Acanthopagrus schlegelii  is a commercially important species in Japanese waters. Assessing its population structure is essential to ensure its sustainability. In the Northwestern Pacific, historical glacial and interglacial periods during the Pleistocene have shaped the population structure of many coastal marine fishes. However, whether these events affected the population of black sea bream remains unknown. To test this hypothesis and to assess the population structure of black sea bream, we used 1,046 sequences of the mitochondrial control region from individuals collected throughout almost the entire Japanese coastal waters and combined them with 118 sequences from populations distributed in other marginal seas of the Northwestern Pacific Ocean. As in other coastal marine fish with similar distribution, we also found evidence that the glacial refugia on the marginal seas prompted the formation of three lineages in black sea bream. These lineages present signatures of population growth that coincided with the interglacial periods of the Pleistocene. While the origin of Lineages B and C remains unclear, the higher relative frequency of Lineage A in the southernmost location suggests its origin in the South China Sea. The non-significant pairwise ΦST and AMOVA of Japanese populations and the presence of these three lineages mixed in Japanese waters; strongly suggest that these lineages are homogenized in both the Sea of Japan and the Pacific Ocean. Our results indicate that the black sea bream should be managed as a single stock in Japan until the strength of connectivity in contemporary populations is further addressed using non-coding nuclear markers.The black sea bream  Acanthopagrus schlegelii  is widely distributed in the Northwestern Pacific Ocean, around the coastal waters of the South China Sea, Korea, and Japan except for the Ryukyu archipelago . Sequences were aligned in Clustal X . Besides, we assessed the number of clusters using a bayesian approach implemented in BAPS v5.2 , were calculated in DnaSP v6 (H), haplotype and nuclear diversity  (S), were also calculated in DnaSP.Neutrality tests and demographic parameters like Tajima’s D , Fu’ Fs D  , and theT = τ∕2u, where u = 2μk and with µbeing the mutation rate and k the alignment length (686 bp here). We consider an average generation time of four years (−8 per site per year (3.6%/Myr) reported for the mtDNA control region of teleost (The time of demographic expansion (T) was calculated from the relationship ur years , and a m teleost .7 generations sampled every 5,000 iterations, and with the first 25% discarded as burn-in. All runs yielded an effective sample size (ESS) of more than 200 for the parameter of interest after burn-in. The skyline plots were generated in Tracer v.1.7.1 (http://tree.bio.ed.ac.uk/software/tracer/).Past dynamics of the female effective population size were observed through Bayesian Skyline Plots (BSPs) calculated in BEAST 2.4.8 . We usedP = 0.006). When Japanese populations are compared with Chinese and Taiwanese population, the Zhenjian population (DJ) from the Northern East China Sea is only significantly different from the Hiroshima (HH) population . From the remaining 45 pairwise comparisons, 31 were significant and their ΦST ranged from 0.036–0.121. While some of the pairwise ΦST comparisons were significant, all of the values were still very low  and Hiroshima (HH) populations ; however, 98.5% of the variation was observed within populations. When only Japanese groups are considered, AMOVA indicates the absence of population genetic structure among Japanese populations  with 99.73% of the variation explained within populations. Similarly, the ΦFST values were significant but very low  and the AMOVA among them had accumulated a variation of 41.29% with an ΦFST of 0.412 at P = 0  = −8545.8301), herein Lineages A, B, and C; in which the frequency of lineage B and C were similar in all marginal seas except in the South China Sea, and lineage A was more abundant in the South China Sea compared with the other locations . Howeverat P = 0 . The TCSh ranged from 0.957–0.995), and the nucleotide diversity was overall low but higher in Lineage A (π = 0.0097) than in the Lineage B (π = 0.0048) and C (π = 0.0043).The genetic diversity indices of each lineage are shown in τ)   were simτ) . OverallPopulation genetic structure of marine fishes are assumed to be very low or inexistent because of absence of physical barriers and wide-range dispersal of larvae by the ocean currents . In the Sardinella zunasi (Mugil cephalus (Ruditapes philippinarum (For black sea bream, BAPS bayesian clustering detected the presence of three different lineages which explained 41.29% of the variation in the AMOVA analyses. The presence of these lineages within each of our geographical locations  also proa zunasi , based tcephalus , based oppinarum , based oTakifugu niphobles in the Japan Sea, East China Sea, Yellow Sea and the Pacific Ocean region, using the mtDNA COI. The frequency of the COI haplotypes in their samples allowed The South China Sea appeared composed mostly by individuals of Lineage A, suggesting the origin of this lineage in this northernmost location of our collections, very likely due to the enclosure of this marginal sea during glacial periods of the Pleistocene . The relThe expansion time calculated the female effective population size of black sea bream lineages coincides with the rise of sea levels during interglacial periods around 300,000 and 150,000 years ago , which mFurther studies of populations genetics in black sea bream will benefit from the use of more polymorphic loci from the nuclear genome. These loci will be useful to assess the contemporary population connectivity of this fish. However, studies such as from In the present study, we assess the population genetic structure of black sea bream and test whether the glacial-interglacial periods of the Northwestern Pacific Ocean played a role in the divergence and distribution of its populations. To test this hypothesis, we used 1,046 sequences of the mitochondrial control region from black sea breams distributed in almost the entire coast of Japanese waters and combined them with 118 sequences from other marginal regions. We observed the presence of three different lineages of black sea bream with signatures of population expansion during interglacial periods of the Pleistocene. The sequences of these lineages are carried by black sea bream populations inhabiting Japanese waters suggesting that, although separated in the past, they have already homogenized. Our results highlight the role of historical climate fluctuations into the genetic structure of coastal marine fishes of the Northwestern Pacific and provide useful information for the fisheries management of black sea bream. Based on our results, we recommend managing black sea bream as a single stock in Japan until more studies using more polymorphic nuclear DNA markers clarify the absence or presence of any contemporary barrier.10.7717/peerj.11001/supp-1Figure S1Click here for additional data file.


Urechis unicinctus is the only Echiurini species distributed in Bohai Gulf of China. The wild populations of this species have sharply declined in China due to overfishing. Over 150 samples from Bohai Gulf were collected in the present study, which were classified into five populations according to their geographic areas and body colors. The genetic diversity and population structure of these populations were investigated by mitochondiral COI and D-loop sequences. The haplotype diversity of U. unicinctus based on COI and D-loop sequences were still high. In addition, the evolution rate of D-loop region could faster than the COI gene of U. unicinctus. Meanwhile, over 99% genetic diversity was contributed by different individuals within populations. Moreover, phylogenetic trees did not show clear geographic or color cluster. Our findings indicated that this species in Bohai Gulf of China should be treated as a whole population. Urechis unicinctus is a benthic invertebrate mainly distributed along the coast of Russian, Japan, Korea, and the Bohai Gulf of China  is a marker mtDNA gene often used to study the genetic diversity and population structure of marine species  is a genetic material that exists in the cytoplasm of eukaryotes and is significantly different from the nuclear DNA in structure and function . All DNA samples were stored at −20 °C for further application.Genomic DNA was isolated from the body wall using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit  according to the manufacturer's instruction. Agarose gel electrophoresis with 1% concentration was used to detect whether DNA was successfully extracted, and then the concentration and purity of each successfully extracted DNA were measured by NanoPhotometerCOI gene and D-loop region were amplified by PCR using the specific primers (The  primers . All PCRCOI (417 bp) and 155 D-loop (163 bp) sequences were obtained respectively in the present study. All sequences were deposited in GenBank with accession numbers MT346032-MT346374. All sequences were aligned and manually corrected using the software ClustalX v1.83  and the fixation index (FST) were conducted to estimate the genetic differentiation among different populations by Arlequin v3.5.2.2 software  to 0.973 (SR), and Pi ranged from 0.010 (SR) to 0.013 (DB and HB) (For detected . Among t and HB) .For D-loop region, 58 polymorphic sites and 52 haplotypes were identified . Among 5U. unicinctus populations were calculated based on COI and D-loop sequences . Meanwhile, significant Tajima's D values were observed in DR, HB, and SR based on COI, and DB, DR, HP, and SR based on D-loop, respectively  and D-loop (-61.319) sequences when all samples were pooled together (The Tajima's ectively . In Fu'stogether .COI gene has been proved to display greater base substitution frequency than other mtDNA genes, so it has been used as a suitable marker for population genetic studies in diverse animals (Du et al. U. unicinctus from seven and three different geographic populations in Bohai Gulf and Laizhou Bay, respectively (Gong et al. U. unicinctus were higher than those of many reported marine invertebrates (Schulze U. unicinctus was investigated for the first time in this study. The Hd and Pi values of U. unicincuts were also higher than those of other marine soft-bodied organisms, such as Asian Green Mussel Pernaviridis (Lau et al. Sepiella japonica (Xia et al. COI and D-loop sequences in U. unicinctus was still high. Moreover, both higher nucleotide diversity and pairwise genetic distance were observed in the D-loop region compared to the COI gene from the U. unicinctus populations detected in the present study (COI gene and D-loop region were representative sequences of the coding and noncoding regions in the mtDNA, respectively. The finding of the present study suggested that the evolution rate of non-coding sequence could faster than the coding region in the mtDNA of U. unicinctus. Schulze . In contnt study . The COIFST value is an effective index to assess the differentiation among populations (Wright FST values based on both of COI and D-loop sequences among five U. unicinctus populations were small and not significant. This result indicated that the genetic differentiation among U. unicinctus populations with different geographic areas and body colors were relatively low. Meantimes, the pairwise genetic distances between different populations were all below 0.1, which suggested a pattern of homogeneity among U. unicinctus populations (Billington and Hebert U. unicinctus populations. All results above informed that the populations of U. unicinctus with different geographical areas and body colors might comprise a panmictic population. Consistent results were also found in previous studies of U. unicinctus in different geographic locations of China (Gong et al. s Wright . In the D and Fu's Fs tests, were used to examine recent population expansion when the null hypothesis was rejected (Zhang et al. D and Fu's Fs values based on the COI and D-loop sequences in all detected U. unicincuts were significantly negative. These results indicated that population expansion event could have occurred in the history of U. unicincuts evolution. In addition, the negative values of neutrality tests also suggested the purifying selection of population. For different populations of U. unicinctus, the significances of Tajima's D and Fu's Fs tests were consistent. However, the results of these tests based on COI and D-loop sequences were different in DB, HB, and HP populations (COI and D-loop sequences of U. unicinctus. It is a pity that we cannot estimate the expansion time and the detail evolution differences of U. unicinctus, and these topics should be further studied.Neutrality tests, such as Tajima's ulations . These rU. unicinctus is still high based on COI and D-loop sequences, in which COI gene represented more haplotype diversity. Moreover, there was no obvious genetic differentiation between different geographic localities and body colors. Our investigations have directive significance for management and resource conservation of this commercial marine species. High genetic diversity can facilitate the selection of germplasm in breeding activities and the development of targeted breeding technologies to improve the nutritional and economic benefits of U. unicinctus. Further study based on more molecular markers and samples is needed to extend the present understanding.The haplotype diversity of Conceptualization, Tong Liu and Wenbo Chen; supervision, Tong Liu and Wenbo Chen; methodology, Hongxin Shang, Tingting Yang, and Lu Wang; formal analysis, Saisai Zhang, Yang Sun and Lin Ma; investigation, Ying Chen and Bin Zhang; writing–original draft preparation, Saisai Zhang and Mo Li.


