cover photo for volume 60, number 1 2010: a model example ...€¦ · & microbial processes and...

Yvan Moenne-LoccozCenter for Microbial EcologyUniversity of LyonVilleurbanne, France

Emmanuel MongodinInstitute for Genome SciencesUniversity of MarylandSchool of MedicineBaltimore, Maryland

Pamela J. MorrisMarine Biomedicine and EvironmentalSciences Center

Medical University of South CarolinaCharleston, South Carolina

Mark MorrisonCSIRO Livestock IndustriesQueensland Bioscience PrecinctSt. Lucia, Queensland, Australia

Gerard MuyzerDepartment of BiotechnologyDelft University of TechnologyDelft, The Netherlands

Toshi NagataCenter for Ecological ResearchKyoto UniversityOtsu, Japan

Hans W. PaerlInstitute of Marine SciencesUniversity of North Carolina at Chapel HillMorehead City, North Carolina

Karsten PedersenDepartment of Cell and Molecular BiologyGoteborg UniversityGoteborg, Sweden

James I. ProsserSchool of Biological SciencesUniversity of AberdeenAberdeen, United Kingdom

Juan L. RamosEEZ-CSICGranada, Spain

Francisco Rodriguez-ValeraDivision de Microbiologia and Evolutionary

Genomics GroupUniversidad Miguel HernandezSan Juan de Alicante, Spain

Bernhard H. SchinkDepartment of BiologyUniversity of KonstanzKostanz, Germany

Stephen SchusterThe Pennsylvania State UniversityUniversity Park, Pennsylvania

Kate M. ScowDepartment of Land, Air, and Water ResourcesUniversity of California at DavisDavis, California

Hauke SmidtLaboratory of MicrobiologyWageningen UniversityWageningen, The Netherlands

Gary StrobelDepartment of Plant SciencesMontana State UniversityBozeman, Montana

Jan Roelof Van Der MeerDepartment of Fundamental MicrobiologyUniversity of LausanneLausanne, Switzerland

Bess B. WardDepartment of GeosciencesPrinceton UniversityPrinceton, New Jersey

K. Eric WommackDelaware Biotechnology InstituteNewark, Delaware

Andre-Denis G. WrightDepartment of Animal ScienceUniversity of VermontBurlington, Vermont

Aims and ScopeMicrobial ecology lies at the heart of functioning for almost every eco-system on the planet, from the deep-sea vents and subsurface systems, tohuman and animal well-being; from pristine marine and terrestrial environ-ments to industrial bioreactor functioning. The journal Microbial Ecologyprovides a dedicated international forum for the presentation of highquality scientific investigations of how microorganisms interact with theirbiotic and abiotic environments, with each other as well as with theirneighbors and hosts, to carry out their diverse functions. Microbial Ecologynow features articles of original research in full paper and note formats, aswell as brief reviews and topical position papers. The Editors encouragesubmissions in the following areas:

& ecology of microorganisms in natural and engineered environments& genomic and molecular advances in the understanding of microbial

interactions and phylogeny& microbial drivers of biogeochemical processes& inter- and intraspecific microbial communication& ecological studies pertaining to animal, plant and insect microbiology anddisease

& microbial processes and interactions in extreme or unusual environ-ments

& microbial population and community ecology& microbial processes and interactions associated with environmentalpollution

& technical and theoretical advances in microbial ecology

COVER PHOTO for Volume 60, Number 1 2010: A model example for the importance ofmicrobes to ecosystem function, reef building corals rely on endosymbiotic dinoflagellates in thegenus Symbiodinium for their survival and growth. The elk-horn coral, Acropora plamata, and thebrain coral, Colpophyllia natans, featured on the cover, represent just a few of the host niches thatare occupied by different species of symbiont. Photograph by Robin T. Smith (see related article onpages 250–263).

HOST MICROBE INTERACTIONS

The Relative Significance of Host–Habitat, Depth,and Geography on the Ecology, Endemism, and Speciationof Coral Endosymbionts in the Genus Symbiodinium

J. Christine Finney & Daniel Tye Pettay &

Eugenia M. Sampayo & Mark E. Warner &

Hazel A. Oxenford & Todd C. LaJeunesse

Received: 16 December 2009 /Accepted: 21 April 2010 /Published online: 26 May 2010# Springer Science+Business Media, LLC 2010

