The genetic signature of modern Europeans is the cumulated result of millennia of discrete small-scale exchanges between multiple distinct population groups that performed a repeated cycle of movement, settlement, and interactions with each other. In this study we aimed to highlight one such minute genetic cycle in a sea of genetic interactions by reconstructing part of the genetic story of the migration, settlement, interaction, and legacy of what is today the Transylvanian Saxon. The analysis of the mitochondrial DNA control region of 13 medieval individuals from Feldioara necropolis  reveals a genetically heterogeneous group where all identified haplotypes are different. Most of the perceived maternal lineages are of Western Eurasian origin, except for the Central Asiatic haplogroup C seen in only one sample. Comparisons with historical and modern populations describe the contribution of the investigated Saxon settlers to the genetic history of this part of Europe. The present-day territory of Romania reunites four distinct historical regions , divided by the Carpathian Mountains Range that arches through the middle of the country. Until their unification into a single country in the 20th century, these provinces evolved rather independently, sometimes at odds with each other, for more than five centuries under different political and religious influences, being alternately challenged by distinct foreign powers . This taRepresenting, today, the central-western region of Romania, Transylvania was, in the Middle Ages, a province with a special identity within the Kingdom of Hungary, whose structure was gradually integrated during the 10th–13th centuries. This integration was carried out in stages and involved mainly two populations, known in historiography as the Szeklers and the Saxons. The first group, already existing in the kingdom since the 10th century, had a predominantly military role in defending the borders . The secThese colonization events imprinted the demographic history of populations from the current territory of Transylvania creating a cultural complex space, but the extent of these influences and other ancestral fine details remain largely unknown. Few studies have explored the genetic features of ethnic groups presently living in Romania in the hope of a better understanding of their biological and cultural identity, connections to other populations, and genetic legacy ,9,10. MoThe purpose of this study is to investigate the maternal  genetic diversity of medieval individuals discovered in Feldioara necropolis, potential members or descendants of a group that attained the farthest German colonization limit , and to The human archaeological remains analyzed in this study were discovered at Feldioara necropolis, also known as Marienburg (Brașov County). The cemetery was located in a square between the evangelical church and the parsonage. The excavations in this area were conducted between 1991 and 1995 , in 1998The routine funerary ritual in this necropolis involved the direct deposition of the adult bodies, without coffins, in graves with a step. This type of grave consists of a first larger rectangular hole continued by a smaller inferior pit outlining the shape and size of the human body, including a niche for the head. The funeral inventory is very poor, consisting of reused Roman coins, six Hungarian anonymous denars, and three lockrings with “S” -shaped ends . While tAll DNA extractions and amplifications of the selected archaeological samples were performed in a sterile environment in laboratories exclusively dedicated to ancient DNA research at the Interdisciplinary Research Institute on Bio-Nano-Sciences, “Babeș-Bolyai” University, Romania. All steps were conducted following precise guidelines and appropriate workflow protocols for aDNA processing previously described . Even thThe biological material used for DNA isolation was represented by dental samples with a very good preservation status for each tested historical individual. When available, multirooted teeth were selected. Prior to DNA extraction, each sample was decontaminated by mechanical removal of the external layer of the sample using a dental micro-drill . Then, the samples were UV-irradiated (254 nm) for 45 min. Routinely, 100 mg of powder was collected from the dentine of the tooth root and then used for aDNA isolation. A negative control  was processed for every batch of three samples during the extraction and DNA amplification steps to monitor potential contamination with exogenous DNA.The DNA extraction was performed in a designated pre-PCR area following a published silica-based protocol , but we The amplification of both hypervariable regions (HVR-I and HVR-II) of the human mitochondrial genome was performed using a set of eight mini-primer pairs, specially designed for highly degraded samples . BesidesThe mtDNA data generated for the individuals sampled in this study were compared to ancient and modern mtDNA sequences deposited in online databases to identify their genetic relatedness. This comparison aims to provide traces of evidence on past migratory routes and geographic origin of the analyzed population. The human mitochondrial sequences retrieved from publicly available data, detailed below, were first manually grouped based on their age , and then according to their geographic origin. The resulting datasets, one consisting of 21 historical populations (including the population considered in this study) and one, including 35 modern Eurasian populations, were used for haplogroup-based analysis, as well as for sequence-based analysis as detailed below. The population from Feldioara were compared to an ancient dataset consisting of 747 sequences of European populations and a Byzantine group : Lombardp-values) for each cluster using 10,000 bootstrap replications. The significance of each cluster in the resulting dendrogram was given as an approximately unbiased (AU) p-value shown at branches.Principal component analysis (PCA) was carried out based on mtDNA haplogroup frequencies of medieval and modern datasets. In total, 33 mitochondrial haplogroups were considered in the PCA of 21 ancient populations , whereasp-values of the historical populations were graphically represented as a level plot . The haplogroup affiliations were corroborated by diagnostic coding region sites which are all detailed in The maternal genetic composition of the analyzed group consisting of 13 samples indicates a high level of heterogeneity since it is comprised of 13 distinct haplotypes. The fact that these individuals do not share the same mtDNA profiles suggests that there are no strong maternal relationships among the analyzed group. The lack of identical maternal lineages might be a consequence of the small sample size, randomly selected from different parts of the necropolis, hardly any from adjacent burials . HoweverThe mitochondrial haplogroup architecture of the analyzed medieval group includes predominantly common west Eurasian clades , but not exclusively, as one of the sequences belonging to a middle-aged female (M26) was classified into haplogroup C, an east Eurasian lineage . The genThe sequence of the M26 sample showed a transition at 16357, characteristic of the C4a2 subclade. This variant is commonly found in the mtDNA pool of modern inhabitants of Siberia (particularly Evenks and Yakuts), while in Eastern Europe it is generally poorly represented ,54. ThisHaplogroup H, the prevailing lineage in the modern gene pool of Europeans, accounts for nearly 40% of the mtDNA variation in the investigated group, being represented by different clades . For most of them, further classification into derivative sub-lineages might have been possible with the aid of whole coding region data, but not for H7f, which is precisely identified due to the presence of the diagnostic mutation 16168T in the reconstructed sequence of the M42a sample. This particular haplotype seems rather scarce in historical samples as it has been revealed only by the mitochondrial signature of a female from Isola Sacra associated with the Imperial Rome . The HV9To gain more clues regarding the genetic affinities between the sampled individuals as a group and other historical and modern populations reported in literature, we conducted haplogroup-based and sequenced-based analyses. The PCA of 21 ancient populations was constructed based on mtDNA haplogroup frequencies , and visp values were visualized on a level plot  . The prep < 0.05) from two modern populations located in Central Asia (Kazakhstan and Kyrgyzstan), while the lowest genetic distances were observed relative to contemporary Slavic populations, mainly from Eastern Europe . The calculated Fst values between pairs of populations and their corresponding p-values, along with reference population information are given in The medieval Feldioara population showed significant differences  dominate among the sampled individuals, the exception being haplogroup C. Based on available published data, the C4a2 variant identified in one Feldioara sample, can be ultimately traced to the Eastern Asian Bronze Age. A possible scenario of the influx of this lineage into Feldioara might be related to the arrival of Hungarian conquerors in the Carpathian Basin at the end of the 9th century. The population genetic comparisons performed in this study potentially reveal some connections to other historical or modern Eurasian population, but these relations should be treated with care due to the low resolution of the emerging pattern shaped by the low samples size and the use of a partial mtDNA genomic marker. A much more complex and accurate picture of ancestry for the Transylvanian region might be provided by future genome-wide studies and increased number of samples.


