drawing values from a prior distribution of parameter values, the observed data are compared to synthetic data based on similarity to summary statistic values, and both parameters and models can be quantitatively compared using posterior distributions and Bayes Factor (BF) scores . The synthetic datasets had population sample sizes matched to the relevant species, migration rate  drawn from a uniform prior distribution (0.001–0.05), and effective population (Ne) size drawn from a log‐uniform prior distribution . For each model, we simulated two million datasets each with 500,000 independent sequences of 10 base pairs, and drew SNPs in equal number to those available in the empirical dataset for each species. We then used arlsumstat in arlequin v3.5 .We set out to test alternative models of gene flow among populations and estimate gene flow rates. We employed an Approximate Bayesian computation (ABC) approach . If two models had high posterior probabilities, goodness‐of‐fit to the observed values was tested for both. The rejection algorithm favors those models which simulate data closest to the observed data, while the neural network attempts to correct for deviation from the simulated data and the observed data. Given the finite number of simulations we employed and the vast state space of the models, we focus on the results from the neural network to provide the most accurate estimate of model parameters. Parameters (population size and gene flow) were estimated under the best fitting model using a tolerance value of .05.ABC analysis was completed using the fastsimcoal2 v2.5  for an independent estimate of parameters under the best fitting model, which can be estimated jointly over multiple populations . Weekly resurveys were conducted 2006–2015  and C. pellucida (FST = 0.037), showed less differentiation than the two short‐winged species, M. boulderensis (FST = 0.081) and A. clavatus (FST = 0.062)  and highest  populations in M. sanguinipes. Mantel tests showed no association with geographical distance for M. boulderensis , M. sanguinipes , and C. pellucida , but isolation by distance was significant for A. clavatus .A total of 18,604 to 29,213 SNPs were identified in the four species (Table C. pellucida) to 10.5% (M. boulderensis) of the genetic variation . There is clear population structure for all populations and species. Comparing species, A. clavatus had the lowest amount of overall admixture, with individuals having, on average, 90.5 ± 13.1% assignment to a single ancestral population. Melanoplus sanguinipes had the highest levels of admixture overall, with an average of 75.5 ± 15.0% assignment to a single ancestral population. Camnula pellucida and M. boulderensis had similar levels of admixture .The first two principal components combined explained 8.0%  tests were found in M. boulderensis for both growth  and nearly significant (α = .10) tests were found for reproductive traits . Significant (α = .05) tests were found in A. clavatus for growth traits , and growth traits were nearly significant in association with genetic distance .We examined the correlation of phenotypic differences among populations on the elevational gradient and a set of predictor variables  using Mantel tests and assuming a linear relationship Table . Notably3.3A. clavatus, the upslope gene flow model  among populations across species, and rates were similar among long‐winged and short‐winged species across methods. While the estimated population size did not vary systematically with elevation in most cases, the A. clavatus upslope model and the M. sanguinipes source‐sink model suggested larger population sizes at lower elevation. Two species had single populations with reduced population sizes, a mid‐elevation site for A. clavatus, and the highest elevation site for C. pellucida.Parameter estimates Table  using ap3.4F3,111 = 4.52, p < .005) with overlap along the elevation gradient increasing in the following order: A. clavatus, M. boulderensis, C. pellucida, and M. sanguinipes. This order corresponds to the earlier season, short‐winged species exhibiting less overlap among sites .Phenological overlap among sites, reflecting the potential for population mixing and gene flow, varies among years and along the elevation gradient  exhibit greater genetic differentiation (in pairwise FST and clustering results) than the long‐winged species (C. pellucida and M. sanguinipes), suggesting reduced dispersal ability. Phenological overlap among populations, an ecological measure of potential gene flow, reinforce these differences among short and long‐winged species.First, genome‐wide genetic data confirm that all four species exhibit population genetic differentiation despite the short geographical distances among sites. That is, although populations along this elevation gradient are in close geographical proximity, their estimated gene flow rates are low, generally in a bidirectional stepping stone pattern. Consistent with our predictions, short‐winged species ” and physiological traits of each species. Clinal divergence with limited gene flow can be consistent with local adaptation, as the strength of selection need not be exceptionally strong to maintain adaptive alleles  across distance and a horseshoe‐shaped pattern in PC1 and PC2 .Across environmental gradients, the amount and direction of gene flow between populations influence the evolutionary processes controlling phenotypic variation. Gene flow is known to facilitate