Cytogenetics of four foam-nesting frog species of the Physalaemus gracilis group (Anura, Leptodactylidae)

: Intending to increase the knowledge about cytogenetics of Physalaemus and the sparsely studied P. gracilis group, we analyzed the karyotypes of P. carrizorum , P. gracilis , P. lisei , and P. sp. aff. gracilis . We studied chromosome morphology, heterochromatin patterns, Ag-NORs location and mapped the repetitive DNA sequence PcP190. All species showed diploid karyotypes composed of 22 bi-armed chromosomes and similar C-bands and Ag-NOR patterns. C-bands were mainly centromeric and pericentromeric; non-centromeric C-bands were detected on the telomeres of pair 1 in P. lisei , although polymorphic, and interstitially on pair 10 of P. gracilis . This last character is useful to distinguish P. gracilis from its sibling species P. sp. aff. gracilis . The Ag-NOR sites were detected on the long arms of chromosome pair 8 but with a variable position among species. Clusters of PcP190 showed centromeric and pericentromeric positions coincident with conspicuous C-bands, on pairs 2 and 9 in P. gracilis and P. sp. aff. gracilis , pair 3 in P. carrizorum , and pair 7 in P. lisei . These results significantly increase the knowledge about Physalaemus cytogenetics and encourage further studies on the satellite PcP190 in other genera of Leiuperinae to better understand its taxonomic distribution and the evolutionary dynamics.


INTRODUCTION
The foam-nesting frog genus Physalaemus is a monophyletic group of Neotropical leptodactylids belonging to the sub-family Leiuperinae that inhabits several South American ecoregions, from the southern part of the Guianas and Venezuela to central Argentina, including a large portion of Brazil, southeastern Colombia, eastern Bolivia, Paraguay and Uruguay (Frost 2019).Current knowledge about the phylogenetic relationships of these frogs arranges them into two major clades.The P. signifer clade comprises the P. deimaticus and P. signifer species groups, plus P. maculiventris and P. nattereri.Additionally, the P. cuvieri clade consists of five species groups: P. biligonigerus, P. cuvieri, P. henselii, P. olfersii, and P. gracilis, and the species P. cicada unassigned to any group (Lourenço et al. 2015).
The cytogenetics of Physalaemus has provided valuable information to elucidate the taxonomic relations and chromosome evolution of these frogs.Only 27 of the 48 currently recognized species have been karyotyped (Quinderé et al. 2009, Tomatis et al. 2009, Vittorazzi et al. 2014a, 2016, Lourenço et al. 2015 and references therein).As a rule, Physalaemus species have diploid karyotypes with 2n = 22, although two different fundamental numbers (FN) can be observed (42 and 44).The karyotypes with FN = 42 have a small telocentric chromosome and are found in all species of the P. signifer clade and P. fernandezae, whereas a FN = 44 is present in all the remaining species of the P. cuvieri clade.
Characteristic of many Physalaemus species is the location of the Nucleolar Organizer Regions (NORs) on small-sized chromosomes (i.e., pairs 8 to 11).However, the homology of this character among species should be interpreted cautiously, since it showed broad intraspecific variability (Ananias et al. 2007a, Quinderé et al. 2009, Vittorazzi et al. 2014a, Nascimento et al. 2019) or could be homoplastic (Lourenço et al. 2015).Within the P. cuvieri clade, most species show NORs on pair 8, which is a plesiomorphic condition of the genus (Lourenço et al. 2015).Similarly, C-bands are other interesting characters that may be phylogenetically informative.For instance, all karyotyped species of the P. cuvieri group share an interstitial Cband on the metacentric pair 5, and almost all species of the P. biligonigerus group share a pericentromeric C-band on the short arm of pair 3 (Vittorazzi et al. 2014a, and references therein).Besides, the satellite DNA (satDNA) sequence PcP190 that was mapped in several species of Engystomops, Physalaemus, and Pseudis is a promissory marker for the establishment of chromosomal homologies and its variation can reveal phylogenetically informative patterns in anurans (Vittorazzi et al. 2011, 2014b, 2016, Targueta et al. 2018, Gatto et al. 2016, 2018, 2019).
