© 2013 The Japan Mendel Society Cytologia 78(3): 223–234 Cytogenetic Comparison and Chromosome Localization of the Nucleolar Organizer Region of Four Grouper Genera (Pisces, Epinephelinae) from Thailand Krit Pinthong1, Bhuvadol Gomontean1, Bungon Kongim1, Suthip Khakhong2, Tawat Sriveerachai3, and Weerayuth Supiwong4* 1 Department of Biology, Faculty of Science, Mahasarakham University, Kantarawichai, Maha Sarakham 44150, Thailand 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Muang 83000, Thailand 3 Phuket Coastal Research and Development Center, Phuket, Muang 83000, Thailand 4 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand Received July 23, 2012; accepted March 11, 2013 Summary We report the first cytogenetic comparison of four grouper genera from Thailand. Kidney cell samples were taken from the blueline hind (Cephalopholis formosa), humpback grouper (Cromileptes altivelis), orange-spotted grouper (Epinephelus coioides), and leopard coralgrouper (Plectropomus leopardus). The mitotic chromosome samples were prepared directly from the kidney cells. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that diploid chromosome numbers of Ce. formosa, Cr. altivelis, E. coioides, and P. leopardus were 2n=48 for all species, and the fundamental numbers (NF) were 52, 52, 50, and 48, respectively. The presence of metacentric, submetacentric, acrocentric, and telocentric chromosomes were 2-0-2-44, 0-2-2-44, 0-0-2-46, and 0-0-0-48, respectively. After the Ag-NOR banding tech- nique, one pair of nucleolar organizer regions (NORs) was observed on the short arm telomeric re- gion of chromosome pair No. 2 in Ce. formosa, on the short arm telomeric region of chromosome pair No. 18 in Cr. altivelis, on the short arm telomeric region of chromosome pair No. 20 in E. coioi- des, and on the long arm subcentromeric region of chromosome pair No. 20 in P. leopardus. The karyo- a t t m type formula could be deduced as Ce. formosa (2n=48): L2+L28+M16+S2 ; Cr. altivelis (2n=48): a t sm t t t a t t t t L2+L24+M2 +M8+S2; E. coioides (2n=48): L28+M2+M16+S2; and P. leopardus (2n=48): L24+M24. Key words Pisces, Grouper, Serranidae, Karyotype, Chromosome. Groupers represent the most important commercial fish species in the tropical and subtropical regions of the world (Heemstra and Randall 1993). Some grouper species (Epinephelus coioides, E. malabaricus, E. akaara, and E. bruneus) have been successfully bred and raised in captivity throughout Southeast Asia (Sawada et al. 2007, Wang et al. 2002). However, the cytogenetic profiles of these species are still not fully characterized. While past studies focused mainly on conventional staining and banding techniques, fluorescence in situ hybridization (FISH) was rarely applied (Ding et al. 2004). Currently, ichthyologists use various methods, including conventional staining, C-banding, Ag-NOR banding, and FISH, to obtain cytogenetic information on fish (Sola et al. 2000, Kavalco et al. 2005); each of these methods elucidates a different aspect of the karyotype characteristics. For exam- * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.78.223 224 K. Pinthong et al. Cytologia 78(3) ple, Ag-NOR staining shows the regions containing the actively transcribed ribosomal RNA genes (rDNA). Because the chromosomal distribution patterns of heterochromatic bands, NORs, and rDNAs revealed by cytogenetic techniques are typically species-specific in fish, they prove to be useful chromosome markers in fish studies, particularly as cytotaxonomical markers in systematic evolutionary studies of closely related fish taxa (Ren et al. 1996, Gornung et al. 2001, Galetti et al. 2006, Wang et al. 2010). The family Serranidae is one of the most important families of marine fish, as many of its spe- cies are of commercial value and present particular biological traits. Serranids have wide variations in size, shape, and color, with species ranging from those that are no longer than 3 cm to those that are more than 2 m long and weight 300 kg (Randall 1995). Sex determination is also peculiar; serra- ninae species are synchronic hermaphrodites (genera Serranus and Hypoplectrus), while groupers Table 1. Review of groupers cytogenetic reports in the subfamily Epinephelinae (genera; Cephalopholis, Plectropomus, Alphestes, Cromileptes, Epinephelus, Mycteroperca). Species 2n Karyotype formula NF NOR-banded References a t t m Ce. formosa 48 L2+L28+M6+S2 52 2 (TR) Present study t t P. leopardus 48 L24+M24 48 20 (SCR) Present study A. afer 48 48a 48 2 Molina et al. (2002) Cr. altivelis 48 2st, 46a 50 2 Takai and Ojima (1995) a t sm t t 48 L2+L24+M2 +M18+S2 52 18 (TR) Present study E. adscensionis 48 48a 48 24 (SCR), 2 (TR) Molina et al. (2002) E. diacanthus 48 2sm, 46a 50 – Natarajan