Coenonympha macromma and C. gardetta, which can be found flying together and hybridizing in narrow contact zones in the southern French Alps. We characterized the genomic composition of individuals, their morphology and their local habitat requirements, within and around a hybrid zone. Genetic diversity analysis at 794 SNPs revealed that all individuals within the hybrid zone were highly admixed, which was not the case outside the hybrid zone. Cline analysis showed that, despite ongoing hybridization, 56 out of 122 loci differentially fixed or nearly so between the two species were impermeable to introgression across the sharp hybrid zone (9 km wide). We also found concordance in cline position and width among genetic, morphological and environmental variation, suggesting a coupling of different reproductive barriers. Habitat characteristics such as the presence of trees and shrubs and the start of the growing season were strongly associated with the genetic variation, and we found evidence of divergence at genetic markers associated with morphology and physiology, putatively involved in visual or environmental reproductive isolation. We discuss the various behavioural and ecological factors that might interplay to maintain current levels of divergence and gene flow between this species pair.Identifying the mechanisms involved in the formation and maintenance of species is a central question in evolutionary biology, and distinguishing the selective drivers of populations’ divergence from demographic processes is of particular interest to better understand the speciation process. Hybrid zones are recognized to provide ideal places to investigate the genetic architecture of speciation and to identify the mechanisms allowing diverging species to maintain their integrity in the face of gene flow. Here, we studied two alpine butterfly species,  Coenonympha gardetta is a typical alpine species, largely distributed at elevations above 1,500 m along the Alps, the French Massif Central and the Balkans, whereas C. macromma is restricted to the south part of the French Alps, where it replaces C. gardetta. The two species are found flying together in narrow zones where their distribution ranges abut. Hybrids are relatively common in these contact zones, which highlights incomplete reproductive isolation between species, even if they remain highly differentiated genetically at a large geographic scale . Indeed, in a previous study  and that it exhibits intermediate wing morphology and ecological preferences (climatic niche). Statistical modelling of different past evolutionary scenarios allowed us to confirm the hybrid origin of C. macromma and estimate its emergence at around 12,000–20,000 years ago. Investigating the patterns of species divergence and its drivers in this clade would further provide more general insights into the process of homoploid hybrid speciation  and C. macromma (south) overlap. Elevation and habitat change markedly along this transect, from mid‐elevation semi‐closed vegetation habitats (i.e. a high coverage by shrubs and trees) at P1, to high‐elevation open vegetation (grasslands) at P11. The 9 sites sampled outside the contact zone have individuals with wing traits characteristic of only one of either parental species . The obtained DNA reads (~100 million of 2 × 125 paired‐end reads) were used to call SNP genotypes with the STACKS pipeline , a minimum coverage of 4 to create a stack (‐m 4 in ustacks function) and a maximum of 6 different nucleotides to merge two different stacks (‐M 6). Highly repetitive stacks and overmerged tags were dropped using both the “Removal algorithm” and the “Deleveraging algorithm.” Furthermore, a maximum of 8 mismatches was allowed for considering two individual tags as the same locus and to merge them in the catalogue (‐n 8 in the cstacks function). In order to evaluate the experimental success for each library and each individual, we used ProcessMyRAD scripts  using a range of M values. By looking at the number of assembled RAD tags across M values, it provides a way to find a value allowing assembling a maximum of loci without stacking too many paralogs. M = 6 was chosen because values above this threshold did not substantially increase the number of assembled loci within individuals . A data set of single nucleotide polymorphisms (SNPs) was produced using a double‐digested restriction site‐associated DNA (ddRAD) sequencing procedure using a modified version of the protocol of Peterson, Weber, Kay, Fisher, and Hoekstra  as descrs Figure .C. gardetta, pure C. macromma and contact zone) (parameters ‐r 0.4 and ‐p 3 in the populations function of STACKS). Only one polymorphic site was randomly kept for each polymorphic RAD fragment to avoid inclusion of physically linked SNPs. Finally, SNPs with a minor allele frequency lower than 1%  were removed from the data set.To avoid an excess of missing data, all further analyses were performed using only RAD fragments genotyped for more than 40% of the samples of each of the three types of population (pure 2.3that is observed heterozygosity (Hobs), gene diversity (Hexp) and Fis, were calculated for populations with at least 5 samples, using the function “basic.stats” of the hierfstat R package . Population structure across the whole study area was assessed using sNMF, a constrained method of genetic clustering  in order to estimate the probability of assignation of each individual to the two parental species genetic groups (C. macromma and C. gardetta). We assumed that the pure parental genetic makeup was correctly inferred in the analysis by using numerous (20 C. macromma and 39 C. gardetta) individuals from species‐specific allopatric populations  and five possible ways to fit tails . To ensure that the likelihood surface was correctly explored, we conducted three independent runs for each model, each run consisting of 20,000 iterations following an initial burn‐in phase of 500 iterations. For each locus, we retained the model with the lowest Akaike information criterium (AIC) and extracted from this model the parameters for cline width (w) and centre (c) estimates. We also used the difference between the observed minimum and maximum allelic frequencies (∆P) to estimate the slope (m) of each cline using the following equation: m = (∆P/w)  on the superimposed coordinates and used the two first PCs to visualize and test for the potential divergence between parental populations of C. macromma and C. gardetta. We kept only the two first PCs for each trait because they gathered in each case a high proportion of the morphological variation (see Results), and we wanted to stay focused on the strongest interspecific morphological divergences between parental species. The trait differences between parental populations were tested using a one‐way MANOVA on the first two PCs, with shape as the dependent variable and species as a factor. A wing size index was also obtained using the sum of the log‐transformed centroid sizes estimated independently for the fore‐ and hindwing landmarks. Difference in size among species was tested with Student's t test with size as dependent variable and species as factor. The generalized Procrustes analysis and the different statistics were performed in R with the packages Rmorph , ade4  remaining from the Procrustes superimposition of the eyespot landmarks as a proxy for eyespot positions, and the loadings of each individual along the first principal component (PC1) remaining from the Procrustes superimposition of the white band landmarks as a proxy for white band shape. PC1 represented most of the variation for the two last traits  and discriminated the parental populations (see Results). The seven models used were derived from the ones described for morphological traits in Derryberry et al. : annual mean temperature, annual precipitation, precipitation seasonality and temperature seasonality. Two habitat descriptors were obtained from the analysis of temporal series of NDVI  rasters from MODIS satellite imagery every 16 days from 2000 to 2015  in a pixel of 250 m × 250 m around each sampled population. For the eleven localities within the contact zone, we re‐estimated the proportion of high vegetation structures by sampling in each site three plots of 10 m2 where at least one Coenonympha individual was found. On these plots, we counted the number of times a tree or a shrub is present at each of the 60 points distributed each 30 cm along the two diagonals. Difference in environmental conditions between species was tested with Student's t test with each of the environmental variables as dependent variable and species as factor, using the environmental values of each pure species locations: 5 sites for C. macromma and 4 sites for C. gardetta . Again, we selected the best‐fit model based on AIC and recovered from it the cline centre, width and slope. The association between environmental variation and genetic composition of individuals was tested through independent linear regression with HINDEX as response variable and each environmental variable as explanatory variable. All the regressions were performed in R, and we recovered the strength of the association represented by the r‐squared of the linear correlation to compare them among variables.33.1M = 6  and uniform distribution of nucleotides along the read Figure . These d6 Figure . A total3.2C. macromma/C. gardetta contact zone were highly admixed, whereas the allopatric populations of each species showed genetically pure individuals  and P7 (0.5)  Table a.Hexp) and heterozygosity (Hobs) within the hybrid zone compared to pure populations of parental species , highlighting the fact that the 122 diagnostic loci, which are highly differentiated between the two species at large geographic scale, also show a strong turnover along the hybrid zone. A total of 56 of these loci even returned a cline with complete change in allelic frequency across the transect (∆p = 1), revealing that 46% of the species‐specific alleles analysed here are not exchanged between the two extremities of the hybrid zone. Furthermore, although 87% of the markers were fixed to 0 on the left side (i.e. “C. macromma side”) of the cline, only the fixed 56 reached 1 on the right tail (i.e. “C. gardetta side”). A subset of 23 loci exhibited a particularly sharp transition along the contact zone with an allelic frequency ranging from 0 to 1 in less than 3,000 m and a slope superior to 0.0005 , the alignment of eyespots and the shape of the white band on the ventral side of the hindwing   Figure . When ans Figure , with nog Figure .r2 = .46) than for eyespots (r2 = .25).Wing size returned a flat cline indicating random variations across the contact zone Figure . In cont3.5C. macromma and C. gardetta . Furthermore, all the environmental variables that distinguish the two parental species showed a strong cline across the contact zone, with ML estimates of cline centre close to those obtained for genetic markers and morphological traits   Figure . The wid44.1C. macromma and C. gardetta in the south of the French Alps . We characterized in this area a set of 11 populations showing a gradient of admixture between pure C. macromma individuals and nearly pure C. gardetta individuals , whereas a few show extremely strong values Figure b. In thePmax = 1 in the right tail of their clines (i.e. “C. gardetta side”), even when scored several kilometres away from the centre . It suggests that a part of C. macromma genetic material can introgress more easily into the C. gardetta genetic background than the reverse. Such asymmetry in the introgression pattern has already been associated with differences in population abundance  that maintain the species integrity despite incomplete isolation and many generations of genetic exchange. We go a step further in dissecting the barrier effects, and found that both intrinsic  and extrinsic (vegetation structure) barriers are probably involved in reproductive isolation between 4.4C. gardetta and C. macromma is partially maintained by divergent environmental requirements, we might expect a disruption of the hybrid zone equilibrium in the next decades. The rise of temperature together with the increase in shrub/tree coverage at high elevations could favour C. macromma over its parental species C. gardetta, pushing the contact zone towards northernmost latitudes, as already reported for many other hybrid zones (Buggs, C. macromma genetic material into C. gardetta genetic pool would threaten the genetic integrity of the species in the future, at least locally (Hohenlohe, Amish, Catchen, Allendorf, & Luikart, This study provides evidence for coincidence of morphological, ecological and genetic clines in a narrow hybrid zone where the overall barrier to gene flow is strong, but not complete. The incompleteness of the reproductive isolation observed in these two alpine taxa highlights the tenuous balance between