Pinniped Karyotype Evolution Substantiated by Comparative Chromosome Painting of 10 Pinniped Species (Pinnipedia, Carnivora)

Numerous Carnivora karyotype evolution investigations have been performed by classical and molecular cytogenetics and were supplemented by reconstructions of the Ancestral Carnivora Karyotype (ACK). However, the group of Pinnipedia was not studied in detail. Here we reconstruct pinniped karyotype evolution and refine ACK using published and our new painting data for 10 pinniped species. The combination of human (HSA) and domestic dog (CFA) whole-chromosome painting probes was used for the construction of the comparative chromosome maps for species from all three pinniped families: Odobenidae– Odobenus rosmarus Linnaeus, 1758, Phocidae – Phoca vitulina Linnaeus, 1758, Pusa sibirica Gmelin, 1788, Erignathus barbatus Erxleben, 1777, Phoca largha Pallas, 1811, Phoca hispida Schreber, 1775 and Otariidae – Eumetopias jubatus Schreber, 1775, Callorhinus ursinus Linnaeus, 1758, Phocarctos hookeri Gray, 1844, Arctocephalus forsteri Lesson, 1828. HSA and CFA autosome painting probes have delineated 32 and 68 conservative autosome segments in the studied genomes. The comparative painting in Pinnipedia supports monophyletic origin of pinnipeds, shows that pinniped karyotype evolution was characterized by slow rate of genome rearrangements (less then one rearrangement per 10 million years), provides strong support for refined structure of ACK with 2n = 38 and specifies plausible order of dog chromosome synthenic segments on ancestral Carnivora chromosomes. The heterochromatin, telomere and ribosomal DNA distribution was studied in all 10 species

New Data on Comparative Cytogenetics of the Mouse-Like Hamsters (Calomyscus Thomas, 1905) from Iran and Turkmenistan.

The taxonomy of the genus Calomyscus remains controversial. According to the latest systematics the genus includes eight species with great karyotypic variation. Here, we studied karyotypes of 14 Calomyscus individuals from different regions of Iran and Turkmenistan using a new set of chromosome painting probes from a Calomyscus sp. male (2n = 46, XY; Shahr-e-Kord-Soreshjan-Cheshme Maiak Province). We showed the retention of large syntenic blocks in karyotypes of individuals with identical chromosome numbers. The only rearrangement (fusion 2/21) differentiated Calomyscus elburzensis, Calomyscus mystax mystax, and Calomyscus sp. from Isfahan Province with 2n = 44 from karyotypes of C. bailwardi, Calomyscus sp. from Shahr-e-Kord, Chahar Mahal and Bakhtiari-Aloni, and Khuzestan-Izeh Provinces with 2n = 46. The individuals from Shahdad tunnel, Kerman Province with 2n = 51-52 demonstrated non-centric fissions of chromosomes 4, 5, and 6 of the 46-chromosomal form with the formation of separate small acrocentrics. A heteromorphic pair of chromosomes in a specimen with 2n = 51 resulted from a fusion of two autosomes. C-banding and chromomycin A3-DAPI staining after G-banding showed extensive heterochromatin variation between individuals

Karyotype Evolution in 10 Pinniped Species: Variability of Heterochromatin versus High Conservatism of Euchromatin as Revealed by Comparative Molecular Cytogenetics

Pinnipedia karyotype evolution was studied here using human, domestic dog, and stone marten whole-chromosome painting probes to obtain comparative chromosome maps among species of Odobenidae (Odobenus rosmarus), Phocidae (Phoca vitulina, Phoca largha, Phoca hispida, Pusa sibirica, Erignathus barbatus), and Otariidae (Eumetopias jubatus, Callorhinus ursinus, Phocarctos hookeri, and Arctocephalus forsteri). Structural and functional chromosomal features were assessed with telomere repeat and ribosomal-DNA probes and by CBG (C-bands revealed by barium hydroxide treatment followed by Giemsa staining) and CDAG (Chromomycin A3-DAPI after G-banding) methods. We demonstrated diversity of heterochromatin among pinniped karyotypes in terms of localization, size, and nucleotide composition. For the first time, an intrachromosomal rearrangement common for Otariidae and Odobenidae was revealed. We postulate that the order of evolutionarily conserved segments in the analyzed pinnipeds is the same as the order proposed for the ancestral Carnivora karyotype (2n = 38). The evolution of conserved genomes of pinnipeds has been accompanied by few fusion events (less than one rearrangement per 10 million years) and by novel intrachromosomal changes including the emergence of new centromeres and pericentric inversion/centromere repositioning. The observed interspecific diversity of pinniped karyotypes driven by constitutive heterochromatin variation likely has played an important role in karyotype evolution of pinnipeds, thereby contributing to the differences of pinnipeds’ chromosome sets

Complex Structure of Lasiopodomys mandarinus vinogradovi Sex Chromosomes, Sex Determination, and Intraspecific Autosomal Polymorphism.

