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Reference:Biol. Hull 191: 144-154.(October. 1996) An Ancient Chemosensory Mechanism Brings New Life to Coral Reefs AILEEN N. C. MTOARKSEES1H*,IKHEANJYIASIHWIABOA2R, AM2ASAANSDUKMEAKBOABTAO3,OKMAOZRUIY2U4KI SHIMOIKE2, , 1MarineBiotechnologyCenter, MarineScienceInstitute, UniversityofCalifornia, Santa Barbara, California 93106;2AkajimaMarineScienceLaboratory, 179Aka, Zamami-son, Okinawa 901-33. Japan;*MarineEcologyResearchInstitute. 4-7-17Arahama, Kasluwazaki, Niigata, 945-03. Japan;andADepartment ofAquaticBiosciences. Tokyo UniversityofFisheries. 4-5-7Konan, Minato-ku, Tokyo 108. Japan Thefirst scleractinians, progenitors ofmodern corals, uted throughout the Indo-Pacific; Cyphastreaand Goni- began toappear240millionyearsago;bythelateJuras- astrea (first appearing in the Oligocene and Eocene, re- sic(150Ma) mostfamiliesofmodern coralshadevolved spectively), although common, are limited to the Indo- andbegunforming reefs (1. 2). Mechanisms controlling Pacific (1, 2). Larvae ofthe acroporids and faviids that the recruitment ofnew corals to sustain these structures we tested are generated by cross-fertilization ofgametes are, however, poorly understood (3). Corals, like many releasedintotheplanktonduringmass-spawningevents, marineinvertebrates, beginlifeassoft-bodiedlarvaethat the dominant form ofsexual reproduction in corals (3). are dispersed in theplankton (3, 4). As thefirst step in We found that larval detection ofsuitable reefsubstrata developing a calcifiedcoralcolony, the larva must settle iscontrolled by chemosensory recognition ofacue asso- outoftheplankton ontoasuitablesubstratum andmeta- ciated with encrusting red algae, among the major ce- morphoseto thesinglecalcifiedpolyp stagecementedto menters ofthe reef. A similar process operates in Carib- the reef(3, 5). Ouranalyses ofthe metamorphic require- bean agariciid species that brood their larvae (5-10). mentsoflarvaeindivergentcoralfamiliessurprisedusby These agariciids are in two genera, Agaricia and Lepto- revealing the existence of a common chemosensory seris. that first appeared in the Miocene and Oligocene. mechanism that is required to bring lan<ae out ofthe respectively(1,2). Ourfindingsthussuggestthatthische- plankton andonto the reef. This mechanism appears to mosensory mechanism is common to at least the Acro- be quite old, predating both thephylogenetic divergence poridae, Faviidae, and Agariciidae three majoranddi- ofthese coralfamilies and the development ofdifferent vergentcoral families. modesofcoralreproduction. In the laboratory at Akajima, Japan, larvae of the We analyzed the requirements for metamorphosis of widely distributed mass-spawning Indo-Pacific corals A larvae from 10 species of Pacific Acropora the acro- Acroporanasutaand digitiferaexhibitastrict require- porid genus with by far the greatest number of known ment for a specific environmental cue: surface contact species (76 in the Indo-Pacific, about one-sixth the esti- with sympatric crustose red algae is required for cue de- mated number ofscleractinian species in that region) tection (Fig. 1A, B). The strength ofthe larval response and three species representing three common genera of (% metamorphosis) to all five crustose red algae tested Pacific Faviidae, the second most speciose scleractinian varies directly with larval age (days post-fertilization). family (1,2). Most, but not all, species ofAcropora and But regardless oflarval age, the response to the seawater Favia (genera that first appear in the