Karyotypic analyses and morphological comments on the endemic and endangered Brazilian painted tree rat Callistomys pictus ( Rodentia , Echimyidae )

The genus Callistomys belongs to the rodent family Echimyidae, subfamily Echimyinae, and its only living representative is Callistomys pictus, a rare and vulnerable endemic species of the state of Bahia, Brazil. Callistomys has been previously classified as Nelomys, Loncheres, Isothrix and Echimys. In this paper we present the karyotype of Callistomys pictus, including CBG and GTG-banding patterns and silver staining of the nucleolus organizer regions (Ag-NORs). Comments on Callistomys pictus morphological traits and a compilation of Echimyinae chromosomal data are also included. Our analyses revealed that Callistomys can be recognized both by its distintinctive morphology and by its karyotype.

Karyotypes of Echimyinae are known only for Pattonomys semivillosus and for some species of Phyllomys, Makalata and Isothrix and their chromosome numbers ranged from 2n = 22 in Isothrix pagurus to 2n = 96 in Phyllomys medius (Table 1).
In a review of the genus Isothrix, Patton and Emmons (1985) followed Cabrera (1961) and Honacki et al. (1982) and kept Nelomys pictus within Isothrix.The same I. pictus was later classified as Nelomys (Emmons and Feer, 1990) and then included in Echimys (Woods, 1993;Emmons and Feer, 1997).Emmons and Vucetich (1998) examined the fossil mandible of one specimen identified as Lasiuromys villosus (a synonym of Isothrix bistriata) by Winge (1888) and several specimens of Nelomys pictus (including the holotype) and concluded that they belonged to the same genus.A comparison of these specimens with the three genera in which Nelomys pictus had been previously included led Emmons and Vucetich (1998) to conclude that N. pictus did not belong to any of them.Considering its distinct morphology, these authors suggested a new genus, Callistomys, to contain N. pictus and Callistomys sp.(formerly identified as Lasiuromys villosus).
Here we report new cytogenetic data on the endemic, rare and endangered Echimyinae Callistomys pictus, including Ag-NOR staining, CBG-and GTG-banding, as well as comments on the external morphology, cranial anatomy and geographical distribution of the species.
The specimen reported herein was incidentally captured in April 2002, preyed and injured by a domestic dog in a cacao plantation at Fazenda Santo Antônio (14°41'46" S, 39°15'22" W), Ilhéus, state of Bahia, Brazil.Ilhéus is the type-locality of Nelomys pictus Pictet, 1843.The animal was sacrificed according to Ethical Issues in the Use of Animals (Colégio Brasileiro de Experimentação Animal, COBEA, 1991), tissues were deposited in the collection of the Instituto de Biociências, Universidade de São Paulo (IBUSP) and the voucher specimen was deposited at the Museu de Zoologia, Universidade de São Paulo (MZUSP), São Paulo, Brazil, under the number MZUSP 31404.
Morphology -We employed Wahlert (1974Wahlert ( , 1983Wahlert ( , 1985) ) and Woods and Howlands (1979) for the nomenclature of cranial foramina.Dental nomenclature followed Iack-Ximenes et al. (2005) which was modified from Lavocat (1976) with further considerations from Butler (1985), Jaeger et al. (1985), Flynn et al. (1986), Jaeger 698Ventura et al. (1989), Bryant andMcKenna (1995), andCandela (1999a;1999b;2002) External morphology: fur includes only soft hairs; hairs brown at base with tip white or black.Pelage is dense and long.Callistomys has a unique color pattern among echimyid rats: body white with black coat, extending from the top of head, nape, and back to the basis of tail.Tail densely haired, black at base and white distally.Limbs broad and stout, hands and feet relatively short but broad.Cranial anatomy: Skull is large and strong, rostrum and nasals medium sized and broad.Lateral wings of frontal well-developed forming a roof over orbital region; postorbital process of zygoma rounded and formed by squamosal.Petrosal bone covered by squamosal posteriorly.Incisive foramina long and fusiform, mostly formed by premaxillar.Septum of incisive foramina wide and long and formed by premaxillar.Palatal region rectangular, long and slender; palatine extending up to M1. Sphenopalatine foramen double.Mesopterygoyd fossae with slit-shaped lateral openings.Alisphenoid region wide; alisphenoid channel not differentiated; buccinator and masticator foramina confluents; foramen ovale medium sized; maxillary vein passes through foramina; transverse canal foramen well-developed.Bullae round, inflated, with tiny stiliform process, tegmen timpani short and wide; external auditory meatus opening in a short and strong tube.Upper molariforms tetralophodonts; anteroloph and the fourth loph (meta-loph+protoloph) connected lingually as well as mesoloph and posteroloph.In young adults and younger specimens hypoflexus and mesoflexus deep, isolates anteroloph protoloph U-shaped from mesoloph posteroloph; but in older ones, the M1-M3 with narrow mure connecting protocone to hypocone.Lower molar trilophodonts and dP4 tetralophodont with metafossetid between first loph (antero-lophid+metalophid) and mesolophid; mesolophid connected by mure to hipolophid; hipolophid and posterolophid connected labially by hipoconid.
