The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the ‘selfing syndrome’, which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood.Daphne kiusiana, endemic to East Asia; plants in the eastern region  exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets.Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period.D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck.Our results suggest that the selfing-associated morphological changes in  Most flowering plants have perfect flowers in where the anthers and stigmas of a single sporophyte are held in close spatiotemporal proximity . AlthougThe selfing species/lineages derived from outcrossing ancestors are often accompanied by a common set of morphological and functional changes to the flowers, termed the ‘selfing syndrome’ , and it Capsella rubella was driven preliminarily by a severe population bottleneck within the last 50 000 years  and var. atrocaulis (found in China and Taiwan). The former has small flowers with calyx tubes 7–8 mm long, whereas the latter has slightly larger flowers with calyx tubes 10–14 mm long  in the populations on offshore islands of southwestern Korea (hereafter referred to as western populations) are much larger than those in southeastern Korea and Kyushu, Japan (referred to as eastern populations) (ast Asia . Two varlations) . These mlations) .Selfing leads to increased homozygosity through the fixation of alleles at gene loci and reduces effective recombination  and diffD. kiusiana, with subcapitate inflorescences focusing on genetic characteristics, palaeodistribution, historical population demography and floral morphological traits between the two geographical populations (west vs. east). We address the following specific questions: (1) What differences do the genetic makeups of these two geographical groups exhibit? (2) What factors promoted the evolution of the eastern populations with inconspicuous  floral traits that have probably been induced by selfing? (3) How are the demographic signatures of the group with these characteristic flowers exhibited? We characterize the genetic composition of D. kiusiana distributed in Korea and Japan using nuclear microsatellites and multilocus-based cpDNA datasets, and examine the associated variations in morphological floral traits. Finally, we employ ecological niche modelling (ENM) and approximate Bayesian computation (ABC) to gain further insights into the historical migration patterns and demographic details of these populations. Our study provides evolutionary insights into how selfing affects the sustainability and adaptive potential of wild plant populations.To our knowledge, the majority of previous population-level studies related to the evolution of the selfing syndrome have been of herbaceous plants. In this study, we aim to elucidate the evolutionary history of a wild shrub, Daphne kiusiana Miq. is commonly used as an ornamental plant because of its graceful inflorescence shape (resembling a bride’s bouquet) and pleasant fragrance , until use. Total genomic DNA was extracted from dried leaf samples using the DNeasy Plant Mini Kit  following the manufacturer’s instructions. The concentration of extracted DNA was determined using a Nano-300 microspectrophotometer  and diluted to 15 ng µL−1 to obtain the same concentration of template DNA in each sample for analysis.Our study was conducted on almost all known populations of D. kiusiana . PCR amplification was performed as follows: initial denaturation at 95 °C for 15 min; 35 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 1.5 min and extension at 72 °C for 1 min; and final extension at 72 °C for 10 min. The PCR products were diluted with ddH2O (1:30), and 1 µL was analysed on an ABI 3730XL sequencer with GeneScanTM-500LIZTM Size Standard (Applied Biosystems). Allele sizes and peaks for each sample were determined in triplicate using Peak Scanner 2 software (Applied Biosystems) to minimize genotyping errors.We used 16 nuclear microsatellite markers  previously developed for nth update = 200; burn-in = 2000). This showed that the null allele frequency of all the loci examined ranged from 0.004 % to 0.015 %. Therefore, we used 16 microsatellite markers for statistical analysis.Before analysing the microsatellite data, we estimated the null allele frequency using INEst  software, which calculates the null allele frequency regardless of the effect of inbreeding  by runniTo ensure that the number of loci was sufficient to analyse the genotype of each individual in the samples, a genotype accumulation curve was applied using the ‘poppr’ (v.2.0.2) package in R . We thenNA), number of private alleles (PA), observed heterozygosity (HO), expected heterozygosity (HE) and inbreeding coefficient (FIS). Allele richness (AR) and genetic differentiation among populations (FST) were determined by calculating the overall fixation index according to the method of Genetic diversity parameters were evaluated for each locus based on simple sequence repeat (SSR) allele frequency data using GenAlEx 6.5 . These iMES’ software (robust multilocus estimation of selfing rates). This technique is relatively insensitive to null alleles and scoring errors, which may otherwise lead to the overestimation of selfing rates  method maximizes the log-likelihood of the multilocus heterozygosity structure of a sample. As a significance test for selfing rate associated with the g2 method, RMES computes the probability that there is no selfing (g2 = s = 0) obtained from 10 000 iterations of a random assortment of single-locus heterozygosities among individuals. A 95 % confidence interval (CI) for selfing rate provided by RMES was used to determine the significance of selfing rate associated with the ML method.To estimate selfing rates at the pooled lineage level, we employed ‘RDetailed methods are provided in We targeted 27 non-coding regions of cpDNA using MPM-seq for all samples. This method can provide noteworthy information faster and more cost-effectively in phylogenetic analyses based on independent sources of genomic data . The rawK = 1 to K = 9 clusters. The optimal number of clusters, K, was determined via the K method, using ‘STRUCTURE HARVESTER’  to determine the genetic structure using the covariance-standardized approach of pairwise Nei’s genetic distances in GenAlEx 6.5. To identify genetic boundaries between the populations, we performed a barrier analysis (DA) and the proportion of shared alleles (Dps). Pairwise genetic distances (DA and Dps) were generated with MSA software using a bootstrap analysis of 1000 replicates.For microsatellite analysis, we used a Bayesian clustering approach implemented in STRUCTURE 2.3 , an ML phylogenetic tree was constructed using Geneious R11.0.5 . We considered two closely related species, Daphne depauperata (MW245833) and D. acutiloba (MW246150), as the outgroups (https://www.ncbi.nlm.nih.gov/). Alignments were performed using ‘MAFFT’ , including D. kiusiana var. atrocaulis (Rehder) F. Maek., and the sample localities in our investigation. A total of 452 occurrence points were spatially filtered with 25 km between each point using SDMtoolbox 2.4  were collected during the peak flowering season from 2016 to 2022 (mainly during February and March 2022). During the field survey, the number of flowers was counted by randomly selecting one inflorescence on each individual. Then, we cut the branched stems, including the inflorescences, and brought them to the laboratory at Chonnam National University for subsequent measurements. For each sample, we imaged the longitudinal sections of the flowers using a digital camera  connected to a stereomicroscope . The length and width of the calyx lobe, length of the calyx tube, length of the pistil, and minimum distance between the stigma and anthers were measured using digital calipers built into iworks 2.0 software (Nahwoo)  . SamplesF test in one-way ANOVA. We also examined the correlations between flower traits and herkogamy in the pooled samples. The degree of herkogamy was estimated as the distance between the shortest stigma and the anther, as their stamens generally consist of two pairs of four. The area of the calyx lobe was roughly estimated by half the length × width. All statistical analyses and significance tests were performed using R v.4.1.2 . The significance of the differences between the two lineages was confirmed by a statistical  v.4.1.2 .Significant differences in genetic diversity were observed between the western and eastern populations .AR = 1.422, HE = 0.095) than in the western lineage . Similarly, the genetic diversity of the pooled eastern lineage  was lower than that of the pooled western lineage  (Genetic diversity was lower in the eastern lineage (= 0.484) .FST values evaluated between the populations within each lineage were higher in the east (mean = 0.712) than in the west (mean = 0.291), and were significant over all loci (P < 0.001) . HMLGs occurred only in the eastern lineage, where one or a few genotypes dominated each population (0.136–1.000), and the observed heterozygosity was extremely low . In addi< 0.001) .2 method and ML method at the lineage level were considerably higher in the east, averaging 92.0 and 91.4 % compared to 17.8 and 12.9 % in the west, respectively  and the eastern lineage (LII). In the STRUCTURE analysis, while the mean likelihood scores increased progressively with K values of 1–9, ∆K values computed for all K classes indicated a strong signal at K = 2 . Fourteen polymorphisms (11 nucleotide substitutions and three indels) were identified, and three haplotypes (D1–D3) were determined. Each population harboured only a single haplotype. Consistent with the results of the microsatellite analysis, the haplotype network showed a linear topology, indicating that D. kiusiana was clustered into two different lineages , a probable bottleneck occurred within LI at 12.1 [4.9–67.1] kya, while within LII at 54.1 [10.4–999.3] kya (TDIV) was estimated to be 21.3 (12.8–70.4) kya  kya .D. kiusiana . Our results suggest that the selfing lineage in D. kiusiana has been largely driven by gradual directional selection towards lower levels of floral visibility and herkogamy for efficient self-pollination in response to historical environmental changes. The transition to selfing accompanied by morphological modifications may have been triggered by severe competitive interactions among/within species. Intrinsic factors of D. kiusiana, such as its life-history traits and niche in the ecological succession, may also have been important in facilitating its evolution. Thus, the selfing syndrome lineage or species may exhibit a demographic signature of a gradually reduced effective population size, as evidence of adaptation.To our knowledge, this is the first study to characterize in detail the evolutionary history and demographic dynamics associated with the selfing syndrome in a wild shrub with subcapitate inflorescences. The eastern lineage (LII) of MES allowed the estimation of selfing rates in our study species and revealed that the relative ratio of LII was significantly higher. We also found that HMLGs at all loci occurred only in LII. One or a few HMLGs dominate each population and coexist with several rare genotypes. This is consistent with the genetic patterns in selfing populations in which a few HMLGs dominate specific habitats (We found that the eastern populations (LII) had significantly higher homozygosity than the western populations (LI). This pattern is typical of the genetic characteristic observed in selfing species/populations  and is chabitats .On the other hand, although it is highly unlikely, as many flowering plants flexibly combine sexual and asexual reproduction systems, our results related to HMLGs may open the possibility for asexual reproduction, such as vegetative propagation and apomixis. However, these plants are inherently heterozygous and consistently maintain their original genetic diversities. Moreover, heterozygosity is expected to increase under clonal propagation owing to the accumulation of mutations over long-term evolutionary progress ; plants would have progressively colonized the ECS shelf and predominated the shrub layers of the forests. Indeed, the abundance of congeneric evergreen shrubs often increases in open areas . ClimateD. kiusiana may have been accelerated by limited pollinators  shows demographic stability, whereas the predominantly outcrossing western lineage (LI) is associated with a severe population bottleneck. In detail, the effective population size of LI remained stable for a relatively long period but showed a dramatic decline during the Holocene. In contrast, the effective population size of LII decreased in a gradual manner during the last glacial period and then remained stable from the LGM to the present (In  present . As the D. kiusiana may have taken place from the onset of the last glacial that could have triggered intense competitive interactions. In this context, functional and morphological changes to the flowers for effective self-pollination may have been almost completed at the LGM. These inferences are consistent with the hypothesis that flower size decreased in the Capsella selfing lineage before its extensive geographical spread (The SRM in LII estimated the event time of the size reduction to be ~54 100 years ago, during the last glacial period (ca. 110–10 kya; mcac142_suppl_Supplementary_Data_S1Click here for additional data file.mcac142_suppl_Supplementary_Data_S2Click here for additional data file.mcac142_suppl_Supplementary_FiguresClick here for additional data file.mcac142_suppl_Supplementary_TablesClick here for additional data file.


Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages.Insecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure.Mosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy–Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (− 0.00457 to 0.04213), Nm values were high (9.4–71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations.Phenotypic insecticide resistance to deltamethrin was observed in An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.Despite a relatively high level of phenotypic variation in the The online version contains supplementary material available at 10.1186/s12936-023-04522-5. With more than 600,000 malaria deaths worldwide in 2020, malaria continues to be a major problem in endemic tropical regions . Vector Anopheles species is frequently observed between populations from different regions of the same country . Five replicates were performed per K clusters. Using an admixture model and correlated allele frequencies, each run was carried out with 1,000,000 iterations after a burn-in period of 100,000 [K value with the highest Ln [Pr (X|K)].Genetic diversity of microsatellite alleles was investigated using  3.5.2.2 . Mosquit 3.5.2.2  and furt2.2 [IS) . Pairs o2.2 [IS) , 57. To parisons , 59. Arlutations , 60–63. ype data . The dis 100,000 . The estAn. funestus s.l., 794 as An. gambiae s.l., 102 as An. rufipes, 1 as Anopheles. tenebrosus, and 1 as Anopheles pharoensis. A total of 254 mosquitoes (5.8%) were excluded for a variety of reasons, including they were missing from the tube, the mosquito was damaged, the tube was wrongly or poorly labeled, a male mosquito was collected, or because a mosquito was identified as other than An. funestus s.l., An. gambiae s.l. or An. rufipes. A total of 168 An. gambiae s.l. and 420 An. funestus s.l. were selected from study sites where at least 30 mosquitoes of each species complex (An. gambiae s.l.) and group (An. funestus s.l.) were available for further molecular analysis. Of these, An. gambiae s.l. mosquitoes were from 5 study sites  to 88.8% (Ilha Josina)  were An. arabiensis .Differences in 30-min mortality between sites were in part explained by differences in species composition. For example, 98% of mosquitoes collected from Buna  were An. funestus s.s. populations across space  to 84.3% (Punguene). Even within the neighbourhoods of Palmeira, significant diversity was observed in 2-h mortality of the An. funestus population, ranging from 9.3% to 86.2%. In addition to An. funestus s.s. and An. gambiae s.l., An. rufipes was collected, with 99 out of the 102 identified originating from Chobela. Of these, all were dead at the 120-min exposure mark in the different exposures: 9 in the 1 × deltamethrin exposure, 51 in the 5 × deltamethrin exposure, and 28 in the 1 × bendiocarb exposure (remaining An. rufipus were identified from control bottles).Species-specific mortality rates were only available at the 2-h exposure mark, since the CDC bottle bioassay was run for the full 2-h exposure, and mosquitoes were killed and stored at this timepoint for future species identification. Significant differences in susceptibility were observed among the An. arabiensis that were tested for the 1014 locus of the kdr gene that confers pyrethroid resistance, 162 (98.8%) were homozygous susceptible and 2 (1.2%) did not amplify. This is consistent with the 100% phenotypic susceptibility to 1X deltamethrin that was observed in this species across study sites, except for 3 de Fevereiro with 87.5% mortality. Of the 420 An. funestus s.s. mosquitoes, 408 had enough DNA extract available for the CYP6P9a gene detection PCR for pyrethroid resistance. 401 (98.3%) were homozygous resistant, one was heterozygous resistant (0.2%), two were homozygous susceptible (0.5%), and four (1.0%) did not amplify.Of the 164 An. arabiensis mosquitoes were analysed across 6 microsatellite loci. All microsatellite loci were polymorphic, of which the number of distinct alleles per locus ranged from 2 (AG3H27) to 18 (AG3H249), with a mean of 8.5  to 30 (FunD), with a mean of 15.8  had significant heterozygote deficiency and did not conform to Hardy–Weinberg equilibrium (P < 0.000197)  . Within populations, only An. arabiensis from Ilha Josina had one significant overall pairwise comparison (P < 0.00341), AG2H164 and AGXH100. The low range of linkage disequilibrium across populations of An. arabiensis indicates no detectable population subdivision. In contrast, for An. funestus s.s., five (33.3%) of the overall pairwise comparisons were statistically significant   Table a. OveralAn. arabiensis and An. funestus s.s. were assessed across respective six loci by AMOVA  were inconclusive for An. arabiensis, where the value of K that showed the highest Ln Pr(X|K) followed by a plateau with increasing K was never reached; The posterior probability estimates were erratic for An. funestus as the value of K increased . These results indicate that the likelihood of an individual mosquito belonging to cluster K are unreliable and not clearly assigned to any one group for both Anopheles species.Bayesian clustering analysis did not reveal population structure for hypothetical An. funestus s.s. and An. arabiensis, in and around Manhiça district revealed marked differences. Whereas An. arabiensis was fully susceptible to 1 × deltamethrin across most study collection sites, An. funestus s.s. showed a considerable range of 1 × deltamethrin resistance. Molecular analysis of genes that confer pyrethroid resistance, kdr for An. arabiensis and CYP6P9a in An. funestus s.s., further supported this phenotypic evidence with 99% homozygous susceptible kdr in An. arabiensis and over 98% homozygous or heterozygous resistant CYP6P9a in An. funestus s.s.. The high level of CYP6P9a resistance in An. funestus s.s. plays an important role in the moderate to high phenotypic resistance observed. It is important to note that known insecticide resistance markers are only the tip of the iceberg. It is highly likely that there are other, currently undetected, genetic mutations underlying the observed variations in resistance in this study [An. funestus s.s. in Zambia, Malawi, and Mozambique showed that barriers to gene flow can play a major role in the underlying genetic differences and varied phenotypic resistance profiles as well as in elucidating resistance mechanisms [An. funestus s.s. and An. arabiensis to characterize gene flow and its role, if any, in the spread of genes that confer insecticide resistance and to understand its implications for vector control interventions in Manhiça district and surrounding areas.Surveillance of malaria vectors encompasses a wide range of activities, from characterizing geographical distribution to investigating population structure. In this study, an initial investigation of insecticide resistance in the main malaria vectors, is study . Previouchanisms , 69. TheAn. arabiensis and An. funestus s.s. as evidenced by highly polymorphic loci, significant departures from Hardy–Weinberg Equilibrium, and moderate inbreeding coefficients (FIS). The results are consistent with previously reported genetic differentiation of An. arabiensis and An. funestus s.s. in eastern and southern Africa, including some areas in Mozambique [An. arabiensis but was high (33%) in An. funestus s.s., suggesting possible population structure for this species across the study collection sites. Although moderate levels of genetic differentiation can be the result of geographic barriers to gene flow, that is likely not the case in this study area where there are no such significant features or large distances separating populations. However, it is noteworthy to point out that Ilha Josina is surrounded by swamps and inaccessible during the rainy season. There are many factors influencing the genetic differentiation in both Anopheles species such as null alleles or grouping of gene pools (Wahlund effect), which may underestimate heterozygosity or indicate population substructure, respectively. Inbreeding or nonrandom mating may also lead to heterozygote deficits. Additional factors may be spatial pooling from different houses or foci within houses and/or temporal pooling over a 2-month sampling period [A moderate level of genetic differentiation was observed across the six loci each for zambique , 70–76. g period . Geneticg period .Anopheles species, but is lower than several other Anopheles population genetics studies that use at least ten loci [An. arabiensis, this weak amplification may also be due to limited usefulness of microsatellite markers designed for its sibling species An. gambiae s.s., which has been previously reported for another sibling species, An. melas [An. arabiensis and An. funestus s.s. had been stored for 3 years and had undergone extensive transport prior to handling and use for laboratory assays, which may have affected specimen integrity and limited successful microsatellite amplification of several loci.This study successfully amplified six microsatellite loci for each ten loci , 76, 78.n. melas . On the ST) and the analysis of molecular variance (AMOVA) confirmed that there is no population structure for An. arabiensis and An. funestus s.s. in the study collection area. The low FST values observed in this study are consistent with other population genetics study for both Anopheles species [Anopheles species populations, supporting the lack of genetic structure, and suggesting each Anopheles species as a single panmictic population in the study area. This genetic variance within populations is higher but consistent with recent findings by Kaddumukasa et al. [An. funestus s.s. Similarly, the estimate for the exchange of genes among populations, also known as Nm, has been reported to be between 3–101 for An. arabiensis and 6–483 for An. funestus s.s., depending on the geographical scale surveyed [m values for An. funestus s.s. of 5.7–101.4 and An. arabiensis of 9.4–56.2 in this study are similar, although given the relatively short distances between populations, the lower Nm values for An. funestus s.s. are particularly interesting. Overall, over these short geographical distances, no significant correlation was found between migration rates and distance between locations . In combination with the moderate genetic differentiation and high linkage disequilibrium recorded for An. funestus s.s., there may be sub-populations that mostly interbreed but overlap, which could explain the phenotypic variation observed in deltamethrin resistance. Finally, for both Anopheles species, Structure analysis showed repeatedly inconsistent run results and no clear assignment to cluster K such that genetic structure could not be determined. In combination with the FST, AMOVA, and Nm, the Structure results provide strong evidence that there is no population structure and that both An. arabiensis and An. funestus s.s. mosquitoes on this small geographical area of maximum 70 km radius are highly admixed.Further investigation of population pairwise comparisons  occurred within populations and other measures of gene flow suggest a single highly admixed population for each Anopheles species. Continued molecular surveillance of An. arabiensis and An. funestus s.s. population dynamics using additional polymorphic microsatellite loci or other molecular tools and focused sampling plan will be critical for assessing local changes in gene flow. The current results suggest that insecticide resistance genes, including future ones to novel active ingredients, may easily spread in the area, which can impact the efficacy of vector control strategies, but also that possible transgenic mosquito release programmes will likely not encounter any challenges in local expansion and coverage.There was no evidence of population genetic structure of Additional file 1. Supplemental file containing supplemental tables S1-S9 and supplemental figures S1-S4.