Psammodromus algirus separated by a 600 m elevational gradient. These populations seem to be differentially adapted to their environments despite showing low levels of genetic differentiation (according to previously studies of mtDNA and microsatellite data). We performed a search for outliers  trying to identify specific loci with FST statistics significantly higher than those expected on the basis of overall, genome‐wide estimates of genetic divergence. We find that local phenotypic adaptation (in terms of a wide diversity of characters) was not accompanied by genome‐wide differentiation, even when we maximized the chances of unveiling such differentiation at particular loci with FST‐based outlier detection tests. Instead, our analyses confirmed the lack of genome‐wide differentiation on the basis of more than 70,000 SNPs, which is concordant with a scenario of local adaptation without isolation by environment. Our results add evidence to previous studies in which local adaptation does not lead to any kind of isolation , but maintains phenotypic divergence despite the lack of a differentiated genomic background.Usually, adaptive phenotypic differentiation is paralleled by genetic divergence between locally adapted populations. However, adaptation can also happen in a scenario of nonsignificant genetic divergence due to intense gene flow and/or recent differentiation. While this phenomenon is rarely published, findings on incipient ecologically driven divergence or isolation by adaptation are relatively common, which could confound our understanding about the frequency at which they actually occur in nature. Here, we explore genome‐wide traces of divergence between two populations of the lacertid lizard  Here, we show that local phenotypic adaptation  is not accompanied by genome‐wide differentiation, even when we maximized the chances of unveiling such differentiation at particular loci with FST‐based outlier detection tests. We believe in the importance of adding evidence to previous studies in which local adaptation does not lead to any kind of isolation, as ecologically‐driven divergences seem to be dominant in related bibliography. The analysis of mitochondrial DNA sequences has shown that these populations present very little genetic differentiation  is the most abundant lacertid lizard in the Mediterranean scrubland and forest habitats of the Iberian Peninsula (it is only absent from the northern Eurosiberian area). It is a medium‐sized species  in which variation in body size is an important indicator of fitness, as it determines survival, male attractiveness, and female fecundity  that can range from small sublabial marks in subdominant males to occupy practically the entire head in the largest males. This visual signaling is complemented by the existence of femoral pores as a means of chemical signaling. The number of femoral pores is higher in males and their activity increases as the breeding season advances in response to increased blood androgen levels , scrub patches where Cistus laurifolius predominates and, to a lesser extent, granite outcrops and grasslands. The average annual temperature is 6.2°C and the average annual rainfall is 1,170 mm. The lowland population is located at El Pardo , a forest of evergreen holm oaks (Quercus ilex) with shrubs of Cistus ladanifer. It is separated from the montane population by 32 km in a straight line, and has an average annual temperature of 12.5°C and an average annual rainfall of 438 mm. Psammodromus algirus is the most abundant lizard species in both locations, but reaches higher densities at higher altitude  and its habitat is composed by deciduous oak forests . Captures were made in the breeding season (April–May) of 2015, and the number of ticks carried by each individual was counted in the field. The lizards were transported to the lab, where all phenotypic traits  were measured, and photographs of the throat in ventral position were taken to quantify sexual coloration. For that purpose, we processed the photographs with Adobe Photoshop CS6 as explained by Llanos‐Garrido et al. . In order to analyze between populations differences, we used two‐way ANOVAs with sex and population as factors and with different morphological measures as the dependent variables. When necessary, we used ANCOVAs to control for the effect of body size. To assess the relationship between tick load and different explanatory variables , analyses were restricted to the Navacerrada population. To summarize variation in male size in this population, we ran a Principal Component Analysis (PCA) that produced a single factor explaining 84.2% of the variation in the data matrix and giving high factor loadings to all size variables  lacked sufficient DNA to be included in our analyses, leaving a final sample size of 39 genotyped lizards. We used the restriction enzyme Pst1 to prepare the libraries for genome‐wide genotyping with GBS (Genotyping by Sequencing). The sequencing was done in an Illumina HiSeq2500 sequencer, and once the sequences were obtained, we characterized single nucleotide polymorphisms (SNPs). For this purpose, we used the UNEAK pipeline, implemented in TASSEL v.5.0  and a locus‐specific effect (α). We selected loci with α > 0, suggesting positive selection, and a false discovery rate (corrected by multiple testing) of q < 0.05.We also applied another filtering step before doing the outlier analysis to minimize the false positive rate. We discarded loci whose minor allele frequencies were <0.05 in any population  and loci that could not be sequenced in at least 75% of the individuals in each population. The resulting database consisted of 6,421 SNPs. To identify SNPs putatively under selection, we performed an outlier analysis with Bayescan v.2.1.  conditions. This was done because the environmental differences described above between the two populations could lead not only to divergent selective pressures in some SNPs, but also to respond to a selective pressure present in one of the populations that is absent in the other .Once this was done, we tried to annotate SNPs with the highest values of 2.5To evaluate environmental variation between El Pardo and Navacerrada Figure , we comp33.1F1,18 = 10.85, p = .004) and that only the largest males could develop a sexual coloration , uncolored males tended to have more ticks than colored ones .We found significant differences between populations that confirmed the results of previous studies: lizards from the lowland population were smaller and had relatively longer legs than those from the montane population; in addition, they never presented tick infestation, despite adult ticks being present in their habitat Table . Sexual 3.2K = 1, cv‐error = 0.382), indicating no apparent genetic structure between both populations. Even when forcing the model toward higher values of K , we still failed to find any signs of between‐populations genetic structure  and did not differ significantly for PC2 .The analysis of genetic structure with admixture yielded a single more plausible model that grouped all samples into a single cluster . However, we explored the loci with largest values of α, the magnitude of the locus‐specific effect of selection, in case they were biologically meaningful. The largest increase in α was concentrated on four maximum  values, two of which could not be annotated at all , which corresponded to the coding region of a repeated transmembrane ankyrin involved in the cellular response to salicylic acid. The other sequence was annotated with a much higher and nonsignificant E‐value (0.6) as the coding region of a nuclear photoreceptor protein. None of these SNPs presented allelic frequencies that deviated significantly from those expected under H‐W conditions after applying the sequential Bonferroni correction , as well as relatively high levels of environmental variation along the cells that an individual would have to cross in a theoretical shortest route migration following valley bottoms  or montane .By using the scree plot criterion, the environmental PCA yielded a single axis  that retained four variables: annual mean temperature BIO1), max temperature of the warmest month (BIO5), mean temperature of the warmest quarter (BIO10), and annual precipitation (BIO12)  than the ones presented here  adapted populations, which remain undifferentiated on a genome‐wide scale, a phenomenon that could be more frequent than previously thought due to publication bias against this apparent lack of positive results ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); software (lead); writing – original draft (lead). Javier Pérez‐Tris: Funding acquisition (lead); project administration ; resources (lead); supervision ; writing – review and editing . José A. Díaz: Formal analysis (supporting); funding acquisition (lead); investigation ; project administration ; supervision ; visualization (lead); writing – review and editing .Supplementary MaterialClick here for additional data file.


Triplophysa yarkandensis) is limited to the Tarim River basin, which is the largest inland river in China. However, the population size of T. yarkandensis has been diminishing, and it is critically endangered in the Tarim River basin due to the gradual depletion of water resources, together with alien invasion and agricultural cultivation in Tarim River. In this study, we adopted the RAD-seq method to investigate the population genetics of T. yarkandensis, and a high degree of genetic variations and significant genetic differentiation was detected among T. yarkandensis populations in the Tarim River basin. The obtained data contribute to understanding the genetic status of T. yarkandensis, and help to provide the scientific management strategies and direct future monitoring and utilization of the genetic resource in Xinjiang region.The distribution of Kashgarian loach , and the pairwise FST values ranged from 0.4579 to 0.8736, indicating high levels of genetic differentiation among these populations. The discriminate analysis of principal components (DAPCs) and neighbor joining (NJ) tree revealed that the nine populations could be separated into two clusters , and modest genetic differentiation between south and north populations was observed, while the individuals from several populations were not clustered together by geographical location. The evidence of two genetic boundaries between south and north populations (except TTM) was supported by barrier analysis. The Bayesian skyline plotting indicated that T. yarkandensis populations in the Tarim River basin had not experienced genetic bottlenecks, and the effective population size remained stable. This study first clarified the genetic diversity and differentiation of T. yarkandensis populations in the Tarim River basin, and it provided valuable molecular data for conservation and management of natural populations.The distribution of  Triplophysa yarkandensis, belonging to Nemacheilidae, is a kind of small economic fish. It is mainly distributed in slow-moving and shallow water habitats. Specifically, the distribution of T. yarkandensis is limited to the Tarim River basin, which is the largest inland river in China. The Tarim River and its tributaries are typical seasonal rivers in an arid area, and it often does not produce runoff. In recent years, climate change and human activities resulted in the water curtailment and river desiccation of the Tarim River basin [T. yarkandensis has been diminishing and it is critically endangered [er basin . As a redangered . ChanginT. yarkandensis plays an important role in the fragile local ecosystem of the Tarim River basin, and previous studies have shown that the origin and evolution of the genus of Triplophysa may be associated with uplifts of the Tibetan Plateau [T. yarkandensis. However, information on population structure and historical population dynamics of T. yarkandensis populations in the Tarim River basin is still lacking. Therefore, evaluating the genetic diversity, population structure and population dynamics of T. yarkandensis is quite essential for the further utilization of genetic germplasm resources and wild population conservation. Plateau ,4,5. Pre Plateau  and repr Plateau , mitocho Plateau ,9 and tr Plateau  of T. yacytb, coi, d-loop), more than thousands of SNPs at genomic level could substantially improve the capacity to unravel genetic diversity and divergence in species with both strong and weak population structure [Misgurnus anguillicaudatus populations in the Yangtze River basin was detected with 2092 high-quality SNPs [Micropterus spp.) [Rhodeus ocellatus kurumeus [Creteuchiloglanis macropterus) [T. yarkandensis in the Tarim River basin. We aimed to (1) use SNP genotyping to investigate fine-scale genetic variations of T. yarkandensis populations; (2) evaluate the population differentiation of the populations in the Tarim River basin and (3) provide genomic evidence on the historical population