We investigated the karyotype of 18 didelphid species captured at 13 localities in the Brazilian Amazon, after conventional staining, C-banding, Ag-NOR and fluorescent in situ hybridization (FISH) using the 18S rDNA probe. Variations were found in the X chromosome, heterochromatin distribution and the 18S rDNA sequence. The main variation observed was in the position of the centromere in the X chromosome of Caluromys philander Linnaeus, 1758 and Marmosa murina Linnaeus, 1758. For both species, the X chromosome showed a geographical segregation in the pattern of variation between eastern and western Brazil, with a possible contact area in the central Amazon. C-banding on the X chromosome revealed two patterns for the species of Marmosops Matschie, 1916, apparently without geographic or specific relationships. The nucleolus organizer region (NOR) of all species was confirmed with the 18S rDNA probe, except on the Y chromosome of Monodelphis touan Shaw, 1800. The distribution of this marker varied only in the genus Marmosa Gray, 1821 [M. murina Thomas, 1905 and M. demerarae Thomas, 1905]. Considering that simple NORs are seen as a plesiomorphic character, we conclude that the species Marmosa spp. and Didelphis marsupialis Linnaeus, 1758 evolved independently to the multiple condition. By increasing the sample, using chromosomal banding, and FISH, we verified that marsupials present intra- and interspecific chromosomal variations, which suggests the occurrence of frequent chromosomal rearrangements in the evolution of this group. This observation contrasts with the chromosomal conservatism expected for didelphids.

Marsupials, Amazon basin, C-band, NORs, 18S rDNA, Chromosomal rearrangements

In the Americas, subclass Metatheria Huxley, 1880 is represented by the three marsupial orders: Didelphimorphia Gill, 1872, Paucituberculata Ameghino, 1894 and Microbiotheria Ameghino, 1889. The largest of the three American orders is Didelphimorphia, which is represented by the family Didelphidae Gray, 1821, whose species are widely distributed throughout the continent. Didelphidae is the only marsupial group present in Brazil. Together with rodents, they make up an important part of the mammalian fauna of the Amazon region (Voss and Jansa 2009, Wilson and Reeder 2011). Currently, 14 genera and 39 species are recorded in the Amazon basin. Although moderate in terms of species richness, didelphids are abundant in the region (Brandão et al. 2015).

Historically, the first cytogenetic data on American marsupials were recorded by Jordan (1911; cited in Reig et al. 1977), on the spermatogenesis of Didelphis virginiana Kerr, 1792. Since then, our knowledge of cytogenetics of American and Australian marsupials has grown significantly. Hayman (1990) reported the karyotype of 178 species of American and Australian marsupials and Svartman (2008) reported 45 karyotypes for American marsupials.

Unlike other mammal orders, such as Rodentia Bowdich, 1821, marsupials show relatively little chromosomal variation (Nagamachi et al. 2015). Chromosomal stability in marsupials was first verified in conventional staining karyotypes that revealed the existence of three main diploid numbers in species from both continents: 14, 18 and 22 chromosomes.

Among all the metatherian families, Macropodidae Gray, 1821 (order Diprotodontia Owen, 1866) is the most diverse in diploid number, varying from 2n=10 to 32. While the American Didelphidae has only the three main diploid numbers, with the most frequent being 2n=14 (Reig et al. 1977, Hayman 1990, Palma and Yates 1996, Carvalho et al. 2002), which has been suggested as the ancestral diploid number of all marsupials (Reig et al. 1977, Westerman et al. 2010). Further comparisons using chromosome banding in American and Australian marsupial species revealed that chromosomal stability is verified not only on the diploid number but also on longitudinal banding patterns that show intense conservation on chromatids (Yonenaga-Yassuda et al. 1982, Rofe and Hayman 1985, Casartelli et al. 1986, Souza et al. 1990, Svartman and Vianna-Morgante 1999).

Limited sampling effort has hampered the estimation of species richness in the Amazon, leaving large gaps in our knowledge of the mammalian fauna (Voss and Emmons 1996, da Silva et al. 2001). Currently, of the 39 species of Amazonian marsupials (Brandão et al. 2015) only 17 have associated cytogenetic data (Nagamachi et al. 2015). However, considering the taxonomic instability of Amazonian marsupials, this representation might not be accurate, since new phylogenetic studies will probably change the current classification of several taxa. Furthermore, the earlier literature often lacks a connection between the karyotype of putative species and the analyzed specimens, making it difficult to verify a posteriori the taxonomic identification attributed to a given karyotype.

