Cardamine impatiens and Cardamine resedifolia, which occupy separate habitats along the elevation gradient. Previous genome-wide analyses revealed the occurrence of distinct selective pressures in the two species, with genes involved in cold response evolving particularly fast in C. resedifolia. In this study we surveyed patterns of molecular evolution and genetic variability in a set of 19 genes, including neutral and candidate genes involved in cold response, across 10 populations each of C. resedifolia and C. impatiens from the Italian Alps (Trentino). We inferred the population structure and demographic history of the two species, and tested the occurrence of signatures of local adaptation in these genes. The results indicate that, despite a slightly higher population differentiation in C. resedifolia than in C. impatiens, both species are only weakly structured and that populations sampled at high altitude experience less gene flow than low-altitude ones. None of the genes showed signatures of positive selection, suggesting that they do not seem to play relevant roles in the current evolutionary processes of adaptation to alpine environments of these species.Species evolution depends on numerous and distinct forces, including demography and natural selection. For example, local adaptation and population structure affect the evolutionary history of species living along environmental clines. This is particularly relevant in plants, which are often characterized by limited dispersal ability and the need to respond to abiotic and biotic stress factors specific to the local environment. Here we study the demographic history and the possible existence of local adaptation in two related species of Brassicaceae,  Changes in population size and population subdivision are common features of most species′ evolutionary history. Depending on the geographic distribution of the populations, species may consist of more or less isolated populations, where neutral and non-neutral forces may have distinct effects. These forces, combined with the degree of inter-population migration and the local effective population size, define the relative role of genetic drift and selection in shaping both intra- and inter-population genetic differentiation and population structure –4. The eFST, allele frequencies) of candidate and putatively neutral loci along the geographic or environmental gradient of interest [Population structure typically results from a combination of demographic  and adaptive factors . Distinguishing the effects of such factors at the genetic level can be rather challenging. One approach relies on disentangling the effects of natural selection, which act at the locus-specific level, from the genome-wide effects of demographic processes. For instance, it is possible to compare the degree and pattern of genetic differentiation . All genes were readily sequenced by 454 pyrosequencing and included five outliers identified by Ometto et al. [Cardamine species.We studied patterns of genetic variability at 19 genes in 60 individuals each of o et al.  as putatC. impatiens we sampled five populations located in the upper margin of the species′ range . For both species, we sampled 6 individuals in each of 10 populations . For C. –2,526 m . AttentiArabidopsis thaliana as involved in stress-responses typical of high altitude habitats; and (iii) eight putatively neutral genes randomly chosen among the C. resedifolia and C. impatiens orthologs that did not display any signature of positive selection in Ometto et al. [Cardamine sequences sequenced by Ometto et al. [A. thaliana orthologues to predict intron/exon boundaries and evaluate putative gene lengths. Primers were then designed to delimit partially overlapping sequences with length compatible with 454 read size . A list of primers and PCR conditions used is provided in We employed a high-throughput approach in which tagged PCR products for each gene and individual (i.e. amplicons) were sequenced at once using the Roche 454 GS-FLX sequencing technology. We sequenced 19 genes, including: (i) four candidate genes for the adaptation to high altitude ; (ii) seo et al.   according to the manufacturer′s instructions. To remove residual enzymatic activity, all steps were followed by purification with SPRI beads (Beckman Coulter). Library preparation for 454 sequencing was carried out using a modified GS FLX Titanium Rapid Library preparation kit. In particular, the A-tailing of the two super-pools was performed in a total volume of 20 μl containing 200 ng of DNA, 1 x RL buffer, 2 mM dATP, and 5 unit of Taq polymerase (Sigma) at 72°C for 20 min; subsequently, 2.5 μl of ATP from the same kit was added for adapter ligation. Library quantitation was performed with the Kapa Library Quantification kit (Resnova). EmPCR library was performed according to the GS FLX Titanium emPCR Method Manual—Lib-L LV protocol for the short super-pool, and according to the Long Fragment Lib-A emPCR Amplification protocol for the long super-pool library. Finally, the two libraries were sequenced separately in a two-region picotiter plate of a GS FLX Titanium instrument (Roche 454 Life Science) using GS FLX Titanium Sequencing kit (Roche) according to the manufacturer′s instructions.The PCR products of each sample were pooled in a short (amplicon size <600 bp) and a long pool (>600 bp). Each of the resulting pools was purified with Agencourt AMPure XP kit (Beckman Coulter), blunt ended, methylated with CpG methyltransferase, and ligated to one of 120 sample-specific custom barcoded adapters (MIDs). Following the quantification with the Quant-iT Picogreen dsDNA Assay kit (Invitrogen) in a Synergy 2 fluorometer (BioTek), pools were normalized and combined into a short and a long super-pool to maximize sequencing efficiency. The two super-pools were then dephosphorylated and digested with the restriction enzyme GS Amplicon Variant Analyzer (AVA) application version 2.3 (Roche) to assign each read to the correct individual/gene assembly. Specifically, AVA associated a read to the appropriate individual based on its MID, and to the appropriate gene based on its match to the A. thaliana reference sequence. This process assigned 460,580 reads to one of the 2,276 assemblies . Assembled reads had mean length ± standard deviation of 395.0 ± 110.6 bp. To improve the alignments, we imported the assemblies and the relevant reference sequences into Geneious v5.3 [We used the ous v5.3 , and re-n of reads covering the polymorphic site, the number k of reads with the minor allele, and the error rate fM. This probability follows a binomial distribution and can be written asfM corresponds to the mean error rate estimated across all regions and polymorphism types described in Fior et al. [fM = 0.024. We then iteratively calculated Pfalse for increasing values of n, whilst setting k equal to the smallest integer following 0.3 ×n. We estimated that a minimum depth of coverage of n = 5 gives a reasonably low probability of a false polymorphism detection of Pfalse = 0.0055. Subsequently, we scanned each assembly for the presence of polymorphic sites covered by at least 5 reads and with the minor allele present in more than 30% of them. To facilitate haplotype reconstruction, we also included sites with insertion/deletion polymorphisms. The script eventually recorded the two alleles, the read they were found in, and the relative position within the assembly. This information was used to reconstruct minimal haplotypes for each read of the assembly. Since minimal haplotypes overlapped only partially, we identified the complete region haplotypes by visually recognizing consistent association between consecutive alleles. We identified polymorphisms in a total of 222 individual/gene combinations , 93 of which in C. resedifolia and 125 in C. impatiens. In 14 assemblies, haplotype phasing was hindered by the presence of a pair of consecutive polymorphic sites not covered by the same reads. We therefore randomly picked one partial haplotype from the two halves marked by the discontinuity and built two putatively complete minimal haplotypes. This reconstruction does not affect measures of gene polymorphism and divergence, nor the population structure and association analyses. Following the minimal haplotype identification, we used Geneious to build the consensus sequence of each assembly by retaining the most common nucleotide observed across reads. Finally, we incorporated the minimal haplotype(s) into the consensus by using the known alleles′ positions, eventually generating two sequences per gene for each individual. All sequences have been deposited in GenBank under the accession numbers KJ427834–KJ432279.Identification of heterozygous sites, characterization of the alleles, and haplotype reconstruction were done using a custom perl pipeline. The identification of heterozygous sites is often complicated by the possible presence of sequencing errors , or error et al. , namely A. thaliana orthologous sequence was added to the alignment to allow the identification of intron/exon boundaries in the Cardamine sequences and trim incomplete codons at the 5′ and 3′ ends of the alignment.We used the MUSCLE algorithm  implemenC. impatiens and C. resedifolia populations. For each species we used as input data a matrix containing all intraspecific variable sites (excluding singletons). We ran the program using the admixture model with uncorrelated allele frequencies and no location information (LOCPRIOR = 0). We run 10 independent replicates with a number K of clusters ranging from 1 to 6, with a burn-in period of 200,000 iterations followed by another 1,000,000 iterations. The putative optimal K was calculated by identifying the value at which the likelihood of the data reaches a plateau and using the approach of Evanno et al. [We used the model-based clustering method implemented in the software STRUCTURE ,39 to ino et al. . Individo et al. .