Haemogogus leucocelaenus, a sylvatic vector of yellow fever virus, were found to vary with the degree of agricultural land use and isolation of fragments of Atlantic Forest in municipalities in the state of São Paulo where specimens were collected. Genotyping of 115 mitochondrial SNPs showed that the populations with the highest indices of genetic diversity (polymorphic loci and mean pairwise differences between the sequences) are found in areas with high levels of agricultural land use (northeast of the State). Most populations exhibited statistically significant negative values for the Tajima D and Fu FS neutrality tests, suggesting recent expansion. The results show an association between genetic diversity in this species and the degree of agricultural land use in the sampled sites, as well as signs of population expansion of this species in most areas, particularly those with the highest forest edge densities. A clear association between population structuring and the distance between the sampled fragments (isolation by distance) was observed: samples from a large fragment of Atlantic Forest extending along the coast of the state of São Paulo exhibited greater similarity with each other than with populations in the northwest of the state.Genetic diversity and population structuring for the species  Tropical forests are home to most of the planet’s biodiversity . Two exaThe intense deforestation observed in tropical forests not only constitutes one of the main threats to species in these areas , but alsHaemagogus and Sabethes. Anthropogenic landscape changes, such as deforestation and changes in land use, are the main reasons for the increase in the incidence of zoonoses, including sylvatic YF [In the particular case of zoonoses caused by pathogens transmitted by mosquitoes, spillover events are largely determined by the overlapping of hosts, vectors and human populations . Sylvativatic YF ,11,12. Ivatic YF .Haemagogus and Sabethes in Central and South America and Aedes (subgenus Diceromyia and Stegomyia) on the African continent [Aedes aegypti, while in the latter, humans are infected sporadically when they enter forested areas where the virus circulates between NHPs and wild mosquitoes [Yellow fever (YF) is an infectious disease  caused bontinent ,15. The squitoes . An intesquitoes .Haemagogus janthinomys (Dyar 1921) and Haemagogus leucocelaenus  [Hg. janthinomys is frequently found infected with YFV in outbreaks of YF in the Southeast, Midwest and North (in the last of which it is endemic), whereas Hg. leucocelaenus is found in the Southeast and South [The species considered the main vectors in Brazil are n, 1924) ,18, bothnd South ,21,22,23An outbreak of sylvatic YF of unprecedented magnitude occurred in Brazil recently, and 2276 human cases and 2978 laboratory-confirmed epizootics were reported by the end of 2021 ,25,26. OHg. leucocelaenus [Alouatta spp.) [In the particular case of deforestation and the consequent habitat fragmentation, the result is not only an increase in edge surface , which icelaenus  and howlta spp.) , but alsta spp.) . At the ta spp.) . Hence, ta spp.) ,33.Another characteristic arising from man-made landscape changes is reduced structural connectivity , which iHg. leucocelaenus, in view of its ecological plasticity to impacted environments.As mentioned earlier, recent studies carried out in areas of the Atlantic Forest have shed light on the influence of forest fragmentation on the dispersion and circulation of YFV ,33. TherHg. leucocelaenus based on specimens collected in Atlantic Forest fragments in different municipalities in the state of São Paulo. Our hypotheses assume different values of genetic diversity associated with land use in the locations sampled, as well as a possible pattern of population structuring associated with the degree of isolation of the fragments (isolation by geographic distance), i.e., the greater the distance between fragments, the greater the genetic distances among specimens. Likewise, the greater the connectivity of the landscape, the lower the genetic differentiation.In light of this, the present study sought to identify population structuring and genetic variability in 2 (24.8 million ha) and has an estimated population of 46,289,333, with a population density of 166.25 inhabitants/km2 [The state of São Paulo extends over 248,219.48 kmants/km2 . Currentants/km2 ,41 , following the manufacturer’s protocol, and quantification was performed by fluorometry with the QuBit dsDNA HS Assay Kit , according to the manufacturer’s protocol.Total genomic DNA was extracted from individual mosquitoes with the DNeasyHg. leucocelaenus. For this, DNA libraries were prepared using one-fifth of the total volume recommended for the Nextera XT Library prep kit (Illumina); after this modification, the full preparation was carried out, according to the manufacturer’s protocol. After being grouped and loaded into two mid-output flow cells, the DNA samples were sequenced in the NextSeq500 (Illumina) platform using 151 single-read cycles.In this study, we employed a low-density genome Illumina sequencing protocol ,44 to obHg. leucocelaenus—NC_057212.1  using Burrows–Wheeler Aligner 0.7.17 [FastQC was used to assess the quality of the sequence reads , and ther 0.7.17 . This opVariant calling was performed with bcftools in the SAMtools 1.10 package , and theThe values for nucleotide diversity within the populations and the analysis of molecular variance (AMOVA)  for the specimens grouped by collection location were estimated with Arlequin 3.5 . For nucequences ,52, bothSTF, following the mathematical model of Weir and Cockerham [p ≤ 0.05) of the mean values of STF based on the permutation test (1000 replicates).Signs of demographic events in the populations (expansion or retraction) were identified with Tajima’s D test for neutrality  and Fu’sockerham . This paockerham , and allvcfR package [pvclust [Data from variant calling were manipulated with the  package  to allow package  and pvcl[pvclust , respectadegenet package was used to perform discriminant analysis of principal components (DAPC) among the samples [adegenet [reshape2 [ggplot2 [ggpubr 0.40 [The  samples . The eigadegenet  and reshreshape2  packages[ggplot2  and ggpuubr 0.40 . All theubr 0.40 .Hg. leucocelaenus, this corresponds to 5.7 km [www.fbds.org.br/, accessed on 10 May 2023) , (5) Non-forest Formation—Native bushy or herbaceous vegetation; and (6) Forestry—Eucalyptus sp. or Pinus sp. plantations.Two buffers with radii of 2850 m and 5700 m, respectively, were defined around each collection point to calculate the landscape metrics. The buffer radius was based on an estimate of the expected dispersal radius of 0.3 to 0.5 times the maximum dispersal distance of a species . In the o 5.7 km . Each ofay 2023) . This waSubsequently, the land-use classes were used to quantify the landscape metrics: Total area (CA) and Percentage of the landscape occupied (PLAND), and the Total Edge (TE) and Density Edge (DE) metrics were also calculated for the Forest Formation class. Regression analysis was performed, using the landscape metrics as the predictor variable and the values for the diversity indices theta S and theta After sequencing, the average depth for each specimen sequenced was approximately 1,300,000 reads, representing an average coverage of 1.23× for the full genome, as expected for a low-density sequencing protocol.Hg. leucocelaenus has not been sequenced to date, we had to use the mitochondrial genome (NC_057212.1). A total of 115 mitochondrial SNPs were genotyped, corresponding to a genotyping rate of 51.11%  were analyzed, the sampled populations were found to have on average 23 loci, with less than 25% of missing data . Pindamonhangaba had the greatest number of these loci , while M = 2.21) .In the neutrality test to identify signs of population expansion or retraction, only São Paulo and Mairiporã exhibited concordance in the statistically significant results for Tajima’s D and Fu’s FS tests; both had negative values, suggesting expansion events. For the other populations, there were no statistically significant results for Tajima’s D test, while for Fu’s FS test, statistically significant negative values were observed for the Rio Preto, Caraguatatuba, Balsamo and Pindamonhangaba populations .STF was recorded between the municipalities of Igaratá and Miracatu (1), followed by Balsamo and São Paulo (0.74982), Balsamo and Mairiporã (0.67021), São Paulo and Rio Preto (0.6422) and Balsamo and Igaratá (0.62904). Generally, the two populations in the São José do Rio Preto mesoregion  had the largest values in most of the comparisons with the other municipalities sampled, and the smallest value for these two populations was precisely when they were compared with each other (0.1099). The smallest value for all the comparisons was recorded between São Paulo and Mairiporã (0.0201)  .STF index and the geographic distances between the collection sites. Geographic distance not only has a strong association with the pattern of genetic structure observed, but also explains by itself around 45% of the variation in the data, suggesting clear isolation by distance . These are closer to each other in the bidimensional space than to the populations in the eastern region of the state, and Caraguatatuba is segregated from the remainder, which are close to each other . This paDiscriminant analysis of principal components once again revealed overlapping of the specimens in the northwest of the state of São Paulo, with a few individuals in the Pindamonhangaba and São Paulo populations showing greater similarity with individuals from Balsamo and Rio Preto. However, most of the specimens in the populations, other than those from Balsamo and Rio Preto, overlap to form practically a single cluster . With thp-value = 0.03417) and accounted for approximately 55% of the observed pattern (M2pi_5700m), while for the theta S index, this area had a less accurate fit  and accounted for approximately 48% of the observed pattern (M2S_5700m)  and Rio Preto , whereas the corresponding figures for Pindamonhangaba were 32% and 50.1%, respectively . InteresS_5700m) .Another characteristic that the municipalities with the highest values for the genetic diversity indices had in common was the low proportion of forest cover. Balsamo and Rio Preto had the lowest values , while the corresponding figures for Pindamonhangaba were 66% and 47.6%, respectively . HoweverHg. leucocelaenus may be occurring as a function of the degree of isolation of the sampled vegetation fragments (isolation by distance) in municipalities in the state of São Paulo. The results show greater genetic diversity (theta S and This study tested the hypothesis that genetic diversity is associated with land use in the areas where samples were collected and that population structuring of The populations with the greatest genetic diversity indices (number of polymorphic loci and mean pairwise differences between the sequences) were found in areas with a high degree of agricultural land use (>32%). When only the populations in the São José do Rio Preto mesoregion were considered, agricultural land use exceeded 75% of the areas sampled, clearly showing there is a strong association between this land-use class and the pattern of genetic diversity in these populations, as shown by the theta Haemagogus, Mucci et al. [Hg. janthinomys/capricornii had multiple blood meals , indicating the influence of areas adjacent