adaptation by increasing the genetic diversity upon which selection can act, but it is more likely to reduce divergent selection on an ecological gradient as it allows an influx of maladaptive alleles to populations along a cline  at upper elevation sites, suggesting a lack of reproduction or limited recruitment in these populations. This is reinforced by observed declines in reproductive potential  with elevation for M. sanguinipes (Levy & Nufio, Gene flow patterns could also be driven by how wind direction facilitates dispersal. In particular, the genetic clustering of A. clavatus and M. boulderensis, could subsequently erode and upslope flow of warm‐adapted alleles could facilitate adaptive responses to climate change. However, climate change could also decrease phenological overlap among populations and exacerbate population differentiation. High‐elevation populations might then contract and experience greater fragmentation (Rubidge et al., Climate change could quickly alter genetic structure in these species. Dispersal may increase as warm days increase the upward flow of air near the ground. Population differentiation, as observed in the alpine populations of None declared.All authors assisted in paper writing. L.B.B., R.A.S., and S.D.S. conceived the study and L.B.B., C.R.N, and R.A.S. collected specimens. R.A.S. and S.D.S. conducted the genetic data collection and analyses, C.R.N. conducted the phenological surveys. Click here for additional data file.


Acrorbis. Only one species, Acrorbis petricola, is currently included within this genus, whose geographical distribution is restricted to three known populations, one in Brazil and the other two in Argentina. Because of habitat specificity and limited geographical distribution, the species is considered vulnerable in Argentina and endangered in Brazil. In this work, we identify five new populations of A. petricola in southern Upper-Paraná Atlantic Forest, exclusively found on waterfalls from the Misiones Province, Argentina. Based on these populations and on specimens of one of the two historical populations from the Misiones Province, we explored the morphological features of shells and reproductive system of specimens from each location and provide the first molecular data on the species. We used DNA sequences from cytochrome c oxidase subunit I (COI) and 16S-rRNA genes to investigate the molecular diversity, genetic distances and genealogical relationships among populations. We verified the existence of intra- and interpopulation morphological variability, with the greatest variation being found in spire, spiral sculpture, penis sheath, flagella, prostatic diverticula and bursa copulatrix. We found interpopulation genetic diversity, with no intrapopulation variation, and identified six geographically structured genetic lineages with maximum genetic distances of up to 2.3%. Different combinations of morphological characters with the same genetic background within each locality were observed. The finding of new populations genetically differentiated not only broadens the known distribution of the species, but also illustrates that waterfall environments in the Atlantic Forest harbour a hidden diversity of Acrorbis that still remains to be discovered. This scenario suggests a complex evolutionary history that needs to be unveiled and taken into account for future development of conservation strategies in this endemic genus.High-energy freshwater environments such as rapids and waterfalls in the Upper-Paraná Atlantic Forest are home to highly endemic minute freshwater snails of the genus  Waterfalls and river rapids are natural laboratories for evolutionary research as they usually host a highly endemic flora and fauna, especially adapted to the constant humid conditions and the force of water, including several molluscan species –4. Such The Upper-Paraná Atlantic Forest, the southwestern and largest ecoregion of the Atlantic Forest, constitutes one of the “hottest hotspots” of biodiversity and endemism worldwide ,7. DespiAcrorbis Odnher, 1937 (Gastropoda: Planorbidae) represent a clear example of endemic micromolluscs occurring in poorly studied high-energy freshwater habitats of the Upper-Paraná Atlantic Forest, for which currently available knowledge is scarce and fragmentary. Odhner [Acrorbis to accommodate Acrorbis petricola Odhner, 1937, an atypical planorbid described from specimens collected in rocky banks of the Ariranha River, Nova Teutônia, Santa Catarina State, Brazil [Acrorbis odhneri Hylton Scott, 1960, a new species was added to the genus from Argentina based on specimens collected in Salto Encantado, a waterfall located in the Salto Encantado Provincial Park, Aristóbulo del Valle municipality, Misiones Province [A. odhneri was regarded as a junior synonym of A. petricola [Acrorbis individuals in the Iguazú National Park, Misiones Province, Argentina, showed some variability in shell morphology and male terminal genitalia in relation to previous descriptions [A. odhneri, that study also suggested that both nominal species were conspecific under the name A. petricola [Snails of the genus . Odhner  introduc, Brazil –20. UndeProvince –24. Subsetricola . A new cAcrorbis is considered to be monotypic [To date, onotypic ,22,24,25onotypic ,24–26. Tonotypic ,22,24,26onotypic ,27. HoweAcrorbis populations occurring at the Upper-Paraná Atlantic Forest. The objectives of this paper are: (i) to confirm the taxonomic identity of the