Pvs230 was studied in Plasmodium vivax parasite isolates from the China–Myanmar border (CMB) and central Myanmar.Sexual stage surface antigens are potential targets of transmission-blocking vaccines (TBVs). The gametocyte and gamete surface antigen P230, a leading TBV candidate, is critical for red blood cell binding during exflagellation and subsequent oocyst development. Here, the genetic diversity of Plasmodium vivax isolates were collected in clinics from malaria-endemic areas of the CMB (143 samples) and Myanmar (23 samples). The interspecies variable part  and interspecies conserved part  of Pvs230 were amplified by PCR and sequenced. Molecular evolution studies were conducted to evaluate the genetic diversity, signature of selection, population differentiation, haplotype network, and population structure of the study parasite populations and publicly available Pvs230 sequences from six global P. vivax populations.π = 0.002) and Myanmar (π = 0.001) isolates. Most amino acid substitutions were located in the IVP and cysteine-rich domain of Pvs230. Evidence of positive selection was observed for IVP and purifying selection for ICP. Codon-based tests identified specific codons under natural selection in both IVP and ICP. The fixation index (FST) showed low genetic differentiation between East and Southeast Asian populations, with FST ranging from 0.018 to 0.119. The highest FST value (FST = 0.503) was detected between the Turkey and Papua New Guinea populations. A total of 92 haplotypes were identified in global isolates, with the major haplotypes 2 and 9 being the most abundant and circulating in East and Southeast Asia populations. Several detected non-synonymous substitutions were mapped in the predicted structure and B-cell epitopes of Pvs230.Limited genetic diversity was observed for the CMB (Pvs230 in global P. vivax populations. Geographically specific haplotypes were identified for Pvs230. Some mutations are located within a potential B-cell epitope region and need to be considered in future TBV designs.We detected low levels of genetic diversity of The online version contains supplementary material available at 10.1186/s13071-022-05523-0. The National Malaria Control Programs (NMCPs) in the Greater Mekong Subregion (GMS) have made substantial strides in malaria control, and in the past 9 years, most of the GMS countries endemic for malaria are making progress in reducing their malaria burden . HoweverTBVs induce antibodies that target the malaria antigens expressed in sexual stages , and are responsible for reducing the oocyst density in the mosquito midgut . Based oPlasmodium vivax has been identified as an orthologue of P230 in Plasmodium falciparum. Although Pfs230 has been studied extensively, limited studies have focused on Pvs230. Genetic diversity studies for vaccine candidate genes in malaria-endemic areas will provide useful information for vaccine design. Studies on asexual-stage vaccine candidate genes, such as ama1, revealed that the polymorphic antigen-induced immune response is allelic variant-dependent, which will limit the efficacy of the vaccine design based on this antigen I–IV) was assembled, excluding the primer sequences. To investigate the global population structure of Pvs230, 108 publicly available Pvs230 sequences were retrieved from GenBank, representing six locations: Papua New Guinea (PNG) (AB574595–AB574615), Thailand (AB574575–AB574594), Central China , Madagascar (AB574548–AB574554), Turkey (AB574528–AB574547), and Brazil (AB574508–AB574527) [Pvs230 gene, including the number of polymorphic sites (S), the total number of mutations (η), the average number of nucleotide differences (k), nucleotide diversity (π), the number of haplotypes (H), haplotype diversity (Hd), and the number of synonymous mutations (SP) and non-synonymous mutations (NS), was performed using DnaSP, version 6.12.03 [π values across the IVP and ICP of Pvs230 were generated using a 90-bp sliding window with 3-bp step size [Raw sequence data of MEGA 7.0 . A 2811- 6.12.03 . Slidingtep size . SequencPvs230 gene, dN–dS, Tajima’s D, Fu and Li’s D* and F*, and McDonald–Kreitman (MK) indices were calculated using DnaSP v6.12.03 [dS) and non-synonymous (dN) mutations were estimated and compared by the Z-test (P < 0.05) in MEGA 7 using the Nei–Gojobori method with Jukes and Cantor correction [dN–dS > 0 implies a positive selection, whereas dN–dS < 0 designates a purifying selection [D test, the departure from neutrality in the nucleotide frequency distributions was determined by comparing the values of θ (estimated nucleotide diversity) derived from π and S [D* and F* tests were used to test the hypothesis that all mutations are selectively neutral, and the mutations in the external branches were compared to the genealogy branches [D and Fu and Li’s D* and F* tests correspond to positive diversifying selection or population structuring due to an excess of variants at intermediate frequencies, whereas a negative value indicates population size expansion and/or negative/purifying selection. Sliding window plots with a window size of 90 and a step size of 3 were also generated for the Tajima’s D and Fu and Li’s D* and F* values, and dN–dS was used to identify regions of Pvs230 under selection [Plasmodium cynomolgi P230 (GenBank accession no. AB574620) was used as the interspecies outgroup for the MK test, which allows for determination of the ratio of synonymous substitutions to non-synonymous substitutions between and within species [P < 0.05). A codon-based test using SLAC (Single-Likelihood Ancestor Counting) [dN–dS indicated high statistical significance (P < 0.05).To test the neutral theory of evolution in the v6.12.03 . The ratrrection . A valueelection . In Taji π and S . Fu and branches . Positivelection . The seq species . An exceounting)  methods ounting)  was usedPvs230 among global isolates was estimated by calculating Wright’s fixation index (FST) using Arlequin software, version 3.5.2.2 [FST values is defined as described previously [P-value < 0.05 was considered significant difference. To investigate the genetic relatedness among the Pvs230 haplotypes, a haplotype network was constructed using the median-joining (MJ) method implemented in the PopART v1.7 software with a haplotype frequency > 1 [Pvs230 sequences (nt 52–2862) from the global isolates [https://itol.embl.de/).The degree of genetic differentiation in  3.5.2.2 . Interpreviously , with noency > 1 . A phyloisolates  was consisolates  with a bisolates . Final tPvs230 haplotypes were investigated using the Bayesian model-based clustering method implemented in STRUCTURE software, version 2.3.4 [K) ranging from 1 to 10. Each run was carried out with a “burn-in” period of 105 and Markov chain Monte Carlo (MCMC) length of 2 × 105 iterations under an admixture model with correlated allele frequencies among populations. The most probable K was inferred by the Evanno et al. method [K, using the greedy algorithm to increase speed, and a run of 1000 random repeats of the data. The graphical depiction of the STRUCTURE results was generated using Distruct software [Population structure and clusters of global isolates based on on 2.3.4 . Ten ind. method  using th. method . CLUMPP . method  was usedsoftware .Pvs230 were predicted using the ABCpred server (www.imtech.res.in/raghava/abcpred) with a threshold of 0.7 and a window length of 16 [https://services.healthtech.dtu.dk/service.php?BepiPred-2.0) with a threshold of 0.5 and peptide length > 10 [Pvs230 protein was predicted using the I-TASSER meta-threading procedure (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) [Pvs230 using PyMOL software .The B-cell epitopes in IVP and ICP of th of 16 , and usigth > 10 . The thrTASSER/) . The ovePvs230 gene was successfully amplified and sequenced from 166 P. vivax samples collected in the CMB area (n = 143) and central Myanmar (n = 23). For the sequenced region (nt 52–2862), there were 30 and 19 polymorphic sites in isolates from the CMB and Myanmar, respectively (Table k) of the CMB isolates for the entire sequence, IVP, and ICP were 5.558, 3.518, and 2.041, respectively  for the entire sequence, IVP, and ICP of Pvs230 among the 143 P. vivax isolates of the CMB isolates were 0.002, 0.006, and 0.001, respectively  and 32 non-synonymous SNPs (NS SNPs)  were clustered within the IVP, resulting in a peak π of 0.026 for this region  and degenerative repeats (DEDGD and DGND) were detected in the IVP of Pvs230 of the CMB and Myanmar isolates, dN–dS, Tajima’s D, Fu and Li’s D* and F*, and MK test were performed. Although the values of Tajima’s D, and D* and F* were not significant for either the CMB or Myanmar isolates, differences in Pvs230 domains existed, including positive values in the IVP for the CMB isolates and negative values in the ICP in both regions. Significant values of dN over dS were observed in the entire Pvs230 sequence and IVP in isolates from both regions, suggesting positive diversifying selection. Sliding window plots depicted significant negative D, D*, and F* values in the IVP of Myanmar isolates. Meanwhile, significant negative D* and F* test values were detected in the ICP of CMB isolates, supporting purifying selection acting on this domain , particularly within IVP , had significantly more non-synonymous than synonymous substitutions expected from the comparison with P. cynomolgi. A similar pattern from the MK test was observed in the Myanmar isolates  between the West Asia (Turkey) and the Oceania (PNG) populations. High FST values (0.261–0.609) were detected between the East Asia  and Southeast Asia (Myanmar) populations, between West Asia and Brazil and other Asia populations, and between Oceania with Asia , Africa, and Brazil populations. A moderate range of FST values (0.173–0.249) were detected when comparing the CMB Myanmar, Oceania, and Africa populations with the Myanmar and West Asia populations, and with South America, Thailand, and Africa populations. FST values among East and Southeast Asia populations approached zero, indicating a high degree of genetic similarity. The lowest FST value (0.038) was observed between CMB and Thailand populations, suggesting extensive gene flow between these populations . Of the 92 haplotypes, 63% (58/92) were single, and 84.8% (78/92) were region-specific. There was also significant haplotype sharing between regions and continents  and N623–K649 , which contained two (V236F and V275M) and one (E631K) polymorphic residue, respectively. Lower nucleotide and haplotype diversity was found in epitopes E419–M448, K693–N764, N792–E818, and K836–S851, while the T333–A354 epitope was conserved, suggesting functional constraints of these regions.The modeled structure of 230 Fig. . The ove230 Fig. . A moderPvs230 are species-specific in Plasmodium. In this study, we found that the presence and extension of the N-terminal tandem repeats in the IVP of Pvs230 differed by geographical origin, with most repeated patterns restricted to the region of origin. We found that replicate 12 (r12) was the most prevalent variant in the CMB isolates and was shared with Central China, Southeast Asia, and Oceania populations. Tandem repeats in some malaria vaccine candidate genes, such as circumsporozoite protein (CSP), differ according to transmission intensity and show an association with the response to treatment [Pvs230 of P. vivax remains elusive.Due to the surface location and functional essentiality in forming exflagellation centers, Pvs230 has been considered a malaria TBV candidate and has undergone extensive studies , 47, 48.reatment , 50, 51.Pvs230, with most variations accumulated in IVP and CRDI–II in ICP, suggesting that these regions are potentially more exposed to the host immune system. Immunological studies indicate that the pro-domain (downstream of the cleavage site in IVP) and CRDI–II domain in ICP could elevate a naturally acquired antibody response in endemic areas [Pvs230 haplotypes in