Lutzomyia umbratilis is the main vector of Leishmania guyanensis in the Brazilian Amazon and in neighboring countries. Previous biological and molecular investigations have revealed significant differences between L. umbratilis populations from the central Brazilian Amazon region. Here, a phylogeographic survey of L. umbratilis populations collected from nine localities in the Brazilian Amazon was conducted using two mitochondrial genes. Statistical analyses focused on population genetics, phylogenetic relationships and species delimitations. COI genetic diversity was very high, whereas Cytb diversity was moderate. COI genealogical haplotypes, population structure and phylogenetic analyses identified a deep genetic differentiation and three main genetic groups. Cytb showed a shallower genetic structure, two main haplogroups and poorly resolved phylogenetic trees. These findings, allied to absence of isolation by distance, support the hypothesis that the Amazon and Negro Rivers and interfluves are the main evolutionary forces driving L. umbratilis diversification. The main three genetic groups observed represent three evolutionary lineages, possibly species. The first lineage occurs north of the Amazon River and east of Negro River, where Le. guyanensis transmission is intense, implying that L. umbratilis is an important vector there. The second lineage is in the interfluve between north of Amazon River and west of Negro River, an area reported to be free of Le. guyanensis transmission. The third lineage, first recorded in this study, is in the interfluve between south of Amazon River and west of Madeira River, and its involvement in the transmission of this parasite remains to be elucidated. Such factors may reduce the dispersal capacity of sandflies, leading them to become isolated populations, with a resulting loss of genetic diversity within populations and the increase in differentiation between them5. The low flight capacity7, characteristic of these insects, and the type of soil used for breeding are also factors that may contribute further to population isolation in consequence favor genetic differentiation and, eventually, speciation. In this context, studying the microevolutionary processes that act at the level of sandflies vector populations is key for the identification of these patterns, which are baselines for the epidemiological studies, surveillance and vector control strategies.The diversification patterns  in phlebotomine Diptera have been linked to multiple factors, such as climate events, geographic barriers , biomes, latitude and altitude, habitat modification and landscape fragmentation caused by anthropogenic actions, among others8, lizards9, frogs10, birds12 and insects14—a scenario referred to as the “river-barrier hypothesis”15. This hypothesis predicts that major Amazonian rivers significantly reduce or prevent gene flow between populations inhabiting opposite banks, so promoting genetic divergence and increasing the opportunity for allopatric speciation. If this is correct, (1) it is expected that sister species will occur on opposite river banks, (2) the genetic similarity between populations will be greater in sites on the same bank that in sites on opposite banks separated by the same distance, and (3) the boundaries of species distributions will coincide with large rivers17.It has been proposed that the major rivers of the Amazonian region have acted, and continue to act, as significant dispersal barriers for a variety of taxa, including primatesLutzomyia (Nyssomyia) umbratilis Ward and Fraiha, 1977 is the main vector of the etiologic agent that causes American Cutaneous Leishmaniasis (ACL), Leishmania (Viannia) guyanensis Floch, 1954, in north South America19. In the Brazilian Amazon, L. umbratilis is highly anthropophilic and has been identified as the main vector of this parasite in the states of Amazonas, Amapá, Pará and Roraima21, an area that together holds > 50% of the cases of human leishmaniasis recorded for the whole Brazilian Amazon21. Lutzomyia umbratilis has also been implicated in the transmission of Le. guyanensis in French Guiana and Suriname23.25. In Brazil, L. umbratilis has been recorded in all states in the northern region24, as well as the states of Mato Grosso and Mato Grosso do Sul 27, and the states of Maranhão, Pernambuco and Alagoas, in the northeastern region30. This vast geographic area, with discontinuous distribution and the presence of geographic barriers, along with the low flight capacity of these insects7, means that L. umbratilis populations could be highly susceptible to evolve into differentiated populations, evolutionary lineages and distinct species.This phlebotomine species occurs in northern South America, including Bolivia, Brazil, Colombia, French Guiana, Guyana, Peru, Suriname and VenezuelaLutzomyia umbratilis was described by Ward and Fraiha31, based on specimens  captured in the Monte Dourado area of the Jari River (north of Amazon River), state of Pará, Brazil. Prior to this L. umbratilis had been wrongly identified as Lutzomyia anduzei Rozeboom 194232 due to their great morphological similarity. The species have overlapping geographic distributions across a large portion of northern South America24. Nowadays, they may be distinguished based on the internal and external genitalia morphology of male and female adults and by molecular markers33.L. umbratilis are often found in rainforest, especially in areas of the high humidity and dim light, as consequence, this species has been recognized as ombrophilous, as is expressed in its name – L. umbratilis. Adults of this species have been captured using aspirators in bases of tree trunks during daytime, as well as with CDC (Center for Disease Control) miniature light traps at ground level, and in the forest canopy at night – a behavior termed acrodendrophily34. Lutzomyia umbratilis seems to be abundant in the central Brazilian Amazon region, tending to decline towards the margins of this region35.In the Brazilian Amazon, adults of 36 reported that the susceptibility of this vector to Leishmania may vary within the central Brazilian Amazon region. These authors reported L. umbratilis populations naturally infected with Le. guyanensis to the north of the Negro and Amazon Rivers; but they did not observe natural infections by Leishmania in this species to the north of the Amazon River and west of the Negro River. Arias and Freitas13 hypothesized that this fluvial system could act as a vicariant barrier to Le. guyanensis transmission. In line with this hypothesis13, a study of experimental infection, performed with two samples of L. umbratilis from opposite banks of these rivers, revealed higher vector competence in a sample from north of Amazon River than in sample from north of the Amazon River and west of the Negro River37, supporting the Arias and Freitas’s hypothesis13. In fact, the presence of cryptic or sibling species complexes in a given region can produce heterogeneous patterns of parasite transmission, because they may differ in vector competence (the intrinsic ability of a vector to transmit a pathogen), host feeding preference, feeding behavior and breeding sites as well as they may either occur sympatrically or have distinct geographical distributions.Arias and FreitasL. umbratilis populations from Manaus and Manacapuru  revealed significant biological differences38. A second study that combined morphology and isozymes analyses of four L. umbratilis populations from this fluvial system showed subtle morphological differences in the immature stage and in female genitalia, whereas the isozymes markers did not reveal any differences among populations studied39. However, isozymes are not appropriated markers to separate species that have recently diverged.A biological study conducted with COI-mtDNA) revealed low and non-significant differentiation between populations situated on the same banks of the Amazon and Negro Rivers, but marked and very significant differences between populations situated on the opposite banks of these rivers40. This pattern resulted in the absence of isolation by distance (IBD). Although all haplotypes were connected in a unique network, the strong bimodality observed with the mismatch analyses, when all samples were analyzed together, was consistent with long-term isolation, suggesting historical isolation between the analyzed populations40. These groups probably attained complete isolation following the most recent formation of the Negro and Amazon Rivers network (~ 2.4 Mya to present), which was considered as the main evolutionary force. In line with these results, another study, using the Barcode region (COI) of these L. umbratilis two groups, also failed to find evidence for haplotype sharing. However, they could be identified by unique fixed mutation, as well as the genetic distance was rather small33. Later, Freitas and others41, using also a COI fragment, compared Manacapuru and Rio Preto da Eva populations, situated on the opposite banks of the same rivers, with a sample of Recife, in the northeast region from Brazil. Consistent with previous studies40, two distinct groups were observed: one group clustered individuals from Rio Preto da Eva (north Amazon River and east Negro River) plus Recife and, another group, clustered only individuals of Manacapuru (interfluve located of the north Amazon River and west of Negro River). This finding indicates that the Recife population may have been derived from populations from the north Amazon River.Unlike the results with isozymes, a phylogeographic structure study using molecular markers  to test the hypotheses: (1) Are the largest rivers in the Brazilian Amazon acting as barriers on the L. umbratilis populations? (2) Are the populations located on the opposite banks of the Amazon and Negro Rivers and in the interfluves genetically different? ; (3) How many lineages or species exist within L. umbratilis complex in the region studied?However, most of these studies are based on samples from a small number of locations. As a result, additional investigations are needed to fully elucidate the number of species in this complex and the role of each one in the transmission of L. umbratilis adults were performed in nine localities within the Brazilian Amazon, with seven localities in the state of Amazonas , one in state of Pará , and one in state of Amapá (Porto Grande/Serra do Navio [PG]). Figure : Cachoeira Porteira , km 43 of BR-174 Highway , Rio Preto da Eva , Manaus , Pitinga  and Porto Grande/Serra do Navio ; (2) north of Amazon River and west of Negro River : Manacapuru  and Novo Airão ; (3) south of Amazon River and west Madeira River : Ramal do Sampaio, in the municipality of Autazes . Table Collection of COI sequences of the individuals from Cachoeira Porteira, km 43 of BR-174 Highway, Rio Preto da Eva, Manaus, Manacapuru and Novo Airão were previously analyzed by Scarpassa and Alencar40, while three new samples  were analyzed in this study using the same COI fragment , using permanent license number 38440-1 awarded to VMS.Sandflies were collected with CDC miniature light traps