Comparative cytogenetic analyses of hatchetfishes Carnegiella marthae and Carnegiella strigata (Gasteropelecidae) from the Rio Negro basin were performed using conventional Giemsa staining, silver (Ag) -staining and C-banding. The diploid chromosome numbers of both species equaled 2n = 50 but their karyotypes were distinct. We found evidence for sex chromosomes in C. marthae since karyotype of males presented 20 M + 12 SM + 4 ST + 14 A and ZZ ST chromosomes while the females presented 20 M + 12 SM + 4 ST + 14 A and ZW ST chromosomes of distinct size. Conversely, C. strigata presented 4 M + 4 SM + 2 ST + 40 A chromosomes without sex chromosome heteromorphism. Karyotypes of both species had two NOR-bearing SM chromosomes of distinct size indicating the presence of multiple NOR phenotypes. The sex chromosome pair had specific C-banding pattern allowing identification of both Z and W. This heteromorphic system has previously been described for the gasteropelecids.
fish cytogenetics; karyotype differentiation; NOR phenotypes; heteromorphic sex chromosome system; cytotaxonomy
Comparative cytogenetics of Carnegiella marthae and Carnegiella strigata (Characiformes, Gasteropelecidae) and description of a ZZ/ZW sex chromosome system
Maria Leandra TerencioI; Carlos Henrique SchneiderI; Maria Claudia GrossI; Adailton Moreira da SilvaII; Eliana FeldbergI; Jorge Ivan Rebelo PortoI
ICoordenação de Pesquisa em Biologia Aquática, Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil
IICentro de Estudos Superiores de Parintins, Universidade Estadual do Amazonas, Parintins, AM, Brazil
Send correspondence to Send correspondence to: Jorge Ivan Rebelo Porto Coordenação de Pesquisa em Biologia Aquática Instituto Nacional de Pesquisas da Amazônia Caixa Postal 478, 69011970 Manaus, AM, Brazil Email: firstname.lastname@example.org
Comparative cytogenetic analyses of hatchetfishes Carnegiella marthae and Carnegiella strigata (Gasteropelecidae) from the Rio Negro basin were performed using conventional Giemsa staining, silver (Ag) staining and Cbanding. The diploid chromosome numbers of both species equaled 2n = 50 but their karyotypes were distinct. We found evidence for sex chromosomes in C. marthae since karyotype of males presented 20 M + 12 SM + 4 ST + 14 A and ZZ ST chromosomes while the females presented 20 M + 12 SM + 4 ST + 14 A and ZW ST chromosomes of distinct size. Conversely, C. strigata presented 4 M + 4 SM + 2 ST + 40 A chromosomes without sex chromosome heteromorphism. Karyotypes of both species had two NORbearing SM chromosomes of distinct size indicating the presence of multiple NOR phenotypes. The sex chromosome pair had specific Cbanding pattern allowing identification of both Z and W. This heteromorphic system has previously been described for the gasteropelecids.
Key words: fish cytogenetics, karyotype differentiation, NOR phenotypes, heteromorphic sex chromosome system, cytotaxonomy.
The family Gasteropelecidae (Characiformes) is a group of Neotropical fishes that includes three genera: Thoracocharax (two species), Gasteropelecus (three species), and Carnegiella (four species). Gasteropelecids inhabit major river basins of Central and South America, except those from southeastern Brazil. Almost all gasteropelecids have commercial value being very popular among fishkeeping hobbyists and mainly the Carnegiella species are widely exported throughout the world. They are commonly known as hatchetfish due to their ability to propel themselves out of the water, flapping their pectoral fins in flight either to evade predators or to catch insects (Weitzman, 1954; Weitzman, 1960; Géry, 1977; Weitzman and Palmer, 2003).
Karyology in gasteropelecids is very scarce and mostly is restricted to the record of chromosome number. Overall, only a single species of each genus (Gasteropelecus sternicla, Thoracocharax stellatus and Carnegiella strigata) has been the subject of cytogenetic studies. Apparently, Thoracocharax and Gasteropelecus possess higher diploid number (2n = 54 and 2n = 52) while Carnegiella possesses lower (2n = 50 and 2n = 48). In addition, a ZZ/ZW sex chromosome system was described for T. cf. stellatus (Carvalho et al., 2002).
