Sex chromosomes in the Vizcacheras’ White-lipped frog, Leptodactylus bufonius (Anura, Leptodactylidae)

Abstract

Cytogenetic analyses were performed on specimens of Leptodactylus bufonius from different localities in Argentina. Mitotic chromosomes were studied with Giemsa and differential staining techniques (Ag-NOR, C-banding, and CMA₃/DAPI) and fluorescence in situ hybridization with the 18S DNAr probe. All specimens showed karyotypes with 2n = 2x = 22 and FN = 44. Secondary constrictions were present in the long arm of chromosome pair 8, coincident with Ag-NOR and hybridization signals of the 18S DNAr probe. The C-banding technique evidenced an important amount of heterochromatin with a sex-linked pericentromeric band in the short arm of chromosome pair 4. This heterochromatic band was heteromorphic in males but present in both homologues of females, and it was CMA₃ positive (DAPI negative) at fluorescence staining. The occurrence of heteromorphic XY sex chromosomes in L. bufonius is the second known case in Leptodactylus and the fifth within the speciose family Leptodactylidae.

Key words
C-banding; cytogenetics; heterochromatin; heteromorphism; XY

INTRODUCTION

The diverse and complex ways of sex determination among different vertebrate taxa constitute an interesting research field that is currently focused on revealing genes and mechanisms involved, as well as chromosome locations (Bull 1983, Valenzuela 2008). To date, this is a poorly studied topic in Anura, and those species with already identified sex chromosomes are just a small proportion as compared with other vertebrate orders (Miura 2017). Besides, the characteristics of such phenomena and associated evolutionary histories are barely known (Schmid 1983, Schmid et al. 2010, Uno et al. 2015).

However, an extraordinary variety of sex chromosome systems were described in anurans (Odierna et al. 2007, Nascimento et al. 2010, Schmid et al. 2010, Saba & Tripathi 2014, Patawang et al. 2014, Gazoni et al. 2018, Sangpakdee et al. 2017). While most species show simple heteromorphic chromosomes (XY, ZW), there are also complex mechanisms, as W0 founded in Leiopelma hochstetteri (Green 1988) or the multiple sex chromosomes observed in Leptodactylus pentadactylus (Gazoni et al. 2018). Other extreme examples are Xenopus tropicalis (Roco et al. 2015) and Glandirana rugosa (Miura & Ogata 2013), in which different sex-determining systems coexist: XY, ZW, and non-differentiated sex chromosomes. Moreover, sex chromosome polymorphisms were also observed in Gastrotheca pseustes, with two different Y-chromosome morphs (Schmid et al. 1990). The impressive variation of sex determination systems in anurans, also reported at different morphological differentiation stages, makes this group an excellent target for studying sex chromosomes origin and evolution in vertebrates.

Most sex chromosomes already identified in anuran amphibians are microscopically indistinguishable (homomorphic) when they are studied with conventional staining techniques (Hillis & Green 1990, Schmid 1983, Schmid & Steinlein 2001, Eggert 2004). Because of this, some researchers employed traditional cytogenetic markers such as C-bands, Ag-NOR, replication banding patterns, and DNA base-specific fluorochromes (see Schmid et al. 2010 for a review). More recently, modern molecular cytogenetic techniques like comparative genomic hybridization and chromosome mapping of repetitive sequences with fluorescent in situ hybridization have been used (Abramyan et al. 2009, Vittorazzi et al. 2014, Gatto et al. 2016, 2018, 2019).

Heterogametic Y or W chromosomes that are morphologically identical to their counterparts (X and Z, respectively) would indicate that they still did not develop supramolecular evident differences at the chromosomal level, representing a primitive stage of evolution of the sex chromosomes (Schmid 1990, Schartl et al. 2016). It has been suggested that this feature could be related to the dynamics of sex-determining genes when they fail to be anchored to a specific chromosome, determining a continuous process of replacement called “turnover of sex-determining genes and sex chromosomes” (Schartl 2004, Sarre et al. 2011, Miura 2017). Furthermore, other processes could be responsible for the prevalence of homomorphic sex chromosomes in anurans like the “fountain of youth” hypothesis (Perrin 2009), in which occasional sex reversion events may occur (XY females or ZW males), enhancing the recombination in sex-specific regions and preventing the accumulation of deleterious mutations.

The family Leptodactylidae currently comprises 231 species (Frost 2021), distributed in three subfamilies: Leptodactylinae, Leiuperinae, and Paratelmatobiinae. Within the first, the Neotropical genus Leptodactylus is a natural group of 82 currently recognized species (Frost 2021), clustered in the L. fuscus, L. latrans, L. melanonotus, and L. pentadactylus groups (Heyer 1969). Leptodactylus bufonius of the L. fuscus group is one of the most abundant species in the semi-arid environments of the American Gran Chaco, and surrounding areas in northern Argentina, Paraguay, southeastern Bolivia, and the states of Mato Grosso and Mato Grosso do Sul in central Brazil (Heyer 1978, Schalk & Leavitt 2017, Brusquetti et al. 2019). Like other species of the L. fuscus group, L. bufonius presents burrowing habits, and males build subterranean chambers in moist soil near streams or ponds for the incubating foam nests produced during amplexus (Gallardo 1964, Maxson & Heyer 1988, Ponssa 2008, Faggioni et al. 2017).