COI gene, 176 specimens were analyzed, with 111 from the study of Scarpassa and Alencar40, and 65 analyzed specifically for the current study. For the Cytb gene, 187 specimens from the nine localities were analyzed in this study  or in sterile water. A small aliquot of this DNA was stored at − 20 °C and used in the amplification by polymerase chain reaction (PCR) of the two analyzed markers. All DNA remaining from the specimens was kept frozen at − 81 °C as voucher DNA in the vector insect collection at the Laboratory of Population Genetics and Evolution of Vector Mosquitoes at the Instituto Nacional de Pesquisas da Amazônia, in Manaus, Brazil. DNA extracted from all individuals was amplified with specific oligonucleotide primers for the two genes, as described below.Total genomic DNA was extracted individually from whole sandflies using the Phenol–Chloroform protocolCOI), was amplified by PCR using the primers described in Zhang and Hewitt44. The PCR reactions was included Platinum Taq DNA Polymerase of High Fidelity (LIFE TECHNOLOGY), and negative control was included for all reactions. The PCR products were electrophoresed in 1% agarose gel stained with Gel Red Nucleic Acid Gel Stain  and visualized under UV light to check the size and quality of expected products. The amplified products were purified using PEG precipitation (20% polyethylene glycol 8000 and 2.5 M NaCl). Both DNA strands were sequenced using the Big Dye Terminator Kit, v. 3.1. The reactions were electro-injected in an automated ABI 3130 xl Genetic Analyzer .A long fragment (1181 bp), representing the variable region of end 3’ of cytochrome c oxidase I gene (Cytb gene was amplified by PCR using the primers described in Coutinho-Abreu and others45. The PCR reactions was included Platinum Taq DNA Polymerase of High Fidelity (LIFE TECHNOLOGY), and a negative control was used in all reactions. The PCR products were electrophoresed in 1% agarose gel stained with Gel Red Nucleic Acid Gel Stain , and visualized under UV light to verify the size and quality of expected products. Amplified products purification and sequencing reactions were carried out as described above.A fragment of 512 bp from the COI and Cytb sequences were automatically aligned using Clustal W, and manually edited in BioEdit v. 7.0.8.046 with the aid of the electropherogram viewer Chromas Lite47. Consensus sequences of both genes were confirmed with the mitochondrial genome of L. umbratilis48, using the BLAST (BASIC LOCAL ALIGNMENT SEARCH TOOL)49, which resulted in 99 to 100% identity. The sequences of this study were mapped between the positions 1759 and 2939 bp for COI, and between the positions 11,241 and 11,752 bp for Cytb of mitochondrial genome of this species48.COI and Cytb datasets were checked for saturation levels against genetic distances using DAMBE50, which allows testing for the presence of any saturation between transition and transversion rates in relation to genetic distances. Results showed no saturation for either marker, indicating the datasets are appropriate for the applied phylogenetic inferences. Haplotypes were determined using DnaSP v. 5.1051 and TCS v. 1.2152. Identical sequences were considered to represent a single haplotype. To analyze population history and haplotype genealogy, haplotype networks were analyzed for the two markers separately. To calculate the maximum number of mutational connections between pairs of sequences, the 95% parsimony criterion was used, with analyses run on TCS v. 1.2152. Using this criterion, the possibility that all samples analyzed formed a single network or two or more disconnected networks was investigated.Both 53 and Bayesian Inference (BI) with the Mr.Bayes v. 3.2.554 and *BEAST v. 2.055 algorithms. Kimura2-Parameters (K2P) distances56 were used for the NJ analysis, with 5000 replicates, while the ML and BI analyses were inferred using the General Time Reversible (GTR) + G + I nucleotide substitution model for COI, and the HKY nucleotide substitution model for Cytb, both previously selected with the Akaike Information Criterion (AIC) via jModelTest57. In the ML analyses, a tree was generated with 2000 replicates. In the BI analyses, for each marker two simultaneous independent runs of the Markov Chain Monte Carlo (MCMC) were performed for 100 million generations, while sampling every 1000 generations with a burn-in of 25%. Bayesian Posterior Probabilities (BPP) were used to assess nodal support.Unfortunately, concatenated analyses with two markers were not possible as the DNAs of the same individuals from Manacapuru and Novo Airão populations did not amplify for both markers used. Thus, the phylogenetic relationships were inferred with all haplotypes for each marker using Neighbor-Joining (NJ) and Maximum Likelihood (ML) in MEGA v. 7.0COI) were used: GMYC 58, bGMYC (with implementation Bayesian of GMYC)59 and ABGD (Automatic Barcode Gap Discovery)60. Delimitation with the GMYC approach is based on assigning branching events to two categories, speciation and coalescent within species. With the assumption that species are monophyletic, a set of most recent common ancestor nodes can be specified that determines the type of branching events. bGMYC model identifies uncertainties in the limits of the species as per the changes in the ramification rates in the phylogenetic tree when distinct populations contain several species. ABGD model allows partitioning of the DNA sequence datasets into clusters of similar taxa, establishing a range of maximum values of intraspecific divergence (P), without an a priori species hypothesis. These analyses were conducted using the bGMYC SPLITS package61 and implemented in the R package, version 3.6.061. The ultrametric trees were generated with *BEAUTi and *BEAST v.1.762 and used in these analyses.To infer the limits between lineages, three models based on single locus data  were also estimated in *BEAST v. 2.0COI and Cytb regions extracted from mitochondrial genome of L. umbratilis from Saint-Georges de l’Oyapock, French Guiana [Accession numbers: KP702938 and KP702939]48. Saint-Georges de l’Oyapock is situated on the border with the state of Amapá, Brazil, and lies north of the Amazon River  was also implemented for the h) and nucleotide (π) diversities, the average number of nucleotide differences (K), number of polymorphic sites (NS), transition and transversion rates (Ts/Tv), and the neutrality tests of Tajima’s D66 and Fu’s Fs tests67 for each sample and total were inferred using DnaSP, v. 5.1051 and Arlequin, v. 3.168. Tajima’s D was run to test strict neutrality, while the neutrality test, Fu’s Fs, was estimated to test population size stability. The latter test is more powerful for detecting population expansion and genetic hitchhiking.The intra-population genetic diversity measures, such as haplotype , the number of net nucleotide substitutions per site between samples (Da), the number of shared polymorphisms between samples pairs (Ss), and a number of fixed differences between samples pairs (Sf) were calculated in DnaSP, v. 5.1051.Using the 70 for both markers. In these analyses, all sequences from both datasets were used and 1–9 clusters were employed , and five independent runs were made. The most probable genetic cluster configuration was prepared by comparing the log-likelihood values of the best models.To check the number of genetic groups obtained in the previous analyses, we implemented the Bayesian Analysis of Population Structure (BAPS) with BAPS softwarep genetic distance. These analyses were calculated in MEGA, v. 7.053, and the standard errors were estimated by bootstrapping with 1000 replicates.The genetic groups retrieved in the haplotypes network and phylogenetic analyses were used to estimate the mean intra- and inter-group genetic distances using the uncorrected-FST values among samples was applied using a Mantel test71 in Arlequin, v. 3.168 with 2000 permutations. Straight-line geographic distances among the localities were estimated using Google Earth.To test the isolation by distance (IBD) hypothesis, the correlation between straight-line geographic distances and L. umbratilis from the Brazilian Amazon were estimated. A total of 176 specimens were analyzed for COI and 187 for Cytb, totaling 363 individuals sequenced  were more common than transversions , with most occurring at the third codon position  for both markers. COI and Cytb had 110 (9.31%) and 41 (8.01%) variable sites, of which 56 (4.74%) and 14 (2.73%) were parsimoniously informative, respectively. The average nucleotide composition for the COI was 39.9% T, 30.7% A (A + T = 70.6%), 15.2% C, and 14.1% G, while for Cytb these values were 39.5% T, 38.5% A (A + T = 78%), 13.5% C, and 8.5% G.The molecular variation and phylogeographic structure of nine natural populations of COI, the 176 individuals generated 89 haplotypes, of which 52 (H1 to H52) had been described by Scarpassa and Alencar40, and 37 being new haplotypes (H53 to H89) identified in this study. Of these, 79 (88.76%) haplotypes were singletons or exclusive to a single sample. Table For Cytb, the 187 sequences generated 36 haplotypes, ranging from two (Autazes) to nine (Cachoeira Porteira) haplotypes per sample  tree topology inferred with the 89 haplotypes from ups Fig. ; howeverups Fig. .Figure 4COI . The second group consisted of two large subgroups: A) the most apical subgroup consisted three clusters: (A1) all haplotypes from the Cachoeira Porteira, BR-174 Highway, Rio Preto da Eva, Manaus plus H18 (shared), H73 and H74 from Pitinga and the Saint Georges l’Oyapock sequence (BPP: 0.96); (A2) all haplotypes from Porto Grande/Serra do Navio (BPP: 0.99); (A3) haplotypes  from Pitinga, which formed sister cluster with Porto Grande/Serra do Navio (BPP: 1.0); (B) the most basal subgroup consisted two clusters (BPP: 0.93): (B1) all haplotypes from Manacapuru and Novo Airão (BPP: 0.98), and B2) haplotypes  from Pitinga (BPP: 0.99), which formed sister cluster with Manacapuru and Novo Airão populations. In this analysis L. anduzei and B. flaviscutellata were used as outgroups.Bayesian Inference (BI) tree based on COI Fig.  retrieveL. umbratilis, respectively  and BI trees inferred with the 36 haplotypes from 55 , in the samples from Manaus, Pitinga, Autazes, Porto Grande/Serra do Navio (Table Fs test was negative and significant in samples from Manaus (P < 0.0001) and Porto Grande/Serra do Navio (P < 0.05), consistent with recent population expansion or positive selection. For Cytb, Tajima’s D and Fu’s Fs tests were negatives and significant (P < 0.05) in samples from Cachoeira Porteira, BR-174 Highway, Rio Preto da Eva and Manaus, favoring the interpretation of recent population expansion or selective sweeps. This finding is compatible with the observed star-shaped haplotype network  to 0.9333 (Rio Preto da Eva versus Autazes).Table Cytb, the results obtained for the six samples  were similar those described for COI by Scarpassa and Alencar40, where low genetic distances were observed between localities situated on the same side of the rivers and within interfluves, whereas high and significant genetic distance