Gladiolus imbricatus in five mountain ranges, including the Tatra, Pieniny, Gorce, Beskid Niski (Western Carpathians) and Bieszczady Mts (Eastern Carpathians), was studied with inter-simple sequence repeat (ISSR) markers. The species is a perennial plant occurring in open and semi-open sites of anthropogenic origin (meadows and forest margins). We checked a hypothesis on the microrefugial character of the plant populations in the Pieniny Mts, a small calcareous Carpathian range of complicated relief that has never been glaciated. Plant populations in the Tatra and Pieniny Mts had the highest genetic diversity indices, pointing to their long-term persistence. The refugial vs. the non-refugial mountain ranges accounted for a relatively high value of total genetic variation . One of the Pieniny populations was of hybridogenous origin and shared genetic stock with the Tatra population, indicating there is a local genetic melting pot. A weak genetic structuring of populations among particular regions was found . This could be an effect of the frequent short-distance and sporadic long-distance gene flow. The dispersal of diaspores between the remote populations in the Western Carpathians and Eastern Carpathians could be affected by the historical transportation of flocks of sheep from the Tatra to Bieszczady Mts.The genetic diversity in 11 populations of  The landscape is dominated by resistance to weathering rocks of Jurassic and Lower-Cretaceous origins, which contribute to the extraordinary richness of the site and microclimate conditions. It is located at a distance of only 20 km from the floristic center of the Tatra Mts. The uniqueness of the flora of the Pieniny Mts underlines the presence of endemics: Neogene Taraxacum pieninicum Pawł. and Pleistocene Erysimum pieninicum (Zapał.) Pawł. and glacial relicts Dendranthema zawadzkii (Herbich) Tzvelev and Juniperus sabina L. It has a rich flora with an estimated ca. 1,100 vascular plant species that constitute nearly 50% of the total Polish plant biodiversity1. Among the Western Carpathian ranges, the existence of an Eemian forest refugium is only postulated in Pieniny2.The Pieniny Mts (Polish Western Carpathians) are a relatively low range (Mt Wysoka 1050 m above sea level) spread on an area of ca. 100 km6. Wallachian settlers inhabited higher areas of the ranges with poorer soils that were unsuitable for cultivation, but enabled the continuation of the livestock and shepherding economy. In this way, Wallachian settlers created a permanent element of the Carpathian landscape. The sites where sheep and cattle were kept in pens and sites around shepherds’ huts are now dominated by anthropogenic, nitrophilous plant communities8.Traditional shepherding in the montane forest zone also led to the origin of unique meadow community Gladiolo-Agrostietum capillaris assoc., growing on the shallow rendzinas of sites of previous lower montane beech forest9. This is an endemic plant association for the Western Carpathians. Extremely floristically rich overall, with 30 to 40 species per square meter and up to 70 species per 100 m210, with the ecological center in the Pieniny Mts. Now, the plant association of the Pieniny Mts is being monitored within a Natura 2000 network of protected areas11. To protect it in the Pieniny National Park, cultural  sheep grazing is performed to increase species richness and maintain the region’s specific character12. The mountain meadows had their best ecological prospects in times of extensive agricultural-pastoral management. However, now that many of the areas that were previously used for economic purposes have been abandoned, the meadows have become rare and vulnerable.It is known from historical records that the Carpathian ranges were colonized by the Wallachian tribes from the Balkans northward, who were accompanied by sheep breeds that can be dated back to the thirteenth and fourteenth centuriesG. imbricatus. Before human activity opened the mountain landscape, it was probably characterized by ecotones between forest and rock sites, forest gaps , glades, rocky hillsides, balks, stony riversides, and alluvial forests10. Currently, G. imbricatus is disappearing in a wide array of plant communities across Central Europe13, including in the Pieniny Mts, where meadows with the character species have diminished from 300 ha in the 1960s to 35 ha in 200014. Long-term regular extensive management is essential for the persistence of these populations15.One of the character species of the plant association is G. imbricatus in the Western Carpathians. Especially, we attempt to (i) identify the region(s) with high genetic diversity of the species represented by their presumable refugial populations, (ii) identify the relationships between the Pieniny Mts and the neighboring Tatra and Gorce Mts (ca. 20 km), as well as the remote Beskid Niski and Bieszczady Mts (ca. 120–250 km), and (iii) discuss the phylogeography of G. imbricatus when compared with other species with a similar ecological profile.The main aim of the present study was to evaluate the molecular diversity of populations of 16 and the ipni.org database.The names of species follow Mirek et al.Gladiolus L. belongs to the Iridaceae family and has two distinct areas of geographic distribution. The main center is in Eastern and Southern Africa (Capensis), where more than 100 species have been described. The second area of distribution is in the southern part of Eurasia, especially in the Middle East and the Mediterranean region. In this area, the number of species is much smaller. Hamilton17 listed six species as indigenous to Europe. European species of Gladiolus are polyploids with a chromosome number from 60 in tetraploids to 180 in dodecaploids, but aneuploids there are known to exist. The evolution of the European Gladiolus species has been affected by hybridization and polyploidy. Gladiolus imbricatus covers Central and Eastern Europe, the Mediterranean, Caucasia, and West Siberia17.The genus G. imbricatus is an anemochore, perennial species, and a bulbo-tuber geophyte. It blossoms in July, pollinated by Lepidoptera. It occurs throughout Poland, but it is a rare species, more often inhabiting mountainous areas. Available historical data suggest that it was common in southern Poland in the nineteenth century21. It occurred in thermophilous oak forests of Potentillo albae-Quercetum, in moist lowland meadows of the Molinion alliance23, and as a weed in oat and barley fields24, probably as a remnant of previous meadow communities18. It was also a co-dominant species in many types of upland pastures and meadows18. This state has persisted until modern times; there has only been a drastic decline in the number of its sites in the last few decades. This process is particularly visible in the central and northern parts of the country, where the appearance and disappearance of new populations is observed26. In southern Poland, population declines and fragmentation are also recorded. As a result, G. imbricatus is now endangered in Poland27. Moreover, it is gradually disappearing in the whole of Europe28. In Poland, the plant species is under law protection.2) to DNA isolation was collected in nine populations in the Western Carpathians and in two populations in Eastern Carpathians , LP (Tatra) S, B (Bieszczady), BN (Beskid Niski Mts) represent wet meadows of Molinion caeruleae alliance  and according to Convention on the Conservation of European Wildlife and Natural Habitats (Bern Convention) (1979). The sampling in the Bieszczady National Park was permitted by document no. 60/17. According to Polish law, it does not require the permission of the relevant institutions when we collect small part of leaves outside the area of National Park. The formal identification and collection of the plant material used in our study by authors: A. Sutkowska (A.S.) and J. Mitka (J.M.). Voucher specimen from the site in Beniowa (Bieszczady), stored in the herbarium of the Bieszczady National Park at ID 1503 . DNA samples of all examined specimens are available from the author (A.S.) upon request.The sampling of 2) without damage symptoms caused by insects and mold. DNA was extracted with Genomic Mini AX Plant (A&A Biotechnology) according to the manufacturer's instructions. The sequences of primers were taken from Stepansky et. al.29 and are shown in Table 30 and optimized by Sutkowska31. Amplification was carried out with 25 µl reaction mixture: 2.5 µl tenfold concentrated reaction buffer supplied by the Taq DNA polymerase manufacturer (Fermentas), 1.5 mM MgCl2, 0.19 mM of each dNTPs (Fermentas), 27 pmol primer, 200 ng template DNA and 1.4 units of Taq polymerase. The annealing temperature for primers ISSR7 was 44 °C, and for ISSR1, ISSR3, ISSR6 was 47 °C. Optimal conditions for the reaction were as follows: initial denaturation: 94 °C—5 min, 42 amplification cycles: denaturation 94 °C—59 s, annealing 44 °C (47 °C)—59 s, polymerization 72 °C—59 s, final polymerization 72 °C—7 min. A negative control reaction without DNA template was included in each amplification. To confirm the results, 50% of the samples were amplified twice.DNA was isolated from fully developed fragments of leaves 4 cm without Products were subjected to electrophoresis on 1.5% agarose gel stained with ethidium bromide (50 µl/100 ml) at 100 V about 1.5 h. PCR products (bands patterns) were observed and archived with Imagemaster VDS (Pharmacia—Amersham).32.For analysis of the length of PCR products, GelScan ver. 1.45 (Kucharczyk TE) software was used. ISSR reproducibility tests included within-plate (n = 12) and between-plate (n = 9) replicates independently analyzed from the DNA extractsHj. Statistics of genetic diversity and population genetic structure were computed after estimating allele frequencies, including the percentage of polymorphic bands (PLP) at 95% criterion. The calculations were performed with AFLP-SURV version 1.033.All the analyses performed were based on the following assumptions: ISSR markers behaved as dominant markers; co-migrating fragments were considered homologous loci; and populations were at Hardy–Weinberg equilibrium, in which case allele frequencies were estimated from Bayesian method with the non-uniform prior distribution of allele frequencies, to calculate expected heterozygosity I) was calculated to provide a relative estimate of the degree of genetic variation within each population, based