Lumbricus rubellus, are commonly found across highly contaminated former mine sites and are considered to have under-gone selection for mitigating metal toxicity. Comparison of adapted populations with those found on less contaminated soils can provide insights into ecological processes that demonstrate the long-term effects of soil contamination. Contemporary sequencing methods allow for portrayal of demographic inferences and highlight genetic variation indicative of selection at specific genes. Furthermore, the occurrence of L. rubellus lineages across the UK allows for inferences of mechanisms associated with drivers of speciation and local adaptation.Populations of the earthworm, Using RADseq, we were able to define population structure between the two lineages through the use of draft genomes for each, demonstrating an absence of admixture between lineages and that populations over extensive geographic distances form discrete populations. Between the two British lineages, we were able to provide evidence for selection near to genes associated with epigenetic and morphological functions, as well as near a gene encoding a pheromone. Earthworms inhabiting highly contaminated soils bare close genomic resemblance to those from proximal control soils. We were able to define a number of SNPs that largely segregate populations and are indicative of genes that are likely under selection for managing metal toxicity. This includes calcium and phosphate-handling mechanisms linked to lead and arsenic contaminants, respectively, while we also observed evidence for glutathione-related mechanisms, including metallothionein, across multiple populations. Population genomic end points demonstrate no consistent reduction in nucleotide diversity, or increase in inbreeding coefficient, relative to history of exposure.L. rubellus appears to provide an exemplary system for exploring drivers for speciation, with a continuum of lineages coexisting across continental Europe, while distinct lineages exist in isolation throughout the UK.Though we can clearly define lineage membership using genomic markers, as well as population structure between geographic localities, it is difficult to resolve markers that segregate entirely between populations in response to soil metal concentrations. This may represent a highly variable series of traits in response to the heterogenous nature of the soil environment, but ultimately demonstrates the maintenance of lineage-specific genetic variation among local populations. The online version of this article (10.1186/s12863-017-0557-8) contains supplementary material, which is available to authorized users. Monitoring life-history parameters among organisms is routinely used as a means of establishing the risks imposed by pollutants on natural populations , 2. In kHeliconius melpomene, which has benefited from high-quality genome assemblies used to resolve population structure, and is underpinned by wing colour [Identification of regions of the genome under selection is empowered with the availability of high-throughput sequencing methods such as Restriction-site Associated DNA sequencing (RADseq), which allows for identification of markers across the genome , 9. Whatg colour , 13. Whig colour . As suchg colour ) can be Lumbricus rubellus. This species is a major terrestrial sentinel that consists of highly divergent lineages [L. rubellus to contaminants, persistent contact with upper-most region of soil and important ecological role in nutrient cycling. Much of the research that uses L. rubellus has focussed upon the effects of heavy metal contamination, and a body of work has built up upon understanding how this species is capable of persisting across highly contaminated former mine sites [L. rubellus, compared to those inhabiting comparatively clean soils [Defining genomic variation associated with local adaptation and speciation is particularly pertinent to the ecotoxicological model earthworm, lineages , 17 and lineages . Its usene sites –21. The ne sites –25. Resian soils –21, thouL. rubellus is complicated because this species is comprised of a number of highly diverse lineages. Across continental Europe, several mitochondrially divergent lineages have been recorded [L. rubellus appearing to be a highly polymorphic species. Alternative analyses focussing on British earthworm lineages have found evidence for behavioural drivers of isolation, postulated to be the result of lineage-specific pheromones [Understanding adaption in recorded , with L.eromones . While veromones , 28, it L. rubellus previously identified at sites that have been historically exposed to heavily contaminated mine soils. Specifically, we defined the discrete nature of earthworm populations at the lineage and at the local level, putting emphasis upon identifying genetic variation that segregates populations relating to soil pollutants at highly contaminated mine sites. Initially, we made use of draft reference genomes from both lineages present in the UK to infer lineage specific associations, thereafter inferring population structure to determine whether any similarities exist in the relative ability of certain genotypes to inhabit specific soil types. Through comparison with proximal populations found on less contaminated soils, we have provided insights into ecological processes derived from segregating patterns of genetic differentiation, while calculation of population genetic statistics will demonstrate the long term effects of environmental contamination upon natural populations. Finally, access to genomic scaffolds has allowed us to determine proximal genes that are likely under selection and therefore surmise adaptive mechanisms and pathways that are shared between lineages.Here, we used RADseq with the aim of identifying the impacts of selection upon genetic variation in populations of n = 40), a former As and Cu mine in Devon, South West England; Carrock Fell  an As and W mine located on the edge of the Lakes District of North West England, and also from Cwmystwyth , which is a former Pb mine located in Mid-Wales. Sample site coordinates are reported in Additional file L. rubellus lineages were sampled from sites using a fork to dig into the epigeic (top 10 cm) of soil, before individuals were removed by hand. After collection, all earthworms were maintained upon native soils until returned to the laboratory. Individuals were rinsed with deionised water and a sample from their posterior was taken using a sterile scalpel blade. Both tail segments and remaining tissues were then frozen in liquid nitrogen and stored at -80 oC for latter processing.Earthworms were sampled in 2010 from three UK sites with known histories of metal pollution as well as local control sites with low soil metal concentrations. The three sites were Devon Great Consols . Only reads that aligned uniquely were used in subsequent analyses. Reads aligned to the lineage B genome  were forwarded to the ref_map package from Stacks, which was run using the options –n 3 and –m 3. The Populations module of Stacks was then run, limiting the output to loci existing in at least 10% of the population with at least 5× coverage, resulting in 219,545 SNPs. The Populations module outputs SNP data in Plink and Structure formats, limited to a single SNP per locus, chosen at random to account for linkage. Inbreeding coefficient and neucleotide diversity were calculated by the populations module of Stacks, while the “--missing” option in Plink v. 1.90b3.29 [Raw sequence data was assessed for quality and processed using Stacks v. 1.30. Briefly, process_radtags was used to demultiplex samples, remove low-quality reads and trim to 90 bp. All samples achieved greater than 100,000 reads following this process, with the average being 2.083 M reads. Reads were aligned to genomes sourced from .90b3.29  was used.90b3.29  to definK populations [K = 1, was used to estimate the allele frequency distribution (lambda). The distinction between lineage A and B (K = 2) was characterised with an initial run of 100,000 burn-in followed by 100,000 data collection. Individuals were split into two groups, representing each of the two lineages, and structure was run again to test for the number of discrete populations therein, testing across runs  implementing values of K from 1 to 7 with 10 replicates of each. Structure harvester was used to identify the most appropriate value of K via implementation of the Evanno method [K where delta K is highest was used in a final run  that was plotted using Distruct2.pl (http://www.crypticlineage.net/pages/distruct.html). The pairwise relationships of all individuals found locally to each other were formally assessed using KING kinship coefficient estimator v. 1.4 [The smartPCA module of EIGENSOFT v. 6.0.1  was usedulations , 38. Foro method , 40. Ther v. 1.4 , which whttps://github.com/cgevans/scikits-bootstrap#egg=Package) in python 3.4, with a bootstrap sample pool of 20,000. To define markers, or associated regions, that are under selection, we used statistical tools for detection of outlier SNPs that deviate from those that are neutrally evolving. PGDspider v. 2.1.0.0 [L. rubellus lineage B genome using biopython scripts (http://biopython.org/). These regions were checked against the nr database using blastx from Blast + v. 2.2.29 [After binning individuals in populations as defined by population structure analyses, we reran the Populations module of stacks to determine lineage and site-specific population genetic end points, which was also used to derive Fisher’s exact test results. One-way ANOVA in Minitab v. 17 was used to determine significant differences for nucleotide diversity and inbreeding coefficient, between lineages and the contamination status of sites. Bootstrapped 95% confidence intervals were calculated using the scikits module  via the Tracy-Widom statistic, when considering all individuals even though each explains only a relatively small proportion of overall variance; though consistent with similar analyses [p < 0.05, Pearson’s correlation coefficient = 0.76 and 0.32 for PC1 and PC2, respectively). Individuals assigned to lineage B possessed a far greater proportion of reads aligning to the lineage B genome (21–43%) over those assigned to lineage A (<12%). Under these assignments, individuals from CF had the highest skew towards a single lineage, with only a single individual from contaminated soils assigned to lineage B (blue), while 27.3% of genotypes attributed to the 5 lineage B individuals from the control population clustered with lineage A (red). Net nucleotide distance between the lineages, as calculated in structure, was 0.126.We used PCA to provide initial insight into the structure of analyses , 46. Plocant P <  × 10−12 L. rubellus lineages, we used PCA to highlight the numbers of discrete clusters forming along significant principle components, before deriving the proportion of an individual’s genotypes that clustered together using the software, Structure. For lineage A, only the first PC was significant  in the statistics defining inbreeding or nucleotide diversity that was specific to lineage or the contamination status of sites, at least with regard to the environmental factors considered , with the marker found to be missing in individuals from lineage A, except for one that was heterozygous and another that was homozygous for the alternative allele.Several scaffolds were found to be likely bearers of pheromones, therefore, those where the top hit achieved an E-value greater than 1 × 10P-value <1 × 10−5 were analysed to determine the functionality of proximal genes. A number of SNPs are associated with scaffolds containing genes for metal binding or transport, as well as stress responses, and are candidates for further analysis regarding biochemical mechanisms associated with detoxification  was observed in lineage A individuals from CWM on a scaffold that sees two SNPs nearby to a PARP gene, involved in DNA repair and programmed cell death. Also on this scaffold is a predicted palmitoyltransferase