Karyotypic diversity in seven Amazonian anurans in the genus Hypsiboas (family Hylidae)

Background Hypsiboas species have been divided into seven groups using morphological and genetic characters, but for most of the species, there is no cytogenetic information available. A cytogenetic analysis using conventional staining, C-banding, silver staining, and fluorescence in situ hybridization (FISH) with telomeric sequence probes were used to investigate the karyotype of seven Amazon species of the genus Hypsiboas belonging to the following intrageneric groups: H. punctatus (H. cinerascens), H. semilineatus (H. boans, H. geographicus, and H. wavrini), and H. albopunctatus (H. lanciformis, H. multifasciatus, and H. raniceps). The aim was to differentiate between the karyotypes and use the chromosomal markers to distinguish between the Hypsiboas groups. The data were compared with a previous phylogenetic proposal for these anurans. In addition, H. lanciformis, H. boans, and H. wavrini are described here for the first time, and we characterize the diploid numbers for H. cinerascens, H. geographicus, H. multifasciatus, and H. raniceps. Results The diploid number for all of the species analyzed was 24, with the exception of Hypsiboas lanciformis, which had 2n = 22 chromosomes. The constitutive heterochromatin distribution, nucleolar organizer region locations, and interstitial telomeric sites differed between the species. A hypothesis that the heterochromatic patterns are evolving is proposed, with the divergence of the groups probably involving events such as an increase in the heterochromatin in the species of the H. semilineatus group. The FISH conducted with the telomeric probes detected sites in the terminal regions of all of the chromosomes of all species. Interstitial telomeric sites were detected in three species belonging to the H. semilineatus group: H. boans, H. geographicus, and H. wavrini. Conclusion The results of this study reinforce the complexity previously observed within the genus Hypsiboas and in the different groups that compose this taxon. More studies are needed focusing on this group and covering larger sampling areas, especially in the Brazilian Amazon, to improve our understanding of this fascinating and complex group.


Background
Hylidae is considered the most diverse family among the anurans, with 936 described species [1], of which about 90 are found in the Brazilian Amazon [2]. Recent cytogenetic studies of species from this family have demonstrated intrapopulational variation, with polymorphisms of the nucleolar organizer regions (NORs) [3], different diploid numbers in the same nominal species [4,5] and intra-generic variations such as the localization of the NORs among species [6].
Based on a compilation of cytogenetic data for the hylids, the majority of the species had a diploid number of 26 [7], although some genus such as the Hypsiboas spp. showed reductions, with the majority having 2n = 24 chromosomes [8][9][10][11][12]. Despite the conserved constant diploid number found in Hypsiboas spp., the karyotypic organization of the species cannot be considered conserved (Table 1)  .
Regarding the karyotypic descriptions available for the species composing the H. albopunctatus group, there is some degree of confusion about the names adopted for the different taxa, resulting in divergences of the cytogenetic information available. Only H. albopunctatus, H. fasciatus, and H. raniceps have been karyotyped [4,[9][10][11]15,31] even though reportedly there was no cytogenetic data available for H. fasciatus [31]. H. multifasciatus was cytogenetically described for the first time by Beçak [31]. However, Beçak [9] did not describe H. multifasciatus, but rather H. bischoffi, which belongs to the H. pulchellus group and is similar to H. multilineatus [34]. This indicates that the H. albopunctatus group may comprise species complexes [34], which would explain why different cytotypes have been described for the same species-such as H. multifasciatus from the states of Amazonas and Goiás in Brazil [12]. A similar situation was observed in H. raniceps, which has been described as having three distinct karyotypic formulas among the individuals encountered in the states of Mato Grosso and Goiás in central western Brazil [4,11], with one additional formula from Amazonas in northern Brazil.
There are both inter-and intraspecific variations in the chromosome formulas in the positions of their nucleolus organizer regions (NORs) and in the distribution of the constitutive heterochromatin [4,8,9,[16][17][18]24,25,27]. Additionally, the species H. albopunctatus demonstrates a reduction in the diploid number, having 2n = 22 chromosomes in addition to the presence of a B chromosome [4,12]. The karyotypic patterns of organization are not established for the groups, and it is impossible to know if there are any cytogenetic features that characterize the Hypsiboas groups or if a concordance between the phylogenetic proposal and the chromosomal patterns