populations using traditional morphological criteria based on shell morphology and morphological features of the reproductive system; (ii) to investigate the morphological variability among localities; and (iii) to explore the genetic background of the populations and to provide the first molecular data for the species. In this process we gained insights into a hidden diversity of Acrorbis in waterfall environments of the Atlantic Forest.In this study, we surveyed waterfall environments in central and southern Misiones Province, Argentina, and identified new Acrorbis petricola in the Misiones Province [Living snails were collected by hand in six waterfall environments in central and southern Misiones Province during mollusc surveys conducted between 2017 and 2018. The waterfall known as Salto Encantado in Salto Encantado Provincial Park was specifically surveyed as it represents the first historical record for Province . PermissLiving specimens were relaxed in water with menthol crystals for 30–60 min and immersed in hot water (80°C) for 30–60 sec. Soft parts were then separated from the shell and preserved in 96% ethanol for anatomical and molecular studies. Shells were firstly cleaned with a fine paint brush in a 10% sodium hypochlorite solution for 10 min to remove vegetation and other encrustations from external shell surface, and then washed with distilled water. Subsequently, shells were cleaned in isopropanol using an ultrasonic cleaner for 5–10 sec, and then air dried .A. petricola from Nova Teutônia, Brazil, and Iguazú National Park, Argentina, housed in the malacological collection at the Museo de La Plata . These samples were included herein in order to explore the genetic background of the other historical records of A. petricola in the Atlantic Forest, including its type locality.Voucher specimens were deposited in the malacological collection of the Instituto de Biología Subtropical (IBS-Ma), CONICET–Universidad Nacional de Misiones (UNaM), Misiones Province, Argentina. In addition, we obtained ethanol preserved tissues of museum specimens of Digital images in dorsal, ventral, and lateral views were obtained for ten specimens from each of the new localities with a Canon 6D camera equipped with a Nikon BD Plan 10X objective. Five shell measurements were taken in that material , namely The anatomy of the reproductive system of three photographed specimens per locality was studied . Specimens were dissected using a Labomed Luxeo 4D stereomicroscope and analysed according to Hubendick , Paraens3 samples of foot muscle of ethanol-preserved individuals with an ADN PuriPrep-T kit  following the manufacturer’s instructions. We selected 30 samples belonging to A. petricola  and the outgroup species Antillorbis nordestensis  and Biomphalaria peregrina . Collection information and GenBank accession numbers for the samples analysed are presented in DNA was extracted from 1 mm16S-rRNA (hereafter 16S) and cytochrome c oxidase subunit I (COI) genes were amplified using the primers 16SF-104 (5′-GAC TGT GCT AAG GTA GCA TAA T-3′) and 16SR-472 (5′-TCG TAG TCC AAC ATC GAG GTC A-3′) for 16S [5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) for COI [16S and COI genes were conducted as in Rumi et al. [A. petricola, the repeated attempts to amplify COI and 16S loci were unsuccessful; consequently, that material was not included in further analyses.Partial sequences of the mitochondrial  for 16S , and the for COI . PCR ampi et al.  and Vogli et al. , respecti et al. , with am16S gene was performed using MAFFT 7 through the MAFFT web-server  [COI sequences were aligned using Clustal X 2.1 [https://www.ncbi.nlm.nih.gov/orffinder/). For 16S, a putative secondary structure model was generated following the template proposed by Lydeard et al. [16S gene predicted for other molluscs. The number of haplotypes (h), number of segregating sites (S), as well as nucleotide (π) and haplotype (Hd) diversities per marker were computed by means of the DnaSP 6.12.01 program [p).Multiple sequence alignment of server/)  with theal X 2.1 , and trad et al.  and contd et al. . This mo program . Nucleot program  using thAcrorbis phylogenetic datasets were analysed separately as 16S (279 bp), COI (655 bp) and concatenated . The ML analysis was conducted with PhyML 3.0 [http://www.atgc-montpellier.fr/) with the Nearest-Neighbor Interchange branch swapping algorithm. Optimal models of nucleotide substitution were selected using the SMS program [https://www.phylo.org/) [COI and concatenated datasets, and GTR+I for 16S sequences) as identified in jModelTest 2.1.7 [Phylogenetic trees were constructed using maximum likelihood (ML), and Bayesian inference (BI). hyML 3.0  via the  program  by means program . For the program  via the lo.org/) , inferrilo.org/) . Nodal slo.org/)  with thest 2.1.7  under thst 2.1.7 , in ordeAcrorbis were recorded in the six waterfall environments surveyed in central and southern Misiones Province, five of these being new localities for the genus . Sequence alignments showed a total of 21 and 14 variable positions for the COI and 16S genes, respectively , although differences found here can be ascribed to intraspecific variation, as the morphology and proportion of flagella are within the intrapopulation variability previously described for specimens from Nova Teutônia [The general morphology of the reproductive system of the specimens from the six records examined here is in good agreement with previous studies