First cytogenetic information for Drymoreomys albimaculatus (Rodentia, Cricetidae), a recently described genus from Brazilian Atlantic Forest

Abstract The recently described taxon Drymoreomys albimaculatus is endemic to the Brazilian Atlantic Forest and its biology and genetics are still poorly known. Herein, we present, for the first time, the karyotype of the species using classical and molecular cytogenetics, which showed 2n=62, FN=62, and interstitial telomeric signals at the sex chromosomes. Nuclear and mitochondrial DNA sequences from the two karyotyped individuals verify the taxonomic identity as the recently described Drymoreomys albimaculatus and confirm the relationship of the species with other Oryzomyini. Additionally, external morphological information is provided.

Recently, Percequillo et al. (2011) described Drymoreomys albimaculatus as a new monotypic genus, endemic to the Brazilian Atlantic Forest and known from a few localities in São Paulo and Santa Catarina states. Phylogenetic analyses based on morphological traits and DNA sequences [1143bp of cytochrome b (Cyt b) and 1235bp of interphotoreceptor retinoid binding protein (IRBP) genes] revealed the placement of D. albimaculatus in the tribe Oryzomyini, raising to 30 the number of extant Oryzomyini genera. According to those analyses, Percequillo et al. (2011) revealed that D. albimaculatus is the sister species of the Andean rat Eremoryzomys polius.
Here, we describe the karyotype of D. albimaculatus for the first time. In order to investigate the molecular identification of the two karyotyped animals, we added its Cyt b and IRBP sequences to the molecular data published by Percequillo et al. (2011). Additionally, we present morphological comments on the specimens.

Sampling
One male and one female were collected with pitfall traps in Santa Virgínia, Parque Estadual da Serra do Mar [45°03.00' to 45°11.00'W (DDM); 23°24.00' to 23°17.00'S (DDM)], state of São Paulo, Brazil. Pelage color and external measurements were taken during the fieldwork. Vouchers of both individuals are deposited in the Coleção de Mamíferos da Universidade Federal do Espírito Santo (UFES) under the catalog numbers UFES 2271 and UFES 2272.

Cytogenetic analyses
Metaphases were obtained in vivo from spleen and bone marrow, according to Ford and Hamerton (1956) with modifications. Conventional Giemsa staining was used to determine the diploid (2n) and the number of autosome arms (FN). GTG and CBG-banding were performed according to Seabright (1971) and Sumner (1972), respectively, with modifications. Fluorescent in situ hybridization (FISH) with a FITC labeled (C 3 TA 2 ) n peptide nucleic acid (PNA) probe (DAKO) was carried out following the recommended protocol (Telomere PNA FISH Kit/FITC, Code No. K5325, DAKO). Mitotic plates were digitally captured with visible light or blue and green filters (emission at 461 and 517 nm, respectively) in an Axioskop 40 epifluorescence microscope (Carl Zeiss) equipped with an Axiocam camera and AxionVision software. Images were overlaid and contrast enhanced with Adobe Photoshop CS5.1.

DNA extraction, amplification, and sequencing
DNA was extracted from liver with Chelex 5% (Bio-Rad) following Walsh et al. (1991). Amplification of an 820 bp fragment of Cyt b and a 782 bp of IRBP was performed with PCR using primers MVZ5 and MVZ16 (Irwin et al. 1991;Smith and Patton 1993), and A1 and F (Stanhope et al. 1992), respectively. Both extraction and PCR controls were used for each amplification. Each PCR mixture had 30 ng of DNA, 25 pmol of each primer, 0.2 mM of dNTP, and 2.52 µL of reaction buffer (50 mM KCl, 2.5 mM MgCl2, 10 mM Tris-HCl; pH 8.8), and 0.2 units of Taq DNA polymerase (Invitrogen) were added to complete 18 µL. Forty amplification cycles were performed in a thermal cycler (Eppendorf Mastercycler ep Gradient, Model 5341). Each cycle consisted of denaturation at 94°C for 30 s, annealing at 48°C for 45 s, and extension at 72°C for 45 s for Cyt b, and denaturation at 94°C for 30 s, annealing at 60°C for 60 s, and extension at 72°C for 180 s for IRBP. A final extension at 72°C for 5 min was performed for both Cyt b and IRBP amplifications. The PCR products were separated using 1% agarose gels in TAE buffer. Nucleotide sequencing was conducted using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Sequences of each animal were aligned with sequences from previously published data deposited on GenBank by Bonvicino and Moreira (2001), Weksler (2003), and Percequillo et al. (2011) using MAFFT ver. 5 (Katoh et al. 2005 under the iterative method of global pairwise alignment (G-INS-i). Our sequences were submitted to GenBank under accession numbers KF031014-KF031017.

