Karyotypic diversification in two Atlantic species of Holocentridae (Pisces, Beryciformes)

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1 CARYOLOGIA Vol. 57, no. 3: , 2004 Karyotypic diversification in two Atlantic species of Holocentridae (Pisces, Beryciformes) T. O.F. Bacurau and W. F. Molina * * Departamento de Biologia Celular e Genética, Universidade Federal do Rio Grande do Norte, Centro de Biociências, Campus Universitário, , Natal-RN. Brazil. Abstract There are few cytogenetic data on marine fish, particularly in some little Orders, such as the Beryciformes, although some emphasis has been placed on some isolated groups. In this study, the karyotypes of Holocentrus ascensionis and Myripristis jacobus species (Holocentridae) were described showing differentiated chromosome characteristics for diploid number, FN and frequency and localization of NORs sites. Such accentuated divergences may reflect the polyphiletism in this group previously suggested by mtdna analyses. Key words: Beryciformes, fish cytogenetics, Holocentrus, Myripristes. INTRODUCTION Karyotype analysis is a valuable tool in identifying chromosome markers to establish phylogenetics relationship and evolution among taxonomic groups. However, most of the cytogenetic data existing on marine fish refers to Perciformes species, where the most frequently observed diploid number is 48 chromosomes. There is a significant lack of studies on groups with a smaller number of representatives. The interpopulational genetic variation in marine species has been shown to be smaller than that found in freshwater fish, due apparently to a greater genetic flow (Lacson 1992; Ward et al. 1994). Thus in the marine environment, bearing in mind the lower capacity to identify effective interpopulational cytogenetic markers, population differences are frequently observed when analyzing samples with a wider geographic distribution (Molina 2000; Sena 2003). Among the Beryciformes, the Holocentridae family is widely distributed geographically and is represented by 65 species, distributed in eight genera. To date there are no chromosome studies among these fishes. The objective of this study was to characterize cytogenetically two Holocentridae species, Holocentrus ascensionis and Myripristis jacobus, from the Northeast coast of Brazil and the isolated St. *Corresponding author: fax , molinawf@yahoo.com.br. Paul Rocks, using conventional staining techniques, Ag-NORs and c-banding. MATERIALS AND METHODS Cytogenetic analyses were set up from fourteen Holocentrus ascensionis specimens, collected in Rio Grande do Norte and Ceará and from sixteen Myripristis jacobus specimens from Rio Grande do Norte, Salvador (northeast coast of Brazil) and the St. Paul Rocks, located approximately 960 km from the Brazilian coast (Figure 1). All individuals were submitted to mitotic stimulation for 24 hours (Lee and Elder 1980; Molina 2002) before obtaining the mitotic chromosomes, using renal tissue, according to the in vitro method (Gold et al. 1990). The nucleolar organizing regions were identified by the Ag-NORs method (Howell and Black 1980), while the constitutive heterochromatin was shown by C-banding (Sumner 1972). The chromosomes were classified according to Levan et al. (1964). RESULTS The analysis of Holocentrus ascensionis (302 metaphases) showed a diploid value of 2n=50 chromosomes (2m+6sm+16st+26a; FN=74) (Figure 2). Ribosomal sites were localized on the telomeric portions of the short arm of the 2 nd submetacentric pair and on the 12 th subtelometric pair. Visible heterochromatic blocks were distributed in the pericentromeric regions and less frequently in the telomeric regions (Figure 2).

2 karyotypic diversification in two atlantic species of holocentridae (pisces, beryciformes) 301 Fig. 1 Karyotype of Holocentrus ascensionis, a. conventional staining with Giemsa; b. C-banding. NORs-bearing pairs (2 nd and 22 th ) are in the box.