The Physalaemus gracilis group is composed of six species (P.barrioi, P. carrizorum, P. evangelistai, P. gracilis, P. jordanensis, and P. lisei) that inhabit southeastern Brazil, northeastern Argentina, and Uruguay (Nascimento et al. 2005, Lourenço et al. 2015, Cardozo & Pereyra 2018).Cytogenetic data for this group is scarce and restricted to P. barrioi (Provete et al. 2012) and P. gracilis (Brum-Zorrilla & Sáez 1968, de Lucca et al. 1974), which have karyotypes of 22 bi-armed chromosomes (FN = 44).In P. barrioi, there is additional information regarding the position of NORs terminal on pair 10 and C-banding pattern that is mostly centromeric and pericentromeric, except for an interstitial band present on the long arm of pair 4 (Provete et al. 2012).
In this paper, we cytogenetically studied four species of the Physalaemus gracilis group: P. carrizorum, P. gracilis, P. lisei, and a different species from Uruguay referred herein as P. sp. aff.gracilis.We provide information concerning Ag-NORs, C-banding patterns, and the chromosomal location of the repetitive sequence PcP190, and discuss our findings under the view of current taxonomy and phylogenetic hypotheses.

MATERIALS AND METHODS
We studied 13 specimens of Physalaemus carrizorum, 4 of P. gracilis, 7 of P. sp. aff.gracilis, and 7 of P. lisei.Mitotic metaphases were obtained from intestines and bone marrow tissue following Schmid et al. (2010).The chromosome number and morphology were studied on preparations stained with buffered Giemsa solution (10%).C-bands and Ag-NOR were obtained according to Sumner (1972) and Howell & Black (1980), respectively.The satDNA sequence PcP190 (Vittorazzi et al. 2011) was mapped to mitotic preparations by in situ hybridizations after Pinkel et al. (1986).Probes consisted of PcP190 sequences amplified from a cloned type 1-PcP190 fragment (KX170909) using a PCR dig Probe Synthesis Kit (Roche) and were detected with Rhodamine conjugated with anti-digoxigenin (Roche).Chromosomes were measured using the software DRAWID (Kirov et al. 2017), excluding secondary constrictions in measurements, and classified either as metacentric (m), submetacentric (sm), and subtelocentric following Green & Sessions (1991).In the karyograms, the chromosome pairs were ordered to reflect our hypotheses of chromosomal homeologies (Lourenço et al. 2015, Targueta et al. 2018)
The Ag-NORs were detected only on the long arms of chromosome pair 8, coinciding with secondary constrictions in Giemsa stained metaphases.They were terminal in Physalaemus sp.aff.gracilis and P. gracilis, but interstitial in P. carrizorum and P. lisei (Figure 1 a,d).Besides, one female of P. sp. aff.gracilis (MNHN 9511) and one male of P. gracilis (SMRP 37.16) showed different sizes for the Ag-NORs between homologues (Figure 1 a,d).
Heterochromatic bands were observed in the centromeric regions and stained more intensely on (peri)centromeres of pair 3 in the four species and pair 7 in Physalaemus lisei (Figure 2a, c, e, g).In Physalaemus sp.aff.gracilis and P. gracilis, the NORs were also associated with C-bands (Figure 2c, e).In P.
gracilis, an additional interstitial C-band was observed in pair 10.Moreover, in P. lisei, we observed polymorphisms for terminal C-bands associated with chromosomes of pair 1.Of the four specimens analyzed by this technique (3♀, 1♂), the three females showed positive C-bands on both homologues in 1q while the male was heterozygous for such band, being present in only one chromosome.On the other hand, two of these females showed additional C-bands in both homologues in 1p (Figure 2i).
FISH with PcP190 probe showed fluorescent centromeric/pericentromeric marks in one chromosome pair of Physalaemus carrizorum and P. lisei, and in two chromosome pairs in P. sp. aff.gracilis and P. gracilis, always associated with heterochromatin but with a different location between species: pairs 2 and 9 in P. gracilis and P. sp. aff.gracilis, pair 3 in P. carrizorum, and pair 7 in P. lisei (Figure 2b, d, f, h).

