Gasterosteus aculeatus. This teleost is a protected species in Portugal and Spain and local extinctions have been reported in both countries during the last decades. Our objectives were (i) to determine whether the Iberian populations of G. aculeatus are unique or composed of any of the major evolutionary lineages previously identified and (ii) to assess the evolutionary potential of these peripheral populations. We genotyped 478 individuals from 17 sites at 10 polymorphic microsatellite loci to evaluate the genetic variability and differentiation of the Ibero-Balearic populations. We also sequenced 1,165 bp of the mitochondrial genome in 331 of those individuals in order to complement the estimates of genetic diversity in the Ibero-Balearic region. We predicted the evolutionary potential of the different sites analysed based on the contribution of each of them to total allelic/mitochondrial diversity. An intraspecific phylogeny at European level was reconstructed using our data from the mitochondrial cytochrome b gene (755 bp) and published sequences. The so-called Transatlantic, European and Mediterranean mitochondrial lineages were found to be present in the Ibero-Balearic region. Their phylogeography suggests a history of multiple colonisations. The nuclear results show, however, a strong correlation between population structure and drainage system. The following basins should be prioritised by conservation policies in order to preserve those populations with the highest evolutionary potential: the Portuguese Vouga and Tagus as well as the Spanish Majorca and Limia. Maintenance of their connectivity, control of exotic species and monitoring of habitat properties are strongly recommended in those areas. Genetic variation alone cannot, however, ensure the persistence of these peripheral southern populations of G. aculeatus. On the one hand, the analysis of a historical sample from Eastern Spain (Penyscola) revealed no genetic erosion, which suggests a fairly sudden extinction of that population. On the other hand, the reintroduction program implemented in the Valencian Community has mostly failed despite our finding of similar level of genetic diversity between the wild source and the captive-bred released individuals.Genetic isolation and drift may imperil peripheral populations of wide-ranging species more than central ones. Therefore, information about species genetic variability and population structure is invaluable for conservation managers. The Iberian populations of three-spined stickleback lie at the southwestern periphery of the European distribution of  The combined effects of isolation and drift may cause peripheral populations of wide-ranging species to be more imperilled than central ones , 2. ThisGasterosteus aculeatus Linnaeus, 1758), a mainly circumboreal-north-temperate fish, widely known as model species in evolutionary biology [The decline experienced by peripheral populations is well exemplified by the three-spined stickleback . For. ForG. a in Spain, and, in application of an analogous transposition of the European Directive, by the Instituto da Conservação da Natureza  and Direcção Geral dos Recursos Florestais  in Portugal. Samples provided by other researchers  were collected following procedures reviewed by their institutions and approved by the competent authorities .Ethics approval of all procedures involving vertebrate animals is legally required under the Spanish legislation , which is a transposition of the European Directive 86/609/EEC. In agreement with article 18 and annexes VII and XI of the aforementioned Royal Decree, all animal procedures performed as part of the experimental work described in this paper have received prior and explicit approval from the competent authorities, defined in article 3e of the Law, and substantiated in the corresponding regulations of the Spanish autonomous communities. Thus, permissions for fieldwork and the concomitant experimental procedures  were issued by the Xunta de Galicia (permit #35/2007), Diputación Foral de Bizkaia (reference number 9621) and Parc Natural de S’Albufera . For comparison purposes, we aimed at using the same set of markers as Mäkinen et al.  and Mäkistn122, (replaced by stn82 in the present work), which failed to amplify for individuals from Guisande (S4) and Rato (S5) during preliminary tests. Microsatellites were amplified as specified in Pérez-Figueroa et al. [We initially genotyped the 478 sampled individuals  using the same 18 nuclear microsatellites used by Mäkinen et al. , except a et al. . PCR procytb) and 409 bp from the control region (cr). PCR amplifications for cytb were performed in a final volume of 20 μL containing 100ng DNA template, 1 X Buffer, 2.5mM MgCl2, 0.2 mM dNTPs, 0.5 μM each primer, 1 U GoTaq Flexi DNA Polymerase (Promega). Thermocycler profile was as follows: initial denaturation ; 35 cycles of denaturation , annealing  and extension ; final extension . PCR amplifications for the cr were similar to those of cytb. The only difference was the annealing temperature: 55°C.We sequenced a total of 1,165 bp of mtDNA in 17–22 individuals chosen at random within each sampling locality. We used the primers described by Mäkinen & Merilä  to obtaicr fragment.PCR products were electrophoresed on 2% agarose gels and visualised under UV light after ethidium bromide staining. Products were purified with MultiScreen kit (Millipore) and used as template for direct sequencing on an ABI Prism 3730xl. DNA sequences were inspected and aligned using SEQSCAPE 2.5 (ABI). Alignments were straightforward, as only a mononucleotide indel was found in the stn38 and stn46) and 54 (locus 1125pbbe) [stn3, stn12 and stn174 to show intermediate alleles (1 bp difference). Therefore, these three loci were excluded from further analyses. The frequency of null alleles was then calculated using Oosterhout’s estimator as implemented in MICRO-CHECKER 2.2.3 [pbbe [stn19, stn21, stn38, stn46, stn57, stn79, stn110, stn163 and stn195 [The number of alleles per locus ranged between six (loci 125pbbe) . Basic dER 2.2.3 . Eight mER 2.2.3 : 1125pbb.3 [pbbe , stn19, d stn195 .Number of alleles observed and averaged over loci, observed and unbiased expected heterozygosities, as well as analyses of Hardy-Weinberg (HW) segregations were computed with GENODIVE 2.0b25 . Alleliccytb and cr fragments, as well as and their frequencies and other standard indices of genetic variation such as the number of segregating sites (S), nucleotide diversity per gene (π), haplotype diversity (Hd) and the average number of nucleotide differences (k) were calculated in DNAsp 5.1 [Haplotypes defined by the NAsp 5.1 . New hapFST/(1- FST)) and geographical distances (raw and log10 transformed). We calculated the matrix of shortest pairwise water distances among sampling sites using the Ruler Path option in Google Earth. For comparative purposes, linear distances were calculated for each pair of sampling locations with Geographic Distance Matrix Generator 1.2.3 [As isolation-by-distance (IBD) scenarios are not suitable for model-based Bayesian clustering of genotypes, we tested the correlation between genetic  ranging from one to 18. We preliminary determined the number of clusters by applying both the methods of Evanno [K-value. Then, we ran DISTRUCT [After ruling out IBD, we used STRUCTURE 2.3.4  and BAPSf Evanno  and Pritf Evanno  as implef Evanno . We usedf Evanno  to permuDISTRUCT  to visuaDISTRUCT , we discK of clusters. Then, the best partition of data into K clusters is identified as the one with the highest marginal log-likelihood. We performed ten independent simulations for each value of K from 1 to 18. Lastly, we performed an admixture analysis based on mixture clustering .With regard to BAPS, we firstly applied a non-spatial genetic mixture analysis  clusteriθ unbiased estimator of FST [D [In the light of the results provided by the clustering algorithms, pairwise differentiation among sampling localities was calculated using the nuclear dataset. Firstly, we calculated the values of the IAM-based r of FST  and theif FST [D  across lcytb and cr was inferred calculating a 95% statistical parsimony network using TCS 1.2 [The phylogeny of the mitochondrial haplotypes resulting from concatenation of  TCS 1.2 . The rescytb+cr concatenated dataset (preliminary Bayesian and Maximum Likelihood trees not shown) and the fact that Malhi et al. [cytb on the Scottish three-spined sticklebacks led us to the use of the cytb for phylogenetic analyses at European level. For this, we gathered a 755 bp cytb dataset of 172 haplotypes by aligning and collapsing our Iberian cytb sequences and the homologous fragments reported in prior literature [G. aculeatus (Accession number AB094627). Due to the difficulty of representing in such a network the actual sample size of some haplotypes, readers are referred to Both the lack of phylogenetic resolution as from the i et al.  only pubterature , 49–54  to produce a single  pool of maximal gene diversity. For this, we set λ = 1 , 1000 individuals and 2000 steps to perform the simulated annealing algorithm. The rationale of this calculation is that maximisation of genetic diversity is equivalent to maximisation of effective population size. In addition, maximising genetic diversity is expected to lead to maximum allelic richness in the long-term [The evolutionary potential of different populations can be predicted from the contribution of each of them to the overall species level allelic diversity , 56. Sucong-term .stn57 to 0.8% at 1125pbbe). We scored a total number of 251 alleles. Each individual resulted in a different multilocus genotype, except for two individuals from S13. These two specimens differed, however, in their genotypes for excluded loci 7033pbbe, stn174 and stn132. The total number of alleles per locus ranged between six (stn46) and 54 (1125pbbe)  . At popu125pbbe) .stn21xstn195 at S16 (Penyscola) and stn19xstn163 at S11 (Tagus), adjusted P-value for 5% nominal level = 0.0014 and 0.0011, respectively) corresponded to different linkage groups according to [We observed two significant deviations from gametic equilibrium at locality level. The two loci involved in each case .No significant deviations from Hardy-Weinberg (HW) equilibrium were detected at the population level, after correcting for multiple tests (adjusted cytb + cr) data matrix contained 331 sequences and defined 48 haplotypes  deposited in FigShare [Hd = 0.953 ± 0.004 and average number of nucleotide differences k = 6.775 ± 3.201. The most diverse localities were the Portuguese S9 and S10 , whereas only one haplotype was found in the following localities: the three Basque sites (S1-S3), S8  and the German S17 (Günz)  .cytb sequences, whereas eight others resulted monomorphic  . Twenty-one substitutions were parsimony informative and most changes (82.14%) corresponded to third codon positions and implied no amino acid replacement. Three Northwestern Spanish localities  were the most diverse as from 17 Günz) .The haplotype we found at the translocated site of Valencia (S14) did not match the one reported at river Orlina , the soucr were the northern Portuguese S9 Mondego and S10 Vouga, followed by S5 Rato and S6 Asma, both at Miño river basin. Again, several localities resulted monomorphic for this marker  . The most diverse localities as from 17 Günz) .p > 0.05) regardless of using Mantel’s or Spearman’s r statistics and raw or log10 transformed geographic distances. Therefore, Bayesian clustering methods were justified. The surveyed localities showed a high degree of nuclear structuring. Actually, there were different levels of organisation present in the genetic structure. Firstly, all but two sampling sites  were differentiated according to the partition of data (K = 17) supported by the highest Pr(K = k) in STRUCTURE  and therefore difficult to notice (sea green colour) at K = 12 clustering pattern was highlighted as relevant by both Evanno’s method and the plateau of L(K)  , S2 Fig.ectively . In addiectively .p < 0.01) as from the analysis based on clustering of 17 groups: Sad9 , Vou06 , Val15  and Gui111 .BAPS also identified 17 distinct clusters regardless of grouping sampling sites or individuals. There were nine perfect matches between sampling locality and cluster . Four inθ values were significantly greater than zero for all pairwise comparisons