3 302 bacurau and molina Fig. 2 Karyotype of Myripristis jacobus, a. conventional staining with Giemsa. The nucleolar organizer pair (1 st pair) is shown in the highlighted box. b. pattern of constitutive heterochromatin distribution. Myripristis jacobus (363 metaphases) presented a karyotype with 2n=48 acrocentrics (FN=48). Ag- NORs were identified on the telomeric region of the long arm of the 1 st chromosome pair. Heterochromatin regions were identified in the pericentromeric regions of all the chromosome pairs. A conspicuous heterochromatin block was observed on the 11 th pair in the telomeric region (Figure 3). DISCUSSION Numerical and structural chromosome patterns detected in Holocentrus ascensionis and Myripristis jacobus show the karyotypic diversity found in the Holocentridae family. Thus while the first species presented 2n=50, with a karyotype showing all the chromosome types and multiple Ag-NORs, the other showed 2n=48a and FN=48, with a unique Ag- NORs and heterochromatin distributed mainly in the pericentromeric regions of all the chromosomes. The chromosome dynamic found in the Holocentridae has also been observed in other groups. In the Melamphaidae family, belonging to the Stephanoberyciformes Order, that is closely related to the Beryciformes, different diploid numbers have been described among the two species analyzed, Melamphaes parvus, 2n=50 and Scopelogadus mizolepis bispinosus, with 2n=46 chromosomes (Ohno 1974). In some cases, marked differences in the numbers and macrostructures can be a reflection of a polyphyletic condition.

4 karyotypic diversification in two atlantic species of holocentridae (pisces, beryciformes) 303 A diploid value with 2n=60 chromosomes is probably a basal condition for the Teleostei (Brum 1995), thus the evolutionary reduction in the chromosome number must have arisen through fusions and deletions until fixing at lower chromosome numbers such as the 48 chromosomes conservatively maintained in the Perciformes. This basal karyotype remained practically unaltered in the marine species of Percomorpha, becoming more derived with wider diploid number according to the group, for increasing diploid values (50, 52, 54) (Brum 1995). The localization of ribosome sites on a single acrocentric pair in Myripristis jacobus, interstitially close to the centromere, has been considered as a common condition for many Perciformes species (Affonso 2000). Thus any variation of this pattern should be considered as a derived condition (Brum 1995, 1996; Ozouf-Costaz et al. 1997; Aguilar and Galetti 1997). The presence of nucleolar organizing regions and divergent karyotypes, found in the Holocentridae, suggest the occurrence of intense chromosome rearrangements (centric fissions and pericentric inversions) in this group. The absence of more cytogenetic data on the family limits the use of the Ag- NORs sites as effective cytotaxonomic markers. However, in Perciformes and correlated groups with many chromosome data, the information from the number and distribution of the Ag-NORs has been effective in understanding the karyotypic evolution as identified in Serranidae, Pomacanthidae, Pomacentridae (Aguilar and Galetti 1997; Affonso 2000; Molina 2000). The heterochromatin patterns observed in Holocentrus ascensionis and Myripristis jacobus shows the presence of heterochromatin segments concentrated in the pericentromeric regions and in some chromosomes in the telomeric region. A condition of low heterochromatin contents normally reduces or eliminates the dynamic to changes given by the heterochromatic segments in the karyotypes of the species (Molina 2000). In an attempt to establish an evolutionary pattern for the fishes, Gosline (1971) divided the Teleostei group into three categories, basal, intermediary and derived groups, taking into consideration the chromosome number and the FN. In this division, the Beryciformes were placed in an intermediary position, while the Perciformes were placed among the derived groups. The data indicate a wide distribution of diploid values with many chromosomes with two arms, mainly in the base group. The cytogenetic characteristics of the intermediary group suggest there is a transition from the basal group with numerical variation about 50 chromosomes to 2n=48 of the derived group. The cytogenetic homogeneity observed in the geographic samples of Holocentrus ascensionis and Myripristis jacobus may be a reflection of genetic flow among them observed by RAPD analysis (Bacurau 2003), due to the prolonged pelagic period of the species (Tyler et al. 1993). The data obtained still do not allow identification of cytogenetics patterns for the Holocentridae. However, in view of the karyotypic diversity observed, the existence of a different situation from Perciformes is plausible, with the absence of a modal diploid number for the family. Analyses of a greater number of species would permit the establishment of chromosome symplesiomorphies that better define the Beryciformes. Acknowledgements The authors are grateful to Dr. Paulo Roberto de Melo Affonso, for assistence during fish collection. 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