Bembidion and complex haplotype networks of Paederus/Paederidus, with some haplotypes being drainage-specific and others found in distant populations, indicate a general lack of isolation by distance. Signs of recent demographic expansion were detected for most RPB with the latest population collapse for some rove beetles. To some extent, migration of examined species has to be limited by watersheds. Observed phylogeographic patterns are essential for correctly understanding RPB meta-population functioning.Riverine predacious beetles (RPB)  are highly diverse and numerous elements of riverine ecosystems. Their historical and contemporary distribution and diversity are highly dependent on natural flow regimes and topography of watercourses. Despite broad knowledge of their ecology, data on population genetic diversity and connectivity are lacking. This study aimed to fill this gap in order to solve two principal hypotheses assuming (i) congruence of phylogeographic patterns observed for RPB indicating that they share a common history and the ecological adaptations to the dynamic environment, (ii) genetic structuration of populations according to river basins. The Carpathian populations of four ground beetles and three rove beetles were examined using cytochrome oxidase and arginine kinase sequencing. There are substantial differences in RPB demographic history and current genetic diversity. Star-like phylogeny of  It is well known how Mediterranean, temperate and boreal taxa responded to the Pleistocene glacial periods, as well as current constraints for European taxa distribution and diversity3. There are several paradigms being well supported by multiple sources of data, which describe refugia identified mostly in the south of the continent, but also local (cryptic) refugia that played a role during various periods for particular taxa being associated with some environments 5. However, some taxa could not be easily assigned to these paradigms, due to their extrazonal distribution. Species inhabiting mountain areas belong to such a non-standard taxa, whose distribution is island-like with more or less isolated populations in mountain ranges. Many mountain species are related to either boreal or arctic populations. Such species were widespread during glacial periods, being currently restricted to warm-stage refugia at high altitudes or latitudes2. Consequently, such a species usually consists of many phylogenetic lineages of divergence, justifying their treatment as evolutionary units or independent taxa. Other species of extraordinary phylogeographic patterns are those inhabiting linear habitats, e.g., along watercourses. River networks often cross various climatic zones but are isolated by watersheds, which greatly affect the phylogeography of taxa occupying running waters, river channels or floodplains7. Moreover, the movement of individuals along watercourses often depends on the water flow, which is unidirectional, and many such species could easily disperse downstream but not hardly upstream8. This has consequences for their distribution, diversity and dynamics9. Probably among the poorest known phylogeography are organisms inhabiting montane river networks, due to their restricted ranges to rivers crossing mountains at selected altitudes10. Therefore, it could be expected that such a species should be restricted to only selected watercourses isolated by mountain ranges and strong unidirectional flow11. This would imply the existence of many phylogenetic lineages and a highly limited gene flow. On the other hand, it could be expected that the high dynamics of montane rivers  had to force some evolutionary adaptations to such an unpredictable environment12. Consequently, species living along montane watercourses should exist in meta-populations, which imply some level of migration enabling re-colonization of areas being inundated13. It is possible that both patterns are not mutually exclusive and some gene flow exists but could be rather restricted within river basins, while not so between watercourses isolated by geographic barriers. Of course, distribution and diversity is different for freshwater taxa (living only in running waters), riverine species (inhabiting river channels), or riparian organisms (associated with floodplains). Moreover, some taxa are dependent on running water their whole life, whereas others only in immature stages, and still others are distributed in the surroundings of watercourses. That different habitat requirements must have a great impact on biogeography.Knowledge on the biogeography of European taxa is the most complete and comprehensive thanks to numerous phylogeographic research on various species14 and crustaceans7. Exemplary studies on insects in the Carpathians are only available for riffle beetles (Elmidae)15. Studies on riverine species by means of taxa spending part of their life in water  and next utilizing surrounding habitats are also infrequent for mountains. Studies on salamanders16, Trichoptera17, Diptera19, and the beetle Carabus variolosus F.20, could be assigned to this type. In particular, riparian taxa meaning those that live along watercourses but are not directly dependent on freshwaters. Research on amphibians21 and a study on the beetle Liparus glabrirostris could be listed here22.Current knowledge on the biogeography of river, riverine or riparian organisms in mountain areas (considering Europe only) is incomplete; it is mostly limited only to selected species and focused on particular areas. The best-known groups are freshwater animals, living their whole lifecycles inside rivers, like fishes22). Some taxa have mixed ancestry, originating from Mediterranean mountains and local refugia, and the pathways of their expansion are complex17. This leads, in some cases, to the formation of hybrid zones across mountain ranges21. Genetic diversity and spatial structure of populations of riverine or riparian species in mountains reflects their history and contemporary gene flow. For many taxa, distinct evolutionary units were reported from particular mountain ranges, some of which occurred to be of taxonomic values19. There are also signs of dispersal being restricted by barriers (more pronounced for freshwater than riparian taxa)6.There are some biogeographic patterns that are common for species living along montane watercourses. When restricting this issue to Central European mountain systems, the following regularities arise. Many species living in or along watercourses survived the Pleistocene in local glacial refugia like the southern slopes of the Alps, Dinaric Mts., the Carpathians  are isolated by high mountains from outer areas, even if these rivers finally meet the sea. The Carpathians are also an interesting area for studying the biogeography of riverine species, as there is great diversity of rivers flowing at various altitudes and being fed mostly by rainfall, which often cause inundations and occasional floods29.The Carpathians are one of the largest mountain systems on the European mainland30. These species prefer the initial conditions of river channels, with scattered pioneer vegetation and many potential prey, such as eggs of insects and other invertebrates, fly larvae, mites or numerous Apterygota, as well as small adult insects31. Such habitat is described as “alpine rivers and the herbaceous vegetation along their banks,” being protected under the EU habitat directive (Habitat Code: 3220). Because of their narrow environmental requirements, large species diversity, numerous populations and relatively easy methods of collection in the field, these beetles have been designated as indicators for river channel quality34. Many studies utilize RPB, mostly Bembidion carabids35, but also other ground and rove beetles36 for the assessment of river channel naturalness or the impact of man-made alterations (like damming or channelization). Knowledge on the details of their biology and ecology is available only for some groups and population dynamics in most species are poorly understood, whereas many aspects of population genetics and phylogeography are almost completely unknown38. Regarding genetic data, only sequences of barcodes are available for some ground and rove beetles (e.g. a barcode library for Bembidion species39). Mitochondrial markers were also available for some taxa being the subject of phylogenetic studies, however mostly in deep phylogenies of beetles up to the level of families37. To our knowledge, there is no study aiming to understand the past and present distribution and diversity of riverine ground or rove beetles using molecular tools.Among the riverine predacious beetles (RPB), the most important ecological groups are ground beetles (Carabidae) and rove beetles (Staphylinidae). Some species belonging to these families are known to be strictly associated with sand, gravel or cobblestone alluvium, however they do not live in the water40 and applied because genetic data on the populations of these beetles should be implemented in their use as indicators of river channel quality. It is unknown which species have greater or lower genetic diversity and how the connectivity of their population functions. This could have consequences for their use as indicators.Filling the gap in the knowledge about the genetic diversity of RPB can be beneficiary for both basic and applied science. Basic because ground and rove beetles are important for understanding the biogeography of riverine species associated with mountain areas as they are top predators in invertebrate communitiesBembidion: B. decorum  (hereafter Bdec); B. varicolor  (hereafter Bvar); B. modestum  (hereafter Bmod); B. punctulatum  (hereafter Bpun), and three species of rove beetles: Paederus limnophilus  (hereafter Plim); Paederidus rubrothoracicus  (hereafter Prub); Paederidus ruficollis  (hereafter Pruf). Specifically, we aimed to describe the history of RPB, and current diversity of populations (intra- and inter-population genetic variability). This study aimed to verify the following hypotheses:There is substantial congruence of phylogeographic patterns observed for RPB, indicating that they share a common history and that their ecological adaptations to the dynamic environment are similar.Populations of RPB are genetically structured according to basins and being isolated by watersheds.Here we used several species of ground and rove beetles as objects of phylogeographic studies on species living along (sub)montane watercourses in the Carpathians – four species of Cox1 and ArgK sequences were 625 bp and 688 bp (Bmod), 637 bp and (Bpun), 636 bp and 674 bp (Bvar), 635 bp and 687 bp (Bdec), 577 bp and 685 bp (Plim), 638 bp and 690 bp (Pruf) and 568 bp and 695 bp (Prub). No indels or stop codons were detected in any sequence.After trimming low quality fragments, the lengths of The overall genetic diversity of all examined beetle species was found to be high  based on both markers is above 0.9 in all taxa) Table . When anCox1 and ArgK) resulted in very complex patterns, with some haplotypes being common for more beetles, and this was more pronounced in the case of the Bembidion than the Paederus/Paederidus taxa . Generally differentiation of populations of row beetles occurred to be lower than ground beetles. Moreover, values of FST and GST indices were larger for Cox1 than for ArgK markers. The highest genetic differences occurred between geographically most distant populations.The heatmaps of That limited spatial structuring was further confirmed by AMOVA and the Mantel test. Molecular variance measured between four major regions of the Carpathians (Western vs. Eastern vs. Southern vs. Apuseni Mts.) revealed a relatively low proportion of variance, which could be attributed among these regions . For Bvar, IBD was also significant but negative indicating that distant populations are even slightly less distinct than adjacent populations . For other species, IBD was insignificant: Bmod , Bdec , Plim , Pruf , and Prub  .The Mantel test showed that IBD is significant but weak only for Bpun  probably started expansion earlier—during LGM. Results for Plim are inconsistent, as mismatch distribution suggest its earlier expansion, whereas the Bayesian skyline plot shows a relatively stable population size. Only for two taxa (Plim and Prub) are there signs of a very recent decline of population sizes, however, confidence intervals are rather wide; therefore, this finding needs to be taken with caution. It seems that the Carpathians were not an important refugium for RPB, contrary to some other species living in watercourses5. The existence of most  diverse populations of RPB in this part of the Carpathian range reflects that common phylogeographic pattern, although rather recent estimates of expansion of these beetles in the Carpathians prevent from drawing conclusions about the role of the southern ranges as their glacial refugia. To solve this issue, further studies including populations from the Balkans, the Dinaric Mts., and the Alps are needed, as it is likely that RPB settled the Carpathians from external refugia. Interesting is that some populations in the Eastern and southern slopes of the Western Carpathians possess also high genetic diversity. This could be explained by the settlement of these areas by diverse populations or by beetles from different sources (various migration routes and/or waves). This issue could not be investigated based on selected markers, and genomic information should be implemented.The southern mountain ranges of the Carpathians harbors, on average, populations having highest values of diversity indices. This is probably an effect of their origin or rather the direction of expansion in the Carpathians from the south to the north during the Holocene period. This is confirmed by the lowest genetic diversity from the outer part of the Western Carpathians, settled being probably at the end of beetle spread in the Holocene period. The Southern Carpathians are known as an important local glacial refugium for temperate taxa in Europe15 or riparian insects 22. For these other animals associated with mountain watercourses, many phylogenetic lineages were determined across the Carpathians, being mostly geographic-specific, which indicate spatial structuring. Mitochondrial haplotype networks obtained for Bembidion beetles, but not for rove beetles, are quite similar to those observed in Elmidae15, for which common haplotypes were found in many sites, indicating limited spatial structuring. RPB seemed to not be clearly structured genetically over the Carpathians. Only a portion of molecular variance could be attributed between regions or basins. Only for two out of the seven taxa, a significant correlation between geographic and genetic distances was detected, however always being very weak. This is caused by many haplotypes found in single localities and some haplotypes being found in several individuals from distantly localized populations . On the other hand, a mapping of more common haplotypes showed that some of them are specific to particular areas of the Carpathians, but they are rarely associated with river basins. In summary, these patterns generally reject the assumption that populations of RPB are genetically structured remaining isolated by barriers (watersheds on mountain ranges) and that beetles found in rivers belonging to the same basin are not (or are less) genetically isolated than beetles captured in different basins. It was suspected that RPB are poor dispersers due to their behavior during inundations—rove beetles escape on the ground , whereas ground beetles fly very short distances (authors’ observations). This assumption is likely wrong and these species are capable of long distance migration, possibly only during some phases of their lifecycle, e.g., emergence after wintering or during mating41. Unfortunately, knowledge on the biology of these species is very limited, with many details on the ecology and biology  remaining unknown42. High genetic diversity and complex phylogeographic patterns suggest that predacious riverine beetles exist in a system of meta-populations, with individuals moving between the occupied sites. It is most probable that some populations are vanishing during floods 41. This could be compared to the genetic diversity of Elmidae15. Subsequently, empty sites are colonized by ground and rove beetle individuals from other areas (rivers). This would explain the missing clear spatial structure of their populations, as gene flow had to be substantial, albeit partially limited by distance. These are just possible explanations that need to be verified. Most likely there are some differences in responses of ground and rove beetles to the changeable environment, which resulted in more complex haplotype networks observed for Paederus/Paederidus than for Bembidion. Likely different mechanisms of isolation are responsible for that, especially as simple isolation by distance is generally absent in RPB, and other isolation models  need to be verified43.To solve this issue examination of loci responsible for environmental adaptations is required44.Highly complex patterns of haplotype networks, both generated from both mtDNA and nuclear genes, are interesting. This is contrary to freshwater invertebrates with known phylogeography in the Carpathians 36. However, their utility as indicators was based solely on their narrow habitat requirements34. Moreover, these species are used as indicators at the level of community. Such a basic or applied use of these beetles is done without knowledge on the details of the history and current diversity of particular species in the sampled area, which could have important consequences for their distribution, density or dynamics. This study brings some novel findings, which could be valuable for the use of predacious riverine beetles as indicators. First of all, it seems that RPB have many commonalities in their recent (Holocene) spread over the Carpathians, which proves that the history of particular taxa should not be issue in their use in community studies for monitoring or conservation purposes, as apparently, all these beetles experienced similar changes in distribution and abundance. Consequently, their present occurrence has to be mostly shaped by contemporary environmental constraints, rather than past history. Moreover, the majority of their populations across the Carpathians possessed high genetic variability and only some areas seemed to be settled by less genetically diverse populations. This information could also be important for comparing biodiversity data from various areas, as it is possible that populations having lower genetic diversity are more prone to being affected by inundations or man-made alterations of rivers. The open question is whether lower genetic diversity is an effect of origin of populations (historic expansion?) or it is a result of some external factors acting recently like river regulation and damming. This problem could be also solved with use of genomic data in conjunction with landscape characteristics (landscape genetics)46.Ground and rove beetles, particularly taxa belonging to the examined genera, are known as excellent indicators of river channel quality26 as far as information about species inhabiting river channels but not living in running waters like crustaceans15 or fishes14. Phylogeographic and demographic data revealed many common features of examined beetles being members of two unrelated groups, but sharing environment and belonging to the same trophic guild. Therefore, Hypothesis 1 was confirmed as there is substantial congruence of phylogeographic patterns observed for RPB, indicating that they share a common history and that their ecological adaptations to the dynamic environment are similar.For the first time, a comprehensive elaboration of the phylogeography of RPB from a mountain range on the example of the Carpathians is presented. Molecular analyses for several species of RPB substantially broaden the knowledge on the biogeography of the CarpathiansHigher genetic diversity was reported mostly from the Southern Carpathians, however many sites in the Eastern and inner Western Carpathians also harbor populations of high variability. Populations of these beetles are not clearly geographically structured. However, some common haplotypes occurred to be widespread in particular regions or basins of the Carpathians, indicating some degree of isolation. This is confirmed by weak isolation by distance reported only for some taxa. It seems that the examined beetles are more capable of greater migration than expected based on their limited flight activity. Therefore, Hypothesis 2 was rejected as populations of RPB are genetically structured according to basins and being isolated by watersheds.The crucial discovery of this study is that species of RPB, regardless of their taxonomic affinity and phylogenetic relations, have many common features describing their phylogeography and demography.Bembidion Latreille, 1802 is the genus which the highest species richness within whole Carabidae family. To date, more than 1200 species have been described47. In Palaearctic region occurring more than 900 species and subspecies, while from Europe so far discovered approximately 400 taxa48. Most species live along banks of running or standing waters, but also on open areas37.Paederus Fabricius 1775 and Paederidus Mulsant & Rey, 1877 are members of the same tribe Paederini Fleming, 1821 . Approximately 650 species of these genera occur in the world, mostly in subtropical and tropical areas49. Only some genera are well represented in Europe, very few in northern parts of the continent and/or preferring mountainous habitats. About five species of the genus Paederidus are currently known from Europe, while Paederus in this area has about 13 representatives. Paederus species preferring wet and humid habitats. They live on the edge of reservoirs and rivers, on mudflats, among sparse vegetation, only a few species reside in dry and warm environments. Paederidus is also most commonly found on the banks of rivers and streams, with a distinct preference for mountain and foothill sites.Bembidion were selected for this study: B. decorum  (hereafter Bdec); B. varicolor  (hereafter Bvar); B. modestum  (hereafter Bmod); B. punctulatum  (hereafter Bpun).Four species of Paederus limnophilus  (hereafter Plim); Paederidus rubrothoracicus  (hereafter Prub); Paederidus ruficollis  (hereafter Pruf).Three species of rove beetles (Staphylinidae) were selected for this study: 40). During the selection of species for the study, we were also guided by the following criteria: (i) all species with mountain-type distribution, known to occur throughout the Carpathian mountain system; (ii) habitat requirements of the species limited to gravel or cobblestone alluvium in (sub)montane river valleys; (iii) numerous populations over the Carpathians; (iv) numerous and rich populations, allowing to catch at least 5–10 specimens from as many species as possible per site. During field sampling (see below), dozens of species of ground and rove beetles were caught and preserved for molecular use. However, most of these taxa were found in only single or several localities from particular parts of the Carpathians, usually as a single individual per site. These species were discarded due to insufficient sampling. Others of a relatively large number of individuals collected occurred to be lowland species with ranges spanning into the Carpathians, and were also discarded.All mentioned species from both families share the same ecological features. Adult and larval forms of all species live in the same environment and are predatory and simultaneously are not dependent on vegetation, which is highly variable and unstable in the environment of dynamic mountain rivers42. Sampling was done during the highest abundance of predacious beetles since end of May to the beginning of July.Beetles were sought and collected across the Carpathians between 2017 and 2021. Unfortunately, due to the Russian invasion of Ukraine, we had to abandon the plan for sampling beetles in the southern part of the Ukrainian Carpathians in 2022 year. That resulted in approx. 