Comparative cytogenetics of Physalaemus albifrons and Physalaemus cuvieri species groups (Anura, Leptodactylidae)

Abstract Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.


Introduction
Currently, the genus Physalaemus Fitzinger, 1826 is classified in the subfamily Leiuperinae Bonaparte, 1850 in the family Leptodatylidae Werner, 1896 (Pyron and Wiens 2011) and is composed of 46 species (Faivovich et al. 2012, Frost 2013. A detailed phylogenetic analysis of the species of Physalaemus is not yet available but some supraspecific groupings have been proposed. Lynch (1970) recognized four species groups: P. pustulosus, P. biligonigerus, P. cuvieri and P. signifier, which was followed until recently. Based on a phenetic analysis of morphometric characters, Nascimento et al. (2005) resurrected Engystomops Jiménez de la Espada, 1872 to include the species previously allocated to the P. pustulosus group (sensu Lynch, 1970), resurrected Eupemphix Steindachner,1863 for the single species E. nattereri Steindachner, 1863 (included in the P. biligonigerus group by Lynch, 1970) and recognized seven species groups of Physalaemus: P. albifrons, P. cuvieri, P. deimaticus, P. gracilis, P. henselii, P. olfersii and P. signifer. Because Eupemphix was paraphyletic with respect to Physalaemus in phylogenetic analyses that included eight (Pyron and Wiens 2011) and five (Faivovich et al. 2012) species of Physalaemus, Faivovich et al. (2012) proposed that Eupemphix is a junior synonym of Physalaemus, but did not allocate P. nattereri to any species group. The monophyly of each of the seven species groups of Physalaemus proposed by Nascimento et al. (2005) remains to be tested and possible synapomorphies of these groups are still to be recognized (see comments in Borteiro and Kolenc 2007, Vera Candioti et al. 2011. According to the taxonomic proposal of Nascimento et al. (2005), P. albifrons (Spix, 1824) was removed from the P. cuvieri group (sensu Lynch 1970) and grouped together with P. biligonigerus (Cope, 1861), P. marmoratus (Reinhardt and Lütken, 1862) and P. santafecinus Barrio, 1965, three species that were previously allocated to the P. biligonigerus group proposed by Lynch (1970). Interestingly, Lobo (1996) indicated that the species of the P. biligonigerus group (sensu Lynch 1970; that included P. marmoratus, as P. fuscomaculatus) shared shovel-shaped metatarsal tubercles with P. albifrons. Otherwise, Vera Candioti et al. (2011) argued that larval oral morphology does not support the reallocation of P. albifrons proposed by Nascimento et al (2005), because the larval oral configuration of P. albifrons is almost identical to that of members of the P. cuvieri species group and differs from that of P. biligonigerus, P. santafecinus and probably P. marmoratus.
Detailed descriptions of the karyotypes of P. biligonigerus and P. marmoratus (as P. fuscomaculatus), which included the identification of the nucleolus organizer regions (NOR) and heterochromatic sites, were already provided (Amaral et al. 2000, Silva et al. 2000. On the other hand, only the chromosome number and morphology are known for P. albifrons (Denaro 1972), and no chromosomal data are available for P. santafecinus.
In the present work, we present a detailed characterization of the karyotype of P. albifrons, describe the karyotype of P. santafecinus and extend the cytogenetic analyses of the P. cuvieri group in order to better characterize the group from which P. albifrons was removed. Our aim is to provide additional evidence that could be used to compare the P. albifrons and P. cuvieri species groups.

