Comparative cytogenetics of the Physalaemus gracilis group (Anura, Leptodactylidae) with characterization of the karyotype of Physalaemus evangelistai Bokermann, 1967

Comparative cytogenetics of the Physalaemus gracilis group (Anura, Leptodactylidae) with characterization of the karyotype of Physalaemus evangelistai Bokermann, 1967 Pedro Henrique Pacheco Mosquini1, Lucas Henrique Bonfim Souza1, Juan Martín Ferro2, Luciana Bolsoni Lourenço1 1 Laboratório de Estudos Cromossômicos, Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas (LabEsC – UNICAMP), 13083-863 Campinas, São Paulo, Brasil 2Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical (CONICET-UNaM), Facultad de Ciencias Exactas Químicas y Naturales, Universidad Nacional de Misiones, Félix de Azara 1552, CPA N3300LQF, Posadas, Misiones, Argentina Corresponding author: Luciana Bolsoni Lourenço ([email protected]) Academic editor: Larissa Kupriyanova|Received 11 September 2025|Accepted 14 October 2025|Published 7 November 2025 https://zoobank.org/474B385C-F102-44BF-B4AD-86D6E04C0C07 Citation: Mosquini PHP, Souza LHB, Ferro JM, Lourenço LB (2025) Comparative cytogenetics of the Physalaemus gracilis group (Anura, Leptodactylidae) with characterization of the karyotype of Physalaemus evangelistai Bokermann, 1967. Comparative Cytogenetics 19: 171–188. https://doi.org/10.3897/compcytogen.19.171637 Abstract The anuran species group Physalaemus gracilis comprises six species, and variation in the location of nucleolus organizer regions (NORs) was observed across the four species that have been karyotyped to date. The NORs are located interstitially on chromosome 8 of P. carrizorum Cardozo et Pereyra, 2018, and P. lisei Braun et Braun, 1977, terminally on chromosome 8 of P. gracilis (Boulenger, 1883), and terminally on chromosome 10 of P. barrioi Bokermann, 1967. To contribute to the comparative analysis of this group, including the assessment of the hypothesis of homology among these NOR-bearing chromosomes, we described the karyotype of P. evangelistai Bokermann, 1967, and expanded the cytogenetic analyses of P. carrizorum, P. lisei, and P. barrioi. We used classical cytogenetic techniques and mapped, by fluorescent in situ hybridization (FISH), two repetitive sequences: the PcP190 satellite DNA and the U2 snRNA gene. Physalaemus evangelistai exhibited a 2n = 22 karyotype, with metaand submetacentric chromosomes, which corresponds to the typical karyotypic configuration of the genus. We found an interstitial heterochromatin DAPI-positive band on the short arm of the NOR-bearing chromosomes 8 of P. evangelistai and P. carrizorum from Palmas-PR, and chromosome 10 of P. barrioi, which corroborates the hypothesis that these chromosomes are homologous. In P. evangelistai, an additional NOR was observed on chromoCompCytogen 19: 171–188 (2025) doi: 10.3897/compcytogen.19.171637 https://compcytogen.pensoft.net Copyright Pedro Henrique Pacheco Mosquini et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE COMPARATIVE Cytogenetics International Journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics A peer-reviewed open-access journal Pedro Henrique Pacheco Mosquini et al. / Comparative Cytogenetics 19: 171–188 (2025) 172 some 9 of females. Moreover, the karyotype of P. carrizorum from Palmas-PR differed from that previously described for P. carrizorum from Misiones, particularly in the number of PcP190 clusters and intrachromosomal position of the NOR on chromosome 8. Specimens from Palmas-PR showed a terminal NOR on chromosome 8 and PcP190 clusters on chromosomes 1 and 3, whereas those from Misiones had an interstitial/pericentromeric NOR on chromosome 8 and a single PcP190 cluster on chromosome 3. Further analyses are still needed to assess whether these cytogenetic differences represent interspecific variation. Keywords C-band, karyotype, Leptodactylidae, NOR, PcP190 