Karyotype characterization and nucleolar organizer regions of marsupial species (Didelphidae) from areas of Cerrado and Atlantic Forest in Brazil

The karyotypes of 23 specimens belonging to 16 species from nine genera of Brazilian marsupials (family Didelphidae) were studied. The animals were collected in eight localities of Cerrado or Atlantic Forest biomes in the states of Goiás, Tocantins and São Paulo. The karyotypes were analyzed after conventional Giemsa staining and silver staining of the nucleolus organizer regions (Ag-NORs). New karyotypic data were obtained for Gracilinanus microtarsus(2n = 14, FN = 24),Marmosopspaulensis(2n = 14, FN = 24),Micoreusparaguayanus(2n = 14, FN = 20) and Monodelphis rubida (2n = 18, FN = 32) and are discussed in detail. The karyotypes of G. microtarsus, M. paulensisand M.paraguayanusinclude three large pairs of submetacentrics (pairs 1, 2 and 3) and a medium-sized metacentric or submetacentric pair 4. Pairs 5 and 6 are small submetacentrics in G.microtarsusand M.paulensis and acrocentrics in M. paraguayanus. M. paulensis presented a single Ag-NOR in pair 6 (6p6p), while M. paraguayanusexhibited multiple Ag-NORs in pairs 5 and 6 (5pq5pq6p6p). There was variation in size and morphology of the sex chromosomes among these species. Monodelphisrubidapresented a karyotype with 2n = 18 and FN = 32 composed of a large submetacentric pair 1, a medium-sized metacentric pair 2 and six pairs of submetacentrics (pairs 3 through 8). The X was a small acrocentric and the Y was dot-like. A single Ag-NOR bearing pair (5p5p) characterized M.rubida.Relevant karyotypic information was obtained for 19 specimens belonging to 12 species collected in areas sampled for the first time [Caluromys lanatus and C. philander (2n = 14, FN = 20), Gracilinanusemiliae (2n = 14, FN = 24), Marmosamurina, Metachirusnudicaudatus and Micoureusdemerarae (2n = 14, FN = 20), Monodelphisamericana(2n = 18, FN = 32) and M.domestica(2n = 18, FN = 20), and Didelphis marsupialis,Philanderfrenata,P.opossumand P.sp (2n = 22, FN = 20)]. Although the karyotypes were relatively conserved with respect to the morphology of the autosomes among species with the same diploid number, some differences regarding FN, sex chromosomes morphology and Ag-NORs patterns were detected.

There are currently 331 recognized species of living marsupials assembled into seven orders, three of which occur in the American continent (Didelphimorphia, Paucituberculata and Microbiotheria). They represent an extreme example of karyotype conservation, which is observed in most living species from the Australian and American continents. Some marsupials present a 2n = 14 karyotype, while others exhibit a karyotype with 2n = 22 (Pagnozzi et al., 2000;Svartman and Vianna-Morgante, 2003). The diploid number of 2n = 18 also occurs, but so far it has only been observed in four species of the genus Monodelphis found in Brazil and Bolivia (Pagnozzi et al., 2002;Carvalho et al., 2002).
The most frequent 2n = 14 karyotype has been traditionally considered ancestral to both American and Australian species (Rofe and Hayman, 1985;Hayman, 1990) and was suggested to have given rise to the highest diploid numbers through a series of centric fissions (Rofe and Hayman, 1985;Metcalfe et al., 2004). An alternative hypothesis claimed that the ancestral karyotype was similar to that with 2n = 22 and that centric fusions played a role on the origin of the derived karyotypes with lower diploid numbers (Svartman and Vianna-Morgante, 1998;Carvalho and Mattevi, 2000).
Karyological studies have demonstrated that the nucleolus organizer regions (NORs) and C-banding patterns represent important cytogenetic markers in conserved karyotypes because they reveal patterns that may characterize different species. According to Svartman and Vianna-Morgante (1999), marsupial karyotypes with the same diploid numbers differed in the amount of pericentromeric constitutive heterochromatin and in the number and distribution of Ag-NORs. These differences among species may be useful in the taxonomy of Didelphidae.
We analyzed 23 specimens belonging to 16 species from nine genera of Didelphidae collected in eight different localities of Cerrado and Atlantic Forest in the states of São Paulo, Goiás and Tocantins (Brazil). The karyotypes were analyzed after conventional staining and after silver staining of the nucleolus organizer regions (Ag-NORs) ( Table 1). We present data on 19 specimens belonging to 12 species collected in areas not previously surveyed (Table 1). We discuss in detail the new karyotypic data obtained for specimens of Gracilinanus microtarsus, Pereira et al.

