Coturnix delegorguei delegorguei) using traditional methods in Western Kenya has been ongoing for generations, yet their genetic diversity and evolutionary history are largely unknown. In this study, the genetic variation and demographic history of wild African harlequin quails were assessed using a 347bp mitochondrial DNA (mtDNA) control region fragment and 119,339 single nucleotide polymorphisms (SNPs) from genotyping‐by‐sequencing (GBS) data. Genetic diversity analyses revealed that the genetic variation in wild African harlequin quails was predominantly among individuals than populations. Demographic analyses indicated a signal of rapid demographic expansion, and the estimated time since population expansion was found to be 150,000–350,000 years ago, corresponding to around the Pliocene–Pleistocene boundary. A gradual decline in their effective population size was also observed, which raised concerns about their conservation status. These results provide the first account of the genetic diversity of wild African harlequin quails of Siaya, thereby creating a helpful foundation in their biodiversity conservation.Hunting wild African harlequin quails ( This study looks into the genetic diversity and demographic history of wild African harlequin quails using mtDNA and genotyping by sequencing data. The results of this work will provide a foundation useful in its protection and biodiversity conservation. However, the Japanese quail (Coturnix coturnix japonica) is the most common reared species in Kenya and other parts of the world, mainly kept for both meat and egg production  and the wild common (C. Coturnix) quail  and 22 domestic Japanese quails from Siaya and Kajiado Counties of Kenya, respectively Figure . This co2.2A 760bp mitochondrial DNA control region fragment was amplified via polymerase chain reaction (PCR) using primers AV1F2 and CR1b .MseI , T4 DNA ligase (NEB), ATP (NEB), and MseI Y adapter N‐containing barcode. The restriction–ligation reactions were heat‐inactivated at 65℃ and then digested with the HaeIII (GGCC) restriction enzyme at 37°C. The restriction ligation samples were purified with Agencourt AMPure XP . Polymerase chain reaction (PCR) was conducted using the purified samples, the Phusion Master Mix  universal and index primers, and i5 and i7 sequences. The PCR products were purified using Agencourt AMPure XP, pooled, and electrophoresed on a 2% agarose gel. A Gel Extraction Kit  was used to isolate 375–400 basepair fragments (with indexes and adaptors). These fragments were purified using Agencourt AMPure XP, and the resulting products were diluted for sequencing. Then, paired‐end sequencing of the selected tags was performed using the Illumina NovaSeq high‐throughput sequencing platform at the Novogene Bioinformatics Technology Company, China.We then constructed the GBS library in accordance using the predesigned scheme. Genomic DNA was incubated at 37℃ with 2.4N), with >50% bases with a Phred quality <5, with >10 nucleotides alignment to the adapter, with >10% mismatches, and that contain the HaeIII enzyme sequence.Raw reads were processed through a series of quality control procedures which involved removing reads with ≥10% unidentified nucleotides (Coturnix Japonica 2.0 genome (Assembly accession number GCF_001577835.1) with the parameters “mem ‐t 4 ‐k 32 ‐M”  version 0.7.17 was used to align the retained reads against the mand Li, . Variantmand Li, .2.5C. japonica and C. coturnix) was estimated using a median‐joining network and a maximum likelihood tree generated with network version 5.0  and principal component analysis (PCA) of the genotyping‐by‐sequencing SNP dataset were used to assess the distribution of genetic variation among wild African harlequin quail individuals and populations. Both tests were performed using SNPRelate R package version 1.22.0 . To investigate the genetic ancestry of the wild African harlequin quail individuals and admixture between the wild and domestic quail species, a maximum‐likelihood‐based clustering algorithm in ADMIXTURE version 1.3.0 was applied  error value was considered most optimal.Molecular variation among populations test (2.6D and Fu's Fs) and mismatch analysis (sum of squares deviation (SSD), raggedness index (rag.), theta0 (θ0), theta1 (θ1), and Tau (τ)) were calculated using Arlequin version 3.5.2.2  was calculated using the equation τ = 2 μLt, where τ is the tau value, μ is the substitution rate, and L is the length of the sequence  and a generation time of 1 year was assumed , which translated to a haplotype diversity of 0.949±0.016, whereas only one haplotype (haplotype 33 farm‐reared) was observed among the 17 domestic Japanese quails mtDNA control region sequences. These generated haplotypes were then used in subsequent analysis.3.2C. japonica and C. coturnix) from GenBank formed three haplogroups  mtDNA haplotype was the chosen outgroup.Out of the 32 wild African harlequin quail mtDNA haplotypes observed, haplotype 1 was the most common and present in 9 individuals. In contrast, the majority of the haplotypes were only present in one individual each (singletons). None of the other reference quail mtDNA haplotypes Table  clusteres Figure . All thes Figure . HaplogrFST value of 0.000075, signifying slight genetic variation among the three sampled populations. Overall, the wild African harlequin and domestic Japanese quails were separated into two distinct clusters.Principal component analysis showed no signs of population structuring in wild African harlequin quails of Siaya County as most individuals were grouped in a single cluster Figure . The firK = 2, which had the least cross‐validation error  and Fu's Fs  values based on the mtDNA dataset were both negative for the wild African harlequin quail populations of Siaya County, indicating a signal of demographic expansion and/or positive selection. This observation was further supported by the unimodal mismatch distribution  and 0.0343 ± 0.00571 (p > .05), respectively. The population expansion size values (θ0 and θ1) were 0.00234 and 1169.888, respectively, also indicating a high population expansion. The tau (τ) value for the estimated age of expansion was 3.58 ± 0.504 (4.88–2.12) at a 95% confidence interval (CI). This translated to an estimated time since population expansion of 257,000 ka (Thousand years ago), 95% CI 150–350 ka, corresponding to around the Pliocene–Pleistocene boundary. The molecular divergence time between the African harlequin, Japanese, and common quails was estimated by the TimeTree database to range between 5.13 and 17.60 Ma (million years ago). However, the estimated median time was 11.03 Ma during the Miocene epoch of the Neogene period.The estimated mean Tajima's n Figure  and the The demographic history and effective population size changes of the wild African harlequin quail of Siaya County were displayed on a stairway plot Figure . The fol4Several genetic diversity studies have been conducted on domestic Japanese and wild common quails using mtDNA and microsatellite markers  conservation status are listed collectively under the “least concern” category according to the International Union for Conservation of Nature (IUCN), more studies aimed at monitoring their distribution and effective population size changes need to be conducted, especially in areas where the harlequin quail is considered as a complementary poultry protein source.The use of mitochondrial DNA and genotyping by sequencing methods provided an overview of the genetic diversity of wild African harlequin quail populations of Siaya County. We did not detect any population structure between individuals in our study regions; however, more study sites are recommended to fully capture individuals that follow different migratory routes. We found no evidence of hybridization between wild African harlequin and domestic Japanese quails in the Siaya region. A noticeable decline in the number of wild African harlequin quails in the region has been a source of concern in line with their conservation status. This study has provided the necessary information required for decision‐making regarding their preservation and utilization as a source of protein. Even though the harlequin quail subspecies ; Methodology ; Validation ; Writing – original draft (lead); Writing – review & editing . Newton O. Otecko: Methodology ; Validation ; Writing – review & editing . Grace Moraa Kennedy: Formal analysis (supporting); Validation ; Writing – review & editing . John Musina: Validation ; Writing – review & editing . Bernard Agwanda: Validation ; Writing – review & editing . Vincent Obanda: Validation ; Writing – review & editing . Jacqueline Lichoti: Supervision (supporting); Validation ; Writing – review & editing . Min‐Sheng Peng: Formal analysis (supporting); Validation ; Writing – review & editing . Sheila Ommeh: Conceptualization (lead); Funding acquisition (lead); Investigation (lead); Methodology ; Project administration (lead); Resources (lead); Supervision (lead); Validation ; Writing – original draft (supporting); Writing – review & editing .This study received ethical clearance from the Kenya Wildlife Service under permit number KWS/BRM/5001 to sample wild African harlequin quails and a “no objection for the research” from the Director of Veterinary Services, Ministry of Agriculture, Livestock and Fisheries in Kenya under permit number RES/POL/VOL.XXVII/162 to sample domestic quails.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Tables S1 and S2Click here for additional data file.


