Aquilegia flavescens × A. formosa population in British Columbia that is disjunct from its parents—the latter species is present locally but ecologically separated, while the former is entirely absent. To confirm hybridity, we used multivariate analysis of floral characters of field-sampled populations to ordinate phenotypes of putative hybrids in relation to those of the parental species. Microsatellite genotypes at 11 loci from 72 parental-type and putative hybrid individuals were analysed to assess evidence for admixture. Maternally inherited plastid sequences were analysed to infer the direction of hybridization and test hypotheses on the origin of the orphan hybrid population. Plants from the orphan hybrid population are on average intermediate between typical A. formosa and A. flavescens for most phenotypes examined and show evidence of genetic admixture. This population lies beyond the range of A. flavescens, but within the range of A. formosa. No pure A. flavescens individuals were observed in the vicinity, nor is this species known to occur within 200 km of the site. The hybrids share a plastid haplotype with local A. formosa populations. Alternative explanations for this pattern are evaluated. While we cannot rule out long-distance pollen dispersal followed by proliferation of hybrid genotypes, we consider the spread of an A. formosa plastid during genetic swamping of a historical A. flavescens population to be more parsimonious.We report the investigation of an  Aquilegia formosa and Aquilegia flavescens, two western North American taxa, in which the latter species is absent both locally and regionally. We present analyses of floral morphology, nuclear admixture and maternally inherited plastid sequence to confirm hybridity and discuss possible origin scenarios. We suggest that A. flavescens may have formerly occupied the region and became extirpated through genetic assimilation by its congener.The outcomes of natural hybridization are incredibly varied, ranging from hybrid speciation to extinction of parental taxa. This research describes the investigation of an ‘orphan’ hybrid population between  Senecio squalidus occurs in the UK without either parent due to human-mediated dispersal of germplasm from a hybrid zone between two species in Sicily , interspecific hybridization is a striking evolutionary phenomenon. Throughout their natural distribution, intergrading floral forms often occur in zones of species range overlap  are hybrids, or rather pink-flowered morphs of otherwise typical A. flavescens  data of the putative hybrids in relation to allopatric parental populations. To assess the direction of hybridization and test hypotheses on the origin of the hybrid population, we compared maternally inherited plastid haplotypes of the hybrid individuals to those of allopatric parental populations as well as local A. formosa populations. As Aquilegia seeds do not disperse far, an A. flavescens maternal origin would support the extirpation of a pre-existing A. flavescens population, possibly through genetic swamping by local A. formosa. Alternatively, an A. formosa maternal origin could support spread of an A. formosa plastid lineage through a contact zone, or long-distance pollen dispersal from A. flavescens. Lastly, we used spatial analysis of herbarium specimen phenotypes to explore evidence for introgression throughout the distribution of these species.A putative hybrid population of  formosa  was orighttp://www.pnwherbaria.org/data.php, 31 June 2017). Measurements focused on known defining species traits and other relevant traits revealed through preliminary inspection of specimens. For each pressed specimen, a single post-anthetic (open) flower was measured for seven continuous traits: corolla width, spur length, anther exsertion, petal lamina length, petal lamina width, sepal length and sepal width [see]. Whether the petal laminae were cleft was recorded as a categorical trait. Herbarium specimens were geolocated according to information from herbarium sheet labels, the BC Geographical Names database  and Google Earth. To explore whether floral morphology corresponds to reported pollination mode, we measured the bill lengths of 10 rufous hummingbird (Selasphorus rufus) and 10 calliope hummingbird  specimens from the UBC Beaty Biodiversity Museum collection, which are the most common hummingbird species with breeding ranges overlapping the ranges of A. formosa and A. flavescens.We measured floral phenotypes of 191 herbarium specimens from the University of British Columbia (UBC) and University of Idaho (ID) Stillinger herbaria. University of British Columbia specimens were measured directly from herbarium sheets; ID specimens were measured via size-scaled photographs accessed through the Consortium for Pacific Northwest Herbaria specimen database , and near Clearwater, BC , and two allopatric populations of A. flavescens on Mt. Kobau, BC  and at Beehive Lakes, Idaho   and montane sagebrush community (Mt. Kobau). The hybrid population was found between 1900 and 2100 m in elevation on an east-facing drainage beneath Porcupine Ridge in the Marble Range . The Aquilegia at this site , and one along the Pavilion-Clinton Highway, ~20 km from the hybrid population . Populations were visited during peak bloom season in the summers of 2017 and 2018, with the exception of the population at Beehive Lakes, in which only several plants remained in bloom, precluding collection of floral phenotype data.Field sites included allopatric populations of 16.65°W) . The habsite see  occurredPlants with at least one post-anthetic flower were sampled in a haphazard fashion at least 1 m apart within the allopatric populations. In the hybrid population we sampled additional plants at the extremes of the present colour variation  to assess whether parental-type colouration correlated with parental-type morphology. For each plant, we photographed the outer face of the sepal whorl for a single flower using a Canon PowerShot sx50 HS digital camera . A second photo was taken of the SpyderCheckr 24 colour card  under the same light conditions for later standardization. We extracted nectar from one or multiple flowers from each plant with glass capillary tubes inserted into the back of the floral nectaries and measured nectar volume in the capillary tube before extruding it onto a percent sugar refractometer  for measurement of sugar concentration . Samples of leaf tissue from each plant were collected in silica gel, and whole plants were then pressed in the field.Aquilegia that are thought to evolve neutrally were chosen from the literature (trnT-trnL region (GenBank: MK228981–MK228983) was amplified with primers trnTUGUF and 5′trnLUAAR (rps16 region (GenBank: MK228978–MK228980) was amplified using primers rps16F and rps16R as described in We extracted DNA according to a 2 % CTAB extraction protocol . MicrosaMASS  implemented in the R package MASS . We usedImages were shot in RAW format and imported into Lightroom™ . Using SpyderCheckr colour correction software , images of the colour card were used to create calibrations that were then applied to corresponding images of sepals in Lightroom. These calibrated images were then imported into Photoshop™  in the Adobe RGB colour space. The magnetic lasso tool was used to select a randomly chosen sepal or all sepals from each flower. We recorded the mean pixel value for each channel  in RGB colour space from this selection, and used the ratio of the mean pixel values of the red and green channels as a measure of the sepal colour variation as perceived by humans . The RGBHo and He). Within populations, we calculated the average Ho, gene diversity (Hs) and inbreeding coefficient (FIS) using the R package hierfstat , number of alleles, and observed and expected heterozygosity , which is implemented in the R package K (the number of genetic clusters) set equal to two. All STRUCTURE runs used the admixture model allowing for correlated allele frequencies, with 100 000 burn-in and Markov chain Monte Carlo repetitions. Results were checked for consistency across 20 runs and final ancestry coefficients of individuals were averaged using the program CLUMPP .Geolocations of herbarium specimens confirmed that the ranges of the parental species overlap in montane regions west of the Rocky Mountains r = 0.85), validating the use of the log R/G metric. Field-collected plants from parental populations exhibited non-overlapping variation in the relative amount of red vs. green reflectance in the sepals  as those of pure A. flavescens (Colour scores (log R/G) were highly correlated with visually assigned integer scores within the colour-variable hybrids  = 0.37, 95 % confidence interval (CI) = 0.28, 0.47; A. flavescens: p = 0), such that the presence of cleft petal laminae is diagnostic of A. formosa.The floral morphology discriminant axis scores of herbarium specimens showed distinct yet overlapping distributions. The mean classification error rate over 1000 iterations of 5-fold cross-validation was 0.08, in accordance