The objective of this study was to assess the genetic diversity andphylogenetic relationship of nine sheep populations, including two famoushigh prolific populations and seven popular mutton populations raised inChina. Overall, these sheep populations in this study exhibited a richgenetic diversity. Both the expected heterozygosity and Nei's unbiased genediversity ranged from 0.64 to 0.75, with the lowest value found in Dorset sheep (DST) andthe highest in Hu sheep (HUS) and Ba Han sheep (BAS). The polymorphic information content (PIC) varied between 0.59 in DST and 0.71 in HUS and BAS. Specifically, forindividual breeds, the small-tail Han sheep (STH) and the four introduced populations did notdisplay the expected diversity; therefore more attention should be paid tothe maintenance of diversity during management of these populations. Theresults of un-weighted pair-group method (UPGMA) phylogenetic tree and structure analysis indicated that thenine investigated populations can be divided into two groups. Suffolk (SUF) and DSTwere clustered in one group, and the other group can be further divided intothree clusters: German Mutton Merino (GMM)–BAS–Bamei Mutton sheep (BAM), HUS–STH and Du Han (DOS)–Dorper (DOP). This clustering result isconsistent with sheep breeding history. TreeMix analysis also hinted at thepossible gene flow from GMM to SUF. Together, an in-depth view of geneticdiversity and genetic relationship will have important implications forbreed-specific management. It provides meat and milk with high-qualityprotein and useful accessory products (wool and skin) for human. Therefore,sheep became an important livestock species and promoted the spread of humanfarming civilization . China has a long history ofsheep domestication  and famous sheep breeds withhigh reproductive performance. For example, small-tail Han sheep (STH) andHu sheep (HUS) had attracted much attention for their excellentcharacteristics of high litter size and year-round estrus . In addition to reproductivetraits, meat production traits are also the focus of attention for sheepfarmers. Since the growth rate and performance of meat production in Chinesesheep populations are relatively low compared with famous mutton sheepbreeds worldwide , Suffolk (SUF), Dorset (DST) and GermanMutton Merino (GMM)), these perfect mutton breeds were introduced to Chinain the past two decades and are now common in many farms of China. However,with the introduction of foreign breeds to improve production performance ofindigenous purebred sheep, the number of indigenous purebred sheep with highfertility is gradually decreasing  in China. Therefore,the evaluation of current genetic diversity situation in these sheeppopulations is very essential for breed protection. In addition, this studyalso involved seven mutton sheep populations raised in China including fourintroduced mutton populations mentioned above and three mutton populationsin Inner Mongolia of China, which is the main producing area of mutton inChina. The genetic diversity assessment for the seven mutton populationswill be helpful for the formulation of plans of further management andutilization for these populations.Microsatellite markers belong to a class of neutral markers, which have richpolymorphism in population and are widely distributed in the genome;therefore they are suitable for genetic diversity analysis. Previous studiesindicated that microsatellite markers had been well used to assess geneticdiversity and population structure of sheep breeds  and goat breeds . Therefore, in this study, apanel of 26 microsatellite markers was employed to evaluate the geneticdiversity, population structure and phylogenetic relationship of the ninesheep populations. The results will provide important information for theimplementation of breed-specific management, propagation or conservationprograms.22.1All experiments involving animals were authorized by the Animal EthicsCommittee of the Institute of Animal Science, Chinese Academy ofAgricultural Sciences (no. IAS2019-49).2.2Nine sheep populations – small-tail Han (STH), Hu (HUS), Suffolk (SUF),Dorset (DST), Dorper (DOP), German Mutton Merino (GMM), Bamei Mutton (BAM),Ba Han (BAS) and Du Han (DOS) – were analyzed in this study (see Table 1 for specific sample information). A total of 5 mL jugular vein blood was collectedfrom 60 individuals (30 ewes and 30 rams) in each population.Then genomic DNA was extracted according to protocol described by Russell. The DNA was dissolved in TE buffer (10 mmol/LTris-HCL (pH 8.0), 1 mmol/L EDTA (pH 8.0)) and stored at 2.3Taq polymerase and 1 All sheep were genotyped using 26 microsatellite markers distributed indifferent chromosomes. Information of 26 microsatellite markers issummarized in Table 2. The primers for the first 24 microsatellite markerswere recommended by the joint ISAG–FAO Standards Committee for analysis of sheepgenetic diversity . The primers for the other twoloci are selected from the report of Davies et al. (1995) andDi et al. (2012). Forward primers were 52.4The genetic diversity parameters of nine sheep populations, including meannumber of effective alleles  to25 (MAF214) (Table 2). The highest effective number of alleles  was built using TreeMix. The residuals from the trees wereinspected to estimate how well the model fit the data .Residuals above or below zero indicate that populations are more or lessclose to each other than they are presented in the tree. Based on this, itis implied that the actual genetic relationships between SUF and GMM andbetween DST and STH were closer than the evolutionary tree shows when nomigration events were allowed . The model with one migrationevent  alluded an obvious gene flow from GMM to SUF. Combiningthe maximum likelihood tree  and residual plot , itwas suggested that DST was more closely related to GMM than presented in thetree. However, when two or more migration events were assumed, thephylogenetic relationship shown in maximum likelihood tree was inconsistentwith true breeding history of these sheep populations; therefore they arenot shown in Fig. 4.4The results of genetic variation analysis give us some hints. Firstly,overall, the nine sheep populations in this study have high geneticdiversity and their mean The results of the UPGMA phylogenetic tree and structure analysis areconsistent with breeding history of these sheep breeds. First of all, SUFand DST were clustered in one group. Since both SUF and DST horn wereoriginally bred in the UK and both of their sire lines are Southdown rams, geneticrelationships between the two breeds were relatively closer and clusteredtogether. Then the other seven sheep populations were clustered into anothergroup. Of them, the three populations  were classed in onesubgroup. The sire line in the initial breeding process of BAM and BAS isGMM and BAM rams, respectively. Therefore, a close genetic relationshipexisted among the three populations. Then, both HUS and STH belong to shortfat-tailed Mongolian sheep . A previous studysuggested that, from the Mongolian Plateau, short fat-tailed sheep migratedsoutheast to the east of China , which is a currentdistribution area of HUS and STH. Therefore, the two breeds were clusteredtogether.The gene flow and genetic exchange in the nine sheep populations wereelucidated by TreeMix analysis. The results implied the possible gene flowfrom GMM to SUF. First of all, the gene flow is probably related to thehistory of human migration in Europe. From the 7th century BCE to the3rd century BCE, Celtic tribes living in Germany had a great westwardmigration, and some of them entered Britain . Then, fromthe fifth century CE, some of the Saxons and Angles of Germanictribes again poured into Great Britain from Germany. Sheep has beenconsidered as an important and easy-to-carry domestic animal for humanseveral thousand years ago; therefore the gene flow between GMM and SUFmight appear following human migrations, commercial trade, and extensivetransport. Secondly, the gene flow was associated with the widespread use ofMerino sires across Europe that commenced after the Middle Ages. Therefore,extensive haplotype sharing between Merino (including GMM) and otherEuropean breeds was observed . Thirdly, previousanalysis on the genetic relationships among world's sheep breeds suggests amajor sheep migration route from South-West Asia to Britain and the Nordicregions, whose end potentially includes the route from Germany to theBritain . Meanwhile, the genome-wide analysis of world'ssheep breeds reveals frequent genetic exchange occurred during thedevelopment of modern sheep breeds . In summary, theseresults can help us further understand the migration history and geneticrelationship among populations for this important livestock species.5As a whole, these sheep populations in this study exhibited rich geneticdiversities. However, specifically for individual breeds, STH and the fourintroduced populations did not display the expected diversity. The resultsof the UPGMA phylogenetic tree and the structure analysis are consistentwith sheep breeding history. TreeMix analysis hinted at a possible gene flowfrom GMM to SUF. Together, these results will have important implicationsfor sheep breed-specific management.