gene (ZDHHC3), known to mediate Ca2+ transport across cell membranes [P = 6.66 × 10−6) in defining individuals between contaminated and control sites, pertinent to the transport of the phosphate analogue, arsenate [P = 2.58 × 10−5) lies near to a metallothionein that has previously been identified in L. rubellus [The lowest embranes . Far fewarsenate , 51. Anorubellus  and is sL. rubellus using a high-density set of SNPs, and are able to clearly distinguish between lineages, which tend to form discrete populations across the UK in most instances. Subsequently, we have demonstrated that populations found on highly contaminated former mine sites are almost indistinguishable from those from nearby, relatively unpolluted soils, except for at a small number of sites across the genome. Many of the most significantly different genetic signatures that segregate between populations and species allow us to gain further insight into mechanisms playing a role in local adaptation and species divergence.Here, we have principally demonstrated that we can observe population structure in Aporrectodea caliginosa that were surveyed using nuclear and mitochondrial markers. Individuals at CF demonstrate relatively low FIS, while subsequent kinship analysis shows that no individuals from CF were as, or more related, than first cousins. We provide further evidence against hybridisation through use of MDS, which incorporates identity by state to clearly differentiate between lineages across the first dimension. While there is mixed evidence both for and against population structure and gene flow across smaller spatial scales [Firstly, we were able to distinguish between lineages through alignment to two voucher draft genomes and make clear genomic distinctions between the two lineages. Giska et al.  demonstrl scales , 56, sucL. rubellus lineages at DGC in an analysis by Kille et al. [In our analysis of SNPS most significantly segregating between the two lineages, a number occur close to genes implicated in processes that have recently been suggested as drivers for lineage differentiation. The most well supported SNP was proximal to a gene controlling histone lysine demethylation, which is one of the most prominent epigenetic mechanisms controlling chromatin structure. DNA methylation is another epigenetic modification that has previously been differentiated among e et al.  . In thiL. rubellus genome [D statistic [Work by Jones et al.  suggestss genome . Indeed,s genome . Overalltatistic , though Soil metal concentrations, both here and in the literature, have demonstrated concentrations of metals at these sites known to be well above those known to cause effects on naïve populations , 60–62. SERCA, that varies between individuals identified as lineage A and a control population. A number of the genes identified during this investigation purport to this hypothesis and are therefore of likely importance to earthworm continuation at this site in both lineages.CWM is the site of a former Pb/Zn mine that discontinued production before 1920 , and thoL. rubellus from DGC and CF to As has been previously demonstrated [L. rubellus [Evidence supporting adaptation of nstrated , 20 and nstrated , 68, liknstrated . Genes, nstrated –72 and rnstrated . In eartrubellus , which wrubellus . SynthesL. rubellus across the UK, in attempts to observe drivers for lineage divergence. Specifically, Spurgeon et al. [L. rubellus originating from mine sites are perturbed in the long term. The fact that we fail to resolve population structure between mine and control sites, or to even see complete segregation at any SNPs, demonstrates that earthworms inhabiting highly contaminated former mine sites are likely derived from standing genetic variation extant among populations in less contaminated soils and may even continue to experience gene flow.Our analysis is likely to be underpowered in our ability to distinguish specific SNPs, particularly for lineage B individuals from CF where we see no major variants. However, selection pressure across sites can vary immensely, given the highly heterogenous nature of soils; therefore, attempting to define the effects of large-scale contamination may not be as simple as detecting resistance for a single toxicant. Work by Spurgeon et al.  and Liebn et al.  found thL. rubellus appears to provide an exemplary system for exploring drivers for speciation, with a continuum of lineages coexisting across continental Europe, while distinct lineages exist in isolation throughout the UK. Further work with this species will provide evidence as to the effect of genetic diversity upon adaptive capability.We’ve used genomic analysis of earthworms in the UK with the view of identifying genomic variation in response to environmental variables. While we can observe population structure and define lineages, it is difficult to resolve markers that segregate entirely between populations in response to soil metal concentrations. This may represent a highly variable series of traits in response to the heterogenous nature of the soil environment, but ultimately demonstrates that natural populations of a cosmopolitan species are generally robust to long term metal contamination. This system demonstrates the importance of determining ecological end points of ecotoxicological models, as their relevance can shift relative to population and evolutionary history. Beyond this, Additional file 1: Figure S1.L. rubellus (n = 128) sampled across from the UK. Triangles represent individuals originating from former mine sites, while circles represent those from nearby control sites. The amount of variance explained by each component is noted on their respective axes. Figure S2. Principle component analysis remaining significant PCs, calculated from RADseq data of populations of L. rubellus belonging to lineage B (n = 66). The amount of variance explained by each component is noted on their respective axes. Circles represent control sites, triangles represent former mine sites. Figure S3. Intra-population estimation of kinship coefficient among L. rubellus, relative to lineage and sample site. A negative kinship coefficient estimation indicates an unrelated relationship. Figure S4. Plot of multidimensional scaling analysis incorporating IBS for populations of L. rubellus, relative to lineage and sample site. The first 6 dimensions are reported and the amount of variance explained by a particular dimension is detailed on their respective axis. Figure S5. Outlier results as calculated by Bayescan when a q-value  of 0.05 is imposed. Each SNP is plotted to infer signals of selection when populations are compared, including lineages A and B (a), as well as lineage-specific populations inhabiting former mine sites and proximal control sites . Figure S6. Scree plot for the proportion of variance explained by principle components 1–15 as determined by PCAdapt. (DOCX 447 kb)Significant principle components (PC3–6) defined by the Tracy-Widom statistic, calculated from RADseq data of all Additional file 2:RADseq Protocol. An explicit description of the RADseq protocol, listing the methodology, reagents and equipment for amplifying and sequencing genomic libraries with an Illumina HiSeq. (XML 63 kb)Additional file 3:Gene annotation data output by blastx as associated with SNPs considered to be most strongly under selection. Tab Additional file 4:Lineage B genome locations of all SNPs, including respective values output by Bayescan, PCAdapt and Fisher’s exact test when comparing populations for signals of selection. (ZIP 479 kb)


Rhododendron aureum growing at the alpine in Changbai Mountain (China) were studied in two types of habitat (in the tundra and in Betula ermanii forest). Identification and delimitation of genets were inferred from excavation in small populations and from amplified fragment length polymorphism (AFLP) markers by the standardized sampling design in large populations. Clonal architecture and clonal diversity were then estimated. For the four small populations, they were monoclonal, the spacer length  was shorter and branching intensity  was higher in the tundra than that in Betula ermanii forest. For the five large populations, they were composed of multiple genets with high level of clonal diversity ; the spatial distribution of genets showed that the clonal growth strategy of R. aureum exhibits both guerilla and phalanx. Our results indicate that the clonal plasticity of R. aureum could enhance exploitation of resource heterogeneity and in turn greatly contribute to maintenance or improvement of fitness and the high clonal diversity of R. aureum increase the evolutionary rates to adapt the harsh alpine environment in Changbai Mountain.Four small oval populations and five large intensive populations of  Plant survival in alpine landscapes is constantly challenged by the harsh and often unpredictable environmental conditions. Life cyClonal plants individuals can be recognized at two different organizational levels: genets and ramets. A genet is a group of genetically identical individuals, which have grown in a given location, all originating vegetatively, not sexually, from a single ancestor. The genet comprised of all tissues originating from one zygote, all genetically identical members of a clone, also called genetics individuals or clones, whereas a ramet is an individual or a potentially independent part of a clonal colony. A ramet is a potentially independent part of a genet. ,8. The sCarex bigelowii Torr. ex Schwein and the alpine Carex curvula All. demonstrated high levels of clonal diversity[Rhododendron ferrugineum L. intermediate to high levels of genetic diversity were detected based on AFLP molecular markers[A high degree of asexual reproduction is often associated with genetic monomorphism,16, howediversity–20. Popur markers. Such a r markers,22,23. Mr markers,25: (i) Although excavation can directly reveal the extent and patterns of clonal plants, excavation is not effective over large areas due to the high cost, root fragmentation and root grafting. Moreover, root fragmentation removes the physical evidence of clonality, root grafting may connect non-clonal genets. Molecular methods are a major advance over phenological or root connectivity studies in the identification of clonal plants, and many studies have successfully used Amplified Fragment Length Polymorphisms AFLPs to identRhododendron aureum Georgi , the target species in this study, is a perennial evergreen dwarf shrub with well-branched trailing stems inhabiting alpine regions of Korea, China, Japan, and the Kamchatka peninsula. This plant is approximately 1 m high, blooms from June to July, and the flowers have a pale yellow color. It has been shown that occupies the snowmelt gradient and especially to dominate in early exposed places[R. aureum exhibits morphological and behavioral adaptations to the alpine environment[Betula ermanii forest of Changbai Mountain, altitude ranging from 1,000 to 2,506 m a.s.l.