dynamics of T. yarkandensis. These data are essential to providing scientific management strategies and direct future monitoring of the genetic resource of T. yarkandensis in Xinjiang region.Over the past years, restriction site-associated DNA sequencing (RAD-seq) has become a powerful and widely-used technique in conservation biology studies, especially for the species without a reference genome ,11,12. Utructure . For insity SNPs , but anoity SNPs . Recentlity SNPs , black bus spp.) , Rhodeuskurumeus  and Nuji® DNA Mini Kit  following manufacturer’s instructions. The RNA pollution was treated using Ribonuclease A . DNA quality was assessed by 1.2% agarose gel electrophoresis, and the DNA concentration was determined by the NanoDrop 2000 spectrophotometer  and Qubit 2.0 .Fish samples were taken from 14 locations in the Tarim River basin using nylon line fishing nets from May to October 2019. In total, 119 specimens were collected from nine sample sites in the Tarim River basin . The taiT. tibetana [RsaI and HinCII) was selected. Genomic DNA was incubated at 37 °C with RsaI, T4 DNA ligase, ATP, and RsaI adapter. Restriction-ligation reactions were heat-inactivated at 65 °C, and then digested for additional restriction enzyme HinCII at 37 °C. The PCR reaction was performed using diluted restriction-ligation samples, dNTP, Taq DNA polymerase and primer containing barcode. Subsequently, the PCR productions were purified and pooled. The pooled sample was incubated at 37 °C with RsaI, T4 DNA ligase, ATP and Solexa adapter. The digested fragment sequences with lengths of 364–464 bp were enriched for library construction following the protocol [The predicted electronic enzymatic digestion was performed using the genome sequences of a closely-related species, tibetana , as a reprotocol . The PCRT. yarkandensis is not available, a de novo orthology search was conducted. Briefly, the retained reads were demultiplexed according to the barcodes using the process_radtags program from the Stacks pipeline. The ustacks program was used to align the individuals into exactly matching stacks with the parameter of M = 4. A catalog of RAD loci was built with cstacks and all individuals in the population were matched against the catalog using the sstacks program. The populations program was used to filter loci . To address linkage disequilibrium (LD) between SNPs and to account for potential ascertainment bias, only one SNP per RAD locus was randomly selected with the populations parameter of “–write_single_snp”. The test of Hardy–Weinberg equilibrium (HWE) for each population was performed with populations program. To eliminate the bias of loci out of HWE, a Perl script “filter_hwe_by_pop.pl” implemented in dDocent program [The raw data generated from the Illumina sequencer were filtered with the fastp program . To conf program  was usedHO) and expected heterozygosity (HE), and nucleotide diversity. Pairwise F-statistics (FST) among the nine populations were determined with Arlequin v3.4.5 [K) was assessed by calculating an entropy criterion with cross-validation using the snmf function. Lower cross-entropy values indicate a better model fit. Meanwhile, population admixture was also evaluated with a Bayesian approach using fastStructure [K value. Meanwhile, we analyzed the genetic structure using the non-model-based multivariate approach DAPC  implemented in the “adegent” package [The obtained loci were used to evaluate population genetic diversity, including major allele frequency, observed heterozygosity (n v3.4.5 . Geneticn v3.4.5  in R. Thtructure . The Cho package . The optFST/(1−FST).We used plink v 1.90  to perfoCyprinus carpio, Danio rerio) in the NCBI NT database, which indicated that the obtained reads were reliable. An average of 10.90 million tags were obtained for each individual. After filtering the stacks with low coverage, 186,838 assembled loci were genotyped with an average coverage per individual of 17.10. According to the filtering criterion that loci presented in >75% individuals in the populations and MAF > 0.05, 129,873 candidate SNP markers were obtained for the subsequent genetic analysis  across all populations ranged from 0.2260 to 0.2604 within nine populations. HO was lowest in WSX (0.2260) and highest in JDT (0.2604). The nucleotide diversity (Pi) among nine populations ranged from 0.2364 to 0.2861, which was highest in KZE. The individual inbreeding coefficients (FIS) of the populations ranged from 0.0236 to 0.0779 (K = 1 ~ 9). The DAPC BIC showed the lowest value with K = 2 (K) was set from 1 to 9. The results revealed that the optimal number of clusters was two (K = 2) as identified by minimum cross-entrop value. In addition, the loci filtered by HWE test were used for the Bayesian analysis using fastStructure (K = 1 ~ 9). The result revealed that the optimal K value was 2 by running the ChooseK.py script, which is consistent with the number of clusters determined with “snmf” and DAPC methods. The average admixture proportions of two clusters for each population are shown in The genetic diversity and allele polymorphism of nine populations were generally consistent. Observed heterozygosity . The association between genetic and geographic distance was tested with Barrier software, with improved Monmonier’s method. The result showed that the genetic boundaries between the populations supported the two boundaries . Then, the population size underwent a steady period. After that, the population size experienced a modest increase 40 thousand years ago.Demographic history of  package . The optT. yarkandensis natural populations. Comprehensive population genetic studies of the T. yarkandensis populations in the Tarim River basin contribute to revealing the genetic diversity of natural populations and evaluating the status of germplasm resource. Actually, most areas of the Tarim River basin belong to a depopulated zone with high altitude. Meanwhile, due to the river diversions and cutoffs of the Tarim River basin, the sample collection was quite difficult for biological researchers. In this study, we spent several months collecting the wild samples of T. yarkandensis for the population genetic study in 2019. Unfortunately, no T. yarkandensis individual was caught in some sampling locations; thus, several populations in the upper reaches of the Tarim River basin were absent in the present study. More populations from other locations  will be collected for systematic evaluation of genetic resource in our future studies.The Tarim River basin is the main distribution area of T. yarkandensis, we selected de novo mapping pipeline with Stacks for SNP identification. For genetic analyses, we used the populations parameter of “–write_single_snp” to trim the outlier SNPs, which could impact the accurateness of genetic structure and genetic differentiation inference. A total of 125,893 high-quality SNPs were detected and used for population genetic analyses in this study. To further eliminate the genetic bias, we filtered the obtained loci using the HWE test, and used the filtered loci to carry out the structure analyses and PCA. Overall, it could be concluded that the nine populations of T. yarkandensis exhibited high genetic diversity, and atypical genetic differentiation was observed among these populations. Particularly, the genetic variations of these populations mainly derived from the among-population variations. We found that the pairwise FST values among these populations were very high, indicating that the genetic exchange among these populations of T. yarkandensis was very limited compared with other small bottom-dwelling fish species, such as M. anguillicaudatus [Given the lack of whole-genome sequences of caudatus . It coulT. yarkandensis were actually less interfered by agricultural activities. In this context, although anthropogenic factors could affect the genetic structure and differentiation of fish populations [T. yarkandensis populations should be monitored in the long term to avoid significant reductions in abundance due to the genetic characterizations, including the high levels of genetic variation originating from among-population variation and the small effective population size. Most importantly, these genetic characterizations together with the fragmented and isolated habits in the Tarim River basin increase the probability of outbreeding depression. The fine-scale genetic differences among populations should be regarded with caution in the conservation of T. yarkandensis. Multi-generational outbreeding depression can reduce fitness to a greater extent than reductions generated by inbreeding depression [According to the geographic locations, we assigned AKT, SWS, WC, JDT, WSX and TTM into south populations, which are distributed in the lower reaches of the Tarim River basin, and DWQ, TKX and KZE were assigned into north populations, which are located in the upper reaches of the Tarim River basin. Based on the results of population structure and the NJ tree, the nine populations were separated into two clusters, but the level of genetic differentiation between two clusters was moderate. Most individuals from south populations were clustered together, and the two clusters could not be completely separated based on the results of genetic admixture and phylogenetic analysis. Notably, most individuals from TTM showed a closer affinity to the north populations. TTM is located in Taitema Lake, which is the terminal lake of the main stream of the Tarim River and the beginning of the Qarqan River. Taitema Lake was dried up and had experienced progressive degradation without water flows over several decades until the ecological water conveyance project (EWCP) was initiated by transferring water from Bosten Lake to the terminal Taitema Lake . We speculations ,36, espeulations ,37, we npression . In thisT. yarkandensis is native to the Tarim River basin, and the wild capture of T. yarkandensis rarely occurred. However, biological invasion of other alien fish species in the Tarim River basin, such as Paramisgurnus dabryanus, Perca fluviatilis, and Ophiocephalus argus, severally impact the native fish biodiversity [Pseudorasbora parva, Carassius auratus, and Hypomesus olidus, which was different to the upper reaches. Therefore, scientific control of alien fish should be implemented to protect the native fish species in the Tarim River basin, especially in the lower reaches. Meanwhile, artificial breeding and releasing could be adopted as conservation and management approaches for T. yarkandensis. Importantly, with the releasing of artificially produced offspring into wild, the genetic consequences of these releases on natural populations should be evaluated to maintain genetic diversity and avoid degeneration of genetic resources in further studies. Overall, comprehensively investigating the genetic diversity and population structure contributed to understanding the genetic status of natural resources of T. yarkandensis in the Tarim River basin, and this study laid a foundation for the conservation and management of natural T. yarkandensis populations.In the past decades, traditional techniques and strategies have been established to monitor biological diversity of fish populations in the wild. With the advance of genome-wide molecular markers, study of fish population genetics is increasingly important and popular in ecological, evolutionary and conservation fields. As mentioned above, iversity . Actualliversity , which bT. yarkandensis were obtained with the Stacks pipeline for population genetic analyses. High levels of genetic diversity were detected among nine populations in the Tarim River basin. The majority of genetic variations originated from among-population variations, and a high levels of genetic differentiation among these populations was observed. The nine populations could be separated into two clusters , and modest genetic differentiation between south and north populations was observed, while the individuals from several populations were not clustered together by geographical location. T. yarkandensis populations in the Tarim River basin had not experienced genetic bottlenecks, and the effective population size remained stable. This study provides useful genetic information for conservation and management of T. yarkandensis populations in the Tarim River basin.In this study, a total of 129,873 genome-wide SNPs of