of Paraense & Deslandes  and ParaParaense  has showcription , based ocription  and Paracription  confirmeTeutônia . In relaA. petricola populations may be indistinguishable based on morphological features alone, our study revealed the existence of interpopulation genetic diversity within the species’ limited distribution range, with absence of intrapopulation genetic variation. The six examined locations were genetically distinct with the presence of a single and unique haplotype per locality, which allows each of them to be recognized as a geographic group. Pairwise genetic distances among the six locations showed similar divergence levels for both markers, ranging from 0.6% to 2.0% for COI, and from 0.4% to 2.3% for 16S. Upon considering the historical population of Salto Encantado as a reference for comparison, Salto Capioví was shown to be the location with the smallest genetic distance for the COI gene, but was the one with the greatest divergence for the 16S gene. Conversely, Salto Chávez exhibited the greatest distance for COI and the smallest divergence for 16S, thus suggesting that both markers are probably evolving at different rates among some of the populations, a finding in need of further investigation. Beyond this, the highest divergences found are within the range of intraspecific variability usually reported in Planorbidae . ,53. A. pples age . Thus, fA. petricola populations in the Misiones Province were found in waterfall environments, such a pattern seems fully consistent with vicariance events. These could be related to climatic and tectonic factors associated with the geological history and geomorphological evolution of the fluvial systems of Misiones during the Cenozoic, where the hydrographic network was completely different from the current one [Acrorbis in the Upper-Paraná Atlantic Forest.For both mitochondrial markers, genetic diversity indices showed high values of mean haplotype and nucleotide diversities. When interpreted in light of the spatial distribution of genetic variation, these findings suggest that despite the short geographical distance between some localities, populations have been genetically isolated for a large amount of time with limited gene flow, a condition attributable to stable, geographically subdivided populations having a long evolutionary history ,56. By crent one ,58. Simirent one ,60). FurAcrorbis that still remains to be discovered in waterfall environments of the Atlantic Forest, which include genetically differentiated populations. The genealogical relationships among haplotypes depict a complex evolutionary history that needs to be unveiled and taken into account for future development of conservation strategies in this endemic genus. Nonetheless, the identification of six geographically isolated evolutionary lineages in the Misiones Province appear to warrant their future recognition as evolutionarily significant units (ESUs) for conservation prioritisation [Acrorbis based on the territorial network of protected areas of Misiones. To date, only Salto Encantado and Salto Capioví populations are included within protected areas, in a provincial and municipal park, respectively. In this context, the finding of Acrorbis populations in the waterfalls known as Salto Chávez, Salto Teodoro Cuenca, Salto Krysiuk and Salto Paca, widely used as ecotourism resources, could serve as the basis for the environmental protection of such areas, as they harbour a unique genetic diversity of A. petricola, which is documented here for the first time.In summary, this study not only broadens the known distribution of the species, but also illustrates a hidden diversity of tisation –63. The tisation . The infS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Aedes aegypti, the major vector of dengue, yellow fever, chikungunya, and Zika viruses, remains of great medical and public health concern. There is little doubt that the ancestral home of the species is Africa. This mosquito invaded the New World 400‐500 years ago and later, Asia. However, little is known about the genetic structure and history of Ae. aegypti across Africa, as well as the possible origin(s) of the New World invasion. Here, we use ~17,000 genome‐wide single nucleotide polymorphisms (SNPs) to characterize a heretofore undocumented complex picture of this mosquito across its ancestral range in Africa. We find signatures of human‐assisted migrations, connectivity across long distances in sylvan populations, and of local admixture between domestic and sylvan populations. Finally, through a phylogenetic analysis combined with the genetic structure analyses, we suggest West Africa and especially Angola as the source of the New World's invasion, a scenario that fits well with the historic record of 16th‐century slave trade between Africa and Americas. It is an invasive species with patterns of global migration that continue today , a dark mosquito breeding in tree holes and preferring blood meals from nonhuman wildlife , is a “domestic” mosquito, breeding in human‐generated containers and preferring humans for blood meals  have been well‐studied and strong genetic structure among and within continents have been documented [e.g.,  are at risk for at least one of the diseases transmitted by this species  chip  estimate the genetic diversity and differentiation among African populations and compare them with Aaa populations outside of Africa, and (c) identify the possible source(s) of the New World