dispersal and selection commonly observed in hybrid zones Abbott, . The Alps Buggs, . More drNone declared.J. Mavarez, L. Després and T. Capblancq designed the study and collected the samples. T. C. performed ddRAD experiments and bioinformatics treatment of the sequences, as well as the genetic, morphologic and environmental analyses. L.D. performed the ncbi blast analysis. T. C., J. M. and L. D. wrote the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Glacial refugia are centers of high biodiversity. Therefore, knowledge on their locations and reactions of associated populations and landscapes to climatic changes is crucial for conservation management. We here investigated the biogeography of a butterfly species linked to open forest habitats. Using mitochondrial and nuclear markers in combination with Bayesian simulations, we analyzed the location and age of potential glacial refugia of the species. We identified five putative refugia in Europe. Considering the ecological needs of our study species, tree density within these refugial areas, in contrast to earlier assumptions, must have exceeded the level of individually scattered trees. Our results also provide evidence that especially the refuge areas in the Carpathians were previously underestimated regarding their age: the refugia in the Southern Carpathians presented suitable conditions throughout several glacial cycles, probably since the Mindel or Riss cycles. Additionally, our analyses provided support for a forest refugium near the Tatra Mountains persisting the last glacial maximum. Our results underline the usefulness of this and probably other butterfly species as indicators of forest refugia. During cold stages, warm-adapted species were restricted to refugia. These species in general retreated towards the equator with the onset of a glaciation; consequently, the Mediterranean peninsulas became important centers of survival and re-colonisation in the western Palearctic4. Various biogeographical studies extended the knowledge about glacial refugia and added Arctic-Alpine and Siberian faunal elements for Europe5. Genetic studies discovered numerous genetic lineages, often with complex structures, and thus led to the exploration of extra-Mediterranean and cryptic refugia in Europe6.Distributions of living organisms are subject to permanent change with glacial and interglacial cycles of the Pleistocene having strong effects on the ranges of animals and plants worldwide, especially in Europe9. In a number of cases, species occupied both classical Mediterranean and extra-Mediterranean refugia13. The majority of these species are those of open land or semi-open landscapes. However, species ecologically linked to closed forest structures so far are rarely detected for these northern refugia. Nevertheless, numerous tree species have survived the last cold phase of the Pleistocene in these extra-Mediterranean refugia, according to studies using subfossil evidence of timber, pollen profiles, genetic analyses, and niche modeling16. These lines of evidence demonstrate that retreat areas for tree species were located especially at the edge of high mountain systems . In particular, the eastern edge of the Alps, the southern slopes of the Southern Carpathians, and parts of the Carpathian Basin appear to have provided suitable conditions for the survival of temperate tree species18. Also on the Balkan Peninsula, trees were probably more widespread under glacial climatic conditions than previously assumed15.Meanwhile, it has been shown for a larger number of temperate species representing a large array of different animal and plant groups that they have survived glacial conditions in northern, often cryptic refugia16. To investigate this question, it is necessary to study species that are dependent on the existence of forest ecosystems. One such species that cannot survive without the existence of forest ecosystems is the Scotch Argus, Erebia aethiops 23. This satyrid butterfly is widespread in the western Palearctic in forested areas and can be found mostly continuously from the French Massif Central through Europe and western Siberia to the western Altai Mountains. In the north, it reaches the plains of northern central Europe only sporadically; in the south, it is missing in Iberia, and the entire south of the Balkan Peninsula. Relatively few populations are known from peninsular Italy. All over this range, the morphological differentiation is weak23; nevertheless, several morphology-based subspecies have been described, e.g. the southern Alps subspecies E. aethiops rubia Fruhstorfer, [1909]. However, many of these morphological lines were synonymized by Varga24. Although E. aethiops does not reach the Atlantic Ocean, an isolated population exists in Scotland, formerly described as subspecies caledonia Verity, [1911]. Further populations isolated from the main distribution area exist in the mountains of northern Turkey and in the Caucasus, which are currently treated as a separate subspecies, E. aethiops melusina Herrich-Schäffer, [1847].All this has significantly changed our understanding of the conditions in Pleistocene Europe. However, most studies have focused on the presence and density of single trees species in an area at a certain time, but not whether they also created the ecological conditions of a forest ecosystemE. aethiops across large parts of the species’ range from its westernmost parts in the Massif Central in France to the eastern Carpathians and the eastern Balkan Peninsula. We supplemented our dataset with available sequences from Genbank and BOLD to account for potential northern and Asian lineages and compared this entire data set with sequence data from other closely related species of the aethiops-group restricted to eastern Asia. We used mitochondrial and nuclear genetic information to estimate divergence times of the main differentiation events of this butterfly depending on forests to address the following questions:E. aethiops originate from and what does this tell us about the persistence of forest ecosystems in Europe?Where did E. aethiops and which conclusions can be drawn from this for the dynamics of forest ecosystems?Which historical processes can explain the current distribution of E. aethiops located and which new insights on the dynamics of forest ecosystems can be derived from this?Where were glacial refugia of To better understand the dynamics of forest ecosystems in Europe during the sequence of several glacial-interglacial cycles, we studied the phylogeography of E. aethiops haplotypes was 0.0091  with an overall mean genetic distance of 0.004 (s = 0.002).Concatenated COI and NDI sequences 1,212 bp) of 133 specimens from 28 populations yielded 34 haplotypes , and the majority of its satellite haplotypes being restricted to the Alps. In the Tatras, another group formed a secondary star-like structure with H1 at its core. However, H1 was also found in Baile Herculane (Romania) and Trigrad (Bulgaria); the latter was dominated by two satellite haplotypes of H1. The common H7 with its two satellites was also restricted to the Tatras and the Romanian Carpathians. Only the haplotypes H5  and H28  were exclusive for the Romanian Carpathians. The three populations sampled in Slovenia had three endemic haplotypes, that were most closely related to each other. This result partially coincided with the results of the STRUCTURE analysis of allozyme data (see below) in the divergence of the Nanos population, but was contradictory for the two populations from the Slovenian Alps (Medvodje and Sija), which, according to the allozyme data, formed a group together with the large majority of populations from the Alps , samples from the western Caucasus represented a different group which genetically is most closely related to individuals from the Urals and the Altai Mountains.The geographically expanded dataset with additional samples from Genbank and BOLD, based exclusively on a 429 bp COI fragment, yielded a considerably simpler haplotype network  and observed heterozygosity (Ho), the percentage of all polymorphic loci (Ptot) and the percentage of loci with the most common allele not exceeding 95% (P95) (see supplementary o). All loci and populations were in HWE. The 14 significant deviations from the HWE (applying to single loci in single populations) were reduced to two after Bonferroni correction . A test of LD revealed deviations in six populations after Bonferroni correction , and the variance between individuals within populations . The unweighted genetic distance26 between all 27 populations varied from 0.015 to 0.219 with a mean of 0.062 (± 0.044 SD) , followed by between population variance  to 0.633 (Col des Aravis vs. Spiazzi) (see supplementary Pairwise genetic distances (derived for K = 5) were the highest for the central Italian Alps group  and an unresolved backbone28. Apart from a limited geographic overlap between E. neriene and E. aethiops in the Altai Mountains, all lineages are distributed strictly allopatrically28.The species E. alcmena, hardly differ suggest an allopatric origin instead of a common speciation origin.Furthermore, the ecologically rather similar species E. aethiops and E. neriene show an ecological character displacement in their limited area of sympatry in elevation levels in the Altai Mountains29, also rejecting the hypothesis of nearby centers of origin for both species. Consequently, we suggest that the origin of E. aethiops is west of the Altai Mountains. Such a western Palearctic origin of E. aethiops was already postulated by Pena et al.30, but based on a dataset not including its sister species analyzed by Nakatani et al.28.Consequently, an evolution in or nearby these species’ current distribution ranges is much more parsimonious than a single speciation cradle in the Altai area with subsequent expansion of these lineages, but later extinction in their center of origin in the majority of cases. The fact that the ecological niches, with the exception of the more thermophilic E. aethiops give additional evidence for a European origin of the species because the COI sequences reported from the Pontic Mountains and Transcaucasia (both northern Turkey) on the one hand and the ones from the Urals to the Sajan Mountains on the other are branching from two opposite sides of the European network. Additionally, the sequences of the individuals from the Russian Caucasus (i.e. north of the main ridge of these mountains) are directly derived from the Ural/Sajan Mountains group, showing its maximum p-distance towards the geographically nearby populations of Transcaucasia.The internal genetic structures of Based on these data, the following evolutionary scenario and range dynamics seem the most likely: a population group expanded from Europe to Asia Minor with subsequent allopatric differentiation in the mountain ranges of northern Turkey. An additional range expansion likely occurred along a north-eastern pathway reaching as far east as the Sajan Mountains as its eastern endpoint. This expansion has given rise to a secondary one in southerly direction, reaching as far south as the Russian Caucasus. This scenario well explains why the most genetically distinct populations of this species are in such close geographic proximity: they represent the two endpoints of an pincer-like expansion rooted in Europe. As the genetic differentiation at the Eurasian level is considerably higher than in Europe alone, the time frame of these shifts must be seen prior to the beginning of the differentiation in Europe (i.e. 560 ky BP), but due to the differentiation depth, the split likely is not older than the onset of the Pleistocene.E. aethiops, detected at the mitochondrial and nuclear level, is between the Central Italian Alps populations and all others. The age of this split was estimated at about 560 ky BP, i.e. approximately at the beginning of the pronounced glacial-interglacial cycles with about 100,000 years per full cycle25. This matches other molecular clock-based estimates of divergence events of other taxa in this region7. Hence, it is likely that the distribution of European E. aethiops was covering at least parts of the Alps and its surrounding areas prior to the onset of the Günz glacial. Due to the climatic deterioration during this ice-age, which was not as severe as the following ones, but nevertheless stronger than before25, this formerly continuous distribution area was apparently split up. Consequently, a previously functional belt of interconnected forest ecosystems at the edge of the Alps must have been disrupted by climatic changes for the first time in the Pleistocene. This apparently has led to the spatial separation into two large population groups of E. aethiops in Europe.When looking at Europe in more detail, the oldest split within 31. The genetic diversity patterns at the mitochondrial and nuclear levels as well as the structure of the