Journal of Heredity Advance Access originally published online on August 26, 2008
Journal of Heredity 2009 100(1):119-122; doi:10.1093/jhered/esn064
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Brief Communications |
The Karyotype of Franciscana Dolphin (Pontoporia blainvillei)
Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, 91501-970, Brazil (Heinzelmann and Andrades-Miranda), Laboratório de Imunogenética, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 43323, lab. 212, Porto Alegre, RS, 91501-970, Brazil (Heinzelmann, Chagastelles, and Chies), Grupo de Estudos de Mamíferos Aquáticos do Rio Grande do Sul (GEMARS), Rua Felipe Neri, 382/203, Porto Alegre, RS, 90440-150, Brazil (Danilewicz)
Address correspondence to L. Heinzelmann at the address above, or e-mail: lari2512{at}yahoo.com.br.
Despite the recent increase in studies on franciscana dolphin (Pontoporia blainvillei) molecular biology, there has been no published karyotype information, as opportunities for sampling live individuals are very rare. In the present study, the diploid number of the species was established from corneal cell cultures of 2 newborn male franciscanas live stranded (2n = 44). From the comparison of the chromosomal number to the cetacean karyotype phylogeny, we suggest that the most parsimonious hypothesis is that the ancestral character state in the group is the diploid number of 42, with an extra chromosome pair appearing independently twice during cetacean evolution, once in the suborder Odontoceti and once in the suborder Mysticeti. This information on chromosomal number may be useful to future genetic mapping projects of the species.
Key Words: pontoporia blainvillei • karyotype • franciscana • corneal • culture cell • cetacean
The franciscana, Pontoporia blainvillei, is a small dolphin endemic to shallow coastal waters of tropical and temperate regions along the coasts of Brazil, Uruguay, and Argentina (Crespo et al. 1998; Siciliano et al. 2002). The species is the only member of the family Pontoporiidae. Due to the dolphin's behavior of avoiding engine boats, its small size, inconspicuous color pattern, and small group size, franciscanas are one of the most difficult coastal cetaceans to be observed in the wild.
This species is probably the most endangered small cetacean of the Southwest Atlantic Ocean, as a result of high levels of entanglement in commercial gillnets (Danilewicz 2007). The population that inhabits southern Brazil and Uruguay suffers a mortality of more than 1000 animals each year (Ott et al. 2002; Secchi et al. 2003) and is currently listed as Vulnerable in the International Union for Conservation Nature (IUCN) Red List of Threatened Species (IUCN 2007).
The franciscana is one of the most studied cetaceans of South America, having many aspects of its biology described (e.g., Pinedo and Hohn 2000; Danilewicz et al. 2002, 2004). Nevertheless, molecular studies have been first published only in the last 10 years with the description of differences in the mtDNA between 2 populations of franciscanas (Secchi et al. 1998). Subsequently, studies applying mtDNA and microsatellite techniques to investigate population structure, social ecology, and phylogeny have been conducted by many authors (Hamilton et al. 2001; Ott 2002; Valsecchi and Zanelatto 2003; Lázaro et al. 2004). Despite these advances in franciscana molecular biology, there has not been published data on the species’ karyotype. The objective of this note is to describe the karyotype of the franciscana. In addition, a comparison with the karyotype of the living cetacean families is provided.
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Two newborn male franciscanas, live stranded in northern Rio Grande do Sul, southern Brazil, in October and November 2006 were sampled before and after demise. The blood collected before death did not result in an appropriate cell culture (data not shown). After death, the animals were preserved at 4 °C until processing. Corneal culture for cytogenetic preparations was obtained according to Duffield et al. (1991), with modifications. The eyes from each animal were harvested 2 days after death, stored in sealed plastic bags, and maintained at 4 °C for 3–5 days before use. The eyes were rinsed with sterile phosphate-buffered saline and, to prevent contamination, the entire corneal layer was excised in 1 ml of normal culture medium (NM) consisting of Dulbecco's Modified Eagle's Medium (Sigma, St. Louis, MO), 3.7 g/l sodium bicarbonate, 2.5 g/l HEPES, 10% fetal calf serum (v/v), gentamicine (0.6 mg/ml), and Fungizone 2.5 µg/ml (Amphotericin B, Gibco, Carlsbad, CA). After 1 h at 37 °C, 5% CO2, the corneas from both animals were minced in small pieces, transferred to 12-well cell culture plates (one piece per well) and maintained at the same conditions but without Fungizone. After 24 h, the corneal pieces were washed with Hanks balanced salt solution–Ca2+ and Mg2+ free (Sigma) and incubated with 0.25% trypsin-EDTA solution (Sigma) for 5 min to facilitate the detachment of cells from tissue. The released cells were centrifuged, resuspended in NM without Fungizone, and transferred to another 12-well cell culture plate. After the adherent cells reached confluence, they were transferred to 6-well cell culture plates (first passage). The culture was expanded in 25 cm2 bottles.