Telomeric sequence mapping
Combining telomeric probes with fluorescence in situ hybridization (FISH) detected sites in the terminal  (Figures 4a and c, respectively), the ITSs were seen on the short arms of both homologs of pair 2 and on the long arms of both homologs of pair 5. The centromeric ITSs in H. geographicus were seen on both homologs of pairs 1 and 5; the ITSs on pair 1 correspond with the NOR sites in this species (Figure 4b). However, ITSs were not found in H. cinerascens, H. lanciformis, H. multifasciatus and H. raniceps.
The diploid number of 22 can be seen in H. albopunctatus [4,9] and H. lanciformis [present work], both of which are in the H. albopunctatus group. Based on the chromosome data of H. albopunctatus, it is possible that an end-to-end fusion occurred in the Hypsiboas ancestral (2n = 24) involving small chromosomes, probably chromosome pairs 11 (NOR region) and 12, because they are similar in length [4]. The same may also have occurred in H. lanciformis, explaining the evolution of the karyotype of this species with its reduced diploid number.
ITSs are repetitive sequences, which can derive from chromosomal rearrangements (centric fusion, in tandem fusion, or inversion) during vertebrate karyotype evolution [38,39] representing the remaining sequences in newly formed chromosomes. Alternatively, ITSs can also result from the amplification of telomeric sequences, be the result of unequal crossing-over and transposition, be sequences introduced by a telomerase error, or be the result of integration between transposons and telomeric sequences [37,38]. ITSs have been found in hylid frogs and in some species of Hyla in North America such as H. chrysoscelis and H. versicolor; these have been attributed to unequal crossing-over during meiosis, submicroscopic deletions, and differential amplifications [37,40]. Telomeric sequences commonly occur outside of the terminal regions in the Hylidae family [37,38] and are present in H. boans, H. geographicus, and H. wavrini belong to H. semilineatus group. These sequences may be the result of chromosomal rearrangements and represent the remains of sequences in newly formed chromosomes, or they may be due to integration between transposons and telomeric sequences as has been observed in other species [37,38]. There is no consensus on the presence or absence of ITSs and their relation to chromosomal rearrangements, because many factors may be involved [41]. However, no ITSs were found in the other four Hypsiboas species analyzed in this study, suggesting the answer may be selection by an unknown agent that may not alter their fitness [37].
The absence of the ITSs in most Hypsiboas species does not necessarily indicate that the hypothesis that Hypsiboas species were derived from a common ancestor with 26 chromosomes is incorrect. Chromosomes derived from fusion events may have small telomeric sites that cannot be easily detected by FISH, or the telomeres could be lost before the fusion or eroded by molecular processes [42]. As there has been no alteration in the basal diploid number of Hypsiboas, the ITSs observed in the chromosomes of H. boans, H. geographicus, and H. wavrini probably reflect non-Robertsonian rearrangements, given that that these three species are not found in the basal group.
Hypsiboas cinerascens (previously Hyla granosa within the Hyla granosa group) belongs to the Hypsiboas punctatus group (monophyletic group fusion between the Hyla punctata and Hyla granosa groups) [13] and displays the basal cytogenetic characteristics of the Hypsiboas, including a diploid number of 24, poorly visible constitutive heterochromatin, and an active NOR on a single chromosome pair [14, present work]. A detailed comparison between the karyotypic patterns of the Hypsiboas punctatus group was not possible, because the data was restricted to the Hypsiboas punctatus diploid number of 24 [14,21,29,30].
The species H. boans, H. geographicus, and H. wavrini (H. semilineatus group) show processes of heterochromatin accumulation or heterochromatization [43][44][45] during their evolution, a characteristic that distinguishes these species from the others in the group. Additionally, H. boans and H. wavrini are phylogenetically related, with similar patterns of constitutive heterochromatin distribution, and the number and localization of the NORs and ITSs. The proximity between H. wavrini and H. boans can also be seen in their morphological and reproductive similarities, which makes it difficult to differentiate between these species in the field [46,47]. Both species have been found in sympatry in Colombia, and though they occupy identical niches, they differ in their vocalization and reproductive periods [48]. However, the karyotype formulas differ between H. boans and H. wavrini, allowing them to be differentiated [present work].
Almost 90% of speciation events are accompanied by chromosome changes [42]. NORs are considered excellent markers in karyotype evolution studies in amphibians [49,50], despite the occurrence of rare variations within the species [51]. Most anuran genera have heteromorphic NORs, and the differences in their size may be due to in tandem duplication or triplication, which can affect one or both DNAr clusters [49]. The duplicated NORs found in H. albomarginatus may have resulted from differential gene activity or be a duplication by mobile elements [47]. In one study, three of four species (H. albomarginatus, H. semilineatus, and H. pardalis) had heteromorphic NORs [17].