fossil record in the and brown algal controls remains nil (0% metamorpho- Eocene and Cretaceous, respectively) are widely distrib- sis). Overall, A. digitifera was siAgnificantly more respon- sivetoinductivealgae than was nasuta(two-wayAN- OVA; coral effect: F == 18.3, df = 1, P = 0.0016). Al- Received28May 1996:accepted7August 1996. though there was no significant difference between *Towhomcorrespondenceshouldbeaddressed. species in their response to these algae (two-way AN- 149 150 A. N. C. MORSE ET AL. UU 1 80 ALGAL CUE FOR CORAL METAMORPHOSIS 151 OVA; coral * algae interaction: F = 0.89, df = 4, P = (dependence on crustose red algae) of cue recognition 0.503), A. digitifera exhibited less variation in response persist for the duration oflarval competence for meta- among algae (one-way ANOVA: F = 0.61, df = 6, P = morphosis(Fig. 1C, D, E). Species-specificdifferences in 0.72) than did A. nasnta (one-way ANOVA: F = 2.9, df theduration ofcompetenceareobvious. The magnitude = 6, P= 0.095). Such patternsare similarto the species- ofthe larval response foreach species isdependent upon specific differences previously found in agariciid cor- larval age (Fig. 1). Larvae exhibited little or no response als(10). to Peyssonnelia sp. at 3 days post-fertilization; respon- Larvae maintained in seawater alone (>30days) con- siveness developed by 5 days, peaked at 7 days, and de- tinue planktonic swimming and never develop beyond clinedthereafteratratesthatarespecies-specific(Fig. 1C, the larval stage illustrated in Fig. 2A (Fig. 1C, D, E). D,E). Metamorphosis is initiated on contact with an inductive Ifthese results reflect larval potential for dispersal in alga. e.g.. Peyssonnelia sp. orany offour morphological the plankton, then the differences in the rates ofdecline forms of Hydrolithon reinboldii; larvae rapidly stop ofcompetenceamongall ofthetestedacroporid species, swimming, their bodies elongate and remain in close with no accompanying loss in stringency or specificity contact with the algal surface, and within a few hours, ofcue requirement (e.g., Fig. 1), suggest species-specific theyround upandcementthemselvestothealgalsurface differences in the windows ofopportunity for successful oradjacentsubstratum. Thefinalstagesofmetamorpho- settlement and metamorphosis on the reef. Assuming sis are marked by the development of 12 radial skeletal that rate ofdecrease ofcompetence with larval age is in- elements, thecalcined septaandcostae (Fig. 2B). Larvae verselycorrelated withthedistance ofdispersal from pa- ofthe mass-spawning congeners A. tennis. A.Jlorida, A. rental colonies (all other chemical and hydrodynamic gemmifera, A.formoxa, A. hyacinthus, A. sp. 1, A. sp.4 factorsbeingequal),A. tennisappearstohavethepoten- and A. sp. 5 all exhibit a similar strict requirement for tial forwidestdispersal and significant recruitment forat contact with eitherorboth Peyssonneliasp. andH. rein- least 5-30 days post-fertilization.A. digitiferaappearsto boldii (Fig. 2C; and A. N. C. Morse el a/.. University of have the potential for somewhat widerdispersal than A. California, Santa Barbara, in prep). Although, in some nasuta. although the majority ofpotential recruits from instances, the brown alga Lobophora variegata pro- both species will be dispersed over similar distances (5- moted first stage elongation ofthe larvae, further devel- 12 days). Although thebulk ofsettlementand metamor- opment rarely ifeveroccurred. When larvae from seven phosis might occur soon after the larvae reach compe- acroporid species (50 larvae/species