on the formula I = -Σ Pi log2 Pi, where Pi was the frequency of each ISSR band. The number of alleles (bands) with nonzero frequency  and the reciprocal of homozygosity  were calculated using POPGENE version 1.3234. As an additional diversity marker, the rarity index DW, corresponding to “frequency-down-weighted marker values” per individual35 was computed using AFLPdat36. High DW values are expected in long-term isolated populations37. Analysis of molecular variance (AMOVA)38 was carried out using the program Arlequin version 3.1140.Shannon’s diversity index 46 performed with the NTSYSpc ver. 2.11 multivariate analysis package47. NMDS displays a monotone relationship to the distances implied by the original data matrix and achieves a much better fit in fewer dimensions than is possible with other ordination methods.The matrix of 77 samples × 167 ISSR products coded as binary (0–1) data was used to calculate Nei and Li (Dice) distances48, applying a Bayesian model-based clustering algorithm for the use of dominant markers49. The numbers of K = 1–5 groups were tested in ten replications per K. A burn-in period 100 000 was applied, followed by a procedure using 500 000 Markov chain Monte Carlo (MCMC) repetitions. The estimation of the optimal number of groups was based on the likelihood of partitions, estimates of posterior probability provided in STRUCTURE output, examined as a function of increasing K48 and ΔK values, estimating the change in the likelihood function with respect to K and estimated as an indicator of the most reliable clustering structure50. The similarity between runs was estimated using the symmetric similarity coefficient51 with the R-script Structure-sum-201136. K = 2 represented an optimal clustering structure.The genetic division among individuals in populations of both species was estimated using STRUCTURE, version 2.3.3In the ISSR analysis of 77 individuals with 4 primer combinations, 165 unambiguous fragments (loci) were selected ranging between 130 and 990 bp. The number of PCR products per individual varied in particular primers within 3–13 Table . The witJU and Pieniny BW. The mean allele number na and the effective number of alleles ne ranged between 1.077–1.440, and 1.078–1.192, respectively. The mean percentage of polymorphic loci (PLP) ranged between 22.6 and 44.0%, and Shannon’s I index was between 0.049 and 0.187. Higher values for genetic variation were recorded in the Tatra Mts, then in the Pieniny Mts. Lower values were found in the Gorce and Bieszczady Mts. The survey of the rarity index DW revealed a similar pattern of variability across regions. Its highest values were found in Tatra JU and Pieniny BW, and the lowest in Beskid Niski BN and Gorce LB.All genetic variation values were consistently highest in populations Tatra G. imbricatus: (1) Pieniny BW, (2) Tatra JU, and (3) the remaining individuals  supported sister group . The second population Gorce LB is presumably of the hybridogenous origin, with the admixture of Tatra JU , Bieszczady B, S, and Gorce LB. Here a reticulation between Pieniny BW and Bieszczady B was found. The second group forms an unresolved cluster with Gorce LU and Tatra LP (80% support). The third group consists of the remaining Pieniny populations TKD, SN, TKG (50% support) and the population from Beskid Niski BN  show the distribution of genetic diversity among populations Fig. . The gen LU Fig. .B shared two unique bands with Pieniny BW and two unique bands (Bieszczady S) with Pieniny TKD. The NJ group Gorce LU/Tatra LP shared two unique bands with Beskid Niski BN. The only population with two private bands was Pieniny TKD , occurred exclusively in two or three geographical regions. The populations from Bieszczady Mts KD Table .G. imbricatus: 37.61% (p < 0.001). The analysis of the genetic structure of the populations in five geographical regions showed small, statistically insignificant, population differentiation in the particular mountain ranges . The test a hypothesis on the genetic divergence of the populations in two presumably refugial ranges: Pieniny and Tatra Mts gave the statistically significant result: 14.12% (p = 0.003) . The latter calcareous mountain range could have formed a local refugium and genetic melting pot for many plant species.Open-site natural vegetation forms peculiar plant communities in high-mountain zones , northern latitudes (arctic tundra belt), and continental climates (steppe formations). Historically, it also occurred in the Carpathian forelands and adjacent lowlands in the Late Vistulian (the Oldest Dryas), forming the tundra and cold steppe formations, according to many pollen spectra. No pollen of Iridaceae was found at that time in PolandG. imbricatus in the Western Carpathians and the flow of alleles between both neighboring and distant populations. A study on G. imbricatus53 also showed the low percentage of polymorphic bands PLP in populations of the species located about 100 km more to the north in Poland (25–26%). Our study showed the value of PLP within 40–55%. Nei’s genetic diversity index Hj was also higher in the Carpathians (0.150–0.194 vs. 0.084–0.100). The among-population component of diversity [analysis of molecular variance (AMOVA)] amounted to 36.6% in the Carpathians and 22.0% in populations located further north in Poland53. The similar genetic diversity indices PLP and Hj found at G. imbricatus in the vicinity of Minsk (Belarus)54, and amounted to 38%, and 0.130, respectively. The reason for such high genetic diversity is unknown; the population was perhaps related to an unrecognized “cryptic” refugium nearby.Our study revealed the high genetic variability of G. imbricatus. The result showed the statistically significant genetic divergence between refugial and non-refugial areas, with the outcome being especially convincing when compared with the non-significant structuring of the populations in five geographic regions of the Polish Carpathians. Populations of the species localized more to the north in Poland seem genetically impoverished in comparison with those occurring in the mountains. Similar results concerning the lowland vs. mountain populations showed studies on forest grass Bromus benekenii55 and wet meadow Primula farinosa56, which could have originated from the glacial refugia, especially those located in the Carpathian refugium57.Generally, the higher genetic diversity of Carpathian populations compared to those in lowland areas could be an effect of their refugial character . For AMOVA, these two geographical regions accounted for 14% (p = 0.003) of the total genetic variance of the Carpathian populations of Melica transsilvanica, studied in the Pieniny Mts and Małopolska Upland (southern Poland)58. The species has a different ecological profile than G. imbricatus; it grows on dry, exposed outcrops in pioneer rupicolous grasslands  and in xerothermic grasslands of the Cirsio-Brachypodion pinnati alliance on the Małopolska Upland. The high level of among-population diversity was linked with a decreased within-population diversity index (PLP 3.7%) and nearly total genetic homogeneity. Taken together, the results of the studies on M. transsylvanica in the Pieniny Mts point to the high geographical isolation of the metapopulation in the region and the dominant self-pollinating system of the species is inducing the lack of gene flow58.A much higher among-population component of diversity (AMOVA) that amounted to 86% displayed a grass, G. imbricatus populations in the Carpathians could be related to the presumably refugial character of some Western Carpathian populations. This has been proven by the occurrence of the private bands, a typical phenomenon in the relict areas59, and the high value of the DW index60. Accordingly, the high genetic diversity could be the effect of the long-time presence of large population numbers in the interglacial15, counteracting the effects of genetic drift or genetic bottleneck that affect smaller populations more heavily. An example is the extant Polish population of Primula farinosa in the Pieniny Mts which probably formed a continuous distribution in the interglacial between the Carpathian Mts and Polish lowlands56. Today, the relict plant population demonstrates a distinct genetic makeup and admixture of various genetic lineages, an example of the Pieniny genetic melting pot. Increased diversity was obtained through the redistribution of genetic information in a given place by dispersal from refugial areas60. Our study of G. imbricatus also provides clear phylogeographic evidence on the existence of a local genetic melting pot in the Pieniny Mts. The Bayesian inference shows the two genetic types of the species in the Polish Carpathians. They formed a hybridogenous population Pieniny BW, the sister group, with a donor population Tatra JU. In the other population, Pieniny TKD, one individual (TKD8) also shows the genetic admixture from Tatra JU. It is compelling evidence of the local short-distance migrations. Moreover, frequent short-distance dispersal could probably prevent the effects of genetic drift (genetic bottleneck) in the distracted and divided populations of southern Poland.The relatively high within-population genetic diversity of the LU) has close genetic links with Tatra LP (NJ), and the second (Gorce LB) with Tatra JU . Interestingly, the most remote populations, Bieszczady B and S, were linked genetically to the Pieniny BW population, as well as with Gorce LU and Tatra LP (a distance of ca. 250 km). The reticulation in NJ and the sharing of the rare specific alleles in such remote populations as Bieszczady B and S, and Gorce/Tatra group LU/LP, together with Pieniny BW, TKD, SN, point to the occurrence of long-distance dispersal. The placement of the population from Beskid Niski BN in one group with Pieniny populations could characterize the stochastic process.The remaining populations in the Pieniny Mts formed a distinct but marginally supported cluster (50%), showing the different histories of their origin. The same concerns apply to two populations in the Gorce Mts. Geographically adjacent, they have not originated from the same genetic stock; the first  by rail from the Tatra Mts to the Bieszczady Mts in the 1960s was the result of overgrazing in the Tatra Mts area, and the administrative decision of the Tatra National Park authorities64. Also, many villages in the Beskid Niski Mts were