Tabanus bromius (Diptera: Tabanidae) is one of the most notable Tabanidae species of veterinary and medical importance distributed throughout the Palearctic region. In this study, we investigate the genetic diversity and the phylogeographic structure of T. bromius sampled from Turkey, Croatia, and Iran.COI) and 1339 bp of the nuclear DNA internal transcribed spacer (ITS) were sequenced from 247 individuals representing 15 populations.For this purpose, a 686-base-pair (bp) fragment of mitochondrial DNA cytochrome oxidase I gene  compared to a low nucleotide diversity (π = 0.020605 for COI gene and π = 0.013667 for the ITS region), present for a high number of singleton and private haplotypes/alleles imply population expansion in the past. The results of phylogenetic analysis led to the uncovering of geographically significant groupings of lineages with regard to the entrance of the species into Anatolia and the location of major geographic barriers. According to current data, the species appears to have entered Turkey from Caucasia and Iran. A molecular clock applied to the COI data suggests that T. bromius diverged from the outgroup species nearly 8.83 million years ago, around the end of the Miocene era.The sequences generated 169 T. bromius populations have undergone a series of expansions and retreats in the past. Our current findings suggest that T. bromius split from outgroups around the Late Miocene. Subsequent diversification events during the climatic and environmental fluctuation times of the Late Pliocene and Early Pleistocene periods also significantly influenced the species, resulting in the formation of some major genetic lineages. The phylogenetic analyses indicate that T. bromius most likely entered Turkey from the Caucasus region and Iran.The results of this study indicate remarkable genetic diversity across the studied range of the species. High haplotype/allele versus low nucleotide diversity and demographic analyses implied that the The online version contains supplementary material available at 10.1186/s13071-021-04970-5. Tabanidae comprises almost 4500 species, showing widespread distribution throughout the world, with about 660 species in the Palearctic region alone , 2. HorsTabanus bromius is an important common pest species in the Palearctic region, including Turkey. The species was first recorded from Turkey in 1957 by Moucha and Chvála [T. bromius from Turkey.Investigating the geographic distribution of widespread pest and vector species is crucial for determining infectious diseases, vector control, and pest management . To dated Chvála , 11. Desd Chvála , there hLocated in the Western Palearctic region, Turkey is one of the most significant places in the world due to its high biodiversity and number of endemic species . The higT. bromius to gain greater insight into this species. Therefore, in the present study, we use cytochrome oxidase I (COI) of the mitochondrial genome and the internal transcribed spacer (ITS) region of the nuclear genome as the combination to (i) reveal the genetic diversity of T. bromius; (ii) determine the distribution of genetic variation across the sampled area; (iii) reconstruct phylogenetic trees to reveal evolutionary relationships of haplotypes/alleles; (iv) estimate divergence of lineages; and (v) reveal possible factors that generate the contemporary phylogeographic pattern in T. bromius. Our results contribute to both the fauna of Tabanidae in Turkey and the vector epidemiology.In recent years, there has been an increase in research involving Turkish fauna to explore the phylogenetic and phylogeographic structure of various animal groups –25. AlthTabanus bromius females were collected from the 13 locations  covering most of the distribution range of the species in Turkey. Fresh specimens of T. bromius were caught using a malaise trap or collected manually while sucking blood from cattle. We also used museum materials registered in the inventory of the Zoology Museum of Eskişehir Technical University, Faculty of Science Department of Biology. Specimens from Iran and Croatia were also included in this study  representing 15 populations  were used to isolate the total DNA. All sampling locations, their abbreviations, and the sample size of the studied populations are given in Table COI gene was amplified using LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) primer pair [COI gene were set as follows: 5 min at 95 °C for initial denaturation; 35 cycles of 35 s at 94 °C for denaturation; 45 s at 42 °C for annealing; 1 min at 72 °C for elongation, and 7 min at 72 °C for final extension. For amplification of the ITS (ITS1-5.8S rRNA-ITS2) region of nuclear DNA, CS249 5′-TCG TAA CAA GGT TTC CG-3′ and FL 5′-GCT GCA CTA TCA AGC AAC-3′ [2 (25 mM), 2.5 µl of each primer (2.5 mM), 2.5 µl bovine serum albumin , 0.5 µl dNTPs (10 mM), 0.2 µl of Taq polymerase (5 U/µl) , and 1 µl of template DNA. All of the PCR products were purified using a GeneJET gel extraction kit  according to the manufacturer’s instructions. The amplicons (COI-ITS) were sent to a company  for sequencing in both directions to reduce the possibility of base-calling errors.A 686 base pair (bp) of the mitochondrial mer pair . AmplifiC AAC-3′ , CAS18sFC AAC-3′  primers COI sequences were genuine mitogenomes in their origin, internal stop codons and nonsense mutations were carefully checked after translating the sequences into amino acids using DnaSP 5.10.1 software [COI haplotypes and MK936073-MK936162 for the ITS1-5.8S-ITS2 alleles). Despite all our efforts, we were not able to amplify the COI gene of 14 of the Turkish museum specimens, and we, therefore, included only 233 COI sequences and 247 ITS sequences in our data analysis. Sequences from five other tabanid species were used as outgroups: Tabanus atratus (GenBank: KM243515), T. rufofrater (GenBank: DQ631993), and T. bifarius [COI haplotypes, T. bifarius-1 and T. bifarius-2 [Chromatograms sent by the company were first visually checked, and subsequently transferred to Geneious 11.2.1 software  for editsoftware . All hapbifarius  for the farius-2  for the π) and haplotype (h) diversity [FST, and their significance was tested by 10,000 permutations. The hierarchical distribution of genetic diversity was calculated as an analysis of molecular variance (AMOVA) with 20,000 permutations, with statistical significance of P ≤ 0.01, using the Arlequin 3.5.2.2 program [T. bromius population. Population demographic analyses and any deviations from neutrality were characterized using mismatch distribution analysis as implemented in DnaSP 5.10.1 [Hri) [D [FS [P ≤ 0.05.Molecular diversity indices, including the number of polymorphic sites (S), nucleotide  , Tajima’[Hri) [D , and Fu’) [D [FS . StatistT. bromius haplotypes/alleles using PAUP* 4.0b10 [COI gene and the nuclear ITS1-ITS2 region, respectively. The divergence time of the COI haplotypes and their confidence intervals were generated using a Bayesian Markov chain Monte Carlo (MCMC) method by applying the GTR + I + G model following the uncorrelated relaxed lognormal clock, as implemented in the BEAST version 1.5.2 [COI data set was calibrated by 2.3% sequence divergence per lineage per million years mutation rate [Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) were used to infer phylogenetic relationships among * 4.0b10  and MrBa* 4.0b10 , respect* 4.0b10 . The JMo* 4.0b10 , 60 was on 1.5.2 . The COIion rate . Both thion rate . The BEAion rate  was usedwww.inkscape.org) to visualize the networks.Since phylogenetic trees may not always resolve evolutionary relationships among sequences due to reticulations , we usedCOI gene of 233 T. bromius specimens from Turkey, Iran, and Croatia generated 169 unique sequences. A total of 220 T. bromius samples from the Turkish localities yielded 157 haplotypes, 10 specimens from Croatia generated nine haplotypes, and three samples from Iran produced three distinct sequences. The haplotypes and their frequencies are shown in Additional file k1 = 5.1 (purines) and k2 = 6.0 (pyrimidines). When the COI sequences were translated into 228 amino acids, only 12 amino acids showed polymorphism. Pairwise differences between the haplotypes ranged between 1 bp (0.1%) and 39 bp (5.9%). Haplotype (h) diversity in T. bromius was h = 0.9909 ± 0.1091, and the nucleotide diversity was π = 0.020605 ± 0.012850. Among all of the COI haplotypes, seven haplotypes were shared between populations, and there were 139 singleton and 23 private haplotypes. In the Turkish populations, there were 128 singleton and 22 private haplotypes. However, in the Croatian population, there were eight singleton haplotypes, and there was only one private haplotype. Of the Iranian specimens, all three produced three unique haplotypes. No haplotypes were shared among Turkey, Croatia, and Iran.All of the sequences of the T. bromius, including the ITS1 + 5.8S rRNA + ITS2 region, was 1339 bp in length. Since the 5.8S region (123 bp) showed no polymorphism, we eliminated it from our data set; therefore, only the ITS1 and ITS2 regions were combined and used to determine T. bromius alleles. Therefore, the concatenated sequence of the ITS1-ITS2 region was 1216 nucleotides in size without any length differences among 247 samples, which were collapsed into 90 unique alleles  showed polymorphism, of which 390 sites (32.0%) were parsimony informative. The average base composition was 43.0% (A), 35.0% (T), 12.4% (G) and 9.4% (C), and the transition to transversion ratio was R = 1.38. The pairwise difference between alleles ranged from 1 bp (0.1%) to 326 bp (3.3%). The average allele diversity was Ad = 0.8193 ± 0.1417 and the nucleotide diversity was π = 0.013667 ± 0.007385. The numbers of singleton and private alleles were high, where 63 alleles were singleton, 23 were private alleles, and only four alleles were shared among the localities. The number of singleton alleles was 59, while the number of private alleles was 21 in the Turkish populations. In the Croatian population, four alleles were determined as singleton alleles, with one private and one shared allele being found. All of the Iranian specimens produced one private and one shared allele. There was no singleton allele in the Iranian population.The amplified size of the nuclear genome of COI haplotypes produced significantly negative Fu’s FS values , implying population expansion. However, Tajima’s D was not significant . Harpending’s raggedness index Hri = 0.0011 and SSD = 0.0038 were low. Individually analyzed populations showed that a number of the Turkish populations, such as Çanakkale , Giresun , Kayseri , Muğla  and Sinop  expanded in the past, while all other remaining populations produced no significant values , implying deviations from neutrality, while Fu’s FS  value was non-significant. Hri (0.0045) and SSD (0.0040) values calculated for all of the alleles were low. When each population was analyzed separately, Fu’s FS values were non-significant for all of the populations, except for Antalya. The Antalya population also suggested population growth by a significantly positive value . The Çanakkale , Elazığ , Eskişehir , Hatay , and Muğla  populations generated significantly negative values  comprised an Iranian haplotype (H113) and Turkish haplotypes from the eastern part of Turkey . The second main clade (Clade B) splits into two subclades (B1 + B2); the subclade B1 with a northern basal haplotype (H21) sampled from the Artvin population, and most of the remaining haplotypes were from the Giresun and Artvin populations. Moreover, the subclade BI is composed of haplotypes representing eastern, central, northern, and western Turkey. The subclade B2 has a basal haplotype (H111) sampled from the Iranian population, with the remaining haplotypes from Croatia, and it is mainly dominated by the western and central Turkish haplotypes.All three analyses conducted for both data sets produced similar tree topologies with different bootstrap/posterior