Abstract Dinoflagellates in the genus Symbiodinium areamong the most abundant and important group of eukaryoticmicrobes found in coral reef ecosystems. Recent analysesconducted on various host cnidarians indicated that Symbio-dinium assemblages in the Caribbean Sea are genetically andecologically diverse. In order to further characterize thisdiversity and identify processes important to its origins,samples from six orders of Cnidaria comprising 45 generawere collected from reef habitats around Barbados (easternCaribbean) and from the Mesoamerican barrier reef off thecoast of Belize (western Caribbean). Fingerprinting of theribosomal internal transcribed spacer 2 identified 62genetically different Symbiodinium. Additional analyses ofclade B Symbiodinium using microsatellite flanker sequencesunequivocally characterized divergent lineages, or “species,”within what was previously thought to be a single entity (B1or B184). In contrast to the Indo-Pacific where host-generalist symbionts dominate many coral communities,

partner specificity in the Caribbean is relatively high andis influenced little by the host’s apparent mode ofsymbiont acquisition. Habitat depth (ambient light) andgeographic isolation appeared to influence the bathymetriczonation and regional distribution for most of theSymbiodinium spp. characterized. Approximately 80% ofSymbiodinium types were endemic to either the eastern orwestern Caribbean and 40–50% were distributed tocompatible hosts living in shallow, high-irradiance, ordeep, low-irradiance environments. These ecologic, geo-graphic, and phylogenetic patterns indicate that most ofthe present Symbiodinium diversity probably originatedfrom adaptive radiations driven by ecological specializa-tion in separate Caribbean regions during the Pliocene andPleistocene periods.

Introduction

Cnidarian animals in obligate symbioses with dinoflagel-lates of the genus Symbiodinium dominate many shallowtropical marine environments, and their communities formthe basis of coral reef ecosystems. As such, their microalgalsymbionts constitute the most abundant eukaryotic microbialgroup living in these biodiverse ecosystems. Symbiodiniumreside in host tissues at millions of cells per squarecentimeter [1] and provide the energy required by reef-building corals to grow, calcify, and reproduce [2, 3]. Theimpact of ocean warming on the stability of coral–microalgalsymbioses has raised the need for understanding the basicecological and evolutionary processes underpinning thesepartnerships. In response to stressors caused by externalenvironmental change, resident symbionts are sometimesdisplaced by stress tolerant species [4]. Determining the

Electronic supplementary material The online version of this article(doi:10.1007/s00248-010-9681-y) contains supplementary material,which is available to authorized users.

J. C. Finney :H. A. OxenfordCentre for Resource Management and Environmental Studies(CERMES), University of the West Indies,Cave Hill Campus, Barbados

D. T. Pettay : E. M. Sampayo : T. C. LaJeunesse (*)Department of Biology, Pennsylvania State University,University Park, PA, USAe-mail: [email protected]

M. E. WarnerCollege of Earth, Ocean, and Environment, University ofDelaware,Lewes, DE, USA

Microb Ecol (2010) 60:250–263DOI 10.1007/s00248-010-9681-y

http://dx.doi.org/10.1007/s00248-010-9681-y

extent to which external environmental conditions influencehost–symbiont specificity may ultimately reveal how flexiblethese associations are to environmental change [5–8].

Molecular genetic analyses of Symbiodinium diversityare beginning to reveal how host–symbiont specificity,barriers to dispersal, and physical environmental factorslimit the distribution of certain partner combinations [e.g.,7, 9–13]. Surveys conducted around the world and/or indifferent reef habitats have sometimes shown significantdifferences in the amount of diversity and relative ecolog-ical dominance among evolutionarily divergent groups ofSymbiodinium (clades A to D) that associate commonlywith cnidarians [7, 14]. In an era of rapid climate change,obtaining a baseline of the ecological and geographicdistributions of symbiont diversity and their host specificityis needed for correctly assessing the responsiveness ofcoral–algal symbioses to sea surface warming throughchanges in partner combinations (discussed in [5, 15]).

The ecological and biogeographic distributions of Sym-biodinium diversity remains poorly characterized for muchof the world. The diversity of these symbionts in theCaribbean are of particular interest for the followingreasons: (1) the region possesses a unique coral faunaseparate from the Indo-Pacific [16] and (2) is a regionwhere coral populations have declined steadily over thepast several decades [17]. One ecologically importantobservation, but which requires further verification, is thatSymbiodinium diversity found among Caribbean hostassemblages is high (relative to the host diversity) incomparison to reefs in the Indo-Pacific [14, 18]. A broadanalysis of Symbiodinium community diversity has onlybeen conducted at one Caribbean location [9]. Otherinvestigations of diversity focused on a small number ofhost taxa usually limited to a particular region [19–21, 22,23, 24, 25] or employed conservative genetic markers thatdo not resolve ecologically relevant operational taxonomicunits [clade-level resolution only, e.g., 26, 27]. The fewgeographic surveys of host–symbiont partnerships suggestthat many symbiont species have limited regional distribu-tions [6, 10, 28, 66]. Therefore, the distributions ofparticular Symbiodinium may serve as proxies for delimit-ing regions of connectivity in the tropical western Atlantic.As such, efforts contributing to a more thorough under-standing of Symbiodinium diversity, ecology, and biogeog-raphy would be of considerable benefit to coral reefresearch.