-6 to account for the uncertainty of the spatial coordinates of individuals. For each species we run five (ten for C. impatiens) replicate analyses, each with a burn-in of 200,000 iterations followed by another 10,000,000 iterations and a thinning of 100. Analyses were done using the uncorrelated frequency model, and the number K of clusters ranged between 1 and 12.In a second approach, the inference of population structure was based on the explicit use of both genetic and spatial information. We used Geneland  to simulFST between populations  and theby DnaSP . Tests w by Jost  using th by Jost . For eacC. impatiens and C. resedifolia. For both species, we simulated four simple demographic history scenarios  framework implemented in the package ABCtoolbox  to infercenarios : constan-9–5×10-7 mutations/site/generation) and the recombination rate (0–1×10-8 recombination events/site/generation). As summary statistics we used the number of segregating sites, S, and the average pairwise difference per base pair, π [et al. [et al. [The four models were described by one to five parameters, including population size and time of population size changes, to which we added priors for the mutation rate , was used to evaluate the linkage disequilibrium index r2 [We estimated basic population genetic parameters and performed neutrality tests with the program DnaSP v5.10 . Levels se pair; ) and θW g sites; ). Divergrsity, H , and thetatistic . The protatistic   for the period 2003–2010, and mean rainfall precipitation for the period 1981–2010 . Mean values were estimated separately for spring and summer, coinciding with the vegetative and reproductive phase of plants , while they were not correlated with geographical coordinates , suggesting that they can impose selective pressure on the sampled populations irrespectively from a possible geographic-based population-structure. Similarly, mean rainfall precipitation values were not significantly correlated with longitude, latitude and elevation , nor were they correlated to mean temperature for a given season . Four independent association analyses (one per climatic variable set as predictor) were performed with the mixed linear model approach [K ranging from 2 to 5. Given the small sample size and in order to minimize type II errors, the whole SNP dataset was used for this analysis. To correct for multiple testing, P values were calculated based on 100,000 permutations of the dataset and further corrected by the number of independent tests .We tested for association between genetic variability and environmental features associated with cold and desiccation stress, i.e. temperature and rainfall precipitation. For each population, we determined mean daily temperature using reconstructed satellite data time series . These samples were all successfully sequenced using the high-throughput sequencing approach, and subsequent haplotype reconstruction resulted in a final dataset of 104–120 haplotypes per gene for each species. The sequenced genomic regions had lengths ranging from 504 to 1,748 bp (mean ± SD = 931 ± 345 bp), and contained up to 8 intronic and 9 coding regions. An overview of gene lengths and organization is presented in Tables Depending on the gene region, we could obtain PCR amplification in 52–60 individuals of both C. impatiens and C. resedifolia in the Trentino region.Our analyses revealed weak population structure for both et al. [K = 2 as the optimal number of clusters  to either a H or L population revealed that such difference was not expected by chance , and suggests some degree of population differentiation along the altitudinal gradient. When each of the five pairs of adjacent H and L-populations was tested separately, the asymmetry was statistically significant for three comparisons (0.0001 < P < 0.05).The approach of Evanno et al. , based oet al. ,39, identers Fig –2c. Suchters Fig  comparedters Fig . SpecifiK = 5, which then can be considered as the best K according to the plateau criterion, with most of the populations mainly belonging to separate clusters. Exceptions were found for few individuals within single populations, and for the Cad population, where all individuals were admixed  than only L-populations (0.262), although the difference was not statistically significant . We also investigated population differentiation using the actual differentiation index Dest [Dest was 0.064, and significantly higher for pairwise comparisons involving only H-populations (0.089) than only L-populations , consistent with larger differentiation between populations found at the upper distribution range limit. The analysis of the isolation-by-distance model rejected an association between genetic (FST) and geographic distance . The correlation with distance remained not significant when using Dest , suggesting that geographic distribution has limited effects on the genetic composition and differentiation of C. impatiens populations.The overall ex Dest , which hWe used the ABC approach to infer the most likely demographic history of the Trentino populations among four model scenarios. Power analysis revealed that the constant population size model (CON), the expansion model (EXP), and the reduction model (RED) could be correctly identified as correct models in our ABC analyses . The REDP = 0.0003; 10,000 randomizations). This asymmetry was confirmed for three out of the five pairs of adjacent H and L-populations (0.0001 < P < 0.04). In C. resedifolia the likelihood of the observed genetic variability did not reach a plateau at the increase of K. Nonetheless, an interesting pattern emerged with K = 3 clusters, which defined a group of four western populations having very similar ancestry, two “central” populations with genetic ancestry similar to the easternmost population, and three eastern populations in which the third cluster contributed heavily. The overall structure pattern was confirmed at K = 4, when a fourth cluster was detected with extremely low frequency (0.001 in a single population), and at K = 5, when an additional cluster separated one of the eastern populations. The spatial structure was strongly supported by the results of the landscape genetics approach of Geneland, which inferred K = 3 clusters describing a longitudinal population subdivision across Trentino  than only L-populations . This difference was confirmed by Dest , and was consistent with less gene flow among populations found at high altitudes than among populations found at low altitude. Interestingly and opposite to what observed for C. impatiens, in C. resedifolia there is isolation-by-distance .The overall differentiation as measured by The ABC approach inferred in the RED model the most likely demographic history of the Trentino populations . PosteriC. resedifolia and C. impatiens (Tables P = 0.09), and levels of nucleotide polymorphism, measured as π and θW, were also similar in C. resedifolia and C. impatiens in both the coding and non-coding regions . Taken together, and assuming equal mutation rates, these results suggest that the two species have comparable effective population size Ne. This conclusion, however, is in part contradicted by two further observations. The first is based on the analysis of haplotype diversity values estimated separately in each of the sampled populations: in this case the across-populations mean was significantly larger in C. resedifolia than in C. impatiens . The contrast to the overall haplotype diversity analysis is consistent with relatively higher within-population and lower-between genetic homogeneity in C. impatiens, as supported by a significantly larger differentiation index Dest in C. impatiens than in C. resedifolia . The second observation is based on the analysis of genetic association between polymorphic sites. The two species had levels of intragenic linkage disequilibrium (ZZ) that were not statistically significant (P = 0.084). However, the degree of linkage disequilibrium r2 over all pairwise comparisons of the concatenated dataset was significantly lower in C. resedifolia than in C. impatiens , suggesting long-range linkage disequilibrium in the latter, possibly associated with higher levels of selfing.Analysis of basic population genetics statistics revealed little differences between patterns of molecular evolution of s Tables  Tables. DT values producing a mean of DT across gene close to zero  levels, which rules against recent, rare, variants typical of recent positive selection , and according to Tajima′s test no gene departed from neutrality in this species. To further test for departures from the neutral model of evolution, we employed the maximum frequency of derived mutations (MFDM) test proposed by Li [P value lower than 0.05 in C. impatiens, which however became not significant after multiple testing correction. In C. resedifolia, no gene departed from neutral expectations according to the MFDM test. Thus, neutrality tests did not provide evidence for the action of positive selection in any of the genes analyzed, suggesting that natural selection may have not occurred in these genes during the recent history of the two species. Consistently, there were no difference in levels of nucleotide polymorphism, haplotype diversity, linkage disequilibrium (ZZ), and Tajima′s D between candidate genes and neutral genes in neither C. impatiens and C. resedifolia , indicating similar rates of evolution in the two classes of genes.The analysis of the polymorphism frequency spectrum (which was limited to the non-coding portions of the genes) revealed fairly large heterogeneity across genes in both species, with both positive and negative Tajima′s o Tables  and 2. Iima test  revealede sweep, ). Rathered by Li . Only a We next investigated whether the joint analysis of polymorphism and divergence data of coding regions provided patterns compatible with the action of positive selection Tables  and 2. TC. impatiens: range of mean precipitation was 213–276 mm in spring and 224–346 in summer; for C. resedifolia: ranges were 200–296 in spring and 235–346 in summer; C. impatiens, a total of 23 SNPs (hereafter referred to as SNPsrain) distributed in 9 genes were significantly associated with variation in mean spring rainfall precipitation. The number of SNPsrain changed depending on the number of genetic clusters K used to define the Q-matrix, but most were confirmed for different K values and all but one were found associated at K = 3. Twelve of these SNPsrain were also significantly associated to variation in mean summer rainfall precipitation. There was no significant enrichment in candidate genes among those harbouring SNPsrain .Populations sampled at their upper and lower altitudinal bounds experience contrasting temperature regimes . HoweverC. resedifolia, where six SNPsrain were associated with mean spring (n = 6) and summer (n = 4) rainfall precipitation and distributed over six genes .The results were very similar in ix genes . All sucC. impatiens, two observations point to a lack of appreciable population structure across the sampling transect. The first is the absence of a consensus in the number of genetic clusters identified by the different approaches used in this study. For instance, STRUCTURE inferred the presence of two genetic clusters, while Geneland detected four clusters  involved in salt stress response  was larger than in H-populations , consistent with a larger effective population size in the former.Compared to  species . The twoW, and longitude in H-populations . This suggests that western H-populations harbor more genetic diversity than eastern populations, possibly because of a longer establishment allowing for higher gene flow with the neighboring populations, thus increasing the heterozygosity of the populations. This hypothesis is in part supported by a phylogeographic analysis of C. resedifolia across its distributional range [et al. [K = 10 where genetic clusters identify sequential populations along the Western, and especially Eastern Alps (which include Trentino). The population structure thus conforms to that of a species experiencing fair degree of gene flow between populations, which are however sufficiently isolated (given their habitat preference) to lead to significant genetic isolation-by-distance. The results of the demographic analysis suggested a quite severe population reduction around 45,000 years ago , thus before the LGM . Therefore, the Trentino population seems to have only marginally suffered the habitat contraction following the LGM, either because this population was sampled very close to the putative southern refugia [C. resedifolia populations may indeed contain diversity from multiple ancestral peripheral populations [The