with some degree of visual overlap of the distributions for each species along the discriminant axis. The loadings of each character onto the discriminant axis see  indicate−4, considerably lower than that of the LDA of herbarium specimen phenotypes. The loadings of traits onto the discriminant axis were similar to those in the herbarium specimen analysis, with the dimensions of the laminae again having the largest loadings. The proportions of field-collected individuals with cleft petal laminae also closely matched those estimated from herbarium specimens . The field-collected parental specimens were thus taken to be representative of the ‘pure’ species floral phenotypes and were used thereafter for direct comparison with the putative hybrid phenotypes.The floral morphology discriminant axis scores of the Marble Range flowers fell largely in between the clusters of the representative parental types , although the 95 % CI for the difference in proportions between the Marble Range population and all A. formosa specimens marginally overlapped zero . Depending on the genetic basis of interspecific differences, the overall intermediacy of hybrid phenotypes could result from intermediacy in additive traits, or from mismatched combinations of non-additive traits from either parental species. We therefore investigated the distribution of each floral character independently in the Marble Range plants in relation to the parental trait distributions. This revealed that the Marble Range floral characters are on average intermediate for all continuous traits, with the exception of sepal length [see]. These results imply an additive genetic basis in the traits governing interspecific differences between the parental species. As parental-type traits should segregate in recombinant hybrids, we also calculated pairwise trait correlations in the hybrids to investigate possible co-segregation of traits . A. flavescens using the methods used here. All flowers sampled in the Marble Range population contained measurable quantities of nectar, with a mean of 3.48 µL . While these values give a general sense of nectar volumes in these populations, meaningful comparison of the hybrid and parental types for this phenotype remains inconclusive, due to the complex dependencies of nectar volume on developmental stage, environmental conditions and nectar removal by visitors.Although we found up to 8 µL of nectar within a single A. formosa and A. flavescens. In the absence of prior information, the STRUCTURE analysis of allopatric specimen genotypes was able to perfectly distinguish species when hybrid genotypes were excluded (not shown) and K was set equal to two. Unweighted paired group method with arithmetic mean and NJ trees also showed strong species clustering when hybrids were excluded (not shown). The genetic data performed more poorly at species discrimination as compared to floral morphology data; cross-validation showed that the DAPC model for allopatric parental specimens had a misclassification rate of 0.04 (vs. 3 × 10−4 for the morphology LDA). To see if this discrepancy could be a result of differences in sample size, we randomly subset the floral phenotype data set to contain the same sample sizes as in the genetic data set and repeated the cross-validation procedure. The classification error rate increased to 8 × 10−4, a value still considerably lower than the classification error rate based on genotype. This observation suggests that the species are more divergent in morphology than in genotype.Analysis of microsatellite data revealed genetic differentiation between A. flavescens and A. formosa . Hybrids also showed intermediate placement between parental clusters in the principal coordinate analysis based on Bruvo’s distance [see] and showed variable placement in the NJ and UPGMA trees [see].All analyses performed suggested that the individuals from the Marble Range population have genetic ancestry from both  formosa  and 5. I formosa . ReplicatrnT-trnL, which is treated as a single mutation in the haplotype network, was predominantly species-specific and contributed to species separation of haplotypes; however, a single yellow-flowered individual at Mt. Kobau notably possessed the A. formosa allele at this site. We searched GenBank records  and determined that the indel sequence is a 5-bp repeat with zero to three repeat units present in different members of the genus. Semiaquilegia adoxoides, sister to Aquilegia , possesses one unit, A. jonesii possesses zero, A. formosa and several congeners possess two, and A. flavescens alone possesses three out of the taxa represented, suggestive of a history of insertion and reversal at this locus.No haplotypes constructed from plastid sequences were represented in members of both species . A singlA. formosa-type plastid haplotype  and spatial proximity to the geographical range centroid of the alternate species relative to that of the same species . Put anoA. formosa both in the adjacent valley and 20 km away along the Pavilion-Clinton Highway, the evidence supports the idea that the hybrids descended from a local A. formosa maternal lineage. Interestingly, an alternative A. formosa haplotype is shared by individuals in both Robert’s Lake and Clearwater, which occur on either side of the Marble Range, over 400 km apart. This does not support an isolation-by-distance pattern and likely reflects a complex phytogeographic history of this species in the mountainous and highly dissected landscape of BC.Given that the haplotype of the Marble Range hybrid plants was shared by Aquilegia and in other members of Ranunculaceae  historical A. flavescens populations occurred in this region, or (ii) long-distance pollen dispersal occurred. Under the first scenario, the hybrids would represent ‘ghosts’ of one or more extirpated A. flavescens populations. Alternatively, under the second scenario, long-distance pollen dispersal could have resulted from hummingbird migrational movements. The ranges of both the rufous hummingbird and the calliope hummingbird overlap with the distributions of A. formosa and A. flavescens. Both species have been directly observed to visit A. formosa , supporting the plausibility of this hypothesis. Under the genetic swamping hypothesis, the high proportion of A. flavescens ancestry can be more easily reconciled, as genetic swamping does not lead to a loss of parental alleles, but only of intact parental genomes , and from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants Program (grant no. RGPIN-2014-05820) to Q.C.B.C.J.S.G. and Q.C.B.C. planned the research. J.S.G. collected herbarium and field data, extracted DNA, performed analyses and wrote the manuscript. D.M.P. assisted with field work and generated molecular data. C.R.B. identified the population and carried out initial field surveys. Q.C.B.C. assisted with field work, suggested analyses, edited the manuscript and provided funding for the study. All authors contributed to and approved the final manuscript.None declared.Supplementary AppendixClick here for additional data file.Supplementary VideoClick here for additional data file.Supplementary Collections BlindClick here for additional data file.


Ovis canadensis) are a managed big game species that suffered widespread population extirpations across western North America throughout the early 1900s. Subsequent translocation programs have successfully re‐established many formally extirpated bighorn herds, but most of these programs pre‐date genetically informed management practices. The state of Nevada presents a particularly well‐documented case of decline followed by restoration of extirpated herds. Desert bighorn sheep (O. c. nelsoni) populations declined to less than 3,000 individuals restricted to remnant herds in the Mojave Desert and a few locations in the Great Basin Desert. Beginning in 1968, the Nevada Department of Wildlife translocated ~2,000 individuals from remnant populations to restore previously extirpated areas, possibly establishing herds with mixed ancestries. Here, we examined genetic diversity and structure among remnant herds and the genetic consequences of translocation from these herds using a genotyping‐by‐sequencing approach to genotype 17,095 loci in 303 desert bighorn sheep. We found a signal of population genetic structure among remnant Mojave Desert populations, even across geographically proximate mountain ranges. Further, we found evidence of a genetically distinct, potential relict herd from a previously hypothesized Great Basin lineage of desert bighorn sheep. The genetic structure of source herds was clearly reflected in translocated populations. In most cases, herds retained genetic evidence of multiple translocation events and subsequent admixture when founded from multiple remnant source herds. Our results add to a growing literature on how population genomic data can be used to guide and monitor restoration programs.Conservation biologists have increasingly used translocations to mitigate population declines and restore locally