The present cytogenetic analysis confirmed the variability of 2n = 24, 2n = 22, 2n = 20, and 2n = 18 within the genus Aplastodiscus, contrary to that is commonly observed in the subfamily Hylinae, in general with invariable 2n = 24 karyotypes [25]. Distinct diploid numbers in Aplastodiscus were originally reported [8] and, more recently, the sample of karyotyped species was enlarged [9, 10]. Taking into account our report on A. callipygius, analysed here for the first time, a total of nine representatives of the genus now have described karyotypes: A. cochranae and A. perviridis (A. perviridis group) with 2n = 24; A. albofrenatus A. arildae A. ehrhardti, and A. eugenioi (A. albofrenatus group) with 2n = 22; A. albosignatus and A. callipygius with 2n = 20; and A. leucopygius with 2n = 18 (A. albosignatus group).
It is important to emphasise that several individuals of these species have been collected in more than one locality and no karyotype intraspecific variation in the chromosome number has been found except, at the first sight, in the sample of A. albofrenatus (2n = 24 in Floresta da Tijuca, Rio de Janeiro, RJ, and 2n = 22 in Boraceia, SP) and A. albosignatus (2n = 20 in Boraceia, SP, and 2n = 18 in Teresópolis, RJ) [8], although this variation was probably consequence of misidentification, according to the author. Later, Carvalho et al. [9, 10], based on the geographical distribution of Aplastodiscus and on the cytogenetic data of some individuals collected in the same or near the localities screened by Bogart [8], concluded that the formerly karyotyped specimens were actually misidentification, and suggested that the animals with 2n = 22, 2n = 20, and 2n = 18 corresponded to A. arildae A. albosignatus, and A. leucopygius, respectively. Additionally, considering that the range of A. perviridis includes the state of Rio de Janeiro [7] and that the diploid number for this species is 2n = 24 [10, present work], the A. albofrenatus from Tijuca Forest, RJ [8], could be A. perviridis or some other species of Aplastodiscus.
Observing the karyotypes analysed so far with standard Giemsa staining, it was noticed that, although the ordering or the nomenclature adopted for each chromosome pair could differed among the authors, the chromosome constitution was equivalent within each group of species. Even presenting distinct diploid numbers, some shared characteristics could be recognized: the first five chromosome pairs in A. perviridis, A. callipygius, and A. leucopygius were equivalent in morphology and relative size; similarity also exists for the first five pairs of A. arildae and A. eugenioi, both exhibiting 2n = 22 karyotypes, but their pair 2 is clearly metacentric and larger than the 2 observed in the three former species; all six species exhibited a subtelocentric medium-sized marker but in distinct positions in the karyograms, that is, in the majority of the species the marker was the pair 6, whereas in A. callipygius and A. leucopygius it corresponded to pair 8. On the other hand, some conspicuous karyotype differences could be pointed out: a progressive reduction in the number of the small pairs, totalling six in A. perviridis, five in A. arildae and A. eugenioi, two in A. callipygius, and one in A. leucopygius; the presence in A. callipygius and A. leucopygius of two large-sized chromosome pairs 6 and 7, not observed in any other karyotype.
Taking into account that 2n = 24 was considered a synapomorphy for Hylinae [5], most probably the ancestor of Aplastodiscus had 24 chromosomes (Figure 7A), and the karyotype constitution would be equivalent to that observed in the related genera Bokermannohyla and Hypsiboas, as well as to that of the 2n = 24 A. cochranae and A. perviridis. Therefore, the chromosome evolution within the genus Aplastodiscus occurred primarily by reducing the diploid number from an ancestor with 2n = 24 due to chromosome fusions. However, replication banding data obtained for the first time in species of Aplastodiscus could not be used for identifying the probable structural rearrangements, although correspondence of banding patterns had been confirmed among some chromosomes.
Other analysis provided additional data on the karyotype variability within the genus Aplastodiscus. Both Ag-impregnation and FISH with an rDNA probe confirmed one pair of NOR-bearing chromosomes for all species. The eventual heteromorphism of Ag-NOR, that is, presence and lack of labelling in metaphases of some of the individuals in all analysed species, was interpreted as result rather from a differential activity than from the deletion in the amount of rDNA repeats, since two FISH signals, equivalent in size in both homologues, were observed in all cases. So, the transcriptional activity of rDNA might be inactivated or to be too low to be detected by silver impregnation in some chromosomes.
In A. perviridis A. arildae A. callipygius, and A. leucopygius, the NOR was located in a homeologous small-sized chromosome, although corresponding to the 11 in the two former species and to 9 in the two latter, due to the reduction in the diploid number. This condition of NOR in one of the smallest chromosome pairs was also observed in other species of Aplastodiscus[9, 10], as well as in the hylids of genera Bokermannohyla Hyla Hypsiboas, and those belonging to Scinax of rubber clade [25–32], and this can be considered a plesiomorphy for the family. These marker chromosome pairs are most probably homeologous, although with non-coincident position in the case of the karyograms of species with the same chromosome number.
In A. eugenioi of the present sample and from the literature [9], the NOR had a derived location, in a medium-pair 6, or pair 7 in the case of A. ehrhardti and A. albofrenatus, but the latter species had an additional NOR site in chromosome 1 [9]. In the three species the medium-sized pairs bearing NOR, referred by us as the 6, were probably the same, and this condition may constitute a synapomorphy. Gross structural rearrangement seemed not to be the mechanism underlying the change of NOR from a small-sized to a medium-sized chromosome, because the chromosome 6 was always recognized as a subtelocentric marker in all species, independently if bearing or not NOR. Minute structural rearrangements, transposition by means of mobile elements or other mechanisms were not discarded, but they were not demonstrated through the used banding techniques. These mechanisms would also explain the change of NOR from the long to the short arms of chromosome 9 in A. leucopygius.