to forest fragments on the feeding habits of this genus. Curiously, the same study failed to identify multiple blood meals for the species Hg. leucocelaenus, which has greater ecological plasticity than Hg. janthinomys/capricornii [Landscape structure, i.e., differences in land use, are directly associated with different patterns in mosquito communities , whose ri et al.  found thricornii , 2010). ricornii ,71.Hg. leucocelaenus, specimens of which are found throughout the São José do Rio Preto region [The results of the present study may, therefore, reflect not only the plasticity of o region , but also region , where to region . Howevero region ,75,76.Regarding demographic events (population expansion and retraction), for six out of eight, or the majority of the populations, the results of at least one of the neutrality tests were negative and statistically significant, but only the results for the São Paulo and Mairiporã populations were concordant between the two tests (Tajima’s D and Fu’s FS). Negative values in these tests generally indicate recent population expansion. Common to the areas where specimens of these two populations were collected are the following factors: (i) they are located in Cantareira State Park (CSP), an important remnant of Atlantic Forest in the state of São Paulo and considered the largest urban forest in the world ; (ii) thHg. leucocelaenus [Hg. leucocelaenus in areas close to the edge of CSP and in locations with different degrees of plant cover [The juxtaposition of urban areas and forests has a direct impact on plant species composition , which ccelaenus . Howevernt cover ,80,81. Ont cover , which cSTF (0.02–0.75), these agree with the findings reported by Alonso et al. [Mansonia spp., which used the same protocol to identify mitochondrial SNPs as in our study. In terms of the degree of population structuring, the specimens collected in the northwest of the state of São Paulo are clearly separated from the populations in the eastern region of the state and also have the largest values of STF in the pairwise comparison with these populations. The smaller values for the populations outside the northwest region indicate greater genetic proximity among these populations and suggest that if there is a physical barrier, this is not a complete obstacle to the dispersal of individuals. In this regard, our results corroborate the idea of isolation by distance, according to which the greater the distance between species, the greater the genetic differentiation observed. In a slightly larger-scale study with populations of Aedes scapularis, another sylvatic species, Petersen et al. [Aedes fluviatilis and Anopheles cruzii) and an invasive species (Ae. aegypti) [Although pairwise comparison of the populations yielded high values of o et al.  in a stun et al.  observedaegypti) ,84,85. NHaemagogus, distances of between 5 and 11 km have been recorded [Hg. leucocelaenus, a distance of 5 km was recorded. However, mosquitoes can cover large distances by passive dispersal [Mosquitoes generally have a dispersal range of around 6 km . For therecorded , and in ispersal , and in ispersal . The greHg. leucocelaenus specimens can travel along the so-called “ecological corridors” found in the vegetation fragments, like those through which YFV spread in recent epizootics [Hg. leucocelaenus aided by a certain degree of landscape connectivity.Likewise, the populations that formed a single clade  and that are separated by distances of between 5 and 230 km are part of, or are located close to, a continuous strip of Atlantic Forest remnant, a large fragment of vegetation extending along the Atlantic coast of the state of São Paulo . This reizootics . In addiizootics . It is, Hg. leucocelaenus.The type of molecular marker used in the present study  has been successfully used for population characterization both on a macro ,91 and mA possible limitation of this study concerns the collection of immature specimens, in particular, in the municipality of Miracatu, where a single collection was performed. Consequently, there is a large chance that the individuals collected constitute the same brood and, therefore, that our results for this area do not accurately reflect the genetic variability of this population.Hg. leucocelaenus mosquitoes was associated with greater agricultural land use in the areas sampled, and signs of population expansion for this species were observed in areas with the largest values of forest edge surface. In addition, clear population structuring associated with the distance separating the fragments sampled (isolation by distance) was observed, and the populations from a large area of vegetation  exhibited greater similarity to each other.Greater genetic diversity in


Prunus sibirica L.) is a woody tree species of ecological, economic, and social importance. To evaluate the genetic diversity, differentiation, and structure of P. sibirica, we analyzed 176 individuals from 10 natural populations using 14 microsatellite markers. These markers generated 194 alleles in total. The mean number of alleles (13.8571) was higher than the mean number of effective alleles (6.4822). The average expected heterozygosity (0.8292) was higher than the average observed heterozygosity (0.3178). Shannon information index and polymorphism information content were separately 2.0610 and 0.8093, demonstrating the rich genetic diversity of P. sibirica. Analysis of molecular variance revealed that 85% of the genetic variation occurred within populations, with only 15% among them. The genetic differentiation coefficient and gene flow were separately 0.151 and 1.401, indicating a high degree of genetic differentiation. Clustering results showed that a genetic distance coefficient of 0.6 divided the 10 natural populations into two subgroups (subgroups A and B). STRUCTURE and principal coordinate analysis divided the 176 individuals into two subgroups (clusters 1 and 2). Mantel tests revealed that genetic distance was correlated with geographical distance and elevation differences. These findings can contribute to the effective conservation and management of P. sibirica resources.Siberian apricot ( Prunus sibirica L.) is a deciduous perennial woody tree species of the genus Prunus in the Rosaceae subfamily Prunoideae1. Given its white or pinkish flowers in spring, ripe yellow fruit in summer, and highly diverse leaf colors in autumn, the tree is considered to have particular ornamental value3. As a pioneer tree species in the restoration of vegetation, P. sibirica can tolerate cold, drought, and barren land and, thus, has high ecological value4. In addition, the seeds of this species contain amygdalin and high-quality oils, which have significant economic value6. Consequently, the government in north China promotes this tree as an economic forest species in semi-arid and arid areas, and breeding improved varieties with excellent characteristics is desirable.Siberian apricot  assess the genetic diversity, differentiation, and structure of P. sibirica and (2) develop strategies to protect P. sibirica germplasm resources. The results of our research will contribute to the effective conservation and management of the wild P. sibirica resources.In view of these considerations, we obtained genetic materials from 176 samples in 10 natural P. sibirica were used in this study. Location information for 176 samples  and purified them by 1% agarose gel electrophoresis. NanoDrop2000 spectrophotometry  was used to test the concentration and purity of the extracted DNA, and the high quality DNA was stored at − 20 °C until use.P. sibirica35. We used 14 pairs of microsatellite markers  initiated with a 105 °C hot lid, (2) initially denatured at 94 °C for 5 min, (3) 35 cycles of 94 °C for 30 s, 55 °C for 30 s , and 72 °C for 30 s, (4) finally extended at 72 °C for 5 min, and (5) stored at 4 °C. The amplified products were visualized by non-denaturing polyacrylamide gel electrophoresis at 220 V for 90 min. After rinsing, silver staining, and development, the gel was imaged using a Bio-Rad gel detection system .In our previous studies, we developed microsatellite markers for 36. POPGENE v 1.32 software37 was used to calculate genetic diversity parameters, including the number of alleles (Na), the number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and Shannon’s information index (I)40. Polymorphic information content (PIC) was determined using Cervus v 3.0 software41.We used Image Lab v 6.1 software to read the gel bands and obtained the raw genotype data after manual correction. The data were converted to different formats for further analysis using GenAlEx v 6.502FST analysis among populations, and principal coordinate analysis (PCoA) were computed by GenAlEx v 6.50243. In addition, we used GenAlEx v 6.502 to mantel test the correlations about genetic distance with geographical distance and genetic distance with elevation differences37.The results of analysis of molecular variance (AMOVA), pairwise 45. This software was performed by the following parameters: (1) used the admixture model to perform a Markov chain Monte Carlo (MCMC) simulation, and set at 200,000 and 100,000 corresponded to the number of repetitions and the length of the burn-in period, and (2) set the K value from 1 to 10 with 10 repetitions. The optimal value of K was estimated using Evanno’s ∆K method and Structure Harvester47. A geographical distribution diagram with the proportion of subgroups membership was plotted using ArcMap v 10.2.Genetic structure was analyzed based on Bayesian clustering using STRUCTURE v 2.3.448. We used the MEGA v 7 software to calculate the Nei’s genetic distances between the 10 natural populations and constructed the UPGMA clustering relationship tree50.We calculated genetic similarity coefficients between 176 individuals using the Simple Matching (SM) algorithm and constructed clustering relationship tree graphs using the unweighted pair group method with arithmetic means (UPGMA), both using Ntsys-pc v 2.10sPrunus sibirica in Kazuo County, Liaoning Province, China.The plant materials used in this article did not involve disputes. Plant materials were collected in compliance with institutional, national, and international guidelines and legislation. The plant material in our collection is preserved in the National Germplasm Repository of P. sibirica samples were amplified by the 14 pairs of microsatellite primers, generating a total of 194 alleles  to 19 (Y65 and X47), with a mean of 13.8571 alleles. The Ne for each locus ranged from 3.2636 (L75) to 9.0865 (P40H), with an average of 6.4822. The Ho varied from 0.0114 (P57H) to 0.6989 (Y5), whereas the He ranged from 0.6936 (L75) to 0.8899 (P40H). And the mean value of Ho (0.3178) was lower than that of the He (0.8292), suggesting a lower level of heterozygosity in P. sibirica. The He value above 0.5 reflected that P. sibirica population was not affected by high-intensity selection and possessed high genetic polymorphism. The I for each locus ranged from 1.5161 (L75) to 2.4459 (P40H), with an average of 2.0610.The extracted DNA of 176 P. sibirica populations are shown in Table Na per population ranged from 3.6429 (R population) to 8.2143 (NZD population), with a mean of 5.8500. The Ne per population ranged from 2.3912 (R population) to 5.3442 (NZW population), with a mean of 3.7703. The I value per population ranged from 0.8769 (R population) to 1.7759 (NZW population), with a mean value of 1.3677. The