Galaxea, Journal of Coral Reef Studies 11: 109-118（2009）

Original paper

Survey of mycosporine-like amino acids in different morphotypes of the coral Galaxea fascicularis from Okinawa, Japan

Irina M. YAKOVLEVA1, 2 and Michio HIDAKA1, *

1 Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan 2 A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia

* Corresponding author: M. Hidaka E-mail: [email protected]

Communicated by Makoto Tsuchiya

Abstract The coral Galaxea fascicularis exhibits marked Introduction variation in polyp color and several color morphs have been described (e.g. brown polyp with green oral disc, Mycosporine-like amino acids (MAAs) are a well- BG; brown with green septal tentacles, Gs; brown with recognized family of secondary metabolites that minimize white fluorescent tentacles, Wt). Color morphs of sessile damage from exposure to solar UV radiation by direct marine inver­tebrate species may exhibit considerable screening (Adams and Shick 1996; Neale et al. 1998). differences in their capacity to absorb UV radiation, al­ MAAs are present in various aquatic organisms, includ- though only a few studies have examined intra-species ing marine invertebrates (reviewed by Shick and Dunlap variation in the UV-absorbing com­pounds, mycosporine- 2002). Sessile marine invertebrates may show great var­ like amino acids (MAAs). The objective of this study iations in color (Wicksten 1989). The color morphs of was to investigate possible differences in the composi- some invertebrate species exhibit considerable differ- tion and quantity of MAAs in the coral tissue and symbio­ ences in their capacity to absorb UV radiation (Gleason tic algae of several G. fascicularis color morphs. Marked 1993; Stoletzki and Schierwater 2005; Hirabayashi et al. differences in the composition and quantity of MAAs 2006). It is likely that the types and quantities of UV- were found between the BG and Gs/Wt morphs of G. absorbing compounds differ among conspecific indivi- fascicularis, both in the coral tissue and freshly isolated duals or colonies. At present, however, only a few studies symbiotic algae. Future studies, especially genetic analy­ have examined variations in MAAs between color mor­ ses of corals and their symbionts, are necessary to un­ phs of a single invertebrate species. Gleason (1993) dem­ derstand whether these observed differences in the com­ onstrated that green morph colonies of the Caribbean position and quantity of MAAs reflect phylogenetic or coral Porites astreoides had greater quantities of asterina- bio­geographical differences among the corals. 330 than did brown morph colonies of the same species and that these differences were positively correlated with Keywords mycosporine-like amino acids, coral, color the differential resistance of these color morphs to incident morph, nematocyst, Galaxea fascicularis UV radiation. The predominance of green P. astreoides 110 Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis colonies in shallow water was accounted for by their lected at Sesoko and five colonies collected at Oku be­ increased resistance to UV radiation. longed to the BG color morph. At Zampa, six Gs morph The scleractinian coral Galaxea fascicularis is known