Odontamblyopuslacepedii, 547-bp fragments of the mitochondrial DNA control region were sequenced. A low level of nucleotide diversity (0.0065 ± 0.0037) and a high level of haplotype diversity (0.98 ± 0.01) was observed. The Maximum Likelihood (ML) and Bayesian Inference phylogenetic trees showed no significant genealogical structure corresponding to sampling locations. The results of AMOVA and pairwise FST values revealed some significant genetic differentiation among populations, and the isolation by distance (IBD) analysis supported that the genetic differentiation was associated with the geographic distances. The demographic history of O.lacepedii examined by neutrality tests, mismatch distribution analysis, and Bayesian Skyline Plots (BSP) analysis suggested a sudden population expansion, and the expansion time was estimated to be around the Pleistocene. We hypothesize that the climate changes during the Pleistocene, ocean currents, and larval dispersal capabilities have played an important role in shaping contemporary phylogeographic pattern and population structure of O.lacepedii.Coupled with geological and geographical history, climatic oscillations during the Pleistocene period had remarkable effects on species biodiversity and distribution along the northwestern Pacific. To detect the population structure and demographic history of  Odontamblyopuslacepedii , commonly referred to as “eel goby” or “worm goby,” is an elongated, mud-dwelling benthic fish   , which iacepedii . Previouacepedii , b, taxoacepedii  and phylacepedii . HoweverThe complex interactions of post geological-history events, life history, and oceanographic condition as evolutionary processes played an important role in shaping population genetic structure and biodiversity of marine fishes . The EasSynechogobiusommaturus  was inferred to promote gene flow among populations, thus having a major effect on its phylogeographic pattern  was carried out in 25 μL reaction mixture containing 17.5 μL of ultrapure water, 2.5 μL of 10×PCR buffer, 2 μL of dNTPs, 1 μL of each primer (5 μM), 0.15 μL of Taq polymerase, and 1 μL of DNA template. The PCR amplification was carried out in a Biometra thermal cycler under conditions referred to PCR product was purified with a Gel Extraction Mini Kit. The purified products were used as the template DNA for cycle sequencing reactions performed using BigDye Terminator Cycle Sequencing Kit, and bi-direction sequencing was conducted on an ABI Prism 3730 automatic sequencer (Applied Biosystems) with the same primers used for sequencing as those for PCR amplification.A 547 bp fragment of mitochondrial DNA control region was amplified using the primers OLF: CGCTGCTTCAAAGAAGGGAGATT (forward) and OLR: CTCCCTTGTCAACTTGCCTTAG (reverse) . The polS), nucleotide diversity  among populations was estimated by Migrate-n version 3.6.11  and refined manually. Molecular diversity indices, such as the number of haplotypes, polymorphic sites  phylogenetic tree was constructed with 1000 bootstrap replications based on distances calculated using the best selected model K2P. The phylogenetic trees were constructed with Odontamblyopusrebecca as the outgroup. The network of haplotypes was constructed using PopART software base on the minimum spanning network (D test of Tajima (1989) and Fs test of Fs has been shown to be especially sensitive to departure from population equilibrium as in case of a population expansion. The method to test demographic expansion is mismatch distribution which is the distribution of the observed number of differences between pairs of haplotypes based on three parameters: τ, θ0, and θ1  (BSP). To obtain the effective convergence, HKY + I + G model, stepwise skyline model and a strict molecular clock with 1×108 iterations for Markov chain Monte Carlo (MCMC) were performed in this study. Tracer 1.7.5 software was used to generate the skyline plot  . The val growth) . The pop growth) . BEAST v growth)  was usedine plot .KX894323–KX894449). Most haplotypes were unique, of which 108 were singletons (haplotypes represented by a single sequence in the sample). Of the remaining 19 haplotypes, 13 were shared among populations, but six haplotypes belonged to one population. The most common haplotype was present in six locations with 22 individuals , accounting for 17.3% of all samples.All sequences were aligned, and 547-bp segment of the control region was obtained for 189 specimens. A total of 83 polymorphic sites were detected and 127 haplotypes were defined (Table π) varied from 0.0037 to 0.0095, while the haplotype diversity (h) ranged from 0.83 to 0.99. The phylogenetic topology based on ML analyses revealed no significant genealogical branches or clusters corresponding to sampling localities  between Log FST and geographic distance, indicating that geographic distance can explain 54% of the genetic variation.The pairwise Odontamblyopuslacepedii for all samples is presented in Fig. Hri = 0.027±0.000, P > 0.05), and SSD , and the evident unimodal mismatch distribution indicated a sudden expansion event. The Tajima’s D and Fu’s Fs tests were negative with significant P values (P < 0.001), which supported the hypothesis of population expansion  was 1.4 × 107 times higher than before expansion (θ0) for O.lacepedii. Bayesian Skyline Plots for all samples showed late Pleistocene demographic expansion   in the Japan-Ryukyu-Guam region, and the oceanic currents might contribute to the dispersal and migration of larvae of this species  analysis of this study supported that the genetic differentiation was associated with the geographic distances. The dispersal ability of marine organisms will weaken as the distance increases, which often leads to the IBD patterns (The population structure in marine species has been assumed to have low genetic differentiation among widespread populations due to their high potential dispersal ability and the absence of obvious geographical barriers . In the  species . The pel species . The eggall area , and theall area . Besidespatterns .Apart from historical events and life history , environOdontamblyopuslacepedii. With modern exploitation and habitat destroyed, O.lacepedii may experience high fishing pressure. The results of the present study have important implications for fisheries management and conservation efforts and for other species with similar life history characters.Historical events of the Pleistocene, ocean currents, and larval dispersal capabilities have played an important role in shaping the contemporary phylogeographic patterns and population structures of


Eleutheronema are the important fishery resources in Indo-Pacific regions and classified as the endangered species with considerable conservation values. Their genetic diversity and population structure remain essentially unknown but are critical for the proper management and sustainable harvests of such important fisheries. Here, the mitochondrial DNA sequences of CO1 and 16s rRNA were determined from 75 individuals of Eleutheronema tetradactylum and 89 individuals of Eleutheronema rhadinum collected from different locations of South China Sea and Thailand coastal waters. Genetic diversity analysis revealed that both E. tetradactylum  and E. rhadinum  had low diversity. Population structure analysis demonstrated the shallow genetic differentiation among the South China Sea populations. The limited communication between China and Thailand populations caused the high genetic differentiation in all groups due to the low dispersal ability. Reconstruction of CO1 phylogenetic relationships and demographic studies across Indo-West-Pacific regions provided strong evidence for a shared common origin or ancestor of E. tetradactylum and E. rhadinum. Eleutheronema rhadinum were further subdivided into two distinct genetic lineages, with Clade A dominantly distributing in Thailand and Malaysia and Clade B distributing in China coastal waters. Phenotypic divergence, characterized mainly by the depth of caudal peduncle and length of caudal peduncle, was also observed for all populations, which was possibly associated with specific local adaptations to environmental changes. Our study suggested a strong need for the development of proper fishery management strategies and conservation actions for the imperiled Eleutheronema species.The threadfin  Eleutheronema belongs to the Family Polynemidae, which consists of three valid species, including three fingers threadfin (Eleutheronema tridactylum), four fingers threadfin (Eleutheronema tetradactylum), and East Asian four fingers threadfin (Eleutheronema rhadinum). Eleutheronema tetradactylum, also known as the Indian salmon due to its wide distribution in the Indo-West-Pacific region, is an important commercial fish species. However, fisheries of E. tetradactylum have drastically decreased over the past decade due to the over-exploitation and possibly water pollution2. The fish has been classified as endangered by the International Union for Conservation of Nature (IUCN). The East Asian four-finger threadfin, E. rhadinum, is endemic to the East Asia region, including China coastal water (South and East China Seas), Japan, Vietnam, and Malaysia3. Especially in the China coastal water, it is a commercially crucial fish for coastal and inshore small-scale fisheries, with high auction prices in local fish markets. In contrast, E. tridactylum is relatively rare but also considered as a valid species.The genera Eleutheronema tridactylum can be easily distinguished from the other two Eleutheronema species by determining the morphological characters such as pectoral fins1. However, identification of E. tetradactylum and E. rhadinum is relatively difficult based on their morphological discrimination by visual check due to their external morphological similarities. Therefore, molecular markers such as mitochondrial DNA sequencing are helpful for accurate species and bloodstock identification4. The cytochrome c oxidase subunit 1 (CO1) gene has been used to discriminate Eleutheronema species, demonstrating the advantages of the molecular marker in identifying and resolving issues in taxonomy and geographical distribution of taxa3. In addition, mtDNA, particularly the CO1 and 16s rRNA genes, have been proven to be the powerful tools for revealing the phylogeographic patterns and genetic diversity.E. rhadinum in the East and South China Seas regions in 2013 was based on the CO1 method5. The results revealed that the populations from Qingdao, Zhuhai, and Zhoushan developed divergent genetic structures and experienced a population expansion. Wang et al.6 used the mtDNA Cytb method to analyze the population diversity of E. tetradactylum in both East and South China Seas, including Zhoushan, Wenzhou, Shantou, and Qionghai, which showed a high level of haplotype diversity and low nucleotide diversity among three populations.The distribution of species is a complex expression of its ecological and evolutionary history, and assessing the population genetic, morphological, and environmental data could provide new insight into the effects of the environment on the population structure of species. A previous study on the population differentiation of E. tetradactylum and E. rhadinum in Southeastern regions, especially the geographical differentiation, is still unknown. This study analyzed the morphological differentiation and population genetic structure of Eleutheronema species with morphometric measurements and CO1 and 16s rRNA sequences newly obtained from 75 E. tetradactylum and 89 E. rhadinum individuals from Southern China and Thailand (west and east) coastal waters. Our study focused on the genetic differences between the China and Thailand populations. The morphological analysis, one of the simplest and most efficient methods to identify fish stock structure and discriminate the species, were conducted by using differences in body measurements and morphological characteristics. We performed the quantitative morphological study to investigate whether allopatric Eleutheronema differed morphologically. Additionally, the newly obtained data were subjected for phylogenetic relationships and historical demography analysis of Eleutheronema species across the Indo-West-Pacific regions by integrating with previously reported data. Assessing the population diversity and differentiation of Eleutheronema species is crucial for evaluating the adaptive ability to the changeable environment and essential for establishing the conservation strategies.To date, knowledge of the population differentiation and genetic diversity of Eleutheronema tetradactylum and 89 Eleutheronema rhadinum individuals were collected, respectively, from three  and four  sites during Nov. 2020 to Jan. 2021  and 16s rRNA (574 bp), PCR was carried out using the previously universal primers for CO1 and 16s rRNA7. The 25-μL PCR reaction system included 2.5 μL of 10 × Buffer, 2.0 μL of dNTPs, 1.0 μL of the primers (0.5 μL forward and 0.5 μL backward), 0.5 μL of template DNA, 0.2 μL of Taq (TaKaRa), and 18.8 μL of ddH2O. The PCR cycle parameters were as follows: 94 °C for 4 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, and 72 °C for 5 min for the final extension. One μL of each PCR product was detected by 1% agarose gel electrophoresis and observed under the UV light. All these qualified PCR products were submitted for sequencing. Species identification was performed based on CO1 and 16s rRNA sequences  method. The confidence levels were assessed using the bootstrap procedure with 1000 replications. All these analyses were performed in MEGA 11 software8.To amplify the partial mtDNA fragments of ces Fig. . In detaT), standard length (LS), head length (LH), body depth (DB), eye diameter (DE), depth of caudal peduncle (DCP), length of caudal peduncle (LCP), length of caudal fin (LCF) and height of caudal fin (HCF). In addition, six indices, including head length/standard length (LH/LS), eye diameter/head length (DE/LH), depth of caudal peduncle/length of caudal peduncle (DCP/LCP), body depth/standard length (DB/LS), length of caudal peduncle/standard length (LCP/LS) and length of caudal fin/length of caudal peduncle (LCF/LCP), were also used to describe the morphological traits.After fish were harvested and photographed, the photographs were processed with tpsDIgv.2.12 software to measure the following morphological indicators: total length , including mouth tip, operculum top, first dorsal fin front, first dorsal finback, second dorsal fin front, second dorsal finback, caudal top, caudal bottom, anal finback, anal fin front, and pectoral fin, were selected Fig. , and theCO1 and 16s rRNA datasets. The