The mandarin vole, Lasiopodomys mandarinus, is one of the most intriguing species among mammals with non-XX/XY sex chromosome system. It combines polymorphism in diploid chromosome numbers, variation in the morphology of autosomes, heteromorphism of X chromosomes, and several sex chromosome systems the origin of which remains unexplained. Here we elucidate the sex determination system in Lasiopodomys mandarinus vinogradovi using extensive karyotyping, crossbreeding experiments, molecular cytogenetic methods, and single chromosome DNA sequencing. Among 205 karyotyped voles, one male and three female combinations of sex chromosomes were revealed. The chromosome segregation pattern and karyomorph-related reproductive performances suggested an aberrant sex determination with almost half of the females carrying neo-X/neo-Y combination. The comparative chromosome painting strongly supported this proposition and revealed the mandarin vole sex chromosome systems originated due to at least two de novo autosomal translocations onto the ancestral X chromosome. The polymorphism in autosome 2 was not related to sex chromosome variability and was proved to result from pericentric inversions. Sequencing of microdissection derived of sex chromosomes allowed the determination of the coordinates for syntenic regions but did not reveal any Y-specific sequences. Several possible sex determination mechanisms as well as interpopulation karyological differences are discussed

Complex Structure of Lasiopodomys mandarinus vinogradovi Sex Chromosomes, Sex Determination, and Intraspecific Autosomal Polymorphism.

The mandarin vole, Lasiopodomys mandarinus, is one of the most intriguing species among mammals with non-XX/XY sex chromosome system. It combines polymorphism in diploid chromosome numbers, variation in the morphology of autosomes, heteromorphism of X chromosomes, and several sex chromosome systems the origin of which remains unexplained. Here we elucidate the sex determination system in Lasiopodomys mandarinus vinogradovi using extensive karyotyping, crossbreeding experiments, molecular cytogenetic methods, and single chromosome DNA sequencing. Among 205 karyotyped voles, one male and three female combinations of sex chromosomes were revealed. The chromosome segregation pattern and karyomorph-related reproductive performances suggested an aberrant sex determination with almost half of the females carrying neo-X/neo-Y combination. The comparative chromosome painting strongly supported this proposition and revealed the mandarin vole sex chromosome systems originated due to at least two de novo autosomal translocations onto the ancestral X chromosome. The polymorphism in autosome 2 was not related to sex chromosome variability and was proved to result from pericentric inversions. Sequencing of microdissection derived of sex chromosomes allowed the determination of the coordinates for syntenic regions but did not reveal any Y-specific sequences. Several possible sex determination mechanisms as well as interpopulation karyological differences are discussed

The Ancestral Carnivore Karyotype As Substantiated by Comparative Chromosome Painting of Three Pinnipeds, the Walrus, the Steller Sea Lion and the Baikal Seal (Pinnipedia, Carnivora).

