Ancient DNA research is revealing a human history far more complex than that inferred from parsimonious models based on modern DNA. Here, we review some of the key events in the peopling of the world in the light of the findings of work on ancient DNA. The human past on many timescales is of broad intrinsic interest, and genetics contributes to our understanding of it, as do paleontology, archaeology, linguistics and other disciplines. Geneticists have long studied present-day populations to glean information about their past, using models to infer past population events such as migrations or replacements, generally invoking Occam’s razor to favor the simplest model consistent with the data. But this is not the most straightforward approach to understanding such events: the obvious way to study any aspect of human genetic history is to analyze population samples from before, during and after the period of interest, and to simply catalogue the changes. Advances in ancient DNA (aDNA) technology are now beginning to make this more direct approach possible, facilitated by new sequencing technologies that are now capable of generating gigabases of data at moderate cost (Box 1). This abundance of data, combined with an understanding of the damage patterns indicative of authentic aDNA, greatly simplify the recognition and avoidance of the bugbear of the field: contamination.Here, we review some of the key events in the peopling of the world in the light of recent aDNA findings, discussing new evidence for how migration, admixture and selection have shaped human populations.For decades, the theories about the origin of modern humans were summarized in two main competing models: multiregional evolution or recent replacement from Africa , 2. GeneThere are hundreds of research papers discussing the out-of-Africa migration using archaeological data, present-day human genetic data or even genetic data from the human microbiome. Most of this work refines the recent replacement model, including suggesting a time-frame for the expansion  as well Neanderthal ancestry in all present-day non-Africans is estimated to be 1.5–2.1 % . The broHomo species. Future findings will likely show that many of the assumptions reported here were simplified and that, even with aDNA, we still have to invoke Occam’s razor to explain the data: that is, until sufficient human fossils have been sequenced.Admixture with Denisovans also occurred, possibly in South-East Asia , and affaDNA evidence has thus supported the replacement model as an explanation for most human variation, but has transformed and enriched this model in ways not anticipated in the earlier debate: first by discovering Denisovans, whose fossil record currently remains unrecognized, and second by revealing the multiplicity of admixture events, which include at least one that cannot be detected in present-day DNA.Europe was first populated by modern humans around 45,000 ya, but (except for some southern areas) was depopulated during the glacial maximum that occurred 25,000 ya and subsequently repopulated as the climate improved, with farming beginning ~8000 ya during the Neolithic transition (Box 3). Decades of debate have been dedicated to understanding the origin of agriculture in Europe, focusing particularly on whether it spread from its place of origin in the Near East by demic diffusion (movement of farmers) or by acculturation of the indigenous hunter-gatherers (movement of ideas). Geneticists first attempted to answer this question by sampling modern populations from Europe and the Near East and then comparing the genetic diversity of classical protein markers (Box 1) between the two regions.Science magazine featured an image by Cavalli-Sforza and colleagues showing maps of Europe constructed with ten loci using multivariate techniques to reveal clines [In 1978, the cover of l clines , which tl clines , 25 and l clines , with pel clines .Recent aDNA studies reveal, however, that populating Europe has been a much more complex process, and that the Neolithic transition (Box 3) was not even the event that most influenced the present-day genetic landscape.The first aDNA complete genome sequence from Europe came from the Tyrolean Iceman; a 5300-year-old (Late Neolithic or ‘Copper Age’) natural mummy discovered in 1991 in the Ötztal Alps. Surprisingly, the Iceman had more genetic affinity to present-day Sardinians than to the present-day populations inhabiting the region where he probably lived , showingIn summary, aDNA findings have provided conclusive evidence for the movement of farmers at the beginning of the Neolithic transition, but also for the incorporation of the hunter-gatherer gene pool, and therefore support what might be called a ‘leaky demic diffusion’ model. In this respect, the new findings merge the previous ideas. But in demonstrating the large genetic contribution of the Yamnaya during the Bronze Age, they again reveal major events that were not anticipated in the earlier genetic debate.Although it has long been accepted that Native Americans’ ancestors migrated from Asia via Beringia (present-day Alaska) to occupy the Americas, much uncertainty has surrounded both their origin within Asia and the number of migrations. Genetic analyses show that Native Americans are most closely related to northeast Asians, but with different skull morphology. In 1996, the finding of a 8340–9200-year-old male human skeleton along the Columbia River shoreline outside Kennewick, Washington State, USA, heightened the debate on the origin of Native Americans. Initial assessment of the skeleton suggested he was anatomically distinct from modern Native Americans and more closely related to circumpacific groups such as the Ainu and Polynesians. Kennewick man, as the skeleton came to be known, was recently sequenced and found to be genetically closer to modern Native Americans than to any other population worldwide, therefore showing continuity with Native North Americans over at least the past eight millennia, despite the difference in morphology .Insights into the Asian origin of the Native Americans came from a genome sequence of a 24,000-year-old boy found in Mal’ta in south-central Siberia . The MalControversy about the origin of Native Americans was recently sparked once more when analysis of present-day DNA from some Amazonians showed a small proportion of ancestry more closely related to indigenous Australians, Papuans and Andaman Islanders than to any present-day Eurasians or Native Americans, suggesting to the authors that there were two founding populations in the Americas . HoweverThe Paleo-Eskimo cultures that settled in Greenland appear to have also originated from a migration from Siberia, but more recently (~5500 ya) and independently of the early migration that gave rise to the majority of Native Americans. Subsequent migration of the ancestors of the Inuit is evident from the genome sequence of ~4000-year-old Saqqaqman .In this example, the aDNA data support and refine the existing models of the early peopling of the Americas, notably by informing on the different independent migrations and by simplifying the interpretation of the morphological differences between the first Americans and later Native Americans, showing these differences to be part of the same genetic continuum.EPAS1, a transcription factor involved in the response to hypoxia, which most probably helps the Tibetans to live at high altitudes [EPAS1. Other examples include Neanderthal alleles that are enriched in genes affecting keratin filaments, which make up most of the outer layer of human skin and produce hair, suggesting that Neanderthal alleles may have helped modern humans to adapt to non-African cold environments [Modern humans have come to inhabit an impressive diversity of ecological niches, many of which required local adaptation for survival. Several genetic signatures of adaptations have been identified by searching for alleles that are at high frequency in specific modern populations relative to other populations. For example, by studying genes that show population-specific allele-frequency differences between Tibetans and Han Chinese, a signal of positive natural selection was detected in ltitudes . Numeroultitudes . This coltitudes ; therefoltitudes . On the ronments . The samAdaptation to non-African environments was also believed to be the cause of human variation in skin color. It was thought that the light skin of Europeans was a Paleolithic adaptation to facilitate vitamin D production in reduced sunlight regions . ConsistThe warming after the last glacial period, followed by the Neolithic transition and the adoption of agriculture, introduced major changes in the lifestyle and diet of human populations. These events are proposed to have triggered new waves of selection that helped humans to adapt to the resulting social and environmental changes. In particular, selection on immune genes was believed to have been increased by the spread of diseases after the Neolithic due to dense settlements and proximity to domesticated animals. aDNA from Mesolithic Europeans shows, however, that adaptive variants associated with pathogen resistance in modern populations were already present in hunter-gatherers before the advent of agriculture . In factaDNA findings are thus now beginning to transform our understanding of recent positive selection in humans, both by introducing new mechanisms such as adaptive introgression, and by showing that our estimates of the timing of selective sweeps derived from models using present-day populations were unreliable, with the consequence that some widely held hypotheses about the selective forces were also unreliable.not parsimonious, and aDNA reveals a much richer history, as in the other examples considered here. In either situation, human evolutionary genetics is moving to a paradigm where we first look for evidence from aDNA and interpret present-day genetic variation in its light.Findings from aDNA research are currently transforming our understanding of human history at an ever-increasing pace. When evolution was parsimonious, aDNA may support the prevailing model, as with the initial peopling of the Americas; but more often, evolution was What are the limits to how far this can go? Very ancient samples more than 100,000 years old and some geographical regions of great interest, such as the Near East and Africa, remain challenging for aDNA research. Both time and poor DNA preservation in hot wet climates