First Report of Chromosome Analysis of Saddleback Anemonefish

First Report of Chromosome Analysis of Saddleback Anemoneﬁsh, Amphiprion polymnus (Perciformes, Amphiprioninae), in Thailand

Alongklod Tanomtong1, Weerayuth Supiwong1*, Arunrat Chaveerach1, Suthip Khakhong2, Tawatchai Tanee3 and La-orsri Sanoamuang1

1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Muang 83000, Thailand 3 Faculty of Environment and Resource Studies, Mahasarakham University, Mahasarakham, Muang 44000, Thailand

Received May 31; accepted October 6, 2012

Summary We report the first chromosome analysis for the saddleback anemonefish (Amphiprion polymnus) from Thailand. Kidney cell samples were taken from 5 male and 5 female fish. The mitotic chromosome preparation was prepared by directly from kidney cells. Conventional and Ag- NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of A. polymnus was 2n=48 and the fundamental numbers (NF) was 96 in both male and female. The types of chromosomes were 6 large submetacentric, 2 large acrocentric, 10 medium metacentric, 12 medium submetacentric, 6 medium acrocentric, 10 small metacentric, and 2 small submetacentric chromosomes. The region adjacent to the telomeres on the short arms of chromosome pair 21 showed clearly observable secondary constriction/NORs. The karyotype formula for A. polymnus could be deduced as: sm a m sm a m sm 2n (48)=L 6 +L2+M10+M 12+M6+S10+S 2

Key words Saddleback anemoneﬁsh, Amphiprion polymnus, Karyotype, Chromosome.

Thailand is one of the world’s richest places for biodiversity, especially for marine fish species. With more than 2,000 species recorded, Thailand has become one of the species diversity cen- ters of the world. Marine fish are especially important as they provide a high quality source of pro- tein as well as food source for people who live near the coast. Because of its attractive color, fish in the subfamily Amphiprioninae play an important role in national economy. There are 2 genera and 29 species in the subfamily Amphiprioninae in Thailand, and 7 species can be reached. For the genus Amphiprion, there are skunk anemonefish (A. akallopisos Bleeker 1853), Clark’s anemonefish [A. clarkii (Bennett 1830)], red saddleback anemonefish [A. ephippium (Bloch 1790)], false clown anemonefish (A. ocellaris Cuvier 1830), pink anemonefish (A. perideraion Bleeker 1855), saddleback anemonefish [A. polymnus (Linnaeus 1758)], and sebae anemonefish (A. sebae Bleeker 1853) (Boonphakdee and Sawangwong 2008). Of about 13,000 marine fish species that have been recorded (Nelson 1994), fewer than 2% of these have been studied cytogenetically (Brum 1996). Although most of these fish share a common environment lacking apparent physical barriers, they do not show a general chromosome evolutionary trend. The diploid chromosome number varies from 2n=22–26, in some species of

* Corresponding, e-mail: [email protected] DOI: 10.1508/cytologia.77.441 442 A. Tanomtong et al. Cytologia 77(4)

Table 1. Review of anemoneﬁshes cytogenetic reports in the subfamily Amphiprioninae

Species (Anemoneﬁsh) 2n NF m sm a t NORs References

Saddleback (A. polymnus) 48 96 20 20 8 ̶ 2 (a) Present study Clak’s (A. clarkii) 48 78 14 16 18 ̶ ̶ Arai and Inoue (1976) 48 86 12 26 10 ̶ 2 (a) Takai and Kosuga (2007) False clown (A. ocellaris) 48 84 14 22 12 ̶ ̶ Arai et al. (1976) Tomato (A. frenatus) 48 92 14 22 8 4 2 (a) Molina and Galetti (2004) 48 86 12 26 10 ̶ 2 (a) Takai and Kosuga (2007)

Remark: 2n=diploid chromosome number, NF=fundamental number, m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, and NORs=nucleolar organizer regions.

