The distribution of mycosporine-like amino acids (MAAs) and the phylogenetic identity of symbiotic...

y and Ecology 337 (2006) 131–146www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolog

The distribution of mycosporine-like amino acids (MAAs) and thephylogenetic identity of symbiotic dinoflagellates 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, USAc 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 species of symbiotic cnidarians that included hydrozoan corals, anemones, gorgonians and scleractinian corals wasconducted 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. Symbiodinium populations wereidentified to sub-generic levels using PCR-DGGE analysis of the Internal Transcribed Spacer 2 (ITS2) region. Regardless of cladeidentity, all Symbiodinium extracts containedMAAs, in contrast to the pattern that has been found in cultures of Symbiodinium, whereclade A symbionts produced MAAs whereas clade B, C, D, and E symbionts did not. Under natural conditions between one and fourMAAs 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 datasuggest that Symbiodinium is restricted to producing five MAAs and there also appears to be a defined order of appearance of theseMAAs: 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 potentiallyproviding protection against the direct and indirect effects of UVR. No depth-dependent changes could be identified due to a highvariability of MAA concentrations when all species were included in the analysis. The presence of at least one MAA in all symbiontand host fractions analyzed serves to highlight the importance of MAAs, and in particular the role of mycosporine-glycine, asphotoprotectants in the coral reef environment.© 2006 Elsevier B.V. All rights reserved.

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

⁎ Corresponding author. Tel.: +52 998 871 0219x66; fax: +52 998871 0138.

E-mail address: [email protected] (A.T. Banaszak).

0022-0981/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2006.06.014

1. Introduction

Environmentally-relevant ultraviolet radiation (UVR,280 to 400 nm) is a key factor in the structure and

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132 A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146

distribution of coral reef communities (Jokiel, 1980; Shicket al., 1996; Dunlap and Shick, 1998; Gleason, 2001).Organisms living in shallow water tropical reef environ-ments are potentially exposed to high UVR doses and doserates due to the low solar zenith angles, the natural thinnessof the ozone layer over tropical latitudes, as well as the hightransparency of the water column (Smith and Baker, 1979;Baker et al., 1980). UVR can penetrate to 25m in coral reefenvironments (Fleischmann, 1989) although this value canchange dramatically depending on the composition of thewater column (Smith and Baker, 1979; Baker et al., 1980).

An important photoprotective response by marineorganisms is the production of mycosporine-like aminoacids (MAAs). Due to the high molar absorptivity ofMAAs (Є ranges from 28,100 to 50,000 mol−1 l cm−1),they absorb very efficiently within the wavelength rangefrom 309 to 360 nm (Dunlap and Shick, 1998). MAAshave absorption bands that are wide at about 40 nmFWHM for asterina-330 (λmax=330 nm), mycosporine-glycine (λmax=310 nm) and palythinol (λmax=332 nm)and 50 nm FWHM for purified shinorine (λmax=334 nm)and for palythine (λmax=320 nm) (Dunlap et al., 1989;Gleason, 1993; Sinha et al., 1998; Adams and Shick,2001), compared to chlorophyll a, which at room tem-perature inmethanol has an FWHMof 23 nm (Balny et al.,1969). Individually, therefore, MAAs provide a widespectral screen against UVR and when found in com-bination the screening potential is even broader.

The combination of efficient absorption of UVR toprevent its absorption by sensitive cellular components,the harmless dissipation of these potentially damagingwavelengths preventing the formation of singlet oxygen orsuperoxide radicals and their photostability indicates thatMAAs are ideal photoprotectants. High energy photonsabsorbed by shinorine and porphyra-334 are rapidlydissipated as heat by internal conversion as indicated bytheir low fluorescence quantum yields and by directphotoacoustic calorimetric determinations (Conde et al.,2004). In addition there is no energy transfer to chlorophylla as well as an absence of free radical or triplet stateformation (Conde et al., 2000, 2003, 2004; Moison andMitchell, 2001, Whitehead and Hedges, 2005).

The effectiveness of MAAs as photoprotectants hasbeen demonstrated in a cultured, bloom-forming dinofla-gellate Akashiwo sanguinea (=Gymnodinium sangui-neum) (Neale et al., 1998) and in the green sea urchinStrongylocentrotus droebachiensis (Adams and Shick,1996, 2001; Lesser et al., 2006). Using biologicalweighting functions for the inhibition of photosynthesisin the bloom-forming dinoflagellate A. sanguinea, Nealeet al. (1998) showed that the presence of high concen-trations of MAAs in high light grown cultures eliminated

the sensitivity of photosynthesis in theUVrange from320to 360 nm whereas in low light grown cultures with lowconcentrations of MAAs, the cultures were highlysensitive within this wavelength range. The absorptionspectrumof theMAAs corresponded almost exactly to themaximal biologically-effective UVR spectrum estimatedfor this dinoflagellate, showing that these optical screensprovide complete protection exactly within the mostdamaging wavelength range (Neale et al., 1998). Thephotoprotective role of MAAs has also been demon-strated for different developmental stages of S. droeba-chiensis. An inverse relationship was shown between theconcentration of MAAs and the first cleavage delay ineggs as well as a reduction in the levels of UV-induceddevelopmental abnormalities in embryos and larvae(Adams and Shick, 1996, 2001). However, sea urchinlarvae with high MAA concentrations suffered somecleavage delays as well as abnormalities (Adams andShick, 1996, 2001) showing that at least during embryo-genesis of sea urchins, MAAs do not provide completeprotection. Accumulation of DNA damage could beresponsible for some of the cleavage delays in embryosexposed to UVR (Lesser et al., 2006).