the global isolates, with varying levels of haplotype diversity among regions. The highest diversity was detected in Asia, where 73 haplotypes were reported, including the two major haplotypes (2 and 9) and several minor haplotypes, indicating a close genetic relatedness between these P. vivax populations, comparable to the analysis of Pvhap2 sequences [Polymorphic sites were distributed unevenly across ic areas , 52, 53.ic areas . Recombiic areas . In our equences . The lowPv230 showed low genetic diversity (π = 0.001–0.002), consistent with a previous report [P. vivax, including Pvs28/25 , Pvs48/Myanmar, ), and Pv, Brazil ). This h evasion . Pfs230  evasion . Althougf Pfs230 , and thef Pfs230 , 61, sugPvs230 showed divergent haplotype distribution patterns in different P. vivax populations. Population differentiation, especially among the Asian populations, was supported by both Wright’s FST statistic and phylogenetic analysis, which indicated population subdivision among the P. vivax populations. The FST estimation between East/Southeast Asia  and West Asia (Turkey) populations was remarkable (0.41–0.558), whereas parasites from the GMS exhibited only slight geographical genetic differentiation. Although antigens under selection may not be suitable markers for population genetic studies, the results from Pvs230 analysis are highly congruent with the population genomics studies conducted using whole genome sequencing data from the GMS [P. vivax populations suggest the lack of gene flow barriers in the GMS, which may be further enhanced by the extensive human migration in this region [P. vivax populations were grouped into three clusters by structure, phylogenetic, and haplotype network analyses, similar haplotypes in the Pvs230 sequences were found in many parts of the world. Therefore, geographical differentiation of the parasite population and conservation of certain Pvs230 haplotypes should be considered for Pv230-based vaccine design.Despite low sequence diversity,  the GMS . These ls region , 64. In Pvs230 gene, and neutrality tests showed that the gene is under positive selection on the IVP and under purifying selection on the ICP. The study revealed continent-specific and shared Pvs230 haplotypes in the global isolates, and further reinforced the observation from genomic analysis, showing little genetic differentiation in the GMS. The presence of dominant haplotypes in the ICP circulating in East and Southeast Asia parasite populations suggests the possibility of developing a universal Pvs230-based TBV in this region.This study revealed limited genetic diversity in the Additional file 1: Table S1. Primers used in Pvs230 amplification and sequencing.Additional file 2: Table S2. The diversity pattern of the N-terminal repeat region in IVP of Pvs230 from global isolates. Related to Fig. Additional file 3: Table S3. Codon-based tests for selection on Pvs230 in China–Myanmar border and Myanmar isolates.Additional file 4: Figure S1. Phylogenetic analysis of Pvs230 sequences from global P. vivax populations. The maximum-likelihood phylogenetic tree reconstructed based on alignment by ClustalW with bootstrap analysis to assess clade support (500 replicates) was shown for Pvs230 global isolates. The global isolates were clustered into three main groups, the branches of which are shown in red (group 1), green (group 2), and blue (group 3).Additional file 5: Figure S2. Median-joining network of Pv230 among eight geographically diverse populations. Haplotypes composed of nucleotide polymorphism in ICP of Pv230 with a frequency > 1 were used to create a median-joining network. Each node represents one haplotype, node size indicates haplotype frequency, and node color corresponds to the country of origin. Line length is proportional to genetic distance.


Lepas anatifera and clarifying the potential roles of temperature maintaining this pattern are crucial for our understanding of the distribution and biodiversity of pelagic macrobenthos. In the present study, mitochondrial cytochrome oxidase subunit I (mtDNA COI) from three South China Sea (SCS) populations and six Kuroshio Extension (KE) region populations of L. anatifera sampled from fixed buoys and genome‐wide SNPs from a subset of populations (two SCS populations and four KE region populations) were sequenced and analyzed for investigating the genetic pattern of the pelagic barnacle. Water temperature was different among sampling sites; in other words, the water temperature decreased with latitude increases, and the water temperature on the surface was higher than in the subsurface. Our result showed that three lineages with clear genetic differentiation were found in different geographical locations and depths based on mtDNA COI, all SNPs, neutral SNPs, and outlier SNPs. Lineage 1 and lineage 2 were dominant in the subsurface populations and surface populations from the KE region, respectively. Lineage 3 was dominant in the SCS populations. Historical events during the Pliocene epoch shaped the differentiation of the three lineages, while, nowadays, temperature heterogeneity maintains the current genetic pattern of L. anatifera in the northwest Pacific. The subsurface populations were genetically isolated from the surface populations in the Kuroshio Extension (KE) region, implying small‐scale vertical thermal heterogeneity was also an important factor maintaining the genetic differentiation pattern of the pelagic species.Macrobenthic invertebrates are ubiquitously distributed in the epipelagic zone of the open ocean. Yet, our understanding of their genetic structure patterns remains poorly understood. Investigating the genetic differentiation patterns of pelagic  Lepas anatifera attached to fixed buoys and moorings set in the Northwest Pacific. Our results showed that there were three genetic‐differentiated lineages in different geographical locations and depths and confirmed that both horizontal and vertical thermal heterogeneity was important for maintaining the genetic differentiation of L. anatifera populations in the open oceans.Macrobenthic invertebrates are ubiquitously distributed in the open oceans; however, we are rarely understanding their phylogeographic patterns in the open ocean. In the present study, we investigated the phylogeographic pattern of  The northward Kuroshio transported a large amount of heat from low latitudes to middle and high latitudes in a very narrow area, and the southward hydrophile transported low temperature and low salt water from high latitudes to low latitudes, resulting in a significant ocean temperature gradient in the Kuroshio Extension region Qiu, . Under tL. anatifera on these moorings and buoys are important for studying the genetic diversity and the factors maintaining the genetic differentiation pattern in the NWP. In the present study, we focus on the genetic differentiation patterns of L. anatifera sampled vertically and horizontally from these buoys and subsurface moorings in both the South China Sea (SCS) and KEC. We aimed to investigate the following questions: (1) whether there is genetic divergence existing among NWP populations for L. anatifera; (2) if there is, what is the role of temperature driving the formation and maintenance of the corresponding pattern? Our results provide new insights into the mechanisms of the formation and maintenance of species' genetic patterns in the NWP. Moreover, our research approach is of general relevance for future studies on phylogeographic patterns and genetic adaptation of species distributed in the epipelagic zone of the open oceans.The pelagic barnacles 22.1L. anatifera were collected from three sites in the South China Sea (SCS) and six sites in the Kuroshio Extension (KE) region  method , the total genomic DNA of the 186 L. anserifera   was used to filter the data. We retained only those loci that were genotyped in at least 60% of samples in a population and unshared SNPs were pruned to reduce haplotype‐wise missing data. VCFtools . DNA was extracted with an SDS (sodium dodecyl sulfate) protocol, and DNA libraries were constructed using NEBNext DNA Library Prep Kits (New England Biolabs). Samples were processed and sequenced using Illumina HiSeq 2500 Rapid Run flow cell (150 base pairs paired‐end reads) at Novogene . The quality of the sequence data was assessed with fastp software , haplotype diversity (h), and nucleotide diversity (πc) for each population using DnaSP 6.0 , observed heterozygosity (Ho), and nucleotide diversity (πG) were calculated based on filtered SNPs mentioned above using the populations program in Stacks v.2.5.1  tree was constructed in MEGA X using the COI sequences of 186 individuals with 1000 bootstrap replicates. To further verify the reliability of phylogenetic tree topology based on COI, SNPs filtered from the GBS  were used to perform phylogenetic analyses under the same settings described above. To better understand the relationships between the lineages in a global context, we also construct an NJ tree using COI sequences of To get a clear genealogical relationship among haplotypes, a minimum‐spanning network (MSN) was constructed by PopART , shared by all loci and a locus‐specific component  shared by all the populations using a logistic regression  using PLINK  >1.5  was performed based on adaptive data sets associated with temperature using the RDA function from the 2.5FST values  ] is linearly correlated with geographic distance. The correlation between geographic distance and genetic distance was detected using the Mantel test in GenAlEx 6.5  model is often used to test whether the migration of populations conforms to the stepping‐stone model Bohonak, . If the K values was predefined from one to nine. The optimal K values were assessed using the cross‐validation (CV) procedure implemented in ADMIXTURE with the ‐‐cv flag. The principal component analysis (PCA) using the R package tidyverse was further applied to investigate the genetic divergence among all individuals.Population structure and individual ancestry were predicted based on all SNPs, neutral SNPs, and temperature‐related outliers, respectively, using ADMIXTURE v.1.3.0  and nucleotide diversity (0.098 ± 0.001) with overall value. The nucleotide diversity values of populations in the KE region (COI: 0.015–0.089) were generally higher than that of the SCS populations (COI: 0.008–0.011)  from 186 individuals attached to subsurface moorings or surface buoys Figure . DuplicaL. anatifera populations. Among them, lineage 2 and lineage 3 clustered together and then clustered with lineage 1 , and also between surface populations  and subsurface populations (KE‐1 and KE‐2) from the KE region (0.380–0.709)  and also within populations    Figure . AMOVA region 0.52–0.917, ) Figure .3.2−1  among the three lineages were shown as follows: 0.185 (0.045) between lineage 1 and lineage 3, 0.144 (0.035) between lineage 2 and lineage 3, and 0.167 (0.042) between lineage 1 and lineage 2. Using 1.4% to 2.33% MyrD and Fu's FS of neutrality test as well as permutation tests with the SSD and HRI statistics . The BayeScan approach was applied to 15,238 SNPs  retained after quality control and 425 SNPs were identified as outliers  at the false discovery rate (FDR) threshold of 0.05. The left loci after removing the 425 loci under selection were further filtered by HWE and LD. After filtering HWE at 10 Bayes factor (BF) greater than 1.5, 226 candidate SNPs were identified as “outlier SNPs” associated with local temperature variables. Among these candidate SNPs, 213 and 59 SNPs were strongly correlated with the average temperature in January (T1) and August (T8), respectively, and 46 SNPs were correlated to both temperature variables. The 226 SNPs significantly associated with temperature variables were also identified as outliers by BayeScan.The BAYENV analysis based on all 15,238 SNPs revealed that outlier loci could be screened based on both temperature variables (T1 and T8) Figure . With thp = .001) with an adjusted coefficient of determination (Radj2) of 0.487, with T1 and T8 accounting for 55.36% and 44.72% of the total explained variance, respectively. The first two axes of the RDA accounted for 99.51% and 0.49% of the genetic variations, respectively .Correlation coefficients between T1 and T8 was 0.89, and the variance inflation factor (VIF) values for the two predictors were below 5, indicating that multicollinearity between the two predictors should not be a problem for the model. For the RDA analysis based on the 226 SNPs associated with local temperature, the RDA model was globally significant  of populations, observed heterozygosity (Ho), and nucleotide diversity (πG) ranged from 0.05676 to 0.06745, 0.04630 to 0.07909, and 0.05962 to 0.07064, respectively  , neutral SNP (5180 SNPs), and outlier SNP (226 SNPs) datasets, respectively. NJ tree constructed based on three SNP datasets also structured three lineages in s Figure , which aPCA analysis revealed that all populations were mainly divided into three groups, consistent with the three lineages identified by the NJ phylogenetic tree Figure . In the The most suitable numbers of ancestral populations for the three datasets were three according to the K values with the lowest cross‐validation error (CV). The results of ADMIXTURE analysis based on three SNP datasets all revealed a genetic structure consistent with the results of PCA Figure : first, 4L. anatifera. Our results also reveal that temperature, especially small‐scale thermal heterogeneity, is an important environmental factor in maintaining the high genetic diversity of pelagic species in the open oceans.Speciation or genetic divergence in the open ocean where presumably the absence of geographic barriers to gene flow has puzzled ecologists and evolutionary biologists. In the present study, we have found genetic differentiation between the SCS and the KE populations, and vertical genetic divergence in the KE populations of the pelagic barnacle 4.1Lepas anatifera exhibits high abundance worldwide  can date back to the Pliocene epoch, and all three lineages have experienced historical expansion during the late Pleistocene interglacial cycles. The temperature‐related outlier SNPs also distinguish the three lineages, especially in the PCA plot based on the outlier SNPs, and lineages from the KE region and SCS were largely separated.The goose barnacle e Young, . In the Hexechamaesipho pilsbryi  region contained haplotypes from all three lineages and potentially act as a transitional population between the Kuroshio Extension Region and the South China Sea. The existence of the lineage of SCS in KE‐6 could be potentially driven by the northward Kuroshio Current as what has been seen in other barnacles  was a significant factor influencing the distribution patterns of putatively adaptive genetic variation among populations. Among the 19 candidate adaptive loci, cuticle protein 6 possibly plays an important role in enhancing stress resistance in the cuticle of L. anatifera  are the most abundant and widespread biofouling taxa globally ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Lisha Hu: Conceptualization ; funding acquisition ; methodology ; visualization ; writing – original draft ; writing – review and editing . Zhaohui Chen: Data curation ; methodology ; resources ; writing – review and editing . Yunwei Dong: Conceptualization ; funding acquisition (lead); project administration ; writing – original draft ; writing – review and editing .The authors have no conflict of interest to declare.Appendix S1Click here for additional data file.


The extant members of the Asian rhinos have experienced severe population and range declines since Pleistocene through a combination of natural and anthropogenic factors. The one-horned rhino is the only Asian species recovered from such conditions but most of the extant populations are reaching carrying capacity. India currently harbours ~ 83% of the global wild one-horned rhino populations distributed across seven protected areas. Recent assessments recommend reintroduction-based conservation approaches for the species, and implementation of such efforts would greatly benefit from detailed genetic assessments and evolutionary history of these populations. Using mitochondrial data, we investigated the phylogeography, divergence and demographic history of one-horned rhinos across its Indian range.st value 0.68–1) corresponding to the states of Assam (n = 28 haplotypes), West Bengal and Uttar Pradesh (both monomorphic). The reintroduced population of Uttar Pradesh showed maternal signatures of Chitwan National Park, Nepal. Mitochondrial phylogenomics suggests one-horned rhino diverged from its recent common ancestors ~ 950 Kya and different populations  coalesce at ~ 190–50 Kya, corroborating with the paleobiogeography history of the Indian subcontinent. Further, the demography analyses indicated historical decline in female effective population size ~ 300–200 Kya followed by increasing trends during ~ 110–60 Kya.We report the first complete mitogenome from all the extant Indian wild one-horned rhino populations . Further, we identified all polymorphic sites and assessed rhino phylogeography  across India. Results showed 30 haplotypes distributed as three distinct genetic clades (FThe phylogeography and phylogenomic outcomes suggest recognition of three ‘Evolutionary Significant Units (ESUs)’ in Indian rhino. With ongoing genetic isolation of the current populations, future management efforts should focus on identifying genetically variable founder animals and consider periodic supplementation events while planning future rhino reintroduction programs in India. Such well-informed, multidisciplinary approach will be the only way to ensure evolutionary, ecological and demographic stability of the species across its range.The online version contains supplementary material available at 10.1186/s12862-022-02045-2. The members of Rhinocerotidae family were once one of the most diverse and widely distributed terrestrial herbivores with complex evolutionary history . By lateThe one-horned rhino, being the only Asian species recovered from severe population decline in the past are critical for the evolutionary potential of this group. With a current population size of ~ 3700 individuals , it retains ~ 96% of the Asian rhino population , 13, 14.In this paper, we investigated the phylogeography and evolutionary history of one-horned rhinos in India (henceforth Indian rhino) as it harbours 83%  of the gJava = 15,514, SAfrica = 10,680, SSumatra = 130, SIndia = 18) and nucleotide diversity  but high haplotype diversity . Both African rhino species (white and black rhino) data were combined for this analyses as no intra-species variation was observed in the available data.Sequencing with 23 primers  values among these three clusters were significantly high ranging from 0.68 to 1  and Uttar Pradesh , Sumatra—0.96 (15 samples), and Java—0.91 (6 samples), respectively], Indian rhino mitogenome showed much lower values for segregating sites and nucleotide diversity  . We have also received one tissue sample from Valmiki National Park (henceforth NP), Bihar forest department assumed to be representing the wild rhinos of Nepal (Letter/no.-1296 dated 16.10.2020). No ethical permissions were required for tissues as they were collected from naturally dead rhinos as well as for dung samples.2 area in India and Nepal [During the 1600s, the one-horned rhinos were distributed throughout the northern Indian subcontinent covering all the major river basins from Pakistan to Indo-Myanmar borders. The species has lost most of its habitat and population size due to a range of anthropogenic interventions  , 9, 14 and Nepal , 14. Betnd Nepal . The mosThe state of West Bengal currently retains ~ 350 rhino individuals distributed between two parks, Gorumara NP  and Jaldapara NP . This population has recovered from a severe population decline of ~ 20 individuals during early 1900s (due to severe habitat loss) . Apart fOverall the sampling strategy in this phylogeography study was to select unique rhino individuals from different parts of the species distribution in India. A total of 160 samples (72 tissues and 88 dung) covering four states Fig.  were useTissue DNA was extracted using already established protocol for Indian rhino mentioned in Ghosh et al. . For dunTo assess genetic variation of the extant rhino populations, complete mitogenome data was generated for representative samples from each park  were aligned and manually screened to identify the segregating sites. Further, a total of 15 primers were designed (multiple primers covering the segregating sites) to amplify all the polymorphic sites as < 500 bp fragments to ensure higher success rate from—poor quality dung DNA samples. These primers were standardised following same protocol described above. For all field collected samples (tissue = 56 and dung = 88) individual identification was performed using a panel of 14 microsatellites (described in ). After Diceros bicornis  were aligned in Mega v7 to generate a complete mitogenome sequence and manually checked to identify any nucleotide ambiguities. Annotation was done using MITOS2 web with default settings and vertebrate mitochondrial genetic code  followedNC012682 ), CeratoNC001808 ), DiceroMFO66643  and RhinFJ905815 , MK90914FJ905815 ). We calFJ905815  for all st and differential hierarchical AMOVA analysis was performed using Arlequin v. 3.0 [Population-wise basic indices of genetic variations  were calculated for concatenated sequence data (2531 bp from seven genes) using DnaSP v.5 followed by a median joining  haplotypn v. 3.0  to confiTo identify the clades, Bayesian phylogeny was constructed with MrBayes v. 3.2.7  using 16Equus caballus, Genbank: NC001640), donkey , Asiatic wild ass  and zebra  as outgroups. GTR + G substitution model was selected through jModelTest v2.1.3 for this multi-species data. Birth–death speciation was considered as tree prior [To estimate divergence among clades, rate of mutation for Indian rhino was calculated using BEAST v.2.3.6 . Analysiee prior , 34 alonee prior , 33. Duree prior , 72;  see prior , 75. ThetMRCA (time to Most Recent Common Ancestor) was inferred using the estimated mutation rate with lognormal distribution under strict molecular clock  , 35. MCMTo estimate past fluctuations in population size, Bayesian skyline analysis (Bayesian skyline plot or BSP) was conducted using concatenated sequence data with monophyletic constraint to the identified maternal clades ensuring phylogenetic construction. Analysis was conducted with multiple datasets  to ascertain any possible impacts of genetic structure in the data . In all Additional file 1: Table S1. Details of the primers used in sequencing Indian rhino whole mitogenome and phylogeography data. Table S2. Sample (both tissue and dung) of Indian rhinos used in this study. A total of 111 individual rhino samples  were used in this study. Out of the 72 tissue samples, 16 samples representing different parks were used to generate the whole mitogenome data. Table S3. Mitogenome organization in Rhinoceros unicornis. Codons respective to each tRNA are mentioned in parenthesis. Table S4. Comparative analysis results of genetic diversity indices among five extant rhino species. Table S5. Details of the variable sites based on concatenated sequence of 2531bp of Indian one horned rhino mtDNA haplotypes.Additional file 2: Figure S1. Whole mitogenome organisation and annotation of Rhinoceros unicornis. Figure S2. Gene-wise comparative analyses of polymorphism indices  for all extant rhino species. Figure S3. Estimation of mitogenome mutation rate for Indian rhino using Caballine  as outgroup. Internal node calibration points are presented in italics (bold). Estimated node ages and branch mutation rates with available reference in literature is marked with * [d with * , 10, 29.