incubated together) tence, these differencesamongspeciesthat recognizethe were given a choice between crustose red algae and same algae may serve to decrease post-settlement com- brown algae, all that had metamorphosed after 4-h petition for space among some ofthe settlers. Variation exposure(70%)werefoundonlyonthecrustoseredalgae among these species in timing ofgamete release during (Peyssonnelia sp. and //. reinboldii} (Fig. 2C); no larval mass-spawning events (11-14), in concert with varia- metamorphosis was detected on brown algae or on the tions in currents and other hydrodynamic factors, may chambersurfaces;control larvae(same numberand spe- additionallycontributetoareduction inthepotential for cies) remained swimming. In a similar experiment, 500 post-settlement interaction. competent larvae from each offiveacroporid species(A. Significantly, we believe, larvae ofthese Pacific acro- nasuta, A. tennis. A.formosa, A. hyacinthus, andA. gem- porids and those in two Caribbean genera, Agaricia and mifera, tested together in a 30-1 flow-through tank)were Leptoseris (Agariciidae), both appear to recognize and presented a choice of algae, live corals, inert substrata require the same class ofalgal cue for the induction of collected from the reef, "fouled" panels, andavariety of larval settlement and metamorphosis. This is in spite of inert materials commonly used as settlement plates; all their very different modes of sexual reproduction and settlement and metamorphosis occurred only on larval development (3). Acroporids participate in mass- coralline algae (A. N. C. Morse el a/.. University ofCali- spawning events during which millions ofgametes are fornia, Santa Barbara, in prep.). released into the plankton for cross-fertilization, fol- The stringency ofthis requirement (no metamorpho- lowed by larval development in the plankton; in con- sisintheabsenceofanexogenouscue)andthespecificity trast, agariciids have evolved the less common mode of prepared from algaeaspreviouslydescribed(6, 7,9);live//. reinboldiispecimensweregroupedaccording tothe surfacecharacteristicsofdifferentgrowth forms(by A. N. C. M.); specimenspreserved in formalin and seawater were dehydrated, embedded in paraffin, sectioned (8-^m thickness), and identified with a light microscope to speciesand genuslevel (by M. B.) Lobophora variegata(Lamouroux). Hydrolithon rcinhnldii(Webervan Bosseet Foslie)Foslie;Pcyx.wnneliasp. [similartoP. obscuraWebervan Bosseand P.comhu-olaPicconeetGrunow(19)]. 152 A. N. C. MORSE ET AL. Figure2. Larval behaviorandearly melamorphicchangesofPacificacroporidcoralsin responsetoa morphogeniccueassociatedwithPacificcrustoseredalgae.(A)Typicalshapeofsoft-bodiedlarva> 3days post-fertilization:.1 tlixiulerularva.8dayspost-fertilization,swimmingnormallyinthewatercolumn.(B) Final stage ofmetamorphosis; formation ofradial skeletal elements (septa and costae) and elevation of centralareaaround mouth:A nii.tiilularva, 8dayspost-fertilization, incubatedwith Peyssonneliasp. (C) Early-stagemetamorphosis(4h)ofamixtureoflarvaeofA naaiilu.A tlixiiilfru. I lennix,A.tormosa.A. geninnterci. I llonila.A s/> 5onwholespecimensof// rcnthoUliiandATS\onncliasp.