among sampling localities. No pairwise comparison resulted in a negative D, as it would be expected for undifferentiated populations.Pairwise differentiation was minimal for the following intrabasin comparisons: Asma-Rato (Miño basin) and Antela-Salas (Limia basin), Castaños-Gobelas (Nervión basin), as well as between the Mediterranean sites of Penyscola and Valencia . The θ vθ values if compared to the Basque Txingudi (S1) or the Galician Guisande (S4), whereas it was most differentiated from Mondego (S9) as from D. The differentiation between Tagus (S11) and Majorca (S15) was also one of the five largest ones revealed by D (The Portuguese river Mira (S13) was very much differentiated, especially if compared to Guisande  or the Basque Castaños (S2). The Portuguese Sado (S12) also showed large led by D .cr) and the cytochrome b (cytb) showed a higher degree of reticulation for the former (cr yielded more haplotypes than the cytb in certain areas (e.g. rivers Tagus (S11) and Mira (S13)). This higher variability resulted in differentiation of Txingudi (S1) from the other two Basque sites, or Majorca (S15) from the other two Mediterranenan localities. The number of haplotypes found in the Portuguese Mondego (S9) and Vouga (S10) was the same for both markers. However, the variability of the cytb was higher in all the Northwestern Spanish samples but S4 and S8 (which showed the same number of variants regardless of the marker). Namely, Antela (S7) harboured seven haplotypes as from cytb, but just two as revealed by cr.The phylogenetic networks obtained for the control region  both frlineages  were prelack Sea , AdriatiAdriatic , 60 and lineages  were abscytb haplotypes into five categories according to their geographic distribution: widespread, disjunct, regional, local and private  private haplotypes. Most of them were terminal variants . Five ofThe loss of allelic diversity was more severe when Majorca (S15), Vouga (S10) and Antela (S7) were removed from the pool of samples (3–4% each). The same result was obtained when analysing the extant Ibero-Balearic sites (S1-S15). The mitochondrial results also revealed that the loss of mitochondrial diversity would be highest without these three localities (6–10%) .cGDpool), regardless of analysing the whole dataset or the 15 Ibero-Balearic extant sites (Tagus (S11) was the sample contributing the most (23%) to a pool with maximal nuclear genetic diversity  and relatively mild conditions occurred during the Last Glacial period (119–8 kya) [G. aculeatus to reach the Western Iberian Margin in several occasions. Indeed, dramatic hydrological changes took place along the Western Iberian Margin during the last 61 kya [River Tagus may be bearing the genetic footprint of two different migration events. On the one hand, the “H31-colonisation” of the Mediterranean, as its most abundant haplotype (the highly divergent and private H84) is phylogenetically closer to H31 than to H4, also present at Tagus but in low frequency . On the 9–8 kya) . Such flt 61 kya , 64. Fort 61 kya . Bearingt 61 kya , the arrThe star-like phylogeny centred in the disjunct haplotype H19 indicated a population expansion in Northwestern Spain , as eighG. aculeatus [G. aculeatus was only eastwards or if any of the haplotypes present in Europe, e.g. H4, dispersed into North America at some point. The phylogeography of the nine-spined stickleback Pungitius pungitius does not support such a westward dispersal [The star-like phylogeny around the disjunct H4 haplotype  indicates transoceanic dispersal. Its derived H29 was private to the American locality of Maine, whereas haplotype H7 was only found in Scotland and the H127-H129 variants were endemic to two geographically close sites in Southwestern Ireland. Transoceanic dispersal has been previously suggested for culeatus , 71. Theculeatus . The queispersal , but furispersal .The three Basque sites shared haplotype H35 with the Western French sites of Borgneuf and Oleron , in agreG. aculeatus in Portugal and Spain are common to distant areas such as North America: introduced species and human-derived changes on water quantity and quality [Gambusia holbrooki, Lepomis gibbosus, Micropterus salmoides) and crayfish (Procambarus clarkii) [G. aculeatus at low latitudes. The three-spined stickleback is likely displacing northwards because of global warming [The threats reported for  quality , 11, 76.clarkii) , 77. Les warming . Thus, p warming , particu warming .G. aculeatus on whose habitats conservation efforts should be focused, as their loss would dramatically impact on the overall allelic and mitochondrial diversity. Prioritisation of highly diverse and effectively large populations  is a better approach to maintain the evolutionary potential of a species. This is because allelic-diversity measures are more correlated with long-term and total response to selection, an observation valid to unlinked neutral markers [Majorca (S15), Vouga (S10) and Antela (S7) were unambiguously indicated by both the nuclear allelic and mitochondrial diversity analyses as the Ibero-Balearic populations of  markers , 56. The markers  in our aG. aculeatus from Majorca contributed the most to the overall nuclear and mitochondrial diversity of this species in the Ibero-Balearic region. This result reinforces the interest in preserving this isolated peripheral population from the loss of genetic diversity due to drift. This insular population was recently postulated as an Evolutionarily Significant Unit (ESU) [We found that it (ESU) : not onlit (ESU) .G. aculeatus and in these two basins is also needed to evaluate their effective population size. River Vouga was free of dams until 2015 when the Ribeiradio—Ermida hydroelectric project  was fully operative. These two dams are in the middle reach of river Vouga, where G. aculeatus is likely absent from. However, such physical barriers are known to cause hydrological alterations  leading to severe changes in river function  [M. salmoides, L. gibbosus, G. holbrooki and P. clarkii are present in the Vouga and Mondego river basins [G. aculeatus was inferred in California between rivers Ventura and Santa Clara (whose mouths are 10 km apart) either because of drainage conjunction during a Holocene lowering of the sea level or because of contemporary and occasional gene flow via ocean dispersal during winter storms [Vouga (S10) and Antela (S7) were the most interesting Iberian sites in terms of prioritising conservation efforts. The loss of both of them would diminish nuclear and mitochondrial diversity in the Ibero-Balearic region about 7 and 20%, respectively. Therefore, fine scale monitoring of uations)  that migr basins , 83. Ourr basins  and ocear basins . Marine r storms . The mour storms .G. aculeatus from Antela (S7) occur in a habitat that suffered drastic human-mediated changes. Not only were two dams built downstream of Antela (see below), but also the original lake was desiccated, artificial channels were constructed, water quality was impaired by pollution and the exotic L. gibbosus and P. clarkii occur in that area. The permanent water body of the Antela Lake is now reduced to a 26 km x 17 m channel, inhabited by the three-spined stickleback, although seasonal floods occur in the surroundings [Ne) of Antela to be intermediate  between the ones obtained for the upper reach of Miño and two of its tributaries (Rato and Guisande) [personal observation). The lack of gene flow between Antela and Salas since 1949  likely contributed to the differentiation obtained between these two sites: their differentiation was lower than the Basque Castaños-Gobelas intrabasin comparison, but similar to the one between Rato and Asma, within the Miño river basin  . A similuisande) . The threr basin . In addiGDpool) was the highest  has come to an end because of the abundance of exotic species. However, the reintroductions at River Bullent  pose a more promising scenario, i.e. a lower impact of exotic species and a better status of other threatened species as Valencia hispanica and Salaria fluviatilis. We recommend a genetic monitoring of the individuals to be released in order to ensure the same level of genetic variability found at the source. Translocations of G. aculeatus proved successful elsewhere [We failed to identify any of the three main genetic factors to extinction risk reviewed by Frankham  at Penysulations , 93. Ove980/90’s . The Cat980/90’s ) and sams et al. , as stn3lsewhere , 85, 94.lsewhere , to physTransatlantic, European and Mediterranean mitochondrial lineages recognised and defined for G. aculeatus by prior literature were present in the Ibero-Balearic region. By contrast, the Black Sea, Adriatic and Irish lineages were absent. Our results suggested that Atlantic three-spined sticklebacks colonised the Iberian Peninsula several times during the end of the Last Glacial period. G. aculeatus showed a strong nuclear population structure highly concordant with the Iberian hydrological pattern. The Portuguese populations of Vouga and Tagus, as well as the Spanish Antela (Northwest Spain) and Majorca should be prioritised by conservation policies. Their loss would severely erode the genetic diversity of G. aculeatus in the Ibero-Balearic region, so maintenance of their connectivity with nearby populations, control of exotic species, and monitoring of habitat properties are recommended in those areas. We would like to highlight the conservation value of the Central and Southern Portuguese populations, not simply because of their genetic and habitat distinctiveness, but rather because of the indication of sporadic coastal dispersal involving Mira and Sado. The site of Penyscola showed an intermediate level of genetic diversity, so that its extinction was likely too abrupt to leave any genetic footprint. Lastly, the ex-situ conservation program implemented in the Valencian Community mostly failed despite ensuring a similar level of genetic diversity between the source and the captive-bred individuals. Environmental changes caused by human activity have been frequently argued to explain local declines and extinctions reported during recent decades, particularly with regard to the influence of exotic species and modification of drainage patterns caused by land-use changes. However, the persistence of peripheral populations at southern latitudes may be also conditioned by the tolerance of G. aculeatus to physiochemical changes related to global warming. Our allelic-diversity results are expected to contain information regarding the evolutionary potential for adaptation to environmental changes. Nevertheless, a deeper knowledge about the relative contribution of extrinsic and intrinsic factors to fitness is needed in order to properly undertake conservation actions that will preserve the three-spined stickleback at low latitudes.The S1 Figcytb data were aligned and collapsed with the homologous fragments reported in Europe by prior literature [G. aculeatus (Accession number AB094627). Readers are referred to Our terature , 49–54. (TIF)Click here for additional data file.S2 FigK); bars correspond to standard deviation, after 20 independent runs. Down: rate of change in the log probability of data between successive K values (∆k). It is worth noting that K may be underestimated if there is hierarchical structure.Up: estimated log probability of data for the different number of inferred clusters ((PDF)Click here for additional data file.S1 Tablek, number of alleles; AR, allelic richness overall populations ; HO, observed heterozygosity; HE, expected heterozygosity. Significance of FIS was obtained after 1,000 randomisations of alleles; *** p ≤ 0.001. Standard deviations are showed in parentheses. The three highest values of AR and HE are marked in bold, whereas the lowest appear underlined.(DOCX)Click here for additional data file.S2 Tablestn3, stn12 and stn174 excluded from this calculation due to presence of intermediate (1 bp difference) alleles.Loci (DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableAbove diagonal: straight line distances. Below diagonal: shortest pairwise water distances among sampling sites using Google Earth. Note that samples from S14  were geographically coded as located at river Orlina , the source of individuals for the translocated specimens founding S14.(DOCX)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.


