Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides New Insights into Sex Chromosome Evolution in Paradoxical Frogs

Abstract

In the frog genus Pseudis, previous works found a sex-linked heteromorphism of the PcP190 satellite DNA in the nucleolus organizer region (NOR)-bearing chromosome pairs of Pseudis bolbodactyla and Pseudis tocantins, which possess a ZZ/ZW sex determination system. A pericentromeric inversion was inferred to have occurred during W chromosome evolution, moving a chromosomal cluster enriched by the PcP190 from the short arm (as observed in P. bolbodactyla) to the NOR-bearing long arm (as observed in P. tocantins). However, whether such an inversion happened in P. tocantins or in the common ancestor of Pseudis fusca and P. tocantins remained unclear. To assess this question, we mapped PcP190 in the karyotype of P. fusca from three distinct localities. Southern blotting was used to compare males and females. The mitochondrial H1 fragment (which contains the 12S ribosomal RNA (rRNA), tRNAval, and 16S rRNA genes) and cytochrome b gene were partially sequenced, and a species tree was inferred to guide our analysis. Pseudis fusca specimens were placed together as the sister group of P. tocantins, but based on genetic distance, one of the analyzed populations is probably an undescribed species. A cluster of PcP190, located in the long arm of chromosome 7, is sex linked in this putative new species but not in the remaining P. fusca. We could infer that the pericentromeric inversion that moved the PcP190 site to the NOR-bearing chromosome arm (long arm) occurred in the common ancestor of P. fusca, the putative undescribed species, and P. tocantins.

Keywords: chromosome evolution; chromosome rearrangements; phylogeny; satellite DNA; sex chromosomes.

Figure 1

Karyotype of a female of Pseudis fusca from Carlos Chagas–MG subjected to Giemsa conventional staining (A), C-banding (B), C-banding followed by DAPI (C), and MM staining (D), and silver impregnation by the Ag-NOR method (E). The inset in (B) shows chromosome pair 1 obtained from another metaphase, in which the terminal C-bands clearly can be seen. Bar: 5 µm.

Figure 2

Pair 7 of the female ZUEC 22078 (upper panel) and the male ZUEC 22077 (bottom panel) of Pseudis fusca from Carlos Chagas–MG sequentially subjected to C-banding, DAPI, and MM staining and silver impregnation (Ag-NOR method). In the ideograms (Id) of pair 7, NORs are indicated by gray circles, centromeres are in black, and adjacent to them is the heterochromatin in green and blue, representing simultaneous DAPI/MM staining. Note that the centromeres are also stained with DAPI and MM, but this is not shown in the ideograms (Id). Bar: 5 µm.

Figure 3

Alignment of the PcP190 satDNA sequences isolated from Pseudis fusca with complete sequences from the PcP-2 group previously isolated from P. tocantins (KX170921, KX170922, KX170923, KX170924), P. bolbodactyla (MH370391, MH370392, MH370395), and P. paradoxa (MH370405 and MH370407). Gray arrows indicate the P190F and P190R primer annealing regions. Sequences that show the central part of the CR with N breaks represent partial monomers.

Figure 4

Representative Southern blot analysis of PcP-2 sequences from two males (M) of Pseudis fusca from Coronel Murta–MG (Pfus (CM)), a male (M) and a female (F) of P. fusca from Salinas–MG (Pfus (Sal)), a male and a female of P. fusca from Carlos Chagas–MG (Pfus (CC)), and a female of P. tocantins as a positive control (Ptoc).

Figure 5

Fluorescent in situ hybridization of PcP190 satDNA in a male karyotype of Pseudis fusca from Coronel Murta–MG (ZUEC 13235) (A), a female karyotype of P. fusca from Salinas–MG (ZUEC 24518) (B), and a female of P. fusca from Carlos Chagas–MG (ZUEC 22078) (C). Bar: 5 µm.

Figure 6

Comparative genomic hybridization in Pseudis fusca from Carlos Chagas–MG. Chromosome preparation stained with DAPI (A) and subjected to CGH (B). (C) Chromosome pair 7 from the metaphase shown in (A,B) stained with DAPI, subjected to CGH, and silver-impregnated by the Ag-NOR method. In the ideograms, the heterochromatic regions are shown in black, the NORs in gray circles, and the region revealed by CGH in dashed green. Bar: 5 µm.

Figure 7

Phylogenetic relationships of Pseudis and Lysapsus inferred by Bayesian analysis of the mitochondrial H1 and cytb fragments. Numbers at the nodes indicate posterior probability values. Pseudis fusca from Carlos Chagas – MG is in bold.

Figure 8

Evolutionary hypothesis for sex chromosome evolution in Pseudis. The cladogram shows the phylogenetic relationships inferred in the present work and the ideograms of the NOR-bearing chromosomes. In the ideograms, the regions of PcP190 satDNA are shown in pink, heterochromatic blocks in black, and NOR in gray circles. Ideograms are based on data from Busin et al. [9,48], Gatto et al. [10,11], and the present study. Character changes in the NOR-bearing chromosomes of Pseudis: (1) paracentromeric inversion that moved the NOR from a distal region to a region closer to the centromere; (2) pericentromeric inversion that moved the PcP190 site from the short arm to the long arm; (3) paracentromeric inversion that moved the NOR to a pericentromeric area and the PcP190 site to the interstitial region in the W chromosome of P. tocantins; and (4) amplification of heterochromatin in the W chromosome of P. tocantins. Blue, orange, and purple arrows or arrowheads represent three alternative hypotheses for the sex-linked heteromorphism observed in P. tocantins, Pseudis sp., and P. bolbodactyla. Arrows indicate acquisition of the sex chromosome heteromorphism related to PcP190 satDNA. Arrowheads indicate loss of sex chromosome heteromorphism related to PcP190 satDNA.