(D)Metamorpho- sisof.I llnniliilarvaein responsetoinductivemoleculespurified from // reinboldiiandcoupledtoresin beads. In (A)and (B) theassayswereasdescribed in Fig. 1. In (C) 20competent larvaeofeach ofseven specieswereincubatedtogetherin200mlFSWwithorwithoutsmallintactspecimensofPeyssonneliasp., Hvdrt>lithii reinhiilt/ii. and l.obupliura variegata In (D) the resin with adsorbed inducer was the same preparationasthatassayedinTableI;larvaewerebatchmatesofthosein(C). sexual reproduction in which cross-fertilization occurs Fragments ofboth the Caribbean and Pacific algal con- internally, and subsequent larval development and geners (Hydrolithonand Peyssonnelia) induced levelsof broodingoccurwithin the maternal polyps. metamorphosis in Agaricia humilis larvae that are not The possibility ofsimilarcue recognition in members significantly different (Table I; one-way ANOVA: F = ofthese two families was first suggested by the fact that 1.59, df= 4, P= 0.198). Moreover, when the same pro- the algal species (Hydrolithon reinboldii and Peysson- cedures developed for biochemical purification, charac- nelia} that are shown here to induce metamorphosis of terization, and coupling (7, 9, 15) ofthe polymeric in- PacificacroporidlarvaehavecongenersintheCaribbean ducer of agariciid larvae from H. boergesenii to a hy- that induce metamorphosis of agariciid larvae (5-10). drophobic resin were applied to H. reinboldii. the This suggestion is confirmed by the demonstration (Ta- purified moleculeadsorbedtoresinwasrecognizedbyA. ble I) that the Pacific alga, Hydrolilhon reinboldii. con- humilis larvae (Table I). Larval settlement and meta- tainsan extractable polymeric morphogen that isappar- morphosis ofat least seven species in two genera ofCar- ently in the same class of molecules as one previously ibbean agriciids is strictly dependent on chemosensory identified in its Caribbean congener, H. boergesenii. recognition ofa uniqueclassofsulfatedglycosaminogly- ALGAL CUE FOR CORAL METAMORPHOSIS 153 TableI batch ofresin-adsorbed morphogen purified from H. re- >/i n'.\i'oHM'otlarvaeoftheCaribbeancoral. Agaricia inboldii(Fig. 2D);when incubatedwith resin lackingthe humilis,loinducersfromCaribbeanandI'aci/iculi^ulcongeners adsorbed molecule, larvaeofthe Pacificcorals remained arrested with no metamorphosis. Settlement and meta- Metamorphosisat48h morphosis in the testedAcroporaspeciesarethusappar- Inducer (mean% std.error) ently induced by chemosensory recognition ofthe same Caribbean classofalgal sulfatedglycosaminoglycan. None 10' + 30: The acroporids and agariciids are members ofthe re- llydrolnhonI'ocrgewui cently defined clade of complex scleractinians (16). In fragments 25' + 30: 62 9.1 experimentsotherwise identicaltothoseabove, larvaeof Peyssonneliasp.fragments 30: 73 16.1 the mass-spawning faviid corals from Akajima Favia CHyoinltrruollitrheisminhuergeseniiinducer 10* favus, Goniastrearetiformis, and Cyphastreasp. mem- on resin 10* 90 6.2 bersofataxonomically distinctcladeofrobustcorals(2, Pacific 16, 17), also exhibited a strict requirement for the same None 10' + 30- class of algal morphogen recognized by the complex Hydfrroalgimiehnotnsreinboldn(form///) 302 53 8.4 coral larvae. Thus, the inducer molecule purified from Hydrolithonreinboldii(formiv) the two Pacific algae, Hydrolithon reinboldii and Peys- fragments 20' +302 56 7.8 sonnelia sp. (each adsorbed to separate batches of hy- Peyssonelliasp.fragments 30: 87 6.7 drophobic resin) induced metamorphosis ofCyphastrea Controlresin 10' sp. larvae. There was no spontaneous metamorphosis in Hydro/iliumreinboldiiinducer thepresenceofresinwith noadsorbedcue. Fragmentsof onresin 10' 30 6.2 the intact alga H. reinboldii induced metamorphosis of CompetentA, humilislarvaewereincubated in 10 ml FSWwith or Cyphastraea sp., whereas seawater alone, dead coral without additions as shown: none = FSW alone; fragments = algae branches, and the brown alga L. variegata produced no preparedaspreviouslydescribed(6. 