Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 www.elsevier.com/locate/jembe

The distribution of mycosporine-like amino acids (MAAs) and the phylogenetic identity of symbiotic in cnidarian hosts from the Mexican Caribbean ⁎ Anastazia T. Banaszak a, , Maria Guadalupe Barba Santos a, Todd C. LaJeunesse b, Michael P. Lesser c

a Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apartado Postal 1152, Cancún, Quintana Roo, 77500, Mexico b Department of Biology, Florida International University, University Park Campus, Miami, Florida, 33199, USA c Department of Zoology and Center for Marine Biology, University of New Hampshire, Durham, New Hampshire, 03824, USA Received 22 May 2006; accepted 10 June 2006

Abstract

A survey of 54 of symbiotic cnidarians that included hydrozoan corals, anemones, gorgonians and scleractinian corals was conducted in the Mexican Caribbean for the presence of mycosporine-like amino acids (MAAs) in the host as well as the Symbio- dinium fractions. The host fractions contained relatively simple MAA profiles, all harbouring between one and three MAAs, principally mycosporine-glycine followed by shinorine and porphyra-334 in smaller amounts. populations were identified to sub-generic levels using PCR-DGGE analysis of the Internal Transcribed Spacer 2 (ITS2) region. Regardless of clade identity, all Symbiodinium extracts contained MAAs, in contrast to the pattern that has been found in cultures of Symbiodinium, where clade A symbionts produced MAAs whereas clade B, C, D, and E symbionts did not. Under natural conditions between one and four MAAs were identified in the symbiont fractions, mycosporine-glycine (λmax =310 nm), shinorine (λmax =334 nm), porphyra-334 (λmax =334 nm) and palythine (λmax =320 nm). One sample also contained mycosporine-2-glycine (λmax =331 nm). These data suggest that Symbiodinium is restricted to producing five MAAs and there also appears to be a defined order of appearance of these MAAs: mycosporine-glycine followed by shinorine (in one case mycosporine-2-glycine), then porphyra-334 and palythine. Overall, mycosporine-glycine was found in highest concentrations in the host and symbiont extracts. This MAA, unlike many other MAAs, absorbs within the ultraviolet-B range (UVB, 280–320 nm) and is also known for moderate antioxidant properties thus potentially providing protection against the direct and indirect effects of UVR. No depth-dependent changes could be identified due to a high variability of MAA concentrations when all species were included in the analysis. The presence of at least one MAA in all symbiont and host fractions analyzed serves to highlight the importance of MAAs, and in particular the role of mycosporine-glycine, as photoprotectants in the coral reef environment. © 2006 Elsevier B.V. All rights reserved.

Keywords: Clade analysis; Photoprotection; Symbiodinium; ; Ultraviolet radiation; UV

1. Introduction ⁎ Corresponding author. Tel.: +52 998 871 0219x66; fax: +52 998 871 0138. Environmentally-relevant ultraviolet radiation (UVR, E-mail address: [email protected] (A.T. Banaszak). 280 to 400 nm) is a key factor in the structure and