may impose insurmountable limits to resolving many questions related to the origin and genetic diversity of our species. Identifying favorable locations within these regions , or releHomo heidelbergensis (‘Sima de los Huesos’) or any sequence data from Homo floresiensis or Homo erectus? Who were the sources of the non-Neanderthal, non-Denisovan archaic admixture already detected? What did Denisovans look like? What were the number, timing and routes of the major expansion(s) of fully modern humans out of Africa? What was the full richness of subsequent human population history and adaptation throughout the world, including episodes that have left no traces in present-day populations? We expect our understanding to be transformed again in these and unforeseen directions, perhaps even before this review is published.In the near future, we look forward to insights into human history ranging from hundreds of thousands of years ago to the past few centuries. Can we obtain nuclear sequences from The distribution of the human blood groups” [Over the past 100 years, the datasets and mathematical methodologies used in population genetics have changed enormously, providing an ever better understanding of human genetic diversity over time and space. In 1954, Arthur Mourant published his ground-breaking book “ groups” , probablThe development of the polymerase chain reaction (PCR) in the 1980s introduced the use of molecular markers to population genetics and allowed, for the first time, the study of evolutionary distances between alleles at a locus. This methodological progress, along with theoretical advances such as identity by descent developed by Gustave Malécot in 1939  and coalThe first widely used molecular markers were variants of the mitochondrial DNA (mtDNA) and the non-recombining region of the Y-chromosome (NRY). mtDNA is inherited maternally and transmitted from a mother to her children, while the NRY is inherited paternally passing down from father to son. These uniparental markers are transmitted from one generation to the next intact (apart from new mutations) and have known mutation rates, allowing straightforward construction of phylogenies and inference of some aspects of population relationships. Uniparental loci are, however, sex-specific and experience strong drift, providing a limited view of the complex human history. For example, Neanderthal mtDNA analysis shows no evidence of admixture with modern humans , althougThe study of genome-wide markers was initiated using microsatellites  but was simplified by the development of single nucleotide polymorphism (SNP) arrays. The effective population size of autosomal variants is expected to be four times that of mtDNA and NRY, making autosomal variants less prone to drift and providing insight further back into human history. Nevertheless, inferences from SNP arrays are limited by ascertainment biases arising from their design, which generally incorporated SNPs that were discovered in a few populations and were inadequate to capture global genetic diversity.The development of next-generation sequencing (NGS) resolved many of the limitations of the previous methodologies by generating gigabases of sequence data from the whole genome, reducing ascertainment biases while increasing the power to detect evolutionary processes. NGS produces large numbers of short sequencing reads. This feature is particularly useful for ancient DNA analysis and has allowed the sequencing of genomes that are tens of thousands of years old, making the direct study of the evolutionary changes over time and space possible. NGS is thus currently revolutionizing the field of population genetics.D and looked at the derived alleles in Neanderthal. The ancestral allele is defined by the Chimpanzee sequence and is labeled A, the derived allele is labeled B. Two possible patterns of SNPs can then be observed and counted: ‘ABBA’ or ‘BABA’. If Human1 and Human2 shared the same history in their relation to Neanderthal, they will not differ in their derived allele frequencies , and D will not differ significantly from zero. Gene flow from Neanderthal to one of the human populations will lead to an increase in the derived alleles that have occurred on the Neanderthal branch in that human population and D will deviate from zero. Green et al. performed the following test: D and found D was always positive from a significant excess of ABBA sites over BABA sites. The test has subsequently become widely used in many different ways.Patterson’s D-statistic is a comparative measure of allele sharing between two populations and an outgroup. It can be used as a formal test for admixture and can provide information about the direction of gene flow. It was first introduced by Green et al.  to show The Neolithic transition or revolution refers to the change in lifestyle of humans from hunting-gathering to agriculture, through domestication of plants and animals, which led to the development of permanent settlements, towns, cities, trade and eventually the rise of civilization. Neolithic transitions occurred independently in several parts of the world; the one that transformed Europe started in the Near East around 10,000 years ago and spread through Europe over the next few millennia. This cultural transition had considerable consequences for human genetic variation by stimulating growth in population size, and triggering multiple expansions and mixtures, as well as adaptation to certain diets and diseases.


Salix dasyclados, S. schwerinii and S. viminalis in the vicinity of the Lake Baikal in Southern Siberia. Individuals were sampled in fourteen populations and classified as pure species or hybrids based on a set of morphological characters. They were then genotyped at 384 nuclear SNP and four chloroplast SSR loci. The STRUCTURE and NewHybrids softwares were used to estimate the frequency and direction of hybridization using genotypic data at the nuclear SNP loci.Hybridization and introgression are said to occur relatively frequently in plants, and in particular among different species of willows. However, data on the actual frequency of natural hybridization and introgression is rare. Here, we report the first fine-scale genetic analysis of a contact zone shared between the three basket willow species, S. viminalis or S. schwerinii and no F1 or F2 hybrids were found. The rest of the genotyped individuals were classified as pure species and formed two clusters, one with S. schwerinii individuals and the other with S. viminalis and S. dasyclados individuals. The two clusters were significantly genetically differentiated, with FST = 0.333 . In contrast, for the chloroplast haplotypes, no genetic differentiation was observed as they were completely shared between the species. Based on morphological classification only 5 % of the individuals were classified as introgressed individuals, which was much less than what was detected using genotypic data.As many as 19 % of the genotyped individuals were classified as introgressed individuals and these were mainly encountered in the centre of the contact zone. All introgressed individuals were backcrosses to We have discovered a new willow hybrid zone with relatively high frequency of introgressed individuals. The low frequency of F1 hybrids indicates that ongoing hybridization is limited, which could be because of the presence of reproductive barriers or simply because the conditions are not favorable for hybridization. We further conclude that in order to get a complete picture of the species composition of a hybrid zone it is necessary to use a combination of morphological characters and genetic data from both nuclear and chloroplast markers.The online version of this article (doi:10.1186/s12862-015-0461-7) contains supplementary material, which is available to authorized users. Hybridization is a phenomenon that happens when mating between individuals from different species generates viable offspring and is a process that is estimated to occur in approximately 25 % of all plant species , 2. Hybret al. [Salix eriocephala and S. sericea with only morphological characters. Therefore, in addition to morphological characteristics, molecular markers provide a good tool for assessing the degree of gene flow and genetic structure of hybrid zones. Due to the contrasting modes of inheritance of chloroplast and nuclear DNA they are expected to generate different patterns of genetic diversity within and among populations [In the past, hybrids were characterized solely based on morphology, however the use of morphological traits alone may not be sufficient to identify all hybrids or introgressed individuals , which wet al.  that faiulations . Thus a ulations .Salix; Salicaceae) is a plant genus with tree and shrub species that are common in riparian habitats worldwide. The genus contains about 400 species [n = 19. However, ploidy levels can vary extensively both within and among species and range from diploids up to dodecaploids [S. fragilis and S. alba in Europe [S. sericea and S. eriocephala in the U.S. [S. caprea, S. cinerea, S. atrocinerea and S. aurita [Willows  [S. viminalis × S. dasyclados it was found that the S. dasyclados parent was hexaploid and the offspring tetraploid [S. dasyclados individuals could be allopolyploids possessing genomes from several species. However, observations indicate that these three species are predominantly diploid.Interspecific crosses between werinii) , 26 has werinii) –33. Intetraploid , indicatSalix dasyclados, S. schwerinii and S. viminalis located in the vicinity of the Lake Baikal in Southern Siberia. The main objectives were to morphologically and genetically characterize individuals in the contact zone and to estimate the frequency and direction of hybridization using 384 nuclear SNP and four chloroplast SSR markers together with a set of morphological characters. Samples were collected in fourteen populations. We specifically asked the following questions: i) Is the contact zone a hybrid zone, that is, do we find hybrids or introgressed inidviduals? ii) If yes, what is the population structure of the hybrid zone? Do we primarily find intermediate F1 hybrids or introgressed individuals? iii) Is introgression uni- or bidirectional? iv) Can morphology be used to predict the structure of the hybrid zone? v) What are the characteristics of the reproductive barriers between the species?In the present study we performed fine-scale genetic analysis of a contact zone with the three species A total of 375 individuals were collected from fourteen populations and based on morphological characterization nineteen individuals (5 %) were classified as hybrids.S. viminalis × S. dasyclados hybrids, seven as S. viminalis × S. schwerinii hybrids and one as either a S. viminalis × S. dasyclados hybrid or a S. schwerinii × S. dasyclados hybrid  , thus they form a representative subset of all collected samples. Two SNPs in S. schwerinii and three in S. viminalis deviated significantly from Hardy-Weinberg equilibrium (p ≤ 0.01)  in S. viminalis, 0.101  in S. schwerinii and 0.333 between the species . When all samples were included in clustering analysis with the STRUCTURE software [K) was estimated to 2, both when the original method from Pritchard et al. [Q-values ≥ 0.9 and 44 of these were assigned to cluster 1 and twenty were assigned to cluster 2 , while eight were classified as S. schwerinii and three as S. viminalis  .Fifteen of the genotyped individuals 19 %) had 9 % had Qds Table . These ids Table . Morphol.9 Table , of whicccmp2 was the most variable with six alleles and ccmp4 the least variable with two alleles, ccmp5 had four and ccmp6 had five alleles. When combined, these alleles formed 24 different haplotypes. When only including the individuals from the Baikal region (N = 50) the PhiPT  was estimated to -0.023 (p = 0.647) in S. viminalis, to 0.107 (p = 0.100) in S. schwerinii and to -0.006 (p = 0. 