The number of taxa analyzed to date is also limited, and existing cytogenetic analyses have been usually restricted only to the diploid and fundamental numbers (Nagamachi et al. 2015). New advances in the taxonomic classification of Amazonian marsupials, complementary techniques of cytogenetic analysis (banding, in situ hibridization), and added sampling efforts (more specimens, new localities) are necessary to improve current knowledge on the cytogenetics of these animals.

In this study, we analyze the main morphological differences in the sex chromosomes and the C-band pattern of 18 didelphid species from the Brazilian Amazon. In addition, we describe for the first time karyotype for six species (Monodelphis touan, Monodelphis aff. adusta, Monodelphis sp., Marmosops impavidus, Marmosops bishopi and Marmosops pinheiroi) and discuss these patterns in a broader geographical context, including other regions of Brazil and South America.

We cytogenetically analyzed 111 individuals in 18 species and 8 didelphid genera, collected in 13 localities in the Amazon (Table 1 and Figure 1). Scientific collecting permits were obtained from the Brazilian Institute of the Environment and Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renováveis – IBAMA), according to SISBIO license numbers: 02005.000642/03-11 (IBAMA); 02000.002336/2003-93 (IBAMA); 02005.002672/04 (IBAMA); 37585-5 (SISBIO); 37592-4 (SISBIO). The specimens were deposited at the Mammals Collection of the National Institute of Amazonian Research (INPA), Manaus, Brazil. Specimens are indicated by species, sampling sites, genus and collector number, followed by INPA collection number (in parentheses) when available,and their field codes are listed bellow. Karyotyped specimens at the figures: Figure 2: a) Marmosa demerarae (RNL 46, boxes: MCA 27); b) Metachirus nudicaudatus (SISTAP-M-302; boxes: SISSIS-M-64); c) Gracilinanus emiliae (SISTAP-M-243); d) Marmosa murina (RNL 69, boxes: CEF 18); e) Caluromys philander (SISTAP-M-244, boxes: CAN 34, SISTAP-M-305); f) Caluromys lanatus (CTGA-M-701); ; g) Marmosops pinheiroi (INPA 5377, boxes: EE 192) (SISTAP-M-278, boxes: EE107, INPA 5408);:. Figure 3: a) Glironia venusta (BAC 80); b) Monodelphis aff. adusta (INPA 5388); c) Monodelphis touan (INPA 5404); d) Monodelphis sp. (CAN 44); e) Didelphis marsupialis (EE 249, boxes: EE174)."

Figure 1.

Sampling sites plotted on the Amazon basin map, Amazonas State: 1 Japurá River, Japurá city 2 Juruá River, Juruá city 3 Negro River, Santa Isabel do Rio Negro city 4 Purus River, Tapauá city 5 Cuieiras River, Manaus city 6 Manaus city, urban área: Federal University of Amazonas’s campus (UFAM) and Isaac Sabá Oil Refinery) 7 Aripuanã River, Novo Aripuanã city 8 Uatumã River, Presidente Figueiredo city 9 Jatapú River, São Sebastião do Uatumã city; Pará State: 10 Trombetas River, Oriximiná city 11 Tapajós River, Aveiro and Santarém cities 12 Jari River, Almeirim city; Rondônia State: 13 Madeira River, Porto Velho city. Geographic coordinates at the Table 1.

Figure 2.

Karyotypes under conventional staining (I), C-band (II), 18S rDNA and Ag-NOR (III), sex chromosomes in the boxes: a Marmosa demerarae b Metachirus nudicaudatus c Gracilinanus emiliae d Marmosa murina, (IV) variations on the 18S sites found in the individuals from Purus River, Tapauá city, Amazonas State e Caluromys philander f Caluromys lanatus g Marmosops pinheiroi.

Figure 3.