Hypsiboas geographicus (H. semilineatus group) had NORs present on the centromeric region of pair 1, while in the other species of this group, the NORs were present in another chromosomes pair, such as pair 11 in H. semilineatus [17]. In H. geographicus, the NOR is in same region as the ITS, and it is possible that both structures are associated with different satellite/repetitive DNA classes, because they are in the centromeric region [52]. Despite some authors being unable to find an association between the NORs (specifically 18S rDNA) and ITSs [37], this would explain the amplification of those telomeric sequences in the interstitial region of the chromosomes. However, the silver nitrate impregnation technique only identifies active sites [53], meaning the possibility of multiple ribosomal sites in these groups or variable chromosomal localization among the species cannot be eliminated. Multiple NOR active sites were observed in H. lanciformis [present work] and H. raniceps [4] (both in the H. albopunctatus group) [13], as well as in H. prasinus (H. pulchellus group) [9,23,27]. The hybridization of 45S ribosomal DNA probes only in H. albopunctatus and H. pardalis indicated the presence of one labeled chromosome pair [14,17]. Since silver associates with nucleolar proteins involved in the transcriptional activity of ribosomal genes from the 45S rDNA cistrons [51,52] and can also impregnate heterochromatic regions rich in acidic residues [54], the multiple NORs present in H. lanciformis and H. raniceps may be indicating a heterochromatic region. However, the position of the NORs varies among the species, and it is possible that the ribosomal genes are changing during the karyotypic evolutionary process [17,48,49]. Despite some authors [17] finding that each monophyletic clade in the Hylidae phylogenetic tree [13] had the ribosomal cistron located in a specific chromosome pair (based on the NOR data), there may be no typical pattern for each group, with the presence of both simple and multiple NORs among the species of those taxa (Table 1).
In addition to the differences in the number and localization of the NORs, two different diploid numbers and different karyotypic formulas were seen in the H. albopunctatus group. As such, in spite of the fact that H. albopunctatus has 2n = 22 chromosomes, other species of the group have a diploid number of 24 [4,9,10,12,15], and this reduction to 22 is not a true characteristic of the group. In addition, despite the decrease to 2n = 22 chromosomes in H. lanciformis, no ITSs were encountered in that species, possibly due to genetic erosion of those sequences. Given that H. lanciformis is typically found in forest fragments and along forest edges [55][56][57] where it would be more susceptible to anthropogenic interactions such as water contamination, which can cause several diseases [58], the lack of ITSs could also be due to selection by an unknown agent that does not prejudice the development of the species [37,41]. These same forces may also be acting on H. albopunctatus, which frequently occurs in disturbed areas [57]; in addition to having 22 chromosome pairs, many individuals of this species have supernumerary chromosomes [4].
Both species, Hypsiboas lanciformis [present work] and H. albopunctatus [4,9], had a reduced chromosome number (2n = 22) relative to their co-generic species (2n = 24) in a phylogenetic tree of the family Hylidae [13]. However, looking at the diploid number data plotted for this tree, it is apparent that chromosome number can either occur independently in these species, is related to their natural history, or is a species characteristic [43]. Thus, they have the same common ancestor, but are grouped in different clades [13].
Different patterns of distribution of the heterochromatin were found in the H. albopunctatus group. H. lanciformis had heterochromatic blocks in the centromeric region of most of its chromosomes [present work], as did H. albopunctatus [4,12]. H. raniceps and H. multifasciatus showed weak heterochromatic blocks distributed in only a few pairs of chromosomes. In addition, there were clear differences in the distribution patterns of heterochromatin among the populations of H. raniceps such as between the individuals from the northern and central regions of Brazil [4,10,11,15]. The variation in the quantity and distribution of the constitutive heterochromatin is an important characteristic that can be used to differentiate between populations based on an epigenetic mechanism [59]. Additionally, heterochromatin is normally rich in repetitive sequences that may have important roles in speciation and/or adaptation, as they are less subject to selective pressurewhich favors the accumulation of differences during evolutive processes [44,60,61].
Differences in genome size are primarily due to events of heterochromatin addition or deletion involving DNA satellite families [62]. The DNA content was 6.61 pg/N for H. lanciformis, while those of H. cinerascens (synonym of Hyla granosa) and Hypsiboas geographicus were 4.53 pg/N and 3.28 pg/N [45], respectively. Heterochromatin was present in the centromeric region in most of the chromosomes of both H. lanciformis and H. cinerascens, but like the DNA content, the quantity of heterochromatin was different relative to others such as H. lanciformis, which had more heterochromatic blocks and a higher DNA content. However, when the C-banding patterns of H. lanciformis and H. geographicus were compared, their heterochromatin was similar, despite the difference in their DNA content. Recent work has demonstrated epigenetic influences on the pattern of heterochromatin distribution in chromosomes [59,63] that could explain the absence of a correlation between the high DNA content and more heterochromatic blocks found in H. geographicus and the number and localization of NORs and ITSs.

Conclusion
The data presented in this study reinforces the complexity previously observed within the genus Hypsiboas and in the different groups that compose this taxon. More studies focusing on this group and covering larger sampling areas, especially in the Brazilian Amazon, are needed to gain a better understanding of this fascinating, but complex group.

Chromosomal analyses
Mitotic chromosomes were obtained from the bone marrow and liver of the hylids after in vitro (1%) and in vivo (0.1%) colchicine exposure [64,65]. Cell division was induced in some specimens by injecting them with biological yeast (0.1 mL per 10 g of animal bodyweight) and maintaining them alive for 48-72 h [35,66].

Classical and molecular cytogenetic analysis
The cell suspensions were analyzed after routine staining with conventional stain (10%), C-banding [54], Ag-NOR staining [53], and FISH [67] with a telomeric probe. The FISH telomeric probe was digoxigenin-labelled by a nick translation reaction using a Roche TM kit and amplified by polymerase chain reactions as previously described [68]. Chromosomes were organized by decreasing size, and the morphology was determined based on the centromere position [69]. Fundamental numbers were determined by conventional staining at metaphase and by exposure to barium hydroxide.
Abbreviations FISH: Fluorescence in situ hybridization; ITS: Interstitial telomeric site; NOR: Nucleolar organizer region.