respective part of the mitochondrial haplotype network indicate a long-lasting persistence in the areas adjoining the Po Valley at low abundance, but largely without strong fluctuations in the total number of individuals. The second (i.e. the widespread) group’s geographic centre of origin cannot be reconstructed as clearly as in the first case, but our data suggest the eastern or south-eastern part of the Alps for several reasons. The genetic diversity at the nuclear level is still highest here today, a centre west of the Central Italian Alps group would not be compatible with the southern Alps group’s rear edge status and, as known from other studies33, the eastern and southeastern Alps probably represent one of the most important centres for glacial survival of species dwelling in forests, next to the Illyrean region, as indicated by the endemic Slovenian and Albanian haplotypes.One of these two groups correspond to the lineage currently found in the Italian parts of the Alps adjoining the Po Valley. However, due to the lineage’s rear edge position, it did not participate in all the following range dynamicsE. aethiops lineage must have taken place from this putative eastern Alpine centre of dispersal. A molecular clock analysis suggests a largely synchronous split at about 400 ky into six lineages. These lineages are also distinguished by the haplotype network, which together with the genetic distances identifies two differentiation levels within these six lineages. Thus, two comparably older lineages are found in Slovenia and Cheile Butii, with the latter located in a deep valley system in the southern Carpathians. All other haplotypes found in the Alps, northern, eastern, and southern parts of the Carpathians, western and eastern parts of the Balkan Peninsula are representatives of the younger differentiation events.All further range dynamics of the widespread 34, and their long-term biogeographical consequences cannot be reconstructed unambiguously. However, a range expansion over the Carpathian region almost certainly must have taken place. It must have been facilitated by a forested link between the north-eastern Alps and the Tatras and adjoining northwestern Carpathian areasduring interglacials18. This connection between these two regions, involving gene flow, is well documented when it comes to mountain species35, but so far is not well-known regarding species intimately linked to forests.The range expansion during the mid-Mindel interstadial, which falls approximately within the time frame at about 400 kyE. aethiops in the Carpathians since then can be explained by two partly diverging lines of evidence. The haplotype tree suggests the existence of several spatially separated glacial refugia in the southern Carpathians both during the Mindel and the Riss ice age. The haplotype network with its few slightly differentiated haplotypes partly contradicts this interpretation and requires a stronger reduction of populations in the Carpathians during these periods. The differentiated haplotype H5, restricted to the southern Carpathian location Cheile Butii, supports this hypothesis. These results are in line with previous findings that prove the survival of trees in the Carpathians during glacial periods18, as well as the existence of extra-Mediterranean refugia of arboreal species there6. However, our results also indicate that the existing communities were not just habitats with single trees, but that they must have represented forest ecosystems, at least locally.The persistence of E. aethiops in this region. The high number of endemic species in Slovenia38 and several phylogeographic studies40 support a comparatively long isolation in Slovenia, also of E. aethiops. In this case, the Slovenian populations, similarly to the Central Italian Alps lineage, must have held a rear edge position, and the colonisation of the western Balkan Peninsula (at least as far south as Albania) must have occurred independently from the eastern Alpine region, without the Slovenian populations having any impact on them. Our data consequently suggests that forest ecosystems were present in Slovenia, just as in the Carpathian region, at least since the Mindel glacial, but probably throughout the entire Pleistocene. This goes beyond the previous knowledge of the uninterrupted existence of trees and arboreal species42. Furthermore, our data provide additional evidence for the importance of the western Balkan Peninsula as a Mediterranean glacial refugium for arboreal biota.The colonisation of the western Balkan Peninsula might also go back to the Günz-Mindel interglacial, but cannot be reconstructed with certainty. Based on the haplotype tree, the expansion to this region should have taken place via Slovenia. In the subsequent glacial period, the link between the Slovenian populations and the eastern Alps should have been disrupted first, followed by the split between the western Balkans and Slovenia. However, the haplotype network and the genetic distances contradict this hypothesis and indicate independent dispersal events regarding the origin of the genetic lineages endemic to these two regions. Based on our mitochondrial data, the Slovenian populations must have become separated from those of the eastern Alps during the Mindel ice-age. This is also supported by our nuclear data. Consequently, the following range dynamics seem plausible: the forest ecosystem on the eastern and south-eastern margin of the Alps that still might have been uninterrupted during the Günz ice age must have become disconnected during the colder Mindel glacial, leading to vicariance in E. aethiops across the Balkan Peninsula, a frequently observed pattern is apparent, i.e. the separation between a western and an eastern Balkan lineage43. Whilst the northward range expansions of typical temperate species originate from their Balkan lineages during transitions from cold to warm periods, the Balkan lineages usually represent the endpoints of colonisations for cold-adapted mountain species, reaching the region from the eastern Alps and the southern Carpathians, respectively44. Consequently, although E. aethiops has to be considered a temperate forest species, it behaves like a mountain taxon in this context. Furthermore, in contrast to boreal taxa, which have been expanding into this region during ice ages, the expansion of E. aethiops most likely took place during warmer periods, in which forests were spreading over large parts of this region, thus facilitating the species’ dispersal.Looking at the phylogeographic structure of E. aethiops, which is in line with the results of our Bayesian Skyline analysis. This hypothesis is also supported by the genetic patterns at the nuclear level, for which no detectable genetic differentiation in the southern Carpathian region exists. The mitochondrial level, however, might largely reflect the Riss glacial refugia, an apparent contradiction, which might be explained by different dispersal potentials of the sexes22, but which needs to be investigated in more depth in future work.According to our genetic data, the colonisation of the before mentioned western Balkan Peninsula from the eastern Alps and the eastern Balkan Peninsula from the southern Carpathians happened within similar time horizons. However, the almost complete lack of differentiation between the eastern Balkans and the southwestern Carpathians (population Baile Herculane) supports the theory of an expansion via the Iron Gate (i.e. crossing of the Danube through the south-western Carpathians) to the Balkan Peninsula, not before the Eem interglacial. During the same time frame as this southern expansion, quick expansions out of the geographically strongly restricted Riss glacial refugia in the Carpathian region must have taken place. These expansions (that must have been associated with the complete reforestation of this region) apparently led to an area-wide distribution of E. aetiops not only from the southern Carpathians, but also from a centre of dispersal in the eastern Alps. Such a mixture of Alpine and Carpathian elements of certain taxa in the Tatras has already been demonstrated several times11. However, the existence of numerous endemic haplotypes and allozyme alleles in the Tatras also suggests that E. aethiops not only colonised this region during the Eem interglacial, but was also able to survive the relatively mild Würm glacial there. A similar situation has been indicated for the eastern Carpathians, but the available data is not as robust as in the case of the Tatras. Our data thus show that not only individual trees must have been present on the southern fringe of the Tatras  during the last glacial period45, but that there were local refugia in which temperate forests have survived permanently.The expansion within the Carpathian region during the Eem interglacial must have covered the entire arc up to the Tatras. However, both nuclear and mitochondrial data show that both the Tatras and the eastern Carpathians were populated by E. aethiops' occurrence is also strongly influenced by its own biogeographic history. Therefore, the species cannot be considered as an indicator species of the entire western Palaearctic forest ecosystem, but within its range it can enrich the knowledge about the history and dynamics of deciduous forest structures. Thus, due to its mobility and sensitive response to environmental changes, it reacts reliably to changes in its ecosystem. Therefore, its recent distribution and genetic structure carries information not only on its own evolutionary history, but also on that of its associated ecosystem.If one considers the species patchy distribution or lack at the Mediterranean peninsulas and the insufficient coverage of the Siberian Taiga, it becomes evident that Erebia aethiops is a univoltine species with overwintering larvae. Eggs are laid on a wide spectrum of different grasses , but also, less commonly, on sedges 46. The caterpillars pupate end of June, resulting in a flight season starting in July and ending at the beginning of September, with the main activity in late July and August.The Scotch Argus E. aethiops individuals representing 35 populations  were sampled from 2002 to 2014 from the Massif Central in France to the Romanian Carpathians and Bulgarian mountain systems  following the manufacturer’s protocol.Erebia48, as well as the NADH dehydrogenase subunit I . COI was amplified with the primer pair LEP-F1 (5′-ATTCAACCAATCATAAAGATATTGG-3′) and LEP-R1 (5′-TAAACTTCTGGATGTCCAAAAAATCA-3′)49 applying the following PCR protocol: 95 °C for 5 min, followed by 38 cycles at 95 °C for 30 s, 49 °C for 90 s, 72 °C for 60 s and terminated with a final extension step at 68 °C for 30 min. NDI was amplified using the primer pair FAW-NDI (5′-TTCAAACCGGTGTAAGCCAGG-3′) and FAW-16S (5′-TAGAATTAGAAGATCAACCAGC-3′)50 and the following PCR protocol: 95 °C for 5 min, 33 cycles at 95 °C for 30 s, 56 °C for 90 s, 72 °C for 60 s and terminated at 68 °C for 30 min. PCR products were visualized on 1.4% agarose gels, stained with GelRed . Amplified products were purified with a mix of FastAP and Exonuclease I  and sent to Macrogen Europe  for sequencing in both directions with the same two primers as were used for PCR.Variation of two mitochondrial genes was assessed for a subset of 133 individuals representing 28 populations covering all eight regions , and the average number of nucleotide differences (k) were calculated with DNAsp v. 655. The concatenated file was used to construct a Minimum Spanning haplotype network56 with PopArt v. 1.757.Sequences were assembled with G58. Published data of Pararge aegeria (GenBank accessions in Table Erebia pronoe were used as outgroups. Partitions and substitution models were estimated with Partitionfinder v. 2.1.159 based on the lowest Akaike Information Criterion (AIC). The HKY model with empirical base frequencies and a Gamma distribution with a category count of 4 was selected. We ran several analyses to select the best fitting tree model. The coalescent constant population model performed best and hence was chosen for the final analysis. The molecular clock was calibrated with a substitution rate of 0.017760. We ran the analyses with 40 million generations sampling every 4,000 iterations. After checking the MCMC chain for convergence in Tracer v. 1.7.161, a burn-in of 10% was applied. Three individual runs were performed and combined using LogCombiner v. 1.8.462. TreeAnnotator v. 2.562 was used to generate a summary tree with common ancestor heights. Figtree v. 1.4.461 was used for visualisation.A Bayesian tree was reconstructed based on the mitochondrial data with BEAST v. 2.563. This analysis was based on the extended COI data set of E. aethiops and further COI sequences of E. neriene, E. niphonica and E. alcema obtained from Genbank and Boldsystems. The additional accession numbers are given in the RASP tree. The individual sequences