results of STRUCTURE and Geneland revealed a population subdivision defining a clear longitudinal pattern of population structure. Notably, this genetic differentiation is accompanied by a significant correlation between population-specific polymorphism, θal range ,67, and  refugia  and thusC. resedifolia no gene departed from neutrality, suggesting that the candidate genes did not undergo episodes of positive selection in the recent history of the species, neither at the species nor at the population level. The association analyses revealed six SNPsrain in as many genes associated with mean rainfall precipitation, one of which was located at a non-synonymous site of the salt stress responsive gene AT1G07890. However, as mentioned above, rainfall data suffer from low resolution, thus preventing robust conclusions about the significance of this result. For instance, the two SNPsrain associated with mean spring rainfall had their minor allele at high frequency in two populations, Rum and Ter, that had the same rainfall values and were assigned to the same cluster in the population structure analysis  regimes, we did not have enough power to detect departures from neutrality due to a partial coverage of the genetic diversity of the species. Our samples cover only a limited portion of the species distribution range, and a broader sampling (including more extreme environments) may provide precious genetic variants involved in local adaptation. In addition, the presence of gene flow among populations may also affect the fixation probability of the beneficial allele by introducing less fit migrant alleles  would definitely increase the chance of finding the genetic basis for local adaptation in these species. Moreover, the landscape genomic analyses may have lacked enough power due to the low number of individuals sampled in each population, although this may not have been a limited factor when contrasting low- versus high-altitude populations. A final possibility rests upon the strong link between efficiency of selection and the demographic processes and properties typical of the two species. Depending on the strength of local selective pressures, local adaptation may in fact be hindered by the reduction in the efficiency of selection due to small effective population size [C. resedifolia lineage may have been captured only by the codon substitution model approach. Additional population genomics approaches will in the future certainly help identifying possible targets of positive selection and local adaptation in the two species.Several reasons may explain the absence of signatures of local adaptation in es (e.g. ). The sees (e.g. ), actualion size . This reion size  or from ion size , which wion size . The reaS1 FigFor each demographic model we report the marginal densities (a) and modes (b), the bandwidth of the kernel (c), and the posterior probabilities of each of the model parameters (d).(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


We used 10 microsatellite loci to investigate the spatial structure, patterns of gene flow and migration routes of Lampetra populations in Europe. We sampled 11 populations including the migratory L. fluviatilis and four resident species, L. planeri, L. alavariensis, L. auremensis and L. lusitanica, the last three endemic to the Iberian Peninsula. In this southern glacial refugium almost all sampled populations represent a distinct genetic cluster, showing high levels of allopatric differentiation, reflecting long periods of isolation. As result of their more recent common ancestor, populations from northern Europe are less divergent among them, they are represented by fewer genetic clusters, and there is evidence of strong recent gene flow among populations. These previously glaciated areas from northern Europe may have been colonized from lampreys expanding out of the Iberian refugia. The pair L. fluviatilis/L. planeri is apparently at different stages of speciation in different locations, showing evidences of high reproductive isolation in the southern refugium, and low differentiation in the north.Ice ages are known to be the most dominant palaeoclimatic feature occurring on Earth, producing severe climatic oscillations and consequently shaping the distribution and the population structure of several species. Lampreys constitute excellent models to study the colonization of freshwater systems, as they commonly appear in pairs of closely related species of anadromous  The Quaternary climatic oscillations and geographic restrictions imposed by the impassable glaciated areas are thought to have had major effects on the evolution and dispersal of various species e.g. ,2). It i. It i2])via sea. These fish breed in fresh water, having ample opportunities to colonize these unexploited systems and establish freshwater isolates [Recently deglaciated regions were relatively inaccessible to freshwater fishes, as they do not normally disperse among river basins; they were, however, easily reached by anadromous fishes (migratory species that reproduce in freshwater before migrating to the sea where they grow to the adult stage), which could access isolated basins isolates . In someisolates ,6. In soisolates . For thiLampetra fluviatilis) and the resident European brook lamprey (Lampetra planeri) are considered paired species. They only occur in European watersheds, extending from southern Norway to the western Mediterranean and the Iberian Peninsula in the south. The three brook lampreys Lampetra alavariensis, Lampetra auremensis and Lampetra lusitanica, which are endemic to western Iberian Peninsula  [L. fluviatilis [L. planeri is found in several river basins, L. alavariensis, L. auremensis and L. lusitanica are confined to one or two basins, and the anadromous L. fluviatilis currently occurs in the Tagus river basin only [L. alavariensis, L. auremensis and L. lusitanica are well supported monophyletic groups, divergent from the present-day L. fluviatilis. However, L. planeri share haplotypes with the parasitic form, implying that their emergence was more recent [L. fluviatilis and L. planeri has thus been considered problematic, as studies using different markers have revealed lack of differentiation between the species  , are appviatilis . In thissin only ,10. The sin only ,11. The e recent ,11. The es (e.g. ), leavin [et al. , using gL. fluviatilis and in four resident species, L. planeri, L. alavariensis, L. auremensis and L. lusitanica. We tested the hypotheses that the Iberia is a source for the northward recolonization by the migratory species, the possible existence of a speciation gradient in Europe, and test for contemporary gene flow among and within species.In this study, we investigate postglacial colonization of European fresh waters by analyzing patterns of genetic differentiation in a group with alternative life histories. We analyzed 10 polymorphic microsatellite loci, coupled with two mitochondrial genes, in the anadromous Travaux publics, Agriculture, Ruralité, Nature, Forêt et Patrimoine, and the German Federal Agency for Nature Conservation, respectively. In Finland, the samples have been collected from commercial lamprey catches, so those animals were caught for human consumption, and no authorization was needed.This study was carried out in strict accordance with the recommendations present in the Guide for the Care and Use of Laboratory Animals of the European Union—in Portugal represented by the Decree-Law n°129/92, Portaria n°1005/92. By the time the experimental work took place, the University’s ethics committee only dealt with research involving humans. Nevertheless, since two authors have an official license for animal experimentation (Category C from FELASA), issued by the Veterinary National Authority (DGV), Portuguese Ministry of Agriculture and Sea, the experiments involving living animals were performed in accordance to international rules regarding animal welfare. Collection of samples in Portugal was carried out without euthanizing the specimens, using the collecting permits provided by the Institute for Nature Conservation and Forestry, I.P. . In Belgium and Germany sampling authorizations are provided by Gouvernement Wallon—-1), a piece of tissue was removed from the dorsal fin. After recovery individuals were released near the capturing sites. Sampled species were the European river lamprey L. fluviatilis, the European brook lamprey L. planeri, and the three recently described Iberian brook lampreys L. alavariensis, L. auremensis and L. lusitanica [L. fluviatilis and L. planeri are found in sympatry (m refer to the migratory species (L. fluviatilis), and by r to resident species . When treated together, populations from Belgium, Germany and Finland are hereinafter referred as “northern populations” and populations from the Iberian Peninsula as “southern populations”. All rivers sampled in the Iberian Peninsula drain to the Atlantic Ocean, rivers Warche (Meuse basin) and Schaale (Elbe basin) drain to the North Sea, whereas rivers Beke (Warnow basin) and Lestijoki drain to the Baltic Sea  fluorescent dyes. Primer sets were grouped into three multiplex reactions  and 1× DreamTaq™ Buffer. PCR conditions were as follows: initial denaturation at 94°C for 1 min, followed by 25 cycles of 30 sec at 94°C, annealing for 30 sec at temperatures ranging from 55 to 60°C and 30 sec at 72°C, and a final extension of 7 min at 72°C. A number of sets of difficult amplification were completed using a Multiplex PCR Kit (Qiagen®) with 5 μl Qiagen Multiplex PCR master Mix, 3 μl RNase-free water, 1 μl Primer Mix (2 μM each primer) and 1 μl of 50 ng μl-1 of genomic DNA, using the following protocol: initial activation step at 95°C for 15 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 57°C for 90 sec and extension at 72°C for 60 sec, and a final extension of 30 min at 60°C. The PCR reactions were conducted on a Bio-Rad® thermal cycler.A total of 415 specimens from 10 sites were used in the analysis, with sample sizes ranging from 29 to 52 . Initialmarinus: ,17; Lethn sp. N: ; Ichthyo fossor: ; Lampetrardsoni:  were scrardsoni: ; Lspn 01ardsoni: ; and Pmani: [μ 5 . These 1eactions , and pol® 310 Genetic Analyzer and fragments were sized with GeneScan™-500 LIZ™ Size Standard. Allele sizes were determined using the software GeneMapper® 3.7 (Applied Biosystems®).Samples were genotyped in an ABI PRISM4 permutations, as implemented in ARLEQUIN 3.11 [sensu Nei 1978) and mean number of alleles across loci (MNA), inferred using GENETIX 4.05.2 [Microsatellite loci were first tested for null alleles, stuttering and large allele dropout using the software MICRO-CHECKER 2.2.3 . Each miUIN 3.11 . GeneticX 4.05.2 , with thX 4.05.2 .FST using the Weir & Cockerham's estimator [4 permutations, as implemented in GENETIX. The distribution of genetic variation was accessed among and within the 11 samples, the sympatric L. fluviatilis and L. planeri, and the genetic clusters attained with population structure analysis, through analysis of molecular variance (AMOVA) [4 permutations.The genetic differentiation among samples was accessed through pairwise stimator , and sig (AMOVA) . These a5, followed by 5 million Markov steps. Using the same parameters, two additional structure analyses were performed, one including solely the eight samples of L. planeri