The community assemblages of Symbiodinium amongreef cnidarians from various reef habitats in Barbadoslocated in the West Indian Province, eastern Caribbean,were compared with similar habitats in Belize found in theCaribbean Province, southern Yucatan peninsula [sensu29]. In each region, cnidarians in the orders Scleractinia,Gorgonacea, Actinaria, Zoanthidea (=Zoantharia), Anthoa-

thecatae, and Corallimorpharia from three depth rangeswere sampled. The dominant resident symbiont wasidentified in these samples using denaturing gradient gelelectrophoresis (DGGE) fingerprinting of the internaltranscribed spacer 2 (ITS2) region of the ribosomal array[9, 30, 31]. In an effort to improve the phylogeneticresolution of ecologically different Symbiodinium [21, 65],the flanker sequences of two microsatellite loci (Si4.86 andSi15) were directly sequenced from samples of Scleractiniacontaining clade B Symbiodinium. These data were thenused to assess the general significance of host identity inthe distribution of different symbiont “species” and toevaluate the extent to which external environmental factorsinfluence the existence of certain host–symbiont partner-ships. To compare the relative importance of geography anddepth (i.e., irradiance) in structuring Symbiodinium, com-munity diversities from shallow, intermediate, and deephabitats were analyzed and combined with similar datacollected from the northern Yucatan peninsula [PuertoMorelos, Mexico; 9].

Materials and Methods

Sample Collection

Biopsies (small fragments, tentacles, oral disks, etc...) fromvisually “healthy” reef cnidarians were taken using SCUBAor by free diving. Samples from Belize were collected inJuly 2002 and August 2003 from reef habitats surroundingthe Smithsonian station on Carrie Bow Caye while samplesfrom Barbados where collected between July 13 and August26, 2005 (Fig. 1). Collections from Barbados were augmentedby samples obtained in November of 2007. To ensure acomprehensive range of hosts were sampled, cnidarians inthe orders Scleractinia (stony corals), Gorgonacea (seafans, rods, and whips), Actiniaria (anemones), Zoanthidea(zoanthids, button polyps), Anthoathecatae (fire corals),and Corallimorpharia (mushroom polyps) were sampledfrom representative shallow (1–5 m; 500–1,200 µE m−2

s−1), intermediate (5–10 m; 200–400 µE m−2s−1) and deep(>10 m; <150 µE m−2s−1) reef environments, as well as asite in Barbados that was adjacent to a rum distilleryoutfall where corals are exposed to high nutrients andtemperatures 1–2°C greater than ambient. Sample sizeswere largely dependent on the cnidarian diversity presentat each location [Table 1; 9]. For stony corals andhydrozoan fire corals, tissue samples were collected usinga small chisel and hammer to remove a fragment(approximately 1–2 cm2) of tissue-covered skeleton. Forother cnidarians a pair of scissors was used to remove thetip of a small branch (gorgonians), oral disk, or tentacles(anemones, corallimorphs, and zoanthids).

Habitat Partitioning Among Caribbean Symbiodinium 251

Extraction of Symbiodinium from Host Tissuesand ITS-DGGE Fingerprinting

Tissue was removed from hard coral fragments using anairbrush, while soft corals were crushed via mortar andpestle, and anemone tentacles or zoanthid/corallimorph oraldisks shredded with scissors and macerated using a FisherScientific Tissuemiser®. Symbiodinium cells were isolatedfrom the cnidarian tissue by centrifugation at 1,000 g(Fig. 2b) and the resulting pellets preserved in 20% DMSO

preservation buffer [32] and stored at −20°C (Fig. 2b). Afew months after sample collection, genomic DNA wasextracted using the Wizard DNA prep protocol (Promega)modified by [14].