Materials and methods
Specimens of P. albifrons, P. santafecinus, P. albonotatus (Steindachner, 1864), P. centralis Bokermann, 1962 andP. cuqui Lobo, 1993 from different localities in Brazil and Argentina were analyzed. For an unequivocal identification of the species, both morphological and acoustic characteristics were utilized. Each specimen's locality and voucher number in the scientific collection where it was deposited are provided in Table 1.
Metaphase chromosome spreads were obtained from cell suspensions of the intestine and testes of animals pre-treated with colchicine (2%) for at least 4 hours (according to Schmid et al. 2010, or adapted from King andRofe 1976). Prior to the removal of the intestine and testes, the animals were deeply anesthetized with lidocaine gel 2%. Chromosomes were conventionally stained with 10% Giemsa and sequentially submitted to C-banding (King 1980) and silver staining by the Ag-NOR method (Howell and Black 1980) or to fluorescence in situ hybridization (FISH) (Viegas-Péquinot 1992) with the rDNA probe HM 123 (Meunier-Rotival et al. 1979). C-banded metaphases from P. albifrons were also stained with DAPI (0.5 mg/mL). For each species, at least 10 metaphases that were submitted to each technique were analyzed. Morphometric analyses were done using the MICROMEASURE v3.3 software (Reeves and Tear 2000). The chromosomes were classified according to the criteria proposed by Green and Sessions (1991).

Results
All of the analyzed individuals had a diploid complement of 22 chromosomes. By comparing all of the karyotypes of Physalaemus to each other, we noted a high interspecific similarity for the first seven chromosome pairs, and the homeology of these chromosomes could be inferred. Therefore, in each karyotype presented here, these chromosomes were ordered in such a way that their numbers could reflect these homology hypotheses even when their sizes suggested a different numbering. However, the smallest chromosomes (pairs 8-11) varied significantly among the species analyzed, and were numbered only by chromosome size. A detailed description of the karyotype of each species is presented below and the Appendix (Additional file 1) present all the karyotypes arranged together. Table 1 summarizes the data on NORs and non-centromeric C-bands.

Physalaemus albifrons
The P. albifrons chromosomes were classified as metacentric (pairs 1, 2, 5, 6, 9 and 11), submetacentric (pairs 4, 7, 8 and 10) or subtelocentric (pair 3, which is at the threshold between submetacentric and subtelocentric classifications) ( Fig. 1a; Table 2). C-banding followed by DAPI staining detected all of the centromeric regions and an interstitial heterochromatic band in the short arm of chromosome 5 as well as pericentromeric bands in the short arm of chromosomes 8 and 9 (Fig. 1b). The Giemsa stained C-banded metaphases showed this same pattern, but after DAPI staining, the bands could be more easily visualized. Chromosomes 3 and 4 were very similar, but chromosome 3 had a slightly smaller centromeric index and a strong centromeric Cband, which extended to the short arm ( Fig. 1b; Table 2). The NORs were located distally in the long arm of chromosome 8 (Fig. 1c) and coincided with the secondary constrictions that were observed in Giemsa-stained meta-Figure 1. Karyotypes of P. albifrons after Giemsa-staining (a) C-banding followed by DAPI-staining (b) and in situ hybridization with a nucleolar rDNA probe (c). In b the inset shows the C-banded chromosome pair 5 stained with Giemsa; in c the inset shows the NOR-bearing chromosome pair 8 after silver staining. Arrows in b point the interstitial C-band in 5p. Bar=10mm. Table 2. Morphometric parameters of the P. albifrons, P. albonotatus, P. centralis, P. cuqui and P. santafecinus karyotypes. The measurements were based on 10 metaphases of each species. CN: chromosome number; CI: centromeric index; SD: standard deviation; RL: relative length. CC: chromosome classification; m: metacentric; sm: submetacentric; st: subtelocentric. * 1 Chromosomes were numbered in order to reflect our hypotheses of homeology for the Physalaemus chromosomes even when their sizes suggested a different numbering. * 2 Value at the threshold between submetacentric and subtelocentric classifications.  (Fig. 1a). In three specimens (ZUEC 17925, ZUEC 12363 and MNRJ 24224), a size heteromorphism was observed between the homologous NORs by FISH with an rDNA probe ( Fig. 1c) and by silver staining (Fig. 1c -inset). In two specimens (MNRJ 24230 and 24232), the NOR-bearing homologous chromosomes 8 were homomorphic. For the remaining specimens, we were not able to determine if a NOR size heteromorphism was present.