extirpated populations. Genetic data can guide the selection of source populations for translocations and help evaluate restoration success. Bighorn sheep ( Historically, bighorn sheep occupied most of the rugged, high‐elevation habitats found from northern Mexico to southwestern Canada  and goats (Capra hircus L.)  and perhaps other unrecognized lineages  populations were found primarily in the Mojave Desert, although a handful of remnant herds persisted in the Great Basin Desert  were translocated into the southern two thirds of the state , California bighorn sheep  into the northwest, and Rocky Mountain bighorn sheep  into the northeast  to generate population genomic data for desert bighorn sheep sampled from the Great Basin and Mojave Deserts to explore the population genetic context and consequences of these translocations. Our goals were to: (a) characterize genetic diversity and differentiation among herds, especially among Mojave source herds; (b) evaluate patterns of admixture in reintroduced herds in light of translocation histories; and (c) quantify the degree of genetic diversity within translocated herds composed of either single or mixed origins relative to source populations. Our results provide a reference for continued translocation decisions and a baseline for understanding how past and future population responses might relate to genetic variation within and among herds. More broadly, our study illustrates how high‐throughput sequencing approaches can be used to illustrate the population genetic context and consequences of translocation activities spanning several decades.22.1We obtained muscle, liver, or blood samples from a hunter harvest return program and a long‐term herd‐monitoring program (NDOW). We maximized spatial coverage of samples to represent as many extant herds as possible, but actively monitored populations or those with higher harvest rates were disproportionally represented. DNA was extracted using Qiagen DNeasy Blood and Tissue Kits and quantified via spectrophotometry with a QIAxpert device .2.2EcoRI and MseI). Next, an Illumina adaptor, an 8–10 bp barcode identifier , and matching sticky ends were attached to the EcoRI cut sites, while only an Illumina sequencing adaptor and the matching sticky ends were attached to the MseI cut sites. Fragments were amplified using PCR, and the products for each individual were randomized into three separate pools. To further reduce complexity, we size selected for fragments ranging from 350 to 450 bp in size using a Pippin Prep device . Further details on the full library preparation protocol are available at Dryad (https://doi.org/10.5061/dryad.25f502n). Each of the three pools was sequenced on four lanes of an Illumina HiSeq 2500 at the University of Wisconsin‐Madison Biotechnology Center.Reduced‐representation libraries for Illumina sequencing were prepared using a GBS protocol , low‐quality reads, and Illumina adaptors using bowtie2_db . A custom Perl script was used to filter reads containing Illumina adaptor fragments, correct single or double bp errors in barcode regions , and parse reads into separate files for each individual . Individuals represented by abnormally low numbers of reads were discarded. Reads from the remaining individuals were aligned to the domestic sheep genome  were identified, and genotype likelihoods were calculated using 2.3N = 303), as well as a subset of four remnant source populations that have never received translocations (N = 55). The four source populations included three remnant Mojave herds  and a suspected remnant Great Basin herd on Lone Mountain (unit 212). Individuals from the Black and Muddy Mountains historically moved freely between these two contiguous mountain ranges and are considered a single population , estimate ancestry coefficients for each individual, and estimate genotype probabilities for each individual at each locus. Importantly, this model utilizes a population allele frequency prior and incorporates information about genotype uncertainty arising from variation in sequencing coverage, sequence error, and alignment error during parameter estimation , with smaller DIC values consistent with better model fit. Analyses for k > 7 in the full dataset and k > 4 in the source dataset failed to consistently converge and had high DIC values, so they were not considered further. To provide MCMC sampling with a starting point and to facilitate chain convergence, we conducted principal component analysis (PCA) on a covariance matrix of the genotype likelihoods and used k‐means clustering based on principal components (PCs) to cluster individuals. Starting membership probabilities to the clusters were then calculated for each individual with linear discriminant analyses (LDA) on all PCs. Both k‐means clustering and LDA were performed using the MASS package .In order to determine the most probable FST , as well as for a subset of populations that have never received translocated individuals  from genotype probabilities. Mean HE was calculated based on allele frequencies using expectations from Hardy–Weinberg equilibrium. In contrast, mean HO was calculated directly from the genotype probabilities, where an individual was scored as heterozygous at a given locus if the genotype probability ranged between 0.9 and 1.1 . Heterozygote deficiency (FIS) was calculated as FIS =1 ‐ (HO/HE), with positive values consistent with lower than expected heterozygosity  and loci with a mean coverage greater than ten were removed . This resulted in a final dataset of 303 individuals and 17,095 SNPs, with a mean coverage of 4.34X per locus per individual.Following initial filtering and the removal of individuals for which few reads were generated, we retained a dataset of 337 individuals with a mean of 2,607,602 reads per individual . Initially, 1,188,258 variant sites were identified, of which 17,922 loci remained after 1) retaining only one variant site per contig, 2) keeping only biallelic SNPs, 3) removing sites with maf <0.05, and 4) excluding sites where more than 40% of individuals did not have at least one read sequenced. In the final round of filtering, individuals with more than 50% missing data were removed (3.1entropy) to investigate potential fine‐scale genetic structure among four source herds  that have never received translocated individuals (Table k) was better supported than models with k = 2 or k = 3 (Table entropy model below).We used a hierarchical Bayesian model  from the three Mojave source populations , while the three Mojave hunt units were less differentiated from one another , the Muddy Mountains , the River Mountains , and the Mormon Mountains , while the maximum FST was found between hunt units 212 and 184  that had never received translocated individuals, the model did explain over 40% of the variation in the data  to 0.201  and ranged from 0.133  to 0.190  than units 212, 269, and 271, which all had roughly equivalent mean HO estimates (HO =0.154 – 0.157). Relative to remnant populations, reintroduced populations did not have reduced levels of HE  or HO . Finally, the number of translocation sources revealed a marginally positive  association with both HE . Individuals from these herds were historically dependent on regular movements and access to the nearby Colorado River for water , where serial reintroductions over the past century resulted in less diverse populations containing only a subset of the source's genetic diversity ; Johnson et al., Tympanuchus cupido (L.); Westemeier et al., Vipera berus (L.); Madsen, Shine, Olsson, & Wittzell, Even prior to the advent of modern sequencing technologies, a pivotal question in restoration genetics was whether translocation programs could establish populations with sufficient levels of genetic diversity. Maintaining diversity could be important for avoiding inbreeding depression and providing sufficient genetic variation for adaptation. Although genetic diversity can be maintained by choosing a large and diverse pool of founders, genetic surveys of potential source individuals prior to translocation are rarely utilized  populations found that natural lakes had lower genetic diversity and higher genetic differentiation than stocked lakes (Valiquette, Perrier, Thibault, & Bernatchez, FST values among remnant source populations were elevated relative to those among re‐established populations (Fig. Although several thousand translocations have been conducted over the past century without genetic monitoring (Laikre et al., ons Fig. . Higher ons Fig. , and whions Fig. . As the 4.5Emerging technologies that allow for increases in the extent of genomic sampling are now reshaping our understanding of the genetic consequences of conservation actions, even for organisms with complicated management histories (Allendorf, Hohenlohe, & Luikart, https://doi.org/10.5061/dryad.25f502n.The GBS library preparation protocol, individual fastq files, and a matrix of genotype probabilities are available from the Dryad Digital Repository:  Click here for additional data file.


