Turtles of the genera Geoemyda and Pangshura (Testudines: Geoemydidae) lack differentiated sex chromosomes: the end of a 40-year error cascade for Pangshura

Abstract

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.

Keywords: Comparative genome hybridization; Evolution; FISH; Karyotype; Microsatellite; Sex chromosomes; Sex determination; Telomeres; Turtles.

Figure 1. Karyograms and C-banded metaphases of Geoemyda japonica (A–D), Geoemyda spengleri (E–H), and Pangshura smithii (I–L).

Please note that microchromosomes are paired according to size for illustration, which does not correspond to actual homology of chromosomes.

Figure 2. FISH with rDNA, (GATA)₈ and telomeric probes in metaphases of Geoemyda japonica (A–H), Geoemyda spengleri (I–P), and Pangshura smithii (Q–X).

Chromosomes are stained blue with DAPI, and the signal of the probe is pseudocolored in red. In CGH, the male genome is stained with FITC (green color) and the female genome with rhodamine (red color). Genomic regions common for both sexes appear yellow due to the combination of green and red color. Chromosomal regions with similar sequence content in both sexes are visualized in yellow. Arrows indicate the chromosome pair 12, with the prominent C-positive block.

Figure 3. The original karyogram of Sharma, Kaur & Nakhasi (1975) (A), their karyogram re-arranged by us (B), and a new karyogram of a female individual from our studied material (C).

Note that the chromosomes misidentified as Z and W in the original study (A) can be autosomal and easily assigned according to size and morphology into the pairs 16–26 and 7–9, respectively, in our new karyogram (C). Numbers in the re-arranged karyogram (B) refer to the original assignment of chromosome pairs by Sharma, Kaur & Nakhasi (1975).

Figure 4. Phylogenetic reconstruction of the sex determination modes in turtles from the family Geoemydidae.

Phylogenetic relationships follow Spinks et al. (2004), Lourenço et al. (2013) and Pereira et al. (2017).