average of He (0.6590) was higher than that of Ho (0.3183). These comparisons indicated that the NZW population shows the highest genetic diversity within the assessed populations, whereas the R population shows the lowest.The genetic diversity parameters of the 10 P. sibirica populations and 85% within them , with the gene flow of 1.401 (Nm > 1), which indicated a moderate interchange of genes among these populations. Paired FST values (Table FST = 0.415 > 0.25) and the lowest differentiated being the LB and LC populations (FST = 0.002 < 0.05).AMOVA showed that 15% of the genetic variation occurred among the 10 em Table . The genes Table  indicateP. sibirica populations  to 0.065 (HW and HZ). With the exception of population R, which showed substantial genetic differentiation from the others, we found a moderate and low degree of genetic differentiation within subgroups (FST < 0.15).A UPGMA dendrogram generated based on Nei’s genetic distances of the 10 ons Fig.  revealedes Table  within sP. sibirica samples can be organized into two subgroups (clusters 1 and 2). The subgroup membership probabilities of 176 individuals from the 10 populations are shown in Fig. The STRUCTURE results revealed that ∆K reached a maximum value when K = 2 Fig. , therebyWith the increase of K value , clusters 1 was separated into smaller subgroups based on the geographical distribution Figs. . AccordiP. sibirica samples could be divided into two subgroups in complete consistency with the structure result. Furthermore, the constructed UPGMA tree clearly shows that P. sibirica individuals can be divided into two subgroups (P1 and P2) under a coefficient of 0.83    Fig.  and elev01) Fig. , respect51. In this study, we obtained discrete allelic data for 176 P. sibirica accessions from 10 natural populations based on analysis using 14 microsatellite markers. We evaluated the genetic diversity of P. sibirica in terms of allelic richness, heterozygosity, and allelic diversity. Compared with previous studies17, the present study significantly extends the sampling area of P. sibirica to the north , with a corresponding expansion of climate type from intermediary and warm to cold temperate zones. The mean genetic parameters of each locus detected in our research  is comparable to that reported by Wang et al.15 . These findings indicate that within the genus Prunus, P. sibirica has a slightly higher level of genetic diversity than apricot (Prunus armeniaca L.) 21, and a significantly higher diversity compared with sweet cherry (Prunus avium L.) (Na = 9.800 and He = 0.700)52, peach (Prunus persica L.) (Na = 6.410 and He = 0.490)53 and alpine plum (Prunus brigantina Vill.) (Na = 5.040 and He = 0.430)54. Moreover, compared with xylophyta in the Rosaceae family, the diversity is lower than that of apple  (Na = 23.060 and He = 0.830)55 and higher than that of pear (Pyrus pyrifolia) (Na = 7.120 and He = 0.780)56. The average genetic parameters of the populations detected in our research  is higher than that of Prunus mira Koehne 57. We speculate that this relatively high level of genetic diversity in P. sibirica is related to their area of distribution. Li et al.13 considered that P. sibirica might accumulate considerable intraspecific genetic variation to adapt to the diverse ecological conditions in its wide range of distribution. Climatic fluctuations over recent millions of years have tended to influence the genetic diversity of species. However, the center of P. sibirica distribution is East Asia, which is generally considered to have been a large-scale refuge during the Pleistocene, characterized by a relatively small range of environmental changes58. We thus speculate that this species may have retained a large proportion of its intraspecific genetic variation. Furthermore, Mehlenbacher et al.1 found that the hybridizations between any two true apricot species were successful, including those among P. sibirica, P. armeniaca, P. mandshurica, and P. mume. Thus, interspecific hybridization may also influence the accumulation of genetic variation in P. sibirica.Genetic diversity reflects the evolutionary characteristics of species and their adaptation to the environment, which is typically determined based on the data of continuous traits or discrete allelic statesP. sibirica distributed in different geographical regions differ significantly in terms of genetic diversity, with the highest levels of genetic diversity being detected in the NZW and NZD populations distributed in the northeastern region of Inner Mongolia, followed by the LB, LK, and LC populations in western Liaoning, and then the HW, HZ, and HL populations in northern Hebei and the NA population in central Inner Mongolia, with the lowest diversity detected in R populations in southern Zabaykalsky Krai. The natural habitat of the NZW and NZD populations is located near the Changbai Mountains overlaps with the natural distribution range of the Manchurian apricot (Prunus mandshuria Skv.), and most germplasms are older and less disturbed by human activities, which may account for higher genetic diversity of these populations. Population R is the only population distributed within the cold temperate zone, and although has lower genetic diversity than other assessed populations, it has accumulated unique genotypes during the process of adapting to its marginal habitats. In addition, germplasm resources in western Liaoning have been widely reported15. In the present study, the ranking of the genetic diversity levels in the Liaoning populations (LC < LK < LB) is consistent with the previous findings15. Contrastingly, Li et al.13 were of opinion that the genetic diversity was higher in the LC population than in the LK population. However, we found the genetic diversity of the NA population with similar geographical distance was relatively low, which is also consistent with the results presented by Chen et al.12 and Wang et al.15.In the present study, we found that the natural populations of 59. This is consistent with our present findings for P. sibirica, the genetic variation of which mainly exists within species and populations. We detected a high degree of genetic differentiation at the species level (0.15 < FST < 0.25), and given because the existing gene flow (Nm = 1.401 > 1), there is a resistance to genetic drift, there was no further differentiation60. Consequently, the genetic structure was relatively stable. Given the extensive and discontinuous distribution of P. sibirica, we speculate that gene flow among populations is maintained by animal-mediated seed dispersal rather than a combination of pollen transmission and insect- and wind-mediated dispersal, which is also consistent with the views of Li et al.13. In the distribution area surveyed in the present study, rodents hoarded P. sibirica seeds at dispersed locations, and exploited these less often or consumed them only in the absence of alternatives, owing to the amygdalin content61. We speculate that the primary mode of gene flow is the moderate exchange of genes between the buried P. sibirica seeds and local populations through pollination after they have developed to reproductive maturity.Woody plants tend to maintain a larger extent of variation within populations than between populations, particularly in the case of those species with a large geographical range, outcrossing, and seed dispersal mediated mainly by wind or animalsP. sibirica consists of two subgroups: the eastern and western subgroups, based on the STRUCTURE, clustering, and PCoA results. We found that whereas there was a moderate and low genetic differentiation among populations within the same subgroup (excepting the R population), there was a high differentiation between the populations belonging to different subgroups, among which the R population of the eastern subgroup and the HL population of the western subgroup were characterized by the highest genetic differentiation (FST = 0.415 > 0.25). Based on the common natural hybridization phenomena among plants of the Li subgenus apricot group1 and the distribution of plants of subgenus apricot in each region, we speculate that western subgroup may have undergone an introgression with P. armeniaca, which has thus influenced the genetic structure of the P. sibirica. Liu et al.14 divided P. sibirica germplasm into two subgroups and similarly believed that the northwestern subgroup was largely derived as a consequence of introgression between the northeastern subgroup and P. armeniaca. Mantel test results revealed the significant influence of geographical distance and elevation distribution in contributing to the genetic structure among P. sibirica in the study area (p < 0.001). Furthermore, the area surveyed in the present study falls within a semi-humid region, and given the high degree of genetic differentiation between populations in the western subgroups region lying in the warm temperate zone and those in the eastern subgroup region lying in the middle and cold temperate zone, we speculate that temperature also influences the genetic structure of P. sibirica.We established that the genetic structure of P. sibirica habitat have been destroyed, and its natural distribution range continues to decline13. To maximize the conservation of P. sibirica genetic resources, we have developed conservation strategies for P. sibirica in the study area depending on our findings. The genetic variation of P. sibirica exists occurs mainly within populations, and thus conservation efforts should focus on maintaining and increasing the genetic diversity level within populations. In this regard, we established that the genetic diversity of the NZW and NZD populations distributed in northern Inner Mongolia was significantly higher than that of other assessed populations, and thus the in situ conservation of these two populations should be given priority22. To protect the existing populations, we recommend the establishment of protected areas or sites to enhance management, which would require cooperation with the local government in charge of forest management. Moreover, to protect the genetic resources of populations with relatively low levels of genetic diversity, we propose to improve the habitats of P. sibirica via complementary silvicultural actions using forest plants from the same or genetically related populations. Furthermore, we have collected genetic materials from the natural population and ex situ preserved in a germplasm conservation base. Lay a solid foundation basis for long-term breeding and genetic resource conservation of P. sibirica.As a consequence of frequent human activities, such as fruit and seed picking, grazing, and deforestation, large areas of Prunus sibirica to assess their genetic diversity, differentiation, and structure. Our findings indicate that P. sibirica has a high level of genetic diversity, particularly within the NZW and NZD populations distributed in northeast Inner Mongolia. At the species level, there is a high degree of genetic differentiation and a relatively stable genetic structure. The genetic structure of P. sibirica can be affected by geographical distance and elevation distribution, with 10 natural populations dividing into two subgroups. This finding will contribute to the conservation research and the rational utilization of P. sibirica.In this research, we used 14 microsatellite markers to analyze samples collected from 176 individuals in 10 natural populations of Supplementary Information.