0022-0981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2006.06.014 132 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 distribution of coral reef communities (Jokiel, 1980; Shick the sensitivity of photosynthesis in the UVrange from 320 et al., 1996; Dunlap and Shick, 1998; Gleason, 2001). to 360 nm whereas in low light grown cultures with low Organisms living in shallow water tropical reef environ- concentrations of MAAs, the cultures were highly ments are potentially exposed to high UVR doses and dose sensitive within this wavelength range. The absorption rates due to the low solar zenith angles, the natural thinness spectrum of the MAAs corresponded almost exactly to the of the ozone layer over tropical latitudes, as well as the high maximal biologically-effective UVR spectrum estimated transparency of the water column (Smith and Baker, 1979; for this , showing that these optical screens Baker et al., 1980). UVR can penetrate to 25 m in coral reef provide complete protection exactly within the most environments (Fleischmann, 1989) although this value can damaging wavelength range (Neale et al., 1998). The change dramatically depending on the composition of the photoprotective role of MAAs has also been demon- water column (Smith and Baker, 1979; Baker et al., 1980). strated for different developmental stages of S. droeba- An important photoprotective response by marine chiensis. An inverse relationship was shown between the organisms is the production of mycosporine-like amino concentration of MAAs and the first cleavage delay in acids (MAAs). Due to the high molar absorptivity of eggs as well as a reduction in the levels of UV-induced MAAs (Є ranges from 28,100 to 50,000 mol−1 lcm−1), developmental abnormalities in embryos and larvae they absorb very efficiently within the wavelength range (Adams and Shick, 1996, 2001). However, sea urchin from 309 to 360 nm (Dunlap and Shick, 1998). MAAs larvae with high MAA concentrations suffered some have absorption bands that are wide at about 40 nm cleavage delays as well as abnormalities (Adams and FWHM for asterina-330 (λmax =330 nm), mycosporine- Shick, 1996, 2001) showing that at least during embryo- glycine (λmax =310 nm) and palythinol (λmax =332 nm) genesis of sea urchins, MAAs do not provide complete and 50 nm FWHM for purified shinorine (λmax =334nm) protection. Accumulation of DNA damage could be and for palythine (λmax =320 nm) (Dunlap et al., 1989; responsible for some of the cleavage delays in embryos Gleason, 1993; Sinha et al., 1998; Adams and Shick, exposed to UVR (Lesser et al., 2006). 2001), compared to chlorophyll a, which at room tem- Many studies provide supporting evidence for the perature in methanol has an FWHM of 23 nm (Balny et al., photoprotective role of MAAs in corals. MAA concentra- 1969). Individually, therefore, MAAs provide a wide tions decrease with depth (Dunlap et al., 1986; Gleason, spectral screen against UVR and when found in com- 1993; Gleason and Wellington, 1995; Shick et al., 1995; bination the screening potential is even broader. Banaszak et al., 1998; Corredor et al., 2000; Lesser, 2000) The combination of efficient absorption of UVR to andintheabsenceofUVR(Banaszak and Trench, 1995; prevent its absorption by sensitive cellular components, Shick et al., 1991). Compared to other phyla, the harmless dissipation of these potentially damaging cnidarians possess the highest diversity of MAAs (Banas- wavelengths preventing the formation of singlet oxygen or zak, 2003). At least 11 different MAAs have been char- superoxide radicals and their photostability indicates that acterized from hard corals alone (Dunlap and Chalker, MAAs are ideal photoprotectants. High energy photons 1986; Dunlap et al., 1986; Gleason, 1993; Gleason and absorbed by shinorine and porphyra-334 are rapidly Wellington, 1995; Shick et al., 1995; Wu Won et al., 1995, dissipated as heat by internal conversion as indicated by 1997; Drollet et al., 1997; Jokiel et al., 1997; Teai et al., their low fluorescence quantum yields and by direct 1997, 1998; Banaszak et al., 1998) with up to 10 MAAs photoacoustic calorimetric determinations (Conde et al., identified in a single coral, Stylophora pistillata (Shick 2004). In addition there is no energy transfer to chlorophyll et al., 1999; Shick, 2004). Various cnidarian species have a as well as an absence of free radical or triplet state been shown to contain MAAs exclusively in the host formation (Conde et al., 2000, 2003, 2004; Moison and component (Banaszak and Trench, 1995; Yakovleva and Mitchell, 2001, Whitehead and Hedges, 2005). Hidaka, 2004), which may be accounted for by diet The effectiveness of MAAs as photoprotectants has (Banaszak and Trench, 1995; Shick, 2004)orduetothe been demonstrated in a cultured, bloom-forming dinofla- presence of bacteria such as Vibrio (Dunlap and Shick, gellate Akashiwo sanguinea (=Gymnodinium sangui- 1998). neum)(Neale et al., 1998) and in the green sea urchin MAAs may also be acquired from symbionts such as Strongylocentrotus droebachiensis (Adams and Shick, has been shown in the association between the upside 1996, 2001; Lesser et al., 2006). Using biological down jellyfish Cassiopeia xamachana and the dinofla- weighting functions for the inhibition of photosynthesis gellate Symbiodinium microadriaticum (Banaszak and in the bloom-forming dinoflagellate A. sanguinea, Neale Trench, 1995). In culture, the photoprotectants are et al. (1998) showed that the presence of high concen- produced by the algae and leaked to the surrounding trations of MAAs in high light grown cultures eliminated medium. While the aposymbiotic scyphistomae stage A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 133 does not contain MAAs, upon infection with symbiotic produced in two species of corals, Platygyra ryukyuensis algae, the ephyrae and adult jellyfish stages contain the and S. pistillata (Yakovleva et al., 2004). same suite of MAAs as the symbiotic algae in culture Although many species, including corals, have been (Banaszak and Trench, 1995). surveyed for the presence of MAAs, few studies to date Due to the inherent difficulties of identifying Symbiodi- restricted to studies in laboratory culture have separated nium spp. the DNA sequence diversity of the small subunit the symbionts from the host tissue and determined the ribosomal DNA gene (SSUrDNA) has been used to assign MAA profiles for both components. The objective of this Symbiodinium into sub-generic lineages (clades) within a study was to determine whether the relationship of MAAs phylogenetic tree (Rowan and Powers, 1991). Originally, to clade identity in field collected organisms is consistent three clades A, B and C were identified and over the last with that found in laboratory cultures by determining the decade the number of recognized clades has increased to MAA distribution in many cnidarian species at one sam- eight (A to H) (Coffroth and Santos, 2005). Many clades pling point. contain numerous ecologically distinctive types character- ized principally through PCR-denaturing gradient gel 2. Materials and methods electrophoresis (DGGE) analyses of the Internal Tran- scribed Spacer (ITS) regions (LaJeunesse, 2002, 2005). 2.1. Collection of samples Clades A through D maintain associations with scleractinian corals. In the Mexican Caribbean based on an extensive Samples of C. xamachana were collected in the sampling of the reefs of Puerto Morelos, approximately 40 Bojorquez Lagoon, Cancun (21°07′N, 86°45′W) whereas distinct types have been found (LaJeunesse, 2002). A few the rest of the samples of symbiotic cnidarians were types, C1, C3 and B1, are generalists, being found in many collected by SCUBA or snorkel on reefs adjacent to Puerto different host taxa, whereas there are many distinctive types, Morelos, Quintana Roo on the northeast coast of the referred to as specialists that populate specific host taxa Yucatan Peninsula, Mexico (20°50′N, 86°52′W). Samples (LaJeunesse, 2002; LaJeunesse et al., 2003). were collected from four sites: the reef crest (1–2 m), the All Symbiodinium belonging to clade A, including shallow lagoon (1–3 m) and the fore reef (3–8m),all S. microadriaticum (Banaszak and Trench, 1995)as located at the “La Bocana Chica” reef and from the fore well as the reclassified S. (=Gymnodinium) linucheae reef (10–15 m) at Petem Pich. Table 1 contains collection (LaJeunesse, 2001) synthesized up to three MAAs whereas depths and sites for the species used in this study. The none of the clades B and C Symbiodinium produced MAAs samples were transported in seawater, covered to prevent in culture (Banaszak et al., 2000). The one representative overexposure to solar radiation and to maintain tempera- of clade E S. “californium”, the symbiont of Anthopleura tures relatively constant and brought to the laboratory elegantissima (LaJeunesse, 2001), did not produce MAAs within 2 h of collection. On arrival, the samples were in culture (Banaszak and Trench, 1995)nordidS. kawagutii maintained in a covered aquarium fitted with flowing (Banaszak et al., 2000) that belongs to clade F seawater and processed within 36 h of collection. (LaJeunesse, 2001). The symbiont of Aiptasia pallida in Gorgonian species were processed immediately due to culture, S. bermudense,producedMAAsonexposureto their inability to withstand confined conditions. Prior to artificial UVR (Lesser, 1996) and although at the time the processing of the samples, the surfaces were cleaned of clade type was not determined, it was later described as contaminants using 45 μm filtered seawater. Due to clade B (Rowan and Powers, 1991; Banaszak et al., 2000). collection permit restrictions, few replicates could be Under the experimental conditions used by Banaszak et al. taken. (2000) however, S. bermudense did not produce MAAs. Mycosporine-glycine is the more frequently observed 2.2. Isolation of Symbiodinium from host tissues and most concentrated MAA among diverse cnidarian species in various field studies (Dunlap and Chalker, 1986; The isolation of Symbiodinium from host tissues Dunlap et al., 1986; Shick et al., 1991, 1995; Gleason, depended on host type. Branches of gorgonian colonies 1993; Gleason and Wellington, 1995; Teai et al., 1997; were cut into 2 to 3 cm fragments and ground in a mortar Banaszak et al., 1998). This MAA has been shown to have and pestle with extraction buffer (EB, 1.2 μm filtered moderate, concentration-dependent antioxidant activity by seawater and 5 mM EDTA). In some cases, the resultant scavenging free radicals in extracts from marine organisms slurry was filtered through a 100 μm mesh to remove (Dunlap and Yamomoto, 1995) and quenching singlet large fragments of host tissue or mucous. oxygen (Suh et al., 2003). It also provides rapid protection Scleractinian and hydrozoan tissues were separated against oxidative stress while antioxidant enzymes can be from the coral skeleton (5 to 50 cm2) using a Water-Pik 134 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146