There is a large amount of information about chromosome data in Leptodactylus that covers near half of its 82 recognized species, with a widespread characteristic karyotype of 2n = 2x = 22 and bi-armed chromosomes (FN = 44) (Brum-Zorrilla & Sáez 1968, Bogart 1974, Heyer & Diment 1974, Silva et al. 2000, 2004, 2006, Amaro-Ghiraldi et al. 2004, 2006, Arruda & Morielle-Versute 2008, de Oliveira et al. 2012, Gazoni et al. 2018, 2021, de Oliveira et al. 2013, Coelho et al. 2016, Gonzalez et al. 2016). To date, there is a single report of chromosomic sex determination in these frogs, described in L. pentadactylus, with a mechanism involving multiple sex chromosomes (Gazoni et al. 2018).

Cytogenetic studies in Leptodactylus bufonius are mainly restricted to the description of its diploid number, based only on few specimens of a small portion of its geographic distribution in Argentina (Barbieri 1950, Brum-Zorrilla & Sáez 1968, Bogart 1974, Heyer & Diment 1974). In the present study, we make a thorough characterization of cytogenetics in this species with the aid of different banding techniques in specimens from several localities in Argentina. We also describe a new case of heteromorphic sex chromosomes in Anura, which constitutes the second known in the genus Leptodactylus.

MATERIALS AND METHODS

We analyzed 41 specimens of Leptodactylus bufonius (21 males, 20 females; Fig. 1, details in Appendix). Mitotic metaphases were obtained from cell suspensions of bone marrow and intestinal epithelium, using the protocol described by Schmid et al. (2010). Slides were conventionally stained with 10% phosphate-buffered Giemsa (pH 6.8). The nucleolar organizer regions (NORs) were detected by Ag-NOR staining according to Howell & Black (1980) and by fluorescent in situ hybridization (FISH) with the ribosomal 18S biotinylated probe (Pinkel et al. 1986). Location of heterochromatin was determined using standard C-banding technique (Sumner 1972), and the composition of heterochromatic AT-rich and GC-rich bands was evidenced, respectively, with the fluorochromes DAPI (4’, 6-diamidino-2-phenylindole) and CMA₃ (Chromomycin A3) (Schweizer 1976). Karyotypes were arranged in decreasing size, according to the nomenclature proposed by Green & Sessions (1991, 2007). Assessment of chromosome size and morphology was performed with DRAWID v0.26 software (Kirov et al. 2017).

Figure 1
Sampling localities of Leptodactylus bufonius analyzed.

RESULTS

The karyotype of Leptodactylus bufonius is composed of 11 pairs of bi-armed chromosomes (2n = 2x = 22; FN = 44), arranged in seven pairs of large and medium-sized chromosomes and four small ones. Pairs 1, 2, 5, 6, 8–11 are metacentric, pair 3 is submetacentric, whereas pairs 4 and 7 are subtelocentric (Fig. 2; Table I). NOR sites were identified in the interstitial region of the long arm of both homologues of pair 8 (Fig. 3), according to silver staining and hybridization signals of the 18S DNAr after FISH experiments, in coincidence with secondary constrictions.

Figure 2
Karyotypes of Leptodactylus bufonius. Female. Conventional staining (a) and C-banding (b). Male. Conventional staining (c) and C-banding (d). Sex chromosomes (pairs 4) are shown in boxes. Bar = 5 µm.

Figure 3
NOR-bearing chromosomes pair in Leptodactylus bufonius (pair 8), characterized by Ag-NOR (a), CMA₃ (b), DAPI (c), 18s DNAr probe (d). Bar = 5 µm.

Table I
Chromosome morphology in Leptodactylus bufonius. Abbreviations: % set = percentage of total complement; CI = centromeric index; SD = standard deviation; M = metacentric; SM = submetacentric; ST = subtelocentric.

Chromosome number	1	2	3	4x	4y	5	6	7	8	9	10	11
% set	15.74	12.25	10.98	5.17	4.99	10.47	10.09	8.51	6.55	5.6	5.13	5.59
CI ± SD	0.478 ± 0.02	0.402 ± 0.04	0.369 ± 0.05	0.239 ± 0.02	0.236 ± 0.02	0.429 ± 0.04	0.429 ± 0.04	0.258 ± 0.04	0.425 ± 0.05	0.433 ± 0.04	0.441 ± 0.04	0.419 ± 0.04
Type	M	M	SM	ST	ST	M	M	ST	M	M	M	M

The C-banding pattern revealed heterochromatin in the centromeric region of all chromosomes, with a large number of additional heterochromatic bands (Fig. 2b and d). Interstitial C-bands were observed on the short arm of pairs 1, 2, 5, 6, 8, and 9, and on the long arm of pairs 3, 5–9, with a conspicuous heterochromatic band on both arms of pair 6. Pericentromeric C-bands were detected on the short arm of pair 7 and the long arm of pairs 4 and 10.