Mustela putorius), a species under threat in several European countries, in the Białowieża Primeval Forest (BPF), Poland, before and after the invasion of the American mink (Neovison vison). Using 11 microsatellite loci and a fragment of the mitochondrial control region we show that levels of diversity changed in the polecat population over 53 generations (over the period 1959–2012) and after the invasion of mink. When compared with other threatened European polecat populations, high levels of diversity are observed in the population in BPF in both periods, as well as in other areas in Poland. Our data shows that genetic structure was not present either before or after the mink invasion in BPF. This would suggest that the polecat population in Poland was not affected by invasive species and other negative factors and would be a potential good source of individuals for captive breeding or genetic rescue conservation management actions in areas where such actions are needed, for example the UK.The introduction and expansion of an invasive non-native species could have important consequences for the genetic patterns and processes of native species, moreover if the new arrival competes strongly for resources and space. This may result in the demographic decline of the native species. Knowing the effects on the levels of genetic diversity and structure in native species is key in terms of their conservation. We analysed temporal (over 50 years) genetic variation of the population of the European polecat ( Invasive non-native species have the potential to cause a decrease in native species number and density in ecosystems mainly by predation or competition . If the Neovison vison, is a small generalist semi-aquatic mustelid that was brought to Europe in the 1920s for commercial fur farming [Mustela putorius, despite being the carnivore species most affected by it [Native to North America the American mink,  farming . Escapee farming . Since t farming . As a co farming , 10, 11. farming –14, lessed by it .The European polecat is a medium-sized mustelid widespread in the western Palaearctic . It inhaThe American mink and polecats’ spatial and trophic niches partially overlap , 28 poteBPF is one of the last non-altered habitats in Europe, with low hunting or poaching pressure on small mustelids, a high density of polecat preys and with no ferrets or European mink living there  . The AmeIn this study we used eleven autosomal microsatellite markers and a fragment of the mitochondrial DNA to investigate the effects of the American mink invasion on the neutral genetic diversity of the polecat population in the Białowieża Primeval Forest. We genotyped the historical  and current  polecat populations in BPF to analyse: i) potential changes in the levels and distribution of genetic diversity due to the American mink invasion, and ii) changes in the demographic history of the polecat population. Additionally, we genotyped samples from other locations in order to understand the current genetic structure of the polecat population in Poland.A total of 92 individuals were collected during the period between 2000 and 2012 from the areas of Białowieża Primeval Forest , Biebrza river basin , Narew river basin , Augustów Forest , Drawa and Warta river basins  and Bug river basin  (contemporary samples) and one with unknown location (OTH) . Muscle All tissue and hair samples were stored at -20°C prior to DNA extraction. Additionally, DNA samples were obtained from 116 museum skulls from BPF stored at the zoological collection of Mammal Research Institute PAS collected over the period 1959–1998, and originated from either road kills or animals shot by hunters. Material from historical samples was obtained from the turbinals in the nasal cave. In the case of BPF, both contemporary and museum samples combined represents a time span of 50 years. See .DNA from fresh tissues  was extracted using the DNA Tissue Kit (QIAGEN) following the manufacturer’s instructions. Samples of the turbinals were extracted from the nasal cave with the help of a stick. Due to the softness of this bone tissue, each sample could be powdered manually inside the tube used for its digestion by using a plastic stick. DNA was extracted using the QIAamp DNA Investigator Kit (QIAGEN) following the manufacturer’s instructions. Bone extractions were carried out in a separate room specially conditioned for working with non-invasive and ancient material. All the manipulations were made in a laminar-flow hood that was UV irradiated previous to and after any manipulation. Negative extraction controls were performed in every extraction of 23 samples to monitor possible contamination. Among the 150 museum samples, 92 rendered enough DNA for the microsatellite study (77%) and 63 were sequenced for the mtDNA.M. lutreola (MLUT25). Twelve additional microsatellite markers developed for the American mink had also been assayed in an initial set of 18 random samples and discarded due to spurious results , five of them were developed for the polecat , and one was developed for the European mink, ® BIO-RAD) thermocycler. PCRs consisted of an initial denaturation step at 95°C for 15 minutes, followed by 30 cycles (33 cycles for museum samples) of 94°C for 30 s, 60°C for 90 s, and 72°C for 60 s, and a final extension at 60°C for 30 min. To monitor the possibility of allelic dropout related to low DNA quality and/or quantity in museum and hair samples these were amplified and genotyped three times each and for each multiplex. A high rate of allelic dropout was observed for the Mvis072 marker in samples from the museum so this marker was excluded from all analyses. A final dataset of eleven microsatellite markers was used for further analysis. Fragments were analysed in an ABI3130xl Genetic Anlyser (Applied Biosystems). Allele scoring was performed with the GeneMarker (v 1.85) software (SoftGenetics). Microsatellite genotypes are provided in With the set of twelve microsatellite that rendered good results, three different multiplex were carried out using a Multiplex PCR Kit (QIAGEN) in final reaction volumes of 5 μl for the fresh samples and 8 μl for samples extracted from turbinals, containing a final concentration 1x QIAGEN Multiplex PCR Kit (Qiagen), (0.2 μM) of each primer, 0.25 μg/ μl BSA (Fermentas) and 30 ng of DNA (for museum samples 2 μl of DNA extraction were used) in a  or 20 μl (museum samples) and comprised 3 μl (fresh samples) and 6 μl (museum samples) of DNA extract (3–10 ng—about 60 ng per reaction), 5 μl (fresh samples) and 10 μl (museum samples) HotStar Taq Master Mix Kit (Qiagen), 0.5 μM of each primer in final concentration. PCR cycling comprised an initial denaturation at 95°C for 15 min, followed by 35 (fresh samples) and 38 (museum samples) cycles at 94°C for 60 s, 56°C for 60 s, 72°C for 90 s and a final extension at 72°C for 10 min. PCR products were sequenced using the BigDye v 3.1 Terminator Cycle Sequencing Kit (Applied Biosystems) and an automated DNA fragment analyser . Sequences were edited, assembled and aligned using the program Sequencher 4.9 (Gene Codes Corporation).A fragment of 640 bp of the mtDNA control region was amplified in forward and reverse orientations using the primers LutF  and Mpu1Only the cohorts defined in BPF and NAR-BIE were considered to have adequate sizes to perform population analyses on them. Samples from AUG, DRA and BUG  were considered only in the analysis of population structure.Diversity. Haplotype and nucleotide diversity (π) and the number of segregating sites (S) were estimated and mtDNA haplotypes identified for the global polecat populations with DnaSp v5 [DnaSp v5 . IndicesDnaSp v5 .Population differentiation and genetic structure. We calculated ΦST and exact test of differences between the population pre and the population post arrival of the American mink  using Arlequin 3.5 [quin 3.5 . In ordePopulation demographic history. Past population dynamics of the polecat was investigated with a Bayesian Skyline Plot model using BEAST v1.8.1 [−8 substitutions/site/generation as estimated for Mustela erminea in [T v1.8.1 . We usedminea in  from a cminea in  showed tDiversity. Observed and expected heterozygosities (Ho and He respectively) and heterozygote deficits, linkage disequilibrium and Hardy-Weinberg (HW) equilibrium were tested for BPF and NAR-BIE with the probability test of Genepop v3.5 [pop v3.5  using a pop v3.5 . Signifipop v3.5 . Geneticpop v3.5 . Levels Population differentiation and genetic structure. We calculated FST and exact test of differences between the population pre and the population post arrival of the American mink using Arlequin 3.5. Using the same scheme of sliding windows as for mtDNA, we calculated pairwise FST value [ST to RST distances as the latter would underestimate differentiation when populations are not highly structured [ST value  among alructured  whereas ructured . Correlaructured .We further investigated the pattern of spatial genetic structure at present using data from microsatellites. For this we conducted a spatial principal component analysis (sPCA) for BPF  and NAR-BIE site  using the Adegenet (version) package  for R. T6 steps after a burn-in period of 105 steps. All chains were run using the F model for correlations of allele frequencies across clusters [We performed a clustering analysis of all the individuals sampled in this study using STRUCTURE v2.3 . This veclusters . Any posclusters . The figclusters .Population demographic history. The occurrence of a genetic bottleneck in the recent past was investigated in the contemporary sample using the program BOTTLENECK [1 and N0, respectively), the mutation rate (μ) and the time since the population started changing (T). We used wide lognormal priors with large variances to affect posterior distributions as little as possible  intervals and plotted the results using the R packages Coda [4 sampling points to build a single, definite posterior distribution. We visualized the trace plots of the MCMC chains and calculated the mean values and the 90% high probability density (HPD) of the posterior distribution for each parameter with the program Tracer 1.7 [TTLENECK . The proTTLENECK  i.e. witTTLENECK . AdditioTTLENECK . We estiTTLENECK , as implTTLENECK . The demTTLENECK , 63. ThiTTLENECK  and the TTLENECK  in the dTTLENECK . It allo used in ). A gene used in . Three iges Coda  and Locfges Coda . Once coacer 1.7 .D and ∏) in the polecat population were 0.82 and 0.0043 before, and 0.67 and 0.0028 after the arrival of the American mink  and 84 were from after the American mink invasion (48 of them from BPF), 17 corresponded to individuals sampled in between (period 1974–1997), and three were from unknown date and/or origin. All sequences were deposited in GenBank under accession numbers OK644317-OK644331 and OM069433—OM069566. The length of the fragment was 532bp excluding the previously described polypyrimiden CnTn tract in the control region of polecats , 69, 70.can mink . This pocan mink . This die was 0.65. The genotyping of the pre-American mink population  with eleven markers rendered a total of 74 alleles. Number of alleles per locus ranged between four and ten and expected heterozygosity He was 0.63. The genotyping of the post-American mink population  with eleven markers rendered a total of 80 alleles. Number of alleles per locus ranged between five and nine and expected heterozygosity He was 0.67. The genotyping of the BPF population after the arrival of the American mink  with eleven markers rendered a total of 73 alleles. Number of alleles per locus ranged between four and nine and expected heterozygosity He was 0.66. Deviation from Hardy-Weinberg equilibrium was not detected in the populations (p > 0.05). When compared to the total post-American mink population, He was lower in historical BPF , and AR was similar . Expected heterozygosity was similar between the pre- and