Posterior probability distributions for migration rates from two-population IM analysis.

<p>Coalescent-based estimates of migration rates (scaled by mutation rate) for three studied species inferred separately from (A–C) nuclear sequence data that included nine nuclear markers, and from (D–H) one mitochondrial marker gene.</p

Probability densities of four parameters in coalescence simulation as functions of length of internode.

<p>Graphical visualization in which (1–P_{Concord-mtDNA}) denotes the probability density of observing discordant mtDNA phylo tree; (P_binom-nuc) denotes the probability density of observing eight topologically concordant nuclear gene trees out nine studied nuclear loci in total; (P_Coal-mtDNA) denotes the cumulative probability of mtDNA coalescence along the internode and (P_Coal-nuc) denotes the cumulative probability of coalescence of nuclear locus along the internode. Note that there is very small intersection of probability densities allowing for observing eight out of nine topologically concordant nuclear loci while having discordant mtDNA tree (see the text for details).</p

Summary of nucleotide variation.

a<p>Data are in the order for <i>C. taenia</i>; <i>C. elongatoides</i>; <i>C. tanaitica</i>; and all three species. <i>Cobitis fahirae</i>, <i>C. vardarensis</i> and <i>C. paludica</i> were sequenced as one individual per species per locus and not summarised. <i>L</i>, sequence length (bp); <i>h</i>, number of haplotypes; <i>S</i>, number of polymorphic sites.</p

Posterior probability distributions for migration rates from three-population IMa2 analysis.

<p>Coalescent-based estimates of migration rates (scaled by mutation rate) for three studied species inferred from (A–C) nine nuclear markers and (D–F) combined mito-nuclear sequence data that included one mitochondrial marker gene and nine nuclear markers.</p

Phylogenetic comparison of gene trees constructed from nuclear and mitochondrial gene markers and mito-nuclear discordance.

<p>Bayesian DNA gene trees constructed from nine nuclear gene markers and one mitochondrial <i>cytb</i> gene marker were rooted with sequences from <i>C. paludica</i>. Haplotype numbers correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080641#pone-0080641-t002" target="_blank">Table 2</a>. Bar represents 0.1 substitution/site. The schematic tree shows the phylogenetic conflict of <i>C. tanaitica</i> topology between mitochondrial and nuclear gene markers.</p

Parameters calculated from alternative tests using sequence data to explain <i>C. tanaitica</i> mito-nuclear discordance.

<p>Contemporary and ancestral population sizes are denoted by (<i>θ_C.tae</i>, <i>θ_C.tan</i>_,<i>θ_C.elo</i>_,<i>θ_C.tae,C.tan</i>, <i>θ_{C.tae,C.tan,C.elo}</i>). Divergence times are denoted by (<i>τ_C.tae,C.tan</i> and <i>τ_{C.tae,C.tan,C.elo}</i>), and interval between those times is denoted by (<i>γ</i>). Migration rates are denoted by (<i>m</i>) with relevant index. All parameters are scaled by mutation rate <i>μ</i>, and can be converted to absolute values using the relations <i>θ</i> = 4<i>Nμ</i> (where <i>N</i> is effective population size), <i>m</i> = m/<i>μ</i> (where m is gene-flow rates per gene copy per generation, <i>τ</i> = t<i>μ</i> (where t is a time of population splitting at <i>τ</i> generations in the past), and <i>γ</i> = t<i>μ</i>. Parameters estimated by BPP program are denoted by ($), those by IM by (#), and those by IMa2 by (@). The parameter <i>γ</i> was calculated from <i>τ</i>s given by BPP and ds programmes. <i>C. taenia</i> (<i>C.tae</i>), <i>C. tanaitica</i> (<i>C. tan</i>), and <i>C. elongatoides</i> (<i>C. elo</i>).</p

Spined loaches (<i>Cobitis</i>) used in this study.

<p>Spined loaches (<i>Cobitis</i>) used in this study.</p

Prior and posterior distributions of parameters in the BPP Bayesian analysis of the nine nuclear loci.

a<p>Priors set with <i>Ne</i> = 225,000 covering Watterson's <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080641#pone.0080641-Watterson1" target="_blank">[91]</a> θ estimate (<i>θ</i>_W = 0.002, <i>Ne = </i>138,889) and from branching event t = 3.31 Mya between European closely related <i>Cobitis</i> species <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080641#pone.0080641-Tang1" target="_blank">[104]</a>. Relatively fast autosomal mutation rate (<i>μ</i>) of 3.6×10⁻⁹ estimated in vertebrates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080641#pone.0080641-Axelsson1" target="_blank">[105]</a> was used to transform prior expectations of <i>θ</i> and <i>τ</i> from absolute estimates of <i>N_e</i> and t. Both <i>τ</i> and <i>θ</i> are measured as the expected number of mutations per site.</p>b<p>Prior for the node age was generated from the Dirichlet distribution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080641#pone.0080641-Yang1" target="_blank">[63]</a>: equation 2).</p><p><i>C. tae</i> = <i>C. taenia</i>, <i>C. tan</i> = <i>C. tanaitica</i>, <i>C. elo</i> = <i>C. elongatoides.</i></p

Photographs, karyotypes and Europe-wide distribution of spined loach sexual species (<i>Cobitis</i>) from this study.

<p>(A,C,E) Photographs (scale bar = 1 cm) and (B,D,F) respective karyotypes of three widespread <i>Cobitis</i> species. Karyograms with diploid chromosome number (2n), metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric chromosomes (a) were modified after Janko et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080641#pone.0080641-Janko2" target="_blank">[50]</a>; (G) Sampling localities of <i>Cobitis taenia</i> (light gray squares; 1–10), <i>C. elongatoides</i> (dark gray squares; 11–20), <i>C. tanaitica</i> (black squares; 21–30), <i>C. paludica</i> (checkered square; 31), <i>C. fahirae</i> (spotted square; 32), and <i>C. vardarensis</i> (reticulated square; 33). Insets show European species distribution with respective markings as given in squares. Note that locality no. 1 is situated more eastward, as marked by the arrow.</p

Mapping of our cytogenetic results onto modified phylogenetic relationships of the Labeoninae based on nuclear and mitochondrial genes by Yang et al. [2] and classification of Tan and Armbruster [1].

The common names of the species are indicated under the scientific name. The position of Epalzeorhynchos munense was manually added to our figure, since it is not included in the [2] phylogeny, thus requiring confirmation.</p