values were detected between localities from opposite sides and between interfluves. But, when the Pitinga, Autazes and Porto Grande/Serra do Navio samples were included in the comparisons, the genetic distance values varied depending on the sample. Higher FST values were observed (0.4870) between Manaus versus Pitinga (same side) than between Manacapuru versus Pitinga samples (0.1559) and Novo Airão versus Pitinga (0.1395), situated on opposite sides of the rivers.For Dxy), the number of net nucleotide substitutions per site between samples (Da), the number of shared polymorphisms between pairs of samples (Ss), and the number of fixed differences between pairs of samples (Sf) for the two genes. For COI, similarly FST statistic, the highest Dxy, Da and Sf values were found in the comparisons between Autazes versus Cachoeira Porteira and between Autazes versus Manacapuru (both with Sf = 15), and between Autazes versus Manaus and between Autazes versus Porto Grande/Serra do Navio (both with Sf = 18). For Cytb, the highest values were between Autazes versus Manacapuru and between Autazes versus Novo Airão (both with Sf = 8).Table COI, and three for Cytb   Figs.  and S4.p distance) was also calculated between lineages retrieved from the haplotype networks  to 2.3% (between lineages IV and VI). Between lineages and L. anduzei and B. flaviscutellata s.s. the mean genetic distances were 5.7% and 11.9%, respectively. Table Cytb genetic distances. These ranged from 0.05% (between lineages I and II) to 1.7% (between lineages II and III). Between the three lineages and L. anduzei genetic distances varied from 4.4% to 4.5%.Genetic distance .A Mantel test was used to analyze the correlation between genetic and geographic distances among samples for each marker. These analyses showed that there was no correlation between genetic and geographic distances for either marker  haplotypes from Pitinga  showed shared haplotypes and low genetic differentiation, with no or few fixed sites, and having most haplotypes clustered within the same haplogroup, as well as in phylogenetic analyses Figs.  reflectiytb Fig.  identifiytb Fig.  providedCOI40, the Cytb analyses also found that populations from Manacapuru and Novo Airão, situated in the interfluve of the north Amazon River and west of Negro River, were genetically similar to each one  of the Amazon River and east of Negro River (Ss = 0 and Sf = 6–7), and between Manacapuru/Novo Airão and Autazes . These findings reinforce the evidences that the Manacapuru and Novo Airão populations could represent a distinct evolutionary lineage  from the remaining specimens studied here. This was shown by a disconnected haplotype network  in the most comparisons  as a porous barrier to gene flow between populations of the opposite sides in this stretch of River. Further investigations, focusing on a broader sampling and the use of genomic approaches, such as RADseq that provide higher resolution to detect genetic structure and heterozygous individuals, will be needed to clarify these findings recorded at Pitinga.Lineage delimitation based on the bGMYC model identified six lineages for udy Fig. , a resulks Figs. , 3 and iks Figs. , 5, 6. Tks Figs.  indicateks Figs. , supportks Figs.  also shoks Figs.  and for ver Fig. a. AnavilOn the other hand, the Lineage 6 (Autazes) was the most isolated from remaining lineages Fig. ; a resulp distances were low between lineages , contrasting with very high FST values between samples 92, Anopheles triannulatus (1.7–2.3%)93 and Anopheles nuneztovari (1.6–2.8%)94 complexes, which characterize these species as of recent evolutionary origin. Thus, the differentiation level observed here is indicative of recent divergence between lineages, supporting our conclusions as reported above. This occurs due to the fact that, in young species, fixed differences may be observed only in genes involved in the speciation process95.The values for uncorrected-es Table . StudiesL. umbratilis populations north of Amazon/east of Negro Rivers and north of the Amazon/west of Negro Rivers revealed appreciable and significant biological differences between them38. Justiniano and others38 compared the first laboratory bred generations of L. umbratilis populations obtained of the opposite banks of the Negro River (Manaus and Manacapuru). Interestingly, these samples showed remarkable differences in their life cycle, fecundity, fertility and adult longevity, with the population from Manaus being more productive and living longer than the population from Manacapuru. Such differences could be due to intrinsic biological features resulting to distinct evolutionary adaptations due to their geographical isolation by the Negro River. On the other hand, Justiniano39 observed discrete morphological differences in the immature stage, and in the number and size of the spines of armature of the female genital atrium between populations of these locations. The results of this study support the previous biological38 and molecular studies41 and, when taken together, it is possible to infer that the populations north and south of the Amazon River and interfluves represent distinct “species”, which could have diverged more biological and molecular levels than morphologic39 level, with implications distinct epidemiologic37. Genomics approach using loci involved in the different biologic aspects of L. umbratilis may clarify this issue. For example, some of these efforts may be focused on the mechanisms that determine the efficiency as vector, as well as to identify specific loci (outliers) that are acting in the most variable adaptability processes of these lineages. In this study, we also recorded a new independently evolving lineage in Autazes. Hence, our findings provide convincing arguments in favor of the hypothesis that what is currently termed L. umbratilis represents, in fact, at least three evolutionary lineages, possibly species, in the studied area.Previous studies conducted with Le. guyanensis transmission is intense, implying that L. umbratilis is an important vector there. The second lineage occurs in the interfluve between north of the Amazon and west of the Negro Rivers , because it occurs in the same bank as the type locality 31, so that the other two taxa therefore represent unrecognized species.Overall, the first lineage occurs north of the Amazon and east of Negro Rivers (Lineage I; Fig. L. umbratilis three lineages in the Brazilian Amazon. The origin of these lineages is most likely to be associated with the formation of the Amazon drainage system and its tributaries. The first and second lineages are on the north of the Amazon/east of the Negro Rivers and north of the Amazon/west of the Negro Rivers, respectively; the third lineage is known from the interfluve between south of the Amazon River and west of the Madeira River. The three lineages may represent three species. However, their confirmation will require detailed examination morphological of immature and adult stage combined with molecular studies to provide diagnostic characters that can allow them to be recognized as valid species. Given the strong evidence of genetic subdivision within the Pitinga population, we do not rule out the possibility that more lineages or species exist in the Brazilian Amazon, as well as in other locations unsampled in northern South America. These findings will aid epidemiological studies, surveillance and vector control programs in these regions, especially north of the Amazon River, where the transmission by Le. guyanensis can be very high.In conclusion, our findings revealed Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Information.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


Technomyrmex brunneus. This species is native to southern Asia and has invaded many islands of the southern Japanese archipelago, allowing us to compare traits among different invasive populations. We find a supercolony in one of the three invasive populations studied, though interestingly, this population did not show the expected pattern of lower genetic diversity. The supercolony population did differ in diet compared to other populations. While it is unclear if variation in these traits is linked, our findings add weight to arguments that plasticity is important in facilitating invasiveness.Invasive ants are a global economic and ecological problem. Understanding what makes them tick is thus an active area of scientific research. Many invasive ant species form large networks of inter-connected colonies (‘supercolonies’) which can span many hundreds of kilometres. Supercolonies are typically a characteristic of invasive populations, and studies have also identified shifts in other traits including diet in invasive populations compared to populations in the native range. This suggests that the ability to plastically change behaviour in this way may facilitate invasiveness. In this study, we assess trait plasticity in the invasive range of the ant Technomyrmex brunneus in Japan to assess trait plasticity in the invasive range of this species. We find evidence for plasticity in social structure among island populations, with a supercolony evident on one of the three islands studied. Interestingly, we found no evidence of lower genetic diversity in this population, though natural isotope data indicate it was operating at a lower trophic level than other populations. These findings add weight to arguments that invasive species may benefit from the capacity to adaptively mould themselves to new ecological contexts.Characters in invasive populations often differ from those in the native range, and the ability to express different characters may enhance invasive potential. Ants are among the most pervasive and damaging invasive species, by virtue of their transportability and broad-ranging ecological interactions. Their success is often attributed to the ability to exhibit different characteristics in invasive populations, including the formation of large, unicolonial associations (‘supercolonies’). It remains unclear, however, if such characteristics are a product or cause of the ecological dominance of invasive ants, and the advancement of our understanding has likely been restrained by the fact that studies to date have focused on a few globally important species with well-established invasions. In this study, we take advantage of an ongoing invasion of the tramp ant  Linepithema humile and the Red Imported Fire Ant Solenopsis invicta exhibit shifts to lower trophic positions in their invasive ranges [Social insects are among the most successful animal invaders and dominate the list of the most invasive terrestrial arthropods ,2,3. Inve ranges ,28. Suche ranges ,28. Mutue ranges ,30,31.L. humile and S. invicta e.g., [Studies on trait plasticity in invasive species have to date have largely focused on well-established populations of major pest species, such as ta e.g., ,32,33 anta e.g., ,34,35. TTechnomyrmex brunneus Forel, 1895 (Hymenoptera: Formicidae: Dolichoderinae) in the southern islands of Japan, to investigate trait plasticity among invasive populations of this species. This species