Heterochromatin heterogeneity and chromosome heteromorphism in Cerdocyon thous (Mammalia, Canidae)

Heterochromatin heterogeneity and chromosome heteromorphism in Cerdocyon thous (Mammalia, Canidae)

Terumi Hatanaka¹, Antonio Junqueira Tambasco² and Pedro Manoel Galetti Junior¹

¹Departamento de Genética e Evolução, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905 São Carlos, SP, Brasil. Fax: [phone], E-mail: [e-mail] Send correspondence to P.M.G.Jr.

²Empresa Brasileira de Pesquisa Agropecuária - Embrapa, Centro de Pesquisa de Pecuária do Sudeste - CPPSE, 13560-970 São Carlos, SP, Brasil.

ABSTRACT

Few cytogenetic studies have been conducted on South American canids. Cerdocyon thous is a representative of the Canidae, living in the forests, open wooded areas and savannahs of South America. Compared to other canid species, C. thous has a large proportion of metacentric and submetacentric chromosomes as well as a large amount of constitutive heterochromatin, especially along the short arm of submetacentric chromosomes. In the present study, different chromosome banding methods were used to characterize the heterogeneous nature of the large heterochromatic segments and to propose an organizational model for this segment that occupies the entire short arm of most two-armed chromosomes. Furthermore, chromosome heteromorphism related to the short arm of large submetacentric chromosome corresponding to no. 3 in male and female karyotypes is described.

INTRODUCTION

Cerdocyon thous is a canid known as bush dog that lives in the forests, open wooded areas and savannahs of South America (Brazil, Colombia, Venezuela, Guyanas, western Bolivia, Paraguay, Uruguay, and northern Argentina). Five subspecies are recognized (Bispal, 1988). C. thous was previously considered to belong to the genus Dusicyon and was classified as D. thous (Clutton-Brock et al., 1976). It was also considered a subgenus of Canis, and cited as C. cerdocyon thous (Van Gelder, 1978). Cerdocyon is currently recognized as a distinct genus (Redford and Eisenberg, 1992). Bush dogs are dark canids with a grizzled-brown or grey pelage. Their legs maybe tawny, underparts brownish-white, and short ears have an ochreous or rufus coloration. The tail is fairly long, bushy and either totally dark or with a black tip (Clutton-Brock et al., 1976).

The accelerated destruction of Brazilian areas occupied by wild canid populations is progressively reducing the habitats of these animals. Lack of genetic and ecological information for carnivores has impaired elaboration of strategies to minimize the impact of environmental deterioration on these populations (Azevedo et al., 1996). Consequently, these studies are important because they may contribute to more appropriate management of these animals.

Wurster-Hill (1973) was the first to describe C. thous from a cytogenetic viewpoint. It has a diploid number of 74 chromosomes. The karyotypic complement of this species consists of a large proportion of submetacentric/metacentric types, and presents the highest fundamental number in the Canidae (Wurster-Hill, 1973). Previous C-banding studies demonstrated large fractions of constitutive heterochromatin along practically the entire length of the short arms of metacentric and submetacentric chromosomes (Wayne et al., 1987). The present study used different chromosome banding methods to obtain a more detailed characterization of the heterochromatin present in the karyotypic complement.

MATERIAL AND METHODS

Two animals from the Municipal Zoo of Limeira, SP, Brazil (a female designated animal no. 1, and a male, animal no. 2), and two from Florestal Park of Ripasa S.A. Celulose e Papel, located in Ibaté, SP, Brazil (two males, animal nos. 3 and 4) were cytogenetically analyzed. Metaphase chromosomes were obtained by peripheral blood lymphocyte culture (Moorhead et al., 1960). Constitutive heterochromatin was detected by the technique of Sumner (1972), and the nucleolar organizer regions (NORs) were identified by silver staining (Howell and Black, 1980). Fluorescent bands were studied using the base-specific DNA-binding fluorochromes mithramycin (MM), chromomycin A₃ (CMA₃) and 4'-6'-diamidino-2phenylindole (DAPI), as suggested by Schmid (1980) and Schweizer (1980). Distamycin A (DA) was used in combination with MM, CMA₃ and DAPI (Schweizer, 1980).