abandoned after the Second World War and the flocks of sheep from the Tatra/Pieniny regions were then introduced there. In our opinion, this could be a crucial factor in determining the close genetic relations between populations in such remote regions.The genetic similarity of the distant populations (Bieszczady vs. Tatra Mts) may be a result of numerous overlapping factors, including multidirectional gene flow in the dispersal history, long-distance dispersal during postglacial recolonization, and survival in several detached refugiaThe similarities among populations in close areas of the Pieniny, Tatra, and Gorce Mts, where sheep flocks were abundant, densely distributed, and interchanged often in historical times, could be explained by short-distance diaspore dispersal.Aconitum species in the Carpathians and the Sudetes63, an introgression was found between species with non-overlapping geographical ranges, probably as a result of seed dispersal with pasturing within a distance of ca. 20 km (Mantel test). It seems to be an average of short-distance seed dispersal in the mountains, in the realm of the traditional agro-pastoral economy.While studying the genetic diversity of Cicerbita alpina across Europe65, the authors found a weak phylogenetic structure of the tall-herb community species among Carpathians regions  . Also, the level of genetic divergence of the species (30%) in the non-hierarchical analysis is lower than an average in the case of mountain species. The result was explained by the biological characteristics of the species: a tall plant (up to 2.5 m), producing a large number of small diaspores over long distances, can promote gene flow between geographically distant regions and result in low parameters of genetic isolation.The lack of genetic differences between populations in different regions is a common phenomenon in phylogeographic studies. For example, in a study on the genetic diversity of a subalpine perennial G. imbricatus could have existed in the Western Carpathians. An example is the population Tatra JU, the most genetically diversified population forming a distinct genetic group among the populations that have been studied. A hybridogenous population was found in the Pieniny Mts, which are generally believed to be a glacial refugium, proving the dispersal from the Tatra Mts and the existence of the local genetic melting pot. The other dispersal from the Tatra Mts refugium was also traced adjacent to the Gorce Mts. The unexpected genetic links found between the remote populations of G. imbricatus, namely the Pieniny/Tatra and Bieszczady populations, could be explained by long-distance dispersal linked with the transportation of sheep flocks between the Tatra and Bieszczady Mts in the second half of the previous century. The migrations of flocks were accompanied by the plants (diaspores), which facilitated the mixing of hitherto isolated populations. In effect, a lowering of the values of genetic divergence between distant populations of G. imbricatus was noted. In this way, the genetic links between plant populations in close or distant mountain ranges could be affected by the recent, stochastic processes leading to the decreasing values of genetic divergence indices. In conclusion, the role of many centuries of agro-pastoral human economic activity in the migration of the plant species seems, in some cases, crucial.Summing up, glacial refugia of


Reconquista by the Catholic kingdoms of the north.Historical records document medieval immigration from North Africa to Iberia to create Islamic al-Andalus. Here, we present a low-coverage genome of an eleventh century CE man buried in an Islamic necropolis in Segorbe, near Valencia, Spain. Uniparental lineages indicate North African ancestry, but at the autosomal level he displays a mosaic of North African and European-like ancestries, distinct from any present-day population. Altogether, the genome-wide evidence, stable isotope results and the age of the burial indicate that his ancestry was ultimately a result of admixture between recently arrived Amazigh people (Berbers) and the population inhabiting the Peninsula prior to the Islamic conquest. We detect differences between our sample and a previously published group of contemporary individuals from Valencia, exemplifying how detailed, small-scale aDNA studies can illuminate fine-grained regional and temporal differences. His genome demonstrates how ancient DNA studies can capture portraits of past genetic variation that have been erased by later demographic shifts—in this case, most likely the seventeenth century CE expulsion of formerly Islamic communities as tolerance dissipated following the  Prehistoric populations have been the focus of most of the ancient DNA (aDNA) work published on Iberia so far, including the study of Mesolithic individuals10, the impact of Neolithic dispersals12, and the incursions of individuals with Steppe-related ancestry at the time of the transition from the Chalcolithic to the Bronze Age15.The location of Iberia, bridging the Mediterranean and the Atlantic, and its proximity to Africa, has allowed contacts with populations of distinct ancestries over time, making the Peninsula a genetic and cultural crossroads. There is both archaeological and direct genetic evidence of contacts between Iberia and North African populations since at least the Late Neolithic6. Although Iberia intensified contacts with North Africa through Phoenician traders, Carthaginians and Roman conquerors16, North-African genetic contribution seems to have been restricted to southern populations until the eight century CE6. It is only with the Islamic conquest of Iberia in 711 CE that records start pointing towards a substantial influx of people from North Africa, involving the culturally and genetically differentiated Arab and Amazigh (Berber) peoples18. Attempts have been made to estimate their contribution to the genetic landscape of medieval Iberia using modern genomes, revealing a faint southwest-northeast pattern of decreasing North African-related ancestry20, which have recently been confirmed by means of aDNA analysis6.aDNA researchers have recently begun to explore in detail historical intervals of known population movements21. Berbers had converted to Islam as a result of the Arab conquest of North Africa in the preceding century and embarked in a slow and complex process of Arabisation that lasted centuries. However, they were far from culturally homogeneous; a deep division existed between nomadic and sedentary Berber groups, and it was the latter who first settled in the rural areas of Spain18. Although Berber numbers in Iberia were likely larger than those of the Arabs, they initially wielded no significant political power, but this changed during the eleventh–thirteenth centuries CE with the establishment of the Almoravid and Almohad Berber empires18.Although Arabs were the urban and political elite during the Umayyad Caliphate, ruling from 711 CE until the end of the Caliphate of Cordoba in 1031 CE, they are thought to have been a minority amongst the new settlers. Berbers formed the bulk of the army who first seized Visigothic Spain in the eighth century CEMoriscos (Muslims forcibly converted to Christianity) persisted in East Iberia  until 1609 CE, when at least one third of the populace was forcibly expelled by the Spanish Crown and relocated to North Africa22. Historical documentation suggests that the population of the eastern Mediterranean provinces of Castellón, Valencia, Alicante, and—to a lesser degree—Murcia and parts of Andalusia (Almeria and Granada), was greatly reduced, with subsequent resettlement from Aragon, Catalonia and Navarre to avoid economic and demographic collapse23. Many surnames currently widespread in the Valencian region are geographically structured and reflect their provenance from the colonizing regions  of Plaza del Almudín in the city of Segorbe   who was buried in the Islamic RY > 0.077; Supplementary Fig. 28). Although U6 in general, and U6a in particular, is present in higher frequencies in North and West Africa30, the complete mitochondrial genome dataset currently available is heavily biased towards Europe, and U6a1a1a, which dates to 3.5 thousand years ago (ka) (maximum-likelihood node estimation based on modern variation), appears to have a more southern European distribution 2  is the earliest documented finding of a U6 lineage in Iberia. Based on the results of our newly generated Iberian mitochondrial dataset , U6a can be found at a frequency of 1.6% in modern mainland Iberian populations, with a peak of 3.6% in the south of Spain   and peaks in modern Berber populations, with frequencies reaching > 80%41, being almost fixed in some groups, such as the southern Moroccan Tachlhit-speakers42 and the Chenini–Douiret and Jradou from Tunisia40. In Europe, it is found mostly in Iberia and Sicily at frequencies < 5%43.We assigned UE2298/MS060 to the Y-chromosome haplogroup E1b1b1b1 (E–M310) , using Iberia_IA, Levant_BA and Morocco_LN/Guanches as reference populations   Fig. .Figure 3f3 runs using different outgroups  consistently show a higher proportion of shared drift with Middle/Late Neolithic, Chalcolithic and Bronze Age Iberian populations, and with the Anatolian Neolithic , consistently generated results close to zero and non-significant (|Z|-score < 3), which might be an indicator that a population genetically close to Morocco_LN contributed to the ancestry of UE2298/MS060 in similar proportions to an Iberian source. We note that we did not observe any major differences in the patterns observed for outgroup-f3 and D-statistics using different approaches to minimise the effects of post-mortem damage   are accepted , whereas models using populations from Catalonia, in the northeast of the Peninsula, are rejected   but not from Islamic_Valencia , in line with the position of UE2298/MS060 in the PCA  and either the Guanches or Morocco_LN (Table D-statistics comparing these two North African populations indicate that UE2298/MS060 is closer to Morocco_LN (|Z|> 3)  range from 26.2 to 27.6‰ , with a mean of 26.8 ± 0.5‰ (1σ). The converted δ18Odw values  fit with the meteoric water values for eastern Iberian coast. The δ18OVSMOW values from both teeth sampled from UE2298/MS060 are consistent with the rest of the population and the small difference in values between the different molars (M1/M2 and M3) provide