[R. aureum is one of the constructive and dominant species on the alpine ecosystem in Changbai Mountain, and it plays an important role in maintaining the ecological balance by preventing and controlling soil erosion. For R. aureum, few seedling establishment has been observed in Changbai Mountain [R. aureum may be similar to R. ferrugineum which is clonal reproduction in alpine[R. aureum.ed places. These avironment. In Chin m a.s.l.. The rec m a.s.l.. The R. Mountain , and we in alpine. HoweverThe aims of this study were (i) to identify the clonal reproduction in candidate monoclonal populations; (ii) to determine the clonal growth strategy; (iii) to define the clonal diversity and spatial distribution of clones in candidate multiclonal populations and (iv) to discuss the effect of clonality on adaptation to alpine environment. These aims were addressed in four candidate monoclonal populations and five candidate multiclonal populations in Changbai Mountain using field investigation and AFLP markers.Betula ermanii forest with altitude range from 1800m to 2600m.This study was conducted on the northern and western slope of Changbai Mountain, which is generally acknowledged as the highest mountain in northeast China and eastern Eurasia. The region is a typical mountain climate. The climate conditions are also distinct in different altitudes. The average annual temperature is generally 3°C–7°C, and annual precipitation is over 600 mm. With relatively high altitude above sea level, the annual precipitation is over 1400 mm. The alpR. aureum were investigated by excavation in the field during the summer of 2015. DX1 and DX2 were located in the Betula ermanii forest; DX3 and DX4 were located in the alpine tundra. The spacer length, branching intensity and branching angle was measured. We have re-transplant the R. aureum back to the original place immediately to reduce the damage to these populations. The leaves from ten ramets in each candidate monoclonal population were sampled and dried directly in silica gel. The candidate multiclonal populations for investigating clonal propagation and clonal diversity was carried out in large and dense populations on flat areas with no intervening larger patches of other species or bare ground. Four rectangular sampling plots and one linear arrangement plot were selected on Changbai Mountain to assess clonal propagation and clonal diversity of the population. CN1, CN2, CN3 was located in the north slope of Changbai Mountain and CW1 and CW2 located in the western slope. Populations CN3 and CW1 were in the Betula ermanii forest, the rest populations were on the tundra. The geographic coordinates for all populations were shown in Table 1. As population CN1 was roughly distributed in a linear arrangement along the terrain, we obtained 36 samples along the terrain. Within the other plots, 35 to 44 leaves (one per branch) of R. aureum were collected at each point of intersection of a 1 m × 1 m grid and immediately preserved in silica gel to prevent DNA degradation.Four candidate monoclonal populations (DX1-DX4) which the shape of the population on the ground is oval  with diameter range from 1.3m to 1.9m of Total DNA was extracted from silica-dried leaves using a Plant Genomic DNA Kit . DNA samples were diluted to 10 ng/μl and then stored at -20°C until further analysis.EcoRI , 1 U of MseI (New England Biolabs (Beijing) LTD), 1μl of 10 × restriction–ligation buffer, 1 U of T4 DNA ligase, 0.2 mM of ATP, 1.0 μM of MseI adapter and 0.1 μM of EcoRI adapter and double-distilled water. The mixture was incubated at 37°C for 8 h, 16°C for 4 h, inactivated at 65°C for 10 min, and stored at 4°C. Pre-amplification was performed in a 25μl solution containing 2.5μl of diluted restriction–ligation product, 0.2 μM of dNTPs, 0.3 μM of each primary amplification primer, 2.5 μl of 10× PCR buffer and 0.5 U of Taq polymerase . The pre-amplification conditions consisted of pre-denaturation for 5 min at 94°C, followed by 30 cycles of denaturation for 30 s at 94°C, annealing for 60 s at 56°C, and elongation for 80 s at 72°C, with a final extension for 10 min at 72°C and storage at -20°C. Products were diluted 1 to 40 (v/v) with ddH2O. The optimized selective amplification PCR reaction system (25 μl) for R. aureum 2 μl diluted pre-amplification product, 0.2 mM dNTPs, 2 μM EcoR I and MseI selective primer, 2.5μl of 10× buffer and 0.5 U of Taq polymerase and double-distilled water. We pre-screened 64 selective primer pairs and chose ten pairs that were reliable for this study. The detail sequences of primers were in S1 Table. The selective PCR reaction had two cycle sets: 13 cycles of 30 s at 94°C, 30 s at 65°C  and 60 s at 72°C, followed by 18 cycles of 30 s at 94°C, 30 s at 56°C and 80 s at 72°C. After selective amplification, the products were mixed 1:1 with a loading buffer , denatured at 95°C for 5 min, and separated on 6% denaturing polyacrylamide gels (6% polyacrylamide and 7 M urea) in 1× TBE buffer at 70 W for 4.5 h. Gels were stained according to the silver staining method[The amplified fragment length polymorphism (AFLP) method developed by Vos et al was perfng method. To testTukey’s LSD post hoc test was used to test the significant differences of spacer length, branching intensity and branching angle between different populations. Statistical analyses were performed using 19.0 SPSS for Windows .I) [PPL), genetic distance and genetic identity. The clonal structure identification followed the method of Mejías and Vonlanthen [AFLP-amplified DNA fragments (bands) were scored as present (1) or absent (0), and a data matrix of the AFLP banding patterns of all populations was assembled for further analysis. The program POPGENE v1.31  was usedI) , percentnlanthen ,40. Basenlanthen  using NTnlanthen .To estimate clonal diversity, four frequently used measures were calculated following Ellstrand and Roose:(1) The number of genets (G)D)[Ni is the number of individual shoots with AFLP phenotype i and N is the total number of samples. This measure describes the clonal heterogeneity in a population; it ranges from 0 to 1. When D = 0, a population is composed of a single clone; when D = 1, a population has no clonal growth.(2) Simpson diversity index (D):D=1−∑(NE)[E = 0, indicating that the entire population has only one clone or the most ramets belong to one clone while the other clone only contain one ramets; when E = 1, there are same ramets of different clones within the population.(3) Fager's evenness (E):E=D−DmiR)[(4) The clonal richness (R):R=G−1N−2, 66.10 ± 16.75°respectively. There are some difference between the different populations on the spacer length and branching intensity, whereas the branching angle was similar among the four populations, showed in Fig 1. In DX1 and DX2, the spacer length was longer, but the branching intensity was lower than that in DX3 and DX4 significantly . As DX1 and DX2 were located in the B. ermanii forest, DX3 and DX4 were located in the alpine tundra; thus monoclonal populations located in the tundra showed the shorter spacer length and higher branching intensity than that the monoclonal populations located in B. ermanii forest and there was no difference of branching angle between the two habitat types .The four small oval populations (DX1-DX4) are indeed monoclone revealed by excavation. The average spacer length, branching intensity and branching angle of the four monoclonal populations is 24.17 ± 9.77 cm, 90.86 ± 50.78 branches/mTable 2). The minimum genetic identity ranged from 0.952 to 0.977 with average of 0.962 and the maximum genetic distance ranged from 0.023 to 0.048 with average of 0.038. Here we set the genetic identity threshold and genetic distance threshold to 0.962 and 0.038 respectively, which means that samples which genetic identity more than 0.962 and genetic distance less than 0.038 were defined as the same clones in multiclonal populations. We have found that the PPL and diameter of monoclonal populations were positively correlated .For the 40 samples from four monoclonal populations, AFLP analysis was used to detect the threshold value which determines ramets belonging to a same clonal lineage. The number of unambiguously scorable fragments generated by ten AFLP primer combinations range from 291 to 292 in 40 individuals from 4 monoclonal populations, molecular size of the fragments ranging from 100 to 1500bp , with high clonal evenness (E = 0.79–0.89). The clonal diversity is similar between the populations growing in the tundra  and the populations growing in B. ermanii forest . Maps showing the spatial distribution of the samples assigned to genets in CN2, CN3, CW1 and CW2 populations are shown in Fig 4. As population CN1 was roughly distributed in a linear arrangement from CN101 to CN136 according to the terrain, we can see the spatial distribution in Fig 3A. The samples belonging to each genet were usually spatially grouped and in many cases, samples belonging to the same genet were separated by other genets. Indicating that the clonal growth strategy of R. aureum exhibits both guerilla and phalanx. Thus the species exhibits a transitional clonal growth form.Based on the genetic identity and genetic distance threshold, the clone identification results are shown in R. aureum. In alpine ecosystems, with increasing altitudes, plant life is challenged by low temperatures, a shorter vegetation period, more snow, and harsher conditions. Life cycles of alpine plants are threatened by a high degree of uncertainty as to whether flowering, fruiting, germination, and establishment can be successfully completed [R. aureum exhibits a high level of clonality and exhibits transitional clonal growth strategy on Changbai Mountain. The study regions have a temperate continental climate with long cold winters, short warm summers, and extremely low mean annual temperatures that restricted the life cycle of R. aureum. Moreover, R. aureum blooms for only about a week, with flowering times ranging from June at low altitudes to August at high altitudes. R. aureum exhibits low selfing ability, and it is pollinated mostly by insects[We predominantly observed clonal propagation on Changbai Mountain of ompleted ,48. Pereompleted ,49. Clonompleted . R. aurey insects; howevery insects. As a reR. aureum to survive since clonal plants can adapt to heterogeneous environments by risk spreading among genets [The spacer length and branching intensity of R. aureum showed significant differences in the tundra and in B. ermanii forest. In the tundra of Changbai Mountain, there are abundant light resources as no high tree cover, the R. aureum populations showed high degree of intensity; whereas in the B. ermanii forest, lighting resources are limited as the cover of B. ermanii, in order to obtain more light resources, the R. aureum populations was sparse. Clonal plasticity could enhance exploitation of resource heterogeneity by clonal plants, and in turn greatly contribute to maintenance or improvement of fitness[R. aureum showed highly plastic changes in morphology of individual ramets that enable effective exploitation of local concentrations of essential resources once they have been located [Clonal propagation is essential for g genets ,52, clong genets –55, forag genets –58, and g genets . The morg genets ,61. The f fitness. Shrubs  located .Table 3) on Changbai Mountain, within-population clonal diversity was rather higher than the average for clonal species  as revealed by Ellstrand [R. aureum, we have found genetic diversity even in populations consisting of a single clone, with variation that was proportional to the population diameter . Somatic mutations and diffuse centromeres can contribute to standing genetic variation in populations. Genetic diversity is key for a species to survive and adapt to changing environments [R. aureum increase the evolutionary rates to adapt the harsh alpine environment in Changbai Mountain.Populations of clonal plants consisting of few genets are subject to similar genetic processes that affect population, such as genetic drift and inbreeding which may lead to loss of genetic variability and may cause inbreeding depression ,64. Thoullstrand . In manyllstrand ,65–67. Illstrand ,68,69. Mllstrand , and thellstrand ,71. Sevellstrand ,72. In Rronments –76 and tR. aureum was widespread in Changbai Mountain reviewed by excavation and AFLP markers. The ramets of monoclonal populations arranged more intensive in tundra than that in the B. ermanii forest. The clonal growth strategy of R. aureum exhibits both guerilla and phalanx. R. aureum showed high level clonal plasticity in different habitats. The plasticity of R. aureum allows them to change their growth form so that they could adapt to alpine heterogeneous habitat. R. aureum exhibit a high level of genetic variation within populations. With the genetically more diverse, R. aureum have ability to buffer the effects of poor environmental conditions in alpine and increase the evolutionary rates to adapt the harsh alpine environment in Changbai Mountain.In our study we have found the clonal reproduction of S1 Table(DOCX)Click here for additional data file.