Bos indicus or “zebu”), descend from a domestication process that took place >8,000 years ago in South Asia. Here we present an intercontinental survey of Y-chromosome diversity and a comprehensive reconstruction of male-lineage zebu cattle history and diversity patterns. Phylogenetic analysis revealed that all the zebu Y-chromosome haplotypes in our dataset group within three different lineages: Y3A, the most predominant and cosmopolitan lineage; Y3B, only observed in West Africa; and Y3C, predominant in South and Northeast India. The divergence times estimated for these three Zebu-specific lineages predate domestication. Coalescent demographic models support either de novo domestication of genetically divergent paternal lineages or more complex process including gene flow between wild and domestic animals. Our data suggest export of varied zebu lineages from domestication centres through time. The almost exclusive presence of Y3A haplotypes in East Africa is consistent with recent cattle restocking in this area. The cryptic presence of Y3B haplotypes in West Africa, found nowhere else, suggests that these haplotypes might represent the oldest zebu lineage introduced to Africa ca. 3,000 B.P. and subsequently replaced in most of the world. The informative ability of Interspersed Multilocus Microsatellites and Y-specific microsatellites to identify genetic structuring in cattle populations is confirmed.All tropically adapted humped cattle ( Genetic studies of cattle domestication have pointed out that modern cattle (Bos taurus and B. indicus) resulted from at least two genetically distinct auroch populations (B. primigenius and B. nomadicus)5. The domestication of B. taurus in the Near East and expansion of taurine cattle into Europe has received particular attention8. However, studies of the domestication and spread of the zebu cattle lineage, B. indicus, are relatively scant.The last two decades of genetic studies on the origin of livestock species have unveiled a remarkable set of new questions regarding the origin and spread of domesticated animals. Research on single versus multiple domestications, interbreeding of wild and domestic animals, and cultural and faunal exchanges is transforming current perspectives on domestication trajectories and biodiversity10. South India may have been an additional centre for cattle domestication12, as may have been Gujarat in western India13. Several areas of domestication or wild capture are consistent with genetic studies, which have identified two major mtDNA haplogroups (or lineages) in zebu cattle: I1 and I24. An extensive survey of modern cattle suggests that the maternal zebu cattle lineage I1 likely originated from the domestication of local wild cattle (Bos namadicus) in northwestern South Asia4. Animals of this lineage may have spread through South Asia  and beyond the Subcontinent eastwards to Southeast Asia and southern China 4. After this initial spread, additional genetic diversity was recruited to domestic herds from South Asian wild cattle populations carrying the haplogroup I2.Archaeological findings suggest that zebu cattle were domesticated 8,000–9,000 years ago (B.P.) and dispersed throughout northwestern South Asia by 6,000 years before present4 and genome-wide14 information studies conducted on B. indicus analyses of Y-chromosome diversity are rare. Seminal studies using Single Nucleotide Polymorphisms (SNPs) identified a single zebu Y-chromosome lineage (Y3)15. Admixture between B. taurus and B. indicus in Africa and differences in diversity parameters assessed using Y-chromosome markers18 have contributed to understand the complexity of domestication processes, the early spread of male zebu cattle in South Asia and the introduction of zebu to Africa through Indian Ocean trade.In comparison with mtDNA19 but present African zebu cattle populations are the result of multiple introductions. Archaeology suggests that most animals came through eastern rather than northern Africa. Pre-Aksumites, Aksumites and their trading partners in Yemen and the Red Sea brought zebu cattle to the Horn of Africa 2,000–1,600 B.P.21 following well known sea trade routes from South Asia to the Gulf of Oman23. Humped cattle followed Sahelian routes, appearing in West Africa by 1,000 B.P.24. The rinderpest panzootics of 1889–1896 are estimated to have annihilated up to 80% of herds in many regions or over 5.2 million African taurine cattle25 and resulted in zebu being massively reintroduced along the eastern coastline of Africa, largely replacing African taurine bulls.Humped zebu-like cattle were introduced to Egypt from the Levant in small numbers and depicted in tombs and temples 3,400–3,000 B.P.26. However, the African zebu and sanga populations (crosses between African taurine and zebu cattle) mainly carry Y-chromosomes of zebu origin17. The absence of zebu mitochondria in African cattle implies that introgression of the zebu lineage into Africa was mostly male mediated. A number of microsatellite-based studies have also shown that zebu introgression into Africa declines from East to West26. Moreover, a study using Y-chromosome markers, found that the zebu-specific Y-chromosome sub-lineage Y3B27 was absent from the extant zebu population from the Indian subcontinent. Very recently, the X-degenerate region within the male-specific part of the bovine Y-Chromosome was resequenced28 allowing to identify two sub-haplogroups within the B. indicus Y3 chromosome: the Y3a sub-haplogroup identified in Chinese cattle and the Y3b sub-haplogroup mainly carried out by Indian zebu sires. However, African zebu was not included in that analysis28. Here, we report on the genetic analysis of a large number of West African and South Asian zebu Y-chromosomes to shed light on the origin and spread of zebu cattle within south Asia and from south Asia to Africa.Genetic data have demonstrated that African cattle mainly carry the maternal T1 taurine mtDNA lineageB. indicus males belonging to 22 cattle populations from Asia and Africa were analysed  and the six Y-chromosome specific microsatellites 32. Two MCMC runs starting from different random trees were completed. Each run consisted of 20 million replications and four chains. All sample points prior to reaching convergence were discarded as burn-in samples. The remaining samples were used to generate a majority rule consensus tree, where the percentage of samples recovering any particular clade represented the clades posterior probability32; and (c) a MJ network connecting different haplotypes was constructed using the program Network 4.5.233 (available at http://www.fluxus-engineering.com/). To avoid reticulation, a reduced median algorithm34 was used to generate an.rmf file to which the median joining network method33 was applied to this file. Following the recommendations of35, the “frequency >1” option was applied to discard singly occurring Y-types. The same weights were assigned to each polymorphism.IMM bands and microsatellite alleles were combined into haplotypes. Observed haplotypes were analysed as follows: (a) Analysis of correspondence was performed using the “Proc Corresp” of the SAS/STAT package ; (b) applying the Bayesian procedures implemented in the program MrBayesh, and its variance, V(h), were calculated according to Nei36 (formulas 8.5 and 8.13 therein). The standard error of h, SE(h), was calculated by taking the 36. The between-populations genetic identity matrix was computed as the average across loci of the term to 36,where xij and yij are the frequencies of the ith allele at the jth locus within the populations x and y, using the program MolKin37. The information provided by the between-population genetic identity was summarized, from the complementary of this matrix, using a PCA. The PC scores and the canonical dimensions computed for each analysed population were used to construct interpolation maps drawn using the Spatial Analyst Extension of ArcView, available at http://www.esri.com/software/arcview/. The Inverse Distance Weighted (IDW) option with a power of two was selected for the interpolation of the surface. IDW assumes that each input point has a local influence that decreases with distance. The sampling area of each population was used as geographic coordinates and interpolation surfaces were divided into eight equal classes.An unbiased estimate of haplotype diversity, 38. We refer only to the initial capture of males as domestication  and refer to later instances of capture and recruitment of wild bulls to herds as introgression event or gene flow. Model 1 assumed an ancestral Y-chromosome population undergoing a bottleneck associated with a single domestication and subsequent population split at the first introduction of zebu into Africa  into three different populations, two of them being merged at 2,000 B.P.; Model 2 assumed three different ancestral Y-chromosome domesticated populations evolving separately during 250,000 years39, with two of them being merged at 2,000 B.P.; Model 3 assumed two ancestral Y-chromosome domesticated populations one of them splitting into two subpopulations at 3,500 B.P.  and received migrants in an introgression event at 3,000 B.P. from the other population of the ancestral lineage. The scenarios modelled represent simple (Model 1) or complex (Models 2 and 3) domestication processes that originated the present admixed zebu Y-chromosome population. Model 3 represents the more complex, and sustained in time, domestication scenario including a dominant ancestral Y-chromosome population which acts as a genetic source of the others. The merging of two of the ancestral populations at 2,000 B.P. represents a secondary, less intense, recruitment of wild oxen, probably out of the Indus Valley, suggested by mtDNA structure4. Current effective population sizes , IMMs mutation rate (ranging from 2 × 10−5 to 2 × 10−4 