post-arrival mink population in BPF  and AR was lower in the historical population  .The genotyping of the whole set of individuals (n = 208) with eleven markers rendered a total of 84 alleles. Number of alleles per locus ranged between five and ten, and global expected heterozygosity H032) see . FinallyMitochondrial DNA. The ΦST value between the pre and post arrival of the American mink populations was low but significant (ΦST = 0.08 p < 0.001). P value for the exact pairwise differences was as well significant (p < 0.001). This pattern was the same when considering only the individuals from BPF in the population post  and a significant p value for the exact pairwise differences (p < 0.001). This difference might be explained by the change in haplotype frequencies. Only nineteen out of 74 pairwise comparisons among cohorts in BPF and NAR-BIE sites were significant after Bonferroni correction for both ΦSTs and pairwise differences . Otherwise, the p value for the exact pairwise differences was not significant (p = 1.00). This pattern was the same when considering only the individuals from BPF in the population post arrival of the American mink (FST = 0.014 p < 0.001), and a very strong non-significant p value for the exact pairwise differences (p = 1.00). The exact test has been shown to be more powerful than the FST test [ST value, suggest that if any differentiation is present it might be subtle. Thirty-five out of 98 pairwise comparisons among cohorts in BPF and NAR-BIE sites were significant after Bonferroni correction  with a 95% of SMM model  or with a 78% of stepwise mutation model (SMM) . The mode shift test showed a normal L-shaped distribution in both cases. The analyses rendered almost identical results for the historical population in BPF. Estimated effective population size for the population pre-dating the arrival of the American mink in BPF  was similar than that of the population after .e of 2,399 individuals . This demographic process occurred around 7.8 ky ago (0.049–4.9x109 HPD 95%). The convergence among MCMC chains and the ESS values behaved better for the Ne estimated, ancestral and present, than for T. This, along with the wide confidence intervals obtained for T, suggests that the latter parameter could not be properly estimated from our data, and both the median and intervals for T should be taken with caution.Using the coalescent-based approach implemented in MSVAR 1.3 we found evidence of a historical decline in the polecat population size. An ancestral population with Ne of 15,382 individuals  suffered a reduction that brought the population to a NThe analyses of the polecat population in BPF before and after the arrival of the American mink, and of other additional populations with both microsatellite and mitochondrial markers suggested an almost absence of effects of the arrival of this non-native, invasive competitor on the genetic patterns of the polecat population in terms of both diversity and structure. This would be in agreement with recent data showing weak evidence that the American mink had a negative impact in the demography of the polecat in Poland .th century [Levels of nuclear diversity in the present polecat population in Poland are in the highest values of the range when compared to other polecat populations  and cons century . Mitocho century  although century . These l century ).e has remained similar. Adult sex ratio (ASR) has proven to be heavily shifted towards males in NE Poland  and in Belarus this shift being up to an increase from 59% to a 95% of males in the population [Ne) and increases the deleterious effects of genetic drift. In the case of the polecat, one male mates with 1–3 females while females usually mate with one male [We were not able to find differences in the levels of diversity between the extant population and the population predating the arrival of the American mink, at any of both nuclear or mitochondrial levels. Although the timeframe considered before and after the arrival of the American mink is not long, it still includes 53 generations, considering one year the generation time of the polecat . Other spulation . Analysipulation . The intpulation . Theory one male . If a sione male , enough one male . This coOn the other hand, the lack of genetic structure observed among populations from diverse areas of Poland, either as a result of a recent common origin of the potential populations or because they are connected by gene flow, would explain why genetic diversity could have been maintained. This lack of structure is remarkable given the geographical distance between the sampling locations (up to 700 km). The sex dispersing in the polecat is the male, while females remain more phylopatric . They die with respect to the nuclear genome. Otherwise, confidence intervals are big enough to infer that the bottleneck happened earlier in time, and the mtDNA fragment analysed is not informative enough to trace it. A bigger battery of nuclear markers as SNPs would be necessary to have a clearer picture on this question.None of the methods used were able to detect a recent bottleneck in the polecat population. MSVAR was able to detect recent population reductions in otter and in the British polecat populations using a similar number of markers , 78, proA review on the polecat status in 34 countries found that populations were declining or suspected to be declining in 20 of them, including Poland . HoweverS1 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableSliding-window cohorts for A- microsatellites and B- mtDNA sequences. Samples were subdivided in cohorts of four years. n indicates the number of individuals in each cohort. Only cohorts integrated by eight or more individuals are listed and considered for the analyses (and thus included here).(XLSX)Click here for additional data file.S3 TableA) Number of alleles, He and Ho values for the eleven microsatellites for all the samples in this study. B) List of microsatellite markers tested but not finally used in this study.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableA- mtDNA haplotypes found in this study. B- Distribution of the Haplotypes in the populations.(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Bulinus truncatus  on a regional (17–150 km) and inter-regional (1000–5400 km) scale. This snail species acts as an intermediate host of Schistosoma haematobium and Schistosoma bovis, which cause human and animal schistosomiasis respectively.Gastropod snails remain strongly understudied, despite their important role in transmitting parasitic diseases. Knowledge of their distribution and population dynamics increases our understanding of the processes driving disease transmission. We report the first study to use high-throughput sequencing (HTS) to elucidate the population genetic structure of the hermaphroditic snail Bulinus truncatus snails were collected in Senegal, Cameroon, Egypt and France and identified through DNA barcoding. A single-end genotyping-by-sequencing (GBS) library, comprising 87 snail specimens from the respective countries, was built and sequenced on an Illumina HiSeq 2000 platform. Reads were mapped against S. bovis and S. haematobium reference genomes to identify schistosome infections, and single nucleotide polymorphisms (SNPs) were scored using the Stacks pipeline. These SNPs were used to estimate genetic diversity, assess population structure and construct phylogenetic trees of B. truncatus.A total of 10,750 SNPs were scored and used in downstream analyses. The phylogenetic analysis identified five clades, each consisting of snails from a single country but with two distinct clades within Senegal. Genetic diversity was low in all populations, reflecting high selfing rates, but varied between locations due to habitat variability. Significant genetic differentiation and isolation by distance patterns were observed at both spatial scales, indicating that gene flow is not strong enough to counteract the effects of population bottlenecks, high selfing rates and genetic drift. Remarkably, the population genetic differentiation on a regional scale  was as large as that between populations on an inter-regional scale. The blind GBS technique was able to pick up parasite DNA in snail tissue, demonstrating the potential of HTS techniques to further elucidate the role of snail species in parasite transmission.HTS techniques offer a valuable toolbox to further investigate the population genetic patterns of intermediate schistosome host snails and the role of snail species in parasite transmission.The online version contains supplementary material available at 10.1186/s13071-022-05445-x. Bulinus truncatus  is one of the main intermediate hosts of Schistosoma haematobium , the causative agent of human urinary schistosomiasis. It is also an intermediate host of the bovine parasite Schistosoma bovis  and different Paramphistoma and Echinostoma species . Bulinus truncatus is a tetraploid species belonging to the monophyletic B. truncatus/tropicus complex . All of the B. truncatus queries but four  were due to the presence of a misidentified/mislabelled sequence of B. tropicus in the reference dataset (GenBank accession KJ157497). We assume that this reference sequence was misidentified as: (i) it was the only B. tropicus sequence in an otherwise mono-specific clade of B. truncatus (83% bootstrap support) and (ii) all other B. tropicus reference sequences were recovered in two clades, one including three specimens from Cameroon (60% bootstrap support) and the other also including B. natalensis and B. nyassanus  were significantly different from zero in all samples, except in the population of Cameroon (CAM_BAK), notably the population exhibiting the smallest sample size . A discontinuous distribution of individual geographic and genetic distances was observed due to the two different spatial scales being sampled , large scale across countries)  Fig. . Positives) Fig. .Fig. 5IsThe midpoint-rooted ML tree reconstructions , indicating relatively high amounts of gene flow on a regional scale of 100 km . In Senegal, the low differentiation might also be a relic of the Diama dam construction (see below) that was followed by a rapid expansion of snail populations, leading to highly inbred and genetically similar snail populations in the lower valley of the SRB, as was found for Biomphalaria pfeifferi in the same area [Fst 0.709–0.894) between these three populations and the Sen_GUEC population only 100 km further upstream, of a magnitude comparable to the genetic differentiation between the Senegalese and Egyptian populations (Fst 0.689–0.751), which are almost 5000 km apart. To interpret this differentiation of the Sen_GUEC population, a few factors influencing gene flow need to be considered. First, high stream velocities in rivers might flush away snails, either through detachment from their substrate or through exceedance of the snail’s tolerance limits, giving rise to gene flow from upstream to downstream populations [Bulinus species [Consistent with previous studies , 58, 59,w et al. , 24 in Came area . Howeverulations , 62. Seculations , 64 or tulations . The comulations , 64, 65. species .Considering these phenomena, the genetic dissimilarity of the Sen_GUEC population could be explained by its location along the Doué River, which discharges in the Senegal River approximately 60 km downstream and originates from the same river approximately 160 km upstream. It is possible that the current from the Doué River is an effective barrier for snails from the lower valley of the SRB to migrate upstream, while snails coming from the higher part of the Senegal River can migrate downstream easily. Furthermore, because it is a tributary of the Senegal River, the Doué River populations contribute relatively little to the gene pool in the lower SRB compared to the populations coming from the upper parts of the Senegal River. A similar