Nototheniidae (Ozouf-Costaz et al. 1997), an Antarctic fish group, to 2n=240–260, in some anadromous Acipenseridae, which show sev- eral micro-chromosomes (Fontana et al. 1997). Most marine fish studied have a diploid complement of 48 acrocentric chromosomes (Sola et al. 1981, Klinkhardt et al. 1995, Brum 1996). There are only 3 species in the subfamily Amphiprioninae that have already been consid- ered through cytogenetic studies. These results show a diploid chromosome number 2n=48 Fig. 1. General characteristics of the saddleback anemonefish (Amphiprion (Arai and Inoue 1976, Arai et al. 1976, Molina polymnus), scale bar indicates and Galetti 2004, Takai and Kosuga 2007) 5 cm. (Table 1). The present study is aimed at the cytogenetic analysis of saddleback anemonefish (Amphiprion polymnus) (Fig. 1). From the present study, we show the standardization of karyotype and idiogram of A. polymnus. This report de- scribes the first chromosome staining by conventional staining and Ag-NOR staining techniques in A. polymnus.

Materials and methods

The A. polymnus samples were obtained from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Thailand. The ﬁsh, 5 males and 5 females of A. polymnus, were transferred to laboratory aquaria and were kept under standard conditions for 7 days prior to the experiments. Chromosome preparation was conducted by squash technique from kidney cell (Chen and Ebeling 1968, Nanda et al. 1995). 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) chromosome were measured and calculated for the length of total arm chromosome (LT, LT=Ls+Ll). Relative length (RL) and centromeric index (CI) were estimated. CI was also computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were used in karyotyping and idiograming.

Results and discussion

According to the results, this is the first report on A. polymnus cytogenetical knowledge. Our results showed that 2n=48 of diploid chromosome number is consistent to the reports of Arai and Inoue (1976), Arai et al. (1976), Molina and Galetti (2004), and Takai and Kosuga (2007) that 2012 Chromosome Analysis of Saddleback Anemonefish, Amphiprion polymnus in Thailand 443 showed 2n=48 of Clark’s anemonefish (A. clarkii), false clown anemonefish (A. ocellaris) and tomato anemonefish (A. frenatus). We found that the fundamental number (NF, number of chromosome arms) of A. polymnus is 96 in both male and female. No strange size chromosomes related to sex were observed. In pomacentrid fish, including anemonefishes, the karyotypes of 30 species has been reported and these are characterized by chromosome numbers varying from 27 to 48 and NF varying from 48 to 86 (Arai and Inoue 1976, Arai et al. 1976, Ojima and Kashiwagi 1981, Takai and Ojima 1987, 1991a, 1991b, 1995, Molina and Galetti 2004, Takai and Kosuga 2007). Takai and Ojima (1987) suggested that karyotypes of pomacentrid fish have diversified from the ancestral karyotype, consisting of 48 acrocentric chromosomes, mainly toward an increase in NF involving pericentric inversions. Therefore, anemonefish with higher NF may be the most advanced group in the karyotypic evolution in family Pomacentridae. The chromosomes of A. polymnus consist of 20 metacentric, 20 submetacentric, and 8 acrocentric chromosomes (Fig. 2). It is different from the reports of Arai and Inoue (1976) that showed the chromosomes of A. clarkii consisting of 14 metacentric, 16 submetacentric, and 18 acrocentric chromosomes. Arai et al. (1976) found the karyotype of A. ocellaris in Japan consisting of 14 metacentric, 22 submetacentric, and 12 subtelocentric/acrocentric chromosomes. Molina and Galetti (2004) reported that the chromosome of A. frenatus from Philippines (Pacific) composed of 14 metacentric, 22 submetacentric, 8 subtelocentric, and 4 acrocrntric chromosomes. Takai and

Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) saddleback anemoneﬁsh (Amphiprion polymnus), 2n=48 by conventional straining technique (scale bars 10 μm). There is no observation of strange size chromosomes related to sex. 444 A. Tanomtong et al. Cytologia 77(4)