mutation/generation) and the size of the initial domesticated populations  were modelled assuming uniform distributed priors. Divergence times were measured in years and transformed to generations for simulations using a generation time of 4.84 years40. For Y-chromosome microsatellites, rapidly mutating markers mutation rate41 (0.008 mutations/generation) was used under a strict stepwise mutation model. The two IMMs, which are considered slow mutation dominant markers27, were simulated as independent markers. Both the IMMs and the six different microsatellite loci typed were simulated using SIMCOAL for 180 individuals. This latter figure coincides with the number of available non-American zebu Y-chromosomes plus those haplotypes that were only observed in American zebu.Three historical scenarios for domestication and the spread of zebu cattle were modelled Fig.  using th42 with 106 simulation replicates generated under each scenario and using four summary statistics, the standard deviation over loci of the number of alleles (Ksd), the mean heterozygosity over loci  of the simulations matching closest the empirical data based on distances between observed and simulated statistics was retained for the estimation of the marginal densities under the General Linear Model (GLM)44 for each evolutionary scenario. These were also used for the assessment of the posterior odds (Bayes factors)45 for each model given the observed data. The ABC model choice was validated using simulated replicates under the three models as pseudo-observed datasets to estimate the power to distinguish between models, the percentage of model misclassification and the posterior probabilities supporting wrong choices43.The posterior probability of each proposed scenario was then calculated using the software ABCtoolboxρ statistic (i.e. the average number of mutations from derived haplotypes) to a haplotype designated as ancestral for the haplogroup46. The average distance to the node of interest (ρ was transformed to absolute time estimates by multiplication (ρ × years per 1 mutation). As a phylogeny-based statistic, ρ offers the advantage of being unbiased by demographic processes. The sampling error of ρ was approximated as 27 and rapid mutating Y-chromosome microsatellites, divergence times were computed assuming the average mutation rate of 0.0008/generation reported in39 for intermediate- rate mutating Y-chromosome markers.Divergence times were estimated via the a and Y3b) within the zebu Y3 Chromosomes by Chen et al.28 made necessary carrying out additional analyses to ascertain their consistency with the variability identified in the current analysis. A 443pb fragment  of the X-degenerate region within the male-specific part of the bovine Y-Chromosome was resequenced for diagnostic purposes. The amplified fragment included three mutations  separating the Chen et al.’s28B. indicus Y-Chromosome sub-haplogroups Y3a and Y3b. A total of 51 sequences were obtained. A complete description of the laboratory methods used is in Supplementary Table The very recent identification of two sub-haplogroups  in zebu cattle. The number of alleles per microsatellite varied from one (BM861) to four , revealing a total of 19 alleles across the six loci here considered (Supplementary Table Sixteen of the bands (seven for UMN2405 and nine for UMN2303) described for the IMMs used in Pérez-Pardal We identified 47 different haplotype combinations resulting from the genotypes of two IMMs and six microsatellites markers. Of these, 29 are unique. The three most frequent haplotypes  were identified in 18 different populations. However, these three haplotypes were neither found in non-Indian Asian samples nor in sires from northeastern India. Haplotype H24 was identified in 23 samples from the six West African populations analyzed . The Y3B included 12 (25%) haplotypes and 24 (10%) samples, while the Y3C included 9 (20%) haplotypes and 38 (15%) samples  on the IMM UMN2303, while family Y3C is defined by the absence of the allele 149 on microsatellite UMN0307 and the predominant presence of band 124 on IMM UMN2303 (Supplementary Table A and Y3B while that between families Y3A and Y3C was slightly lower (0.87). Correspondence analysis separated the haplotypic families Y3B and Y3C on Dimension 1 (X-axis) while the main haplotypic family (Y3A) was differentiated on Dimension 2 (Y-axis). The network obtained using the whole dataset was highly reticulated  network and the correspondence analyses concur in the identification of three Y3 haplotypic families Fig. . The maises Fig.  identifyies Fig. . The Y3AA haplotypes) from those of Southern India and Yemen (where family Y3C haplotypes are present). The second PC contrasts the genetic variability of the Yemen, and southern and northeastern India populations with those samples from West Africa in which haplotypes belonging to family Y3B are frequent , indicating that two of the descendants of these domesticated populations would share recent history but a third population would remain isolated  until the present. This fact leaded us to retain the hypothesis underlying Model 2 as more plausible.A and to the “African” haplotypic family Y3B identified here can be included into the Chen et al.’s28B. indicus Y-Chromosome sub-haplogroup Y3b, identified in zebu sires of Indian origin, while the sequences assigned to the current the haplotypic family Y3C carried mutations g.3631254a > g, g. 3631400t > c and g. 3631401t > g and, therefore, are consistent with the definition of the Chen et al.’s28B. indicus Y-Chromosome sub-haplogroup Y3a, mainly identified in Chinese zebu sires.Resequencing 15. All zebu cattle Y-chromosome haplotypes fall within a distinct Y3 haplogroup. More specifically, zebu cattle Y-chromosomes can be classified into three different Y3 haplotypic families  was not required. In all instances, veterinarians followed standard procedures and relevant national guidelines to ensure appropriate animal care. Semen doses were routinely collected by different companies not directly related to our research project.Supplementary InformationSupplementary Dataset 1


Much of animal-associated microbiome research has been conducted in species for which little is known of their natural ecology and evolution. Microbiome studies that combine population genetic, environment, and geographic data for wild organisms can be very informative, especially in situations where host genetic variation and the environment both influence microbiome variation. The few studies that have related population genetic and microbiome variation in wild populations have been constrained by observation-based kinship data or incomplete genomic information. Here we integrate population genomic and microbiome analyses in wild threespine stickleback fish distributed throughout western Oregon, USA. We found that gut microbiome diversity and composition partitioned more among than within wild host populations and was better explained by host population genetic divergence than by environment and geography. We also identified gut microbial taxa that were most differentially abundant across environments and across genetically divergent populations. Our findings highlight the benefits of studies that investigate host-associated microbiomes in wild organisms.  Microbiome research is now a major area of research in ecology and evolution , which iDespite this previous research, we still do not fully understand how host genetics and environmental factors interact to influence the structure and function of an individual animal’s microbiome ,10. DuriGiven the extensive effects the microbiota can have on the host, it is not surprising that the host has evolved to modulate the microbiota. The innate and adaptive immune systems evolve to monitor and alter the abundances of microbes . Like otThe majority of the research on host-microbiome associations has focused on model organisms in the laboratory. Studying microbiome associations in wild hosts could augment and expand what is learned in lab settings by incorporating natural genetic variation interacting with ecologically relevant environmental variation, often in environments in which the hosts evolved. A historical problem has been a lack of the genetic and genomic tools for such studies on non-model organisms, but this problem is quickly receding because of advances in genomic sequencing technology ) . These tGasterosteus aculeatus) ), na.rm = na.rm/length(x), where x is ASV counts. The “estimateDispersions” function was used to estimate dispersions. We tested gut ASVs for significant differential abundance both between inland and coastal freshwater populations and between estuary and freshwater populations using the DESeq function with the default Benjamin–Hochberg multiple-inference correction and with the fitType parameter set to ‘local’. Only ASV differential abundances that were statistically significant (p ≤ 0.05) were considered in our biological interpretation of these results. In order to determine how gut microbiome compositional variation among threespine stickleback in Oregon was influenced by host population genetic structure and environment, we first looked broadly at what bacterial groups were present in our gut samples. Successively, we quantified AVS abundance differentiation across the major geographic partitions. We did this by converting the phyloseq to a DESeq dataset using the phyloseq_to_deseq2 function in the DESeq2 v1.18.1 R package . The funST in pairwise comparisons between coastal and inland fish populations, was on average two-fold greater (~0.242) than between inland (~0.125) and four-fold greater than that among coastal populations (~0.063)  . We found that gut communities varied in terms of both alpha and beta diversity among individual fish, as well among populations on average . CoastalST , than by environment  and sample period   than by