and L. fluviatilis (K between 1 and 9), and the other including the four samples from the North (K between 1 and 5). These allow detection of further structure in these populations, if present, that otherwise would be hidden due to the high differentiation among the five species, and between the northern and southern samples. The optimal K, and clustering achieved, was inferred using the protocol defined by Evanno et al. [The Bayesian model-based clustering approach implemented in STRUCTURE 2.2  was usedo et al.  as impleo et al. , and tako et al.  was used0) immigrants from unsampled populations . We useations’, , and the. method  to compuPatterns of differentiation were visualized by principal coordinates analysis (PCoA), a multivariate technique that allows to find and plot the major patterns within a multivariate dataset, like multiple loci and multiple samples. This analysis was computed using GenAlEx 6.5 ,36.L. fluviatilis and L. planeri from Portugal. NewHybrids uses a Bayesian approach to identify different categories of hybrid individuals through the computation of the posterior probability that individuals fall into different hybrid  or pure parental categories. It uses the allele frequencies of multilocus genotypes and a Markov Chain Monte Carlo procedure. Simulations were performed with a burn-in period of 105, followed by a sampling period of 105 Markov steps. A threshold of posterior probability > 50% was set up to classify an individual as belonging to a certain category.The software NewHybrids 1.1  was usedm) between migratory populations were inferred using a Bayesian assignment test-based method in the program BAYESASS 3.0.1 [7 MCMC iterations (discarding the first 106 iterations as burn-in) were performed, and samples were collected every 2000 iterations. The convergence and stability of the MCMC algorithm was checked by visual inspection of plotted posterior parameter estimates using the software Tracer 1.6 [Estimates of recent migration rates m) betwee5 simulations, and the distribution of allele frequency classes was examined for a deviation from the normal L-shaped distribution [M ratio (M = k/r) statistic test as implemented in M_P_VAL [k is the number of alleles present at a given microsatellite locus and r is the overall range in allele size. In recently reduced populations M is expected to be smaller than in populations at equilibrium, since the loss in any allele will contribute to a reduction in k, whereas only a loss of the smallest or largest alleles will contribute to a reduction in r, and thus k is expected to decrease more quickly than r. Reductions in population size were considered significant if less than 5% of the replicates are below the observed M value. Following Garza & Williamson (2001) [ps = 0.9, Δg = 3.5 and three values of θ , where θ = 4Neμ , ps is the proportion of one-step mutations, and Δg is the average size of non one-step mutations.Demographic signatures of recent bottlenecks were tested using the heterozygosity excess method implemented in BOTTLENECK 1.2.02  under thribution . Past re M_P_VAL , where kn (2001) , we usedPopulations LIS and OES were not included in the demographic analysis because they present only one polymorphic locus.For a better understanding of the historical processes that may underlie present population structure, we performed a phylogenetic analysis of mtDNA sequences of the populations included in this study, and additional populations from across the species distributional range.m, BEKEr, ELBEm, WARCr) were amplified and sequenced for both cytochrome b and ATPase 6/8 mitochondrial genes, following the protocol in Mateus et al. [L. planeri and L. fluviatilis. MtDNA sequencing of 44 individuals from LESTm, BEKEr, ELBEm and WARCr revealed a total of 14 new haplotypes (H83 to H96), which combined with the 52 haplotypes from GenBank [A total of 44 samples from northern Europe . One sequence each of the species ctively; ,11).The phylogenetic relationships among mtDNA haplotypes were reconstructed using PAUP*  by the nμ 5 in SPLr and NABr; Iun 10 in SPLr and BEKEr; Iun 5 in ELBEm and BEKEr; and Iun 14 in SPLr and BEKEr. Significant null alleles’ signature is related with heterozygote deficit and therefore with deviations from Hardy–Weinberg equilibrium, as seen in For microsatellites, signs of null alleles were detected with MICRO-CHECKER for a few situations across loci and populations: Pmar, SPLr and SADOr showed three unique alleles each, BEKE two, and SFL one (r and OESr) to 3.8 (ELBEm), mean allelic richness (AR) from 1.08 (LISr) to 2.62 (ELBEm), and expected heterozygosity (He) from 0.0239 (LISr) to 0.4417 (NABr) (The summary statistics of the genetic diversity indices for each locus and sample are provided in  SFL one . The mea7 (NABr) .FST = 0.498, P<0.001) with pairwise FST values ranging from 0.0114 (ELBEm-LESTm) to 0.8915 (OESr-ESMr), all being significant  (A considerable level of genetic differentiation among samples was observed (average  P<0.05) .P<0.001); AMOVA between the sympatric paired L. fluviatilis/L. planeri from the Tagus basin revealed that 31.64% of the variance was significantly (P<0.001) explained among species; and AMOVA among the eight genetic groups attained with STRUCTURE .AMOVA analysis among the 11 samples indicated that 48.34% of genetic variance occurred among samples  WARCr, 3) ESMr, 4) LISr+OESr, 5) NABr, 6) SPLr, 7) SFLm and 8) SADOr  and L. fluviatilis from Tagus , with a greater number of admixed individuals in the anadromous populations (LESTm and ELBEm) (r) are distinct and constitute a distinct genetic cluster with high proportion of membership  constitute two distinct genetic clusters  that present a few admixed individuals between them. SPLr also shows some evidence of admixture with ESMr (L. planeri and L. fluviatilis (m+BEKEr+ELBEm) is well supported.The STRUCTURE analysis using the microsatellite data revealed that the 11 samples are grouped in eight distinct genetic clusters: 1) LEST8) SADOr . The fird ELBEm) . Most inp 0.949; . This isith ESMr . When STviatilis , K = 5, viatilis , K = 2, r, ESMr, NABr and SADOr) 100% of the individuals were assigned to their correct source population  and the resident L. planeri from Warnow (BEKEr) had individuals assigned among the three populations, in agreement with STRUCTURE, in which the three populations form a distinct genetic cluster (see above). The sympatric L. fluviatilis and L. planeri (SFLm and SPLr) had almost all individuals assigned correctly (96% and 98%), and small percentages (4% and 2%) assigned between them. LISr and OESr showed 73% and 97%, respectively, of correctly assigned individuals, and the remaining were assigned also among each other (P of 0.01 for first-generation (F0) immigrant analysis, a total of seven individuals had a probability below the threshold value, but all were assigned to a sampled location: one individual of LESTm was assigned to BEKEr; one of BEKEr was assigned to ELBEm; one individual of ELBEm was assigned to BEKEr and another to WARCr; one individual of WARCr was assigned to ELBEm; one individual of SPLr was assigned to SFLm; and one individual of SFLm was assigned to LESTm.Individual assignment tests were applied to further investigate the genetic distinctiveness of the populations. In four populations of resident species , revealed the existence of mainly six distinct clusters, NABBEm+SFLm . These rThe neighbour-joining phylogenetic tree attained from mtDNA sequences is characterized by low structuring, with many basal polytomies . Only thL. planeri and L. fluviatilis was investigated using sympatric populations, in this case L. planeri and L. fluviatilis from the Tagus basin, Portugal , using NewHybrids. In this analysis, each individual was assigned a posterior probability (p) of belonging to one of the six different genotype classes resulting from two generations. From the 52 samples of L. planeri, 49 (94%) were classified as being pure L. planeri using the posterior probability threshold of 0.5, 20 of which showing p > 0.99 and 25 showing 0.8 < p < 0.99  with posterior probability of 0.664, and one individual was classified as being pure L. fluviatilis (posterior probability = 0.537) (L. fluviatilis were identified as such (pure L. fluviatilis), from which 40 exhibit p > 0.99, and 5 showing 0.8 < p < 0.99. No F1 or backcross hybrids were found in any of the species (The existence of putative hybrids between p < 0.99 . For thi= 0.537) . All the species .m) among migratory populations were estimated using BAYESASS. This analysis was performed for the three anadromous populations included in the study, i.e., L. fluviatilis from Tagus (SFLm), from Elbe (ELBEm) and from Lestijoki (LESTm). The proportion of individuals derived from their own location was high in SFLm (m = 0.979) and in LESTm (m = 0.968), and relatively low in ELBEm (m = 0.705) (m = 0.286) was detected from LESTm into ELBEm. SFLm is the most isolated population, with the highest proportion of non-immigrants (m = 0.979) and low migration rates (m≤0.02) in both directions (Recent migration rates (= 0.705) . Accordirections , Table 6r), which showed significant (P<0.05) heterozygote excess according to the three mutational models tested, and a shift in the distribution of allele frequency classes from the expected L-shaped distribution (r) also presents significant (P<0.01) heterozygote excess according to IAM, but no deviation from the expected L-shaped distribution , as the M ratio was significantly smaller than the equilibrium expectation (P<0.05) for all the prebottleneck θ values considered. None of the remaining populations presented signs of reduction in population size , haplotypic richness increases from north to south.The colonization processes that took place after the glacial periods, when populations from the southern Mediterranean peninsulas expanded north across Europe, shaped, together with recent processes, the biodiversity of current taxa. Southern populations isolated in refugia and sub-refugia accumulated variation through the ice ages, and the founders that rapidly moved northward during interglacials only represented a subsample of the southern diversity . This pan et al.  for the  [et al.  for Nortn et al.  and Spic [et al.  found thet al. [Barbus sclateri [Squalius aradensis [Pseudochondrostoma polylepis [et al. [This study is another evidence of the Iberian Peninsula as a glacial refugium, as proposed by Taberlet et al. , and possclateri , Squaliuradensis  and Pseuolylepis . Likewis [et al.  with AtlL. planeri from Warche in Belgium, which constitutes a distinct genetic cluster with high proportion of membership. This population seems to be facing a genetic bottleneck . Nabão (NABr) population presented significant heterozygote excess according to the infinite allele model (IAM). These results, however, were not corroborated by mtDNA data, where Fu’s Fs [D [r calculated in the present study, and NABr from Mateus et al. [versus the recent history retrieved from microsatellite loci. This would mean that populations historically not showing evidence for contraction in population size (as attained from mtDNA data), may have faced a recent genetic bottleneck, as revealed by microsatellite data. Grouping of Iberian populations by STRUCTURE reflects in general their specific status, with the