371) between the species. Thus, the chloroplast haplotypes did not display any significant genetic differentiation, either within or between the species .Seventy individuals originating from Europe and the Baikal region were successfully genotyped with the four chloroplast SSR markers. As expected, one allele per locus and individual was found for each of the markers. A total of seventeen alleles were detected and Salix dasyclados, Salix schwerinii and Salix viminalis are phenotypically similar and several morphological characteristics are needed to distinguish them from each other. Given that the three species recently diverged, it is possible that they would hybridize where they come into contact in nature, unless some strong reproductive barriers have evolved. To study this, individuals from all three species were sampled in fourteen populations in a region in Southern Siberia where the distributions of the species meet and overlap. 81 % of the genotyped individuals were assigned to one of two clusters; one composed of S. schwerinii and one of S. viminalis and S. dasyclados individuals. Relatively high level of genetic differentiation was observed between the clusters (FST of 0.333), suggesting that they are largely reproductively isolated. However, 19 % of the genotyped individuals were identified as hybrids, which shows that the species boundaries are partly permeable. Both the STRUCTURE and the NewHybrid analyses showed that none of these individuals were intermediate F1 hybrids but backcrosses to either S. schwerinii or S. viminalis. This observation demonstrates that introgression is bidirectional in this willow hybrid zone. Furthermore, the absence of F1 hybrids is striking and is best explained by the presence of pre- and/or postmating reproductive barriers that form reproductive isolation between the species [S. schwerinii and S. viminalis display asynchronous flowering in the region of sampling. However, at our field site outside of Uppsala in Sweden , S. schwerinii plants flower considerably earlier than S. viminalis plants, bearing in mind that those S. viminalis plants originate from Central Europe and not from the region near Lake Baikal. Possibly, postmating barriers are operating together with premating barriers to produce strong reproductive isolation, as premating barriers alone are often permeable and leaky [The three closely related basket willow species  species . Current species  and as w species , 43, botnd leaky . Howevernd leaky  were conBetula spp. [Quercus spp. [S. viminalis and S. schwerinii individuals from Europe and Siberia, respectively, i.e. long way from the hybrid zone, divergence was estimated at 600,000 ya [An interesting observation was that chloroplast haplotypes were shared between the species and no genetic differentiation was found, even when including samples from Europe. This could indeed be the result of homoplasy, however as extensive haplotype sharing has been reported in other willow , 45, 46 ula spp.  and Quercus spp.  using encus spp. . In a pr0,000 ya . The effThe chloroplast and nuclear DNA gave contrasting results as there was no significant genetic differentiation with the chloroplast haplotypes, while with the nuclear genotypic data there was relatively high level of genetic differentiation. This pattern is a likely consequence of the differences in intraspecific gene flow that the nuclear and the chloroplast DNA experience . Since nS. schwerinii were in fact introgressed individuals. This demonstrates the difficulties and uncertainties in detecting hybrids solely based on morphology, which could be particularly difficult at low levels of introgression. Another surprising finding was that individuals classified as S. dasyclados clustered with individuals classified as S. viminalis. As we were unable to distinguish the species from each other with this large set of markers, one conclusion could be that the sampled individuals should be regarded as one and the same species. Still, they display enough morphological differences to be classified as different species by trained botanists. In order to solve this, more samples need to be analysed, both genetically and morphologically.Compared to the 19 % of the genotyped individuals that were assessed to be of hybrid origin, only 5 % were classified as hybrids using traditional morphological characteristics. Most noteworthy was that eight individuals that were classified as pure S. viminalis and S. schwerinii. This was further supported by the lack of intermediate F1 hybrids, which could be a consequence of some of reproductive barrier(s). In addition we found that introgression was bidirectional so that backcrosses happen with both S. schwerinii and S. viminalis. Another surprising observation was the genetic similarities between S. dasyclados and S. viminalis, which leads us to doubt the taxonomic status of S. dasyclados.We have discovered a new hybrid zone between three willow species with rather high frequency of introgressed individuals, which were identified both with morphological characters and genotypic data. We found extensive sharing of chloroplast haplotypes between the species and a large number of shared nuclear polymorphisms, a pattern that is expected from introgression but also from ancestral polymorphisms that remain in the populations due to incomplete lineage sorting. We also demonstrated rather high levels of reproductive isolation between S. dasyclados, S. schwerinii and S. viminalis and putative hybrids were collected from plants in the field in July 2012. Samples were collected in fourteen different populations , 0.9 mM of a mix of dNTPs , 0.1 μM of each primer  and 0.25 U Taq HotStar Plus . The PCR program included 95 °C for 5 min, then 30 cycles at 95 °C for 30 s, Tm for 30 s, 72 °C for 30 s, then 8 °C on hold. Tm was 58 °C for ccmp2, ccmp4 and ccmp6 and 56 °C for ccmp5. The forward primers were fluorescently labeled at the 5´end; ccmp2 with 6-FAM, ccmp4 with NED, ccmp5 with VIC and ccmp6 with PET. Allele sizes were determined with the PeakScanner TM Software v1.0 . In addition to individuals sampled in the fourteen populations in Southern Siberia another twenty samples, morphologically classified as S. viminalis were scored with the chloroplast SSRs; four originated from western Russia and sixteen originated from Europe. Two chloroplast regions were sequenced and analysed for sequence variation; an intron in the tRNA-Leu gene (trnL) amplified with primers c and d [rbcL gene (Rubisco) amplified with primers carbcLF and carbcLR [2 , 0.8 mM of a mix of dNTPs , 0.2 μM of each primer  and 1.5 U Amplitaq GOLD . The PCR program included 94 °C for 5 min, five cycles of 94 °C for 30 s, 65-60 °C for 30 s, (- 1 °C every cycle), then 25 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s, then 72 °C for 10 min and finally 10 °C on hold. Amplification was determined by agarose gel electrophoresis. PCR products were cleaned and sequenced at Macrogen Inc.  on both strands using the forward and reverse PCR primers as sequencing primers. The chromatograms were visually inspected and edited using Lasergene SeqMan vs 9.1.0 (DNASTAR).Genomic DNA from dried leaf tissue samples was isolated using the DNeasy Mini Plant Kit . Genotypes were determined at 384 previously developed single nucleotide polymorphism (SNP) loci  with theocations . Allelesocations . The chl c and d  and a fr carbcLR . The PCRFST both within and between S. schwerinii and S. viminalis populations using Arlequin v. 3.5 [S. schwerinii and S. viminalis were estimated. For these analyses, individuals were grouped based on morphological characteristics and the hybrids and S. dasyclados were not included.The R package  adegenetn v. 3.5 . Shared K-value (i.e. the number of clusters) was set from one to eight, a value within this range being expected to be the most likely result. The length of the burn-in period was set to 10,000 and the Monte Carlo Markov Chain (MCMC) model after burn-in was run for an additional 100,000 iterations. For each K, 40 replicates were run and runs with an outlier value of lnPD were removed . The optimal value of K was determined by examination of the L(K) and Evanno’s ∆K statistics [Genetic differentiation was investigated using Bayesian cluster analyses with the STRUCTURE software v 2.3.2.1 –38. An aatistics  using thatistics .PhiPT within and between S. schwerinii and S. viminalis was assessed using GenAIEx v 6.5 [www.esri.com.For each of the four chloroplast SSR markers, allele numbers and size ranges were assessed. Alleles were combined in haplotypes for every individual. Ex v 6.5 , 60. HapQ-value from STRUCTURE, which shows the proportion of an individual’s genome that originates from each of the K clusters, was used as hybrid index  values from the different runs which were then averaged across the 5 runs.Hybrids and backcrosses were identified using the nuclear genotypic data and the softwares STRUCTURE –38 and Nt et al. ). Thus, The data set supporting the results of this article is archived in LabArchives (doi: 10.6070/H48P5XJ2).