environment  and sample period . In addition, we found that environment  better predicted beta diversity in terms of Weighted UniFrac distance to centroid than FST  and sample period   and weighted UniFrac distance to centroid . The random effects of sample period were completely negligible for all diversity metrics we tested  . In addi = 0.64) . Lastly,e tested . ST: 2R = 0.080, p < 0.001) explained the most variation relative to the other factors , population genetic heterozygosity , environment , and river mile  explained similar portions of variation in gut microbiome in terms of Bray–Curtis dissimilarity among fish gut communities. FST strongly covaried with both geographic distance  and river miles , as well as population heterozygosity . The remaining majority of gut microbiome variation among these six wild fish populations remained unexplained by the factors we accounted for in this study (~71%).Permutational multivariate analysis of variance (PERMANOVA) showed that population genetic divergence  based on relative abundance in the metacommunity . These g2fold > 20) in fish guts in the Proteobacteria (inland) and on the Fermicutes (coast). Nine phyla were enriched in fish guts in both inland and coastal populations. In contrast, Tenericutes were enriched only in inland freshwater fish, and Fusobacteria, Spirochaetes, Nitrospirae, Ignavibacteriae, Lentisphaerae, and Cloacimonetes were enriched only in coastal freshwater fish (Mycoplasma penetrans [Fusobacterium varium and Treponema spps.), but the rest were taxa typically associated with lake and sludge communities.Looking more finely in a subset of 1100 of the total 16,530 bacterial ASVs and 16 of the total 23 phyla, we found a significant difference in abundance between regions of ASVs a. Nearlyter fish a. The Teenetrans ,54,55. E2fold > 20) in estuary fish (one Spirochaetes) and freshwater . Seven phyla were enriched in gut microbiomes in both environments. In contrast, the Spirochaetes were enriched in only estuary fish, and the Fusobacteria, Gemmatimonadetes, and Verrucomicrobia were enriched in only freshwater fish (Fusobacterium varium and Treponema spps.), and the remainder were not well matched with bacteria that had known origins.Comparing differential abundance between environments, we found significant differences in a subset of 404 ASVs comprising 11 phyla b. More tter fish b. The SpWe have come to appreciate the importance of the host-associated microbiome in vertebrate health and disease. This current understanding is due to recent rapid advances in this field, primarily using experimental vertebrate models . However, despite our awareness and efforts, we still lack a complete understanding of the relative roles of environment and host genetic variation in host microbiome assembly. Studies of microbiomes of hosts in the wild can help fill this knowledge gap. Wild hosts can possess incredible genetic variation and are found among diverse environments, and we often know much about their natural evolution and ecology, yet little work in this field has taken advantage of this. In addition, studies that have been done using wild populations have not broadly considered the host genome. Rather, they have focused narrowly on specific genes or small regions of the host genome deemed important by experimental work. This is an important issue to address in order to understand how experimental research should be interpreted and to advance the field.Here, we addressed this problem by collecting both host population genomic and microbiome data from wild populations of threespine stickleback fish that inhabit various environments in Oregon. We hypothesized that if host genetics played an important role in gut microbiome assembly, then, with a robust level of population genetic resolution, we would find a pattern in the gut metacommunity that reflected the genetic relationship among populations of wild hosts. We selected six populations of stickleback situated in Oregon among three watersheds over a range of geographic distances (~12–80 km apart as a crow flies) and across two distinct environments (estuary and freshwater). Doing so allowed us to measure the relative degree to which each factor explained variation in gut microbiome among hosts. Using several hundred single nucleotide polymorphisms randomly spread throughout the genome, we first confirmed a strong genetic differentiation between inland and coastal populations, as previously seen in Oregon ,37. In aOur results provide evidence that genome-wide genetic variation among host populations can influence divergence in the gut microbiome. Our findings agree with findings in other systems that relatively more similar gut microbiomes exist among related as compared to unrelated individuals ,21,23,58t = −1.23, df = 94, p = 0.22). It was also previously suggested that if high allelic heterozygosity in MHCII genes was a proxy for high host genetic heterozygosity, then higher host population heterozygosity may have explained lower beta diversity in Canadian stickleback populations [We found that both alpha and beta diversity varied among wild stickleback fish populations in Oregon. This variation in gut diversity was primarily predicted by population genetic divergence among these populations. In addition, we found that population genetic divergence better explained a partition in gut microbiome composition in Oregon than environment, geography, sample period, and host population genetic heterozygosity. This evidence supports previous findings that inner-host genetics have a more important role in the assembly of the gut-associated microbiome in wild hosts than geography and environment. In previous work conducted on Canadian stickleback populations, gut alpha diversity was negatively correlated with heterozygosity in Major Histocompatibility Complex class II (MHCII) alleles . In congulations . In contulations . These “Even though host population genetic divergence best predicted gut diversity, environment also explained a portion of the overall variation in the gut microbiome. This was corroborated by our finding that environment best predicted gut beta diversity (weighted UniFrac distance). In addition, stickleback populations found in estuaries had on average higher beta diversity and lower alpha diversity than freshwater populations. This greater inter-individual variation in estuary fish could have been due to the exposure of estuarian fish to a more diverse pool of microbes. Stickleback acquire their microbes in part through diet, sediment, and water sources ,36. ThesGiven that population genetic divergence explained a portion of gut metacommunity variation, we wanted to know which microbial taxa contributed to significant differential abundance between inland and coastal freshwater fish. We found that a small subset of the gut metacommunity was significantly differentiated in terms of abundance across the main divergence in host population genetic structure in Oregon. These ASVs represent a group of bacteria that could strongly interact with the host in terms of genome-wide allele variation by being heritable and important in host fitness. This notion seemed plausible considering the history of threespine stickleback. These ASVs, if isolated, could be used to address the question of differential colonization in lab fish raised free of microbes, a benefit of work in a system that exhibits both natural variation and that can be manipulated in the laboratory. 2fold > 20 suggesting a strong enrichment due to environment-related processes. We speculate that a strong association of some taxa in the gut can be specific to the environments in which hosts are found. This specificity could be due to the sorting of the microbial pool in the environment. However, it has been reported that microbes in the environment do not predict what is found in the stickleback gut [It is unclear if host-environment interactions can influence the gut microbiome indirectly via the host. There was a subset of phyla comprised of ASVs that were significantly enriched in both or only one of these environments. A smaller subset of these was enriched logback gut . FurtherWe also observed that estuary and freshwater fish had different gut communities in terms of the relative abundance of major phyla present in the gut, as was the case in another study in Canada . The FirUsing comparative studies to document patterns of microbiome variation in natural populations of organisms that vary in terms of host genetics and environments is very important in providing foundational knowledge of potential mechanisms affecting host-microbe relationships in the wild. However, we acknowledge that the design of this study is limited by the periodicity of the collections and that the observed effect of genetic divergence could be explained by the combination of environment, sampling period, and geographical location. Therefore, as is the case with many comparative studies, these results are preliminary and further validation is needed through manipulative experiments. Such studies should include an expansion of populations collected during the same time frame and studies that bring wild populations into the lab and grown under common conditions to reduce the influence of environment. We studied stickleback gut microbiomes in the wild and found that host population genetic divergence (based on 391 randomly selected genome-wide SNPs) explained gut microbiome partitioning, in terms of both diversity and composition, better than geography, environment, sample period, and population genetic heterozygosity. In addition, geography, environment, sample period, and population genetic heterozygosity equally influenced the gut microbiome. Lastly, we found that relatively small subsets of the gut microbiome in wild stickleback in Oregon uniquely differentiated in terms abundance across different factors.