Table 1 Table 1 (continued) Collection information for the species sampled in this study Host species Nichupte Lagoon Rear Channel Petem Host species Nichupte Lagoon Rear Channel Petem sampled Pond (1–3m) Zone (5–10 m) Pich sampled Pond (1–3m) Zone (5–10 m) Pich (1–2.5 m) (12–14 m) (1–2.5 m) (12–14 m) Meandrinidae Scyphozoa Dendrogyra 1.5 Cassiopeia 0.5 cylindricus xamachana Dichocoenia 8 shade (n=3) stokesi C. xamachana 0.5 Meandrina 212 light (n=3) meandrites Hydrozoa Poritidae Millepora 17 Porites astreoides 17 alcicornis P. colonensis 7 M. complanata 17 P. divaricata 1 (brown) Scleractinia P. divaricata 1 Acroporidae (yellow) Acropora 5 P. furcata 1 1.5 7 cervicornis Siderastreidae A. palmata 1 Siderastrea radians 1 Agariciidae (n=3) Agaricia agaricites 7 Astrocoeniidae f. agaricites Stephanocoenia 2.5 12 (brown) michelini A. agaricites 7 Actinaria f. agaricites Aiptasia tagetes 0.5 (yellow) A. tagetes (pallida) 0.1 A. agaricites 1.5 Condylactis 1 f. danai gigantea A. agaricites 7 Lebrunia danae 1 f. purpurea Gorgonacea A. fragilis 8 Briareum 7 A. humilis 1 asbestinum A. tenuifolia 1.5 Eunicea mammosa 7 Leptoseris 7 Gorgonia flabellum 7 cucullata Muricea muricata 7 Leptoseris sp. 6 Muriceopsis flavida 7 Caryophylliidae Plexaura 1.5 Eusmilia fastigiata 2.5 7 homomalla Faviidae Pseudoplexaura 7 Colpophyllia 1.5 7 flagellosa natans P. wagenaari 1.5 Diploria clivosa 2.5 1 Pterogorgia citrine 7 D. labyrinthiformis 7 Zoanthidea D. strigosa 2.5 Palythoa 1.5 7 Favia fragum 1 caribaeorum Manicina areolata 2.5 P. grandis 7 Montastraea 7 Zoanthus sociatus 7 annularis Corallimorpharia M. cavernosa 1 Discosoma sp. 7 M. faveolata 2.5 7 Ricordea florida 1.5 7 (n=3 at 2.5 m) Mussidae Isophyllastrea 7 or airbrush (Paasche Millenium, USA) with EB into a rigida Isophyllia sinuosa 1 plastic zip-lock bag to collect the disrupted tissue. The Mycetophyllia 7 plastic bag was sealed and shaken vigorously for 1 to lamarckiana 3 min to disrupt polysaccharides when there were large Mycetophyllia 7 amounts of mucous. The disrupted tissue was then sp. juvenile homogenized with a Tissue-Tearor. A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 135