These programs are unlikely to succeed if captive populations do not recover from the severe bottleneck imposed when they are founded with a limited number of individuals from remnant populations. In small captive populations allelic richness may be lost due to genetic drift, leading to a decline in fitness. Wild populations of the Hawaiian tree snail Achatinella lila, a hermaphroditic snail with a long life history, have declined precipitously due to introduced predators and other human impacts. A captive population initially thrived after its founding with seven snails, exceeding 600 captive individuals in 2009, but drastically declined in the last five years. Measures of fitness were examined from 2,018 captive snails that died between 1998 and 2012, and compared with genotypic data for six microsatellite loci from a subset of these deceased snails (N = 335), as well as live captive snails (N = 198) and wild snails (N = 92). Surprisingly, the inbreeding coefficient (isF) declined over time in the captive population, and is now approaching values observed in the 2013 wild population, despite a significant decrease in allelic richness. However, adult annual survival and fecundity significantly declined in the second generation. These measures of fitness were positively correlated with heterozygosity. Snails with higher measures of heterozygosity had more offspring, and third generation offspring with higher measures of heterozygosity were more likely to reach maturity. These results highlight the importance of maintaining genetic diversity in captive populations, particularly those initiated with a small number of individuals from wild remnant populations. Genetic rescue may allow for an increase in genetic diversity in the captive population, as measures of heterozygosity and rarified allelic richness were higher in wild tree snails.As wild populations decline,  Hermaphroditic molluscs with high fecundity and the ability to self-fertilize may purge deleterious alleles and recover from a severe bottleneck Achatinella consists of at least 41 species of hermaphroditic tree snails with long life spans, late maturity, and single, live births Achatinella, and resulted in the declaration of all remaining species in the genus as Endangered The genus A. lila, with less than 200 known individuals remaining over less than five percent of its historic range (isF) over generations. We also compared genetic variation in wild A. lila with the captive population to provide long-term management recommendations.One species of special concern is ic range . A captiA. lila was founded in 1997 at the endemic Hawaiian tree snail captive-breeding facility at the University of Hawai‘i at Mānoa with seven adult snails collected and housed under USFWS permit PRT-826600. Sexual maturity is indicated by a lipped shell, or thickened edge of the shell aperture, at around 15 mm shell length in this species The captive population of Cladosporium sp.) originally isolated from a native snail-host plant Captive snails were reared in environmental chambers with controlled temperature and humidity designed to mimic wild conditions A. lilat = −0.49, P = 0.63). Birth dates, combined with shell length at the time of death, were used to calculate juvenile survival (snails surviving more than one year beyond their birth date) for the years 1998 through 2012. Based on published growth curves Demographic data were collected from 2018 snails preserved between 1998 and 2013, 198 living snails in 2013, and from facility records. Using shell growth curves generated in captive Achatinella lila lives on native plants in forest dominated by O‘hia (Metrosideros polymorpha) in the northern Ko‘olau Mountain Range on the island of O‘ahu, Hawai‘i. Its known range currently extends two kilometers north to south along a constricted elevation of 700–850 m on the steep windward, moist slopes, where it co-occurs with sister species A. sowerbyana, A. byronii, and A. bulimoidesOnly non-lethal, well-established, previously published methods were used for tissue collection Each sample was genotyped at eight previously identified polymorphic microsatellite loci A. lila  was calculated as 5.59 years according to the equation M – 1 + (1/(1-v) 1 offspring born in years 1997 to 2002, F2 offspring born in years 2003 to 2008, and F3 offspring born in years 2009 to 2014. Snails brought into captivity in 1997, all of which died by the end of 2002, were referred to as the “founder” generation. Statistical tests were performed using the statistical package JMP (10.0). Fecundity was compared among generations using a Kruskall-Wallis nonparametric test followed by a nonparametric Wilcoxon test between each pair of generations. Changes in juvenile survival and survival to maturity over time were assessed using logistic regression analysis. Adult survival, or the proportion of adults surviving each year, was tested over time using linear regression analysis. Changes in survival to maturity among generations were tested using chi square analysis. Errors are reported as standard deviation.The total captive population, including both deceased and living snails between 1997 and 2013, was divided into founder, F3 generation, whose members were not old enough to have reached sexual maturity at the time of sampling, genetic measures were compared between deceased and living snails. Individual heterozygosity, calculated as the proportion of heterozygous loci, was arcsine transformed and compared among groups in the program JMP (10.0) using a t-test or an ANOVA, as appropriate, depending on the number of groups compared. Arlequin isF), observed heterozygosity (OH), expected heterozygosity (EH), and genetic diversity.Within generations born in captivity, we compared genetic measures for those that reached sexual maturity with those that did not reach sexual maturity. For the FIS, length, and arcsine-transformed individual heterozygosity (proportion of heterozygous loci) were tested with linear regression analysis using the statistical package JMP version 10.0.To assess the relationship between individual fitness and heterozygosity, parentage was assigned for live-sampled captive offspring (<15 mm shell length) for all potential parents (adults ≥ 15 mm shell length) within the same cage, using the program Cervus version 3.03 STF) was assessed between the captive live population and the wild population with the software package Arlequin RA) for a minimum sample size of n = 86 gene copies (43 diploid snails) was calculated using HP Rare, a program that implements rarefaction to control for sample size variation among groups Population differentiation  adults  generations  and F21X = 177.4, P <0.0001), juvenile survival , and adult survivorship  significantly declined over time, but the magnitude of decline varied among generations. The proportion of snails surviving to maturity did not significantly differ between the F1 (0.53) and F2 (0.50) generations . Juvenile survival was above 60 percent for the F1 (67%) and F2 (65%) generations, but declined to 27 percent in the F3 generation . Adult survivorship was highest from 1998 to 2002 (0.84±0.10), declined somewhat from 2003 to 2008 (0.59±0.09), and was lowest from 2009 to 2013 .All measures of survival including survival to maturity (N = 335) of 2018 captive snails preserved between 1998 and 2012 were evaluated for genetic diversity measures, along with all living captive snails (N = 198), and wild snails from the southern (N = 5) and northern (N = 87) field sites .Approximately 17 percent (ld sites . Rarifield sites . AllelicisF) decreased over time in snails reaching maturity, and snails that did not reach maturity had higher inbreeding values in each generation than those that did reach maturity  or F2  generations. In contrast, living snails from the F3 generation did have significantly higher heterozygosity than F3 deceased snails , but linear regression equations indicated that snails were likely to have approximately one less offspring for every 0.2 decrease in individual heterozygosity .The inbreeding coefficient . Wild snails (IS(wild)F = 0.224, P <0.001) had a similar inbreeding coefficient to live captive snails born in 2012 and 2013 (IS(captive juvenile)F = 0.244, P <0.001). Values for the Garza-Williamson bottleneck index were similar in wild snails (0.65±0.20) and live captive adults (0.64±0.23).The live-sampled captive snail population displayed significant genetic differentiation from the wild-sampled snail population . Furthermore, measures of fitness were correlated with measures of heterozygosity. Snails with higher heterozygosity were more fecund and more likely to survive to sexual maturity.Measures of fitness in the founding adults were comparable to wild populations of closely related tree snail species. The founders of the captive Captive population allelic richness declined significantly over time, and is now lower, though not significantly so, in captive snails than in wild snails. A loss of allelic richness may interact with a number of other factors leading to the observed fitness declines in this captive population. Loss of genetic diversity is associated with an increased risk of mortality from multiple stressors A. lila and determined to be nonsignificant Several potential stressors that may be responsible for the captive population decline have been examined. Density effects on fecundity were tested in A. lila tested. Bottleneck effects may not be observed until several generations after a bottleneck occurs, so these values are not likely due to the bottleneck at the initiation of the captive population. However, the documented history for achatinellid species indicates they have been in severe decline due to predation, habitat fragmentation, and over-collection in the wild for at least the last half century, if not longer Euglandina rosea, or the Rosy Wolf Snail, that has devastated Hawaii's endemic snails A. sowerbyana also indicated the wild populations have experienced bottlenecks A. lila relative to known historical distributions , all from isolated individuals. Rates of self-fertilization likely vary among species in the subfamily Achatinellinae. Previous studies suggest a mixed-mating system A. lila, or this species may have a higher intrinsic rate of self-fertilization than close relatives. Unfortunately, populations where some self-fertilization occurs may be at an additional disadvantage, building up mildly deleterious alleles through self-fertilization, and expressing them in outcrossing individuals Self-fertilization could also inflate the inbreeding coefficient in this hermaphroditic species. Self-fertilization has only rarely been observed in A. lila both show significant departure from Hardy-Weinberg expectations, as tested with the ISF statistic, there is no population available to provide a baseline for comparison. Therefore, we cannot say with certainty whether cumulative inbreeding, self-fertilization, or some other explanation is responsible for the significant ISF values observed in this study, particularly the high inbreeding coefficient observed in the founding snails. The presence of null alleles, or alleles that don’t amplify due to mutations in the flanking primer region but are nonetheless present, can also artificially raise the inbreeding coefficient. Null alleles can be statistically detected when amplification rates are lower than expected for a particular locus. Prior to analysis, we discarded one locus due to a high probability of null alleles, as well as a second locus due to minimal polymorphism, and have high confidence that the presence of null alleles is minimal in the remaining six loci.Since the only two remaining wild populations of Three strategies have been suggested to retain fitness and genetic diversity in captive populations: optimize heterozygosity through molecular tracking and managed breeding; minimize inbreeding through tracking individuals and their offspring; or create inbred pairings to purge deleterious alleles M) were nearly identical in the 2013 wild (0.65±0.20) and live (0.64±0.23) captive populations, suggesting the wild population has also experienced a recent bottleneck. These M values are also consistent with those found in other species with a known history of demographic reduction, which ranged from 0.60–0.69 A. lila have been largely attributed to heavy predation, but populations devastated by predation and over-collection may also be experiencing bottleneck effects. Low density and fragmentation in the remaining wild populations may decrease mating options, increase inbreeding and rates of self-fertilization, and further decrease the likelihood of recovery.Garza-Williamson bottleneck-index values (A. lila will soon be placed into a newly-built predator exclosure near the southern sampling location (Lessons learned from the patterns of demographic and genetic change in the captive population will be used to better manage wild populations. Wild location . This ex