Myrceugenia correifolia is a rare arboreal species that grows in deep canyons and hilltops of the Coast Range of north-central Chile between 30° and 35°S. In the northern edge of its distribution M. correifolia grows in small patches of fog-dependent forest surrounding by xeric vegetation. These forest formations are thought to be remnants of an ancient and continuous rainforest that according to some authors became fragmented during aridization of the Neogene (Neogene relict) and to others during warm-dry cycles of the Pleistocene . Here we asked whether the northernmost populations of M. correifolia are Neogene relicts, glacial relicts, or the result of a recent northward colonization. To answer this question we examined genetic diversity and population divergence of M. correifolia using microsatellite markers, tested various competing population history scenarios with an approximate Bayesian computation (ABC) method, and complemented these data with ecological niche modeling (ENM). We detected three genetic clusters with a distinctive latitudinal pattern  and high levels of differentiation (FST = 0.36). Demographic inference supported an admixture event 31 kya between two populations that diverged from an ancient population 139 kya. The admixture time coincides with the beginning of a period of wet conditions in north-central Chile that extended from 33 to 19 kya and was preceded by dry and cold conditions. These results suggest that increased precipitation during glacial periods triggered northward expansion of the range of M. correifolia, with subsequent admixture between populations that remained separated during interglacial periods. Accordingly, ENM models showed that suitable habitats for M. correifolia in north-central Chile were larger and less fragmented during the Last Glacial Maximum than at present, suggesting that northernmost populations of this species are glacial relicts.Rare species frequently occur in areas with microclimatic conditions that are atypical for their regions, but that were more common in the past, and that probably have operated as climatic refugia for a long time.  Species with limited geographic distributions are considered more vulnerable to climatic changes and land use conversion than widespread species, and therefore are of central concern in conservation biology . Rare spThe Mediterranean region of central Chile, one of the world’s plant diversity hotspots, has high levels of richness and endemism . Many trAextoxicon punctatum, distributed from 30° to 43°S  approach to test different historical scenarios. Finally, we used ecological niche modeling (ENM) to identify the climatic niche of this species under current environmental conditions and to predict its potential distribution during the LGM and mid-Holocene. In this way, we explore the “bioclimatic” connectivity of Coast Range summits during glacial and interglacial periods. We combined this information to assess whether the northernmost populations of M. correifolia can be considered: (1) relicts of Neogene that became gradually fragmented by the progressive aridization of north and central Chile; (2) relicts of range expansion during wet conditions of the last glacial period that became fragmented during aridization of Holocene; or (3) the result of a recent northward colonization (this is during Late Holocene). If this species persisted in north-central Chile isolated for a long time, we expect to find strong genetic divergence between northern and southern populations. In contrast, if M. correifolia colonized northern areas more recently, we expect to find low levels of populationdivergence, a south-to-north decline in genetic diversity and isolation by distance.Here we examined genetic diversity and population divergence of the rare Mediterranean species M. correifolia from nine sites located between 30°39′S and 34°41′S in the Coast Range of Chile, covering most of the current distribution range of the species (Table 1). A random sample of 15 individuals was collected from each site (when available).We collected plant material of TM Software version 1.0 (Applied Biosystems).Genomic DNA was extracted from each sample using the DNEasy Plant mini kit . A total of 124 individuals were genotyped for eight microsatellite loci , using the primers and protocol described by N), number of rare alleles (AR), expected heterozygosity (He), observed heterozygosity (Ho), and deviations from Hardy–Weinberg using GenAlEx version 6 (FIS) and its 95% confidence interval (CI) using the R package diveRsity (Table 1). Analyses were performed in R  and assign probabilistically each individual to these clusters without a priori knowledge of putative populations. Ten independent runs were carried out for K ranging from 1 to 9 using a Markov Chain Monte Carlo run length of 500,000, a burn-in of 50,000 and an admixture ancestry model assuming correlated allele frequencies. To determinate the most likely number of clusters (K) we used the rate of change in Ln P(D) (the log probability of data) between successive K values as suggested by FST values for all pair of sites in GenAlEx, and then performed a Mantel test with geographic distances. In order to discount those clusters obtained by STRUCTURE resulted from simple IBD, we conducted a partial Mantel test between genetic distances and clusters using geographic distances as covariate  and when all populations were pooled .Genetic differentiation within and among populations was measured by analysis of molecular variance (AMOVA)  using Geovariate . Given tovariate , we examovariate . We perfM. correifolia we used coalescent-based ABC implemented in DIYABC v2.0 . The third scenario includes an admixture event that resulted from range expansion of two populations that diverged from an ancient population. This scenario is based on the hypothesis that increased precipitation during glacial periods triggered range expansion of M. correifolia, with subsequent admixture between populations that remained separated during the last interglacial period (LIG). We set uniform priors for all demographic parameters. The microsatellite loci were divided into two groups: (1) all dinucleotide repeat loci and (2) the single trinucleotide repeat locus. We ran 4 × 106 simulated datasets for each scenario to estimate posterior probabilities using logistic regression. We selected the scenario with the highest posterior probability and estimated the values of the associated demographic parameters.To reconstruct the history of population divergence of ABC v2.0 . This anM. correifolia from a total of 18 unique-georeferenced species records. We obtained the occurrences from national herbaria: Universidad de Concepción (CONC); Museo Nacional de Historia Natural (SGO), online database Global Biodiversity Information Facility (GBIF); Cooperative Taxonomic Resource for American Myrtaceae (CoTram) and our own field work. We modeled the climatic niche employing the maximum entropy method (2). For the LGM and mid-Holocene model projections we used the pre-industrial calibrated Model for Interdisciplinary Research on Climate . Regression analyses revealed a significant effect of latitude on allelic richness , Shannon genetic diversity index , He , but not on Ho . Three genetic clusters were detected in M. correifolia by Structure analysis with a distinctive latitudinal pattern: (1) north, with the sites Fray Jorge and Talinay; (2) center, with the sites Santa Inés, Zapallar, and Qda. Córdova; and (3) south, with the remaining sites . Most individuals (94%) were assigned to a single genetic cluster (80 of inferred ancestry) and AMOVA indicated high differentiation among regions, with 36% of the variation explained by differences among them and only 8% by differences among populations. Divergence between north and central clusters (FST = 0.22) was weaker than the barrier between central and south clusters (FST = 0.31). The Mantel test between pairwise FST values and geographic distances revealed a strong and significant relationship between geographic and genetic distance , suggesting a significant IBD pattern . A significant association between genetic distances and clusters  was also detected when geographic distance was included as a covariate, indicating the presence of additional barriers to gene flow.Figure 1). This scenario had a probability of 0.53 with a 95% CI ranging between 0.51 and 0.56 that did not overlap with that of scenario 2  and scenario 1 . According to scenario 3, admixture occurred 6,330 generations ago  from two populations that diverged from an ancient population 27,800  generations ago. The rate of admixture was higher between north and central populations (r = 0.64) than between the central and southern groups (1 -r = 0.36). The effective population size was greater in the central group  than in northern  and southern groups .DIYABC analyses indicated that the most likely scenario is scenario 3, which describes an admixture event that resulted from range expansion of two populations that diverged from an ancient population . Paleoclimatic models suggested that areas of high and moderate suitability were larger and less fragmented during the LGM than at present. The potential distribution of M. correifolia during mid-Holocene was similar to the present one, but with a larger area of high suitability, extending between 33° and 34°S .The ENM model for M. correifolia, one of the dominant trees of these fog-dependent forests, suggest that divergence of its northernmost populations located in the emblematic Fray Jorge–Talinay National Park was not as ancient as previously proposed.Fog-dependent forests that today grow isolated on tops of the Coast Range of Central Chile are thought to be remnants of an ancient and continuous rainforest that according to some authors became fragmented during aridization of the Plio-Pleistocene transition , and to M. correifolia, with subsequent admixture between populations that remained separated during interglacial periods. According to DIYABC analysis, admixture occurred 6,330 generations ago between two populations that diverged from an ancient population 27,800 generations ago. The generation time of natural populations of M. correifolia remains unknown, but there are data for individuals grown in the National Botanical Garden of Chile , indicating that they can reach reproductive maturity at 5 years. Assuming a generation time of 5 years, admixture would have occurred 31 kya and ancient divergence at 139 kya. Admixture time