aligned CO1 and 16s rRNA sequences were subjected to DnaSP v510 to identify the polymorphic sites and estimate haplotypes data, especially for the number of private haplotypes unique to each population, respectively. The mismatch distribution analysis (MDA) was used to infer the demographic stability of phylogenetic clades and species using DnaSP software. The Tajima’s and Fu’s Ts tests were performed on the Arlequin program11. The TCS haplotype network was constructed to estimate the gene genealogies using the statistical parsimony approach at the population level in PopART12. Bayesian skyline plot (BSP) was used to examine the historical demographic fluctuation using the BEAST 2.6.0 program13.Standard measures of genetic diversity were evaluated based on 14 based on CO1 and 16s rRNA haplotype datasets. The outgroup species used were Megalobrama amblycephala and Scophthalmus maximus for the CO1 and 16s rRNA datasets. As listed in Table CO1 sequences of E. tetradactylum and E. rhadinum, retrieved from the public NCBI database, were used as the final nucleotide sequence alignment datasets for phylogenetic analyses and divergence time estimation. The target sequences were the haplotypes samples from India, Malaysia, and Vietnam for CO1 of E. tetradactylum and E. rhadinum16.A maximum-likelihood tree was constructed for phylogenetic analyses under the GTR + I + G model in IQtreeCO1 sequences was conducted in BEAST 2.6.0. Program with a lognormal relaxed molecular clock algorithm under the calibrated-Yule model and gamma distribution under the HKY model. Posterior distributions of parameters were estimated using 1,000,000 Markov-chain-Monte Carlo (MCMC) generations sampled every 1000 generations, with a 20% burn-in in the TreeAnnotator 2.6.0 program17. The consensus tree was visualized in the FigTree program (https://github.com/rambaut/figtree/releases).For divergence time estimation, the BEAST analysis based on All experimental protocols were approved by the Research Committee of City University of Hong Kong. All methods were carried out in accordance with the relevant guidelines and regulations of the City University of Hong Kong.CO1 sequences was successfully amplified and sequenced from 75 individuals of E. tetradactylum and 89 individuals of E. rhadinum from different sites. Based on the CO1 analysis, we detected 5 and 16 haplotypes, respectively, from E. tetradactylum and E. rhadinum  and PA (0.197) than ZS (0.105). In E. rhadinum, CO1 haplotypes varied from 3 (JH) to 8 (ZZ). The haplotype diversity was the highest in ZZ (0.663). The populations of ZJ and ZZ showed the statistically negative Tajima’s D value, which could signify the demographic expansion. The MDA revealed similar results  was also successfully sequenced from 75 and 89 individuals of E. tetradactylum and E. rhadinum  and nucleotide diversity (π = 0.00170) were found in populations SA. Overall, the populations from Thailand showed higher genetic diversity than the China population both for E. tetradactylum and E. rhadinum.The mitochondrial um Table b. No hapE. tetradactylum and E. rhadinum between China and Thailand populations, as shown in Fig. E. tetradactylum, 5 haplotypes were closely related to a small number of mutation steps, and the Hap_1 was likely the most primitive haplotype, which evolved into others. In E. rhadinum, 16 haplotypes were distributed between the two branches, including China and Thailand branches. Only the Hap_7 was shared in ZJ and SA of the Thailand branch. One (hap_1) in E. tetradactylum and two (Hap_2 and Hap_8) in E. rhadinum were used as the central radiation distribution for most haplotypes. Other haplotypes were formed by a small number of mutations of these haplotypes. As shown in the TCS network of 16s rRNA haplotypes, the Hap_1 in E. tetradactylum and Hap_4 in E. rhadinum were the most primitive haplotype, which showed central radiation distributions. Also, in E. rhadinum, the haplotypes of China and Thailand populations were divided into two branches; only Hap_2 was shared in ZJ and SA.The TCS network was constructed to identify the phylogenetic relationships in E. tetradactylum, the average Fixation index (Fst) between PA and the other two sites was 0.81344 in ZS (p < 0.05) and 0.73738 in ZJ (p < 0.05). In E. rhadinum, high value of Fst was observed between SA and other three China population, including ZJ , JH  and ZZ . Overall, pairwise Fst comparisons revealed that E. tetradactylum and E. rhadinum in China and Thailand were significantly differentiated, indicating low population connectivity among these sites. However, no significant population differentiation was observed within China populations both in E. tetradactylum and E. rhadinum, which indicated that all China populations did not diverge at the subspecies level. The greater genetic differentiation coefficient means less gene flow among populations. Therefore, due to the gene flow among the China populations, a low level of genetic divergence among different China populations was observed.The level of population genetic differentiation between China and Thailand populations was also evaluated Table . In E. t19. The present study based on mtDNA gene sequences aimed to investigate the genetic diversity of Eleutheronema species. The results revealed remarkably low haplotype diversity and nucleotide diversity in all populations. The E. tetradactylum showed low levels of variability, which might reflect the impact of human activities, including marine pollution and overfishing20. Although E. rhadinum showed relatively higher genetic diversity than E. tetradactylum, its genetic diversity levels were still remarkably lower than other marine organisms distributed in the China coastal water23. Since E. tetradactylum was classified as endangered by the IUCN, more attention should be paid to conserve the imperiled E. tetradactylum and E. rhadinum. Additionally, the low genetic diversity of ZS strongly indicated the necessities to strengthen the genetic management of artificial breeding populations to ensure that artificial breeding populations have a higher level of genetic diversity. A low level of genetic divergence among different populations in China suggested genetic similarities in the Chinese coastal water. Generally, genetic homogeneity in marine fishes can be attributed to the absence of barriers between ocean basins and adjacent continental margins. The ocean currents in the China sea could facilitate the dispersal of marine larvae, and may be responsible for the genetic homogeneity. A previous study indicated that the dispersal of E. tetradactylum was sufficiently low, which had lower swimming performance and poor orientation and can be effortlessly hindered by the geographical barrier24. Thus, limited communication between China and Thailand populations caused the high genetic differentiation in all groups. Our results implied that the limited ecological population connectivity of local China populations might permit self-recruitment rather than passive dispersal. Therefore, low genetic diversity and shallow population structure of E. tetradactylum and E. rhadinum resulted in a serious concern about fisheries management and conservation of these two Eleutheronema species.Genetic diversity could directly reflect the potential and ability of species to adapt to environmental changesE. tetradactylum and E. rhadinum in China and Thailand with those elsewhere in the West-Pacific region, we used our newly generated CO1 sequences and publicly available CO1 DNA sequences from NCBI’s GenBank database. We retrieved 9 haplotypes from 17 sequences for E. tetradactylum and 8 haplotypes from 12 sequences for E. rhadinum from the public database , Malaysia, and Thailand (Satun province). On the other hand, the haplotypes of Clade B were from China  and Vietnam. Thus, CO1 haplotypes of E. rhadinum collected from China and Thailand could be allocated into Clade A and B. Haplotypes from the Clade A and B coexisted only in population ZJ. Additionally, a star-like pattern appeared in the haplotype network in Clade B of E. rhadinum, suggesting the signature of demographic expansion in the process of dispersal. Moreover, some low frequency haplotypes were also identified in Clade B, that may have originated as a result of adaptation to the conditions in this area26. However, these populations of E. tetradactylum did not go through recent population expansion as star-like topology was the result of population expansion27.To determine the phylogenetic relationships of se Table . Two hapees Fig. a, E. tetses Fig. b, E. tetCO1 haplotypes of E. tetradactylum and E. rhadinum, respectively. All these clades in the CO1 data showed negative values in Tajima’s D and Fu’s Fs, but only the Tajima’s D values were significant. By testing the species itself, the same pattern disappeared, and the value of Fu’s Fs was only significant in E. rhadinum. Based on the mismatch distribution analyses (MDA) on CO1 for each species  analyses based on d B Fig. b. In E. E. tetradactylum and E. rhadinum, since E. rhadinum had long been recognized as a junior synonym of E. tetradactylum28. Moreover, the results indicated the probability that two slightly different genetic groups existed within E. rhadinum due to genetic breakdown, including Clade A and B. Similar patterns were also observed in the population of Sardina pilchardus, and the causes for the isolation of the population may be related to oceanographic barriers29. Individuals belonging to Clade A were dominantly distributed in Malaysia and Thailand and may not have the opportunity of demographic expansion on the Malay Peninsula. Also, previous study indicated that Malay Peninsula played a role in shaping the contemporary genetic structure among populations of Pampus chinensis30. However, demographic expansion occurred in Clade B, which mainly included China populations and was consistent with previous population structure analysis that E. rhadinum in the East and South China Seas developed divergent genetic structures and experienced a population expansion5. To better understand the population dynamics of Eleutheronema species, whole genome resequencing analysis needs to be conducted in further population genetic studies of adaptation and natural selection of Eleutheronema species.Overall, the newly obtained and previously reported data were applied to estimate the phylogenetic relationships and demographic analysis, which provided strong evidence for a shared common origin or ancestor of E. tetradactylum and E. rhadinum, the PCA of all 6 morphometric variables extracted 3 principal components , explaining 81.27% and 81.05%of the total variance, respectively  and DB/LS (0.72021). In E. rhadinum, the 2 variables with high loadings on PC1 were DCP/LCP (0.56505) and LCF/LCP (0.52844). Strong loading of characters involved in DE/LH (0.67347) and LH/LS (0.69152) was also respectively observed in the case of PC2 and PC3.In this study, principal component analysis (PCA) based on 6 morphometric variables among six populations showed certain degrees of overlap, and PC1 and PC2 showed 41.4% and 21.6% of the total variance, respectively Fig.  and S6. p value of the permutation test was used to describe the shape variations among populations. Results of CVA and grouping of 6 populations in the two canonical variables for each species are shown in Fig. E. tetradactylum and E. rhadinum were clearly separated into distinct clusters in PA and ZZ. E. rhadinum from both geographical regions (JH and SA) was not clearly isolated along the first two canonical variate axes. Moreover, E. tetradactylum and E. rhadinum from ZJ also showed certain degrees of overlap .Subsequently, CAV based on the lap Fig.  by pairw31. Our study revealed the phenotypic divergence of Eleutheronema species among diverse populations, characterized mainly by the depth of caudal peduncle and length of caudal peduncle, indicating the evolution in the caudal peduncle which is associated with swimming behaviors in deeper waters32. The caudal fin was also among the variables with high loadings on the PC1, suggesting evolution in the caudal area, likely associated with the consequence of phenotypic plasticity in response to hydrological conditions. A high morphological divergence was also reported in Eleutheronema collected from China, suggesting rapid and apparently adaptive morphological divergence of Eleutheronema species in response to changes in China coastal water33. However, some morphometric and meristic characteristics were not distinct between E. tetradactylum and E. rhadinum in Zhanjiang (ZJ), suggesting that E. tetradactylum and E. rhadinum shared a common ancestor, and the phenotypic modifications might be mainly due to the adaptation to local habitats of Zhanjiang. The morphological analysis also revealed that different local environmental conditions of China and Thailand coastal water might have influenced the Eleutheronema differently, as evidenced by morphological modifications to better adapt and survive in the local ecosystem. Overall, the morphological divergence among the different populations in China and Thailand coastal water reflected the geographic isolation underlying the population structure and specific local adaptations to environmental changes.To adapt to changeable environments, fish modify their morphology and physiology, and the phenotypic plasticity results in morphological divergence which may, in some instances, be involved in response to different environmental conditionsEleutheronema species in China and Thailand coastal waters. Low genetic diversity and shallow population structure were observed in E. tetradactylum and E. rhadinum, which suggested the need for considerable fisheries management and conservation. The phylogenetic relationships and population genetic structures provided strong evidence for a shared common origin or ancestor of E. tetradactylum and E. rhadinum. In Indo-West-Pacific regions, the E. rhadinum populations were likely subdivided into two genetic lineages, including Clade A and B. The Thailand and Malaysia populations, which belonged to Clade A, may have no demographic opportunity during dispersing. However, demographic expansion occurred slightly in Clade B, mainly distributing in the China's coastal water. Our present study provided important understanding of the genetics and morphology of E. tetradactylum and E. rhadinum and have implications for future conservation and management efforts for Eleutheronema species.The present genetic and morphological data provided novel genetic information on the population genetic structure and demographic history of Supplementary Information.