The ITS2 region was analyzed using denaturing gradientgel electrophoreses in combination with DNA sequencingof dominant bands (Fig. 2c). This technique resolves manyecologically and physiologically distinct Symbiodinium [9,13, 25, 30, 33, 34], while being sufficiently conserved toenable comparison among communities from distant geo-graphic areas [28]. The ITS2 was amplified using theprimers: ITS2 clamp (5′CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGG-GATCCATATGCTTAAGTTCAGCGGGT-3′) and ITSintfor2 (5′GAATTGCAGAACTCCGTG-3′) with a touch-down polymerase chainreaction (PCR) protocol following [35]. Amplified PCRproducts were analyzed on denaturing gradient gels (45–80%) as previously described by [9].

Distinct ITS2-DGGE profiles (fingerprints) were char-acterized by sequencing of the dominant bands (Fig. 2c).Bands were excised them from the gel, eluted in DNAasefree water overnight, reamplified with a reverse primerlacking the GC-clamp, and directly sequenced [9]. Finger-prints and their corresponding sequences were comparedagainst a reference database from diversity analyses con-ducted at other Caribbean locations [6, 9]. Newly charac-terized fingerprints diagnostic of an ecologically distinctsymbiont were given an alphanumeric name. The capitalletter of these taxonomic designators refers to the particularclade of Symbiodinium and the numbers and lower caseletters refer to bands diagnostic of the symbiont’s ITS2-DGGE fingerprint (Fig. 2c).

Microsatellite Amplification and Sequencing

The microsatellite loci Si15 [36] and Si4.86 [21] wereamplified and the flanker regions directly sequenced fromsamples of scleractinians containing Symbiodinium B1 todetermine the extent to which these might be furtherpartitioned phylogenetically [21].

Phylogentic and Statistical Analyses

Phylogenetic relationships based on sequences generatedfrom ITS2-DGGE analyses of clade B and C diversityfound in each reef system were reconstructed usingmaximum parsimony PAUP, version 10.4b [37]. Informa-tive sequence gaps as a fifth character state were included,delayed transformation was chosen for character stateoptimization, and no model of molecular evolution wasassumed. Previous comparisons of several phylogeneticmethods show that maximum parsimony reconstructionsare consistent with maximum likelihood and Bayesian

Barbados

Stann Creek

Belize

sampling sites

10 km

5 km

Nnorth

Yucatan

south Yucatan

15˚

30˚

15˚

30˚

Figure 1 Sampling sites from Barbados, eastern Caribbean, andCarrie Bow Caye on the Mesoamerican reef, western Caribbean

252 J. C. Finney et al.

Tab

le1

Symbiod

inium

diversity

inho

stassemblages

from

reefsin

Belize,

western

Caribbean

andBarbado

s,easternCaribbean

Shallo

w(1–5

m)

Interm

ediate

(6–1

0m)

Deep(>10

m)

Hosttaxa

Belize

Barbado

sBelize

Barbado

sBelize

Barbado

s

Hyd

rozoa(firecoralsandhy

droids)

Milleporaalcicornis

B1d

(1)

B1(1);B23

(1)

B37

(2)

B23

(3)

Milleporacomplan

ata

A4a

(1);B(1)

B1(1);B31

a(2)B32

(1);B1d

ay(1);

B1m

(2)

B1(1)

B1(1)

Milleporasqua

rosa

B1(3);B31

(1)

B23

(1)

B1(3);B23

(1);B30

(1)

Myrionemasp.

A4a

(1)

Scleractin

ia(corals)

Acrop

oracervicornis

A3(1)

A3(3),D1a

(2)

Acrop

orapa

lmata

A3(1)

A3(4)

A3(1)

Aga

riciaag

aricites

C3a

(1)

C3(2);C3q

(5)

C3(1);C3a

(1)

C3(3);C3q

(2);C3/B1

(1)

C3(2);C52

(1)

C3b

(5),C3q

(1);C3/B1(1)

Aga

riciafrag

ilis

C3(3)

C3/B1(1)

Aga

riciahu

milis

C3a

(1)

C3(1)

C3/B1(1)

C3b

(1);C3/B1(1)

Aga

ricialamarcki

C3b

(1)

Aga

riciatenu

ifolia

C3a

(3)

C3a

(2)

C3a

(2)

Aga

riciaun

data

C3(1)

Colpo

phyllia

natans

B1(1);D1a

(1)

B6(1)

B1(2)

B6(2)

B1(2);D1a

(1)

Con

dylactisgiga

ntea

B1(2)

Dendrog

yracylin

dricus

B1(2)

B1k

(2)

Dicho

coenia

stokesii

B1(1)

B1(2)

B1(1)

