The order Perissodactyla, the group of odd-toed ungulates, includes three extant families: Equidae, Tapiridae, and Rhinocerotidae. The extremely rapid karyotypic diversification in perissodactyls has so far prevented the establishment of genome-wide homology maps between these three families by traditional cytogenetic approaches. Here we report the first genome-wide comparative chromosome maps of African rhinoceroses, four tapir species, four equine species, and humans. These maps were established by multidirectional chromosome painting, with paint probes derived from flow-sorted chromosomes of Equus grevyi, Tapirus indicus, and Ceratotherium simum as well as painting probes from horse and human. The Malayan tapir (Tapirus indicus), Baird's tapir (T. bairdii), mountain tapir (T. pinchaque), lowland tapir (T. terrestris), and onager (E. hemionus onager), were studied by cross-species chromosome painting for the first time. Our results, when integrated with previously published comparative chromosome maps of the other perissodactyl species, have enabled the reconstruction of perissodactyl, ceratomorph, and equid ancestral karyotypes, and the identification of the defining evolutionary chromosomal rearrangements along each lineage. Our results allow a more reliable estimate of the mode and tempo of evolutionary chromosomal rearrangements, revealing a striking switch between the slowly evolving ceratomorphs and extremely rapidly evolving equids.

Recent molecular and morphological studies place Artiodactyla and Cetacea into the order Cetartiodactyla. Within the Cetartiodactyla such families as Bovidae, Cervidae, and Suidae are well studied by comparative chromosome painting, but many taxa that are crucial for understanding cetartiodactyl phylogeny remain poorly studied. Here we present the genome-wide comparative maps of five cetartiodactyl species obtained by chromosome painting with human and dromedary paint probes from four taxa: Cetacea, Hippopotamidae, Giraffidae, and Moschidae. This is the first molecular cytogenetic report on pilot whale, hippopotamus, okapi, and Siberian musk deer. Our results, when integrated with previously published comparative chromosome maps allow us to reconstruct the evolutionary pathway and rates of chromosomal rearrangements in Cetartiodactyla. We hypothesize that the putative cetartiodactyl ancestral karyotype (CAK) contained 25-26 pairs of autosomes, 2n = 52-54, and that the association of human chromosomes 8/9 could be a cytogenetic signature that unites non-camelid cetartiodactyls. There are no unambiguous cytogenetic landmarks that unite Hippopotamidae and Cetacea. If we superimpose chromosome rearrangements on the supertree generated by Price and colleagues, several homoplasy events are needed to explain cetartiodactyl karyotype evolution. Our results apparently favour a model of non-random breakpoints in chromosome evolution. Cetariodactyl karyotype evolution is characterized by alternating periods of low and fast rates in various lineages. The highest rates are found in Suina (Suidae+Tayasuidae) lineage (1.76 rearrangements per million years (R/My)) and the lowest in Cetaceans (0.07 R/My). Our study demonstrates that the combined use of human and camel paints is highly informative for revealing evolutionary karyotypic rearrangements among cetartiodactyl species.

The genus Microtus consists of 65 extant species, making it one of the rodentia genera with the highest number of species. The extreme karyotype diversification in Microtus has made them an ideal species group for comparative cytogenetics and cytotaxonomy. Conventional comparative cytogenetic studies in Microtus have been based mainly on chromosomal banding patterns; the number of Microtus species examined by molecular cytogenetics-cross-species chromosome painting-is limited. In this study, we used whole chromosome painting probes of the field vole Microtus agrestis to detect regions of homology in the karyotypes of eight Microtus species. For almost all investigated species, species-specific associations of conserved chromosomal segments were revealed. Analysis of data obtained here and previously published data allowed us to propose that the ancestral Microtus species had a 2n = 54 karyotype, including two associations of field vole chromosomal segments (MAG 1/17 and 2/8). Further mapping of the chromosome rearrangements onto a molecular phylogenetic tree allows the reconstruction of a karyotype evolution pathway in the Microtus genus.

Cross-species chromosome painting has become the mainstay of comparative cytogenetic and chromosome evolution studies. Here we have made a set of chromosomal painting probes for the field vole (Microtus agrestis) by DOP-PCR amplification of flow-sorted chromosomes. Together with painting probes of golden hamster (Mesocricetus auratus) and mouse (Mus musculus), the field vole probes have been hybridized onto the metaphases of the tundra vole (Microtus oeconomus). A comparative chromosome map between these two voles, golden hamster and mouse has been established based on the results of cross-species chromosome painting and G-banding comparisons. The sets of paints from the field vole, golden hamster and mouse identified a total of 27, 40 and 47 homologous autosomal regions, respectively, in the genome of tundra vole; 16, 41 and 51 fusion/fission rearrangements differentiate the karyotype of the tundra vole from the karyotypes of the field vole, golden hamster and mouse, respectively.