Taking into account that understanding the genetic features of gasteropelecids might be of fundamental importance either for future plans related to conservation genetics or even to reveal aspects related to their evolution, this paper describes and compares the karyotypes of two Carnegiella species with other gasteropelecids and reports the occurrence of a ZZ/ZW sex chromosome system.
Cytogenetic analyses were performed on 22 specimens of blackwinged hatchetfish C. marthae (10 males and 12 females) and on 25 specimens of marbled hatchetfish C. strigata (10 males and 15 females) sampled in the middle Rio Negro basin, Amazonas, Brazil. Specimens were collected from opposite tributaries as follows: in Rio Preto (left margin 0° 9' 10" S, 63° 52' 58" W) three females and three males of C. marthae and five females and four males of C. strigata; in Igarapé Zamula (left margin 0° 50' 12" S, 62 45' 58" W) six females and four males of C. marthae and five females and three males of C. strigata; in Rio Unini (right margin 01° 42' 04" S, 61° 38' 21" W) three females and three males of C. marthae and five females and three males of C. strigata. The fish were registered and deposited in the collection of the Fish Genetics Laboratory at the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Amazonas, Brazil. Chromosomal preparations were obtained from kidney cells, following the airdrying technique of Bertollo et al. (1978). Constitutive heterochromatin was identified according to Sumner (1972) and nucleolar organizing regions (NORs) were detected using the technique described by Howell and Black (1980). Whenever possible, slides were stained sequentially with Giemsa, Cbanding and silver nitrate solution (AgNOR) according to the procedures described in Centofante et al. (2002). Chromosome morphology was classified as metacentric (M), submetacentric (SM), subtelocentric (ST), and acrocentric (A). We scored metacentric, submetacentric and subtelocentric chromosomes as biarmed and acrocentric as uniarmed to determine the chromosome arm number or fundamental number (FN).
We found that C. marthae had a modal chromosome number of 2n = 50, FN = 86 (20 M + 12 SM + 4ST + 14A) without population differentiation. However, different karyotypes were observed between the sexes since karyotype of males presented ZZ ST chromosomes while the females presented ZW ST chromosomes of distinct size. Heterochromatin was located in the centromeric and terminal regions in the majority of the chromosomes. The sex chromosome pair showed a specific Cbanding pattern allowing identification of both Z and W. The Z sex chromosome presented a large block of heterochromatin in the terminal position and the W showed heterochromatin in centromeric and terminal positions. AgNORs were analyzed in 41 metaphases. Interphasic nuclei showed a minimum of one and a maximum of three nucleoli and a maximum of two chromosomes bearing the NORs. After Giemsa/silver nitrate sequential staining the AgNORs were identified on the terminal position of the short arm of nonhomologous submetacentrics (pairs 11 and 15), suggesting multiple NORs (Figure 1).
Carnegiella strigata has a modal chromosome number of 2n = 50, NF = 60 (4 M + 4 SM + 2 ST + 40 A) without evidence of sexual dimorphism or population differentiation for this species. The constitutive heterochromatin appeared distributed mainly at the centromeric and terminal positions. AgNORs analyzed in 52 metaphases and interphasic nuclei showed a minimum of one and a maximum of three nucleoli, and a maximum of two chromosomes bearing the NORs. After Giemsa/silver nitrate sequential staining the AgNORs were identified on the terminal position of the short arm of nonhomologous submetacentrics (pairs 3 and 4), again suggesting multiple NORs (Figure 2).