Physalaemus albonotatus
The P. albonotatus chromosomes were classified as metacentric (pairs 1, 2, 5, 6, 8, 9, 10 and 11), submetacentric (pairs 4 and 7) or subtelocentric (pair 3, which is at the threshold between submetacentric and subtelocentric classifications) ( Fig. 2a; Table  2). Curiously, chromosome 5 was larger than chromosomes 3 and 4 in some of the analyzed metaphases (as seen in Figure 2b). Heterochromatin was detected in the centromeres of all chromosomes and interstitially in the long arm of chromosome 2 and in the metacentric chromosome 5 (Fig. 2b). Only two C-banded chromosome pairs 5 were good enough to be measured. Therefore, we tentatively assigned the interstitial C-band of chromosome 5 to its short arm, but further analyses are necessary to test this hypothesis. Chromosomes 3 and 4 were very similar, but chromosome 3 had a slightly smaller centromeric index and a strong centromeric C-band, which extended to the short arm ( Fig. 2b -inset; Table 2). Silver staining detected NORs distally in the long arm of chromosome 8 adjacent to a faint C-band and in both arms of chromosome 9 (Fig. 2d). The NOR in the long arm of chromosome 9 apparently coincided with a C-band (Fig. 2d). All of these NORs could be seen as secondary constrictions in Giemsa-stained metaphases (Figs. 2a and 2c).

Physalaemus centralis
The P. centralis chromosomes were classified as metacentric (pairs 1, 2, 5, 6, 8, 9, 10 and 11) or submetacentric (pairs 3, 4, 7 and 8) ( Fig. 3a; Table 2). A secondary constriction was detected in the pericentromeric region of the long arm of chromosome 9 and coincided with the NOR that was recognized by silver staining (Fig. 3a -inset). A NOR size heteromorphism was observed in all of the P. centralis specimens analyzed. C-bands were present interstitially in the long arm of chromosome 2, in the short arm of chromosome 5, in the long arms of chromosomes 8 and 9, in the pericentromeric region of the short arm of chromosome 10, and in all of the centromeres (Fig. 3b). Chromosomes 3 and 4 were very similar, but chromosome 3 had a slightly smaller centromeric index and a strong centromeric C-band, which extended to the short arm ( Fig. 3b; Table 2).
In three specimens, a heteromorphic chromosome pair 8 composed of homologues with different morphologies and C-banding patterns was observed (Figs. 3a and 3b). While one chromosome 8 showed a conspicuous interstitial C-band that sometimes could be seen as two heterochromatic blocks (chromosome 8a in Fig. 3b), its homologue had no observable interstitial heterochromatic block (Fig. 3b). In the ZUEC 13696 specimen, the pericentromeric C-bands in the long arms of the homologous chromosomes 2 were heteromorphic in size. Additionally, the homologue that had the smaller pericentromeric C-band also had an additional and conspicuous terminal C-band in the long arm ( Fig. 3b -inset).

Physalaemus cuqui
The P. cuqui chromosomes were classified as metacentric (pairs 1, 2, 5, 6, 8, 9, 10 and 11), submetacentric (pairs 4 and 7) or subtelocentric (pair 3, which is at the threshold between submetacentric and subtelocentric classifications) ( Fig. 4a; Table 2). Heterochromatic bands were observed interstitially in the long arm of chromosome pair 2, in the metacentric chromosome pair 5 and in the centromeric regions of all of the chromosomes (Fig. 4b). Only one C-banded chromosome pair 5 could be measured. Therefore, as well as for P. albonotatus, we tentatively assigned the interstitial C-band of chromosome 5 of P. cuqui to its short arm, but further analyses are necessary to test this hypothesis. Chromosomes 3 and 4 were very similar, but chromosome 3 had a slightly smaller centromeric index and a strong centromeric C-band, which extended to the short arm ( Fig. 4b; Table 2).
In three specimens, the Ag-NORs were located in the long arm of chromosome pair 8 and in the short and long arms of chromosome pair 9 (LGE 1635-1636, MLP DB 4973) (Fig. 4c -left), but only one chromosome 9 was silver-stained in the MLP DB 5560 specimen (Fig. 4c -middle). Additionally, one specimen (MLP DB 6480) showed an additional Ag-NOR in the short arm of one chromosome 4 ( Fig. 4cright). These Ag-NORs were coincident with the secondary constrictions visualized in Giemsa-stained metaphases (Fig. 4a).