Decalobanthus boisianus is a native plant of Hainan Island, China, which has caused considerable damage to tropical forest ecosystems in recent decades. Understanding the genetic diversity and structure of this species can facilitate uncovering the molecular mechanism of its invasive ability. Here, we collected 77 individuals of D. boisianus spanning 8 distribution areas with a gradient of human disturbance intensity  to assess patterns of genetic diversity and structure using inter simple sequence repeat (ISSR) markers. We found that a total of 220 loci were scored with 13 primers using ISSR methods, and that 198 loci were polymorphic. The genetic diversity of D. boisianus among these eight forests decreased with increasing human disturbance intensity. Over 70% of the total genetic variation was present within populations, while less than 30% of variation was found among populations. There was a high gene flow (1.27) among them due to a lack of effective geographic barriers. The mean Nei's genetic distance of D. boisianus populations was found to be relatively small , and the average genetic similarity of the eight populations was high . Our findings indicate that the genetic diversity of D. boisianus correlated to human disturbance density, and that D. boisianus populations in Hainan Island have frequent gene exchange. We suggest that reduce deforestation to decrease human disturbance may be a good way to prevent the invasion of D. boisianus. R. Simões & Staples is a liana that distributed in Hainan Island and Guangzhou City. It also has three varieties including Decalobanthus boisianus var. boisiana, Decalobanthus boisianus var. fulvopilosa, and Decalobanthus boisianus var. sumatrana, which distributed in Yunan and Guangxi Province of China, as well as Laos, Vietnam, Malaysia, Kalimantan Island, and Sumatra Island  A. R. Simões & Staples which is also a harmful weeds, Decalobanthus borneensis (Merr.) Simões & Staples, Decalobanthus eberhardtii (Gagnep.) Simões & Staples, and Decalobanthus pacificus (Ooststr.) Simões & Staples, ect. D. boisianus is adapted to a wide range of ecological conditions, due to its ability to undergo both sexual and clonal reproduction , moderate disturbance intensity group , and high disturbance intensity group .The eight forests are located in Wuzhishan National Nature Reserve (WZS), Bawangling National Nature Reserve (BWL), Yinggeling National Nature Reserve (YGL), Diaoluoshan National Nature Reserve (DLS), Jianfengling National Nature Reserve (JFL), Limushan Nature Reserve (LMS), Baishiling Nature Reserve (BSL), and Ganshiling Nature Reserve (GSL) Figure . We took2.2DNA was extracted from our 77 samples using the Plant DNA Isolation Kit . We followed the manufacturer's protocol using 25–30 mg of dried leaf material. DNA quality and quantity were determined visually by comparisons with the DNA marker DL2000 on 1.0% (W/V) agarose gel electrophoresis. Samples were then stored at −20°C prior to PCR amplification.http://www.biotech.ubc.ca/services/naps/primers/Primers.pdf). An initial experiment was performed to determine the suitable primer and reaction conditions. One sample randomly selected from each eight D. boisianus populations to test the preliminary number of polymorphic loci with 100 primers and to optimize the reaction and amplification procedure for PCR. Finally, we obtained optimal reaction and amplification procedures as well as 13 primers that produced a high number of variable and readable loci . Polymerase chain reaction started with an initial activation step at 94°C for 4 min, followed by 45 cycles of denaturation at 94°C for 45 s, annealing at 52–54°C for 50 s  in 20 μl reaction volumes containing 2 μl of DNA (50 ng/μl), 2 μl of primer (10 μM), 6 μl of ddHThe amplified products were separated on a 1.5% (W/V) agarose gel in 1× TAE buffer staining by GoldView  with a setup of 100V, 200 mA and 50 min and then visualized and photographed under ultraviolet light. A 2000 bp DNA ladder  was used to estimate the molecular weights in the Alphalmager—2200 UV Transilluminator .2.3Only bands that were clear and reproducible were used to construct data matrices. The amplified bands were scored as presence (1) or absence (0) of a particular band for each primer. These data were then used to assemble the data matrix of ISSR phenotypes.2.4D. boisianus populations across different human disturbance intensity using ANOVA analysis.We calculated the proportion of polymorphic loci (PPL), Shannon's diversity index (I), Nei's gene index (H), the observed number of alleles (Na), and the effective number of alleles (Ne) to assess levels of genetic diversity among our populations using the POPGENE program v. 1.32  represents the portion of the total genetic diversity found among populations Nei, , and theons Nei, . Populatons Nei, .D. boisianus with a Bayesian model‐based cluster analysis, using the STRUCTURE program version 2.3.4  among populations were estimated by using toolbox in ArcGIS 10.3  according to the coordinate (the longitude and latitude) of each population. A Mantel test was performed to assess isolation‐by‐distance with 1,000 random permutations, to test the significance of the relationship between ɸst and geographic distances. The distribution of whole genetic diversity was estimated using the molecular variance (AMOVA), taking populations and individuals as different levels. The significance of ɸst genetic differentiation was assessed with 1,000 random permutations. The principal coordinate analysis (PCoA) was carried out for the genetic relationships among populations. These analyses above  were carried out using the GenALEx6.5 program .A total of 220 unique loci ranging in size from 150 to 1800 bp were scored from ISSR marker products. From these, 198 (90.00%) were polymorphic Table . A mean 3.2D. boisianus populations collecting from the eight forest sites, the mean values of the proportion of polymorphic loci, the observed number of alleles (Na), the effective number of alleles (Ne), Shannon's diversity index (I), and Nei's gene index (H) were 48.18%, 1.48 ± 0.11, 1.25 ± 0.04, 0.23 ± 0.04, and 0.15 ± 0.03, respectively (Table Among the eight D. boisianus population in WZS), moderate intensity group , and high‐intensity group , respectively  = 18.89, p = 0.003; PPL: F = 18.89, p = 0.003; Na: F = 19.08, p = 0.003; Ne: F = 23.05, p = 0.002; I: F = 21.34, p = 0.002; and H: F = 24.54, p = 0.001).We also found the genetic diversity varies significantly with human disturbance intensity, with the high, medium, and low genetic diversity observing in low‐intensity group (3.3t) and gene diversity within population index (Hs) were 0.21 ± 0.03 and 0.15 ± 0.01, respectively. The genetic differentiation index among the eight populations (Gst) was 0.28. AMOVA analysis showed that a relatively large proportion of genetic variation (75.67%) occurred within the eight D. boisianus populations, whereas only 24.33% of genetic variation was observed among the eight populations , while the genetic distances among the eight D. boisianus populations were relative low (0.07), indicating the close relationships among the eight D. boisianus populations  indicated that there were no significant correlations between Nei's genetic distances and geographic distances.The mean value of genetic similarity among the eight ns Table . The ManD. boisianus populations can be clustered into three groups, including group 1 , group 2 (populations in JFL and GSL) and group 3 (population in YGL). These three groups, moreover, were confirmed using UPGMA dendrogram  but the maximum one was obtained at K = 3 Figure a, suggesm Figure .44.1D. boisianus populations significantly increase with the decrease in human disturbance intensity  = 18.89, p = 0.003; PPL: F = 18.89, p = 0.003; Na: F = 19.08, p = 0.003; Ne: F = 23.05, p = 0.002; I: F = 21.34, p = 0.002; H: F = 24.54, p = 0.001), which proves our hypotheses. Human disturbance may influence the balance between sexual and clonal reproduction of D. boisianus populations  among the eight populations result from the high dispersal ability of D. boisianus to against barriers throughout the expansion of this species   and AMOVA values (24.88%) indicated that there was a large proportion of genetic variation (over 70%) present within the eight ) Nybom, , too. HeD. boisianu populations are categorized to three different groups  with genetic distances of this species, some nearby populations have clustered together, such as WZS with LMS and BSL, JFL with GSL populations. This implied that the horizontal expansion of clones might also lead to gene flow among nearby populations of that species and made their genetic relationships closer.UPGMA dendrogram, Bayesian analysis and PCoA analysis all show that the eight  Figures , and 4,  Figures , suggest5D. boisianus was correlated to the human disturbance intensity, which has been proved to be a major factor governing plant invasion. High dispersal ability and adaptive potential of D. boisianus may be important factors that were attributed to its large distances expansion and cause serious damage to the ecosystems. So far, artificial removal and chemical controls have been implemented to kill and prevent D. boisianus in the study areas, but were seldom effective. We suggest reducing deforestation to decrease human disturbance to forests for one hand. In addition, effective efforts should be taken to remove D. boisianus, and restore the secondary forests.Generally, we found that genetic diversity of Huan Jiang, Wenxing Long, Hui Zhang, Chengneng Mi, Tao Zhou, and Zongzhu Chen conceived and designed the experiments; Huan Jiang and Wenxing Long performed the laboratory work; Hui Zhang and Zongzhu Chen analysed the data; Huan Jiang, Wenxing Long, Hui Zhang, and Zongzhu Chen wrote the paper. Click here for additional data file.