200 km gap in sampling design. Although samples already collected in Eastern Carpathians in Poland, Slovakia, Ukraine and Romania represent well this part of beetle ranges. Individuals were sampled from river alluvia using of exhauster. These beetles usually hide under cobblestones or inside gravel. To facilitate finding of individuals the water was flushed on the ground, what forced beetles to move – individuals run from the area being wet as the result of natural escape behaviorIn total we sampled beetles for molecular use on 159 sites  from 111  main rivers and their smaller but always named tributaries , 126 (Bdec), 117 (Bpun), 72 (Bmod), 118 (Prub), 105 (Pruf), 108 (Plim), from 19, 31, 28, 32, 29, 27 and 30 sites, respectively.Individuals were immediately preserved in 96% ethanol and further stored at -22ºC for molecular use. Before DNA extraction, each individual was determined to the species level.Cox1 was amplified using primer pairs: B1490-Bcoi2R37, LCO1490-HCO219850 or Paed-F2-Paed-R251. Nuclear protein-coding gene – Arginine Kinase (ArgK) was amplified using primers AK168F and AK939R52. The reagent concentrations used for the amplifications and PCR cycling profiles of both markers were as in Kolasa et al.51. After purification, the PCR fragments were sequenced using a BigDye Terminator v.3.1. Cycle Sequencing Kit (Applied Biosystems) and run on an ABI 3100 Automated Capillary DNA Sequencer. Sequences (haplotype only) are deposited in GenBank under accession numbers provided in Appendix 1.Whole beetle bodies were used for DNA extraction using a Nucleospin Tissue kit (Macherey–Nagel). https://blast.ncbi.nlm.nih.gov/)53. All sequences occurred to belong to the species assigned to the species based on their morphology (data not shown). Moreover, all newly generated sequences were aligned with selected sequences of other Bembidion or Paederus/Paederidus species, and maximum-likelihood trees were generated using IqTree server (http://iqtree.cibiv.univie.ac.at/)54. In all cases, sequences were grouped consistently according to the morphological identification of beetles  (data not shown).Prior to intraspecific analyses, all newly generated sequences were compared with available resources in GenBank using Basic Local Alignment Search Tool  or according to river basins , haplotype diversity (Hdiv), nucleotide diversity (πdiv), as well as inter-population indices like FST, GST and NST were computed using the program DnaSP v. 557, separately for both markers. The values of the latter indices were next shown in matrix heathmaps considering the geographic groups according to Table http://www.heatmapper.ca).Standard genetic indices for populations such as haplotype number  was conducted in ARLEQUIN v. 3.559 was performed in ARLEQUIN v. 3.558, using pairwise FST values (FST / (1-FST)) and straight-line geographic distances in kilometers (log(km)).An isolation by distance (IBD)60 were reconstructed separately for each marker in PopArt61, and the same was also done for the combined sequences. Distribution of haplotypes over the Carpathians (separately for each marker and species) was visualized using QGIS ver. 3.28.9 (http://qgis.org) and CorelDraw ver. 18 (http://coreldraw.com). All maps were created using QGIS based on elevation raster downloaded from the WorldClim database  and shapefile "Hydrography of Europe", downloaded from http://www.efrainmaps.es. Carlos Efraín Porto Tapiquén. Geografía, SIG y Cartografía Digital. Valencia, Spain, 2020. All source files used for visualization of data are freely available.Neighbor-joining (NJ) haplotype networksCox1 due to lacking reliable estimates of mutation rates for the ArgK gene in beetles.Demographic analyses were done solely on 63 D test and Fu64 Fs were used for verifying neutrality of examined sequences and to detect signs of past demographic changes in the examined populations.Two statistical tests Tajima65 was calculated in ARLEQUIN v. 3.5 only for Cox1 in order to examine the demographic history, and specifically, test for historical  expansions of populations of the species. The probable time of expansion (how long ago the expansion occurred) was estimated by the parameter τ. Due to lack of calibration events based on (sub)fossil materials for examined species, the mutation rate was estimated in range of 0.0158–0.0196 divergence lineages per million years according to Papadopoulou et al.66.A mismatch distribution (MD)67. To investigate the posterior probability distribution of effective population size (Ne), the best-fit model of nucleotide substitutions and their parameter values as priors were estimated without the assumption of any particular demographic model68. The Cox1 and ArgK nucleotide substitution rates were set to the average value commonly found in beetles65 with a strict molecular clock. Markov chain Monte Carlo sampling was run for 10 million generations with parameters sampled for every 5.0 × 104 generation. The initial 10% of the run was discarded as burn-in. We determined the effective sample size for the posterior distribution of estimated parameter values using Tracer v. 1.669.Additionally, a coalescent-based Bayesian skyline plot (BSP) was generated using BEAST v. 2.6.5Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


Lepus tolai Pallas, 1778), L. t. lehmanni inhabiting Northern and Northwest Xinjiang and L. t. centrasiaticus inhabiting Central and Eastern Xinjiang using SNP of specific-length amplified fragment sequencing (SLAF-seq) and four mitochondrial DNA (mtDNA). Our results showed a relatively high degree of genetic diversity for Tolai hares, and the diversity of L. t. lehmanni was slightly higher than that of L. t. centrasiaticus, likely due to the more favorable ecological environment, such as woodlands and plains. Phylogenetic analysis from SNP and mtDNA indicated a rough phylogeographical distribution pattern among Tolai hares. Strong differentiation was found between the two subspecies and the two geographical groups in L. t. centrasiaticus, possibly due to the geographical isolation of mountains, basins, and deserts. However, gene flow was also detected between the two subspecies, which might be attributed to the Tianshan Corridor and the strong migration ability of hares. Tolai hare population differentiation occurred at approximately 1.2377 MYA. Population history analysis based on SNP and mtDNA showed that the Tolai hare population has a complex history and L. t. lehmanni was less affected by the glacial event, possibly because its geographic location and terrain conditions weaken the drastic climate fluctuations. In conclusion, our results indicated that the joint effect of ecological environment, geographic events, and climatic factors might play important roles in the evolutionary process of L. t. lehmanni and L. t. centrasiaticus, thus resulting in differentiation, gene exchange, and different population history.The main topic of population genetics and evolutionary biology is the influence of the ecological environment, geographical isolation, and climatic factors on population structure and history. Here, we estimated the genetic diversity, genetic structure, and population history of two subspecies of Tolai hares ( L. europaeus) by isolating populations in the distinct refugia where they adapted and differentiated in allopatry, leading to genome incompatibilities (L. yarkandensis) during the Quaternary climate oscillations, providing a suitable environment for maintaining the relatively high genetic diversity of this species  has sandy yellow, brownish gray, or gray dorsal pelage with a dark ripple (L. capensis) or Brown hares, but studies based on molecular biology  is a high throughput, high-resolution, and low-cost marker development technology that has emerged recently . This teCOI, ND4, CYTB, and D-LOOP) to evaluate the genetic diversity of two subspecies of Tolai hare in different habitats and reveal the effects of ecological environment, geographical isolation, and climate change on population structure characteristics and population demography history. This study will help to understand the evolutionary history of this species and provide essential data to maintain the biodiversity and stability of the Xinjiang ecosystem.This study used SLAF-seq to identify genome-wide SNP markers and combined them with four mtDNA markers , three from Tarbagatay Prefecture, 13 from Bortala Mongol Autonomous Prefecture (including 12 from Jinghe and one from Wenquan), and six from Ili. Twenty-five L. t. centrasiaticus samples included 11 from Dabancheng, two from Tuokexun, and 12 from Kumul. The samples were divided into northern, northwest, central, and eastern groups, and the geographical details of the sampled populations are shown in Muscle or skin tissue samples were collected from 106 Tolai hares from 12 geographic populations in northern, northwestern, central, and eastern Xinjiang from 2008 to 2019 . The 81 Muscle samples were preserved in sterile tubes with anhydrous alcohol at -80°C until total genomic DNA extraction using a DNA tissue extraction kit. Genomic DNA integrity was determined using 1.0% agarose gel electrophoresis.Oryctolagus cuniculus) OryCun 2.0 genome  served as the reference genome for genome electronic enzyme digestion prediction. The selection principle for the enzyme digestion scheme was as follows: the proportion of restriction fragments located in the repetitive sequence was as low as possible, the restriction fragments were distributed as evenly as possible across the genome, the length of restriction fragments was highly consistent with the experimental study system, and the number of restriction fragments obtained matched the expected number of tags. A RsaI-EcoRV-HF® restriction enzyme was used to digest the genomic DNA. From the constructed SLAF library (Oryzasativa ssp. japonica genome (http://rapdb.dna.affrc.go.jp/) was selected as the positive control for sequencing, and SOAP v2 software  and GC content. All sample reads were mapped to the OryCun 2.0 genome sequence using BWA v0.7.5a-r405 . Then, wCOI, ND4, CYTB, and D-LOOP fragments of mitochondrial DNA were amplified. COI PCR primers were 5′-AGGAACAGCCCTYAGTCT-3′  and 5′-GGTGGGCTCAAACAATAA-3′  (ND4 were 5′-GCAAAGAATCATTACTACGCAAA-3′ (F) and 5′-TTGCGACGATTACTAAGGCTA-3′ (R) (CYTB were 5′-CGAACCCCACAAAACCAATTAC-3′ (F) and 5′-GGTGAGTTGATCTCCGTTTCTG-3′ (R), CYTB primers were designed by ourselves based on the published mitochondrial genome of Tolai hare. PCR primers for the D-LOOP were 5′-CAGAGATGGAGATYAACTC-3′ (F) and 5′-GCATGGGCTGATTAGTCAT-3′ (R)  . PCR priA-3′ (R) . PCR priT-3′ (R) . The PCRT-3′ (R) .