is endemic to east, southeast, and south Asia and invasive in Japan, Korea, Borneo and New Guinea [T. brunneus is present as an invader in the southern-most mainland island, Kyushu, the oceanic islands of Ogasawara, and most, if not all, of the island chain extending from Kyushu to Taiwan. Reports of this species in Japan extend as far back as the early 1900s  when it was reported in Kyushu [T. brunneus, polydomous (multiple nest) colonies can consist of millions of adults, and may include both ergatoid (wingless) queens and males, as well as the usual alate (winged) sexual forms [T. brunneus in at least one invasive population in Japan appears to exhibit supercoloniality [In this study, we take advantage of the ongoing invasion of w Guinea . In Japan Kyushu . It was n Kyushu  and in tn Kyushu , while in Kyushu  more recn Kyushu . The disal forms ,42. Thisal forms . It is aal forms , the sugoniality , while tTechnomyrmex brunneus ants were sampled from three island populations in the sub-tropical region south-east of the main islands of Japan between latitude 26 and 33° N  by far the largest of the three, and Chichijima 23.45 km2, 27°07’44″ N, 142°21’78″ E) considerably more isolated than both Okinawa and Hachijojima . These islands also differ considerably in invasion age: T. brunneus was established in Okinawa by the 1980s [nd 33° N : Hachijond 33° N . Climatehe 1980s , in Ogashe 1980s .Ants were sampled using forceps from foraging trails or the upper parts of nests by removing the surface of logs of lifting rocks and transferred directly to 99% ethanol for genetic analysis or transferred to tubes with wet tissue for later behavioural observation. To assess how traits which might be associated with invasiveness vary among these populations, we assessed the following characteristics for ants in each location: supercolony status, trophic niche, population genetic structure, and morphological diversity. Details are provided for each of these factors below. In most cases, a random sub-sample of all colonies was used in each analysis, though samples from the four sites in north-eastern Okinawa were from traps and thus could not be used for isotope or behavioural analyses. Details on allocation of colonies to analyse are summarised in Ants typically display high levels of aggression between colonies, and the absence of inter-colony aggression can be used as an indicator of supercoloniality . Behavio13C to 12C in parts per million, expressed as δ13C) and nitrogen  can be used to infer tropic niche, as the ratio of different isotopes of carbon can help discriminate among different sources of primary production (sugars), while that of nitrogen isotopes can be used to infer trophic position, because the δ15N of a consumer is typically enriched by ~3.0‰ for each trophic level [3 were taken from the upper mineral soil layer (5–15 cm) within 1 m of each ant collection site and kept refrigerated until analysis. Ants were removed from alcohol within five days of the collection as extended storage in alcohol can influence isotopic measurements [15N/δ13C notation, as per million deviations relative to international standards. The analytical error during the overall process of mass spectrometry was less than 0.2 ‰. Baseline corrected values of δ15N and δ13C were calculated for each sampling site as ant values—soil values.Natural isotope ratios of carbon  using t Matsuki  except f Matsuki  to ident Matsuki , removinadegenet [AN), allelic richness (AR), expected heterozygosity (EH), observed heterozygosity (OH), and inbreeding coefficient (ISF). Isolation by distance was assessed using a Mantel test implemented in the ade4 package of R [poppr package of R [STF in hierfstat [pegas package of R [STF and ISF, we determined 95% confidence intervals (CIs) of estimates using 10,000 bootstraps and considered values significantly different to zero if CIs did not span zero. Finally, we used DAPC  implemented in adegenet to describe genetic clusters of individuals using K-means clustering of principal components. We ran K-means clustering with different numbers of clusters (K = 1–10) and identified the optimal number of clusters as the value of K with the lowest BIC.We used the R package adegenet  to quantage of R . Geneticage of R . We alsoage of R  FST in hierfstat . We asseage of R , correctage of R . For bothttp://imageJ.nih.gov/ij/; accessed on 20 January 2020) based on photographs taken using a Canon EOS Kiss X9 digital camera attached to Nikon AZ100 stereomicroscope. We measured five morphological characters for five workers from each of 10 colonies from each population: Head Width (HW): maximum width of head including eyes, measured in full-face view; Head Length (HL): maximum length of head in full-face view; Tibia Length (TL): maximum length of the hind femur, measured in anterior view; Mesosoma Length (ML): diagonal length of the mesosoma in profile from the pronotum to the posterior basal angle of the propodium; Scape Length (SL): maximum length of antennal scape  and thus reduce colony efficiency, which relies on this diversity ,65,66,67al scape . These clmtest [multcomp package [factoextra [Statistical analyses were performed in R v. 4.0.2 . For agglmtest  on model package . Isotopectoextra . PCA facctoextra .2 = 161.09, p < 0.001; mean aggression score: χ2 = 158.01, p < 0.001). Pairwise comparisons indicated no difference between Okinawa and Chichijima for number of interactions or aggression score , while Hachijojima differed significantly from both Okinawa and Chichijima for number of interactions  and mean aggression score . There was no influence of distance between colony pairs on aggression within islands overall  and no interaction between distance and island .We found clear differences in intra-colony aggression between populations. Aggressive behaviours were not observed in Hachijojima, with all assays obtaining a score of 0 (no aggression), while we found consistently high aggression, with scores of 2 or 3, in Okinawa and Chichijima . Both th2 = 23.59, df = 2, p < 0.001) but not for carbon . Post-hoc tests indicated that δ15N values for Hachijojima were significantly lower than both Chichijima  and Okinawa , but that there was no difference between the latter two sites , while Hachijojima exhibited a slightly lower range of values for δ13C than either Okinawa or Chichijima.Baseline-corrected isotope values of ants varied significantly between islands for nitrogen . AMOVA indicated that 66.8% of the variation was within individuals, 23.8% within populations and 9.4% between populations, with this differentiation significant at all levels (p = 0.01 in each case). The DAPC analysis inferred that the optimal clustering was obtained with K = 2, and we summarise these results and those for K = 3 in K = 2 analysis segregated largely into Chichijima and (Okinawa + Hachijojima) clusters. When considering a K of 3, the pattern of clustering indicated that Chichijima samples were separated from other populations, while admixture occurred between populations from Hachijojima and Okinawa. Overall, these data indicate that there was low genetic variation within all populations, and no indication that the supercolony population in Hachijojima was structured differently to other sites. Island populations were genetically distinct from each other, though there was a higher affinity between Okinawa and Hachijojima than either of these islands and Chichijima.We found no significant pattern of isolation by distance (IBD) at any site, though inbreeding coefficients were significantly different from zero at all sites, and all sites also exhibited deviations from HWE at some loci. Pairwise We found only minor variation in morphological characters between populations . CompariTechnomyrmex brunneus in the southern islands of Japan. Genetic analyses indicated that, while all populations were genetically distinct, the Hachijojima and Okinawa populations were genetically more closely associated with one another than either population was with the Chichijima population. This is consistent with the relative isolation of Chichijima, which is part of the Ogasawara archipelago, an isolated oceanic island chain ~1000 km from the Japanese mainland and ~1500 km from Okinawa. In contrast, both Hachijojima and Okinawa are of continental origin and connected by numerous other intermediate islands. Nonetheless, the differences in life-history characteristics we identified were not consistent with the genetic relationships between populations. Our results indicate the population in Hachijojima constitutes a supercolony, confirming earlier reports [In this study, we compare the life-history characteristics of three invasive populations of the ant  reports , while wT. brunneus in Japan seem typical of invasive ants in exhibiting low genetic diversity and a lack of genetic isolation by distance (IBD) [Supercolonies can arise because of a breakdown of the recognition system ants use to discriminate ‘friends’ (nestmates) from ‘foes’ (non-nestmates) ,33,72. Tce (IBD) ,24,80. Tce (IBD) ,41 and iL. humile [S. invicta [Formica paralugubris were both up-shifted and down-shifted compared to native range populations [S. invicta [Technomyrmex ants [13C values observed in this population. This might also help explain the extremely high densities reported in this population, as carbohydrate supplementation can fuel colony growth, worker activity and aggression in ants, thus enhancing competitive performance [Anoplolepis gracilipes . Previo invicta ), possib invicta ,82,83. Imex ants ,84. The formance ,31,81. Wformance , and theOkinawa: ,86. An iOkinawa: ,29. ThisT. brunneus in Japan, by comparing social structure, trophic niche, population genetics, and morphology among three invasive populations. We find evidence of plasticity in trophic niche and social structure, with a supercolony and use of a lower trophic niche in Hachijojima. This supercolony was not associated with lower genetic diversity, suggesting an ecological basis for this difference. While it is possible that there were causal links between supercolony status and the differences in trophic level, we cannot rule out other influences such as differences in the timing of invasion and/or differing ant communities between sites. These data lend support to the idea that plasticity is common in invasive species and may be a facilitator of invasiveness [T. brunneus in the native range are needed to establish if the traits exhibited within invasive populations in Japan are pre-adaptive or the result of ongoing evolution during the invasion process [T. brunneus and other ongoing invasions will help develop our understanding of the degree to which dietary shifts and supercoloniality can independently or collectively facilitate invasiveness in ants.In this study, we assessed inter-population plasticity in traits likely to be associated with invasiveness in siveness ,10,11. Wsiveness ,88, a su process ,16,87,89 process ,42 may v