Physalaemus santafecinus
The P. santafecinus chromosomes were classified as metacentric (pairs 1, 2, 3, 5, 6, 8, 9, 10 and 11) or submetacentric (pairs 4 and 7) ( Fig. 5a; Table 2). The NORs were located distally in the long arm of chromosome 9 (Fig. 5a -inset). C-bands were detected in all the centromeric regions. Additionally, pericentromeric C-bands were present in the short arms of chromosomes 1 and 2 and in the short arm of chromosome 8. Small C-bands were also detected proximally in the long arms of chromosomes 4 and 7 and distally in the long arm of chromosome 1. A conspicuous C-band was observed in the short arm of chromosome 3, which was almost entirely heterochromatic. Terminal faint C-bands could be seen in all of the chromosomes (Fig. 5b). When the Ag-NOR method was performed on C-banded metaphases, we could undoubtedly recognize the chromosome 9 as the NOR-bearing chromosome while chromosomes 8 had strong pericentromeric C-bands (data not shown).

Discussion
To date, 23 of the 46 species of Physalaemus were karyotyped and all of them have 2n=22 (Beçak 1968, Beçak et al. 1970, Denaro 1972, De Lucca et al. 1974 Silva et al. (2000), and a reference to the P. atlanticus Sazima, 2004 andP. moreirae (Miranda-Ribeiro, 1937) karyotypes in the discussion of Ananias et al. (2007)], as does P. nattereri (Beçak 1968, Lourenço et al. 2006, Ananias et al. 2007) and P. fernandezae (Müller, 1926)  ). The karyotypes of the remaining species of Physalaemus, including the species of the P. cuvieri and the P. albifrons groups that we focused on in our present investigation, have FN=44 and a biarmed chromosome 11. Considering the close phylogenetic relationship inferred for P. nattereri and P. signifer (Pyron and Wiens 2011, Faivovich et al. 2012, Fouquet et al. 2013, which was the only species of the P. signifer group already included in phylogenetic analyses, it is possible to suppose that the telocentric chromosomes 11 of P. nattereri and P. signifer have the same origin. On the contrary, the similar chromosomes 11 of P. fernandezae and the P. signifer group probably result from a homoplasy ).