Locally adapted breeds of livestock are of considerable interest since they represent potential reservoirs of adaptive fitness traits that may contribute to the future of sustainable productivity in a changing climate. Recent research, involving three hill sheep breeds geographically concentrated in the northern uplands of the UK has revealed the extent of their genetic diversity from one another and from other breeds. Results from the use of SNPs, microsatellites, and retrovirus insertions are reviewed in the context of related studies on sheep breeds world-wide to highlight opportunities offered by the genetic resources of locally adapted hill breeds. One opportunity concerns reduced susceptibility to Maedi Visna, a lentivirus with massive impacts on sheep health and productivity globally. In contrast to many mainstream breeds used in farming, each of the hill breeds analyzed are likely to be far less susceptible to the disease threat. A different opportunity, relating specifically to the Herdwick breed, is the extent to which the genome of the breed has retained primitive features, no longer present in other mainland breeds of sheep in the UK and offering a new route for discovering unique genetic traits of use to agriculture. The importance of locally adapted livestock breeds is becoming recognized through their contribution to food security in marginal land areas of the world. Reviews by the FAO and other international agencies highlight the crucial significance of these breeds, both as sustainable resources of food but also as living reservoirs of biodiversity, providing genetic adaptive fitness traits to improve mainstream agriculture FAO, .In the UK, the locally adapted hill breeds of sheep are typically farmed on Disadvantaged or Severely Disadvantaged Areas for agricultural production Defra, . These bArchive.defra.gov.uk/foodfarm/farmanimal/diseases/atoz/fmd/2001; www.royalsoc.ac.uk (2004). Policy document 25/04 ISBN 0854036105 Royal Society Infectious Disease in Livestock Inquiry: Follow Up review]. Losses to the Herdwick sheep breed were of particular concern, which led to the setting up of the first national gene bank for sheep and a government commitment to survey the precise level of geographical concentration that existed amongst commercially farmed regional breeds.The extent of the breed's geographical concentration, leads to their susceptibility and disproportionate losses if an infectious disease enters their region. This issue was first recognized in the UK during the Foot and Mouth Disease (FMD) epidemic of 2001 which started in the northwest of England and disproportionately affected livestock farmed in that region [The Sheep Trust, a national UK charity, working with Sheep Breeder Associations (SBAs) and more than 1000 breeders, geo-referenced individual flocks of 12 regional breeds . Historically, sheep numbers increased as a reflection of headage payments in the EC's Common Agricultural Policy (CAP) and this undoubtedly led to some overgrazing of the uplands. Changes to CAP then switched to subsidy payments based on land area rather than numbers of animals farmed. This is leading to major reductions in hill sheep numbers, since farmers are no longer being paid on flock sizes. In some areas, agri-environment schemes to promote rejuvenation of the vegetation are also decreasing sheep numbers further with zero stocking of the hills for certain times of the year.Endangerment of the genetic resources of livestock may arise through disease but significantly, for the hill breeds of sheep throughout Europe, changes in the policies of national governments and EU regulations are also leading to new risks (www.gov.uk/government/uploads/system/uploads/attachment_data/file/254127/uk-breeds-at-risk.pdf). However, it is also crucial to determine the genetic distinctiveness of each breed. This is an indicator of the biodiversity deserving protection and is also a potential route to identifying adaptive fitness traits that can underpin the future sustainability and security of food production.Recognition of risk to a sheep breed's genetic resources through geographical concentration has recently been accepted in policies for breed protection and conservation  using a candidate gene approach, as well as the increasingly widespread application of the http://www.herdwick-sheep.com; www.dalesbredsheep.co.uk; www.roughfellsheep.co.uk). Equal numbers of registered rams from each of the breeds comprised the populations selected because provenance, familial origins and lack of relatedness could be confirmed. Also, it was reasoned that rams contribute the major genetic resource to a breed, whilst forming only a small fraction of the total breed numbers.A selection of these approaches was applied to study the genetic resources of the three geographically concentrated hill breeds, each farmed in adjacent regions of the UK northern uplands.  a major disease, reducing sheep health and productivity as well as lifespan. Studies by Heaton et al. , 2013 haTMEM154 alleles was 0.51 across 74 sheep breeds world-wide , stringently accredited for the absence of MV, represent a major component of the mainstream sheep industry. Genetic resistance of the hill breeds to lentivirus infections offers an alternative route to achieve an infection-free status for the national flock and an important reason to protect and continue to make use of the genetic resources of these locally adapted breeds.In the UK, flocks of continental breeds such as Texels  was weakly positive in all cases, with the lowest value of 0.059 in Herdwick and highest in Rough Fell (0.162). These values are within the range previously found for two other northern UK breeds with substantially greater population sizes  genome. In contrast, R0 was absent from Rough Fell and Dalesbred, both of which contained abundant retrotypes more typical of other UK breeds.The presence of R0 individuals retained in the present day Herdwick population, provides an indicator of origins of domestication as well as farming practices over the centuries. The data suggest Herdwicks originate from a common ancestral founder flock to other “primitive” breeds retaining the R0 retrotype. Within the UK only the North Ronaldsay of the Orkney Islands are known to have R0's and to an extreme proportion. Elsewhere, substantial levels of R0's were found in populations in Sweden, Finland and Iceland , none of which were shown to have the rare retrovirus insertions (Chessa et al., A second surprising feature shared both by the Herdwicks and the Rough Fell, was the presence of two extremely rare retrovirus insertions in their populations. These insertions were so rare that they were not included in the global retrotype classification and in fact in that earlier study, the pair was only found in Texel breed populations (Chessa et al., Genetic diversity in farm animals is becoming recognized as a highly significant resource. With the rapid progress in DNA sequencing technologies and the availability of SNP chips for many major species, genetic breeding is increasingly being used for improvement. As yet however, many of these practical applications continue to focus on production traits and the mainstream breeds of livestock.Predicted impacts of climate change, increases in food requirement for the world population and economic costs of farming are all contributing to renewed interest in the genetic resources of those breeds that are locally adapted to their farmed habitats. This interest is of relevance to improving a sustainable form of agricultural productivity in both the developed and developing worlds. Low inputs with good feed conversion, hardiness to harsh environments, enhanced resistance to parasites and pathogens, and low management costs are all characteristics of local breeds when farmed extensively in the habitats to which they are adapted FAO, .In this context, locally adapted sheep breeds are production systems of our food on land that often cannot support any other forms of agriculture. Their ability benefits current food security and provides opportunities for future farming systems that will be required to maintain productivity in the predicted harsh conditions of climate change. Despite the benefits offered by these breeds, the very real risk of their genetic erosion requires urgent action before these living reservoirs of adaptive fitness traits become lost forever.The first step is to analyze their genetic distinctiveness to demonstrate to policy makers the biodiversity that the sheep breeds contain. The three UK breeds recently studied have not been analyzed previously, but extend earlier molecular studies of other locally adapted and mainstream breeds. A surprisingly broad genetic range was observed for the hill breeds, even though they were farmed in close proximity within adjacent regions of the northern uplands of England.OvineSNP50BeadChip and global collaborative networks such as the International Sheep Consortium, will provide many more opportunities for large-scale genetic characterization of sheep breeds. Already there are examples of those technologies used to explore traits relevant to adaptation, such as growth characteristics (Zhang et al., Next generation sequencing, the use of the OvineSNP50BeadChip has recently been applied in a large project studying 32 sheep breeds adapted to a wide spectrum of different regional climates (Lv et al., However, many more data are required linking the different phenotypic traits for potential use in genetic improvement to geographies and the specific ecologies and parasite challenges to which the livestock are adapted. Recent advances in landscape genomics and the bringing together of large data sets combining phenotypic recording, DNA analyses and environmental parameters are providing a new platform to achieve this holistic understanding (Joost et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Barbara Bernhardt discusses how the increasing importance of genomics in the clinic will change the role of genetic counselors. Barbara A Bernhardt, MS, CGC Figure  is a genGenetic counseling is ‘the process of helping people understand and adapt to the medical, psychological and familial implications of genetic contributions to disease’ . TraditiIn the United States and Canada, genetic counselors hold a masters degree from one of 35 accredited genetic counseling training programs. The didactic and clinical training components of these programs are aimed at supporting the development of competencies in four broad areas: genetics expertise and analysis; interpersonal, psychosocial and counseling skills; education; and professional development and practice. The current 3,500 trained genetic counselors in North America are board certified through the American Board of Genetic Counseling. In addition, there is currently licensure of genetic counselors in 14 US states.In the past, genetic testing was used to look for variants in single genes that are associated