Many studies provide supporting evidence for thephotoprotective role of MAAs in corals. MAA concentra-tions decrease with depth (Dunlap et al., 1986; Gleason,1993; Gleason and Wellington, 1995; Shick et al., 1995;Banaszak et al., 1998; Corredor et al., 2000; Lesser, 2000)and in the absence of UVR (Banaszak and Trench, 1995;Shick et al., 1991). Compared to other invertebrate phyla,cnidarians possess the highest diversity of MAAs (Banas-zak, 2003). At least 11 different MAAs have been char-acterized from hard corals alone (Dunlap and Chalker,1986; Dunlap et al., 1986; Gleason, 1993; Gleason andWellington, 1995; Shick et al., 1995; WuWon et al., 1995,1997; Drollet et al., 1997; Jokiel et al., 1997; Teai et al.,1997, 1998; Banaszak et al., 1998) with up to 10 MAAsidentified in a single coral, Stylophora pistillata (Shicket al., 1999; Shick, 2004). Various cnidarian species havebeen shown to contain MAAs exclusively in the hostcomponent (Banaszak and Trench, 1995; Yakovleva andHidaka, 2004), which may be accounted for by diet(Banaszak and Trench, 1995; Shick, 2004) or due to thepresence of bacteria such as Vibrio (Dunlap and Shick,1998).

MAAs may also be acquired from symbionts such ashas been shown in the association between the upsidedown jellyfish Cassiopeia xamachana and the dinofla-gellate Symbiodinium microadriaticum (Banaszak andTrench, 1995). In culture, the photoprotectants areproduced by the algae and leaked to the surroundingmedium. While the aposymbiotic scyphistomae stage

133A.T. Banaszak et al. / Journal of Experimental Marine Biology and Ecology 337 (2006) 131–146

does not contain MAAs, upon infection with symbioticalgae, the ephyrae and adult jellyfish stages contain thesame suite of MAAs as the symbiotic algae in culture(Banaszak and Trench, 1995).

Due to the inherent difficulties of identifying Symbiodi-nium spp. the DNA sequence diversity of the small subunitribosomal DNA gene (SSUrDNA) has been used to assignSymbiodinium into sub-generic lineages (clades) within aphylogenetic tree (Rowan and Powers, 1991). Originally,three clades A, B and C were identified and over the lastdecade the number of recognized clades has increased toeight (A to H) (Coffroth and Santos, 2005). Many cladescontain numerous ecologically distinctive types character-ized principally through PCR-denaturing gradient gelelectrophoresis (DGGE) analyses of the Internal Tran-scribed Spacer (ITS) regions (LaJeunesse, 2002, 2005).CladesA throughDmaintain associationswith scleractiniancorals. In the Mexican Caribbean based on an extensivesampling of the reefs of Puerto Morelos, approximately 40distinct types have been found (LaJeunesse, 2002). A fewtypes, C1, C3 and B1, are generalists, being found in manydifferent host taxa,whereas there aremany distinctive types,referred to as specialists that populate specific host taxa(LaJeunesse, 2002; LaJeunesse et al., 2003).

All Symbiodinium belonging to clade A, includingS. microadriaticum (Banaszak and Trench, 1995) aswell as the reclassified S. (=Gymnodinium) linucheae(LaJeunesse, 2001) synthesized up to threeMAAswhereasnone of the clades B and C Symbiodinium producedMAAsin culture (Banaszak et al., 2000). The one representativeof clade E S. “californium”, the symbiont of Anthopleuraelegantissima (LaJeunesse, 2001), did not produce MAAsin culture (Banaszak andTrench, 1995) nor didS. kawagutii(Banaszak et al., 2000) that belongs to clade F(LaJeunesse, 2001). The symbiont of Aiptasia pallida inculture, S. bermudense, produced MAAs on exposure toartificial UVR (Lesser, 1996) and although at the time theclade type was not determined, it was later described ascladeB (Rowan and Powers, 1991; Banaszak et al., 2000).Under the experimental conditions used byBanaszak et al.(2000) however, S. bermudense did not produce MAAs.

Mycosporine-glycine is the more frequently observedand most concentrated MAA among diverse cnidarianspecies in various field studies (Dunlap andChalker, 1986;Dunlap et al., 1986; Shick et al., 1991, 1995; Gleason,1993; Gleason and Wellington, 1995; Teai et al., 1997;Banaszak et al., 1998). ThisMAA has been shown to havemoderate, concentration-dependent antioxidant activity byscavenging free radicals in extracts frommarine organisms(Dunlap and Yamomoto, 1995) and quenching singletoxygen (Suh et al., 2003). It also provides rapid protectionagainst oxidative stress while antioxidant enzymes can be

produced in two species of corals, Platygyra ryukyuensisand S. pistillata (Yakovleva et al., 2004).

Although many species, including corals, have beensurveyed for the presence of MAAs, few studies to daterestricted to studies in laboratory culture have separatedthe symbionts from the host tissue and determined theMAA profiles for both components. The objective of thisstudy was to determine whether the relationship ofMAAsto clade identity in field collected organisms is consistentwith that found in laboratory cultures by determining theMAA distribution in many cnidarian species at one sam-pling point.