Karyotypes under conventional staining (I), C-band (II), 18S rDNA and Ag-NOR (III), sex chromosomes in the boxes: a Glironia venusta bMonodelphis aff. adustac Monodelphis touan d Monodelphis emiliae e Monodelphis brevicaudata f Monodelphis sp. g Didelphis marsupialis.

All voucher specimens: Glironia venusta Thomas, 1912: (BAC 80) – Caluromys philander Linnaeus, 1758: Tapajós River (male: SISTAP-M-297; SISTAP-M-305; SISTAP-M-318; SISTAP-M-382; female: SISTAP-M-244); Trombetas River (female: CTGA-M-652); Purus River (female: CAN 34); Manaus (female: MSN 01); (female: BAC 102) – Caluromys lanatus Olfers, 1818: Japurá River (female: CTGA-M-701) – Marmosops sp. Matschie, 1916: Aripuanã River (female: MCA 3; MCA 7; MCA 8; MCA 26; MCA 31; MCA 35; male: MCA 4; MCA 16; MCA 38; MCA 39); Jari River (female: TAG 3459; RNL 70); Juruá River (male: EE 107; EE 139; female: EE135); Cuieiras River (female: EE 198; EE 211; male: EE 192; EE 201; EE216) – Marmosops bishopi Pine, 1981: Negro River (male: SISIS-M-127); Purus River (male: SISPUR-M-135; SISPUR-M-157; SISPUR-M-160; SISPUR-M-164; SISPUR-M-135; CAN 30; CAN 51; female: CAN 48) – Marmosops pinheiroi Pine, 1981: Tapajós River (male: SISTAP-M-237; SISTAP-M-278; female: SISTAP-M-268; SISTAP-M-277) – Marmosops parvidens Tate, 1931: Trombetas River (male: CTGA-M-501; CTGA-M-516; CTGA-M-531; CTGA-M-532; CTGA-M-551; CTGA-M-555; CTGA-M-581; CTGA-M-600; female: CTGA-M-533) – Marmosops impavidus Tschudi, 1845: Purus River (male: SISPUR-M-149) – Marmosops cf. pakaraimae Voss, Lim, Díaz-Nieto et Jansa 2013: Japurá River (male: SISJAP-M-705) – Marmosa murina Linnaeus, 1758: Jari River (male: RNL 45); Uatumã River (male: CEF 4; CEF 8; CEF 18; CEF 27; CEF 28; CEF 32; female: CEF 16; CEF 34; CTGA-M-8; CTGA-M-22; CTGA-M-41;), Negro River (male: SISIS-M-57; SISIS-M-63); Trombetas River (female: CTGA-M—519); Purus River (male: CAN 43); Japurá River (male: CTGA-M-708) – Marmosa murina Linnaeus, 1758: Aripuanã River (female: MCA12, Japurá River (male: SISJAP-M-764)- Gracilinanus emiliae Thomas, 1909: Tapajós River: (male: SISTAP- M-245; SISTAP- M-343; SISTAP- M-344; SISTAP- M-345) – Marmosa demerarae Thomas, 1905: Aripuanã River (female: MCA 27; MCA 36; MCA 58; MCA 65; male: MCA 21; MCA59); Jari River (female: RNL 31; RNL 48; male: RNL 30; MCA 32; MCA 46; MCA 49; MCA 58; MCA 61; MCA 64; MCA 66; MCA 67) Juruá River (female: EE136; male: EE 143); Manaus (female: EE 149: EE 150; EE 151; EE 154; EE 158; EE 159; EE 169; EE 222; EE 228; 229; EE 234; male: EE 157; EE 167; EE 170; EE 176; EE 189; EE 194; EE 196; EE 202; EE 215; EE 220; EE 235); Cuieiras River (female: EE 193; EE 219); Tapajós River (female: SISTAP-M-229; SISTAP-M-241; SISTAP-M-321; SISTAP-M-333; SISTAP-M-369; male: SISTAP-M-267; SISTAP-M-279; SISTAP-M-322); Trombetas River (female: CTGA-M-579; CTGA-M-590; CTGA-M-622; CTGA-M-667; CTGA-M-672; male: CTGA-M-535; CTGA-M-539; CTGA-M-557; CTGA-M-558; CTGA-M-572; CTGA-M-573; CTGA-M-578; CTGA-M-580; CTGA-M-613); Negro River (female: SISIS-M-85; SISIS-M-110; SISIS-M-117; SISIS-M-128; male SISIS-M- 86); Purus River (female: SISPUR-M-145; CAN 25; CAN 31; CAN 50: male: SISPUR-M-144; SISPUR-M-147; SISPUR-M-148) – Monodelphis aff. adusta Thomas, 1897: Madeira River (male: MCA 15) – Monodelphis touan: Jari River (male: TAG 2731; RNL 68) – Monodelphis sp. Burnett, 1830: Purus River: (male: CAN 44) – Monodelphis emiliae Thomas, 1912: Aripuanã River (female: MCA 31) – Metachirus nudicaudatus Geoffroy et Saint-Hilaire, 1803: Jari: River (RNL 47); Cuieiras River: (female: EE 200); Tapajós River (female: SISTAP-M-230; SISTAP-M-230; male: SISTAP-M-251; SISTAP-M-269); Trombetas River: (female: CTGA-M-655); Jatapú River: (female: CTGA-M-52; CTGA-M-58); Negro River: (female: SISIS-M-64; SISIS-M-78; male: SISIS-M-84; SISIS-M-116); Purus River: (male: CAN 33) – Didelphis marsupialis Linnaeus 1758: Jari River: (female: RNL 44; RNL 53; RNL 59; male: RNL 52; RNL 55; RNL 62; RNL 63); Manaus: (female EE 174; EE 197; EE 204; EE 224; EE 227; EE 246; EE 250; EE 155; EE 155; EE 173; EE 183; EE 190; EE 203; EE 205; EE 206; EE 223; EE 232; EE 233; EE 237; EE 247;EE 248; EE249; EE 190); Uatumã River (female: CEF 5; male: CEF 13); Trombetas River (female: CTGA-M-594; CTGA-M-606; male: CTGA-M-607); Purus River (male: SISPUR-M-185); Negro River (male: SISIS-M-73):Tapajós River (female: SISTAP-M-324; SISTAP-M-346; SISTAP–M-347;male: SISTAP-M-243); Japurá River: (male: CTGA-M-732).

