New Insights into the Sex Chromosome Evolution of the Common Barker Frog Species Complex (Anura, Leptodactylidae) Inferred from Its Satellite DNA Content

Abstract

Satellite DNAs (satDNAs) play a crucial role in understanding chromosomal evolution and the differentiation of sex chromosomes across diverse taxa, particularly when high karyotypic diversity occurs. The Physalaemus cuvieri-Physalaemus ephippifer species complex comprises at least seven divergent lineages, each exhibiting specific karyotypic signatures. The group composed of Ph. ephippifer, Lineage 1B of 'Ph. cuvieri' (L1B), and a lineage resulting from their secondary contact is especially intriguing due to varying degrees of sex chromosome heteromorphism. In this study, we characterized the satellitome of Ph. ephippifer in order to identify novel satDNAs that may provide insights into chromosomal evolution, particularly concerning sex chromosomes. We identified 62 satDNAs in Ph. ephippifer, collectively accounting for approximately 10% of the genome. Notably, nine satDNA families were shared with species from distantly related clades, raising questions about their potential roles in anurans genomes. Among the seven satDNAs mapped via fluorescent in situ hybridization, PepSat3 emerged as a strong candidate for the centromeric sequence in this group. Additionally, PepSat11 and PepSat24 provided evidence supporting a translocation involving both arms of the W chromosome in Ph. ephippifer. Furthermore, a syntenic block composed of PepSat3, PcP190, and PepSat11 suggested an inversion event during the divergence of Ph. ephippifer and L1B. The variation in signal patterns of satDNAs associated with nucleolar organizer regions (NORs) highlights the complexity of NOR evolution in this species complex, which exhibits substantial diversity in this genomic region. Additionally, our findings for PepSat30-350 emphasize the importance of validating the sex-biased abundance of satDNAs.

Keywords: chromosomal homologies; chromosomal rearrangements; nucleolar organizer region; repetitive elements; satellitome.

Figure 1

Karyotypes hybridized with the PepSat3-152 probe. (A) Specimens of Physalaemus ephippifer from Santa Bárbara, Pará, Brazil. (B) Samples from Lineage 1B (L1B) of ‘Ph. cuvieri’, from Urbano Santos, Maranhão, Brazil. (C) Individuals from São Pedro da Água Branca (SPAB), Maranhão, and Dom Eliseu (DE), Pará, both localities being within the known range of the CZ Pep-L1B. Scale bar = 5 μm.

Figure 2

Karyotypes hybridized with the PepSat11-237 satDNA probe. (A) Specimen from Santa Bárbara, Pará, Brazil. The inset displays the heteromorphic sex chromosome pair Z and W from a female from the same locality. (B) Female from Urbano Santos, Maranhão, Brazil. The inset shows pair 9 from a male from the same site. (C) Sample from São Pedro da Água Branca (SPAB), Maranhão. The inset highlights the female Z and W chromosomes marked with the probe. The dashed inset shows the same chromosome pair from the karyogram hybridized with both PcP190 1a and PepSat11-237 probes, where both signals are adjacent, with PcP190 located closer to the centromeric region. Scale bar = 5 μm.

Figure 3

Chromosomes hybridized with the PepSat24-620 probe. (A) Female Physalaemus ephippifer from Santa Bárbara, Pará, Brazil. The left insets show the sex chromosomes of two additional specimens mapped with the same probe, while the right inset highlights the W chromosome marked with both PepSat11-237 (green) and PepSat24-620 (red). Note that signals are located in opposite arms of the chromosome. (B) Chromosome pair 9 of both male and female from Urbano Santos, Maranhão, Brazil. (C) Z and W chromosomes of specimens from CZ Pep-L1B. Scale bar = 5 μm.