were assigned occurrences coded as consecutive letters . Based on the results of the BioGeoBears model test, an S-DEC analysis was performed with the maxarea = 6 setting.The same protocol was followed to obtain an input and consensus tree for a reconstruction of ancestral area analysis with RASP v. 4.2racer v. 1.7.1. We used the substitution rate estimated by Papadopoulou et al.60.To model the demographic history of the populations, a Bayesian Skyline plot was generated with BEAST. Bayesian skyline analyses were run with the coalescent tree prior, in a single run with 40 million generations, log parameters were sampled every 4,000 iterations. The effective sample size and female population size was evaluated using Te; Ho), total number of polymorphic loci (Ptot) and the percentage of polymorphic alleles per locus with the most common allele not exceeding 95% (P95)) were estimated with G-Stat v. 364. Tests on Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were performed with Arlequin v 3.565. A sequential Bonferroni correction66 was applied. A non-hierarchical and a hierarchical genetic variance analysis (AMOVA) were performed with Arlequin to attribute the genetic variation to the following levels: among populations, among individuals within populations, and within individuals. Additionally, pairwise FST values were estimated with Arlequin. A Neighbor Joining (NJ) phenogram67 based on Nei’s genetic distance26 and 1,000 bootstrap replicates was generated with Phylip v. 3.6768. Structure v. 2.3.469 was used to infer the number of genetic clusters without a priori definition of populations. To ensure a better delimitation of the individual groups, which were suggested by Structure, each population was examined for a change in the main allele. We tested for genetic clusters (K) from two to 27 . Ten replicates were carried out for each K, with a burn-in of 100,000 and 500,000 MCMC replicates performed thereafter. Due to the un-reliability of logarithmic probabilities, an ad hoc method was used and the ∆K value, based on the change of the logarithmic probability of successive K values, was determined70. The high ∆K value for K = 2 was ignored a suggested by Hausdorf and Hennig27. Additionally, a Geneland71 analysis was conducted with ten runs, each of 250,000 generations, in an uncorrelated frequency model with the number of classes treated as unknown and variable along the MCMC chain.Alleles were labeled according to their relative mobility, starting with “1” for the slowest. Their frequency, the total number of alleles per locus (A) and further genetic diversity parameters (i.e. expected and observed heterozygosity (HSupplementary information.


Undaria pinnatifida provides such an opportunity because it has been cultivated in its native range in Northeast Asia but also introduced to four other continents in the past 50 years. Here we present the results of a genome-wide analysis of natural, cultivated and introduced populations of U. pinnatifida to elucidate human-driven evolutionary change. We demonstrate that these three categories of origin can be distinguished at the genome level, reflecting the combined influence of neutral (demography and migration) and non-neutral (selection) processes.Human activity is an important driver of ecological and evolutionary change on our planet. In particular, domestication and biological introductions have important and long-lasting effects on species’ genomic architecture and diversity. However, genome-wide analysis of independent domestication and introduction events within a single species has not previously been performed. The Pacific kelp  Human-driven evolutionary changes are captured by comparison of the genomes of natural, cultivated and introduced populations of a globally distributed Pacific kelp. These processes leave footprints in the genomes of many species that are cultivated5, domesticated7 or transported across different biogeographic regions 9. In-depth examination and understanding of these changes is an important research area, because of their considerable ecological, economic and health implications10.Unprecedented evolutionary experiments have resulted from the spread of humans on our planet. Initially considered anecdotal and rare12. As far back as 12,000 years ago, and the transition in human behaviour from food gathering to cultivation13, agricultural societies depended on the domestication and diversification of wild species, notably through selective breeding, hybridization or inbreeding15. As a result of the application of these methods, there was selection for so-called ‘domestication phenotypes’. These traits can arise through conscious selection  or unintended selection , challenging the clear discrimination of traits directly selected during domestication and other traits.Domestication is a form of co-evolution between a species  and another species it controls (in terms of growth and reproduction) for its own benefit. This process has shaped the evolution of hundreds of plants and animals17. These human-driven migrations bring into contact populations or species that have evolved in isolation in their respective native range, exacerbating evolutionary changes, notably through admixture and hybridization20, as well as by selective pressure on the introduced species in its new range21. Biological introductions thus represent a fascinating opportunity to understand major evolutionary processes, such as genotype by environment interactions22 or speciation dynamics23.In a globalized environment, where borders are crossed legally and illegally every day by hundreds of thousands of humans and goods, plant and animal species are dispersed knowingly or unwittingly around the world. Since the end of the twentieth century, nonindigenous species have become a major concern in our societiesUndaria pinnatifida (Harvey) Suringar  provides such an opportunity. In its native range of Northeast Asia, this brown edible seaweed was exploited for centuries before being cultivated, and its farming represents 6.9% of worldwide seaweed production24. The transition from the harvesting of natural populations to the cultivation on ‘long lines’ happened during the 1950s25 with the development of seaweed cultivation techniques and their application to U. pinnatifida in Japan, then Korea and finally China26. During this period, farmers selected desired phenotypes and only recently were breeding techniques used to develop cultivars28. Parallel to cultivation, U. pinnatifida has been intentionally and unintentionally transported by humans across the planet. Since its first report outside its native range , U. pinnatifida has become established along the coastlines of 14 countries across 4 continents32. This kelp presents the rare characteristic of being independently cultivated in its native range and introduced in four continents outside its native range. This situation contrasts with other well-studied cases such as Oryza33 and Sorghum34 in which domestication preceded escape to the wild. Recent studies have demonstrated the importance of genome-wide analyses, based on whole-genome sequencing data, to understand human-driven evolutionary change, notably with respect to domestication7, with fewer studies examining invasive species9, and none addressing both aspects at the same time. Here we report the genome sequence of a Korean cultivar of U. pinnatifida, sequenced independently from the genome sequence of the Chinese gametophyte35. On the basis of whole‐genome sequencing of multiple individuals sampled in native, cultivated and introduced populations, we compared genome architecture across these different categories. We argue that the differences observed among them probably result from the combined influence of demography and selection.Only a handful of case studies offer the opportunity to simultaneously address human-driven evolutionary change due to domestication and introduction. The Pacific kelp U. pinnatifida sporophyte harvested in November 2015 in Wando, Korea and generated a nuclear genome assembly using PacBio long reads with ~100× sequence coverage  into 30 linkage groups corresponding to the number of chromosomes in U. pinnatifida37  repeated elements, of which at least 19.14% are transposable elements, representing 121 Mb of the genome  reduced gene density in Kr2015 when compared to E. siliculosus . Therefore, organization of brown algal chromosomes appears to be similar to that in fungi41, but different from that in plants42. This differential insertion of repeated elements does not appear to have disturbed the gene order: synteny is largely conserved between U. pinnatifida Kr2015 and E. siliculosus  and U. pinnatifida (30 chromosomes) may be explained by 4 splitting and fusion events involving 5 and 7 chromosomes, respectively  and 1,130,417 high-quality insertions or deletions (indels) shared across the 9 populations  and phylogenetic reconstruction revealed a clear segregation of individuals according to their geographic locations  best explaining the genetic variance , as observed in France47.Time  appears to have little to no influence, because populations remain stable over time. For instance, the individuals sampled in 1987 and 2017 in the introduced population of Wellington  were indistinguishable in the three analyses , particularly in selfing species such as U. pinnatifida47.When compared to those of populations from its native range, the characteristics of the French and New Zealand 51, it has been hypothesized that the introduction occurred as a result of aquaculture in France and shipping in New Zealand. In France, U. pinnatifida would have been first introduced in the Thau Lagoon (Thau population here) with Pacific oyster imports from Asia, and then transported to Brittany . Two sequential founder events thus probably occurred, from the same source in the native range49. Conversely, repeated introductions probably occurred with shipping in New Zealand, leading to a moderate decrease in genetic diversity when compared to the native range and the French populations, as found using mitochondrial haplotype analysis49. This scenario was further supported by individuals sampled in Wellington in 1987 at the time of the first report of this alga in New Zealand52. These seaweeds had a genetic diversity that was slightly higher than in the French population  in accordance with expectations of the cultivation process (with some exceptions53). This observed discrepancy might be explained by the difference in the scale of the cultivation in these two countries. In France, U. pinnatifida cultivation remains limited to a few farms, whereas in Korea, its culture averages 0.5 million wet weight tonnes annually26. Owing to this large scale, the fertilization of culture ropes is carried out in large indoor pools in which multiple sporangia of individuals with valued phenotypes are placed together ) could also produce patterns of genetic diversity that resemble selective sweeps. Furthermore, the putative functions of loci implicated in putative selective sweeps can be overinterpreted61, and in the absence of other evidence , they should be considered with caution. We identified putative selection signals associated with cultivated or introduced populations of U. pinnatifida by using a decorrelated composite of multiple signals (DCMS)62 method on three different statistics calculated in 50-kb windows along the genome  were enriched in several biological processes such as glycolipid biosynthesis and cytokinetic process  in genomic regions under putative selection63, but interestingly, different pathways were recovered in the two species. More generally, the biological process selected during cultivation in the two species differed greatly . Similarly, with the exception of one copy, peptidase s8 and s53, which were proposed as blade length and width quantitative trait loci in S. japonica64, were not detected by the DCMS analysis  in Wellington Harbour between 1987 and 2017 did not reveal the enrichment of a particular biological function  was assessed by alignment of the proteins encoded in the genomes of E. siliculosus and S. japonica and core eukaryotic genes , France (introduced populations) and New Zealand (introduced populations) was extracted, and for each individual, ~30× coverage of short-read data were generated , π, fixation index (FIS), LD (estimated from r2) and ROH  were estimated (D and population differentiation (FST). For each window and each statistic, a P value was determined and these were used to calculate the DCMS value of each window (The population structure was investigated with PCA, phylogenetic tree reconstruction and admixture analyses . For eacstimated . Detectih window .Further information on research design is available in the Supplementary InformationSupplementary Figs. 1–17 and Note.Reporting SummaryPeer review informationSupplementary TablesSupplementary Tables 1–19.