RESULTS

All animals studied presented a diploid number of 74 chromosomes. Of these, 15 pairs were meta/submetacentric, and the remaining 21 were acrocentric. The sexual X chromosome was large and submetacentric and the Y small and acrocentric (Figure 1). The fundamental number (FN) was 106. A clear difference in short arm size in the large submetacentric pair (no. 3) was detected in three (nos. 1, 2 and 4) of the four animals studied.

Figure 1 -
Giemsa karyotype of Cerdocyon thous (male animal no. 3). The female sex chromosomes (XX) (animal no. 1) are also indicated in the top right corner.

At least four NOR-bearing chromosomes were detected by silver staining. The Ag-NOR sites were located in the telomeric region of the long arm of one large submetacentric and three acrocentric chromosomes (Figure 2).

Figure 2 -
Partial metaphase of Cerdocyon thous (male animal no. 3) showing the positive Ag-NORs (arrowheads).

The heterochromatin regions detected by C banding were quite abundant. Large heterochromatin blocks occupied the entire length of the short arm of all submetacentric chromosomes. Other C⁺ bands were located in the centromeric region of some chromosomes, including the X chromosome. The Y was almost fully heterochromatic (Figure 3).

Figure 3 -
Karyotype of Cerdocyon thous (male animal no. 3) showing the positive C-banding pattern. The male sex chromosomes (XY) are indicated in the top right corner.

DAPI staining revealed negative regions predominantly in the centromeres of most chromosomes. Some ill-defined Q-like bands were detected along the chromosome arms. The heteromorphism observed in chromosome pair no. 3 consisted of a DAPI-positive region detected at the end of the short arm of one of the homologues, but absent in the other (Figure 4).

Figure 4 -
a: DAPI-stained karyotype of a Cerdocyon thous female (animal no. 1), b: detail of chromosome pair no. 1 stained with Giemsa (on the left) and heterochromatin detected by C banding (on the right). The female sex chromosomes (XX) are indicated in the top right corner.

Mithramycin and chromomycin A₃ staining revealed a pattern almost opposite to that of DAPI. Strongly fluorescent, large MM⁺ blocks were located in the centromeric regions and extended to the paracentromeric portion of all submetacentric chromosomes. In some of these chromosomes, a small, less intensely MM-stained portion was observed at the end of the short arm, possibly corresponding to DAPI-positive regions (Figure 5).

Figure 5 -
Cerdocyon thous karyotype (female animal no. 1) bearing DA/DAPI fluorescent bands. The female sex chromosomes (XX) are indicated in the top right corner.

Use of distamycin A counterstain in combination with DAPI revealed fluorescent bands in most of the C⁺ band regions, except for two pairs of acrocentric chromosomes, one metacentric pair and the sex chromosomes. Heteromorphism related to the size of DA/DAPI⁺ blocks was also observed in chromosome pair no. 1 of the female karyotype (animal no. 1) (Figure 6).

Figure 6 -
Karyotype of Cerdocyon thous (female animal no. 1). DA/CMA₃ staining. The female sex chromosomes (XX) are indicated in the top right corner.

At the end, at least four types of heterochromatin were detected in the karyotype of C. thous: 1) C band⁺, DA/DAPI⁺, DAPI^- and DA/MM⁺ present in the centromeres of submetacentric chromosomes and chromosome pairs 1, 16, 17 and 18; 2) C band⁺, DA/DAPI⁺, DAPI⁺ and weaker DA/MM detected in paracentromeric regions of submetacentric chromosomes; 3) C band⁺, nonfluorescent DA/DAPI, DAPI^- and DA/MM⁺ revealed in the centromeres of chromosome pairs 2, 19, 21 and 22, and finally 4) C band⁺, not differentiated by DA/DAPI, DAPI or DA/MM, present in sex chromosomes.