exception of LISr and OESr that were grouped together, but not grouped with the other population of L. planeri from Portugal (SPLr). Those two populations present very low levels of genetic diversity, having one single polymorphic locus and no private alleles. In general, the alleles present in those populations are rather common, most of the times having a frequency of more than 50% in other populations, namely SPLr. The differentiation of those populations from SPLr seems, therefore, to reflect this lack of diversity, which statistically makes them unique. Those populations may be facing a genetic bottleneck, but this analysis could not be performed due to the existence of a single polymorphic locus.Demographic analyses revealed consistent signs for recent contraction of population size in the population from Meuse , unlikeL. fluviatilis from the Iberian Peninsula (the SFLm genetic cluster). Because contemporary gene flow from south to north and vice-versa is apparently happening in very small proportions  share haplotype 62 with L. fluviatilis from the Tagus watershed (Iberian Peninsula)  see , and is The repeated emergence of resident forms from ancestral migratory ones in different locations and times is a phenomenon very well known in lampreys –7,60, anL. alavariensis, L. auremensis and L. lusitanica are well supported species, while the brook L. planeri shares haplotypes with the parasitic form, which is evidence that its emergence was more recent [L. planeri and L. fluviatilis the scenario is different, as levels of genetic differentiation vary among populations. Sympatric L. fluviatilis and L. planeri from the Tagus basin present significant genetic differentiation and almost no signal of hybridization. These results are in agreement with the previous work using restriction site-associated DNA (RAD) sequencing [L. fluviatilis and L. planeri, however, indicate that these species are grouped in a single cluster  and may be experiencing, or have experienced until recently, gene flow. This scenario is most likely explained by the postglacial colonization of northern habitats by a southern L. fluviatilis-type and, consequently, later emergence of northern populations, as explained below. Differentiation between resident and anadromous populations may, therefore, be an ongoing process in many locations, where speciation is still underway. This scenario was also found for the same species in France, where variable levels of divergence were found among sympatric population pairs [Ichthyomyzon unicuspis) and northern brook (Ichthyomyzon fossor) lampreys from the Great Lakes, suggesting the existence of ongoing gene flow between them at least in this region [Previous data on mtDNA suggested that the three Iberian brook lampreys e recent ,11. Presquencing  that sugon pairs . This mas region .m and LESTm, which is corroborated by the assignment tests  and adjacent coastal area. Lampetra fluviatilis migrants have been separated on the basis of size into “typical” and “praecox” forms, whose mean lengths are approximately 30 and 24 cm, and mean weights about 53 and 22 g, respectively [et al. [BAYESASS revealed high recent gene flow between the migratory northern populations ELBEests see . In contdings of , who sugectively . The sizectively . The pop [et al.  for this [et al. , which iLampetra, placing the Iberian Peninsula as a major source of postglacial colonization for this genus. We suggest the existence of a speciation continuum between L. fluviatilis and L. planeri, i.e., different populations of this pair likely represent differentiation at different stages, as result of the different timing of colonization and refugial persistence. This hypothesis should be further investigated with the addition of more samples, namely of sympatric populations.The use of microsatellites, coupled with information of mtDNA data, has proved effective in unravelling the patterns of colonization in European L. fluviatilis from the Iberian Peninsula has limited dispersion movements, resembling the “praecox” form described for this species by Abou-Seedo and Potter (1979).Gene flow between migratory populations is high in northern regions, but low between northern populations and the southern Iberian population. We hypothesise that the migratory S1 FigPlots represent the log probability along iterations, where the burn-in iterations are indicated in light grey and sample iterations in black; and the Bayesian posterior density of the parameter estimates for a) the three migratory populations and b) all the 11 populations. As should be expected in a case of convergence, the log probability oscillates around a plateau, and the oscillations are quite regular, i.e., there are no persistent lows or highs  in the plot.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableP<0.05; **, P<0.01; ***, P<0.001; n, sample size; †, private allele with frequency >50%.Sample acronyms correspond to locations as in (DOCX)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Rhynchocypris oxycephalus is a cold water fish with a wide geographic distribution including the relatively warm temperate regions of southern China. It also occurs in second- and third-step geomorphic areas in China. Previous studies have postulated that high-altitude populations of R. oxycephalus in southern China are Quaternary glacial relics. In this study, we used the mitochondrial gene Cytb and the nuclear gene RAG2 to investigate the species phylogeographical patterns and to test two biogeographic hypotheses: (1) that divergence between lineages supports the three-step model and (2) climatic fluctuations during the Quaternary resulted in the present distribution in southern China.R. oxycephalus in matriline C, with the subsequent warming resulting in its split into submatrilines C1 and C2, leaving submatriline C2 as a relic in southern China.Phylogenetic analysis detected three major matrilines ; with matrilines B and C being further subdivided into two submatrilines. Based on genetic distances and morphological differences, matriline A potentially represents a cryptic subspecies. The geographic division between matrilines B and C coincided with the division of the second and third geomorphic steps in China, suggesting a historical vicariance event. Pliocene climatic fluctuations might have facilitated the southwards dispersal of R. oxycephalus. Notably, no genetic diversity was detected in several populations, all of which possessed unique genotypes. This indicates the uniqueness of local populations and calls for a special conservation plan for the whole species at the population level.Our study demonstrates that geological events (three steps orogenesis) and climate fluctuations during the Pliocene were important factors in shaping phylogeographical patterns in The online version of this article (doi:10.1186/s12862-014-0225-9) contains supplementary material, which is available to authorized users. Both geological events and climatic fluctuations usually influence genetic variation distribution in populations and species. The uplifting of mountain systems and the development of river systems may form vicariance between populations, resulting in genetic diversification and even speciation -5. ChinaGekko swinhonis) coincides with a boundary consisting of the Qinling and Taihang Mountains, suggesting a historical vicariance pattern . The nuclear Recombinase Activating Gene 2 (RAG2) encodes components of the recombinase involved in immunoglobin recombination and T-cell receptor genes and appears as conserved single copies in all vertebrates examined to date [RAG2 sequences  were obtained from a subset of samples consisting of 127 individuals with the primers RAG2-108f (5′-CCV ARA CGC TCA TGT CCA AC-3′) and RAG2-1324r (5′-TGG ARC AGW AGA TCA TKG C-3′) [Taq DNA polymerase. Sterile water was added to reach the final volume. Reactions took place in the following program: an initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52–58°C for 45 s, extension at 72°C for 1 min, and a final extension at 72°C for 8 min. PCR products were sent to commercial sequencing companies (Shanghai Sangon Biotech Inc.) for purifying and then sequenced in both directions with BigDye™ Terminator Cycle Sequencing Kit on an ABI PRISM® 3730 DNA Analyzer . The same PCR primers were used for sequencing.Total genomic DNA was extracted from muscle tissue following the salt-extraction methods of Tang et al. . The mit CCG-3′) . Two Cyt to date ,30. PartKG C-3′) . AmplifiCytb and 127 RAG2 sequences have been newly sequenced in this study. Multiple alignments of sequences were performed using CLUSTAL X [A total of 753 LUSTAL X  and subsLUSTAL X . The nucLUSTAL X . SequencLUSTAL X . NuclearLUSTAL X , for whiLUSTAL X . RecombiLUSTAL X . IdenticLUSTAL X . Newly dRAG2 alleles under the same settings as for Cytb, except for model of substitution (GTR+I+R). Furthermore, the Network 4.6.0.0 program [Phylogenetic relationships among mitochondrial haplotypes were reconstructed using Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) approaches. Selection of the best-fit nucleotide substitution models was based on codon position using the Akaike Information Criterion in jmodeltest 2.0 ,41. For  program  was emplCytb data, a range of mutation rates (1–2% per my) were adopted, as they have been widely employed for mitochondrial Cytb gene analysis in cyprinid fish [th tree, and the first 10% of trees sampled were treated as burn-in. The effective sample size for parameter estimates and convergence was checked with Tracer 1.5 [For divergence time estimation, the molecular clock was evaluated using a two-cluster test in the LINTREE program . Sequencnid fish -50. Two acer 1.5 . FigTreeacer 1.5  was usedCytb data between populations having more than 10 samples in ARLEQUIN 3.5 [Diversity estimations for both pooled and individual population datasets were calculated in DnaSP, including the number of haplotypes (n), haplotype diversity (h), and nucleotide diversity (π). The mean divergence among haplotypes and divergence between matrilines were calculated by an uncorrected p-distances model as implemented in MEGA, standard errors were estimated by bootstrapping using 10,000 replicates. Pairwise population differentiation was estimated by computing pairwise Φst for the QUIN 3.5 . To inveThe plausibility of an isolation-by-distance scenario was explicitly tested by Mantel’s tests in ARLEQUIN to analyze the relationships between genetic  and geographical (straight line differences) distances between sampling localities.Cytb data to trace the demographic history of matrilines B1, B2, C1, and C2. Matriline A was excluded form demographic analysis because of the small sample size. First, Tajima’s D [Fs [Three methods were used with the jima’s D  and Fu’s’s D [Fs  were use’s D [Fs  were cal’s D [Fs . The sig’s D [Fs  were impR. oxycephalus and R. lagowaskii [R. oxycephalus and the Xinyang  population using digital calipers . Variation of these key morphometric characteristics among the three matrilines  was subsequently assessed by ANOVA. Tukey’s Honest Significant Difference was employed for post-hoc pairwise comparisons. The sample sizes for morphological analysis were 30, 170, and 433 for matrilines A, B, and C, respectively.The ratio of caudal peduncle depth to head length (CPD/HL) and caudal