Gesneriaceae with 14 genera and 23 species, amongst which two species and one variety are recognised in the genus Oreocharis. These three Oreocharis taxa are all Hainan-endemics and show a complex geographical distribution pattern with considerable morphological intermixtures. In this study, we combined DNA (nuclear ITS sequences and cpDNAtrnL-trnF and ycf1b) to evaluate genetic delimitation for 12 Oreocharis populations from the island, together with morphological similarity analysis using 16 morphological traits. The results showed Hainan Oreocharis taxa were monophyletic with relative low genetic diversity within populations, highly significant genetic differentiation amongst populations and a significant phylogeographical structure. The 12 populations formed three genetically distinct groups, roughly correspondent to the currently recognised two species and one unknown lineage. The PCA analyses of morphological traits indicate three distinctive groups, differing mainly in petal colour and corolla shapes. The roles of river and mountain isolations in the origin and distribution of these three lineages are discussed.Hainan Island harbours an extraordinary diversity of  As a biodiversity hotspot in the world (2) and contains almost twice the number of Gesneriaceae species than Sri Lanka , which is twice the size of Hainan Island  and eight species (including one variety) are endemic  and all are endemic to the island . We aim to determine (1) whether or not the currently recognised three species or variety can be supported by genetic data (2) what factors  are responsible for the evolution and maintenance of these Hainan-endemic Oreocharis?Here, we sampled 12 populations of Oreocharis taxa covering all the suitable habitats of the genus on the island were collected, including populations DW (Dongwu in Bawangling), DE (Donger in Bawangling), FT (Futou in Bawangling), NG (Nangao), HM (Mt. Houmi), JF (Mt. Jianfeng) and CH (Chahe at the foot of Mt. Jianfeng) from O.dasyantha, populations QX (Mt. Qixian), WZA (Wuzhi A in Mt. Wuzhi) and WZB (Wuzhi B in Mt. Wuzhi) from O.flavida and populations YG (Mt. Yingge) and LM (Mt. Limu) from unidentified Oreocharis sp.  sequence, the ITS region comprising spacer 1, the 5.8S gene and spacer 2 (cpDNA) intron–spacer region trnL-trnF , 0.5 μl each 5 μM primer, 0.5 μl DNA template and 0.5 μl 5 U/μl Taq polymerase (Aidlab Biotechnologies Co. Ltd). PCR was conducted in a 2720 Thermal cycler  and Veriti 96-Well Thermal Cycler . The PCR programme for nrDNA and trnL-trnF was designed for an initial denaturation at 94 °C 5 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C, 1 min at 72 °C and with a final extension of 10 min at 72 °C. Amplification of ycf1b used the following protocol: 4 min at 94 °C, 35 cycles of 30 s at 94 °C, 40 s at 58 °C and 1 min at 72 °C, ending with 10 min at 72 °C. All the PCR products were checked by electrophoresis. Then purification and sequencing of PCR products were finally sequenced by an ABI 3730 DNA Analyzer based on the BigDye Terminator Cycle Sequencing Ready Kit  in BGI (Beijing Genomics Institution), the chemistry and primers being used above in BGI.One nuclear ribosomal DNA   analysis was conducted using MEGA v.6.5 (BS) replicates. Bayesian Inference (BI) analysis was conducted using MrBayes version 3.1.2 (MCMC) was analysed for 10 million generations and sampling every 10000 generations for two independent Bayesian runs. The first 2500 trees  were discarded as burn-in and the remaining trees were summarised in a 50% majority-rule consensus tree with the posterior probabilities (PP). The mean and posterior of each branch were visualised by FIGTREE v.1.4.2  . In addiGA v.6.5  with theon 3.1.2 . The Mar v.1.4.2 . SequenccpDNA sequences obtained were evaluated with the software BioEdit (MK587942–MK588003). Subsequently, we combined the two no-coding cpDNA regions as a single locus by the programme SequenceMatrix v.1.7.8 , nucleotide diversity (π) and measures of DNA divergence (K) values were analysed by the programme DNASP v. 6.12.01 , nucleotype/haplotype diversity , total gene diversity index (STN) and genetic differentiation index within populations (STG) were measured using Haplonst  was conducted to estimate genetic variation which was assigned within and amongst populations using GENALEX v. 6.503  that was larger than 200 for each parameter. To further explore the relationships amongst unique nucleotypes, genealogical relationships were inferred from Median-Joining network (MJ) of NETWORK v. 5.001 (http://www.fluxus-Engineering.com/).Prior to Bayesian analysis, the optimal model of nucleotide substitution was detected for each gene using MRMODELTEST v. 2.3 , based oAIC . Two indv. 1.7.1  by checkNJ) tree was selected to build the phylogenetic relationship of Oreocharis taxa populations in Hainan Island by MEGA v.6.5, with Kimura two-parameter model  was set from 1 to 10 and each K run 10 times. Each run comprised a burn-in period of 1 × 105 interactions with 1 × 105MCMC steps after burning. The most suitable value of K was determined from Structure Harvester (http://taylor0.biology.ucla.edu/structureHarvester/) by using ΔK and the log-likelihood value. Finally, the result from programme STUCTURE for the best K value was drawn in CLUMPAK server (http://clumpak.tau.ac.il/index.html).A Bayesian clustering approach conducted in STRUCTURE v. 2.3.4  was usedIBD) for each population was tested by a Mantel test in GENALEX between pairwise genetic distance (uncorrected sequence divergence (Dxy) for nuclear DNA and cpDNA) and geographical distance.To detect whether there was local genetic variation under geographically limited dispersal, isolation by distance  corolla colour , (ii) corolla shape and type , (iii) corolla width , (iv) corolla mouth width , (v) floral tube length , (vi) sepal length  and (vii) number of petals .Five stamen traits were included in the analyses: (i) anther position , (ii) stamen type , (iii) pollen presentation , (iv) anther shape  and (v) hair on filament .Two stigma characters and two leaf traits were also included in the analyses: (i) location of stigma , (ii) number of stigma , (iii) serration of leaf edge  and (iv) leaf epidermal hair in abaxial side . Measurements were taken with a rectilinear scale and rounded to the nearest 0.1 mm.PCA) was conducted in SPSS v. 19.0  test between the ITS1/2 and trnL-F (p > 0.05).The combined ITS1/2 and BI and ML analysis showed Hainan Oreocharis taxa being monophyly with PP (posterior probability) = 0.79 and BS  = 38% , is the sister to Hainan Oroecharis taxa in the current tree with relatively high support (Appendix Both the Appendix . In addiAppendix .bp). A total number of 56 polymorphic sites were present, of which 48 were parsimony-informative, which allowed the identification of 16 different nucleotypes from a size of 238 samples  with a significant rate of homogeneity (P = 1) in the congruency test, indicating that there was no significant difference in the laboratory between the two cpDNA regions. The alignment contained 55 polymorphic sites and 8 indels .The combined alignment of the two ls Table . A totalHd) and nucleotide diversity (Pi) for each population are summarized in Table nrDNA and cpDNA. Generally, except for population WZB, YG and LM presented high genetic diversity in both nrDNA and cpDNA, nuclear gene ITS in population CH and chloroplast gene trnL-F and ycf1b in population DE, NG and HM showed variable genetic diversity, the rest of the populations having very low nucleotide and haplotype diversity  was not significantly greater than the genetic differentiation index within populations , revealing that Hainan Oreocharis taxa have no correspondence between haplotype comparability and geographic distribution  was partitioned amongst populations, which was higher than the variation (1% and 3%) within populations, based on the ITS and cpDNA data, respectively, revealing highly significant genetic differentiation amongst populations   Figs , 3. The ncy Figs , 3b. TheNJ tree, based on nrDNA and cpDNA, suggested 12 populations were clearly clustered into three major groups, which well corresponded to the three defined Oreocharis taxa in Hainan Island, i.e. O.dasyantha (includes O.dasyanthavar.ferruginosa), O.flavida and Oreocharis sp. Additionally, the analyses also presented a close relationship between O.flavida and Oreocharis sp., then with O.dasyantha.The results of the cpDNA  than for nuclear DNA , the relationship between genetic and geographical distance for 12 populations was significant both in nuclear DNA and cpDNA ; (2) campanulate, zygomorphic, orange flower with included stamen and stigma ; (3) thin tubular, zygomorphic yellow flower with included didynamous stamens (populations YG and LM of Oreocharis sp.). The corolla colour and corolla shapes may play a key role in ordinations of morphological traits with high values of 45.41% and 34.04%, followed by location of stigma and length of the corolla tube with the values 9.293% and 5.272%.According to the floral syndromes, the Principal Component Analysis of 16 floral characters of Hainan ers Fig. : (1) tubOreocharis taxa are monophyly  and an unknown species showed clear geographical isolations associated with three different river ranges. O.dasyantha are found mostly in the watershed of the Changhua River (the secondly largest river on the island), O.flavida distributes in Mt. Wuzhi and Mt. Qixian, the upper reaches of the Wanquan River. The unknown species is restricted in the upper reaches of the largest river on the island, i.e. Nandu River. We concluded that these three groups may have evolved and maintained largely through allopatric differentiations.Most ty Table  and overty Table  and a siOreocharis populations in Hainan Island were restricted in > 1000 m high-elevation mountains with massive humidity, such that the island-like habitat became fragmented caused by a deep and wide valley in the complicated mountains system, which resulted in blocking of gene flow of Oreocharis populations with weak seed dispersal ability even at the fine scale  W. T. Wang. Our results can also be explained by the isolation of Changhua River . Besides two species (includes one variety) currently recognised in Hainan Island, populations from Mt. Limu and Mt. Yingge should be treated as a new species or subspecies, which is still in need of further illumination.According to morphological traits, all the 12 ups Fig. . Such di