ADH) and lactase persistence (LCT), were not identified, while the higher/lower frequencies of derived mutations were strongly correlated with the genetic affinity to East Asian/Western Eurasian populations. Our attested complex population movement and admixture in the agriculture and pasture interlaced area played an important role in the formation of modern Mongolians.Mongolians dwell at the Eastern Eurasian Steppe, where is the agriculture and pasture interlaced area, practice pastoral subsistence strategies for generations, and have their own complex genetic formation history. There is evidence that the eastward expansion of Western Steppe herders transformed the lifestyle of post-Bronze Age Mongolia Plateau populations and brought gene flow into the gene pool of Eastern Eurasians. Here, we reported genome-wide data for 42 individuals from the Inner Mongolia Autonomous Region of North China. We observed that our studied Mongolians were structured into three distinct genetic clusters possessing different genetic affinity with previous studied Inner Mongolians and Mongols and various Eastern and Western Eurasian ancestries: two subgroups harbored dominant Eastern Eurasian ancestry from Neolithic millet farmers of Yellow River Basin; another subgroup derived Eastern Eurasian ancestry primarily from Neolithic hunter-gatherers of North Asia. Besides, three-way/four-way qpAdm admixture models revealed that both north and southern Western Eurasian ancestry related to the Western Steppe herders and Iranian farmers contributed to the genetic materials into modern Mongolians. ALDER-based admixture coefficient and haplotype-based GLOBETROTTER demonstrated that the former western ancestry detected in modern Mongolian could be recently traced back to a historic period in accordance with the historical record about the westward expansion of the Mongol empire. Furthermore, the natural selection analysis of Mongolians showed that the Major Histocompatibility Complex (MHC) region underwent significantly positive selective sweeps. The functional genes, alcohol dehydrogenase ( The vast Eurasian steppe zone stretching from Hungary in the west to Mongolia and northeastern China in the east has witnessed a dynamic demographic history. Ancient DNA findings from Western Eurasian Steppe showed the massive continental-scale steppe population migrations, admixture, and turnover since the Early Bronze Age . Both arLCT/MCM6) leading to the negligibly low frequency of derived mutations conferring lactase persistence indicates that animal husbandry for livelihood was adopted in the Eastern Steppe by local hunter-gatherers instead of causing by massive populations movements and turnover in Mongolian . Informed consent was obtained from all participants included in the study.Genotyping was performed on the Illumina arrays covering genome-wide 600,000 SNPs designed to identify all known paternal Y chromosome and maternal mtDNA lineages. We first analyzed the relatedness of individuals measured by IBD (identified by descent) segments using KING software ; unrelatf3 in the form of f3  and f4 statistics using qp3Pop and qpDstat in the ADMIXTOOLS package  on the merged dataset using the smartpca built-in EIGENSOFT package . Modern  package . We addeIGENSOFT  with fstIGENSOFT . We condf3-statistics in the form f3 using the qp3pop program with default parameters in ADMIXTOOLS to explore the potential admixture surrogates showing significantly negative f3 value. For modeling f4 statistics-based admixture and estimating ancestral proportions in Mongolian, we applied qpWave  is consistent with being a clade—since separation from the ancestors of a set of outgroup populations. qpAdm (A and PX divided by their standard errors (Z=(PA-PX)/√ἀA2+ ἀ X2, where ἀA and ἀ X are the corresponding jackknife standard errors)  . Therefo errors) . To unde errors) .via heatmap, clustering dendrogram, and PCA.Bayesian clustering implemented in FineSTRUCTURE was used to reconstruct polygenetic relationships and further identify population structure. To reduce the computational burden, we randomly sampled 10 to 20 individuals in a large reference group. We first phased genome-wide dense SNP data using the SHAPEIT2 version  and thenWe performed a GLOBETROTTER  analysisvia DAVID Bioinformatics Resources (http://geneatlas.roslin.ed.ac.uk/) and GTEx , respectively.The integrated haplotype score (iHS) and XP-EHH analysis were conducted to identify recent natural signatures of positive selective sweeps in the Mongolian population using the R packaged rehh2 . The SNPesources  and searWe generated and filtered 39 unrelated Mongolian individuals from Inner Mongolia Autonomous Region and merged the data with that published on modern and ancient populations in Eurasia to obtain a comprehensive population profile. In a principal component analysis (PCA) of Eurasian individuals, modern and ancient Eastern and Western Eurasian populations were separated into PC1 and PC2 split Eastern Eurasians along a north-south cline with Tungusic and Mongolic speakers who also connecting with the west-east Eurasian cline . MongoliA model-based populations clustering analysis using ADMIXTURE showed a similar pattern . Overallf4 statistics of all individuals  analysis and pairwise ividuals . Taking f4 reflecting as no significant Z  showed that Mongolian_inner possessed the most shared ancestry with modern Han groups and Mongolian_outer had strong genetic drift with Tungusic populations, while Mongolian_mid shared closer genetic affinity with Han and Tungusic populations. The genetic affinity profile also demonstrated that, in outgroup f3   , Mongoli; Mbuti) . When in; Mbuti) .f4 statistics test in the form of f4    (The genetic differentiation and affinity profile among three Mongolian subgroups was further certified by an_sub2) , showingan_sub2) . The resan_sub2) , f4(Mla_HGDP) , and fian_NHT)  did provf4 (|Z| < 3) and f4  ( part of |Z| < 3) , Mongolian_BX that showed the genetic homogeneity with Mongolian_outer/Mongol/Mongolia_Medieval, and Buryat that showed the genetic continuity with Mongolia_Medieval . We obta|Z| < 3) . In addiThe phased Mongolian and Eurasian populations data were also used to conduct haplotype-based fineSTRUCTURE and the finer-scale population structure of Mongolian was further comprehensively characterized. The inferred polygenetic tree showed that Mongolian_inner clustered with Mongola_HGDP and Mongolian_BCET, one part of Mongolian_mid clustered with Mongola, and the others clustered with Mongol, while Mongolian_outer was clustered with Mongolian_BX and Mongol . Besidesf3) tests to characterize the admixed gene pools of three Mongolian subgroups. Testing all possible pairs of 115 present-day “source” groups and 117 ancient “source” groups, we detected highly significantly negative f3 statistics , supporting the qualitative impression of east-west admixture from PCA and ADMIXTURE analyses. To highlight the difference among Mongolian subgroups, we looked into f3-results with representative reference pairs comprising ancient Eurasians . Farmer-related ancestry was the best representation of Eastern Eurasian ancestry for Mongolian_inner compared to Ulchi; farmer-related and Neolithic hunter-gatherers–related ancestry (Ulchi is regarded as the most genetic homogeneous population with Neolithic hunter-gatherers of DevilsCave) both represented ancestries related to Eastern Eurasian well in Mongolian_mid and Mongolian_outer. Considering the admixture events and sources that we observed in Mongolian subgroups, we applied qpWave/qpAdm to validate different proposed admixture scenarios and ancestral proportions. In the two-way mixture model of Western Steppe populations and Eastern Eurasians (χ2 p ≥ 0.05), while the model of Eastern Eurasian simply represented by Neolithic hunter-gatherers  or millet farmers (YR_LN) and farmers in West Liao River (WLR_MN) mostly failed, indicating that Neolithic hunter-gatherers, millet farmers, and Western Steppe populations contributed to the formation of Mongolian population together and the gene flow from the population related to millet farmers into the gene pool of Mongolian continued to today. The ancestral proportion of Western Steppe in those Mongolian subgroups was distinct, showing the parallel genetic makeup of Mongolian_outer and Mongolian_BX harboring a higher level of Western Steppe ancestry , and the proportion of the ancestry in Mongolian_inner, Mongola_HGDP, and Mongolian_BCET were similar , the proportion in Mongolian_mid intermediated between Mongolian_inner and Mongolian_outer, coinciding with the population structure mentioned above. A more complex three-way model of YR_LN + Mongolia_N_North + Russia_Sintashta_MLBA fitted all Mongolian groups (χ2 p ≥ 0.05)   but showWe reconstructed the deep demographic history using qpGraph. Mbuti, Denisovan, Onge and Tianyuan were included to explore the basal model; Early Bronze Age Afanasievo and Chemurchek, Neolithic hunter-gatherers in Mongolia Plateau, millet farmers (YR_LN), Tibetan Plateau, and Iron Age Hanben were used as ancestral source proxies from Western Eurasian, Mongolia, millet farmers in Yellow River, and southern populations. We found that Mongolian subgroups could be modeled as the mixture of EBA_Chemurchek (34–37%) derived from Western Steppe herders (47–55%) and Mongolia’s Neolithic hunter-gatherers related ancestry and Han-related ancestry (63–66%) . Our qpGThe ALDER method based on weighted linkage disequilibrium statistics also provided evidence of population structure within Mongolians . ALDER dWe further performed haplotype-based GLOBETROTTER to obtain a high-resolution characterization of the admixture landscaped of three Mongolian subgroups. All targets showed robust signals of west-east admixture . The wesWe assigned 39 mitochondrial genomes based on 4,198 maternal lineage-informative SNPs and 33 Y-chromosomal genomes based on 22,512 paternal lineage-informative SNPs . The mat10p (iHS) score in the Major Histocompatibility Complex (MHC) region, indicating that genes in this region might experience strong positive selection, which has been already found in previous studies. In addition, in the gene TRPM1 located in chromosome 15, more than 30 SNPs showed strong selection signatures (−log10p > 4), which indicated significant enrichment of selection in this genomic region. The EDAR gene (rs922452) was identified with higher |iHS|, which has shown the strong signatures of positive selection in East Asians (ADH) gene cluster was not identified. The derived allele frequencies of the ADH gene family in those Mongolian subgroups, however, were higher and associated with the genetic affinity to Han   . In addi < 0.01)  showed oresponse . Furtherresponse .LCT/MCM6, frequency < 0.07143), which showed a strong positive correlation with the genetic affinity to Western Eurasian  (FADS) gene family which plays vital role in the biosynthesis of polyunsaturated fatty  . Given t < 0.05)  intermeded fatty  has beenf4 suggested that our focused Mongolian existing population stratification was genetically separated into three subgroups. Overall, even though three Mongolian subgroups had a closer genetic relationship with Tungusic populations, which might result from Altaic-speaking populations—the common ancestor