with a specific condition, whose presence is suspected because of clinical presentation, family history or ethnicity. Recent technological advances, including next generation sequencing and genome-wide single nucleotide polymorphism (SNP) microarrays, have provided opportunities to test quickly, accurately and cheaply for variants in multiple genes simultaneously or for variants across the entire genome. These technologies have enabled new genomic applications in areas such as prenatal testing, carrier screening, newborn screening, diagnostic testing, predictive testing for common complex conditions, pre-symptomatic testing and pharmacogenetics . In addiAs increasing amounts of genomic data become available, there will be a need to educate both clinicians and the public about the implications of the data for patients and their family members. These data will also raise important ethical questions relating to areas such as privacy and confidentiality, reproductive choices, and personal autonomy. Genetic counselors will be called upon to provide consultation for individual patients and clinicians to help with the interpretation of genomic data and to discuss options for acting upon the data.The rapid advances in genomics will lead to an increase in the volume of test results and a need to understand the processes by which genetic variants are classified and interpreted.Genetic counselors will be challenged to remain current in their knowledge of new technologies and their awareness of resources that are available to evaluate the implications of genomic variants.The development of new genomic tests and therapies has led to high public and patient expectations for improved diagnostic capabilities and treatments. Tempering public expectations and educating patients about the limitations of genomic technologies will become an important role for genetic counselors, and will influence how we obtain informed consent and discuss test results with patients . BecauseIn the past, genetic counselors have had the luxury of spending an entire session discussing a patient’s susceptibility to and personal experiences with a single condition, such as breast cancer or cardiomyopathy. With the advent of genome-wide testing, many secondary findings may be available to be returned to patients, and genetic counselors will need to find new ways to streamline both pre-test education and counseling as well as post-test results disclosure. As some secondary findings are likely to be medically actionable, genetic counselors will need to gain expertise about the underpinnings of behavioral change as they increasingly engage with patients in health-promotion activities.Given the public’s low level of genetics literacy in general, genetic information has traditionally been viewed as complex. With the advent of genomic testing, the information being returned to families is becoming even more complex because there is more of it, much of it is probabilistic, and some has uncertain implications. Moreover, some of the information may be unanticipated and might relate to conditions of which families have no prior experience, as would be the case with incidental findings from whole-exome sequencing. Effective pre-test genetic counseling should prepare families for the possible disclosure of such information, and additional visits or other follow-up may be needed to promote optimal coping and understanding. But, as genomic testing becomes more routine, especially as it moves out of the genetics clinic and is offered by non-geneticist physicians, there may be less emphasis on pre-test counseling and post-test follow-up because of constraints on physician time. Families may respond by requesting genetic counseling or by turning to information available on the internet to help them make sense of genomic test results.We’re just now beginning to gather data on how families understand and act on complicated genomic information. There is some anecdotal evidence that families may ascribe more importance to incidental findings than is warranted. In an attempt to make sense of uncertain information, families may also begin a search for additional information, or might choose to classify an uncertain finding as either pathogenic or benign . Within As genomic testing diffuses into clinical care, I suspect that the expertise and contributions of genetic counselors will be increasingly recognized and valued. Genetic counselors will be the primary professionals called upon to educate primary-care providers and specialists, and to assist them with ordering genomic testing and with interpreting and understanding test results.After genetic testing for susceptibility to breast and ovarian cancer became feasible in the mid-1990s genetic counselors were integrated into oncology services. As more genomic testing has become feasible for other groups of diseases, including neurologic, cardiologic and psychiatric diseases, genetic counseling services are being sought by these specialties. I would envision that, in the future, genetic counselors will be an integral part of many multidisciplinary teams. Because of this, genetic counselors will need to become familiar with the impact on patient management of results from a broader array of genetic tests.As genomic research using human subjects expands, genetic counselors will be included in research teams to assist with obtaining informed consent and in returning results. Genetic counselors also contribute to research on the process of providing genomic services and assessing outcomes, especially those relating to genetic counseling.Accurate data are not available to determine whether the current supply of genetic counselors is adequate to meet demand. Anecdotally, over the past few years, it appears that there are more positions available than there are trained counselors to fill them, although the number of training slots has increased about 25% over the past five years. In the near future, growth in the supply of genetic counselors will be limited by the number and size of training programs. In recognition of the need to increase the number of genetic counselors as the scope of practice expands, the National Society of Genetic Counselors has established a workforce growth working group to develop a strategic plan for increasing both the number and diversity of trained genetic counselors.Increased hiring of genetic counselors has been tempered by inconsistent reimbursement for genetic counseling services resulting from a combination of factors, including lack of licensure, credentialing, insurance reimbursement and even billing for services provided. The National Society of Genetic Counselors has been addressing this issue and there has been gradual improvement; but it is probably correct to say that, at present, genetic counseling services are rarely self-sufficient.The future of genetic counseling will depend on how quickly and how broadly genomic testing technologies are incorporated into clinical care. This adoption in turn will be predicated on studies documenting that new genomic tests improve patient outcomes and reduce costs. In addition, implementation will be driven by patient and healthcare providers’ demand for the technologies, and payers’ willingness to reimburse for genomic testing and counseling.In the past century, genetic counselors were primarily a part of a team, including a clinical geneticist, that provided services in a genetic clinic. Service-delivery models are likely to change as genomics is integrated into primary and specialty care. To improve access, telemedicine counseling opportunities will increase as will the need for e-learning approaches. Because most genetic counselors are drawn to the profession because of an opportunity to provide one-on-one counseling to families, either as a part of a team or independently, such services are likely to persist so long as families and providers find value in them. But in addition to serving individual patients and families, in the future, I envision that genetic counselors will be serving the provider community and the healthcare system by taking on the role of gatekeeper to genomic tests.Moving forward, there will be many new and diverse opportunities for genetic counselors as researchers, consultants and educators. I see an incredibly exciting but somewhat uncertain future for genetic counseling. Because of our organization through the National Society of Genetic Counselors and through our alliances with other professional and lay organizations, we are well positioned to chart our own future course. The author declares that she has no competing interests.


Cobitis fish species, C. lutheri in the Dongjin River, and C. tetralineata in the Seomjin River in Korea. The construction of water canals about 80 years ago has unidirectionally introduced C. tetralineata into the native habitat of C. lutheri, and then these species have hybridized in the main stream section of the Dongjin River. According to the divergence population genetic analyses of DNA sequence data, the two species diverged about 3.3 million years ago, which is interestingly coincident with the unprecedented paleoceanographic change that caused isolations of the paleo-river systems in northeast Asia due to sea-level changes around the late Pliocene. Multilocus genotypic data of nine microsatellites and three nuclear loci revealed an extensively admixed structure in the hybrid zone with a high proportion of various post-F1 hybrids. Surprisingly, pure native C. lutheri was absent in the hybrid zone in contrast to the 7% of pure C. tetralineata. Such a biased proportion must have resulted from the dominant influence of continually introducing C. tetralineata on the native C. lutheri which has no supply of natives from other tributaries to the hybrid zone due to numerous low-head dams. In addition, mating experiments indicated that there is no discernible reproductive isolation between them. All the results suggest that the gene pool of native C. lutheri is being rapidly replaced by that of continually introducing C. tetralineata through a hybrid swarm for the last 80 years, which will ultimately lead to the genomic extinction of natives in this hybrid zone.River connections via artificial canals will bring about secondary contacts between previously isolated fish species. Here, we present a genetic consequence of such a secondary contact between  A number of hybridizations have been reported across a wide taxonomic range of animal species  are well known to undergo natural hybridizations with closely related species . In the Dongjin River, possible habitats were investigated along the mainstream of the river, where the hybrid individuals were previously observed  and three nuclear DNA loci   of Taq DNA polymerase, 1 μL of genomic DNA, and 12.88 μL of distilled water). Each amplification reaction was performed under the following conditions: initial denaturation at 94°C for 2 min, followed by denaturation at 94°C for 40 sec, annealing at different temperatures depending on the marker  for 40 sec, and elongation at 72°C for 1 min, which was repeated for 35 cycles in the first PCR and 30 cycles in the second PCR, with a final elongation step at 72°C for 5 min. All PCR products were sequenced using an ABI 3730xl automatic sequencer . To solve the phase problem of heterozygotic nucleotide sites of diploid DNA sequences, DNA sequences were processed with PHASE program .In the  Palumbi  and H164 Palumbi . The pro Palumbi . The seq Palumbi , in a 25Cobitis species were estimated on the basis of four independent genetic loci and their molecular substitution rates that was calibrated according to a recently discovered fossil from east Asia. The population genetic divergence model IMa2 (Hey Nm), and splitting time between C. lutheri in the Mangyeong River  and C. tetralineata in the Churyeong Stream . To certify whether our data satisfies the assumptions of this IMa2 model, recombination tests of the three nuclear