Dynamics of tandemly repeated DNA sequences during evolution of diploid and tetraploid botiid loaches (Teleostei: Cobitoidea: Botiidae)

<div><p>Polyploidization has played an important role in the evolution of vertebrates, particularly at the base of Teleostei–an enormously successful ray-finned fish group with additional genome doublings on lower taxonomic levels. The investigation of post-polyploid genome dynamics might provide important clues about the evolution and ecology of respective species and can help to decipher the role of polyploidy <i>per se</i> on speciation. Few studies have attempted to investigate the dynamics of repetitive DNA sequences in the post-polyploid genome using molecular cytogenetic tools in fishes, though recent efforts demonstrated their usefulness. The demonstrably monophyletic freshwater loach family Botiidae, branching to evolutionary diploid and tetraploid lineages separated >25 Mya, offers a suited model group for comparing the long-term repetitive DNA evolution. For this, we integrated phylogenetic analyses with cytogenetical survey involving Giemsa- and Chromomycin A₃ (CMA₃)/DAPI stainings and fluorescence <i>in situ</i> hybridization with 5S/45S rDNA, U2 snDNA and telomeric probes in representative sample of 12 botiid species.</p><p>The karyotypes of all diploids were composed of 2n = 50 chromosomes, while majority of tetraploids had 2n = 4x = 100, with only subtle interspecific karyotype differences. The exceptional karyotype of <i>Botia dario</i> (2n = 4x = 96) suggested centric fusions behind the 2n reduction. Variable patterns of FISH signals revealed cases of intraspecific polymorphisms, rDNA amplification, variable degree of correspondence with CMA₃⁺ sites and almost no phylogenetic signal. In tetraploids, either additivity or loci gain/loss was recorded. Despite absence of classical interstitial telomeric sites, large blocks of interspersed rDNA/telomeric regions were found in diploids only.</p><p>We uncovered different molecular drives of studied repetitive DNA classes within botiid genomes as well as the advanced stage of the re-diploidization process in tetraploids. Our results may contribute to link genomic approach with molecular cytogenetic analyses in addressing the origin and mechanism of this polyploidization event.</p></div

List of analyzed species of Botiidae, sample sizes and distribution areas.

<p>List of analyzed species of Botiidae, sample sizes and distribution areas.</p

CMA₃/DAPI staining in selected botiid species.

<p>Mitotic metaphases of (<b>A</b>) <i>B</i>. <i>almorhae</i>, (<b>B</b>) <i>L</i>. <i>guilinensis</i>, (<b>C</b>) <i>L</i>. <i>microphthalma</i>, (<b>D</b>) <i>S</i>. <i>pulchra</i>. For better contrast, pictures were pseudocoloured in red (for CMA₃) and green (for DAPI). Open arrows indicate CMA₃-positive sites whose interindividual site-number variability is depicted in insets. Bar = 10 μm.</p

Phylogenetic relationships and karyotype characteristics of inspected botiids.

<p>2n, karyotype description, NF and idiograms showing chromosomes bearing 45S (red), 5S (green) rDNA and U2 snDNA (violet) sites are plotted onto phylogenetic tree obtained by Bayesian analysis based on the mitochondrial (<i>cyt b</i>) and nuclear (<i>RAG1</i>, <i>IRBP</i>) genes. Polymorphic rDNA sites are in brackets. The asterisk denotes species' karyotypes already published in Bohlen et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195054#pone.0195054.ref049" target="_blank">49</a>], with the st and a chromosome pairs being here scored together in one st-a category.</p

Karyotypes of botiid species after Giemsa staining.

<p>(<b>A</b>) <i>B</i>. <i>almorhae</i>, (<b>B</b>) <i>B</i>. <i>udomritthiruji</i>, (<b>C</b>) <i>Ch</i>. <i>macracanthus</i>, (<b>D</b>) <i>L</i>. <i>elongata</i>, (<b>E</b>) <i>L</i>. <i>microphthalma</i>, (<b>F</b>) <i>P</i>. <i>fasciatus</i>, (<b>G</b>) <i>S</i>. <i>superciliaris</i>, (<b>H</b>) <i>Y</i>. <i>lecontei</i>. Bar = 10 μm.</p

rDNA FISH in selected botiid species.