probability values. Therefore, only Bayesian consensus trees were provided here for both the es Figs. , 5. The T. bromius diverged from the outgroup haplotypes around 8.83 million years ago (MYA). The separation of the most basal haplotype (H44) from the main clade was around 6.00 MYA (Clade A). A small clade formed by Sinop haplotypes  located in northern Turkey diverged from the main clade nearly 4.30 MYA. The Iranian and eastern Turkish haplotypes  appear to have diverged from each other around 3.10 MYA. The separation of the B clade into the subclades B1 + B2 was about 2.73 MYA. The subclade B1 has a northern basal haplotype (H21) from the Artvin population. The remaining haplotypes are from certain Black Sea localities, in addition to the central, northern, and western Turkish populations. One of the Iranian haplotypes (H113), along with the Hakkari haplotypes (from eastern Turkey) formed a small clade. This haplogroup diverged from other eastern (Elazığ and Bitlis) haplotypes around 1.86 MYA. The remaining haplotypes from Iran separated from the Turkish and Croatian haplotypes around 2.44 MYA. In subclade B2, the Iranian  and Turkish haplotypes  split from other Turkish haplotypes about 1.75 MYA. All of the Croatian haplotypes were grouped with a Turkish haplotype (H168) from the Thracian part of Turkey (the European part of Turkey), which was placed at the basal part of subclade B2. The subclade separation around 1.82 MYA implies early Pleistocene diversification. Further splitting events continued throughout the Pleistocene period generating intermediate to shallow splits creating small T. bromius haplogroups.Application of a molecular clock showed that the ingroup haplotypes of COI gene , being the most frequent and most common allele shared among the Artvin, Çanakkale, Eskişehir, Kayseri, and Sinop populations of T. bromius, produces a star-shaped structure. A22 is also a shared allele among Turkey , Croatia, and Iran. It is remarkable that the A43 allele (n = 1 from Eskişehir) is connected to eight alleles, including the southern, northern, eastern, and western Turkish populations, and Croatia.A minimum spanning network of the ITS region is given in Fig. FST values show significant genetic differentiation for the COI data set . Iran also produces the next highest statistically significant FST value with another northern Turkish population (from Artvin) . In addition, the Iranian population is genetically different from other Turkish populations . On the other hand, Iran-Hakkari and Iran-Bitlis populations, which are geographically relatively close to each other, produce low FST values . For the Croatian population, the highest FST is observed in comparisons between Croatia and Artvin , and Giresun , respectively. Pairwise comparisons also show that the highest differentiation between the Turkish populations is between Çanakkale and Artvin , and between Çanakkale and Giresun , respectively.The pairwise FST analysis , Iran , Çanakkale , Antalya , Croatia  and Kayseri . For the Croatian population, the highest genetic differentiation was observed between Croatia and Çanakkale , and Bitlis , respectively. For the Iranian population, the highest FST value was between Iran and Çanakkale . The lowest genetic differentiation  was between the Hakkari and Iranian populations, which are geographically in proximity compared to other populations.Genetic differentiation between populations, using the ITS data sets, was also employed through pairwise P ≤ 0.01), followed by “among populations“  and “among groups“  for the COI data set when all populations are grouped under three clusters . The genetic differentiation between the groups is FST = 0.54, P ≤ 0.001. On the other hand, the ITS data set shows that 65.2% of the genetic variation is within populations and 34.7% is among populations when each locality is considered as a separate group. The genetic differentiation between the groups is FST = 0.34, P ≤ 0.001  is distributed among the Black Sea region, western, and central Anatolia. The presence of the A15 allele only in the western part of a major mountain range known as the Anatolian Diagonal dividing Anatolia into East and West [T. bromius might have also been affected by this barrier. Moreover, the absence of shared haplotypes/alleles between the eastern and western sides of the mountain line supports the importance of the Anatolian Diagonal on T. bromius. In fact, it has been shown that the Anatolian Diagonal plays a key role in the distribution of species and genetic lineages, not only in plants [T. bromius in Turkey is further supported by the pairwise FST analysis predicting the levels of genetic differentiation of populations and reveals significant differences from each other in many populations. Iran is one of the entrance doors into Turkey of T. bromius and the population representing this region is quite different from the Black Sea populations (Giresun and Artvin), while they differ slightly from the eastern populations (Hakkari and Bitlis). Another entrance gate, the Black Sea region (Artvin), has an extremely high differentiation rate with its population of Çanakkale and Croatia (representing the European region) proposing the presence of geographic separation of lineages after entering into Turkey [The frequency and geographic distribution of and West  suggestsn plants , 75, butn plants , 76–78. o Turkey , 68, 71.T. bromius, the mismatch analysis of the COI data, where all of the populations included in the analysis generated a bimodal profile with Hri = 0.0011 and SSD = 0.0038, but were not statistically significant, indicates population expansion [FS (−23.7386) is significantly negative, indicating an excess of rare haplotypes over what would be expected under neutrality in an expanding population is also implied by the mismatch distribution. Negative and non-significant Tajima’s D imply that populations of T. bromius did not deviate from neutrality. In our results, although Tajima’s D and Fu’s FS value seem to conflict, the reason is different statistical power, because Fu’s FS is more powerful and more sensitive than Tajima’s D to population size changes [Hri = 0.0045 and SSD = 0.0040, but it was non-significant, indicating population expansion. For the ITS1-ITS2 region, it can be seen that the species has a stable population distribution. This is also supported by the negative significant Tajima’s D = −1.9563 value implying population expansion. Overall, the mismatch distribution analysis and other population demographic analyses, in addition to the combination of high haplotype and low nucleotide diversity of T. bromius populations for both the COI gene and the combined ITS1-ITS2 regions, suggest rapid demographic expansion from a small population in the past [The effects of historical factors can be traced by employing several population demographic analyses . For T. xpansion . Fu’s FS changes . On the the past .T. bromius species in the Palearctic region, and is not focused on any assessment of phylogeography. Our study species, T. bromius, shows a cosmopolitan distribution in the Palearctic region. The species has been recorded from all European countries [T. bromius and other Tabanus species [Horseflies are a cosmopolitan group spread across all continents and are reported to be of Pangea origin. It is thought that they emerged in the late Triassic period just before the drifting of the Laurasia and Gondwana continents . Based oountries   is around 6.00 MYA, coinciding with the end of the Miocene period. In the ingroup, an eastern haplotype from Elazığ (H54) split from its sister clade almost 4.30 MYA around the Early Pliocene period. Similarly, the estimated divergence time of the two main clades (Clade A and Clade B) was approximately 3.10 MYA, which indicates the Late Pliocene period, and ongoing diversification events have continued from the beginning of the Pleistocene period until the present day. This coincides with the effects of glacial and interglacial changes in the Quaternary (in Pleistocene) [T. bromius species.Our current findings indicate that e period . It seemstocene) , 94, 95.stocene) . Such distocene) . These mstocene) . It appeT. bromius suggest that the species most likely entered Anatolia through both the Caucasus region and Iran. The lineage that entered through Iran has shaped the eastern populations  up to the Anatolian Diagonal, and the lineage that entered through the Caucasus spread westward through the Black Sea region and crossed the Anatolian Diagonal. Thereafter, Central Anatolia , south of Turkey , and Western Anatolian populations  were relatively separated in the western part of Anatolia. Furthermore, the placement of the haplogroup, consisting of the Thracian and Croatian populations in the same clade, suggests that the species provided a genetic source to the Croatian population. This suggests that the Central European T. bromius populations may have originated from Thracian populations or that a portion of the T. bromius European populations originated from the lineage from Turkey. The Black Sea region populations (Artvin and Giresun) are in subclade B1; it may be correct to assume that the collected samples are related to the distribution in a narrow area north of the North Anatolian mountain range. This is also supported by the co-grouping of Black Sea populations in one of the three-star phylogenies in the haplotype network. The Sinop population haplotypes present an interesting pattern due to phylogenetic trees, suggesting that the Sinop population is interacting with other populations. Similar results have been observed for T. bifarius [T. bromius species in countries such as Russia and Ukraine, it may be assumed that a lineage in the north of the Black Sea (from the Caucasian), which was a freshwater lake at that time, reached Europe. Moreover, these results agree with our results that the Turkish T. bromius populations originated in the Caucasus region (Artvin) and Iran.Our phylogenetic analyses of bifarius . AntalyaT. bromius populations show quite remarkable genetic diversity. Demographic analyses and high haplotype/allele versus low nucleotide diversity imply that the T. bromius populations might have undergone a series of expansions in the past. The T. bromius haplotypes split from outgroup species around the Miocene period, and current results clearly indicate that the structuring of T. bromius genetic lineages was significantly influenced by climatic and environmental fluctuations during the Late Pliocene and Early Pleistocene periods. The phylogenetic analysis findings show that the species appears to have entered Turkey from Caucasia and Iran, and that the presence of geographic barriers, such as the Anatolian Diagonal, separates geographically important lineage groupings. Larger sampling across the western Palearctic region and a more detailed study are necessary to draw a general conclusion regarding the phylogeographic structure of T. bromius.In conclusion, the Additional file 1: Table S1. PCR cycles for the ITS (ITS1-5.8S rRNA-ITS2) region.Additional file 2: Table S2. COI haplotypes and their frequencies in populations.Additional file 3: Table S3. ITS1-ITS2 alleles and their frequencies in populations.Additional file 4: Figure S1. Mismatch distribution profile of each population obtained using the COI haplotypes. The observed distribution is represented by a red line (Obs), and the expected by a green line (Exp).Additional file 5: Figure S2. Mismatch distribution profile of each population obtained using the combined ITS1-ITS2 alleles. The observed distribution is represented by a red line (Obs), and the expected by a green line (Exp).Additional file 6: Table S4. COI gene FST analysis results showing pairwise genetic variations between populations. *: P ≤ 0.01, -: not significant.Additional file 7: Table S5. ITS1-ITS2 region FST analysis results showing pairwise genetic variations between populations. *: P ≤ 0.01, -: not significant.