Chromosome pair 4 showed a pericentromeric C-band on the long arm of both homologues, although a sex-biased heteromorphism was observed for the presence of a pericentromeric C-band on the short arm of this pair. Over 25 specimens analyzed (14 males, 11 females), this C-band was present on both homologues in females but only in one of the homologues in males (Fig. 2, in boxes; Fig. 4).

Figure 4
Ideogram and sex chromosomes (pairs 4) from five females (a) and five males (b) of Leptodactylus bufonius, after C-banding.

Additionally, three other polymorphic variations in the C-banding pattern were detected in individuals of both sexes and from different localities, without defined geographical patterns (Fig. 5). First, a polymorphism for the presence of an interstitial C-band was identified in chromosome pair 2. This condition was detected in homozygosis in a male (LGE 12949; Fig. 5a) and in heterozygosis in three males (LGE 12163, LGE 12948, and LGE 13439; Fig. 5b) and two females (LGE 12046 and LGE 13437). The remaining specimens did not present interstitial bands in this pair (homozygous condition, without bands; Fig. 5c). A second polymorphism was detected for the presence of an additional interstitial C-band on the short arm of pair 6, detected in homozygosis in four males (LGE 10085, LGE 12163, LGE 12949 and LGE 13439, Fig. 5d) and two females (LGE 9264 and LGE 10084). Finally, a polymorphism consisted of the presence of a pericentromeric band on the longs arms of pair 7, detected only in homozygosis in three males (LGE 12944, LGE 13264, and LGE 13391; Fig. 5f) and three females (LGE 8098, LGE 9264, and LGE 10084) of the sample.

Figure 5
Chromosome polymorphisms for the presence of additional C-bands in Leptodactylus bufonius. Polymorphic C-bands in homozygosis (a, d, and f), heterozygosis (b), and chromosomes without additional bands (c, e, and g). Bar = 5 µm.

The CMA₃/DAPI fluorochromes staining evidenced CMA₃ positive (DAPI negative) heterochromatin in the interstitial region of the short arms of pairs 1–6 and 8 (including the heteromorphic band in pair 4), in the long arms of pairs 3 and 5, 7–8, and in the pericentromeric region of pairs 4 (Fig. 6). The conspicuous heterochromatic band observed on the short arm of pair 6 was characterized as CMA₃ negative and DAPI negative. A CMA₃ positive (DAPI negative) bright fluorescent band was detected in the interstitial position of pair 8, in coincidence with secondary constrictions (Fig. 3b and c). Similarly, the heteromorphic band observed on pair 4 was characterized as CMA₃ positive (DAPI negative). Although the CMA₃ bands were conspicuous, the negative DAPI bands depended on the state of condensation of the chromosomes.

Figure 6
CMA₃/DAPI banding patterns in Leptodactylus bufonius. Mitotic metaphases of a female (a, b) and male (c, d), stained with CMA₃ and DAPI fluorochromes (left and right, respectively). Sex chromosomes are indicated with arrows. Bar = 5 µm.

DISCUSSION

The 2n = 2x = 22 and FN = 44 reported here for Leptodactylus bufonius agrees with previous data (Barbieri 1950, Brum-Zorrilla & Sáez 1968, Bogart 1974, Heyer & Diment 1974), and the morphology of chromosomes is in concordance with that reported by Bogart (1974, Fig. 2). The 2n = 2x = 22 is the widespread chromosome number in Leptodactylidae (Tomatis et al. 2009, Targueta et al. 2010, Gazoni et al. 2012, Vittorazzi et al. 2014, Lourenço et al. 2015, Cardozo et al. 2016 and cites therein), and could be considered the plesiomorphic condition for Leptodactylus. In this genus, 34 species have been cytogenetically analyzed (Brum-Zorrilla & Sáez 1968, Bogart 1974, Heyer & Diment 1974, Silva et al. 2000, 2004, 2006, Amaro-Ghiraldi et al. 2004, 2006, Arruda & Morielle-Versute 2008, de Oliveira et al. 2012, Gazoni et al. 2012, 2018, 2021, de Oliveira et al. 2013, Coelho et al. 2015, Gonzalez et al. 2016). Only two of them have a different basic chromosome number: L. brevipes (2n = 2x = 20; Gazoni et al. 2012, 2021) and L. silvanimbus (2n = 2x = 24; Amaro-Ghiraldi et al. 2006), which would represent autapomorphic character states.