The metaphases were obtained from bone marrow by in vivo method according to Ford and Harmerton (1956). Each animal received 1mL/100g weight of a 0,0125% colchicine solution for 30 minutes, the cells were exposed for 20 minutes to a 0,075M KCl solution, fixed 3:1 in methanol and acetic acid and stored at -20 °C. The C-band and Nucleolus Organizing Regions (NORs) patterns were determined according to the techniques described by Sumner (1972), and Howell and Black (1980), respectively. Chromosome pairing considered morphology in decreasing order of size and the chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a) according to the ratio of chromosome arms and the position of the centromere, according to Patton (1967). 18S rDNA sequences were mapped by fluorescence in situ hybridization (FISH) according to Pinkel et al. (1986), whose probe was obtained by polymerase chain reaction (PCR) using the following primers designed by Gross et al. (2010): 18SF (5’-CCG CTT TGG TGA CTC TTG AT-3’) e 18SR (5’-CCG AGG ACC TCA CTA AAC CA-3’) and labeled with digoxigenin (DIG-Nick translation, ROCHE) or Biotin (Bio-Nick translation, ROCHE), following manufacturer’s instructions.

Among the 18 species analyzed, 11 showed 2n=14; six 2n=18 and one 2n=22 chromosomes (Table 1).

In the species with 2n=14, we observed a very similar structure among the autosomes. These karyotypes include six autosome pairs (Fig. 2), three large submetacentric pairs, one metacentric pair and two small pairs that varied in morphology in the different species, resulting in differences in the chromosomal formulas and fundamental numbers (FNa). FNa=20, with formula 2m+6sm+4a+XX/XY, was recorded in Marmosa demerarae (Fig. 2a-I) and Metachirus nudicaudatus Geoffroy an Saint-Hilaire, 1803 (Fig. 2b-I). FNa=22, with formula 2m+6sm+2st+2a+XX/XY, was present in Gracilinanus emiliae Thomas, 1909 (Fig. 2c-I), Marmosa murina (Fig. 2d-I), Caluromys philander (Fig. 2e-I) and Caluromys lanatus Olfers, 1818 (Fig. 2f-I). FNa=24, with formula 6m+6sm+XX/XY, was recorded in species of the genus Marmosops including M. bishopi (Pine, 1981), M. pinheiroi Pine, 1981, M. parvidens Tate, 1931, M. impavidus Tschudi, 1845, and M. cf. pakaraimae Voss, Lim, Díaz-Nieto et Jansa 2013. The five species of Marmosops presented similar karyotypic characteristics (Fig. 2g-I – only M. pinheiroi shown).