We investigated the evolutionary history of the striped field mouse to identify factors that initiated its past demographic changes and to shed light on the causes of its current genetic structure and trans-Eurasian distribution. We sequenced mitochondrial cyt b from 184 individuals, obtained from 35 sites in central Europe and eastern Mongolia. We compared genetic analyses with previously published historical distribution models and data on environmental and climatic changes. The past demographic changes displayed similar population trends in the case of recently expanded clades C1 and C3, with the glacial (MIS 3–4) expansion and postglacial bottleneck preceding the recent expansion initiated in the late Holocene and were related to environmental changes during the upper Pleistocene and Holocene. The past demographic trends of the eastern Asian clade C3 were correlated with changes in sea level and the formation of new land bridges formed by the exposed sea shelf during the glaciations. These data were supported by reconstructed historical distribution models. The results of our genetic analyses, supported by the reconstruction of the historical spatial distributions of the distinct clades, confirm that over time the local populations mixed as a consequence of environmental and climatic changes resulting from cyclical glaciation and the interglacial period during the Pleistocene. Spermophilus citellus, European hamster Cricetus cricetus, common vole Microtus arvalis, several mice species) from the Asian steppes to central Europe [Over the Pleistocene and Holocene, the global environment has undergone cyclical large-scale shifts that have affected ecosystems, species’ spatial structures and population trends ,2,3,4. Tl Europe ,6,7. Thel Europe ,9.Whether or not a species can adapt to environmental changes strongly affects its vulnerability, thus influencing its genetic structure and shaping its distribution and spatial diversity ,11. For A. sylvaticus [A. uralensis, very quickly becoming a dominant element of the small mammal community [Currently, we still observe progressive expansion of the striped field mouse (SFM) in Europe , the Siblvaticus ,20 and oommunity .Apodemus were used for assessing demographic and environmental factors that affected their populations during the Plio-Pleistocene [Mus enabled researchers to reconstruct the historical backdrop of shrinking forests and expanding grasslands [Additionally, small mammals constitute a suitable model group for reconstructing the history of ecosystems and biota affected by glacial and interglacial cycles ,22,23. Wistocene , and theasslands ,26. Apodemus agrarius), hereafter SFM, is one of the most widespread rodents with an Eurasian distribution extending from eastern Asia to central Europe [The striped field mouse  and Mongolia. The material included hair roots and tissue samples taken from museum specimens and root hair samples were collected from SFMs captured in Poland in wooden live capture traps between 2014 and 2015. The captured animals were marked and then released back into the wild. The sampled hair roots were stored in a freezer at −20 °C, and the tissue samples were stored in 96% ethanol until DNA extraction using a Sherlock AX DNA isolation kit . The procedure for catching and collecting the animals was approved by the Second Local Ethics Committee for Animals, Wrocław University of Environmental and Life Sciences .We analyzed the mitochondrial DNA variability of SFM using cytochrome b (cyt b) sequences from Eurasia, including newly sequenced specimens from Europe and Mongolia and other sequences from GenBank . Apodemus, in each PCR reaction [We used two universal primers L14724 and H15915 for genotyping cyt b in the genus reaction . Each PCreaction  and aligreaction , implemereaction . Rattus and R. norvegicus  as an outgroup. To select the best-fit model of nucleotide substitution, we used the Bayesian Information Criterion (BIC) in MEGA 7, with the one partition consisting of three codons (positions 1–3) of cyt b [We generated phylogenetic trees using Bayesian inference in BEAST ver. 1.8.4 , employiof cyt b . The TN9of cyt b . SeveralAVE , 1000 permutations were used. Correlations among geographical and genetic distances (IBD) were estimated with the Mantel test, using AIS. For all the regions  and clades designated by phylogenetic analysis, we calculated several parameters of genetic diversity using DnaSP ver. 5  and ArleWe used three different approaches to analyze the demographic history of the SFM based on cyt b sequences. The first uses the mismatch distribution (MMD) of pairwise differences ,42, anal2 statistic [Apodemus populations with divergences of 130 ka or older [We also tested the demographic history using three neutrality tests: Tajima’s D test , Fu’s Fstatistic , determitatistic . The statatistic . The evotatistic . To calctatistic . After ttatistic , and theor older ,50.The second method of demographic analysis we used was the Bayesian skyline plot method (BSP), implemented in BEAST software ver. 1.8.4 ,51. A poTo corroborate the results with the past expansions reconstructed by BSP analysis, we tested several distinct demographic scenarios that could have occurred during the last glaciation and postglacial periods using an approximate Bayesian computation (ABC) framework in DIYABC ver. 2.1.0 . The ABChttp://www.worldclim.org, date of access: 29 April 2019), with a resolution of 2.5′ [To estimate the historical distribution range of the SFM during the Last Glacial Maximum, we employed environmental niche modelling (ENM), using the maximum entropy method implemented in MaxEnt software ver. 3.4 . MaxEnt  of 2.5′ . To avoiThe estimated areas of the species’ distribution during the LGM can be interpreted, especially in the temperate climate zone, as the refugia that members of the species would most likely have used under the most unfavorable climatic conditions. All the analyses were performed to reconstruct the historical distributions of SFMs across the whole of Eurasia as well as the identified clades  with the confirmed strong signal of a recent expansion during the last glaciation modelled separately.This analysis resulted in 184 newly sequenced specimens: 153 from central Europe , representing 59 haplotypes, and 31 specimens from eastern Mongolia, representing 19 haplotypes. Additionally, 134 haplotypes (289 sequences) were obtained from GenBank . All of p < 0.05) and from the far east Russia (All of the SFM specimens had a mean haplotype diversity (Hd) of 0.98 and nucleotide diversity (π) of 0.013. The Asian populations had the highest nucleotide diversity (π = 0.0156) and the average number of nucleotide differences (k = 17.804), with particularly small values for China (π = 0.011 and k = 12.849) and Mongolia (π = 0.0115 and k = 13.097). The Asian SFM specimens from China and Mongolia exhibited a higher genetic diversity than those from western Europe . Clades C1 and C3 had higher IBD signals , as well as Eurasia; after excluding the island populations from Jeju Island and Taiwan , however, Europe did not exhibit the IBD pattern , though Asia did . The AAIA indicated significant allelic aggregation in the Eurasian population  and in clades C1  and C3 , suggesting a non-random distribution of genotypes. The GLSI revealed a cline of genetic diversity of SFM from eastern Asia to Europe, with the highest diversity in Asia, and decreasing diversity towards the east and north-east in clade C3 , i.e., positive relationships between pairwise genetic and geographical distances, for the entire region studied  showed that the differences between the clades accounted for 68% of the total variance , with thBoth the spatial and sudden demographic expansion models, as well as the neutrality tests, displayed the population growth of the SFM, as indicated by the strong signal of a recent SFM expansion in Eurasia, Asia and Europe . This siThe sudden and spatial models estimated similar expansion times of the SFM in Eurasia . The models also estimated similar distributions of pairwise differences . The Asi4) at 97,300  generations ago  and at 63,500  generations ago  for clade C1, with estimated population declines (t2) during and after the late Glacial at 20,500  generations ago (about 13–41 ka) and 13,400  generations ago , for clades C3 and C1, respectively  expansion initiated during the early Weichselian (MIS 5) or earlier, and the postglacial population decline . The besectively .The best scenario in the second model a, with rAn analysis confirmed the first expansion of SFM during the middle and late Weichselian (MIS 4–2) and postglacial population decline preceded the second expansion during the middle/late Holocene (~5–7 ka).2) 218,000 generations ago (~300 ka), preceding the split of ancestral  Korean population (t1) ~189,000  generations ago  (The reconstruction of SFM history in east Asia in the context of its colonization of the Korean peninsula (Model 3) included the specimens from China and Korean peninsula (clades C3 and C5) supporting scenario 4 with ancestral population represented by specimens from Korean remote islands and the Korean peninsula from the mainland China population diverged (t–508 ka)  distribu–508 ka) .The fitted model of SFM distribution, with two distinct clades (C1 and C3) highlighted during the LGM, indicated that these two populations had partially overlapped environmental niches . The recAnalysis of climatic factors (variables) used in the reconstruction of current and past ENMs indicate the very high impact of temperature-related variables on prediction in the case of clade C1 (with >60% contribution), compared to other, strictly Asiatic clades (C3–C5) for which the precipitation-related variables had an most important impact in model building (with >50% contribution) .The phylogenetic structure of Asian populations, with monophyletic clades representing the island populations from Taiwan and Jeju Island, supports prior analyses ,74,75. TThe genetic divergence between the European and Asian mainland populations was smaller than the divergence between the mainland and island populations of SFM, but equilibrium models of isolation by distance showed that genetic variance between sampling locations increased with their geographic distance. The westward significant decrease in genetic diversity observed in Europe supports the hypothesis of a relatively recent expansion into western Asia and Europe . The farCurrent knowledge of the demographic history of the SFM, especially in the context of its westward expansion into Europe through Siberia, is still insufficient. The demographic analyses of the whole sample demonstrated that the general SFM population showed a tendency to grow, as indicated by both the spatial and sudden demographic expansion models and confirmed by the neutrality tests. The indices of demographic history based on neutrality tests indicated signs of expansion for only two genetic clades, C1 and C3 . NeverthThe two methods used in this study to estimate expansion time, mismatch analysis (MMA) and Bayesian skyline plot (BSP) gave different results of estimated expansion time for the Eurasian specimens of the SFM . There aApodemus from previous publications, we chose the molecular clock calibration technique [The demographic history of SFM, reconstructed using the BSP method, showed an early, long period of stability followed by rapid population growth during the last glaciation . This seechnique ,50. The The south-eastern Asian clade C3, probably distributed along the Korean peninsula and adjacent areas, diversified relatively recently. The first Korean fossil records, identified solely in the upper Pleistocene and Holocene deposits, indicate a recent expansion to the Korean peninsula  Figure b. 4 plants to the dominance of grassy C3 plants [Artemisia [1, ~300 ka), and preceded the divergence of the highly supported strictly Korean (peninsulan) population during the MIS 7/MIS 8—glacial/interglacial transition  (The reconstructed past demographic trends (BSP) of clade C3, with population growth during the lower (MIS 5a–d) and middle Weichselian (MIS 3–4), showed a strong correlation of the SFM’s history with sea level changes after the last interglacial c. This s3 plants . During rtemisia , with bortemisia . The sceA. argenteus and A. speciosus colonized the Japanese islands no later than the middle Pleistocene, when the Tsushima land bridge was formed [A. peninsulae, colonized only the Hokkaido island during the upper Pleistocene glaciations, when the land bridge between Hokkaido and Sakhalin was formed. In both examples presented above, the colonization of the Japanese Archipelago can be placed before the SFM expansion into the Korean peninsula and far east Russia region. The presence of harvest mouse Micromys minutus in the Japanese Archipelago and more recent (~300 ka) colonization of southern islands  is still discussed and requires an explanation [Results of recent and glacial ENMs indicate the presence of habitats potentially suitable for SFM in south Japan . Neverths formed . The Korlanation . The moslanation , an unli−2/Ma) evolutionary rate estimates by the MMD. The recent westward expansion of SFM to central Europe has not been tracked in detail to date. A preliminary analysis based on the short genetic distance between specimens from Europe and the Russian far east permitted an approximation of the duration of the SFM’s westward expansion; according to Suzuki et al. , it was To understand why the last