coincides with the beginning of a period of wet conditions in north-central Chile that extended from 33 to 19 kya and that was preceded by dry and cold conditions . However, this result is in line with several studies showing that general circulation models tend to underestimate the magnitude of LIG warming respect to proxy reconstructions  is somewhat earlier than the beginning of the LIG. This period, extending between 115 and 130 kya, was characterized by global warming, sea level rise and increased summertime-insolation . The incructions . For theructions .Figure 5). We detected a genetic barrier at 31.5°S separating Fray Jorge and Talinay from the remaining sites of M. correifolia. A similar disjunction has been reported for D. winteri  than the central  and northern groups . Reduction in genetic diversity southward has been documented in species from north-central .Structure analysis also revealed a second barrier at 33.5°S, separating the southern group from the other populations of -central  and sout-central , and hasFP conceived and designed the study, analyzed and interpreted data, and wrote the paper. CI, MC, and GP obtained and analyzed microsatellite data. PM performed niche modeling analysis. LH designed the study and interpreted data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Isatidae L. is a complex and systematically difficult genus in Brassicaceae. The genus displays great morphological polymorphism, which makes the classification of species and subspecies difficult as it is observed in Isatis glauca Aucher ex Boiss. The aim of this study is characterization of the genetic diversity in subspecies of Isatis glauca Aucher ex Boiss. distributed widely in Central Anatolia, in Turkey by using Amplified Fragment Length Polymorphism (AFLP) technique.Eco RI-Mse I primer combinations produced 805 AFLP loci, 793 (98.5%) of which were polymorphic in 67 accessions representing nine different populations. The data obtained by AFLP was computed with using GDA  and STRUCTURE (version 2.3.3) software programs for population genetics. The mean proportion of the polymorphic locus (P), the mean number of alleles (A), the number of unique alleles (U) and the mean value of gene diversity (He) were 0.59, 1.59, 20, and 0.23 respectively. The coancestry coefficient (ϴ) was 0.24. The optimal number of K was identified as seven. The principal component analysis (PCA) explained 85.61% of the total genetic variation.Eight different Isatis glauca ssp. populations showed a high level of genetic diversity, and the AFLP analysis revealed that high polymorphism and differentiated subspecies could be used conveniently for population genetic studies. The principal coordinate analysis (PCoA) based on the dissimilarity matrix, the dendrogram drawn with UPGMA method and STRUCTURE cluster analysis distinguished the accessions successfully. The accessions formed distinctive population structures for populations AA, AB, E, K, and S. Populations AG1 and AG2 seemed to have similar genetic content, in addition, in both populations several hybrid individuals were observed. The accessions did not formed distinctive population structures for both populations AI and ANP. Consequently, Ankara province might be the area, where species Isatis glauca Aucher ex Boiss. originated.The online version of this article (doi:10.1186/1999-3110-54-48) contains supplementary material, which is available to authorized users. Isatis glauca Aucher ex Boiss., is a diploid biennial or perennial herbaceous species with a chromosome number set of 14 (2n = 28). Isatis glauca has four recognized subspecies, glauca, galatica Yıldırımlı, sivasica (Davis) Yıldırımlı and iconia (Boiss.) Davis. The distribution of this polymorphic species in Turkey is represented in Figure Isatis L., species grown in the Eastern and South Eastern Anatolia and across the Eastern Mediterranean region were made by Mısırdalı  of those pollen grains were tricolpate , 129 (16.33%) were syncolpate, 87 (11.01%) were stefanocolpate and 69 (8.73%) were dicolpate. Gilbert et al.  by using AFLP. Al-Shehbaz et al. (Brassicaceae (Cruciferae). Kızıl . Spataro et al.  by using ISSR markers. All these studies reveal that although there are many reports on morphological and genetic variation in the Brassicaceae family, very few studies are found on morphological or genetic variation in Isatis glauca Aucher ex Boiss.Taxonomic and cytological investigations on the ısırdalı  . Kızıl  reportednd Zarre  studied i et al.  examinedo et al.  displayend Negri  analysedu et al.  assessedu et al.  presenteu et al.  showed ti et al.  analyseda et al.  detectedI. glauca, subspecies populations from Central Anatolia in Turkey. AFLP method was used to estimate genetic diversity (He), population structure and genetic differentiation between populations using coancestry coefficient ϴP (= FST). In addition, the study also aimed to find the effects of climate , and geography  on the genetic diversity of nine I. glauca populations.The aim of this study was to analyse genetic diversity and population structure using molecular marker techniques among the I. glauca subspecies populations collected from Ankara/Ayas (AA), Ankara/Beytepe (AB) valley, Ankara/Incek (AI), Ankara/Golbasi1 (AG1), Ankara/Golbasi2 (AG2), Ankara/Polatlı (ANP), Eskisehir (E), Konya (K) and Sivas (S)  and 1 μL Eco R I adaptor (5 pmol/L = 5 μM). The RLMM was prepared by using 1.1 μL aliquots T4 DNA ligase buffer 10x, 4.267 μL a-RLMM, 0.05 μL Mse I (20 U/μL) enzyme, 0.05 μL Eco R I (100 U/μL) enzyme, and 0.033 μL T4 DNA ligase enzyme (30 weissU/μL). 96 well PCR microtiter plates were used for 67 samples. For each sample a 5.5 μL RLMM aliquot was dispensed into the wells and the 5.5 μL of the diluted sample genomic DNA (50 ng) was added. PCR microtiter plates were short spinned in a centrifuge (Sigma 1–14) and left for incubation (Techne TC-412) at 37°C for 6 hours.The aliquots of the restriction-ligation master-mix (a-RLMM) were prepared in a 2 mL Eppendorf tube without enzymes and the T4 DNA ligase buffer 10x. In a- restriction-ligation master-mix (RLMM) for one sample, the following were added; 0.617 μL H2O, 1.1 μL NaCl (0.5 M), 0.55 μL BSA 10× (1 g/L), 1 μL Eco R I and Mse 1 adaptors, that contained one selective nucleotide. Preselective PCR, reaction consisted of 2 μL (10X) PCR buffer, 1.6 μL (2 mM) MgCl2, 1.2 μL (2.5 mM) dNTPs, 1 μL (5 pmol) Eco R I primer, 1 μL (5 pmol) Mse I primer, 0.2 μL (1 μ/μL) Taq DNA polymerase, 9 μL ddH2O, and 4 μL diluted restriction-ligation products. The diluted restriction-ligation products were kept at −20°C until used. Preselective amplification for 20 cycles was carried out through PCR. Initial extended denaturation was carried out at 94°C for 2 min, followed by denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 2 min, and the final extension at 60°C for 30 min.Before the preselective PCR amplification, the 5-μL restriction-ligation products were diluted by adding 95 μL TE (1X). For the preselective PCR amplification, the primers, were complementary to the sequences of the Eco R I and Mse I primers, which contained three selective nucleotides. For the fluorescent-based fragment analysis, the Eco R I selective primers of 39, 36, 39, and 36 were labelled with FAM-1, FAM-2, FAM-3, and FAM-4 respectively, while the Eco R I selective primers 33, 36, 33, and 36 were labelled with VIC-1, VIC-2, VIC-3, and VIC-4 fluorescent dye. Mse I primers were used with the unlabelled ones. The selective PCR amplification used in this study is given in Additional file Selective PCR amplification was carried out using 10 μL preselective PCR amplification products diluted in 190 μL TE (1X). The diluted preselective PCR amplification products were kept at −20°C until used. The selective PCR amplification was done with Capillary electrophoresis of selective amplification products were performed by using ABI 3130 × l . For capillary electrophoresis; 1 μL selective PCR amplification products were denatured using 0.2 μL LIZ-500 size standard at 95°C for 5 min followed by heat shock applied by chilling on ice at 0°C for 5 min. Then, permanently denatured single stranded DNA was loaded on ABI 3130 X l. After capillary electrophoresis, AFLP fragments were scored into a binary data matrix as 1 (present) or 0 (absent) using Gene Mapper 4.0 software package (Applied Biosystems).I. glauca ssp. populations, we used the software program STRUCTURE  by evaluating K = 1–9. For parameter set, in our model, we selected with admixture as ancestry model and the allele frequencies were assumed to be correlated, since it is more reasonable to assume common ancestry of such closely related populations. The length of burn-in Markov Chain Monte Carlo (MCMC) replications was set to 10000 and data were collected over 100000 MCMC replications in run, based on previous literature suggesting that this level is sufficient  developed by Evanno et al. , a software package for Windows . PCoA is a scaling or ordination method that starts with a matrix of similarities or dissimilarities between a set of individuals. The method aims to produce a low-dimensional graphical plot of the data in such a way that distances between the points in the plot are close to the original dissimilarities. For PCoA, the dissimilarity matrix values were used to ordinate 67 accessions representing nine Turkish s Figure  was conss Figure  genetic s Figure  version s Figure .Figure 3P, A and He, and N respectively, as well as geographical  and climatic  data , this GST is identical to Wright’s  (Hartl and Clark ϴ (= FST) was measured for every locus and for all loci in each population.Genetic differentiation between populations is often estimated with the Nei’s coefficient GST Nei  for domiWright’s  FST  as dependent and the environmental variables  as independent.Multiple regression analysis (MR) (SPSS version 11 for Windows) was conducted with AFLP diversity and allele diversity in nine populations. MR employed the genetic variables  and the eight polymorphic primer sets selected and applied for all of the 67 accessions. The data were obtained using selected eight primer combinations in both pre-scanning and applied through all accessions including those eight accessions were compared to evaluate the reproducibility of AFLP markers. The percent of reproducibility was calculated for each primer combination. Average percent of reproducibility was calculated as 90.62% that ranged 86.38% (E36XM35) to 97.78% (E36XM34) among the primer combinations.Eco