BACKGROUND: Loaches of the family Nemacheilidae are one of the most speciose elements of Palearctic freshwater ichthyofauna and have undergone rapid ecological adaptations and colonizations. Their cytotaxonomy is largely unexplored; with the impact of cytogenetical changes on this evolutionary diversification still unknown. An extensive cytogenetical survey was performed in 19 nemacheilid species using both conventional (Giemsa staining, C- banding, Ag- and Chromomycin A3/DAPI stainings) and molecular (fluorescence in situ hybridization with 5S rDNA, 45S rDNA, and telomeric (TTAGGG)n probes) methods. A phylogenetic tree of the analysed specimens was constructed based on one mitochondrial (cytochrome b) and two nuclear (RAG1, IRBP) genes. RESULTS: Seventeen species showed karyotypes composed of 2n = 50 chromosomes but differentiated by fundamental chromosome number (NF = 68-90). Nemachilichthys ruppelli (2n = 38) and Schistura notostigma (2n = 44-48) displayed reduced 2n with an elevated number of large metacentric chromosomes. Only Schistura fasciolata showed morphologically differentiated sex chromosomes with a multiple system of the XY1Y2 type. Chromomycin A3 (CMA3)- fluorescence revealed interspecific heterogeneity in the distribution of GC-rich heterochromatin including its otherwise very rare association with 5S rDNA sites. The 45S rDNA sites were mostly located on a single chromosome pair contrasting markedly with a pattern of two (Barbatula barbatula, Nemacheilus binotatus, N. ruppelli) to 20 sites (Physoschistura sp.) of 5S rDNA. The cytogenetic changes did not follow the phylogenetic relationships between the samples. A high number of 5S rDNA sites was present in species with small effective population sizes. CONCLUSION: Despite a prevailing conservatism of 2n, Nemacheilidae exhibited a remarkable cytogenetic variability on microstructural level. We suggest an important role for pericentric inversions, tandem and centric fusions in nemacheilid karyotype differentiation. Short repetitive sequences, genetic drift, founder effect, as well as the involvement of transposable elements in the dispersion of ribosomal DNA sites, might also have played a role in evolutionary processes such as reproductive isolation. These remarkable dynamics of their genomes qualify river loaches as a model for the study of the cytogenetic background of major evolutionary processes such as radiation, endemism and colonization of a wide range of habitats.

Department of Ecology Faculty of Science Charles University Prague Viničná 7 128 44 Prague 2 Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Prague Viničná 5 128 44 Prague 2 Czech Republic

Laboratory of Fish Genetics Institute of Animal Physiology and Genetics Czech Academy of Sciences Rumburská 89 Liběchov 277 21 Czech Republic

Research Institute for Limnology University of Innsbruck Mondseestraße 9 A 5310 Mondsee Austria

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Small Body, Large Chromosomes: Centric Fusions Shaped the Karyotype of the Amazonian Miniature Fish Nannostomus anduzei (Characiformes, Lebiasinidae)

Using Species Groups to Approach the Large and Taxonomically Unresolved Freshwater Fish Family Nemacheilidae (Teleostei: Cypriniformes)

Against the mainstream: exceptional evolutionary stability of ZW sex chromosomes across the fish families Triportheidae and Gasteropelecidae (Teleostei: Characiformes)

A supernumerary "B-sex" chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus

Multiple sex chromosomes in teleost fishes from a cytogenetic perspective: state of the art and future challenges

Measurement of Chromosomal Arms and FISH Reveal Complex Genome Architecture and Standardized Karyotype of Model Fish, Genus Carassius

Present and Future Salmonid Cytogenetics

Chromosomes of Asian cyprinid fishes: Variable karyotype patterns and evolutionary trends in the genus Osteochilus (Cyprinidae, Labeoninae, "Osteochilini")

Patterns of Sex Chromosome Differentiation in Spiders: Insights from Comparative Genomic Hybridisation

Taxonomic Diversity Not Associated with Gross Karyotype Differentiation: The Case of Bighead Carps, Genus Hypophthalmichthys (Teleostei, Cypriniformes, Xenocyprididae)

An Insight into the Chromosomal Evolution of Lebiasinidae (Teleostei, Characiformes)

Centric Fusions behind the Karyotype Evolution of Neotropical Nannostomus Pencilfishes (Characiforme, Lebiasinidae): First Insights from a Molecular Cytogenetic Perspective

Deciphering the Evolutionary History of Arowana Fishes (Teleostei, Osteoglossiformes, Osteoglossidae): Insight from Comparative Cytogenomics

Comparative Cytogenetics and Neo-Y Formation in Small-Sized Fish Species of the Genus Pyrrhulina (Characiformes, Lebiasinidae)

Deciphering the Origin and Evolution of the X1X2Y System in Two Closely-Related Oplegnathus Species (Oplegnathidae and Centrarchiformes)

Genomic Organization of Repetitive DNA Elements and Extensive Karyotype Diversity of Silurid Catfishes (Teleostei: Siluriformes): A Comparative Cytogenetic Approach

Chromosomes of Asian cyprinid fishes: cytogenetic analysis of two representatives of small paleotetraploid tribe Probarbini