© 2013 The Japan Mendel Society Cytologia 78(3): 223–234

Cytogenetic Comparison and Chromosome Localization of the Nucleolar Organizer Region of Four 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 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 ( formosa), (Cromileptes altivelis), orange-spotted grouper ( coioides), and leopard coralgrouper ( 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 , 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 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, , 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 () 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

Table 1. Review of groupers cytogenetic reports in the subfamily Epinephelinae (genera; Cephalopholis, Plectropomus, , Cromileptes, Epinephelus, ).

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 (), 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. formosa and P. leopardus, not previously reported, consist of 48 chromosomes as well (Fig. 2). Previous cytogenetical reports on the genus Epinephelus have shown remarkable numerical (2n=48) and structural chromosome homogeneity with several acrocentric chromosomes and a common heterochromatin distribution at the centromeric or pericentromeric position (Molina et al. 2002). The fundamental numbers (NF, chromosome arm number) of Ce. formosa, Cr. altivelis, E. coioides, and P. leopardus are 52, 52, 50, and 48, respectively. Although most Serranid species have an NF of 48 with only acrocentric chromosomes in the complement, the number of chromo- some arms in the genus Epinephelus varies from 48 to 62. Under the assumption that species with a larger NF are more advanced in evolutionary terms (Ghigliotti et al. 2007), the most recent species within the genus Epinephelus would be E. fario, NF=62; E. merra, NF=62 (Zheng et al. 2005) and E. lanceolatus, NF=56 (Jiun and Mei 2009). However, despite differences in NF, all Epinephelus species share the same diploid chromosome number (2n=48). Such changes in chromosome arm number appear to be related to the occurrence of pericentric inversions, which are among the most common modifications contributing to karyotypic rearrangement in fish and other vertebrates (King 1993, Galetti et al. 2000, Wang et al. 2010). The presence of biarmed chromosomes in the karyotypes of Cr. altivelis, NF=50 (Takai and Ojima 1995); E. diacanthus, NF=50 (Natarajan and Subrahmanyan 1974); E. tauvina, NF=50 (Rodríguez-Daga et al. 1993); E. sexfasciatus, NF=50 (Chen et al. 1990); E. fuscoguttalus, NF=50 (Liao et al. 2006), E. coioides, NF=50 (Shifeng et al. 2010); and E. faveatus, NF=50 (Magtoon and Donsakul 2008) represents a derived karyotypic feature that could have arisen in a common ances- tor as a result of pericentric inversions in an acrocentric chromosome pair. This type of rearrange- ment, together with Robertsonian rearrangements (chromosome fusion or fission) and reciprocal translocations, has been recognized as being responsible for some of the important changes leading to the present karyotypic diversity of the order (Pisano and Ozouf-Costaz 2003, Eler et al. 2007, Wang et al. 2010). The chromosome types and sizes of Ce. formosa consist of two large acrocentric, 28 large telocentric, 16 medium telocentric, and two small metacentric chromosomes; those of Cr. altivelis 2013 Cytogenetic Comparisons of Four Genera in Grouper Species 227

Fig. 2. Metaphase chromosome plates and karyotypes of the blueline hind, Cephalopholis formosa (A.), humpback grouper, Cromileptes altivelis (B.), orange-spotted grouper, Epinephelus coioides (C.), and leopard coralgrouper, Plectropomus leopardus (D.) by a conventional straining technique. All species share the karyotype composed of 48 chromosomes. Scale bars indicate 5 μm. consist of two large acrocentric, 24 large telocentric, two medium submetacentric, 18 medium telo- centric, and two small telocentric chromosomes; those of E. coioides consist of 28 large telocentric, two medium acrocentric, 16 medium telocentric, and two small telocentric chromosomes; and those of P. leopardus consist of 24 large telocentric, and 24 medium telocentric chromosomes. Cytogenetical analyses of 10 Epinephelinae species (Klinkhardt 1998) revealed a common chromo- somal pattern that supports the monophyly of the group. This asynchrony between morphological traits and karyotypical pattern evolutionary rates suggests that the speciation process in some marine groups was mainly established by pre-zygotic mechanisms of reproductive isolation (Molina et al. 2002). From the Ag-NOR banding technique, this cytogenetic study of four grouper species found that Ce. formosa (2n=48) has NORs on the short arm telomeric region of chromosome pair No. 2, Cr. altivelis (2n=48) has NORs on the short arm telomeric region of chromosome pair No. 18, 228 K. Pinthong et al. Cytologia 78(3)