R I and Mse I primer combinations generated 805 fragments. The primer combination E36xM34 produced the highest number (194) of fragments, while the primer combination E36xM33 produced the least number (32) of fragments (data not shown). The primer combination E36xM34 displayed the highest number (510) of fragments in population AA in total of eight individuals, while the primer combination E39xM41 displayed the least number (81) of fragments in population AI in total of six individuals. Average number of fragments per population was calculated according to total present fragment numbers produced by eight primer combinations in all individuals of each population and ranged 252.17 (in population AI) to 314.75 (in population AA).In AFLP analysis, eight Eco R I and Mse I primer combinations produced 805 fragments (loci), 793 (98.5%) of which were polymorphic and 12 (1.5%) were monomorphic. The proportion of the mean number of the polymorphic loci (P = 0.59), the mean number of alleles (A = 1.59) and the mean value of the genetic diversity (He = 0.23) for nine populations, are represented in Table P = 0.68), the highest number of alleles (A = 1.68), and the highest value (He = 0.26) of genetic diversity (Table P = 0.50) and the lowest number of alleles (A = 1.5), while population S has the lowest genetic diversity value (He = 0.20).In AFLP analysis, eight ϴ (= FST) or genetic differentiation between the populations is 0.24  (Table GD = 0.24) was observed between population AA and population S, while the lowest genetic distance (GD = 0.04) was observed between AG1 and AG2 (Data not shown).The mean value of the coancestry coefficient l) Table . The higI. glauca ssp. populations, 19 unique alleles were observed  for STRUCTURE in order to determine the number of subpopulations identified the optimal value for  for STRU7 Figure  develope7 Figure . This meAccessions formed distinctive population structures for populations AA, AB, E, K, and S. Populations AG1 and AG2 seemed to have similar genetic content, in addition in both populations several hybrid individuals were observed. Populations AI and ANP did not display distinctive population structures. In these populations, all individuals, except one individual in population AI, were seemed to be hybrids. In general, all populations have hybrid individuals.The hybrid individuals detected in this study is an indication of gene flow between populations. Particularly, extensive gene flow was observed between AG1, AG2, AI, and ANP. It is noteworthy that there is a population (light green colour) found in hybrid individuals, although there is no such a distinctive population among the populations analysed in this study. This might be because of there is no any sample from this population among the analysed populations or that might be extinct in the evolutionary history of subspecies populations. Therefore, some individuals are still carrying on the genetic material on their genetic background.I. glauca ssp. sivasica) clustered in the second main group. In the first main group, population AA  separated clearly from rest of the populations in the first subgroup. In the second subgroup populations clustered into further subgroups. The populations AG1 (I. glauca ssp. glauca) and AG2 , populations AB (I. glauca ssp. glauca) and ANP , and population AI (I. glauca ssp. glauca) clustered together in the second subgroup. In third subgroup, population K  and E  clustered together.In the dendrogram, there are two main groups Figure . All theP, A, He, and AL  displayed 11.96% of the variance, while the second axis (Eigen vector) and the third axis (Eigen vector) showed 9.41% and 7.85% of the variance respectively (data not shown). The codes of 67 accessions are plotted according to the first and the second axis that corresponded to different regions, from where nine glauca ssp. populations.We employed the multiple regressions (MR) to determine AFLP allelic diversity. The results of MR analysis showed that the combination of altitude, latitude, longitude, temperature, humidity, and rainfall accounted for a considerable proportion of the variation observed in AFLP fragments diversity Table . When coI. glauca subspecies in agreement with Gilbert et al. . According to fragment analysis, the highest number of present fragments were observed in population AA from Ankara/Ayaş. STRUCTURE analysis results indicated that accessions formed a distinctive and stable population structure for population AA. Although, high number of fragments were amplified, low level of genetic diversity was observed. There might be low or no gene flow with other populations due to geographic distance or some other reasons. Therefore, observed homozygosity might be higher than the other populations analysed in this study. We also considered that anthropogenic stresses might have important role on the level of genetic diversity observed in population AA. There is a paper factory in the area and also the plants generally grow on the highway road sides. These factors might cause to reduce genetic diversity in the population. On the other hand amplification of higher number of present fragments might refer that accesions from the population AA that still contain genetic upload of the original population of ssp. I. glauca as in the case of I. tinctoria  of unique alleles, which had frequencies higher than 0.05. The population S is geographically distant from the rest of the populations analysed in this study. Therefore, existed unique alleles in populations will drive genetic differentiation between populations as it was observed between population S and the other populations in this study.Isatis L. is economically important plant especially for natural dyestuff beside its medicinal uses. The unique alleles found in I. glauca ssp. populations as molecular markers might be an indication of desirable traits, which can be used in the future breeding programs of Isatis L. plant species.Unique alleles have potential as favourable genes for tolerance to severe conditions, especially at higher altitudes . This might show that formation of I. glauca ssp. galatica. Some genotypes from Ankara/Ayaş (pop AA) migrated through Ankara/Polatlı (population ANP) and Eskişehir (population E)  Figure . On the I. glauca ssp. galatica. The gene flow rates between the population ANP and the populations AG1, AG2, and AI seemed to be considerably high. Therefore, in populations AI and ANP all individuals except, one in pop AI, were hybrids.The populations from Eskişehir (E) and Konya (K) have distinctive population structures now and they can be recognised as the varieties of LA and AL, mainly contributed to the second and third components. The sum of those components is 48.55%. In addition, multiple regression analysis displayed that when all environmental factors combined they had great effect on the genetic data . These results displayed that natural selection might play important role in genetic diversity of I. glauca ssp. populations.The mutations and recombinations generates variation, which underpin genetic diversity in a population. However, selection, genetic drift and gene flow may play an important role in the genetic diversity of particularly in small size populations. The selection might be natural or artificial as it was observed in cultivated crop plant species Suneson . DespiteI. glauca subspecies were quite small (data not shown). Plants usually grow on the highway road sides, which negatively effect the population size because, the construction of new highways or extending road widths, remove some plant genotypes from those places or they have to move inner sides or different niches . Anthropogenic stressess may result in genotypes, which have desirable traits such as drought tolerance, climatic changes, etc. to be disappeared and may cause to the genetic drift or bottleneck effect in the germplasms of I. glauca subspecies populations.During the field studies, it was noticed that population sizes of the Isatis L. was economically an important plant during the Roman times in both Europe and Asia  was used for large scale indigo production in Europe. However, according to some recent phytochemical studies  and Konya (K) were recognised as the varieties of v.Isatis glauca Aucher ex Boiss. originated.Ankara province might be the area, where species vi.Isatis glauca species and its taxonomy. Consequently, the subspecies and their varieties will be allocated taxonomically in appropriate way.The genetic information obtained in the present study will assist to acquire more information about the population genetic structure, the basis for speciation or subspecies formation in The main conclusions of this study are as followed;EG is doing her PH.D at Hacettepe University in Ankara, Turkey.ÖÖ is an assistant professor at Hitit University, Faculty of Art and Science, Department of Biology, Çorum, Turkey.ŞY is a professor at Hacettepe University, Faculty of Science, and Department of Biology in Ankara, Turkey.Additional file 1: Table S1: Accession numbers (AN), population codes (PC), the province, where the accessions were collected and subspecies name of the accessions analysed in this study. Accession numbers were given by Professor Dr. Şinasi Yıldırımlı. First two letters represent the population code, which the accession belongs to it. The first number represent collection year and the second number(s) represents the accession number assigned. (DOCX 25 KB)Eco R I E, and Mse I M). (DOCX 19 KB)Additional file 2: Table S2: The primers and adaptors used in this study . (DOCX 18 KB)Additional file 3: Table S3: Primer combinations used in this study; Additional file 4: Table S4: Selective PCR amplification program, used in this study. (DOCX 18 KB)Additional file 5: Table S5: Climatic  data of the places, where the populations were collected (Population code PC). (DOCX 19 KB)Isatis glauca subspecies populations. (DOCX 19 KB)Additional file 6: Table S6: Private alleles observed in studied nine Turkish P, average number of allele A, average number of allele per polymorphic locus A P, genetic diversity He, temperature T, rainfall RA, humidity HU, altitude AL, latitude LA and longitude LN). (DOCX 20 KB)Additional file 7: Table S7: Component matrix of variables and their contributions to principal components (Abbreviations: Number of polymorphic locus Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Authors’ original file for figure 7Below are the links to the authors’ original submitted files for images.