Tarentola mauritanica, using simple sequence repeats. By analyzing the genetic diversity of populations in different regions, we found that Morocco is the genetic diversity hotspot for the species, followed by the Iberian Peninsula. However, historical gene flow estimates identified the Iberian Peninsula, not Morocco, as the primary contributor of colonizing individuals, along with continental Italy to a lesser extent. Currently, mainland Italy is the main source of introduced individuals, likely due to the plant nursery trade. The study suggests that human-facilitated introductions from various geographical origins, with numerous regions colonized through continental Italy during two distinct periods, are responsible for the recurrent entry of individuals belonging to the European lineage of T. mauritanica into the Mediterranean and Macaronesia. These findings can inform better monitoring surveys and conservation programs by identifying putative current colonization routes of alien species.In this study, we aimed to understand the contemporary and ancient colonization routes of the Moorish gecko,  Moreover, human globalization is leading to an increase in the rate of species’ translocation to areas outside their native geographical range3. Curiously, reptiles seem to be particularly prone to biological introductions, either by being one of the most often introduced animal groups, or by being notably sensitive to the impacts of alien species4.It is widely known that the introduction of species into new localities may have considerable ecological and evolutionary consequences for the native species and host ecological communities5. Ultimately, this knowledge can have conservation implications by aiding in the design of monitoring and inspection programs for particular colonization routes or expected propagule size7.Therefore, determining the routes of introduction—the geographic pathways of the propagules between the source and the introduced populations—provides not only information about the history of the colonization process, but also an understanding of the origin and construction of the genetic composition of the newly established populations10. Hence, the need to use faster evolving genomic regions such as simple sequence repeats (SSRs) or commonly referred to as microsatellites. Extensive genotyping endeavours come with a high price tag and yield reduced benefits when conducting typical genetic analyses13. Consequently, research teams frequently resort to analysing a more limited set of genetic markers. Research conducted in various species, including fruit flies11, fish15, birds16, amphibians17, wild boars18, felids19, and beetles20, has shown that using a set of 6–14 microsatellite loci provides sufficient information to detect even subtle population structure.Unfortunately, we are many times faced with very low genetic diversity and shallow divergence patterns when studying introduced populations originated from either human introductions or very recent natural colonization events, precluding the assessment of the ultimate geographic origin of the source e.g.,Tarentola mauritanica  has a widespread geographic distribution across Southern Europe, the Maghreb region of North Africa, namely Morocco, Algeria and Tunisia, some Macaronesian islands, and is also present in several localities of the American continent21 , ending up with a total of 8 microsatellite loci for further analyses.Out of the 11 genotyped microsatellite markers, three of them were removed from further analyses  and the Iberian Peninsula are clearly the regions harbouring the populations containing the uppermost number of alleles and allelic richness, in contrast to Greece and the Balearic Islands Table  and S3. ica Fig. .Table 1G45’s method, STRUCTURE identified that the best K was K = 4 , surpassed by two introduced geckos, Hemidactylus mabouia, and Tarentola mauritanica57, one snake , and one allochthonous skink . Hence, it is clear that although largely sedentary, reptiles are frequently introduced by humans, many times during transport of building material, soil or cultivated plants4.The Balearic Islands appear as a striking example of a region marked by several faunal introductions across time; this archipelago that once harboured substantial levels of endemicity, now hosts more alien than native reptile and amphibian taxa reviewed inTarentola geckos in particular, which are primarily a North African clade, have naturally reached long distances such as many Macaronesia islands but also Cuba and the Bahamas, most likely by rafting on buoyant vegetation, at least 23 Mya42. Nevertheless, the current geographic distribution of T. mauritanica is partly the result of recurrent anthropogenic introductions e.g.,60. Their success as human-assisted colonizers is somewhat associated with the synanthropic habits of this species61, allied with their relatively small size and cryptic nature.T. mauritanica, harbouring most of the mitochondrial DNA diversity as well43. Although, the mtDNA results from Belluardo et al.44 suggest Italy as the centre of diversification of the European lineage of T. mauritanica, we have to acknowledge that their North African sampling was very limited and, possibly unable to tackle its underlaying genetic diversity. Moreover, all population structure analyses presented here support Morocco and the Iberian Peninsula as two divergent clusters with very little gene flow between them or with the other remaining groups , which highlights the importance of the Iberian Peninsula and Italy. This was the time of the Roman Empire (753 B.C. to A.D. 476) when cities were being developed, and communication networks expanded, which have clearly favoured the translocation of highly anthropophilic species68. In fact, during this historical time there was a peak of introduced reptile and amphibian species in the Iberian Peninsula68, a region known to be one of the most important gold suppliers during the Roman Empire69. Hence, these results together with the distribution of the genetic diversity support the Iberian Peninsula as the most likely entering point of the European lineage of T. mauritanica from North Africa, and from there to the remaining geographic regions.Considering the distribution of the genetic diversity but mostly of the evolutionary history of this European lineage, a bigger contribution of Morocco as a source of introduction was expected Fig. . Indeed,71. The study from Belluardo et al.44 identified the presence of a common widespread mitochondrial 16S haplotype in Sardinia, Sicily, Morocco, Algeria and Tunisia. Additionally, fossil remains of T. mauritanica dated from the Late Pleistocene-Holocene were found in Sicily (San Vito lo Capo)66. However, the origin of both Sardinian and Sicilian populations remains unknown, and an excellent opportunity for future studies.Very importantly, we need to acknowledge Sardinia and Sicily as major sampling gaps in this study. Apart from being the two biggest islands in the Mediterranean, they had many historical connections with North Africa including herpetological species exchangeTarentola geckos42. Accumulating evidence is strongly identifying the “plant nursery trade”  as one of the main forms of reptile introduction into new territories4, since these animals often use plants and trees for refuge and thermoregulation e.g.,73. This kind of human-mediated trade in the Mediterranean has been responsible, for instance, for the introduction of the Italian wall lizard, Podarcis siculus e.g.,74, the brahminy blind snake, Indotyphlops braminus75, and the colubrid snakes Hemorrhois hippocrepis, Malpolon monspessulanus, and Zamenis scalaris53, outside their native geographic ranges. Curiously, many of these introductions result from the trade of old olive trees transported from Italy78. Indeed, the olive tree trade appears as a modern vector for bioinvasions across the Mediterranean for a wide spectrum of alien species, Tarentola mauritanica included, having been recorded in Catalonia (Spain) around olive trees brought from Italy77, but also in Lake Garda, Northern Italy79, and Beaugeay, on the Atlantic coast of France80. Hence, previous studies together with the results obtained here, point to the plant nursery trade from Italy as a putative modern route responsible for the introduction of T. mauritanica across a wide geographic range. Additionally, the Mediterranean Basin has a very intense maritime traffic , which is thought to be the main driver for the introduction of Italian Moorish gecko individuals in the islands of Corfu30 and Lesvos31 in Greece. Overall, the colonization of the European lineage of T. mauritanica in the Mediterranean and Macaronesia seems to result from a combination of several human-mediated introductions from multiple geographic sources, with many regions having been colonized by Italy in at least two different periods far apart in time. Most importantly, this study has identified and highlighted the most likely current colonization routes for this lineage, which will hopefully help authorities in the design of better monitoring and inspection conservation programs.Currently, Italy seems to be the main source of Moorish gecko introduced individuals, although most of the gene flow takes place within each geographic region, contrary to the long-distance colonization typical in Tarentola mauritanica collected across 44 Mediterranean (some islands included) and Madeira populations were used, with each location represented by at least five individuals. Genotypes from all Italian populations were retrieved from Belluardo et al.44. All individuals included in this study belong to the mitochondrial European clade83. Tissue from tail tip muscle was collected from each individual and preserved in 96% ethanol. More details on the localities and origin of the samples are presented in Table In this study, 555 individuals of 84. 11 microsatellite loci were genotyped  from a battery developed specifically for T. mauritanica 85. All loci were amplified according to the described conditions in Arranz et al.85. All amplifications were performed including negative controls. PCR products were separated by size on an ABI3130xl genetic analyser using the 350ROX size standard. Allele sizes were determined using GENEMAPPER v.6.086 and checked manually.Genomic DNA was extracted using a standard high-salt protocol87. Linkage disequilibrium (LD) and deviations from Hardy–Weinberg equilibrium (HWE) were tested with “GENEPOP on the Web” 89. We applied the False Discovery rate90 to correct p values (p < 0.05) from HWE and LD multiple exact tests, using the package fdrtool91 in R92.The presence of possible null alleles, allele scoring errors due to stuttering and large allele dropout was evaluated using MICRO-CHECKER v2.2.3To avoid the computer intensive assessment of such a large number of distinct populations, these were grouped into nine geographical regions , which were considered in some of the posterior analyses (see population structure results that support this).exp and Hobs, respectively), using the R diveRsity package and the divBasic function93. The rarefied allelic richness (R-Ar) per population was calculated with the R package hierfstat94, which accounts for variation in sample size (each sample included a minimum of five loci). To identify geographic changes in genetic diversity, the rarefied allelic richness was spatially interpolated using the Inverse Distance Weighting method (IDW) in QGIS v.3.16.11 “Hannover”95.Basic microsatellite diversity was evaluated separately for each population based on the number of alleles per locus (Na), expected and observed heterozygosity  were estimated using 1000 permutations, and pairwise estimates were considered significant when 95% CIs did not overlap zero. These analyses were applied to all 44 populations and also to the nine defined geographic regions.Population differentiation was assessed by means of pairwise F99. Ten independent runs were performed for a number of clusters (K) ranging between 2 and 8. Runs consisted of a burn-in period of 104 iterations, followed by 106 MCMC reps, correlated allele frequencies, admixture model, and no prior information regarding population of origin. The best K was identified using STRUCTURE HARVESTER Web v0.6.94100, and deemed as the best K describing the observed genetic data. Graphical plotting of STRUCTURE results was implemented in the online software CLUMPAK101, and geographic plotting of ancestry coefficients using the R package mapplots102.Genetic structure was assessed using a Bayesian clustering analysis, implemented in STRUCTURE 2.3.4tess3r103; and a discriminant analysis of principal components DAPC;104.Additionally, two other methods were implemented as alternative and complementary approaches to assess the genetic diversity; a Bayesian Clustering algorithm using tessellations and Markov models for spatial population genetics under the R package tess3r algorithm implements a new version of the program TESS105, based on geographically constrained matrix factorization and quadratic programming techniques. The new algorithms are several orders faster than the Monte-Carlo algorithms implemented in previous versions of TESS.The The DAPC summarizes the data to minimize genetic differentiation within previously defined groups, while maximizing it between groups, and it does not consider HWE or linkage equilibrium as necessary conditions. For this analysis, we assumed four genetic clusters based on the Bayesian Information Criterion (BIC) and ΔK see “” section106 and BayesAss v3.04 (BA3) , respectively.Historical and contemporary migration rates were estimated among the major nine geographic regions with MIGRATE v3.7.2e generations in the past107. The data were assumed to follow a Brownian motion mutation model. The FST calculation method was used to generate starting values for both θ and M. Uniform priors were specified for both parameters with a minimum of 0, mean of 50, maximum of 100, and a delta of 10. The Bayesian method was implemented to infer θ and M, specifying two independent runs, static heating with four chains , a sampling increment of 20, 50,000 recorded steps, and a burn-in of 10,000. Convergence was assessed by examination of ESS values with a target of at least 1000.MIGRATE uses the coalescent in a Bayesian or maximum likelihood framework to calculate two parameters from the data, θ and M, where θ represents the effective population size (4Neμ for nuclear DNA), and M the mutation-scaled immigration rate (m/µ). This coalescent-based approach is most suitable for estimating migration rates over thousands of years or approximately 4N8 iterations and discarding of the first 106 iterations as burn-in. The delta values DA, DF, and DM were set to 0.4, 0.5, and 0.4, respectively. Convergence of the chains was validated using Tracer v1.7.1108.Unlike MIGRATE, BAYESASS does not assume genetic equilibrium and is therefore, more suitable for inferring contemporary (over the past few generations) processes. The model in BAYESASS assumes linkage equilibrium between loci but allows for deviations in Hardy–Weinberg proportions by introducing an additional inbreeding (F) parameter. Several analyses with different starting seeds were performed and each Markov chain Monte Carlo run involved 10circlize109.Graphical plotting of migration rates among geographical regions was performed with the R package https://www.up.pt/portal/pt/conhecer/organizacao/comissao-de-etica/). Codes of all issued sampling permits are provided in Table All methods were carried out in accordance with relevant guidelines and regulations. The entire experimental protocol was approved by the Ethics Committee of the University of Porto (Supplementary Information.


