Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei)

<div><p>Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (<i>Cobitis</i>). We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA). Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in <i>Cobitis</i>. Asexual animals described in other case studies reproduce ameiotically, while <i>Cobitis</i> hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.</p></div

Ultrametric phylogenetic tree demonstrating estimated speciation times of parental species and the Hybrid clade I.

<p>Species-specific karyotypes arranged from Giemsa stained chromosomes are shown along the right side of cladogram. Confidence intervals of nodes of interest are in grey colour. TT, <i>C</i>. <i>taenia</i>; NN, <i>C</i>. <i>tanaitica</i>; EE, <i>Cobitis elongatoides</i>. Chromosomes were arranged in a decreasing size order and classified in four morphological groups: metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a).</p

Representative karyotypes of hybrid biotypes after GISH and/or DAPI/Giemsa staining.

<p>(A) EN hybrid metaphase with hybridization pattern of <i>Cobitis elongatoides</i> gDNA in green, <i>C</i>. <i>tanaitica</i> gDNA in red. (B) ET hybrid with <i>C</i>. <i>elongatoides</i> in red, <i>C</i>. <i>taenia</i> in green. (C) EEN hybrid with <i>C</i>. <i>elongatoides</i> in green, <i>C</i>. <i>tanaitica</i> in red. (D) EET hybrid with <i>C</i>. <i>elongatoides</i> in red, <i>C</i>. <i>taenia</i> in green. (E) ENN hybrid with <i>C</i>. <i>elongatoides</i> in red, <i>C</i>. <i>tanaitica</i> in green. (F) ETT hybrid with <i>C</i>. <i>elongatoides</i> in red, <i>C</i>. <i>taenia</i> in green. Capital letters represent haploid genome sets: E, <i>C</i>. <i>elongatoides</i>; N, <i>C</i>. <i>tanaitica</i>; T, <i>C</i>. <i>taenia</i>. Chromosomes were arranged in a decreasing size order and classified in four morphological groups: metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). Probes labelled with biotin-16-dUTP were detected with FITC-streptavidin (green signals on chromosomes); probes labelled with digoxigenin-11-dUTP were detected with anti-digoxigenin-rhodamin (red signals on chromosomes). To visualize the morphology of chromosomes DAPI (A, B, C, D) or Giemsa (E, F) stained karyotype was used. Captured DAPI stained karyotypes were inverted. Bars equal 5 μm. Detail information about individuals used is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146872#pone.0146872.s004" target="_blank">S1 Table</a>.</p

Observed and simulated distribution of clonal age estimators.

<p>(a) Correlation between the observed values of <i>dist.bp</i> and Tomiuk and Loeschcke’s <i>I</i>; (b, d) Distributions of Hartigan’s <i>D</i> values calculated from simulated <i>dist.bp</i> histograms at every 200^th generation of the simulation. Each value was calculated from a 50∶50 mixture of sympatric and allopatric clones. Arrows indicate the observed value; frequency distributions of the observed values of <i>dist.bp,</i> and <i>I</i>, respectively (c, e).</p

Europe-wide distribution, reproductive pathways and contact zone in Odra R. basin of studied species.

<p>(a) Distribution area of <i>C. tanaitica</i> (blue circles), <i>C. taenia</i> (red area), and <i>C. elongatoides</i> (yellow area), with the directions of postglacial colonization of Europe by <i>C. taenia</i> (red arrows) and <i>C. elongatoides</i> (yellow arrow). Secondary contact zones are indicated by zigzag symbols, and the dispersal of clonal lineages is indicated by dotted arrows (modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045384#pone.0045384-Janko2" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045384#pone.0045384-Culling1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045384#pone.0045384-Janko7" target="_blank">[64]</a>); (b) inferred reproductive pathways in <i>Cobitis</i> complex. Sperm at straight arrow indicates true fertilization, whereas sperm at round arrow indicates a triggering the egǵs development without paternal genetic contribution to the offspring; (c) Sampling sites in the Odra R. hybrid zone. For each locality, the presence of <i>C. taenia</i> and <i>C. elongatoides</i> is indicated by red and yellow dots, respectively and we indicate co-occurring hybrid biotypes, and their number in parentheses, if more than one. For each biotype on a given sample site, we list the presence of clones (MLL) and their absolute frequencies in parentheses, if more than one.</p

Histograms of the frequency distribution of pairwise distances in bp among genotyped individuals.

<p>(a–b) Data from <i>C. elongatoides</i> and <i>C. taenia</i>; (c–d) Distributions from observed (natural) and simulated diploid hybrids; (e–f) distributions from the observed (natural) and simulated triploid hybrids.</p

Dynamic Formation of Asexual Diploid and Polyploid Lineages: Multilocus Analysis of <em>Cobitis</em> Reveals the Mechanisms Maintaining the Diversity of Clones

<div><p>Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the <em>Cobitis taenia</em> hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically) reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal <em>Cobitis</em> hybrids, our data thus provide the most compelling evidence that 1) the origin of asexuality is causally linked to interspecific hybridization; 2) successful establishment of clones is not restricted to one specific ploidy level and 3) the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.</p> </div

Unrooted statistical parsimony networks of haplotypes belonging to <i>C. taenia</i>-like (T) (upper panel) and <i>C. elongatoides</i>-like (E) (lower panel) clades (sensu [<b>13</b>]).

<p>White circles denote haplotypes found in sexual individuals only, dark grey circles denote those found in hybrids only, and light grey circles denote haplotypes shared by both hybrid and sexual individuals. The sizes of haplotypes are proportional to their frequency. Small blank circles represent missing (unobserved) haplotypes. Newly sequenced haplotypes are in bold. Rectangles delimit the hybrid clades I and II.</p

The plots of <i>dist.mut</i> and <i>dist.bp</i> values as a function of true age.

<p>Each gray line tracks the evolution of one clonal lineage over the time. Simulations are shown for the 2 values of mutation rate (µ) under SSM mutation model. At selected times, we represent the boxplots showing the median of simulated values of <i>dist.mut</i> and <i>dist.bp</i>. as well as first and third quartile; whiskers represent 1.5 times the interquartile range.</p