peduncle length to depth (CPL/CPD) are key morphometric variables for discrimination between gowaskii ,60. We mCytb, we obtained 755 R. oxycephalus sequences from 36 populations, including two sequences from GenBank. The sequence alignment provided a data matrix of 1,097 bp, of which 289 bp (26.3%) were parsimony informative. Base frequencies were unequal . Strong compositional biases against G existed at the second and especislly at the third position (just 10.1%). A total of 124 haplotypes were identified among the 755 R. oxycephalus sequences  yielded congruent topologies and they consistently supported three matrilines  had significantly different substitution rates compared with the other sequences. Therefore, these three sequences were excluded from further analysis. A strict molecular clock was conducted for the remaining samples  and nucleotide (π) diversities in all samples were 0.9770 ± 0.0010 and 0.0523 ± 0.0006, respectively. The number of haplotypes (n), h, and π varied among populations . All pairwise Φst values were statistically significant, varying from 0.099 to 1.000 (P <0.001 to P = 0.027). Genetic divergence varied from 6.5 to 7.4% among matrilines A, B, and C, and from 5.5 to 5.6% between submatrilines B1-B2 and C1-C2 . However, some populations  were genetically distinct despite their geographical proximity. These populations are distributed on opposite sides of the same mountains.A significant positive correlation was found between geographical distance and genetic divergence among populations . For CPD/HL, there was no significant difference between matrilines A and B . However, significant pair-wise distinctions were detected between matrilines A and C and between matrilines B and C . For CPL/CPD, matriline A values differed significantly from those of B and C , but there was no significant difference between matrilines B and C .The ANOVA analysis showed that two key taxonomic characteristics varied significantly among the three matrilines  and C (C1 and C2). The nDNA phylogenetic and network analysis revealed a deep genetic divergence analogous to mtDNA . The results revealed that matriline A is a distinct monophyletic group and forms a sister relationship with R. oxycephalus. Furthermore, samples from Xinyang , representing matriline A in our samples, are morphologically distinct from the other populations in taxonomically relevant ratios. Therefore, we suggest that matriline A represents a cryptic subspecies, which may be geographically restricted to Japan and parts of China including the Huaihe River. Although Japan is currently separated from eastern China, studies have proposed that these regions most likely had the same freshwater ichthyofauna during the Miocene and Pliocene [There is a deep genetic divergence between matriline A and the other matrilines for s Figure . We alsoPliocene .Cytb sequences analysis carried out in our study revealed three distinct evolutionary lineages in R. oxycephalus. Do these major lineages correspond to two or three distinct species? Compared with the general definitions of five different groups of candidate species [p-distances between the R. oxycephalus lineages reached a value associated with subspecies divergences (including matrilines B and C). Additionally, morphological differences (CPD/HL) were also observed between the major lineages . However, the relationship between matrilines B and C is not completely supported by the RAG2 gene data, but matriline A is indeed distinct from the others (as discussed in the previous paragraph). Therefore, the hypothesis of matrilines B and C being distinct subspecies or even species requires further research.The  species , all uncR. oxycephalus divergence is likely associated with the orogenesis of China’s three steps. Geological evidence indicates that recent large-scale intense uplift of the Qinghai-Tibetan Plateau took place during the Pliocene 3.6 mya [Cytb gene sequences, He and Chen [Gekko swinhonis) lineages also coincides with the Qinling and Taihang Mountains boundary, suggesting a historical vicariance pattern. The orogeny of the Qinling Mountains may have launched the independent lineage divergence [The deep divergence observed between matrilines B and C likely reflects long-term isolation and ecological differences between high- and low-elevation regions, which is probably associated with China’s complex geological history. As indicated in Figure  3.6 mya , contrib 3.6 mya . During  3.6 mya ,67. Baseand Chen  suggestevergence . Thus, bR. osculus) in western North America [Pachytriton), all major intraspecific lineages originated before the Quaternary period and pre-Quaternary climate change played an important role in the formation of regional biodiversity [There is a 5.5% mtDNA difference between submatrilines C1 and C2  parsimony informative sites and the most variable sites occurred in different haplotypes. AMOVA analysis revealed that 96.75% of the genetic variation was attributed to differentiation among populations. In contrast, genetic diversity within populations was very low and 12 populations possessed a single haplotype despite typically analyzing over 20 fish per population. These populations may have had extremely small founding populations. A similar phenomenon has been reported in Quasipaa boulengeri, a montane stream-dwelling frog, where 85% of the haplotypes only occurred in a single location and 16 populations out of 45 had zero haplotype and nucleotide diversity [Chinese iversity . High leR. oxycephalus was geographically restricted to local montane areas. Approximately 97% of the haplotypes are unique for their respective populations. The majority of genetic variance was seen at the population level and was correlated with geographic distance; more distant sites within the hydrological network were more genetically differentiated. In addition to complex topological changes, this pattern may have resulted from the isolation of populations because of specific habitat requirements. Rhynchocypris oxycephalus mainly occurs in the clear cold permanent headwaters of montane streams. This specific ecological requirement results in small population sizes and restricts gene flow among populations (each population possesses unique haplotypes). Therefore, these populations most likely survive in isolated habitats and differentiate through genetic drift and selection.The genetic structure of a species is greatly influenced by the palaeogeographical history of the region that comprises its geographical range. In the present study, mtDNA analysis suggested that et al. [R. oxycephalus. The geographical distribution of this species is expected to contract with progressing climate change and become severely limited, particularly in south-eastern China. Anthropogenic threats, such as deforestation, illegal fishing, and habitat destruction  may represent major threats to this species. These factors place R. oxycephalus at risk for extinction.The Chinese minnow currently has a wide geographical distribution in East Asia and to date is ranked as ‘not threatened’. However, individual populations are small because they are restricted to stream headwaters and may be sensitive to climate change and anthropogenic threats. Using ecological niche modeling, Yu et al.  predicteR. oxycephalus has important implications for the conservation of this species. However, many R. oxycephalus populations inhabit areas that are not protected. Our study may serve as a guide for monitoring and developing a conservation strategy for this species. Within this framework, our analyses suggest that the three matrilineal ranges  be considered individual Management Units. All of these require protection because of their genetic uniqueness, which includes exclusive mtDNA haplotypes and private nuclear alleles.Genetic variability is widely recognized as an important component of natural biodiversity. The maintenance of high genetic diversity could prevent the loss of a species evolutionary potential . The disR. oxycephalus will be reduced if these populations become extinct. Climate change projections indicate severe threats to R. oxycephalus and to these southern populations in particular [Additionally, the southern populations including those from the Yangtze River, Zhejiang Province, and the Min River demand special attention. This species is geographically restricted to local streams and many of these populations possess negligible genetic diversity. Overall genetic diversity in rticular . ConsequR. oxycephalus phylogeographic patterns in China. The results support the hypothesis that geological events (three steps orogenesis) have driven divergence between matrilines B and C. Based on the molecular clock, it is unlikely that climate fluctuations during the Quaternary facilitated the southwards dispersal of R. oxycephalus in matriline C and established relics in south China as postulated by Zhang and Chen [Cytb and RAG2 gene data; analysis with additional markers would greatly enhance our understanding of this species.This study assembles a large dataset to infer and Chen . Pliocen


The main cattle breed in Korea is the brown Hanwoo, which has been under artificial selection within a national breeding program for several decades. Varieties of the Hanwoo known as Jeju Black and Chikso were not included in the breeding program and remained isolated from the effects of recent artificial selection advancements. We analysed the Jeju Black and Chikso populations in regards to their genetic variability, state of inbreeding, as well as level of differentiation from the mainland Hanwoo population.Ne) of only 11 and 7, respectively. Despite a small Ne, higher than expected heterozygosity levels were observed (0.303 and 0.306), however, lower allelic richness was found for the two island populations (1.76 and 1.77) compared to the mainland population (1.81). The increase in heterozygosity could be due to environmental disease challenges that promoted maintenance of higher genetic variability; however, no direct proof exists. Increased heterozygosity due to a first generation crossing of genetically different populations is not recorded. The differentiation between the Korean populations had FST values between 0.014 and 0.036 which is not as high as the differentiation within European beef or dairy cattle breeds (0.047–0.111). This suggests that the three populations have not separated into independent breeds.Jeju Black and Chikso were found to have small estimated effective population sizes  contains supplementary material, which is available to authorized users. Recent developments in genome-wide data collection have enabled researchers to construct world-wide patterns of ancestry and admixture in domesticated cattle , 2. WhilCattle bones found on Jeju Island were most closely related to Jeju Black suggesting that the progenitors of these cattle inhabited the island already 1100–2000 years ago . This tiFirst records of Chikso cattle can be dated back to a picture on an ancient tomb mural from AD 357 . The Chikso cattle nowadays comprise about 4000 animals of which 3000 exist on the mainland and another 1000 animals on Ulleung Island.Both Jeju Black and Chikso were classified as endangered by the National Report on the State of Animal Genetic Resources of the Republic of Korea (2004). However, exact numbers of total or effective population sizes are not recorded and studies on the genetic divergence of the breeds as well as their genetic variability and inbreeding are sparse –11. MainBesides the varieties of Hanwoo within Korea, Chinese Yanbian cattle have been shown to be genetically highly similar to Hanwoo  but haveHere, we provide important information (based on genome-wide markers) about the state of the Jeju Black and Chikso populations in regards to their genetic variability, state of inbreeding, as well as level of differentiation from the mainland Hanwoo population. Results of this work should be of value for practical decision making on how to best conserve these populations.Ne), are strongly associated with the genetic variability and the ability of a population to react to environmental changes. To get a better understanding of the size and structure of the Korean cattle populations we estimated Ne based on the linkage disequilibrium (r2) between the genome-wide markers. The effective population sizes for Jeju Black and Chikso were 11 and 7, respectively, whilst it was 97 for the mainland Hanwoo , Jeju Special Self-Governing Province Community, ), and arFurther, we estimated the effective population size for five European taurine breeds as well as a Brahman population which were much lower than previously reported , 18. NevAR) estimates also confirmed a reduction in genetic variability for the two island populations . In total, 1531 protein coding genes were found in Chikso, 2519 genes in Jeju Black, and 10,282 genes in Hanwoo cattle.As expected from the larger number of genes for the Hanwoo breed, more gene sets were identified as significantly enriched and mostly just reflect the unbalance in the number of genes in the various processes. Thus, most genes were unsurprisingly involved in general processes such as regulation of transcription, proteolysis or cell adhesion Table . NeverthFST values did not yield any significant differences between Hanwoo and Chikso, or Jeju Black and Chikso which confirms the closeness of these populations. One region on chromosome 7  despite the large geographic area and distance that these cattle inhabit. From a Korean production perspective Yanbian is of interest because it is an ideal population to provide insights into the effects of the breeding program on the genetic architecture of the Hanwoo . Yanbian show higher levels of genetic diversity than Hanwoo which could be due to the intensive selection on the latter but this is potentially confounded with unaccounted crossing of Yanbian with other European breeds – Yanbian had lowest FST values with European breeds compared to the other Korean breeds (Table The divergence between Yanbian and Hanwoo is very low and not significant (FST 0.036) compared to the divergence to the mainland Hanwoo population at FST 0.016 and FST 0.014  or dairy breeds . No ethics statement was required for the collection of other animal samples used in this study as data was provided by secondary sources.n = 20) were collected from the Gyeonggi and Gyeonbuk provinces; and brown Hanwoo (n = 40) cattle were sampled from the Hanwoo Improvement Centre of the National Agricultural Cooperative Federation in Seosan (Chungnam province).Cattle were sampled from research stations on mainland Korea and Jeju Island. On Jeju Island, 20 Jeju Black cattle were sampled at the Subtropical Animal Experimental Station. Chikso cattle  and one indicine breed (n = 19 Brahman) were sampled from Australian populations and included in the analysis for comparison . Quality control was performed with snpQC  and the Ne) for each breed was estimated with the LDNe program [r2 as linkage disequilibrium according to Hill [Ne was estimated per chromosome and then averaged across the entire genome. The mating system was chosen to be at random even though this is not fully realistic for livestock populations.The effective population size  and private allelic richness (pAR) were estimated with the HP-Rare v1.0 program [FIS and FST), estimated according to Weir and Cockerham [StAMPP package in R [FST values were achieved by 100 bootstrapping replicas.Allelic richness  carriedFinally, we used ADMIXTURE 1.23  to prediGeneCodis program (release 3) [2 test was used to compute P-values which were corrected for multiple testing through false-discovery rate [The numbers of fixed and segregating markers were assessed and the overlap of markers between breeds calculated. A gene enrichment analysis for segregating markers that were unique for each of the Korean breeds was carried out. Genes including a direct marker or genes within one Mb in either direction of a marker  were regarded as adaptive variation whereas all other markers were regarded as neutral variation. The lease 3) –46 was uery rate .http://dx.doi.org/10.5061/dryad.p2b3b.Datasets will be made available upon acceptance of this paper and before publication. Genotypic data of the Korean cattle breeds are deposited with Dryad:


Geneclass and Structure) were used to infer translocation events based on nuclear data whereas mitochondrial data were used for validation of these inferences. Using Geneclass, we found translocation events in a majority of populations. Additional cases of translocation and many instances of admixture were identified using Structure, thanks to the clear-cut ancestral genetic structure detected in this species. In particular, a strong divide between Alpine and Central European populations, also apparent at mitochondrial markers, helped uncover details on translocation events and related processes. Translocations and associated admixture events were found to be heterogeneously distributed across the species range, with a particularly high frequency in Central Europe. Furthermore, translocations frequently involved multiple geographic sources, some of which were over-represented. Our study illustrates the importance of range-wide investigations for tracing translocations back to their origins and for revealing some of their consequences. It provides some first clues for developing suitable conservation and management strategies.In contrast to biological invasions, translocations of individuals within a species range are understudied, due to difficulties in systematically detecting them. This results in limited knowledge about the corresponding processes and uncertainties regarding the status of extant populations. European larch, a forest tree whose fragmented native distribution is restricted to the Alps and to other Central European mountains, has been massively planted for at least 300 years. Here we focus on the genetic characterization of translocations having taken place within its native range. Microsatellite variation at 13 nuclear loci and sequence data of two mitochondrial DNA fragments were analyzed on the basis of a comprehensive range-wide population sample. Two complementary methods ( Translocations are defined as intentional or accidental anthropogenic movements of living organisms from one place to another , S1 TextS1 Text. Differentiating native from non-native material can provide critical information for the conservation of genetic resources , 9, 10. a priori a native reference sample completely exempt of translocations, as forest history cannot in most cases be reconstructed in detail at the individual stand level [Among the few existing studies, some have focused on admixture between native and non-native stocks of fishes for conservation purposes by relying on Bayesian approaches –16. Thesnd level , 10, 20.Larix decidua Mill.) is a good candidate to study within-range translocations due to its restricted pollen dispersal ability that should result in high population structure [Larix decidua is found at high altitudes in the Alps and in other Central European mountains  as well as in the Polish lowlands  or a “manie du mélèze” (French) [European forest trees represent particularly good models to investigate within-range translocations. Historical records indicate that forest reproductive material  has been traded across trees’ native ranges for centuries. However, the overall impact is not clear, as native material might or not be better adapted than non-native one. Moreover, the onset of massive plantations is often quite recent and admixture proceeds on a rather long time scale due to the long generation times of trees , 20–22. tructure , 24. Itstructure , 26, whi(French) –31. Repo(French) . A conse(French) ). In conL. decidua, to the point that we can safely reconstruct the ancient genetic structure and identify native material? Are translocations more frequently detected in some parts of the range than in others? Have some seed sources been overrepresented among the transferred material? Have multiple geographic sources been used in plantations? Have translocations been followed by admixture? To answer these questions, we rely on molecular markers and population genetic methods (Geneclass and Structure [The present study focuses on the following questions: Can translocations be accurately identified in tructure –35) to sLarix decidua, in particular in Central Europe, is difficult due to its long plantation history. With permission of the Nordwestdeutsche Forstliche Versuchsanstalt (NW-FVA), we relied on a comprehensive collection of populations sampled in 1957–1958 within the native range to establish an international provenance trial , 36Larixl stands . Moreove variety  listed ad before .We genotyped 24 individuals per population at 13 highly variable microsatellite loci combined in two multiplexes . GenotypatpA) from eight individuals per population. Primer sequences for UBC460 are provided in [atpA (L3atp-2-1 5’- GCGGCTGCCTATAGATACGA-3’ and L3atpf1/1 5’- GCTACCGAGGCAGATATGGA-3’) are based on published sequences [atpA) cycles of denaturation at 94°C for 1 min, annealing at 65°C (UBC460) or 57°C (atpA) for 45 s and elongation at 72°C for 2 min 50 s (UBC460) or 1 min 50 s (atpA). The cycles were followed by a final elongation step at 72°C for 10 min. PCR products were sent to LGC Genomics  for clean-up and sequencing. Sequence editing and alignment were performed using CodonCode Aligner Version 4.0.3. Haplotypes were detected after concatenating both sequenced regions and their relative frequencies were calculated using GenAlEx 6.41 [atpA region contained a minisatellite with two different repeat motifs and a variable number of repeats. This variation was coded into single nucleotide positions to compute a minimum spanning network using TCS 1.21 [We sequenced two mtDNA regions  was set from one to nine, with 10 runs for each K value. We used a burn-in of 200,000 and 1,000,000 iterations and explored both the no-admixture and the admixture model. To determine the most likely value of K, we computed the posterior probabilities and the second order rate of change L”(K) with Structure Harvester [R package APE [Bayesian cluster analysis relied on ture 2.1 , 35. Thearvester , 47. We kage APE . In addikage APE  to identStructure results) that do not belong to the most frequent ancestral group. By so doing, we assume that (i) prior to translocations a strong ancestral genetic structure existed, with no or limited admixture before the onset of translocations by humans, that (ii) native individuals of each population originate from a single