glaciation allowed the transcontinental dispersion of the SFM to the west, we can analyze the species’ requirements in the context of environmental and climatic changes throughout its history. If we consider the range of glaciers during the penultimate  glaciation (the most extensive one in Eurasia), we see that expansion during this period was unlikely . The glaThe SFM inhabits mostly open and semi-open habitats  across northern Eurasia ,103,104.The reconstructed vegetation of eastern Europe and Siberia during the middle and late Holocene showed that forest biomes at that time covered a noticeably larger area than they did during the glacial period ,111. TheResearchers still do not agree on when the SFM first appeared in Europe. Most studies, usually based on the dating of scarce fossilized remains, emphasize that a crucial factor in the species’ expansion into Europe was the post-Neolithic deforestation during the Holocene ,113,114.Our reconstruction of the historical distribution of the SFM has confirmed that potential refugia might have existed in south-western and southern Europe  . Most ofPoaceae and Artemisia as the most common species), scrublands (with Juniperus and Betula) and boreal woodlands (with Pinus and Betula) [This deforestation, a consequence of climate changes during the LGM, and the resulting rapid reduction of the species’ range, probably explains the decline in the population growth rate during MIS 2, reaching the early and middle Holocene. The observed bottleneck of the Eurasiatic population C1, falling on late Glacial/early Holocene (~12–38 ka), is similar to the estimated time of disjunction between European and east Asiatic populations, dated on the early Holocene (<12 ka) or pre-LGM period (~39 ka), depending on the used molecular clock calibration ,29. The  Betula) ,116. Dur Betula) .During the late stages of the Holocene (mostly the late Holocene), the natural environment in Europe started to change, an effect of increasing human activity, which significantly affected biotopes ,114,118.Mustela eversmanii [Spermophilus citellus [Cricetus [Sicista subtilis [Saiga tatarica [During glaciation, semi-open and open biomes dominated in northern Eurasia, thereby contributing to rapid expansions of other steppe species, which increased their western ranges during this period. Examples include ersmanii , Spermopcitellus , CricetuCricetus , Sicistasubtilis  and Saigtatarica , all of The methods implemented in this work can help to understand the reasons for recent population declines and to better reconstruct the history and demography of other widely distributed species, with still poorly recognized history, especially in the context of past climatic and environmental changes, that could shape their current genetic and population structure and initiated large-size expansion.The reconstruction of the SFM’s historical demographic trends show that its history, in terms of its population and overall Eurasian range, was closely related to environmental and climatic fluctuations during the upper Pleistocene and early Holocene. The analysis of the SFM demographic history showed two populations that recently expanded: one from south-eastern Asia and the far east of Russia to the Korean Peninsula, and the other that expanded across the northern hemisphere as far as Europe. In the case of the south-east Asian population, the reconstructed demographic trends were correlated with sea level changes and the formation of favorable biomes on new land bridges formed by the exposed sea shelf. The westward expansion of the northern population, from north-eastern Asia through Siberia to central Europe, began after the last interglacial period. Having occurred during a period of relatively stable and warm climate in the second phase of the last Glacial period, the population growth of the SFM could be associated with the favorable environmental and climatic conditions at that time. In both cases, our results support the role of climate-induced environmental changes during the upper Pleistocene and early Holocene, as an important factor shaping history of the species. These results can help to understand the factors that influenced Pleistocene expansion and Holocene population declines of some other mammals, such as the European hamster, European ground squirrel, Steppe polecat or Saiga antelope, which had wider ranges during the upper Pleistocene than they have now.The second important issue in this work is the recognition of the impact of man-made large-scale environmental changes during the Holocene on the species demographic history. In the case of the studied rodent, the Holocene expansion in Europe is considered to be the species’ second expansion after the population decrease during the Pleistocene–Holocene transition, affected by postglacial changes in northern environments. In the case of SFM, this recent expansion can be associated with the middle and late glacial human-induced changes in European biota. The research techniques used in this work, combining the analysis of genetic material with modelling of predictive past species distribution and a review of habitat changes over the upper Pleistocene and early Holocene constitute an effective method of reconstructing the history of SFM in relation to past climatic conditions, and identify the environmental factors that influenced past demographic trends and could have initiated large-scale or regional expansions, or bottleneck events, within site-specific populations.


Carex buxbaumii and C. hartmaniorum are sister species of the clade Papilliferae within the monophyletic section Racemosae. An unambiguous identification of these species is relatively difficult due to the interspecific continuum of some morphological characters as well as the intraspecific variability. The study was aimed at determining the range of variability, both morphological and genetic, within and between these two closely related and similar species.Carex populations was assessed in the presence of eight Inter Simple Sequence Repeat (ISSR) primers.The sedges were collected during botanical expeditions to Armenia, Estonia, the Netherlands, and Poland. The morphological separation of the two species and their populations was tested using the Discriminant Function Analysis (DFA). The genetic variability of the 19  C. hartmaniorum are, morphologically and genetically, more homogenous than the populations of C. buxbaumii. Compared to C. hartmaniorum, C. buxbaumii usually has wider leaf blades, a shorter inflorescence, a lower number of spikes which are shorter, but wider, and longer bracts and utricles. The AMOVA showed a larger variation between the populations of C. buxbaumii, representing 25.65% of the total variation in the taxon. Two populations of C. buxbaumii (from Poland and Estonia) are separated from the remaining populations, both genetically and morphologically; their individuals show shorter utricles and glumes, compared to the typical specimens of C. buxbaumii, and correspond with the morphology of putative infraspecific taxa described by Cajander (var. brevisquamosa and var. confusa).Results of the study indicate a considerable genetic affinity between the two sedge species (mean Si = 0.619). However, the populations of C. buxbaumii requires further studies throughout the distribution range of C. buxbaumii, addressing habitats, morphology and genetics (including a chromosome count or a combination of different genetic methods), particularly as the variability in C. buxbaumii may be associated with the species’ polyploid origin.The taxonomic status of the putative infraspecific taxa within  Carex buxbaumii Wahlenb. and C. hartmaniorum A.Cajander belong to the section Racemosae G.Don  which counts over 60 species worldwide, growing mainly in wetlands but also on dry plains in the Arctic and alpine tundra of North America and Eurasia, rarely in South America  as well as in North America and the northern part of South America  in 1935 . Thus, for more than 130 years, they were treated as a single species. Owing to the morphological similarity, Papilliferae T.V.Egorova within the section Racemosae. Recent morphological studies and molecular phylogenetic analyses have confirmed the affinity between C. buxbaumii, C. hartmaniorum and C. adelostoma V.I.Krecz., and indicated their close relationship with C. holostoma Drej. . Samples within a population were collected 2–20 m apart from each other to reduce the chance of collecting individuals from the same clone. The samples were collected from an area usually 2–8 ha in size. In the Netherlands, C. buxbaumii (population 10) individuals were highly scattered, for which reason the specimens were collected from a larger area (more than 10 ha).Sedges were collected during botanical expeditions to Armenia, Estonia, the Netherlands, and Poland ; Table 1The herbarium material was deposited at the Herbarium Stetinensis (SZUB) and the Herbarium of Estonian University of Life Sciences (TAA).Carex hartmanii does not comply with the International Code of Nomenclature for algae, fungi, and plants  and Carl Hartman (son). Thus, in accordance with article 60.8(b) and Note 4 of the International Code of Nomenclature , from dried leaves of three plants from each population. The magnetic bead-based DNA extraction method was applied and the monocots protocol was followed . The conCarex taxa (The ISSR (Inter Simple Sequence Repeat) method is based on inter-microsatellite sequence polymorphism and has been frequently used to assess genetic variability of different plant species, including closely related rex taxa .The ISSR analysis was run according to a modified method of C. buxbaumii and C. hartmaniorum were significant. The Kruskal-Wallis test and Dunn’s test of multiple comparisons were used to pinpoint those populations differing significantly from others. Morphological separation of the specimens was tested using Discriminant Function Analysis (DFA). The data used in DFA were standardized so that each variable would have a mean of 0 and a standard deviation of 1. Calculations were performed using the software package Statistica v. 13.1 for Windows , the non-parametric Mann–Whitney U test was used to examine whether morphological differences between  Windows .pi log2pi, while the gene population diversity index is based on the formula h =1 −∑pi2, where pi is the frequency of the i th allele. The value of the Polymorphism Information Content (PIC) for a dominant marker system was calculated as PIC = 1 - p2 - q2, where p is band frequency and q is no-band frequency  run with the aid of the GenAIEx 6.5 software  and the Principal Component Analysis (PCA). As the PCA is relatively objective and provides a reasonable indication of relationships, it was used to confirm the similarity of the grouping obtained with the UPGMA dendrogram. These two analyses were carried out using the Statistica v. 13.1 for Windows software package  and per cent contribution (%P) of polymorphic loci were computed, as were two indices of genotypic diversity: the Shannon index (I) and the gene population diversity h; . The Sharequency . The valrequency . The intsoftware . A genetsoftware . The fol package .U test, C. buxbaumii and C. hartmaniorum differ significantly in terms of nine characters analysed , bract length (BL), and utricle length (UL), whereas the second function is determined mostly by the culm height (CH) and utricle length (UL). The first two discriminant functions collectively explain 84% of the variance   .C. buxbaumii populations which were significantly different from others studied  and bract length (BL) separates most of the specimens of population 16 from populations 14 and 18 .A total of 128 DNA amplification products were obtained in the presence of 8 ISSR primers; the products showed a wide size range, from 2500 bp (in the presence of primer 840) to 180 bp (primer 813), 87.5% of the products being polymorphic . The mic8, anchored at the 3′  ending. Their efficiency in the detection of polymorphism among the sedges studied was confirmed by PIC values, which were higher than those obtained for other primers. The average PIC for all the sedge populations studied was not overly high (0.236). The least-informative ISSR starters were 888, M21 and 813, the PIC of which did not exceed 0.2.The Amplification Effectiveness Index (AEI), which informs on the average number of polymorphic amplification products obtained in the presence of a single primer, was about 14 . The higC. buxbaumii populations  and the lowest level of genetic variability were typical for the Polish populations, opposed to the populations sampled in the Netherlands, which showed a relatively high variability. Particularly low values were shown by population 3  and 14 . On the other hand, a considerable genetic variability, as determined from allele frequencies, was found in genotypes of C. buxbaumii, particularly in the Dutch population 10 (h =0.603) and the Polish