and Asia invasion.To address this challenge, we leverage a high‐throughput species‐specific genotyping Note on nomenclature: The subspecies designations Aedes aegypti formosus and Ae. aegypti aegypti were formally recognized by Mattingly  and mixed populations in West Africa which may represent the initial differentiation of Aaa  genotyped using the Axiom_aegypti1 genotyping array  including both Aaf and Aaa samples (as well as Ae. mascarensis). We further filtered this dataset eliminating highly linked loci using the—indep option  of plink  and discriminant analysis of principal components (DAPC), using the R packages LEA  between all pairs of populations and their significance (significance level of 0.05) were calculated in Arlequin v3.5.2.2 . The partitioning of the genomic variation was evaluated in the following levels: 1) Africa/outside Africa, 2) West Africa/East Africa, 3) domestic/peridomestic/sylvan populations, and 4) between the African countries. Details on the grouping for the AMOVA analyses are provided in Table The partitioning of the genomic variation among and within populations was evaluated through a hierarchical analysis of molecular variance, AMOVA  and genetic distance matrices, for all the African populations, we performed a Mantel test with 999 permutations using the “ade4” package in R v.3.4.4 (R Core Team 2018).2.5Ae. mascarensis individuals as an outgroup and six samples of Aaa outside Africa  to confirm the distinctiveness of the Aaa lineage from all the African lineages  analysis, as implemented in RaxML  and the African dataset are shown in Figure broad dataset  and 0.31 toward the South Africa‐Kenya cluster .The results of the fastSTRUCTURE analyses on the t Figure , K = 3 sAe. mascarensis from Mauritius, cluster together. Three additional African populations .Interestingly, the Indian Ocean island samples, Reunion and Focusing on the continental African dataset Figure , it becobroad dataset  signs of isolation by distance (IBD) among the African populations Figure . This is3.3The rooted ML phylogenetic tree is presented in Figure 4Ae. aegypti has two major genetic groups. These two groups generally correspond to the described subspecies, Ae. aegypti formosus (Aaf) in Africa and Ae. aegypti aegypti outside Africa (Aaa) with Aaa being monophyletic , given the geographic closeness of the two islands, ~120 km apart. Ae. aegypti and Ae. mascarensis can hybridize and produce fertile offspring  and Johannesburg, South Africa (AFS) separated by ~3,000 km Figures , and 5, While all evidence point to a single domestication event leading to Aaa outside of Africa, there are secondary, independent domestications taking place within Africa. Genetic patterns suggest that populations in human habitats in Africa today do not have a single origin, and often mix with nearby peridomestic or sylvan populations [as also seen in microsatellite data  forms a monophyletic group. Aaa outside Africa (New World and Asia) also forms a monophyletic group implying a single origin  toward the New World genotype  would have been introduced to those ships and they may have been already semidomesticated in the towns or coastal villages of West Africa . Thus, these “proto‐Aaa” mosquitoes could survive the long voyage between West Africa and New World. Interestingly, during the early period of slave trade, 1500‐1650, ~70% of the trade was carried out by Portugal (Eltis & Richardson, Using genetic data, the time of origin of Aaa in the New World has been estimated to be ~400‐500 years ago (Crawford et al., Ae. aegypti in Africa has taken on new importance. After decades of low levels, yellow fever has been resurging in Africa (Kraemer et al., From a public health perspective, None declared.PK carried out part of the molecular laboratory work, the data analyses, and drafted the manuscript. BE carried out the largest part of the molecular laboratory work and drafted an earlier version of the manuscript. AG‐S carried out part of the molecular work and revised the manuscript critically for important intellectual content. BK, MM, JL, GLG, DA, CP, AB, JP, CAS, and ADT provided samples and revised the manuscript critically for important intellectual content. JRP conceived, designed, and coordinated the study, edited the manuscript, and obtained funds for the research. All authors gave final approval for publication.The data used are available in vector.base.org under the project ID VBP0000295


Aphelinus certus. We addressed several questions about local adaptation: Do parasitoid populations differ in host ranges or in levels of parasitism of aphid species within their host range? Are differences in parasitism among parasitoid populations related to geographical distance, suggesting clinal variation in abundances of aphid species? Or do nearby parasitoid populations differ in host use, as would be expected if differences in aphid abundances, and thus selection, were mosaic? Are differences in parasitism among parasitoid populations related to genetic distances among them? To answer these questions, we measured parasitism of a taxonomically diverse group of aphid species in laboratory experiments. Host range was the same for all the parasitoid populations, but levels of parasitism varied among aphid species, suggesting adaptation to locally abundant aphids. Differences in host specificity did not correlate with geographical distances among parasitoid populations, suggesting that local adaption is mosaic rather