colonies and eight Wt morph colonies were collected. BG to exhibit intraspecific variation in both skeletal morph­ and Gs color morphs were always found in sunlight- ology and color (Chevarier 1971; Veron and Pichon 1980; exposed areas, whereas Wt color morphs grew in shaded Hidaka and Yamazato 1985). Several color morphs have habitats. All collected colonies were kept in an outdoor been described based on polyp color (e.g. brown polyp tank supplied with running unfiltered seawater for 2-3 with green oral disc, BG; brown with green septal ten­ days prior to analysis. During this period, the seawater tacles, Gs; brown with white fluorescent tentacles, Wt). temperature in the tank was 27-29℃. The tank was shaded Colonies of G. fascicularis are also divided into two mor­ by black plastic mesh. Individual polyps (~2 cm in length) photypes based on the shape of their tentacular micro- were taken from each of the colonies, blotted, and used for basic p-mastigophore (MpM) nematocysts (Hidaka 1992), observations of nematocyst type and MAA analyses of although mixed-type colonies exist in which tentacles coral tissues or freshly isolated zooxanthellae. Between contain both types of nematocysts (Wewengkang et al. five and eight colonies of each color morph from each site 2007). Recently, Watanabe et al. (2005) found that G. were used in the analyses. fascicularis populations from Okinawa consist of two types, one with a long and the other with a short non- Preparation of freshly isolated zooxanthellae for MAA coding region in their mitochondrial DNA. These mito­ analysis chondrial variants generally correspond with the different Coral polyps were rinsed gently with 0.45 µm filtered tentacular nematocyst phenotypes. Thus, it is likely that seawater (FSW) and then coral tissue was removed from there are at least two types of G. fascicularis, which can the coral skeleton using a Water-Pik with FSW (Johannes be distinguished by their mitochondrial DNA and tenta­ and Wiebe 1970). To remove the mucus produced during cular nematocysts. the isolation procedure, coral blastate was first filtered In the present study, we compared the composition through a 350 µm nylon mesh. The blastate was then and abundance of MAAs among Galaxea fascicularis homogenized with a potter homogenizer. Following cen­ color morphs collected from three sites at Okinawa Island, trifugation at 1,500×g for 5 min, filtration through 40 µm Japan, to study whether different color morphs are char­ mesh was performed. The algal pellet was washed five acterized by unique complements and concentrations of times by centrifugation [1,500 g for 5 min; 2,000 g for 10 MAAs. Since zooxanthellate cnidarians may acquire MAAs min (twice); and 3,000 g for 15 min (twice)]. This method from their symbionts (Banaszak and Trench 1995), we of isolating and cleaning zooxanthellae yields intact algal also attempted to examine the occurrence of MAAs in cells free from visible contaminations. Freshly isolated zooxanthellae isolated from different G. fascicularis color zooxanthellae were adsorbed on Millipore filters (0.45 morphs and compared the composition of host tissue µm) using a syringe and were then processed for MAA MAAs with that of their symbionts. analysis.