7,9);controlresin = hydrophobic metamorphic response. Similarly, Favia favusand Gon- interaction chromatography (HIC) resin (9. 15) with no adsorbed chemicalinducer;induceronresin = HICresinwithadsorbedinducer iastrea retiformis metamorphosed in response to frag- purifiedfromtheindicatedalga(9, 15).n = thetotal numberoflarvae ments ofintact Peyssonnelia sp., but were unresponsive tested ineach condition in replicatetests(5 larvae/test)performed ei- to L. variegata and seawater alone. Relatively few of ther in May 1995 ('), May 1996 (:), orearlier(*, data from 9). Meta- these faviid larvaewereavailable, which limitedthe test- morphosis means development to the single-polyp stage (refs. 5, 15; ing ofthe other algae and purified inducers adsorbed to ayfsnredos.mFiBpgar.roe2onBdt)ea.ldPc.e-o1rlcohenunimteiaslgiiensstlwhaeerrvleaaebaorwrceasrtieonreoybtrtreaaainrnsiefndogrbfmayecidslipftooyrnatstataUnteCiosStuBisc.alruealnnedaaelsr-e roenssitnrawtietthheeascthricnogreanlcsypeacnidess.peTchifeicrietsyulotfs,thheowreevqeuri,redmeemn-t conditionspreviouslydescribed(6. 7, 9):H. boergesenii andPeysson- ofthese robust corals for the same class ofalgal inducer neliasp.werecollectedfromBonaire.NetherlandsAntilles;H.reinbol- (A. N. C. Morse el ai. University ofCalifornia, Santa dii (forms /'/'/& iv) and Peyssonnelia sp. came from Akajima, Japan. Barbara, in prep.). tFhreagimnedunctesrosfwaellrealpguareiwfeierdefprreopmarferda,gmsehnitpspeadn(d-a2d0sCo)r,besdtotroedH(I-C80rCes)i;n The algal-cue-dependent settlement and metamor- byproceduresdeveloped with H. boergesenii(7,9, 15). Resinassays: phosisofagariciidsdescribed herehasbeen showntoop- 20 mgresinwith orwithoutadsorbed inducerfrom H boergeseniior erate effectively in the ocean as well as in the laboratory from// reinboldii(formiv). (9), and to contribute to substratum specificity ofaga- riciid recruitment in the natural environment (6). We further suggest that this mechanism may enhance the can (7, 9) that is associated with the cell walls ofa num- probability of successful reproduction of coral species ber ofCaribbean crustose red algae (5, 7-9, 15). Enzy- thatdependoncross-fertilization. Inmass-spawningcor- matic and biochemical analyses demonstrated that this als the success of cross-fertilization of gametes in the inductive polymer has a molecular weight of 5-10 Kd plankton depends on a rapid encounter with gametes and does not induce metamorphosis in coral larvae that from anothercolonyofthesamespecies,andthusonthe are not inducedby the intactparental algae (7). Both the propinquityofreproductivecoloniesofthesamespecies. biochemical characterization of the inducer isolated Similarly,specieswithinternalcross-fertilizationdepend from an inductive Pacific alga and its recognition byA. on the transfer of sperm from one colony to another. humilis larvae indicate that this morphogen belongs to Field manipulationofcolonydistancesinAgariciahum- the same class of algal cell-wall polysaccharide as the ilis revealsthat colonies must be < 2 m from their near- compound obtained from Caribbean red algae. As we est neighbor for successful production of larvae (P. T. show here, acroporid larvae assayed at Akajima meta- Raimondi and A. N. C. Morse, UniversityofCalifornia, morphosed in response to both H. reinboldii fragments Santa Barbara, in prep.). and intact algae (Figs. 1, 2B, C), as well as to this same Theevidencepresented hereindicatesthat representa- 154 A. N. C. MORSE KT AL live species of three very large families of corals, the references therein in Eco.