pattern has been observed in Egypt, where populations in the lower Nile River are genetically similar to each other but are distinct from populations upstream . FinallyB. truncatus and B. globosus, respectively, and indicates that  gene flow is not strong enough to completely counteract the effects of high selfing rates and genetic drift in B. truncatus [.A clear IBD pattern and positive spatial autocorrelation is present, both on a regional scale and on an inter-regional scale, even after omitting the Sen_GUEC population from the regional analysis. This finding is consistent with the results from microsatellite studies by Viard et al.  and Nyakruncatus , 68.B. truncatus strongly influences the population genetic diversity. In this study we found an excess of homozygotes and low levels of genetic diversity in all populations, which is consistent with previous studies on highly selfing freshwater snails  compared to the snail, resulted in the massive outbreak of intestinal schistosomiasis [The hermaphroditic nature of ls e.g. , 69, 70), 70B. trls e.g. , 71, 72), 72B. trls  might be attributed to the temporary habitats in both countries. The Egyptian snails were sampled inside irrigation canals that dry up frequently, while the Senegalese snails originate from the Senegal, Lampsar and Doué River that are prone to large debit fluctuations [B. truncatus and the ability to quickly (re)colonise previously dried-out habitats [B. truncatus populations [Viard et al. , 66 showtuations  and fromhabitats , 79 resuhabitats , 80, 81.B. truncatus populations  comprised only about 3–4% of the total snail biomass, in our samples up to 9% of total reads could be attributed to schistosome parasites. Although the percentage of parasite biomass in a snail cannot directly be related to the percentage of parasite reads in that sample, this still shows that even blind HTS techniques can detect parasite DNA in snail tissue without prior amplification rounds, opening up new avenues for future research on the importance of genetics in determining snail-parasite compatibility.The GBS technique was able to confirm snail infections in samples that were previously identified to be infected during shedding experiments in the field, but it could also detect prepatent infections as it detected schistosome DNA in two samples that were negative after shedding. Although Preston et al.  showed tB. trunatus clades could be identified, each comprising snails from a single country but with two distinct clades within Senegal. The genetic diversity within populations was low because of high selfing rates, but varied between geographic locations, probably due to habitat variability. Considerable genetic differentiation at both a regional and an inter-regional scale was observed, mainly due to the clear differentiation of the Sen_GUEC population from the other Senegalese populations. However, a clear IBD pattern remains present on both spatial scales, even after the Sen_GUEC population was omitted from the analysis. This indicates that regional gene flow is not strong enough to counteract the effects of bottlenecks, high selfing rates and genetic drift. The genetic differentiation patterns observed in B. truncatus are remarkably close to the patterns observed in S. haematobium in the area, which could explain the differences in parasite susceptibility of snails from the lower and middle valley of the SRB. HTS techniques offer a valuable toolbox to further investigate these patterns.In this study, five distinct Additional file 1: Table S1. Sample list and information.Additional file 2: Table S2. Best close match DNA barcoding identification (1% distance threshold) of vouchers considered in this study.Additional file 3: Figure S1. Neighbour Joining (NJ) tree representing K2P genetic distances among vouchers considered in this study (blue dots) and 181 public reference sequences (http://www.boldsystems.org/) from 14 Bulinus genera .Additional file 4: Figure S2. Percentage of reads assigned to Schistosoma haematobium contamination per snail specimen.Additional file 5: Figure S3. Log-likelihood probability values (LnP(D)) and ∆K  as obtained in STRUCTURE with K ranging from 1 to 8 .Additional file 6: Figure S4. Maximum likelihood tree reconstruction based on 763 SNPs recovered in the 100% of specimens considered in this study (see text for explanations).Additional file 7: Figure S5. Maximum likelihood tree reconstruction based on 6577 SNPs recovered in at least 70% of specimens considered in this study (see text for explanations).


Wolbachia, are common in insects and other invertebrates but infection dynamics across species ranges are largely under studied. Specifically, we lack a broad understanding of the origin of Wolbachia infections in novel hosts, and the historical and geographical dynamics of infections that are critical for identifying the factors governing their spread. We used Genotype-by-Sequencing data from previous population genomics studies for range-wide surveys of Wolbachia presence and genetic diversity in North American butterflies of the genus Lycaeides. As few as one sequence read identified by assembly to a Wolbachia reference genome provided high accuracy in detecting infections in host butterflies as determined by confirmatory PCR tests, and maximum accuracy was achieved with a threshold of only 5 sequence reads per host individual. Using this threshold, we detected Wolbachia in all but 2 of the 107 sampling localities spanning the continent, with infection frequencies within populations ranging from 0% to 100% of individuals, but with most localities having high infection frequencies (mean = 91% infection rate). Three major lineages of Wolbachia were identified as separate strains that appear to represent 3 separate invasions of Lycaeides butterflies by Wolbachia. Overall, we found extensive evidence for acquisition of Wolbachia through interspecific transfer between host lineages. Strain wLycC was confined to a single butterfly taxon, hybrid lineages derived from it, and closely adjacent populations in other taxa. While the other 2 strains were detected throughout the rest of the continent, strain wLycB almost always co-occurred with wLycA. Our demographic modeling suggests wLycB is a recent invasion. Within strain wLycA, the 2 most frequent haplotypes are confined almost exclusively to separate butterfly taxa with haplotype A1 observed largely in Lycaeides melissa and haplotype A2 observed most often in Lycaeides idas localities, consistent with either cladogenic mode of infection acquisition from a common ancestor or by hybridization and accompanying mutation. More than 1 major Wolbachia strain was observed in 15 localities. These results demonstrate the utility of using resequencing data from hosts to quantify Wolbachia genetic variation and infection frequency and provide evidence of multiple colonizations of novel hosts through hybridization between butterfly lineages and complex dynamics between Wolbachia strains.Infections by maternally inherited bacterial endosymbionts, especially  Wolbachia  how Wolbachia infects novel hosts and (2) the historical biogeography of infection dynamics within and among host lineages.The endosymbiotic bacteria in the genus olbachia  have beel attack . The manWolbachia infections occur in more than half of insect species   loci  loci . PresencLST loci . HoweverWolbachia resequencing data from phylogeographic and population genomics studies of host species  is known as the Karner blue butterfly and is a federally listed endangered species . These populations also possessed a mitochondrial haplotype identical to a haplotype found in L. melissa (the proposed source of the infection), but distinct from haplotypes found in the eastern portion of their range (east of Lake Michigan)  sequence reads from 2,377 butterflies of the genus  to 2018 . These d studies .Wolbachia sequence reads found among GBS reads from North American Lycaeides butterflies to address the following questions: (1) how does variation in detection thresholds of sequence depth and sequence length influence Wolbachia infection frequency estimation? (2) how does the frequency of infection  vary across host populations, lineages, and geography? and (3) how are Wolbachia genotypes and groups of genotypes (which we equate to strain types) distributed across geography and host taxonomy? We use the answers to these questions to construct hypotheses about the history and biogeography of infections in Lycaeides. We also discuss the opportunities and limitations to using Wolbachia reads present in resequencing data as an inexpensive tool for understanding Wolbachia dynamics in natural populations. We also argue that similar data from other host taxa might contribute to the growing understanding of the evolution and history of Wolbachia-host interactions.We used We extracted genomic DNA, generated GBS libraries, and sequenced these libraries following the methods described in Lycaeides butterflies harbor Wolbachia . We interpreted this as a possible indication that butterflies from different localities, or taxa, harbored a diversity of Wolbachia strains and that different reference genomes might yield better assemblies for some strains and therefore also some host localities or taxa.Though we knew from PCR-based surveys that Wolbachia reference genomes representing each of supergroups A, B, and F  out of the 1,651 scaffolds in the L. melissa genome, and not from any of the larger chromosomal level (23 autosomes and Z sex chromosome) scaffolds. Based on the length (similar to size of other Wolbachia genomes) and mapping metrics of this region, we believe this scaffold to be the genome of Wolbachia infecting the host butterfly individual used in the genome assembly. We then pursued a second assembly in which we used this Scaffold 1260 as a species-specific reference Wolbachia genome for further analysis.Because nuclear integration of e genome . Upon quWolbachia reference genomes. Here we used the supergroup A, B, and F genomes described above plus the Scaffold 1260 from the Lycaeides reference genome. The pan-genome was constructed by first annotating the representative Wolbachia genomes using prokka , and then combining the files to produce a reference pan-genome using the Roary , reads were aligned using bowtie2 software  from samtools view -F 2432  for infection that maximizes concordance of infection status with results from the previous PCR-based amplification study in these same butterfly species.We first quantified the number of mapped reads and the length of mapped reads in an individual’s sam files were each compressed into bam files (using samtools) for more efficient downstream analyses. We then performed variant calling and genotyping on the sorted and indexed bam files from the previous step using the bcftools mpileup command (skipping indels) followed by the bcftools call and view commands  and retained only bi-alleleic sites (–ploidy 1–variants-only -m2 -M2 -v snps). The choice to employ a haploid model was based on preliminary analyses from a diploid model. Given the existence of more than 1 major Wolbachia strain and sympatry among strains in some instances (see Results), it is possible that individual host butterflies could contain multiple infections . However, models for variant calling with higher ploidy  compromised our ability to call variants as haplotypes because phasing of alleles at multiple sites was not possible. Therefore, we employed the haploid model to produce useful haplotypic data. This undoubtedly prevented discovery of additional haplotypes in individuals with multiple infections, but did produce population genetic data for those individuals with single infections. The raw vcf file was then filtered to only keep sites with a maximum missingness of 25% using vcftools . We retained 1,277 individuals  with haplotypes of length 115 bp.The alleles in each individual from this haplotypes . Strain types, and haplotypes within strains, were then mapped onto the geographical and taxonomic distributions of the host butterflies. Thus, the distribution of Wolbachia strains and haplotypes  and used BEAST v2.6.3 to estimate Bayesian Skyline Plots  to understand Lycaeides individuals sequenced, a total of 3,727,714,988 sequence reads were generated . From the Wolbachia mapping protocol described in the Materials and Methods section and on filtering for reads with mapping quality . We found that results from this threshold matched very well with results from a previous PCR amplification study . A thres0 bp see . Similar0 bp see . HoweverThe numbers of infected individuals were not substantially changed by varying the minimum number of reads required to diagnose infected individuals for most localities. The exceptions where prevalence did vary with different thresholds were localities for Karner Blue butterflies in the eastern portion of their range (Indiana Dunes (5), Allegan (6), Saratoga (7)) and several of the Sierra Nevada hybrid lineage localities .  