than clinal, with a spatial scale of less than 50 kilometers. We sequenced and assembled the genome of A. certus, made reduced‐representation libraries for each population, analyzed for single nucleotide polymorphisms, and used these polymorphisms to estimate genetic differentiation among populations. Differences in host specificity correlated with genetic distances among the parasitoid populations.Parasitic wasps are among the most species‐rich groups on Earth. A major cause of this diversity may be local adaptation to host species. However, little is known about variation in host specificity among populations within parasitoid species. Not only is such knowledge important for understanding host‐driven speciation, but because parasitoids often control pest insects and narrow host ranges are critical for the safety of biological control introductions, understanding variation in specificity and how it arises are crucial applications in evolutionary biology. Here, we report experiments on variation in host specificity among 16 populations of an aphid parasitoid,  We use these data to address several questions about local adaptation: Do parasitoid populations differ in host range  or host preference ? Is interpopulation variation related to geographical distance, reflecting clinal variation in the abundances or quality of aphid species? Or, is interpopulation variation independent of geographical distance, as would be expected if differences in aphid abundances or quality, and thus selection, were mosaic? Are interpopulation differences in parasitism related to genetic differentiation, suggesting local adaptation? If so, is the variation associated with genes having different functions?Here, we report on geographical variation in host use among 15 populations of an aphid parasitoid, A. certus population when exposed to a taxonomically diverse set of seven aphid species. We sequenced reduced‐representation genomic libraries for each population to find single nucleotide polymorphisms (SNPs) for analysis of genetic differences. To provide a framework for determining homology among SNP loci, we sequenced and assembled the genome of A. certus. We tested the relationships between differences in parasitism versus geographical and genetic distances among these populations. Lastly, we investigated the functions of genes located near reduced‐representation loci with versus without SNPs. While we expected that SNP variation might be in linkage disequilibrium with genes affecting host specificity , we did not expect there to be a causal relationship between SNP polymorphisms and differences in host specificity.To answer these questions, we measured parasitism by each 22.1Aphelinus comprises more than 90 species  and are weak fliers  mature eggs , an invasive pest with which A. certus may have been introduced  from 15 populations in 12 locations in Asia, and we also collected one invasive population in eastern Pennsylvania  at 20°C, 50%–70% relative humidity, and 16:8 hr (L:D) photoperiod. When experiments were conducted, the parasitoids had been in culture 6–44 generations.During exploration for parasitoids to introduce against the soybean aphid, we collected ia Table . The paria Table , each cuAphis glycines Matsumura on Glycine max (L.) , Aphis gossypii (Glover) on Gossypium hirsutum L. , Rhopalosiphum maidis (Fitch), Rhopalosiphum padi (L.), and Schizaphis graminum (Rondani) on Hordeum vulgare L. ; in the tribe Macrosiphini, we used Diuraphis noxia (Kurdjumov) on H. vulgare and Myzus persicae (Sulzer) on Raphanus sativus L. . These aphids span the phylogeny of aphids reported as hosts of the A. varipes complex, of which A. certus is a member. Also, these aphid–plant combinations provide contrasts of aphids in the same versus different genera on the same versus different plant species. Six of these aphid species have distributions that overlap the geographical range of the populations we studied  or aphids protected by defensive endosymbionts , and differences in parasitism among aphid species on different plant species . The experimental unit for these analyses was a female parasitoid exposed to a single aphid species. Some dependent variables had non‐normal distributions with variances proportional to means, so we used the appropriate distributions  for the dependent variables. We chose the distribution that gave highest model probability calculated from the residual deviance divided by residual degrees of freedom compared to a chi‐square distribution  to test the effects of parasitoid population and the interaction between aphid species and parasitoid population on the number of parasitized (mummified) aphids, adult emergence rates (proportion of parasitized aphids from which adult wasps emerged), and the proportion of males among adult parasitoid progeny. To examine the role of plant species, we used GLM to test differences in parasitism among aphid species on the same plant species  were not used, and after removing these replicates , we had data for 6–14 females  from 16 parasitoid populations on each of the seven species of aphids for a total of 1,163 females. To reduce inadvertent laboratory selection, we tested the populations within three years of collection. We also used GLM to test the effects on parasitism of generations in culture and the interaction between aphid species and generations in culture. Because of space constraints, we were able to measure responses of a maximum of 120 females on the