Fig. 3. Metaphase chromosome plates and karyotypes of the blueline hind, Cephalopholis formosa (A.), humpback grouper, Cromileptes altivelis (B.), orange-spotted grouper, Epinephelus coioides (C.), and leopard coralgrouper, Plectropomus leopardus (D.) by the Ag-NOR banding technique, All species share the karyotype composed of 48 chromosomes. Arrows indicate the nucleolar organizer regions (NORs) (scale bars=5 μm).

E. coioides (2n=48) has NORs on the short arm telomeric region of chromosome pair No. 20, and P. leopardus (2n=48) has NORs on the long arm subcentromeric region of chromosome pair No. 20, which is a pair of the marker chromosomes (satellite chromosomes) (Figs. 3 and 6). In all species of the genus Epinephelus investigated to date, the NOR-bearing chromosome pair No. 24 is conserved. However, in E. alexandrines, E. guaza (Martinez et al. 1989), E. awoara (Hong and Yang 1988), E. caninus (Rodríguez-Daga et al. 1993), E. fasciatomaculatus, E. fasciatus (Li and Peng 1994), E. marginatus (Martinez et al. 1989, Aguilar and Galetti 1997, Sola et al. 2000), E. adscencionis (Molina et al. 2002), and E. malabaricus (Zou et al. 2005), NORs are located in the subcentromeric region of this chromosome pair, while in E. guttatus (Medrano et al. 1988), E. malabaricus (Ueno and Jarayabhand 1991, Hu et al. 2004), E. bruneus (Guo et al. 2008), and E. coioides (Shifeng et al. 2010), NORs are located on its short arms. Thus, in the genus Epinephelus, 2013 Cytogenetic Comparisons of Four Genera in Grouper Species 229 the NOR-bearing chromosome pair is involved in pericentric inversion (Sola et al. 2000). By comparing the 29 species of the subfamily Epinephelinae whose NOR location have been investigated, three cytotypes of NOR distribution patterns were observed: cytotype I, where only one pair of NORs is located in the subcentromeric region of acrocentric chromosome pair No. 24; cytotype II, where one pair of NORs is located in the subcentromeric region of acrocentric chromo- some pair No. 24, and an extra pair of smaller NORs is located on another pair of chromosomes; and cytotype III, where only one pair of NORs located on the short arms of biarmed chromosome pair No. 24 (Wang et al. 2010). This phenomenon is not unusual; similar occurrences have been described in other organisms and attributed to structural changes such as duplications and/or deletions (Cross et al. 2006, Fujiwara et al. 2007). Therefore, both the diversified types of NOR locations and NOR size variations indicate that certain chromosome rearrangements must have occurred in the NOR-bearing chromosome pairs during karyotype evolution of the genus Epinephelus (Wang et al. 2010). Within the order Perciformes, the presence of a single NOR pair at an interstitial position seems to be the most frequent situation, especially in species with conserved karyotypes (Galetti et al. 2006). If we assume that the presence of a single NOR is an ancestral trait of the family Serranidae (Molina et al. 2002), the presence of multiple NOR sites in the genus Epinephelus, as found in E. adscencionis (Molina et al. 2002), E. marginatus (Sola et al. 2000), E. malabaricus (Zou et al. 2005), and E. bruneus (Guo et al. 2008) would indicate that these species are more recently derived than the species with a single NOR site (Wang et al. 2010). The four grouper species from Thailand demonstrated that the chromosome markers on chro- mosome pair No. 1 are the largest telocentric chromosomes. The important karyotype feature is the asymmetrical karyotype. The largest chromosome is three times larger than the smallest chromo-

Table 2. Mean short arm chromosome length (Ls), long arm chromosome length (Ll), total arm chromo- some length (LT), relative length (RL), centromeric index (CI), and standard deviations (SD) of RL and CI from 20 metaphases of the blueline hind (Cephalopholis formosa), 2n=48.

