﻿First karyotype description of the species of Adenomera Steindachner, 1867 (Anura, Leptodactylidae) in the “ thomei” clade

﻿Abstract The genus Adenomera Steindachner, 1867 currently comprises 29 nominal species, some of which are suggested to be cryptic species complexes. The present study was carried out with specimens of the “thomei” clade that encompasses three taxa distributed in the Atlantic Forest biome: Adenomerathomei Almeida et Angulo, 2006, Adenomera sp. L., and Adenomera sp. M. We used classical cytogenetics to describe the diploid number and karyomorphology of these three species collected in two different locations in the state of Bahia, Brazil. Our results revealed the diploid number 2n = 24 (FN = 34) with two pairs of metacentric chromosomes (pairs 1 and 5), three pairs of submetacentric chromosomes (pairs 2, 3, and 4), and seven pairs of telocentric chromosomes (pairs 6, 7, 8, 9, 10, 11, and 12). Further morphological, bioacoustic, and cytogenetic data (C-banding and AgNor) are needed to better delineate the lineages within the “thomei” clade.


Introduction
The genus Adenomera Steindachner, 1867 currently comprises 29 described species that are distributed from tropical South America to the east of the Andean region (Carvalho et al. 2021). Due to the history of systematic reviews and the complex taxonomy of this group, taxonomic knowledge has not kept pace with the knowledge on its phylogeny (Duellman 2005;Menin et al. 2008;Fouquet et al. 2014). Out of the several hurdles for taxonomic studies on this genus, we highlight the high intra and interspecific similarities and the presence of cryptic species complexes (Fouquet et al. 2014). The difficulty increases when studies use only molecular data, disregarding other characteristics and making the interpretation of results less accurate (Pyron and Wiens 2011;De Sá et al. 2014).
Cytogenetic studies on the genus Adenomera date from the 1970s (Bogart 1970(Bogart , 1974 when the karyotypes of Adenomera andreae (Müller, 1923), Adenomera hylaedactyla (Cope, 1868), Adenomera lutzi Heyer, 1975, and Adenomera marmorata (Steindachner, 1867) were described. However, the volume of cytogenetic information for the genus has not significantly advanced over these five decades. Campos et al. (2009) described the karyotypes of individuals from western São Paulo associating them with the nominal species Adenomera aff. bokermanni Heyer, 1973, A. hylaedactyla, and A. marmorata. Additionally, the karyotype of Adenomera diptyx (Boettger, 1885) was described by Zaracho and Hernando (2011). Thus, there is cytogenetic information for only five species among the 29 species described for this genus. Therefore, the small number of described karyotypes makes it difficult to both understand the chromosomal evolution of the genus and to better delimit species (Campos et al. 2009;Zaracho and Hernando 2011).
Among the clades within the genus Adenomera, the species of the thomei clade, Adenomera thomei Almeida and Angulo (2006), Adenomera sp. L, and Adenomera sp. M, are restricted to the Atlantic Forest in Brazil. Adenomera thomei was described from specimens collected in a cocoa plantation in the municipality of Linhares in the state of Espírito Santo (Almeida and Angulo 2006). Currently, there are records of this species also in the states of Rio de Janeiro, São Paulo, Minas Gerais, and Bahia (Almeida and Angulo 2006;Fouquet et al. 2014). The specific boundaries among these lineages are unclear mainly due to the lack of information on Adenomera sp. L and Adenomera sp. M, both of which are found only in the southern region of the state of Bahia (Fouquet et al. 2014). Knowledge on the bioacoustics, morphology, and cytogenetics for representatives of this clade is scarce (Angulo et al. 2003;Angulo 2004;Duellman 2005) and thus far it has not been used to distinguish between these two lineages.
Karyotypic information associated to DNA sequence data has helped clarify the taxonomy and systematics of some Brazilian anuran groups (Lourenço et al. 2008;Targueta et al. 2010;Suárez et al. 2013;Lourenço et al. 2015;Ferro et al. 2018;Marciano-Jr et al. 2021). To date, all information available regarding cytogenetic data within Adenomera populations is taxonomically inconclusive (e.g., Campos 2009). Nevertheless, these chromosome data provided support on taxonomic decisions on a broad study of species delimitation of Adenomera marmorata, which included DNA sequence, morphological, and bioacoustic data (Cassini et al. 2020). Thus, it is clear that further cytogenetic studies on the genus Adenomera will allow more robust conclusions regarding this taxonomically challenging group. The objective of this study was to describe for the first time the karyotype of Adenomera species of the "thomei" clade from different locations in southern Bahia and compare the chromosomal patterns among the specimens.