Bombus terrestris is used worldwide for crop pollination. Despite its positive impact on crop yield, it has become a widespread threat to biodiversity due to its interactions with local bumblebee populations. Commercial subspecies introduced to the Iberian Peninsula since the 1990s without any regulation have colonized the environment, with evidence of naturalization and introgression with the endemic subspecies Bombus terrestris lusitanicus. We have used mitochondrial and nuclear genetic data to describe the current genetic diversity of the Iberian population and to estimate the expansion of commercial bumblebees. Samples from the natural distribution range of the commercial subspecies, the natural intergradation area between the two subspecies and from a period prior to the use of commercial colonies  have been used for comparison. Our results show that the mitochondrial haplotype of the commercial breeds has spread throughout the territory, which, together with subtle changes observed in the nuclear genetic diversity of the populations, indicates that hybridization and consequent introgression are occurring in most of the peninsula. It is, therefore, necessary to improve the existing legislation concerning the management and exportation of commercial bumblebees to conserve locally adapted populations.The bumblebee  In the last decades, given their economic and environmental importance, concern for the welfare of pollinators has risen3. Many studies have explored the main potential causes of their decline4. In addition to those identified as major causes , anthropic translocations of domesticated taxa like honey bees or bumblebees within their natural ranges are receiving more attention7. Such human-mediated movement may harm other pollinators through competition for resources and the spread of parasites . More research on these effects is needed to encourage regulatory authorities to implement scientifically supported conservation measures.In the context of the current human-induced biodiversity crisisBombus terrestris  began to be used commercially in greenhouses for crop pollination in the 1980s in Belgium, and the subspecies B. t. terrestris and B. t. dalmatinus Dalla Torre, 1882 are now widely used in artificial pollination11. Commercial breeds can escape from greenhouses into natural habitats when they seek floral resources12 or nesting places and can colonize suitable environments due to their high thermic resistance, generalist diet and wide climatic tolerance14. The exportation of Bombus terrestris outside its Palearctic distribution range has made this bumblebee an invasive species16, driving native species to local decline or extinction due to competition, pathogen spillover and reproductive interference17.The West Palearctic bumblebee species B. terrestris was classified as an invasive species in 2006 and its importation is now severely restricted17. In Argentina, the importation of B. terrestris and B. impatiens Cresson, 1863 is also strongly regulated to protect the native South American species B. dahlbomii Guérin-Méneville, 183516, although commercial species can still spread from Chile15. Within the natural distribution range of the species, specific regulations to protect endemic subspecies of B. terrestris have also been approved, as commercial breeds could become a potential competition to the wild populations based on their characteristics 8. Norway and the Canary Islands have strict importation policies on foreign bumblebees11, while in the United Kingdom the trade of non-native subspecies is restricted, favouring the use of the endemic subspecies B. t. audax 9. However, no regulation has been enacted to protect the endemic subspecies B. t. lusitanicus  in the Iberian Peninsula, where there is a high density of greenhouse agriculture that use bumblebees as managed pollinators  to identify differential changes in the genetic diversity of populations. Male diploidy rates (2n♂) were also determined to estimate the inbreeding in bumblebee populations35. In haplodiploid species such as B. terrestris, male diploidy is caused by homozygosis at the sex determination locus (sl-CSD) in diploid individuals, which would develop into females if they were heterozygous36. The presence of diploid males implies a reduction in the number of workers, which can negatively affect the fitness of the colony28. Although male diploidy occurs naturally37, high rates have been linked with declines in effective population size and low genetic diversity38 and therefore would be expected to appear at a lower rate in hybrid populations.Both mitochondrial and nuclear markers were analysed to estimate genetic diversity parameters and clustering patterns across B. terrestris populations from a broader perspective on both spatial  and temporal (before and after the 1990s) scales, taking into account the dispersion of commercial breeds  into the wild and potential introgression events between wild and commercial populations. We hypothesize that, due to the dispersion of commercial subspecies from greenhouses, hybridization with the wild populations of the endemic subspecies is not limited to the south and east of the Iberian Peninsula39 and introgression is occurring across the territory. These events would have a more pronounced impact on the genetic diversity of the populations in the south of the peninsula, where many bumblebee breeding companies provide pollination services to the high quantity of greenhouse crops that depend on bumblebees40.This research aims to study the genetic diversity and structure of the Iberian B. lucorum  and one B. soroeensis ), and 31 from pinned collections had low-quality DNA extractions. Of the remaining 555 individuals , 487 were morphologically identified as B. t. lusitanicus (87.75%), 58 as B. t. terrestris (10.45%) and 10 as morphological hybrids (1.8%). Within the Iberian sampling, B. t. terrestris individuals and most hybrids were found in the south of the peninsula and in the Pyrenees , by sequencing the Bter_rrnL fragment in 33 individuals (5.95%) and by the 16S fragment in 89 individuals (16.04%) from previous studies39.Mitochondrial haplotype information was obtained for the 555 morphologically described individuals. The mitochondrial haplotype was determined by the B. t. lusitanicus individuals with the central-European haplotype (discrepant hybrids) were found in 12 of the 17 Iberian locations sampled (0% to 44%) and the Pyrenees (18.74%). 61.1% of the naturalized B. t. terrestris found in the south of the peninsula bore the Iberian haplotype. Higher percentages of discrepant hybrids were found in the two intergradation areas: the Pyrenees  and the southern peninsula , while three individuals from France presented the Iberian haplotype (REF_FR: 13.04%) and no hybrids were found in the Belgian reference group. In the historical population, the central-European haplotype was found only in one individual close to the intergradation area of the Pyrenees (Iberian haplotype in REF_SP80: 97.73%).Two haplotypes were detected: a haplotype more frequent in the Spanish and Portuguese populations  and one more frequent in the Belgian and French populations   and the historical reference (REF_SP80) at low frequencies (Oosterhout > 0.25%). Most loci presented a statistically significant deviation from HWE expectations due to heterozygote deficit. None of the microsatellite loci analysed showed significant linkage disequilibrium.Microsatellite information was only considered from locations with more than ten genotyped individuals .B. t. lusitanicus and 17 from Belgium and France (REF_BL and REF_FR) as reference B. t. terrestris by the assignment test using GenAlEx. The presence of hybridization was corroborated with microsatellite analysis. The GeneClass assignment test using Nei’s standard distance41 and the Bayesian method42 allowed us to observe introgression in all the Iberian localities analysed  were classified as reference 2 = 0.593; p = 0.033) between the percentage of hybrids found by mitochondrial haplotype and by nuclear genetic parameters  values varied from 0.630 to 0.716 . The fixation index (Fis) showed signs of inbreeding in both the Iberian and the historical reference populations.Allelic richness (Ar) ranged from 4.27 to 4.63 across the Iberian populations . The results after random removal (hIN/rOUT)  suggest that the decrease of HE in hOUT might not be due to the mere removal of individuals  were compared with those obtained after the removal of individuals labelled as hybrids  in each location  Table . The Wills Table , but to 2 = 0.012; p = 0.741) and were likely affected by other variables. However, the variation in HE due to the presence of hybrid individuals (ΔHE = HEhIN − HEhOUT) does depend significantly on the percentage of hybrids present in each location   present , indicate that the mitochondrial haplotype associated with central European populations of B. terrestris has expanded beyond both the natural intergradation area  and the anthropic one , reaching most of the north–south gradient with some exceptions in the northwest. These results contrast with the haplotype distribution in historical populations  in which all individuals presented the Iberian haplotype, except one individual detected close to the natural intergradation area in the Pyrenees, where natural hybridization occurs. These results support the idea that the endemic Iberian haplotype was predominant in the territory and that, due to hybridization and introgression events with naturalized commercial breeds, the central European haplotype has recently spread into the environment. This change in the haplotype distribution of the peninsula is a first signal of how this expansion is modifying the genetic pool of the populations, which may soon develop into losses of local adaptation45. Another hypothesis to explain the present genetic structure could be migration, although unlikely. In the context of climate change, the only plausible hypothesis is a migration from the Southern area to the north1. However, based on our knowledge, the European subspecies was never recorded in the Southern region of Spain and we did not detect any individuals with the central European haplotype in the historical data set but one, close to the natural intergradation area in Pyrenees.This research aimed to study the genetic diversity of the Iberian B. t. lusitanicus in the Iberian Peninsula. Previous records of B. t. terrestris and hybrid individuals have occurred only in the south39 and west24 (putative genetic hybrids) of the Iberian Peninsula, whereas we have found evidence of introgression in most of the territory , as B. terrestris dispersion can be assisted by strong winds to cross long distances33. On the other hand, in the most northwestern localities sampled , the central European haplotype was not found, which suggests that these populations of B. t. lusitanicus are less affected by introgression, probably due to a lower density of greenhouses in the area, as well as a larger distance from the two main areas of intergradation40 29. Furthermore, the use of old samples as historical references in population genetics will be especially important in subsequent years, as we monitor the expected changes in the distribution of not only B. terrestris subspecies but also other organisms that are affected by climatic change and anthropisation. The distribution range of B. t. lusitanicus, instead of shifting or reducing, is expanding, both in altitude and latitude46, while the populations of B. t. audax in the UK have become bivoltine7. Given the subspecies’ similar resistance to heat stress14, further changes due to climatic change are expected to occur. Even under these conditions, although we must assume that hybridization may play a key role in evolution48, there is a consensus that human-induced introgression of non‐indigenous organisms harms native gene pools50.This study confirms that the inclusion of samples from old collections is crucial to assess the evolution of the genetic