Mus musculus spp.) that strike this balance between data density and laboratory efficiency. Here we evaluate the utility of a custom targeted capture genotyping-by-sequencing approach to support research on plaguing house mouse populations in Australia. This approach utilizes 3,651 hybridization capture probes targeting genome-wide SNPs identified from a sample of mice collected in grain-producing regions of southeastern Australia genotyped using a commercially available microarray platform. To assess performance of the custom panel, we genotyped wild caught mice (N = 320) from two adjoining farms and demonstrate the ability to correctly assign individuals to source populations with high confidence (mean >95%), as well as robust kinship inference within sites. We discuss these results in the context of proposed applications for future genetic monitoring of house mice in Australia.The management of invasive species has been greatly enhanced by population genetic analyses of multilocus single-nucleotide polymorphism (SNP) datasets that provide critical information regarding pest population structure, invasion pathways, and reproductive biology. For many applications there is a need for protocols that offer rapid, robust and efficient genotyping on the order of hundreds to thousands of SNPs, that can be tailored to specific study populations and that are scalable for long-term monitoring schemes. Despite its status as a model laboratory species, there are few existing resources for studying wild populations of house mice ( Invasive alien pest species pose significant threats due to persistent and widespread impacts on endemic biodiversity, human health, and economic enterprise. The management of invasive species has been greatly enhanced over the last several decades by the application of population genetic analyses, which can provide valuable information regarding pest population structure, invasion pathways, and reproductive biology . More reDespite these potential benefits, the application of genomic approaches to problems in invasive species management is often hindered by constraints of available SNP genotyping techniques. While whole-genome (re)sequencing provides the most comprehensive view of genetic variation and has become increasingly accessible in recent years, it is still often prohibitively expensive for mammalian or larger-sized genomes and for long-term monitoring programs that involve widespread and repeated sampling over time, and resulting datasets place large demands on computation and data storage infrastructure. In comparison, reduced-representation sequencing approaches  offer gMethods that allow effort to be targeted on specific genomic regions of interest  can greatly increase sequencing and bioinformatic analysis efficiency compared to whole-genome resequencing . HybridiMus musculus spp.) in southeastern Australia that are notable for aperiodic eruptions in population numbers that can lead to ‘mouse plagues’ [Here we present the development and evaluation of a targeted capture SNP genotyping approach for population genetic analyses of invasive house mice  and all protocols were reviewed and approved by the CSIRO Wildlife, Livestock and Laboratory Animal, Animal Ethics Committee . All mice were live-trapped, and released at the point of capture as soon as possible following visual confirmation of no adverse effects.The use of animals in this study was carried out in strict accordance with the Australian code for the care and use of animals for scientific purposes  at two agricultural study sites in the Adelaide Plains of South Australia  along with an additional 24 individuals from other sites in southeastern Australia: Northern Mallee, Victoria ; the Yorke Peninsula, South Australia , and near Murrumbateman, New South Wales  . These sGenomic DNA was isolated using column-based methods  following the manufacturer’s recommended protocol. DNA concentration was quantified with a Qubit 3 fluorometer (Thermo Fisher Scientific Inc.) and purity was assessed by inspecting the A260/A280 ratio for each sample on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.).2 = 0.5) in PLINK v1.9 [In order to assess genetic variation across all study sites, we selected a subset of purified DNA samples (N = 72) that included 48 samples randomly selected from the two South Australia sites along with all 24 samples from the other sites. These samples were submitted to Neogen, Inc.  for genotyping on the GigaMUGA  IlluminaINK v1.9 . To evalINK v1.9  and visuth percentile. This list of target SNPs was submitted to the oligonucleotide provider  for design of complementary probes (based on the mm10 reference genome) and final screening for any predicted off-target activity. 5’-biotinylated oligonucleotide probes (120 nucleotides length each) for the final panel design  on the To evaluate the performance of our custom panel, we genotyped 320 individuals  following the manufacturer’s recommended guidelines . Briefly, genomic libraries were prepared from 100 – 120ng of genomic DNA per sample using Lotus DNA Library Prep Kits  at 0.4x the standard reaction volume for cost savings. Each sample was subject to a single enzymatic preparation step that included enzymatic fragmentation at 32°C (to target a 200 bp average insert size), followed by end-repair and A-tailing at 65°C. Illumina-compatible stubby adapters were subsequently ligated to each sample, followed by PCR library amplification (9 cycles) with unique dual-indexed primers , to minimize effects of index-hopping. Libraries were quantified in triplicate by qPCR  then combined in equimolar concentrations into pools of 16 samples each in preparation for target capture. Pooled libraries were hybridized to probes in the presence of universal blocking oligos  and mouse Cot-1 DNA  to minimize off-target binding, and fragment capture was performed with washed streptavidin beads. Post-capture library amplification was carried out using high-fidelity PCR  with xGen Library Amplification primers (IDT Ltd.) for 10 cycles, followed by clean-up using AMPure XP bead reagent  at 1.5X reaction volume. Mean fragment length of captured pooled libraries was assessed via automated electrophoresis using an Agilent 2200 TapeStation system  with high sensitivity D1000 reagents, and library quantification performed using qPCR  in triplicate at two concentrations for each library after serial dilution. These pools were then pooled together in equimolar concentrations to generate either 48-plex  or 96-plex  final sequencing libraries, which were sequenced on a MiSeq instrument  for 300 cycles (2 x 150 bp paired end) using v2 Micro and v2 standard flowcell kits, respectively, along with a 5% spike-in of PhiX control library.http://www.bioinformatics.babraham.ac.uk/projects/fastqc), followed by trimming and filtering of adapter sequences using BBTools (http://bbtools.jgi.doe.gov). Sequences were mapped to the mm10 reference genome using BWA-MEM2 [Quality checks of raw sequence data were carried out using FastQC  using the concordance command in SnpSift v5.1d .To characterize genetic variation and assess the ability to discriminate fine scale population genetic structure with the hybridization sequencing dataset, we performed PCA and DAPC on the Adelaide Plains (AP1 and AP2) samples (N = 320) using adegenet in R. These study sites are located in close proximity (~5km) on adjoining farms within homogenous cropping habitat with no obvious barriers to mouse movement. We reasoned that these sites would thus provide a challenging scenario to test the ability of the custom genetic marker panel to detect fine scale genetic differentiation and assign samples to source populations under conditions with probable gene flow, and at a scale that is likely relevant for farm-level management decisions. As the goal was to visualize genetic differentiation (as opposed to inferring genetic clusters de novo), DAPC was carried out with samples assigned a priori to populations, which has been shown to yield robust estimates of genetic distance . AdditioPairwise kinship coefficients among all mice in these study populations were estimated using PC-Relate  as impleQuality assessment of GigaMUGA genotypes resulted in the exclusion of two samples (≥10% missing SNP genotypes) and 8,918 markers (genotypes missing for ≥10% of samples). For initial assessment of genetic analyses that focused on autosomal loci, markers were further filtered for a minimum minor allele frequency of 0.05  and linkage disequilibrium pruning , resulting in a final dataset of 49,058 markers . PCA of these genotypes  identifiVKORC1  except a single individual that had a missing genotype at one locus.Of the 70 individuals retained in the analysis, all were homozygous for the reference allele at both SNPs in Results of the DAPC for the GigaMUGA dataset are summarized in Inspection of individual marker loadings on the discriminant functions identified a panel of 3,783 targeted SNPs that were submitted for 1x tiling oligonucleotide probe design. Quality assessment of this initial panel identified 132 sequences that were predicted to have moderate to high off-target activity and were thus excluded. The final pool of 3,651 markers included targets distributed across all 19 autosomes, with an average of 176.4 (S.D. = 45.3) markers per chromosome, along with 282 and 17 markers on the X and Y chromosomes, respectively . Probes Analysis of mapped sequencing reads indicated successful capture of the target loci , with deFor the 47 samples that were genotyped with both the GigaMUGA and the hybridization capture sequencing panel, a total of 3,562 SNPs (97.6% of the targeted loci) were retained for comparison after initial quality filtering (see above). For these markers, we observed strong concordance between datasets, with 98.9%  of called genotypes identical, whereas only 0.66%  were discordant . Analysis of the genomic distribution of these additional SNPs indicated the vast majority (96.1%) occurred within 150bp of a target SNP , suggestResults of PCA and DAPC for the sequencing dataset are provided in r = 0.894, t(1079) = 65.47, p < 0.001).The mean pairwise kinship coefficients among the 47 individuals in the reduced sequencing dataset was relatively low , suggesting the majority of individuals were unrelated to one another, though we detected four incidences of first degree relatives , 15 second degree relatives , as well as 15 third degree relatives . Estimates were strongly correlated with the values obtained from the full GigaMUGA genotypes  for the same samples , that lThe PCA of population genetic structure with the GigaMUGA dataset revealed a hierarchical pattern of population differentiation, with genetic distances among population clusters largely mirroring the geographic distances among populations. This result is consistent with a pattern of isolation by distance (IBD), which is supported by previous studies of wild mice in agricultural settings  though mIn designing the custom hybridization capture panel presented here, we sought to include markers from the GigaMUGA that were likely to be informative across our study populations, and at a scale that would suit library preparation in a single 96-well plate format followed by sequencing on a commonly accessible benchtop sequencing instrument. Our assessment of the resulting sequencing dataset indicated a high proportion of successful target capture, with little off-target activity, and overall strong concordance with the GigaMUGA genotypes. The additional SNPs identified in the sequencing data may have some utility for applications that do not require markers to be in linkage equilibrium or the conversion of linked biallelic SNPs into multiallelic ‘microhaplotypes’ for improved power in some analyses . EncouraOverall, our assessment of a commercially available SNP array suggests that, while the technology performs well and may provide a quick ‘off-the-shelf’ genotyping solution, the design focus on genotyping laboratory lines has likely created efficiency trade-offs for studies of wild mouse populations as a large majority of markers appear irrelevant to variants segregating in the wild. As an alternative, researchers interested in wild populations of other model organisms may consider the approach demonstrated here of utilizing genotyping arrays for marker discovery, which subsequently informs design of a custom targeted sequencing panel. While the taxonomic range of commercially-available genotyping arrays is relatively limited see , some stM. m. domesticus lineage, suggesting that analysis of wild populations that might vary in subspecies ancestry could lead to erroneous estimates of allelic diversity, and thus any population genetic inference based on such statistics should be interpreted with caution [M. m. domesticus ancestry [In comparison to other genotyping-by-sequencing methods that query similar numbers of SNPs such as ddRADseq , per sam caution , 29. We ancestry , 55.In contrast to genotyping arrays, which typically require only basic processing of tissues prior to submission to a genotyping service provider, hybridization capture protocols involve considerably more handling time for sequencing library preparation, target capture, final purification and quantification. Moreover, downstream analysis of sequencing datasets may require a greater degree of bioinformatic processing compared to analysis of array-derived genotypes. In our study, some cost efficiencies were gained by preparing libraries at 0.4X the standard reaction volume and performing sequence capture on multiplex pools of samples. At higher scales of sample throughput, some of the most labor intensive aspects might be alleviated by the use of liquid handling robots where appropriate , and seqIn conclusion, here we have presented an evaluation of two methods for generating high-density SNP datasets in the context of applied population genetics. The most suitable method for a given study will ultimately depend on the relative constraints in terms of time, project budgets, and available expertise in molecular laboratory techniques and bioinformatics. Continual optimization of such protocols will be a key in furthering the integration of genetic insights into management of invasive and invasive pest species.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


Falagoniamexicana is an aleocharine distributed from northern Mexico to Guatemala and El Salvador. It is associated with Attamexicana ants and lives within their piles of waste or external debris. The phylogeography and historical demography of 18 populations from Mexico, Guatemala, and El Salvador were studied. The data set encompasses a 472 bp fragment of the COI. Results suggest that F.mexicana was originated during Middle Pliocene (ca. 0.5 Mya), starting its diversification at the Upper Pleistocene and Holocene. Populations were recovered forming at least four main lineages, with a significant phylogeographic structure. Evidence of contemporary restricted gene flow was found among populations. The historical demography suggests that the geographic structure is due to recent physical barriers  rather than ancient geological events. Also, recent geological and volcanic events in the east of the Trans-Mexican Volcanic Belt and the Sierra Madre Oriental might be responsible for the restricted gene flow among populations. Skyline-plot analyses suggested that a demographic expansion event took place at the end of the Late Quaternary glacial-interglacial cycles. Falagoniamexicana Sharp, 1883 is a species of Coleoptera that belongs to the subfamily Aleocharinae (Staphylinidae) .linidae) . Particurocesses , 1978, aand ants , as is t America , 2012. BFalagonia Sharp, 1883 included two species: F.mexicana and F.crassiventris Sharp, 1883. Currently, F.crassiventris is located in the genus Pseudofalagonia Santiago-Jiménez, 2010; therefore, Falagonia is now a monotypic genus  epipharynx with a spinose process on the apico-medial margin; 2) with a pair of setae on the sensory area of ligula; 3) with simple punctures on elytral disc; 4) without tuberculate punctures on elytral disc; 5) tergite IV of male with a protuberance on each side of midline; 6) tergite VIII of male without lobes on posterior margin; and 7) tergite VIII of female without lobes on posterior margin. It is important to highlight that the specimens of F.mexicana are very homogeneous, except for some variation in size (4–7 mm); in color ; in the length of the α-sensilla of the epipharynx, as well as the length of the spines of the spinose process on the apico-medial margin of the epipharynx. Moreover, they present secondary sexual dimorphism. Males have a protuberance on each side of the midline of tergite IV, and a protuberance on the midline of tergite VIII.The genus ic genus . At a phF.mexicana has been recorded in a wide altitude range . This species has been recorded in diverse types of vegetation  ants. The reddish color F.mexicana allows it to blend in with the debris. Apparently, despite having developed wings, this species does not fly. Flight intercept traps have been placed near the debris and we have not been able to catch them. Instead, by placing pit-fall traps in the vicinity of the detritus we have obtained some specimens. The nests of A.mexicana with its associated debris seems to be islands in the geographic distribution of F.mexicana, due to their presumed inability to fly and limited long-distance movement. Therefore, geographical barriers have a great effect in reducing gene flow between its populations, as well as restrictions on its geographical distribution. Physical barriers can lead to genetic isolation when they remain for an extended period of time, which is essential for lineage divergence .The sampling was carried out in 18 localities from El Salvador, Guatemala, and Mexico  of the Cytochrome Oxidase subunit I (COI) mitochondrial gene. This fragment is located between positions 250 and 720 of the COI gene. This gene was selected since it presents an adequate mutation rate for this kind of studies , 3 µL of dNTP’s (8mM), 3 µL of each primer (10mM), 0.2 µL of Taq polymerase (5 U / µL) and 9.8 µL of ddH2O adding to a final volume of 30 µL. The PCR protocol consisted of an initial denaturation for 180 s at 94 °C; 35 cycles of 30 s at 94 °C as denaturing temperature, 30 s at 45 °C for primer annealing and 90 s at 74 °C for extension; followed by one final extension cycle of 300 s at 72 °C. PCR products were visualized in a 1% agarose gel stained with ethidium bromide and purified using the GeneJET Genomic DNA Purification Kit . The PCR samples were sent to the Macrogen company (South Korea) for sequencing. Sequences were assembled with the software SEQUENCHER v. 5.2.4 (Gene Codes Corporation). The alignment of the sequences was trivial; therefore, it was done manually using the software MEGA v. 7 (OQ608846–OQ608984).From total genomic DNA, we obtained the sequence for a fragment of 472 base pairs  was conducted. This was performed with the aid of the software SAMOVA v. 1 (FCT (AMOVA) was carried out including every population, followed by an analysis between the statistically significant regions identified by the SAMOVA. This was conducted with 1000 permutations using the software ARLEQUIN v. 3.5 (H), haplotype diversity (Hd), nucleotide diversity (π) (S)  , and seg (π) (S) . These v (π) (S) . This anK v. 4.5  using thPseudofalagonia sp. (acc. num. OL764948.1), a species closely related to F.mexicana, was used as outgroup. The Markov Monte Carlo Chains (MCMC) were run for 10 million generations sampling trees and parameters every 1000 generations. The consensus tree was obtained with their respective posterior probabilities after discarding the initial 25% of the stored trees (ML) optimality criterion was carried out using the software RAXML v. 8  for the COI was used to date the genealogy of Falagonia. This rate between taxa is based on the substitution rate estimated by COI gene of the coleopteran mitochondrial genome. Alternatively, a more conserve substitution rate of 2.0 (10-2 subs/site/My/lineage) was used  and posterior intervals spanning the 95% of the highest posterior density. This analysis was repeated four times and the trees resulting from each run were combined using the software LOGCOMBINER v. 1.10.4 . The 55 remaining characters correspond to variable sites . This locality had nearly 30 times the nucleotide variation found in Flor de Chiapas . The lowest value for haplotype diversity was at Lachiguiri (Hd = 0.2), while the highest value  was at Acahuizotla (Guerrero), Neblinas (Queretaro) and Tepexco (Puebla). The global Fu’s F value  is consistent with a significant demographic expansion history. However, none of the individual populations showed statistically significant values for this descriptor.Genetic variation descriptors Table  show thaSAMOVA (k change from 2 to 15) showed a gradual increase in the FCT values  and Acahuizotla-Tepexco. The Mantel test revealed a significant positive correlation , which suggests isolation by distance. The AMOVA showed also significant genetic structure . Approximately 77% of molecular variance occurs among populations, while the remaining 23% is explained by the genetic differences within populations  to a continuous exchange of individuals between Xalapa-La Orduña, and Xalapa-Cerro Colorado.The es Table . The higns Table . PopulatML , and a “big clade” (PP = 1) that encompasses the populations from Tierras Altas de Chiapas (TAC) to Sierra Madre Oriental (SMO). This “big clade” presents a substructure recovering a monophyletic group formed by Valles Centrales de Oaxaca (SMS: VCO) which is the sister group to the clade containing the rest of the sampled populations.The best-fitting nucleotide substitution model was TrN+G+I. The model’s specific parameters were: base frequency = 0.3175, 0.1536 0.1489, 0.38; nst = 2; ts:tv ratio = 4.7931; Gamma shape = 0.5600; ncat = 4; and pinvar = 0.7490. The trees recovered by Bayesian  the skyline-plot showed a demographic increment process that started approximately 4,000 years ago  suggesting effective gene flow. Possibly, these haplotypes are ancestral that gave rise to the more recent ones. Gene flow is supported by the values obtained for migrants per generation (Table M = 0.113–0.643) suggest the existence of potential geographic barriers. The Isthmus of Tehuantepec could be such barrier. The gene flow is a complex process as suggested by the Mantel´s test. The mutation pattern observed in haplotypes H24 and H19 suggests that they might mediate the inferred gene flow. They are intermediate haplotypes between those from Sierra Madre Oriental, Tierras Altas de Chiapas, Tlalixtac, and Yautepec. There is a negative relationship between Hd and sample size  as well as in the east (Flor de Chiapas) of the Isthmus. However, the number of segregated sites and nucleotide diversity differ greatly within these two populations. The population of Lachiguiri has maintained gene flow with the Candelaria Loxicha and Yautepec populations  and in the Pacific, and the most recent lineage to the south of the Trans-Mexican Volcanic Belt, on the Coastal Plain of the Gulf of Mexico. FST for the bark beetle Dendroctonusmexicanus, reporting an allopatric fragmentation event among the populations to the north of the Trans-Mexican Volcanic Belt, and the Sierra Madre del Sur. They assumed a long-distance colonization process with continuous migration between