We observed three different morphologies for X chromosome: metacentric in G. emiliae and Marmosops spp. (Fig. 2c-I and 2g-I); submetacentric in the only female of C. lanatus (Fig. 2f-I); and acrocentric in M. demerarae and M. nudicaudatus (Fig. 2a-I and 2b-I). In Caluromys philander and Marmosa murina, we observed an intraspecific variation in the structure of the X chromosome, acrocentric and submetacentric, both in specimens from the same and different localities (Fig. 2e-I and 2d-I).

The bare-tailed woolly opossum (C. philander) has X chromosome either acrocentric or submetacentric, with females being either homozygous or heterozygous carriers of the heteromorphic X (Fig. 4). In the murine mouse opossum (Marmosa murina), the metacentric or submetacentric X was found in individuals throughout the Brazilian Amazon, except in the Purus River (Fig. 5, locality 4); it was also found in individuals from two localities in central Brazil (Fig. 5, localities 25 and 26). These are situated at the southern limits of the distribution of M. murina and both, the submetacentric X and the acrocentric X, are sympatric at locality 26. Furthermore, in the northern Amazon in Colombia, Venezuela and Peru, the X chromosome is metacentric (Fig. 5, localities 18, 19 and 20) (Reig et al. 1977, Carvalho et al. 2002). The acrocentric X was found in the Purus River (Fig. 5, locality 4), and in central, southeastern and northeastern Brazil (Fig. 5, localities 16 and 22-28) (Souza et al. 1990, Palma and Yates 1996, Carvalho et al. 2002).

Figure 4.

Geographic locations of Caluromys philander individuals and its sexual chromosomes morphology data in South America. Literature data represented by empty circles and present work represented by full circles: (● 14) Venezuela, Reig et al. 1977; (○ 4) Purus River; (○ 6) Manaus city, urban área: Federal University of Amazonas’s campus (UFAM); (● 15) Manaus REMAN (Isaac Saba Oil Refinery), present work and Souza et al. 2013; (○ 10) Trombetas River; (○ 11) Tapajós River; (○ 12) Jari River, Souza et al. 2013; (● 16) Pernambuco State, Souza et al. 1990; (● 17) São Paulo State, Svartman and Vianna-Morgante 1999 and Pereira et al. 2008. m = metacentric; sm = submetacentric; a = acrocentric; d = dot-like.

Figure 5.

Geographic locations of Marmosa murina individuals and its X chromosome morphology data in South America. Literature data represented by empty circles and present work represented by full circles: (● 18) Villa Vivêncio, Colômbia, Hayman and Martin 1974; (● 20) Loreto-Peru, Reig et al. 1977; (● 19) Bolívar, Venezuela, Reig et al. 1977; (○ 3) Negro River; (○ 2) Juruá River; (○ 4) Purus River; (○ 8) Uatumã River; (○ 10) Trombetas River; (○ 12) Jari River; (● 16) Pernambuco State, Souza et al. 1990; (● 21) Tartarugalzinho, Amapá State State, Carvalho et al. 2002; (● 22) Vila Rica Mato Grosso State, Pagnozzi et al. 2002; (● 23) UHE Peixe Angical,TO, Pereira et al. 2008; (● 24) Tocantins State, Lima 2004; (● 25) Uruaçú, Goiás State , Carvalho et al. 2002; (● 26) Colinas do Sul, Goiás state; (● 27) UHE Corumbá IV Luziania, Goiás state Pereira et al. 2008; (● 28) Pacoti, Ceará State, Pagnozzi et al. 2002; (● 29) Espírito Santo State, Paresque et al. 2004. m = metacentric; sm = submetacentric; a = acrocentric.