DISCUSSION

Chromosome heteromorphism in Cerdocyon thous

The Giemsa-stained karyotype of C. thous was first described by Wurster-Hill (1973). With a diploid number of 74 chromosomes, the karyotype of this species consists of a large proportion of meta/submetacentric chromosomes. This sets C. thous apart from other canids endemic to South America, such as Chrysocyon brachyurus (Newnham and Davidson, 1966, Wurster-Hill and Centerwall, 1982; Hatanaka, 1996, among others) and Speothos venaticus (Wurster-Hill and Centerwall, 1982), in which acrocentric chromosomes predominate. Thus, C. thous has the highest fundamental number in the family Canidae (Wurster-Hill, 1973).

Different chromosome banding methods detected heteromorphism in chromosome pair no. 3 in three of the animals. The heteromorphism was characterized by a prominent difference in size of the short arm of the homologues of this chromosome pair. Results obtained with base-specific fluorochromes strongly suggested the absence of a DAPI-positive region at the end of the short arm in one chromosome of this pair. Three alternative hypotheses may explain this heteromorphism: 1) deletion of a fraction of the short arm of a large submetacentric chromosome, 2) the opposite, i.e., regional duplication of a segment of the short arm, and 3) translocation that may also involve another chromosome not identified in the karyotypic complement.

Intraspecific polymorphisms among canids have been described for the fox species, Alopex lagopus, in which diploid numbers of 48, 49 and 50 chromosomes have been found (Gustavsson and Sundt, 1965; Mayr et al., 1986; Swintonski and Gustavsson, 1991, among others). Heterochromatin heteromorphisms have also been observed in this species (Mayr et al., 1986). In fact, chromosome polymorphisms are more common than originally thought among mammals, although few reports have connected them to phenotypic effects (Henricson and Bäckström, 1964; Gustavsson, 1969, among others). The heterochromatin heteromorphism described here in C. thous seems to be frequent in these animals, and it could be detected in high percentage (75%) of the sampled animals. Therefore, it is not known whether it is represented only in captive populations or if it also appears in wild populations.

Constitutive heterochromatin and karyotype differentiation in C. thous

C. thous had more heterochromatic regions demonstrated by C banding than other canid species such as Chrysocyon brachyurus (Hatanaka, 1996) and Canis familiaris (Pathak et al., 1977, 1982; Stone et al., 1991; Hatanaka, 1996, among others). The presence of heterochromatic short arms in most two-armed chromosomes in the complement of C. thous may explain the high fundamental number (FN = 106) of this species (Wurster-Hill, 1973; Wayne et al., 1987; present study). A similar situation was described for rodents of the genus Peromyscus (Pathak et al., 1973). In this genus, all species present a diploid number of 48, but the fundamental number varies from 56 to 96. C banding reveals extensive variation of small heterochromatic arms along the two-armed chromosomes. In a study of two extreme cases of variation in chromosome arm number in P. crinitus (eight heterochromatic arms) and P. eremicus (48 heterochromatic arms), the authors noted that if these heterochromatic arms were removed, euchromatin length on the long arm would be similar for the two species. In this case, the number of heterochromatic arms determines the great variability in fundamental number.

Thus, even though the heterochromatin segment apparently plays a minor role in the chromosome evolution of C. brachyurus and C. familiaris (Hatanaka, 1996), C. thous seems to be different. Development of large heterochromatin blocks on the short arm of various chromosomes appears to be an important trend for stabilizing newly formed chromosomes in some centric fissions (Imai, 1988, among others).

Other canids, such as Alopex lagopus, also show a large amount of heterochromatin, preferentially located in centromeric regions, and occasionally extending to paracentromeric regions (Mäkinen, 1985). In this species, most of the Cband⁺ heterochromatic chromosome arms showed medium fluorescence with chromomycin A₃. However, some presented bright CMA₃⁺ bands, indicating the presence of heterogeneous heterochromatin (Mayr et al., 1986). When stained with DA/DAPI, most of these chromosome arms showed uniformly weak fluorescence and DA/DAPI⁺ heterochromatin was observed in only three chromosome pairs. Similarly, heterogeneous heterochromatin has been repeatedly demonstrated in primates (Schmid et al., 1986; Pieczarka et al., 1996), birds (Mayr et al., 1989), amphibia (Schmid, 1980) and fish (Souza et al., 1996).