The soft-bodied species, scyphozoans, actiniarians, scribed in LaJeunesse (2002). The reaction products were corallimorphs and zoanthids, were first cleaned of debris run on an 8% polyacrylamide denaturing gel containing a by hand. The oral discs, including , were gradient of 3.15 M urea/18% deionized formamide to removed with a razor blade, chopped into small pieces, 5.6 M urea/37% deionized formamide and separated by resuspended in EB and homogenized with a Tissue electrophoresis for 9.5 h at 150 Vat a constant temperature Tearor (Biospec Products, Inc., USA) or glass grinder in of 60 °C along with ITS 2 standards as described in the case of actiniarian anemones. LaJeunesse (2001, 2002). All samples were loaded with a To separate the symbionts from host tissues, the 2% Ficoll loading buffer (2% Ficoll-400, 10 mM Tris– slurries were centrifuged for 5 min at 1000×g in a HCl pH 7.8, 1 mM EDTA, 1% bromophenol blue). The swinging bucket rotor in a clinical centrifuge (Centra gel was stained in 1× TAE and Syber Green (Molecular CL2, IEC, USA). The supernatant, containing host tissue Probes, Eugene, Oregon) for 25 min using the manufac- as determined by light microscopy at 200× magnification, turer's specifications, and the gel was then photographed. was frozen at −70 °C, lyophilized (Labconco, USA) and If new fingerprint profiles were identified, diagnostic stored at −20 °C for MAA and protein analysis. The bands were excised from the denaturing gel, eluted and pellet, containing mostly symbionts, was resuspended in reamplified with primers lacking the GC-clamp. Positive EB and homogenized and 0.01% (v/v) Triton X-100 was reactions were purified using a Promega Wizard PCR- added and gently mixed. The slurry was centrifuged using prep DNA kit (Promega, Madison, Wisconsin) and di- a fixed angle rotor for 2 min at 925×g, and the supernatant rectly sequenced and analyzed on an Applied Biosystems discarded. This step removed any remaining host cell 310 genetic analyzer (Division of Perkin Elmer, Foster debris as determined by observation with a light City, California) and the sequences compared against a microscope at 200× magnification. The pellet was database of established types (cf. LaJeunesse, 2005). resuspended immediately in EB, mixed well and centrifuged under the same conditions. The wash step 2.4. Absorbance spectra of coral fragment extracts was repeated two more times. The final pellet was centrifuged with a fixed angle rotor at 4500×g for 2 min in Absorption spectra of methanolic extracts of coral a microcentrifuge (Costar 10MVSS, Corning-Costar, fragments were recorded between 250 and 750 nm using USA). A small portion of the resulting pellet was stored an Aminco DW2 spectrophotometer controlled by an in a high salt (NaCl-saturated), DMSO (20%), EDTA Olis data collection system and methanol as a reference. (0.25 M) preservation buffer (Seutin et al., 1991). The remainder of the pellet was frozen at −70 °C, lyophilized 2.5. Determination of MAA concentrations and stored at −70 °C for MAA and protein analysis. For all symbiont and host samples the extraction 2.3. DNA extraction and DGGE analysis and analysis of MAAs were performed according to the procedures in Dunlap and Chalker (1986) as The Wizard DNA-prep protocol (Promega, Madison, modified by Shick et al. (1992). The lyophilized WI), modified by LaJeunesse et al. (2003), was used to samples were resuspended in 100% HPLC grade extract nucleic acids. Approximately 10 to 30 mg of methanol and extracted at 4 °C overnight. The extracts material was vortexed with glass beads and 600 μlNuclei were centrifuged at 1000×g for 2 min and the Lysis Buffer (Promega). The lysate was incubated with supernatant was used for MAA and protein analysis. 0.1 mg ml−1 proteinase K for 1 to 2 h at 65 °C. Protein MAAs were separated by reverse-phase, isocratic high Precipitation Buffer (250 μl; Promega) was then added performance liquid chromatography (HPLC) on a and the extract was incubated on ice for 15 min. After Brownlee RP-8 column (Spheri-5, 4.6 mm centrifugation for 5 min at 12,000 rpm, 600 μlofsuper- ID×250 mm), which was protected with an RP- natant was transferred and 700 μl isopropanol (100%) and 8 guard column (Spheri-5, 4.6 mm ID×30 mm). The 30 μlNaAc(3M,pH5.6)wereadded.Followingin- mobile phase consisted of 40% to 55% methanol (v/v), cubation on ice for 10 min, the precipitated DNA was 0.1% glacial acetic acid (v/v) in water and run at a centrifuged and the pellet washed with 70% ethanol. The flow rate of 0.6 ml min− 1. MAA peaks were detected DNA was centrifuged again for 5 min, dried and resus- by absorbance at 313 and 340 nm. Standards were pended in 70–80 μl water. The final eluate from each available for the following seven MAAs: mycosporine- sample was diluted 1:10 and 1 μl was used as a template glycine, shinorine, porphyra-334, palythine, asterina- for amplification. The internal transcribed spacer 2 region 330, palythinol and palythene. Peak identities were (ITS 2) was then amplified for DGGE analysis as de- confirmed by co-chromatography with standards and 136 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 with the ratios of 313 to 340 nm absorbances. Peaks 1.80%) and porphyra-334 (0.18% to 0.85%). For one were integrated and the quantification of individual species, Ricordea florida from 7 m depth, mycosporine- MAAs was accomplished using HPLC peak areas and glycine accounted for 60.72% of the total MAAs, with calibration factors as determined by analysis of the 39.19% by porphyra-334 and the remainder (0.08%) seven standards. MAAs were normalized to soluble made up of shinorine. There is no relationship between protein as determined from an aliquot of the methanol- how many MAAs are present within the Symbiodinium extracted sample using the procedure of Bradford fraction and those present in the host fraction, except that (1976) in kit form (Bio-Rad, Inc.). Concentrations are mycosporine-glycine occurs in all symbionts and hosts expressed in nmol MAA mg protein− 1. (Table 2) and there is no relationship between the presence of MAAs in the symbionts and clade 3. Results designation (Table 2). The proportion of MAAs within the symbiont In total, up to four MAAs, mycosporine-glycine, fraction relative to the host fraction was very variable shinorine, porphyra-334 and palythine were quantifi- ranging from 2.75% of the total holosymbiont MAAs ably detected in Symbiodinium (Table 2). The symbiont in M. areolata from 2.5 m depth to 69.70% of the total fraction of Manicina areolata contained large amounts holosymbiont MAAs in Condylactis gigantea from of mycosporine-2-glycine although due to the lack of a 1 m depth (Fig. 1). The total concentration of MAAs standard this MAA could not be quantified. Forty-eight for all of the species sampled (Fig. 2A), for the samples of corals contained one MAA only, always symbiont fraction (Fig. 2B) and for the host fraction mycosporine-glycine, in the symbiont fraction. Fifteen (Fig. 2C) does not vary with depth. In individual coral samples contained mycosporine-glycine as the major species, which were sampled at different depths, there component (greater than 50% of the total concentration) is a trend for decreasing MAA concentration but the and in two samples mycosporine-glycine was found lack of replicate within species samples prevents any with less than 50% of the total concentration (48.29% in statistically significant conclusions. On a reef-wide Porites divaricata from 1 m depth and 16.61% in Pa- basis at any depth there is a wide variability in the total lythoa caribaeorum from 1.5 m depth). The second MAA concentrations found within the symbionts and major component in the Symbiodinium fraction was within their hosts (Fig. 2A–C). Absorption spectra of shinorine, found in 17 samples (51.71% in P. divaricata methanolic extracts of a selection of species collected from 1 m depth to 0.09% in Gorgonia flabellum from between 2 and 3 m depth (Fig. 3A, B) highlight this 7 m depth) followed by porphyra-334 in 7 samples variability with pronounced peaks in the UV region in (38.70% in Discosoma sp. to 0.30% in Montastraea some species (Fig. 3A) and shoulders or indistinct annularis both from 7 m depth) and palythine in 3 peaks in other species (Fig. 3B). samples (46.45% in P. caribaeorum from 1.5 m depth In Table 3, the number of MAAs and their identities and 0.20% in G. flabellum from 7 m depth). The for Symbiodinium freshly isolated from a wide variety concentrations of total MAAs ranged from 5.94 to of hosts are shown based on the number of MAAs that 365.63 nmol MAA mg protein− 1 in the symbionts is produced. In some cases, Symbiodinium does not isolated from the hosts M. areolata and Stephano- contain MAAs even though the host fraction does, coenia michelini, respectively, both of which were although this does not apply for any of the Symbiodi- collected at 2.5 m depth. nium analyzed in the present study. The rest of the MAA concentrations ranged from a minimum of Symbiodinium are separated based on the number of 34.74 nmol MAA mg protein− 1 in the host fraction of MAAs that they produce. Symbiodinium that contain Agaricia fragilis collected at 8 m depth to a maximum one MAA always contain mycosporine-glycine. Those of 604.85 nmol MAA mg protein− 1 in the host fraction that contain two MAAs contain mycosporine-glycine of Agaricia agaricites f. danai collected at 1.5 m depth. and shinorine, with one exception, the symbionts of M. Between one and three MAAs (mycosporine-glycine, areolata that contain mycosporine-glycine and mycos- shinorine and porphyra-334) were detected in the porine-2-glycine. Symbiodinium that contain three cnidarian host species (Table 2). In 59 of the 68 species MAAs contain mycosporine-glycine, shinorine and only mycosporine-glycine was detected and in the porphyra-334 with one exception in P. caribaeorum remaining 9 species, mycosporine-glycine was the where the third MAA is palythine rather than predominant MAA. In eight of these nine species, porphyra-334. The symbionts of Dendrogyra cylindri- mycosporine-glycine accounted for 97.35% to 99.68% cus and G. flabellum contain four MAAs, mycospor- of the total MAAs followed by shinorine (0.18% to ine-glycine, shinorine, porphyra-334 and palythine, the A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 137

Table 2 Identification of host species, clade designation of symbiont isolated from the host species and the identity of mycosporine-like amino acids found in the symbiont fractions and in the host fractions Host species sampled Clade Number of % MG % SH % PR % PI Number of % MG % SH % PR % of % of MAAs designation MAAs in MAAs MAAs in in host of symbiont symbiont in host symbiont species Scyphozoa Cassiopeia xamachana A1/B1 2⁎ 64.94 35.06 1 100.00 51.26 48.74 shade (n=3) C. xamachana high A1 2⁎ 50.03 49.97 1 100.00 18.55 81.45 light (n=3)

Hydrozoa Millepora alcicornis A4a 1 100.00 1 100.00 11.00 89.00 M. alcicornis n.d. 1 100.00 n.d. M. complanata B1 2 65.96 34.04 1 100.00 15.07 84.93 M. complanata n.d. 1 100.00 n.d.

Acroporidae Acropora cervicornis A3 1 100.00 1 100.00 13.81 86.19 A. palmata A3 1 100.00 1 100.00 19.91 80.09

Agariciidae Agaricia agaricites C3a 1 100.00 2 99.68 0.32 14.69 85.31 f. agaricites (brown) A. agaricites C3a 1 100.00 3 99.33 0.49 0.18 4.28 95.72 f. agaricites (yellow) A. agaricites f. danai 2 71.87 28.13 2 99.53 0.47 4.35 95.65 A. agaricites f. purpurea 1 100.00 3 97.35 1.80 0.85 25.45 74.55 A. fragilis 1 100.00 1 100.00 47.91 52.09 A. humilis C3a 1 100.00 1 100.00 22.47 77.53 A. tenuifolia C3a 1 100.00 1 100.00 10.51 89.49 Leptoseris cucullata C3 1 100.00 1 100.00 12.89 87.11 Leptoseris sp. 1 100.00 1 100.00 25.80 74.20

Caryophylliidae Eusmilia fastigiata B1 1 100.00 1 100.00 27.79 72.21 E. fastigiata B1 1 100.00 1 100.00 14.04 85.96

Faviidae Colpophyllia natans B6 3 62.15 27.79 10.07 1 100.00 39.91 60.09 C. natans 1 100.00 1 100.00 22.45 77.55 Diploria clivosa B1 1 100.00 1 100.00 17.42 82.58 D. clivosa 1 100.00 1 100.00 12.39 87.61 D. labyrinthiformis B1 1 100.00 1 100.00 23.33 76.67 D. strigosa B1 1 100.00 1 100.00 25.04 74.96 Favia fragum B1 3 94.66 4.74 0.60 1 100.00 14.69 85.31 Manicina areolata B1 1⁎⁎ 100.00 1 100.00 2.75 97.25 Montastraea annularis B1 3 99.21 0.49 0.30 1 100.00 18.88 81.12 M. cavernosa C3 1 100.00 1 100.00 10.91 89.09 M. faveolata lagoon (n=3) D1a 1 100.00 1 100.00 15.53 84.47 M. faveolata C7 1 100.00 n.d. n.d.

Mussidae Isophyllastrea rigida C3 2 94.48 5.52 1 100.00 28.83 71.17 Isophyllia sinuosa C3 1 100.00 1 100.00 18.05 81.95 Mycetophyllia C3 1 100.00 1 100.00 15.67 84.33 lamarckiana Mycetophyllia C? 1 100.00 1 100.00 23.12 76.88 sp. juvenile (continued on next page) 138 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146

Table 2 (continued) Host species sampled Clade Number of % MG % SH % PR % PI Number of % MG % SH % PR % of % of MAAs designation MAAs in MAAs MAAs in in host of symbiont symbiont in host symbiont species Meandrinidae Dendrogyra cylindricus B1 3 54.32 19.64 25.61 2 99.16 0.84 15.12 84.88 Dichocoenia stokesi B1 1 100.00 1 100.00 13.60 86.40 Meandrina meandrites B1 1 100.00 1 100.00 19.37 80.63 M. meandrites B1 1⁎ 100.00 1 100.00 33.51 66.49

Poritidae Porites astreoides A4a 1 100.00 1 100.00 28.89 71.11 P. astreoides 1 100.00 1 100.00 12.59 87.41 P. colonensis C1a 1 100.00 1 100.00 12.95 87.05 P. divaricata (brown) C9 1 100.00 2 99.13 0.87 28.33 71.67 P. divaricata (yellow) 2⁎ 48.29 51.71 1 100.00 22.05 77.95 P. furcata shallow 1m A4/B1 2⁎ 65.79 34.21 2 98.62 1.38 19.14 80.86 P. furcata 1 100.00 1 100.00 18.19 81.81 P. furcata C4 n.d. 1 100.00 n.d.