Marmosops paulensis, Micoreus paraguayanus and Monodelphis rubida.
Mitotic preparations were obtained from bone marrow and spleen following routine protocols. After Giemsa staining, at least 15 metaphases/individual were analyzed to establish the diploid (2n) and fundamental numbers (FN = number of autosome arms) and the chromosomes morphology. Metaphases were photographed with a 100X objective under a Leica CW 4000 or Zeiss Axiophot photomicroscope equipped with image capture systems and softwares for chromosome analyses (Karyo, Leica and Ikaros Karyotyping System, MetaSystems). After silver staining (Howell and Black, 1980), the Ag-NORs were analyzed in the maximum number of cells possible.
Three diploid numbers were found in the studied Didelphidae: (

I) Karyotypes with 2n = 14
Gracilinanus microtarsus and Marmosops paulensis presented karyotypes with 2n = 14/FN = 24 composed of three pairs of large submetacentrics (pairs 1, 2 and 3), a medium-sized submetacentric pair 4 and two pairs of small submetacentrics (pairs 5 and 6); the X chromosome was a small metacentric ( Figure 1A and B). The Y chromosome was acrocentric in Marmosops paulensis ( Figure 1B). The karyotypes reported here for G. microtarsus and M. paulensis are very similar, despite the difference in the size of the X chromosome, larger in G. microtarsus than in M. paulensis. (Figure 1A and B).
The karyotype of a female Gracilinanus microtarsus from Biritiba-Mirim, São Paulo (Table 1), differed from specimens previously collected in the state of Rio Grande do Sul and also from individuals of G. agilis and G. emilae trapped in Goiás and Minas Gerais (Carvalho et al. 2002;this work). While pair 4 was submetacentric and the X chromosomes were metacentric in our specimen of G. microtarsus, pair 4 was metacentric in the specimens of Gracilinanus previously reported and the X chromosome was a submetacentric in G. microtarsus and G. agilis. Although marsupial karyotypes have been traditionally considered conservative, the present results indicate that some degree of interspecific karyotype variation occur in Didelphidae. In Gracilinanus, Ag-NORs were located on the short arms of pair 6 in specimens of G. microtarsus and G. emilae, but there are no data for G. agilis (Carvalho et al. 2002).
The karyotype of Marmosops paulensis is reported herein for the first time. The submetacentric pair 4 of M.
paulensis differed from the same pair described as metacentric in specimens of M. incanus from Espírito Santo and Bahia (Paresque et al., 2004;Pagnozzi et al., 2002) and in M. parvidens from Mato Grosso (Pagnozzi et al., 2002). The X and Y chromosomes of M. paulensis are metacentric and acrocentric, respectively, as were those observed in M. parvidens (Pagnozzi et al., 2002) and M. incanus (Paresque et al., 2004). The Ag-NORs were located at the telomeres of the short arms of pair 6 in M. paulensis (Figure 2A; Table 1). This result agrees with those reported by Svartman and Vianna-Morgante (2003) and Carvalho et al. (2002) for Marmosops incanus and M. parvidens from Goiás and Bahia, Brazil.
Micoureus paraguayanus exhibited 2n = 14 and FN = 20 and a karyotype with three pairs of large submeta-Karyotypes and NORs in Brazilian marsupials centrics (pairs 1 through 3), one pair of medium metacentrics (pair 4) and two small acrocentric pairs (pairs 5 and 6; Figure 1C). The X chromosome was a small acrocentric. This karyotype is identical to that previously described for specimens of M. demerarae collected in the Amazon and in the states of Pernambuco, Mato Grosso, Bahia and São Paulo (Casartelli et al., 1986;Souza et al., 1990;Pagnozzi et al., 2000;Svartman and Vianna-Morgante, 1999). A karyotype with 2n = 14 and FN = 24 was reported in five individuals identified as M. demerarae trapped in localities of Goiás and Rio Grande do Sul (Carvalho et al. 2002). We believe that the difference in the reported FNs is due to distinct degrees of chromatin condensation of the short arms of pairs 5 and 6, which were considered as biarmed by Carvalho et al. (2002).
According to Voss and Jansa (2003), the specimens identified as Micoreus demerarae collected in Rio Grande do Sul and analyzed by Carvalho et al. (2002) were misidentified and were actually M. paraguayanus. We cannot discard the possibility that other previously karyotyped specimens of M. demerarae were also misidentified. Nevertheless, the present paper is the first to describe the karyotype and the Ag-NORs distribution in a specimen identified as M. paraguayanus.
Micoureus paraguayanus was the only species from our sample with more than two Ag-NORs. Besides the Ag-NORs at pair 6, this species also had Ag-NORs at the telomeres of both chromosome arms of pair 5 and there was variation in the number of Ag-NORs per cell. Out of ten cells analyzed, two presented five Ag-NORs: on the telomeres of the long arms of one homologue of pair 5, on both telomeres (long and short arms) of the other element of pair 5 and on the telomeres of the short arms of pair 6 (5q5pq6p6p). The remaining eight cells presented six Ag-NORs (5pq5pq6p6p) ( Figure 2B; Table 1). This 5pq5pq6q6q Ag-NORs pattern was also reported in specimens of M. demerarae from Rio Grande do Sul (Carvalho et al., 2002) and by Svartman and Vianna-Morgante (2003), who described six positive signals after FISH with a ribosomal probe and after silver staining in a single specimen of M. demerarae from an unknown Brazilian locality. It is important to point out that the two specimens of M. demerarae herein studied were collected in a locality not previously sampled (Biritiba-Mirim) and exhibited a single pair with an Ag-NOR (pair 6; Table 1), differing from the multiple Ag-NORs (four to six sites) reported in the literature for specimens collected in the Amazon (Casartelli et al., 1986) and in the states of Pernambuco (Souza et al., 1990), Goiás and Rio Grande do Sul (Carvalho et al., 2002). Nevertheless, we analyzed the Ag-NORs in only six cells (Table 1) and more cells have to be analyzed in order to confirm this result.