satellite DNA Introduction Karyotype comparisons can be highly informative for distinguishing certain clades, as they provide characters related to diploid number, chromosomal morphology and size, the distribution of repetitive elements, and other traits that may be specific to distinct lineages (Dobigny et al. 2004; Guerra 2008; Mezzasalma et al. 2023). The Neotropical genus Physalaemus Fitzinger, 1826 represents an interesting clade in terms of chromosome evolution, and karyotype data have proven informative for comparing phylogenetically related groups within this genus (see Tomatis et al. 2009; Vittorazzi et al. 2014b; Lourenço et al. 2015; Ferro et al. 2022; Souza et al. 2025 and references therein). In addition, this genus is characterized by a high frequency of chromosomal polymorphisms and a large number of cryptic species (e.g., Barrio 1965; Cassini et al. 2010; Nascimento et al. 2019), which often hinder species identification based solely on morphological characteristics. The anuran genus Physalaemus, which belongs to the family Leptodactylidae and comprises 50 species, is distributed from southern Venezuela and the Guianas to eastern Bolivia, Paraguay, southeastern Colombia, Uruguay, and a large portion of Brazil (Frost 2025). Phylogenetic inferences divide this genus into two major clades: the Physalaemus signifer clade and the Physalaemus cuvieri clade (Lourenço et al. 2015). The latter clade encompasses five species groups—P. gracilis, P. biligonigerus, P. cuvieri, P. henselii, and P. olfersii—as well as the species Physalaemus cicada Bokermann, 1966, which has not been allocated to any group (Lourenço et al. 2015). Species of Physalaemus exhibit karyotypes with 2n = 22 and fundamental numbers (FN) of 42 or 44 (see discussion in Tomatis et al. 2009; Lourenço et al. 2015). The karyotypes of all karyotyped species of the P. signifer Clade have FN = 42 and are characterized by the presence of a telocentric chromosome pair 11, which was hypothesized to be a synapomorphy of this clade (Lourenço et al. 2015). Another notable cytogenetic feature in this genus is a heterochromatic band on the short arm of chromosome 5, which may represent a synapomorphy of the Physalaemus cuvieri group (Vittorazzi et al. 2014b; Lourenço et al. 2015). The Physalaemus gracilis group is composed of six species: P. lisei Braun et Braun, 1977, P. gracilis (Boulenger, 1883), P. evangelistai Bokermann, 1967, P. carrizorum Comparative cytogenetics of the Physalaemus gracilis group 173 Cardozo et Pereyra, 2018, P. jordanensis Bokermann, 1967, and P. barrioi Bokermann, 1967 (Lourenço et al. 2015; Cardozo and Pereyra 2018). Four out of these six species have had their karyotypes previously described (Brum-Zorrilla and Saez 1968; De Lucca et al. 1974; Provete et al. 2012; Ferro et al. 2022), while the karyotypes of P. evangelistai and P. jordanensis remain unknown. Across the karyotyped species, variation in the position of the nucleolus organizer region (NOR) is observed: it is interstitial on chromosome 8 of P. carrizorum and P. lisei, terminal on chromosome 8 of P. gracilis, and terminal on chromosome 10 of P. barrioi (Brum-Zorrilla and Saez 1968; De Lucca et al. 1974; Provete et al. 2012; Ferro et al. 2022). Given that these NOR-bearing chromosomes are also similar in morphology and size, Ferro et al. (2022) proposed that they are homologous. However, the absence of further cytogenetic markers still prevents a comprehensive evaluation of this hypothesis. Other interesting chromosomal markers observed in the P. gracilis group are sites enriched with the PcP190 satellite DNA (satDNA) (Vittorazzi et al. 2011), which is a repetitive DNA sequence widely found in anurans, reported in seven families of Hyloidea to date (Vittorazzi et al. 2011, 2014a; Gatto et al. 2016, 2018; Targueta et al. 2018; Gatto et