Analysis of MAAs Materials and methods Coral polyps were extracted with 5 ml of 70% absolute methanol (MeOH) of high performance liquid chromato­ Collection of corals graphy (HPLC) grade for 16 h at 4℃. Similar extraction Colonies of different Galaxea fascicularis (Linnaeus procedures were used on isolated, cleaned zooxanthellae. 1767) color morphs were collected from depths of 0.5-1 Methanol-extracted polyps and filters with algal cells m during low tide at three sites off Okinawa Island (the were stored at −20℃ for later analysis of total protein northern reef of Sesoko Island, Oku, and Zampa) in content, following the procedure of Bradford (1976) with August 2002. Seven of the G. fascicularis colonies col­ bovine gamma globulin as a standard. The UV spectra of Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis 111 methanol extracts were measured using a spectrophoto­ Shape of tentacular nematocysts meter (Hitachi U-2000A) for the presence of absorption External tentacles, which extend outwards from the peaks in the wavelength range 250-400 nm. Preliminary polyp, were isolated under a dissecting microscope, and comparison of absorption values between extracts obtained microbasic p-mastigophore (MpM) nematocysts in the from the wash supernatant and filtered cells revealed that acrosphere region were observed under a differential in­ less than 4% of MAA was lost due to filtration. Methanol terference contrast (DIC) microscope. Colonies of G. extracts were then centrifuged for 5 min at 3,000 g to re­ fascicularis were classified based on the shape of their move particles and passed through two C-18 Sep-Pak Plus MpM nematocysts­ as described previously (Hidaka 1992; cartridges (Water) in series to remove chromatographically Weweng­kang et al. 2007). intractable material. The supernatant was then evaporated to dryness at 30℃ using a centrifugal vacuum evaporator (CVE-100, EYELA) and temporarily stored at −20℃ Results before analysis. MAA analysis was performed by slightly modifying Complements and concentrations of MAAs in the the procedures described by Dunlap and Chalker (1986) tissue and freshly isolated symbiotic algae of Galaxea and Shick et al. (1992). Using a Shimadzu HPLC system, fascicularis color morphs MAAs were separated by reverse-phase isocratic HPLC HPLC separations of methanolic extracts of the coral on a Nucleosil 100 C8 column (25 cm, 4.6 µm) protected tissue and freshly isolated symbiotic algae from different with a Nucleosil C8 guard (4.6 mm i.d.×10 mm length) G. fascicularis color morphs collected from different sites filled with the same material, in an aqueous mobile phase of Okinawa, Japan, are shown in Fig. 1. Collectively, coral of 0.1% acetic acid and 15% methanol. The flow rate was tissue of the examined color morphs contained eight 0.79 ml min−1, and the detection of peaks was by UV ab­ MAAs in quantifiable amounts, but only three of these sorbance at 313 and 330 nm. Individual peaks were iden­ (shinorine, mycosporine-glycine, and palythine) occurred tified by online absorption spectra, retention time, and in all colonies of the color morphs examined. Mycosporine- co-chromatography with prepared standards from the 2-glycine and asterina-330 occurred only in the coral tis­ zoanthid Palythoa tuberculosa (mycosporine-glycine, sue of BG morph colonies collected at Sesoko and Oku palythene, and palythinol), the sea anemone Anthopleura (Fig. 1A, B), while Gs morph and Wt morph colonies uchidai (mycosporine-2 glycine), and the red algae collected at Zampa contained three other compounds, Porphyra sp. (shinorine and phorphyra-334), Chondrus porphyra-334, palythinol, and palythene (Fig. 1C, D). The crispus (palythine, asterina-330), and Mastocarpus stel­ extracts of symbiotic algae isolated from BG morph latus (shinorine). Peaks were baseline corrected and in­ colonies collected at Sesoko Island and at Oku did not tegrated before concentrations were calculated using avail­ contain measurable levels of MAAs (Fig. 1E, F), whereas able extinction coefficients (Shick et al. 1992; Karsten et extracts of the algae isolated from Gs and Wt morph al. 1998). The MAA concentration in each sample was colonies collected at Zampa contained MAAs in quan­ normalized to the amount of protein in the coral fragments tifiable amounts (Fig. 1G, H). The symbiotic algae isolated or in the isolated algal cells. from Gs morph colonies contained three MAAs: shinorine, Similarity a phenogram was constructed for the four G. mycosporine-glycine, and palythine (Fig. 1G), while those fascicularis populations based on the proportional con­ isolated from Wt morph colonies contained only shinorine centrations (average of five to eight colonies for each (Fig. 1H). population) of the eight MAAs that were present at least The mean total and individual concentrations of MAAs in two populations (Table 1). Similarity was determined in the coral tissue and symbiotic algae of Galaxea fasci­ by cluster analysis (Statistica 6.0, StatSoft Inc.). cularis color morphs are presented in Table 1. The total MAA content in coral tissue did not differ significantly [Tukey-Kramer HSD test (TKH), P＞0.05] among the 112 Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis

Fig. 1 HPLC chromatograms (detector at 313 nm) of methanolic­ extracts of the coral tissue (A-D) and freshly isolated symbionts­ (E-H) of Galaxea fascicularis color morphs collected from different sites of Okinawa, Japan. The color morphs and site of collection are shown in each figure. The following mycos­porine-like amino acids (MAAs) were identified: shinorine, SH; porphyra-334, PR; mycosporine-2 glycine, M2G; mycosporine­ -glycine, MG; palythine, PT; palythinol, PL; asterina-330, AS; palythene, PN Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis 113

BG morph from Sesoko, its counterpart from Oku, and the morph colonies was porphyra-334, followed by palythine Gs morph from Zampa. However, Wt morph colonies and palythinol. In Wt colonies, palythine replaced por­ from Zampa contained significantly less MAA than did phyra-334 as the second most predominant MAA whereas colonies of the other color morphs (TKH, P＜0.05). The porphyra-334 was present as a minor component. difference was mainly due to the decreased concentra- Total MAA concentrations in symbiotic algae isolated tion of mycosporine-glycine, which was the most abun­ from the Gs and Wt morphs, the only color morphs in dant MAA in the coral tissue of the three examined G. which zooxanthellae contained detectable amounts of fascicularis color morphs. Mycosporine-glycine always MAAs, were only about 10% and 3% of that of the coral accounted for more than half of the total MAA pool tissue, respectively (Table 1). However, the concentrations (Table 1). The concentrations of other MAAs were highly of shinorine in coral tissue and isolated algae were similar variable among the color morphs studied. In BG morph for both Gs and Wt morphs. The abundance of this MAA colonies collected at Sesoko, mycosporine-2 glycine and was significantly higher in the symbionts of Gs morph palythine were the next most abundant MAAs followed in colonies than in those isolated from Wt morph colonies order of decreasing concentration by asterina-330 and (TKH, P＜0.01). Mycosporine-glycine contributed most shinorine, whereas in colonies collected at Oku, the second highly to the MAA pool in symbiotic algae isolated from most abundant MAA was mycosporine-2 glycine fol­ Gs morph colonies. lowed by asterina-330, with palythine and shinorine being Further analyses of the percent composition (molar present at very low concentrations (Table 1). In contrast, proportions) of specific MAAs in the coral tissue indicated the second most abundant MAA in the coral tissue of Gs obvious differences between the examined color morphs

Table 1 Mycosporine-like amino acid (MAA) concentrations in the coral tissue and symbiotic algae (zooxanthellae) of color morphs of Galaxea fascicularis collected at different sites of Okinawa, Japan

SH, shinorine; M2G, mycosporine-2 glycine; PR, porphyra-334; MG, mycosporine-glycine; PT, palythine; AS, asterina- 330; PL, palythinol; PN, palythene. Values are given as mean±standard deviation (n＝5-8) and expressed as nmol per mg protein. 114 Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis of Galaxea fascicularis (Fig. 2). BG morphs collected The phenogram based on the proportional complements from Sesoko and Oku had similar molar proportions of of MAAs in the coral tissues showed that BG morph four out of the five MAAs commonly present in their colonies collected from Sesoko and Oku were closely tissue, with marked differences only in palythine, the related to each other and formed a clade (Fig. 3). The Gs proportional concentration of which was considerably and Wt morphs collected from Zampa formed another higher in colonies collected from Sesoko (ANOVA, F(1,10) clade, although they were less similar than were the BG ＝51.402, P＜0.001). Both Gs and Wt morphs had sig­ morphs from Sesoko and Oku. The relatively large dis­ nificantly (TKH, P＜0.05) smaller proportions of mycos­ tance between the Gs and Wt morphs was mainly due to porine-glycine and three-fold larger (TKH, P＜0.05) the exceptionally low concentrations of porphyra-334 in proportions of shinorine than did BG morph colonies. the Wt colonies. Despite the similarities in relative abundance of mycos­ porine-glycine, shinorine, and palythene between the Gs Nematocyst types of different G. fascicularis color and Wt morphs (TKH, P＞0.05 for all three MAAs), morphs porphyra-334 accounted for about 26% of the total MAAs BG morph colonies from Sesoko and Oku had type I in the Gs morph but was scarcely present in the Wt morph. (S) microbasic p-mastigophores (MpM) and belonged to The proportions of palythine and palythinol were also morphotype S. Both Gs and Wt morph colonies collected different between Gs and Wt morphs (TKH, P＜0.05). from Zampa had MpM nematocysts of type II (H) and