All the sampled species of Aplastodiscus had similar heterochromatin distribution, with C-bands in the centromeres and at the NOR sites. Our data on A. perviridis A. arildae, and A. eugenioi differed from the C-banding pattern of the corresponding species previously analysed [9, 10], that demonstrated additional secondary C-positive regions in some chromosome pairs. This might be indicative of population difference or even be result of technical procedures. In spite of the apparent uniformity in the C-banding, an unequivocal molecular heterogeneity of the heterochromatin was revealed by CMA3 staining and FISH with a telomeric probe. In fact, the centromeric heterochromatin of the chromosomes of A. perviridis was GC-rich repetitive region, as shown by its bright fluorescence after CMA3 staining. On the other hand, the hybridisation of the telomeric probe outside of the ends of all chromosomes in A. arildae and A. eugenioi, and chromosome 3 in A. leucopygius, indicated the presence of repeats similar to (TTAGGG)n in the centromeric region. Another possible type of centromere repetitive region corresponded to that of the chromosomes of A. callipygius and A. leucopygius, since neither the base-specific fluorochromes nor the telomeric probe yielded a fluorescent labelling.
Occasionally, interstitial hybridisation of the telomeric probe may represent true vestiges of telomeres, corroborating structural rearrangements occurred during chromosome evolution, as described in rodents [33, 34]. Nevertheless, this possibility was excluded in the Aplastodiscus species [9, present work], and in other frogs presenting ITS [35, 36]. Regardless, the presence of repetitive DNA bearing telomere-like sequences outside the telomeres might represent an additional cytological marker for species or even species groups.
The meiotic analysis in A. arildae A. callipygius, and A. leucopygius confirmed the occurrence of multivalent chromosome pairing, as described in A. albofrenatus and A. arildae[9]. While in our sample of A. arildae and A. callipygius a clear tetravalent pairing was seen, in A. leucopygius the tetravalent figure was not characteristic, because the involved chromosomes formed two recognizable bivalents. In all these three species, the chromosomes of the largest pair were involved in the tetravalent.
In vertebrates, including frogs [37–40], rings or chains of meiotic multivalents have been reported. The most illustrative case among animals was described in Ornithorhynchus anatinus[41], in which the multivalent formation was attributed to sequential reciprocal translocations. The same occurred in one specimen of the frogs Haddadus binotatus[39] and Leptodactylus pentadactylus[40], which presented meiotic chain and several odd heteromorphic chromosomes in their karyotypes.
In our study there was no evidence of reciprocal translocation to explain the tetravalent formation, unless it involved minute segments, not detected by the used banding techniques. Another explanation would be the non-chiasmatic ectopic pairing between terminal repetitive sequences of non-homologous chromosomes, proposed by Schmid et al. [12] as a reasonable alternative for similar cases described in the literature [9, 37, 38]. Our data gave no support to any of these hypothesis.
Our cytogenetic analysis on Aplastodiscus and the comprehensive comparative analysis allowed us to consider the following possible homeologies: chromosomes 1, 4, and 5 of A. perviridis, A. arildae, A. callipygius, and A. leucopygius; the chromosome 2 of A. perviridis, A. callipygius, and A. leucopygius with the chromosome 3 of A. arildae and A. eugenioi; the chromosomes 3 of A. perviridis, A. callipygius, and A. leucopygius; the chromosomes 6 and 11 of A. perviridis, A. arildae, and A. eugenioi with the chromosomes 8 and 9, respectively, of A. callipygius and A. leucopygius; the chromosomes 7, 8, 9, and 10 of A. perviridis, A. arildae, and A. eugenioi; and the chromosome 12 of A. perviridis with the chromosome 10 of A. callipygius. The corresponding chromosome 2 of A. arildae and A. eugenioi, and the chromosomes 6 and 7 of A. callipygius and A. leucopygius were interpreted as resulted of rearrangement. Based on these presumed data, the chromosome evolution in the genus Aplastodiscus from an ancestor with 2n = 24 was outlined. Nevertheless, two evolutionary pathways were proposed: one involving two fusions events, in which participate the small elements 7, 8, 9, and 10, giving rise to two new large-sized pairs 6 and 7, as in the karyotype with 2n = 20 of A. callipygius and with 2n = 18 of A. leucopygius; and the other, fusion involving the small chromosome 12 and the large chromosome 3, giving rise to the metacentric pair 2, as in the karyotypes with 2n = 22 of A. arildae and A. eugenioi. This hypothesis is supported by our present cytogenetic data, but undoubtedly, other resolute approaches (e.g., chromosome painting, gene linkage, among others) are still necessary in order to confirm the chromosome evolution within the genus Aplastodiscus.
Another achievement of the present study was the confirmation, by means of chromosome analysis, of the relationships among species or species groups of Aplastodiscus, as shown in the adapted phylogenetic tree based in Wiens et al. [6], and shown in Figure 7B. Including the known diploid numbers of all karyotyped species, the two pathways in the chromosome evolution were well visualised, and the cytogenetic data gave support to the molecular phylogeny and distribution of the species in the known groupings. Certainly, further species sampling, especially of those that have never been karyotyped, will be of great interest to confirm or not the relationships within the genus Aplastodiscus.