In the present study, the analysis of C band⁺ results, especially of submetacentric chromosomes, allowed us to propose a model for the organization of this chromosome segment (Figure 7), in which DA/DAPI⁺ and DA/MM⁺ bands are interspersed in the heterochromatin. In a less probable alternative hypothesis, this heterochromatin might have peculiar organizational characteristics leading to a positive response to both types of fluorochrome.

Figure 7 -
Organizational model for the C band⁺ segment, especially with reference to submetacentric chromosomes. a: C band, b: DAPI stain, c: DA/CMA₃ stain and d: DA/DAPI stain (solid dark for positive and dotted line for negative bands).

ACKNOWLEDGMENTS

The authors are indebted to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and to IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) Grant No. 315/95 for the collection of the material used in the present study. The authors also thank Fernando S. Magnani, Rogério R. Paschoal and Ana Rita C. Salles from the Parque Ecológico, Antonio T. Viana from São Carlos, Danielo Castigioni Mazon from the Municipal Zoo of Limeira, and Ripasa (Ibaté, SP). Publication supported by FAPESP.

RESUMO

Os estudos citogenéticos em canídeos sul-americanos, de uma forma geral, são raros. Cerdocyon thous é um representante da família Canidae e habita florestas, matas abertas e savanas da américa do Sul. O seu cariótipo é formado por uma grande proporção de cromossomos metacêntricos e submetacêntricos quando comparado com outras espécies desta família e possui também uma grande quantidade de heterocromatina constitutiva, especialmente ao longo do braço curto dos cromossomos submetacêntricos. No presente estudo, diferentes métodos de bandamentos cromossômicos permitiram verificar o caráter heterogêneo dos grandes segmentos heterocromáticos e a proposição de um modelo de organização deste segmento que ocupa todo o braço curto da maioria dos cromossomos de dois braços. Além disso, é descrito um heteromorfismo cromossômico relacionado ao braço curto de um grande cromossomo submetacêntrico, equivalente ao no. 3 do cariótipo de animais de ambos os sexos.

(Received June 18, 1997)