Chromosome Chromosome Chromosome Ls Ll LT CI RL pairs size type

1 0.000 0.708 0.708 1.000±0.000 0.027±0.001 Large Telocentric 2* 0.168 0.510 0.679 0.751±0.011 0.026±0.001 Large Acrocentric 3 0.000 0.659 0.659 1.000±0.000 0.025±0.001 Large Telocentric 4 0.000 0.640 0.640 1.000±0.000 0.024±0.001 Large Telocentric 5 0.000 0.620 0.620 1.000±0.000 0.023±0.001 Large Telocentric 6 0.000 0.604 0.604 1.000±0.000 0.023±0.001 Large Telocentric 7 0.000 0.590 0.590 1.000±0.000 0.022±0.001 Large Telocentric 8 0.000 0.574 0.574 1.000±0.000 0.022±0.001 Large Telocentric 9 0.000 0.565 0.565 1.000±0.000 0.022±0.001 Large Telocentric 10 0.000 0.559 0.559 1.000±0.000 0.021±0.000 Large Telocentric 11 0.000 0.554 0.554 1.000±0.000 0.021±0.000 Large Telocentric 12 0.000 0.552 0.552 1.000±0.000 0.021±0.000 Large Telocentric 13 0.000 0.547 0.547 1.000±0.000 0.021±0.000 Large Telocentric 14 0.000 0.540 0.540 1.000±0.000 0.021±0.000 Large Telocentric 15 0.000 0.535 0.535 1.000±0.000 0.021±0.001 Large Telocentric 16 0.000 0.510 0.510 1.000±0.000 0.020±0.001 Medium Telocentric 17 0.000 0.508 0.508 1.000±0.000 0.019±0.001 Medium Telocentric 18 0.000 0.505 0.505 1.000±0.000 0.019±0.001 Medium Telocentric 19 0.000 0.499 0.499 1.000±0.000 0.019±0.001 Medium Telocentric 20 0.000 0.492 0.492 1.000±0.000 0.019±0.001 Medium Telocentric 21 0.000 0.484 0.484 1.000±0.000 0.018±0.001 Medium Telocentric 22 0.000 0.465 0.465 1.000±0.000 0.018±0.001 Medium Telocentric 23 0.000 0.440 0.440 1.000±0.000 0.017±0.001 Medium Telocentric 24 0.138 0.193 0.335 0.577±0.017 0.013±0.001 Small Metacentric

Remark: * =NOR-bearing chromosome (satellite chromosome). 230 K. Pinthong et al. Cytologia 78(3)

Table 3. Mean short arm chromosome length (Ls), long arm chromosome length (Ll), total arm chromo- some length (LT), relative length (RL), centromeric index (CI), and standard deviations (SD) of RL and CI from 20 metaphases of the humpback grouper (Cromileptes altivelis), 2n=48.

Chromosome Chromosome Chromosome Ls Ll LT CI RL pairs size type

1 0.000 0.664 0.664 1.000±0.000 0.028±0.000 Large Telocentric 2 0.134 0.503 0.637 0.784±0.044 0.027±0.000 Large Acrocentric 3 0.000 0.599 0.599 1.000±0.000 0.026±0.000 Large Telocentric 4 0.000 0.582 0.582 1.000±0.000 0.025±0.000 Large Telocentric 5 0.000 0.569 0.569 1.000±0.000 0.024±0.000 Large Telocentric 6 0.000 0.560 0.560 1.000±0.000 0.023±0.000 Large Telocentric 7 0.000 0.546 0.546 1.000±0.000 0.023±0.000 Large Telocentric 8 0.000 0.538 0.538 1.000±0.000 0.023±0.000 Large Telocentric 9 0.000 0.534 0.534 1.000±0.000 0.023±0.000 Large Telocentric 10 0.000 0.529 0.529 1.000±0.000 0.022±0.000 Large Telocentric 11 0.000 0.525 0.525 1.000±0.000 0.022±0.000 Large Telocentric 12 0.000 0.516 0.516 1.000±0.000 0.022±0.000 Large Telocentric 13 0.000 0.511 0.511 1.000±0.000 0.022±0.000 Large Telocentric 14 0.000 0.476 0.476 1.000±0.000 0.021±0.000 Medium Telocentric 15 0.000 0.472 0.472 1.000±0.000 0.020±0.000 Medium Telocentric 16 0.000 0.456 0.456 1.000±0.000 0.019±0.000 Medium Telocentric 17 0.000 0.442 0.442 1.000±0.000 0.019±0.000 Medium Telocentric 18* 0.141 0.266 0.408 0.654±0.000 0.018±0.000 Medium Submetacentric 19 0.000 0.395 0.395 1.000±0.000 0.017±0.000 Medium Telocentric 20 0.000 0.389 0.389 1.000±0.000 0.017±0.000 Medium Telocentric 21 0.000 0.381 0.381 1.000±0.000 0.016±0.000 Medium Telocentric 22 0.000 0.370 0.370 1.000±0.000 0.016±0.000 Medium Telocentric 23 0.000 0.363 0.363 1.000±0.000 0.016±0.000 Medium Telocentric 24 0.000 0.300 0.300 1.000±0.000 0.013±0.000 Small Telocentric