from Lycaeides localities had infection frequencies that exceeded 90% of individuals, with 85 of the 107 sampled localities showing greater than 90%  . At the species or lineage level, most infections rates are greater than 94%  (infection rates: 0–0.5%), 1 population of L. ricei from the Marble Mts. in California (38) (infection rate: 58%), a small number of L. melissa populations, mostly in the western Great Basin  (infection rates: 80–88%), 1 population of the hybrid lineages in the Jackson area (at Swift Creek (92) [infection rate: 75%]), in the Sierra Nevada  (infection rates: 15–75%) and in the White Mountains  (infection rates: 87–89%)  . In poputhan 94% . The exc 87–89%) .vcf file with 115 variable sites and 1,277 individuals was used for population genetic analyses. Based on a haplotype network analysis with 95% statistical parsimony and PCoA of pairwise distances among haplotypes, we found that 1,267 out of 1,277 genotyped individuals carried Wolbachia haplotypes from 1 of 3 major haplotype networks  ). The highest strain diversities were observed in the western Great Basin L. melissa populations and in some of the localities of hybrid lineages of Lycaeides (The filtered networks  that cornetworks . We consd wLycC) . Each ofd wLycC) . The strd wLycC) . In addid widely . Butterfycaeides .Wolbachia strain wLycA was observed in 992 individuals and was the most frequent strain. Among the 19 haplotypes within strain wLycA, haplotypes A1 and A2 were observed in 936 individuals . Though these 2 haplotypes were differentiated by a single mutational step  where A1 was observed; A1 was also observed in the 4 L. samuelis localities sampled in Wisconsin (1–4) , in the Jackson hybrids (82–92), and hybrid lineages in the Warner Mountains (93–94), Jarbidge Mountains (97), and at Steens Mountain (95)  for which L. anna is the maternal parent) (L. melissa and L. idas at Dubois (85) (L. ricei population at Cave Lake (37), and in two L. melissa localities in the Rockies   . Both A1 .wLycB was observed in 103 individuals and included 44 haplotypes. Strain wLycB haplotypes occurred most frequently in the populations of L. melissa in the western Great Basin (68–78) (“L. melissa—West” in wLycB haplotypes in these western populations. The other haplotypes occur in low frequency in these L. melissa West populations and at County Line (106), part of the hybrid lineage in the White Mountains, and in 2 L. idas populations , 1 population of L. melissa East (56), the Big Lake (33) population of L. ricei, and 3 populations of L. idas (Spruce Barley (30), Garnet Peak (21), and Hayden Valley (25); Strain wLycC was observed in 172 individuals and included 3 haplotypes. Strain wLycC haplotypes were confined to L. anna populations (8–13) and hybrids in the Sierra Nevada and White Mountains . These hybrids have mixed ancestry from both L. melissa and L. anna and the latter is presumed to be the maternal lineage based on patterns of mtDNA variation  and Marble Mt.s (38) L. ricei populations, which are the southern-most sampled L. ricei localities and adjacent to the range of L. anna  that were observed as singletons, 5  in 4 localities in the western Great Basin  (L. melissa West), 1 (D1) in the County Line (106) hybrid population, 2 (G1 and G2) in the Marble Mountains (38), 1 (I1) at Wheatland , and 1 (J1) at Yuba Gap   .wLycA and wLycC, and about 400 for strain wLycB . Strain wLycA  shows a constant scaled population size of 0.02 stretching into the very distant past. Strain wLycB , the hybrid lineages in the White Mountains in California (106), and Jackson, Wyoming area (83–84)) seems to have existed at much higher population sizes (wLycA) in the distant past, but has experienced a growth phase starting 0.003 time units in the past and has grown up to wLycC  has a very small and constant population size  (5–7), but Karner blue populations in the western portion of the range (1–4) do not exhibit the same reduction in estimated prevalence with increasing minimum reads threshold. Similarly, the other localities that show the decline in numbers of infected individuals with increasing minimum reads threshold occur in the hybrid lineage of Lycaeides in the Sierra Nevada , yet other hybrid lineages do not show a similar pattern. It is not immediately obvious why these localities differed in their apparent sensitivity to the minimum reads threshold. The overall number of sequence reads per individual could affect the probability of detection, but while the eastern Karner localities have lower median number of reads compared to the total set of 2,377 individuals, the Sierran hybrid populations have more reads per individual than the overall median . Alternatively, it is possible that there is variation in Wolbachia densities within individuals among localities that influences detection probability  were ultimately acquired by Lycaeides in North America. Analysis of Wolbachia infections from Lycaeides from Europe and Asia, or from associated parasites or parasitoids, might shed light on the origins of North American infections. However, our survey of geographic patterns of population genetic variation in combination with inference of demographic histories of the 3 major strains suggest that transmission of infection within North American Lycaeides butterflies occurred primarily through introgressive acquisition. We provide an overview of these patterns.Population genetic analyses of Wolbachia substitution rates over long timescales  exhibit just 1 mutational difference , the contemporary hybrid zone between L. melissa and L. idas at Dubois (85), or localities at or near range boundaries, such as Cave Lake (37)). The confinement of these haplotypes largely within 2 Lycaeides species seems compatible with the hypothesis of cladogenic acquisition in the ancient past through a common ancestral lineage of L. idas and L. melissa, followed by independent divergence of the 2 lineages. Alternatively, the distribution of haplotypes A1 and A2 might be consistent with introgression from 1 of the species into the other accompanied by mutation. Further, the exceptions to the distributional pattern (e.g. hybrid lineages and a hybrid zone) appear to be examples of introgressive acquisition of strain wLycA haplotypes outside of L. melissa and L. idas. Thus, there is perhaps more support for introgressive acquisition of wLycA haplotypes, though cladogenetic acquisition cannot be ruled out. Evidence for multiple modes of Wolbachia transmission in natural populations is also found in the Drosophila  for which L. anna is the presumed maternal lineage  and Shovel Creek (39). Thus, as with strain wLycA haplotypes, wLycC haplotypes appear to have spread to a limited extent outside of a Lycaeides species range via introgression among lineages, specifically in this case from L. anna to nearby populations of L. ricei.A similar demographic history of constant population size over time is seen in strain  strains . Strain  lineage   , Deer Mt. Road (76), and Gardnerville (78)). However, wLycB haplotypes were observed in other widely distributed places and other Lycaeides taxa including: L. idas in Alaska (30) and Montana (21), L. ricei at Cave Lake (37), L. melissa in central Nevada (56), in the Jackson hybrid lineage (84), the hybrid lineage in the White Mountains of California (106), and the putative hybrid lineage at Hinkley in northern Nevada (96). The relative rarity of this strain, its recent population expansion , that was sampled over multiple years and strain wLycB appears to have increased in frequency from 2011 to 2018  relatively recently  . In thesxpansion , coupledulations . The con to 2018 . The stuLycaeides butterflies each contained a dominant (most frequently observed) major strain . This pattern is consistent with interspecific transfer from L. melissa to L. samuelis  and cytochrome oxidase II (COII) genes, wLycA here. Another mtDNA lineage (lineage II) co-occurred with the first lineage and was detected in populations of L. melissa from the western Great Basin and from the hybrid population in the White Mt.s , corresponding to the distribution of wLycB. Lastly, the third mtDNA lineage (lineage I) was observed in L. anna, hybrid lineages derived from L. anna in the Sierra Nevada and adjacent L. ricei localities, corresponding to wLycC. The close geographical correspondence of major Wolbachia lineages observed here and previous mtDNA haplotype distributions suggest that the expected disequilibrium between Wolbachia strains and mtDNA can be detected using GBS data.The distribution of ilibrium . Direct Wolbachia infection frequencies and genetic variation using GBS data from host Lycaeides butterflies suggests that this approach could be applied in other systems. Given the quantity of resequencing data generated recently, it might be possible to rapidly survey Wolbachia and other endosymbiont infections in a wide variety of host organisms and answer broad questions about the history, geography and mode of acquisition of infections. However, resequencing methods do not specifically target Wolbachia genomes and there exist several limitations. The sequence reads from Lycaeides GBS data did not map to any of the multilocus sequence typing (MLST) loci (Wolbachia supergroups) and connect studies phylogenetically from surveys of GBS data without further sequencing. Additionally, the stochasticity inherent in the methods for resequencing data, combined with the sparseness of endosymbiont sequence reads from host organisms, presents some challenges. Stochasticity arising from library preparation and from the sequencing of these multiplexed genomic libraries, among other possible sources of stochasticity, creates variation in sequence depth across fragments and individuals. This variation can contribute to false negatives for infection detection. Given variation in sequencing effort across studies, we note that the threshold for infection detection (here we used a minimum of 5 sequence reads) will need to be carefully examined for each investigation. False positives from GBS data seem less likely than false negatives compared to PCR-based methods for infection detection, though contamination of samples is an important consideration for both PCR-based and GBS survey methods. The usefulness of resequencing data for population genetics investigations of endosymbionts will be facilitated by the development of new methods for detecting infection and for genotyping that can, for example, more fully account for uncertainty and accommodate the possibility of multiple infections within individuals. Despite these limitations, the use of resequencing data can cheaply and relatively easily facilitate surveys of endosymbiont infection and population genetics.Our survey of ST) loci  and it sjkac213_Supplemental_MaterialClick here for additional data file.