same date. To avoid confounding the effects of population differences with differences in dates tested, we spread the replicates for each population over multiple dates and tested several populations on each date. The seven populations collected in 2002–2003 were all tested together on five dates in 2004–2005. The three populations collected in 2004–2005 were all tested together on four dates in 2006. The two populations collected in 2006 were tested together on three dates in 2007. The remaining four populations, collected in 2001, 2007, 2008, 2010 , were each tested on one or two dates. For analysis of adult emergence rate, we only used replicates where at least four aphids were mummified, and for analysis of sex ratio, we only used replicates where at least four adult parasitoids emerged, so the replicate numbers were lower for these variables.Using Spearman's rho in the cor.test function in the stats package in R, we tested the correlations between adult emergence rates and (a) the proportion of male progeny and (b) the number of parasitized aphids. If females prefer aphid species on which their progeny are fittest, one would expect a correlation between these performance measures and parasitism rates, such that proportion males would be lowest and adult emergence rates would be highest for the aphid species with the highest parasitism.2.5A. certus. Because the population of A. certus in North America has only very recently established and thus is unlikely to have differentiated from Asian populations, we excluded it from this analysis. We calculated great‐circle distances between collection sites using latitudes and longitudes for each site  using the adonis function in the vegan R package . The sequencing library consisted of DNA from 24 male progeny from a single female extracted using the Qiagen DNeasy Blood and Tissue Kit . The DNA was used to make a genomic library with the Illumina TruSeq DNA LT Library Preparation Kit , and paired‐end sequencing was conducted on an Illumina HiSeq 2500 at the Delaware Biotechnology Institute, Newark, Delaware.To determine orthology of SNP loci and find genes in http://www.clcbio.com) running on the bioinformatics cluster at the Delaware Biotechnology Institute, Newark, Delaware. We trimmed the Illumina sequences with a quality limit of 0.05 and an ambiguous limit of five nucleotides. After trimming, the read lengths averaged 144 nucleotides and the paired‐distances were 208–518 nucleotides. For assembly, we used a bubble size of 50, a word size of 25, and minimum contig length of 200 nucleotides.Quality trimming and de novo assembly were done with the CLCBio Genome Workbench  using BLASTP  and one frequent cutter (CviQI with a 4 bp recognition site) , which together determined the number of unique locations of fragments across the genome and the lengths of these fragments. Custom adaptors, with barcodes for each population that also served to register clusters on the Illumina HiSeq during sequencing, were ligated onto the fragments using T4 ligase . The ligates were pooled and purified using Agencourt AMPure XP beads . The purified ligate was separated into 10 aliquots that were amplified in separate PCRs to both increase copy number at each locus and add more adaptor sequence for sequencing. The adaptors were designed so that the only fragments that amplify would have the rare‐common combination of cut sites. After PCR, the products were pooled and then size‐selected (300–350 bp) using the BluePippin system . After quantification with qPCR, the resulting fragments were sequenced for ~100 nucleotides in single‐end reads an Illumina HiSeq 2500  at the Delaware Biotechnology Institute.To generate data for phylogenetic and population genetic analyses, we used next‐generation sequencing of reduced‐representation genomic libraries to genotype single nucleotide polymorphisms (SNPs) among the 16 g et al. . Brieflyhttps://github.com/UD-CBCB/RedRep. Briefly, sequences were deconvoluted by barcode using custom scripts and the FASTX‐Toolkit . Custom scripts and CutAdapt  or nongenic and then categorized genic loci based on SNP content (defined as “with SNP” or “without SNP”). We compared the functions of genes near reduced‐representation loci with versus without SNP loci. To determine the function of these genes, we compared their amino acid sequences to those of proteins in the RefSeq database  using BLASTP, and we conducted domain analyses with InterProScan . Parasitism differed among populations for all aphid species except D. noxia, which was rarely parasitized by any parasitoid population  before parasitism was measured. However, neither generations in culture nor the interaction between aphid species and generations in culture affected parasitism . Nor was A. glycines among the most favored aphid species: Only two populations parasitized A. glycines more than other aphid species, while M. persicae and R. padi were the most parasitized aphid species in four and five populations, respectively. Excluding D. noxia, the aphid species with maximum and minimum parasitism varied among parasitoid populations, with five of the six aphid species having maximum and minimum parasitism by females from at least one parasitoid population. Parasitism varied among the aphid species on H. vulgare , from little or no parasitism of D. noxia by any population to maximum parasitism of R. padi by five populations  with very different chemistries .Parasitoid population and the interaction