al. 2019). In the analyzed species of the P. gracilis group, Ferro et al. (2022) identified a small number of chromosomal clusters of PcP190 per karyotype, distributed in apparently species-specific patterns. While in P. lisei a cluster of PcP190 was mapped to the (peri)centromeric region of chromosome 7, in P. carrizorum it was found on chromosome 3, and in P. gracilis, on chromosomes 2 and 9 (Ferro et al. 2022). To enhance our understanding of chromosomal homologies and evolutionary relationships within the P. gracilis group, we described the karyotype of P. evangelistai, presented new data for P. carrizorum from a newly sampled locality, and conducted a comparative analysis of repetitive DNA clusters among the species with available chromosomal data in this species group. Material and methods Species, chromosome preparations, and conventional techniques We analyzed samples of P. barrioi, P. carrizorum, P. evangelistai, P. gracilis, and P. lisei (Table 1). Physalaemus evangelistai specimens and P. carrizorum samples from Palmas, state of Paraná, were collected under the authorization of the Chico Mendes Institute for Biodiversity Conservation/Biodiversity and Information System (ICMBio/SISBIO - permit number 32483). The sampling sites and voucher numbers in scientific collections are provided for all analyzed specimens in Table 1. Chromosome preparations were obtained from cell suspensions deposited in the scientific collection “Shirlei Maria Recco Pimentel” (SMRP), housed at LabEsCUNICAMP. These suspensions were previously prepared from intestinal and testicular tissues, following, respectively, the protocols of King and Rofe (1976), with modifications outlined by Gatto et al. (2018), and Schmid (1978), replacing the hypotonic KCl Pedro Henrique Pacheco Mosquini et al. / Comparative Cytogenetics 19: 171–188 (2025) 174 solution with cold distilled water. All experiments were conducted in accordance with relevant guidelines and approved by the Committee for Ethics in Animal Use of the University of Campinas (CEUA/UNICAMP). Mitotic metaphases were stained with 10% Giemsa, C-banded following Sumner (1972), with modifications described in Siqueira et al. (2008), and silver-impregnated using the Ag-NOR method following Howell and Black (1980). Some C-banded metaphases were sequentially stained with 4’-6 diamidine-2-phenylindole (DAPI, 0.5 µg/mL) and Chromomycin A3 (CMA3, 0.5 mg/mL) after removal of Giemsa-staining in 50% acetic acid for five minutes. Mitotic metaphases were observed using an Olympus BX-60 microscope (Olympus, Tokyo, Japan). Images were captured with a Quick Start FL-20 camera, using Mosaic 2.4 (Tucsen Photonics, Fuzhou, China). Brightness and contrast adjustments were made using Adobe Photoshop 2021 (Adobe Systems, San Jose, CA, USA). Isolation and analysis of nucleotide sequences of PcP190 satellite DNA Genomic DNA samples were obtained from liver tissue of one specimen of P. evangelistai (CFBH 45518) and one specimen of P. lisei (ZUEC 24003). DNA extraction was performed following the TNES method as employed by Medeiros et al. (2013), or alternatively using the QIAamp Fast DNA Tissue Kit (QIAGEN, Hilden, Germany), Table 1. Sampling sites and voucher numbers in zoological and cytogenetic collections of the analyzed specimens. MG – Minas Gerais state, PR – Paraná state, RS – Rio Grande do Sul state, SP – São Paulo state, CFBH – Coleção de Anfíbios Célio Fernando Baptista Haddad, Universidade Estadual Paulista, Rio Claro, SP, Brasil. LGE – Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical (CONICET-UNaM), Posadas, Misiones, Argentina. ZUEC – Museu de Diversidade Biológica, Universidade Estadual de Campinas, Campinas, SP, Brasil. SMRP – Collection of tissue and chromosome preparation Shirlei Maria Recco Pimentel, LabEsC-UNICAMP, Campinas, SP, Brasil. 1Specimen collected by Provete et al. (2012). 