The information involving differential staining techniques in Leptodactylus bufonius is provided herein for the first time. NORs in the pair 8 of this species are in a similar position than most of the Leptodactylus already studied and is considered the plesiomorphic state in the genus (Silva et al. 2000, Amaro-Ghilardi et al. 2004, 2006, Arruda & Morielle-Versute 2008, de Oliveira et al. 2012, Gazoni et al. 2012, Coelho et al. 2016). The few known exceptions were observed in L. mystacinus (pair 4 or 8; Amaro-Ghilardi et al. 2006, Silva et al. 2006), L. natalensis (pair 7; Gazoni et al. 2021), L. petersii (pair 4; Amaro-Ghilardi et al. 2006, Gazoni et al. 2012, 2021), L. rhodomystax (pair 3; Gazoni et al. 2012), and L. brevipes (pair 4; Gazoni et al. 2012, 2021).

The C-banding in Leptodactylus bufonius seems not to be typical of Leptodactylus, where karyotypes of the species usually bear poor amounts of heterochromatin (Silva et al. 2000, 2006, Amaro-Ghilardi et al. 2004). Variation in the distribution of heterochromatic bands other than centromeric has only been reported for L. latrans (Silva et al. 2000, Amaro-Ghilardi et al. 2004), L. fuscus (Silva et al. 2000), and L. petersii (Coelho et al. 2016).

Furthermore, the C-banding technique evidenced the presence of a chromosome sex-determination system of the type XY in chromosome pair 4. In all analyzed females, this pair was indistinguishable regarding C-bands, while males presented a pericentromeric C-band in the short arm of only one of the homologues. Similar heteromorphisms in terms of the amount of heterochromatin were described for Gastrotheca walkeri and G. ovifera, and species of the genus Eupsophus, in which the Y chromosome lacks heterochromatin and differs in size with its homologue (Iturra & Veloso 1981, Cuevas & Formas 1996, Schmid et al. 2002).

According to several cytogenetic and molecular studies about the evolution of sex chromosomes, the morphological differentiation of heteromorphic sex chromosomes may initiate with heterochromatin accumulation (Ray-Chaudhuri et al. 1971, Jones 1984, Schmid & Steinlein 2001). Therefore, Leptodactylus bufonius and other above-mentioned anuran species do not fit in this evolutionary model, as the Y or W chromosomes do not present large amounts of heterochromatin. To explain this, Schmid et al. (2002) suggested that the presence of a smaller amount of heterochromatin in chromosome Y compared to the chromosome X could be due to deletion rather than accumulation. They further suggest that the heterochromatinization of the Y or W chromosome is possibly not the only evolutionary way that originates the morphological differentiation of sex chromosomes. There are some examples in anurans that may confirm this assumption. For instance, in Rana japonica, there is a single block of heterochromatin in the pericentromeric region of the long arm of the X chromosome that is absent in the Y chromosome (Miura 1994), and in Eupsophus migueli, the Y chromosome does not show heterochromatic regions at all (Iturra & Veloso 1989). Similarly, in the North American teiid lizard Aspidoscelis tigris there is less pericentromeric heterochromatin in the Y than the X chromosome (Bull 1978).

Among Leptodactylidae, there are only a few reported cases of sex chromosomes. In the Engystomops petersi species complex (as Physalaemus petersi in Lourenço et al. 1998, 1999, and Engystomops petersi and E. freibergi in Targueta et al. 2010), different types of heteromorphic XY chromosomes were detected. In this example, it is noteworthy that in some specimens from Acre, Brazil, the X chromosome contained interstitial heterochromatic segments, absent in the Y chromosomes, while in other individuals from the same population, a terminal NOR in the long arm of the Y chromosome can be observed. Furthermore, some individuals of this species from Puyo, Ecuador, only presented XY chromosomes with different morphology. In Physalaemus ephippifer, ZW chromosomes were identified by an additional segment, which comprises a distal NOR and an adjacent terminal C-band in the short arm of the W chromosome (Nascimento et al. 2010). Heteromorphic XY chromosomes were also described for Pseudopaludicola saltica (Duarte et al. 2010). In Leptodactylus, sex chromosomes were confirmed only for L. pentadactylus (Gazoni et al. 2018), which presents the largest number of sex chromosomes found among vertebrates. Males of this species show a chromosome ring consisting of 12 elements, resulting from multiple translocation events. Barale et al. (1990) also reported an XY system for L. macrosternum (as L. chaquensis) and proposed a sex heteromorphism regarding a pericentromeric C-band in the first chromosome pair. However, these results differ from those obtained by Gazoni et al. (2012), who ruled out the occurrence of sex chromosome differentiation in this species.

A recent phylogenetic analysis of Leptodactylus recovered L. bufonius as the sister taxon of L. troglodytes, L. cupreus, and L. mystacinus + L. apepyta, in a clade that is sister of all remaining species of the L. fuscus group (de Sá et al. 2014, Schneider et al. 2019). In this group, the C-banding pattern is known only for L. mystacinus, L. gracilis, L. plaumanni, L. fuscus, and L. notoaktites (Silva et al. 2000, 2004, 2006, Arruda & Morielle-Versute 2008, de Oliveira et al. 2013), in which sexual chromosomes have never been detected. The large number of Leptodactylus species of which C-banding patterns are unknown does not allow us to determine whether the presence of sex chromosomes in L. bufonius corresponds to an autapomorphy or if it is a more extended condition in the L. fuscus group or the entire genus.