The gasteropelecids are characterized by a nonconservative karyotype, with diploid number varying between 2n = 48 and 2n = 54 constituted by a large number of subtelocentric and acrocentric chromosomes (Carvalho et al., 2002). The two Carnegiella species studied here presented very distinct karyotypes. Carnegiella strigata presented many uniarmed chromosomes while C. marthae has mainly biarmed chromosomes demonstrating that several chromosomal rearrangements have occurred in the course of their karyoevolution. Previous cytogenetic studies conducted in C. strigata found different haploid numbers. In this study C. strigata presented a diploid number (2n = 50) that corroborates the findings of Hinegardner and Rosen (1972) who found a haploid number of n = 25, but did not corroborate the findings of Scheel (1973) who found a haploid number of n = 24. Unfortunately we have not yet been able to determine the karyotypes of Carnegiella myersi and Carnegiella shereri, and this is necessary to better understand chromosome evolution in the genus.
Although considered a less speciose fish family, when compared to other characiforms, the relationships within the Gasteropelecidae and their relationship with other characiforms are not fully resolved. Weitzman (1960) putatively considered Thoracocharax the least specialized genus while Gasteropelecus and Carnegiella would be more closely related to each other, with Carnegiella considered a neotenic form derived from Gasteropelecus. If this is true, then a scenario for chromosome evolution in gasteropelecids would consider 2n = 54 chromosomes a plesiomorphic character, as found in Thoracocharax and Gasteropelecus, and giving rise to 2n = 50 chromosomes of Carnegiella. It appears that chromosomal evolution in each genus followed its own path, basically reducing chromosome number. However, further studies will be necessary since Thoracocharax securis, Gasteropelecus maculatus, Gasteropelecus levis, C. myersi and C. schereri were not yet cytogenetically studied.
Regarding sex chromosomes, it is well known that Neotropical fish display an amazing variety of sex chromosome systems ZZ/ZW being the most common (MoreiraFilho et al., 1993). When the heteromorphic ZZ/ZW system was discovered in the gasteropelecid T. cf. stellatus (Carvalho et al., 2002) it was noticed that the ZW chromosomal pair presented both different morphology and different sizes (a large SM and a small A). In the genus Carnegiella, C. marthae presented its ZW chromosomal pair as subtelocentrics of distinct sizes (a large and a small). Thus, since we are reporting the second ZZ/ZW sex chromosome system for gasteropelecids, the occurrence of a ZZ/ZW system in two out of three gasteropelecid genera indicates that sex chromosome differentiation is not an isolated case as previously thought.
The evolution of sex chromosomes has been discussed at length and it is possible that cytogenetic differences result from structural rearrangements, such as inversions, translocations, duplications, and addition of heterochromatin (Ayling and Griffin, 2002). Considering that less than half of the gasteropelecids were cytogenetically analyzed, there is no a priori reason to assume that the ZZ/ZW sex chromosome system has a common origin in this clade.
With respect to the nucleolar organizing region, it was evident that there are two positive AgNORs occurring on nonhomologous chromosomes. This leads us to putatively suggest a case of multiple NORs for both Carnegiella species. Variation in copy number of the rDNA repeating units or even inactivation of transcription in some rDNA clusters may explain this behavior. Heterozygous combinations where only one of the homologues bears NORs is not uncommon in cytogenetics literature on fish, as described for the Neotropical fish groups Curimatidae (Feldberg et al., 1992), Serrasalminae (Nakayama et al., 2002) and Tetragonopterinae (Gross et al., 2004). However, an analysis of nucleolus organizer regions (NORs) by Fluorescence In Situ Hybridization (FISH) with 18S rDNA will be needed to confirm how many ribosomal cistrons are in fact involved in the organization of NORs.
Overall, the present results should be useful for further cytotaxonomic and cytosystematic studies within the family Gasteropelecidae taking into account the level of chromosomal differentiation between species, the second description of a ZZ/ZW sex chromosome system in gasteropelecids, the evidences for multiple NORs in Carnegiella and the fact that the study samples did not differ across the tributaries in the Rio Negro basin.
The authors wish to thank CNPq/PRONEX for financial support, Turkys Aquarium and K2 Tropical Fish for providing some specimens and Richard Carl Vogt for the English review of the manuscript. M.L.T. and A.M.S received Technological Development (DTI) scholarships while C.H S and M.C.G received M.Sc. scholarships from CNPq.
Received: August 22, 2006; Accepted: April 18, 2007.
Associate Editor: Pedro Manoel Galetti Junior
License information: This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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22 Aug 2006