Figure 5. Giemsa-stained (a) and C-banded (b) karyotypes of P. santafecinus.
In a, the inset shows the NOR-bearing chromosome 9 after silver staining. Bar=10mm.
The karyotype of P. santafecinus described here is very similar in chromosomal size and morphology to those of P. biligonigerus, P. marmoratus and Physalaemus sp. aff. biligonigerus (Amaral et al. 2000, Silva et al. 2000. The chromosomes classified by Amaral et al. (2000) as 4 and 5 probably correspond to chromosomes 5 and 4, respectively, of the karyotype of P. biligonigerus described by Silva et al. (2000) and of the P. santafecinus karyotype. Such a discrepancy emerges, however, from the use of different criteria for the numeric classification of the chromosomes rather than from a real divergence between the karyotypes.
A remarkable characteristic of the P. santafecinus karyotype that is shared with the karyotypes of P. biligonigerus, P. marmoratus and Physalaemus sp. aff. biligonigerus is a conspicuous C-block on the short arm of chromosome 3 (3p) ( Table 3). This large heterochromatic C-block is not detected in P. albifrons or in any species of P. cuvieri group. Instead, a small C-band pericentromerically located on 3p was already detected in the karyotypes of the species currently allocated to the P. cuvieri group that were already studied by C-banding [i.e., P. albifrons, P. albonotatus, P. centralis, P. cuqui (present work), P. ephippifer (Steindachner, 1864) (Nascimento et al. 2010) and one of the populations of P. cuvieri Fitzinger, 1826 that was studied cytogenetically by Quinderé et al. (2009)]. Although the pericentromeric C-band in 3p of P. ephippifer could be easily observed, it was also much smaller than those observed in P. santafecinus, P. biligonigerus, P. marmoratus and Physalaemus sp. aff. biligonigerus. In the latter four species, the larger size of this C-band probably explains the larger size of 3p in these karyotypes. A small pericentromeric C-band that extend from the centromere to the short arm of chromosome 3 was also present in P. barrioi Bokermann, 1967(Provete et al., 2012, P. olfersii and P. feioi (as P. olfersii; Milani et al. 2010), which are the species of P. gracilis group (P. barrioi) and P. olfersii group (P. olfersii and P. feioi) already studied by C-banding.
Interestingly, a large 3p showing a large C-band was also observed in P. nattereri (Lourenço et al. 2006, Ananias et al. 2007), a species previously allocated to the P. biligonigerus group by Lynch (1970). Although a rigorous phylogenetic analysis of the Physalaemus genus is not yet available, in recent phylogenetic inferences P. nattereri was recovered as the sister species of P. signifer and was not closely related to P. biligonigerus (Pyron and Wiens 2011, Faivovich et al. 2012, Fouquet et al. 2013. In this phylogenetic context the most parsimonious hypothesis is to consider the large heterochromatic region in chromosome 3 of P. nattereri to be homoplastic with respect to the large heterochromatic region in chromosome 3 of the P. santafecinus, P. biligonigerus, P. marmoratus and Physalaemus sp. aff. biligonigerus karyotypes. This hypothesis is particularly plausible if we consider the evolutionary dynamics of satellite DNAs, which are the principal components of heterochromatin (reviewed in Charlesworth et al. 1994). The copy number of satellite DNA repeats can vary dramatically, as they are frequently involved in unequal crossing over and other events as rolling circle replication and conversion-like mechanisms (reviewed in Charlesworth et al. 1994, andin Ugarkovic andPlohl 2002).
On the other hand, the available data do not prevent the large C-band found on 3p of P. santafecinus, P. biligonigerus and P. marmoratus from being a synapomorphy of this group of species, which could have arisen from the amplification of a small C-band. Table 3. Comparison of chromosome 3 of species of P. cuvieri (left column) and P. albifrons (right column) groups. Black areas in the ideograms represent C-bands. * 1 Based on Silva et al. (1999) and Quinderé et al. (2009). * 2 Nascimento et al. (2010). * 3 Based on Amaral et al. (2000). Despite the proposals of Lynch (1970) and Nascimento et al. (2005) disagree with regard to the relationships of these three species with other Physalaemus species, the close relationships of P. santafecinus, P. biligonigerus and P. marmoratus was considered in both studies. A phylogenetic analysis designed to study the relationships in the genus Physalaemus, however, is crucial to test this hypothesis. Also, further molecular characterization of the heterochromatic bands on 3p could help to provide additional evidence of the inferred heterochromatin amplification process. In addition to the large C-band in 3p, the karyotype of P. santafecinus is also similar to those of P. biligonigerus P. marmoratus and Physalaemus sp. aff. biligonigerus (Amaral et al. 2000, Silva et al. 2000 based on the presence of several telomeric C-bands and a pericentromeric C-band in the short arm of chromosome 8 as well as the NOR location. In all of these karyotypes, the NOR-bearing chromosome is small and metacentric, and it was classified as chromosome 9 in the karyotype of P. santafecinus (described here) and in the karyotypes of P. biligonigerus, P. marmoratus and Physalaemus sp. aff. biligonigerus described by Amaral et al. (2000). However, in the karyotype of P. biligonigerus described by Silva et al. (2000), the NOR-bearing chromosome was considered to be chromosome 8, which has a conspicuous pericentromeric C-band. Because Silva et al. (2000) apparently did not perform sequential C-banding and Ag-NOR in order to properly identify the NOR-bearing chromosome in C-banded metaphases, it is likely that the NOR-bearing chromosome is chromosome 9 in the C-banded karyotype shown by those authors.