Kosuga (2007) reported karyotypes of A. （A） clarkii and A. frenatus consisting of 12 metacentric, 26 submetacentric, and 10 acrocentric chromosomes. The present study, the first cytogenetic report of A. polymnus, was accomplished using Ag-NOR staining technique. The objective of this technique is to reach out the nucleolar organizer regions (NORs) which represent the lo- cation of genes (loci) that function in ribosome synthesis (18S and 28S ribosomal RNA) (Sharma et al. 2002). In addition, the short arm near telomere of largest acrocentric chromosome pair 21 showed clearly observable NORs （B） (satellite chromosomes) (Fig. 3). It is quite consistent to the reports of Takai and Kosuga (2007), who reported karyotypes of A. clarkii and A. frenatus which showed clearly observable 1 pair of NORs on acrocentric chromosomes, and Molina and Galetti (2004), who reported that A. frenatus showed 1 pair of NORs-bearing on acrocentric chromosomes. Normally, most fish have only 1 pair of small NORs on chromosomes. If some fish have more than 2 NORs, this may be caused by the Fig. 3. Metaphase chromosome plates of translocation between some parts of chromo- male (A) and female (B) saddle- somes which have NOR and another chromo- back anemonefish (Amphiprion some (Sharma et al. 2002). polymnus), 2n=48 by Ag-NOR The chromosome of mitotic metaphase banding technique, scale bars in- dicate 10 μm. The region adjacent cells and the karyotypes of A. polymnus by to the short arms near telomere of conventional staining and Ag-NOR staining largest acrocentric chromosome techniques are shown in Figs. 2 and 3. The pair 21 showed clearly observable chromosomes length in centimetres of 20 cells nucleolar organizer regions (satellite chromosomes). (males and females) in mitotic metaphase was measured. The mean length of short arm chromosome (Ls), long arm chromosome (Ll), total length of arm chromosome (LT), relative length (RL), centromeric index (CI), and type of chromosome were presented in Table 2. The idiogram of A. polymnus shows the gradually decreasing length of the chromosomes. The A. polymnus demonstrated that the chromosome markers are the chromosome pair 1 is the largest metacentric chromosomes. An important karyotype feature of A. polymnus is the asymmetri- cal karyotype, which was found in 3 types of chromosomes (metacentric, submetacentric, and acrocentric chromosomes). The largest chromosome is 3 times larger than the smallest chromosomes. Figures 4 and 5 shows the idiogram of the A. polymnus from conventional staining and Ag-NOR banding techniques. The karyotype formula for A. polymnus is as follows: sm a m sm a m sm 2n (diploid) 48=L 6 +L 2+M10+M 12+M6+S10+S 2 2012 Chromosome Analysis of Saddleback Anemonefish, Amphiprion polymnus in Thailand 445

Table 2. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL) and centromeric index (CI) from 20 metaphases of male and female saddleback anemoneﬁsh (Amphiprion polymnus), 2n=48

Chromosome pairs Ls Ll LT CI RL Chromosome size Chromosome type

1 3.3883 3.4914 6.8797 0.5075 0.0428 Medium Metacentric 2 3.2664 3.4914 6.7578 0.5166 0.0421 Medium Metacentric 3 2.7461 3.5849 6.3310 0.5662 0.0394 Medium Metacentric 4 3.0352 3.2215 6.2568 0.5149 0.0390 Medium Metacentric 5 2.2467 3.2390 5.4858 0.5904 0.0342 Medium Metacentric 6 2.6041 2.7322 5.3364 0.5120 0.0332 Small Metacentric 7 2.2467 2.9893 5.2361 0.5709 0.0326 Small Metacentric 8 2.1430 2.9394 5.0824 0.5784 0.0316 Small Metacentric 9 1.8069 2.5728 4.3798 0.5874 0.0273 Small Metacentric 10 2.0254 2.2611 4.2865 0.5275 0.0267 Small Metacentric 11 3.8488 7.0098 10.8586 0.6455 0.0676 Large Submetacentric 12 3.2761 5.8562 9.1323 0.6413 0.0569 Large Submetacentric 13 2.7322 5.4115 8.1437 0.6645 0.0507 Large Submetacentric 14 2.4796 4.8841 7.3638 0.6633 0.0459 Medium Submetacentric 15 2.3807 4.6786 7.0593 0.6628 0.0440 Medium Submetacentric 16 2.2439 4.7298 6.9737 0.6782 0.0434 Medium Submetacentric 17 2.2467 4.5116 6.7583 0.6676 0.0421 Medium Submetacentric 18 2.0309 4.6171 6.6480 0.6945 0.0414 Medium Submetacentric 19 1.9056 4.4457 6.3513 0.7000 0.0395 Medium Submetacentric 20 1.6839 3.5114 5.1953 0.6759 0.0323 Small Submetacentric 21* 1.5099 8.5609 10.0707 0.8501 0.0627 Large Acrocentric 22 1.2495 5.6309 6.8804 0.8184 0.0428 Medium Acrocentric 23 1.5225 5.1864 6.7089 0.7731 0.0418 Medium Acrocentric 24 1.4719 4.9512 6.4231 0.7708 0.0400 Medium Acrocentric

Remark: *=NOR-bearing chromosome.