\y.sient\ of the World, Vol. 25, Coral Acroporidae, Faviidae, and Agariciidae, have evolved Reels Z. Duhinsky,ed. ElsevierSciencePublishers,Amsterdam. 4 Babcock,R.C.,andA.J.Ileyward. 1986. Larvaldevelopmentof similar morphogenic requirements and chemosensory certaingamete-spawningscleractiniancorals. CoralReefs5: 111- signal recognition systems for larval recruitment from 1 16. the plankton tothereef. Thismechanism isindependent 5. Morse,A.N.C. 1991. Howdoplanktoniclarvaeknowwhereto oftheirgeographic historiesand oftheirmodesofsexual settle?Am.Scientist79: 154-167. reproduction. Our findings indicate that species in these 6. MCoonrtsreo,lD.ofE.l,arNv.alHomoektearm,orAp.hNo.siC.sMaonrdser,ecarnuditRm.eAn.tJiennsseynm.pa1t9r8i8.c three families evolved chemosensory receptors that rec- agariciidcorals.J. Exp. Mar. Biol Ecol. 116:193-217. ognize the same class ofrequired chemical morphogen. 7. Morse, D. E., and A. N.C. Morse. 1991. Enzymaticcharacter- Unless this mechanism arose independently multiple ization ofthe morphogen recognized by Agaricia hinnilis (scle- times, theresultssuggestan adaptation ofacommon an- ractiniancoral)larvae.Biol. Bull. 181: 104-122. cestor. Our own findings, coupled with recent revisions 8. Minovresret,ebAr.atNe.lCa.rv1ae9.92P.p.R3o8l5e-4of03algianePilnatnhte-ArneicrmuailtmInetnetraocftmiaonrsinien oftheevolutionary historiesofscleractinian coralsbased theMarineBenthos. D. M. John. S. J. Hawkins,and H. H. Price, on both molecular phylogenetic analyses (of both eds.SystematicsAssoc.SpecialVol.46.Clarendon Press,Oxford. nuclear and mitochondria! genes) and morphometric 9. Morse, D. E., A. N. C. Morse, P. T. Raimondi, and N. Hooker. and palaeontological studies (2, 16. 17, 18), suggest that 199-4. Morphogen-based chemical flypaper forAxark-ia luimilix corallarvae.Biol Bull 186: 172-181. this common mechanism is relatively old. It appears to 10. Morse, A. N. C. 1992. Unique patternsofsubstratum selection predate not only the phylogenetic and geographic diver- bydistinctpopulationsofAfturiciahniniliscontributetoopportu- genceofthecoralswestudied, but alsotheemergence, at nisticdistributionwithintheCaribbean.Proc. 7lh Inll ('oralReef 240 Ma, ofthe mineralizedcoral skeleton (16, 18). Symp Vol. 1:501-502. 11. Harrison, P. I.., R. C. Babcock, G. D. Bull, J. K. Oliver, C. C. Wallace,andB.L.Willis. 1984. Massspawningintropicalreefs. Acknowledgments Science223: 1 186-1 1X9. 12 Babcock, R.C.,G. Bull, P. L. Harrison,A.J.Heyward,J. K.Oli- We thank M. Hatta and T. Sugiyama for assistance ivenrg.sCo.fC1.05Waslcllaecrea,ctainnidanB.coLr.alWislpleicsi.es19o8n6.theSyGnrecahtroBnaorurisesrpRaewenf-. with gamete fertilization, and M. H. Carr, L. A. Espada Mar. Biol.90: 379-394. and A. Stewart-Oaten forassistance with statistical anal- 13. Hayashibara,T.,K.Shimoike,T.Kimura,S.Hosaka,A.Heyward, Gysaeisn.esW,eA.thKaunrkisA,.aAnldldDr.edMgeo,rsJ.eCfoonrnheelllp,fuEl.sDueglgoenstgi,onSs. sP.paHwanrirnigsoant,AKk.ajKiumdao,Isalnadnd,M.OkOimnoarwia., 1J9a9p3a.n. MPaatrt.erEncsolo.fPcroorga.l Ser. 101:253-262. forthemanuscript.Theprojectwasmadepossiblebythe 14 Shimoike, I., T. Hayashibara, T. Kimura, and M. Omori. 1992. interest and generous support of S. Hosaka, and by Observationsofsplitspawning\nAcroporaspp.atAkajimaIsland, grants to A. M. from NSF, Division ofOcean Sciences, Okinawa.Proc. 1th. Inll CoralReefSymp Vol. 1:484-488. the NOAA National Undersea Research Program, and 15. 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