Siderastreidae Siderastrea radians B5 1 100.00 1 99.82 0.18 51.58 48.42 (n=3)

Astrocoeniidae Stephanocoenia A3 1 100.00 n.d. n.d. michelini S. michelini 1 100.00 1 100.00 65.73 34.27

Actinaria Aiptasia tagetes B1 1⁎ 100.00 1 100.00 14.13 85.87 A. tagetes (pallida) B1 3 66.85 9.38 23.77 1 100.00 52.73 47.27 Condylactis gigantea A3 1 100.00 1 100.00 69.70 30.30 Lebrunia danae C1 1 100.00 1 100.00 35.28 64.72

Gorgonacea Briareum asbestinum B19 1 100.00 1 100.00 62.92 37.08 Eunicea mammosa Gorgonia flabellum B1 4 98.45 0.09 1.26 0.20 1 100.00 44.47 55.53 Muricea muricata Muriceopsis flavida 1 100.00 1 100.00 34.33 65.67 Plexaura homomalla B1 1 100.00 1 100.00 27.95 72.05 Pseudoplexaura flagellosa Pseudoplexaura wagenaari Pterogorgia citrina n.d. 1 100.00 n.d.

Zoanthidea Palythoa caribaeorum C1 3⁎ 16.61 36.95 46.45 1⁎ 100.00 51.12 48.88 P. caribaeorum 2 55.88 44.12 1 100.00 11.63 88.37 P. grandis C3 2⁎ 51.73 48.27 1 100.00 24.36 75.64 Zoanthus sociatus A4/A3/ 1 100.00 1 100.00 28.28 71.72 B1/C1

Corallimorpharia Discosoma sp. 3 56.51 4.78 38.70 1 100.00 13.82 86.18 Ricordea florida 1 100.00 3 60.72 0.08 39.19 11.48 88.52 R. florida C3c 1 100.00 1 100.00 7.94 92.06 Also listed are the % contributions of MAAs from the symbiont versus the host fractions to the total MAA pool. MG=mycosporine-glycine, SH=shinorine, PR=porphyra-334, PI=palythine. ⁎Indicates that the sample contained trace amounts of many other MAAs, ⁎⁎indicates that the sample contained mycosporine-2-glycine, n.d. indicates not determined. A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 139

flagellates A. sanguinea (=G. sanguineum) and Gym- nodinium (=Gyrodinium) cf. striatum (Litchman et al., 2002). Mycosporine-glycine is also the only MAA capable of functioning as an antioxidant (Dunlap and Yamomoto, 1995) and it was the only MAA found with an absorption peak within the UV-B range. Due to the high light environment within which the corals from this study were collected, mycosporine-glycine is likely to be functioning in a dual role, as an antioxidant as well as a photoprotectant in adult coral colonies in a similar

Fig. 1. The concentrations of total MAAs in the host versus the symbiont fractions for cnidarians from different depth ranges. Diamond shapes indicate samples collected from Bojorquez lagoon at a depth of less than 0.5 m. Samples collected from “La Bocana Chica” at the reef crest or reef lagoon at depths of 1–2.5 m are indicated by square shapes and triangle shapes indicate samples collected at depths of 7 to 8 m from the fore reef. Circles indicate samples collected at the fore reef located at Petem Pich at a depth of 10 to 15 m. maximum number present in the samples. There is no relationship between the number or concentration of MAAs and the clade designation of the Symbiodinium analyzed.

4. Discussion

Mycosporine-like amino acids were ubiquitous in our survey of the symbiotic cnidarian community from the Puerto Morelos barrier reef off the coast of Mexico. All of the cnidarians examined contained a simple MAA profile of primary MAAs and no host-modified second- ary MAAs were detected (sensu Shick, 2004). Raven (1991) calculated that 19% of the total cellular energy budget is required to synthesize a concentration of 100 mol m− 3 of an MAA such as palythine. Despite the cost involved in producing MAAs, the presence of photoprotectants is obviously vital as at least one MAA, principally mycosporine-glycine, was present in every sample of host tissue and the symbiotic dinoflagellates contained within them. The use of mycosporine-glycine over other MAAs has the advantage in that it limits the use of nitrogen in a nitrogen-limited environment (Gleason, 1993) that can otherwise be used for chlorophyll, growth and repro- duction. Less efficient repair and decreased MAA concentrations as a result of nitrogen limitation relative to controls resulted in significantly increased sensitivity Fig. 2. The total MAA concentrations in the symbiont fractions (A), to inhibition of photosynthesis by UVR in the dino- the host fractions (B) and the holosymbiont (C) as a function of depth. 140 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146

Fig. 3. Absorption spectra of methanolic extracts of various species of symbiotic cnidarians collected from 2 to 3 m in the Puerto Morelos back reef lagoon. Samples that contain an obvious peak in the UV region are shown in (A). Circles: Siderastrea siderea (λmax =322 nm), squares: Siderastrea radians (λmax =322 nm), diamonds: Discosoma sanctithomae (λmax =338 nm) and triangles: Porites divaricata (Pd, λmax =309 nm). Spectra that contain a shoulder or weak peak in the UV region are shown in (B). Circles: Porites furcata, squares: Porites colonensis, diamonds: Diploria labyrinthiformis and triangles: Agaricia humilis. manner to that suggested for the larvae of A. agaricites commonly associate with clade B and C symbionts (Gleason and Wellington, 1995). and to a lesser extent with clade A (LaJeunesse, 2002; MAA production and release by symbionts was LaJeunesse et al., 2003). Clade D is not common to recently correlated with clade A Symbiodinium under most coral taxa, and has only been identified in exposure to UVR in culture whereas no MAAs were Montastraea faveolata from the region where we detected in isolates representing clades B, C, E and F conducted our survey (LaJeunesse, 2002, LaJeunesse, (Banaszak et al., 2000). Under natural conditions in unpublished data). Based on the phylogenetic posi- the Mexican Caribbean, all cnidarian symbionts, tions of the four clades of Symbiodinium (Coffroth regardless of clade designation, contain at least one and Santos, 2005) that produce MAAs in the Mexican and, in some cases, up to four different MAAs (Table Caribbean, it is highly likely that all clades of Sym- 3). Unlike in the Indo-Pacific, Caribbean corals biodinium can produce MAAs under natural A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 141