2. Materials and methods

2.1. Collection of samples

Samples of C. xamachana were collected in theBojorquez Lagoon, Cancun (21°07′N, 86°45′W) whereasthe rest of the samples of symbiotic cnidarians werecollected by SCUBAor snorkel on reefs adjacent to PuertoMorelos, Quintana Roo on the northeast coast of theYucatan Peninsula,Mexico (20°50′N, 86°52′W). Sampleswere collected from four sites: the reef crest (1–2 m), theshallow lagoon (1–3 m) and the fore reef (3–8 m), alllocated at the “La Bocana Chica” reef and from the forereef (10–15 m) at Petem Pich. Table 1 contains collectiondepths and sites for the species used in this study. Thesamples were transported in seawater, covered to preventoverexposure to solar radiation and to maintain tempera-tures relatively constant and brought to the laboratorywithin 2 h of collection. On arrival, the samples weremaintained in a covered aquarium fitted with flowingseawater and processed within 36 h of collection.Gorgonian species were processed immediately due totheir inability to withstand confined conditions. Prior toprocessing of the samples, the surfaces were cleaned ofcontaminants using 45 μm filtered seawater. Due tocollection permit restrictions, few replicates could betaken.

2.2. Isolation of Symbiodinium from host tissues

The isolation of Symbiodinium from host tissuesdepended on host type. Branches of gorgonian colonieswere cut into 2 to 3 cm fragments and ground in a mortarand pestle with extraction buffer (EB, 1.2 μm filteredseawater and 5 mM EDTA). In some cases, the resultantslurry was filtered through a 100 μm mesh to removelarge fragments of host tissue or mucous.

Scleractinian and hydrozoan tissues were separatedfrom the coral skeleton (5 to 50 cm2) using a Water-Pik

Table 1Collection information for the species sampled in this study

Host speciessampled

NichuptePond

Lagoon(1–3 m)

RearZone(1–2.5 m)

Channel(5–10 m)

PetemPich(12–14 m)

ScyphozoaCassiopeia

xamachanashade (n=3)

0.5

C. xamachanalight (n=3)

0.5

HydrozoaMillepora

alcicornis1 7

M. complanata 1 7AnthozoaScleractiniaAcroporidaeAcropora

cervicornis5

A. palmata 1AgariciidaeAgaricia agaricites

f. agaricites(brown)

7

A. agaricitesf. agaricites(yellow)

7

A. agaricitesf. danai

1.5

A. agaricitesf. purpurea

7

A. fragilis 8A. humilis 1A. tenuifolia 1.5Leptoseris

cucullata7

Leptoseris sp. 6CaryophylliidaeEusmilia fastigiata 2.5 7

FaviidaeColpophyllia

natans1.5 7

Diploria clivosa 2.5 1D. labyrinthiformis 7D. strigosa 2.5Favia fragum 1Manicina areolata 2.5Montastraea

annularis7

M. cavernosa 1M. faveolata

(n=3 at 2.5 m)2.5 7

MussidaeIsophyllastrea

rigida7

Isophyllia sinuosa 1Mycetophyllia

lamarckiana7

Mycetophylliasp. juvenile

7

Table 1 (continued )

Host speciessampled

NichuptePond

Lagoon(1–3 m)

RearZone(1–2.5 m)

Channel(5–10 m)

PetemPich(12–14 m)

MeandrinidaeDendrogyra

cylindricus1.5

Dichocoeniastokesi

8

Meandrinameandrites

2 12

PoritidaePorites astreoides 1 7P. colonensis 7P. divaricata

(brown)1

P. divaricata(yellow)

1

P. furcata 1 1.5 7SiderastreidaeSiderastrea radians

(n=3)1

AstrocoeniidaeStephanocoenia

michelini2.5 12

ActinariaAiptasia tagetes 0.5A. tagetes (pallida) 0.1Condylactis

gigantea1

Lebrunia danae 1GorgonaceaBriareum

asbestinum7

Eunicea mammosa 7Gorgonia flabellum 7Muricea muricata 7Muriceopsis flavida 7Plexaura

homomalla1.5

Pseudoplexauraflagellosa

7

P. wagenaari 1.5Pterogorgia citrine 7

ZoanthideaPalythoa

caribaeorum1.5 7

P. grandis 7Zoanthus sociatus 7

CorallimorphariaDiscosoma sp. 7Ricordea florida 1.5 7


or airbrush (Paasche Millenium, USA) with EB into aplastic zip-lock bag to collect the disrupted tissue. Theplastic bag was sealed and shaken vigorously for 1 to3 min to disrupt polysaccharides when there were largeamounts of mucous. The disrupted tissue was thenhomogenized with a Tissue-Tearor.


The soft-bodied species, scyphozoans, actiniarians,corallimorphs and zoanthids, were first cleaned of debrisby hand. The oral discs, including tentacles, wereremoved with a razor blade, chopped into small pieces,resuspended in EB and homogenized with a TissueTearor (Biospec Products, Inc., USA) or glass grinder inthe case of actiniarian anemones.

To separate the symbionts from host tissues, theslurries were centrifuged for 5 min at 1000×g in aswinging bucket rotor in a clinical centrifuge (CentraCL2, IEC, USA). The supernatant, containing host tissueas determined by light microscopy at 200×magnification,was frozen at −70 °C, lyophilized (Labconco, USA) andstored at −20 °C for MAA and protein analysis. Thepellet, containing mostly symbionts, was resuspended inEB and homogenized and 0.01% (v/v) Triton X-100 wasadded and gentlymixed. The slurry was centrifuged usinga fixed angle rotor for 2 min at 925×g, and the supernatantdiscarded. This step removed any remaining host celldebris as determined by observation with a lightmicroscope at 200× magnification. The pellet wasresuspended immediately in EB, mixed well andcentrifuged under the same conditions. The wash stepwas repeated two more times. The final pellet wascentrifuged with a fixed angle rotor at 4500×g for 2min ina microcentrifuge (Costar 10MVSS, Corning-Costar,USA). A small portion of the resulting pellet was storedin a high salt (NaCl-saturated), DMSO (20%), EDTA(0.25 M) preservation buffer (Seutin et al., 1991). Theremainder of the pellet was frozen at −70 °C, lyophilizedand stored at −70 °C for MAA and protein analysis.