P. cuvieri
In contrast to P. santafecinus, P. biligonigerus, P. marmoratus and Physalaemus sp. aff. biligonigerus, the telomeric C-bands could not be detected in the karyotype of P. albifrons. Additionally, the NOR in P. albifrons was detected interstitially in the long arm of the submetacentric chromosome 8. This NOR-bearing chromosome very closely resembles the NOR-bearing chromosome found in some populations of P. cuvieri (Silva et al. 1999, Quinderé et al. 2009) as well as in P. albonotatus and P. cuqui (present work). The P. albifrons karyotype presented here is very similar to the Giemsa-stained karyotype described for this species by Denaro (1972). However, the chromosome classified by Denaro (1972) as No. 11 is probably the one we classified as No. 8, and the secondary constriction observed by Denaro (1972) is likely to be the site recognized as NOR by silver impregnation in the present work.
Despite the similarity between the NOR-bearing chromosome of P. albifrons and those of some species of the P. cuvieri group, it would be premature to consider this a synapomorphy of P. albifrons and species of the P. cuvieri group because the evolutionary divergence of this character (i.e., NOR location) has not yet been elucidated. We cannot discard the possibility that the NOR found in P. albifrons and in some P. cuvieri species is plesiomorphic with respect to the other NOR sites found in Physalaemus species. This interpretation derives from the fact that the NOR-bearing chromosome 8 found in other leiuperines, as Pleurodema diplolister (Peters, 1870) (Lourenço et al., 2006), resembles that of P. albifrons and some P. cuvieri species group and could constitute the same state of character.
Another chromosome feature found in P. albifrons that was also detected in species of the P. cuvieri group was the interstitial C-band in chromosome 5 (Table 4). This C- Table 4. Comparison of chromosome 5 of species of P. cuvieri (left column) and P. albifrons (right column) groups. Black areas in the ideograms represent C-bands. * 1 C-band was tentatively assigned to the short arm (see text for details). * 2 Based on Silva et al. (1999) and Quinderé et al. (2009). * 3 Nascimento et al. (2010). * 4 Based on chromosomes described as No. 3 by Amaral et al. (2000).

P. erikae
No C-banding data P. fischeri No C-banding data P. kroyeri No C-banding data band was observed in all of the species of the P. cuvieri group already analyzed by the C-banding technique, including P. cuvieri (Silva et al. 1999, Quinderé et al. 2009), P. ephippifer (Nascimento et al. 2010), P. albonotatus (present work), P. centralis (present work) and P. cuqui (present work). However, this band was not detected in the Cbanded karyotypes of the other three species currently allocated in the P. albifrons group (Amaral et al. 2000, Silva et al. 2000 or in species of the P. henselii group ), the P. olfersii group (Milani et al. 2010) and the P. gracilis group (Provete et al. 2012). Based on these data, the interstitial C-band in the medium-sized chromosome classified as No. 5 is a putative synapomorphy of P. albifrons and the species of the P. cuvieri group. However, because of the small size of this C-band, which could make its detection by the C-banding technique particularly difficult, and because of the dynamics of the satellite DNA sequences, which are subject to recurrent amplification/deletion events, this hypothesis must be taken with caution. A comprehensive phylogenetic study of the genus Physalaemus and a molecular characterization of this interstitial C-band would allow this hypothesis to be properly evaluated.
In conclusion, we were not able to recognize any chromosomal character that would support the reallocation of P. albifrons from the P. cuvieri group to the P. albifrons group together with P. biligonigerus, P. marmoratus and P. santafecinus.
Interestingly, in addition to the data regarding chromosomal characteristics, larval morphology also does not seem to support the composition of the P. albifrons group. Physalaemus biligonigerus, P. santafecinus and P. marmoratus have a similar larval oral disc configuration (LTRF 2/2, with a dorsal gap in the marginal papillae) that differs considerably from that of P. albifrons, whose oral disc is almost identical to that of the tadpoles of the P. cuvieri group and is thus characterized by an LTRF 2/3 with dorsal, ventrolateral and ventral gaps in the marginal papillae (Vera Candioti et al. 2011). During embryogenesis of the oral disc of Physalaemus, ventrolateral gaps appear in the marginal papillae, apparently in all species of the genus (see Vera Candioti et al. 2011). The ventrolateral gaps persist only in the tadpoles of P. cuvieri species group [except P. fischeri (Boulenger, 1890) and P. cicada Bokermann, 1966], in P. riograndensis Milstead, 1960 (P. henselii group) and in P. albifrons (see Vera Candioti et al. 2011). On the other hand, ventral gaps develop only in tadpoles of P. albifrons, in species of P. cuvieri group (except P. fischeri) and in two species of the P. henselii group [P. henselii (Peters, 1872) and P. fernandezae (Müller, 1926)]. Among the leiuperines, the ventrolateral gaps were only observed in some species of Pseudopaludicola (see Vera Candioti et al. 2011), and although its presence during development appears to be plesiomorphic for Physalaemus, its persistence in larval stages is a putative synapomorphy of the P. cuvieri group (including P. albifrons). Finally, the internal oral morphology of tadpoles of P. albifrons differs from that of P. biligonigerus, P. marmoratus and P. santafecinus based on the presence of three lingual papillae, which is a characteristic shared with some species of the P. cuvieri group (Oliveira et al. 2010).