At the population level, African introgression decreased the prevalence of specific anosmia/hyposmia to this odorous compound. Taken together, these findings validate the conjecture that in addition to cultural exchanges, genetic transfer may also influence the sensory perception of the population in contact.While recent advances in genetics make it possible to follow the genetic exchanges between populations and their phenotypic consequences, the impact of the genetic exchanges on the sensory perception of populations has yet to be explored. From this perspective, the present study investigated the consequences of African gene flow on odor perception in a Malagasy population with a predominantly East Asian genetic background. To this end, we combined psychophysical tests with genotype data of 235 individuals who were asked to smell the odorant molecule beta-ionone (βI). Results showed that in this population the ancestry of the OR5A1 gene significantly influences the ability to detect βI. At the individual level, African ancestry significantly protects against specific anosmia/hyposmia due to the higher frequency of the functional gene (OR ratios = 14, CI: 1.8–110,  Olfactory perception varies considerably from one individual to another, to such an extent that the same odorous stimulus can trigger various hedonic reactions, ranging from disgust to pleasure ,2. This Education, age, sex, context, experience, as well as the physical and cultural environment play an important role in the development of inter-individual diversity of perception ,7,8,9,10Therefore, genetic differences between populations could also induce differences in odor perception at the population level. Notably, the functional form of the OR5A1 gene is the minor allele in Asia, whereas it is the most common allele in Africa . TheoretHowever, to test whether gene flow can influence population odor perception, two important prerequisites must be demonstrated. (i) The first is to show that variants such as rs6591536 have a similar impact on individual βI-perception across different human populations and especially in admixed populations with significant biological and cultural differences from the populations where the genotype/phenotype relationship has been identified. The association between rs6591536 and βI-anosmia/hyposmia has only been published by Jaeger et al. 2013), and it is well known that only a limited number of genetic associations can be generalized and replicated in other human populations , and it . Indeed,This paper proposes to assess these two prerequisites in a mixed population living in the central highlands of the island of Madagascar that shows a primary ancestry of East Asian origin (>65%). Archaeological, ethnographic, and genetic data converge on the same observation that the colonization of the central highlands of Madagascar was a recent event by a population primarily of Indonesian origin but with great cultural and genetic enrichment from African populations ,9. SpeciUniversity students originating from Antananarivo participated in the study. Inclusion criteria were: (i) at least three of their four grandparents originating from the Antananarivo area; (ii) age between 18 and 33 years old; (iii) smoking fewer than five cigarettes a day; (iv) no subjects to allergies and nasal polyposis. Participants were also asked to attend the testing sessions without wearing any perfume, aftershave, face cream, or deodorant, and to fast for at least one hour prior to the test. On the basis of kinship coefficients estimated by King software , one cas® . The language used in the written documents  was French, but participants were allowed to answer in French or Malagasy.All experimental procedures were performed with 15 mL standardized glass vials , containing a scentless polypropylene fabric  to optimize evaporation and air/oil partitioning, embedded either with a solution of mineral oil mixed with a specific concentration of βI, or only with mineral oil. All tests were performed in a regular classroom at the University of Antananarivo. Food grade βI (CAS: 14901-07-6) was obtained from Sigma-Aldrich FranceThe study was conducted in two steps: perceptual evaluations and detection thresholds (step 1) and genotyping (step 2).Step 1: Perceptual evaluations and detection thresholdn = 235) to sniff a vial containing a 20,000 ppm solution of βI for three seconds. Then, they were asked to rate the odor using the following 7-point scales: Hedonic ; Intensity (1: absent (no odor), 2: very low, 3: low, 4: medium strong, 5: strong, 6: very strong, 7: extremely strong); Familiarity ; Edibility . Finally, participants were asked to freely identify and/or describe the odor.The odor perception test was performed by asking all participants (−7 mol/L to 2.05 × 10−2 mol/L (0.02 ppm to 2000 ppm). The participant’s task was to sniff each of the four vials in a row for three seconds with both nostrils, and to state which vial differed from the other three vials . They evaluated whether they perceived nothing, a slight odor, or a strong odor in the designated vial. Each concentration was presented only once, and concentrations were presented in ascending order. We identified the individual detection threshold for βI as being the lowest concentration of βI that the individual identified correctly as well as all subsequent highest concentrations. This method is based on the principle that an individual who is able to detect the odor at a given concentration will not make any mistakes at higher concentrations. Since 25% of the individuals might choose the last correct vial by chance only, we used the concentration threshold of 2.05 × 10−3 to discriminate between sensitive and insensitive individuals.One month later, an odor detection test was performed on 206 participants among 235 include initially using a sequence of six 4-alternative forced-choice tests, following the procedure described in Rouby et al. (2011) . The smeStep 2: Genotypinghttps://people.virginia.edu/~wc9c/KING/manual.html, accessed on 1 July 2021).After detection tests, participants gave a saliva sample for genotyping. rs691536 was genotyped for all individuals using Illumina HumanOmni2.5-8 (Omni2.5) BeadChip and analyses were performed using PLINK 1.9 and R. KING software used to check family relationship by estimating kinship coefficients . The release of the odorant is done simply by rubbing the reserve of printed microcapsules [Participants were asked to smell a second odorant molecule tetrahydrothiophene  a very common odorant molecule in the participants’ environment. The task consists of an identification performance based on a 4-choice test . As stated by Rouby et al. “capsules .−6. We continued through the following steps using 2,262,562 sites. Using Plink 1.07 [We perform a GWAS analysis for ßI perception in the Malagasy cohort following the recommendation presented in Marees, et al. . Based oink 1.07 , we perfhttps://genome.weizmann.ac.il/horde/, accessed on 1 July 2021 [From the HORDE database (uly 2021 ), we lisuly 2021 ,30,31. WLocal ancestry assignment across the genome was performed based on HumanOmni2.5-8 data using the ELAI algorithm  and unphp-value > 0.5). This result suggests that the sample is from an unstructured population, which is adequate for the proposed study.Among 235 participants from the highlands of Madagascar, genotyping of SNP rs6591536 revealed 97 individuals homozygous with an AA genotype, 105 individuals heterozygous with an AG genotype, and 33 individuals homozygous GG . The A ap > 0.223).We found no difference in terms of the beta-ionone threshold of perception per age-class . All groups had a very low level of anosmia to the tetrahydrothiophene. Of the 206 participants selected, 96.61% (N = 199) recognized the smell of gas. Of the seven participants unable to recognize the odor, three were AA (on rs6591536) and the remaining four carried the G allele. We did not exclude any individuals based on this analysis since the 3% who did not identify the smell of gas might be specifically anosmic or hyposmic to tetrahydrothiophene.The general anosmia test performed on a tetrahydrothiophene molecule identification (gas) did not reveal any significant difference between the genotype groups  before assessing their detection threshold with six 4-alternative forced-choice tests, following the procedure described in Rouby et al. .−1 mol/L, 20,000 ppm), Malagasy participants (N = 235) evaluated βI as pleasant but only as moderately intense and familiar   with genotype as a between-subject factor showed that there were significant perceptual differences between AA, AG, and GG carriers  A. In parp-value = 0.01) but the difference was not significant compared to the GG group  . Because of the 4-alternative forced-choice used in this test, 25% of individuals could have chosen the correct vial at any concentration purely by chance; therefore, it cannot be ruled out that the 25% of AA individuals who chose the correct vial did so solely by chance.The association between the ability to detect βI and rs6591536 genotype was assessed using a detection score test with βI concentrations between 2.05 × 10N = 206) A. Regard−3 mol/L A. In conp-value < 2.2 × 10−16, GG vs. AA: D = 0.88 p-value = 1.4 × 10−14), while there was no significant difference between AG and GG  confirming again the recessive nature of the allele A.When computing the individual score detection for βI (see Methods), we observed that GG and AG exhibited a significantly lower detection scores than AA individuals .We then tested, as observed by Jaeger et al., whether the G and A variants of rs6591536 can confer a 100-fold difference in sensitivity in the population. The majority of carriers of the functional form of the olfactory receptor (variant G) are able to detect concentrations of βI at 2.05 × 10p-value = 2 × 10−48; 95%). We also confirm Jaeger et al.’s result showing that the rs6591536 genotype is a reliable predictor of sensory acuity. Indeed, we found that 96.6% of the hyposmic/normosmic phenotype can be predicted by this variant which is strikingly similar to the result obtained by Jaeger et al. (96.3%). These results show that the perceptual ratings and threshold test by genotype distribution in Madagascar highland population is consistent with the populations studied