2.3. DNA extraction and DGGE analysis

The Wizard DNA-prep protocol (Promega, Madison,WI), modified by LaJeunesse et al. (2003), was used toextract nucleic acids. Approximately 10 to 30 mg ofmaterial was vortexed with glass beads and 600 μl NucleiLysis Buffer (Promega). The lysate was incubated with0.1 mg ml−1 proteinase K for 1 to 2 h at 65 °C. ProteinPrecipitation Buffer (250 μl; Promega) was then addedand the extract was incubated on ice for 15 min. Aftercentrifugation for 5 min at 12,000 rpm, 600 μl of super-natant was transferred and 700 μl isopropanol (100%) and30 μl NaAc (3M, pH 5.6) were added. Following in-cubation on ice for 10 min, the precipitated DNA wascentrifuged and the pellet washed with 70% ethanol. TheDNA was centrifuged again for 5 min, dried and resus-pended in 70–80 μl water. The final eluate from eachsample was diluted 1:10 and 1 μl was used as a templatefor amplification. The internal transcribed spacer 2 region(ITS 2) was then amplified for DGGE analysis as de-

scribed in LaJeunesse (2002). The reaction products wererun on an 8% polyacrylamide denaturing gel containing agradient of 3.15 M urea/18% deionized formamide to5.6 M urea/37% deionized formamide and separated byelectrophoresis for 9.5 h at 150Vat a constant temperatureof 60 °C along with ITS 2 standards as described inLaJeunesse (2001, 2002). All samples were loaded with a2% Ficoll loading buffer (2% Ficoll-400, 10 mM Tris–HCl pH 7.8, 1 mM EDTA, 1% bromophenol blue). Thegel was stained in 1× TAE and Syber Green (MolecularProbes, Eugene, Oregon) for 25 min using the manufac-turer's specifications, and the gel was then photographed.If new fingerprint profiles were identified, diagnosticbands were excised from the denaturing gel, eluted andreamplified with primers lacking the GC-clamp. Positivereactions were purified using a Promega Wizard PCR-prep DNA kit (Promega, Madison, Wisconsin) and di-rectly sequenced and analyzed on an Applied Biosystems310 genetic analyzer (Division of Perkin Elmer, FosterCity, California) and the sequences compared against adatabase of established types (cf. LaJeunesse, 2005).

2.4. Absorbance spectra of coral fragment extracts

Absorption spectra of methanolic extracts of coralfragments were recorded between 250 and 750 nm usingan Aminco DW2 spectrophotometer controlled by anOlis data collection system and methanol as a reference.

2.5. Determination of MAA concentrations

For all symbiont and host samples the extractionand analysis of MAAs were performed according tothe procedures in Dunlap and Chalker (1986) asmodified by Shick et al. (1992). The lyophilizedsamples were resuspended in 100% HPLC grademethanol and extracted at 4 °C overnight. The extractswere centrifuged at 1000×g for 2 min and thesupernatant was used for MAA and protein analysis.MAAs were separated by reverse-phase, isocratic highperformance liquid chromatography (HPLC) on aBrownlee RP-8 column (Spheri-5, 4.6 mmID×250 mm), which was protected with an RP-8 guard column (Spheri-5, 4.6 mm ID×30 mm). Themobile phase consisted of 40% to 55% methanol (v/v),0.1% glacial acetic acid (v/v) in water and run at aflow rate of 0.6 ml min−1. MAA peaks were detectedby absorbance at 313 and 340 nm. Standards wereavailable for the following seven MAAs: mycosporine-glycine, shinorine, porphyra-334, palythine, asterina-330, palythinol and palythene. Peak identities wereconfirmed by co-chromatography with standards and


with the ratios of 313 to 340 nm absorbances. Peakswere integrated and the quantification of individualMAAs was accomplished using HPLC peak areas andcalibration factors as determined by analysis of theseven standards. MAAs were normalized to solubleprotein as determined from an aliquot of the methanol-extracted sample using the procedure of Bradford(1976) in kit form (Bio-Rad, Inc.). Concentrations areexpressed in nmol MAA mg protein−1.

3. Results

In total, up to four MAAs, mycosporine-glycine,shinorine, porphyra-334 and palythine were quantifi-ably detected in Symbiodinium (Table 2). The symbiontfraction of Manicina areolata contained large amountsof mycosporine-2-glycine although due to the lack of astandard this MAA could not be quantified. Forty-eightsamples of corals contained one MAA only, alwaysmycosporine-glycine, in the symbiont fraction. Fifteensamples contained mycosporine-glycine as the majorcomponent (greater than 50% of the total concentration)and in two samples mycosporine-glycine was foundwith less than 50% of the total concentration (48.29% inPorites divaricata from 1 m depth and 16.61% in Pa-lythoa caribaeorum from 1.5 m depth). The secondmajor component in the Symbiodinium fraction wasshinorine, found in 17 samples (51.71% in P. divaricatafrom 1 m depth to 0.09% in Gorgonia flabellum from7 m depth) followed by porphyra-334 in 7 samples(38.70% in Discosoma sp. to 0.30% in Montastraeaannularis both from 7 m depth) and palythine in 3samples (46.45% in P. caribaeorum from 1.5 m depthand 0.20% in G. flabellum from 7 m depth). Theconcentrations of total MAAs ranged from 5.94 to365.63 nmol MAA mg protein−1 in the symbiontsisolated from the hosts M. areolata and Stephano-coenia michelini, respectively, both of which werecollected at 2.5 m depth.

