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Journal of Experimental Marine Biology and Ecology 407 (2011) 97–100

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Journal of Experimental Marine Biology and Ecology

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Molecular cytogenetic analysis of Haemulidae ﬁsh (Perciformes): Evidence of evolutionary conservation ☆

Clóvis Coutinho Motta Neto a, Marcelo Belo Ciofﬁ b, Luis Antônio Carlos Bertollo b, Wagner Franco Molina a,⁎ a Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil b Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil article info abstract

Article history: Several fish species belonging to the family Haemulidae present a karyotype consisting of 48 acrocentric Received 9 May 2011 chromosomes (FN=48), and apparently similar chromosomal microstructure, especially in genus Haemulon, Received in revised form 13 July 2011 representing a striking example of intrafamiliar chromosomal conservation. In this study, a more detailed Accepted 18 July 2011 cytogenetic analysis of the species Conodon nobilis and Pomadasys corvinaeformis was performed using C-banding, Available online xxxx Ag-NOR, DAPI/CMA3 staining, in situ digestion by distinct endonucleases and double-FISH to map the 18S and 5S ribosomal genes. Both species showed a similar karyotypic macrostructure with 2n=48 acrocentric Keywords: Marine fish chromosomes and active ribosomal sites at interstitial position on long arms of chromosomal pair 18 and 24 + - Repetitive sequences in P. corvinaeformis and C. nobilis, respectively. These sites were the only CMA3 /DAPI regions in the karyotype. FISH mapping Digestion with restriction enzymes revealed a low number of digestion sites in the heterochromatic segments of Chromosomal evolution both species. The data indicate some degree of interspecific evolutionary diversification At the microstructural level, incorporated in a general pattern of extensive karyotypic conservatism. Thus, the interspecific reproductive isolation leading to phyletic diversification apparently occurred without the contribution of conspicuous karyotypic changes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction to ﬁve different genera (Pomadasys, Conodon, Orthopristis, Haemulon and Anisotremus) indicate that a basal karyotype, with 2n=48 In some ﬁsh groups, the evolutionary changes in morphology appear acrocentric chromosomes, has been largely maintained (Accioly and to be greater than those seen in their karyotypic structure (Molina et al., Molina, 2007; Motta Neto et al., 2011; Nirchio et al., 2007). This 2002). This contrast is found in some Perciformes species, making them karyotype is also present in most families of this order. Thus, to better an appropriate model for evolutionary cytogenetic analyses. In determine the degree of chromosomal conservatism present in this particular, many groups of this order, which is the largest order of family, a more detailed cytogenetic analysis of species belonging to two vertebrates, share a conserved chromosomal structure (i.e., they exhibit Haemulidae genera, the barred grunt Conodon nobilis (Linnaeus, 1758) karyotypic stasis) (Accioly and Molina, 2008; Galetti et al., 2000; Motta and the roughneck grunt Pomadasys corvinaeformis (Steindachner,

Neto et al., 2011; Rocha and Molina, 2008). 1868), was performed using C-banding, AgNO3 staining, CMA3/DAPI However, this general karyotype conservation among Perciformes staining, in situ digestion by distinct restriction endonucleases (AluI, has not yet been well established once the karyotype stasis might be EcoRI, PstIandTaqI) and double-FISH to map the 18S and 5S ribosomal related to the absence of high-resolution cytogenetic analyses. genes. This approach provided an in-depth characterization of the Therefore, microstructural karyotypic variation that would represent karyotype of these fish, particularly with respect to the distribution of RE chromosomal diversification and genomic restructuring can be over- digestion sites throughout the chromosomes. looked (Mazzei et al., 2006; Nirchio et al., 2007; Pisano et al., 2003; Pisano and Ozouf-Costaz, 2000). 2. Materials and methods Haemulidae is one of the largest Perciformes families (Nelson, 2006). Cytogenetic analyses performed on 14 species of this family belonging The C. nobilis sample (N=16) included eight males, seven females and one immature individual. The P. corvinaeformis sample (N=15) included seven males, six females and two immature individuals. All fish ☆ This paper has not been submitted elsewhere in identical or similar form. in this study originated from the coastal city of Natal (5°13′1.73″S 35°9′ ⁎ Corresponding author at: Departamento de Biologia Celular e Genética, Centro de 57.85″W), State of Rio Grande do Norte, on the northeastern Brazilian Biociências, Universidade Federal do Rio Grande do Norte, Campus Universitário, s/n, coast. Lagoa Nova, CEP 59078–970, Natal, RN, Brazil. Tel.: +55 84 321119209; fax: +55 84 32153346. The specimens were subjected to mitotic stimulation in vivo for 24 h E-mail address: [email protected] (W.F. Molina). by the injection of attenuated antigenic compounds (Molina, 2001;

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Molina et al., 2010), anesthetized with clove oil (Eugenol) and sacriﬁced. Mitotic chromosomes were obtained by interrupting the mitotic cycle in vitro using fragments of the anterior kidney (Gold et al., 1990). Heterochromatic regions and nucleolar organizer regions (NORs) were identiﬁed using C-banding and silver staining methods (Howell and Black, 1980; Sumner, 1972), respectively. Staining with the