Remark: * =NOR-bearing chromosome (satellite chromosome).

Table 4. Mean short arm chromosome length (Ls), long arm chromosome length (Ll), total arm chromo- some length (LT), relative length (RL), centromeric index (CI), and standard deviations (SD) of RL and CI from 20 metaphases of the orange-spotted grouper (Epinephelus coioides), 2n=48.

Chromosome Chromosome Chromosome Ls Ll LT CI RL pairs size type

1 0.000 0.492 0.492 1.000±0.000 0.028±0.002 Large Telocentric 2 0.000 0.474 0.474 1.000±0.000 0.027±0.001 Large Telocentric 3 0.000 0.451 0.451 1.000±0.000 0.026±0.001 Large Telocentric 4 0.000 0.439 0.439 1.000±0.000 0.025±0.001 Large Telocentric 5 0.000 0.432 0.432 1.000±0.000 0.025±0.001 Large Telocentric 6 0.000 0.422 0.422 1.000±0.000 0.024±0.001 Large Telocentric 7 0.000 0.418 0.418 1.000±0.000 0.024±0.001 Large Telocentric 8 0.000 0.410 0.410 1.000±0.000 0.023±0.001 Large Telocentric 9 0.000 0.402 0.402 1.000±0.000 0.023±0.001 Large Telocentric 10 0.000 0.394 0.394 1.000±0.000 0.022±0.001 Large Telocentric 11 0.000 0.390 0.390 1.000±0.000 0.022±0.001 Large Telocentric 12 0.000 0.383 0.383 1.000±0.000 0.022±0.001 Large Telocentric 13 0.000 0.379 0.379 1.000±0.000 0.022±0.001 Large Telocentric 14 0.000 0.349 0.349 1.000±0.000 0.020±0.001 Large Telocentric 15 0.000 0.328 0.328 1.000±0.000 0.019±0.001 Medium Telocentric 16 0.000 0.322 0.322 1.000±0.000 0.018±0.001 Medium Telocentric 17 0.000 0.316 0.316 1.000±0.000 0.018±0.001 Medium Telocentric 18 0.000 0.311 0.311 1.000±0.000 0.018±0.001 Medium Telocentric 19 0.000 0.307 0.307 1.000±0.000 0.018±0.001 Medium Telocentric 20* 0.074 0.228 0.302 0.755±0.000 0.017±0.001 Medium Acrocentric 21 0.000 0.306 0.306 1.000±0.000 0.018±0.001 Medium Telocentric 22 0.000 0.299 0.299 1.000±0.000 0.017±0.001 Medium Telocentric 23 0.000 0.285 0.285 1.000±0.000 0.016±0.001 Medium Telocentric 24 0.000 0.191 0.191 1.000±0.000 0.011±0.002 Small Telocentric

Remark: * =NOR-bearing chromosome (satellite chromosome). 2013 Cytogenetic Comparisons of Four Genera in Grouper Species 231

Table 5. Mean short arm chromosome length (Ls), long arm chromosome length (Ll), total arm chromo- some length (LT), relative length (RL), centromeric index (CI), and standard deviations (SD) of RL and CI from 20 metaphases of the leopard coralgrouper (Plectopomus leopardus), 2n=48.