The karyotype was prepared from the third to the fifth passages to avoid possible aneuploidy events induced by long-term culture maintenance. Metaphase cells were collected by the addition of colchicine 0.1 µg/ml for 2 h at 37 °C. Cells were resuspended using 0.075 M KCl hypotonic solution for 10 min at 37 °C and fixed in ethanol:glacial acetic acid solution (3:1). From each preparation, a minimum of 15 chromosome spreads were analyzed to establish the diploid numbers.
GTG and CBG bands were performed according to the methods of Seabright (1971) and Sumner (1972), respectively. Four complete sets of chromosomes from each individual were measured for relative total length, and arm ratios were computed (Table 1). The numbers of autosomal arms was designed as FNa. The photomicrographs were obtained using FUJI Neopan ISO 100 under a Zeiss microscope. The karyotype groups were assembled in a fashion similar to the published karyotype for the Inia geoffrensis (Hsu and Benirschke 1967), and the chromosome group classification followed Levan et al. (1964).
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In order to provide a figure of the evolution of karyotype pattern in cetaceans, information on the karyotype was collated by the compilations presented by Arnason (1974) and O'Brien et al. (2006) and applied to the complete cetacean phylogeny proposed by Price et al. (2005). In this comparison, the gray whale (Eschrichtius robustus) was nested together with the other balaenopterids because the support for a monophyletic clade comprising the traditional families Eschrichtiidae and Balaenopteridae is considered high (Price et al. 2005).
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The modal diploid and autosomal arm numbers of P. blainvillei were 44 and 74, respectively. The karyotype has 16 large to small bi-armed and 5 median and small acrocentric pairs (Figures 1 and 2). The sex pair is metacentric, the X chromosome being median (between pairs 10 and 11) and the Y is the smallest chromosome, as expected from mammal karyotypes. It was possible to identify all chromosomes pairs from one franciscana using G-banding (Figure 1). Compared with other cetaceans, franciscana's karyotype possesses large amounts of C-heterochromatin and showed heteromorphism between homologous chromosomes (Figure 1 box b). The C bands were detected in the terminal regions in the short arms of chromosome pairs 1 to 9 (in the long arm of pair 5), in the proximal regions in the long arm of pairs 2 and 7, and in the pericentromeric regions of all the acrocentrics (Figures 1 and 3). In the X chromosome, the C band was observed in the terminal region. This banding was not detected in pairs 11 to 16 and chromosome Y.
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The order Cetacea is currently recognized to contain about 90 species divided in 14 families: Balaenidae (right whales), Neobalaenidae (pygmy right whale), Eschrichtiidae (gray whale), Balaenopteridae (rorqual whales), Physeteridae (sperm whale), Kogiidae (pygmy and dwarf sperm whales), Platanistidae (Ganges and Indus river dolphins), Ziphiidae (beaked whales), Lipotidae (baiji), Pontoporiidae (franciscana), Iniidae (Amazon river dolphin), Monodontidae (beluga and narwhal), Delphinidae (dolphins and small toothed whales), and Phocoenidae (porpoises) (Rice 1998). Cetacean karyotypes range from 2n = 42 to 2n = 44, with a clear prevalence of the last pattern (Arnason 1974). The karyotype of 2 cetacean families—Neobalaenidae and Platanistidae—has never been reported.
The phylogenetic and cytogenetic data compiled in this study (Figure 4) may throw some light on karyotype evolution in cetaceans. The most parsimonious hypothesis is that the ancestral character state in the group is the diploid number 42. An extra chromosome pair appeared independently twice along the cetacean evolution, once in the suborder Odontoceti and once in the suborder Mysticeti (positions 4 and 5, respectively, in Figure 4). It would be interesting to investigate the karyotype of the family Platanistidae in order to corroborate the hypothesis that the extrapair of chromosomes in the toothed whales appeared in position 4 of the phylogeny. Otherwise, the diploid number 44 would have evolved from position 3 and a reversion of this character state would have occurred in the family Ziphiidae.
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Opportunities for sampling live franciscanas and obtaining quality tissues for cell cultures are very rare. The dolphins are seldom seen alive and are not kept in captivity. The technique utilized in our study proved to be useful as an alternative in cytogenetic studies of a species from which it is difficult to collect samples from live animals. We are aware that the cytogenetic data presented here are limited because they are restricted only to 2 animals, and we hope to add to this data in further studies. Nevertheless, as there is a scarcity of opportunities to sample franciscanas and a lack of knowledge on its karyotype, even this preliminary information is significant. Moreover, the information presented here may be useful for future genetic mapping projects for the franciscana.
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The Conselho Nacional de Desenvolvimento Científico e Tecnológico of the Brazilian Government (CNPq) (141610/2003-4 to L.H. and 142863/2006-8 to P.C.C.). PRO-DOC Protax CAPES/CNPq/MCT to J. A.-M.
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We would like to thank Dr Andrés Delgado Cañedo, Dr Eduardo Secchi, and Andrea Adornes Conrado for support in collecting samples and in the cytogenetic preparations. To Ricardo Hegenbart for all of their expert technical assistance in provided the images of this manuscript. Previous drafts of the manuscript were improved by insightful comments from Dr Márcio Borges Martins and 2 anonymous reviewers.
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Corresponding Editor: Jill Pecon-Slattery
Received January 29, 2008
Accepted July 24, 2008
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