Rangifer. The results are interpreted in the context of conservation concerns and debates on the origin of domestic animals. The study is located in Northeastern Zabaĭkal'e, Russia at approximately 57 degrees North latitude. Blood and skin samples, collected from wild and domestic Rangifer, are analyzed for their mtDNA and microsatellite signatures. Local husbandry traditions are documented ethnographically. The genetic data are analyzed with special reference to indigenous understandings of the distinctions between local domestic types and wild Rangifer. The genetic results demonstrate a strong differentiation between wild and domestic populations. Notably low levels of mtDNA haplotype sharing between wild and domestic reindeer, suggest mainly male‐mediated gene flow between the two gene pools. The nuclear microsatellite results also point to distinct differences between regional domestic clusters. Our results indicate that the Evenki herders have an effective breeding technique which, while mixing pedigrees in the short term, guards against wholesale introgression between wild and domestic populations over the long term. They support a model of domestication where wild males and domestic females are selectively interbred, without hybridizing the two populations. Our conclusions inform a debate on the origins of domestication by documenting a situation where both wild and domestic types are in constant interaction. The study further informs a debate in conservation biology by demonstrating that certain types of controlled introgression between wild and domestic types need not reduce genetic diversity.This study investigates the genetic effect of an indigenous tradition of deliberate and controlled interbreeding between wild and domestic  GeneticRangifer tarandus, are a species commonly considered to be in an early phase of domestication and which often coexist with wild forms  and stretches of taiga. Both wild and domestic reindeer herds coexist today in the region. It is likely that this coexistence has persisted for a long time. Zabaĭkal'e has been suggested to be one possible origin point for reindeer husbandry Wiklund, , with OkRangifer, despite local breeding practices that tolerate or even to encourage controlled inter‐breeding. We hypothesize further that their use of strict criteria to control genetic introgression through culling or castration is key to maintaining this divide. A strong genetic difference between the wild and domestic populations would therefore document their pastoral skill in maintaining and controlling specific reindeer pedigrees. Establishing the identity and level of distinctiveness of wild and domestic herds is further important to settle an old debate on whether or not a domesticated harness reindeer constitutes a distinct type of reindeer, compared to wild reindeer.In this article, we test the hypothesis that Zabaĭkal Evenkis are able to enforce a strict genetic separation of wild and tame 22.1The reindeer in this study were sampled between 2012 and 2014 from wild and domestic regional populations around the settlements of Chapo‐Ologo and Ti͡ani͡a and the encampment at Nomama. We have included also data from a previous study of an Evenki herd at Lake Nichatka sampled in 2001 and 2002 . In contrast to all the other sites, the extended family at Lake Nichatka never had participated in collectivization. Their kinship networks for the most part looked northwards the Bodaĭbo district of Irkutsk 2.2Rangifer from local herdsmen at Chapo‐Ologo, Tia͡nia͡, and Nomama. A total of 117 domestic animals were documented of which 101 were used in this study. Fifty‐six wild Rangifer were described of which 49 samples were used here. The descriptions of domestic animals were structured around a set of open‐ended questions eliciting the life history of a particular animal. The interviews, at minimum, affixed the age, sex, place of birth, and ownership status of each animal. Beyond this we photographed each animal and recorded detailed observations of the reindeer type, its pelage, its role in the social organization of the herd, peculiarities of its behavior, and how it might have been harnessed or tamed for other activities. The latter information was important for eliciting what qualities the herders valued in a domestic animal. We recorded information in the Russian, I͡Akut, and Evenki languages in order to capture details which may translate poorly. Information on wild Rangifer was confined to visual assessments of the age, sex, and qualities of the hunted wild animal.For this study, we collected detailed pedigrees and descriptions of wild and domestic The previously published set from Lake Nichatka are documented only by sex and type (wild/domestic) with some specific biographical information on individual animals available in Abe . Of the 2.3DNA was obtained from hair follicles, skin samples, or from FTA cards designed for forensic work , 0.5 U/μl AmpliTaq DNA polymerase (Applied Biosystems), and dH2O to make up the remaining volume. The samples were cleaned for unincorporated primers and nucleotides using Illustra ExoProStar  diluted 10 times. Cycle sequencing was performed in a 10 μl reaction volume, using BigDye v3.1 sequencing kit (Applied Biosystems) following the manufacturer's recommendations. Purification was carried out using standard EDTA/EtOH precipitation. Capillary electrophoresis and data analysis was performed with an ABI 3130xL‐ or 3500xL instrument (Applied Biosystems). All sequences were sequenced in both directions, and the consensus sequences were aligned by ClustalW  long fragment from the mitochondrial control region (CR) was amplified using the forward primer RtCRF (5′‐AAT AGC CCC ACT ATG AGC ACCC‐3′) , gene diversity, and nucleotide diversity were calculated in DnaSP  to examine genetic differentiation among populations. We also used Arlequin to perform analysis of molecular variance (AMOVA) , among populations within groups (FSC), and among groups (FCT). The AMOVA was run with all samples, imposing no hierarchical structure, and with the populations divided into two groups, separating between domestic and wild populations. Significance associated with the fixation indexes was evaluated through random permutation procedure using 10,000 permutations.For both the CR and microsatellite data, we used Arlequin v.3.5 , a model with uniform priors, admixture, correlated allele frequencies, 20,000 burn‐ins, and 200,000 Markov Chain Monte Carlo (MCMC) iterations was run ten times. For each K‐value, average posterior probability among runs and standard deviation (SD) was calculated. Clumpp  as well as in the microsatellites . Our results show a higher significance level in the mtDNA dataset compared to the microsatellites. This may be explained by the clonal/maternal mode of inheritance of mtDNA resulting in an effective population size that is one‐fourth of nuclear DNA. Smaller population size makes mtDNA more sensitive to population changes as it is more exposed to genetic drift , except between the regional domestic herd from Ti͡ani͡a and the domestic herds from Nomama and Nichatka (p = .06), and between the wild herd from Chapo‐Ologo and the wild herds from Lake Nichatka (p = .09) and Ti͡ani͡a (p = .208). Pairwise genetic differentiation calculated from the CR data show no or low levels of significance, except for the regional population of Nichatka wild reindeer which is significantly different from all other populations (p < .001)  Table . For thep = .028) of the variance can be attributed to differences between wild and domestic, while 93.60% (p < .001) of the variation is found within populations in the microsatellite data. A similar result was evident for the mtDNA data where 4.43%  of the variance is found between wild and domestic, while 83% (p < .001) is found within populations.An AMOVA analysis was run on the entire dataset without imposing any hierarchical structure. It showed that most of the genetic variation is found within populations, rather than among populations, in both datasets Table . When grK = 1 to K = 5 to assess the mean likelihood of there being separate populations within the dataset. The analysis suggested an apparent main structure at K = 2  by individuals from one of the eight populations Table . Except To further test the genetic structure among the domestic reindeer, we re‐ran the STRUCTURE analysis with the 101 domestic samples alone. This analysis supported a separation into three domestic genetic clusters Figure a with ea3.2Zabaĭkal Evenkis traditionally hold reindeer in small local herds scattered across the region ranging in size from a dozen to two hundred head by extended families of four to twelve individuals. The herd structure varies by the needs of each family and by season, but approximately up to 40% of the herd might be breeding females. The balance would be nonbreeding juveniles or calves, with a handful of males kept as breeding bulls and another dozen males castrated and kept for use as transport reindeer Figure . They gebai͡unchikar. The local herdsmen therefore seek to keep their camps near groups of wild forest reindeer, which are an important source of food and skins for clothing and equipment. The wild populations are described as scattered, small, localized groups of ten to twenty animals with a range that intersects with the domestic populations. They do not migrate more than 30 km over the course of a year and alternate their pastures between high alpine meadows and taiga meadows in between mountain ridges  and are rarely slaughtered for food  and domestic Rangifer (oron) are marked semantically as different types. The local practice of deliberately interbreeding domestic cows with wild bulls to produce bai͡unchikar is noted in the literature but has never been described in detail. The first European to mention the practice was Leopol'd Shrenk —our study as whole suggests the reverse: that the genetic integrity of both the wild and domestic populations is maintained. This could be explained by the fact that domestic Rangifer are not held in the same way as pigs. It is not sufficient that they tolerate humans and forage independently close‐to‐hand. Reindeer are held in a highly nuanced state of training where they can be harnessed and ridden—a behavioral quality unlikely to be expected of a pig.Another prominent study of the genetics of animal domestication by Larson and Burger  suggestsRangifer in Alaska (Roffler, The notably low CR haplotype sharing between wild and domestic reindeer, indicate very low levels of female mediated gene flow. This is consistent with female philopatry and male‐biased dispersal commonly seen among most mammal species (Greenwood, Roffler, . In nortRoffler, . Female Roffler, . FurtherThis study was originally designed to test the hypothesis that careful husbandry could successfully keep wild and domestic populations genetically separated despite living in close proximity and despite deliberate attempts to let certain female reindeer to interbreed. The genetic results indicate this is indeed the case. Our results further suggest that it is possible to distinguish local domestic clusters, which may serve as signatures of particular breeding traditions. This finding may be important to bolster local claims to the pedigree status of their herds which could give them access to Russian state funding to preserve pedigree types (Zabrodin & Borozdin, Rangifer stressing the importance of strict selection and introgression along the male line.This study suggests that it is possible to distinguish regional domestic herds genetically despite complex social histories of collectivization and privatization. The status of the Nomama population is a particularly interesting case in point. Despite significant influxes of reindeer purchased from neighboring regions, and Chapo‐Ologo in particular, the Nomama population continues to distinguish itself from other groups by its low level of genetic diversity within the mtDNA and a strongly different microsatellite signature. The mtDNA signature supports an interpretation that there may have been a founding by a small number of females—perhaps those that oral accounts suggested were lassoed from the free‐ranging feral remnants of the defunct state farm. Their distinct microsatellite signature suggests that the founding population may have been augmented by hybridization with local feral reindeer. Further, the proportion population membership in Table Up until now, the classic literature on the origin of reindeer husbandry and of reindeer pedigrees has relied upon the classification of saddling and harnessing technologies as a proxy for grouping together distinct indigenous pastoral traditions. The reindeer bodies themselves be they wild or tame were assumed to be standard. This study suggests that in this region of southeastern Siberia, perhaps one of several “hearths” of reindeer husbandry, wild and domestic types are understood to be phenotypically and behaviorally different by local herders, and that this difference extends to the distinct genetic signatures in each regional population. Unlike in other models of the origin of animal husbandry, this study suggests that a respect for difference between the populations leads to a breeding strategy which enhances the genetic diversity of the species as a whole.None declared.Genetic mtDNA data generated for this study is available on GenBank. Reference numbers are in Supplementary Table  Click here for additional data file.