2Material analyzed by Ferro et al. (2022) and reanalyzed in this work. 3The sample LGE 24608 corresponds to the same specimen labeled as LGE 24602 in Ferro et al. (2022), where the number was a typographical error. Species Locality Voucher number in zoological collection Chromosome preparation accession number Physalaemus barrioi São José do Barreiro, SP, Brasil (22°43'33"S, 44°37'16"W) 1 ZUEC 18146 ♂SMRP 303.1 Piraquara, PR, Brasil (25°29'47"S, 48°58'54"W) LGE 15329 LGE 15329 Physalaemus carrizorum Palmas, PR, Brasil (26°33'21"S, 51°39'41"W) ZUEC 24848 ♂, ZUEC 24853 ♂SMRP 551.1, SMRP 551.2 Arroyo de los Muertos, Misiones, Argentina (27°22'12"S, 54°24'30"W) 2 LGE 24608 ♂ 3LGE 24608 3 Physalaemus evangelistai Salinas, MG, Brasil (16°5'55"S, 42°12'22"W) CFBH 45513 ♂, CFBH 45514 ♀, CFBH 45515 ♂, CFBH 45516 ♂, CFBH 45517 ♂, CFBH 45518 ♀ SMRP 554.1, SMRP 554.2, SMRP 554.3, SMRP 554.4, SMRP 554.5, SMRP 554.6 Physalaemus gracilis Gravataí, RS, Brasil (29°48'9"S, 50°55'50"W) 2 LGE 28043 ♂SMRP 37.19 Physalaemus lisei Gramado, RS, Brasil (29°21'8"S, 50°53'7"W) ZUEC 24003 ♂SMRP 37.15 Comparative cytogenetics of the Physalaemus gracilis group 175 following the manufacturer’s instructions. The PcP190 satDNA was isolated by PCR using the specific set of primers P190F and P190R (Vittorazzi et al. 2011). The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA), following the manufacturer’s instructions. Samples of the purified products were sequenced by the sequencing facility of the Human Genome and Stem Cell Research Center/IB-USP. The obtained sequences were edited using the BioEdit Sequence Alignment Editor v.7.7.1 (Hall 1999) and compared with sequences available in the NCBI database using BLASTn. After BLASTn indicated similarity between the isolated sequences and those of PcP190 type 1a (see Gatto et al. 2016 for details), we aligned the obtained sequences with all complete PcP190 1a monomers, available in the NCBI database. We excluded monomers derived from P. albonotatus (Steindachner, 1864), which has a 7 bp deletion. The average p-distance value was calculated using the MEGA v.11.0.13 software (Tamura et al. 2021), treating alignment gaps and missing data as pairwise deletions. Alignment figures were generated using Geneious v.7.1.3 (https://www.geneious.com). Fluorescent in situ Hybridization (FISH) of PcP190 Satellite DNA and U2 snRNA gene PcP190 satDNA and U2 snRNA gene sequences were obtained from cloned fragments available in the SMRP collection, previously isolated from Physalaemus cuvieri Fitzinger, 1826 (KM361675.1, Vittorazzi et al. [2014a]) and P. ephippifer (Steindachner, 1864) (PQ200500, Souza et al. [2025]), respectively. The PcP190 satDNA was labeled using a PCR Dig Probe Synthesis Kit (Roche) with the universal primers SP6 and T7. The U2 snRNA gene probe was obtained using the primers U2-F and U2-R, designed by Souza et al. (2025). The resulting probes were hybridized with chromosome preparations following the protocol by Viegas-Péquignot (1992). The probes were detected using an anti-digoxigenin-rhodamine antibody (0.06 µg/mL), the chromosomes were counterstained with DAPI (0.5 µg/mL), and the preparations were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Microscope analyses and image acquisition were performed as mentioned above. Results The karyotype of Physalaemus evangelistai The P. evangelistai karyotype had a diploid number of 22 and a fundamental number of 44 (Fig. 1). Chromosome pairs 1, 5, 6, and 10 were metacentric, while pairs 2–4, 7–9, and 11 were submetacentric (Fig. 1A–D). Small heterochromatin blocks were revealed by C-banding in the centromeric region of all chromosomes (Fig. 1C). All these bands were also evidenced by DAPI in metaphases previously subjected to C-banding (Fig. 1D). An interstitial C-band was found on the long arm of chromosome 8, which Pedro