• Azevedo, F.C.C., Marinho-Filho, J.S. and Felipe, M.S.S. (1996). Variabilidade genética e conservaçăo do gato-do-mato pequeno (Felis tigrina) no Brasil. In: III Congresso de Ecologia do Brasil, Brasília, pp. 49.
• Bispal, F.J. (1988). A taxonomic study of the crab-eating fox, Cerdocyon thous in Venezuela. Mammalia 52: 181-186.
• Clutton-Brock, J., Corbet, G.B. and Hills, M. (1976). A review of the family Canidae with a classification by numerical methods. Bull. Br. Mus. 2: 117-199.
• Gustavsson, I. (1969). Cytogenetics, distribution and phenotypic effects of a translocation in Swedish cattle. Hereditas 63: 68-169.
• Gustavsson, I. and Sundt, C.O. (1965). Chromosome complex of the family Canidae. Hereditas 54: 249-254.
• Hatanaka, T. (1996). Contribuiçăo ŕ citogenética de Caninae. Master's thesis, Universidade Federal de Săo Carlos, Săo Carlos, SP.
• Henricson, B. and Bäckström, L. (1964). Translocation heterozygosity in a boar. Hereditas 52: 166-170.
• Howell, W.M. and Black, D.A. (1980). Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: 1-step-method. Experientia 36: 1014-1015.
• Imai, H.T. (1988). Centric fission in man and other mammals. In: The Cytogenetics of Mammalian Autosomal Rearrangements (Daniel, A., ed.). Alan R. Liss Inc., New York, pp. 551-582.
• Mäkinen, A. (1985). The standard karyotype of the blue fox (Alopex lagopus L.). Hereditas 103: 33-38.
• Mayr, B., Geber, G., Auer, H., Kalat, M. and Schleger, W. (1986). Heterochromatin composition and nucleolus organizer activity in four canid species. Can. J. Genet. Cytol. 28: 744-753.
• Mayr, B., Lambrou, M., Schweizer, D. and Kalat, M. (1989). An inversion polymorphism of a DA/DAPI-positive chromosome pair in Anas platyrhynchos (Aves). Cytogenet. Cell Genet. 50: 132-134.
• Moorhead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M. and Hungereford, D.A. (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20: 613-616.
• Newnham, R.E. and Davidson, W.M. (1966). Comparative study of the karyotypes of several species in Carnivora including the giant panda. Cytogenetics 5: 152-163.
• Pathak, S. and Wurster-Hill, D.H. (1977). Distribution of constitutive heterochromatin in carnivores. Cytogenet. Cell Genet. 18: 245-254.
• Pathak, S., Hsu, T.C. and Arrigui, F.E. (1973). Chromosomes of Peromyscus (Rodentia:Cricetidae). IV. The role of heterochromatin in karyotype evolution. Cytogenet. Cell Genet. 12: 315-326.
• Pathak, S., Tuinen, P.V. and Merry, D.E. (1982). Heterochromatin, synaptonemal complex, and NOR activity in the somatic and germ cells of a male domestic dog, Canis familiaris (Mammalia, Canidae). Cytogenet. Cell Genet. 34: 112-118.
• Pieczarka, J.C., Nagamachi, C.Y., Barros, R.M.S. and Mattevi, M.S. (1996). Analysis of constitutive heterochromatin by fluorochromes and in situ digestion with restriction enzymes in species of the group Callithryx argentata (Callithrichidae, Primates). Cytogenet. Cell Genet. 72: 325-330.
• Redford, M.H. and Eisenberg, U.F. (1992). Mammals of the Neotropics 2. The University Chicago Press, Chicago, pp. 430.
• Schmid, M. (1980). Chromosome banding in Amphibia. IV. Differentiation of GC - and AT-rich chromosome regions. Chromosoma 77: 83-103.
• Schmid, M., Haaf, T., Scheres, J.M.J.C. and Wensing, J.A.B. (1986). Heterochromatin in the chromosomes of gorilla: characterization with distamicyn A/DAPI, D287/170, chromomycin A₃, quinacrine and 5-azacytidine. Cytogenet. Cell Genet. 41: 71-82.
• Schweizer, D. (1980). Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58: 307-324.
• Souza, I.L., Moreira-Filho, O. and Galetti Jr., P.M. (1996). Heterochromatin differentiation in the Characidae fish Astyanax scabripinnis Braz. J. Genet. 19: 405-410.
• Stone, D.M., Jacky, P.B. and Prieur, D.J. (1991). The Giemsa banding pattern of canine chromosomes, using a cell synchronization technique. Genome 34: 407-412.
• Sumner, A.T. (1972). a simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res. 75: 304-306.
• Swintonski, M. and Gustavsson, I. (1991). Centric-fusin translocation and whole-arm heterochromatin in the karyotype of the blue fox (Alopex lagopus L.): synaptonemal complex analysis. Cytogenet. Cell Genet. 57: 1-8.
• Van Gelder, R.G. (1978). A review of Canid classification. Am. Mus. Novit. 2646: 1-10.
• Wayne, R.K., Nash, W.G. and O'Brien, S.J. (1987). Chromosomal evolution of Canidae I. Species with high diploid numbers. Cytogenet. Cell Genet. 44: 123-133.
• Wurster-Hill, D.H. (1973). Chromosomes of eight species from five families of Carnivora. J. Mammal. 54: 753-760.
• Wurster-Hill, D.H. and Centerwall, W.R. (1982). The interrelationships of chromosome banding patterns in canids, mustelids, hyens and felid. Cytogenet. Cell Genet. 34: 178-192.

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  June 1998

History

• Received
  18 June 1997