MAA concentrations ranged from a minimum of34.74 nmol MAA mg protein−1 in the host fraction ofAgaricia fragilis collected at 8 m depth to a maximumof 604.85 nmol MAA mg protein−1 in the host fractionof Agaricia agaricites f. danai collected at 1.5 m depth.Between one and three MAAs (mycosporine-glycine,shinorine and porphyra-334) were detected in thecnidarian host species (Table 2). In 59 of the 68 speciesonly mycosporine-glycine was detected and in theremaining 9 species, mycosporine-glycine was thepredominant MAA. In eight of these nine species,mycosporine-glycine accounted for 97.35% to 99.68%of the total MAAs followed by shinorine (0.18% to

1.80%) and porphyra-334 (0.18% to 0.85%). For onespecies, Ricordea florida from 7 m depth, mycosporine-glycine accounted for 60.72% of the total MAAs, with39.19% by porphyra-334 and the remainder (0.08%)made up of shinorine. There is no relationship betweenhow many MAAs are present within the Symbiodiniumfraction and those present in the host fraction, except thatmycosporine-glycine occurs in all symbionts and hosts(Table 2) and there is no relationship between thepresence of MAAs in the symbionts and cladedesignation (Table 2).

The proportion of MAAs within the symbiontfraction relative to the host fraction was very variableranging from 2.75% of the total holosymbiont MAAsin M. areolata from 2.5 m depth to 69.70% of the totalholosymbiont MAAs in Condylactis gigantea from1 m depth (Fig. 1). The total concentration of MAAsfor all of the species sampled (Fig. 2A), for thesymbiont fraction (Fig. 2B) and for the host fraction(Fig. 2C) does not vary with depth. In individual coralspecies, which were sampled at different depths, thereis a trend for decreasing MAA concentration but thelack of replicate within species samples prevents anystatistically significant conclusions. On a reef-widebasis at any depth there is a wide variability in the totalMAA concentrations found within the symbionts andwithin their hosts (Fig. 2A–C). Absorption spectra ofmethanolic extracts of a selection of species collectedbetween 2 and 3 m depth (Fig. 3A, B) highlight thisvariability with pronounced peaks in the UV region insome species (Fig. 3A) and shoulders or indistinctpeaks in other species (Fig. 3B).

In Table 3, the number of MAAs and their identitiesfor Symbiodinium freshly isolated from a wide varietyof hosts are shown based on the number of MAAs thatis produced. In some cases, Symbiodinium does notcontain MAAs even though the host fraction does,although this does not apply for any of the Symbiodi-nium analyzed in the present study. The rest of theSymbiodinium are separated based on the number ofMAAs that they produce. Symbiodinium that containone MAA always contain mycosporine-glycine. Thosethat contain two MAAs contain mycosporine-glycineand shinorine, with one exception, the symbionts of M.areolata that contain mycosporine-glycine and mycos-porine-2-glycine. Symbiodinium that contain threeMAAs contain mycosporine-glycine, shinorine andporphyra-334 with one exception in P. caribaeorumwhere the third MAA is palythine rather thanporphyra-334. The symbionts of Dendrogyra cylindri-cus and G. flabellum contain four MAAs, mycospor-ine-glycine, shinorine, porphyra-334 and palythine, the

Table 2Identification of host species, clade designation of symbiont isolated from the host species and the identity of mycosporine-like amino acids found inthe symbiont fractions and in the host fractions

Host species sampled Cladedesignationof symbiont

Number ofMAAs insymbiont

% MG % SH % PR % PI Number ofMAAsin host

% MG % SH % PR % ofMAAs insymbiont

% of MAAsin hostspecies

ScyphozoaCassiopeia xamachana

shade (n=3)A1/B1 2⁎ 64.94 35.06 1 100.00 51.26 48.74

C. xamachana highlight (n=3)

A1 2⁎ 50.03 49.97 1 100.00 18.55 81.45

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

AcroporidaeAcropora cervicornis A3 1 100.00 1 100.00 13.81 86.19A. palmata A3 1 100.00 1 100.00 19.91 80.09

AgariciidaeAgaricia agaricites

f. agaricites (brown)C3a 1 100.00 2 99.68 0.32 14.69 85.31

A. agaricitesf. agaricites (yellow)

C3a 1 100.00 3 99.33 0.49 0.18 4.28 95.72

A. agaricites f. danai 2 71.87 28.13 2 99.53 0.47 4.35 95.65A. agaricites f. purpurea 1 100.00 3 97.35 1.80 0.85 25.45 74.55A. fragilis 1 100.00 1 100.00 47.91 52.09A. humilis C3a 1 100.00 1 100.00 22.47 77.53A. tenuifolia C3a 1 100.00 1 100.00 10.51 89.49Leptoseris cucullata C3 1 100.00 1 100.00 12.89 87.11Leptoseris sp. 1 100.00 1 100.00 25.80 74.20

CaryophylliidaeEusmilia fastigiata B1 1 100.00 1 100.00 27.79 72.21E. fastigiata B1 1 100.00 1 100.00 14.04 85.96

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

MussidaeIsophyllastrea rigida C3 2 94.48 5.52 1 100.00 28.83 71.17Isophyllia sinuosa C3 1 100.00 1 100.00 18.05 81.95Mycetophyllia

lamarckianaC3 1 100.00 1 100.00 15.67 84.33

Mycetophylliasp. juvenile

C? 1 100.00 1 100.00 23.12 76.88

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Host species sampled Cladedesignationof symbiont

Number ofMAAs insymbiont

% MG % SH % PR % PI Number ofMAAsin host

% MG % SH % PR % ofMAAs insymbiont

% of MAAin hostspecies

MeandrinidaeDendrogyra cylindricus B1 3 54.32 19.64 25.61 2 99.16 0.84 15.12 84.88Dichocoenia stokesi B1 1 100.00 1 100.00 13.60 86.40Meandrina meandrites B1 1 100.00 1 100.00 19.37 80.63M. meandrites B1 1⁎ 100.00 1 100.00 33.51 66.49

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

SiderastreidaeSiderastrea radians(n=3)

B5 1 100.00 1 99.82 0.18 51.58 48.42

AstrocoeniidaeStephanocoeniamichelini

A3 1 100.00 n.d. n.d.