fluorochromes CMA3 and DAPI was performed according to the method described by Schweizer (1980). DNA restriction digests using AluI, EcoRI, PstIandTaqI were performed. The chromosomal digestion patterns, using 0.3 units/μL of each enzyme, were obtained using the following treatment protocol: AluI (AG/CT) at 37 °C for 3 h, EcoRI(GAATTC)at 37 °C for 10 h, PstI (C TGCA/G) at 37 °C for 8 h, and TaqI(TCGA)at65°C for 8 h. After each treatment, the slides were washed in distilled water, stained with 5% Giemsa diluted in phosphate buffer (pH 6.8) for 10 min and air dried. Fluorescent in situ hybridization (FISH) was performed on mitotic chromosome spreads according to Pinkel et al. (1986). The 5S and 18S rDNA sequences were detected by double-FISH analysis. The two ribosomal sequences were isolated from the Hoplias malabaricus Fig. 1. Karyotypes of C. nobilis (a and b) and P. corvinaeformis (c and d). The chromosomes, (Teleostei, Characiformes) genome. The 5S rDNA repeat copy included which were analyzed by Giemsa staining (a and c) and C-banding (b and d), showed 120 base pairs (bp) of the 5S rRNA encoding gene and 200 bp of the heterochromatic blocks at pericentromeric region of all chromosomes in both species. non-transcribed spacer (NTS) (Martins et al., 2006). The 18S rDNA The nucleolar organizing pairs are highlighted. Bar=5 μm. probe corresponded to a 1,400 bp-segment of the 18S rRNA gene, obtained via PCR from nuclear DNA (Cioffi et al., 2009). The 5S rDNA species make it difficult to establish precisely homologies for all partial sequence were cloned into plasmid vectors and propagated in chromosomes. DH5α E. coli competent cells (Invitrogen, San Diego, CA, USA). The 5S rDNA probe was labeled with biotin-14-dATP by nick translation 4. Discussion according to manufacturer's recommendations (BioNickTM Labeling System; Invitrogen, San Diego, CA, USA). The 18S rDNA was labeled by The karyotypes of C. nobilis and P. corvinaeformis comprised nick translation with DIG-11-dUTP, according to manufacturer's 2n=48 acrocentric chromosomes, regarded as a plesiomorphic trait instructions (Roche, Mannheim, Germany). for Perciformes (Galetti et al., 2000). In addition, these karyotypes Approximately thirty metaphases were analyzed for each specimen possibly represent a synapomorphy for Clupeomorpha and Euteleos- to confirm the modal diploid number. The best metaphases were tei (Brum and Galetti, 1997). photographed using an Olympus BX50 epifluorescence photomicro- Interstitial NOR sites in a single acrocentric pair were observed and scope, equipped with an Olympus DP70 digital image capture. The given that they are also present in other Haemulidae species (Motta metaphase images were captured using CoolSNAP-Pro software (Media Neto et al., 2011), they may represent a conserved condition for this Cybernetic). The chromosomes were classified and sorted according to fish family, as well as for others from the same order, such as the nomenclature developed by Levan et al. (1964). Serranidae (Aguilar and Galleti, 1997), Sciaenidae (Feldberg et al., 1999) and Lutjanidae (Rocha and Molina, 2008). This condition may result in a decreased evolutionary dynamism of these sites (Accioly 3. Results and Molina, 2008), accounting for their stability in many Perciformes species. The presence of ribosomal sites in the pair 18, as observed in The two species had a similar karyotype macrostructure, with P. corvinaeformis and other Haemulidae genera, such as Anisotremus 2n=48 acrocentric chromosomes, thereby presenting a number of arms or fundamental number (FN) of 48. Conventional Giemsa staining revealed a conspicuous secondary constriction proximal to the centromere in one of the homologs of chromosome pair 24 in C. nobilis and in both homologs of the pair 18 in P. corvinaeformis. The correspondence between these regions and ribosomal sites was confirmed by silver nitrate staining (Ag-NORs) and hybridization with 18S rDNA probes (Figs. 1a, c and 2b, d). The Ag-NORs sites (Fig. 1a, c) + - were the only chromosomal regions that exhibited a CMA3 /DAPI pattern (Fig. 2a, c). C-banding revealed reduced heterochromatic blocks at centromeric and pericentromeric regions of all chromosomes in both species (Fig. 1b, d). The mapping of the 18S and 5S rDNA sequences by double-FISH showed an absence of synteny between the ribosomal subunits in both species. While the 18S rDNA sites were located near the centromere in chromosome pairs 24 and 18 of C. nobilis and P. corvinaeformis, respectively, the 5S rDNA sites were located at terminal region of the long arms in chromosome pair 5 in both species. An additional site was located at centromeric region of chromosome pair 10 in C. nobilis. A low number of digestion sites seem to be present in heterochro- Fig. 2. Karyotypes of C. nobilis (a and b) and P. corvinaeformis (c and d) after staining with matic regions of both species (Fig. 3), yielding a faint staining pattern fluorochromes CMA3 and DAPI (a and c) and double-FISH with 18S (red signals) and 5S when compared to C-banding. The restriction enzyme digestion + + - (green signals) rDNA probes (b and d). The GC (CMA3 /DAPI ), 5S and 18S sites are patterns and the karyotype symmetry of chromosome pairs in these highlighted. Bar=5 μm. Author's personal copy

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leading to the chromosomal homeostatic state apparently preserved in Haemulidae. In fact, more detailed cytogenetic data in this family indicate some degree of interspeciﬁcevolutionarydiversiﬁcation at the microstructural level, incorporated in a general pattern of extensive karyotype conservatism.

Acknowledgements

The authors wish to thank CNPq (Proc. 556793/2009-9) and FAPESP (Proc. 2009/14881-3) for ﬁnancial support and for the graduate scholarship granted to CCMN (Proc. 579522/2008-3), UFRN for

Fig. 3. Chromosomal digestion patterns in C. nobilis—Cn (a–d) and P. corvinaeformis—Pc providing the appropriate conditions required to perform this study (e–h) with EcoRI, AluI, PstI and TaqI. The arrows indicate heterochromatic regions and to José Garcia Jr. for the taxonomic identiﬁcation of specimens. [RH] presenting non-digested sites to each restriction nuclease. Bar=5 μm. References

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