Distribution -Callistomys pictus occurs in Bahia State, Brazil, and most records are from the municipality of Ilhéus and nearby areas (Moojen, 1952;Emmons and Vucetich, 1998;Vaz, 2002Vaz, , 2005)).C. pictus was recently recorded in Serra da Jibóia, Elisio Medrado, Bahia State, about 150 km North of Ilhéus (Encarnação et al., 2000).Two specimens were collected by Auguste de Meuron, a tobacco dealer, and the locality was labeled as "Brésil".De Meuron lived in the city of Salvador, formerly known as Bahia, a name now used for the state.In view of the specimens from other species (P.pattoni, T. setosus) sent by de Meuron to the MHNN, it is possible that the specimens of Callistomys pictus came from Salvador (MHNN: 94.2464A and 94.2463) or from somewhere in the neighbourhood.Callistomys pictus is endemic to the Atlantic forest of the state of Bahia, Brazil, where it is also found in cacao plantations shaded by native trees.C. pictus is either locally rare or difficult to capture with traditional live trapping methods, since ecological studies with a high sampling effort carried out in different habitats in South Bahia did not record the species (Pardini, 2004;Moura RT, 1999, MSc Dissertation, Universidade Federal de Minas Gerais, BH, Brazil).Nevertheless, Callistomys pictus seems to be more common in cacao plantations in Ilhéus, where local people reported frequent sightings of the animal.
Cytogenetics -Metaphases from the male of Callistomys pictus were obtained from bone marrow and spleen 40 min after an in vivo subcutaneous injection of 0.1% colchicine.Conventional staining with Giemsa, Ag-NORs staining (Howell and Black, 1980), GTG-(Seabright, 1971) and CBG-banding (Sumner, 1972) were carried out following standard cytogenetic procedures.
The karyotype of Callistomys pictus revealed 2n = 42 and FN (number of autosomal arms) = 76 (Figure 1A) and consisted of 18 pairs of meta or submetacentric autosomes decreasing in size (pair 1 to 16, 18 and 20) and two pairs of small acrocentrics (17 and 19).The X and Y chromosomes were, respectively, medium and small acrocentrics; the X being perfectly distinguishable as a medium sized acrocentric.GBG-banding allowed the recognition of all autosome pairs and the sex chromosomes (Figure 1B).CBGbanding evidenced small heterochromatic blocks in the pericentromeric regions of some autosomes and in the sex chromosomes (Figure 2A).The single Ag-NOR was detected in a secondary constriction at the long arm of pair 13 (Figure 2B).
Callistomys pictus, the only living species of the genus, differs from other extant Echimyidae in many major cranial characters and could represent the last survivor of an old clade of Echimyinae.Other species of this genus represented by a single fossil from the Upper Pleistocene-Recent collected at Lapa do Capão Seco, Lagoa Santa, Minas Gerais State, Brazil, was recognized (Emmons and Vucetich, 1998).A cladistic analysis based on morphological data of Echimyidae placed Callistomys in the basal position of the Dactylomyinae/Echimyinae clade (Carvalho and Salles, 2004).The authors found a  slightly distinct topology when fossil taxa were included: Callistomys, Maruchito trilofodonte Vucetich et al., 1993 (a fossil echimyid genus from Middle Miocene), and the Dactylomyinae/Echimyinae formed a basal politomy.These results suggest that Maruchito and Callistomys can be related, as previously proposed by Emmons and Vucetich (1998).Callistomys occurs in the Bahia State of Brazil and is restricted to the Atlantic Forest in a few localities from Ilhéus to Elísio Medrado.Subfossil specimens from Minas Gerais were collected by Lund and the genus does not seem to presently occur near Lagoa Santa (Emmons and Vucetich, 1998), suggesting a recent reduction in its geographic distribution (Figure 3).Records of Callistomys pictus from Lagoa Santa are restricted to Pleistocene fossil specimens.
The taxonomy of Echimyinae is still very complex and the diploid numbers can be helpful in species identification since karyotypes seem to be important diagnostic markers (Table 1), except for Isothrix bistriata, I. negrensis and Echimys sp. with 2n = 90/92.Diploid numbers within Echimyinae are usually high, with the exception of Isothrix pagurus and I. sinnamariensis, with 2n = 22 and 2n = 28, respectively (Patton and Emmons, 1985;Vié et al., 1996), and of the odd 2n = 42 and FN = 76 described herein for Callistomys pictus.
Our results show that the karyotype associated to the restricted geographical range and the unique set of morphological traits are useful in identifying Callistomys pictus.Our data bring important new information thus reducing the knowledge gap of Brazilian biodiversity and, most importantly, contributing to improve conservation and management initiatives.This is specially important for species like Callistomys pictus, which was included as vulnerable in the red list of endangered species published by the Brazilian Institute for Environment (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, IBAMA) in 2003.In fact, as pointed out by Costa et al. (2005), the major threat to endangered small mammals is the scarcity of scientific knowlegde about their distribution, systematics, taxonomy and natural history, since most of them are rare and poorly known and very few sites of Brazil have been adequately surveyed.
. Besides the specimen from Fazenda Santo Antônio, Ilhéus, state of Bahia, eight other specimens of Callistomys pictus were examined, as follows: Brazil: