Derived Cytogenetic Traits, Multiple Nors and B Chromosomes in the Compact Karyotype of Canthigaster Figueiredoi (Tetraodontifor

Marine Genomics 3 (2010) 85–89

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Marine Genomics

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Derived cytogenetic traits, multiple NORs and B chromosomes in the compact karyotype of Canthigaster ﬁgueiredoi (Tetraodontiformes)

Pablo Ariel Martinez ⁎, Washington Candeia de Araujo, Wagner Franco Molina

Universidade Federal do Rio Grande do Norte (UFRN), Departamento, de Biologia Celular e Genética, Centro de Biociências, Lagoa Nova s/n, CEP 59078-970, Natal – Rio Grande do Norte – Brasil article info abstract

Article history: The Tetraodontiformes are one of the main irradiation lineages of Teleosts representing a highly specialized Received 22 June 2010 group. Among its families, Tetraodontidae has the lowest DNA/cell content of the vertebrates, and for this Received in revised form 21 July 2010 reason has been used as a model group for genomic evolution studies. Seeking to widen the cytogenetic Accepted 22 July 2010 database of this family, we performed a chromosomal analysis of the species Canthigaster ﬁgueiredoi using

conventional staining, Ag-NORs, C-banding, base-specific fluorochromes DAPI-CMA3, and in situ hybridiza- Keywords: tion with 18 S rDNA probe. This species has 2n=36 (10 m+6sm+20a; FN=52). Multiple Ag-NORs Tetraodontidae (CMA+) sites were detected on the four chromosome pairs. Heterochromatic blocks (CMA+) were present in B chromosomes 3 3 Pufferfish the centromeric regions of most of the pairs, extending on the short arm of some chromosomes. A GC- Heterochromatic polymorphism positive polymorphic heterochromatic region was identified in some of the individuals, in one or both of the homologs of the 6th pair. The presence of heteromorphic B microchromosomes was detected in the karyotype of one female, exhibiting intra-individual variation of 0–3 Bs. The occurrence of heterochromatic polymorphisms, multiple NORs, and B chromosomes in C. figueiredoi are very infrequent events in marine fish. They are probably associated to the accentuated restructuring and genomic reduction suffered by this family. © 2010 Elsevier B.V. All rights reserved.

1. Introduction may possibly play an important role in speciation in marine environments, through the reproductive isolation of populations. Neotropical fish have demonstrated high karyotypic diversity, Despite the large number of living species, the appearance of including variations in diploid number, presence of sex chromosomes, cytologically different sex chromosomes in marine fish has been B chromosomes, natural triploids, heterochromatin polymorphism, as rare (Morescalchi et al., 1992). Nevertheless, simple and multiple sex well as nucleolus organizer region (NORs) polymorphism (Galetti, 1998). chromosome systems have been described in the Tetraodontiformes In the wide diversity of marine fish, the Order Tetraodontiformes (Sá-Gabriel & Molina, 2004). B chromosomes are also associated to stands out as being one of the main irradiation lines of the teleosts the dynamic conditions of the karyotype. Alternatively called (Lauder & Liem, 1983; Nelson, 2006), as well as being a highly supranumerary or accessory chromosomes, they are dispensable specialized group. The family Tetraodontidae has the lowest DNA elements present in some individuals or populations (Beukeboom, content per cell among the vertebrates (Neafsey & Palumbi, 2003; 1994). Their possible origin is associated to A chromosome rearrange- Noleto et al., 2009). The overall karyotypic patterns of this group are ments, exhibiting from that point on, and their own evolutionary enormously varied, consisting of representatives that exhibit Perci- pathway (Beukeboom, 1994). They have been described for a large of formes-like traits (karyotype composed of 48 acrocentric chromo- number of plant and animal species (Jones & Rees, 1982; Camacho et somes), such as those found in Triacanthidae (Choudhury et al., 1982), al., 2000), and have been observed in more than 40 freshwater fish as well as more derived karyotypes with diploid numbers ranging species (Oliveira et al., 2000). B chromosomes seem to be less from 28 to 52 in the other families (Balistidae, Ostraciidae, Tetra- frequent in marine fish and have been described in Upeneus parvus odontidae and Diodontidae)(Galetti et al., 2006). In addition to (Perciformes) (Pauls et al., 1996), Sphoeroides spengleri and Chilo- numerical and structural chromosomal variations, the specific condi- mycterus spinosus (Tetraodontiformes) (Alves et al., 2008; Noleto, tions present in the karyotype of a number of species, such as different 2009), among others. sex chromosomes, are indicators of karyotypic diversification and Among the Tetraodontidae, the subfamily Canthigasterinae is the most derived group, and despite their unique biological traits, cytogenetic data is scarce. This study presents the karyotypic ⁎ Corresponding author. Departamento de Biologia Celular e Genética, Centro de fi Biociências, Universidade Federal do Rio Grande do Norte, Brasil. Tel.: +54 8487285054. characterization of Canthigaster gueiredoi, using conventional stain- E-mail address: [email protected] (P.A. Martinez). ing, silver impregnation, C-banding, and staining by DAPI/CMA3