Karyotype evolution in Carnivora is thoroughly studied by classical and molecular cytogenetics and supplemented by reconstructions of Ancestral Carnivora Karyotype (ACK). However chromosome painting information from two pinniped families (Odobenidae and Otariidae) is noticeably missing. We report on the construction of the comparative chromosome map for species from each of the three pinniped families: the walrus (Odobenus rosmarus, Odobenidae-monotypic family), near threatened Steller sea lion (Eumetopias jubatus, Otariidae) and the endemic Baikal seal (Pusa sibirica, Phocidae) using combination of human, domestic dog and stone marten whole-chromosome painting probes. The earliest karyological studies of Pinnipedia showed that pinnipeds were characterized by a pronounced karyological conservatism that is confirmed here with species from Phocidae, Otariidae and Odobenidae sharing same low number of conserved human autosomal segments (32). Chromosome painting in Pinnipedia and comparison with non-pinniped carnivore karyotypes provide strong support for refined structure of ACK with 2n = 38. Constructed comparative chromosome maps show that pinniped karyotype evolution was characterized by few tandem fusions, seemingly absent inversions and slow rate of genome rearrangements (less then one rearrangement per 10 million years). Integrative comparative analyses with published chromosome painting of Phoca vitulina revealed common cytogenetic signature for Phoca/Pusa branch and supports Phocidae and Otaroidea (Otariidae/Odobenidae) as sister groups. We revealed rearrangements specific for walrus karyotype and found the chromosomal signature linking together families Otariidae and Odobenidae. The Steller sea lion karyotype is the most conserved among three studied species and differs from the ACK by single fusion. The study underlined the strikingly slow karyotype evolution of the Pinnipedia in general and the Otariidae in particular

Ancestral carnivore karyotype (ACK).

<p>Numbers on the left are HSA and CFA chromosomes. Numbers on the right of each ancestral chromosome are ACK chromosomes according to nomenclature of Murphy et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref026" target="_blank">26</a>] followed by cat (FCA) homology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref013" target="_blank">13</a>]. MFO chromosome numbers are indicated right below the ancestral chromosome. Big numerals below correspond to Nash et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref047" target="_blank">47</a>] ACK nomenclature. The proposed ancestral order of syntenic segments corresponding to dog chromosomes is based on parsimony analyses of chromosome painting data of canid painting probes in Carnivora and two outgroup species: pig and human, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref053" target="_blank">53</a>]. We exchanged ACK11 and ACK12 relative to ACK published in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref011" target="_blank">11</a>] to match chromosome order [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147647#pone.0147647.ref047" target="_blank">47</a>] and to be arranged according to the chromosome size in cat and stone marten karyotypes.</p

Karyotype Evolution in 10 Pinniped Species: Variability of Heterochromatin versus High Conservatism of Euchromatin as Revealed by Comparative Molecular Cytogenetics

Pinnipedia karyotype evolution was studied here using human, domestic dog, and stone marten whole-chromosome painting probes to obtain comparative chromosome maps among species of Odobenidae (Odobenus rosmarus), Phocidae (Phoca vitulina, Phoca largha, Phoca hispida, Pusa sibirica, Erignathus barbatus), and Otariidae (Eumetopias jubatus, Callorhinus ursinus, Phocarctos hookeri, and Arctocephalus forsteri). Structural and functional chromosomal features were assessed with telomere repeat and ribosomal-DNA probes and by CBG (C-bands revealed by barium hydroxide treatment followed by Giemsa staining) and CDAG (Chromomycin A3-DAPI after G-banding) methods. We demonstrated diversity of heterochromatin among pinniped karyotypes in terms of localization, size, and nucleotide composition. For the first time, an intrachromosomal rearrangement common for Otariidae and Odobenidae was revealed. We postulate that the order of evolutionarily conserved segments in the analyzed pinnipeds is the same as the order proposed for the ancestral Carnivora karyotype (2n = 38). The evolution of conserved genomes of pinnipeds has been accompanied by few fusion events (less than one rearrangement per 10 million years) and by novel intrachromosomal changes including the emergence of new centromeres and pericentric inversion/centromere repositioning. The observed interspecific diversity of pinniped karyotypes driven by constitutive heterochromatin variation likely has played an important role in karyotype evolution of pinnipeds, thereby contributing to the differences of pinnipeds&rsquo; chromosome sets

GTG-banded karyotype of the Steller sea lion (<i>Eumetopias jubatus</i>, 2n = 36) with the assignment of homology to human (HSA), dog (CFA) and stone marten (MFO) chromosomes.

<p>The square denotes the centromere position on corresponding chromosome. *—segments were not painted by any dog probe.</p

Examples of localization of dog (A-C) and human (D-F) painting probes with GTG-banding of the same metaphase to the right.

<p>(A) CFA1/26 on EJUB chromosomes; (B) CFA 21+23/18 on OROS chromosomes; (C) CFA16/21+28 on PSIB chromosomes; (D) HSA 19/3 on OROS chromosomes; (E) HSA12/16 on PSIB chromosomes; (F) HSA7/1 on OROS chromosomes.</p