Fig. 2 Proportional concentrations (molar fraction) of the mycosporine-like amino acids represented in the coral tissue of color morphs of Galaxea fascicularis collected from different sites of Okinawa, Japan; n＝5 for each group. Vertical bars represent ±SD. SH, shinorine; PR, porphyra-334; M2G, mycosporine-2 glycine; MG, mycosporine-glycine; PT, palythine; PL, palythinol; AS, asterina-330; PN, palythene; n.t. – no traces of MAAs. The color morphs and site of collection are shown in each figure Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis 115 belonged to morphotype H. also be classified into two groups based on allelic differ­ ences in a nuclear microsatellite locus. These two groups are highly correlated with morphotypes S and H as well Discussion as mitochondrial haplotypes Long and Short (Abe et al. 2008a). Furthermore, partial reproductive isolation was The present results show that different color morphs of observed between morphotypes S and H (Abe et al. Galaxea fascicularis had different compositions of MAAs. 2008b). Thus, the two clades detected using the pheno­ BG morph colonies from Sesoko and Oku (about 50 km gram might be genetically differentiated, and the differ­ apart) had similar MAA complements, which were very ences in MAA complements between the two clades may different from those of Gs and Wt morphs from Zampa reflect genetic distance between the colonies. In addition, (about 27 km from Sesoko). Gs and Wt morph colonies the lack of the secondary MAAs, palythinol and palythene, from Zampa contained six MAAs but only three (mycos­ in the Gs/Wt clade and asterina-330 in the BG clade may porine-glycine, shinorine, and palythine) of them were indicate metabolic differences in the host as previously common between BG and Gs/Wt morphs. These differ­ shown for genotypically different colonies of Stylophora ences are clearly reflected in the phenogram based on the pistillata (Shick 2004). However, the possibility that geo­ proportional complements of MAAs in the G. fascicularis graphic origin may also be of importance in MAA sig­ tissues, which shows two distinct clades (Fig. 3). nature differences cannot be ruled out in this study. The two clades corresponded with morphotypes S and Hunter (1985) found that not only the relative abun­ H based on tentacular nematocysts: BG morph colonies dance of MAAs but also their composition varied among from Sesoko and Oku belonged to morphotype S, and Gs morphotypes of the Hawaiian coral Porites compressa. and W morph colonies from Zampa belonged to morpho­ She suggested that MAA diversity might be caused by type H. Morphotypes S and H generally correspond to modification of their molecular structure by the animal mitochondrial haplotypes Long and Short, respectively tissue and, thus, the MAA complement may reflect genetic (Watanabe et al. 2005). Galaxea fascicularis colonies can differences among colonies. Shick et al. (2002) have shown that the complements of MAAs in zooxanthellate species of sea anemones from the genus Anthopleura are more closely associated with their phylogenetic position than with diet, environmental factors, or endosymbiont type. Conversely, the complement and abundance of MAAs may be regulated by environmental factors, including light intensity, depth, and water motion (reviewed by Shick and Dunlap 2002). Differences in MAA complements might also be due to differences between color morphs in the source of MAAs, since animals cannot synthesize MAAs because they lack enzymes involved in the shikimic acid pathway (Herrmann and Weaver 1999). Marine in­ vertebrates acquire MAAs from their diet (Banaszak and Trench 1995; Shick 2004), from bacteria (Dunlap and Fig. 3 Similarity phenogram of five individuals of each color morph of Galaxea fascicularis collected from different sites of Shick 1998; Yakovleva and Baird 2005), or from algal Okinawa, Japan, based on proportional concentrations (molar endosymbionts (Banaszak and Trench 1995; Shick 2004). fraction) of the mycosporine-like amino acids (MAAs) shown in Thus, it is still unclear whether intraspecific differences Fig. 2. Similarity was determined by cluster analysis and is ex­ in the composition and abundance of MAAs are due to pressed as linked Euclidean distance. The color morphs and site of collection are shown together with morphotype based on genetic distances between hosts, or whether such differ­ tentacular nematocysts (S or H) ences reflect different sources of MAAs or other factors 116 Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis such as environmental conditions. shinorine, and palythine, whereas those isolated from Wt In our study, Gs and Wt morph colonies from Zampa morph colonies contained only shinorine. This suggests contained the same MAA complements, but Gs morph that different color morphs of G. fascicularis harbor colonies had almost twice as many MAAs than did Wt zooxanthella genotypes with different predilections for morph colonies and this difference was mainly due to MAA synthesis. However, this remains to be confirmed higher amounts of mycosporine-glycine and porphyra- by genetic analysis of algal symbionts. It is also possible 334 in Gs morph colonies. Since Gs and Wt colonies were that, in some combinations of host and algal genotypes, present in sunlight-exposed and shaded areas, respectively, most MAAs produced by zooxanthellae might be quickly the amount of solar radiation may be a causative factor in translocated to their hosts, leaving low or trace amounts of the large MAA concentration difference between these MAAs within the algal cells (Banaszak and Trench 1995; color morphs. Mycosporine-glycine is known to be rapidly Shick 2004). Nevertheless, further study is necessary to accumulated by many marine invertebrates in response to elucidate why zooxanthellae isolated from the BG morph UV radiation (reviewed by Shick and Dunlap 2002). In did not contain detectable amounts of MAAs. turn, mycosporine-glycine can give rise to other primary The composition and quantity of MAAs in the algae MAAs, such as porphyra-334, by forming imines with and the host tissue were markedly different. This suggests threonine (Shick 2004). Jokiel and York (1982) reported a that algae alone may not determine the MAA complement significant loss of mycosporine-glycine and porphyra-334 of the host tissue but that MAAs may come from other from the coral Pocillopora damicornis after prolonged sources, such as diet or symbiotic bacteria (Dunlap and shielding from UV light. Similar results have been re­ Shick 1998; Yakovleva and Baird 2005). Another pos­ ported for the reef anemone Phyllodiscus semoni (Shick sibility is that some precursors of MAAs, such as de­ et al. 1991). Therefore, it is likely that high concentrations oxygadusol, are translocated from algal symbionts to the of these two MAAs in Gs morph colonies reflect the ac­ host and the host (or associated bacteria) modifies them to climation of these colonies to high solar radiation. In con­ synthesize secondary MAAs (Shick and Dunlap 2002; trast, low levels of MAAs in Wt morph colonies might Shick 2004). be due to the restriction of this color morph to sheltered In conclusion, the present study showed that color microhabitats. We expect that the decreased concentration morphs of the coral Galaxea fascicularis had different of mycosporine-glycine, the progenitor MAA (Shick and compositions and concentrations of MAAs. The pheno­ Dunlap 2002) possessing moderate antioxidant activity gram based on the proportional complements of MAAs (Dunlap and Yamamoto 1995), in Wt morph colonies may showed two clades, which corresponded with two mor­ render them more susceptible to environmental stresses photypes based on tentacular nematocysts, suggesting (elevated temperature and UV radiation), although this that MAA composition reflects phylogenetic differences should be confirmed by future studies. among the morphs. The composition and quantity of Zooxanthellae associated with different G. fascicularis MAAs may also be affected by environmental factors. color morphs showed different patterns of MAA pro­ Further studies are necessary to investigate how the duction. Zooxanthellae isolated from BG morph colonies genetic differentiation and geographic origin of corals did not contain detectable amounts of MAAs, while affect MAA signature differences. zooxanthellae isolated from Gs and Wt morph colonies contained detectable amounts of MAAs which were among the five primary Symbiodinium MAAs present in Acknowledgments Symbiodinium in culture (Banaszak et al. 2000; Shick 2004) or isolated from cnidarian hosts (Banaszak et al. This study was partly supported by Grant-in-Aid for 2006). However, while the same MAA complement was Scientific Research on Innovative Areas “Coral reef sci­ found in the host tissue of Gs and Wt morphs, zooxanthellae ence for symbiosis and coexistence of human and eco­ from Gs morph colonies contained mycosporine-glycine, system under combined stresses” (No. 20121002) of the Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis 117