population 11 (h =0.596). Within C. hartmaniorum, a relatively large h was shown by the Estonian population 5 (h =0.501) and the Armenian population 9 (h =0.489). The sedge populations showed similar values of the Shannon Index (I), which varied within a narrow range from 0.285 (population 10) to 0.366 (population 19). Overall, the genetic variability of both species as expressed by I, was low (0.338) (A higher number of polymorphic loci about 64%) was identified in the ulations . At the  (0.338)  and 6.4% was idC. buxbaumii populations: Dutch population 10 and Estonian population 19) to 0.885 (for two C. hartmaniorum populations: Polish population 4 and Estonian population 8) (C. hartmaniorum genotypes. The lowest Si within the taxon (0.656) was found for two Estonian populations (5 and 7). The Dutch population 10 and the Polish population 15 proved the most different from the other ones, with the average Si of 0.619 for the two species.The genetic similarity index (Si) calculated for the two species was shown to vary within a relatively wide range, from 0.467 (for two ation 8) . A higheC. buxbaumii; the between-populations and within-population variability accounted for 25.65 and 74.35% of the differences, respectively.As shown by AMOVA, the species studied showed small, albeit statistically significant, genetic differences (13.21%), explained in 17.86 and 68.93% by the variability between all the populations and between their specimens, respectively . A higheC. hartmaniorum only. Among them, the closest genetic affinity is seen between three pairs of populations: the first pair consists of populations 4 (Poland) and 8 (Estonia), the second contains populations 1 (Netherlands) and 9 (Armenia), the third being formed by populations 2 and 3 (both from Poland). The second sub-cluster (b) contains seven populations of C. buxbaumii, including four from Poland  and three from Estonia . In both Carex species, the Estonian and Polish genotypes showed a high genetic similarity. The second major cluster (II) groups three populations genetically divergent from the remaining sedges studied: the Dutch population 10 as well as the Polish population 15 and the Estonian population 16, the last two forming a pair in the dendrogram.The genetic similarity measure metric developed was used, as a matrix, to construct a relevant UPGMA dendrogram . Based oC. hartmaniorum specimens grouped together, relatively close to the origin. This is indicative of a low genetic diversity between the C. hartmaniorum populations, as opposed to the populations of C. buxbaumii, which were divided into four groups. Only one group, formed by the Polish population 14 and the Estonian population 18, was located on the same side of the first  axis together with C. hartmaniorum genotypes. The remaining three groups were located on the opposite side of the horizontal axis which explained more variance between the objects (37.8%) compared to the second axis (15.2% of the variance explained). The Dutch population 10 and the Polish population 15 were placed in the upper part of the vertical axis, in a considerable distance from the Estonian populations 17 and 19  or Next Generation Sequencing (NGS)  .C. hartmaniorum observed in our study could be associated with environmental effects, and, to a lesser extent, with genetic factors. Phenotypic plasticity in plants is considered to be one of the ways plants respond morphologically and/or physiologically to environmental changes , the most morphologically and genetically divergent among the C. hartmaniorum populations studied, grows in a secondary dune valley slightly overblown with sand, with a strongly fluctuating water level . Lower temperatures in the mountains limit cell divisions and result in a smaller size of the plant 8, effectively differentiated between the populations studied, as shown by the two multivariate statistical methods used (UPGMA and PCA).The populations of C. buxbaumii populations was generally similar  to the grouping produced by the morphological data. The agreement between grouping with the DFA as well as the PCA and UPGMA was found between the Polish (11 and 13) and Estonian (17 and 19) populations. Their morphological characters were very similar, as were their genetic traits. Moreover, the DFA and PCA showed two populations (14 from Poland and 18 from Estonia) to distinctly differ from the remaining specimens. These populations showed the largest phenotypic and genetic differences with respect to the Dutch population (10). The relatively high genetic variability in the latter, compared to a fairly low genetic variability observed in the Polish and Estonian populations, could have resulted from a sampling effect .The genetic distance-based grouping of the brevisquamosa and confusa, with the utricle and glume size as diagnostic characters. According to brevisquamosa and confusa are usually smaller (3–3.5 mm) than those in the typical variety . The apex of the glume is usually acuminate or aristate with an awn; in the typical variety, the awn is by brevisquamosa and var. confusa it is by 1/5 to brevisquamosa) or slightly longer (var. confusa) than the utricle brevisquamosa and var. confusa as well as intermediate ones, but they all were distinctly different, morphologically and genetically, from the typical form. The morphological distinctness of the specimens from those populations was already visible during the fieldwork, especially in the case where they were growing close to one another. That was the case with two Polish populations: 13  and 14 (specimens similar to var. brevisquamosa and var. confusa). Both populations were growing in a calcareous meadow, about 50 m from each other, which may suggest similar habitat conditions at the sites of occurrence. On the other hand, effects of (micro)habitat conditions on morphology of those specimens cannot be ruled out. In nature, intraspecific differences may be underlain by numerous mechanisms, including local adaptations, phenotypic plasticity, parental conditions, and selection  corresponds to the description of varieties election . As showphotypes . In addiphotypes . Generalphotypes . Accorditegy see  and deveCarex buxbaumii has a wide distribution range, for which reason the distinctness of those populations may disappear when samples are collected from other geographic areas and populations from the entire range are compared.Studies on plant genetic variability revealed a connection between the genetic variation within a species and its geographic range. Those species with a restricted geographic distribution usually show a lower level of genetic variability, compared to widely distributed species, due to effects of either directional selection promoting adaptation to local environments or random processes such as inbreeding, genetic bottlenecks, or drift acting in small populations e.g., . TherefoRacemosae as a product of polyploidy, and suggest a polyploid origin of C. buxbaumii. Clandestinae and Racemosae; in the latter, the genome size and the chromosome counts in C. buxbaumii and C. adelostoma were almost twice those in C. aterrima, C. parviflora, C. atrata, C. norvegica and C. hartmaniorum. Moreover, the species were referred to as having different cytotypes; mainly 2n = 100, 102, 106 for C. buxbaumii, and 2n = 52, 68 for C. hartmaniorum   . In automosomes) .C. buxbaumii populations than in the C. hartmaniorum ones we studied. The evolutionary success of polyploids is considered to be associated with new phenotypes which exceed the range of their diploid progenitors . Hybridization, i.e., the emergence of a taxon resulting from cross-breeding of different species, and introgression, i.e., the movement of genes from one species into the gene pool of another by back-crossing an interspecific hybrid with one of its parents, are common in Carex . These aC. hartmaniorum belongs  or the latter itself have been involved in the formation of C. buxbaumii. Results of our study point to a considerable genetic affinity between the two sedge species, as evidenced by the mean Si of 0.619. In addition, some populations of C. buxbaumii were genetically more similar to C. hartmaniorum than to conspecific populations. The UPGMA dendrogram constructed based on the genetic similarity metric grouped most C. buxbaumii populations together, in a joint major grouping, with C. hartmaniorum. Although the two sedges were assigned to two separate sub-groups (a and b), this may be taken as a joint origin of some alleles. Unfortunately, our study, by focussing primarily on the assessment of morphological and genetic variability of the two sedge species, does not lend itself to resolving this question, and the consideration presented above are exclusively hypothetical. Resolving the question would call for continuation and a detailed phylogenetic revision based on, inter alia, high-throughput sequencing technologies and analyses of phylogenetic trees. The challenges associated with such approaches involve, among others, finding genetic markers with enough phylogenetic signal and discordance among gene trees inferred from individual markers, which may differ significantly from the species or population tree. Such discordance may originate from hybridization among fully differentiated species with subsequent fixation of loci or incomplete random sorting of alleles due to short intervals between divergence events (ILS) (It is probable that the lineage to which ts (ILS) .C. buxbaumii studied were, morphologically and genetically, more heterogeneous than the populations of C. hartmaniorum. Two populations of C. buxbaumii (from Poland and Estonia) were separated from the remaining populations, as their individuals showed shorter utricles and glumes, compared to the typical specimens of C. buxbaumii, and corresponded with the morphology of putative infraspecific entities described by C. buxbaumii is wide, therefore the differences between these populations may become blurred when sampling is extended to other geographic areas and when populations from throughout the range are examined.The populations of C. buxbaumii requires further studies throughout the distribution range of C. buxbaumii, including a chromosome count and genome size, particularly as the variability in C. buxbaumii may be a result of the species’ polyploid origin.The taxonomic status of the putative infraspecific taxa within C. hartmaniorum and C. buxbaumii, but ought to be used in a more general discussion on polyploidy and its importance in the evolution of plants.The results obtained in this study motivate us to continue the study and to take a more comprehensive approach to the variability observed in the sedges investigated. Habitat-oriented, morphological and genetic analyses, the latter including the chromosome number and combinations of various genetic techniques, should not only expand the knowledge on the variability within 10.7717/peerj.11372/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11372/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11372/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11372/supp-4Supplemental Information 4Me, median; range, minimum and maximum values; IQR, interquartile range; V, coefficient of variation.Click here for additional data file.10.7717/peerj.11372/supp-5Supplemental Information 5p ≤ 0.05) have been marked with bold; CH, Culm height; LW, Leaf width; BL, Bract length; IL, Inflorescence length; NS, Number of female spikes; USL, Uppermost spike length; USW, Uppermost spike width; LSL, Lowest spike length; LSW, Lowest spike width; UL, Utricle length; UBL, Utricle beak length; GL, Glume length; 10, 11, 13, 14, …, number of C. buxbaumii populations  have been marked with bold; CH, Culm height; LW, Leaf width; BL - Bract length; IL, Inflorescence length; NS, Number of female spikes; USL, Uppermost spike length; USW, Uppermost spike width; LSL,- Lowest spike length; LSW, Lowest spike width; UL, Utricle length; UBL, Utricle beak length; GL, Glume length; 1, 2, 4, 5, …, number of C. hartmaniorum populations (see p, significance level; significant differences ( ions see .Click here for additional data file.10.7717/peerj.11372/supp-7Supplemental Information 7Large boxes indicate 25–75% of the interquartile ranges of values, small black boxes—the medians, circle—outlier values, asterisks—extreme values. The populations are numbered according to Click here for additional data file.10.7717/peerj.11372/supp-8Supplemental Information 8Large boxes indicate 25–75% of the interquartile ranges of values, small black boxes—the medians, circle—outlier values, asterisks—extreme values. The populations are numbered according to Click here for additional data file.10.7717/peerj.11372/supp-9Supplemental Information 9Large boxes indicate 25–75% of the interquartile ranges of values, small black boxes—the medians, circle—outlier values, asterisks—extreme values. The populations are numbered according to Click here for additional data file.


FST). This visualizes the structure and the ages of populations and helps infer the history of range expansion, encountered environmental changes, and selection on multiple traits. Next, we further investigate the inferred history using an admixture graph that describes the population split and admixture. Finally, principal component analysis of the selection on edge‐by‐trait (SET) matrix identifies multitrait coadaptation and the associated edges of the admixture graph. We introduce a newly defined factor loadings of environmental variables in order to identify the environmental factors that caused the coadaptation. A numerical simulation of one‐dimensional stepping‐stone population expansion showed that the exploratory analysis reconstructed the pattern of the environmental selection that was missed by analysis of individual traits. Analysis of a public dataset of natural populations of black cottonwood in northwestern America identified the first principal component (PC) coadaptation of photosynthesis‐ vs growth‐related traits responding to the geographical clines of temperature and day length. The second PC coadaptation of volume‐related traits suggested that soil condition was a limiting factor for aboveground environmental selection.During the process of range expansion, populations encounter a variety of environments. They respond to the local environments by modifying their mutually interacting traits. Common approaches of landscape analysis include first focusing on the genes that undergo diversifying selection or directional selection in response to environmental variation. To understand the whole history of populations, it is ideal to capture the history of their range expansion with reference to the series of surrounding environments and to infer the multitrait coadaptation. To this end, we propose a complementary approach; it is an exploratory analysis using up‐to‐date methods that integrate population genetic features and features of selection on multiple traits. First, we conduct correspondence analysis of site frequency spectra, traits, and environments with auxiliary information of population‐specific fixation index ( During the process of range expansion, populations encounter a variety of environments and respond to the local environments by modifying their mutually interacting traits. To capture the history of their range expansion with reference to the series of surrounding environments and to infer the multitrait coadaptation, we propose a complementary approach; it is an exploratory analysis using up‐to‐date methods that integrates population genetic features and features of selection on multiple traits. Another approach is to infer the positive selection on a trait during its evolutionary history by estimating the change in allele frequencies of associated SNPs beyond the level of genetic drift. So‐called “genome scan methods” consider geographically structured populations and detect SNPs related to environmental variables, traits, and phenotypes  on a selection on edge‐by‐trait (SET) matrix. The biplot identifies the principal multitrait coadaptation, and the relevant edges of the admixture graph describe the history of population splits and admixture. To identify the environmental factors that caused the coadaptation, we newly define the factor loadings of the environmental variables based on the factor loadings of the traits and the current among‐population correlations, which are overlaid on the biplot. Once we gain insight into the pattern of multitrait coadaptation and their driving force of environmental factors, we in turn confirm the SNPs controlling the traits to examine the genetic changes that enabled the adaptations. The R codes for our representation method and simulations of population colonization used in this study are available in the Supporting Information. This approach also accepts SNP genotype data and reads Genepop format , which are unique to some populations. By adding a crude measure of their ages as below, the plot provides a crude sketch on the history of range expansion of the populations, the environmental change they experienced and the associated traits adaptation. For SNP allele frequencies, we used the frequencies of derived alleles to infer signatures of environmental adaptation. However, it is difficult to know which allele is derived in data without information on the states in closely related species. In this paper, we adopted an ad hoc approach of using the frequencies of minor alleles as a substitute and expected that the frequencies of most derived alleles would still be low. This simple assignment of derived alleles may have errors, but we hoped that it would capture the SNPs with enhanced allele frequencies that facilitated adaptation to the local environments. To distinguish the association on the basis of positive and negative correlations, we introduced two types of environmental variables: the original environmental value itself and the sign‐reversed value of the original value. Genes and traits that had a positive/negative correlation with the original environmental factors were connected to the original/sign‐reversed environmental variables.FST)  in population i (i=1∼K), and M~lB is the between‐population‐pair matching average over population pairs  linearly arrayed populations, Pop1, …, Pop25 , which were collected from various regions over a range of 2500 km near the Canadian–US border at a latitude of 44′–59′ N, a longitude of 121′–138′ W, and an altitude of 0–800 m  and the traits showed negative correlation . Consistent with the environmental values of the populations, six populations, Pop5 to Pop10, were located near E1. Pop15 to Pop25 were located near E2 in order.Figure 5 Figure . In our Appendix , the env1 and E2)  Figure . Respond5 Figure . Note th3.23.2.1FST values . s27 is characterized by warm temperature (MWMT), whereas i15 and i16 are characterized by high altitude (+ALT) and dry conditions year‐round , as shown in the center of Figure The color gradient of population‐specific FST values than those of IBC ; this means that the seasonal variation of day length is small in the southern area, with mild temperatures  and wet conditions year‐round , as plotted in Figure DAY) was correlated with abaxial stomata density, which indicated that strong southern sunlight stimulates photosynthesis and requires many stomata. Mild temperatures  were correlated with genes associated with growth traits: GH3.9  was correlated with a gene related to water conditions, HRA1 , which means that day length varies greatly from season to season, and low temperatures , as described in Figure Populations in NBC had large population‐specific 3.2.2FST values and high genetic diversity observed in i15 and i16 were due to admixture. Therefore, population s27 was tentatively set as a root in the following analysis. We estimated the history of population splits and admixture by applying TreeMix to the population‐specific genotype frequencies of the 34,131 SNPs. The populations expanded their distribution to the coastal region (s20–s26) and extended the range to northward (s17–19). Some extended further northward (n1–n12) and others extended southward  and toward inland   and the associated climatic gradient of temperature and length of daylight  were the major environmental factors that drove both first and second PC multitrait coadaptation Figure . It was 4A population evolves in space and time and responds to variable environments. From its birth, a population may continuously change its distribution range, and the initial localities may have occasionally been exposed to unprecedented environmental stress. In these localities, individuals and populations can acclimate to such environmental stresses by phenotypic plasticity in a short amount of time, and the populations can adapt by changing their geographical distribution or genomes over a long amount of time. We focused on the latter and aimed at understanding the whole history of the populations by conducting an exploratory analysis of multitrait coadaptations. The whole scheme of the analysis has multiple layers. In the first layer, we generated the features that are used as inputs for the second layer of analysis. The features are population‐wide SNP site frequency spectra, genome‐wide association with traits and environments, and polygenic adaptations mapped on the admixture graph. These features were obtained by ever‐evolving population genetic and quantitative genetic procedures. The exploratory analysis is the second layer of analysis incorporating these features.FST values. To interpret the adaptations, we referred to the first layer analysis of an association study by searching for the genes that are associated with the environmental variables surrounding the characteristic groups of populations.To reveal the whole scenario of historical change of geographical distribution and adaptation to the new environments, we conducted correspondence analysis that visualizes the population structure in relation with the SNPs, environmental variables, and trait variables. From the biplot, the history of range expansion and differentiation was inferred by the population‐specific To understand the multitrait coadaptation, we analyzed the correlations among the estimated history of positive selections that increased or decreased the trait values. The estimates of the selections were obtained by the first layer analysis in which the admixture graph was constructed and represented the history of population differentiation and admixture; then, the positive selection parameters were mapped on the admixture graph based on the spatial allele frequencies of the SNPs associated with the traits.We aimed at showing, through the numerical simulation and analysis of empirical data, that the complexity of populations’ life history can be interpreted well solely by integrating the information of among‐population genetic difference and genome‐wide association with multiple environments and multiple traits. A multilayered approach may be a practical choice. However, our approach is still in its infancy. One direction of future study is to include latent variables that are interpreted as key elements of environmental selection and adaptation. Another direction is quantifying the pattern of adaptation. A natural framework is a Bayesian approach that uses the features provided by the first layer analysis as the prior information.As a final remark, we note that our analysis uses populations as units of analysis. However, populations are often defined by poststratification of the sample. In the case of natural populations of black cottonwood data, we adopted the population assignment provided by the original dataset. The power of our exploratory approach depends on the accuracy of the assignment of the individuals to local populations. Consequently, individual‐level analysis also deserves consideration.The authors declare that they have no conflict of interest.Reiichiro Nakamichi: Data curation (lead); Formal analysis (lead); Methodology (supporting); Software (lead); Writing – original draft . Shuichi Kitada: Conceptualization (supporting); Investigation (lead); Supervision (supporting); Writing – original draft . Hirohisa Kishino: Conceptualization (lead); Funding acquisition (lead); Methodology (lead); Project administration (lead); Supervision (lead); Writing – original draft .Fig S1‐S5Click here for additional data file.Table S1‐S6Click here for additional data file.Appendix S1‐S3Click here for additional data file.