ancestral group, and that (iii) native individuals still represent the majority of individuals. We used simulations to study risks associated with that procedure.In each population, we consider as resulting from translocation events all purebred individuals . We then compared summary statistics before and after excluding these individuals, focusing on allelic richness NA, gene diversity HS, inbreeding coefficient FIS and fixation index FST measured using FSTAT version 2.9.3.2 [hS and the coefficient of genetic differentiation GST [Permut (http://www.pierroton.inra.fr/genetics/labo/Software/).We combined data from  2.9.3.2  with 15,ion GST  using PeStructure in each region using a z-test. Then, to investigate the origin of the material used for translocation, we compared the genetic composition of non-native individuals (recent migrants detected by Geneclass and non-native purebreds detected by Structure) and native individuals . In this analysis, to avoid including false positives, admixed individuals identified by Structure were not included unless they had been identified by Geneclass as being recent migrants. Finally, to study if translocations had involved multiple geographic sources, we counted the number of genetic clusters identified by Structure in populations with versus without evidence for translocation. An individual was considered to belong to a given nuclear cluster if its ancestry was mainly found in this cluster . In populations without evidence for translocation, we considered all clusters except one (the native one); in populations with evidence for translocation, we considered all clusters except two, the native and the most frequent non-native one. We used a Wilcoxon rank-sum test based on the test statistic W to compare the mean number of extra-clusters between these two groups of populations [To study if the two regions  had experienced asymmetric exchanges of genetic material, we compared the proportion of translocation events identified with ulations .Structure in the Alpine versus the Central European region using a z-test. Second, we tested if translocations eventually result in admixture using two different approaches. In a first approach, we predicted that the proportion of admixed individuals should be higher in populations in which translocation events have been detected than in the remaining populations, as contact between populations necessarily precedes admixture. To test this expectation, populations were grouped in two categories: populations with evidence for translocation and populations without such evidence, as explained above. Mean admixture level was then compared between these categories , using a one-sided t-test. In a second approach, we relied on mitochondrial data to study if translocations eventually result in admixture. As individuals of non-native origin should typically be present at low frequency and will then be more likely to mate with local native individuals, whereas local native individuals should mostly mate with each other, the proportion of individuals with admixed genomes should be higher among individuals exhibiting non-native mitochondrial haplotypes than among individuals possessing native haplotypes. These two proportions were measured in each region  and compared using a z-test.First, we compared the proportion of admixture events identified with Larix decidua individuals from 45 populations were successfully genotyped at 13 microsatellite loci. We detected on average 22 alleles per locus. Sequencing of the two mtDNA fragments in 363 individuals from 43 populations led to the detection of 22 mtDNA haplotypes. The observed molecular variation was a combination of nucleotide substitutions, insertions/deletions as well as copy number variation of the minisatellite (Larix sibirica samples are characterized by a single private haplotype (H23) that has a central position in the network. The L.decidua haplotypes form two groups (i.e. lineages), each composed of two frequent haplotypes and some less frequent ones. In lineage 1 , haplotypes H16 and H18 were found in 58% of the individuals. In lineage 2 , haplotypes H9 and H10 occurred in 28% of the individuals  recent migrants in 33 of the 40 native populations  using the model without admixture  and group 2 made of clusters 5–7 (corresponding to cluster 3 in the solution at K = 3). These two groups were also distinguished on the first axis of the principle coordinate analysis, which explains 38% of the variation, compared to 23% for the second axis  presumably non-native individuals distributed in 38 of the 40 populations. There were only 30 individuals identified by both methods . In particular, using Geneclass, we identified 49 migrants not detected with Structure. As expected, Geneclass was less efficient at detecting migrants in populations characterized by high rates of admixture . Note however that a few populations still had comparatively high heterozygote deficit after removing individuals considered to be non-native  than in the original one . In fact, after trimming, most populations became dominated by a single genotypic cluster. Two large regions of the range, the Alps and Central Europe, turned out to be composed of individuals characterized by reciprocally exclusive nuclear clusters and mitochondrial lineages: all populations of the Alpine region were dominated by individuals with nuclear genotypes assigned to cluster group 1 and characterized by mitochondrial lineage 1, and all populations from Central Europe were dominated by individuals with nuclear genotypes assigned to cluster group 2 and characterized by mitochondrial lineage 2, making it possible to compare in detail the translocations across these two regions.By combining results from both dmixture . Geograpin situ and ex situ in provenance tests, indicating that the collection mode did not strongly influence results. The proportion of individuals considered to originate from translocation events was not significantly different in the Alpine and in the Central European region . Considering results from both Geneclass and Structure, we found that one of the four Alpine clusters (cluster 2) was over-represented in non-native material from Central Europe , suggesting that when translocations occur in a given population, they often involve material from more than one source.We found evidence for translocation in larch populations collected both ope 50%; . This clope 50%; . Genotypope 50%; . In contz = -1.5, p < 10–5). Furthermore, mean admixture values were significantly higher in populations with evidence for translocation than in populations without evidence for translocation . Similarly, in populations from Central Europe, 65% (13/20) of the individuals with mtDNA haplotypes from lineage 1 were admixed compared to only 25% (29/118) for individuals with mtDNA haplotypes from lineage 2 .The proportion of admixed individuals was significantly lower in Alpine populations than in Central European ones . The adStructure [Hybridlab, [The other method used to identify translocations relies on the extant range-wide genetic structure identified using tructure . We showbridlab, ) that asThe two approaches used to detect translocation and admixture events turned out to be complementary as they identified largely non-overlapping sets of presumably non-native individuals. Once all these individuals had been removed, a clear-cut geographic pattern emerged. In particular, all populations from the Alps turned out to be genetically different from Central European populations. Hence, we were not compelled to resort to post hoc arguments to dismiss populations from our reference set. In fact, some populations from Central Europe had a very high proportion of non-native genotypes, and the pre-plantation genetic structure that we inferred is probably still not completely devoid of exotic material, as shown by the persistence of relatively high heterozygote deficit in some populations.L. decidua seeds from the Tyrol had been intensively used in plantations [We found that translocations had preferentially involved one of the four Alpine genetic clusters. In the populations sampled outside of the species range, genotypes assigned to this cluster were also particularly frequent. This finding is in agreement with historical information indicating that ntations . In contntations .Our results also show that translocations had typically involved multiple geographic sources. This result is reminiscent of earlier studies in oaks based on chloroplast DNA, where planted stands were shown to have much greater diversity than native ones, likely as a consequence of the use of mixed seed lots during plantations . AnotherL. decidua in Central Europe is more fragmented than in the Alps and populations there are of rather small size, making them more susceptible to genetic swamping [Lärchenmanie” by uncovering its irreversible long-term consequences through a modification of the genetic structure of the species, including in areas where threatened gene pools have been recognized (cf. L. decidua variety polonica). Finally, the results for populations sampled beyond the native distribution range show that we are now in the position to start investigate their make-up and subsequent history.We found strong support for a link between translocation and admixture, as expected if admixture events had taken place subsequently to translocations, once planted trees have become mature. First, we found an increased proportion of admixed individuals in populations where one or more non-native purebred genotypes had been detected, pointing to the recent onset of admixture between native and non-native trees. Second, individuals with non-native mitochondrial haplotypes also had elevated rates of admixture. Although some admixed individuals are likely false positives, the previous findings as well as the high proportion and broad distribution of admixture suggest that in many places at least one generation has elapsed since the first trees were introduced, ruling out seed lot contamination in plantations as the main source of translocation events. The particularly high admixture rate detected in Central Europe makes sense as the distribution of swamping , 5. Transwamping –31. HencL. decidua, we could now in principle identify canker-sensitive Alpine material introduced across the range and native Polish populations threatened by introgression, using greater sample sizes. While further progress should be achieved through the development of new demo-genetic methods that simultaneously identify introductions, admixture events and their timing, existing population genetic tools can already be used more widely, thereby revealing the frequency and prevalence of translocations across the globe as well as their consequences.In conclusion, our study illustrates the importance of investigating translocations across species range to efficiently and accurately trace non-native material of different geographic origins and to uncover native material as well as species ancient history. In the future, such approaches will provide essential information for conservation of genetic resources. For instance, in S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(XLSX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.S8 Fig(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.