Chromosomal analysis of Physalaemus kroyeri and Physalaemus cicada (Anura, Leptodactylidae)

Abstract All the species of Physalaemus Fitzinger, 1826 karyotyped up until now have been classified as 2n = 22. The species of the Physalaemus cuvieri group analyzed by C-banding present a block of heterochromatin in the interstitial region of the short arm of pair 5. Physalaemus cicada Bokermann, 1966 has been considered to be a member of the Physalaemus cuvieri species group, although its interspecific phylogenetic relationships remain unknown. The PcP190 satellite DNA has been mapped on the chromosomes of most of the species of the Physalaemus cuvieri group. For two species, Physalaemus cicada and Physalaemus kroyeri (Reinhardt & Lütken, 1862), however, only the chromosome number and morphology are known. Given this, the objective of the present study was to analyze the chromosomes of Physalaemus cicada and Physalaemus kroyeri, primarily by C-banding and PcP190 mapping. The results indicate that Physalaemus kroyeri and Physalaemus cicada have similar karyotypes, which were typical of Physalaemus. In both species, the NORs are located on the long arm of pair 8, and the C-banding indicated that, among other features, Physalaemus kroyeri has the interstitial band on chromosome 5, which is however absent in Physalaemus cicada. Even so, a number of telomeric bands were observed in Physalaemus cicada. The mapping of the PcP190 satellite DNA highlighted areas of the centromeric region of the chromosomes of pair 1 in both species, although in Physalaemus kroyeri, heteromorphism was also observed in pair 3. The cytogenetic evidence does not support the inclusion of Physalaemus cicada in the Physalaemus cuvieri group. In the case of Physalaemus kroyeri, the interstitial band on pair 5 is consistent with the existence of a cytogenetic synapomorphy in the Physalaemus cuvieri species group.

For Physalaemus cicada and P. kroyeri, the available cytogenetic data are restricted to the chromosome number and morphology (De Lucca et al. 1974). Given this, the objective of the present study was to evaluate the chromosomal features of these two species, in particular the presence of an interstitial heterochromatic band on chromosome 5, which is recognized as a chromosomal synapomorphy in the P. cuvieri group (Vittorazzi et al. 2014b, Lourenço et al. 2015.
The animals were collected with permission of the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA/SISBIO -Process number 10678-2, 20336-1 and 33133-1). For the subsequent techniques, all samples were extracted from euthanized specimens using anesthetic application to the skin (5% Lidocaine) to minimize animal suffering, according to recommendations of the Herpetological Animal Care and Use Committee (HACC) of the American Society of Ichthyologists and Herpetologists (available in http://www.asih.org), and approved by SISBIO/Institute Chico Mendes de Conservação da Biodiversidade as a condition for the concession license.

Chromosome preparation and staining
The metaphases were obtained from intestinal cells of the specimens treated with 2% colchicine for at least 4 hours (following Schmid et al. 2010, or adapted from King andRofe 1976). The chromosomes were stained with Giemsa (10%) and then C-banded (King 1980). The slides were then processed using the Ag-NOR method (Howell and Black 1980) or stained with DAPI (0.5 μg/mL) or mithramycin (0.5 mg/mL). Chromosomal morphometrics were obtained using the MICROMEASURE v3.3 software (Reeves and Tear 2000) and the classification was based on the criteria of Green and Sessions (1991).

Extraction, isolation, cloning and sequencing of the DNA
The genomic DNA of Physalaemus kroyeri and P. cicada was extracted from samples macerated in TNES buffer (50 mM Tris pH 7.5; 400 mM NaCl; 20 mM EDTA; and 0.5% SDS), following Medeiros et al. (2013). Samples of the genomic DNA of P. cicada and P. kroyeri were submitted to a PCR using the primers P190F (AGA CTG GCT GGG AAT CCC AG) and P190R (AGC TGC TGC GAT CTG ACA AGG) (Vittorazzi et al. 2011) for the isolation of the PcP190 satellite DNA. The resulting sequences were purified and ligated to the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA). The recombinant vectors were used to transform E. coli bacteria of the JM109 lineage using a TransformAid™ Bacterial Transformation kit (Fermentas, Burlington, Ontario, Canada), following the maker's recommendations. The procedures for the selection of the recombinant clones and the extraction of the plasmidial DNA were those proposed by Sambrook et al. (1989).
To sequence the fragments, samples of the amplified PCR products were treated with a BigDye Terminator kit (Applied Biosystems, Foster City, California, USA). After precipitation and drying, the products of this reaction were resuspended in loading dye (1:5 Blue-Dextran-EDTA/Formamide), denatured for 3 minutes at 94°C and analyzed in an ABI 3730XL automatic sequencer.
All the cloned fragments were sequenced, although for the comparative analyses, only the complete PcP190 sequences were used. It is important to note that the partial units were not noticeably different in their composition from the complete sequences.