Dactylogyrus vistulae. The analyzed parasite specimens were collected from 13 cyprinoid species from 11 sites in the Apennine and Balkan peninsulas. A total of 159 specimens were genotyped at each of the loci and the number of alleles per locus ranged from 2 to 16, with a mean number of 6.958 alleles per locus. Exceptionally high genetic diversity was observed among D. vistulae individuals in the southern Balkans (mean NA per locus = 3.917), suggesting that generalist D. vistulae expanded from the south to the north in the Balkans and later into central Europe. The initial clustering analysis divided all investigated specimens into three major clusters; however, the results of the subsequent analyses revealed the existence of various subpopulations, suggesting that the population structure of D. vistulae is associated with the diversification of their cyprinoid hosts. In addition, the partition of the parasite population was observed in regions of the sympatric occurrence of two host species, indicating that these hosts may represent a barrier for gene flow, even for generalist parasite species.Approaches using microsatellite markers are considered the gold standard for modern population genetic studies. However, although they have found application in research into various platyhelminth taxa, they remained substantially underutilized in the study of monogeneans. In the present study, a newly developed set of 24 microsatellite markers was used to investigate the genetic diversity of the generalist monogenean species  D. vistulae is associated with the diversification of their cyprinoid hosts. In addition, a partition of the parasite population was observed in regions of the sympatric occurrence of two host species, indicating that these hosts may represent a barrier to gene flow, even for generalist parasite species.The application of a newly developed set of 24 microsatellite markers revealed a strong genetic structure among generalist monogenean parasites. The initial clustering analysis divided all investigated specimens into three major clusters; however, the results of the subsequent analyses revealed the existence of various subpopulations, suggesting that the population structure of  According to the checklist compiled by Gibson et al. . The biHoffman, . Each cyDactylogyrus vistulae Prost, 1967, is the most striking example of generalist Dactylogyrus, evidenced from 24 cyprinoid species of six genera in the Balkans and Central Europe  representing different populations, that is, parasite populations associated with different host species or parasite populations associated with geographically distant populations of the same host species. Moreover, Benovics et al.  or, alternatively, indicate secondary contacts between the hosts. Microsatellite markers are still underutilized in this platyhelminth group, and, to date, only one study designing primers for polymorphic repeats in gyrodactylids has been conducted by Faria et al.  to test their functionality with respect to revealing interpopulation genetic variability in order to investigate the geographical structure of the populations of this species.Over the last few decades, approaches using microsatellite markers  have become the gold standard for most population genetic studies. These highly polymorphic short tandem repeats are, due to their unique characteristics , usually applied to infer gene flow rate, hybridization, or mating pattern on the intra‐ and interpopulation levels. Studies investigating interpopulation variability in monogeneans are often conducted using either morphometrics . Due to the focus of the paper on southern European regions with an intertwined geographic history, data on the D. vistulae population from the Czech Republic were omitted from further analyses. Genomic DNA was isolated using DNEasy Blood & Tissue Kit . Subsequently, library preparation using KAPA LTP Preparation Kit  and sequencing on an Illumina Miseq platform (Illumina) using Miseq Reagent Kit v2 (Illumina) were provided by CEITEC . The QDD , and 0.2 µM of each of the forward and reverse primers. The conditions of the PCR were as follows: initial denaturation at 94°C (2 min), 35 cycles of denaturation at 94°C (60 s), annealing at 51–63°C (30 s), an extension at 72°C (2 min), and a final extension at 72°C (10 min). The PCR products were visualized by 2% agarose gel electrophoresis. Microsatellites that produced unreliable bands, or positive bands for the control sample represented by DNA of the fish host, were further excluded. The remaining microsatellites were tested under the same PCR conditions as described above, with the forward primer labeled by fluorescent dye  and analyzed on an ABI PRISM 3130 Genetic Analyzer  using 500 LIZ Size Standard . Genotypes were scored using GeneMapper Software version 4.0 . Finally, 24 polymorphic microsatellites were pooled into the five multiplex PCR sets. PCRs were carried out in a total volume of 10 µl using the Qiagen Multiplex PCR Kit  with 35 cycles and an annealing temperature of 52°C  and used in this study. Their species identities were evaluated by amplification of the partial genes coding 18S rDNA and 28S rDNA and entire ITS1 region , the number of effective alleles (Ne), Shannon's diversity index, the observed (HO) and expected (HE) heterozygosity, the fixation index , FST , and the Nei‐distances  were computed using the program GenAlEx v 6.5  was tested within the range of 1 to 15. Basically, K range was estimated on the basis that each parasite population was associated with an individual collection site . However, to test whether further fragmentation occurs between sympatric hosts, or even within a single host population, the number of K was increased to 15. A series of ten independent runs with 107 Markov chain Monte Carlo (MC2) iterations, the initial 106 iterations discarded as a burn‐in, were conducted for each tested K. The most optimal K  was selected using the Structure Harvester , and su33.1A), number of alleles per population (NA), and observed and expected allele heterozygosity per population , is presented in Table D. vistulae individuals parasitizing northwestern Greek Squalius prespensis (site G1). Individuals from this site exhibited the highest mean heterozygosity across all loci (HO = 0.453), the highest number of alleles per individual (NA = 3.917), and the highest mean number of effective alleles (Ne = 2.662). The descriptive statistics indicating mean values for basic population parameters for all loci in respect to the collection sites are reported in Table FST were computed for each population pair and are reported in Table FST ranged from 0.103 to 0.959, and the greatest distances were observed between population C4 and the following populations: B1, B2, C1, C2, and C3. A similar pattern was revealed for the C5 population.From all candidate loci, which were selected according to the results of PCR amplification and sequencing tests, 24 were detected to be polymorphic and were chosen for further population analyses. The genetic diversity of each locus per collection site, including range of alleles, total number of alleles per locus . The first one encompassed individuals collected from sites in Bosnia and Herzegovina (B1–B4), and southern Croatia (C1–C3). The second cluster encompassed all individuals from Greek sites G1 and G2. Finally, the third cluster encompassed individuals collected from sites in central Croatia (C4 and C5), and individuals parasitizing T. muticellus in Italy (termed as I1b). The D. vistulae individuals parasitizing S. squalus from the same site in Italy (I1a) were genetically ambiguously associated with all three clusters. Gradually increasing the number of estimated K first separated Greek D. vistulae individuals into two clusters, each associated with the respective collection site (G1 and G2), and later separated the Apennine population parasitizing T. muticellus (K = 5). In addition, the existence of three subpopulations was suggested among D. vistulae from sites B1–B4 and C1–C3; however, the results from both clustering analyses clearly document gene flow between these populations representing seven collection sites in Bosnia and Herzegovina, and Croatia (K = 8). At the higher estimated K (<7), clustering analysis divided the individuals from Italy into two separate subpopulations—each associated with the respective host species. The separation of I1 subpopulations also indicates genetic drift, whereas the observed heterozygosity in population I1b (HO = 0.004) is significantly lower in comparison to population I1a .The suitability of s et al. . Even thD. vistulae. We found low allele variance in all studied markers at ten out of the 12 sites in the northeastern peri‐Mediterranean, where D. vistulae was collected from 13 host species. High allelic diversity was observed in individuals from the two remaining sites (two parasite populations); Aoos in northwest Greece, and the Cerfone Stream (Tiber drainage) in Italy.We used 24 microsatellite markers to investigate the molecular diversity of populations of the generalist parasite D. vistulae in Aoos as a result of high abundance of suitable hosts in the region  in the Peloponnese peninsula. The other factor that potentially impacted the formation of parasite populations in northwestern Greece is the historical formation of the Dessaretes lake system, which occurred in this region and played an essential role in the speciation of cyprinoids in the Balkans. This large connection of water bodies originated in the Pliocene and covered the area of all the present Great Lakes of the Balkan Peninsula, that is, Ohrid Lake (located on the border of Albania and North Macedonia), Prespa Lake , Mikri Prespa Lake (Albania and Greece), and Maliq Lake   shows no or very little molecular divergence between species (or populations) living on both sides of the Adriatic Sea  dispersed together with other cyprinoids and their parasites from the Balkans to Apennine Peninsula  than to the population from T. muticellus  follows the range of the Padano‐Venetian district, which covers the region from the River Vomano in central Italy to the River Krka in Dalmatia. The range of this district essentially corresponds with the aforementioned Po basin during the glacial period. Moreover, these two Telestes species also represent a distant lineage from remaining congeners in the Balkans, whose ancestor diverged in the middle of the Miocene  and T. turskyi (C3) into individual subpopulations; however, it also clearly indicated gene flow between D. vistulae populations sampled in Bosnia and Herzegovina, and those from Croatia. Both S. tenellus and T. turskyi are highly endemic with a strongly limited distribution range. While S. tenellus is primarily endemic to the Cetina River, some populations can also be found in nearby karstic streams; for example, T. turskyi is restricted to the Čikola River . However et al.  or digenr et al.  have so r et al. , and migr et al. . HoweverThe authors declare that they have no competing interests.Michal Benovics: Conceptualization ; Data curation ; Funding acquisition (supporting); Investigation (lead); Methodology (supporting); Project administration (supporting); Software ; Writing‐original draft (lead). Lenka Gettová: Conceptualization ; Data curation ; Investigation (supporting); Methodology (lead); Software ; Supervision (supporting); Validation (supporting); Writing‐review & editing (supporting). Andrea Šimková: Conceptualization ; Funding acquisition (lead); Project administration (lead); Supervision (lead); Writing‐review & editing (lead).https://doi.org/10.5061/dryad.cnp5hqc5p.This article has earned an Open Data badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at