no indication of movement between early childhood and adolescence. Overall, there is no evidence that UE2298/MS060 was an immigrant in East Spain, on the basis of his oxygen values.In order to assess whether or not UE2298/MS060 was likely to have spent their childhood in the local region, we performed stable oxygen analysis on eight individuals from Plaza del Almudín. Tooth enamel carbonate data is presented in Supplementary Table 18OVSMOW = 30.6; > 1.5 times the interquartile range above quartile 3)47, and possibly a migrant from a warmer climate, with a δ18Odw value similar to Africa or the Near East48. Detailed results and discussion of oxygen analysis can be found in Supplementary Note 2.By contrast, one other individual reported here (MS075) seems to be an outlier , FRUITS 49, supports the observation that C4 plants likely played a substantial part in the diet of some individuals and that marine fish consumption was variable  and marine protein (mean: 2.4 ± 2.4% or 0–4.8% of the diet) compared to the rest of the population analysed. On the other hand, he seems to have the highest levels of mammal and C3-plant consumption amongst the analysed individuals  of marine fish consumption amongst the individuals studied here 30, but has also been found in Moroccan remains associated with Iberomaurusian culture, and in the Moroccan Early Neolithic site of Ifri n’Amr or Moussa32 , or derives from a population that moved into Iberia but retained its genetic identity. A second scenario is that he descends from pre-Islamic Iberian genetic diversity. Finally, the third scenario is that he is the result of admixture between Iberian and North African sources.6). The nearest temporal proxy available are the Guanches (from the seventh–eleventh centuries CE), who originated in the Maghreb but have been isolated in the Canary Islands since at least the early Iron Age. D-statistics, however, suggest that UE2298/MS060 is genetically closer to Morocco_LN than to the Guanches (Supplementary Table qpAdm rejects the hypothesis that UE2298/MS060 directly descends from a population resembling either the Guanches or Morocco_LN (Supplementary Table 48). Nevertheless, one should note that aDNA sampling in North Africa is sparse and limited to a few individuals from very specific sites and periods, and we cannot rule out that a population with a similar genetic composition to that of UE2298/MS060 existed in the region around this period.The first scenario would imply that pre-Islamic populations in North Africa would be genetically similar to UE2298/MS060 , with a slight southwest-to-northeast declineD-statistics support this scenario, with tests comparing Morocco Late Neolithic and Iberian populations from different periods not showing him to be significantly closer to one or the other 27 fits the historical narrative of Berber settlement in the region of Sharq al-Andalus18. Considering the genetic evidence, together with the stable isotope results and the historical accounts of intermarriage between local individuals and the North African newcomers, and in agreement with recent aDNA evidence from Iberia6, this third scenario seems the most plausible to explain the ancestry patterns seen in his genome.The third scenario would be that the genetic variation seen in UE2298/MS060 was a result of admixture between Amazigh people who migrated from North Africa to Iberia, and the local population inhabiting the Peninsula, at some point during either the Islamic conquest, the Caliphate period, or the Berber empires. This would explain UE2298/MS060's intermediate position in the PCA and ternary plot (supervised ADMIXTURE) Fig. . D-statiher Fig. . We showher Fig. . The fac33 and the Late Neolithic Moroccan population from Kelif el Boroud site2. There is high differentiation between present-day North African populations and ancient North African individuals available to date , or in a larger previously published HVS-I database (n = 123)56. This absence might be an echo of the brutality of the decree of expulsion of Moriscos (Muslims forcibly converted to Christianity), which may have effectively erased the population carrying North African-related ancestry that lived in the region in the preceding centuries. They were replaced by settlers from regions further north with little North African-related ancestry20. This is in sharp contrast with regions of the Crown of Castilla, where historical sources claim there was better integration of the Morisco identity into the general population, and where no mass deportations were recorded: the frequency of U6, M1 and L lineages are higher in these regions today   Fig. . This pa57. A contemporary individual, MS075, is evidence of continued movement during Berber rule (Supplementary Note 2).This study emphasises the importance of immigration during the Islamic period. In contrast to Andalusia, the region of Valencia is not geographically close to the Maghreb, and was under Islamic rule for a shorter time, but nonetheless developed strong links with the Arab–Berber world during the Islamic period6 show a similar trend of admixture in Islamic Spain. The heterogeneity of genomic patterns that is now being uncovered by aDNA studies emphasises the need for much more detailed, high-resolution fine-scale studies. More individuals and a wider diversity of sites across the Peninsula should be studied to explore the population dynamics during the Islamic period in more detail and assess potential fine differences between geographical regions and periods, and between urban and rural societies.UE2298/MS060 is a single, low-coverage sample and although the results cannot be extrapolated to the population as a whole, recently published results27   to screen for endogenous aDNA content, and later sequenced three additional libraries (one of which was non-USER treated) in half an Illumina HiSeq4000 lane (100 cycles) . We performed oxygen analysis and ZooMS  at the University of York, and dietary isotope analysis of carbon and nitrogen at the Research Laboratory for Archaeology, University of Oxford. Further details of ancient DNA, stable isotope and ZooMS analyses can be found in Supplementary Methods.58, and merged paired-end reads and removed sequencing adapters using leeHom59. We mapped reads both to the human genome reference  instead of chrM) and to only the rCRS with BWA v.0.7.5a-r40560aln (using the optimized settings for aDNA mapping61) and samse. We performed quality control of the alignment with QualiMap v.2.262 and confirmed aDNA authenticity by checking contamination estimates (schmutzi63 and ANGSD64) and post-mortem damage patterns  soft-clipping the terminal 3 base pairs of sequencing reads using the trimBam option in bamUtil package v. 1.0.1466, to control for potential reference bias resulting from downscaling base quality scores that could influence formal tests of admixture. We merged all libraries using picard MERGESAM (https://github.com/broadinstitute/picard). Detailed methods and parameters can be found in Supplementary Methods.We assessed raw read quality with FastQC v.0.11.567 to classify mtDNA haplogroups. We performed Y-chromosome haplogroup classification using Yleaf68, and checked mutations against the ISOGG  SNP index (as of June 2018). We used pathPhynder44 (https://github.com/ruidlpm/pathPhynder) to investigate the affinity of individual UE2298/MS060 with present-day Y chromosomes46. More details on the analysis of uniparental markers of UE2298/MS060 can be found in Supplementary Methods.We used HaploGrep 2.0https://reich.hms.harvard.edu/) using samtools mpileup and pileupCaller (https://github.com/stschiff/sequenceTools). We used convertf and mergeit (both included in EIGENSOFT v.7.2.1 package69) to merge and convert files when necessary.We called pseudo-haploid autosomal SNPs  of ~ 600 k autosomal SNPs was performed using smartpca (EIGENSOFT v.7.2.1) to project 336 ancient samples  in PLINK v.1.0770. The LD pruned dataset (~ 450 k SNPs) was used to run ADMIXTURE v.1.3.071 for post-Iron Age Iberian individuals (shown to display different levels of North African and Levantine-associated ancestries6) in supervised mode for K = 3 (with parameters: --cv and --seed time), using Iberia_IA, Morocco_LN/Guanches and Levant_BA as reference populations (Supplementary Table We filtered the ancient dataset for positions in linkage disequilibrium (LD) using the command 72), and ran the tests in the two datasets . We examined patterns of allele sharing between UE2298/MS060 and present-day and ancient populations using outgroup-f3 statistics, as implemented in qp3Pop, testing three outgroups  to account for deeply divergent human ancestry. We computed D-statistics (using chimpanzee and Mbuti as outgroups) with qpDstat to untangle Iberian and North African-related contributions. For a more refined analysis, we ran a test with the formula D. In order to investigate admixture proportions in the genome of UE2298/MS060, we ran qpAdm (ADMIXTOOLS v.4.1), using allsnps: YES and testing 1- and 2-way models. Following 2-way qpAdm results, we ran a D-statistics test in the form D. All plots were created with RStudio73. Detailed methods and parameters can be found in Supplementary Methods.We added outgroups to the ~ 1.2 M SNP dataset for the formal tests of admixture  with Illumina MiSeq paired-end sequencing (size of fragment: 150 bp) . A detailed description of the long-range PCR protocol, sequencing and data analysis can be found in Supplementary Methods.We performed a reassessment of phylogeographic patterns of mtDNA haplogroup U6 based on a total of 330 modern (35 of which are newly published here) and 32 ancient sequences (including UE2298/MS060) . For the present-day dataset written informed consent was obtained from all sample donors. The research was performed in accordance with the relevant guidelines and regulations and was approved by the University of Huddersfield’s School of Applied Sciences Ethics Committee, the Ethical Committee of the University of Santiago de Compostela, the Ethics Committee for Clinical Experimentation of the University of Pavia and the Ethics Committee of the University of Minho. Portuguese modern samples (PT-codes) were collected among army volunteers, upon approval of the Portuguese Army Chief of Staff, and were fully anonymized with the single purpose of use for population studies.Supplementary Information.Supplementary Figure S4.Supplementary Tables.