S. michelini 1 100.00 1 100.00 65.73 34.27

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

GorgonaceaBriareum asbestinum B19 1 100.00 1 100.00 62.92 37.08Eunicea mammosaGorgonia flabellum B1 4 98.45 0.09 1.26 0.20 1 100.00 44.47 55.53Muricea muricataMuriceopsis flavida 1 100.00 1 100.00 34.33 65.67Plexaura homomalla B1 1 100.00 1 100.00 27.95 72.05Pseudoplexauraflagellosa

Pseudoplexaurawagenaari

Pterogorgia citrina n.d. 1 100.00 n.d.

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

B1/C11 100.00 1 100.00 28.28 71.72

CorallimorphariaDiscosoma sp. 3 56.51 4.78 38.70 1 100.00 13.82 86.18Ricordea florida 1 100.00 3 60.72 0.08 39.19 11.48 88.52R. 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-glycineSH=shinorine, PR=porphyra-334, PI=palythine. ⁎Indicates that the sample contained trace amounts of many other MAAs, ⁎⁎indicates that thsample contained mycosporine-2-glycine, n.d. indicates not determined.


s

,e

Fig. 2. The total MAA concentrations in the symbiont fractions (A),the host fractions (B) and the holosymbiont (C) as a function of depth.

Fig. 1. The concentrations of total MAAs in the host versus thesymbiont fractions for cnidarians from different depth ranges.Diamond shapes indicate samples collected from Bojorquez lagoonat a depth of less than 0.5 m. Samples collected from “La BocanaChica” at the reef crest or reef lagoon at depths of 1–2.5 m areindicated by square shapes and triangle shapes indicate samplescollected at depths of 7 to 8 m from the fore reef. Circles indicatesamples collected at the fore reef located at Petem Pich at a depth of 10to 15 m.


maximum number present in the samples. There is norelationship between the number or concentration ofMAAs and the clade designation of the Symbiodiniumanalyzed.

4. Discussion

Mycosporine-like amino acids were ubiquitous in oursurvey of the symbiotic cnidarian community from thePuerto Morelos barrier reef off the coast of Mexico. Allof the cnidarians examined contained a simple MAAprofile of primary MAAs and no host-modified second-ary MAAs were detected (sensu Shick, 2004). Raven(1991) calculated that 19% of the total cellular energybudget is required to synthesize a concentration of100 mol m−3 of an MAA such as palythine. Despite thecost involved in producing MAAs, the presence ofphotoprotectants is obviously vital as at least one MAA,principally mycosporine-glycine, was present in everysample of host tissue and the symbiotic dinoflagellatescontained within them.

The use of mycosporine-glycine over other MAAshas the advantage in that it limits the use of nitrogen in anitrogen-limited environment (Gleason, 1993) that canotherwise be used for chlorophyll, growth and repro-duction. Less efficient repair and decreased MAAconcentrations as a result of nitrogen limitation relativeto controls resulted in significantly increased sensitivityto inhibition of photosynthesis by UVR in the dino-

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

Fig. 3. Absorption spectra of methanolic extracts of various species of symbiotic cnidarians collected from 2 to 3 m in the Puerto Morelos back reeflagoon. Samples that contain an obvious peak in the UV region are shown in (A). Circles: Siderastrea siderea (λmax=322 nm), squares: Siderastrearadians (λmax=322 nm), diamonds: Discosoma sanctithomae (λmax=338 nm) and triangles: Porites divaricata (Pd, λmax=309 nm). Spectra thatcontain a shoulder or weak peak in the UV region are shown in (B). Circles: Porites furcata, squares: Porites colonensis, diamonds: Diplorialabyrinthiformis and triangles: Agaricia humilis.


manner to that suggested for the larvae of A. agaricites(Gleason and Wellington, 1995).

MAA production and release by symbionts wasrecently correlated with clade A Symbiodinium underexposure to UVR in culture whereas no MAAs weredetected in isolates representing clades B, C, E and F(Banaszak et al., 2000). Under natural conditions inthe Mexican Caribbean, all cnidarian symbionts,regardless of clade designation, contain at least oneand, in some cases, up to four different MAAs (Table3). Unlike in the Indo-Pacific, Caribbean corals

commonly associate with clade B and C symbiontsand to a lesser extent with clade A (LaJeunesse, 2002;LaJeunesse et al., 2003). Clade D is not common tomost coral taxa, and has only been identified inMontastraea faveolata from the region where weconducted our survey (LaJeunesse, 2002, LaJeunesse,unpublished data). Based on the phylogenetic posi-tions of the four clades of Symbiodinium (Coffrothand Santos, 2005) that produce MAAs in the MexicanCaribbean, it is highly likely that all clades of Sym-biodinium can produce MAAs under natural