in the Jaeger (2013) study where the G allele dominantly confers the sensitivity to ß-ionone.Using this definition, forty percent of the Malagasy students appeared to have a reduced sensitivity to βI. A total of 95% of AA carriers were hyposmic while 98.9% of AG and 92.9% of GG were normosmic ; thus, bp > 0.9553 Mann–Whitney test).When controlling for age and sex difference in βI detection scores, no significant difference has been found . It should be noted that other nearby SNPs including some on olfactory receptors are also associated with B-ionone perception, but more weakly .To assess whether the African genetic introgression influenced the perception of βI in the highland of Madagascar, we evaluated the association between βI detection and the ancestry of the OR5A1 gene by applying the ELAI algorithm on OR5A1 locus . On averIn this paper, we aimed to investigate the impact of intercontinental gene flow on βI perception in an admixed population such as the Malagasy population. Our results show that the perception scores and detection test according to the distribution of genotypes in the highland population of Madagascar are consistent with the populations studied in Jaeger’s (2013) study where the G allele dominantly confers sensitivity to ß-ionone. The strong genotype/phenotype association between rs6591536/ßI found by Jaeger et al. (2013) is then confirmed in a Malagasy population. We also showed that in the Malagasy population studied, the frequency of the “insensitive” genotype is lower than that of the “sensitive” genotype due to African gene flow.The phenotyping of individual perception was performed according to the reliable and rapid olfactory test developed by Rouby et al. in European populations  . This poThe extreme similarity between our results and those of Jaeger et al. is surprising, especially considering the extreme differences in genetics, culture, and lifestyle between the study populations, Malagasy and New Zealanders, and Southeast Asian populations. The Malagasy populations are the result of a deep and ancient mixture of Southeast Asian and African island gene flows, which has resulted in uncommon genetic and cultural diversity ,36. MultOne could argue that cultural factors could modulate the genotype/phenotype relationship. Indeed, it is known that repeated exposure to odors can modify sensitivity to them (as in olfactory training) . The MalMany factors can influence the individual phenotype such as sex, genotype, and health status. In the present article, we show that despite all these theoretical possibilities, the studied OR5A1 polymorphism explains 95% of the variability. For example, sex does not influence the threshold of detection of the molecule’s perception. Thus, despite the fact that women are underrepresented in our sample, the computed prevalence of anosmia/hyposmia should be representative of the prevalence encountered in the general population. However, it has to be kept in mind that our study is based on a homogenous sample of young adults (students), and it has been shown that a decrease of the human olfactory perception along with aging . TherefoThe fact that the Malagasy population is genetically and culturally different from the two populations tested in New Zealand and that it comes from an Asian/African mixture suggests that this association is likely to be found in most populations of the world. The global distribution of the rs6591536 polymorphism can therefore be considered a good proxy for predicting the diversity of ßI perception of current world populations; based on the available genomic dataset, a large variation in the proportion of ßI-insensitive individuals worldwide can be expected . βI is pOur results demonstrate that it is possible to track the effect of migration and gene exchange on the sensory perception of a population. These results are largely based on the reliability of the estimation of local ancestry. The reliability of the determination of local ancestry depends mainly on the differentiation of the chromosome fragments, which depends on two parameters (1) the age of the mixture and (2) the age of the separation between the two source populations. The older the separation and the more recent the mixing, the more reliable the local ancestry algorithms are. In the case of the Malagasy population, the admixture is relatively recent (1000 years) and the split extremely old . The two source populations living in Southeast Asia and East Africa are therefore extremely differentiated, with almost no gene flow between these two periods. Therefore, algorithms can reliably assign to one of two sources (Asian versus African sources) each locus, in consequence, the results of the different local ancestry estimators are very consistent  This sugTracking the allelic frequency of rs6591536 in the past population could be an indicator of the sensory phenotype of different migrants who settled in Madagascar and elsewhere. For the present study, populations had sustained contact across the Indian Ocean as early as 4000 BC, which is one of the earliest long-distance maritime contacts . These cIn conclusion, the use of bioinformatics tools such as local ancestry  or ADMIX


Oncorhynchus Spp.) that has provided rich genetic baseline data of allozymes, microsatellites, and single‐nucleotide polymorphisms (SNPs) across the Pacific Rim. Here, we analyzed published data sets for adult chum salmon (Oncorhynchus keta), namely 10 microsatellites, 53 SNPs, and a mitochondrial DNA locus  in samples from 495 locations in the same distribution range . TreeMix analysis of the microsatellite loci identified the greatest convergence toward Japanese/Korean populations and suggested two admixture events from Japan/Korea to Russia and the Alaskan Peninsula. The SNPs had been purposively collected from rapidly evolving genes to increase the power of GSI. The largest expected heterozygosity was observed in Japanese/Korean populations for microsatellites, whereas it was largest in Western Alaskan populations for SNPs, reflecting the SNP discovery process. A regression of SNP population structures on those of microsatellites indicated the selection of the SNP loci according to deviations from the predicted structures. Specifically, we matched the sampling locations of the SNPs with those of the microsatellites and performed regression analyses of SNP allele frequencies on a 2‐dimensional scaling (MDS) of matched locations obtained from microsatellite pairwise FST values. The MDS first axis indicated a latitudinal cline in American and Russian populations, whereas the second axis showed differentiation of Japanese/Korean populations. The top five outlier SNPs included mtDNA3, U502241 (unknown), GnRH373, ras1362, and TCP178, which were identified by principal component analysis. We summarized the functions of 53 nuclear genes surrounding SNPs and the mtDNA3 locus by referring to a gene database system and propose how they may influence the fitness of chum salmon.Genetic stock identification (GSI) is a major management tool of Pacific salmon , U502241 (unknown), GnRH373, ras1362, and TCP178, which significantly differentiated in Japanese/Korean populations. The functions of the outlier SNPs related to fitness, namely endurance exercise training, oxygen consumption, reproduction, and growth. GSI has been a major management tool of Pacific salmon  . The data consisted of the allele frequencies at 14 loci from chum salmon populations at 381 localities in a distribution range   Table  (Beacham5) Table . These dOke_U401‐220, Oke_GHII‐2943, Oke_IL8r‐272, and Oke_U507‐87, to avoid pseudo‐replication following the original study. A total of 53 nuclear SNPs and a combined mtDNA locus (mtDNA3) were included in our analysis . The mtDNA3 locus included three gene loci, Oke‐Cr30 and Oke‐Cr386 (mtDNA control region), and Oke‐ND3‐69 (mtDNA NADH‐3).SNP genotypes were retrieved from the Dryad data repository  from a 14‐microsatellite locus panel were found in Japanese, Russian, and representative North American populations  to estimate pairwise FST between populations. Because only allele frequency data could be accessed, we used the “read.frequency” function and computed pairwise FST values averaged over loci using the “GstNC” function in the R package FinePop v1.5.1 on CRAN.We used microsatellite allele frequencies  as the explained variation measure, which was computed in the R function as Ck=∑j=1kλj/∑j=1Kλj, where λj is the eigenvalue and jλ = 0 if jλ < 0. Following the original study  analysis was performed on the pairwise L^ + 2 × s, where L^ is the maximum composite likelihood and s is the number of parameters to be estimated, which, in this study, was three per migration event, namely, migration edge, branch length, and migration weight. For example, for the model with two migration events, the number of parameters was six. TreeMix maximizes composite likelihood and the 2 × log likelihood ratio is expected to follow an approximate χ2 distribution with three degrees of freedom. Because TreeMix calculates composite likelihoods, the penalty in AIC needs to account for the correlation in the likelihoods. To avoid unconscious bias toward selection of parameter‐rich models, we kept all the candidate models except those where the AIC values were decisively different.To infer demographic history with admixture, we applied TreeMix  values for the data sets. For microsatellites, we computed He for each population at each locus based on the allele frequencies. We then averaged He values over loci. For the 53 nuclear SNPs, we converted the original genotype data to genepop format , where He0,i = /(max He−min He). This conversion represents the standardized magnitude of an He value at the sampling point, with a continuous color gradient ranging from blue to red for the lowest and highest He values, respectively. Population‐specific He values of microsatellites and SNPs were grouped by seven geographical areas: Japan/Korea, Russia, Alaskan Peninsula, Western Alaska, Yukon/Tanana/Kuskokwim, SEA/BC, and WA.Sampling points were visualized by a color gradient of population‐specific He values between all pairs of the groups were tested by one‐way ANOVA, where p‐values were adjusted for the multiple comparisons using the “TukeyHSD” function in R.Differences in average 