turtles demonstrate variability in sex determination and, hence, constitute an excellent model for the evolution of sex chromosomes. notably, the sex determination of the freshwater turtles from the family chelidae, a species-rich group with wide geographical distribution in the southern hemisphere, is still poorly explored. Here we documented the presence of an XX/XY sex determination system in seven species of the Australasian chelid genera Chelodina, Emydura, and Elseya by conventional (karyogram reconstruction, c-banding) and molecular cytogenetic methods (comparative genome hybridization, in situ hybridization with probes specific for GATA microsatellite motif, the rDNA loci, and the telomeric repeats). the sex chromosomes are microchromosomes in all examined species of the genus Chelodina. In contrast, the sex chromosomes are the 4 th largest pair of macrochromosomes in the genera Emydura and Elseya. their X chromosomes are submetacentric, while their Y chromosomes are metacentric. The chelid Y chromosomes contain a substantial male-specific genomic region with an accumulation of the GATA microsatellite motif, and occasionally, of the rDNA loci and telomeric repeats. Despite morphological differences between sex chromosomes, we conclude that male heterogamety was likely already present in the common ancestor of Chelodina, Emydura and Elseya in the Mesozoic period.Amniotes possess two major sex determination systems: genotypic sex determination (GSD) and environmental sex determination (ESD). In GSD, the sex of an individual is determined by its sex-specific genotype, i.e. the combination of sex chromosomes. On the contrary, in ESD, the sex of an individual is influenced by environmental conditions and there are no consistent genotypic differences between sexes. The most well studied type of ESD is the temperature-dependent sex determination (TSD), where the sex of the individual is influenced by the temperature during a sensitive period of embryonic development (the definitions follow Johnson Pokorná & Kratochvíl 1 ). Three amniote lineages, the geckos (infraorder Gekkota), the dragon lizards (family Agamidae) and the turtles (order Testudines), show extensive variability of sex determination systems, and closely related species have either GSD or ESD 1-4 , making them excellent groups for exploring the evolution of sex determination.Turtles include 361 currently recognized extant species 5-7 . Unfortunately, the sex determination system is known in only approximately 24% of all species, and sex chromosomes have been up to now reported for only 20 species 4,[8][9][10] . Phylogenetic reconstruction of sex determination systems suggested that ESD is ancestral in turtles and sex chromosomes, and thus GSD, evolved at least five times independently. In the suborder Cryptodira, XX/ XY sex chromosomes have been reported for Siebenrockiella crassicollis (family Geoemydidae) 4,11,12 and for the genera Staurotypus (family Kinosternidae) 13 and Glyptemys (family Emydidae) 14,15 . In contrast, ZZ/ZW sex chromosomes are...

For a long time, turtles of the family Geoemydidae have been considered exceptional because representatives of this family were thought to possess a wide variety of sex determination systems. In the present study, we cytogenetically studied Geoemyda spengleri and G. japonica and re-examined the putative presence of sex chromosomes in Pangshura smithii. Karyotypes were examined by assessing the occurrence of constitutive heterochromatin, by comparative genome hybridization and in situ hybridization with repetitive motifs, which are often accumulated on differentiated sex chromosomes in reptiles. We found similar karyotypes, similar distributions of constitutive heterochromatin and a similar topology of tested repetitive motifs for all three species. We did not detect differentiated sex chromosomes in any of the species. For P. smithii, a ZZ/ZW sex determination system, with differentiated sex chromosomes, was described more than 40 years ago, but this finding has never been re-examined and was cited in all reviews of sex determination in reptiles. Here, we show that the identification of sex chromosomes in the original report was based on the erroneous pairing of chromosomes in the karyogram, causing over decades an error cascade regarding the inferences derived from the putative existence of female heterogamety in geoemydid turtles.

Telomeres are nucleoprotein complexes protecting chromosome ends in most eukaryotic organisms. In addition to chromosome ends, telomeric-like motifs can be accumulated in centromeric, pericentromeric and intermediate (i.e., between centromeres and telomeres) positions as so-called interstitial telomeric repeats (ITRs). We mapped the distribution of (TTAGGG)n repeats in the karyotypes of 30 species from nine families of turtles using fluorescence in situ hybridization. All examined species showed the expected terminal topology of telomeric motifs at the edges of chromosomes. We detected ITRs in only five species from three families. Combining our and literature data, we inferred seven independent origins of ITRs among turtles. ITRs occurred in turtles in centromeric positions, often in several chromosomal pairs, in a given species. Their distribution does not correspond directly to interchromosomal rearrangements. Our findings support that centromeres and non-recombining parts of sex chromosomes are very dynamic genomic regions, even in turtles, a group generally thought to be slowly evolving. However, in contrast to squamate reptiles (lizards and snakes), where ITRs were found in more than half of the examined species, and birds, the presence of ITRs is generally rare in turtles, which agrees with the expected low rates of chromosomal rearrangements and rather slow karyotype evolution in this group.

The Asian box turtle genus Cuora currently comprises 13 species with a wide distribution in Southeast Asia, including China and the islands of Indonesia and Philippines. The populations of these species are rapidly declining due to human pressure, including pollution, habitat loss, and harvesting for food consumption. Notably, the IUCN Red List identifies almost all species of the genus Cuora as Endangered (EN) or Critically Endangered (CR). In this study, we explore the karyotypes of 10 Cuora species with conventional (Giemsa staining, C-banding, karyogram reconstruction) and molecular cytogenetic methods (in situ hybridization with probes for rDNA loci and telomeric repeats). Our study reveals a diploid chromosome number of 2n = 52 chromosomes in all studied species, with karyotypes of similar chromosomal morphology. In all examined species, rDNA loci are detected at a single medium-sized chromosome pair and the telomeric repeats are restricted to the expected terminal position across all chromosomes. In contrast to a previous report, sex chromosomes are neither detected in Cuoragalbinifrons nor in any other species. Therefore, we assume that these turtles have either environmental sex determination or genotypic sex determination with poorly differentiated sex chromosomes. The conservation of genome organization could explain the numerous observed cases of interspecific hybridization both within the genus Cuora and across geoemydid turtles.

This Article contains an error in the order of the Figures. Figures 4 and 5 were published as Figures 5 and 4 respectively. The correct Figures 4 and 5 appear below as Figs. 1 and 2. The Figure legends are correct.