and Subrahmanyan (1974) E. tauvina 48 2sm, 46a 50 – Rodríguez-daga et al. (1993) E. awoara 48 48a 48 24 (SC) Hong and Yang (1988) E. guttatus 48 48a 48 24 (SA) Medrano et al. (1988) E. guaza 48 48a 48 24 (SCR) Martinez et al. (1989) E. alexandrinus 48 48a 48 24 (SCR) Martinez et al. (1989) E. sexfasciatus 48 2sm, 46a 50 – Chen et al. (1990) E. caninus 48 48a 48 24 (SCR) Rodríguez-Daga et al. (1993) E. fasciatomaculatus 48 48a 48 24 (SCR) Li and Peng (1994) E. fasciatus 48 48a 48 24 (SCR) Li and Peng (1994) E. marginatus 48 48a 48 24 (SCR), 2 (TR) Sola et al. (2000) 48 48a 48 24 (SCR) Martinez et al. (1989) 48 48a 48 24 (SCR) Aguilar and Galetti (1997) E. malabaricus 48 48t 48 24 (TR) Ueno and Jarayabhand (1991) 48 48t 48 24 (TR) Hu et al. (2004) 48 48a 48 24 (SCR), 5 (?) Zou et al. (2005) E. akaara 48 5st, 43a 48 – Wang et al. (2004) E. fario 48 4 m, 6sm, 4st, 34a 62 – Zheng et al. (2005) E. merra 48 4 m, 6sm, 4st, 34a 62 – Zheng et al. (2005) E. moara 48 48a 48 – Guo et al. (2006) E. fuscoguttalus 48 2sm, 46a 50 – Liao et al. (2006) 48 2st, 46t 50 – Wei et al. (2009) E. bruneus 48 2m, 4sm, 42t 54 2, 24, 9 (SA) Guo et al. (2008) E. coioides 48 2sm, 46a 50 24 (SA) Shifeng et al. (2010) t a t t 48 L28+M2+M16+S2 50 20 (TR) Present study E. ongus 48 48a 48 – Rishi and Haobam (1984) E. lanceolatus 48 8sm, 40t 56 – Jiun and Mei (2009) E. faveatus 48 2m, 46a 50 – Magtoon and Donsakul (2008) M. acutirostris 48 48a 48 – Galetti et al. (2006) M. rubra 48 48a 48 – Aguilar et al. (1998) Remarks: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, st=subtelocentric, SC=the secondary constriction in the sub- centromeric region, SA=short arm, SCR=subcentromeric region, TR=telomeric region, and – =not available. 2013 Cytogenetic Comparisons of Four Genera in Grouper Species 225 and allies Ephinephelinae (genera Alpbestes, Epinephelus, Mycteroperca, and Cephalopholis) present asynchronic hermaphroditism (Ohno 1974). Among the 300 plus species that make up the family Serranidae, about half belong to the subfamily Epinephelinae, grouped in 15 genera as 159 species (Heemstra and Randall 1993). Karyological information exists for only 27 species of the subfamily (16.98%), and all share the “ancestral” teleost karyotype composed of 48 uniarmed chromosomes (Galetti et al. 2000, Arai 2011) (Table 1). In the present work, cytogenetical analysis by conventional staining and Ag-NOR banding techniques was carried out on four wild species from the Andaman Sea, including the blueline hind (Cephalopholis formosa), humpback grouper (Cromileptes altivelis), orange-spotted grouper (Epinephelus coioides), and leopard coralgrouper (Plectropomus leopardus) (Fig. 1) in order to: 1) describe in detail the karyotypic features of the four grouper species and 2) explore the chromo- somal evolutionary history of relationships within the groupers. Fig. 1. General characteristics of the blueline hind, Cephalopholis formosa (A.), humpback grouper, Chromileptes altivelis (B.), orange spotted grouper, Epinephelus coioides (C.), and leopard coralgrouper, Plectopomus leopardus (D.) according to Heemstra and Randall (1993). 226 K. Pinthong et al. Cytologia 78(3) Materials and methods Four wild Ce. formosa, Cr. altivelis, E. coioides, and P. leopardus individuals (body weight ranging from 200 to 1,000 g) were collected from the coastal waters of Phuket Province and Phang Nga Province, Andaman Sea, Thailand (two localities). The fish were not sexed due to the fact that under natural conditions, Ce. formosa, Cr. altivelis, E. coioides, and P. leopardus are synchronic hermaphrodites. All specimens were maintained in aerated, flowing seawater aquaria until analysis. Mitotic chromosome samples were prepared from the anterior kidney after in vivo treatment using a method modified from Chen and Ebeling (1968) and Gold et al. (1990). The chromosomes were stained with 10% Giemsa’s for 30 min and identified for NORs by Ag-NOR staining (Howell and Black 1980). The length of short arm (Ls) and long arm (Ll) chromosomes were measured, and the length of the total arm chromosome was calculated (LT, LT=Ls+Ll). The relative length (RL) and centromeric index (CI) were estimated. CI was also computed to classify the types of chromo- somes according to Chaiyasut (1989). All parameters were used in karyotyping and idiograming. Results and discussion Cytogenetic comparisons was carried out on specimens of Ce. formosa, Cr. altivelis, E. coioides, and P. leopardus from two localities in the Andaman Sea to deepen the knowledge of the chromosome component of the species. The karyotypes of Cr. altivelis and E. coioides were found to consist of 48 chromosomes as reported previously (Takai and Ojima 1995, Shifeng et al. 2010), and the karyotypes of Ce.