Table 3 The identity of mycosporine-like amino acids (MAAs) and clade designation of freshly isolated Symbiodinium from the host species as well as the number of MAAs present in the host, derived from this study as well as data from the literature Symbiont species Clade Identity of MAAs Host species No. MAAs in host References Symbionts that do not contain MAAs Symbiodinium sp. A Corculum cardissa 4 Carlos et al. (1999), Ishikura et al. (1997) Symbiodinium sp. A, C Fragum unedo 4 Carlos et al. (1999), Ishikura et al. (1997) Symbiodinium sp. A, C Hippopus hippopus 4 Carlos et al. (1999), Ishikura et al. (1997) Symbiodinium sp. A, C Tridacna crocea 4 Carlos et al. (1999), Ishikura et al. (1997) Symbiodinium sp. A, C Tridacna derasa 4 Carlos et al. (1999), Ishikura et al. (1997) Symbiodinium sp. C a Galaxea fascicularis 5 LaJeunesse et al. (2003), Yakovleva and Hidaka (2004) Symbiodinium sp. C a Pavona divaricata 4 LaJeunesse et al. (2003), Yakovleva and Hidaka (2004) Symbiodinium sp. C a Montipora digitata 7 LaJeunesse et al. (2003), Yakovleva and Hidaka (2004) S. californium E Anthopleura elegantissima 6to7 Stochaj et al. (1994), Banaszak and Trench (1995)

Symbionts that contain 1 MAA Symbiodinium sp. A MG Acropora cervicornis 1 This study Symbiodinium sp. A MG Acropora palmate 1 This study Symbiodinium sp. A MG Aiptasia pallida 1 This study Symbiodinium sp. A MG Millepora alcicornis 1 This study Symbiodinium sp. A MG Stephanocoenia michelini 1 This study Symbiodinium sp. A, B MG Porites astreoides 1 This study Symbiodinium sp. A, C MG Condylactis gigantea 1 This study Symbiodinium sp. A, B, C MG Porites furcata 1 This study Symbiodinium sp. A, B, C MG Zoanthus sociatus 1 This study Symbiodinium sp. B MG Briareum asbestinum 1 This study Symbiodinium sp. B MG Dichocoenia stokesi 1 This study Symbiodinium sp. B MG Diploria clivosa 1 This study Symbiodinium sp. B MG Diploria labyrinthiformis 1 This study Symbiodinium sp. B MG Diploria strigosa 1 This study Symbiodinium sp. B MG Eunicea clavigera 1 This study Symbiodinium sp. B MG Eusmilia fastigiata 1 This study Symbiodinium sp. B MG Meandrina meandrites 1 This study Symbiodinium sp. B MG Montastraea annularis 3 This study Symbiodinium sp. B MG Muricea muricata 1 This study Symbiodinium sp. B MG Muriceopsis flavida 1 This study Symbiodinium sp. B MG Plexaura homomalla 1 This study Symbiodinium sp. B MG Pseudoplexaura flagellosa 1 This study Symbiodinium sp. B MG Pseudoplexaura wagenaari 1 This study Symbiodinium sp. B MG Pterogorgia citrina 1 This study Symbiodinium sp. B MG Siderastrea radians 2 This study Symbiodinium sp. C MG Agaricia agaricites f. agaricites 2 This study Symbiodinium sp. C MG Agaricia agaricites f. danai 2 This study Symbiodinium sp. C MG Agaricia fragilis 1 This study Symbiodinium sp. C MG Agaricia humilis 1 This study Symbiodinium sp. C MG Agaricia tenuifolia 1 This study Symbiodinium sp. C MG Lebrunia danae 1 This study Symbiodinium sp. C MG Leptoseris cucullata 1 This study Symbiodinium sp. C MG Montastraea cavernosa 1 This study Symbiodinium sp. C MG Mycetophyllia lamarckiana 1 This study Symbiodinium sp. C MG Mycetophyllia sp. Juvenile 1 This study Symbiodinium sp. C MG Porites divaricata 2 This study Symbiodinium sp. C MG Ricordea florida 3 This study Symbiodinium sp. D MG Montastraea faveolata 1 This study (continued on next page) 142 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146

Table 3 (continued) Symbiont species Clade Identity of MAAs Host species No. MAAs in host References Symbionts that contain 2 MAAs Symbiodinium sp. A, B MG, SH Porites furcata 2 This study Symbiodinium sp. B MG, SH Millepora complanata 2 This study Symbiodinium sp. B MG, M2G Manicina areolata 1 This study Symbiodinium sp. C MG, SH Agaricia agaricites f. purpurea 3 This study Symbiodinium sp. C MG, SH Isophyllastrea rigida 1 This study Symbiodinium sp. C MG, SH Palythoa caribaeorum 1 This study Symbiodinium sp. C MG, SH Palythoa grandis 1 This study Symbiodinium sp. C MG, SH Porites colonensis 1 This study Symbiodinium sp. C MG, SH Porites divaricata 1 This study

Symbionts that contain 3 MAAs Symbiodinium sp. A MG, SH, PO Aiptasia tagetes 1 This study S. microadriaticum A MG, SH, PO Cassiopeia xamachana 3 Banaszak and Trench (1995) Symbiodinium sp. B MG, SH, PO Colpophyllia natans 1 This study Symbiodinium sp. B MG, SH, PO Favia fragum 1 This study Symbiodinium sp. C MG, SH, PA Palythoa caribaeorum 1 This study