In our study case, the use of conventional banding techniques was sufficient to detect an XY sex chromosome system in Leptodactylus bufonius. However, a more exhaustive characterization is necessary with more resolutive techniques like fluorescent in situ hybridization or comparative genome hybridization.

ACKNOWLEDGMENTS

The authors thank to the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and ANPCyT PICTs 2013-404, 2015-0813, 2015-0820, 2015-2381 and 2017-2437 for financial support. We also thank Claudio Borteiro for important observations in a first draft of the of the manuscript and Andrea Dallagnol, Mónica Soliz, Alberto Taffarel, Jimena Grosso, Grisel Navarro, Francisco Brusquetti, Orlando Escalante, Ana Sofía Duport Bru, Federico Marangoni, Julián Lescano, Emiliano Galli, María Laura Ponssa, María José Tulli, Claudio Borteiro, Daiana Paola Ferraro, Francisco Kolenc, Erica Kubish, Carina Nieto, Marcelo Tedros their help in the fieldwork and provision specimens.

REFERENCES

• ABRAMYAN J, EZAZ T, GRAVES JAM & KOOPMAN P. 2009. Z and W sex chromosomes in the cane toad (Bufo marinus). Chromosome Res 17(8): 1015-1024.
• AMARO-GHILARDI RC, RODRIGUES MT & YONENAGA-YASSUDA Y. 2004. Chromosomal studies after differential staining and fluorescence in situ hybridization using telomeric probe in three Leptodactylus species (Leptodactylidae, Anura). Caryologia 57(1): 53-65.
• AMARO-GHILARDI RC, SKUK G, DE SÁ RO, RODRIGUES MT & YONENAGA-YASSUDA Y. 2006. Karyotypes of eight species of Leptodactylus (Anura, Leptodactylidae) with a description of a new karyotype for the genus. Phyllomedusa 5(2): 119-133.
• ARRUDA MP & MORIELLE-VERSUTE E. 2008. Cytogenetic and random amplified polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia). Genet Mol Res 7: 161-176.
• BARALE GD, PINNA SENN E & LISANTI JA. 1990. Polimorfismo de bandas C ligado al sexo en Leptodactylus chaquensis (Anura Leptodactylidae). Rev UNRC 10(1): 51-53.
• BARBIERI FD. 1950. Observaciones sobre los cromosomas y espermatozoides en algunos batracios del género “Leptodactylus”. Acta Zool Lilloana 9: 455-463.
• BOGART JP. 1974. A karyosystematic study of frogs in the genus Leptodactylus (Anura: Leptodactylidae). Copeia 1974: 728-737.
• BRUM-ZORRILLA N & SÁEZ FA. 1968. Chromosomes of Leptodactylidae (Amphibia, Anura). Cell Mol Life Sci 24(9): 969.
• BRUSQUETTI F, NETTO F, BALDO D & HADDAD CBF. 2019. The influence of Pleistocene glaciations on Chacoan fauna: genetic structure and historical demography of an endemic frog of the South American Gran Chaco. Biol J Linn Soc 126: 404-416.
• BULL J. 1978. Sex chromosome differentiation: an intermediate stage in a lizard. Can J Genet Cytol 20(2): 205-209.
• BULL J. 1983. Evolution of sex determining mechanisms. San Francisco: The Benjamin/Cummings Publishing Company, 316 p.
• CARDOZO DE, BOERIS JM, FERRO JM, BORTEIRO C, KOLENC F, SUÁREZ P, NETTO F, BRUSQUETTI F & BALDO D. 2016. Evidence for independent instances of chromosome number reduction in the genus Pseudopaludicola (Anura: Leptodactylidae). Salamandra 52(1): 11-22.
• COELHO AC, DE MATTOS TL, VIANA P, TERENCIO ML, SCHNEIDER CH, MENIN M & GROSS MC. 2016. Intra-generic and interspecific karyotype patterns of Leptodactylus and Adenomera (Anura, Leptodactylidae) with inclusion of five species from Central Amazonia. Genetica 144(1): 37-46.
• CUEVAS CC & FORMAS JR. 1996. Heteromorphic sex chromosomes in Eupsophus insularis (Amphibia: Anura: Leptodactylidae). Chromosome Res 4(6): 467-470.
• DE OLIVEIRA B, MOARES MS, DERTÔNIO D, FARIA RG & PANTALEÃO SM. 2013. Cytogenetic study of Leptodactylus fuscus and L. latrans (Anura: Leptodactylidae) from the semi-arid Brazilian Caatinga scrublands. Phyllomedusa 12(2): 125-133.
• DE OLIVEIRA HHP, SOUZA CCN, RIBEIRO CL, BASTOS RP, DA CRUZ AD & SILVA DM. 2012. Citogenética Comparativa das Famílias Leptodactylidade e Hylidae do Cerrado Goiano. Estudos (Goiânia) 39(2): 123-131.