Lutzomyia longipalpis is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis in the Neotropical realm. Its taxonomic status has been widely discussed once it encompasses a complex of species. The knowledge about the genetic structure of insect vector populations helps the elucidation of components and interactions of the disease ecoepidemiology. Thus, the objective of this study was to genotypically analyze populations of the Lu. longipalpis complex from a macrogeographic perspective using Next Generation Sequencing. Polymorphism analysis of three molecular markers was used to access the levels of population genetic structure among nine different populations of sand flies. Illumina Amplicon Sequencing Protocol® was used to identify possible polymorphic sites. The library was sequenced on paired-end Illumina MiSeq platform. Significant macrogeographical population differentiation was observed among Lu. longipalpis populations via PCA and DAPC analyses. Our results revealed that populations of Lu. longipalpis from the nine municipalities were grouped into three clusters. In addition, it was observed that the levels of Lu. longipalpis population structure could be associated with distance isolation. This new sequencing method allowed us to study different molecular markers after a single sequencing run, and to evaluate population and inter-species differences on a macrogeographic scale. Lutzomyia longipalpis species, which was described by Lutz and Neiva . The results suggested that geographic distance significantly contributes to the genetic differentiation observed in the We obtained similar results depending on the approaches used to estimate the clusters, considering that PCA and DAPC revealed three clusters Figs  and 3.Lu. longipalpis in the epidemiological chain of visceral leishmaniasis, the knowledge about the taxonomic status of this species has increased considerably in recent years, producing several studies in Latin American countries [Studies about the genetic structure of natural populations of vector insects have many applications in evolutionary biology and conservation, an essential aspect to understand the ecoepidemiology of the diseases . Due to ountries .Over the past 30 years, more than 100 articles have been published using molecular markers to relate or separate sand flies populations, species, groups or genera . Differehigh-throughput genotyping (Next Generation Sequencing—NGS). This technique allows the analysis of large-scale molecular markers, as well as improving inferences about the genetic variability of populations [The increase of the molecular studies has encouraged the enhancement of DNA sequencing technologies. One of the most relevant approaches within ecological and population genetics was the development of ulations , kinshipulations  and undeulations .Lu. longipalpis populations, using the sequencing with the Illumina MiSeq platform, in three markers, simultaneously.There is much interest in applying NGS for sequencing directed to specific genes and in large numbers of individuals . ConsideIt is noteworthy that our sequences generated by the Illumina MiSeq platform of each individual are the product of more than a thousand direct and reverse sequences per fragment. Thus, possible sequence interpretation errors could be detected and filtered, since thousands of sequences are considered. It was different if we used Sanger’s sequencing when only a single sequence is present. Shokralla et al.  comparedLu. longipalpis subpopulations, according to the classification proposed by Wright [Estimating genetic differentiation among populations (FST) is fundamental in genetic studies to understand population demographic history . Our resy Wright , Hart any Wright .Genetic differentiation results from several evolutionary processes, such as gene flow, natural selection, and isolation by geographic distance. The last one is a vicarious process capable of causing speciation in different populations of the same species. This event occurs because geographic space leads to environmental variation , 55, 56 Lu. longipalpis could be associated with distance isolation. This test showed a significant (p<0.00001) positive correlation (r(AB) = 0.9381417) between the genetic distances (FST) and geographic (Km). This result shows that, although there was little genetic differentiation between the individuals, there is a strong isolation by distance between the nine analyzed populations that covered a distance variation of up to 3,334 km.We used the Mantel test in order to evaluate the hypothesis that the levels of population structure of Lu. longipalpis genetically divergent, consistent with geographic distance [Lu. longipalpis, suggesting that this fact is due to the limited capacity of locomotion of the sandflies and caused by the innumerable geographic and climatic barriers that could limit or even prevent gene flow among populations. This hypothesis may explain the distance isolation found in our results, considering that the specimens were collected at geographic distances higher than the study by Soto et al. [Our results confirm a study of genotyping based on microsatellites, which showed Brazilian populations of distance . Based odistance  also obso et al. . FurtherEvaluating the isolation by geographic distance is fundamental. This phenomenon causes, through the time, a new characteristic in one of the populations like, for example, a new sound of courtship, causing the break of the gene flow . This evLutzomyia longipalpis populations analyzed in this study were submitted to PCA to verify possible clusters by similarity of frequencies of the examined alleles. Jombart, Devillard and Balloux [a priori definition of clusters to study population structures. However, PCA has been widely used for genetic analysis as an alternative to Bayesian clustering algorithms. In addition, PCA . This analysis confirmed the clusters demonstrated by the PCA, validating its results. The use of different approaches to analyzing clusters in population genetics is extremely important, providing less biased data evaluation. An advantage of DAPC in relation to other clustering approaches is the possibility of generating a graphical representation of the relationship between inferred clusters [Due to the possibility of bias introduced by the absence of ing DAPC , Lu. lonclusters , as obseLu. longipalpis present morphological variation and differ in the number of spots present in the abdominal tergites, a characteristic observed by Mangabeira [Brazilian populations of ngabeira , when hengabeira , 39.Lu. longipalpis populations were grouped morphotype 1S of the North and Northeast of the country: Palmas (TO), Belém (PA), Jequié (BA) and Governador Valadares (MG). In the green cluster, Lu. longipalpis populations were grouped of the Northeast morphotype 2S of Recife and Fortaleza. Finally, in the blue cluster, Lu. longipalpis specimens were grouped of the Midwest and South of the country—Campo Grande (MS), Miranda (MS) and São Borja (RS). Lutzomyia longipalpis populations of Miranda (MS) and São Borja (RS) present morphotype 1S, while the population of Campo Grande was composed by specimens morphotypes 1S and 2S.In the DAPC , the popLu. longipalpis populations 1S (one spot) and 2S (two spot) are in recent process of speciation (0.22 to 1.02 million years ago) [Lu. pseudolongipalpis [Lu. longipalpis populations.Molecular studies presuppose that ars ago) , 63. Thigipalpis , which ilovesongs” by Lu. longipalpis males during mating [Lu. longipalpis populations are caused by failures during intercourse between specimens that produce different types of copulatory lovesongs. This acoustic signal may play an important role in species recognition, acting as a reproductive barrier and reducing gene flow [lovesongs are controlled by genes such as period (per) and cacophony (cac). These genic regions are analyzed in the present study, since they play an important role in species recognition.In addition to phenotypic differences, a behavioral aspect studied is the production of “g mating , 66, 67.ene flow , 65, 66.Lu. longipalpis, there are also differences considering the main component of sex pheromones. In Brazil, the populations of Lu. longipalpis produce four different chemotypes [Lutzomyia longipalpis in Brazil presented in the Spiegel et al. [Lu. longipalpis populations of Recife (PE) and Fortaleza (CE) produce cembrene-1, whereas the populations of São Borja (RS), Campo Grande (MS) and Miranda (MS) produce 9-methylgermacrene-B. This fact could justify these clusters found in our results and be the factor responsible for the reproductive isolation of the species.Beyond the phenotypic and behavioral differences found in the Brazilian populations of mbrene-2), 68. Thil et al.  researchLu. longipalpis, we used genetic markers directly involved in the speciation process. Therefore, we chose to amplify and analyze two nuclear regions: the IVS6 region of the cacophony gene and the period gene. The IVS6 region of the cacophony gene encodes the α-1 subunit of a voltage-dependent calcium channel. While the gene period is involved in the circadian rhythm and controls the species-specific differences in locomotor and mating activities [Studying complex of species, such as tivities , 19, 21.Lu. longipalpis complex using the per gene are consistent with those obtained in studies of pheromones and copulatory lovesongs [per gene may result in reproductive isolation.The results obtained in studies of ovesongs . Bauzer ovesongs  identificac gene can be used as an excellent molecular marker in population genetics studies, because this genomic region presents an intron with high variability and divergence between species. Bottechia et al. [per gene, suggesting that the IVS6 region may be more affected by introgression.Lins et al.  demonstra et al.  observedLu. longipalpis complex. The mtDNA presents maternal origin, evolves rapidly and does not recombine, being an adequate target to trace genealogy and evolutionary history. The 12S region of mtDNA was used by Beati et al. [Lutzomyia of Colombia and Peru, and identified different species of the genus in those countries.Another target of studies has been the mtDNa, as the 12S region, to evaluate the differentiation of populations of the i et al.  when anaLu. longipalpis belong to a complex of species. This premise can be carry out after analyzing morphological and sexual pheromone studies [lovesongs [In our conception and according to our results, the claim of Souza, Brazil and Araki  is corre studies , 68, 69,ovesongs , 66, 70 ovesongs , 45, 71.Lutzomyia longipalpis genetic structure showed similar patterns according to the approach used, since both PCA and DAPC identified three populations. Thus, the use of different approaches to analyze clusters in population genetics was useful to provide a less biased data evaluation.Lu. longipalpis complex genetic structures can provide details on population differentiation and contribute to understand the processes of divergence and speciation, mechanisms responsible for the heterogeneity of vector capacity and competence, as well as vector susceptibility to infectious agents or insecticides. Thus, the evaluation of the population genetics of this vector can help to plan control measures appropriate to the real conditions of each transmission area of this important endemic in public health.Studies about the