FST) and nucleotide sequence divergence (DA), revealed a low genetic differentiation in native Ethiopian horse populations. However, Kundido feral and Borana domestic horses were slightly diverged from the rest of the Ethiopian horse populations. We also tried to shed some light on the matrilineal genetic root of native Ethiopian horses from a network constructed by combining newly generated haplotypes and reference haplotypes deposited in the GenBank for Eurasian type Turkish Anatolian horses that were used as a genetic conduit between Eurasian and African horse populations. Ninety-two haplotypes were generated from the combined Ethio-Eurasian mtDNA D-loop sequences. A network reconstructed from the combined haplotypes using Median-Joining algorithm showed that haplotypes generated from native Ethiopian horses formed separate clusters. The present result encourages further investigation of the genetic origin of native African horses by retrieving additional mtDNA sequences deposited in the GenBank for African and Eurasian type horses.Matrilineal genetic diversity and relationship were investigated among eight morphologically identified native Ethiopian horse populations using polymorphisms in 46 mtDNA D-loop sequences (454 base pairs). The horse populations identified were Abyssinian, Bale, Borana, Horro, Kafa, Kundido feral horses, Ogaden and Selale. Mitochondrial DNA D-loop sequences were characterized by 15 variable sites that defined five different haplotypes. All genetic diversity estimates, including Reynolds’ linearized genetic distance, genetic differentiation ( Equus were the last of the common Old World livestock to be domesticated   4] who r


Feng Shui woodlands are naturally or artificially formed green areas in southern China. They are precious for maintaining ecosystem balance in modern semiurban environments. However, they are generally small and geographically isolated from each other, and the status of genetic diversity of the plant species within them has been almost neglected. Therefore, we studied the genetic diversity of the endangered Erythrophleum fordii in eight Feng Shui woodlands  in Guangzhou, a large city in southern China, using microsatellites. For comparison, one population with 33 individuals sampled in a nature reserve was also studied. Although our results indicate that significant demographic declines occurred historically in E. fordii, such declines have not resulted in consistent reductions in genetic variation over generations in Feng Shui populations in the recent past, and the levels of genetic variation in these populations were higher than or comparable to the genetic variation of the population in the nature reserve. In addition, our parentage and paternity analyses indicated widespread and potential long‐distance pollen flow within one Feng Shui woodland, indicating the presence of an unbroken pollination network, which would at least partially alleviate the genetic erosion due to habitat fragmentation and the unequal gene contributions of E. fordii parents to their progenies when favorable recruitment habitats are absent under most of the parent trees. Overall, our results suggest that E. fordii in Feng Shui woodlands may not be driven to extinction in the near future. Nevertheless, uncontrolled fast urban development with a lack of awareness of Feng Shui woodlands will cause the local extinction of E. fordii, which has already happened in some Feng Shui woodlands. Feng Shui woodlands conservation but also urban reforestation to balance species conservation and economic developments.It is the first report about genetic sustainability of tree species under urbanization and habitat fragmentation in China. The results will be combined with the one of the other regions to give a general conclusion of tree species' genetic health under urbanization and guide not only  For convenience, we will use the abbreviations of the village names  in the other seven woodlands and DH Mountain are unknown. According to records, E. fordii was planted in SKY village in 1368 A.D. by a troop stationed there that used this tree to make arrows. However, in TB village, one E. fordii tree is tagged with a plate which estimates it to be more than 200 years old , juvenile (1 cm ≤ DBH < 20 cm), and seedling (DBH <1 cm).E. fordii population in it is believed to be well preserved. According to Zhu et al. . In this study, we resampled 33 individuals with DBH >30 cm and genotyped them using not only the previously isolated loci, but also our newly developed ones . The digested DNA fragments were then ligated to adaptors and PCR amplified. Approximately 300–500 bp fragments were subsequently selected and sequenced on Illumina HiSeq X Ten genetic analyzer (Illumina) to produce 150 bp paired end sequencing reads. After sequencing, we obtained a total of 49,971,692 bp of raw reads for one individual and 39,494,352 bp for the other. The raw sequence data are available in the NCBI SRA database with accession numbers SRX5010692 and SRX5010693. Filtering for PCR duplicates and low‐quality reads resulted in 16,662,656 and 10,993,602 bp of useful reads for the two individuals, respectively. These reads were assembled using Rainbow 2.0.4  using GenAlEx 6.501  using GENEPOP 4.3  and private allelic richness (AP) results difficult to compare among populations or life stages, we used the ADZE 1.0 program , while for life stage comparison within population we computed AR and AP using the smallest sample of life stages in each population. Nonparametric Wilcoxon tests were used to test for differences in diversity estimates  between populations and life stages within population. The Wilcoxon tests were performed with wilcox.test function in R software and one‐sided p‐values (“less” or “greater”) were reported.We first estimated null allele frequencies at 20 loci in each population with INEST v2.2 under the individual inbreeding model (IIM) with default parameters  among all pairs of loci. The levels of significance for HWE and LD were adjusted by using the sequential Bonferroni correction Holm, . At the Pparent‐pair)Na following Dow and Ashley . After determining the power of exclusion for the microsatellite loci, using the allele frequency data calculated from all the samples in this population, we ran a simulation to estimate the critical Delta scores necessary for parentage assignments at a 95% confidence level. The simulation parameter values were as follows: 100,000 tests, 18 for the candidate parents (the adults in our samples of TB village), 0.9 for the proportion of candidate parents sampled, 1 for the proportion of loci genotyped, 18 for the minimum genotyped loci, self‐fertilization was allowed, and the default setting were used for the other parameters. Although we could identify the parents at a 95% confidence level for most of the seedlings, there were some seedlings that we could not identify the parents of. Considering the restricted seed dispersal ability of E. fordii for the rest of the seedlings, we assumed the mother tree to be the nearest tree to the seedlings geographically, and then used the same program to perform paternity analysis. In this analysis, we used the same simulation parameters as in the above parentage analysis. After parentage and paternity analyses, the actual pollen immigration rate was estimated as number of seedlings with undetermined paternity/total number of seedlings. The effective pollination neighboring area (Aep) was calculated by Aep = 2πσ2, where σ2 was the variance of the pollen dispersal distance , the pollen immigration rate (mp), pollen dispersal distance (dp), the seed immigration rate (ms), and seed dispersal distance (ds). If initial parameter values were far from the true values, the maximum likelihood algorithm could fail to reach convergence. After trying different initial parameters, the final parameter settings used for estimation were as follows: exponential‐power dispersal kernel for both seed and pollen, genotyping error rates 0.01 for all loci, seed immigration rate 0.01, average seed dispersal distance 4.57, shape parameter of seed dispersal kernel 0.42, pollen immigration rate 0.134, average pollen dispersal distance 160, shape parameter of pollen dispersal kernel 1.6, selfing rate 0.12, and default for the other parameters.We also estimated the pollen immigration using the spatially explicit neighborhood model , prior_THETA_max = 10, prior_GSM_min = 0.1 , prior_GSM_max = 0.8, the repeat size for each locus was specified, and all other options and priors were set to default values.To infer the population demographic history, we used DIYABCskylineplot 1.0.1  using a 5 × 105 Markov Chain Monte Carlo (MCMC) iterations after a burn‐in period of 5 × 105 on total multiloci genotypes of adults without prior concerning of their origin populations. The choice of the probable K value was made both as recommended in STRUCTURE user's manual and by ΔK method  in all populations, and locus EF‐28 displayed null allele frequency higher than 0.05 in five populations  were not significantly different between populations except for WYG and SKY villages in HE value and WYG village and DH Mountain in AP value (Table f) in the populations ranged from −0.1647 to −0.0134, and almost all the populations showed a significant deviation from zero due to heterozygosity excess except LT village and DH Mountain  than in seedlings, but the populations of WYG, ZL, and SKY villages had the highest in seedlings, the youngest generation  of the 257 seedlings in the population of TB village at a 95% confidence level. We assigned a mother trees for 49 of the 51 remaining seedlings according to their geographic position. Paternity analysis then successfully identified the pollen donating trees for 12 of the 49 at the 95% confidence level. Therefore, the parents of 218 seedlings were identified. The distances of the parent–offspring pairs detected ranged from 2.275 to 457.959 m with mean of 160.085 m  was 11.241 ha.The combined nonexclusion probabilities across the 19 microsatellite loci for the first parent, second parent, and parent pairs were 0.0407078, 0.00204951, and 0.00003035, respectively. The combined nonexclusion probability of identity was 1.654 × 10SE 0.021), the pollen immigration rate 0.134 (SE 0.023), the mean pollen dispersal distance 220.910 m (SE 24.836), the seed immigration rate 0.010 (SE 0.007), and the mean seed dispersal distance 4.666 m (SE 4.724).For the population of TB village, the neighborhood model showed the self‐fertilization rate was 0.122  confirmed the admixture. However, to our surprise, these sources could not be from nearby populations we sampled. In ancient times, without efficient transportation tools, the troop stationed in SKY that planted the trees might not have been able to introduce E. fordii individuals from places far away. Since there are no the other E. fordii populations geographically closer to the population of