B1(2);B20

(1)

B1(2);C81

/B1(1)

Diploriaclivosa

B1(2)

B1(6);D1a

(1)

B1(2)

B1(1)

Diplorialabrynthifo

rmis

B1(2)

B1(1);C81

(1);D1a/

C34

(1)

C3(1),D1a

(2);C81

/D1a

(1)

Diploriastrigo

saB1(3)

B1(2);B24

(1)

B1(3)

B1(1);B38

(1);

C3L

(1)

B1(4),B35

(1);C3(1)

Eusmiliafastigiata

B1(1);B1e

(1)

B1(1)

B1(5)

Favia

frag

umB1(3)

B1(2);C3(1)

B1(2)

B1(1)

C81

(2)

Isop

hylla

strearigida

B1(2)

Isop

hylliasinu

osa

B1(2)

B1(5)

Leptoseriscuculla

taC3(2)

C3(1)

C3(1)

Mad

racisdecactis

B7(1)

B7(3)

B71)

B7(1)

B7(8)

Mad

racisform

osa

B7(3)

Mad

racismirab

ilis

B7(1)

B7(4)

B7(3)

B7(1)

Mad

racisph

arensis

B7(1)

B7(3)

Man

icinaareolata

B1(2);C53

(1)

B1(1)


Tab

le1

(con

tinued)

Shallo

w(1–5

m)

Interm

ediate

(6–10m

)Deep(>10

m)

Hosttaxa

Belize

Barbado

sBelize

Barbado

sBelize

Barbado

s

Meand

rina

meand

rites

B1(1)

B1(3)

B1(4)

B1(1)

B1(5)

Milleporasqua

rrosa

B1(3);B31

a(1)

B23

(1)

B1(3);B23

(1);B30

(1)

Mon

tastraea

annu

laris

D1a

C7a/B1(1);C7a/B1j

(2)

C7a

(2)

Mon

tastraea

faveolata

B17

(2);D1a

(1)

B1j/C7a

(1);C7a

(2)

C7(1)

B1j/C7a

(2);C7a/B1(1)

C7(1)

C7a

(9);D1a

(1);C7a/D1a/B1(1)

Mon

tastraea

fran

ksi

C7a

(2)

Mon

tastraea

cavernosa

C3(2);C3p

(1);

C3e

(1)

C3(1)

C3e

(1);C3g

(1)

C3(2);C3o

(2)

Mussa

angu

losa

C11

(2)

C11

(1)

Mycetop

hylliaalicea

C49

(1)

Mycetop

hylliada

nae

C48

(1)

Mycetop

hylliaferox

C11

(2)

C83

(2)

Mycetop

hyllialamarckiana

C49

(1)

C11

(1)

Mycetop

hylliareesi

C3c

(3)

Mycetop

hyllia

C48

(2)

Scolym

iasp.

C3(1)

C11

(4)

D1a

(1)

Siderastrearadian

sB5(5)

B5(1),C46

a(1)

A3(1);C3(1)

A3(1);C3(1);D1a

(1);D1a/B1(1)

Siderastreasiderea

C1(1);C3(1),D1a

(1)

C3(1);D1a

(1);D1a/C3(1)

C46

a(1)

C3(2);D1a

(1)

C3(1)

C3(1);D1a

(1);C81

/D1a

(1)

Stepha

nocoenia

intersepta

C16

(2);C54

(1)

C3(4)

A3(1),C3(2)

Gorgo

nacea(sea

fans,seawhips)

Briareum

asbestinium

B33

(1)

B19

(1)

B19

(1);C3L

(1)

C1(1)

Erythropo

dium

cariba

eorum

C3(1)

B1(2);C1(1)

C3(1)

B1(2);C3(1)

Eun

icea

mam

mosa

B1(1)

Eun

icea

spp.

B1g

(1)

Gorgo

niafla

bellu

mB1(1)

B1(1)

Gorgo

niamariae

B1(1)

B1(1)

B1(1)

B1(1)

Gorgo

niaventalina

B1(1)

B1(1)

B1(2)

Muricea

elon

gata

B1(1)

Muricea

spp.

B1(3)

Plexauraflexuosa

B1(1)

Plexuaranu

tans

B19

(1)

Plexuarasp.