Table 3The identity of mycosporine-like amino acids (MAAs) and clade designation of freshly isolated Symbiodinium from the host species as well as thenumber 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 MAAsSymbiodinium 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 6 to 7 Stochaj et al. (1994), Banaszak

and Trench (1995)

Symbionts that contain 1 MAASymbiodinium sp. A MG Acropora cervicornis 1 This studySymbiodinium sp. A MG Acropora palmate 1 This studySymbiodinium sp. A MG Aiptasia pallida 1 This studySymbiodinium sp. A MG Millepora alcicornis 1 This studySymbiodinium sp. A MG Stephanocoenia michelini 1 This studySymbiodinium sp. A, B MG Porites astreoides 1 This studySymbiodinium sp. A, C MG Condylactis gigantea 1 This studySymbiodinium sp. A, B, C MG Porites furcata 1 This studySymbiodinium sp. A, B, C MG Zoanthus sociatus 1 This studySymbiodinium sp. B MG Briareum asbestinum 1 This studySymbiodinium sp. B MG Dichocoenia stokesi 1 This studySymbiodinium sp. B MG Diploria clivosa 1 This studySymbiodinium sp. B MG Diploria labyrinthiformis 1 This studySymbiodinium sp. B MG Diploria strigosa 1 This studySymbiodinium sp. B MG Eunicea clavigera 1 This studySymbiodinium sp. B MG Eusmilia fastigiata 1 This studySymbiodinium sp. B MG Meandrina meandrites 1 This studySymbiodinium sp. B MG Montastraea annularis 3 This studySymbiodinium sp. B MG Muricea muricata 1 This studySymbiodinium sp. B MG Muriceopsis flavida 1 This studySymbiodinium sp. B MG Plexaura homomalla 1 This studySymbiodinium sp. B MG Pseudoplexaura flagellosa 1 This studySymbiodinium sp. B MG Pseudoplexaura wagenaari 1 This studySymbiodinium sp. B MG Pterogorgia citrina 1 This studySymbiodinium sp. B MG Siderastrea radians 2 This studySymbiodinium sp. C MG Agaricia agaricites f. agaricites 2 This studySymbiodinium sp. C MG Agaricia agaricites f. danai 2 This studySymbiodinium sp. C MG Agaricia fragilis 1 This studySymbiodinium sp. C MG Agaricia humilis 1 This studySymbiodinium sp. C MG Agaricia tenuifolia 1 This studySymbiodinium sp. C MG Lebrunia danae 1 This studySymbiodinium sp. C MG Leptoseris cucullata 1 This studySymbiodinium sp. C MG Montastraea cavernosa 1 This studySymbiodinium sp. C MG Mycetophyllia lamarckiana 1 This studySymbiodinium sp. C MG Mycetophyllia sp. Juvenile 1 This studySymbiodinium sp. C MG Porites divaricata 2 This studySymbiodinium sp. C MG Ricordea florida 3 This studySymbiodinium sp. D MG Montastraea faveolata 1 This study

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Symbiont species Clade Identity of MAAs Host species No. MAAs in host References

Symbionts that contain 2 MAAsSymbiodinium sp. A, B MG, SH Porites furcata 2 This studySymbiodinium sp. B MG, SH Millepora complanata 2 This studySymbiodinium sp. B MG, M2G Manicina areolata 1 This studySymbiodinium sp. C MG, SH Agaricia agaricites f. purpurea 3 This studySymbiodinium sp. C MG, SH Isophyllastrea rigida 1 This studySymbiodinium sp. C MG, SH Palythoa caribaeorum 1 This studySymbiodinium sp. C MG, SH Palythoa grandis 1 This studySymbiodinium sp. C MG, SH Porites colonensis 1 This studySymbiodinium sp. C MG, SH Porites divaricata 1 This study

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

Symbionts that contain 4 MAAsSymbiodinium sp. B MG, SH, PO, PA Dendrogyra cylindricus 2 This studySymbiodinium 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 allsamples examined, it appears that the presence of thehost influences the ability of the symbiont to produceMAAs but whether the host provides a stimulus forthe algae to produce MAAs or a substrate for MAAsynthesis by the algae is unknown. Alternatively, thehigh light environment found within the relativelyclear waters of this coral reef result in the induction ofMAAs whereas the light conditions used in the cultureexperiment were insufficient to result in MAAproduction by clades B, C, E and F Symbiodinium(Banaszak et al., 2000).

The freshly isolated symbiotic algae contained amaximum of fourMAAs, mycosporine-glycine, shinorine,porphyra-334 and palythine, the sameMAAs produced bySymbiodinium under culture conditions (Banaszak et al.,2000). A fifth MAA, mycosporine-2-glycine, was alsofound in Symbiodinium isolated from M. areolata. Sym-biodinium species appear to be limited to producing thesefiveMAAs (Table 3, Shick and Ferrier-Pagès, unpublisheddata in Shick, 2004). Mycosporine-glycine, shinorine,porphyra-334 and mycosporine-2-glycine are the primarySymbiodiniumMAAs, as defined by the different temporalpatterns of accumulation under artificial UVR inS. pistillata relative to secondary MAAs (Shick, 2004).Based on an analysis of the species studied here undernatural conditions, palythine should also be included as aprimary Symbiodinium MAA because it is present in

samples collected in the field (Table 3) as well as inSymbiodinium grown under cultured conditions in thepresence of UVR (Banaszak et al., 2000). By contrast,free-living species of dinoflagellates such as A. sanguinea(=G. sanguineum) contain these MAAs as well as paly-thene (λmax=360 nm) (Neale et al., 1998; Litchman et al.,2002) and Alexandrium species that also contain asterina-330, palythinol, palythenic acid, usujirene and palytheneand other unnamed MAAs (Carreto et al., 1990, 2001).