populations of those geographic provinces. Another study by Dendroctonusapproximatus beetle found differentiation between populations in the group of Sierra Madre Oriental-Sierra Madre del Sur with respect to the Sierra Madre Occidental-Trans-Mexican Volcanic Belt group. This divergence was estimated to have occurred 195,000 years ago in the late Pleistocene. They concluded that the populations of that species represent a model of geographic range expansion and contraction, with subsequent restricted gene flow among putative Pleistocene refuges. Apparently, a rapid genetic structure in space and time for Coleoptera, and possibly for insects in general, is feasible in the mountainous regions of Mexico. It is important to continue with studies like these that will allow us to reinforce possible dominant evolutionary patterns in Neotropical insects.The es Table  and the VA Table  revealedF.mexicana occurred during the Middle Pliocene, followed by a radiation that begun in the Upper Pleistocene that persisted during the Holocene. Such time coincide with several of the glacial-interglacial cycles that took place during those periods  a cladogenetic event occurred separating the FDC clade (east of the Isthmus of Tehuantepec) from its sister group, b) the SMS: VCO clade (west of the Isthmus) differentiated from the TAC clade, and c) the SMO clade (northwest of the Isthmus) split from the TAC- SMS: VCO clade. All this could be regulated by continental uplift and sea level oscillations, or glacial-interglacial cycles.The Isthmus of Tehuantepec is a geographical barrier for gene flow between the east and west regions. Many vicariance events have been invoked for different lineages . The IstFalagonia, the dry and harsh climatic conditions of this area might create a barrier for this species. The northern area of the Isthmus is more humid due to the influence of the Gulf of Mexico. Until a century ago the predominant vegetation was tropical evergreen forest. In the past this vegetation was a continuum ecosystem from northern Veracruz to the northeast of Chiapas . SMO clade to finally establish during the Pleistocene, once geological, geographic, and climatic conditions were more stable. More recently, the volcanic activity to the east, at Chiconquiaco-Palma Sola, during the Quaternary, apparently constituted another barrier to the F.mexicana populations from the northeast of Mexico. The rocks of the Xalapa Monogenetic Volcanic Field, which comprises more than 59 volcanoes of the Quaternary, fall into three groups, the oldest with a little more than 2 Mya, others between 250 to 400 Kya, and others less than 100 Kya  constitute the glacial cycles, while the stages where the climates are equal to or warmer (2–3 °C higher) than the present, are the interglacials  occurred more recently. Other data obtained from the mtDNA from a variety of insect groups has revealed a general pattern where population expansions occurred during the Pleistocene (The population expansion processes inferred for glacials . Glacialglacials . Such drglacials . During glacials . Falagonistocene .F.mexicana are unknown; therefore, there is a lack of information about its natural history, behavior, and ecology. So far, the adults of this species have been only observed and collected in association or near by the nests of Attamexicana. F.mexicana with A.mexicana. The ant species of the tribe Attini depend strictly for their feeding on the cultivation of the Leucoagaricusgongylophorus fungi. Such agricultural practice creates particular micro environmental condition inside the ant´s nest as well as the continuous production of foliar debris. Temperature fluctuations had a harsh effect on insect population perhaps partly due to thermoregulation in conjunction with body mass, playing an important role at the level of ecological niche and geographic distribution patterns. F.mexicana can use the nests of A.mexicana as a refuge to regulate their body temperature and, at the same time, be able to capture prey. Therefore, the external debris of A.mexicana may historically constitute a refuge without abrupt temperature changes, allowing this species to occupy higher altitudinal and wider latitudinal ranges in different vegetation types.The larvae of Falagoniamexicana is a species whose origin can be dated during the Pleistocene and diversified in at least four main haplogroups since then. Geographic barriers such as the Isthmus of Tehuantepec, Trans-Mexican Volcanic Belt and Sierra Madre Oriental have played a major role in the evolution of this species. Even though geographic regions define the haplogroups, evidence of contemporary restricted gene flow was found, which indicates that this lineage is in the process of genetic differentiation. The differentiation among populations was supported by significant values of pairwise Fst, and the global genetic structure revealed by the AMOVA. Also, a significantly positive correlation between genetic and geographic distances, suggesting isolation by distance is a process promoting its genetic structure. Finally, climatic, and volcanic events that occurred during the late Quaternary Period indubitably have shaped the distribution, genetic structure, and demography history of this elusive insect.


Triplophysa species in the Qilian Mountains using the mitochondrial DNA gene (COI), three nuclear genes  and 11 pairs of nuclear microsatellite markers. We collected 11 species of genus Triplophysa living in the Qilian Mountains, among which Triplophysa hsutschouensis and Triplophysa papillosolabiata are widely distributed in the rivers on the northern slope of the Qilian Mountains. There was a high degree of lineage differentiation among species, and the genetic diversity of endemic species was low. The different geographical groups of T. papillosolabiata presented some allogeneic adaptation and differentiation, which was closely related to the changes in the river system. Except for the population expansion event of T. hsutschouensis during the last glacial period of the Qinghai‐Tibet Plateau (0.025 MYA), the population sizes of other plateau loach species remained stable without significant population expansion. Starting from the east and west sides of the Qilian Mountains, T. hsutschouensis, and T. papillosolabiata showed two species colonization routes in opposite directions. The geological events of the uplift of the Qinghai‐Tibet Plateau and the climatic oscillation of the Quaternary glaciation had a great influence on the genetic structure of the plateau loach in the Qilian Mountains, which promoted the genetic differentiation of the plateau loach and formed some unique new species. The results of this study have important guiding significance for fish habitat protection in the Qilian Mountains.In recent years, the influence of historical geological and climatic events on the evolution of flora and fauna in the Tibetan Plateau has been a hot research topic. The Qilian Mountain region is one of the most important sources of biodiversity on the Qinghai‐Tibet Plateau. Many species existed in the region during the Pleistocene glacial oscillation, and the complex geographical environment provided suitable conditions for the survival of local species. The shrinkage, expansion, and transfer of the distribution range and population size of species have significant effects on genetic diversity and intraspecific differentiation. To reveal the effects of geological uplift and climate oscillation on the evolution of fish populations in the Qilian Mountains, we investigated the genetic structure, phylogenetic relationship, and phylogeographical characteristics of genus  Triplophysa species in the Qilian Mountains, and the genetic diversity of endemic species was low. The different geographical groups of T. papillosolabiata presented some allogeneic adaptation and differentiation, which was closely related to the changes in the river system. Except for the population expansion event of T. hsutschouensis during the last glacial period of the Qinghai‐Tibet Plateau (0.025 MYA), the population sizes of other plateau loach species remained stable without significant population expansion. Starting from the east and west sides of the Qilian Mountains, T. hsutschouensis and T. papillosolabiata showed two species colonization routes in opposite directions.There was a high degree of lineage differentiation among the  This genus is widely distributed in the Tibetan Plateau and surrounding water systems and is a special group of loaches adapted to the cold environment of the Tibetan Plateau  populations in the Qilian Mountains, in order to: (1) estimate species diversity and species geographic distribution of the plateau loach fish; (2) reveal the implicit diversity of widely distributed species in the region; and (3) reconstruct the biogeographic history of the plateau loach fish population in the Qilian Mountains. Finally, we tested whether the species differentiation of the plateau loach in the Qilian Mountains was driven by specific geological events and whether the species distribution was likely to coincide with the changes in water systems in the Qilian Mountains. Therefore, we expected to suppose that the genetic diversity of the Plateau loach was related to the geological changes in the region.In this study, we collected and analyzed data from the plateau loach fish (genus 22.1Hedinichthys yarkandensis (Day 1877) were collected from the Shule River for further analysis. These specimens were captured using a cage net. Morphological identification of fishes is mainly based on taxonomic books  was used to search microsatellite loci from the genomic data of T. siluroides (NCBI taxonomy ID: 422203). The search conditions were as follows: the length of the microsatellite repeat unit was 3–6 bp, the number of repeat units was >5, and the flanking sequence of microsatellite loci was >300 bp. Using genomic DNA from 5 individuals of each species as a template, 100 pairs of microsatellite primers were screened. The fluorescence labeling technique was used to detect microsatellite labeling polymorphism, ABI 3730XL DNA analyzer  with a GeneScan‐500Liz size standard was used for capillary electrophoresis, liz‐500 was used as reference, GeneMarker V2.2.0 software (http://www.softgenetics.com) was used for microsatellite labeling typing. Eleven pairs of highly specific and polymorphic microsatellite primers were obtained for subsequent experimental analysis , Bayesian (BI), and neighbor‐joining (NJ) methods. jModelTest Version 0.1.1 software  was used to construct the phylogenetic tree using the neighbor‐joining method, and the support rate of nodes in the phylogenetic tree was estimated by 1000 self‐propagation repetitions. The Markov chain Monte Carlo (MCMC) method of MrBayes 3.2.6 software  were discarded, the remaining trees were reserved to build a majority principle consensus tree, and the Bayesian Posterior Probability (BPP) of the evolutionary tree was calculated.Using  Posada,  was used Posada, . To findhttp://tree.bio.ed.ac.uk/software/figtree/). Due to the lack of fossil evidence of the genus Triplophysa, this study used the mutation rate of mitochondrial genes of the genus Cobitis Linnaeus 1758, in which the rate of molecular evolution per million years per lineage per point was 0.68%, to estimate the differentiation time of the loach genus   was estimated for each species in DnaSP 4.2 software   was used to reconstruct the historical population size dynamics of each species . The population was divided into three distribution areas according to water system: A: Shiyang River system; B: Heihe River system; C: Shule River system. The Bayesian final tree was used for BBM (Bayesian Binary MCMC) analysis, all parameters were kept at default values, and JC + G model was used for calculation. In the Bayesian analysis, 10 Markov chains are used to carry out two independent operations. The algebra of Markov chains is 50 million, and samples are taken every 100 generations. The first 10 million generations are discarded in the final analysis, and the results of the remaining 40 million generations are used to estimate the probability of existence or absence on each node.The ancestral distribution of 2.5http://genepop.curtin.edu.cn). The number of alleles (A), average number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), and expected heterozygosity (He) were calculated in GENETIX software .All loci and groups of Hardy–Weinberg equilibrium (HWE) deviation were detected using the Markov chain Monte Carlo (MCMC) method in GENEPOPV. 