2.6https://www.genecards.org/) and published literature.To compare the genetic differentiation of SNP allele frequencies with the population structure inferred using the microsatellite markers, we analyzed 10 microsatellite loci and 53 nuclear SNPs loci plus the combined mtDNA3 locus. The mtDNA3 locus had five alleles, and the major allele (second one) was used for the meta‐analysis. The functions of all 54 analyzed gene loci were confirmed by referring to the GeneCards database system  of the MDS of the pairwise FST estimated using the microsatellite allele frequencies as:pj(i) is the allele frequency of SNP i  in population j . The explanatory variables mds1j and mds2j are the 1st and 2nd MDS coordinates of pairwise FST values estimated from the microsatellite allele frequencies for the matched populations j . These explanatory variables are population‐specific (J = 114). The regression analysis was performed for each marker , and 54 sets of regression coefficients  were obtained. The p‐values for the coefficients were adjusted for the multiple comparison  by the method of Benjamini and Hochberg , where jp0 = (jp−min p)/(max p−min p). This conversion represents the standardized allele frequency values at the sampling point, with a continuous color gradient ranging from blue to red for the lowest and highest allele frequencies, respectively.To identify genes that best characterize geographical areas, we conducted a principal components analysis (PCA) for the allele frequencies of the 53 nuclear SNPs and mtDNA3 locus using the “prcomp” function in R. Finally, we visualized the geographical distributions of the identified highly differentiated genes using boxplots and geographical maps of the allele frequencies and 33.1FST values based on the microsatellite allele frequencies averaged 0.019 ± 0.010 . The MDS of pairwise FST values , substantial gene flow between American and Asian populations was identified for the microsatellites . Large He values were found in Japanese, Russian, and Alaskan populations, whereas He values were slightly smaller in SEA/BC, WA, and Canadian Yukon populations . Japan and WA populations had the smallest He values. The He values were similar in populations from Western Alaska, Yukon, Alaskan Peninsula, and Russia , locus names, and raw data locus names used in our analysis Table . Five ofq‐values, showing that most of SNPs were differentiated beyond the neutral population structure. The top five outlier loci  was significantly larger in Japanese/Korean populations than it was in Western Alaska for mtDNA3  and TCP178 loci , whereas it was similar in Western Alaska for U502241 , and ras1362 . GnRH373 had smaller He values in Japanese/Korean populations (0.33) than in Western Alaskan populations (0.40) .The allele frequencies of the top five outlier loci were distinct in Japanese/Korean populations in the distribution range Figure . The maj4FST identified the north–south cline in American and Russian populations, and mds2 clearly showed divergence of Japanese/Korean populations, in agreement with the original study  in the early 1980s.mtDNA3  and may indicate a common effect. The original study  is a key regulator of vertebrate reproduction, including that of salmonids ; Data curation (lead); Formal analysis ; Funding acquisition ; Investigation (lead); Methodology ; Project administration (lead); Resources ; Software ; Supervision (lead); Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing . Hirohisa Kishino: Conceptualization ; Data curation (supporting); Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration (supporting); Resources ; Software ; Supervision (supporting); Validation ; Visualization ; Writing‐original draft ; Writing‐review & editing .Fig S1‐S10Click here for additional data file.Table S1‐S3Click here for additional data file.


Diorhabda carinata, D. carinulata, D. elongata, and D. sublineata), introduced from six localities across Eurasia to control the invasive shrub tamarisk (Tamarix spp.) in western North America. We assembled a de novo draft reference genome and applied RADseq to over 500 individuals across laboratory cultures, the native ranges, and the introduced range. Despite evidence of a substantial genetic bottleneck among D. carinulata in N. America, populations continue to establish and spread, possibly due to aggregation behavior. We found that D. carinata, D. elongata, and D. sublineata hybridize in the field to varying extents, with D. carinata × D. sublineata hybrids being the most abundant. Genetic diversity was greater at sites with hybrids, highlighting potential for increased ability to adapt and expand. Our results demonstrate the complex patterns of genomic variation that can result from introduction of multiple ecotypes or species for biocontrol, and the importance of understanding them to predict and manage the effects of biocontrol agents in novel ecosystems.With the global rise of human‐mediated translocations and invasions, it is critical to understand the genomic consequences of hybridization and mechanisms of range expansion. Conventional wisdom is that high genetic drift and loss of genetic diversity due to repeated founder effects will constrain introduced species. However, reduced genetic variation can be countered by behavioral aspects and admixture with other distinct populations. As planned invasions, classical biological control (biocontrol) agents present important opportunities to understand the mechanisms of establishment and spread in a novel environment. The ability of biocontrol agents to spread and adapt, and their effects on local ecosystems, depends on genomic variation and the consequences of admixture in novel environments. Here, we use a biocontrol system to examine the genome‐wide outcomes of introduction, spread, and hybridization in four cryptic species of a biocontrol agent, the tamarisk beetle ( In a typical biocontrol program, highly host‐specific natural enemies (agents) are collected in their native range and introduced into a novel environment to control invasive pests  identify genetic variation indicating ancestry of the four species, including two ecotype pairs within species, to be able to track their distribution across the introduced range, (2) quantify prevalence and levels of hybridization in the introduced range, and (3) examine the consequences of population bottlenecks and hybridization on genome‐wide diversity across broad, landscape‐wide expansion fronts and the hybrid zone. We predicted that admixture among populations and hybridizing species would lead to an increase in genetic variation, while isolation of disconnected patches or range expansion would lead to a decrease. We aim here to illustrate how building the molecular genetic foundation to monitor and predict evolution in introduced species can improve our understanding of mechanisms and consequences of range expansion and hybridization in novel environments.1.1Diorhabda spp. (Coleoptera: Chrysomelidae), a leaf beetle released to control invasive woody shrubs of the genus Tamarix , provides a system in which questions relating to the genomic consequences of invasion can be addressed. Tamarisk is native to North Africa and Eurasia and has become invasive in riparian areas across the western United States and northern Mexico. Stands of tamarisk can form dense monotypic thickets that cause substantial economic and environmental damage including increased fire intensity and frequency , and Chilik, Kazakhstan ; D. sublineata, from Sfax, Tunisia ; and D. elongata from Crete  and Possidi Beach, Greece ecological speciation  known to nest in the shrub, a new challenge has been presented to coordinate riparian restoration efforts with declining density of tamarisk brought about by biocontrol with rapidly evolving tamarisk beetles  haplotypes and morphological markers. These conventional and accessible methods have generally been in agreement for species identification of expanding populations  and once at the twelfth generation (G12). We used an inbred line to reduce heterozygosity and facilitate genome assembly , where We combined two sequencing approaches for reference genome assembly. First, we extracted genomic DNA from the head, thorax, and dissected testes of the G5 male and constructed a library for whole‐genome shotgun sequencing (WGS) using the NEBNext Ultra II DNA Library. This WGS library was sequenced in one lane of a MiSeq platform (Illumina) using v3 reagents to produce paired 300‐bp reads, resulting in approximately 10.4 million read pairs. Second, we conducted 10× Chromium sequencing, which produces long‐distance synthetically linked reads  build a reference panel of parental species and ecotypes, (2) characterize their distributions and hybridization in N. America, and (3) examine genomic consequences of range expansion and hybridization. For (1), we collected e Figure  and Chine Figure . We weres Figure .In total, we sampled 566 beetles, from 37 locations and two laboratory cultures, for population genomic analysis. At each site, we sampled beetles from trees within a 1‐km radius and limited the collection of beetles to no more than 5 individuals per tree where possible. We could not find adults at 19TX and instead collected third‐instar larvae, which were reared to the adult stage under laboratory conditions. Individuals for 31NV, 32UT, 33NV, and 34UT were sampled from the second generation of laboratory cultures established from these sites. All samples were adult beetles transferred as live individuals to coolers with dry ice or immediately to a −80°C freezer. All samples were stored at −80°C until DNA extraction.To prepare restriction site‐associated DNA sequencing (RADseq) libraries, DNA was extracted from individual beetles using a Qiagen DNeasy Blood and Tissue Kit following the manufacturer's protocol. The abdomens of all individuals were removed to avoid DNA of developing embryos, gut microbes, or consumed plant material and allow for later morphological characterization. Samples were treated with 4 μl RNase A (Qiagen) to eliminate RNA contamination. DNA sample concentration was quantified for each individual by fluorometric quantification .SbfI following the protocol described by , below which admixture identification could be unreliable and due to technical biases , we compiled published data  above the 0.067 threshold, then re‐filtered SNPs as above in the Stacks populations module. We then reran Structure for each subset of individuals. This secondary analysis also provided a check for sensitivity of population genetic statistics and ancestry assignment to unbalanced sampling in the global Structure analysis . We classified sampled localities as “hybrid” if the majority of individuals had ancestry from more than one species. We visualized the distributions of q‐values (“hybrid index”) for pairs of inferred ancestral taxa to estimate the degree of back‐crossing among pairs . We present π and FIS calculated from only variant sites. To estimate the proportion of private alleles within each population, we used ADZE v. 1.0  without losing populations with too many missing loci (25% missing data threshold). We present the proportion of private alleles rarified to a standardized sample size of 4. Standard errors in these analyses were calculated across RAD loci.To examine the consequences of population bottlenecks and hybridization on genome‐wide diversity (3), we quantified genomic differentiation among all individuals  within and across sites using population‐based measures. We used the populations module of Stacks to calculate π (nucleotide diversity) and FIS, and proportion of private alleles) were statistically different between hybrids versus pure species and native versus introduced, we performed a one‐way ANOVA in R. We grouped samples within localities according to Structure results using the threshold described above. Native range populations included 46CH, 43GR, 44GR, 41 CR, 37CR, 39CR, and 38CR. Although differing sample sizes between localities could have impacted estimates of private alleles  native range D. elongata, and (c) the suspected hybrid zone. After characterizing IBD, we tested for a linear relationship between latitude of collection site and Chilik ancestry for the D. carinulata SNP dataset and, separately, D. sublineata ancestry using the global SNP dataset.To quantify isolation by distance (IBD), we calculated the distances between the latitude and longitude coordinates of each sampling locality using the haversine formula and constructed a pairwise distance matrix Sinnott, . Then, uD. carinulata, we tested for the signal of asymmetrical range expansion with two origins (1WY and 34UT) across all of introduction sites of D. carinulata. Although 1WY is not the source population for D. carinulata released in Nevada, Colorado, and Utah, it is the best representative population for Fukang releases , a total length of 375.976 Mbp, and more complete, single‐copy orthologs according to BUSCO with 98.2% complete , 1.1% fragmented, and 0.7% missing.Using SPAdes, we first obtained 21,901 scaffolds with an N50 of 55.67 Kbp, an L50 of 1790, a total length of 368.964 Mb, and a BUSCO score of 83.9% complete single‐copy orthologs of the 1367 genes in the BUSCO Insecta set (86.2% of those represented a single time and 0.7% duplicated), 1.8% fragmented, and 14.3% missing. We improved this assembly by incorporating the 10× Chromium synthetic long reads with the LINKS + ARCS hybrid de novo genome assembly approach. This de novo draft 3.2D. carinulata were 72.77% on average, but we noted two distinct peaks near 75% and 100% that reflected species assignments  method; Figure K = 4–10 were identical across replicates after aligning clusters, that is, with low variation among replicates and no additional clusters were recovered, except for hierarchical substructure  and D. elongata (44GR‐41CR).Structure analysis suggested differential establishment, range expansion, and admixture among all six source populations from the four  Figures . At K = D. carinulata and D. elongata , possibly a migrant  was between 0.07 and 0.12 and allele frequency divergence between the two clusters was about 0.006–0.008, supporting low differentiation between these ecotypes. Some individuals we identified as hybrids with other Diorhabda species (presented below) showed ancestry from a third cluster  and 28NM (G48_rep). G48_rep was assigned largely to D. sublineata ancestry with sublineataq = 0.912, while G5_rep was assigned to trispecific ancestry with carinulataq = 0.664, sublineataq = 0.071, and carinataq = 0.265 , near where all four species were released Figures . We idens Figure a, whereas Figure b,c. The 5 Figure .D. sublineata in the global SNP dataset  than either the sites with pure individuals (mean = 0.0454) or within the native range  was significantly greater at sites with hybrids (mean = 0.0863) than in either the sites with pure individuals (mean = 0.0335) or from the native range (mean = 0.0381), supporting the prediction that hybridization could increase genetic diversity Figure . The proa Figure . Sites wr = 0.4481 (p = 0.1011) for D. carinulata, Mantel's r = 0.08 (p = 0.49167) for native range D. elongata, and Mantel's r = 0.5108 (p < 0.005) across the suspected hybrid zone. Only the hybrid zone had a positive IBD signal significant at α = 0.05. Pairwise comparisons across introduced D. carinulata ecotypes and native range D. elongata had greater FST values and were further apart . Origin 1 was estimated to be near 1WY , Origin 2 near the Hoover Dam , and the origin of their union in central Nebraska . The directionality index (ψ) was greatest between 32UT (an introduction site of the Chilik ecotype) and 1WY (a Fukang introduction site) at 0.144, supporting two distinct introduced populations, and the least non‐negative between 2CO and 18TX (0.0142). Orienting ψ from greatest to least generally reflected suspected expansion fronts within and among the two ecotypes . This work represents a crucial foundation to advance evolutionary applications by describing the current distribution of species and ecotypes in the introduced range, interspecific hybridization, and impacts on genome‐wide diversity. We discuss these results in the context of eco‐evolutionary processes, highlight implications for the tamarisk biocontrol program in N. America, and discuss opportunities for further study of this system to improve our understanding of contemporary evolution and biocontrol of invasive plants.To better understand the genomic consequences of multi‐species introductions and range expansion, we used reference‐based population genomics in an exemplar biocontrol system, 4.1Diorhabda populations and spread of these beetles from release sites across the introduced range. Among many original release sites and along suspected colonization routes, individual ancestry assignments were largely composed of the species or population that had been released in the location, suggesting that the extant populations at these release sites remained stable for several generations and naturally expanded along riparian corridors in a predictable pattern. The Fukang release of D. carinulata in Lovell, WY (1WY), appeared geographically stable, with uniform ancestry , spread naturally to 12AZ along the Virgin River corridor, and also shows uniform ancestry , New Mexico (28NM), and Texas  and intermediate distribution of individual q‐values among D. carinata × D. sublineata hybrids in particular suggest a lack of reproductive barriers or even increased fitness relative to parental species, while the distributions of q‐values among the other pairwise hybrids were extreme and could indicate pre‐ or postzygotic barriers to hybridization  and western KS (6KS), as well as natively allopatric D. sublineata and D. elongata in eastern NM  with any of the other species. Our hybridization results reflected the results of previous laboratory crosses, with little to no mating success between D. carinulata and those species, although D. carinulata males could reproduce with females of the other species  and the role of those barriers in the introduced range present would improve our understanding of the stability of cryptic species broadly and in biocontrol  and Texas  exist primarily in N. America as hybrids , compared with Ophraella communa, which was not intentionally released and is spreading adventively (Müller‐Schärer et al., D. carinulata reference genome, including annotation, would greatly improve our ability to identify SNPs, structural variants, and genes associated with traits related to efficacy and safety as a biocontrol agent, for example host choice and diapause induction. Still another opportunity is presented by coupling genomic studies of biocontrol agents with genomic resources from the invasive pests (Lee et al., The draft assembly of Diorhabda. More generally, we highlight possible predictors of human‐mediated translocations and range expansion outcomes, including native range species boundaries and dispersal behavior, and show how genomic tools in a biocontrol system can test these predictions.The application of genomic approaches in biocontrol systems has the potential to improve both our understanding of contemporary evolutionary processes and management of invasive species and conservation (Leung et al., The authors declare no conflict of interest.Supplementary MaterialClick here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Indigenous cattle have extraordinary adaptation capability to diverse environments under low input production system. However, the population size is declining rapidly in Bangladesh due to massive imports of high yielding dairy breeds. The genetic diversity measures are important for assessing population architecture as well as for development of conservation strategies. The aim of this study was to investigate genetic variability and population structure of indigenous cattle genetic resources of Bangladesh using Illumina Bovine SNP50K BeadChip genotyped data. Similar to other zebu populations, low genetic diversity measures were found in Bangladeshi cattle populations. Our findings revealed their distinct genetic structure but showed low levels of genetic differentiation among the six indigenous cattle populations. Moreover, admixture and phylogenetic analysis highlighted historical gene flow among the studied populations. Altogether, our findings provide a comprehensive genomic information on indigenous cattle populations of Bangladesh that could be utilized in their future conservation and breeding research.Understanding the genetic basis of locally adapted indigenous cattle populations is essential to design appropriate strategies and programs for their genetic improvement and conservation. Here, we report genetic diversity measures, population differentiation, and structure of 218 animals sampled from six indicine cattle populations of Bangladesh. Animals were genotyped with Illumina Bovine SNP50K BeadChip along with genotyped data of 505 individuals included from 19 zebu and taurine breeds worldwide. The principal component analysis (PCA) showed clear geographic separation between taurine and indicine lineages where Bangladeshi indigenous cattle clustered with South Asian zebu populations. However, overlapped clusters in PCA, heterozygosity estimates, and Neighbor-Joining phylogenetic tree analysis revealed weak genetic differentiation among the indigenous cattle populations of Bangladesh. The admixture analysis at K = 5 and 9 suggests distinct genetic structure of the studied populations along with 1 to 4% of taurine ancestry. The effective population size suggested a limited pool of ancestors particularly for Sahiwal and North Bengal Grey cattle. In conclusion, these findings shed insights into the genetic architecture of six indigenous cattle populations of Bangladesh for the first time and suggested as distinct gene pools without potential admixture with zebu or taurine populations. Indigenous cattle are an important livestock species in smallholder farming system of Bangladesh. They have potential contributions in rural livelihoods, nutrition security, organic agriculture, and socio-cultural and historic events. Moreover, rural farmers prefer indigenous cattle due to their better reproducibility and adaptation under low-input management practices ,2. In BaIn Bangladesh, the indigenous cattle alone predominated until the late seventies of the twentieth century. Upgradation of indigenous cattle varieties has been practiced for the last five decades to boost up milk production through the introduction of high yielding temperate and tropical dairy breeds . HoweverGenetic diversity illustrates the key aspects of differentiation among the individuals of a population that exists either at phenotypic or DNA level . The meaFor this study, blood samples were collected from institutional, private, and farmers’ herds under the approval of Ethical Committee of Bangladesh Agricultural University, Bangladesh (no. 1218/BAURES/2020/ESRC/AH/10).n = 89), MC (n = 26), PC (n = 45), NBG (n = 21), and DES (n = 22) are distributed in different agro-ecological regions, while SL (n = 15) breed are being predominantly available in milk potential areas. Blood sampling was performed from government or institutional herds , university managed herd , private dairy farm , as well as from 37 smallholders’ farms . The concentration and purity of isolated DNA were measured by the Nanodrop spectrophotometer  prior to genotyping.A total of 240 blood samples were collected from six zebu cattle populations of Bangladesh . Among ts’ farms . Precaut® software  plugin PLINK v.1.9 was employed for genotypes calling. SNP filtering was performed using PLINK v.1.9 [DNA samples were genotyped using Illumina bovine SNP50 v.3 BeadChip with the help of the commercial genotyping service provider . The cattle SNP50 chip possesses 53,218 SNPs that uniformly span over the entire bovine genome. Genome StudioNK v.1.9  based onN), observed (Ho) and expected (He) heterozygosity were estimated using R package hierfstat [To assess the within population genetic diversity, the proportion of polymorphic loci (Pierfstat . The disST), and maximum likelihood phylogenetic tree construction. Two different datasets were used in PCA and admixture analysis, 17,477 SNPs that passed the quality control threshold from 723 individuals belong to 25 global cattle populations .The following approaches were employed to investigate the population structure among the indicine cattle populations of Bangladesh, as well as to assess their relationships with other cattle breeds distributed globally, principal component analysis (PCA), admixture analysis, pairwise genetic differentiation  between two loci. SNPs spanning from 0 to 500 Kb distance were included in this step. The effective population size (Ne) was calculated at different generations from the resulted LD value according to the equation suggested by Sved [Linkage disequilibrium (LD) was used to examine the recombination events of linked SNPs in each population and was measured as the correlation coefficient (r by Sved . The est by Sved  with defN), varying from 0.668 in SL to 0.905 in RCC. The highest proportion of SNPs that belong to MAF category of ≤0.05 was found in RCC (0.272), whereas the lowest proportion was shown in Sahiwal breed (0.120), with overall mean of 0.197 across populations. The highest observed heterozygosity was observed in RCC (0.250 ± 0.180), while the lowest was in PC (0.211 ± 0.166), indicating higher diversity in RCC compared with other indigenous cattle populations. However, small differences were observed regarding heterozygosity measures (Ho and He) among the studied cattle populations. Minor allele frequency distributions in six oindigenous populations under study based on different categories are shown in After quality control procedures, 218 samples and 35,960 SNPs were remained in the final dataset for downstream analysis of Bangladeshi cattle populations. Twenty-two animals were excluded from the analysis due to low call rate (<0.90). The genetic diversity measures are given in The genetic structure among the indigenous cattle population of Bangladesh and 19 selected cattle breeds across the world were assessed through PCA using 17,477 SNPs. Cattle populations were separated at sub-species and region level . The PC1ST values in upper and lower diagonals, respectively among the 25 cattle breeds or populations  was obtained between Bangladeshi indigenous cattle populations and taurine cattle breeds of Africa (ND and OUL), East Asia , and Europe . However, Central Asian (YBH and MG) and European beef breeds (BMA and SGT) showed moderate genetic differentiation (pairwise FST = 0.08 to 0.11 and Nei FST = 0.08 to 0.14) with respect to Bangladeshi indicine populations.The heat map shows Nei genetic distance and pairwise Fulations . The lowinter se breeding. When another 11 European dairy and beef breeds  each possess 17,477 SNPs included with Bangladeshi indigenous cattle populations, K = 9 was found as the optimum K value  that separates Bangladeshi Indigenous cattle from NEL cattle . The opt K value . The adm K value . However K value . However2 for six zebu cattle populations separately. The r2 values both at shorter and longer genetic distances varied among the cattle populations. The most rapid LD decaying pattern was observed in shorter distances of up to about 30 kb (2 in 200 to 500 Kb fragments) were 0.12, 0.08, 0.09, 0.07, 0.06, and 0.04 for SL, DES, NBG, PC, MC, and RCC, respectively. The effective population size was evaluated based on LD estimates (r2) from the recent generation to 10,000 generations ago , Ho (0.30 and 0.39), and He (0.24 to 0.38) compared to the present findings as reported by Sharma et al. [On the other hand, the estimated heterozygosity values of this study (average Ho = 0.22 ± 0.17 and He = 0.19 ± 0.12) were in agreement with most of the previous studies involving different indicine populations. Sharma et al.  and Chaga et al. , and Upaa et al. .Several statistical approaches have been utilized to infer relationships among the cattle populations as well as to assign individuals in their respective populations. The principal component analysis (PCA) using the genotyped dataset of this study along with 19 worldwide distributed cattle populations’ data revealed that Bangladeshi indicine cattle clustered with South Asian zebu cattle and a bit away from African zebu populations. Earlier SNP-based studies in cattle and sheep demonstrated that first (PC1) and second (PC2) components of PCA clustered breeds or populations were based on their geographic origin . LikewisST and Nei genetic distance  breeds [ST estimate between Kankrej and Malvi cattle breeds of India (0.013) and is similar to our results. Earlier FST estimates by Sharma et al. [In our study, the clustering pattern in PCA B, pairwidistance , and popdistance  indicateto 0.012  using mi) breeds , between) breeds . In addi) breeds  reporteda et al.  observeda et al. . This phSimilar to the PCA, consistent results were also obtained from the admixture analysis. Among the zebu populations, the BRM and NEL clearly separated from six Bangladeshi cattle starting at K = 3. The distinct genetic composition of BRM and NEL might be their geographic isolation and long-term selection for desired traits . However2 value, a slower LD decay across the distance in SL cattle indicates its heterozygosity deficit compared to other five zebu populations. The lowest LD in RCC reflects a more diverse population with weak directional selection. In addition, the sharp decline of LD in short distance (2 estimates. For instance, a small sample size leads to bias LD estimation and therefore, a minimum sample size of 55 was suggested for accurate LD estimation based on r2 values [Based on the average rdistance A is also2 values . The low2 values .ST values. A certain amount of zebu and taurine admixture in the investigated populations might be due to their common ancestral origin and the cattle breeding history of Bangladesh. The lack of well-defined and overlapped clusters in PCA and phylogenetic tree highlight the historical gene flow among themselves and the absence of strong directional selection over the generations. Taken together, our findings reported herein the basic features of genomic architecture of Bangladeshi indigenous cattle populations that will aid in their future conservation and genetic improvement strategies and programs.This study provided an important glimpse of genetic diversity, population differentiation, and structure among indigenous cattle of Bangladesh for the first time using high density genome-wide SNP data. In this study, we found relatively low genetic diversity measures that were comparable to other zebu populations worldwide. Furthermore, Bangladeshi cattle population did not differentiate well as independent breeds, revealed by their low F