The data in Table 3 suggest that there is an order ofappearance of MAAs in Symbiodinium: mycosporine-glycine followed by shinorine then porphyra-334 andpalythine. The shikimate pathway is the most likelypathway for the production of MAAs (Chiocarra et al.,1979; Grant et al., 1980; Shick et al., 1999) andmycosporine-glycine is the probable precursor that, byforming imines with serine and threonine, producesshinorine (also known as mycosporine-glycine-serine)and porphyra-334 (also known as mycosporine-glycine-threonine), respectively (Grant et al., 1985; see Fig. 11 inShick, 2004). Via the reduction of the ketone inmycosporine-glycine to an imine, palythine, also knownas iminomycosporine-glycine (Chiocarra et al., 1980), canbe formed (Whitehead et al., 2001).

There is no relationship between the number oridentity of MAAs in the symbiont population relative tothat present in the host tissues (Table 2) nor is there arelationship between the concentration of MAAs in the


symbiont and the host fractions (Fig. 1). The hostfractions contained a maximum of three MAAs,mycosporine-glycine, shinorine and porphyra-334. Inthe cases where the hosts contain fewer MAAs than thesymbiont fraction, it is possible that not all of the MAAsthat are produced by the symbiont are translocated to thehost species or that the MAAs are present in the host butat levels below detection. However, when the hostspecies contains more MAAs than the symbiontfraction, the host may accumulate MAAs from itsexternal diet as has been shown in other symbiotic andnonsymbiotic species (Shick et al., 1992; Banaszak andTrench, 1995; Adams and Shick, 1996; Carroll andShick, 1996; Carefoot et al., 1998, 2000; Mason et al.,1998; Newman et al., 2000; Whitehead et al., 2001) butthere is no transfer of these MAAs to the symbionts.Alternatively, mycosporine-glycine could be translo-cated by the symbionts and, once within the host tissuebioconverted by bacterial or acid hydrolysis (Dunlapand Shick, 1998; Shick et al., 1999; Whitehead et al.,2001; Shick, 2004) thus incrementing the number ofMAAs within the host tissues relative to that of thesymbiont population.

Changes in the concentrations of MAAs with depthhave been found in many coral species (Dunlap andChalker, 1986; Dunlap et al., 1986; Gleason, 1993;Gleason and Wellington, 1995; Shick et al., 1995; Jokielet al., 1997; Teai et al., 1997, 1998; Banaszak et al., 1998),and the trend is also seen here (data not shown), althoughthe absence of replicates precludes statistical analysis.However, a wide range of concentrations of total MAAsin the holosymbiont (Fig. 2A), symbiont (Fig. 2B) and thehost (Fig. 2C) are foundwhen all species are considered atany particular collection depth. This is supported by theabsorbance spectra of the methanolic extracts for variousspecies collected within a small depth range showing thatthe shapes of the spectra are remarkably different (Fig.3A, B). Although this type of analysis is complicated bythe package effect, that is that MAAs are highly packagedin intact dinoflagellate cells resulting in relatively flatspectral absorption in the MAA absorbing region despitehigh concentrations of these compounds (Laurion et al.,2003), there are clear differences in the absorbanceprofiles at any particular depth.

The presence of photoprotective pigments, other thanMAAs, in the host tissue and the modification of thelight environment by the coral skeleton may playimportant roles in modulating MAA production. Host-derived photoprotective pigments such as pocilloporinsand fluorescent proteins may result in attenuation of thelight environment within the holosymbiont (Salih et al.,2000; Dove et al., 2001). In contrast, the morphology of

the coral skeleton appears to play an important role inthe amplification of the internal light field and increasesthe number of photons delivered to the residentsymbiont population (Enriquez et al., 2005). Differencesin the production of host-derived photoprotectivepigments and the large species-specific variability inthe amplification of the internal light fields found incorals may account, in part, for the high variability of theMAA concentrations found at any particular depth.

Thermal tolerance (Iglesias Prieto et al., 1992) as wellas photoacclimatory (Iglesias-Prieto and Trench, 1997a,b)and photoadaptive abilities (Iglesias-Prieto et al., 2004)are physiological attributes that also limit the distributionof Symbiodinium and may further complicate thebathymetric distribution of MAAs on a reef-wide basis.While it is clear that there are physiological differencesbetween Symbiodinium, it is not clear that these arenecessarily clade-specific. To date the only characteristicthat can be related to clade identity is the ability of Sym-biodinium clade A in culture to produce MAAs whereasother clades do not. Under natural conditions thisrelationship does not hold. While it has been suggestedthat clades A and B Symbiodinium are more common toshallow water corals (high light environments) whilemembers of clade C predominate in hosts existing atdepths below 10 m (low light environments) (Baker andRowan, 1997; LaJeunesse, 2002) this relationship doesnot appear to hold for clade C in the Indo-Pacific(LaJeunesse et al., 2003). The role that the physiologicaldifferences of Symbiodinium play in the ecology andevolutionary biology of symbiotic associations is yet to befully explored but requires attention due to the increasingnumber of bleaching events (loss of symbiotic algae fromhost tissue) on a global scale.

Acknowledgements

Funding for this study from the Instituto de Ciencias delMar y Limnología to ATB is gratefully acknowledged.MGBS was funded by a student fellowship from theConsejo Nacional de Ciencias y Tecnología of Mexico.E. Jordán identified the gorgonian species. R. Iglesias-Prieto contributed comments to improve the manuscript.[SS]

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