fluorochromes, revealing aspects of its heterochromatic fraction and 8th, and 11th chromosome pairs (Fig. 1a), a condition that was describing B chromosomes. confirmed by in situ hybridization with 18 s rDNA probes (Fig. 1d). C-banding identifies heterochromatic regions in most of the 2. Material and methods centromeric regions, as well as in the nucleolus organizer regions. Furthermore, a conspicuous heterochromatic block was localized on The specimens of Canthigaster figueiredoi (Moura & Castro, 2002) the long arm in the telomeric region (Fig. 1b), in one of the homologs (n=11; 4 females, 7 juveniles) were collected on the coast of Bahia of the 6th chromosome pair in the karyotype of females. Juveniles (12o58′S, 38o31′W), NE of Brazil. They underwent mitotic stimulation whose sex could not be defined exhibited only one or two homologs using the technique proposed by Molina (2001), for 24 to 48 h. All bearing the heterochromatic block. When submitted to fluorochrome specimens were then anesthetized with clove oil (Eugenol) and staining with DAPI/CMA3, this heterochromatic region proved to be sacrificed for extraction of the cephalic kidney. Mitotic chromosome rich in GC bases (Fig. 1c) and was heteropycnotic in the karyotype of preparations were obtained according to the methodology described two individuals, even under the conventional Giemsa staining by Gold et al., (1990). The silver impregnation technique developed (Fig. 1a). by Howell & Black (1980) was used to detect active ribosomal sites. Heteromorphic B microchromosomes were found in one of the Heterochromatic patterns were determined using the C-banding females analyzed. These microchromosomes showed individual method (Sumner, 1972), while fluorochrome staining with DAPI/ variation of 0 to 3 Bs. Among the cells analyzed, 47% did not present

CMA3 followed the Schweizer (1980) protocol. In situ fluorescent any B chromosomes, 33% had one, 14% two and 6% three B hybridization was based on the procedure adopted by Pinkel et al., chromosomes. Structurally, the B chromosomes were small differ- (1986) with slight alterations. The 18 S rDNA probes were obtained by ent-size acrocentric chromosomes that were shown to be hetero- polymerase chain reaction (PCR) amplification of the DNA of chromatic when submitted to C-banding (Fig. 2) and did not exhibit Prochilodus argenteus (Hatanaka & Galetti, 2004) and were marked ribosomal sequences (Fig. 1d). with biotin-11-dATP, using nick translation, and the BionickKTM Labeling System kit (Gibco.BRL), following the manufacturer specifi- 4. Discussion cations. The metaphases were photographed with a DP70 digital camera system coupled to an Olympus BX50 epiflorescence micro- Previous cytogenetic analysis conducted in species of the genus scope using DPController 1.2.1.108 (Olympus) software. The chromo- Canthigaster showed lower diploid numbers than the other members somes were classified as to centromere position into metacentric (m), of the order, such as C. coronata, with 2n=28 (Arai, 1983), C. rivulata, submetacentric (sm), subtelocentric (st) and acrocentric (a). 2n=34 (Arai & Nagaiwa, 1976) and in the present study, C. figueiredoi with 2n=36 chromosomes. This data reinforces the phylogenetic 3. Results proposals based on molecular data indicating that the subfamily Canthigasterinae is one of the most derived of the Tetraodontiformes Cytogenetic analyzes in C. figueiredoi showed a diploid number (Holcroft, 2005). + + + of 2n=36 chromosomes, with a karyotype formula composed of Ribosomal sites exhibiting Ag-NORs ,C , CMA3 profile, and with 10sm+6st+20a (FN=52). Ag-NOR sites were located on 3rd, 4th, signs of hybridization with 18 S rDNA probes, have been frequently

Fig. 1. Karyotypes of Canthigaster ﬁgueiredoi arranged from Giemsa-stained (a) and C-banded chromosomes (b). The nucleolar organizer pairs are boxed. In (c) and (d), respectively, the DAPI/CMA3 and 18 S rDNA sequences. Note the conspicuous terminal heteromorphic or homomorphic heterochromatic block on the long arms of the pair 6. Bar=5 μm. P.A. Martinez et al. / Marine Genomics 3 (2010) 85–89 87