Symbionts that contain 4 MAAs Symbiodinium sp. B MG, SH, PO, PA Dendrogyra cylindricus 2 This study Symbiodinium sp. B MG, SH, PO, PA Gorgonia flabellum 1 This study Only studies in which the symbiont fraction was analyzed separately from the host fraction have been included. MG=mycosporine-glycine, SH=shinorine, PR=porphyra-334 and PI=palythine. a Clade of species not determined in study but likely to be C based on clade determination of related species. conditions. Due to the presence of MAAs in all samples collected in the field (Table 3)aswellasin samples examined, it appears that the presence of the Symbiodinium grown under cultured conditions in the host influences the ability of the symbiont to produce presence of UVR (Banaszak et al., 2000). By contrast, MAAs but whether the host provides a stimulus for free-living species of dinoflagellates such as A. sanguinea the algae to produce MAAs or a substrate for MAA (=G. sanguineum) contain these MAAs as well as paly- synthesis by the algae is unknown. Alternatively, the thene (λmax =360nm)(Neale et al., 1998; Litchman et al., high light environment found within the relatively 2002)andAlexandrium species that also contain asterina- clear waters of this coral reef result in the induction of 330, palythinol, palythenic acid, usujirene and palythene MAAs whereas the light conditions used in the culture and other unnamed MAAs (Carreto et al., 1990, 2001). experiment were insufficient to result in MAA The data in Table 3 suggest that there is an order of production by clades B, C, E and F Symbiodinium appearance of MAAs in Symbiodinium:mycosporine- (Banaszak et al., 2000). glycine followed by shinorine then porphyra-334 and The freshly isolated symbiotic algae contained a palythine. The shikimate pathway is the most likely maximum of four MAAs, mycosporine-glycine, shinorine, pathway for the production of MAAs (Chiocarra et al., porphyra-334 and palythine, the same MAAs produced by 1979; Grant et al., 1980; Shick et al., 1999) and Symbiodinium under culture conditions (Banaszak et al., mycosporine-glycine is the probable precursor that, by 2000). A fifth MAA, mycosporine-2-glycine, was also forming imines with serine and threonine, produces found in Symbiodinium isolated from M. areolata. Sym- shinorine (also known as mycosporine-glycine-serine) biodinium species appear to be limited to producing these and porphyra-334 (also known as mycosporine-glycine- five MAAs (Table 3, Shick and Ferrier-Pagès, unpublished threonine), respectively (Grant et al., 1985; see Fig. 11 in data in Shick, 2004). Mycosporine-glycine, shinorine, Shick, 2004). Via the reduction of the ketone in porphyra-334 and mycosporine-2-glycine are the primary mycosporine-glycine to an imine, palythine, also known Symbiodinium MAAs, as defined by the different temporal as iminomycosporine-glycine (Chiocarra et al., 1980), can patterns of accumulation under artificial UVR in be formed (Whitehead et al., 2001). S. pistillata relative to secondary MAAs (Shick, 2004). There is no relationship between the number or Based on an analysis of the species studied here under identity of MAAs in the symbiont population relative to natural conditions, palythine should also be included as a that present in the host tissues (Table 2) nor is there a primary Symbiodinium MAA because it is present in relationship between the concentration of MAAs in the A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146 143 symbiont and the host fractions (Fig. 1). The host the coral skeleton appears to play an important role in fractions contained a maximum of three MAAs, the amplification of the internal light field and increases mycosporine-glycine, shinorine and porphyra-334. In the number of photons delivered to the resident the cases where the hosts contain fewer MAAs than the symbiont population (Enriquez et al., 2005). Differences symbiont fraction, it is possible that not all of the MAAs in the production of host-derived photoprotective that are produced by the symbiont are translocated to the pigments and the large species-specific variability in host species or that the MAAs are present in the host but the amplification of the internal light fields found in at levels below detection. However, when the host corals may account, in part, for the high variability of the species contains more MAAs than the symbiont MAA concentrations found at any particular depth. fraction, the host may accumulate MAAs from its Thermal tolerance (Iglesias Prieto et al., 1992)aswell external diet as has been shown in other symbiotic and as photoacclimatory (Iglesias-Prieto and Trench, 1997a,b) nonsymbiotic species (Shick et al., 1992; Banaszak and and photoadaptive abilities (Iglesias-Prieto et al., 2004) Trench, 1995; Adams and Shick, 1996; Carroll and are physiological attributes that also limit the distribution Shick, 1996; Carefoot et al., 1998, 2000; Mason et al., of Symbiodinium and may further complicate the 1998; Newman et al., 2000; Whitehead et al., 2001)but bathymetric distribution of MAAs on a reef-wide basis. there is no transfer of these MAAs to the symbionts. While it is clear that there are physiological differences Alternatively, mycosporine-glycine could be translo- between Symbiodinium, it is not clear that these are cated by the symbionts and, once within the host tissue necessarily clade-specific. To date the only characteristic bioconverted by bacterial or acid hydrolysis (Dunlap that can be related to clade identity is the ability of Sym- and Shick, 1998; Shick et al., 1999; Whitehead et al., biodinium clade A in culture to produce MAAs whereas 2001; Shick, 2004) thus incrementing the number of other clades do not. Under natural conditions this MAAs within the host tissues relative to that of the relationship does not hold. While it has been suggested symbiont population. that clades A and B Symbiodinium are more common to Changes in the concentrations of MAAs with depth shallow water corals (high light environments) while have been found in many coral species (Dunlap and members of clade C predominate in hosts existing at Chalker, 1986; Dunlap et al., 1986; Gleason, 1993; depths below 10 m (low light environments) (Baker and Gleason and Wellington, 1995; Shick et al., 1995; Jokiel Rowan, 1997; LaJeunesse, 2002) this relationship does et al., 1997; Teai et al., 1997, 1998; Banaszak et al., 1998), not appear to hold for clade C in the Indo-Pacific and the trend is also seen here (data not shown), although (LaJeunesse et al., 2003). The role that the physiological the absence of replicates precludes statistical analysis. differences of Symbiodinium play in the ecology and However, a wide range of concentrations of total MAAs evolutionary biology of symbiotic associations is yet to be in the holosymbiont (Fig. 2A), symbiont (Fig. 2B) and the fully explored but requires attention due to the increasing host (Fig. 2C) are found when all species are considered at number of bleaching events (loss of symbiotic algae from any particular collection depth. This is supported by the host tissue) on a global scale. absorbance spectra of the methanolic extracts for various species collected within a small depth range showing that Acknowledgements the shapes of the spectra are remarkably different (Fig. 3A, B). Although this type of analysis is complicated by Funding for this study from the Instituto de Ciencias del the package effect, that is that MAAs are highly packaged Mar y Limnología to ATB is gratefully acknowledged. in intact dinoflagellate cells resulting in relatively flat MGBSwasfundedbyastudentfellowshipfromthe spectral absorption in the MAA absorbing region despite Consejo Nacional de Ciencias y Tecnología of Mexico. high concentrations of these compounds (Laurion et al., E. Jordán identified the gorgonian species. R. Iglesias- 2003), there are clear differences in the absorbance Prieto contributed comments to improve the manuscript. profiles at any particular depth. [SS] The presence of photoprotective pigments, other than MAAs, in the host tissue and the modification of the References light environment by the coral skeleton may play important roles in modulating MAA production. Host- Adams, N.L., Shick, J.M., 1996. 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