Uncovering the genetic basis of species diversification is a central goal in evolutionary biology. Yet, the link between the accumulation of genomic changes during population divergence and the evolutionary forces promoting reproductive isolation is poorly understood. Here, we analysed 124 genomes of crow populations with various degrees of genome-wide differentiation, with parallelism of a sexually selected plumage phenotype, and ongoing hybridization. Overall, heterogeneity in genetic differentiation along the genome was best explained by linked selection exposed on a shared genome architecture. Superimposed on this common background, we identified genomic regions with signatures of selection specific to independent phenotypic contact zones. Candidate pigmentation genes with evidence for divergent selection were only partly shared, suggesting context-dependent selection on a multigenic trait architecture and parallelism by pathway rather than by repeated single-gene effects. This study provides insight into how various forms of selection shape genome-wide patterns of genomic differentiation as populations diverge. Genetic changes accumulate as populations diverge and new species emerge. Here, Vijay et al. resequence the genomes of crow populations at various stages of genetic differentiation, and shed light on the evolutionary processes acting during the origin and hybridization of crows in Europe and Asia. ST, are candidates for being involved in reproductive isolation6The question of how new species originate and adapt to novel environments has been the foundation to the field of evolutionary biology. Rapid development of high-throughput sequencing technologies during the last decade has opened new avenues to address the genetic basis of adaptation and speciation in natural populations. By studying patterns of genetic variation across the genomes of multiple individuals sampled in their natural environment, evolutionary processes governing the accumulation of genetic differences between incipient evolutionary lineages can be caught in the act134Yet, local peaks of genomic differentiation need not necessarily arise as a result of divergent selection advancing reproductive isolation. There is increasing evidence that genomic regions with elevated differentiation can emerge by processes unrelated to speciation9101211Discerning evolutionary processes leading to varying levels of differentiation across the genome is a challenging task. A promising way forward is to sample several populations at various stages of population divergence1641820Corvus (corone) spp. crow species complex at various stages of genetic differentiation  spp.=corone/cornix/orientalis/pectoralis hereafter). Described in detail almost 100 years ago24corone, orientalis) occur both east and west of populations of a black-and-grey pied phenotype (mostly cornix), with hybrid zones in Europe and Siberia  has shown that only a small number of narrow genomic regions, associated with the striking difference in pigmentation pattern, exhibited resistance to gene flow and suggests a simple, yet multi-locus genetic architecture for the trait17C. brachyrhynchos , population structure was dominated by deep divergence among  Spanish corone and eastern Russian orientalis populations—neither reflecting subspecific delineation nor plumage colouration (ST 0.27–0.47) supports a monophyletic origin of the Eurasian species complex from a black ancestor in East Asia. Concordant demographic histories suggest shared ancestry of all Eurasian populations followed by divergence during the late Pleistocene  segregating across all populations, 11.8% were shared with the American crow. Superimposed on a weak isolation-by-distance (IBD) pattern across the Palearctic , and showed a range of admixture proportions (0.532–0.886) attesting to ongoing backcrossing  for 50 kb windows across the genome was observed for all 54 population comparisons (ST') were significantly clustered into distinct ‘outlier peaks' with mean sizes ranging from 117 to 295 kb . With progressing levels of overall differentiation, peaks crystallized more clearly, as evidenced by an increase in autocorrelation (Moran's I) between windows and co-variation in differentiation profiles (Strong heterogeneity in genetic differentiation as measured by Fparisons . Windowsprofiles . This poprofiles :ρ (=4eNr) and LD were positively correlated ‘among' all possible population pairs supporting the assumption of broad-scale recombination rate conservation expected across this short evolutionary timescale; and hence fulfilling a central precondition for shared linked selection across populations.First, genome-wide profiles of the population recombination rate π was correlated ‘among' populations, even when controlling for the effect of μ (approximated by synonymous substitution rate). ‘Within' populations, ρ (and resulting LD) correlated strongly with π (=4eNμ). With little evidence for recombination-associated mutation (and hence r∼μ)  along the genome being reduced in regions of low recombination. π was most reduced in low recombination regions associated with a high density for targets of selection measured as the density of coding sequence, a signature expected for background selection  ref.  and ρ geπ and ρ (and positively with LD). This was true for FST between population pairs, but also for lineage-specific differentiation as estimated by the population branch statistic (PBS) removing effects due to shared ancestry and pseudo-replication. While a correlation with π is expected by the fact that FST is a relative differentiation measure sensitive to population diversity, a correlation with ρ is not expected under neutrality and requires the assumption of linked selection locally reducing effective population sizes in regions of low recombination.Third, measures of genetic differentiation were negatively correlated with ST among all population pairs and for lineage-specific PBS estimates. Moreover, FST co-varied negatively with net divergence between populations (Dxy) suggesting that linked selection acted already in the ancestral populationFourth, differentiation landscapes co-varied positively both for FIn line with theoretical predictions1419ST' from any of these five comparisons is taken as a conservative estimate of the strength of linked selection. Subtracting maximum FST' from orthologous windows of the focal comparisons (ST') measuring relative excess differentiation in s.d. units. Windows classified as outliers (>99th percentile) for FST', but not ΔFST', were interpreted as genomic regions subject to shared selection pressures , and lineage-specific differentiation  were elevated in at least one of the contact zones 2) 2) ref. .Focusing on genes located in ‘contact zone peaks', in genomic regions flagged by divergent local phylogenies ‘cacti', , and thocorone–cornix contact zone. Parallel evolutionary dynamics along the European hybrid zone were indicated by congruent differentiation landscapes across the central and southern part of the hybrid zone, Germany with Poland/Sweden  and even higher for ΔFST' (r=0.94), suggesting a stronger signal for peaks associated with gene flow across the hybrid zone. In all, 58% of all flagged genes were shared and both comparisons contained the same set of melanogenesis-related genes in a major >2 Mb peak on chromosome 18 , additional loci potentially affected by positive selection were identified. Importantly, outlier genes with described functionality in the melanogenic pathway such as cornix–orientalis hybrid zone were least clustered, formed smaller contiguous peaks  with low relative amplitudes emerged, on chromosome 21 and the Z-chromosome  compared with allopatric controls  that were shared across all contact zone comparisons. While caution is warranted for single SNP outlier tests, this number significantly exceeded the random expectation of flagging the same site in three contact zones . Within this shared selected gene set, six genes had a functional link to the melanogenic pathway, including TRPM1, KCTD5, MMP17 and INPP4B detected by nSL, GREB1 identified with XP-EHH and FREM2 detected by iHS.While no candidate gene was shared among all contact zones based on the FAXIN2, LEF1, LRP5/6), a central modulator of the microphthalmia-associated transcription factor (MITF) that has been functionally implicated in the phenotypic differences between European crowsMore generally, pigmentation candidate genes from across all contact zones contained at least one gene associated with the Wnt signalling component of the melanogenesis pathway  spp. species complex from populations across its geographical distribution (spp. referred to by their (sub)species designation corone/cornix/orientalis/pallescens/pectoralis) and sampled 11 hybrids from within the European and Siberian hybrid zones  and Eurasian jackdaw (Corvus monedula) were added as outgroup taxa. Information on taxonomic status of the species complex, and details on samples and sampling locations are given in the We sampled a total of 107 morphologically pure individuals of the Eurasian id zones . We furtflow see . Two indCorvus (corone) spp. population samples and three females out of six American Crows. For details on sex determination see Where possible, (homogametic) male individuals were chosen to guarantee equal sequencing coverage for autosomes and the Z-chromosome, and to avoid systematic biases arising due to coverage differences for population genetic analyses. Yet, due to sample limitations, a total of 23 (out of 118) heterogametic females were included in the http://www.bioinformatics.babraham.ac.uk/projects/fastqc).We extracted genomic DNA using either a standard phenol-chloroform protocol or the Qiagen DNeasy blood and tissue kit. Subsequently we prepared one paired-end library per individual  with Illumina TruSeq DNA Sample Prep Kit v2 according to the manufacturer's instructions. All libraries were sequenced on an Illumina HiSeq2000 machine. Average raw read sequence coverage including the published data was 12.5 ×  assuming a genome size of 1.21 Gb  was subsequently used to assign readgroup information containing library, lane and sample identity. To enhance the alignments in regions of insertion–deletion polymorphisms, we additionally performed local realignment using GATKpicard. Bam files were merged at the individual level, validated, and duplicate reads removed in picard before variant discovery.Raw reads were mapped to the hooded crow reference assembly7.8 ref.  with defHaplotypeCaller in GATK version 3.3.0. Variant detection on autosomal regions utilized one group comprised of all male and female samples under the diploid default setting (−ploidy 2). Z-linked chromosomal regions were analysed separately for male and female groups, by specifying the ploidy level for male samples as ‘−ploidy 2' and for heterogametic female samples as ‘−ploidy 1'.Variant discovery was performed for the 118 crow samples including hybrids together and separately for the 6 American crow samples using et al.HaplotypeCaller in GATK version 3.3.0. using default settings. Variant quality score recalibration, a post-discovery error modelling algorithm implemented in GATKGATK. In a final filtering strategy, variants within regions with annotated repeat content were removed  using the Median Joining method based on the maximum-parsimony algorithmConsensus sequence of mitochondrial genomes was extracted from a subset of individuals specified in corone/cornix/orientalis/pectoralis crow mitochondrial genomes using maximum-likelihood procedures  spp. complex, we utilized a network reconstruction method that incorporates incompatible and ambiguous signals inherent within a genome. A split decomposition network was reconstructed using the program SplitsTree on the autosomal variant data setpectoralis.To investigate the evolutionary relationships among the Corvus brachyrhynchos).We further used a statistical model by Pickrell and Pritchardet al.Eigensoft, which pruned the initial variant set to 16,064,921 SNPs.Principle component analysis was performed on all SNPs following the approach by Patterson ST/1-FST) among populations. For population pools from different sampling locations , the average longitude and latitude values weighted by sample size were used as geographic location. Population pairs with high negative residuals were taken to be genetically more similar than expected by geographic distance as across the entire species complex. A Mantel test was used to assess statistical significance of the correlation between geographic distance and genetic distance, while controlling for pseudo-replication among all possible population comparisons. In the main text, we report the correlation for the entire nuclear genome; correlations for sex chromosome and autosomes were near identical . For details on analytical procedures see We quantified the relationship between geographical and genetic distance (measured as FADMIXTURE version 1.23 (ref. c.) spp. complex. Analyses were run on a total of 15,937,359 segregating autosomal sites, including only variants with <10% missing genotypes. We investigated between 1 and 10 possible population clusters, performing 200 bootstrap resampling iterations to estimate the s.e.We first performed admixture analyses as implemented in the software program .23 ref.  on the sST , each including only variants with a minor allele frequency greater than 5%. Only the latter data set converged on the expected clustering signal of K=2, and accurately assigned the hybrid individuals as admixed. The other data sets likely swamped the diagnostic signal with the overwhelming number of lowly differentiated background variants. Results are therefore reported for the subset of 20,549 and 29,072 SNPs from highly differentiated regions between the parental populations in the Italian and Russian hybrid zone, respectively .To test for gene flow between our three focal population comparisons across morphological contact zones we used the four taxon ‘ABBA-BABA' test. The D-statistic as defined in the study by Durand −9 per generation and a generation time of 5.79 years , nucleotide diversity (π), Tajima's D, Fay and Wu's H, and Fu and Li's D. As the ancestral reference genome sequence we used the consensus sequence from jackdaw, rook and American crow as described above.For each population we estimated the unfolded site frequency spectrum (SFS) using the Bayesian method implemented in the software package 588 refs , 54. We r2 value per window, averaging over all pairwise SNP combinations. All filtering and calculation of LD was performed with vcftoolsr2 is influenced by sample size  and scaffolds inferred to be sex-linked due to the expected difference in recombination in sex chromosomes. We extracted SNPs with a minor allele frequency above 0.1 and calculated the mean ρ per 50 kb window we used LDhelmetρ per bp for the SNPs in every window, from which we then calculated the weighted mean. We approximated the required mutation matrix from zebra finch substitution rates following Singhal et al.