of Tungusic and Mongolian provided by linguistic information, there were differences in sharing genetic affinity with Eurasian populations among Mongolian subgroups. The grouped Mongolian subpopulations showed significant distinction of genetic affinity with previously studied Mongolians of Inner Mongolia Autonomous Region and Mongols. That was, Mongolian_inner had a similar genetic profile with Mongola_HGDP and Mongolian_BCET showing the most shared ancestry with modern Han groups, while Mongolian_outer genetically closed to Mongols showed the higher genetic difference with Sino-Tibetan and southern East Asian populations and lower genetic difference with populations harboring Western Steppe pastoralists related ancestry than Mongolian_inner and Mongolian_mid, and the genetic profile of Mongolian_mid intermediated between Mongolian_inner and Mongolian_outer. The f4, f4, and f4, qpWave homogenous test did further provide evidence of the genetic structure and the diverse sharing genetic affinity to modern/ancient Mongolians among three Mongolian subgroups.We provided newly generated genome-wide SNP data of the Mongolian population from the Inner Mongolia Autonomous Region and performed a comprehensive population genetic analysis to investigate the genetic origin and admixture history. Findings from IBD segments among pairwise individuals, approximate ancestral composition differences from ADMIXTURE result, and pairwise f3 tentatively suggested that the differentiated genetic profile of Mongolians might be the results of various ancestral sources and proportions: the Eastern Eurasian including Neolithic hunter-gatherers related ancestry , millet farmers related ancestry (represented by YR_LN), and relative low proportion of ancestry related to Western Steppe herders contributed to the gene pool of modern Mongolian, in agreement with previous studies  from a gene pool was similar to contemporary Tungusic speakers from Amur River Basin, suggesting a genetic connection among the speakers of languages belonging to the Altaic macrofamily  . The genThe additional ancestral source related to populations of Central Asia (Caucasus/Iranian Plateau/Transoxiana regions) flowed into Eastern Eurasian initiated in the Early Iron Age along the Inner Asian Mountain Corridor/the Tian Shan Mountains, which is detected in the Iron Age groups such as TianShan Saka, Mongolia_Chandman_IA . This geThe Eastern Steppe has served as a crossroad for human population migration and cultural exchanges: the eastward expansion of Western Steppe herders since the Bronze Age ; the WSHADH) gene cluster that underwent regional selective sweeps in East Asia (ADH genes in three Mongolian subgroups showed a strong correlation with the genetic affinity to Han, indicating the possibility of introducing genes into Mongolians. The fact that Mongolians started milk consumption in the Late Bronze Age (The detailed population origin and admixture history provide clues to understanding natural selection and functional genes. In Mongolians, we detected the strong selection signal from the MHC region, which is a key point of the human immune response. Gene enrichment analysis also supported the most enrichment related to the human immune response in terms of cellular component and molecular function. The positive selective sweeps in this region have been already identified in Han populations . Howeverast Asia  was not onze Age  suggesteonze Age . The derf-statistics demonstrated that Mongolian subgroups possessed different Chinese Mongolian/Mongols/Tungusic/East Asian affinities, indicating successful population migration in a frontier city. The successfully fitted four-way admixture model revealed that Eastern Eurasian ancestry included Northeast Asian Neolithic hunter-gatherers related ancestry and East Asian millet farmers related ancestry and Western Eurasian ancestry included Western Steppe herders related ancestry and small Iran-related ancestry. Furthermore, the natural selection analysis of Mongolian showed that the MHC region underwent significant positive selective sweeps and the functional ADH and LCT were not identified. This study characterized the complex population admixture history of Chinese Mongolians, which shed light on the intensified interaction and mixture history of farmers and pastoralists in the boundary between agriculture of contemporaneous imperial Han and pastoral husbandry of herders. Moreover, it revealed intricate genetic structure in a frontier industrial city. The genetical structure of populations inspired that the regional positive selection with allele frequency change might be associated with the genetic affinity. It will be extremely important to expand the set of available ancient and modern genomes across the Eastern Steppe to fully reveal the population structure and history of the Eurasian Steppe and further investigate the local natural selection of functional genes.We generated genome-wide data from 42 Mongolians of the Inner Mongolia Autonomous Region. We first identified a significant genetic differentiation among Mongolians, who were structured into three distinct genetic clusters harboring various Western and Eastern Eurasian ancestries. Findings based on the


By comparing spatial geographical structures of host populations with that of their symbionts light can be shed on their biological interactions, and the degree of congruence between host and symbiont phylogeographies should reflect their life histories and especially dispersal mechanisms.Portunus pelagicus, and its symbiotic pedunculate barnacle Octolasmis angulata from six location sites representing three geographic regions  along the Vietnam coastline. High levels of congruence in their phylogeographic patterns were expected as they both undergo planktonic larval stages.Here, we analyzed the genetic diversity and structure of a host, the blue swimming crab, O. angulata populations showed higher genetic diversity in comparison with their host P. pelagicus . Pairwise Fst and AMOVA analyses showed a more pronounced population structure in the symbiotic barnacle than in its crab host. The DAPC analyses identified three genetic clusters. However, both haplotype networks and scatter plots supported connectivity of the host and the symbiotic barnacle throughout their distribution range, except for low subdivision of southern population. Isolation by distance were detected only for the symbiont O. angulata , while dbMEM supported spatial structure of both partners, but only at MEM-1 .Based on the COI mtDNA markers,  The 3,260 km-long coastline in Vietnam is divided into the Gulf of Tonkin ecoregion in the North (South China Sea province), the central coast and the southeast coast (southern Vietnam ecoregion) and the Gulf of Thailand ecoregion in the South (Sunda Shell province) . In thesPortunus pelagicus, is common at all three locations where it is an economically important species for local fisheries. Throughout its entire distribution range, from the Indo-Pacific to the coast of Africa  interacting species can potentially alter the diversity, function and community dynamics of their hosts  Is there correlation between the phylogeography of the host and the symbiont? (2) Does isolation by distance affect the host-symbiont populations? (3) Is the spatial population structure of the host related to infestation rate of the symbiont?This study aims to investigate the population genetic structure of the swimming crab Portunus pelagicus (n = 160) were collected from fisherman at six locations representing three populations: north (Hai Phong-HP (n = 32) and Quang Ninh-QN (32), central (Khanh Hoa-KH (32) and Phu Yen-PY (32), and south (Kien Giang including Phu Quoc-PQ (16) and Rach Gia-RG (16)) of the Vietnamese coastline (Octolasmis angulata) were collected and identified following O. angulata‘s individuals (n = 32) were chosen from each location . Tissue samples (Chelipeds from crabs and whole body of barnacle) were taken from fresh specimens and preserved in 95% ethanol immediately after sampling.The blue swimming crabs oastline . The craDNA was extracted with DNeasy Tissue Kit  according to the manufacturer’s protocol. Part of the COI mtDNA region was amplified using primers LCO1490 and HCO2198 . PCR reap-values underwent FDR  correction  and nucleotide diversity per site using DnaSP 5.10.01 . Pairwisrrection  to avoidhttp://www.fluxus-engineering.com) by using the median joining algorithm (find clusters function was used to detect the number of clusters in the population (the lowest associated Bayesian Information Criterion (BIC). This analysis provides a graphic description of the genetic divergence among populations in multivariate space.Haplotype networks were drawn with NETWORK 10.1.0.0  using mantel tests in the “vegan” package  implemenn = 160) represented geographical defined populations  were categorized by their intensity: no infection (0), light , medium , and heavy . The hierarchical infestation of O. angulata were plotted against the P. pelagicus populations to investigate the correlation between symbiont infestation pattern and host distribution.The crab individuals  revealed 89 haplotypes. Haplotype number and diversity within sites ranged from 27 and 0.868 ± 0.037 (north) to 49 and 0.963 ± 0.015 , both sites for 64 individuals, 17 haplotypes/32 individuals and 0.835 ± 0.065 (south). Nucleotide diversity from 0.011 (south), 0.013 (north), to 0.019 . The southern population showed lowest genetic diversity, and the central population showed the highest genetic polymorphism in terms of haplotype and nucleotide diversity, and other parameters . The haplotype diversity and nucleotide diversity of the pooled population of barnacles were 0.991 ± 0.002 and 0.002, respectively , while no difference was found between the northern and the central, or the northern and the southern population (P > 0.05) , and Fst between the north and the central was smaller than those between north-south, and center-south populations  . Among tulations .Hierarchical results of AMOVA  revealedP. pelagicus revealed no population differentiation among the three populations  (The haplotype network of ulations . Only onulations . The hapulations . One hapctively)  and 1D.find clusters function indicated three clusters in both the host and the symbiotic barnacle populations  of O. angulata DAPC plots . This implies strong gene flow between crab populations along the Vietnamese coastline. The symbiotic barnacle, however, deviated from this pattern and isolation by distance was detected  . From MEM 2–6, no positive spatial genetics were detected (P > 0.05)  for the  = 0.05) . dbMEM a = 0.05)  and 2D,  > 0.05)  and 2E.The symbiont infestation rates against host distribution are presented in P. pelagicus, and its common symbiotic barnacle, O. angulata. In general, insignificant genetic structure was detected , suggesting high gene flow among the sampled crab populations along a more than 2,000 km long stretch of the Vietnamese coastline. The DAPC analysis did not indicate a spatial genetic pattern as no completely isolated cluster among three clusters was