SKY village than the populations we sampled, a possible explanation is that the source population(s) of SKY village have been extinct under urbanization. Nevertheless, this hypothesis needs further studies with more extensive sampling than ours in the future.We observed that among all the populations, SKY harbored the highest genetic diversity but not the highest number of private alleles Table . A possif values), Duminil et al.  of E. suaveolens, regardless of the population densities , which indicates that their populations had high selfing and inbreeding rates. Our Feng Shui populations of E. fordii did not seem to have suffered from inbreeding because all life stages had near zero f values  of 0.144 and 0.122, and mean pollen dispersal distance (dp) of 160.085 and 220.910 m based on parentage assignments and neighborhood model, respectively, in the population of TB village. Effective pollination neighborhood (Aep) for E. fordii was 11.24 ha between seed trees, equivalent to a circle with a radius of 189.21 m around a seed tree. Hence, considering the very limited seed immigration rate of 0.010 and dispersal distance of 4.666 m we observed, long‐distance gene flow in E. fordii is primarily by pollen dispersal. As both mp and dp are directly related to the sizes and degrees of isolation of areas, they should vary among species and populations within species with similar pollination insects , Manoel et al.  wood demands due to long periods of war and poverty. However, the results from skyline plots Figure  indicatex‐axis  when the skyline plot results suggest the most recent ancestral decline with a high magnitude started  in China , and more records of its use have been found since the Song dynasty (established in 960 A.D.)  Zhou, . One fam4.4E. fordii individuals at short distances. Furthermore, they reveal clearly disproportionate contributions of adults to the recruit landscape due to absence of suitable recruitment environment under some adults, which could remain across reproductive cycles if present unfavorable conditions for seedling establishment continue. Together with substantial self‐fertilization which is often associated with isolated and fragmented small populations (Cheptou, Hargreaves, Bonte, & Jacquemyn, Our results show that despite moderate gene flow via pollen, the limited seed dispersal distance may result in significant relatedness among E. fordii should include Feng Shui woodlands as high genetic diversity harbored in most them and contain many trees as many trees as possible. The mean pollen dispersal distance suggests that seed trees must be separated by at least 220 m. In addition, since the gene pool of the population of SKY village is different from the others, seeds collected from it should be separated and only mixed with those from other populations after being sure no outbreeding depression effects happening.Seed collection for ex‐situ conservation of 5Feng Shui woodlands, as part of CPFs, are valuable supplements for urban forests in southern China, especially with the present policy aiming to build national forest cities, because they are more natural and have higher species diversities than modern artificial green lands (Ye et al., Feng Shui woodlands in China, most of the plant species in CPFs are not considered being threatened in the near future. However, monitoring their gene pools is essential to prevent genetic erosion caused by anthropogenic effects. Therefore, an extension of this study to other species with different life stages and different landscape configures in CPFs would be recommended to know their gene flow and how such flow determines the microevolutionary changes in them.E. fordii may have suffered serious demographic declines before large‐scale human settlements in southern China and has not recovered at the present time due to consistently high demands for its high quality wood. However, parentage analysis indicated that its pollen‐mediated gene flows were not severely affected within the disturbed suburban areas, and genetic diversity was stably maintained across different generations. A previous simulation study of E. fordii also indicated that its longevity with iteroparity provided the potential to maintain genetic diversity in small isolated populations (Zhu et al., Feng Shui woodlands, the major threat to the long‐term adaptation and evolution of E. fordii is the lack of suitable regeneration habitats. The maintenance of large and continuous populations to guarantee high gene flow is also required for long‐term species sustainability.Overall, our results suggest that The authors declare no competing interests.WZF conceived and designed the project and carried out the laboratory procedures and data analyses. WZF and LHL carried out the field collections. All authors contributed to writing the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Rhododendron rex subsp. rex Lévl. is an endangered species endemic to the southwest of China. Although the natural populations of this species are facing continuous decline due to the high frequency of anthropogenic disturbance, the genetic information of R. rex subsp. rex is not yet elucidated. In the present study, 10 pairs of microsatellite markers (nSSRs) and three pairs of chloroplast DNA (cpDNAs) were used in the elucidation of the genetic diversity, population structure, and demographic history of 11 R. rex subsp. rex populations. A total of 236 alleles and 12 haplotypes were found. A moderate genetic diversity within populations , high historical and low contemporary gene flows, and moderate genetic differentiation  were detected among the R. rex subsp. rex populations. Genetic and geographic distances showed significant correlation (p < 0.05) determined by the Mantel test. The species exhibited a conspicuous phylogeographical structure among the populations. Using the Bayesian skyline plot and species distribution models, we found that R. rex subsp. rex underwent a population demography contraction approximately 50,000–100,000 years ago. However, the species did not experience a recent population expansion event. Thus, habitat loss and destruction, which result in a population decline and species inbreeding depression, should be considered in the management and conservation of R. rex subsp. rex.Genetic diversity is vital to the sustainable utilization and conservation of plant species.  Rhododendron is the largest woody plant genus in Ericaceae, containing more than 1000 recognized species, of which 567 species representing six subgenera are known from China , R= 0.754) , R. ripe= 0.800) , and R.= 0.815) , but has= 0.500) . Meanwhi= 0.620) . Inconsiast Asia , but a l0.00240) . The valto cpDNA ,72. Theriversity ,69. As iulations . Thus, ty plants .STF value of R. rex subsp. rex indicated that a moderate genetic differentiation among populations occurred. A total of 83.75% genetic variation occurred within R. rex subsp. rex populations with regard to nSSR markers, whereas 83.53% variation was partitioned among populations with regard to cpDNA sequences. This discordance should be affected by dispersal mechanisms among populations of plant species in Rhododendron [Rhododendron species. Various insect vectors evolved longer dispersal distance for pollen, whereas the seeds dispersed by wind traveled less than 10 m albeit in open landscapes [R. rex subsp. rex.The odendron ,75. Insendscapes . Meanwhindscapes . In genendscapes . TherefoR. rex subsp. rex were grouped into three groups, and the correlation between genetic and geographic distances was significant (p < 0.05). Phylogenetic trees and genealogical haplotype networks based on cpDNA sequences showed that three reciprocally lineages were detected. This species possessed unique genetic lineages and endemic cpDNA haplotypes in its separate refuge populations. The closely related haplotypes H1, H2, and H3 were distributed in populations BJS, BCL, and DLT; H4, H5, H6, H10, and H11 occurred primarily in populations LJS, JZS, LZS, QLB1, QLB2, and QLB3; H7, H8, H9, and H12 were only detected in populations GDX and YS.On the basis of genetic structure analysis by SSR data, the populations of R. rex subsp. rex natural populations. In addition, gene flow is a fundamental micro-evolutionary force, influencing genetic differentiation among populations [R. rex subsp. rex is lower than that of the related species of R. protistum var. giganteum [R. rex subsp. rex populations. Moreover, breeding system is an important factor for the genetic differentiation and structure of a species [Rhododendron species [R. rex subsp. rex is yet to be explored. Based on the positive value of fixation indices (isF) and the phenomenon of all populations deviated from HWE in the present study, we can reasonably speculate that inbreeding is present in populations of R. rex subsp. rex. Hence, the mating system and its influences on genetic differentiation and structure must be further elucidated in R. rex subsp. rex.Habitat dislocation and overexploitation accelerate the generation of genetic differentiation among populations ,13. In tulations ,79. The  species ,22,69. A species ,81, the R. rex subsp. rex experienced a notable reduction approximately 50,000–100,000 years ago. This supposition is supported by the GWI values, which are lower than the critical Mc indices (0.68). Microsatellite-based bottleneck analysis indicated that no recent bottleneck event occurred in the natural populations of R. rex subsp. rex. Therefore, the population demographic contraction detected in the R. rex subsp. rex might have been the result of climate oscillations, and the finding is consistent with the results obtained from other species, such as Cycas simplicipinna [R. rex subsp. rex. We speculate that the populations of R. rex subsp. rex might have survived in situ rather than migrating long distances to suitable habitats and that evolutionary adaptation might have occurred in the cold environment. The current existing populations of R. rex subsp. rex were limited in distribution at 2400–3400 m elevation, and this condition might partly support our speculation. In addition, the complex topology of physical environmental condition in southwest China might cause geographical barriers between population migrations. This scenario was also found in the population demography of Leucomeris decora [Exploring the historical demography of a species can facilitate our knowledge of its ancient evolutionary environment . Quaternicipinna . Typicalicipinna . Howevers decora .R. rex subsp. rex populations. A moderate genetic diversity, a high genetic differentiation, and a conspicuous geographical structure were detected in R. rex subsp. rex. The species possessed unique genetic lineages and endemic cpDNA haplotypes in its separate refuge populations. In addition, we found that R. rex subsp. rex experienced a population contraction approximately 50,000–100,000 years ago based on the comprehensive analysis of demographic history. Furthermore, no recent population expansion occurred in this species. Hence, the conservation of R. rex subsp. rex should focus on habitat destruction and loss, resulting in a population decline and inbreeding depression within populations. Furthermore, all the remnant adult trees of R. rex subsp. rex should receive priority protection for the maintenance of its genetic diversity. This research exhibited tremendous ecological value for the future conservation and sustainable utilization of R. rex subsp. rex and other similar plants, which are subjected to climate oscillation, inbreeding depression, overexploration, and habitat destruction.The present study firstly investigated the genetic diversity, population structure, and demographic history of 11 remnant