ρ as inferred from LD estimates (scaffold_78 with the main FST peak in the Eurasian population). Both matrices yielded qualitatively the same results (Pearson correlation of ρ estimates r=0.99).To generate an estimate of the population-scaled recombination rate HierFstat R packageST was estimated for each segregating site of sufficient sequence coverage . Autosomal and Z-linked variants were treated separately. Before merging male and female variants on Z-linked scaffolds, male diploid calls were haploidized. Window-based FST estimates in increments of 50 kb windows across the genome were then calculated by averaging the variance components across all segregating sites within that window.We calculated hierarchical F-statistics as implemented in the ST estimates from HierFstat, we additionally used methods specifically designed for low-to-medium-coverage sequencing data yielding highly correlated estimates ))+(−log(1-FST(Pop1_Pop3)))−(−log(1-FST(Pop2_Pop3)))/2.In addition to pairwise FDxy) . We therefore applied SAMtools ‘mpileup'Custom scripts were used to calculate the average number of nucleotide substitutions (xy) ref.  on the bcorone–cornix, cornix–orientalis, orientalis–pectoralis) to determine what percentage of outlier loci were in the shared set of SNPs between all three contact zones. In the populations bordering the corone–cornix contact zones all 78,542 outlier variants (100%) also segregated across all contact zones, while only 59,240 (69%) and 40,233 (48%) in cornix–orientalis and orientalis–pectoralis zones, respectively. This yields an expected number of outliers shared among all populations as 0.1 × 1 × 0.1 × 0.69 × 0.1 × 0.48 × 5,447,980∼1.8.We calculated haplotype statistics reflecting the decay of LD for the adjacent populations spanning each of the three contact zones to infer recent signatures of selection65Gallus gallus), and collared flycatcher  and crow (Corvus (corone) cornix). For each 50 kb window, substitution rate estimates were averaged across all genes present in that window. The mean genome-wide substitution rate (dS) was 0.079. Substitution rates did not differ between regions of elevated differentiation between taxa  and the genome-wide average in regions outside ‘peaks' . See We estimated substitution rate at fourfold degenerate sites as a proxy for mutation rate to assess to potential impact of mutation rate on patterns of genetic differentiation. Substitution rate estimates were based on a total of 5,012 reliable 1:1:1: gene-alignments between coding regions of canonical transcripts of chicken  or by common selection pressures shared among all population comparisons ST distribution (FST') expressing genome-wide differentiation in units of s.d. relative to the mean (ST' hereafter). Windows classified as outliers for FST', but not ΔFST', are interpreted as genomic regions subject to selection pressures shared across the entire species complex  independent of specific evolutionary processes acting on any of the target populations. Windows classified as outliers by both approaches were considered to be unique to each focal comparison in genomic position  and/or relative amplitude . These ‘unique' outliers were investigated in more detail for gene content, and were contrasted to background genome-wide non-peak regions as well as common peaks for a set of informative summary statistics such as nucleotide diversity (π), net nucleotide divergence (Dxy), Fay and Wu's H, and haplotype statistics  outliers, and genes in regions of divergent cacti, were enriched for particular classes of genes. For gene ontology and KEGG pathway analysis, we first identified chicken orthologs for which gene ontology and KEGG annotations exist, and subsequently tested for enrichment using the goseq R/Bioconductor package26We tested whether genes located in genomic windows identified as (FAll sequencing data were deposited in the sequence read archive (SRA) of the National Center for Biotechnology Information (NCBI) database under project number PRJNA192205 and in the European Nucleotide Archive (ENA) database under accession id PRJEB9057.How to cite this article: Vijay, N. et al. Evolution of heterogeneous genome differentiation across multiple contact zones in a crow species complex. Nat. Commun.7, 13195 doi: 10.1038/ncomms13195 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-8, Supplementary Tables 1-14, Supplementary Note 1 and Supplementary References


Panthera leo) has raised conservation concerns. In the Savé Valley Conservancy (SVC), in the Lowveld of Zimbabwe, lions were presumably reduced to approximately 5 to 10 individuals. After ten lions were reintroduced in 2005, the population has recovered to over 200 lions in 2016. Although the increase of lions in the SVC seems promising, a question remains whether the population is genetically viable, considering their small founding population. In this study, we document the genetic diversity in the SVC lion population using both mitochondrial and nuclear genetic markers, and compare our results to literature from other lion populations across Africa. We also tested whether genetic diversity is spatially structured between lion populations residing on several reserves in the Lowveld of Zimbabwe. A total of 42 lions were genotyped successfully for 11 microsatellite loci. We confirmed that the loss of allelic richness  has resulted in low genetic diversity and inbreeding. The SVC lion population was also found to be genetically differentiated from surrounding population, as a result of genetic drift and restricted natural dispersal due to anthropogenic barriers. From a conservation perspective, it is important to avoid further loss of genetic variability in the SVC lion population and maintain evolutionary potential required for future survival. Genetic restoration through the introduction of unrelated individuals is recommended, as this will increase genetic heterozygosity and improve survival and reproductive fitness in populations.The rapid decline of the African lion ( Panthera leo). The main threats to lion populations include habitat loss, persecutions (often to protect livestock), the depletion of the natural prey base, and, in some cases, poorly managed trophy hunting [In an increasingly human dominated and fragmented landscape, biodiversity has a changing face. Not surprisingly, an increasing number of species are adversely affected and show signs of negative population growth. A recent study by Ceballos and co-workers  reported hunting , 3.The rapid decline of the African lion has raised conservation concerns. Population size has decreased from roughly 75,000 individuals to 32,000 individuals in only two decades –6, with Southern African lion populations are declining at lower rates than elsewhere in Africa, due to lower human population densities, healthier prey populations, more support  for conservation management, and the more common use of park fences . Lion de2 [2) [Zimbabwe still supports relatively stable (in some areas even increasing) lion populations, as the probability of extinction is considered low in the majority of areas that contain lions . Althoug2 . In Buby2 [2) , 15. Sin2 [2) ). The ne2 [2) . Prior t2 [2) . The oriPuma concolor, where the population increased by three-fold after heterozygosity levels recovered subsequent to the introduction of eight unrelated females [Although the increase of lions in the SVC and BVC seems promising, a question remains whether the populations are genetically viable, considering their small founding population. Furthermore, to ensure a genetically healthy lion population, Björklund  recommen females .In this study, we address two main questions. First, we document the genetic diversity in the lion populations sampled during this study  using neutral genetic markers, both mitochondrial and nuclear, and compare our results to literature from other lion populations across Africa. We use the term ‘population’ as the localized, breeding group of lions in each reserve. We pay special attention to the number of haplotypes (indicative of the number of founding lineages), as well as allelic richness and heterozygosity (indicative of recent population trends). Although only adaptive diversity is directly related to genetic potential  , 25, neuSecondly, we tested whether similar or closely related genetic lineages are present in the different conserved areas. As genetic diversity is a key component of conservation management and essentially reflect the future viability of wildlife populations , 27, 28,The study was approved by the University of Johannesburg’s Ethical Committee. Samples were collected in Zimbabwe and taken to South Africa under import permit 13/1/1/30/2/0-2015/01/003889 and CITES permit ZW/0081/2015 & ZW/0082/2015. Samples were collected opportunistically when lions were fitted with radio-collars, removed from illegal snares, or found dead in the field. When an individual was found dead, a piece of tissue (approximately 5 by 5 cm) was clipped from the ear. When lions had to be immobilized for de-snaring or radio-collaring, 2 ml of blood was drawn from the leg. For immobilization, darts were fired from a maximum distance of 30 m  to reduce the possibility of physical injury. Injury resulting from darting is very rare, and always minor, although dart wounds are routinely cleaned and flushed with antibiotics as a precaution. A combination of Medetomidine (Dormitor), a sedative in the Imidazole group (α-2 agonists) and Ketamine (a short-acting cyclohexylamine knock-down drug) was used as a sedative and analgesic. Blindfolds and ear plugs were used throughout the handling process to minimise any external stimuli.Felis catus [A total of 42 samples were collected , and analysed on an ABI 3730 DNA Analyzer (Life Technologies).We conducted sequence-based genotyping for the mitochondrial cytochrome-ribed in ). The PCHO) and expected (HE) heterozygosity, as well as genetic distances (FST), was calculated using Arlequin 3.5 [R) was calculated for all individuals within each population using maximum likelihood estimates of relatedness calculated with ML-Relate [Microsatellite loci were scored using Geneious 6.1.5 (Biomatters Ltd.) and tested for linkage disequilibrium (using log-likelihood ratio G-statistics) and deviation from Hardy-Weinberg equilibrium in FSTAT 2.9.3.2 . We usedquin 3.5 . STRUCTUquin 3.5  was usedquin 3.5  as implequin 3.5 . CLUMPAKquin 3.5 . PairwisL-Relate .For the mitochondrial data, chromatograms were checked using Geneious 6.1.5 (Biomatters Ltd.) and aligned using Clustal W as implemented in the Geneious software. Sequences were deposited in the GenBank database under accession numbers MG490402 (BVC1), MG490403 (BVC2) and MG490404 (SVC1). For the assessment of overall spatial structure we used NETWORK  to creatHE of 0.66, and GNP by an HE of 0.53. Eleven private alleles are present in the BVC, and five in the GNP population. Inbreeding is also apparent in the SVC (FIS = 0.171), yet is found to be higher in the BVC lion population (FIS = 0.208).A total of 42 lions were genotyped successfully for 11 microsatellite loci (see P = 0.45) when compared to the GNP (AP = 0.64) and BVC (AP = 1.25). However, the