Material and methods
Cytogenetic analysis was performed using 12 specimens of two species in the "thomei" clade collected in three sites in the state of Bahia (BA) ( Table 1) under the SISBIO license 62181. The specimens were taken to the Tropical Herpetology Laboratory at the Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brazil. We identified the specimens collected in the municipality of Ilhéus as Adenomera cf. thomei, since the bioacoustic data showed the same pattern as that recorded for populations in the "thomei" clade. We followed the protocol of Schmid (1978) with modifications. In the present study, a 2% colchicine solution (0.1 ml/10 g of weight) was used during 4-6 h. Subsequently, the specimens were sacrificed with lidocaine gel at a concentration of 5% spread over the entire body. The vouchers were fixed in 10% formaldehyde for 24 hours, kept in 70% alcohol, and deposited at the UESC Herpetological collection.
Chromosomal preparations were obtained from intestinal cells. The intestinal epithelium was kept in a hypotonic solution (0.075 M KCL) for 40 minutes and fixed in CARNOY solution (3:1 methanol: acetic acid). Then, the cell suspensions were placed on the surface of a slide and dried at room temperature in the dark. To determine chromosome composition and the fundamental number (FN), cells were stained with 3% Giemsa for 10 minutes. Chromosomes were classified according to Green and Sessions (1991) as metacentric (M), submetacentric (SM), subtelocentric (ST), and telocentric (T) ( Table 2).
Results obtained were compared with cytogenetic data available in the literature. The images were captured and analyzed using an Olympus BX-51 microscope, a Q-Capture Pro image capture camera, and the Image Pro Plus software. We used Adobe Photoshop CC 2019 for the analysis and arrangement of the karyotype in descending order.
The karyotypes obtained in this study and those already published for the genus Adenomera are shown in Table 2 with their respective diploid number, fundamental number, and karyomorphology.

Discussion
The number of cytogenetic studies on anurans has grown in recent years (e.g., Ferro et al. 2018;Gazoni et al. 2018;Marciano-Jr et al. 2021); however, information for some families and/or genera is still scarce. The genus Adenomera comprises common and abundant species, some of which often occur syntopically (Cassini et al. 2020), but cytogenetic data for the entire genus are still scant compared to other anuran genera. So far, only six of the 29 described species have been karyotyped. Campos (2009) analyzed four populations of Adenomera from the state of São Paulo and identified two species, Adenomera marmorata and Adenomera aff. bokermanni with distinct karyotypes. Adenomera marmorata shows a variation in chromosome pair 12, which is metacentric in the populations of the state of São Paulo. Thus, Campos et al. (2009) hypothesized that it is an interpopulation variation, which was later confirmed by Cassini et al. (2020) in a taxonomic study on the group that integrated DNA sequences, morphology, and bioacoustics. The specimen identified by Campos et al. (2009) as A. aff bokermanni was collected in the municipality of Santa Branca in the state of São Paulo, which is outside the current distribution of A. bokermanni, which is restricted to the southern region of the state of Paraná (Cassini et al. 2020).
The specimens analyzed in the present study were cytogenetically similar to those of A. marmorata and A. aff. bokermanni (Campos et al. 2009). Campos et al. (2009) found an unusual diploid number (2n = 23) when they described the karyotype of A. aff. bokermanni, Voucher -CFBH 11531, and concluded that it was most likely an indicative of a centric fusion involving the telocentric chromosome pairs 7 and 9. The authors Figure 1. Karyotype of "thomei" clade specimens with conventional Giemsa staining a Adenomera cf. thomei from Ilhéus, Bahia, Brazil b, c Adenomera sp. L from Igrapiúna, Bahia, Brazil. All specimens showed the following karyomorphology: pairs 1 and 5 metacentric, 2-4 submetacentric, and 7-2 telocentric. Scale bar: 5 μm. stated that it is not possible to determine with certainty whether the differences in chromosome pairs 7 and 9 correspond to a variation restricted to the specimen analyzed. Therefore, chromosome pairs 7 and 9 will not be used for comparison in our analyses. The chromosomes of pair 8 in the specimens analyzed in the present study are telocentric, whereas those of A. aff. bokermanni are subtelocentric (Campos et al. 2009).
Furthermore, the specimens of Adenomera cf. thomei (Ilhéus, BA) and Adenomera sp. L (Igrapiúna, BA) in the present study showed a karyotype (2n = 24 -FN = 34) identical to that of the specimen CFBH1512 from Santa Branca (SP) and the specimen CFBH 1713 (Adenomera sp. J). Moreover, no bioacoustic, molecular (DNA), or morphological data are available for the Ilhéus population and a taxonomic review including all species within the clade is needed to shed light on their specific limits.
Comparative cytogenetics can be considered an important tool for recovering phylogenetic relationships and confirming taxonomic identity (e.g., Baker 1970;Silva et al. 2004;Aguiar Jr et al. 2007;Urdampilleta et al. 2013;Cassini et al. 2020). The results presented here will contribute to expand the information on the taxonomy and phylogeny of the "thomei" clade and consequently lead to the delimitation of its taxa.