II) Karyotype with 2n = 18
The karyotype of Monodelphis rubida had 2n = 18 and FN = 32, and the eight pairs of autosomes included one large pair of submetacentrics (pair 1), one medium-sized metacentric pair (pair 2) and six pairs of submetacentrics (pairs 3 through 8). The X chromosome was a small acrocentric and the Y was dot-like ( Figure 1D).
Although all the species of Monodelphis already described presented karyotypes with 2n = 18, there was variation in the FNs among and within species. For instance, Pereira et al. karyotypes with FN = 20 and FN = 28 (sampled in localities of Goiás) were both described for specimens of M. domestica (Svartman and Vianna-Morgante, 1999;Carvalho et al. 2002, respectively). The lack of information on the collection sites for the specimens analyzed by Svartman and Vianna-Morgante (1999) prevents further considerations of a possible geographical variation within this species. A fundamental number as high as 30 was described in specimens of M. domestica from Espírito Santo (Paresque et al., 2004), M. kunsi collected in localities of Góias and M. brevicaudata from Roraima and Pará (Carvalho et al., 2002). A karyotype similar to that found in M. rubida (present work), with exclusively biarmed chromosomes and FN = 32, also characterizes M. dimidiata from Rio Grande do Sul (Carvalho et al., 2002) and specimens of M. americana from São Paulo (Biritiba Mirim; Table 1) and from Espírito Santo (Paresque et al., 2004).
M. rubida exhibited a single Ag-NOR on the telomeres of the short arms of pair 5 ( Figure 2C; Table 1). An identical pattern was found in two species of Monodelphis (M. kunsi and M. dimidiata) from Goiás and Rio Grande do Sul, but a different pattern was reported in M. brevicaudata (from the states of Roraima and Pará) and M. domestica (Goiás), in which Ag-NORs were observed on the telomeres of the short arm of the X chromosome (Carvalho et al., 2002). Moreover, in specimens of M. domestica analyzed by Svartman and Vianna-Morgante (2003) four NORs (5p5p, XpXp) were reported in females and three (5p5p, Xp) in males.
A significant variation is observed in the FNs reported for specimens of Monodelphis domestica from different localities in Brazil. Karyotypes with 2n = 18 and FN = 20 occurred in specimens from two localities of Cerrado in Goiás and Tocantins (present study, Table 1) and in specimens analyzed by Vianna-Morgante (1999, 2003; without information on locality). Karyotypes with FN = 28 and FN = 30 were described for specimens from Cerrado of Goiás (Serra da Mesa and Ipameri; Carvalho et al., 2002) and areas of Atlantic Forest in Espírito Santo (Paresque et al., 2004), respectively. The reports of FNs higher than 20 is due to subtelocentric chromosomes being considered as biarmed by several authors and also due to differences in chromatin condensation. The standardization of chromosome nomenclature in marsupial species would prevent the artificial differences that make up for the confusing literature, especially for species in which there is no clear evidence of geographical variation.
In this study we described the karyotypes of three species of Didelphidae from Brazil (Marmosops paulensis, Monodelphis rubida and Micoreus paraguayanus) and a new karyotype for Gracilinanus microtarsus. Our work also increased the number of localities surveyed for 12 marsupial species. Although there was conservation of the autosomes morphology among karyotypes of species with the same diploid number, some differences regarding FN, morphology of the sex chromosomes and Ag-NORs distribution were detected, indicating that marsupial karyotypes are less uniform than believed.