diversity of local populations, as has been done before in other species of the genus B. terrestris populations, which leads to a reduced population structure in a continuum that is not only peninsular but also continental52. When comparing the Iberian populations with their historical reference, only slight variations can be observed in the allelic frequencies, although the data show an overall decrease in allelic richness and expected heterozygosity values. Conversely, the Wilcoxon test suggests that hybridization and introgression events are affecting the native populations by increasing the genetic diversity of those populations with hybrid individuals, which is expected from the admixture of different gene pools45. Given the low structure of bumblebee populations, the effect of this change is difficult to measure, but if this dynamic continues, the loss of endemicity and increased homogenization of European populations will be recurring concerns in the future.As previous studies on the genetic diversity of the species have reported, the heterozygosity values obtained in this analysis, the assignment test (GeneClass) and the clusters inferred (Structure and DAPC) suggest an intense gene flow among 35, although it has been previously discussed that B. terrestris is able to withstand inbreeding in its populations56. In this sense, B. terrestris does not show mating preferences between related and unrelated individuals35, so there are no mechanisms preventing diploidy or even triploidy in males53. However, these results should be taken into account in future studies on the species, as the ploidy values obtained in this study were higher than those of populations of endangered species such as B. florilegus Panfilov, 1956 (2.7%)54 or B. muscorum Linnaeus, 1758 (5%)55.The analysis of genetic diversity in the Iberian Peninsula showed the lowest values in the central peninsular populations , where the highest number of diploid males and one triploid male were found. These results could represent a sign of a potential threat of inbreeding depression in the populationsB. terrestris can escape from greenhouses24 and the great expansion capacity of the managed populations in the environment15, we emphasize the importance of the propagation of the commercial B. terrestris subspecies from greenhouses across the Iberian Peninsula as a driver of population change. Moreover, the inadequate management of colonies by breeding companies and farmers after colony use due to a lack of information about the consequences of their dispersal in the environment is another little-addressed factor contributing to the emergence and dispersion of commercial breeds  in the environment. Some of the mispractices that occur in the territory include the outdoor use of colonies, incomplete removal or abandonment of colonies24 and attempts to manage the nesting of commercial queens in the environment . To avoid escapes, bumblebee nests should be placed only inside greenhouses and destroyed at the end of the crop pollination campaign before new sexual individuals emerge. The arrival of commercial breeds of B. terrestris into new environments and their subsequent colonization is guaranteed unless stricter regulations on their transport and management are adopted16, both outside and within the species’ natural distribution range13. To our concern, there is still no legislation regarding the management of bumblebee colonies for agricultural use in the Iberian Peninsula beyond the advice and instructions of the provider companies. Because the Iberian Peninsula is a large producer and exporter of fruits and vegetables, and therefore an intensive user of commercial pollination services, we endorse stricter legislation on the importation of foreign subspecies and support for companies breeding local subspecies11, as the breeding of B. t. lusitanicus does not involve additional costs compared with other subspecies of B. terrestris . Such regulations are already in effect in other regions within the natural distribution range of the species, such as the United Kingdom, Norway and the Canary Islands. Authorization of the importation of exotic taxa should only be legalised after studying the survival rate, expansion capacity and environmental impact of the managed populations. It is important to coordinate an update of the existing legislation with more active dissemination to first-hand users on the correct management of commercial breeds and the benefits of using local subspecies.Given that 38. To determine the impact of the introduction of commercial bumblebees, a set of individuals collected in Spain in the 1980s before the use of bumblebee colonies started in the peninsula in the 1990s, was included in the analyses. In the current sampling we considered as a population the set of individuals sampled within a radius of 10 km based on the maximum flight distance recorded for B. terrestris  taking into account geographic barriers (e.g. if mountains were present).Individuals were collected along the Iberian Peninsula, the Pyrenees, north of France, and Belgium Fig. . The samB. t. terrestris. As a historical reference, 44 individuals collected in Spain before the introduction of commercial B. terrestris39 were included  in a north–south gradient in 17 locations of the Iberian Peninsula (Supplementary Table ed Table  online.20 (i.e. ferruginous brown coloration only present in B. t. lusitanicus).Individuals from the Iberian Peninsula, Belgium and France were net-sampled and maintained individually in absolute ethanol at -20 ºC until processed. Individuals from the Pyrenees and the historical reference sampling were obtained from the pinned collections of the University of Mons and the Complutense University of Madrid (UCME), respectively. A first subspecies identification of individuals was performed through their morphological characters.58 protocol. Concentration and purity of DNA extractions from pinned individuals were quantified, as DNA obtained from antique individuals presents a lesser quality59.Total DNA was extracted from one leg of each individual following Ivanova et alB. terrestris individuals was obtained with the RFLP approach on the NADH dehydrogenase subunit 2 gene (nad2) following Cejas et al.23. To improve the amplification success, Bovine Serum Albumin  was added to the PCR reactions.Haplotype information of 59. Therefore, for the Pyrenean individuals, a new primer set (F: 5’-TCAACATCGAGGTCGCAATCA-3’ and R: 5’-TGGCTGCGGTATAATTGACTGT-3’) was designed with Primer360 to obtain a shorter fragment (388 bp) within the 16S ribosomal RNA (rrnL) gene. The same PCR conditions were used as in Hines et al.61. Amplicons were sequenced in Secugen . We added the haplotype information from the samples processed in Cejas et al.39 by sequencing the 16S fragment as described in Hines et al.61.DNA is often highly fragmented in samples from old collections, making it difficult to amplify larger sequencesloci designed for B. terrestris62 were amplified in all individuals with two multiplex sets following Cejas et al.63: RB1 , and RB2 . PCR products were sent to the Servei Central de Suport a la Investigació Experimental  for capillary electrophoresis on an ABI Prism 3700 (Applied Biosystems). Allele scoring was performed using GENEMAPPER 4.8 (Applied Biosystems) by comparing alleles with an internal size standard . A second allele scoring quality control was made manually to avoid the wrong assignation as alleles of fluorescence peaks during the automated allele scoring process. Individuals with missing data > 30% were discarded. Siblings were inferred with Colony 2.0.6.464 with a Full-Likelihood analysis under default parameters for haplodiploid species. We retained only one individual from every kinship with a greater probability of 70%.Ten microsatellite 65 to find the proportion of hybrid individuals among subspecies in the Iberian populations, using Nei’s standard distance41 and a Bayesian method42 with MCMC method66 as the criteria for computation. For these analyses, the purest individuals from reference populations  detected in the assignment test (assignation by logarithmic likelihood) in GenAlEx 6.567 were selected as reference groups. The assignment of the Iberian samples was evaluated.The individual assignment test was performed with the program GeneClass 2.0hIN). The same genetic diversity parameters were estimated again after removing all detected hybrids  and naturalized B. t. terrestris individuals (dataset recorded as hOUT). Same remarks were taken into consideration for the French population, (Supplementary Table rOUT).To evaluate the potential effect of the hybridization and introgression between populations, genetic diversity parameters were calculated (1) in Iberian locations with an appropiate number of samples , (2) in the Iberian Peninsula as one population (SP + PT), and (3) in the four reference populations  and estimates of linkage disequilibrium (LD) were calculated for the Iberian and reference populations using Genepop69. The level of significance for the analyses was adjusted with Bonferroni corrections . Observed and expected heterozygosity (HO and HE) were estimated with GenAlEx 6.5. Allele richness (Ar) and private allelic richness (pAr) were calculated with Hp-Rare 1.170, and rarefacted to a sample size of 10 gene copies. The inbreeding coefficient (Fis) was obtained with FSTAT 2.9.471.To avoid bias due to the haplodiploidy of the species, only genotypes of female individuals were considered. Micro-Checker 2.2.3hIN and hOUT were compared with the non-parametric Wilcoxon signed-rank test for related samples using package stats 3.6.0  in R72. For this test, data was paired according to their label and ranked by their absolute differences. The level of significance for the analysis was adjusted under Bonferroni corrections . Wilcoxon signed-rank test was repeated against the random removal of individuals (datasets hIN and rOUT), in order to investigate whether the significance of the results was due to the removal of individuals itself or to the identity of those excluded. The Iberian Peninsula as a population (SP + PT) was not included in this analysis to avoid oversampling.The percentage of hybrid individuals found in each location was compared with the values of expected heterozygosity (HE) and with the change in heterozygosity (ΔHE), after removing the hybrids (hOUT─hIN), to estimate the effect of introgression on the genetic diversity of the populations through linear regressions in R with package stats v3.6.272.Obtained parameters from datasets 73 with a burn-in period of 100,000 steps and 1,000,000 MCMC repetitions. Potential clustering values (K) 1 to 10 were tested under 10 iterations each. Structure Harvester42 was used to select the best fitting K by calculating ΔK74. Software Clumpak75 was used to reach a consensus between the different iterations of each selected K, following the Markov clustering algorithm. Furthermore, a graphical representation was printed with Distruct 1.176.A clustering analysis was carried out on Structureadegenet 2.1.177. Function find.clusters was used to identify the number of genetic clusters according to the Bayesian Inference Criterion (BIC). Nevertheless, from a biogeographic perspective, other K numbers were also analysed. Finally, populations were selected as clusters to investigate their identity and relationship. The number of principal components for the analysis was selected after α-score estimations to explain the data while avoiding overfitting. Results were graphically represented in a scatter plot.Discriminant analyses of principal components (DAPC) was performed using R  package ♂) was assessed for each sampling location. Males with two or more diploid loci were considered as diploids. Diploidy proportion was calculated following Zayed et al.34 by dividing the number of diploid males between the total number of sampled male individuals in the location.The proportion (ϕ) of diploid males (2n Supplementary Information 1.Supplementary Information 2.