Fluorescent in situ Hybridization (FISH)
The labeling of the isolated PcP190 satellite DNA probes used in this analysis was based on PCR amplification in the presence of Digoxigenin-11-dUTP with a DIG Probe Synthesis PCR (Roche, Pensberg, Bavaria, Germany). The probes were mixed with salmon DNA (1 ng/μL of probe) and precipitated with ethanol. All the resulting DNA was dissolved in a hybridization buffer at pH 7 composed of deionized formamide (50%), 2x SSC, phosphate buffer (40 mM), Denhardt's solution, SDS (1%) and dextran sulfate (10%).
The hybridization method used was that described by Viegas-Péquignot (1992), with adaptations for the detection of the Digoxigenin-11-dUTP, which was based on the anti-digoxigenin antibody conjugated with rhodamine (Roche, Pensberg, Bavaria, Germany).
Areas of heterochromatin were detected in the centromeric regions of all the chromosomes, in the pericentromeric region of the long arm of the chromosomes of pair  6, adjacent to the NOR of the chromosomes of pair 8, and interstitially on one of the arms of the metacentric chromosomes of pair 5 (Figure 1c,d). While pair 5 is metacentric, the interstitial C band is located on the arm that appears to be slightly larger. It was also possible to observe a positive mithramycin band together with the NOR (Figure 1d).
Regions of constitutive heterochromatin were detected in the centromeres of all the chromosomes, in the proximal region of the long arm of the chromosomes of pair 2, in the pericentromeric region of the long arm of the chromosomes of pair 4, in the telomeric regions of both arms of the chromosomes of pairs 1, 2, 5, 6 and 7, and a similar pattern, but restricted to the long arms of pairs 3, 4, 9, 10 and 11 (Figure 2b,c). A large block of heterochromatin can be observed on the long arm of pair 8 ( Figure  2b), coinciding with the NOR which was also strongly stained by mithramycin in Cbanded metaphases (Figure 2c -inset).When C-banding was followed by DAPI staining, all the centromeric and telomeric C-bands were revealed as well as a band adjacent to the NOR (Figure 2).

PcP190 satellite DNA
After cloning, sequencing, and the search for similar sequences using the BLASTn tool in GenBank, it was possible to conclude that the sequences obtained with the primers P190F and P190R belong to the PcP190 satellite DNA family, which was first identified in Physalaemus cuvieri (Vittorazzi et al. 2011).
It was possible to clone three fragments of the PcP190 satellite DNA of Physalaemus kroyeri, all of which contain a complete repeat unit of this satellite DNA, of 190 bps (Figure 3). The mean similarity between these fragments was 95%, and when compared with the PcP190 sequences of P. cuvieri (Vittorazzi et al. 2011), the similarity was 93%. Five complete sequences of the PcP190 were obtained from P. cicada, of which, one was 189 bps in length, two were 192 bps long, and two were 200 bps. These differences in the size of the P. cicada result from a polymorphic region of 20 bps, characterized by substitutions and indels (Figure 3). The mean similarity of the P. cicada sequences was 88%, decreasing to 78% in comparison with P. cuvieri (Vittorazzi et al. 2011). The sequences obtained for P. kroyeri and P. cicada were 79% similar, on average.
In the karyotype of Physalaemus kroyeri, the PcP190 satellite DNA was detected in the centromeric region of pair 1. In two of the three individuals analyzed, in addition,  the PcP190 was also detected in the centromeric region of one of the chromosomes of pair 3 (Figure 4a). In P. cicada, the PcP190 was detected in the centromeric region of pair 1, in the individuals from both Limoeiro and Poço Redondo (Figure 4b).
Comparing the karyotypes of Physalaemus kroyeri and P. cicada with one another and the karyotypes described for other Physalaemus species, it is possible to infer homologies in the first seven pairs of chromosomes. This is because the morphology of pairs 1-7 is highly similar in the karyotypes analyzed, despite some differences in size (e.g., P. albonotatus in Vittorazzi et al. 2014b). Even so, it is possible that some of these inferences are erroneous, given that some pairs of chromosomes (pairs 3 and 4, for example, and 5 and 6) are very similar. On the other hand, the recognition of homologies in pairs 8 to 11 is hampered by the fact that these chromosomes are all very small and similar in morphology, except for the telocentric chromosomes classified as pair 11 in the species of the P. signifer clade and P. fernandezae (see Lourenço et al. 2015 and references therein).
In the karyotype of Physalaemus kroyeri, the NOR is located interstitially on the long arm of the chromosomes of pair 8, a situation also observed in P. albifrons (Vittorazzi et al. 2014b), which are sister species inferred by Lourenço et al. (2015), and in some populations of P. cuvieri (Quinderé et al. 2009). The results of the present study permit the differentiation of the karyotypes of P. albifrons and P. kroyeri by the presence of interstitial bands of heterochromatin on the long arms of pairs 6 and 8 in P. kroyeri, which are absent in P. albifrons (Vittorazzi et al. 2014b), and an interstitial band on the short arm of pair 8 in P. albifrons, which was absent in P. kroyeri. One other difference between the two species can be observed in pair 1, in which PcP190 satellite DNA is present in P. kroyeri, but not in P. albifrons (Vittorazzi et al. 2014a).
The interstitial C band in the metacentric pair 5 is present in all the species of the Physalaemus cuvieri group karyotyped up until now, which Vittorazzi et al. (2014b) proposed as a potential cytogenetic marker for the P. cuvieri group, confirmed by the phylogenetic analysis of Lourenço et al. (2015). The results of the present study also indicate that the marker is present in P. kroyeri, another species of the P. cuvieri group.
While the chromosome pair 5 of Physalaemus kroyeri is classified morphologically as metacentric, the arm on which the interstitial band is located is slightly larger, which calls into question the 5p position of this band in the other species of the P. cuvieri group. This difference may have resulted from some structural modification of the chromosome, such as a pericentric inversion, amplification of part of this arm, or a deletion on the opposite arm. Whatever the case, the difference in the position of this interstitial band does not alter its status as a chromosomal synapomorphy in the P. cuvieri group.
The absence of this interstitial band of heterochromatin on chromosome 5 in the karyotype of Physalaemus cicada, keeps the interpecific relationships of this species in doubt. While P. cicada has been considered to be a member of the P. cuvieri group, based on its morphological similarities (Lynch 1970, Nascimento et al. 2005, Lourenço et al. (2015) found no support for this arrangement in their phylogenetic analyses.