• DE SÁ RO, GRANT T, CAMARGO A, HEYER WR, PONSSA ML & STANLEY E. 2014. Systematics of the Neotropical Genus Leptodactylus Fitzinger, 1826 (Anura: Leptodactylidae): Phylogeny, the relevance of non-molecular evidence, and species Accounts. S Am J Herpetol 9: S1-S128.
• DUARTE TC, VEIGA-MENONCELLO ACP, LIMA JF, STRÜSSMANN C, DEL-GRANDE ML, GIARETTA AA, PEREIRA EG, ROSSA-FERES DC & RECCO-PIMENTEL SM. 2010. Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P. saltica. Hereditas (Lund) 147(2): 43-52.
• EGGERT C. 2004. Sex determination: the amphibian models. Reprod Nutr Dev 44(6): 539-549.
• FAGGIONI G, SOUZA F, UETANABARO M, LANDGREF-FILHO P, FURMAN J & PRADO C. 2017. Reproductive biology of the nest building vizcacheras frog Leptodactylus bufonius (Amphibia, Anura, Leptodactylidae), including a description of unusual courtship behaviour. Herpetol J 27: 73-80.
• FROST DR. 2021. Amphibian Species of the World: an Online Reference. Version 6.0 (14 March). Available from: <http://research.amnh.org/herpetology/amphibia/index.html>. American Museum of Natural History, New York, USA.
  » http://research.amnh.org/herpetology/amphibia/index.html
• GALLARDO JM. 1964. “Leptodactylus prognathus” Boul. y” L. mystacinus”(Burm.) con sus respectivas especies Aliadas:(“Amphibia, Leptodactylidae” del grupo “Cavicola”). Rev Mus Argent Cienc Nat 9: 91-121.
• GATTO KPG, BUSIN CS & LOURENÇO LB. 2016. Unraveling the sex chromosome heteromorphism of the paradoxical frog Pseudis tocantins. PloS ONE 11(5): e0156176.
• GATTO KPG, MATTOS JV, SEGER KR & LOURENÇO LB. 2018. Sex chromosome differentiation in the frog genus Pseudis involves satellite DNA and chromosome rearrangements. Front Genet 9: 301.
• GATTO KPG, SEGER KR, GARCIA PCDA & LOURENÇO LB. 2019. Satellite DNA Mapping in Pseudis fusca (Hylidae, Pseudinae) Provides new insights into sex chromosome evolution in paradoxical frogs. Genes 10(2): 160.
• GAZONI T ET AL. 2020. Revisiting the systematics of the Leptodactylus melanonotus group (Anura: Leptodactylidae): redescription of L. petersii and revalidation of its junior synonyms. Zool Anz 290: 117-134.
• GAZONI T, GRUBER SL, SILVA APZ, ARAÚJO OGS, NARIMATSU H, STRÜSSMANN C, HADDAD CFB & KASAHARA S. 2012. Cytogenetic analyses of eight species in the genus Leptodactylus Fitzinger, 1843 (Amphibia, Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations. BMC Genet 13(1): 109.
• GAZONI T, HADDAD CFB, NARIMATSU H, CABRAL-DE-MELLO DC, LYRA ML & PARISE-MALTEMPI PP. 2018. More sex chromosomes than autosomes in the Amazonian frog Leptodactylus pentadactylus. Chromosoma 127(2): 269-278.
• GONZÁLEZ H, CORREA M, MÁRQUEZ M, MORENO A & BELLO Y. 2016. Cariotipo de Leptodactylus insularum (Anura: Leptodactilidae (Barbour, 1906) presente en un fragmento de bosque seco tropical de la región caribe colombiana. Rev Colombiana Cienc Anim Recia 8: 151-158.
• GREEN DM. 1988. Cytogenetics of the endemic New Zealand frog, Leiopelma hochstetteri: extraordinary supernumerary chromosome variation and a unique sex-chromosome system. Chromosoma 97(1): 55-70.
• GREEN DM & SESSIONS SK. 1991. Nomenclature for chromosomes. In: AMPHIBIAN CYTOGENETICS AND EVOLUTION. Green DM and Sessions SK (Eds). San Diego: Academic Press, p. 431-432.
• GREEN DM & SESSIONS SK. 2007. Karyology and cytogenetics. Amphibian Biology 7: 2756-2841.
• HEYER WR. 1969. The adaptive ecology of the species groups of the genus Leptodactylus (Amphibia, Leptodactylidae). Evolution 23: 421-428.
• HEYER WR. 1978. Systematics of the fuscus group of the frog genus Leptodactylus (Amphibia, Leptodactylidae). Nat Hist Mus Los Angel Cty Sci Ser 29: 1-85.
• HEYER WR & DIMENT MJ. 1974. The karyotype of Vanzolinius discodactylus and comments on usefulness of karyotypes in determining relationships in the Leptodactylus complex (Amphibia, Leptodactylidae). Proc Biol Soc Wash 87: 327-336.