DISCUSSION
Previous reports on cytogenetics of the Physalaemus gracilis group only referred to the chromosome number of specimens from  Moreover, within the higher taxon Hyloidea, this character state is shared by the families Brachycephalidae, Craugastoridae (Schmid et al. 2010, and references therein), Bufonidae (Baldo et al. 2012, and references therein), and Odontophrynidae (Ananias et al. 2007b, Rocha et al. 2017, andreferences therein).It is remarkable that in some recent phylogenomic analyzes, all these taxa were recovered within the major clade Commutabirana that includes the superfamily Brachycephaloidea (Brachycephalidae, Craugastoridae, Eleutherodactylidae) and the families Allophrynidae, Bufonidae, Centrolenidae, Dendrobatidae, Leptodactylidae, and Odontophrynidae (Feng et al. 2017, Streicher et al. 2018), and in which the state x = 11 optimizes as a synapomorphy.Alternatively, in a more inclusive phylogenetic hypothesis recently proposed by Jetz & Pyron (2018), x = 11 optimizes as a synapomorphy for a minor clade that excludes Brachycephaloidea.
As previously stated, among the Physalaemus karyotypes, are observed two distinct FN, FN = 44 and FN = 42 (Vittorazzi et al. 2016, and references therein).Karyotypes composed of only bi-armed chromosomes (FN = 44) are present in species of the P. cuvieri clade but also among the other genera of leiuperines Edalorhina, Engystomops, Pleurodema, and Pseudopaludicola (Barrio & Rinaldi de Chieri 1970, Lourenço et al. 2000, 2006, Targueta et al. 2011, 2018, Cardozo et al. 2016, 2018.Conversely, karyotypes with one small pair of telocentric chromosomes (FN = 42) were reported for species of the P. signifer clade and P. fernandezae (P.cuvieri clade), and with one small or medium pair in some species of Engystomops (Tomatis et al. 2009, Lourenço et al. 2015, Targueta et al. 2018).The similar FN observed in these three taxa likely corresponds to homoplasy, and the character state FN = 42 could be considered a synapomorphy of the P. signifier clade (Lourenço et al. 2015).It should be stressed that the relation between P. fernandezae and P. henselii, and the remaining species of the P. cuvieri clade is poorly supported (Lourenço et al. 2015) and, in this regard, future   Lucca et al. 1974).Moreover, in the P. biligonigerus group, which is phylogenetically closely related to the P. gracilis and P. olfersii groups, all species show distal Ag-NORs in pairs 8 or 9 (Amaral et al. 2000, Silva et al. 1999, Tomatis et al. 2009, Vittorazzi et al. 2014a).In this sense, NORs in pair 3 would be an apomorphy of a less inclusive clade within the P. olfersii group, pending the study of this marker on P. lateristriga and P. soaresi to confirm this assumption.Besides, the similar size and morphology of chromosome pairs 8-10 in species of the P. biligonigerus and P. gracilis groups allow us to assume that NOR-bearing chromosomes of these species are homeologous, with the caveat that this hypothesis should be tested with the aid of other chromosome markers.Moreover, Lourenço et al. (2015) stated that the NORs on pair 8 would represent a plesiomorphic condition for Physalaemus, as it is shared by other genera of Leiuperinae and Leptodactylinae that also present 2n = 22 karyotypes (Bogart 1974, Lourenço et al. 2000, 2008, 2015, Coelho et al. 2016).
Two specimens, one of Physalaemus gracilis and one of P. sp. aff.gracilis showed heteromorphisms for Ag-NOR size.This sort of variation is frequently reported as a source of polymorphisms in anuran cytogenetics and may be the consequence of different mechanisms such as unequal meiotic exchanges or tandem duplication of rDNA (Schmid 1982, Schmid et al. 2010).
All species in the Physalaemus gracilis group have similar patterns of (peri)centromeric C-bands.Besides, P. sp. aff.gracilis and P. gracilis show heterochromatin associated with the NOR sites, and P. barrioi and P. gracilis have interstitial C-bands on pairs 4 and 10, respectively (Provete et al. 2012, this study).In P. gracilis, the C-band on chromosome pair 10 has an additional taxonomic value as it allows us to distinguish P. gracilis from its sibling species P. sp. aff.gracilis.Finally, in Physalaemus lisei, the detection of conspicuous telomeric C-bands varies among specimens for both arms (1p and 1q) and, although the only male studied by this technique had a particular pattern not observed in females, with a single C-band on one homologue of 1q, it is still necessary to study more males in order to exclude that this last polymorphism is sex-biased.