Opisthopappus is an endemic genus of the Taihang Mountains that includes only two spatially partitioned species Opisthopappus longilobus and Opisthopappus taihangensis. For this study, the mechanisms behind the genetic variations in Opisthopappus populations were investigated.Climate heterogeneity not only indirectly shapes the genetic structures of plant populations, but also drives adaptive divergence by impacting demographic dynamics. The variable localized climates and topographic complexity of the Taihang Mountains make them a major natural boundary in Northern China that influences the divergence of organisms distributed across this region. Opisthopappus populations or two species. All studied populations were divided into two genetic groups with the differentiation of haplotypes between the groups. At approximately 17.44 Ma of the early Miocene, O. taihangensis differentiated from O. longilobus under differing precipitation regimes due to the intensification of the Asian monsoon. Subsequently, intraspecific divergence might be induced by the dramatic climatic transformation from the mid- to late Miocene. During the Pleistocene period, the rapid uplift of the Taihang Mountains coupled with violent climatic oscillations would further promote the diversity of the two species. Following the development of the Taihang Mountains, its complex topography created geographical and ecological heterogeneity, which could lead to spatiotemporal isolation between the Opisthopappus populations. Thus the adaptive divergence might occur within these intraspecific populations in the localized heterogeneous environment of the Taihang Mountains.Using SNP and InDel data coupled with geographic and climatic information, significant genetic differentiation was found to exist either between Opisthopappus species in the Taihang Mountains. The results provide useful information for us to understand the ecology and evolution of organisms in the mountainous environment from population and species perspective.The localized environmental events through the integration of small-scale spatial effects impacted the demographic history and differentiation mechanism of The online version contains supplementary material available at 10.1186/s12862-021-01790-0. Understanding the processes that drive differentiation between populations and elucidating the mechanisms that underlie the origins and maintenance of genetic variations are major aims and fundamental tasks in evolutionary biology –5, whichDuring this process, intensifying climate change has left an indelible imprint on the composition and divergence of populations or species , 16, whiThe Taihang Mountains, with a north–south orientation , are a prominent natural boundary in Northern China , 22, whiPyrus betulaefolia exhibits abundant genetic diversity and variation, which might have been derived from the diversified environments of different populations located in the Taihang Mountains [Episyrphus balteatus presented a non-obvious phylogeographical structure, which resulted from invalid geographical barriers of the Taihang and Yashan Mountains, where its population division was driven by the climatic changes following the uplift of the Taihang and Yashan Mountains [Climatically, the southern region is home to a warm temperate semi-humid climate with a mean annual temperature of 12.7 °C and precipitation of 606.4 mm, while the northern region has a temperate continental monsoon climate with a mean annual temperature of ~ 10 °C and precipitation of 700 mm. The topographic complexity of the Taihang Mountains coupled with increasing climate variability have significantly impacted many organisms , 24. Beiountains . In contountains . The Taiountains , 24.Opisthopappus), which has been listed as a second-class protected plant in China [Opisthopappus taihangensis and Opisthopappus longilobus [O. taihangensis and O. longilobus, the morphological distinctions are primarily manifested in the leaves and bracts. For the former, there is appressed puberulent on both surfaces of the leaves, two pinnatisect stem leaves, and no bracteal leaves. For the latter, which has hairless leaves, there is one pinnatisect, except the basal stem leaves, and a pair of bracteal leaves beneath the involucres. Furthermore, several other categories of morphological differentiation have been observed, such as the leaf pinnatisect, sparsely pubescent or glabrous surfaces, stoma size and density, pollen colpus depth, and ostiole density [As an important perennial herbal germplasm resource of Asteraceae  estimate the effects of geographical and climatic variables; (iv) identify the possible key environmental factors that drive this differentiation. These results evaluated the demographic dynamics of Opisthopappus during the evolutionary process, explored the underlying mechanisms of inter-/intra-species differentiation, and provided some clues for the investigation of additional plant species in the Taihang Mountains.For the present study (according to the hypothesis above), the roles and influences of environmental factors on species and population differentiation were investigated through the combination of geographic and climatic data, using single nucleotide polymorphisms (SNP) and insertion-deletion (InDel) markers of nuclear genes developed by Chai et al.  based onO. longilobus and 28 haplotypes (H48–H75) in O. taihangensis. No shared haplotypes were detected between O. longilobus and O. taihangensis. For O. longilobus, the H5 haplotype was the most widely distributed, which was shared by three populations. Five haplotypes  were shared by two populations, whereas the other 41 haplotypes were distributed only among a single population , followed by H56 (shared by five populations), H53 (shared by four populations), and H51, 54, 55, 60 (shared by two populations). The remaining 20 haplotypes were detected only in a single population.Among the SNP primers, eight pairs produced repeatable, clear, and stable bands. The total length of eight SNP combination segments was 1921 bp, which contained 1870 conservative sites and 51 polymorphic sites. Based on one hundred twenty sequences, seventy-five haplotypes were identified . This indicated that there were significant phylogeographic structures in the Opisthopappus genus.The value of the genetic differentiation coefficient Opisthopappus  was 0.99327 and the total nucleotide polymorphism (π) was 0.00308 according to the SNP data. As relates to the InDel data, the Nei’s gene diversity index (H), polymorphic loci ratio (PPL), and Shannon’s polymorphism information index (I) were 0.1695, 76.92%, and 0.2777, respectively. For O. taihangensis, the genetic indices, Hd, π, H, PPL and I, were 0.95399, 0.00178, 0.1592, 73.85% and 0.2585 respectively , 15% of the molecular variations within the populations , and only 5% of the molecular variations between populations within species . For the InDel data, the genetic variation distribution trend was similar to the SNP sequences. The results verified that molecular variations existed primarily between the two Opisthopappus species (Table Significant genetic variations occurred either between es Table . Based oO. longilobus and O. taihangensis) clusters.A maximum likelihood (ML) phylogenetic tree was constructed based on eight SNP combination fragments |/sd(L(K))) attained a maximum value, K = 2 was taken on both SNP and InDel data. The most significant possibilities were gathered into two groups  represented a 91.1% variation of the total genetic components. The first three linear discriminant functions (LDs) explained 56.9%, 7.5%, and 7.5% of the eigenvalues of the remaining PCs. All O. longilobus, whereas the other contained the H48-H75 haplotypes from O. taihangensis. Further, the haplotype network . Within O. taihangensis, the differentiation time was at 12.82 Ma (95% HPD: 10.96–14.79 Ma) between haplotypes, while the haplotypes of O. longilobus were differentiated at ~ 13.67 Ma (95% HPD: 11.82–15.58 Ma). From the later Miocene to Pliocene, the intraspecies divergence continuously occurred, where the approximate more recent differentiation time of the intraspecific haplotypes for both species was during the Quaternary Era .At the Miocene-Oligocene boundary , the Opisthopappus genus and two species had experienced a recent expansion based on significantly negative Fu's Fs  values and a non-significant sum of square deviation (SSD) and raggedness index (Rag) values (P > 0.05).The neutral test and mismatch distribution analysis (MDA)  and logistic regression tests . Furthermore, a 95% CI of the PP for this scenario did not overlap with other scenarios under logistic estimation.According to ABC analysis, there was a significant difference between the observed and simulated data based on the posterior distributions of all scenarios. Scenario 3, namely, O. longilobus and O. taihangensis were 2.21E + 04 (95% CI 4.22E + 03–1.24e + 05) and 7.40E + 04 (95% CI 1.36E + 04–3.51E + 05).The PCA of the posterior distributions of model checking analysis revealed that the summary statistics from the observed data produced eigenvectors that were within, or at the margins of 1000 simulated pseudo-observed data sets (PODs), which indicated that scenario 3 was generally suitable for the observed data Fig. . For SceNm O. longilobus → O. taihangensis = 0.3813 (95% CIs 0–1.8790) and Nm O. taihangensis → O. longilobus = 0.7860 (95% CIs 0–3.1748). The mean contemporary gene flow (m) between the two species was also low. Migration occurred from O. longilobus to O. taihangensis and in turn was 0.0059 (95% CIs 0–0.0173) and 0.0052 (95% CIs 0–0.0156), respectively. However, the migration rates within each species were relatively high (O. longilobus: 0.9941 (95% Cis 0.9827–1.0055), O. taihangensis: 0.9948 (95% CIs 0.9844–1.0052)) as estimated by BAYESASS.The historical gene flow generated using MIGRATE were low between the two species, One-way ANOVA following the extraction of bioclimatic variables found that most of them, distributed along the two species were significantly different, including Mean diurnal range (bio2), Isothermality (bio3), Temperature seasonality (bio4), Min temperature of coldest month bio6), Temperature annual range (bio7), Mean temperature of driest quarter (bio9), Mean temperature of coldest quarter (bio11), Precipitation of wettest month (bio13), Precipitation of driest month (bio14), Precipitation seasonality (bio15), Precipitation of wettest quarter (bio16), Precipitation of driest quarter (bio17), and Precipitation of coldest quarter (bio19) , and genetic and environmental distances . The partial Mantel tests also detected significant correlations between genetic and environmental distances conditioned on geographic effects .Significant associations between geographical and environmental distances appeared  across all populations of 2 = 0.2944, P = 0.001) and environmental  distances significantly related to the genetic distance. The joint effects of both the geographical and environmental distances also significantly impacted the genetic distance .Similar results, which revealed that