Fig. 2. Metaphase plates of a female Canthigaster figueiredoi showing one (a), two (b) or three (c) heteromorphic B chromosomes. In (d), heterochromatic pattern present in B chromosome. Bar=5 μm. reported in fish (Amemiya & Gold, 1986; Phillips et al., 1988; Sola et The repetitive DNA sequences account for a substantial fraction of the al., 1992), and specifically in some species of Tetraodontiformes, such genome of many eukaryotes (Charlesworth et al., 1994)andmaybe as Sphoeroides greeleyi, S. testudineus and Cyclichthys spinosus (Noleto dispersed or in tandem repetitions. The size of the repetition units of the et al., 2007). Their repetitive sequences, which were found adjacent or different satellite DNA families is quite variable, oscillating from several interspersed throughout the NORs, have been shown to favor base pairs to hundreds of nucleotides (Koehler et al., 1997). This class of rearrangements involving nucleolus organizer pairs (Vicari et al., DNA is found mainly in centromeric and telomeric regions (Franck et al., 2003). The increased number of copies in these sites is promoted by 1991), as well as associated to heterochromatic regions (Stephan & Cho, frequent unequal changes between adjacent repetitive sequences. The 1994). It is considered that both structural and compositional rearrange- association of tandem repeat genes, such as histones or rDNA, with ments and heterochromatinization are the first steps in differentiating sex heterochromatic regions is exhibited by a number of eukaryotes, such chromosomes (Ohno, 1974), which can be identified at several as Drosophila (Fitch et al., 1990), Xenopus (Pardue et al., 1973) and the differentiation stages (Cano et al., 1996; Bertollo et al., 1997). salamander Notophthalmus (Stephenson et al., 1981). The presence of The fact that females of C. figueiredoi are heterozygotous in terms this chromosomal trait in lower and upper teleosts is characteristic of of heterochromatic polymorphism in the telomeric region of the long convergent evolution. arm on the 6th chromosome pair, suggests a possible relation with a In most species of Tetraodontiformes, simple Ag-NOR sites were ZZ-ZW sex-determination system, an uncommon condition in marine reported (Sá-Gabriel & Molina, 2004; Noleto et al., 2007; Fischer et al., fish, but previously reported for Tetraodontiformes. Simple XX-XY 2000a). Given the cytogenetic data available for Tetraodontiformes, systems have been described in this order for the balistids multiple Ag-NOR sites are a derived and unusual condition (Molina & Rhinecanthus verrucosus and R. aculeatus (Ojima, 1985), an XX-XO Bacurau, 2006). Two main mechanisms have been proposed to system in the triacanthids Triacanthus brevirostris (Choudhury et al., explain the intra and interspecific variations in the number of NORs: 1982), and multiple X1X1X2X2-X1X2Y systems in the monacanthids i) ectopic recombination between the terminal regions of chromo- Stephanolepis cirrhifer (Murofushi, 1980) and S. hispidus (Sá-Gabriel & somes (Hanson et al., 1996; Pedrosa-Harand et al., 2006) and ii) rDNA Molina, 2004). In Tetraodontidae there is a record of X1X1X2X2-X1X2Y transposition acting as mobile elements, varying in size, number, and sex chromosomes in Arothron nigropunctatus (Ojima, 1985). Given the position in the chromosomes (Shishido et al., 2000; Cai et al., 2006; examples existing for this order, where the systems described show Datson & Murray, 2006). male sex determination, the presence of ZW chromosomes in C. 88 P.A. Martinez et al. / Marine Genomics 3 (2010) 85–89

figueiredoi would corroborate the lability of sex determination in fish such as the presence of heterochromatic polymorphisms, multiple (Devlin & Nagahama, 2002; Mank et al., 2006). NORs and B chromosomes are rare events in marine fish, but largely The occurrence of B chromosomes in marine fish has been rare, present in C. figueiredoi. This seems to reflect the marked restructuring however, the present study is the third report in the family and genomic reduction that took place during the karyotypic Tetraodontidae. This condition may be associated to the marked evolution of this family. restructuring (Mandrioli & Manicardi, 2001; Sá-Gabriel & Molina, 2005) and selective genomic reduction that this family was submitted to (Neafsey & Palumbi, 2003), showing approximately the same Acknowledgements complement of genes as other vertebrates (Brenner et al., 1993). The genomic reduction in this group was presumably caused by the loss of The authors wish to thank the Conselho Nacional de Desenvolvi- repetitive DNA or of other non-codifying sequences (Neafsey & mento Científico e Tecnológico (CNPq — Proc. 556793/2009-9) and Palumbi, 2003), resulting in similar-size exons and a marked the Programa de Pós-graduação em Ecologia of the Universidade difference in intron size compared to humans (Jaillon et al., 2004). Federal do Rio Grande do Norte, for their support. We would also like It is widely accepted that B chromosomes derive from A to thank Dr. José Garcia Jr. for the identification of Canthigaster chromosomes (Jones & Rees, 1982). From this perspective the origin figueiredoi. of B chromosomes can be considered as a subproduct of normal karyotypic evolution, possibly derived from fragments of centric fusions or amplifications of fragments from the paracentric regions of References A chromosomes (Camacho et al., 2000). 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