CONCLUSION
Some studies have demonstrated an extensive taxonomic distribution regarding the PcP190 sequences, a satDNA probably derived from 5S rDNA (Vittorazzi et al. 2011).Its presence was confirmed for the families Leptodactylidae (Engystomops, Leptodactylus, and Physalaemus), Hylodidae (Crossodactylus), and Hylidae (Pseudis).This satDNA marker was previously mapped on the karyotypes of 8 species of Engystomops, 7 species of Physalaemus, and 7 species of Pseudis (Vittorazzi et al. 2011, 2014b, 2016, Gatto et al. 2016, Targueta et al. 2018, and references therein;Gatto et al. 2019).As a generality, the chromosome position of PcP190 is biased towards the centromeres or proximally associated with C-bands (Vittorazzi et al. 2014b).Exceptions are the sex chromosomes of Physalaemus ephippifer and species of Pseudis, in which PcP190 clusters were detected in interstitial heterochromatic bands, suggesting a possible role in the differentiation of sex chromosomes (Vittorazzi et al. 2014b, Gatto et al. 2016, 2019).In a similar way to what is observed in other species of Physalaemus, all four species in the P. gracilis group analyzed in this work have variable PcP190 location.It is worth noting that its presence on pair 3 is a recurrent feature shown by 7 of 11 studied species in the genus: P. albifrons, P. albonotatus, P. carrizorum, P. centralis (in addition to several pairs), P. cuvieri (in addition to several pairs), P. kroyeri (in addition to pair 1), and P. ephippifer (in addition to the W chromosome) (Vittorazzi et al. 2011(Vittorazzi et al. , 2014b(Vittorazzi et al. , 2016, this study), this study).This feature was also reported in almost all studied species of Engystomops (6 of 8 species), excepting E. coloradorum and E. "magnus" (see Targueta et al. 2018).The recurring occurrence of PcP190 in pair 3 of both Engystomops and Physalaemus is a promising informative marker to establish homeology.However, in order to obtain a better picture of this character distribution within Leiuperinae, a more considerable amount of data has to be collected in the remaining groups of the P. cuvieri clade (i.e., P. biligonigerus, P. henselii, and P. olfersii groups), the P. signifer clade, Edalorhina, Pseudopaludicola, and Pleurodema (for details about the phylogenetic relationships of these taxa, see Lourenço et al. 2015).Moreover, given the similarity between PcP190 and 5S nucleotide sequences, further data should also include information about the chromosomal mapping of 5S rDNA.
interpretations of the transformation observed in P. fernandezae would change as more information would become available.Almost all karyotyped species of the Physalaemus gracilis group show the NORs in chromosome pair 8, excepting P. barrioi in which this marker is present in pair 10 (Provete et al. 2012).In the P. olfersii group, a clade phylogenetically closely related to the P. gracilis group, the Ag-NORs are present in pairs 3 and 7 in P. feioi (as P. olfersii from the locality of Viçosa, state of Minas Gerais, Brazil, Milani et al. 2011), 3 and 4 in P. olfersii (in specimens from Teresópolis, state of Rio de Janeiro, Brazil, Milani et al. 2011), and possibly in pair 11 in P. soaresi inferred by the presence of secondary constrictions (de
, regardless of chromosome size.Collected specimens are housed in the herpetological collections of the Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical, Posadas, Misiones, Argentina (LGE), and Museo Nacional de Historia Natural, Montevideo, Uruguay (MNHN), and the Collection of tissue and chromosome preparation "Shirlei Maria Recco Pimentel", Department of Structural and Functional Biology at the Biology Institute of the University of Campinas, São Paulo, Brazil.The collections of specimens were approved by the Ministerio de Ecología y Recursos Naturales Renovables (Argentina, MEyRNR 061/2015, 073/2016, 035/2017 and 047/2018), Instituto Chico Mendes de Conservação da Biodiversidade and Sistema de Autorização e Informação em Biodiversidade (Brazil, ICMBio/SISBIO 32483-3), and División Fauna (Uruguay, MGAP Res 195/06).Protocols of euthanasia and preservation of specimens were performed with the approval of the Ethics Committee in Animal Use (CEUA UNICAMP 4802-1 and MNHN 1/2019).The sex and collection information of studied specimens are detailed below.

Table I .
Chromosome measurements of species of the Physalaemus gracilis group.The chromosome percentage is relative to the haploid set.Centromeric index ± Standard Deviation.Chromosome morphology: metacentric (m); submetacentric (sm); subtelocentric (st).