between aphid species and parasitoid population strongly affected parasitism . However, more than 90 percent of the combinations of aphid species and parasitoid populations showed mean emergence rates ≥0.80. Differences in emergence rates were only found for three aphid species .Parasitoid population and the interaction between aphid species and parasitoid population affected the emergence rates of adult progeny of A. certus progeny , but only one aphid species, A. glycines, showed significant variation in proportion of males  for two to six A. certus populations depending on the aphid species, and female‐biased  for one A. certus population on R. padi, with R. maidis being the only aphid species with no biases , indicating that female A. certus laid more fertilized eggs in aphids that they parasitized more.Parasitoid population and the interaction between aphid species and parasitoid population affected the proportion of males among 3.2A. certus populations ranged from 35 km to 2,574 km. The closest populations were the two from Inner Mongolia, and the most distant were those from Inner Mongolia and Japan. A network graph of the similarity in parasitism among A. certus populations suggested some geographical partitioning . Furthermore, differences in parasitism among populations did not correlate with geographical distance between populations .Geographical distances between the Asian 3.3A. certus genome was 290 Mb long, which is 20% smaller than the 361 Mb genome size estimated from flow cytometry , we found homologs for 78%  of these genes in the RefSeq database.With 30 Gb of paired‐end Illumina data and 103x coverage, our assembly of the 3.4A. certus populations and mapping the sequences onto our draft assembly of the A. certus genome yielded 18 K‐163 K reduced‐representation loci, depending on the population .Host plant species did not affect which aphid species were parasitized by these parasitoid populations. The differences in parasitism among aphid species on Aphis glycines would select for greater use of Aphis glycines. However, parasitism was not affected by generations in culture or the interaction between aphid species and generations in culture. Henry et al. , and few generations in culture before testing. Our rearing procedures were designed to maintain existing genetic variation  Many genes across the genome are involved, each having small effects, and our limited sample captured enough of them to explain 14 percent of the variance in parasitism—in this case, any randomly selected set of ~800 genes would explain a similar amount of variation; or (b) a few genes are involved, each with large effects, and our limited sample fortuitously hit a few of these genes, which together explained 14 percent of the variation in parasitism—in this case, a different set of genes might well explain zero percent of the variance. With the available information, we cannot distinguish between these hypotheses. However, we had hoped that analysis of differences in the functions of genes with versus without SNP loci near them might reveal candidates for local adaptation in parasitism. However, we found no enrichment or depletion of the functions of the proteins coded by the two sets of genes.Low dispersal rates and high levels of genetic differentiation suggest that gene flow is restricted among these populations of 5Aphelinus certus, showed that although the parasitoid populations did not differ in host range , they did differ in levels of parasitism of the aphid species tested. Interpopulation differences in parasitism could not be explained by the geographical distance between populations, nor did clustering of populations by parasitism patterns correlate with geographical regions. Instead, we found that interpopulation differences in parasitism were associated with patterns of genetic differentiation, as measured by FST for SNPs distributed across the genome. Of the 760 genes we examined, those with SNPs did not differ in function from those without SNPs. This suggests that interpopulation variation in the sequence or expression level of genes with the same basic functions may be responsible for local differences in host specificity, which stands in contrast to our initial expectation that genes in a limited set of functional categories would be involved in local adaptation. In any case, this is one of the few studies of local differentiation in patterns of parasitism that provides evidence that local adaptation has an underlying genetic basis. Future studies will be needed to determine whether such striking levels of association between variation in host specificity and genetic differentiation are common to other insects that display local variation in host specificity.Parasitic wasps are among the most species‐rich groups on Earth, and a major cause of this diversity may be adaptation to local variation in the presence or abundance of host species. Little is known about local patterns of parasitoid host use, and the research presented here is the largest such study conducted to date. Our research on geographical variation in host use among 16 populations of an aphid parasitoid, FST estimates are archived on Ag Data Commons (DOI: dx.doi.org/10.15482/USDA.ADC/1503305). DNA sequence data are archived in the NCBI Sequence Read Archive .Data from the host specificity experiments along with the The authors declare no conflict of interest related to this manuscript. Click here for additional data file.