• HILLIS DM & GREEN DM. 1990. Evolutionary changes of heterogametic sex in the phylogenetic history of amphibians. J Evol Biol 3(1-2): 49-64.
• HOWELL WT & BLACK DA. 1980. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Cell Mol Life Sci 36(8): 1014-1015.
• ITURRA P & VELOSO A. 1981. Evidence for heteromorphic sex chromosomes in male amphibians (Anura: Leptodactylidae). Cytogenet Cell Genet 31(2): 108-110.
• JONES KW. 1984. The evolution of sex chromosomes and their consequences for the evolutionary process. In: CHROMOSOMES TODAY 8. Bennett MD, Gropp A and Wolf U (Eds). London: George Allen & Unwin, p. 241-255.
• KIROV I, KHRUSTALEVA L, VAN LAERE K, SOLOVIEV A, MEEUS S, ROMANOV D & FESENKO I. 2017. DRAWID: user-friendly java software for chromosome measurements and idiogram drawing. Comp Cytogenet 11(4): 747-757.
• LOURENÇO LB, RECCO-PIMENTEL SM & CARDOSO AJ. 1998. Polymorphism of the nucleolus organizer regions (NORs) in Physalaemus petersi (Amphibia, Anura, Leptodactylidae) detected by silver staining and fluorescence in situ hybridization. Chromosome Res 6(8): 621-628.
• LOURENÇO LB, RECCO-PIMENTEL SM & CARDOSO AJ. 1999. Two karyotypes and heteromorphic sex chromosomes in Physalaemus petersi (Anura, Leptodactylidae). Can J Zool 77(4): 624-631.
• LOURENÇO LB, TARGUETA CP, BALDO D, NASCIMENTO J, GARCIA PC, ANDRADE GV, HADDAD CFB & RECCO-PIMENTEL SM. 2015. Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences. Mol Phylogenet Evol 92: 204-216.
• MAXSON LR & HEYER WR. 1988. Molecular systematics of the frog genus Leptodactylus (Amphibia: Leptodactylidae). Fieldiana Zool 41: 1-13.
• MIURA I. 1994. Sex chromosome differentiation in the japanese brown frog, Rana japonica-1-Sex-related heteromorphism of the distribution pattern of constitutive heterochromatin in chromosome No. 4 of the Wakuya population. Zool Sci 11(6): 797-806.
• MIURA I. 2017. Sex determination and sex chromosomes in Amphibia. Sex Dev 11(5-6): 298-306.
• MIURA I & OGATA M. 2013. Change of heterogametic sex from male to female: Why so easy in the frog? Chromosome Sci 16 (1-2): 3-9.
• NASCIMENTO J, QUINDERÉ YRSD, RECCO-PIMENTEL SM, LIMA JRF & LOURENÇO LB. 2010. Heteromorphic Z and W sex chromosomes in Physalaemus ephippifer (Steindachner, 1864) (Anura, Leiuperidae). Genetica 138(11-12): 1127-1132.
• ODIERNA G, APREA G, CAPRIGLIONE T, CASTELLANO S & BALLETTO E. 2007. Cytological evidence for population-specific sex chromosome heteromorphism in Palaearctic green toads (Amphibia, Anura). J Biosci 32(4): 763-768.
• PATAWANG I, TANOMTONG A, PHIMPHAN S, CHUAYNKERN Y, CHUAYNKERN C, PHAENGPHAIREE P, KHRUEANET W & NITHIKULWORAWONG N. 2014. The Identification of Sex-Chromosomes and Karyological Analysis of Rice Frog, Fejervarya limnocharis (Anura, Ranidae) from Northeast Thailand. Cytologia 79(2): 141-150.
• PERRIN N. 2009. Sex reversal: a fountain of youth for sex chromosomes? Evolution Int J Org Evolution 63(12): 3043-3049.
• PINKEL D, STRAUME T & GRAY JW. 1986. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci 83(9): 2934-2938.
• PONSSA ML. 2008. Cladistic analysis and osteological descriptions of the frog species in the Leptodactylus fuscus species group (Anura, Leptodactylidae). J Zool Syst Evol Res 46(3): 249-266.
• RAY-CHAUDHURI SP, SINGH L & SHARMA T. 1971. Evolution of sex chromosomes and formation of W chromatin in snakes. Chromosoma 33: 239-251.
• ROCO ÁS, OLMSTEAD AW, DEGITZ SJ, AMANO T, ZIMMERMAN LB & BULLEJOS M. 2015. Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis. Proc Natl Acad Sci 112(34): E4752-E4761.
• SABA N & TRIPATHI NK. 2014. Preliminary cytogenetic study and report of ZZ/ZW sex chromosomes in the bullfrog, Hoplobatrachus tigerinus (Anura, Amphibia) from high altitude area of Jammu and Kashmir, India. Nucleus 57(1): 55-59.