Henrique Pacheco Mosquini et al. / Comparative Cytogenetics 19: 171–188 (2025) 176 was DAPI-negative, CMA3-positive, and colocalized with the NOR (Figs 1D, E, 3B). In some metaphases, a small C-band was noted on the short arm of chromosome 8, particularly after DAPI staining (Figs 1E, 3A). In all analyzed specimens, chromosome 8 exhibit an interstitial NOR on the long arm (Figs 1E, 3B), coinciding with a secondary constriction observed in Giemsastained metaphases (Figs 1E, 3B). However, variation in NOR size allowed the identification of two morphotypes: 8a, bearing a small NOR, and 8b, carrying a large NOR (Fig. 1E). Three males (CFBH 45513, CFBH 45515, and CFBH 4516) were heterozygous with respect to this condition (8a8b), while one male (CFBH 45517) was homozygous for the smaller NOR (8a8a). Both females (CFBH 45514 and CFBH 45518) were homozygous for the smaller NOR (8a8a). In addition to chromosome pair 8, one homologue of chromosome pair 9 also carried a NOR in both analyzed females (Fig. 1E), coinciding with a secondary constriction in Giemsa-stained metaphases and being DAPI-negative and CMA3-positive (Fig. 1E). The NOR-bearing chromosome 9 (morph 9b) was larger than its counterpart (morph 9a), which lacks a NOR. All four analyzed males had no NOR on this chromosome pair. Physalaemus carrizorum from Palmas-PR presented a karyotype with FN = 44 and 2n = 22 (Fig. 2A–C). DAPI-positive C-bands were detected in all centromeric regions (Fig. 2B, C). Heterochromatic bands were also present pericentromerically on the short arm of chromosome 3 and on the long arm of chromosome 7, interstitially Figure 1. Chromosomes of Physalaemus evangelistai A female karyotype stained with Giemsa B–D male karyotype sequentially subjected to Giemsa-staining (B), conventional C-banding (C), and DAPI-staining (D) E chromosomes 8 and 9 subjected to Giemsa-staining, conventional C-banding, C-banding with DAPIand CMA₃-stainings, and silver-impregnation using the Ag-NOR method. Scale bars: 5 µm. Comparative cytogenetics of the Physalaemus gracilis group 177 on the short arm of chromosome 8, and terminally on the short arm of chromosome 3 (Fig. 2B, C). This terminal C-band was larger in one homologue of chromosome pair 3 and more noticeable in C-banded metaphases stained with Giemsa (Fig. 2C). Chromosome 8 had a terminal NOR on the long arm (inset in Figs 2C, 3A), coinciding Figure 2. Karyotype of Physalaemus carrizorum from Palmas-PR A–C Metaphase of male ZUEC 24853 after Giemsa-staining (A), C-banding with DAPI-staining (B), and C-banding with Giemsa-staining (C). In the inset in C, NOR-bearing chromosomes after the Ag-NOR method. Scale bars: 5 µm. Figure 3. NOR-bearing chromosomes of Physalaemus carrizorum (A), P. evangelistai (B), and P. barrioi (C), sequentially subjected to Giemsa-staining, C-banding and DAPI-staining, and the Ag-NOR method. Arrowheads indicate an interstitial DAPI-positive C-band present in all species. Note that, although less evident, it is also present in the NOR-bearing chromosome of P. barrioi. Pedro Henrique Pacheco Mosquini et al. / Comparative Cytogenetics 19: 171–188 (2025) 178 with a secondary constriction observed in Giemsa-stained metaphases (Figs 2A, 3A). This chromosome also presented a small interstitial C-band on the long arm adjacent to the NOR, barely seen in some metaphases (Fig. 2C). Additionally, the reanalysis of P. barrioi specimens allowed us to identify a faint DAPI-stained C-band on the short arm of the NOR-bearing chromosome 10, in a position similar to that observed in the NOR-bearing chromosomes 8 of P. evangelistai and P. carrizorum (Fig. 3). Chromosomal mapping of PcP190 satDNA and the U2 snRNA gene in the P. gracilis group The PcP190 satDNA nucleotide sequences isolated from specimens of P. evangelistai and P. lisei (GenBank accession numbers PV583974 and