3.4 . However, T. tenuis is only distributed in the Hei and Shule Rivers, and no specimens have been collected in the Shiyang River. Triplophysa siluroides and T. scleroptera are mainly distributed in the Datong and Huangshui Rivers, the outflow rivers south of the Qilian Mountains. Triplophysa stoliczkae and T. robusta are mainly distributed in the tributaries of the Yellow River, and a few specimens were also collected in the Shiyang River system.A total of 548 specimens of plateau loach 3.2T. shiyangensis and T. robusta had the highest haplotype diversity , while T. hsutschouensis had the lowest haplotype diversity (0.167). The highest nucleotide diversity was found in T. shiyangensis (0.00636), and the lowest was found in T. tenuis (0.00033).The mitochondrial COI gene dataset from the Qilian Mountains contained 557 sequences, which produced 79 haplotypes Table . T. shiyT. papillosolabiata, respectively. The lowest value was 1 in the Datonghe River population of T. scleroptera. The average heterozygosity of the Danghe River population of T. papillosolabiata was highest (0.647), and that of the Tianzhu population of T. scleroptera was lowest (0.043). The average expected heterozygosity was consistent with the average observed heterozygosity. The highest value of polymorphism information content was 1.056 in the Changma population of T. papillosolabiata, and the lowest value was 0.148 in T. siluroides. The PIC of the five population of T. hsutschouensis in Shiyang River was the highest (0.460–0.704), followed by the Heihelinze population (0.466), and the PIC of the two population of T. hsutschouensis in Shule River was the lowest (0.305–0.398). The PIC of the two population of T. papillosolabiata in Shule River was the highest (0.948–1.056), followed by the two population in Hei River (0.850–0.941), while the Xidahe River population and Sugan lakes populations were lower .The 11 pairs of microsatellite loci showed rich genetic diversity in each species. The highest numbers of effective and observed alleles were 2.571 and 4.727 in the Changma population of 3.3K value was 14, T. papillosolabiata with high genetic diversity was further divided into 5 populations with obvious genetic differentiation. The first population (CSC1) included individuals from the Changma and Danghe sections of the Shule River. The second population (CSC2) consisted of samples from the Babaohe section of the upper Heihe River. The third population (CSC3) was composed of individuals from Linze section of lower reaches of the Heihe River and the Yanglong section of the Taolai River, a tributary of upper reaches of the Heihe River. The fourth (CSC4) was made up of the Suganhu population. The fifth population (CSC5) was composed of individuals from Shiyang River, but this population was divided into the sympatric distribution of T. wuweiensis in the optimal structure model.The optimal structure model separated the genetic variation into 14 clusters, and the other acceptable models were 4 and 11 clusters, respectively Figure . The speFst) and AMOVA were analyzed based on COI gene sequences between the five populations of T. papillosolabiata and 10 other species (Tables p > .01), but the statistical tests of Fst values between the other two species showed extremely significant. The Fst values between the CSC2 and CSC3 and CSC2 and CSC5 populations were negative, and the Fst values between the CSC4 and CSC2 populations were the largest (0.88387). The Fst value between the CSC2 population and T. strauchii was the largest (0.99470), followed by that between the CSC2 population and T. tenuis (0.99397). The Fst value between T. stoliczkae and T. shiyangensis was the lowest (0.86247), except for the populations of T. papillosolabiata. The results of AMOVA . The AMOVA . The genetic differentiation index analysis based on nuclear genes between species revealed significant genetic differentiation between 11 species pairs, except that T. leptosoma and T. papillosolabiata shared the same haplotype based on sRH1 and Myh6 gene sequences , Bayesian inference (BI), and neighbor‐joining (NJ) methods, but the support values of some nodes were different. Therefore, only the NJ phylogenetic tree is given in this study Figure . The phyT. leptosoma and T. papillosolabiata shared a single allele.The haplotype evolutionary network diagram constructed based on the COI gene sequences clearly shows the haplotype relationships of 11 plateau loach species Figure . The two3.5T. tenuis, T. siluroides, T. stoliczkae, T. wuweiensis, T. strauchii, T. scleroptera, and T. leptosoma showed a single peak. The Tajima's D and Fu's FS tests of the 6 species of loach were not significant (p > .05), but those of T. tenuis were extremely significant (p < .05). The inconsistent results of this analysis may be due to the small number of haplotypes in the six species, and the small number of nucleotide variation sites may lead to the false appearance of a single peak in the analysis of nucleotide mismatch distribution. These results indicated that the six species, except T. tenuis, did not deviate significantly from the population expansion model. The nucleotide mismatch analysis of different geographic populations of T. tenuis showed a single peak only in Changma population and Danghe river population, but the Tajima' D and Fu's FS tests were not significant (p > .10), indicating that all populations of T. tenuis did not deviate significantly from the population expansion model , and no population expansion events were detected. In contrast, the BSP diagram of each species showed that only T. hsutschouensis had experienced a population expansion event since approximately 0.025 MYA, while the population expansion event of T. tenuis was not obvious. Other species have maintained stable population sizes in recent history without significant population expansion , and the maximum estimated time to the most recent common ancestor of T. wuweiensis, T. leptosoma, and T. papillosolabiata was 6.8741 MYA (approximately the Late Pliocene). The estimation of the time from the earliest to the most recent common ancestor of loach species in the Qilian Mountains suggests that species differentiation occurred in the Late Pliocene and early Pleistocene.Based on the mitochondrial gene evolution rate of 0.68% per million years, it is estimated that the time from the progenies to the most recent common ancestor of the plateau loaches in the Qilian Mountains is between 0.9559 and 19.4165 MYA Figure . The timTriplophysa hsutschouensis based on BBM showed that the ancestral distribution region at node 15 included several possible distribution regions, among which the proportion of ancestral distribution area of Shiyang River system is dominant . The main morphological difference between T. hsutschouensis and T. robusta lies in the different degrees of body surface scales. The body surface of T. hsutschouensis is almost bare and scaleless, which can reduce the waste of body temperature in cold areas , followed by the Heihelinze populations in the Hei River (0.466) and the two populations in Shule River (0.305–0.398). The genetic diversity of different geographical populations of T. hsutschouensis showed an obvious decreasing trend from the Shiyang River to the Hei and Shule Rivers. In the analysis of mitochondrial gene markers, although some populations have only one haplotype or a small population can reduce the genetic diversity of the population  on the Tibetan Plateau. This is consistent with the dispersal pattern of the sympatric species Schizopygopsis chilianensis , followed by the two population in Hei River (0.850–0.941), while the Xidahe River population and Sugan lakes populations were lower . The genetic diversity of populations in different river systems showed an obvious decreasing trend from Shule River to Hei River and Shiyang River. T. papillosolabiata colonized from west to east along the northern Qilian Mountains from the Shule River as a starting point. Triplophysa leptosoma was only collected in the Changma section of the upper reaches of the Shule River. Combined with the data of nuclear and mitochondrial genes, T. leptosoma was considered to be a new species that differentiated from T. papillosolabiata, and the nuclear genes (Myh6 and sRH1) of the two species were not fully differentiated.Wu & Wu, . The inler Zhao, . AccordiT. hsutschouensis colonized from east to west, while T. papillosolabiata colonized from west to east along the northern slope of the Qilian Mountains. The effects of ice age and geographical isolation promoted the genetic differentiation of plateau loach and even formed some endemic species , but T. hsutschouensis does not show a similar phenomenon, which may be related to the strong exotic evolution ability of the plateau loach  have long dried up. The distribution areas of 5Triplophysa in the Qilian Mountains by integrating mitochondrial gene, nuclear gene, and microsatellite data. There is a high degree of lineage differentiation among the species, and T. leptosoma is a new species undergoing differentiation. The tectonic events of the uplift of the Qinghai‐Tibet Plateau and the repeated climatic fluctuations during the Quaternary glaciation had a great influence on the genetic structure of the plateau loach in the Qilian Mountains, which promoted the genetic differentiation of the plateau loach and led to the formation of some endemic species. T. hsutschouensis colonized from east to west, and population expansion occurred during the last glacial period of the Qinghai‐Tibet Plateau, while T. papillosolabiata colonized from west to east along the northern slope of the Qilian Mountains. There were two kinds of colonization paths in opposite directions in the Qilian Mountains, which formed the present species distribution pattern. In addition, habitat conservation is necessary for species with low genetic diversity and narrow distribution (T. wuweiensis and T. leptosoma) to ensure the persistence of genetic diversity. Combined with genetic evidence from other species in the region, our results suggest that different biogeographic patterns emphasize the importance of the Qilian Mountains as a research hotspot for the genetic diversity of some plant and animal groups.In this study, we evaluated the genetic differentiation of the genus Yanyan Du: Formal analysis ; investigation ; resources ; software ; writing – original draft (lead). Yanping Zhang: Investigation . Zhongyu Lou: Formal analysis ; software . Tai Wang: Data curation (lead); investigation ; methodology ; writing – original draft ; writing – review and editing .This work was supported by the National Natural Science Foundation of China (Project 31460560); Innovation Base and Talent Program of Gansu Province (No.21JR7RA720). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in the writing of the manuscript.The authors declare that they have no competing interests.