detected. In contrast, all populations of the barnacle symbiont, O. angulata, demonstrated significant structure differences . However, neither haplotype network nor the DAPC analysis showed an obvious trend of population isolation. The southern population indicated a slight separation from the central and the northern populations, while more pronounced genetic mixing was observed between the central and the northern populations.Our population genetic analyses demonstrated differences in population structure pattern between the crab, P. pelagicus populations in Vietnam. A potential cryptic species complex of P. pelagicus  to exclusively parasites  and the taxon, Cirripedia, is often used as model for ecological and evolutionary research  is a common and widely distributed species in Indo –West Pacific water , Vietnam, and eastern Thailand. Here the barnacle populations are under the complex influence of the SC Warm Current, the mixing of Kuroshio Current, and the Southeastern Vietnamese Offshore (SVO) Current , no population differentiation was detected by Peltogasterella gracilis, is widely distributed throughout Korea, forms a single metapopulation with populations from Japan, although on different hermit crab species , the well-mixed East Sea, and the Pacific Ocean in the central regions , and the slight splitting of the southern population  in Vietnam. Additionally, upwelling and anticyclonic/cyclonic eddies along the south and central coasts , have a greater dispersal potential than larvae of O. angulata, in which IBD apparently influences the population structure along the Vietnamese coastline. A greater dispersal potential could be achieved if larvae of C. testudinaria had a longer larval development time  and had morphologically more efficient swimming appendages. Rather counter intuitively, though, it turns out that C. testudinaria has an approx. three times faster larval development time (9 days) , but like other lepadomorph barnacles   than O.  31 days ! The lararnacles , greatlyarnacles . It is carnacles . It thusP. pelagicus and O. angulata undergo long planktonic larval phases lasting for several weeks; 31 days in O. angulata  can complete its lifecycle within the intermolt phase of even juvenile crabs with short molting intervals  as their settlement substrates where their survival relies on the ability to complete a life cycle within a single intermolt period of its host. Interestingly, Octolasmis) the cyprides are known to collect on crabs that are approaching ecdysis and to postpone settlement until the crab has completed moulting”. Successful dispersal of symbiont and parasitic species without or with restricted free-living stages often rely on the migratory capability of their hosts. Barnacles, however, possess extended pelagic larval stages  and its acanthocephalan parasite (Profilicollis novaezelandensis), although the modes of dispersal in the two species are different (planktonic drift versus bird-mediated). The obscure biogeographic pattern of O. angulata due to mixed overlapping genetic clusters from the Haplotype network and the DAPC analysis, and supported local genetic differences  calls for further studies.Due to the differences in life history and dispersal mode, it should be expected that the host exhibits a weaker population structure than the barnacle (limited by adult sessile phase). This prediction is supported by the current study. Additionally, isolation by distance  seems to have contributed to local genetic differentiation between widely separated Octolasmis appears to be a facultative rather than an obligatory association  and are the key drivers of ecological function and evolutionary processes . A largeociation , and theociation , especiaociation .O. angulata is an extremely common symbiont, occurring on up to 90% of individuals in the host populations, and with intensities of more than 1,000 individuals  on crustacean hosts . The facividuals , may indetention . In contetention  may suppP. pelagicus and the symbiotic barnacle, O. angulata along the Vietnamese coastline. However, in contrast to the host, we found indications of differentiation in the symbiont populations between the three sampling areas, particular in the southern population.Based on COI mtDNA markers, connectivity was observed in both the swimming crab, 10.7717/peerj.11671/supp-1Supplemental Information 1P. pelagicus (B) and O. angulata (D).Each line represents one substitution; small unshaded circles indicate additional substitutions separating two haplotypes. The size of the circle is representative for the frequency of the haplotypes. Filled patterns correspond to geographic affinity of haplotypes. (B and D): Pie charts presents percentage of haplotypes contributed to the network following sampling sites for Click here for additional data file.10.7717/peerj.11671/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11671/supp-3Supplemental Information 3The measurements of swimming crab (Portunus pelagicus) following the geographic locations, and the symbiotic status (number of symbionts), and the common symbiotic barnacle Octolasmis angulata .Click here for additional data file.


Rosa damascena Mill is a well-known species of the rose family. It is famous for its essential oil content. The aim of the present study was to assess the genetic diversity and population structure of a mini core collection of the Iranian Damask rose germplasm. This involved the use of universal rice primers (URP) and start codon targeted (SCoT) molecular markers.Fourteen URP and twelve SCoT primers amplified 268 and 216 loci, with an average of 19.21 and 18.18 polymorphic fragments per primer, respectively. The polymorphic information content for URR and SCoT primers ranged from 0.38 to 0.48 and 0.11 to 0.45, with the resolving power ranging from 8.75 to 13.05 and 9.9 to 14.59, respectively. Clustering was based on neighbor-joining (NJ). The mini core collection contained 40 accessions and was divided into three distinct clusters, centered on both markers and on the combination of data.Cluster analysis and principal coordinate analysis were consistent with genetic relationships derived by STRUCTURE analysis. The findings showed that patterns of grouping did not correlate with geographical origin. Both molecular markers demonstrated that the accessions were not genetically diverse as expected, thereby highlighting the possibility that gene flow occurred between populations.The online version contains supplementary material available at 10.1186/s43141-021-00247-7. Rosa has 200 species and covers more than 18,000 cultivars [Rosa damascena, while Iran is usually referred to as a source of diversity in this respect [Rosa damascena has particular genotypes and cultivars which are noteworthy for their medicinal properties and oil [As a large genus in the Rosaceae family,  respect . Rosa da and oil –6. Since and oil . AccordiRosa genus. Several molecular assays have been used in recent years to test the genetic variation of various rose plants [For the classification and recognition of rose genotypes, chemotaxonomic studies are often addressed in a large variety of different phenolic structures and isozyme markers –11. Nonee plants –22. In tIn the plant genome, the SCoT marker mechanism relies on the start codon (ATG) which has a short conservation around it . These mMI) and the polymorphism information content (PIC). Comparing the ability of marker techniques can assist researchers in selecting the required markers in the amplification of genome fragments, thereby being more effective in using these markers for potential breeding studies [Kang et al.  used a p studies .Rosa damascena accessions from Iran and to demonstrate the effectiveness of different marker systems.This study aimed to investigate genetic variation in different In total, 40 Damask rose genotypes were collected from five regions in Iran Fig. , Table 12O, 10 μl master mix 2XPCR , 2 μl isolated DNA per sample (50 ng/μl), and 1.5 μl per primer (10 pmole/ml). Each PCR cycle ran on initial denaturating at 94 °C for 5 min, 35 denaturation cycles at 94 °C for 45 s, with a primer annealing time of 45 s , total amplified bands (TAB), percentage of polymorphism bands (PPB), resolving power (Rp), polymorphism information content (PIC), and marker index (MI). PIC was calculated based on the formula given by Anderson et al. [The amplified fragments were scored as absent (0) or present (1) in each sample. Screening the primers involved using several discriminatory criteria, including the number of polymorphic bands , effective number of alleles (Ne), and total number of alleles (Na) [H), and Shannon’s information index (I) [K) and the ratio of individual assignment out of each population. For each ‘K’ varying from 1 to 10, the analysis was repeated ten times, and the initial burn-in period was set to 100,000 followed by 100,000 Markov Chain Monte Carlo (MCMC) iterations. Finally, the DK was calculated by STRUCTURE HARVESTER, an online program [Molecular variation analysis (AMOVA) operated via GenAlEx ver. 6.5 to classify genetic diversity . For eacles (Na) , Nei’s [les (Na)  gene divndex (I) . Then, Jndex (I) . The neindex (I) . The genndex (I) . It esti program .Rosa damascena was tested using 14 URP and 12 SCoT primers. Table In this analysis, the genetic polymorphism of Rosa damascena populations were detected by molecular variance analysis (AMOVA) (Table GST)/ gene flow (Nm) equaled 0.117/3.773, 0.185/2.197, and 0.14/2.86, respectively, for URP, SCoT, and the combined data. Genetic diversity per population varied considerably (Table Na) occurred in region I (1.96) and region II (1.94). In region IV and region II, the highest number of effective alleles (Ne = 1.60 and 1.58) were detected, as well as the highest value of Nei’s gene diversity (He = 0.34) and Shannon’s information (I = 0.51). Thus, the highest polymorphic loci (%) (PPL = 97.39%) occurred among the population of region I. According to the SCoT results, the populations of region I and region II had the largest Na (1.97 and 1.92), Ne (1.62 and 1.61), H (0.36 and 0.35), and I (0.53 and 0.52) values, respectively. Such populations also showed high PPL values (98.15 and 94.44%). In addition, region II appeared in the combined data analysis (SCoT + URP) and displayed the highest values for I (0.52), H (0.35), and Ne (1.60). The highest values of these parameters  occurred in the population of region I.Variations among and within the A) Table . The resRosa damascena Mill. The highest distance coefficient (0.872) was observed between accessions Minab (16) (region II) and Barzok1 (9) (region I), whereas the lowest distance (0.180) was identified between two accessions of region V, Khoramabad (39) and Borujerd (40). According to SCoT data, however, the estimated Jaccard’s distance coefficient ranged from 0.107 to 0.785, with an average of 0.602. The largest distance (0.785) was recorded between accessions Barzok2 (11) (region I) and Tabriz (34) (region IV), while the smallest distance (0.107) was observed among two accessions from region III, Semnan1 (26) and Semnan2 (27). The pairwise