Plasmodium falciparum and Plasmodium vivax. Indigenous malaria caused by P. vivax was eliminated in Hainan in 2011, while imported vivax malaria remains. However, the geographical origin of P. vivax cases in Hainan remains unclear.Hainan Province, China, has been an endemic region with high transmission of P. vivax isolates (n = 45) were collected from Hainan Province, and the 6 kb mitochondrial genome was obtained. Nucleotide (π) and haplotype (h) diversity were estimated using DnaSP. The numbers of synonymous nucleotide substitutions per synonymous site (dS) and nonsynonymous nucleotide substitutions per nonsynonymous site (dN) were calculated using the SNAP program. Arlequin software was used to estimate the genetic diversity index and assess population differentiation. Bayesian phylogenetic analysis of P. vivax was performed using MrBayes. A haplotype network was generated using the NETWORK program.Indigenous and imported FST values in Hainan exceeded 0.25, suggesting strong differentiation among most populations except in Southeast Asia. Most Hainan haplotypes were connected to South/East Asian and China’s others haplotypes, but less connected with populations from China's Anhui and Guizhou provinces. Mitochondrial lineages of Hainan P. vivax belonged to clade 1 of four well-supported clades in a phylogenetic tree, most haplotypes of indigenous cases formed a subclade of clade 1, and the origin of seven imported cases (50%) could be inferred from the phylogenetic tree, but five imported cases (42.8%) could not be traced using the phylogenetic tree alone, necessitating epidemiological investigation.In total, 983 complete mitochondrial genome sequences were collected, including 45 from this study and 938 publicly available from the NCBI. Thirty-three SNPs were identified, and 18 haplotypes were defined. The haplotype (0.834) and nucleotide (0.00061) diversity in the Hainan populations were higher than China’s Anhui and Guizhou population, and the majority of pairwise P. vivax populations.Indigenous cases in Hainan display high genetic (haplotype and nucleotide) diversity. Haplotype network analysis also revealed most haplotypes in Hainan were connected to the Southeast Asian populations and divergence to a cluster of China’s other populations. According to the mtDNA phylogenetic tree, some haplotypes were shared between geographic populations, and some haplotypes have formed lineages. Multiple tests are needed to further explore the origin and expansion of The online version contains supplementary material available at 10.1186/s12936-023-04520-7. Malaria was one of the most important infectious diseases in China, where it had a wide geographical distribution . Before Anopheles dirus and Anopheles minimus, and the characteristics of forest goers also facilitate malaria transmission  [For calculation and further analysis, sequences obtained in this study and 938 sequences publicly available from the NCBI  were estimated using DnaSP version 5.6. The number of synonymous nucleotide substitutions per synonymous site (dS) and number of nonsynonymous nucleotide substitutions per nonsynonymous site (dN) were calculated using the SNAP program (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html) [Nucleotide diversity (π) and haplotype diversity (AP.html) .FST and ΦST values were carried out by permutation analyses using 1,000 permutations with an assumption of no difference between populations. The P value was calculated as the proportion of permutations resulting in an FST or ΦST value higher than or equal to the observed value. Analysis of molecular variance (AMOVA) was used to evaluate the extent to which sequence variation was partitioned among populations and geographical areas.Arlequin 3.5 software was used to estimate genetic diversity indices and to assess population differentiation . PairwisP. vivax using MrBayes version 3.2.1 with two runs of four chains each [Bayesian phylogenetic analysis was carried out for ins each , three hhttp://www.fluxu-engeneering.com) with the default parameters and transversions weighted twice as high as transitions [P. vivax mt genomes, and the same colour code described above was used to show the geographic origins of the samples.Median-joining phylogenies were generated using Network version 4.6 . The 22 SNPs and 11 indels defined a total of 19 mt genome haplotypes, including 8 haplotypes in indigenous cases, 9 haplotypes in imported cases and 2 shared haplotype  was moderate, higher than China's Anhui population (0.584) and China's Guizhou population (0.734), but lower than China's regions (0.875). The nucleotide diversity in the Hainan population was 0.00061 ± 0.00004, which was higher than that in China’s Guizhou and Anhui populations and nearly higher than that in China’s other regions and South and West Asia. Furthermore, the haplotype diversity in China (0.918) was also moderate, lower than that in Southeast Asia and South and West Asia (0.972–0.945) and higher than in Africa, Latin-America, Oceania and Korea (0.801–0.899) (Table Before comparing the haplotype and nucleotide diversities of FST (based only on haplotype frequency) and ΦST (based on genetic distance) were estimated to further determine population differentiation for each population by mtDNA sequences. The majority of pairwise FST values from Hainan were greater than 0.25 (ranging from 0.15 to 0.75), suggesting strong population differentiation among most populations except those from Southeast Asia, which showed moderate genetic differentiation . Compared with Southeast Asia, Hainan was less distinct, with FST values of 0.1588 and ΦST values of 0.0466 in the world. In the Chinese P. vivax populations, which included Anhui, Guizhou and other populations, genetic differences between populations were strong  (Table 5) Table . Thus, cP. vivax mt genomes from 45 newly collected Hainan samples as well as 938 previously described isolates from 10 populations revealed a total of 592 haplotypes.The obtained 45 sequences referred to 30 indigenous cases, 14 imported cases and one unclassified case. Sequentially, 30 indigenous cases collected from 2009 to 2011, 14 imported cases from 2011 to 2020 and 1 unclassified case in 2010. Analysis of To characterize the frequencies and relationships of different haplotypes, a minimum spanning tree was constructed. This haplotype network clearly showed geographical clustering of the haplotypes Fig. . For theP. vivax belonged to clade 1, which are most widely spread across the Bayesian phylogenetic tree  comprised all haplotypes: clade 1 , clade 2 , clade 3 (110 haplotypes from AM and 55 from SE), and clade 4 (19 haplotypes from SE and 8 from KO). Hainan’s mt lineages of Plasmodium species were reported, with P. falciparum being the most prevalent, followed by P. vivax [P. vivax should be given greater attention, although indigenous cases have been eliminated in 2021.China officially achieved the elimination of malaria on June 30, 2021 . Five PlP. vivax . These iP. vivax . Those iP. vivax . Therefopvmsp1 and pvcsp [P. vivax populations. Consistent with high malaria endemicity in Myanmar, haplotype diversity was also high (0.85 ± 0.057) and comparable to that in other highly endemic areas of the world [P. vivax population was also found in tropical countries where Anopheles dirus and Anopheles minimus act as primary vectors. In the Greater Mekong Subregion, complex vector species relates to transmission intensity, and the latter further lead to high diversity in the parasite population. Historically, the Guizhou and Anhui populations were defined as temperate P. vivax malarial populations with characteristics of a long incubation period, seasonal transmission by Anopheles sinensis or Anopheles anthropophagus and a mechanism of adaptation to a cold climate [P. vivax, which is transmitted by An. dirus and An. minimus throughout the year. The haplotype diversity of Anhui’s and Guizhou’s populations in China was lower or similar to that of other populations in China.According to the results of this study, indigenous cases in Hainan display high genetic diversity in terms of haplotype diversity and nucleotide diversity, as reported in nd pvcsp . The mt he world . We thushe world . This ph climate . HoweverP. vivax populations, especially those on different continents [FST and ΦST statistics revealed significant differentiation between Hainan and other P. vivax populations in China. Interestingly, three major genotypes in China were also observed: Hainan, Guizhou and Anhui, and others. Generally, old  populations are more genetically diverged than young populations [P. vivax populations is thought to be the result of ancient hominid geographical expansion [In a finite population, a pattern of genetic isolation by geographic distance is generally expected , and thintinents , 35. In ulations . The genxpansion . Geneticxpansion . In thisP. vivax from temperate regions in East and Southeast Asia [Most mtDNA haplotypes from Hainan population were unique but related, suggesting that they might be descendants from the same lineage(s). Haplotype network analysis suggested Southwest Asia as the root or origin of the parasite populations, which is also verified by research on ast Asia . The locast Asia . Haplotyast Asia . Furtherast Asia , may repIn the mtDNA phylogenetic tree, some haplotypes were found to be shared by different geographic populations, which made it difficult to trace the origins of geographic populations with missing travel histories, although most haplotypes formed lineages undergoing population expansion. The result from origin tracing is consistent with imported malaria cases in the USA, which show some closely shared haplotypes and chaos in lineages from different geographic populations . Thus, mP. vivax population structure by focusing on Hainan populations in China. Analysis of the complete mt genomes from 45 clinical samples confirmed that P. vivax displays extensive genetic diversity and that natural populations are clearly structured. Most of indigenous case have formed a unique lineages and two haplotypes were shared with Southeast Asia. While most mtDNA haplotypes from Hainan were related to Southeast Asian haplotypes, they were different from those collected in the centre of China, suggesting that Southwest Asia is the root and that local population subdivision occurs. Multiple tests are needed to further explore the origin and expansion of P. vivax populations.The present study addressed extant Additional file 1: Primer sequences. Primers used for amplifying and sequencing the mitochondrial genome of Plasmodium vivax.Additional file 2: Alignment of the complete mt genomes identified 33 SNPs from Hainan.Additional file 3: The calculation for covariance components within population, among population/within group, and among populations.Additional file 4: Haplotypes in all geographical location.