The Maniq and Mlabri are the only recorded nomadic hunter-gatherer groups in Thailand. Here, we sequenced complete mitochondrial (mt) DNA genomes and ~2.364 Mbp of non-recombining Y chromosome (NRY) to learn more about the origins of these two enigmatic populations. Both groups exhibited low genetic diversity compared to other Thai populations, and contrasting patterns of mtDNA and NRY diversity: there was greater mtDNA diversity in the Maniq than in the Mlabri, while the converse was true for the NRY. We found basal uniparental lineages in the Maniq, namely mtDNA haplogroups M21a, R21 and M17a, and NRY haplogroup K. Overall, the Maniq are genetically similar to other negrito groups in Southeast Asia. By contrast, the Mlabri haplogroups (B5a1b1 for mtDNA and O1b1a1a1b and O1b1a1a1b1a1 for the NRY) are common lineages in Southeast Asian non-negrito groups, and overall the Mlabri are genetically similar to their linguistic relatives (Htin and Khmu) and other groups from northeastern Thailand. In agreement with previous studies of the Mlabri, our results indicate that the Malbri do not directly descend from the indigenous negritos. Instead, they likely have a recent origin  by an extreme founder event  from an agricultural group, most likely the Htin or a closely-related group. The phenotypic resemblances of SEA negritos and African pygmies initially suggested a separate origin in Africa, although convergent evolution now seems more likely . Hunting-gathering was the mode of subsistence traditionally practiced by most SEA negritos, whose communities are distributed in three regions: Andaman Islands, Peninsular Thailand and Malaysia, and the Philippines5. Several genetic studies have been conducted in some of these relic populations and indicated deep ancestry13 which could link these people with the ancestral groups who migrated from Africa into SEA during the late Pleistocene14. However, the hunter-gatherer groups of Thailand are relatively under-studied compared to those from other SEA regions.The indigenous peoples of Southeast Asia (SEA) are often referred to as “negritos” because of their differences in phenotypic appearances, i.e. on average shorter stature, frizzier hair, and darker skin color17 and resemble nearby hunter-gatherer populations in Malaysia, e.g. Jehai and Batek18. For example, the Maniq language belongs to the northern Aslian group of Austroasiatic languages, as do the languages of the Jehai and Batek19, although this most likely reflects a later introduction by agriculturalists21. A previous study of sequences of the first hypervariable region (HVR) of the mtDNA control region reported a relatively large divergence of the Sakai (Maniq) from southern Thailand from other Thai groups22. Analyses of mtDNA from prehistoric human remains in southern Thailand suggested that contemporary SEA negritos are descended from pre-Neolithic populations23.There are two hunter-gatherer groups in Thailand, the Maniq and the Mlabri. The Maniq, also known as Sakai or Kensui, have a census size of ~250 individuals25. This, however, is disputed by Waters (2005)26 who believed that the Mlabri have had contact and trade with neighboring ethnic groups, and thus they are not hunter-gatherer in the strict sense. The conflicting theories as to how they sustain their livelihood make it interesting to study their origin and lineages. With a census size of only ~130 individuals16, the Mlabri live in a mountainous area of northern Thailand and speak a language belonging to the Khmuic branch of Austroasiatic languages. Khmuic languages are also spoken by their neighbors, Htin  and Khmu, who are also the other hilltribes in northern Thailand. Preceding genetic studies reported extremely reduced genetic diversity in the Mlabri and suggested a cultural reversion from agriculture to hunting and gathering27 and genetic affinities between the Mlabri and the Mal, a Htin subgroup28.The other hunter-gatherer group, the Mlabri, are characterized by a nomadic traditional lifestyle, in which they construct shelters from the leaves of banana trees that they move out when the banana leaves turn yellow. Their neighbors call them “Phi Tong Luang” which means the “Spirits of the Yellow Leaves”. Although they do not exhibit the typical “negrito” phenotypes, their mode of subsistence is hunting and gathering30. However, such comparisons are complicated by the different molecular methods that have been typically employed to assay variation . Advances in next-generation sequencing make it feasible to obtain mtDNA and NRY sequences that allow directly comparable, unbiased inferences about female vs. male demographic history32. Here, we present and analyze complete mtDNA genome sequences and ~2.364 Mbp of NRY sequence from the Maniq and the Mlabri  have been useful in illuminating differences in the female vs. male demographic history of human populationsbri Fig. . These th) = 0.29 ± 0.12; mean number of pairwise differences (MPD) = 0.29 ± 0.32; nucleotide diversity (π) = 0.00002 ± 0.00002; segregating site (S) = 1) with just one polymorphic site (nt 43) defining two haplotypes. With 5 haplotypes defined, the Maniq showed higher diversity values  than the Mlabri. However, both of them have lower genetic diversity values than other populations from Thailand  with mean coverages ranging from 277× to 1342×. Extraordinarily low mtDNA diversity was observed in the Mlabri : 1.71–2321.88; Figure 34 while R21 is newly-reported in populations from Thailand. The networks and maximum clade credibility (MCC) trees of M21a and R21 indicate a close relatedness of the Maniq sequences with those from the Jehai , while the Maniq M17a sequences cluster with sequences from the Laotian and Austroasiatic-speaking Blang  observed in the Maniq. When combined with sequences from other populations, the respective estimated ages of M17a, M21a and R21 are ~23.52, ~18.44 and ~8.83 kya Figure . The ageang Fig. .Φst distances indicates that the studied groups have large genetic distances from each other and from most other populations  plot based on ons Fig. . In the ons Fig. , the Mlaons Fig. .Figure 3h = 0.7778 ± 0.1374; MPD = 17.46 ± 8.94; π = 7.39 × 10−6; S = 37) than the Maniq, which show just one haplotype . It should be noted that these four unrelated sequences were collected from three different locations   for ~2.364 Mb of the NRY, with mean coverages ranging from 10× to 25×. In contrast to the mtDNA results, the Mlabri have higher NRY diversity  observed in the Maniq  were mentioned as unpublished data in a previous study13, which also found M21a and R21 to be common lineages, along with several minor SEA-specific haplogroups , indicating some SEA influences. In contrast, we did not detect any SEA-specific mtDNA haplogroups  in the Maniq, but instead we found M17a, which has been reported at low frequency in MSEA38 and ISEA39. Moreover, we detected Y chromosomal haplogroup K in the Maniq, which occurs elsewhere in populations of Papua New Guinea41, the Philippines30 and Australia42. Haplogroup K is an ancient lineages that is dated here to ~41.10 kya, which is younger than previous estimates in Aboriginal Australians (~48.40 kya)42. Overall, the Maniq display basal genetic lineages and closest genetic affinities with indigenous groups in Malaysia or ISEA, supporting a common ancestry of the Maniq with other SEA negrito communities. The reduced genetic diversity (and population size) of the Maniq probably occurred as a consequence of agricultural expansions to this region. The archaeological evidence in southern Thailand suggests that the Neolithic period of the Malay Peninsula started around 4 kya20 and after the expansion of agriculturalists who also brought Austroasiatic languages to this region, the Proto-Aslian groups were fragmented21, probably leading to gradual isolation.The SEA negritos are of interest because they are characterized by basal genetic lineages that likely reflect the earliest dispersal of modern humans from Africa27. We further attempted to identify the closest living relatives of the Mlabri, as these might be their source population. We found close genetic relationships in the NRY lineages between the Mlabri and various Htin subgroups as well as the Khmu, in agreement with an oral tradition, which indicates that the Htin are the ancestors of the Mlabri27. A shared common ancestor of these Khmuic-speaking groups might be another explanation. However, the Mlabri mtDNA lineage is most closely related to the Seak, Soa and Bru groups from northeastern Thailand  and the Ethics Commission of the University of Leipzig Medical Faculty. We obtained saliva samples from 11 Maniq from four different bands in three provinces, i.e. Pabon District (site 1: 3 samples) and Kong-Ra District (site 2: 1 sample), Phatthalung Province; Palien District, Trang Province (site 3: 2 samples); and La-Ngu District, Satun Province (site 4: 5 samples)  and aligned the sequences to the Reconstructed Sapiens Reference Sequence (RSRS)47 using MAFFT 7.27148.Twenty-nine complete mtDNA sequences (18 Mlabri and 11 Maniq) were generated from genomic libraries prepared with double indices and enriched for mtDNA as described previously49 and using genome sequences incluldedin SGDP Panel A50 and Panel B51. Sites were kept that were included in the UCSC table browser CRG Align 100 mappability track of the human reference genome hg19 . In order to obtain confident Y haplogroup calls later, we selected regions with at least one ISOGG SNP (version 8.78) and then applied the following filter criterion: a region was removed when its length was lower than 500 bp and its repeat count of 24-mers was higher than 10 in an average window of 120 bp using the CRG Align 24 mappability track from the UCSC table browser. A total of 2,264 regions with a combined length of 2,364,049 bp were included into the capture probe set, which included 2,071 ISOGG SNPs (version 11.144).We designed a capture probe set for the NRY following a previous study50. We enriched the libraries for the NRY regions via in-solution hybridization-capture51 using our probe set and the Agilent Sure Select system  and sequenced on the Illumina HiSeq. 2500 platform, generating paired-end reads of 125 bp length. Standard Illumina base-calling was performed using Bustard. We trimmed Illumina adapters and merged completely overlapping paired sequences using leeHOM52 and de-multiplexed the pooled sequencing data using deML53.Genomic libraries were prepared for each sample using a double indices schemehg19 using BWA’s aln algorithm54 and all sequence pairs that aligned to the NRY region which are previously defined were retained49. Duplicate reads were removed using PicardTools MarkDuplicates, indel realignment was performed using GATK IndelRealigner55 and base quality were re-calibrated using GATK BaseRecalibrator. We identified single nucleotide variants (SNVs) using GATK UnifiedGenotyper v3.3-0 across all samples simultaneously setting the parameter ploidy to 1 and using DBSNP build 138 as a prior position list. The identified SNVs were further filtered as previously described56. We imputed all samples with missing genotype information at any of these variant sites using BEAGLE57.The sequencing data were aligned against the human reference genome 58 for mtDNA and yHaplo59 for the NRY. Summary statistics for the studied populations and genetic distances (Φst) among the studied and compared populations were computed by Arlequin 3.5.1.360. Comparative data for the mtDNA genome sequences were selected from the literature encompassing East Asia66, Island Southeast Asia69, Taiwan70, India71, Andaman Islands72, Australia75 and New Zealand76. Further information on the populations used for mtDNA comparison are listed in Table 78 were included for comparison; additional details are provided in Table Haplogroup assignment was performed by HaplogrepΦst distance matrices of both mtDNA and the NRY were used to construct MDS plots and NJ trees79 by STATISTICA 13.0  and MEGA 7.080, respectively. Median-joining networks81 of haplogroups without pre- and post-processing steps were performed by Network (www.fluxus-engineering.com) and visualized in Network publisher 1.3.0.0.The 82. We ran jModel test 2.1.783 for selecting the suitable models in each run during the creation of the input file of BEAST via BEAUTi v1.8.082. For mtDNA, the BEAST calculation was done with the data partitioned between coding and noncoding regions with mutation rates of 1.708 × 10−8 and 9.883 × 10−8, respectively35. For the NRY, we used a mutation rate of 8.71 × 10−10 84, and the BEAST input files were modified by an in-house script to add in the invariant sites found in our dataset. After BEAST runs, we used Tracer 1.6 to check the results and TreeAnnotator v1.6.2 and FigTree v 1.4.0 to assemble and draw the Bayesian MCC trees, respectively. The Bayesian MCMC estimates (BE) and 95% HPD intervals of haplogroup coalescent times were calculated using the RSRS for rooting the mtDNA tree and African sequences belonging to haplogroup B2 (GS000035246-ASM) for rooting the NRY tree32.BEAST 1.8.0 was used to construct MCC trees by haplogroup, based on Bayesian Markov Chain Monte Carlo (MCMC) analyseshttps://www.ncbi.nlm.nih.gov/genbank/) with accession numbers MG672483-MG672511 and the Y chromosomal dataset are available from the corresponding author on request.The mtDNA dataset generated during the current study are available in GenBank (Supplementary Information