genes were conducted using SITES  were calculated with BEAST  were obtained from Kim et al. (Misgurnus (M. anguillicaudatus and M. mizolepis) were also acquired in the present study through PCR amplification following the methods described previously , according to the oldest fossil record of C. longipectoralis, using a lognormal distribution with a mean of 1.5, a standard deviation of 1.3, and an offset of 17.5 ) or L-type (from C. lutheri). For this grouping, unrooted neighbor-joining trees of haplotypes from the Dongjin River were built by Geneious tree builder , including haplotypes of the populations of C. lutheri and C. tetralineata from nonhybridized allopatric localities. Minimum spanning trees of the haplotypes from the Dongjin River were constructed for each DNA marker using Arlequin and HapStar (Teacher and Griffiths Hd), Tajima's D  that we developed and characterized in a previous study on C. lutheri were used for their high variability , 0.05 U of Taq DNA polymerase, and 10–50 ng of template. The samples were genotyped using a model 4300 automatic sequencer (LI-COR). To improve the resolution of genotypic analysis, the haplotype data of the three nuclear genes  were included after converting them into multilocus genotype data using PGDSpider  were calculated using STRUCTURE  with the scale of the species (K = 2) and with the scale of the populations (K = 3–6). Additionally, pairwise FST was calculated among the nonhybridized allopatric populations  based on the multilocus genotypic data using Arlequin program. To identify various classes of hybrids  in the Dongjin River, the probability of each distinct hybrid class for each individual was calculated through one million sweeps using NewHybrids, under the framework of Bayesian model-based clustering via Markov chain Monte Carlo simulation .Based on the Cobitis individuals were obtained in their mating season to investigate the reproductive abilities of hybrid individuals. Pure C. lutheri, pure C. tetralineata, and C. lutheri–C. tetralineata hybrids were, respectively, collected from the Jeongeup Stream of the Dongjin River, the Churyeong Stream of the Seomjin River, and the main Dongjin River in the locality of Sanoe-myeon, based on their morphological characteristics , (2) female C. lutheri and male C. tetralineata (LL ♀× TT ♂), (3) female hybrids and male C. tetralineata (TL ♀× TT ♂), and (4) female hybrids and male C. lutheri (TL ♀× LL ♂). Two additional mating pair groups were tested as controls: (1) female and male C. tetralineata (TT ♀× TT ♂), and (2) female and male C. lutheri (LL ♀× LL ♂). To obtain completely mature eggs and sperms for artificial fertilization, Ovarprim  was injected into both male and female adult fish. Twelve hours after the injections, mature eggs and sperms were obtained from females and males, respectively, by pressing their abdomens. The eggs were artificially fertilized and bred by spreading sperms in Petri dishes of 15-cm diameter incubated at 25°C. Subsequently, the development of the embryos was observed and photographs of the developmental process were acquired using a dissecting microscope  with a digital camera (LEICA DFC290). In addition, six indices of early life history characteristics were measured for the six mating pairs: mature eggs size, swelling eggs size, hatching larvae size, hatching rates, abnormality rates, and early survival rates. The rates of abnormality and early survival were determined 5 days after the hatching of the larvae. The values of each index were statistically compared among the mating groups by analysis of variance (ANOVA) and Tukey's HSD tests using SPSS version 21.0 (SPSS Inc.).Sexually mature cs Figs. , 2. FourC. lutheri and C. tetralineata, we estimated population demographic parameters for the Mangyeong and Seomjin River populations  of 82,000–218,000) that was almost three times higher than that of C. tetralineata . The splitting time between C. lutheri and C. tetralineata was estimated to be approximately 3.3 MYA (95% CI of 0.9–6.6). The population migration rates per generation (2Nm) between C. lutheri and C. tetralineata were very close to zero . The full model of five demographic parameters (θ1θ2θAm1 m2) was not rejected by the three nested models . All the P-values were near one for each alternative nested model.To infer the divergence process of the allopatric ons Fig. . We usedons Fig.  were empC. lutheri and C. tetralineata in the Dongjin River  was initially investigated with the haplotypes of one mitochondrial and three nuclear genes. The haplotypes of the populations were clearly divided into two groups (L-type and T-type), which represented two genetic lineages corresponding to the pure allopatric species, C. lutheri and C. tetralineata, respectively  of the T-type was relatively lower than that of L-type except for the SH3PX3 gene, although the proportion of T-type was higher than that of L-type at all loci . This neutrality also holds for the introduced T-type group within the hybrid zone except for Ptr gene.The genetic pattern of hybrids between ci Table . Both TaC. lutheri and C. tetralineata from the closest neighboring rivers of the Dongjin River. When we assumed K to be equal to two (K = 2) from the perspective of interspecific hybridization, the individuals of the hybrid zone  clearly exhibited variously admixed ancestries between the two species, C. lutheri and C. tetralineata  among the hypothetical number of populations (K = 2–6), which corresponds to the number of sampled localities representing the investigated rivers or streams (n = 6). This model suggested that the nonhybridized allopatric populations of each species were genetically subdivided into several groups . In the hybrid zone of the Dongjin River, three genetic groups were found to be admixed with each other. One of these groups was identical to the genetic group of C. lutheri from locality 2, and another was consistent with the genetic group of C. tetralineata from locality 7. The last group was found only in the populations from the Dongjin River.The structure analyses showed the highest posterior probability under the six-population model  and 68 F2 descendants (F2), with high posterior probabilities. No individuals were identified as being pure C. lutheri (P0), backcrossed with C. lutheri (B0) or F1 hybrids (F1). The individuals categorized into P1 or B1 were highly corresponded with Cyt b gene of C. tetralineata, while F2 hybrids were relatively unbiased for the Cyt b .After confirming the intensive interspecific hybridization, we characterized the individuals of the Dongjin River by fitting them into the proposed six classes of hybrid after two generations of hybridization using NewHybrids. To minimize misidentification, we used a reliable threshold posterior probability of 0.50 for NewHybrids  . However, Cyt b, ENC1, and SH3PX3 did not show such significance (Table S1). Therefore, it appears that the significance of Ptr gene is caused by stochastic sampling variance of genetic locus or partly founder effect of the introduced individuals from a small source population. The gene pool of C. tetralineata in the Dongjin River has been maintained by its propagation in the non-native habitat and the constant supply of its original genomes from the Seomjin River as well. Genotypic assignments of the hybrid populations indicated that these introduced individuals originated from a single subpopulation of the Seomjin River, the Oknyeodong Stream  and pairwise FST between the nonhybridized allopatric populations of C. lutheri consistently showed apparent genetic differentiation among populations  during the Pleistocene glacial periods  and others were identified as F2 hybrids (F2) or backcrossed individuals with C. tetralineata (B1), without F1 hybrids being observed  are protected from the hybrid swarm by the low-head dams. If pure C. lutheri were to flow into the hybrid zone from the upper or lower region of the river, there would be continuous inputs of both pure Cobitis species into the hybrid zone of the river. This would, then, generate a more general hybrid zone structure that typically shows a cline of various traits across a geographically narrow zone between two parental species  in the United States has been replaced to a considerable extent with that of introduced African honey bees (A. mellifera scutellata) through the creation of hybrid swarms in less than 50 years  also demonstrated such rapidity, as it took only approximately 20 generations for alleles from non-native tiger salamanders (A. mavortium) to become fixed in the genome of the pure population following the introduction of A. mavortium into the Salinas Valley in the United States  due to the disconnections of paleo-river systems driven by paleoceanographic changes in the northeast Asia in the late Pliocene. Population genetic analyses indicate that the native C. lutheri gene pool in the hybrid zone is likely being replaced by the introduced C. tetralineata gene pool via a hybrid swarm. The constant introductions of C. tetralineata from the neighboring Seomjin River through two water canals seem to greatly contribute to the rapid replacement of the original gene pool of C. lutheri in the hybrid zone. It is carefully anticipated that the genomes of native C. lutheri in the hybrid zone will ultimately go extinct unless the canals or low-head dams along the Dongjin River are eliminated. As the case of semi-isolated C. lutheri, secondary contacts and hybridizations between freshwater fish species that are abruptly brought about by man-made alterations will impact more greatly on small and isolated populations.The present study involving


Felis s. silvestris, is subject to intensive conservation measures, including captive breeding and reintroduction. We here present the first systematic genetic analysis of the captive population of Felis s. silvestris in comparison with a natural wild population. We used microsatellites and mtDNA sequencing to assess genetic diversity, structure and integrity of the ex situ population. Our results show that the ex situ population of the European wildcat is highly structured and that it has a higher genetic diversity than the studied wild population. Some genetic clusters matched the breeding lines of certain zoos or groups of zoos that often exchanged individuals. Two mitochondrial haplotype groups were detected in the in situ populations, one of which was closely related to the most common haplotype found in domestic cats, suggesting past introgression in the wild. Although native haplotypes were also found in the captive population, the majority (68%) of captive individuals shared a common mtDNA haplotype with the domestic cat (Felis s. catus). Only six captive individuals (7.7%) were assigned as wildcats in the STRUCTURE analysis (at K = 2), two of which had domestic cat mtDNA haplotypes and only two captive individuals were assigned as purebred wildcats by NewHybrids. These results suggest that the high genetic diversity of the captive population has been caused by admixture with domestic cats. Therefore, the captive population cannot be recommended for further breeding and reintroduction.Studies on the genetic diversity and relatedness of zoo populations are crucial for implementing successful breeding programmes. The European wildcat,  Due to the ongoing threats to biodiversity, species conservation is still a challenging task The influence of captive breeding on the genetic diversity of endangered species has already been subject to a number of theoretic and genetic studies Felis silvestris silvestris) is an ideal study object for such an analysis. It is still rather common in Europe and classified as Least Concern on the IUCN Red List of Threatened Species The European wildcat  sequencing in order to assess the genetic variability, structure and integrity of the ex situ population. We also analysed samples from a wild population to evaluate if the genetic variability in the captive population is