geographic and environmental distances influenced genetic distances, were obtained by multiple matrix regression with randomization (MMRR) analyses. The effects were that both the geographic . The ANOVA indicated that the PC2 of bioclimatic variables significantly explained the genetic components with the highest explanatory proportions .The full RDA model, including geographic distribution and climatic factors, explained 72.42%  and PC3 , had significant F statistics. PC2 correlated significantly with the ordination axis1 of dbRDA, while PC3 was significantly correlated with axis2. The high adjusted R2 indicated that PC2 and PC3 were sufficiently explained by the two dbRDA axes. Consistent influence estimates were obtained even when the order of predictors was altered; thus, the two variables were highly relevant for explaining the genetic differentiation of the populations across the two species. However, the adjusted R2 of PC1 might be too small to be meaningful in either dbRDA or GLM.The distribution of the PC1-3 of climatic variables along the ordination axis was further examined by GLM (Table 4). The PC2  began to lift during the Eocene (50–55 Ma) and then experienced different stages of growth to attain its current elevation –70. The Opisthopappus genus began to differentiate from O. longilobus to O. taihangensis , the O. longilobus or O. taihangensis began to undergo intraspecific differentiation , but the diverse haplotypes in each species  period, the continuously cooled global climate and the progressive extended QTP uplift brought the topographic and vegetational changes in China , 80. In O. longilobus and O. taihangensis became ever more fragmented, where originally large and continuous populations may be separated into multiple smaller subpopulations. Thus, the most recent diversification of haplotypes within species occurred during the Pleistocene of the Quaternary [Toward the emergence of the Taihang Mountains the stage was being set for a neotectonics movement, from the late Miocene to early Pliocene . The intaternary , which aO. taihangensis and O. longilobus exhibited signs of recent expansion during the evolutionary process  at Shanxi Normal University. The voucher specimens were deposited in the herbarium of College of Life Science, Shanxi Normal University (No:20170105030–20170105050).O. longilobus and thirteen populations of O. taihangensis were sampled, which covered the Opisthopappus distribution ranges  for sequencing.The SNP and InDel primers , 1 µL primer S (10 µM), 1 µL primer A (10 µM), and 5 µL ddHPrior to population genetic analysis, the partition homogeneity test (PHT) were initially conducted by PAUP  to identHd), and nucleotide diversity (π) were calculated using DNASP 5.10 [GST and NST differentiation parameters were examined by PERMUT 2.0 [The haplotypes, haplotype frequencies, haplotype diversity (ASP 5.10 . The genRMUT 2.0  based onH), Shannon’s information index (I), and the percentage of polymorphic loci (PPL), were calculated by POPGENE 1.31 [FST, FCT, and FSC values [For the InDel data, the genetic characteristics, Nei's gene diversity index  was estimated through 10 independent runs using 500,000 step Markov chain Monte Carlo (MCMC) replicates, following a 1,000,000-step burn-in for each run to evaluate consistency. The best grouping number was evaluated by ΔK [Cluster analysis based on the maximum likelihood (ML) method and Nei’s genetic distance, respectively, was performed using MEGA 7.0 . Bayesiaed by ΔK  in STRUCed by ΔK . These 1ed by ΔK  and the ed by ΔK .O. taihangensis and O. longilobus. Kruskal–Wallis tests for the first two principal components (PCs), and the first two linear discriminants (LDs) of DAPC, were conducted to examine the genetic differentiation between the populations and species.To test the genetic differentiation between populations or species, a discriminant analysis of principal components (DAPC) was implemented by the function dapc in the R package ‘adegenet’ , which iOpisthopappus haplotypes. BEAST 1.84 [Chrysanthemum indicum, belonging to the same subtribe of Chrysantheminae with Opisthopappus (holding identified genomic information) was selected as the outgroup in BEAST analysis. The haplotype sequence of each primer was aligned to the NT (Nucleotide Sequence) database followed by manual splicing. Owing to the absence of the record of the Opisthopappus fossil data, the divergence time of Chrysanthemum and Opisthopappus (25.40 Ma) referred to the Time Tree website (http://www.timetree.org/) was adopted as a prerequisite for calibrating the age of most recent common ancestor (tMRCA).A network relationship was generated through the median-joining method in POPART 1.7 , to inveAST 1.84  was empl–9 (5.1 and 7.1 × 10–9) for the nuclear DNA of the Asteraceae species was employed according to the present study [7 times by sampling every 80,000 generations. TRACER 1.5 [The Akaike Information Criterion (AIC) with a “greedy” algorithm in PartitionFinder 2.1.1  was emplnt study . Markov ACER 1.5  was usedD [FS [To assess whether the species had experienced a significant expansion, we utilized ARLEQUIN 3.5  to calcuD  and Fu’sD [FS  values. O. longilobus and O. taihangensis were differentiated from a common ancestral population during the same period. Scenario 2: O. taihangensis was an ancestral population, and O. longilobus was differentiated from O. taihangensis. Scenario 3: O. longilobus was the ancestral population, and O. taihangensis was differentiated from O. longilobus.Approximate Bayesian computation (ABC) analysis, provided by DIY-ABC 2.1.0 , enabledEach scenario was performed with 1,000,000 simulations and six summary statistics  were selected. The substitution rates of nuclear genes were the same as those used in the BEAST analysis. To identify the best-supported scenario under direct and logistic approaches, we selected 1% of the simulated datasets closest to the observed data to evaluate model accuracy and estimate the relative posterior probability (PP) with 95% confidence intervals (95% CI) for each scenario. Further, the parameters including effective population size and divergence generation was estimated under the optimal scenario. The goodness of fit of the best supported scenario was evaluated by the option ‘model checking’ with principal component analysis (PCA). To estimate type I and II errors on the power of model selection, we assessed confidence in scenario choice with 500 simulated pseudo-observed data sets (PODs) for the seven plausible scenarios.Opisthopappus species by MIGRATE-N 3.6 [4 trees) and three long chains (105 trees) with 104 trees discarded as an initial burn-in’ and astatic heating scheme at four temperatures . To ensure the consistency of estimates, we repeated this procedure five times and reported average maximum-likelihood estimates with 95% confidence intervals. The parameters θ and M were estimated using a Bayesian method, which could be employed to estimate the number of migrants per generation (Nm) into each population using the Eq. 4Nm = θ*M.Additionally, the historical and contemporary gene flow were estimated within the two TE-N 3.6  and BAYETE-N 3.6 , respectWhen estimating the contemporary gene flow using BAYESASS 3.0, the parameters were examined including migration rates (m), allele frequencies (a) and inbreeding coefficients (f) to ensure that the optimal acceptance rates of the three parameters fell within the 20–60% range. Ten independent runs were executed to minimize the convergence problem. The result with the lowest deviance was adopted according to the method of Meirmans , where thttp://www.worldclim.org/) with a resolution of 30 arc-sec (~ 1 × 1 km) and extracted using the R package ‘raster’ [O. longilobus and O. taihangensis, after which the PC1–PC3 were reserved for further analysis.Nineteen bioclimatic variables (Bioclim) representing Grinnellian niches , 111, wh‘raster’ . SubsequTo test how the geographical and environmental differences impacted genetic differentiation, the Mantel test, partial Mantel test, and Barrier analysis were applied in this study. Further, a multiple matrix regression with randomization (MMRR) was performed to explore whether the genetic distance responded to variations in geographic and/or environmental distances.FST distance calculated in ARLEQUIN 3.5 was used as the genetic distance. The geographic distance was estimated using the GENALEX 6.5 according to three-dimensional factors . The environmental distance was calculated using the Euclidean distance with PASSAGE 2.0 based on the first three PCs [Pairwise hree PCs .2) and their significance were determined based on 9,999 random permutations. Scatterplots to reveal the relationships between genetic, environmental, and geographic distances were conducted using GraphPad Prism 8 [The Mantel test was performed in the R package ‘vegan’ , whereas Prism 8 .The biogeographic boundaries between population pairs were calculated by the Monmonier’s maximum-difference algorithm in BARRIER 2.2  based onIn addition, distance based redundancy analyses (dbRDA) were performed to elucidate whether the climatic variables conditioned on the geographic distribution explained the genetic differentiation of the populations using the R package ‘vegan’. Firstly, a distance-based principal coordinate analysis (PCoA) of the genetic data at the species level was performed to generate several principal coordinates (PCs) using the R package ‘ape’ . Next, tAdditional file 1: Fig. S1. Phylogenetic relationships between O. longilobus and O. taihangensis. (A): Individual ML clustering of Opisthopappus. Blue branches presented individuals of O. longilobus and red branches presented individuals of O. taihangensis. (B): UPGMA clustering for 24 populations of Opisthopappus based on Nei’s genetic distance. Blue for populations of O. longilobus and red for populations of O. taihangensis.Additional file 2: Fig. S2. Structure analysis from K = 3 to K = 6 for SNP and InDel, respectively.Additional file 3: Fig. S3 The Kruskal–Wallis test of the first two principal components and the first two linear discriminants of the genetic variation revealed significant genetic divergence between species but no or little population differentiation within species. (A–D): Comparisons between species. (E–H): Comparisons among populations.Additional file 4: Fig. S4. Haplotypes network of Opisthopappus. 47 haplotypes (H1-H47) were detected in O. longilobus and 28 haplotypes (H48-H75) in O. taihangensis. No shared haplotypes were detected between O. longilobus and O. taihangensis. The color of each haplotype corresponded to Fig. lotypes H-H47 wereAdditional file 5: Table S1. The results of neutrality tests (Tajima’s D and Fu’s FS tests) and mismatch distribution analyses.Additional file 6: Table S2. ANOVA analysis for the nineteen bioclimatic variables grouped by two different species.Additional file 7: Fig. S5. The average temperature of every month (A) and the average precipitation of every month (B) of the studied populations of the distribution of Opisthopappus.Additional file 8: Table S3. Information of primer pairs.