Athene cunicularia), a species that colonized South American cities just a few decades ago. We assembled a high-quality genome of the burrowing owl and re-sequenced 137 owls from three urban–rural population pairs at 17-fold median sequencing coverage per individual. Our data indicate that each city was independently colonized by a limited number of founders and that restricted gene flow occurred between neighbouring urban and rural populations, but not between urban populations of different cities. Using long-range linkage disequilibrium statistics in an approximate Bayesian computation approach, we estimated consistently lower population sizes in the recent past for the urban populations in comparison to the rural ones. The current urban populations all show reduced standing variation in rare single nucleotide polymorphisms (SNPs), but with different subsets of rare SNPs in different cities. This lowers the potential for local adaptation based on rare variants and makes it harder to detect consistent signals of selection in the genome.When a species successfully colonizes an urban habitat it can be expected that its population rapidly adapts to the new environment but also experiences demographic perturbations. It is, therefore, essential to gain an understanding of the population structure and the demographic history of the urban and neighbouring rural populations before studying adaptation at the genome level. Here, we investigate populations of the burrowing owl ( UrbanizColonization of urban habitats by animals and plants is ideal to study recent adaptive and non-adaptive processes at the genome level. The habitat of city centres and suburban areas and their historical development appear to be similar across a wide geographical range and thus represent repeated natural experiments to study evolution in action ,7. BeingTurdus merula; [Junco hyemalis; [Myiopsitta monachus; [Urban colonization by birds has occurred on an ecological time scale, dating back more than 150 years  to onlyyemalis;  or the monachus; ). Becausonachus;  and can onachus; .Athene cunicularia) in recently colonized Argentinian cities in comparison to neighbouring rural populations. Burrowing owls, a species typically associated with open grasslands in North and South America, have been reported as breeding birds in South American suburban areas for a few decades [Cynomys sp.) in North America or the plains viscachas (Lagostomus maximus) in South America, whose burrows are used for nesting [Here, we analyse the population structure and demographic history of burrowing owls  what is the population structure of urban and rural populations of burrowing owls? We expected a weak structure given the supposedly recent urban colonization and the high dispersal ability of birds; (ii) were the different cities independently colonized, or were they colonized from a single source population that spread across all cities? (iii) what are the contemporary gene flow patterns among neighbouring populations? (iv) does the demographic history differ between the young urban populations and the rural populations? We discuss the implications of our results in terms of our ability to detect local adaptation and signatures of selection in the burrowing owl genome.2.(a)et al. [a priori seven sampling sites for the three urban–rural comparisons , Bahia Blanca rural , Sierra de la Ventana urban (SVurban) and rural , Tandil urban (TAurban) and rural . From each of these sampling sites, we randomly selected 20 breeding individuals from 2012 to 2016, excluding first-degree relatives, except for SVrural where we only sampled 17 individuals . For the largest and more intensively monitored city (Bahia Blanca) we sampled 20 individuals from each of two spatially separated suburban areas (BB1urban and BB2urban) to test the robustness of demographic inference based on simulations of 20 individuals (see below). Both urban sites were matched with BBrural. Below, we refer to each sample site as a ‘population'. For the reference genome, we used an additional blood sample from an adult male from the rural area around Bahia Blanca. All blood samples were stored in ethanol before analysis in the laboratory.As part of a long-term study, a population of burrowing owls has been individually marked, monitored and blood-sampled since 2006 in and around Bahia Blanca, Argentina ,15. Betwet al. . ‘Urban parisons : Bahia B(b)We extracted DNA from the reference male blood sample using a standard phenol–chloroform protocol. The sample was used to construct a 500 bp insert Illumina Truseq2 paired-end sequencing library, a Nextera gel-free mate-pair library (insert size peak at 2500–3500 bp) and a 10 000 bp Nextera size-selected mate-pair library. DNA sequencing was performed using a 2 × 100 bp sequencing protocol on an Illumina Hiseq2500 system at the Sequencing Core Facility of the MPI for Molecular Genetics in Berlin.After adaptor removal and quality clipping as well as removal of PCR-duplicated read pairs, we assembled the sequencing reads using the IDBA assembler  followedWe annotated protein-coding sequences by alignment of all avian protein sequences of the NCBI protein database  against the burrowing owl genome using SPALN v. 2.1.2 .(c)We extracted DNA from all blood samples from the urban and rural populations with the Blood QuickPure kit (Macherey-Nagel) applying a pre-digestion with Proteinase K in Digsol buffer. All 137 birds were individually sequenced with the Illumina HiSeq 2500 technique using a 200- to 300 bp insert paired-end library with 125 bp read length at the Sequencing Core Facility of the MPI for Molecular Genetics in Berlin.owtie2 [For each individual, we mapped reads against the reference genome using Bowtie2 . We callowtie2 . The sinowtie2 . Additio(d)For each individual, we calculated overall heterozygosity  using vcftools . The samFST values [FST values by randomly permuting individuals between the populations 1000 times and comparing the observed FST value with the simulated FST values. The between-population genetic variance  and its significance were estimated in an analysis of molecular variance (AMOVA) and a distance-based redundancy analysis (dbRDA) framework using the R packages pegas and vegan, respectively [To assess population structure, we calculated T values  between T values ,25. We uectively ,27. For ectively . To avoiWithin each population, we tested for ‘isolation-by-distance' using Mantel tests between identity-by-state values  and geodesic distances  implemeNe) within each population. First, we simulated 200 000 datasets of 20 individuals using the software package PopSizeABC [−9 and a recombination rate of 2 × 10−8. We chose those values as representative for the unknown rates in the burrowing owl because they lie between the genome-wide average values for chicken and passerines [We used an approximate Bayesian computation (ABC) method to estimate ancestral dynamics of effective population sizes  and average genotypic LD values of 20 distance bins ranging from 50 kb to 17 Mb, whereby we excluded SNPs with minor allele frequency less than 0.15. We included long-range LD values to estimate recent population sizes because theory predicts that LD between markers of more than 10 Mb distance on chromosomes with a recombination rate of 2 cM Mbions ago ,36. Secoions ago . These sd et al. , the pre3.(a)Gallus gallus chromosomes or linkage groups. We annotated 17 858 protein-coding gene models in the genome assembly, which were on average 22.6 kb long and comprised 35% of the genome size (including introns). These gene models comprised 157 528 protein-coding exons covering 2.3% of the genome size. The burrowing owl genome browser can be found at http://public-genomes-ngs.molgen.mpg.de and is publicly available.Our assembly of the burrowing owl  comprised 1144 Mb in 34 184 contigs with an N50 length of 64.1 kb. Scaffolding and splitting potential mis-assemblies resulted in scaffold N50 length of about 6.4 Mb . After the final reference-assisted scaffolding step, the scaffolds were placed into 447 superscaffolds with an N50 length of 42.1 Mb. Ninety-nine per cent of the burrowing owl assembly (or 58 of the largest scaffolds) were assigned to t-test: t131 = −0.34, p = 0.73). After quality-control and singleton-filtering and after excluding Z-chromosomal SNPs, the total dataset contained 11 114 442 SNPs (on average 1 SNP per 100 bp).Sequencing coverage of the 137 burrowing owls ranged from 8.7-fold to 42.5-fold (median: 17.0-fold) and did not differ between urban and rural birds ((b)t-test: t3 = −4.96, p = 0.016; t-test: t3 = 0.43, p = 0.70; Overall, there were fewer SNPs in the urban populations than in the nearby rural populations  for all pairwise comparisons . We found the lowest FST values among the rural populations (mean FST = 0.003), intermediate values for the urban-rural pairs (mean FST = 0.014) and the highest values between the urban populations . The genetic variance between urban and rural habitats is small, but significant . The genetic variance between rural populations is similar or somewhat higher , but the highest variance occurs between urban populations .urban and BB2urban, although they show different clustering on the third PC . A few individuals from the rural populations lie further from the centre on the line of their respective urban population , despite the strong difference in geographical scale  between urban and rural populations Ne) over the last 1250 years (500 generations) show clear and consistent differences between urban and rural populations, although the credibility intervals still overlap given the limited number of simulations . Whereas urban population size estimates decreased from approximately 100 000 to approximately 100 over the last 100 years, the rural population size estimates decreased only little or not at all over the same period. We thus combined the accepted ABC simulations of all urban populations and all rural populations and plotted median and credible intervals for the pooled groups . A similar difference in behaviour following urbanization, namely increased sedentariness and reduced migratory behaviour, has also been reported for the European blackbird [Chamaea fasciata; [Melospiza melodia; [Both rural and urban owl populations show signs of genetic isolation-by-distance at the individual level, indicating spatial restriction to gene flow even within the predefined populations. Note, however, that the spatial scale over which this isolation occurs differs: the strength of isolation-by-distance , thes major; ) and thevanicus; ). In ourlackbird ,45. Noteasciata; ) and themelodia; ).et al. [At the between-population level, our results suggest that the genetically different urban populations of burrowing owls evolved independently under random genetic drift processes following limited founder populations and limited subsequent immigration. If so, the effective population size of urban populations should decrease from the moment of colonization onwards. We showed that the ABC approach proposed by Boitard et al.  using LDet al. , are limet al. ,49. The et al. [Martinez et al.  hypotheset al. ,51. HoweOur data are consistent with the general pattern that urban populations are smaller and more genetically unique (owing to isolation with random genetic drift) than rural ones . Genetic