genetic distance coefficient was measured based on combined data and revealed a wide spectrum of 0.153–0.789, while averaging at 0.631 among all 40 accessions. The largest distance (0.785) was recorded between accessions Barzok2 (11) (region I) and Tabriz (34) (region IV), while the smallest distance (0.107) was observed among two accessions from region III, Semnan1 (26) and Semnan2 (27). The pairwise genetic distance coefficient was identified among Mashhad ardehal (2) and Barzok1 (9) from Esfahan province (region I), whereas the least distance belonged to the Lorestan province (region V) (Khoramabad and Borujerd) (data not shown).The Jaccard distance coefficient pairs of accessions were estimated by binary data from URP and SCoT primers. In URP, the genetic distance of a pairwise pattern varied from 0.180 to 0.872 with an average of 0.659 among different pairs of landraces of Rosa damascena accessions. A dendrogram was created using SCoT, URP, and combined data (SCoT + URP). The dendrogram classified all accessions into three major clusters  method for the 40 ers Fig. A–C. Accoers Fig. A. In SCoers Fig. B. Accorders Fig. C. The fir = 0.49) between distances revealed by SCoT and URP data for all 40 accessions across five collected regions.Mantel correlation test showed a low and statistically nonsignificant correlation , SCOT (B), and the combined data (C), the first three principal coordinates explained 36.69, 37.34, and 33.85% of molecular variations, respectively. The PCoA biplots showed that all accessions displayed a scattered distribution in the plot, although this did not follow their origins Fig. .Fig. 3PK = 1 to K = 10, and with URP primers, probabilities were most precisely derived at K = 3. Out of the 40 accessions, subgroup 1 included 12 accessions from Kermanshah (4), Esfahan (2), Gilan (4), and Kerman (2), as well as subgroup 2 which comprised all accessions from Markazi, Tehran, Hormozgan, East Azerbaijan, as well as some accessions from Esfahan (5), Fars (2), and Kerman (1) populations. Twelve accessions of Semnan (2), Lorestan (2), Esfahan (5), Kerman, Gilan, and Fars (1) populations were classified into subgroup 3  comprised accessions from Gilan (5), Kermanshah (4), Esfahan (3), Semnan (2), Kerman (2), and Fars (1) populations. Subgroup 2 (15) comprised accessions from the Esfahan (7), Tehran (2), Tabriz (2), Lorestan (2), and Kerman (1) populations; subgroup 3 (8) had accessions from the Esfahan (2), Hormozgan (2), Markazi (2), Fars (1), and Kerman (1) populations. With 26 polymorphic URP and SCoT primers, four different subgroups were achieved  of a parameter represents the amount of polymorphism of a marker, as this can vary from zero to half. The larger the value, the greater the number of alleles and the higher the frequency of polymorphisms for that position in the study population. In the present study, the relatively high PIC and MI values for the URP primers provided an estimation of the discriminating ability of the URP marker systems [Rosa damascena populations with close kinship ratios.URP markers showed higher values of  systems . They shGST) and gene flow (Nm) variables backed up these findings. As a result, the GST values for URP, SCoT, and combined data were 0.117, 0.185, and 0.148, respectively, revealing that genetic variation among populations is relatively low. The indirect estimate of gene flow (Nm) via GST was 3.77 (URP), 2.19 (SCoT), and 2.86 (combined data). The total number of migrants per generation exceeds two. Here, genetic differences may be partly due to gene flow, as the populations of this species are significantly affected by genetic drift. Also, local populations are different if Nm < 1 [Nm occurred in populations, and thus, gene flow prevented drastic genetic differences among gemmates. Population size and the spread of alleles among various regions can add details to this finding [R. damascena accessions from Iran using 31 RAPD. The authors reported that the genetic variation within the collected populations was more than the variation among them. Similar results were achieved in the present study; however, the variation within the populations with both markers was higher than the previous study.According to the AMOVA, 96% and 90% of genetic variations were revealed by the URP and SCoT markers, respectively, which were partitioned within populations, suggesting that the observed variation within genotypes was higher than among them Table . Interpof Nm < 1 . High va finding . Kiani e finding  studied Ne, Na, I, PPL, and He) were reported for region I and region II populations using SCoT and combined data. In the URP marker system, region I had the highest polymorphism percentage (PPL) and the highest number of alleles (Na), whereas precision of genetic diversity was provided by Shannon’s information index and Genetic Diversity Index for populations of regions II and IV. As divergent populations, regions I and II could be selected according to SCoT and combined data, while regions II and IV could be selected according to the URP data. A larger genetic variability here may reflect the population’s frequent allelic variation, while weather conditions can affect ultimate variation among the populations [R. damascena in Turkey, via RAPD and AFLP techniques. Genetic uniformity existed among R. damascena cultivars.Table ulations . Furtherulations . Also, tulations , an extrulations , 57, 58.ulations  and Baydulations  studied R. damascena plants in Turkey can be derived from the same original genotype by vegetative propagation. Rusanov et al. [In the current study, spatial distribution did not align with genetic relationships, based on the neighbor-joining cluster analysis Fig. . For exav et al.  reportedv et al. . Usuallyv et al. .In the current research, the PCO analysis confirmed the results of cluster analysis. Genetic proximities were visually depicted by PCO among populations. In URP and SCoT, genetic difference and geographical distance were not clear-cut. Besides, phenotypic traits have high correlations in some occasions, while the first two components justify more than 90% of the changes. Meanwhile, molecular markers could not justify the higher values of variance of the primary variables by several of the main components. In investigating the genetic diversity using molecular data, the markers should have a uniform and appropriate distribution in the genome so that they can be sampled from the entire genome. As shown in Fig. K and the number of subpopulations (∆K) were identified. In both marker systems, the best level of population classification were K = 3 and in the combined data system K = 4. In this clustering, it was found that the different populations of R. damascena can group into one cluster, such as cluster ‘BI’ in the SCoT marker system, in which seven populations were grouped from five regions and different altitudes. Moreover, our results showed that clustering by both markers and combined markers made similar classifications of the populations of Kermanshah (region V) and Gilan (region IV), thereby assigning them to the same subgroup, while Hormozgan (region II) and East Azerbaijan (region IV) were classified together in a subgroup. If genotypes or cultivars gather into one category from different areas, it may mean that they have the same genetic heritage [R. damascena populations throughout Iran. It seems that due to its high tolerance to drought, this crop is one of the most suitable species in arid provinces of the country. Due to a decrease in agricultural water resources and rainfall, its cultivation can replace many agricultural products which have high water requirements. In addition, the ability of this plant to adapt well to different climates and soil conditions in Iran has made farmers inclined to introduce it to other regions in the country. While the genetic origin of these plants is the same in different regions, the obvious difference may be attributed to the climate in which they emerge. Inter and intraspecific variation can be affected by temperature and rainfall [The neighbor-joining cluster analysis was confirmed by the Bayesian clustering algorithm through STRUCTURE analysis in comparing the 40 accessions  heritage , 64. Thiheritage . The genrainfall .R. canina in Brandenburg (Germany). Fifty-five genotypes were classified into twelve subgroups. They attributed the high genetic variation to the outcrossing, seed dispersal system and polyploidy within the R. canina populations. The level of genetic variation is affected by breeding system, life cycle, seed dispersal, and geographic distribution which are important factors among populations. Rose species are known to be outcrossing, but there is little evidence on their outcrossing frequencies [Rosa canina L. via ISSR markers suggested that geographical distance is effective in causing allelic gaps among genotypes, and ecological conditions could cause genetic variation in R. canina [Roses usually cross-pollinate and are self-incompatible which makes them more genetically diverse between and within populations –69. Jurgquencies . Contrar. canina .K = 1 to 10 (the number of populations), many populations in the existing germplasm are not completely separated based on the regions from which these genotypes originated or were collected (Fig. K population. In fact, the formation of different subgroups in population structures depends on the frequency of allelic differences between the genotypes that make up the population. Most of the genotypes were not attributed completely to subgroups, thereby indicating that many genotypes have intermediate genetic traits of various subgroups, as a matter of genetic variation in this research.The results of model-based clustering was based on the Bayesian statistical index, assuming that the Ancestry model is Admixture type and the allelic frequency model is of continuous type. While also assuming a range of ted Fig. . The mixR. damascene populations by the two marker systems. The mean values of PIC for URP and SCoT markers were 0.42 and 0.37, respectively, indicating the efficiency of the two markers in detecting polymorphism among the studied samples. Also, the results confirmed the efficiency of combined data in estimating the genetic diversity among the populations. The used marker systems showed a comprehensive pattern of the genetic diversity among the Iranian R. damascene populations, which could provide a future insight into Damask rose breeding programs.Crop improvement is influenced by information about the degree and distribution of genetic variation, as well as relationships between breeding materials. The results of the present study revealed a high level of polymorphism in the Iranian Additional file 1: Supplementary Table1. Genotype classification. Classification of genotypes into different groups according to the figure Additional file 2: Supplementary Table2. Pairwise genetic distance coefficients. Jaccard distance coefficients based on SCoT and URP dataAdditional file 3: Supplementary figure1. Mantel correlation. The correlation between SCoT and URP data for all 40 accessions across five regions