comparable to the diversity found in the wild.Here, we present the first systematic genetic analysis of the European captive population of We contacted 124 zoos, 64 of which confirmed keeping European wildcats. We obtained 80 samples  from 30 zoos spanning six European countries  from a natural wild population in the Harz mountains (Germany). The hair samples were collected in the context of a radio telemetry study (M. Götz). The tissue samples were from a monitoring program for roadside casualties. Both studies were carried out with the permit of the Landesamt für Umweltschutz Sachsen-Anhalt which included the collection of samples from European Wildcats  following the manufacturer's instructions . For the hair samples, we extracted DNA with a modified Chelex 100 protocol, using a 10% Chelex solution with addition of 7 µl Proteinase K (18 mg/ml) per sample and overnight lysis 2+). The 5′-end of each forward primer was labelled with a fluorescent dye, either 5-FAM, TAMRA or JOE. The products were genotyped on a MegaBACE 1000 automated DNA sequencer . Fragment lengths were determined using Fragment Profiler 1.2 (Amersham Biosciences). To minimize genotyping errors due to low DNA concentrations , we applied a multiple tube approach as recommended by Taberlet et al. The six samples from the wild caught individuals in Saarland and Rhineland-Palatinate were not genotyped, as the samples size from these populations was too small for population genetic analysis. They served as reference samples for the mtDNA analysis (see below). Therefore only 194 of the 201 samples were genotyped at ten di-nucleotide repeat microsatellite loci  characterized in the domestic cat For 70 wild, 66 captive and 33 domestic individuals we sequenced the mitochondrial DNA fragment NADH dehydrogenase subunit 5 (ND5) based on the study by Driscoll et al. tructure 2.3.4 icro-Checker 2.2.3 As the integration of hybrids and genetic sub-groupings were unknown, we first searched for genetic structure in our samples using Sest, FST and RST using GenAlEx 6.501 ST is still a useful measure if the population split is rather recent (e.g. for comparing differentiation among captive populations), while RST is more useful if the split is deep and if it is likely that mutations have already contributed to population differentiation (e.g. for comparing wildcats and domestic cats) est estimates for correlation with RST and FST, we found a strong positive correlation between Dest and FST  but a negative correlation with RST . Therefore, only the results of FST and RST are provided. For the analyses of population differentiation, we excluded all individuals which could not clearly be assigned to a cluster at K = 2 based on qi>0.8 and where thus assigned as hybrids  for the captive, domestic and wild samples as well as for the genetic clusters in Fstat. GenAlEx was used to determine the expected (He) and observed (Ho) heterozygosity for each locus and each population and to test for deviations from Hardy-Weinberg equilibrium (HWE). Furthermore, we tested the number and frequency of private alleles for domestic cats and wildcats  and analysed the number of alleles shared with captive cats as well as their mean frequencies.For comparing genetic diversity, we calculated the mean number of alleles, allelic richness and inbreeding coefficients (Fe) for each the captive population, domestic population and the wild population (excluding the hybrids) were assessed in ONeSAMP e were 2 and 500, respectively. The captive and wild samples were tested for genetic signatures of population bottlenecks using the Wilcoxon signed rank statistic implemented in Bottleneck 1.2.02 Effective population sizes . Sequences were deposited in GenBank under the accession numbers KM246612-KM246625. The identification of haplotypes was carried out in DnaSP 5 tructure was six . In the STRUCTURE analysis, we found a clear distinction between individuals from the captive  showed a higher differentiation to the captive clusters  than the domestic cluster . For the domestic cluster, the highest differentiation occurred to the wild cluster . The nested AMOVA based on FST revealed that most of the genetic variation occurred within individuals (80%). Yet, a statistically significant portion was explained by the difference between the six genetic clusters (12%) and between the captive, domestic and wild samples (7%). In the RST-based AMOVA, the population level (i.e. the clusters) explained no variation at all, but the three categories  explained 7% of the variation. With RST only 14% of the genetic variation occurred within individuals. The private alleles analysis revealed that captive cats shared 30 private alleles only with domestic cats  and six only with wildcats (8.6%±4.2% SE).Genetic differentiation based upon FWhen analysing the data in NewHybrids, only two captive individuals were assigned as purebred wildcats, whereas most captive cats were either assigned as F2 hybrids  or backcrosses with domestic cats . The projected inertia of the PCA was 15.7% for the first axis and 6.9% for the second axis. The first axis separated the domestic cats , respectively. The captive clusters had a higher allelic richness and higher expected and observed heterozygosities than the wild cluster . There wThe captive population showed significant evidence of a recent bottleneck under both IAM and TPM, but not under SMM. For the domestic population there was only significant evidence of a bottleneck under the IAM, whereas the wild population showed no signs of a bottleneck . When thThe mean relatedness between dyads was r = −0.044±0.007 (mean ± SE) in the wild population and r = −0.042±0.004 in the captive population. The mean individual inbreeding coefficients were 0.149±0.019 in the wild and 0.139±0.019 in the captive population. In both populations most individuals (61%) had an individual inbreeding coefficient <0.125. The number of individuals with an inbreeding coefficient between 0.125 and 0.25 was 18% and 14% for the wild and captive population respectively and the number of individuals with values above 0.25 was 23% and 25%.Within the breeding pairs we found a mean r of 0.068±0.059. Most breeding pairs (47%) had a low relatedness r≤0.05. However, there were five breeding pairs which showed relatedness values above the half-sib level (0.25) with two of them almost reaching the full sib level (0.5).F. s. silvestris  who shared haplotypes with domestic cats, confirming that hybridization still occurs in the wild but at a low frequency. Hybridization with domestic cats has been detected in several free-ranging populations of the European wildcat Our results show that the captive population of the European wildcat represents a hybrid swarm. Only six captive individuals (7.7%) were assigned as wildcats in the STRUCTURE analysis (at K = 2), two of which had domestic cat mtDNA haplotypes, and only two individuals were clearly assigned in the analysis with NewHybrids (one with a domestic cat mtDNA haplotype). Altogether, 68% of the captive population possess domestic cat mtDNA haplotypes. Therefore, the captive population cannot be recommended for further breeding and reintroduction. As mtDNA is maternally inherited, the presence of domestic cat haplotypes in two thirds of the ex situ population does not allow any conclusions on the total amount of domestic cat nuclear introgression into the captive wildcat gene pool. Furthermore, the amount of introgression from male domestic cats remains unknown, as no Y-chromosome markers have been studied. However, the European wildcat is still widespread and it is possible to obtain new founders from the wild. Particularly the large populations in the Hunsrueck Mountains  currently show no indication of past introgression, but more detailed studies are required for any final conclusions. Individuals of hybrid origin cannot be recommended for breeding. This leaves many zoos with the difficult decision of what to do with the individuals which do not qualify as "pure" wildcats. One might question whether the introgression affecting the captive population occurred recently or if it reflects another trace of an ancient introgression event. Hertwig et al. It is very likely that the reintroduced populations of the European wildcat in parts of Germany  represent hybrid swarms as well as they stem from the captive population studied. Removing the reintroduced populations from the wild is not feasible as the administrative effort and costs for such a project would be far too high and it is very unlikely that all individuals could be caught and screened genetically. Moreover, it remains unknown to what extent the reintroduced wildcat populations originated from the captive population or immigrated from wild populations. A genetic survey of the reintroduced populations is therefore needed to assess their status. As the European wildcat currently expands its range (for example in Germany) ex situ conservation programmes Outbreeding is a major threat for the success of ex situ population of F. s. silvestris just by chance (due to the missing network of holders), and might have possibly helped to maintain the strong genetic diversity, it is likely that this diversity has mainly been caused by admixture with domestic cats. Increased genetic diversity caused by admixture of several evolutionary lineages has also been reported from invasive non-native populations The current plan to establish a studbook for the European wildcat also requires a coordinated breeding strategy. The most widely accepted management strategy is minimizing kinship The importance of genetic assessments of captive populations has already been recognized decades ago The present study shows that genetic analyses provide valuable insight into the status of wild and captive populations. Using two different marker systems  helps to unravel introgression patterns. Future studies should also integrate Y-chromosome markers to detect potential male introgression. The integration of hybrids into ex situ populations can have drastic effects on the genetic composition of captive populations. Hybridisation with closely related species or domesticated congeners represents a severe threat to the conservation of endangered species both in situ and ex situ File S1Text S1, Permits for sampling. Text S2, List of participating institutions for genetic analyses. Figure S3, Plots of LnP(D) and delta K  for the microsatellite data obtained with STRUCTURE Harvester. Table S4, Sampling regime. 1) Most juveniles of sampled mothers and siblings were excluded from sequencing. Their haplotypes were inferred using their maternal relatives (number behind slash). 2) One captive individual was sampled twice and thus the duplicate sample was excluded. 3) The DNA quality of the sample from a preserved captive specimen was not sufficient for sequencing. Table S5, Information on the samples used in this study. The table contains all samples which were used in this study, together with their origin, the detected mtDNA haplotypes, the assignments to the STRUCTURE clusters at K = 2, K = 3 and K = 6 as well as the most probable assignment by NewHybrids. At K = 2 all individuals which qualify as hybrids based on qi <0.8 are marked with an *. These individuals were excluded for the analyses of population differentiation. For reasons of data protection the origin of the ex situ samples can only be given as encrypted information. Remarks: 1) DNA quality was too low for genotyping; 2) individual was classified as morphologically suspicious; 3) known hybrid; 4) domestic cat ; 5) single samples from wild populations as reference for sequencing, but not suitable for population genetic approaches; 6) zoo located next to wild population in the Harz mountains; 7) foundling within region of the wild population Rhineland Palatinate; 8) the cat was transferred to a new zoo which also took a sample, the duplicate sample was excluded from analysis.(DOCX)Click here for additional data file.