Phylogenetic analyses
We performed maximum likelihood (ML) and Bayesian analyses using concatenated Cyt b-IRBP data set. For both analyses we used gene-specific unlinked models. The best-fitting model of nucleotide substitution for each gene was selected using the Akaike information criterion in accordance with the procedure outlined by Posada and Buckley (2004), and implemented in jModelTest, version 0.1.1 (Posada 2008). The maximum-likelihood trees were calculated using RAxML (Stamatakis 2006). The statistical support for the nodes was estimated by the nonparametric bootstrap, with 1000 pseudoreplicates (Felsenstein 1985). Bayesian analysis was performed using Mr-Bayes 3.04b (Ronquist and Huelsenbeck 2003). Markov chains were started from a random tree and run for 1.0 x 10 7 generations, sampling every 1000th generation. The stationary phase was checked following Nylander et al. (2004). Sample points prior to the plateau phase were discarded as burn-in, and the remaining trees were combined to find the maximum a posteriori probability estimated of the phylogeny. Branch support was estimated by Bayesian posterior probabilities (BPP). Two simultaneous analyses were performed to ensure convergence on topologies.

Cytogenetic analyses
The animals showed 2n=62, FN=62, and the autosome set composed of 29 acrocentric pairs decreasing in size, and one small metacentric pair (Fig. 1A). The X is a large submetacentric, and the Y is a large submetacentric slightly smaller than the X (Fig. 1A). CBG-banding revealed pericentromeric constitutive heterochromatic blocks in all autosomes and in the long arm of Y (Fig. 1B). GTG-banding allowed the identification of almost all autosomic pairs, the X chromosome exhibited two interstitial bands at the long arm while a conspicuous pattern in the Y was not found (Fig. 1C). FISH detected telomeric signals at the ends of all chromosomes and additional telomeric sequences were found in the pericentromeric region of both X and Y chromosomes (Fig. 1D).

Phylogenetic analyses
The model selected for the phylogenetic analyses (ML and Bayesian) was GTR +I + Γ for each gene. The best ML tree had a -ln likelihood score of -22,345.02. The Bayesian analysis recovered a consensus topology similar to the best ML tree and the results recovered the four well-supported clades A, B, C, and D (Fig. 2) previously reported  Table 1), although the lowest diploid number (Nectomys palmipes, 2n= 16, Barros et al. 1992) does not appear in the figure. by Weksler (2006) and Percequillo et al. (2011). In both phylogenetic analyses, Santa Virgínia specimens (UFES 2271 and UFES 2272) clustered with high statistical support to the recently described D. albimaculatus (Fig. 2, grey area).