The Y chromosome was acrocentric in G. emiliae, Marmosops spp., M. demerarae and M. nudicaudatus (Fig. 2c, g, a, b), and dot-like in C. philander and M. murina (Fig. 2e, d).

Among the species with 2n=18 chromosomes, FNa=20 was recorded in Glironia venusta Thomas, 1912, with formula 2m+2sm+2st+10a+XX/XY (Fig. 3a-I), FNa=30 was recorded in four species of the genus Monodelphis Burnett, 1830: M. aff. adusta Thomas, 1897 (Fig. 3b-I), M. touan (Fig. 3c-I), M. emiliae Thomas, 1912 (Fig. 3d-I), and M. brevicaudata Erxleben, 1777 (Fig. 3e-I) with formula 2m+2sm+8st+2a+XX/XY, and FNa=32 in Monodelphis sp. (Fig. 3f-I), with formula 2m+2sm+10st+2a+XX/XY. We observed two X chromosomes morphologies: acrocentric in M. aff. adusta, M. touan and M. brevicaudata (Fig. 3b, c, e), and submetacentric in Monodelphis emiliae and Monodelphis sp. (Fig. 3d, f). The Y chromosome was acrocentric in M. touan and Monodelphis sp., and dot-like in M. aff. adusta and M. brevicaudata.

Didelphis marsupialis was the only species that presented 2n=22 chromosomes and FNa=20, with formula 20a+XX/XY (Fig. 3g-I), with acrocentric X and Y.

The position of the heterochromatin on the 2n=14 species was centromeric, being conspicuous in M. demerarae (Fig. 2a-II), M. nudicaudatus (Fig. 2b-II), G. emiliae (Fig. 2c-II), M. murina (Fig. 2d-II), and M. pinheiroi (Fig. 2g-II). Caluromys philander and C. lanatus exhibited tenuous heterochromatin, with additional telomeric heterochromatin in C. philander chromosomes (Fig. 2e-II and 2f-II). The X chromosome in C. philander was entirely heterochromatic, except for a distal band in the long arms (Fig. 2e-II); in M. demerarae it was also entirely heterochromatic, except for a proximal euchromatic band in the long arms (Fig. 2a-II); in M. murina (Fig. 2d-II), M. nudicaudatus (Fig. 2b-II) and G. emiliae the heterochromatin was centromeric (Fig. 2c-II).

Two C-band patterns were present in the X chromosome for species of Marmosops. In pattern 1, X was entirely heterochromatic except for a proximal band in the long arms (Fig. 2g – box); in pattern 2, the heterochromatin was in the short arms and the centromere (Fig. 2g – box). Both patterns were present in M. parvidens and M. bishopi, while only pattern 1 was observed in M. cf. pakaraimae, M. impavidus and M. pinheiroi (Table 2). The Y chromosome was entirely heterochromatic in all species.

In the species with 2n=18 chromosomes, the heterochromatin was centromeric in G. venusta (Fig. 3a-II), M. aff. adusta (Fig. 3b-II), M. touan (Fig. 3c-II) and M. emiliae (Fig. 3d-II). The Y chromosome was entirely heterochromatic in M. adusta (Fig. 3b-II) and M. touan (3c-II). It was not possible to determine the C-band pattern in Monodelphis sp. and M. brevicaudata.

NORs confirmed by FISH using the 18S rDNA probe were present in the short arms of pair 6 in all 2n=14 species and G. venusta (2n=18). In M. demerarae and M. murina sites were also detected in the terminal position of the long arms of pair 5 (Fig. 2, a-III e d-III). In M. emiliae (2n=18) the NOR was positioned on the short arms of pair 7 (Fig. 3d-III), and in M. touan in the X and Y chromosomes, although no 18S site was detected in Y (Fig. 3c-III). Only Monodelphis brevicaudata exhibited multiple NORs (Fig. 3e-III), whose sites were in the terminal region of the long arms of pair 7 and the short arms of X and Y.

In D. marsupialis, both the 18S rDNA probe and silver were detected in three chromosome pairs. In two pairs, the sites were located in the terminal region of the long arms, while in one pair they were bitelomeric (Fig. 3g-III). However, in regards to activity, there was a variation of four to eight markings.