π), observed heterozygosity (Ho), excepted heterozygosity (He), and the polymorphism information content (PIC) were calculated using Power-marker v3.25 . mtDNA-based phylogenetic analysis was conducted on the Cipres using the Bayesian Inference (BI) method by MrBayes 3.2.7a (O. cuniculus) genome was used as the outgroup (GenBank accession: GCF_000003625.3 for SNP and AJ001588.1 for mtDNA). ModelFinder was used to identify the best fit base-pair substitution model according to the Bayesian information criterion (BIC) using Phylosuite v1.2.2  method and Maximum-Likelihood (ML) method. The NJ phylogenetic tree was performed in Mega X  with 1,0s 3.2.7a  with fivs 3.2.7a  with 1,0e v1.2.2 , and thee v1.2.2 . The pope v1.2.2 . Treemixe v1.2.2  was usede v1.2.2 . We perfe v1.2.2 .Lepus , the divergence time of L. americanus (approximately 8.6 MYA), the divergence time of L. europaeus (approximately 1.84 MYA)  were found using modelFinder in Phylosuite v1.2.2 . SMC++ v1.15.5  , and Tol.84 MYA) . Best sue v1.2.2 , and uncr v1.7.2  was usedr v1.7.2  was used v1.15.5  was usedn ver3.5 . The misn ver3.5 . To estin ver3.5  with mtDr v1.7.2 .The rabbit genome served as the reference genome for predicting electron enzymes and identified a restriction fragment length of 314–344 bp, ultimately defined as a SLAF tag. Sequencing results for the positive control indicated that the efficiency of paired-end comparison was 96.84%, and the enzyme digestion efficiency of the control was 94.74%, indicating that the process was normal and reliable.High-throughput sequencing of the SLAF library yielded 226.85 Mb of high-quality clean data after strict filtration  ranged from 0.0444 (TC population) to 0.0823 (YL population) across the five geographic populations of Tolai hare in Xinjiang and averaged 0.05926 per population . In general, the genetic diversity indices of L. t. lehmanni  were slightly higher than those of L. t. centrasiaticus  (Nucleotide diversity (pulation . The aveectively . The nor 0.2906) .L. t. centrasiaticus, except for three individuals from the ALT population of L. t. lehmanni. The third branch consisted mainly of samples from the northern and northwestern groups, including samples from the ALT, TC, and YL populations of L. t. lehmanni, and one sample from the DBC population. The clustering relationships among populations were also evident in the PCA, in which L. t. centrasiaticus individuals were clustered, and L. t. lehmanni individuals were clustered together except for individuals from the TC population . Most samples in L. t. centrasiaticus, and three ALT individuals formed another ancestral cluster (shown in red in the image). In addition, fifteen samples have mixed lineages.To evaluate population structure, the ADMIXTURE was performed . Since tFST = 0.0765, p < 0.01). When individuals were pooled into the northern group and northwest group, only 6.57% (p < 0.01) of the variability was partitioned among groups, and differentiation among groups was 0.0657 (p < 0.01). When individuals were pooled into the L. t. lehmanni and L. t. centrasiaticus, only 6.57% (p < 0.01) of the variability was partitioned among populations, and differentiation among subspecies was 0.0657 (p < 0.01).Genetic differences among the geographical groups were examined using AMOVA , and theTo further infer population gene flow, we performed a Treemix analysis based on SNPs . Three mCOI, ND4, CYTB, and D-LOOP) with an alignment length of 2,885 bp in 106 Tolai hare individuals resulted in 99 haplotypes. Nucleotide diversity ranged from 0.0295 (eastern group) to 0.0449 (northwest group)  was slightly higher than that of L. t. centrasiaticus (0.0355)  . The π v(0.0355) . The totL. t. centrasiaticus individuals from the central and eastern groups. Each individual from the QH, ALT, and FH populations were included. The second cluster was comprised of L. t. lehmanni individuals from the northern and northwest groups, and two individuals from the DBC population. In addition to the evolutionary tree, a median-joining network was constructed to further elucidate the phylogenetic relationships among haplotypes , and differentiation was 0.2600 (p < 0.01)  than when individuals were pooled into the central and eastern group  , and FST was 0.2376 (p < 0.01).Genetic differentiation among the groups was next examined using AMOVA. The results showed that when pooling individuals into northern, northwest, central, and eastern groups, the genetic variation among groups was 26.00% ( < 0.01) . Genetic < 0.01) . In addiL. t. lehmanni and L. t. centrasiaticus occurred at approximately 1.1898 MYA  over time based on SNPs. The dynamics of historic Ne for L. t. lehmanni and L. t. centrasiaticus are shown in L. t. centrasiaticus decreased in the last glacial maximum (LGM), while L. t. lehmanni population remained relatively stable. Both subspecies expanded during the interglacial period after the last glacial period (LGP), especially L. t. lehmanni. About 0.001–0.0015 MYA, however, populations of both subspecies declined. In addition, the Tajima’s D, Fu’s Fs, mismatch distribution, and EBSP based on mtDNA were used to analyze the population history of Tolai hares. The neutrality test results showed that Tajima’s D and Fu’s Fs values for L. t. lehmanni were 2.06761 (p = 0.9800 > 0.05) and -7.73931 (p = 0.0480 < 0.05), and -0.00167 (p = 0.5720 > 0.05) and -3.95338 (p = 0.0310 < 0.05) for L. t. centrasiaticus. The mismatch distribution analysis revealed that L. t. lehmanni and L. t. centrasiaticus showed multi-peak curves  than that of other reported species, including rabbits  based on mtDNA was also relatively high compared to other Lepus taxa, such as brown hares . Most samples from the same or adjacent regions clustered together, and their clustering relationships were consistent with their geographic distribution, indicating a rough phylogeographic distribution pattern. This was also supported by the network analysis  (Shinisaurus crocodilurus)   , and Lizdilurus) , our com019 MYA) . Other s019 MYA) , implyinL. t. centrasiaticus.Tolai hare differentiated at 1.2377MYA, and the divergence between subspecies occurred between 1.1183 and 1.1898MYA, later than the formation time of the Tianshan Mountains and the Turpan-Hami Basin . TherefoHowever, the limitations of the SLAF-seq and the mtDNA fragments used in population history analysis should also be considered. In the future, whole genome resequencing and complete mitogenome sequencing will be performed to obtain more reliable results on the Tolai hare population history.L. t. lehmanni and L. t. centrasiaticus. Thus, it is due to the different geographical and geological conditions resulting in a relatively steady climate, making L. t. lehmanni stable and less affected by LGM compared to L. t. centrasiaticus.This paper used SNP and mitochondrial markers to explore the effects of geological formation and environmental and climate factors on the Tolai hare. We found that Tolai hares have a high genetic diversity due to their strong adaption and migration ability. In addition, geological events such as the formation of mountains, basins, and other geographical factors led to differentiation and gene flow between subspecies


Pseudopungtungia nigra Mori, 1935 is an endangered fish endemic to Korea. It lives in the narrow basin of the Geumgang River, Mangyeonggang River, and Ungcheoncheon Stream, which flow into the West Sea of Korea. One population of P. nigra in Ungcheoncheon Stream has been locally exterminated once; it is now inhabiting the upper reaches of the dam through a restoration program. Efforts to identify and understand the genetic structure of these populations are important for conservation planning. Here, we analyzed genetic diversity using 21 microsatellite markers for 9 populations. The mean number of alleles ranged from 4.4 to 8.1, mean allelic richness ranged from 4.6 to 7.8, mean observed heterozygosity ranged from 0.519 to 0.702, and mean expected heterozygosity ranged from 0.540 to 0.763. All groups had recent and historical bottlenecks . Three groups  had significant inbreeding index values, suggesting that they were engaged in inbreeding. We observed a moderate level of genetic differentiation between MG and the rest of the population . The genetic structure exhibited a fitting constant K = 2, along with separation between MG and the remaining populations. With respect to genetic flow, YD (2019), OC, CG, and ND shifted to the UC population (0.263 to 0.278). The genetic flow of each population was transferred only within the population; there was no gene flow among populations, except for the Ungcheoncheon Stream population. This study shows that the Ungcheoncheon Stream population needs conservation efforts to increase its genetic diversity, and the Geumgang River populations needs a conservation plan that considers the possibility of conservation and evolution through gene exchange among the populations.The black shinner  Because of human activities and the expansion of living areas toward rivers, numerous freshwater fish have become endangered via habitat fragmentation (e.g. by dams and weirs), pollution, and overfishing6. In particular, fish living in rivers are sensitive to the effects of physical, chemical, and biological factors on aquatic environments,rapid changes to environmental conditions increase the possibility of species extinction9. Therefore, restoration plans have been established to prevent the extinction of endangered species13.Biodiversity has exhibited greater decline in freshwater ecosystems than in terrestrial and marine ecosystems12. However, the need for genetic management has been continuously advocated for the efficient restoration of endangered species15. Accordingly, genetic technologies have been used and improved in conservation planning in recent decades, but the application of genetic management remains insufficient because of awareness and cost issues17.In the past, genetic management was not considered when designing restoration plans26.Microsatellites have specific sequence repeats, are widely distributed throughout the genome, and have high polymorphism; thus, they are widely used in conservation studies of endangered species to investigate genetic diversity, genetic structure, bottlenecks, and genetic flow among populationsPseudopungtungia nigra Mori, 1935 is an endangered species endemic to Korea, which belongs to the Cypriniformes order, Cyprinidae family, and Gobioninae subfamily. It is found only on the Korean Peninsula and is narrowly distributed in the water systems of the Geumgang River, Ungcheoncheon Stream, and Mangyeonggang River28. The black shinner is highly regarded as an ornamental fish because of its streamlined body and black spot pattern on the tail, it is at risk of overfishing because of human activities. Considering its narrow distribution and risk of overfishing, the black shinner has been designated as first-class endangered wildlife in Korea29. The fish mainly lives in rocky or stony places with good water quality,it breeds by brood parasitism in the spawning grounds of Coreoperca herzi and is thus found together with C. herzi27.The black shinner P. nigra habitat was identified prior to the construction of the Boryeong dam (1992), but it was not found anywhere upstream or downstream after the construction of the dam; thus, it is considered extinct in this region (Ministry of Environment30). In contrast, the construction of the Daecheong and Yongdam dams in the Geumgang River water system, where P. nigra is widely distributed, has led to habitat fragmentation; the construction of continuous weirs continues to cause increasing