Lycaon pictus, APD) are highly endangered, with fewer than 7000 remaining in nature. Captive breeding programs can preserve a genetically diverse population and provide a source of individuals for reintroductions. However, most programs are initiated from few founders and suffer from low genetic diversity and inbreeding. The aims of this study were to use molecular markers to assess genetic variation, inbreeding, and relatedness among APDs in the North American captive population, to use these data to realign studbook records, and to compare these data to wild populations and to the European captive population to facilitate the development of a global management plan. We sequenced mitochondrial and major histocompatibility (MHC) class II loci and genotyped 14 microsatellite loci from 109 APDs from 34 institutions in North America. We identified three likely studbook errors and resolved ten cases of uncertain paternity. Overall, microsatellite heterozygosity was higher than reported in Europe, but effective population size estimates were lower. Mitochondrial sequence variation was extremely limited, and there were fewer MHC haplotypes than in Europe or the wild. Although the population did not show evidence of significant inbreeding overall, several individuals shared high relatedness values, which should be incorporated into future breeding programs.African painted dogs ( Lycaon pictus), APD, also known as the African wild dog or Cape hunting dog, is a highly endangered canid species facing mounting pressures across its extant populations. The global free-ranging population is estimated at approximately 6600 adults, of which only 1400 are mature, i.e., capable of reproducing during any given breeding season, and their numbers are declining [The African painted dog  population of APD was established in 1955, when the first pair of dogs was imported to the San Diego Zoo  is maintf = 0.0312), and effective population size (eN = 26.452), and predicted the loss of gene diversity over time [A Population Analysis & Breeding and Transfer Plan  was conver time . The curver time  represenver time . While tA potential source of error in the PABTP analyses  comes frThe utility of this integrated approach has been demonstrated in the European population of APD. Marsden et al.  combinedAlthough studbooks are utilized in all managed ex situ populations of APD, to date, molecular genetic analyses have been conducted only in the European population. However, effective implementation of a global management plan will require the integration of both genetic data and studbook information from holding institutions in each country. It is also important that similar molecular markers are examined in each population to ensure that the data are directly comparable among populations. The primary aims of this study were (i) to use molecular markers to assess genetic variation, inbreeding, and relatedness among APD in the NA captive population, (ii) to use these genetic data to inform and, where necessary, realign studbook pedigree records, particularly in cases of uncertain or unknown parentage, and (iii) to compare these genetic data to those obtained previously for wild populations ,18,30,31From 2012 to 2017, we sent genetic sampling kits to 37 SSP institutions holding APD in NA, with a request to collect whole blood samples opportunistically during routine veterinary procedures. Samples from 109 APD  were subsequently obtained from 34 institutions, representing 80% of the current population. Blood was collected on FTA Elute collection cards  according to the manufacturer’s instructions, and FTA cards were stored and shipped at room temperature. DNA was extracted from the FTA Elute cards according to the manufacturer’s instructions, eluted into 180 μL 1× Tris–EDTA, and stored at either 4 °C or −20 °C. We included negative controls with DNA extractions to monitor for contamination.To enable comparison with previous work ,30,31,32We amplified a 308 base pair (bp) fragment of the mitochondrial D-loop control region I from each sample using canid-specific primers, ThrL and DLH, which were redesigned specifically for APD . Each 25Endangered species research has increasingly included assessments of variation at the MHC loci due to the adaptive role that the MHC plays in the immune response to pathogens ,37,38,39Sequences were manually edited, trimmed to 340 bp, and aligned by eye using CodonCode Aligner (CodonCode Corporation). To facilitate reconstruction of phased haplotypes, we cloned the DLA-DRB1 locus from ten heterozygous individuals. We ligated amplified sequences into pJet1.2/blunt cloning vectors using the CloneJET PCR cloning kit (ThermoFisher Scientific) and subsequently transformed them into DH5α chemically competent cells (ThermoFisher Scientific), according to the manufacturer’s instructions. We randomly selected ten clones from each individual and sequenced them in both directions at the Pennsylvania State University Genomics Core Facility, University Park, PA. Resulting sequences were manually edited and aligned using CodonCode Aligner (CodonCode Corporation).To assess neutral genetic diversity, we genotyped each individual at 14 physically unlinked microsatellite loci, which we amplified in five multiplexes: (1) Pez12, FH2054, FH2611, (2) Pez8, FH2010, (3) FH3399, FH3965, (4) FH2785, FH2658, Pez15, and (5) CXX155, CXX250, CXX263, and CXX366 ,45,46,47https://doi.org/10.5061/dryad.xksn02vgp).Allele sizes were called using GeneMarker software , based on a known DNA size standard . Each locus was amplified independently two to four times to confirm each genotype, and putative homozygotes were accepted only if the same single allele was observed in at least three independent PCRs. Thereafter, a consensus multilocus genotype was generated for each individual. Consensus microsatellite genotypes were submitted to Dryad (k), and haplotype (gene) diversity (dH). Haplotype frequencies were estimated with Arlequin v. 3.5 [We estimated the most appropriate model of nucleotide substitution for the D-loop sequences using jModelTest 2 ,50. Thisn v. 3.5 . D-loop n v. 3.5  with thon v. 3.5 ,30,31, and the average number of pairwise nucleotide differences per site (k) using DnaSP v. 6.12 [D* statistic, which compares the number of singleton mutations to the total number of variants in a genomic region [OH) and expected heterozygosity (EH) in Arlequin v. 3.5 [ISF (ISF = 1-O/HEH), which is the proportion of genetic variance in a subpopulation that is found within an individual. Negative ISF values (indicative of an excess of observed heterozygotes) and values close to zero are expected in outbred populations and under random mating, respectively, whereas values close to one suggest inbreeding [We estimated the most appropriate model of nucleotide substitution for the DLA-DRB1 sequences using jModelTest 2 ,50. This v. 6.12 . We also v. 6.12 , which cc region . We estin v. 3.5 , and we breeding .(ID)P, the probability that two unrelated individuals drawn at random from a population will have the same multilocus genotype) and the probability of identity for siblings ((ID)sibP, the probability of observing identical multilocus genotypes in siblings). We quantified genetic diversity within the NA population of APD by estimating the average number of alleles per locus (AN) in GenAlEx, and the average number of alleles per locus adjusted for sample size  using FSTAT [OH), and mean expected heterozygosity (EH) based on Hardy–Weinberg assumptions using Arlequin v. 3.5 [IS (FISF = 1 − O/HE).HWe used MicroChecker  and Geneng FSTAT , as welln v. 3.5 . We alson v. 3.5  to estimr) using both the maximum-likelihood method employed by ML-Relate [r, is the probability that two individuals share an allele that is identical by descent, i.e., they inherited the same allele from a common ancestor. Unrelated individuals have r values close to zero, half-siblings share 25% of their genes (r ≈ 0.25), whereas full siblings and parent–offspring dyads share on average 50% of their genes (r ≈ 0.5). Values of r closer to 1 indicate higher than expected levels of relatedness, suggesting that those individuals are inbred, i.e., their parents were closely related. We estimated the most likely relationships between individuals  using ML-Relate, and implemented 100,000 randomizations to estimate 95% confidence intervals for putative relationships. We then compared both r-values and putative relationships between pairs of individuals identified by ML-Relate with those listed in the studbook. ML-Relate does not identify grandparental or avuncular relationships (r ≈ 0.25), or first cousins (r ≈ 0.125). Instead, these would likely be categorized as half-siblings or unrelated based on their r-values. We therefore considered these two ML-Relate categories to be consistent with studbook records for individuals that were first cousins or that shared a grandparental or avuncular relationship. We compared pairwise relatedness (estimated by ML-Relate) among relationship categories recorded by the studbook  using a one-way ANOVA and a post hoc Tukey honestly significant difference (HSD) test.We estimated pairwise relatedness . The average number of nucleotide differences, k, was 1.473. There were only three polymorphic sites, all transitions, in the 308 bp fragment, resulting in only two haplotypes, both of which have previously been identified in APD [The D-loop sequences had low nucleotide diversity (π(JC) = 0.00483 ± 0.00016 SD) and haplotype (gene) diversity  diversity, dH, was 0.796 ± 0.025. Both observed (OH) and expected heterozygosity (EH) were moderately high  and were not significantly different from each other (p = 0.279). The fixation index (ISF = 1 − O/HEH) was −0.093. Significantly positive values for both Tajima’s D  and Fu and Li’s D*  suggested that this region may be under balancing selection [The DLA-DRB1 locus was more variable than the D-loop as there were 30 polymorphic sites in the 340 bp fragment . In the translated amino acid sequence, there were 27 segregating sites. Nucleotide diversity, π, was 0.036 ± 0.001, the average number of pairwise nucleotide differences, election .The DLA-DRB1 haplotypes formed two divergent allele lineages (A and B) in the minimum spanning network . Haploty(ID)P = 1.9 × 10−14; 14 loci (ID)P = 3.5 × 10−15) and the probability of misidentifying siblings as the same individual (13 loci (ID)sibP = 6.2 × 10−6; 14 loci (ID)sibP = 2.9 × 10−6) were extremely low. The average allelic richness (SR) for all individuals (n = 109) sampled was 6.366 ± 2.598 SD (EH) and observed (OH) heterozygosities across all loci were both moderately high . The mean overall coefficient of inbreeding (ISF) was −0.108 ± 0.141 SD. Similar analyses limited to the same 10 loci used by Marsden et al. [t-test, p ≥ 0.147); thus, we present analyses of the full dataset. None of the loci showed significant evidence of null alleles, but several pairs of loci displayed significant linkage disequilibrium (LD). Multiple loci also deviated from Hardy–Weinberg equilibrium (HWE) expectations due to an excess of heterozygotes. However, these results are not unexpected because, while HWE and LD analyses assume independent lineages, this captive population consists of closely related family lines that, by definition, violate the assumptions of HWE and of an LD model. Furthermore, heterozygote excess can be indicative of a population bottleneck [A complete 14-locus genotype was obtained for 91 individuals (83.5%). A further 16 individuals 14.7%) were genotyped at 13 loci, and 2 individuals (1.8%) were genotyped at 12 loci. The probability of identity  was low . However, these included ten pairs of individuals that had relatedness values between 0.713 and 0.850, suggesting that their parents were closely related. Nine of these pairs were classified as full siblings in the studbook, and one pair shared an avuncular (uncle–niece) relationship. As described in the Methods, we considered ML-Relate categories of half-siblings and unrelated to be consistent with studbook records of first cousin, grandparental, or avuncular relationships. Allowing for these ambiguities, 96.4% of the relationships (5675 of 5886 possible pairwise comparisons) identified by ML-Relate were