Interspecific comparison in the P. cuvieri group
Some of the species in the P. cuvieri group are sibling species with important intraspecific morphological variation. Therefore, the identification of these species that is based exclusively on their morphology is sometimes very difficult. Occasionally, species misidentification has occurred, for example, among P. cuvieri, P. albonotatus, P. cuqui and P. centralis (Barrio, 1965). Our results revealed conspicuous cytogenetic differences among most species of the P. cuvieri group. The exception is the great similarity between the karyotypes of P. albonotatus and P. cuqui. Additionally, the karyotypes of the species analyzed here were distinguished from the previously analyzed karyotype of P. cuvieri. The interspecific variation described in this work regarding heterochromatin and NOR distribution is of fundamental importance for the comparative analysis of the P. cuvieri species group.
An interstitial C-band was observed near the centromere in the long arm of chromosome 2 of P. albonotatus, P. centralis and P. cuqui; whereas in the karyotype of P. ephippifer (Nascimento et al. 2010) there is an interstitial C-band in the short arm of chromosome 2. A corresponding interstitial C-band in the short arm of chromosome 2 was reported in P. cuvieri populations from Rio Claro (Silva et al. 1999) and Palmeiras (Quinderé et al. 2009). If these heterochromatic bands were homeologous, it is conceivable that rearrangements (mainly pericentric inversions) involving chromosome 2 might have occurred during the divergence of these species. Interestingly, the present work reports evidence of a rearrangement involving chromosome 2 in P. centralis. In the ZUEC 13696 specimen of P. centralis, heteromorphism for the intrachromosomal location of heterochromatic regions in the chromosome pair 2 suggested that paracentric inversion might have been involved in this chromosomal rearrangement.
Despite the overall similarity in chromosomal morphology among the species currently allocated to the P. cuvieri group, chromosome pairs 8 and 9 differ greatly. The differences in these chromosomes probably arose from the distinct locations of the NOR in these karyotypes, as these rDNA genes occupy different sites in pairs 8 and/ or 9 of these species. The observed pattern of NOR occurrence can be helpful in distinguishing the analyzed species of the P. cuvieri group. Noticeably, a pericentromeric NOR site was found exclusively in the P. centralis karyotype. However, the NORbearing chromosomes (chromosome pairs 8) from the species P. cuvieri (Silva et al. 1999, Quinderé et al. 2009), P. albonotatus, P. cuqui and P. albifrons are quite similar and their homeology could be possible. Otherwise, the evolutionary relationship of this chromosome with the other NOR-bearing chromosomes found in species of P. cuvieri, P. albifrons and other species groups remains unclear, and further studies are necessary to elucidate the rearrangements that give rise to the great diversification of the NOR-bearing chromosomes in this genus.