PcP190 satellite DNA
It was possible to recognize PcP190 satellite DNA in both Physalaemus kroyeri and P. cicada, as found in a number of other Physalaemus species, such as P. cuvieri, P. centralis, P. albonotatus, P. albifrons, P. ephippifer, P. marmoratus and P. nattereri (Steindachner, 1863), as well as members of other leptodactylid genera, such as Pleurodema diplolister (Peters, 1870), Leptodactylus latrans (Steffen, 1815) and Crossodactylus gaudichaudii Duméril & Bibron, 1841 (Vittorazzi et al. 2014a) and in the hylid genus Pseudis (Gatto et al. 2016). This sequence is well conserved, and appears to have an ancient origin in the anurans (Vittorazzi et al. 2014a, Gatto et al. 2016. In Physalaemus cicada, both the sequences and the location of the PcP190 in the karyotype provide interesting insights into the comparison of this species with those of the P. cuvieri group. On average, the PcP190 of the species of this group are 90% similar to one another (Vittorazzi et al. 2014a), although this falls to 78% in the comparison with P. cicada. The chromosomal mapping of these sequences in P. cicada is also distinct from that of the P. cuvieri group, given the lack of a PcP190 site in pair 3, which is characteristic of all the species of the P. cuvieri group analyzed to date (Vittorazzi et al. 2011(Vittorazzi et al. , 2014a. These differences may reflect a more distant phylogenetic relationship between P. cicada and the species of the P. cuvieri group. However, we must consider that given family of satellite DNA may present a different number of repetitions, even in closely-related species, given that the evolutionary dynamics of these sequences favors their continuous amplification and deletion in the genome. This is covered in the original proposal for a DNA satellite library (Fry andSalser 1977, Meštrović et al. 1998), which indicated that different families of satellite DNA coexist in a genome, but that new families may arise continually through the restructuring of the distribution and quantity of the older sequences.

Conclusion
The interstitial heterochromatic band on the metacentric chromosome 5, considered to be a cytogenetic synapomorphy of the Physalaemus cuvieri species group was found in P. kroyeri. In contrast, this marker was absent in P. cicada, which did not support the inclusion of P. cicada in the P. cuvieri species group.

Contribution of the authors
SEV developed the study, collected P. kroyeri and P. cicada, ran the analyses and drafted the manuscript. MS collected P. kroyeri and RGF collected P. cicada, both these authors revised the manuscript. SMRP and LBL developed and coordinated the study and revised the manuscript.