<p>28S rDNA (red, arrows) and 5S rDNA (green, arrowheads) probes mapped on mitotic chromosomes of (<b>A</b>) <i>B</i>. <i>almorhae</i>, (<b>B</b>) <i>B</i>. <i>udomritthiruji</i>, (<b>C</b>) <i>Ch</i>. <i>macracanthus</i>, (<b>D</b>) <i>L</i>. <i>guilinensis</i>, (<b>E</b>) <i>P</i>. <i>fasciatus</i>, (<b>F</b>) <i>S</i>. <i>pulchra</i>. Chromosomes were counterstained with DAPI (blue). Note the presence of double sites of the 5S rDNA on one chromosomal pair in <i>B</i>. <i>almorhae</i> (<b>A</b>-inset) and two chromosomal pairs in <i>B</i>. <i>udomritthiruji</i> (<b>B</b>). Polymorphic rDNA sites in <i>L</i>. <i>guilinensis</i> (<b>D</b>), <i>P</i>. <i>fasciatus</i> (<b>E</b>) and <i>S</i>. <i>pulchra</i> (<b>F</b>) are boxed. Bar = 10 μm.</p

Distribution areas of the investigated species of Botiidae.

<p>1 –<i>B</i>. <i>almorhae</i>, 2 –<i>B</i>. <i>dario</i>, 3 –<i>B</i>. <i>udomritthiruji</i>, 4 –<i>Ch</i>. <i>macracanthus</i>, 5 –<i>L</i>. <i>elongata</i>, 6 –<i>L</i>. <i>guilinensis</i>, 7 –<i>L</i>. <i>microphthalma</i>, 8 –<i>P</i>. <i>fasciatus</i>, 9 –<i>S</i>. <i>pulchra</i>, 10 –<i>S</i>. <i>superciliaris</i>, 11 –<i>S</i>. <i>zebra</i>, 12 –<i>Y</i>. <i>lecontei</i>.</p

5S rDNA and U2 snDNA dual-colour FISH in selected botiid species.

<p>U2 snDNA (red, arrows) and 5S rDNA (green, arrowheads) probes mapped on mitotic chromosomes of (<b>A</b>) <i>B</i>. <i>udomritthiruji</i>, (<b>B</b>) <i>L</i>. <i>microphthalma</i>, (<b>C</b>) <i>Y</i>. <i>lecontei</i>. Chromosomes were counterstained with DAPI (blue). Note the significant spreading of 5S rDNA sites in <i>Y</i>. <i>lecontei</i> (<b>C</b>; 24 signals—arrowheads). The metaphase spread of <i>B</i>. <i>udomritthiruji</i> displays incomplete chromosome set (2n = 97), however the number of two U2 snDNA-bearing chromosomes was consistently observed on all other (including complete though less representative) metaphases in our dataset and with respect to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195054#pone.0195054.g005" target="_blank">Fig 5B</a>, the number of 5S rDNA-bearing chromosomes is also complete. Bar = 10 μm.</p

PNA FISH with telomeric probe in selected botiid species.

<p>Mitotic metaphases of (<b>A</b>) <i>B</i>. <i>almorhae</i>, (<b>B</b>) <i>B</i>. <i>udomritthiruji</i>, (<b>C</b>) <i>Ch</i>. <i>macracanthus</i>, (<b>D</b>) <i>L</i>. <i>guilinensis</i>, (<b>E</b>) <i>P</i>. <i>fasciatus</i>, (<b>F</b>) <i>S</i>. <i>pulchra</i>. For better contrast, pictures were pseudocoloured in green (telomeric repeat probe) and red (DAPI). Open arrows point to chromosomes with remarkable large-sized telomeric signals (<b>C-F</b>). Bar = 10 μm.</p

Macrogen_sequence_files

File produced during the sequencing of the PCR products used for FISH of 5S and 28S rDNA. File names contain "5S" or "28S" depending on what product they refer to