Chromosome Chromosome Chromosome Ls Ll LT CI RL pairs size type

1 0.000 0.677 0.677 1.000±0.000 0.026±0.001 Large Telocentric 2 0.000 0.651 0.651 1.000±0.000 0.025±0.001 Large Telocentric 3 0.000 0.634 0.634 1.000±0.000 0.025±0.001 Large Telocentric 4 0.000 0.622 0.622 1.000±0.000 0.024±0.001 Large Telocentric 5 0.000 0.600 0.600 1.000±0.000 0.023±0.001 Large Telocentric 6 0.000 0.592 0.592 1.000±0.000 0.023±0.001 Large Telocentric 7 0.000 0.582 0.582 1.000±0.000 0.023±0.000 Large Telocentric 8 0.000 0.573 0.573 1.000±0.000 0.022±0.000 Large Telocentric 9 0.000 0.562 0.562 1.000±0.000 0.022±0.000 Large Telocentric 10 0.000 0.553 0.553 1.000±0.000 0.021±0.000 Large Telocentric 11 0.000 0.547 0.547 1.000±0.000 0.021±0.000 Large Telocentric 12 0.000 0.541 0.541 1.000±0.000 0.021±0.000 Large Telocentric 13 0.000 0.525 0.525 1.000±0.000 0.020±0.000 Large Telocentric 14 0.000 0.518 0.518 1.000±0.000 0.020±0.000 Large Telocentric 15 0.000 0.514 0.514 1.000±0.000 0.020±0.000 Large Telocentric 16 0.000 0.507 0.507 1.000±0.000 0.020±0.000 Medium Telocentric 17 0.000 0.499 0.499 1.000±0.000 0.019±0.000 Medium Telocentric 18 0.000 0.493 0.493 1.000±0.000 0.019±0.001 Medium Telocentric 19 0.000 0.486 0.486 1.000±0.000 0.019±0.000 Medium Telocentric 20* 0.000 0.477 0.477 1.000±0.000 0.019±0.001 Medium Telocentric 21 0.000 0.470 0.470 1.000±0.000 0.018±0.000 Medium Telocentric 22 0.000 0.460 0.460 1.000±0.000 0.018±0.000 Medium Telocentric 23 0.000 0.431 0.431 1.000±0.000 0.017±0.001 Medium Telocentric 24 0.000 0.389 0.389 1.000±0.000 0.015±0.001 Small Telocentric

Remark: * =NOR-bearing chromosome (satellite chromosome).

Fig. 4. Idiogram showing lengths and shapes of Fig. 5. Idiogram of the blueline hind, Cephalopholis chromosomes of the blueline hind, Cephalopholis formosa (A.), humpback grouper, Cromileptes formosa (A.), humpback grouper, Cromileptes altivelis (B.), orange-spotted grouper, Epinephelus altivelis (B.), orange-spotted grouper, Epinephelus coioides (C.), and leopard coralgrouper, coioides (C.), and leopard coralgrouper, Plectropomus leopardus (D.) by a conventional Plectropomus leopardus (D.) by a conventional staining technique. All species share the karyotype staining technique. All species share the karyotype composed of 48 chromosomes. Arrows indicate composed of 48 chromosomes. the nucleolar organizer regions (NORs). 232 K. Pinthong et al. Cytologia 78(3)

Fig. 6. Examples of five cells of the nucleolar organizer regions (NORs) (satellite chromosomes) of the blueline hind (Cephalopholis formosa), humpback grouper (Cromileptes altivelis), orange-spotted grouper (Epinephelus coioides), and leopard coralgrouper (Plectropomus leopardus) in Thailand by Ag-NOR banding techniques. Arrows indicate the telomeric NORs and subcentromeric NORs. some. The data from the chromosomal checks on mitotic metaphase cells of Ce. formosa, Cr. altivelis, E. coioides, and P. leopardus are shown in Tables 2–5. Figures 4 and 5 show the idiogram from conventional staining and Ag-NOR banding techniques. In order to gain a more thorough under- standing of the chromosomal evolutionary history of relationships within the groupers, further cytogenetic investigations of the remaining species in this subfamily is required. The karyotype formulas could be deduced as follows.

a t t m Ce. formosa (2n=48): L2+L28+M16+S2 a t sm t t Cr. altivelis (2n=48): L2+L24+M2 +M8+S2 t a t t E. coioides (2n=48): L28+M2+M16+S2 t t P. leopardus (2n=48): L24+M24

Acknowledgments

This work was financially supported in part by a full scholarship from the Ministry of Science and Technology (MOST) and the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant; ATRC-R5304. Special thanks to Dr. Jolyon Dodgson for kindly checking the manuscript.

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