consistent with studbook records. The 3.6% that were inconsistent with studbook relationships were generally misclassified by ML-Relate due to unexpectedly high or low relatedness values between pairs of individuals. Overall, based on relationships identified by the studbook, mean relatedness , between full siblings , and between half-siblings  was similar to expected levels for each relationship category; however, the range of relatedness values was broad in each case. In contrast, mean relatedness between dogs sharing avuncular relationships  was higher than expected. Although avuncular relationships are expected to have relatedness values similar to those of half-siblings (approximately r = 0.25), relatedness between avuncular dogs in the APD was significantly higher than those identified as half-siblings in the studbook , but their relatedness did not differ from those listed as full siblings . Furthermore, first cousins are expected to have low relatedness values of approximately r = 0.125, but relatedness between dogs identified in the studbook as first cousins was higher than expected  and did not differ from that between half-siblings . These higher than expected relatedness values could be due to inbreeding, or errors in studbook records, or a combination of both.ML-Relate and Coancestry produced similar pairwise relatedness estimates. The overall Pearson’s correlation coefficient between methods was 0.959; therefore, only ML-Relate results are reported here. Average relatedness between all sampled APD  had similar, positive LOD scores and Delta values close to zero (meaning that neither could be excluded as the true mother), DLA-DRB1 haplotypes of both candidate mothers, the offspring, and the sire were known and were used to exclude one putative dam. The non-excluded candidate dam was consistent with the studbook record in both cases.r = 0–0.31) between the offspring and the parent listed in the studbook, and between the offspring and putative full siblings, than between the offspring and the alternative candidate parent (r = 0.35–0.54). Additionally, the combinations of dam and sire listed in the studbook were not possible based on the inheritance of DLA-DRB1 haplotypes identified in the offspring  than the NA population. Although the census size of the European population according to Species360 ZIMS (n = 247) is nearly twice that of the NA population, the estimated effective size of the European population was only 1.33–1.35× larger than that of the NA population, depending on the mating system assumed.We estimated the contemporary effective size of the NA population of APD as 17.7 (95% CI 14.7–21.3), assuming that the population is managed in a way that approximates random mating . This esListed as endangered by the International Union for Conservation of Nature , APD popThe PABTP  estimateAlthough several studies have examined the genetic diversity of APD in wild and managed populations in southern africa and east africa ,32,74,76Marsden et al.  identifin = 44) are descended from two female founders captured in 1998 in Botswana [n = 57) are descended from two females from South Africa, and 12% (n = 8) are descended from two females from Namibia [The high frequency of the Z1 haplotype in the NA population (~41%) is likely the consequence of a founder effect compounded by subsequent unequal founder contributions to subsequent generations. According to the AZA studbook , all theBotswana , where ZBotswana ,31, but  Namibia . Reprodu Namibia ,31 but w Namibia , as well Namibia ,77,78.OH = 0.67–0.84, EH = 0.66–0.86; east Africa range OH = 0.54–0.91, EH = 0.57–0.88), and to Europe  [ISF = 0.01) and for wild populations (ISF = −0.08), and suggests an outbred population [Balancing selection at the DLA-DRB1 locus appears to have resulted in more polymorphic sites and higher nucleotide and haplotype diversity than at the noncoding mtDNA D-loop sequences. Although nucleotide diversity was lower than that reported for wild populations in southern Africa and east Africa, which ranged from π = 0.051 to 0.076 , observe = 0.84) ,30,32. T = 0.84) ,30 for Epulation .n = 15, 54%) and Namibia , two were caught in Botswana, and three have unknown origins. The high frequency of the DLA-DRB1 haplotype B6 in the NA population, as well as the presence of haplotypes A2 and B11, which have not been found in east Africa [Previous work on APD  has showt Africa , are conEH = 0.73), but observed heterozygosity (European OH = 0.76) and allelic richness (European SR = 4.8) were both higher in the NA than in the European population [EH = 0.504 [AN = 3.4–4.4) and low expected heterozygosity (EH = 0.504–0.655). Tensen et al. [SR = 2.66–3.06) and expected heterozygosity (EH = 0.514–0.577) in the Lowveld region of South Africa, although they found higher genetic diversity in the nearby Kruger National Park . In contrast, Marsden et al.’s [SR = 4.31; EH = 0.61; OH = 0.62) to Hwange in Zimbabwe . Tensen et al. [SR and EH in these populations, which was supported by high private allelic richness in Kruger National Park [Both expected and observed heterozygosities at microsatellite loci were lower than the gene diversity (89.84%) estimated from the studbook for the PABTP . Howeverpulation . The NA  = 0.504 ), as wel = 0.504  examinedet al.’s ,74 estimn et al. , which inal Park ,30,74 arnal Park ,30,74 arNe) in captive and wild populations of APD using the linkage disequilibrium method of Waples and Do [Ne of the European population  is slign nature  but are .0–21.5; ). We notA primary goal of captive breeding programs is to maximize the preservation of the genetic diversity of the species as a whole, which requires that founders be sourced from a variety of populations and that the captive population is not dominated by a minority of genetic lineages from a single region . FurtherISF = −0.05) and for wild populations (ISF = −0.06) [ISF are also consistent with the low mean kinship (MK = 0.1016) and inbreeding coefficient (f = 0.0312) estimated for the PABTP [Overall levels of relatedness  and inbr= −0.06) . Mean rehe PABTP , althoughe PABTP . Neverthhe PABTP .r-values. In some cases, this could be due to erroneous parentage assignments (discussed below). We identified three cases in the studbook in which individuals assumed to be half-siblings were in fact full siblings, or individuals recorded as having an avuncular relationship were in fact parent and offspring. This could explain some pairs of half-siblings that shared r-values up to 0.58  and between nine pairs of full siblings (r = 0.713–0.840), suggesting that these individuals are inbred, i.e., share parents that are closely related. The studbook [r = 0.75 and 0.79) were full siblings themselves. Furthermore, the mean relatedness , she shared very high r-values with presumptive aunts and uncles (r = 0.56–0.85), suggesting that her parents were closely related . Furthermore, this female was heterozygous B6/B11 at the DLA-DRB1 locus, and the B11 haplotype was found in only 4% of sampled individuals (colored red in Over 90% of the parent–offspring trios predicted by Cervus, and over 96% of the pairwise relationships predicted by ML-Relate, were consistent with studbook records for those individuals, confirming the full sibships and parent–offspring relationships recorded in the pedigree. Furthermore, we were able to resolve with high confidence 10 of 11 documented cases of uncertain paternity. These occurred in two families, and, in both cases, we found that only one of the two possible males sired all the offspring within that family. However, we also identified several likely errors in the studbook records for both dams and sires, which have implications for future breeding and transfer planning, particularly as such errors may lead to underestimation of relatedness and inbreeding in the population inferred in the absence of genetic data. This problem is not unique to the AZA studbook, with similar discrepancies having been documented in the APD studbook in Europe , as wellIn conclusion, our analyses demonstrate the important role of direct genetic information in managing captive populations, with the goal of maximizing genetic diversity, minimizing inbreeding, and supplying individuals for reintroduction into the wild. Our analyses confirmed the majority of relationships described in the AZA studbook, but documented significant skew in the reproductive contribution of founders, leading to high levels of inbreeding in some lineages and loss of founder diversity, particularly at D-loop and MHC loci. We were also able to identify some errors in the studbook and clarify most instances of uncertain parentage, and we recommend that updates be made to both the studbook and 360Species ZIMS database in accordance with our findings. Remaining uncertainty stems mainly from a lack of full sampling of the NA population of APD; we encourage full participation of NA institutions housing APD in genetic analyses as a means of achieving the goals of captive breeding of APD in NA. In our study, microsatellites proved the most informative markers, followed by MHC, whereas D-loop provided the least information. Therefore, if time and funds are limited, we recommend that other institutions focus primarily on genotyping the same microsatellite markers, which would facilitate comparisons among studies of wild and captive populations. If additional data are required—for example, to help resolve uncertain parentage—sequencing of a nuclear locus could also be employed. Use of the DLA-DRB1 locus used here would facilitate comparisons among studies; however, other than finding that this locus is likely under balancing selection, it provided no further information about adaptability, for which it has purportedly been examined in APD  and othe


Biological invasions, the establishment and spread of non‐native species in new regions, can have extensive economic and environmental consequences. Increased global connectivity accelerates introduction rates, while climate and land‐cover changes may decrease the barriers to invasive populations spread. A detailed knowledge of the invasion history, including assessing source populations, routes of spread, number of independent introductions, and the effects of genetic bottlenecks and admixture on the establishment success, adaptive potential, and further spread, is crucial from an applied perspective to mitigate socioeconomic impacts of invasive species, as well as for addressing fundamental questions on the evolutionary dynamics of the invasion process. Recent advances in genomics together with the development of geographic information systems provide unprecedented large genetic and environmental datasets at global and local scales to link population genomics, landscape ecology, and species distribution modeling into a common framework to study the invasion process. Although the factors underlying population invasiveness have been extensively reviewed, analytical methods currently available to optimally combine molecular and environmental data for inferring invasive population demographic parameters and predicting further spreading are still under development. In this review, we focus on the few recent insect invasion studies that combine different datasets and approaches to show how integrating genetic, observational, ecological, and environmental data pave the way to a more integrative biological invasion science. We provide guidelines to study the evolutionary dynamics of invasions at each step of the invasion process, and conclude on the benefits of including all types of information and up‐to‐date analytical tools from different research areas into a single framework. BecauseGenome‐scale datasets have triggered the development of new analytical methods able to deal with large amount of information. Here, we provide an overview of these methods for addressing (i) population structure and colonization routes, (ii) demographic inferences, (iii) local adaptation, and (iv) landscape genetics, the type of data used and the software names, and their utility and their limits Table . These mWhile HTS was revolutionizing population genomics, geographic information systems were revolutionizing ecology. Species distribution models . We discuss the