Leishmania donovani species complex is the causative agent of visceral leishmaniasis, which cause 20–40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 384 publicly available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 24 genes with robust signatures. These include zeta toxin, nodulin-like, and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further study.The  SignificanceLeishmania donovani species complex are globally distributed, with major foci in East Africa, the Indian subcontinent and Brazil. Although global genetic diversity has been described, there has been very little focus on selective pressures in this species. We used a data set of 477 sequenced isolates to search for signals of balancing selection in populations covering these foci, identifying 24 genes with robust signatures of balancing selection. By identifying genes that appear to be subject to strong selection this study contributes to our understanding of the genetic diversity in this parasite, and parasites in general.Protozoan parasites of the Leishmania parasites cause the neglected infectious disease leishmaniasis in over 80 countries. Visceral leishmaniasis (VL) is the most severe form of the disease, caused by Leishmania donovani and Leishmania infantum. Annual cases of VL are estimated at a minimum of 50,000, with a fatality of ≥95% if untreated, and occur primarily in the Indian subcontinent (ISC), Bangladesh, Sudan, South Sudan, Ethiopia, and Brazil  has been studied in iversity , as do piversity . BS signiversity . Given tne cells , BS may ne cells . In all Leishmania species. Here, we use genome data from 477 clinical isolates from the L. donovani species complex (L. infantum or L. donovani) from East Africa, the ISC, and Brazil to identify five populations that are well-represented by genome data. Using a variety of metrics, we search for signals of BS within these populations. We identify multiple strong signatures of BS. Signatures are generally unique to a single population consistent with adaptive divergence between populations.Thus far, there have been no published studies of BS in L. donovani species complex covering the main global foci of East Africa, the ISC, Brazil, and Europe. We utilized 229 L. donovani isolates from the ISC (L. donovani isolates from Ethiopia (L. infantum isolates from Brazil (L. donovani isolates from a variety of locations including Sudan (14 strains), France (6), and Israel (10)  and insertion/deletion polymorphisms (indels). In this data set of 477 isolates, we identify 339,367 SNPs and 14,383 indels.In this study we used population-scale genomic data from  the ISC , 43 L. dEthiopia , 25 L. im Brazil , and 87 ael (10) . Additio, Brazil . This prn = 44).We used the ADMIXTURE clustering tool  to assigFST) values range from 0.27 to 0.90 . In contrast, both East African populations and the older population from the ISC2 show higher genetic diversity, and are likely to have been maintained as larger populations for longer periods of time.Phylogenetic analysis provides some qualitative insight to the history of these species C and D. L. infantum populations  populations contain more indel polymorphisms , with anulations .D, whereas the Tajimas’s D is strongly skewed to negative values in the emerging ISC2 population. To select groups of strains that will approximate panmictic populations, we used the ADMIXTURE analysis to identify isolates that were confidently assigned to one population (n = 433), rather than being interpopulation hybrids. This selection resulted in 59 isolates from East Africa (two populations of 41 and 18), 226 from the ISC (two populations of 211 and 15), and 127 from BM, of which 93 were newly sequenced here . We find scant evidence for shared BS from Betascan1* scores. We define Betascan1* outlier genes as those in the upper 5% of Betascan1* scores for their population. There was little overlap in these outliers; 701 genes are outliers in at least one population, only 42 of these (6%) were outliers in two or more populations  were outliers in more than one. However, because the NCD2 metric measures the similarity of allele frequencies to a target frequency . We found that outliers from EA1 do not show significantly elevated Betascan1* scores in any other population. We repeated this analysis for the 5% highest outliers from ISC1 and from BM  and Tajima’s D (NCD2 or Betascan* and π or Tajima’s D). This intersection identified 38 genes  cosegregating sites and show higher levels of statistical linkage than genome-wide distributions, consistent with expectations for genes that are subject to BS  outliers for at least one test (>99.5 percentile) and 2) outliers (95 percentile) for either Tajima’s D and/or nucleotide diversity . Details of the method are described in Our manual vetting of BS candidates retained 24 genes , of whicLeishmania. The zeta toxin domain protein  encodes a flagellum attachment zone protein essential for host interaction (FLAM3 protein contains a clustered mitochondria (CLU) domain and a domain of repeats  and >5 variants per gene (Tajima’s D and π metrics). The low-diversity Brazil and ISC1 populations contain far fewer regions that satisfy these criteria. However, strong population bottlenecks would enhance the loss of polymorphic sites by drift, ablating strong BS signatures. For example, 99% of 1 kb genomic windows in our BM population contain <5 segregating sites, which will reduce the scale of Betascan* and NCD2 metrics. This could result in a loss of power to detect long-term BS. Only two populations (EA1 and ISC2) appear to have relatively stable population sizes and sufficient nucleotide diversity to identify BS candidates using our pragmatic methods  of selection, rather than a lack of power to detect targets in some populations. Several observations led us to this view. Tajima’s D is not at all correlated between populations (data not shown), but this might be expected if most genes are dominated by drift. The high FST between populations, and relatively few shared polymorphisms are consistent with this. The initial 24 candidate BS genes that were discovered in the EA1 population, did not have statistically higher Tajima’s D (as a group) in any other population . All methods were performed according to the approved guidelines and regulations. A written informed consent was obtained from all study participants or their legal guardians.Bone marrow aspirates were obtained from the routine diagnosis of patients admitted to the Natan Portella Tropical Diseases Institute in Teresina-PI, Brazil. Aspirates were inoculated into a mixed culture medium NNN  containing 2 ml of Schneider’s medium supplemented with 10% fetal bovine serum, 2% urine and penicillin 10,000 U/ml, and streptomycin 10 mg/ml. The positive isolates in mixed media were expanded in Schneider’s liquid medium under the same conditions mentioned above. Extraction of DNA from the parasites was performed after washing to remove culture medium, using Qiagen Blood and Tissue kit was used according to the manufacturer’s recommendation.Genome sequencing was performed on Illumina HiSeq 2500 machines (or similar) to produce paired end 150 nt reads. The majority (95%) of the samples were sequenced to provide mapped read coverage of ≥30× . Raw sequencing reads were submitted to NCBI’s sequencing read archive under the BioProject accession PRJNA702997.L. donovani complex data were downloaded in FASTQ format from the European Nucleotide Archive (ENA: https://www.ebi.ac.uk/ena). Full list of strain names/ENA numbers in L. donovani reference genome (strain BPK282A1) was downloaded from TriTrypDB (version November 2019). Strain reads were mapped to the reference using bwa v.0.7.17  accepting calls with a minimum alternative allele read count ≥5. We accepted calls discovered by both methods, merged all VCFs and regenotyped with Freebayes. The regenotyped VCF was sorted with Picard SortVcf (https://broadinstitute.github.io/picard/) and indexed with GATK IndexFeatureFile. SNP hard-filtering was performed with BCFtools (https://samtools.github.io/bcftools/) on biallelic variants only, to remove sites with any of the following: DPRA < 0.73 or > 1.48; QA or QR < 100; SRP or SAP > 2,000; RPP or RPPR > 3,484; PAIRED or PAIRPAIREDR < 0.8; MQM or MQMR < 40. As chromosome 31 is generally supernumerary, we specified DPB < 3,0401 or > 121,603 to be removed, and for remaining chromosomes, DPB < 182,99 or > 73,197 (<0.5× or >2× median DPB). Biallelic indels were filtered to remove sites with any of the following: DPRA < 0.73 or > 1.48; QA or QR < 100; SRP or SAP > 2,000. VCF annotation was performed with the snpEff v.4.3 package  HaplotypeCaller v.4.1.0.0  using th package  using thWith this variant filtering we observed a correlation between MAF and read coverage at SNP sites . ModelinL. infantum isolate from Brazil . This produced an alignment of 283,378 sites. IQ-TREE v.1.5.5 (http://tree.bio.ed.ac.uk/). Treefiles are available in VCF containing all variants from all 477 isolates was converted to PHYLIP format using vcf2phylip (available at  v.1.5.5  was used v.1.5.5  approximr2 > 0.5) in 2 kb windows with a step size of 1 with PLINK v.1.9 , mixed ancestry strains were removed from population VCFs. Population-specific VCFs were filtered with VCFtools v.0.1.15  to removls of BS . VariantL. donovani were extracted from the annotation .gff, downloaded from TriTrypDB (version November 2019) with BEDtools v.2.27.1  and the nonadmixed BM L. infantum population (127 isolates) was used in the analysis for NCD2: Fixed difference sites were encoded as MAF = 0. Fixed differences were determined using bcftools isec called on VCFs of all L. donovani populations and L. infantum containing fixed variants, resulting in 76,284 fixed difference sites. Our target frequency of 0.5 (tf) and Equation 2 of Ztf-IS scores, with the exception of using the SD for each number of informative sites (IS) rather than simulated SD. P values for each window were calculated by assigning a rank based on Ztf-IS score and dividing by the total number of scanned windows.The NCD2 test used Equation 1 provided by https://github.com/grenaud/glactools) We performed the test on each individual population.The Betascan1* test , using dBetascan1* and NCD2 scores were calculated in windows around each variant site. To obtain values for each gene, we used the maximum Betascan1* score for all variants within the gene and the minimum NCD2 score within each gene (because low NCD2 scores are indicators of BS). Following the recommendations of L. donovani BPK282A1 reference genome were downloaded from TriTrypDB. GO enrichment analysis was performed using the PANTHER service on tritrypdb.org. Proteins that were classed as “hypothetical” or of “unknown function” were BLASTed against the nonredundant protein sequences (nr) database of NCBI to obtain possible identity by shared homology, and to determine conservation across trypanosomes.Gene Ontology (GO) descriptions and gene details for the Genome Biology and Evolution online.evab265_Supplementary_DataClick here for additional data file.