PV983975, respectively) were 87.5% similar to each other. Based on a comparison with PcP190 sequences available in GenBank, both sequences were assigned to the type 1a of PcP190 satDNA, as they were highly similar to previously described sequences of this type (Suppl. material 1). The mean p-distances between the PcP190 sequences of P. evangelistai and P. lisei and the previously described PcP190 1a sequences, excluding primer regions, were 0.0781 and 0.126, respectively (Suppl. material 1). Hybridization of the PcP190 satDNA probes revealed clusters of this sequence in the (peri)centromeric regions of the long arm of chromosome 1 and short arm of chromosome 3 in P. carrizorum from Palmas (Fig. 4A) and P. barrioi (inset in Fig. 4A), while in P. evangelistai only the short arm of chromosome 1 exhibited cluster of this sequence (inset in Fig. 4A). Chromosome mapping of the U2 snRNA gene revealed terminal clusters on the short arm of chromosome 6 in P. barrioi, P. carrizorum, P. evangelistai, and P. lisei (Fig. 4B). Discussion The diploid number of P. evangelistai (2n = 22) is consistent with that reported for all other Physalaemus species karyotyped to date (Perkins et al. 2019), including members of the P. gracilis group (Provete et al. 2012; Ferro et al. 2022). Additionally, P. evangelistai has a FN of 44, a trait shared by all cytogenetically analyzed species within the P. cuvieri Clade, except P. fernandezae (Müller, 1926), which has a FN of 42 due to its smallest chromosome pair being telocentric (Tomatis et al. 2009; Lourenço et al. 2015). All P. evangelistai specimens had a NOR-bearing chromosome pair 8, and an additional NOR was observed on one of the homologues of chromosome pair 9 in the two females analyzed. This additional NOR was not observed in any of the four males analyzed, nor in any other karyotyped species of the P. gracilis group (Provete et al. 2012; Ferro et al. 2022, Fig. 5), which may suggest the presence of a ZZ/ZW sex determination system in this species. NORs have previously been identified as distinguishing markers between sex chromosomes in some anuran species, including Physalaemus ephippifer (Nascimento et al. 2010), Gastrotheca riobambae (Schmid et al. Comparative cytogenetics of the Physalaemus gracilis group 179 1983), Buergeria buergeri (Schmid et al. 1993), Hyla femoralis (Schmid and Steinlein 2003; Wiley 2003), and Engystomops petersi (Targueta et al. 2010). In the cases of B. buergeri (Schmid et al. 1993) and H. femoralis (Schmid and Steinlein 2003; Wiley 2003), a NOR was the sole cytogenetic feature differentiating the sex chromosomes, being found on chromosome Z in B. buergeri, colocalized with heterochromatin, and on chromosome X in H. femoralis. On the other hand, it is worth noting that polymorphisms related to the presence/absence of NORs are relatively common among anurans (e.g., Medeiros et al. 2003; Barth et al. 2009; Zaleśna et al. 2017, among several others). Therefore, the limited size of the P. evangelistai sample analyzed here, particularly with respect to females, prevents us from determining whether the observed heteromorphism is sex-linked or represents an autosomal polymorphism. Further studies involving a larger number of individuals would provide valuable insights into this issue. When comparing NOR locations across the P. gracilis group, some interesting patterns emerge. As in P. evangelistai, chromosome 8 carries a NOR in all the remaining species of the P. gracilis group, except P. barrioi, in which the NOR-bearing chromosome is chromosome 10 (Provete et al. 2012; Ferro et al. 2022, Fig. 5). In the karyotype description of P. barrioi, the first species of this group to have its chromosomes studied, the authors based the numerical classification of the chromosomes on their Figure 4. Chromosome mapping of PcP190 satDNA (in red) and U2 snRNA gene (in green) in