Ministry of Education, Culture, Sports, Science and the principle of protein-dye binding. Anal Biochem 72: Technology (MEXT), Japan and the 21st Century Centers 248-254 of Excellence (COE) Program of the University of the Chevalier JP (1971) Les scleractiniaires de la melanesie francaise Ryukyus. The authors thank the late Dr. Toshiki Watanabe (Nouvelle-Caledonie, Iles Chesterfield, Iles Loyaute, for information on the mitochondrial haplotypes of the Nouvelles-Hebrides). 1ere Partie. Exped Recifs Coralliens Nouvelle-Caledonie, Fond. Singer-Polignac, Paris 5: 5- color morphs. The authors also thank the staff of Sesoko 307, pl 1-38 (Galaxea: pp 58-85, pl 3-9) Station, Tropical Biosphere Research Center, where part Dunlap WC, Chalker BE (1986) Identification and quantification of this study was conducted. MH would like to express his of near-UV absorbing compounds (S-320) in a hermatypic hearty thanks to the late Prof. Kiyoshi Yamazato who scleractinian. Coral Reefs 5: 155-159 taught him about the fascinating life of corals and who Dunlap WC, Shick JM (1998) Ultraviolet radiation-absorbing gave him the opportunity to study coral biology 30 years mycosporine-like amino acids in coral reef organisms: a ago. bio-chemical and environmental perspective. J Phycol 34: 418-430 Dunlap WC, Yamamoto Y (1995). Small-molecule antioxidants References in marine organisms: antioxidant activity of mycosporine- glycine. Comp Biochem Physiol B112: 105-114 Abe M, Watanabe T, Suzuki Y, Hidaka M (2008a) Genetic and Gleason DF (1993) Different effects of ultraviolet radiation on morphological differentiation in the hermatypic coral green and brown morphs of the Caribbean coral Porites Galaxea fascicularis in Okinawa, Japan. Plankton Benthos astreoides. Limnol Oceanogr 38: 1452-1463 Res 3: 174-179 Herrmann KM, Weaver LM (1999) The shikimate pathway. Abe M, Suzuki Y, Hayakawa H, Watanabe T, Hidaka M (2008b) Annu Rev Plant Physiol 50: 473-503 Breeding experiments of the hermataypic coral Galaxea Hidaka M, Yamazato K (1985) Color morphs of Galaxea fascicularis: partial reproductive isolation between colo­ fascicularis found in the reef around the Sesoko Marine nies of different nematocyst types, and enhancement of Science Center. Galaxea 4: 33-35 fertilization success by the presence of parental colonies. Hidaka M (1992) Use of nematocyst morphology for taxonomy Fish Sci 74: 1342-1344 of some related species of scleractinian corals. Galaxea 11: Adams NI, Shick JM (1996) Mycosporine-like amino acids 21-28 provide protection against ultraviolet radiation in eggs of Hirabayashi S, Kasai F, Watanabe MM, Hirose E (2006) Con­ the green sea urchin, Strongylocentrotus droebachiensis. tents of ultraviolet-absorbing substances in two color Photochem Photobiol 64: 149-158 morphs of the photosymbiotic ascidian Didemnum molle. Banaszak AT, Trench RK (1995) Effects of ultraviolet (UV) Hydrobiologia 571: 419-422 radiation on marine microalgal-invertebrate symbiosis. II. Hunter CL (1985) Assessment of clonal diversity and population The synthesis of mycosporine-like amino acids in response structure of Porites compressa (Cnidaria, Scleractinia). to exposure to UV in Anthopleura elegantissima and Cas­ Proc Fifth Int Coral Reef Congr 6: 69-74 siopeia xamachana. J Exp Mar Biol Ecol 194: 233-250 Johannes RE, Wiebe WJ (1970) A method for determination of Banaszak AT, LaJeunesse TC, Trench RK (2000) The synthesis coral tissue biomass and composition. Limnol Oceanogr of mycosporine-like amino acids (MAAs) by cultured, 21: 540-547 symbiotic dinoflagellates. J Exp Mar Biol Ecol 249: 219- Jokiel PL, York RHJ (1982) Solar ultraviolet photobiology of 233 the reef coral Pocillopora damicornis and symbiotic Banaszak AT, Santos MGB, LaJeunesse TC, Lesser MP (2006) zooxanthellae. Bull Mar Sci 32: 301-315 The distribution of mycosporine-like amino acids (MAAs) Karsten U, Franklin L A, Luning K, Wiencke C (1998) Natural and the phylogenetic identity of symbiotic dinoflagellates ultraviolet radiation and photosynthetically active radiation in cnidarian hosts from the Mexican Caribbean. J Exp Mar induce formation of mycosporine-like amino acids in the Biol Ecol 337: 131-146 marine macroalga Chondrus crispus (Rhodophyta). Planta Bradford MM (1976) A rapid and sensitive method for the 205: 257-262 quantification of microgram quantities of protein utilizing Neale PJ, Banaszaak AT, Jarriel CR (1998) Ultraviolet sun­ 118 Yakovleva and Hidaka: Survey of mycosporine-like amino acids of Galaxea fascicularis