Pulsatilla patens populations revealed by neutral SSR markers and putatively adaptive ISJ markers (intron-exon splice junction).Research into the protection of rare and endangered plant species involves genetic analyses to determine their genetic variation and genetic structure. Various categories of genetic markers are used for this purpose. Microsatellites, also known as simple sequence repeats (SSR), are the most popular category of markers in population genetics research. In most cases, microsatellites account for a large part of the noncoding DNA and exert a neutral effect on the genome. Neutrality is a desirable feature in evaluations of genetic differences between populations, but it does not support analyses of a population’s ability to adapt to a given environment or its evolutionary potential. Despite the numerous advantages of microsatellites, non-neutral markers may supply important information in conservation genetics research. They are used to evaluate adaptation to specific environmental conditions and a population’s adaptive potential. The aim of this study was to compare the level of genetic variation in P. patens and three P. patens populations from the nearby region of Vitebsk in Belarus. A total of 345 individuals were examined. Analyses were performed with the use of eight SSR primers specific to P. patens and three ISJ primers.The experiment was conducted on 14 Polish populations of eH = 0.609, eH = 0.145, respectively). An analysis of molecular variance (AMOVA) revealed that, the overall genetic diversity between the analyzed populations defined by parameters STF and ΦPT for SSR (20%) and ΦPT for ISJ (21%) markers was similar. Analysis conducted in the Structure program divided analyzed populations into two groups (SSR loci) and three groups (ISJ markers). Mantel test revealed correlations between the geographic distance and genetic diversity of Polish populations of P. patens for ISJ markers, but not for SSR markers.SSR markers revealed a higher level of genetic variation than ISJ markers (The results of the present study suggest that ISJ markers can complement the analyses based on SSRs. However, neutral and adaptive markers should not be alternatively applied. Neutral microsatellite markers cannot depict the full range of genetic variation in a population because they do not enable to analyze functional variation. Although ISJ markers are less polymorphic, they can contribute to the reliability of analyses based on SSRs. According to recent estimates, endangered plant species account for 6% of globally identified flora . For thrH, fixation index isF, pairwise STF). They are used to describe past and present evolutionary processes in a given population . Molecular markers are also used to evaluate historical and geographic similarities between groups , are the most popular category of markers in population genetics research . MicrosaMicrosatellites are found in transcribed regions, including in protein coding genes and expressed sequence tags (EST) . They maNyholmiella obtusifolia , revealed that over 73% of them contain partial exonic and intronic regions . The ISJ amplified bands are treated as single dominant loci and scored either present or absent.An interesting alternative to the DNA markers discussed above are semi-specific Intron-exon splice junction (ISJ) markers, based on sequences commonly found in plants and indispensable for post-transcription DNA processing . ISJ marTherefore, ISJ markers can be used to investigate adaptation processes and evolution of adaptive traits. Adaptive markers undergo selection and are used mostly in analyses of evolutionary potential, including local adaptations and speciation . AdaptivPolygonatum Mill. (Asparagaceae) (Aneura Dumort. (Aneuraceae) (Chamaedaphne Moench (Ericaceae) (Oryza L. (Poaceae) (Sphagnum L. (Sphagnaceae)  , Aneura uraceae) , Chamaedicaceae) , Oryza LPoaceae)  and Sphagnaceae) .Despite numerous advantages of microsatellites, non-neutral markers may supply important information in conservation genetics research. They are used to evaluate adaptation to specific environmental conditions and a population’s adaptive potential, which is particularly valuable in studies of endangered species. Selected loci are likely to group populations based on adaptively relevant ecological variables, whereas neutral loci are likely to group populations phylogenetically based on geographic proximity ; therefoP. patens populations revealed by ISJ and SSR markers; (b) detect of presence of outlier loci in the data sets; (c) examine the influence of outlier loci on the genetic structure.The aim of this study was to: (a) compare the values of genetic diversity parameters and genetic structure of In particular, we hypothesized that the ISJ markers will be less polymorphic than SSR markers. We expect that the grouping populations based on ISJ markers will be different from groupings based on SSR markers.Pulsatilla patens populations colonizing the west edge of its geographic range. Eastern pasqueflower (Pulsatilla patens (L.) Mill.) is a long-lived monoecious perennial herb classified as a hemicryptophyte , 1 μL BSA, 1.0 μM of each primer and 1 U RedTaq polymerase . Reactions were performed under the following thermal conditions: (1) initial denaturation—4 min at a temperature of 94 °C; (2) denaturation—45 s at 94 °C; (3) annealing—50 s at 52 °C for Pul05, Pul06, Pul07 primers, at 55 °C for Pul02 primer and at 58 °C for Pul01, Pul04, Pul10, Pul11 primers; (4) elongation–90 s at 72 °C; (5) final elongation—7 min at 72 °C. Stages 2–4 were repeated 40 times.SSR analyses were performed with the use of eight primers specific to . patens  and desc2 and 1 U Taq polymerase (OpenExome). Thermal conditions were different in denaturation (60 s at 94 °C) and annealing (90 s at 49 °C) stages.ISJ analyses were performed using three primers . The reaHaeIII DNA size marker (Qiagen) for SSR markers, or the 15–3,000 bp alignment marker (Qiagen) and the 100–2,500 bp DNA size marker (Qiagen) for ISJ markers. Standard OM500 settings were used as the electrophoresis program.PCR products for ISJ and SSR markers were separated in the QIAxcel capillary electrophoresis system, using the Qiaxcel High Resolution Kit; with the 15–500 bp alignment marker (Qiagen) and pUC18/The size of the obtained SSR and ISJ bands were determined by using BioCalculator softwere (Qiagen).Allele frequency in each amplified ISJ locus was identified in view of band presence or absence. It was assumed that every observed band resulted from the amplification of a single locus, therefore, the number of observed bands corresponded to the number of investigated loci. Only allele “1” (band present) or allele “0” (band absent) were observed in every locus. The presence of null alleles was checked using the MICRO-CHECKER 2.2.3 program . For eacA), the number of effective alleles (eA), observed heterozygosity (oH), expected heterozygosity (eH), fixation index isF, the number of loci (N), the number of polymorphic loci (n) and the percentage of polymorphic loci (P), were generated by use of GenAlEx v. 6.501 software  was performed to test correlation between measured parameters and the populations size. Tests of deviation from Hardy–Weinberg proportions at each SSR locus Hardy–Weinberg equilibrium (HWE) and of linkage disequilibrium between pairs of loci were tested in FSTAT v.2.9.3  was conducted to assess the partitioning of the genetic variance within and among populations. AMOVA was performed using STF and ΦPT values for SSR markers and ΦPT values for ISJ markers. The possible correlations between genetic and geographic distance (isolation by distance) (STF/(1 − STF) with geographic distance (km) in a Mantel test   were esttel test . Coordin v.6.501 .STF distance matrix (for SSR) and ΦPT distance matrix (for ISJ) was carried out in GenAIEx 6.501 software  based on the pairwise software . The anaStructure v. 2.3.4 software . Three ISJ markers revealed altogether 75 loci  and eH = 0.145 when estimated with ISJ markers    and eH was highest in populations PA  and PO .The average expected heterozygosity reached ll loci) . A detai= 0.763) . For ISJeH calculated for outlier ISJ loci was twice as high compared to the results for all loci  and ΦPT for ISJ (21%) markers was similar  revealed differences in their extreme values for both categories of applied markers  and BB (Biebrza)–NSz (Szczytno) (STR = 0.024). The highest level of genetic differentiation were noted between: populations NP (Parciaki)–BL (Białowieża) STF = 0.475), and populations GW (Gostynin-Włocławek)–BL (Białowieża) (STR = 0.901), and populations GW (Gostynin-Włocławek)–NS (Strzałowo) (STR = 0.901).An analysis of genetic diversity parameters ( markers  and S6. PT) was noted between populations KO (Kolimagi)–NM (Myszyniec) .For ISJ markers the highest level of similarity . The presence of such correlations was noted for ISJ markers based on parameter ΦPT .The above differences were validated by the results of the Mantel test which did not reveal correlations between the geographic distance and genetic diversity of Polish populations of Both categories of markers revealed together 14 specific alleles in analyzed populations in each case. SSR markers amplified eight specific alleles and ISJ markers–six specific allels . This alStructure program revealed a different number of clusters for studied populations  the analysis conducted in the ulations  and S7. i K = 4;  and neuti K = 4; .Pulsatilla patens populations as inferred by the Bayesian approach. Two main groups of populations were found only for analysis conducted for all SSR loci and neutral SSR loci  Delile. The cited authors relied on SSR and EST-SSR markers and found that SSR revealed a higher number of alleles per locus (A = 2.077–4.044) and higher expected heterozygosity (eH = 0.293–0.485) than EST-SSR . eH = 0.675–0.718, oH = 0.644–0.736) than for adaptive EST-SSR markers . Microsatellites revealed higher heterozygosity than ISJ markers , but theIt is worth to mention that distinctive differences in genetic variation of the studied populations were noted between the applied markers. The populations, where the analyzed parameters reached the highest and lowest values did not correspond for both categories of markers. This could suggest that the ISJ markers used in this study were not selectively neutral. The observed differences could be attributed to the fact that neutral markers are affected by genetic drift, whereas adaptive markers by selection. In the studied populations, the balance between selection and genetic drift is probably disrupted, as demonstrated by the differences in the values of genetic variation parameters.At present, the exclusive use of neutral markers in evaluations of genetic variation is often questioned , in partP. patens. The distribution of genetic variation was generally similar for both categories of used markers. The absence of distinctive differences between the analyzed populations can be attributed to the fact that until recently, Eastern pasqueflower had continuous geographic range in Eastern and Central Europe  were noted in different population pairs . A higher level of genetic diversity, estimated based on putatively non-neutral ISJ markers (ΦPT = 0.209), is consistent with the results obtained by STF = 0.068 for SSR, STF = 0.016 for EST-SSR). Nevertheless, when two SSRs suspected of undergoing directional selection were eliminated, the value of STF (SSR STF = 0.011) dropped below that noted for EST-SSR loci.Despite the fact that genetic diversity parameters revealed highly extreme values between the studied populations, the average values of those parameters were moderate . It could also point to differences in the evolution of the analyzed genome fragments. According to Structure program is not suitable for grouping populations based on adaptive loci. According to the cited authors, the algorithm implemented in the program assumes Hardy–Weinberg proportions and linkage equilibrium which are not appropriate for loci under selection  or three groups (ISJ loci) which, however, differed in the populations that created them  and 4D. requency  and 3D. requency ; Fig. 3.ISJ loci  and 4D aISJ loci  and 4F.  results . A singlelection .P. patens are undergoing isolation by distance. The Mantel test based on SSR markers did not reveal correlations between the geographic distance and genetic diversity of Polish populations of P. patens. A statistically significant correlation was noted for ISJ markers. Despite the above, the estimated value of the test was low (R2 = 0.144), which indicates that geographic distance was weakly correlated with genetic diversity. The Mantel test produced different results for SSRs and putatively adaptive ISJ markers, which confirms that the diversification process has only just begun in P. patens populations and is probably associated with the ongoing isolation of localities.The results of this study do not unambiguously support the conclusion that Polish populations of P. patens populations  and functional genomic  markers and observed that neutral markers amplified more specific alleles (78) than functional markers (63).As mentioned before, genetic data that undergo selection reveal population diversity more quickly than neutral data . Despiteulations . HoweverMicrosatellites have an advantage over ISJ markers in that they support evaluations of inbreeding. Inbreeding decreases the level of genetic variation . InbreedIdentification of outlier loci is one of a key steps in understanding the evolutionary process, because those loci are responsible for genetic variants that affect fitness in different environment . OutlierBayeScan program confirmed that both of the analyzed markers were subject to selection. Four of the eight evaluated SSR loci were outlier loci  based on all studied SSR loci; (B) based on neutral SSR loci; (C) based on outlier SSR loci; (D) based on all studied ISJ loci; (E) based on neutral ISJ loci; (F) based on outlier ISJ loci.Click here for additional data file.10.7717/peerj.2504/supp-8Supplemental Information 8Populations with Q < 0.75 for each clusters are not included.Click here for additional data file.10.7717/peerj.2504/supp-9Supplemental Information 9Click here for additional data file.