species of the Physalaemus gracilis group A chromosomes of P. carrizorum from Palmas, P. barrioi, and P. evangelistai hybridized with PcP190 probe B mapping of the U2 snRNA gene on chromosomes of P. lisei, P. barrioi, P. carrizorum from Palmas, and P. evangelistai. Chromosomes 1 and 3 of P. barrioi shown in the figure were obtained from different metaphases and subsequently arranged in the figure. Pedro Henrique Pacheco Mosquini et al. / Comparative Cytogenetics 19: 171–188 (2025) 186 Milani M, Cassini CS, Recco-Pimentel SM, Lourenço LB (2010) Karyotypic data detect interpopulational variation in Physalaemus olfersii and the first case of a supernumerary chromosome in the genus. Animal Biology Journal 2: 21–28. Nascimento J, Quinderé DR, Recco-Pimentel SM, Lima JRF, Lourenço LB (2010) Heteromorphic Z and W sex chromosomes in Physalaemus ephippifer (Steindachner, 1864) (Anura, Leiuperidae). Genetica 138: 1127–1132. https://doi.org/10.1007/s10709010-9501-9 Nascimento J, Lima JD, Suárez P, Baldo D, Andrade GV, Pierson TW, Fitzpatrick BM, Haddad CF, Recco-Pimentel SM, Lourenço LB (2019) Extensive cryptic diversity within the Physalaemus cuvieri–Physalaemus ephippifer species complex (Amphibia, Anura) revealed by cytogenetic, mitochondrial, and genomic markers. Frontiers in Genetics 10: 719. https:// doi.org/10.3389/fgene.2019.00719 Perkins RD, Gamboa JR, Jonika MM, Lo J, Shum A, Adams RH, Blackmon H (2019) A database of amphibian karyotypes. Chromosome Research 27: 313–319. https://doi. org/10.1007/s10577-019-09613-1 Provete D, Garey MV, Toledo LF, Nascimento J, Lourenço LB, Rossa-Feres DC, Haddad CFB (2012) Redescription of Physalaemus barrioi (Anura: Leiuperidae). Copeia 2012: 507–518. https://doi.org/10.1643/CH-10-142 Schmid M (1978) Chromosome banding in Amphibia. Chromosoma 66: 361–388. https:// doi.org/10.1007/BF00328536 Schmid M, Steinlein C (2003) Chromosome banding in Amphibia. XXIX. The primitive XY/ XX sex chromosomes of Hyla femoralis (Anura, Hylidae). Cytogenetic and Genome Research 101: 74–79. https://doi.org/10.1159/000073421 Schmid M, Haaf T, Geile B, Sims S (1983) Chromosome banding in Amphibia. VIII. An unusual XY/XX-sex chromosome system in Gastrotheca riobambae (Anura, Hylidae). Chromosoma 88: 69–82. https://doi.org/10.1007/BF00329505 Schmid M, Ohta S, Steinlein C, Guttenbach M (1993) Chromosome banding in Amphibia. XIX. Primitive ZW/ZZ sex chromosomes in Buergeria buergeri (Anura, Rhacophoridae). Cytogenetics and Cell Genetics 62: 238–246. https://doi.org/10.1159/000133486 Silva AP, Haddad CF, Kasahara S (1999) Nucleolus organizer regions in Physalaemus cuvieri (Anura, Leptodactylidae), with evidence of a unique case of Ag-NOR variability. Hereditas 131(2): 135–141. https://doi.org/10.1111/j.1601-5223.1999.00135.x Siqueira S, Aguiar Jr O, Strüssmann C, Del-Grande ML, Recco-Pimentel SM (2008) Chromosomal analysis of three Brasilian “eleutherodactyline” frogs (Anura: Terrarana), with suggestion of a new species. Zootaxa 1860(1): 51–59. https://doi.org/10.11646/ zootaxa.1860.1.4 Souza LHB, Silva BC, Pompeo JN, Gatto KP, Lourenço LB (2025) Chromosome homologies and polymorphisms in a Neotropical species complex of frogs revealed by the U2 snRNA gene. Genome 68: 1–11. https://doi.org/10.1139/gen-2024-0131 Souza LHB, Pierson TW, Tenório RO, Ferro JM, Gatto KP, Silva BC, Andrade GV, Suárez P, Haddad CFB, Lourenço LB (2024) Multiple contact zones and karyotypic evolution in a neotropical frog species complex. Scientific Reports 14: 1119. https://doi.org/10.1038/ s41598-024-51421-z Comparative cytogenetics of the Physalaemus gracilis group 187 Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research 75(1): 304–306. https://doi.org/10.1016/0014-4827(72)90558-7 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution 38(7): 3022–3027. https://doi.org/10.1093/ molbev/msab120 Targueta CP, Rivera