number of alleles found in our study populations seem to be average when compared across Africa  held overall when allelic richness was estimated using the rarefaction method (to compensate for different samples sizes). The SVC population returned a value of 2.28, GNP lions 2.84, and the BVC population 3.29. When the rarefaction method was used to estimate private allelic richness , the SVCrica see . When obR = 0.14, similar to GNP lions (R = 0.13), which is most typical for third-order relatives  and more typical for second-order relatives, which means there are generally more kin-linked individuals in this population.Pairwise relatedness between SVC individuals of uncertain parentage is = 0.125; , 49). Th= 0.125; . AmongstOnly three mitochondrial DNA haplotypes, based on the 456 bp segment, are found for all 42 lions from the Zimbabwe Lowveld area. Two of these are unique to the BVC, and the other haplotype is shared amongst 35 individuals in the SVC and GNP, which makes the SVC and GNP populations completely monomorphic for the mitochondrial DNA segment see . It furtST = 0.22; P < 0.001). There are some indications of population differentiation between the three lion populations in the region, with the highest genetic distance  found between the BVC and SVC (FST = 0.32) and the lowest between the BVC and GNP (FST = 0.12) . The effect of inbreeding, and subsequent loss of alleles, is unlikely to have caused the genetic distinction of the SVC population, as the similarly inbred BVC population still clusters together with GNP lions. It could indicate that the SVC lions have been completely isolated during their recent population recovery, while the BVC and GNP lions still had some levels of population admixture.To test for geographic population structure, we compared the allele frequencies in the SVC lion populations with BVC and GNP. STRUCTURE analyses suggest K = 2 as the most likely number of genetic clusters (Ln P(K) = -857,6; ΔK = 887,9), in which the SVC clusters separately from the BVC and GNP populations . Very feLions are one of the most iconic species on earth, but are experiencing significant declines in population numbers and available habitat and consequently have become vulnerable to the negative effects of genetic drift and inbreeding , 19. OneIS values in this population were considered to be caused by the Wahlund effect rather than a small number of founders [IS = 0.228) was found in Hluhluwe-Emfolozi National Park , which is an isolated population that was founded by only a handful of lions [The lion population in the Savé Valley Conservancy in Zimbabwe today numbers more than 200 individuals, but was founded by only approximately 15 individuals. The nearby Bubye Valley Conservancy contains a population of 500–550 lions, which originated from 13 translocated individuals. Reserves and protected areas in Zimbabwe are fenced, with increasing human settlements between reserves. Taken together, questions emerged regarding the genetic diversity of the lions in the SVC and BVC and the possible differentiation between lion populations in the area due to effects of genetic drift. Indeed, we confirmed the loss of allelic richness and low levels of heterozygosity –53. Whenfounders . A more of lions . Even thof lions .R = 0.14) was typical of second-order relatives, and this value was lower than the relatedness found in the Etosha lion population . Intere = 0.21; . It is p = 0.21; , 57, tak = 0.21; , which m = 0.21; , 53. HowThe genetic erosion in the SVC and BVC lion populations is likely to progress in the future, due to the isolated nature of the reserves and limited space to allow for further population growth . To mainP) are also good predictors of population founding events [The SVC lion population is genetically differentiated from the BVC and GNP populations. Although inbreeding and subsequent loss of alleles may cause populations to cluster as distinct lineages in cluster analysis , this dog events , and theb sequences suggest that GNP lions originate from the same historic population. The haplotype found in this area occurs across a wide range in southern Africa [It is unlikely that the observed population structure is a result of the translocation of ten lions into the conservancy in 2005, which could have been the case if the immigrants were genetically divergent. However, private allelic richness is low in the SVC and cytochrome-n Africa . The mitn Africa , which iFrom a conservation perspective, it is important to avoid loss of genetic variation in lion populations and maintain evolutionary potential required for future survival. A large population size and the stimulation of gene flow by dispersal is normally advised to achieve this , 59, 68,S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Nyctalus lasiopterus), their relation to a new colony still in formation, and the impact of this ongoing process on the regionwide genetic makeup. Nuclear differentiation among colonies was relatively low and mostly nonsignificant. Mitochondrial variation followed this pattern, contrasting with findings for other temperate bat species. Our results suggest that this may indicate a recent population expansion. On average, female giant noctules were not more closely related to other colony members than to foreign individuals. This was also true for members of the newly forming colony and those of another, older group sampled shortly after its formation, suggesting that contrary to findings for other temperate bats, giant noctule colonies are not founded by relatives. However, mother–daughter pairs were found in the same populations more often than expected under random dispersal. Given this indication of philopatry, the lack of mitochondrial differentiation among most colonies in the region is probably due to the combination of a recent population expansion and group formation events.For elusive mammals like bats, colonization of new areas and colony formation are poorly understood, as is their relationship with the genetic structure of populations. Understanding dispersal and group formation behaviors is critical not only for a better comprehension of mammalian social dynamics, but also for guiding conservation efforts of rare and endangered species. Using nuclear and mitochondrial markers, we studied patterns of genetic diversity and differentiation among and within breeding colonies of giant noctule bats ( Studying natural populations in their habitat can prove difficult using traditional methods such as mark‐recapture and radiotelemetry  social bonds can supersede kin relations in the choice between emerging groups  within “Maria Luisa Park” (MLP) in the city of Seville . This was the larger of the two colonies, with an estimated 500 bats roosting there in 2007  located in the gardens of the zoo of Jerez de la Frontera ; this colony had an estimated population of 100–150 females. In contrast to these seminatural colonies, the fourth population is found in a large natural Mediterranean mixed oak forest in “Los Alcornocales Natural Park” (ANP) around 100–150 km southeast of Seville  and has an estimated size of several thousand individuals that were sampled at different localities.We sampled a total of 215 individuals present in four maternity colonies in southern Andalusia, Spain. The breeding colony in Doñana National Park (DNP) is located around a group of bat boxes in a small stand of mainly Eucalyptus trees near the marshes at the mouth of the Guadalquivir River . Two breeding colonies of O; N = 23), consisting of samples collected between 2003 and 2005; and (2) the Doñana “recolonization” group , sampled after the yet unexplained three‐year breakdown (2007–2009), during the subsequent recolonization process from 2010 to 2013. For both DO and DR, we selected only females that were registered breeding in the colony during more than 1 year.Samples consisted of wing punch biopsies , haplotype diversity (Hd), nucleotide diversity (π), and the number of segregating sites (S) were calculated using DNASP v. 5.10.1  and expected (He) heterozygosities, as well as deviations from Hardy–Weinberg equilibrium (HWE) were performed in CERVUS v. 3.0.6 , both among colonies and for matrilines , were estimated using ML‐Relate  and of mother–daughter pairs within our data set, but also to examine the distribution of these dyads across colonies. Assignments inconsistent with mitochondrial haplotypes were excluded. For each colony, we estimated the proportion of close associations out of all possible pairs of individuals (% r > 0.25), as well as the proportion (%) of females with at least one close relative within the colony.Pairwise and mean relatedness values (O (first colonization attempt of Doñana), followed by ANP . The median‐joining network showed a star‐shaped structure around the two most frequent haplotypes . Out of the 11 microsatellites, four  deviated significantly from HWE and were excluded from the genetic structure analysis. Selection acting on Nle11 could not be ruled out (P < 0.05), further supporting its exclusion. For estimating relatedness, we resorted to Wagner et al.'s (2006) method, implemented in ML‐Relate, and kept all 11 loci.All microsatellite loci were polymorphic, with an average of 12 alleles, and all were in linkage equilibrium. ɸST averaged 0.11 ± 0.12 (range 0–0.36). This value was due mainly to ANP, which differed significantly from all other colonies  and the three other colonies were on average low (0.015 ± 0.01), ranging between 0 and 0.035  differed slightly from those based on FST, yet both measures were significantly correlated . Nevertheless, no pairwise comparisons based on DEST were significant.Mitochondrial differentiation according to O or DR were included in the analysis , whether DNP was included or not. Among‐colony variation (among‐group component) showed a slight decrease when either Dis Table . Again, is Table . Nuclearis Table .R, H2) to 0.345 , although the latter consisted of only two females. Of the four haplotypes found in DR, one was carried by only one female and two by unrelated females , while in the latter, 9 of 12 dyads involved females from MLP . The number of mother–daughter pairs was uncorrelated with variation in sampling year for each colony , but increased significantly with the number of samples of each colony .Mean pairwise relatedness within colonies was very low  could also result in similar differences.We genotyped bats from three consolidated colonies and a recently colonized site (with two colonization events) and assayed variation both at nuclear and mitochondrial loci and levels of differentiation among the colonies. Haplotype diversity was highest in the Dr > 0.25) and mother–daughter pairs across the region. The number of females with at least one close relative in the same colony was unexpectedly high for some sites, particularly for the colony in the city park of Seville (MLP). However, it is the number of parental associations found within our complete data set and encompassing the whole area studied that stands out the most with 57.1% of parental associations found to be intracolonial. The complementary 42.9% of these involved females from separate colonies, suggesting still, relatively frequent movements and thus significant gene flow between the colonies. A recent study revealed a negative correlation between wing loading, migration tendency, and the magnitude of genetic differentiation among bat populations . This hypothesis is in agreement with the star‐like distribution of the haplotype network. Nevertheless, the presence of private haplotypes in all new colonies points to the possibility of genetic additions from other colonies (or regions) apart from an ANP source. In summary, it seems likely that the lack of structure found is mostly due to recent demographic changes, not yet counteracted by the structuring effect of philopatry.We studied two consecutive colonization attempts of DNP by giant noctules in relation to the three closest known colonies of the species. We found considerable colocalization of female relatives, pointing to a high degree of philopatry and indicating that reported movements do not necessarily result in stable relocations. The lack of differentiation among all the colonies (except for ANP) could be due to the fact that these are too young for any differentiation to become apparent at the mitochondrial level. The formation of new groups or colonies involves the sampling of alleles from one or more parent groups. The degree to which founding individuals are related to one another will influence the genetic variation of the newly formed groups, and consequently the amount of among‐group variation at the population level Storz . If the E. fuscus) by Metheny et al. .Mitochondrial DNA sequences have been uploaded to GenBank . Microsatellite genotypes, sample ID and location, R scripts for Appendix S1. Detailed description of DNA extraction, purification, sequencing and genotyping.Table S1. Summary statistics and PCR specifications for microsatellite loci.Table S2. Pairwise DEST values among populations based on microsatellite data.Click here for additional data file.