In the last decade, advances in systematic and taxonomic studies of the family Didelphidae introduced changes in the taxonomy and nomenclature of several of its taxa (Jansa and Voss 2000, Voss and Jansa 2003, Voss and Jansa 2009, Rossi et al. 2010, Gutiérrez et al. 2010). We used the phylogenetic tree of Jansa and Voss (2014) to map the cytogenetic data of the 18 species we have analysed in order to gain an understanding of chromosome evolution in the group. This work represents the most updated phylogeny of the intergeneric relationships of didelphid marsupials, making our interpretation of the cytogenetic data more integrative than a mere consideration of chromosomal data, and more accurate in light of an independently generated phylogenetic hypothesis.

The autosome structure observed here corroborates karyotypic conservation in the diploid number and chromosomal formula (NFa) as previously described in the didelphid species Didelphis marsupialis, Marmosa demerarae, Metachirus nudicaudatus, Monodelphis touan (previously named M. brevicaudata), Monodelphis aff. adusta (previously named as M. cf. emiliae) and for species of Marmosops (Reig et al. 1977, Yonenaga-Yassuda et al. 1982, Casartelli et al. 1986, Hayman 1990, Souza et al. 1990, Palma and Yates 1996, Svartman and Vianna-Morgante 1998, 1999, Carvalho et al. 2002).

Although didelphids are generally considered to have a conserved karyotype, by comparing the karyotypes among different genera, it was possible to associate them with certain species due to the presence of diagnostic characters. For example, M. demerarae and M. murina differ in their FNa, morphology, and sex chromosome size. In species of the genus Monodelphis, morphological variation in chromosomes was restricted to pair 6, which grants an FNa varying between 30 (as observed in M. aff. adusta, M. touan and, M. brevicaudata) and 32 arms (Monodelphis sp.). However, the same does not occur for the genus Marmosops, in which the five species analysed, present a very similar chromosome macrostructure.

The genus Marmosa has a complex taxonomy and recently underwent great taxonomic changes, with all species of Micoureus, formerly treated as a separate genus, now considered as a subgenus of Marmosa. Considering the taxonomic instability in Didelphidae, with individuals being reclassified, and some complex of species being divided into two or more valid taxa, even purportedly karyotyped species may in fact have their karyotypes still unknown. Thus, our knowledge as to how many and which species among didelphids were karyotyped remains unstable. A revision of the literature for species with reported karyotypes is required.

Dutrillaux (1979) suggested that a small sample size would be inadequate for the knowledge of species karyotypes. Heeding this admonition, we used a relatively large number of individuals for each species analysed to uncover a range of variations that most likely would not have been detected had we used fewer individuals per species. The use of integrative analyses and new methodologies, such as taxonomy, phylogeny, and molecular cytogenetics could improve our understanding of the significance of these chromosomic variations. However, for the Amazon region, a significant limitation for cytogenetic studies is still the restricted collection effort, the vast geographical extent of the region and the difficulty of access to remote areas.

The cytogenetic data presented here shows that didelphid marsupial karyotypes present intraspecific variation in the morphology of sex chromosomes and in chromosomic markers (C-band and NOR) and present some geographic variation in the distribution of these features for several species. Furthermore, there are many areas in the Amazon, including the transition zone between the Amazon and the Cerrado biomes, which do not have cytogenetic records for any didelphid species. This situation seriously undermines our understanding of the significance of the recorded variation, whether it is part of a continuous gradient, or whether it represents intraspecific gradations, or whether it is related to new lineages or cryptic species still uncovered. Thereby, despite the chromosomal stability related to diploid numbers and chromosomal formula in marsupials across continents, didelphids present some intra- and interspecific chromosomal variations, probably related to frequent chromosomal rearrangements. Additional systematic sampling and analyses will be required for a better understanding of the karyotypic evolution of this group.

This work was supported by Fundação de Amparo a Pesquisas do Estado do Amazonas (FAPEAM), Coordenação de Aperfeiçoamento Pessoal (CAPES), the SISBIOTA/Rede BIOPHAM (CNPq); and Pró-Amazônia (CAPES). We thank Dra. MC Gross e Dr. CH Schneider who cooperated with research development and G.H. Shepard Jr. for reviewing the English of the manuscript.