fragmentation. Accordingly, a release project for restoration was conducted to prevent the extinction of fish in the upper stream of Gapcheon, a tributary of the Geumgang River water system, and in the Ungcheoncheon Stream downstream of the Boryeong dam (Ministry of Environment30). However, the population was not restored because of failed habitat adaptation, except in a narrow portion of the Ungcheoncheon Stream in the upper reaches of the Boryeong dam. The Ungcheoncheon Stream population is presumed to have used the Geumgang River water system population for restoration, but this speculation cannot be confirmed because there is no official record. Reintroductions are important tools for the recovery of endangered species31. Effective reintroduction programs in endangered fish populations require extensive knowledge of their genetic structure32. Genetic diversity and bottlenecks in reintroduced populations must be carefully considered for species restoration34. Although restoration was successful, factors such as genetic diversity were not considered in the restoration program at the time. Generally, conservation programs for endangered species have many issues to consider related to genetic factors, such as inbreeding in declining populations36. In the absence of prior knowledge regarding genetic background, perturbations of the genetic structure of the restoration population may have adverse effects38. The identification of genetic diversity and genetic structure in the Ungcheoncheon Stream population restored by reintroduction can provide important insights for conservation planning. Thus, there is a need to clarify the origin during efforts to conserve the genetic diversity of the Ungcheoncheon Stream population.In the Ungcheoncheon Stream water system, a 40. Habitat fragmentation limits gene flow, thereby increasing genetic inbreeding41. Increased inbreeding causes inbreeding depression, and positive feedback from inbreeding depression increasingly accelerates population extinction44. Currently, the population of P. nigra is in a state of habitat fragmentation related to the construction of dams and weirs; it has already been exterminated once in the Ungcheoncheon Stream water system. Because the restored Ungcheoncheon population is isolated, it is likely to experience inbreeding depression in the absence of outside intervention. Genetic studies of some populations in the Geumgang and Mangyeonggang Rivers were conducted using amplified fragment length polymorphism markers to establish a restoration plan45. However, there is a lack of information regarding genetic diversity and structure of the overall population of P. nigra; such information is necessary for comprehensive conservation management plans. Moreover, efficient conservation planning requires genetic studies of restoration and isolated populations.Genetic diversity is the driving force that allows species to adapt to their environment and maintain their evolutionary potentialP. nigra.In the present study, genetic diversity was analyzed for the Geumgang River water system population, Ungcheoncheon Stream population (restored population), and Mangyeonggang River population; the Yudeungcheon Stream (2012) and Mangyeonggang River (2008) populations were also subjected to genetic monitoring analyses. Additionally, the genetic structure was evaluated to determine the distributions of genetic differentiation and variation at the species and population levels; the genetic flow was analyzed to provide information regarding the origin of the restored Ungcheoncheon population (UC). Overall genetic information regarding this species will provide the basis for conservation and management plans for endangered P. nigra Mori, 1935 is an endangered species in Korea and was collected with permission from the Geum River Basin Environmental Office  and Jeonbuk Regional Environmental Office of the Ministry of Environment . The sampled areas were collected as shown in Fig. P. nigra samples, we used pectoral fin tissue to obtain at least 30 DNA samples , expected heterozygosity (HE), and observed heterozygosity (HO) using CERVUS software (Ver. 3.0)48. Allelic richness 49 to correct for population-level differences. Analyses of population inbreeding coefficient (FIS and Hardy–Weinberg equilibrium (HWE deviations were performed using GENEPOP software (Ver. 4.2)50 and ARLEQUIN software (Ver. 3.5)51.Genetic diversity was measured by number of alleles 51. To determine the size of the effective population, LDNE software57 was used for linkage disequilibrium estimation.Two methods were used to estimate bottlenecks. The first method used BOTTLENECK software (Ver. 1.2.02)58 in the Genclass 2 software (Ver. 2.0)59. Genetic differentiation 51. For cluster analysis, principal coordinates analysis (PCoA) based on genetic distance was performed using GenAlEx software (Ver. 6.50)60.Genetic distance was analyzed using Nei’s minimum distance method61 using a Bayesian method of model-based clustering. The assignment test was performed via clustering between individuals, the no-admixture method, an appropriate model, was applied to unmixed water systems. To determine the most appropriate population, the population constant (K) was determined in the range of 1–10. Clustering was performed repeatedly with a burn-in length of 50,000; Markov chain Monte Carlo analysis was repeated with 100,000 iterations. To estimate the population constant (K), cluster results corresponding to the appropriate K values were analyzed with STRUCTURE HARVESTER62. Discriminant analysis of principal components (DAPC) was conducted to analyze gene clusters using the Adegenet package in R (Ver. 2.1.3)63, on a non-model basis without prior information about populations.Genetic structure analysis was performed with STRUCTURE software (Ver. 2.3)64. The Markov chain Monte Carlo method was used for 10,000,000 iterations, with a burn-in length of 1,000,000.Migration rates among populations were measured using BayesAss software (Ver. 3.0.4)65. An approximate Bayesian computational approach (ABC) was used to evaluate the posterior probabilities of past scenarios. Five populations from the Geumgang River water system were combined and divided into three groups, the Mangyeonggang River population and the UC population, to evaluate origin scenarios for reintroduction (UC) history .065. An All experimental protocols were approved by the designated Soonchunhyang University and the permission committee of the Ministry of Environment, Korea . We declare that all methods were performed in accordance with Soonchunhyang University and the Ministry of Environment guidelines and regulations. In addition, all experiments were performed in accordance with ARRIVE related guidelines and Soonchunhyang University regulations. Samples by population were collected according to the guidelines of Soonchunhyang University and the Ministry of Environment, Korea.66, 25 microsatellite loci were successfully amplified and used to establish at least four markers per multiplex PCR set. The multiplex PCR assays consisted of seven-loci (Set1) and six-loci  reaction systems. Information and product sizes for the four multiplex PCR sets are presented in Supplementary Table Among the developed microsatellite markersP. nigra is shown in Table AR) ranged from 4.6 to 7.8, mean observed heterozygosity (HO) ranged from 0.519 to 0.702, and mean expected heterozygosity (HE) ranged from 0.540 to 0.763; these results indicated moderate genetic diversity. Among the nine populations, the CG, OC, and ND populations had high genetic diversity compared with the other populations, whereas the MG (2019 and 2008) populations had low genetic diversity. The YD (2019) population had increased genetic diversity compared with the previously sampled YD (2012) population ; the MG (2019) population also had higher genetic diversity than the previously sampled MG (2008) population . Notably, the UC population, a population restored after extinction in the Ungcheoncheon Stream, had moderate genetic diversity (HO = 0.659).Genetic diversity information for nine populations of P < 0.05). The range of inbreeding coefficients was − 0.019 to 0.102 in the entire population, with high inbreeding signals for the OC (0.102), YD (0.083), and UC (0.096) populations (P < 0.001).Analysis using Fisher’s exact test revealed that all populations except the YD (2012) and MG (2008) populations deviated from HWE (P < 0.05). The MG (2019) and ND population bottlenecks were statistically significant (P < 0.05) in both the infinite allele model and stepwise mutation model. Mode-shift analysis revealed evidence of a bottleneck in the MG (2008) population.Recent bottleneck estimations showed that all populations experienced bottlenecks in the infinite allele model . Effective population size ranged from 52 (MG 2008) to 301 (OC) (Table Ne did not reach a size sufficient to maintain the minimum viable population (Ne < 1000).The M-ratio, representing historical bottlenecks, ranged from 0.440 to 0.482 in all populations; Garza and WilliamsonTable PCoA and DAPC plots showed that the nine populations were divided into two groups, similar to the results of genetic differentiation Fig. . The firK) for all populations was determined to be K = 2, according to the Bayesian clustering method in STRUCTURE , the Geumgang River population , UC population, and Mangyeonggang River population [MG (2019 and 2008)] were separated and AMOVA was performed. AMOVA of the entire group divided the nine populations into two groups; the within-group variation was 86.23%, and the between-group variation was 11.26%. A significant genetic difference was present between the two groups at FST = 0.060 (P < 0.001).Based on these overall results . The genetic variation between the Geumgang River and UC populations was lower than that between the MG and Geumgang River and UC populations, suggesting that the genetic variation between the Geumgang and UC populations was not large.When the Geumgang River and Ungcheoncheon Stream water systems were regarded as three groups, the within-group variation was 96.77%, and the between-group variation was 1.29% Table . There wGenetic flow was plotted using BayesAss software Fig. . The higThe genetic flow among populations, except for gene flow to the UC population, ranged from 0.003 to 0.031; these findings demonstrated that most gene flow occurred within each population, rather than among populations Fig. . OverallP. nigra . Similar to the present study, high genetic diversity was observed in the freshwater and endangered fishes Percocypris pingi, yellow catfish (Pelteobagrus fulvidraco), roughskin sculpin (Trachidermus fasciatus), and golden mahseer (Tor putitora)70. Genetic diversity allows species to adapt and survive in changing environments by counteracting the effects of genetic drift71. Species with high genetic diversity are likely to maintain their high genetic diversity, even if they are endangered, unless the population size declines through events such as habitat destruction68. Therefore, it is presumed that high genetic diversity is maintained at the species level due to the wide distribution and high abundance of Geumgang River populations compared to the other populations.Generally, endangered species tend to have low genetic diversity because of various factors such as genetic drift or inbreeding45 also supported these findings. Kim et al.45 found that MG populations have low genetic diversity and require genetic management. Because the MG population is geographically independent from the Geumgang River water system population and has a narrow geographical distribution compared with the Geumgang River water system population, it has a potential risk of genetic diversity loss. Therefore, for genetic management, the population should be augmented by breeding genetically identical MG populations.We found high genetic diversity at the species level, but low genetic diversity in the MG (2019 and 2008) populations. The amplified fragment length polymorphism results of Kim et al.P. nigra in the Ungcheoncheon Stream water system has been previously extinct and subsequently restored. There is no official record of the breeding population used to restore the Ungcheoncheon Stream water system, although the Geumgang River water system population was reportedly used. Therefore, the high genetic diversity of the UC population may be related to this aspect of restoration. A possible conservation strategy would be to augment populations with high genetic diversity. Although the UC population is genetically diverse because it lives in two small regions, the long-term effects of inbreeding cannot be ignored. Therefore, population augmentation is necessary to increase the genetic diversity of the population and reduce inbreeding.In contrast, the UC population did not exhibit low genetic diversity similar to the Mangyeonggang River water system, although it is also a geographically independent population. 72. In the present study, all populations of P. nigra had recently experienced bottlenecks; the estimated M-ratio was below the critical value (M = 0.68), confirming signs of historical bottlenecks in all populations. The populations of P. nigra may have been affected by anthropogenic activities, such as the construction of dams and weirs in their habitat, which may have caused recent bottlenecks. However, the analysis of bottlenecks can produce biased results due to differences in population size, gene flow, number of samples, and number of loci. Thus, a larger number of samples should be analyzed using a more reliable assay. However, the indications of bottlenecks in the present study suggest that conservation efforts are needed in current populations.The M-ratio and heterozygosity methods can detect genetic bottlenecks over long and short periods of time, respectivelyGenetic monitoring of the YD and MG populations revealed increased genetic diversity over time. These results may have been biased because of the small number of samples. The consequences of these biases can be minimized with more sampling strategies and should be carefully considered for future results. Because the monitoring of populations with low genetic diversity is important for species conservation, continuous genetic monitoring must be conducted to conserve and enhance the gene pool.73.Hardy–Weinberg equilibrium assumes that the number of frequencies remains constant from one generation to the next. This principle is based on five assumptions: no mutations occur, no migration occurs, the population is infinitely large, mating is random, and natural selection does not occur73. OC, YD, and UC have a significant FIS, indicating that random breeding is unlikely to have occurred. Therefore, deviations from the HWE may occur. Inbreeding increases the probability of lethal genes because of genetic homozygosity and genetic diversity loss, hindering population maintenance44. Because genetic diversity is high, short-term effects may not be significant, however, continuous inbreeding causes inbreeding depression, resulting in the loss of genetic diversity and hindering active adaptation to environmental changes14. MG (2018) found that FIS appears as a negative coefficient, indicating the possibility of outbreeding. This may have caused the deviation from the HWE. JJ ND, and CG could not find allelic stuttering, excessive heterozygosity, or homozygosity causing HWE deviation. A sufficient sample size is needed to prevent errors due to these HWE dropouts; therefore, it is necessary to obtain samples from different groups and ages at a single time point.Deviations from HWE can affect these assumptions and microsatellite alleles vary considerably in size, leading to scoring errors due to limitations of markers and sizing techniques72. In this study, all populations of P. nigra had recently experienced bottlenecks; the estimated M-ratio was also below the critical value (M = 0.68), confirming signs of historical bottlenecks in all populations. All populations of P. nigra may have been affected by anthropogenic activities such as the construction of dams and weirs in their habitat, which may have caused recent bottlenecks. However, the detection of bottlenecks can have biased results related to population size, gene flow, number of samples, and number of loci. Thus, there is a need to analyze a larger number of samples for a more reliable assay. However, indications of bottlenecks in this study suggest that conservation efforts are needed in current populations.The M-ratio can detect genetic bottlenecks over a longer period of time, whereas heterozygosity methods can detect bottlenecks over shorter intervalsNe) randomly fluctuates according to population survival and reproduction, such that smaller population size increases the likelihood of extinction over time (Harmon and Braude74). Populations with a small effective population size have a higher proportion of deaths attributable to the same individual, compared with populations that have a large population size. Thus, the loss of genetic diversity is greater in populations with small effective population sizes. Geographically isolated populations increase rates of inbreeding. Importantly, inbreeding causes inbreeding depression; to avoid this, an Ne value of > 100 (N < 5 generations) has been suggested75. In the present study, the Ne value was < 100 in the UC and CG groups; thus, efforts to avoid inbreeding depression are needed. In particular, because the UC population exhibits a significant inbreeding coefficient, an active conservation strategy is required. Although the UC population is highly genetically diverse, it inhabits two small regions in the water system; therefore, the long-term effects of inbreeding cannot be ignored. Therefore, population expansion is necessary to increase the genetic diversity of the population and reduce inbreeding.Effective population size , JJ, and CG] of the Geumgang River water system are connected and exhibit minimal genetic differentiation from each other. Notably, despite its geographical independence, the UC population exhibited minimal genetic difference from the Geumgang River water system populations. This observation was supported by the AMOVA findings of very low genetic variation between the Geumgang River and Ungcheoncheon Stream water system populations.The UC population has a history of reintroduction after extinction in an area. At the time of restoration, it was reintroduced using fish from the Geumgang River water system population, although this approach has not been confirmed by official records. ABC analysis strongly supported a scenario in which the UC population originated from the Geumgang River population.Additionally, we observed gene flow among the seven populations, which supported our hypothesis regarding the origin of the UC population (restoration population). The observed gene flow moved from the four Geumgang River water system populations , CG, and ND) to the UC population. Only one Geumgang River water system population, the JJ population, did not show evidence of migrating to the UC population, suggesting that the UC population was derived from the other four populations.78. However, no downstream unidirectional gene flow was observed in this study, although this may be related to sampling bias. In the present study, the JJ, ND, CG, and YD population habitats were tributaries of the main Geumgang River,upstream gene flow was blocked by dams and weirs. Despite this blockage of upstream flow, downstream movement remains possible. BayesAss software only detects migrations of recent generations; thus, the small number of samples may have hindered the detection of obvious gene shifts. Although each population within the Geumgang River water system could move freely before interruption by dams and weirs, the habitat fragmentation caused by their creation may accelerate population extinction14. Therefore, considering the results of inbreeding and effective population size among fragmented populations, conservation should be prioritized.With the exception of the UC population, gene flow existed only within each group; there was almost no gene flow among the UC, MG, and JJ populations. The five populations in the Geumgang River water system showed no evidence of genetic connectivity despite geographical connection. A possible explanation is that gene flow among these populations has been interrupted through the construction of dams and weirs, which presumably caused habitat fragmentation Fig. . An alteP. nigra lives in the Geumgang River, Mangyeonggang River, and Ungcheoncheon Stream water system in Korea; it practices brood parasitism in the spawning grounds of C. herzi. P. nigra cannot reproduce where C. herzi does not live79. Dams and dikes have been constructed in all habitats of this species, and it has already been exterminated once in the Ungcheoncheon Stream water system because of anthropogenic activities. Therefore, a reintroduction strategy was used to restore the Ungcheoncheon Stream water system population, resulting in successful restoration.70. Despite these efforts, the lack of genetic information regarding the UC population has prevented effective conservation planning. Additionally, the genetic structures of the Geumgang River and Mangyeonggang River water system populations were not identified, hindering the selection of populations for restoration and the identification of populations that require conservation. Lack of prior knowledge regarding these genetic structures is a potential threat that could hasten species extinction through delayed conservation management strategies67.Endangered species conservation is intended to increase effective population size by maintaining genetic diversity and improving gene flowP. nigra genetic diversity and population structure46. In the present study, the genetic structures of the nine populations were determined to provide basic information for use in the selection of priority conservation populations. The UC, OC, and YD populations have high genetic diversity, but the inbreeding issue should be addressed. Additionally, the CG, JJ, and ND populations have a disconnected genetic flow, indicating that conservation efforts require improvement. Because the MG population is a genetically differentiated population, strategies are needed to increase the size of the current effective population and preserve its habitat. Additionally, genetic markers such as single-nucleotide polymorphisms and larger sampling strategies can improve assessments of genetic structure and genetic diversity in newly discovered populations, facilitating the formulation of appropriate conservation plans. Ultimately, the management of inbreeding issues and maintenance of appropriate genetic diversity are essential for the continued conservation of P. nigra; our results provide the basic information needed for efficient conservation efforts. To restore the UC population, fish were artificially propagated using the Geumgang River water system population, which was the most representative population; however, source of the Geumgang River water system population is unknown. Therefore, microsatellite markers were developed to identify Supplementary Tables.