• SANGPAKDEE W, PIMPHAN S, TENGJAROENKUL B, PINTHONG K, NEERATANAPHAN L & TANOMTONG A. 2017. Cytogenetic study of three microhylid species (Anura, Microhylidae) from Thailand. Cytologia 82(1): 67-74.
• SARRE SD, EZAZ T & GEORGES A. 2011. Transitions between sex-determining systems in reptiles and amphibians. Annu Rev Genomics Hum Genet 12: 391-406.
• SCHALK CM & LEAVITT DJ. 2017. Leptodactylus bufonius. Cat Am Amphib Reptil 905: 1-22.
• SCHARTL M. 2004. Sex chromosome evolution in non-mammalian vertebrates. Curr Opin Genet Dev 14(6): 634-641.
• SCHARTL M, SCHMID M & NANDA I. 2016. Dynamics of vertebrate sex chromosome evolution: from equal size to giants and dwarfs. Chromosoma 125(3): 553-571.
• SCHMID M. 1983. Evolution of sex chromosomes and heterogametic systems in Amphibia. In: MECHANISMS OF GONADAL DIFFERENTIATION IN VERTEBRATES. Berlin: Springer, p. 13-22.
• SCHMID M, FEICHTINGER W, STEINLEIN C, NANDA I, MAIS C, HAAF T, VISBAL GARCIA R & FERNÁNDEZ BADILLO A. 2002. Chromosome banding in Amphibia XXII. Atypical Y chromosomes in Gastrotheca walkeri and Gastrotheca ovifera (Anura, Hylidae). Curr Opin Genet Dev 96(1-4): 228-238.
• SCHMID M & STEINLEIN C. 2001. Sex chromosomes, sex-linked genes, and sex determination in the vertebrate class Amphibia. In: GENES AND MECHANISMS IN VERTEBRATE SEX DETERMINATION. Basel: Birkhäuser, p. 143-176.
• SCHMID M, STEINLEIN C, BOGART JP, FEICHTINGER W, LEÓN P, LA MARCA E, DIAZ LM, SANZ A, CHEN SH & HEDGES SB. 2010. The chromosomes of terraranan frogs. Insights into vertebrate cytogenetics. Cytogenet Genome Res 568: 130-131.
• SCHMID M, STEINLEIN C, FRIEDL R, DE ALMEIDA CG, HAAF T, HILLIS DM & DUELLMAN WE. 1990. Chromosome banding in Amphibia XV. Two types of Y chromosomes and heterochromatin hypervariabilty in Gastrotheca pseustes (Anura, Hylidae). Chromosoma 99(6): 13-423.
• SCHNEIDER RG, CARDOZO DE, BRUSQUETTI F, KOLENC F, BORTEIRO C, HADDAD C, BASSO NG & BALDO D. 2019. A new frog of the Leptodactylus fuscus species group (Anura: Leptodactylidae), endemic from the South American Gran Chaco. Peer J 7: e7869.
• SCHWEIZER D. 1976. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58(4): 307-324.
• SILVA APZ, GARCIA PC, MARTINS VG, BACCI M & KASAHARA S. 2004. Chromosomal and molecular analyses of Leptodactylus gracilis gracilis, L. gracilis delattini, and L. plaumanni (Anura, Leptodactylidae): taxonomic implications. Amphib-Reptilia 25(2): 185-196.
• SILVA APZ, HADDAD CFB, GALASSI GG & KASAHARA S. 2006. Multiple nucleolus organizer regions in Leptodactylus mystacinus (Amphibia, Anura) and comments on its systematic position in the L. fuscus group based on cytogenetic and molecular analyses. Genetica 127(1-3): 35-44.
• SILVA APZ, HADDAD CFB & KASAHARA S. 2000. Chromosomal studies on five species of the genus Leptodactylus Fitzinger, 1826 (Amphibia, Anura) using differential staining. Cytobios 103(402): 25-38.
• SUMNER AT. 1972 A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75: 304-306.
• 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. Mol Phylogenet Evol 54(3): 709-725.
• TOMATIS C, BALDO D, KOLENC F & BORTEIRO C. 2009. Chromosomal variation in the species of the Physalaemus henselii group (Anura: Leiuperidae). J Herpetol 43(3): 555-561.
• UNO Y, NISHIDA C, TAKAGI C, IGAWA T, UENO N, SUMIDA M & MATSUDA Y. 2015. Extraordinary diversity in the origins of sex chromosomes in anurans inferred from comparative gene mapping. Cytogenet Genome Res 145(3-4): 218-229.
• VALENZUELA N. 2008. Sexual development and the evolution of sex determination. Sex Dev 2(2): 64-72.
• VITTORAZZI SE, LOURENÇO LB & RECCO-PIMENTEL SM. 2014. Long-time evolution and highly dynamic satellite DNA in leptodactylid and hylodid frogs. BMC Genet 15(1): 111.

Publication Dates

• Publication in this collection
  04 June 2021
• Date of issue
  2021

History

• Received
  9 Apr 2019
• Accepted
  13 June 2019