M, Souza MB, Recco-Pimentel SM, Lourenço LB (2010) Cytogenetic contributions for the study of the Amazonian Engystomops (Anura, Leiuperidae) assessed in the light of phylogenetic relationships. Molecular Phylogenetics and Evolution 54(3): 709–725. https://doi.org/10.1016/j.ympev.2009.10.018 Targueta CP, Rivera M, Souza MB, Recco-Pimentel SM, Lourenço LB (2010) Cytogenetic contributions for the study of the Amazonian Engystomops (Anura; Leiuperidae) assessed in the light of phylogenetic relationships. Molecular Phylogenetics and Evolution 54: 709– 725. https://doi.org/10.1016/j.ympev.2009.10.018 Targueta CP, Vittorazzi SE, Gatto KP, Bruschi DP, Veiga-Menoncello ACP, Recco-Pimentel SM, Lourenço LB (2018) Anuran cytogenetics: an overview. In: Norris N, Miller C (Eds) An Essential Guide to Cytogenetics. Nova Science Publishers, New York, 1–64. Tomatis C, Baldo D, Kolenc F, Borteiro C (2009) Chromosomal variation in the species of the Physalaemus henselii group (Anura: Leiuperidae). Journal of Herpetology 43(3): 555–560. https://doi.org/10.1670/08-122R1.1 Ugarković Đ, Plohl M (2002) Variation in satellite DNA profiles—causes and effects. The EMBO Journal 21: 5955–5959. https://doi.org/10.1093/emboj/cdf612 Viegas-Pequignot (1992) In situ hybridization to chromosomes with biotinylated probes. In: Willernson D (Ed.) In situ hybridization: a practical approach. Oxford University PressIRL Press, Oxford, 137–158. Vittorazzi SE, Lourenço LB, Del-Grande ML, Recco-Pimentel SM (2011) Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae). Cytogenetic and Genome Research 134(2): 101–107. https://doi.org/10.1159/000325540 Vittorazzi SE, Lourenço LB, Recco-Pimentel SM (2014a) Long-time evolution and highly dynamic satellite DNA in leptodactylid and hylodid frogs. BMC Genetics 15: 111. https:// doi.org/10.1186/s12863-014-0111-x Vittorazzi SE, Quinderé YRSD, Recco-Pimentel SM, Tomatis CG, Baldo JD, Lima JRF, Ferro JM, Lima JD (2014b) Comparative cytogenetics of Physalaemus albifrons and Physalaemus cuvieri species groups (Anura, Leptodactylidae). Comparative Cytogenetics 8(2): 103–123. https://doi.org/10.3897/compcytogen.v8i2.6414 Vittorazzi SE, Lourenço LB, Solé M, Faria RG, Recco-Pimentel SM (2016) Chromosomal analysis of Physalaemus kroyeri and Physalaemus cicada (Anura, Leptodactylidae). Comparative Cytogenetics 10(2): 311–323. https://doi.org/10.3897/CompCytogen.v10i2.9319 Wiley JE (2003) Replication banding and FISH analysis reveal the origin of the Hyla femoralis karyotype and XY/XX sex chromosomes. Cytogenetic and Genome Research 101: 80–83. https://doi.org/10.1159/000073422 Zaleśna A, Florek M, Rybacki M, Ogielska M (2017) Variability of NOR patterns in European water frogs of different genome composition and ploidy level. Comparative Cytogenetics 11(2): 249–266. https://doi.org/10.3897/CompCytogen.v11i2.10804 Pedro Henrique Pacheco Mosquini et al. / Comparative Cytogenetics 19: 171–188 (2025) 188 ORCID Pedro Henrique Pacheco Mosquini https://orcid.org/0000-0002-7027-1522 Lucas Henrique Bonfim Souza https://orcid.org/0000-0001-7178-7355 Juan Martín Ferro https://orcid.org/0000-0003-0287-5567 Luciana Bolsoni Lourenço https://orcid.org/0000-0001-6602-6850 Supplementary material 1 Alignment of PcP190 satDNA monomers Authors: Pedro Henrique Pacheco Mosquini, Lucas Henrique Bonfim Souza, Juan Martín Ferro, Luciana Bolsoni Lourenço Data type: tif Explanation note: PcP190 sequences are identified by their GenBank accession numbers. Colored sites indicate nucleotides that differ from the most common sequence observed in the majority of samples. Primers (P190F and P190R) are indicated by the dark arrows. The dark green region corresponds to the conserved region, while the light green corresponds to the hypervariable region of PcP190 satDNA. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/compcytogen.19.171637.suppl1