Pachyptila desolata) populations are separated by over 5000 km, whereas those of the blue petrels  and thin-billed prions (P. belcheri) show considerable overlap. Therefore, we expected the breeding populations of blue petrels and thin-billed prions to show high connectivity despite their geographical distance, and those of Antarctic prions to be genetically differentiated.In seabirds, the extent of population genetic and phylogeographic structure varies extensively among species. Genetic structure is lacking in some species, but present in others despite the absence of obvious physical barriers (landmarks), suggesting that other mechanisms restrict gene flow. It has been proposed that the extent of genetic structure in seabirds is best explained by relative overlap in non-breeding distributions of birds from different populations. We used results from the analysis of microsatellite DNA variation and geolocation (tracking) data to test this hypothesis. We studied three small (130–200 g), very abundant, zooplanktivorous petrels , each sampled at two breeding populations that were widely separated (Atlantic and Indian Ocean sectors of the Southern Ocean) but differed in the degree of overlap in non-breeding distributions; the wintering areas of the two Antarctic prion  in these pelagic seabirds.Microsatellite (at 18 loci) and cytochrome In line with other Southern Ocean taxa, geographic distance did not lead to genetic differences between widely spaced populations of Southern Ocean petrel species.The online version of this article (doi:10.1186/s12862-017-1008-x) contains supplementary material, which is available to authorized users. Pachyptila belcheri on New Island, Falkland Islands). Thus, genetic methods are required.The Southern Ocean  is characterized by an environmental gradient from subantarctic to polar waters, and the fauna reflect adaptations to the thermal and latitudinal structure in this region e.g. , 2). Sea. Sea2]).Pygoscelis adeliae, [Thalassarche chrysostoma, [Diomedea exulans, [T. melanophris displays genetic structure despite the absence of physical barriers, potentially because of its stronger association with continental shelf habitats; this conclusion was based on the comparison with the closely-related grey-headed albatross, which forages mainly at frontal systems which are circumpolar [Several studies that have analysed patterns of genetic diversity in seabird species with circumpolar distributions in the Southern Ocean, have found little genetic and phylogeographic structure despite the large distances between colonies . In concumpolar . Similarcumpolar ; skuas [cumpolar ). Alterncumpolar . To explcumpolar  suggestePachyptila belcheri), and other Pachyptila species . Here we use this newly developed marker set, together with tracking (geolocation) data to test the hypothesis that genetic structure is dependent on the non-breeding distribution pattern in thin-billed prions, Antarctic prions Pachyptila desolata and the closely-related blue petrel Halobaena caerulea. These small petrels have wide breeding distributions, including islands in different ocean basins, and are wide-ranging from subtropical to Antarctic waters [Due to their high mutation rates, microsatellite loci (i.e. regions across the genome where short nucleotide sequences are repeated in tandem) provide powerful tools in population genetics. They allow the testing of hypotheses related to within- and between-population genetic variability and the estimation of other demographic parameters such as gene flow and effective population size , 14. Recc waters , 16–19, Blue petrels, thin-billed prions and Antarctic prions breed on islands in the Southern Ocean, and in each species, we sampled two large breeding populations, one in the south-west Atlantic Ocean  and the other in the Indian Ocean (Kerguelen Islands) . The thrN = 34) and New Island, Falkland/Malvinas Islands ; Antarctic prions at Île Verte, Kerguelen  and Bird Island, South Georgia ; and blue petrels at Île Mayès (N = 30) and Bird Island (N = 19). Blood (0.2–0.4 ml) was sampled by puncture of the wing vein and collection using heparinized capillaries, or using 0.3 ml syringes, and immediately transferred to FTA classic cards . These breeding colonies represent major breeding sites of all three species, with estimates at each site of several million birds for prions, and >100,000 individuals for the blue petrel [During 2010–2012, blood samples were obtained from adult birds breeding in colonies of thin-billed prions located at Île Mayès, Kerguelen . We analysed distribution data from one non-breeding season per bird. Processing of geolocator data was carried out as described previously [Data on non-breeding distributions were obtained using 1 g leg-mounted geolocators, or Global Location Sensors , as described previously , 18, 27.eviously . The noneviously . Kernel eviously . The oveFIS) and its significance were calculated for each population using GENEPOP Genetic structure was analysed using population-model based, and non-model based approaches. We used ARLEQUIN 3.5  to perfob (cyt b) gene was amplified using specific primers (CytB_Pri_F: 5′ -CTAGCTATACACTACACCGC-3′ and CytB_Pri_R: 5′ -CTAGTTGGCCGATGATGATG-3′) [b). Sequences were assembled and aligned in CLC Main Workbench 6.9.2 .An 889-bp fragment of the cytochrome GATG-3′) . PCRs wePachyptila turtur sample was included as an outgroup. The best-fit model of nucleotide substitution for each of our sequence alignment was determined using Bayesian Information Criterion scores and corrected Akaike Information Criterion values in MEGA 6.06 [b trees were inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model [FST) following Weir & Cockerham [FST values were tested with 1000 permutations.A Fairy prion EGA 6.06 . The cytno model  in MEGA,no model . Geneticockerham  in ARLEQockerham . The sig2) of the overall distribution  used by individuals of both populations. The ranges of the tracked adult thin-billed prions from Kerguelen and Falkland/Malvinas overlapped from February to August  of the overall distribution  used by individuals of both populations  based on 25 polymorphic microsatellites suggested a deviation from Hardy-Weinberg equilibrium due to an excess of homozygotes in all six populations. To test if this was due to non-random mating , or theFIS) was higher in the Atlantic Ocean populations for all three species .Bayesian clustering methods did not identify population genetic structure for any of the tested species. For all three species, the Evanno method estimated the highest likelihood LnPr(X|K) for K = 2. STRUCTURE also revealed homogenous distributions of individual genotypes, where all individuals were assigned to approximately equal proportions of the inferred clusters Fig. . The graFST = 0.008, P < 0.001), than those of blue petrels , and thin-billed prions .In addition, classical estimators of population differentiation were very low between Atlantic and Indian Ocean populations for all species. AMOVA results indicated a higher population differentiation between the populations of Antarctic prions  values in blue petrels, and small but significant between-population values in Antarctic prions. However, the more structured cyt b sequences between thin-billed prion populations were manifested as larger and significant FST values using AMOVA  in thin-billed prions, to more pronounced overlap but for a shorter time  in blue petrels. Based on this and on the hypothesis of Friesen et al. , we woulIn contrast, high levels of gene flow in all three species meant that these differences in relative segregation of non-breeding ranges were barely detectable through standard Bayesian and multivariate analyses of population genetic structure. Only model-free analyses of hierarchical genetic structure through AMOVA indicated low, but significant levels of population differentiation in both nuclear and mitochondrial marker sets between Atlantic and Indian Ocean populations of Antarctic prions, in line with expectations. In the blue petrel, which showed the highest spatial overlap between populations in the non-breeding season, there was very little between-population differentiation at both molecular levels, which was also in line with our hypotheses.Diomedea spp. [Eudyptes moseleyi and E. chrysocome [The lack of population genetic structure of the species studied here is similar to results observed in other Southern Ocean seabirds that feed in pelagic waters e.g. , 9. The dea spp. ; northerrysocome ).Within our three study species, we found considerable admixture and evidence for high migration rates Table . The lacFS for thin-billed and Antarctic prions, as well as Tajima’s D for thin-billed prions at the Falklands and Antarctic prions at Kerguelen (Table b networks in the prions and blue petrels are compatible with a restriction to glacial refugia and subsequent population expansions when the species recolonized other subantarctic islands. A similar scenario has been postulated for other Southern Ocean seabirds, including king penguins Aptenodytes patagonicus [Aptenodytes forsteri [The mitochondrial data in the present study also allow an analysis of population demographic history. Negative Fu’s en Table , and theen Table . Naturalagonicus , Emperorforsteri  and pygoforsteri .In summary, our study highlights the low genetic structure of three abundant pelagic seabirds which show varying degrees of spatio-temporal segregation at sea between different breeding populations. In line with several other Southern Ocean seabirds, geographic distance between breeding colonies in these petrels does not generate genetic differences, regardless of the degree of overlap in non-breeding distributions. Thus, our study did not support the hypothesis of Friesen et al.  who sugg