limits and advantages of each of these approaches, from data collection to analytical methods, highlight their complementarity, and identify gaps in our knowledge that need to be filled in order to reach a fully integrative understanding of biological invasion. We structured our review according to the chronological steps of the invasion process although we are aware that assigning the invasive steps can be challenging or may vary in space for a given species.Here, we provide an overview of the demo‐genetic and adaptive processes involved at each step of biological invasions, highlighting the approaches and type of information that are needed to fully describe the invasion process, including historical records (detection time series), demographic inferences , phenotypic measures , environmental characterization of source versus invaded areas (niche comparisons), identification of traits and genes involved in adaptation to the novel environment, and their evolutionary history  and then to Europe  identify the most likely source population(s) (colonization routes), (ii) describe the introduction dynamics: levels of genetic diversity in introduced populations and their source(s) or bottleneck intensity (demographic inferences), and (iii) examine the relationship between introduction modalities, the changes in demographic features, and the successful establishment (phenotypic experiments) Box .BOX 2H. axyridis has been intentionally introduced for biological control establishing wild populations in America, Europe, and Africa. Historical data on biocontrol practices and the reconstruction of colonization routes from genetic data demonstrated a bridgehead scenario, involving the colonization of NAM from both Eastern and Western Asia (admixture event 1) that served as the source for the South American (SAM), South African (SAF), and European (EU) invasions  reveals a reduced number of haplotypes among invasive populations as compared to the native ones  the introduction history and better understand the role of admixture (heterosis) and inbreeding (purge of genetic load). F1 hybrids display higher  and/or spread farther or faster (geographic distance or occupied area) than low‐diversity ones? Concerning the evolution of fitness in introduced populations, the genetic load can be estimated from genomic data  only successfully established populations are observed, and (ii) demographic parameters such as population growth or bottleneck intensity are hardly measurable in the field. For example, quantifying population growth in natural populations would require tedious MRR experiments as soon as new arrivals have been detected and over several generations and then compare the outcome of each detection with the reconstructed invasion history (number of sources and founding diversity). Experimental introductions manipulating the genetics and demography simultaneously have shown that establishment success  increases with the density and/or diversity of founders but depends on the species  and Japan (JAP) (admixture event 1) that served as the source for the initial colonization of North Italy (NIT), and independent introductions from CHI in Albania  and Central Italy (CIT) (admixture event 2)  to human‐modified  environments within its native range, and artificial containers are the mode of its introduction worldwide. Because the realized niche also includes biotic interactions, the absence of parallels between climatic distribution in invaded and source ranges may reflect competitive interactions setting each range limits  before introduction by studying adaptive variation within the source range  and insecticide resistance alleles being preferentially transferred from a species to another  or using estimation of genomic admixture rates as a component of demographic history (colonization routes). However, a large number of tools, based on genetic differentiation, local ancestry inference, or phylogenetic relationships, have been specifically developed to detect recent introgression and to determine the population of origin of introgressed fragments Table . Introgr4.3The functional relevance of genes identified using landscape genomics remains unknown until the observed genetic changes have been linked to phenotypic changes of the selected trait. Establishing this causative link represents one of the greatest challenges in invasion genetics. First, it requires experimental settings in order to disentangle phenotypic plasticity from adaptive evolution in natural populations. The role of phenotypic plasticity in biological invasions has been reviewed elsewhere  as compared to neutral genetic differentiation (FST) and phenotypic differentiation (QST) has been largely used to detect the loci targeted by selection . This approach requires identifying the core and edge populations, which is difficult in the absence of dated introductions but genetic diversity gradient along the expansion axis can inform about the location of ESI  and regional  models and multiple environmental predictors (climate and land use) can significantly improve invasive range predictions especially for ongoing expansion violating the equilibrium assumption of SDM  , as demonstrated by landscape genetics surveys in At the local scale, the genetic structure of populations can partly reflect natural dispersal, which can be influenced by geographic distance, by environmental barriers, or by resistance of land‐cover features. For example, testing land‐cover features as a barrier or corridor for gene flow between populations, Sherpa et al.  demonstr5.4Callosobruchus maculatus and Tribolium castaneum . Historical data are still under‐represented in invasion studies because early detection of introductions and careful monitoring of further expansion are challenging and costly. However, SDM might not be conclusive if based on a limited number of occurrences. Participative citizen science projects represent an interesting alternative to monitoring structures, and will potentially provide huge occurrence datasets, providing the invasive species is easily identified, fueling distribution and evolutionary models  and of changes overtime in human activities in ecological studies would allow to go a step further in our capacity to predict future invasive range expansions. Developing new methods explicitly incorporating different types of data rather than performing separately different types of analyses represents the future challenge in invasion science.We thank Nicolas Bierne and three anonymous reviewers for helpful comments on a previous version of the manuscript.The authors declare that they have no conflict of interest.Appendix S1Click here for additional data file.Table S1Click here for additional data file.


Abrothrix hirta, distributed from 35° S in Chile and Argentina to northern Tierra del Fuego. Based on 119,226 transcriptome-derived SNP loci from 46 individuals of A. hirta, we described the geographic distribution of the genetic diversity of this species using a maximum likelihood tree, principal component and admixture analyses. We also addressed the demographic history of the main intraspecific lineages of A. hirta using GADMA. We found that A. hirta exhibited four allopatric intraspecific lineages. Three main genetic groups were identified by a Principal Component Analysis and by Ancestry analysis. The demographic history of A. hirta was characterized by recent population stability for populations at the northernmost part of the range, while southern populations experienced a recent population expansion.Genetic information on species can inform decision making regarding conservation of biodiversity since the response of organisms to changing environments depend, in part, on their genetic makeup. Territories of central-southern Chile and Argentina have undergone a varying degree of impact during the Quaternary, where the response of local fauna and flora was rather species-specific. Here, we focus on the sigmodontine rodent  Decision making aimed to conserve biodiversity requires information on several aspects of the biological system to be protected. In particular, genetic information can be key when conservation efforts are made to ensure the survival through time of natural populations and the processes in which they are involved . The resThe biota of southern South America (south of 38°S), roughly corresponding to Patagonia, has been subjected to marked climate changes, glaciations, volcanism, tectonics, and seashore shifts over the past thousands of years . Those hAbrothrix hirta is a sigmodontine rodent that was revalidated as a distinct species when it was removed from the synonymy of Abrothrix longipilis , PolyATract mRNA Isolation System II, and TruSeq RNA Sample Preparation Kit, respectively, following 1. Thresholds for minimum quality and read length were set to 20 phred and 25 bp, respectively. Reads derived from ribosomal RNA (rRNA) were excluded from the dataset; these reads were identified mapping trimmed reads using Bowtie version 2.2.6  contains 119,226 SNP loci from all 46 sampled individuals of Abrothrix hirta (A. hirta (Abrothrix longipilis (UACH 8097), A. manni (UACH 7876), and A. sanborni (UACH 8322); the last three were used to form the outgroup, since they are the most closely related species to A. hirta  was calculated using dataset 2, where a single value was obtained for each locality group (G1–G7). To calculate Pi, the average number of nucleotide differences between two randomly chosen samples from within groups of populations was estimated using PopGenome ; this nuAbrothrix hirta, a maximum likelihood tree was inferred using dataset 3 and IQ-TREE  conducted with Plink 1.9 , calculated using dataset 2, and geographic distances among one representative of each group of localities and estimated in decimal degrees using the package sp .5. A projection of 8, 8, 8 was used to generate a joint sfs file, which was the input file for the demographic analysis using GADMA  implemented by GADMA was used to find the best-fit parameter values in order to automatically infer the best demographic model from the joint sfs data. Composite Likelihood Akaike Information Criterion (CLAIC) was used to choose the best model considering the three populations in one.Here the units of analysis were the four uncovered main lineages (see below). One of these  of Fst between pairs of groups (G1-G7) and their geographic distances  in the dataset is K = 3, which yielded the lowest value of cross-entropy . A schemA. hirta for which more than one specimen was included in the analysis, involves periods of population size stability and change, as well as events of gene flow between lineage pairs; it is described below in six-time intervals  remained stable, while in the south (ca. 43°S–54°S), populations have expanded. The Maule and Valdivian lineages maintained their effective population size over the last ca. 15 Ky . In a neOligoryzomys longicaudatus  are included under the synonymy of A. hirta. Our geographic coverage is limited and as such, it does not allow a proper association between available names and our identified lineages. Further studies on morphological and genetic variation are required to address this issue.The genetic patterns depicted in this study identified distinct lineages  among whPhylogeographic and population genomic studies provide the way to uncover the geographic distribution of genetic diversity and the demographic history of target species. As early stated by Abrothrix hirta in Argentina is considered as of low concern  repository, accession numbers The collection of some Chilean specimens was authorized by the Servicio Agrícola y Ganadero ; collection procedures were approved by the Universidad Austral de Chile (permits 04/11 and 296/2017). Other studied specimens were already housed in museum collections.LV conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and tables, authored and reviewed drafts of the manscript, and approved the final draft. GD’E conceived and designed the experiments, authored and reviewed drafts of the manscript, and approved the final draft. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BP declared a past co-authorship with one of the authors, GD’E, to the handling editor.