screens in Gymnodinium sanguineum (Dinophyceae): differentiation in the Caribbean sea anemone Condylactis mycosporine-like amino acids protect against inhibition of gigantea. Mar Biol 147: 744-754 photosynthesis. J Phycol 34: 928-938 Veron JEN, Pichon M (1980) Scleractinia of Eastern Australia. Shick JM (2004) The continuity and intensity of ultraviolet Part III. Families Agariciidae, Siderastreidae, Fungiidae, irradiation affect the kinetics of biosynthesis, accumulation, Oculinidae, Merulinidae, Mussidae, Pectiniidae, Caryo­ and conversion of mycosporine-like amino acids (MAAs) phylliidae, Dendrophylliidae. Aust Inst Mar Sci Monogr in the coral Stylophora pistillata. Limnol Oceanogr 49: Ser 4 442-458 Watanabe T, Nishida M, Watanabe K, Wewengkang DS, Hidaka Shick JM, Dunlap WC (2002) Mycosporine-like amino acids M (2005) Polymorphism in the nucleotide sequence of a and related gadusols: biosythesis, accumulation, and UV- mitochondrial intergenic region in the scleractinian coral protective functions in aquatic organisms. Annu Rev Galaxea fascicularis. Mar Biotech 7: 33-39 Physiol 64: 223-262 Wewengkang DS, Watanabe T, Hidaka M (2007) Studies on Shick JM, Lesser MP, Stochaj WR (1991) Ultraviolet radiation morphotypes of the coral Galaxea fascicularis from Oki­ and photo oxidative stress in zooxanthellate Anthozoa: nawa: polyp color, nematocyst shape, and coenosteum the sea anemone Phyllodiscus semoni and the octocoral density. Galaxea JCRS 9: 49-59 Clavularia sp. Symbiosis 10: 145-173 Wicksten MK (1989) Why are there bright colors in sessile Shick JM, Dunlap WC, Chalker BE, Banaszak AT, Rosenzweig marine invertebrates? Bull Mar Sci 45: 519-530 TK (1992) Survey of ultraviolet radiation-absorbing Yakovleva IM, Baird AH (2005) Ontogenetic change in the mycosporine-like amino acids in organs of coral reef abundance of mycosporine-like amino acids in non- holothuroids. Mar Ecol Prog Ser 90: 139-148 zooxanthellate coral larvae. Coral Reefs 24: 443-452 Shick JM, Dunlap WC, Pearse JS, Pearse VB (2002) Mycos­ porine-like amino acid content in four species of sea anem­ Received: 23 September 2009 ones in the genus Anthopleura reflects phylogenetic but Accepted: 21 October 2009 not environmental or symbiotic relationships. Biol Bull 203: 315-330 Ⓒ Japanese Coral Reef Society Stoletzki N, Schierwater B (2005) Genetic and color morph