Figure 4

Karyotypes mapped with the PepSat16-147 (A–C) and PepSat25-282 (D–F) probes. (A) Female Physalaemus ephippifer from Santa Bárbara, Pará, Brazil. The filled arrowhead on the Z chromosome indicates the only signal of this probe that does not coincide with NORs. (B) Female of Lineage 1B (L1B) of ‘Ph. cuvieri’, collected in Urbano Santos, Maranhão, Brazil. The top inset shows chromosomes from a less compacted metaphase hybridized with the same probe; note that on chromosome 8, the probe signal does not extend across the entire secondary constriction (empty arrowhead). (C) Female metaphase of a specimen of the CZ Pep-L1B lineage, from São Pedro da Água Branca, Maranhão, Brazil. This specimen possesses only one evident NOR-bearing homologue of chromosome 8 (the one on the left in the shown chromosome 8 pairs). Note that the short arm of the W chromosome lacks a cluster of this satellite DNA, although it carries a terminal NOR. The probe signal observed on chromosome 3 is an artifact caused by overlap with the W chromosome in the metaphase spread. The dashed inset exhibits the same chromosome pairs 7, 8, and 9 (ZW) from the karyogram with enhanced brightness for better visualization of the weak signals on chromosome pairs 7 and 8, as well as the difference in intensity compared to the sex chromosomes. (D) Male Ph. ephippifer from Santa Bárbara, Pará, Brazil. (E) Female from Lineage 1B (L1B) of ‘Ph. cuvieri’ from Urbano Santos, Maranhão, Brazil. (F) Male specimen from the CZ Pep-L1B, collected in Dom Eliseu, Pará. Insets in (A,C,D) show NOR-bearing chromosomes from other metaphases hybridized with the probes, followed by Ag-NOR staining. The bottom inset in (B), as well as the insets in (E,F), display the same chromosomes from the karyogram after the Ag-NOR assay. Note that the heteromorphisms in NOR size (for example, in pair 8 in (E)) and/or number (for example, the ZZ pair in (F)) correspond to heteromorphisms revealed by the probes, except for chromosome pair 8 of L1B (shown in (B)), in which the PepSat16-147 cluster is adjacent to the NOR constriction (inset in (B)). Scale bar = 5 μm.

Figure 5

Karyotypes of Physalaemus ephippifer from Santa Bárbara, Pará, Brazil, hybridized with the PepSat30-350 probe. The first karyogram and the inset represent the individuals used for Illumina sequencing in this study (i.e., specimens SMRP 252.156 and 252.166). Notably, these individuals exhibit differences in the number of satDNA clusters on chromosome 5, as indicated by the arrowheads. However, upon analyzing a larger sample, we identified this variation as a polymorphism not linked to sex, as the male specimen SMRP 252.147 also exhibits this signal. Scale bar = 5 μm.

Figure 6

Schematic representation of the Physalaemus ephippifer supercluster generated by RepeatExplorer analysis, showing two clusters corresponding to 40S rDNA sequences, connected to three additional clusters previously mapped in Ph. ephippifer, namely, PepBS [27], PepSat16-147, and PepSat25-282 (this study). In the top left, the sex chromosomes of Ph. ephippifer are shown with DAPI staining, highlighting the secondary constrictions (empty arrowheads). The two other insets in the bottom right depict the chromosomal mappings of PepSat16-147 and PepSat25-282 on the sex chromosomes of Ph. ephippifer, coinciding with NOR regions (filled arrowheads). Scale bar = 5 μm.

Figure 7

Cladogram representing our hypothesis of chromosomal evolution for chromosome pair 9 of Physalaemus ephippifer, Lineage 1B (L1B) of ‘Ph. cuvieri’, and CZ Pep-L1B. Ideograms and chromosome images illustrate the main features of these chromosomes, and numbers 1-3 indicate possible evolutionary changes. All FISH images were generated using digoxigenin-labeled probes and rhodamine-conjugated anti-digoxigenin, and artificial colors were assigned to the satDNA probes using Adobe Photoshop 2021. Asterisks (*) indicate that the pericentric inversion involving the syntenic block composed of PepSat3- PcP190-PepSat11 may have occurred either in the lineage of Ph. ephippifer or in L1B. Arrowheads indicate a polymorphic PepSat11 cluster of the Z chromosome of Ph. ephippifer, which can be absent. The bottom right panel includes a representation of the hypothetical recombination between the W chromosome of Ph. ephippifer and chromosome 9 of L1B (gray rectangle), originally proposed by Souza et al. [11], which may have given rise to the W chromosome of the CZ Pep-L1B. Gray blocks indicate Zqper/8p probe signals, as reported in [11]. Note that, according to this hypothesis, the NOR on Wp of CZ Pep-L1B would have originated from Wq of Ph. ephippifer.