Coturnix japonica) is an important poultry species owing to their high economic efficiency and biological advantages. The genetic diversity of farm quail populations has rarely been studied.The Japanese quail (This study aimed to assess the genetic diversity of farm quail populations and their genetic relationships, which could provide important information for designing breeding programmes to maintain egg and/or meat production efficiency.Molecular phylogenetic and STRUCTURE analyses were conducted for seven farm populations and six laboratory lines using 50 microsatellite markers previously developed by us.Ne, He and STF values, enabled the construction of reliable phylogenetic trees and STRUCTURE plots.The genetic diversity within each farm population was relatively high despite long‐term breeding within closed colonies. However, the genetic variation between populations was absent. Twenty highly polymorphic markers, selected based on In the farm populations analysed in the present study, gene flow between genetically distant populations is needed to restore genetic diversity between farm populations, which could exploit heterosis and decrease the risk of inbreeding depression. Our findings demonstrate that these markers are useful for examining the genetic structure of farm quail populations. We conducted genetic monitoring for six farm populations in Aichi prefecture, Japan, and five laboratory lines, whose breeding histories are well recorded, using 48 microsatellite markers. We estimated the genetic diversity of the farm populations and their genetic relationships by molecular phylogenetic and STRUCTURE analyses, which provided important information to obtain suitable genetically distinct candidates for cross‐mating to restore genetic diversity among populations. Because of their high economic efficiency, rapid sexual maturation and resistance to environmental stresses and disease, Japanese quail have attracted increasing attention as poultry species, second to the chicken, especially in developing countries  per marker was counted using MICROSATELLITE ANALYSER 4.05 , observed heterozygosity (Ho), expected heterozygosity (He), fixation indices  and the chi‐square statistic for Hardy‐Weinberg equilibrium (HWE) were calculated for each marker using GENALEX 6.5  was estimated based on the Expectation Maximisation (EM) algorithm  in each population was calculated using MICROSATELLITE ANALYSER 4.05  between individuals within a population determined using the relatedness analysis conducted with GENEALEX 6.5  and presenting null allele frequencies less than 0.1  using 48 markers, excluding two monoallelic markers see the  section,33.1NA = 23) and the lowest for NGJ0007, NGJ0022 and NGJ0044 (NA = 3) (Table Ne was the highest for NGJ0050 (4.76) and the lowest for NGJ0042 (1.33). The PIC ranged from 0.27 (NGJ0044) to 0.87 (NGJ0018). The He and STF values ranged from 0.27 (NGJ0039) to 0.77 (NGJ0050) and from 0.05  to 0.63 (NGJ0039), respectively. Significant deviation from the HWE was found for 10 markers . Null allele frequencies ranged from 0 (NGJ0044) to 0.35 (NGJ0039).Two markers, NGJ0023 and NGJ0045, were monoallelic Table , and the3) Table . The Ne MNA) ranged from 2.82 for the DY line to 5.52 for the Farm A line. The AR ranged from 2.78 (DY) to 4.90 (Farm D). The lowest Ne and Ho values were observed for the DY line , whereas the highest values were observed for Farm D . Six farm populations from Aichi prefecture (Farms A‒F) showed ISF values of 0.10 on average, ranging from 0.08 (Farm D) to 0.13 (Farm F). The Saitama population had a marginally higher ISF value (0.12). The MGD values for Farms A‒F (66.51 on average) were similar to those obtained for the Saitama population (67.01), but were relatively lower (51.87 on average) in four AARC laboratory lines , as were the ISF values (0.02 on average). These results suggest that genetic diversity was not especially low in all the farm populations from Aichi prefecture (Farms A‒F) compared with the farm population from Saitama prefecture. However, genetic variability in the DY and four AARC laboratory lines was substantially lower due to their small population sizes; the DY line has maintained as a closed population which are generally composed of about 30 males and 50 females per generation, and each of four AARC lines has consisted of around 150 males and 350 females per generation.Genetic diversity indices for each population are shown in Table 3.2Three phylogenetic trees for 13 populations based on Da Figure , Dps Fi, and Dc K = 2 only separated farm and farm‐derived populations  and the rest (four AARC lines and the wild‐derived line) (data not shown), which was not informative for our research. Therefore, we constructed the STRUCTURE plot with the second highest K value (K = 5) because it was more informative. All six farm populations from Aichi prefecture (Farms A‒F) clustered together (shown in orange)  Figure  and were3.3Ne and He values were the same  and the STF value (FST‐20), both of which covered 15 chromosomes. We note that FST‐20 included four markers with STF values less than 0.10 . Because the Dc genetic distance produced the most reliable tree with the 48 markers (see the details in the Discussion), the phylogenetic analyses with NeHe‐20 and FST‐20 were performed using the Dc genetic distance. The NeHe‐20 and FST‐20 phylogenetic trees were largely similar , Toyokawa (Farm E) and Tahara (Farm F) in Aichi prefecture was as high as that observed in the farm population from Saitama prefecture. Furthermore, their 4.2The phylogenetic trees constructed with the 48 microsatellite markers showed very close genetic relationships among six populations from Aichi prefecture (Farms A–F). Moreover, the close relationship of the DY line with the six farm populations shown in the Da and Dc phylogenetic trees was compatible with the breeding history of the DY line that originated from a farm population in Toyohashi in 1963. The AARC‐WW line is a direct descendant from the wild‐derived line introduced from the NILGS. The AARC‐B line was established by crossing the wild‐derived line with a laboratory line introduced from Shinshu University, Japan, whereas the AARC‐C line was established by crossing the wild‐derived line with commercial quail. Consequently, these three lines share a common ancestor (the wild‐derived line), which explains their close genetic relationships. The AARC‐BB line descended from the NIES‐Br line originating from a meat‐type quail population with brown plumage that was imported from Brazil and maintained at the National Institute of Environmental Studies (NIES), Japan. STRUCTURE analysis of 15 laboratory lines in our previous study  that produced reliable phylogenetic trees and STRUCTURE plots. The STRUCTURE plots constructed with NeHe‐20 and FST‐20 may be more informative than that produced with 48 microsatellite markers. First, the Farm C population showed partial genetic affinity with the AARC‐C line, supporting that sires from the AARC‐C line were introduced into Farm C in 2012; and second, the AARC‐WW and wild‐derived lines, which have the same origin, were grouped into a single genetic cluster that did not include the AARC‐BB line. These detailed results were not obtained by analyses with the 48 markers. Carlsson , presenting high Ne, He and STF values and low null allele frequencies, are effective for estimating the genetic diversity of farm quail populations and their genetic relationships.Microsatellites are one of the most popular markers for estimating the genetic diversity in animal and plant populations. Microsatellite markers for Japanese quail were developed comprehensively in previous studies (Kayang et al., Carlsson  demonstrg et al. , when 185Japanese quail are expected to become more widespread in developing countries as a useful source of animal protein. When breeding programmes are formulated to improve egg and/or meat production efficiency of poultry, genetic assessment of farm populations will be important to avoid the loss of genetic diversity or the accumulation of homozygosity that could result in decreased productivity. In this study, using the 50 microsatellite markers, we performed a range of molecular phylogenetic and STRUCTURE analyses that clearly demonstrated the breeding histories and genetic diversities of both the laboratory lines and farm populations of quail from the Aichi prefecture, Japan. The two laboratory quail lines, the AARC‐BB and AARC‐WW, were found to be genetically relatively distant from the farm populations. Based on our data, the Aichi Agricultural Research Center has started to introduce the two laboratory lines into farm populations in Toyahashi city. Furthermore, 20 highly polymorphic microsatellite markers selected from among the 48 markers also accurately estimated the genetic diversity of the quail populations and their genetic relationships, indicating that these microsatellite markers are useful for the genetic assessment of farm quail populations.Mitsuo Nunome: Data curation; Formal analysis; Investigation; Methodology; Resources; Validation; Visualization; Writing‐original draft. Rie Yoshioka: Data curation; Investigation; Resources. Takuro Shinkai: Data curation; Formal analysis; Investigation; Validation; Visualization. Katsutoshi Kino: Data curation; Project administration; Resources; Supervision; Writing‐review & editing. Yoichi Matsuda: Conceptualization; Project administration; Resources; Supervision; Writing‐original draft; Writing‐review & editing.https://publons.com/publon/10.1002/vms3.328.The peer review history for this article is available at Table S1Click here for additional data file.