Discussion
Phylogenetic analyses (ML and Bayesian) recovered the four clades A, B, C, and D (Fig.  2) recovered by Weksler (2006) and Percequillo et al. (2011). In both phylogenetic reconstructions, Santa Virgínia specimens were recovered with high statistical support in clade D, confirming their identity as D. albimaculatus (Fig. 2, grey area), and consistent with Percequillo et al. (2011). Our analyses also recovered D. albimaculatus as the sister species of Eremoryzomys polius and both species diverged early in the clade D (Fig. 2).
The diploid number of D. albimaculatus corroborates the pattern found for the majority of the Oryzomyini species, in which karyotypes present relatively high chromosome number and predominantly acrocentric pairs. The typical heterochromatic pattern of sex chromosomes is also found in most of the oryzomyine species and it is an essential condition for the recognition of the Y (Fig. 1B).
A compilation of karyological studies in representative species of clade D is presented in Table 1. Notably, cytogenetic data in Oryzomyini has increased considerably in the last decades, mainly because the karyotype has become a valid tool for identifying species of this group. Although several species still remain without karyotypic information (e.g., Eremoryzomys polius, Tanyuromys aphrastus, Nesoryzomys swarthy), the diploid number within clade D varies from 16 in Nectomys palmipes (Barros et al. 1992) to 62 in D. albimaculatus. As D. albimaculatus exhibited the highest diploid number reported hitherto and diverged early in clade D, karyotype evolution in this clade based on the phylogeny (Fig. 2), apparently exhibits a trend toward a decrease in the diploid number. This hypothesis could imply chromosomal plasticity in low 2n ratios as suggested by Gardner and Patton (1976). In this sense, tandem fusions have perhaps played significant role in clade D, resulting in the lower diploid numbers. Robersonian rearrangements could have occurred in this group as well, since some species of clade  Table 1). Non-Robertsonian mechanisms such as pericentric inversions, unequal translocations, or whole-arm heterochromatin addition or deletion could also be invoked in those cases of changes in FN but not in 2n (e.g., Sigmodontomys alfari, and Melanomys caliginous, Table 1).
Species of clade D present sex and supernumerary chromosomes easily identifiable with classical cytogenetic approaches, and some species exhibit sex chromosomes with polymorphisms/heteromorphisms and interstitial telomeric signals (ITS; Table 1, Fig.  1D). Thus, this clade is an excellent model to study origin, evolution, and chromatin composition of these chromosomes. For instance, a superficial morphological comparison among sex chromosomes from Table 1 could suggest the occurrence of pericentric inversions, or whole-arm heterochromatin additions or deletions.

Comments on external morphology and natural history
The specimens collected were medium sized (male body mass: 46.5 g, head and body length: 115 mm, and tail length: 142 mm; female body mass: 57 g, head and body length: 127 mm, and tail length: 170 mm). Tail was longer than head and body, and was a uniform color on both sides. Male hind foot was short (25 mm, 22% of head and body length) and ears were small (16 mm; 14% of head and body length). These external morphological measures overlapped with those of the Drymoreomys albimaculatus holotype (Percequillo et al. 2011). Dorsal pelage was reddish-brown; ventral pelage was predominantly grayish. Samples exhibited the pattern of short hind feet consistent with Oecomys. Fore and hind feet digits were covered by silvery-white hairs and the dorsal surface of hind feet were covered by brown hairs forming a patch, in a more conspicuous pattern than the observed for Rhipidomys. Thus, some external morphological traits were similar to those described for Rhipidomys and Oecomys as reported Percequillo et al. (2011). Nevertheless, our samples exhibited the characteristics of the D. albimaculatus holotype that differentiate it from Rhipidomys, such as the shorter, thinner, and sparser mystacial vibrissae and presence of gular to pectoral patches of white hair. Additionally, we detected that, contrary to what is found in Rhipidomys, a tuft of hairs on the tail's end is absent in our samples. On the other hand, several anatomical traits that distinguish the Drymoreomys albimaculatus holotype and species of Oecomys were observed in our samples, such as the plantar surface of pes covered with squamae; dorsal surface of pes with dark patches of brown hairs and the ventral pelage with gular and thoracic white patches (Percequillo et al. 2011). Percequillo et al. (2011) reported that most of the Drymoreomys specimens were collected in pitfall traps; in the present work, the animals were also collected in the same way. These reiterate the importance of further fieldwork effort, with different collecting methods in order to increase the spectrum of small mammals collected. Consequently, our knowledge of small mammal biodiversity will be improved as a whole, which will allow improvements in relevant laws and policies for biodiversity protection.