Fig. 5. Idiogram of chromosomes of the saddleback anemoneﬁsh (Amphiprion polymnus), Fig. 4. Idiogram showing lengths and 2n=48 by Ag-NOR banding technique. shapes of chromosomes of the sad- The arrow indicates nucleolar organizer dleback anemoneﬁsh (Amphiprion region (satellite chromosome) to the short polymnus), 2n=48 by conventional arm near telomere of largest acrocentric staining technique. chromosome pair 21. 446 A. Tanomtong et al. Cytologia 77(4)

Acknowledgments

This work was supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant; ATRC-R5304 and by grant Faculty of Science, Khon Kaen University.

References

Arai, R. and Inoue, M. 1976. Chromosomes of seven species of Pomacentridae and two species of Acanthuridae from Japan. Bull. Natl. Mus. Nat. Sci. Ser. A. (Zool) 2: 73–78. ̶, ̶ and Ida, H. 1976. Chromosomes of four species of coral fishes from Japan. Bull. Nat. Mus. Nat. Sci. Ser. A. (Zool) 2: 137. Boonphakdee, C. and Sawangwong, Y. 2008. Genetic diversity in the pink skunk anemonefish (Amphiprion perideraion) based on cytochrome b and ITS-1 using PCR–RFLP. In: Watabe, S. (ed.), WFC 2008 5th World Fisheries Congress, Yokohama. Brum, M. J. I. 1996. Cytogenetic studies of Brazilian marine fish. Rev. Bras. Genet. 19: 421–427. Chen, T. R. and Ebeling, A. W. 1968. Karyological evidence of female heterogamety in the mosquito fish, Gambusia affi- nis. Copeia 1: 70–75. Chaiyasut, K. 1989. Cytogenetics and Cytotaxonomy of the Family Zephyranthes. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok. Fontana, F., Rossi, R., Lanfredi, M., Arlati, G. and Bronzi, P. 1997. Cytogenetic characterization of cell lines from three sturgeon species. Caryologia 50: 91–95. Howell, W. M. and Black, D. A. 1980. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: 1014–1015. Klinkhardt, M. B., Tesche, M. and Greven, H. 1995. Database of Fish Chromosome. Westrap Wissenschaften, Magdeburg. Molina, W. F. and Galetti, P. M. 2004. Karyotypic changes associated to the dispersive potential on Pomacentridae (Pisces, Perciformes). J. Exp. Marine Biol. Ecol. 309: 109–119. Nanda, I., Schsrtl, M., Fiechtinger, W., Schlupp, I., Parzefall, J. and Schmid, M. 1995. Chromosomal evidence for laboratory synthesis of triploid hybrid between the gynogenetic teleost Poecilia Formosa and its host species. J. Fish Biol. 47: 619–623. Nelson, J. S. 1994. Fish of the World. 3rd edition. John Willey and Sons, New York. Ojima, Y. and Kashiwagi, E. 1981. Chromosomal evolution associated with Robertsonian fusion in the genus Dascyllus (Chrominae, Pisces). Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 57B: 368–370. Ozouf-Costaz, C., Pisano, E., Thaeron, C. and Hureau, J. C. 1997. Antarctic fish chromosome banding: Significance for evolutionary studies. Cybium 21: 399–409. Sharma, O. P., Tripathi, N. K. and Sharma, K. K. 2002. A review of chromosome banding in fishes. In: Sobti, R. C. (ed.). Some aspects of chromosome structure and functions. Narosa Publishing House, New Delhi. Sola, L., Cataudella, S. and Capanna, E. 1981. New developments in vertebrate cytotaxonomy III. Karyotype of bony fish: A review. Genetica 54: 285–328. Takai, A. and Kosuga, S. 2007. Karyotypes and banded chromosomal features in two anemonefishes (Pomacentridae, Perciformes). Chrom. Sci. 10: 71–74. ̶ and Ojima, Y. 1987. Comparative studies of karyotypes and distribution of nucleolus organizer regions in pomacentrid fishes. I. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 63B: 17–20. ̶ and ̶ 1991a. Comparative studies of karyotypes and distributions of nucleolus organizer regions in pomacentrid fishes. 2. Cytobios 65: 199–205. ̶ and ̶ 1991b. Karyotypes and nucleolus organizer regions in two pomacentrid fishes. Chrom. Inform. Serv. 50: 28–30. ̶ and ̶ 1995. Chromosome evolution associated with Robertsonian rearrangements in Pomacentrid fish (Perciformes). Cytobios 84: 103–110.