B19

(1)

Poritesastreoides

A4(7);A4a

(1)

C1a-j(1)

C1a-j(1)

C80

(2);A4/B1(1);B1/C3(1);C3/

B1(2);

A4/C3/B1(1);A4a/C3/B1(1)


Tab

le1

(con

tinued)

Shallo

w(1–5

m)

Interm

ediate

(6–1

0m)

Deep(>10

m)

Hosttaxa

Belize

Barbado

sBelize

Barbado

sBelize

Barbado

s

Poritesdivaricata

A3(3)

C4(1)

C82

(1)

Poritespo

rites(and

Porites

furcata)

A3(1),C4(2)

A4p

roites(2);C82

(2);C82

/A4p

orites

(1)

C4(2)

C82

(2);C82

b(2)

C4(3);C47

(1)

C82

(1)

Pseud

oplexaurasp.

B1i

(1)

B1L

(1)

B1(1)

Pseud

opterogo

rgia

american

aB1(1)

B1i

(1)

B1(1)

Pseud

opterogo

rgia

bipinn

ata

B1(1)

B1(1)

Pseud

opterogo

rgia

sp.

B1(1)

B1(3)

Pterogo

rgia

citrina

B1(1)

B1m

(1)

Pterogo

rgia

guad

alup

ensis

B1(1)

Actinaria

(anemon

es)

Bartholom

eaan

nulata

C1(2)

Epicystiscrucifer

B1(2)

Lebruniada

nae

C1(1)

C1(1)

C1(1)

Sticod

actyla

helia

nthu

sA4a

(1)

A4(1);A4/B1(1)

Zoanthidea(buttonpo

lyps)

Palytho

acariba

eorum

C1(2)

C1(1);C1p

(1)

C1(1)

C1(1);D1a

(1);C1/D1a

(3);D1a/

C1(1)

Palytho

agran

dis

C1(1)

Parazoa

nthu

ssp.

B1(1)

B2(1)

Zoa

nthu

spu

lchellu

sA4(2)

Corallim

orph

arias(m

ushroo

mpo

lyps)

Ricordiaflo

rida

C1(1)

C3(1)

The

depths

ofho

stcollectionaregivenin

parenthesesforshallow,interm

ediate,anddeep

reef

habitats.The

identifierforeach

symbion

trefers

totheevolutionarily

divergentclade(upp

ercase

letter),theITS2-DGGE

fing

erprinttype,includ

ingthedesign

ations

ofon

eor

moredo

minantintragenom

icsequ

encesin

theribo

somal

array(num

bers

andlowercase

letters).

Num

eralsin

parenthesesindicate

thenu

mberof

colonies

inwhich

aparticular

symbion

twas

foun

d.Twoor

moretypesseparatedby

aforw

ardslashindicate

that

thesymbion

tsco-occurredin

thesample


analyses [6, 7]. To assess statistical significance of internalbranching, 1,000 bootstrap replicates were performed. ITS,Si15, and Si4.86 sequences were deposited in Genbank(Supplementary Tables S1 and S2).

Statistical analyses implemented using the softwarePRIMER (V. 6) were performed on presence/absence dataof Symbiodinium diversity characterized from host commu-nities sampled in shallow, intermediate, and deep environ-

ments from Belize and Barbados, as well as frompreviously published data on diversity from shallow anddeep environments near Puerto Morelos, Mexico, locatedon the northern Yucatan Peninsula [9]. Similarity indiceswere calculated between communities using Bray–Curtiscoefficients where similarity is assigned to the presence,and not the absence of an ITS-2 DGGE type [38].

Results

The Ecological and Geographic Distributionof Symbiodinium Diversity

Thirty-eight Symbiodinium types based on ITS2-DGGE“genotyping” were characterized from Barbados and 36from Belize (Table 1). In total, 62 Symbiodinium types werecharacterized from 285 cnidarians representing 34 genera(approximately 54 species) from Barbadian reefs and 191colonies comprising 41genera (approximately 63 species)from Belizian reefs, with only 20% of these symbiontsoccurring in both Belize and Barbados (Table 1). Membersof clades B and C were ecologically dominant in bothCaribbean reef communities and found in numerous hosttaxa occurring at all depths and found in association withcnidarians in the orders Scleractinia, Gorgonacea, Actiniaria,Zoanthidea, Corralimorpharia, Anthoathecatae, and Corrali-morpharia (Tables 1 and 2; Supplementary Fig. S1). Thenumber of distinct Symbiodinium from each clade wassimilar in both regions (17 vs. 14 clade B and 16 vs. 19clade C for Barbados and Belize, respectively; Fig. 3;Supplementary Table S3). Four Symbiodinium in clade Aand Symbiodinium trenchi (formerly D1a), the sole repre-sentative of clade D, were also identified. A significantproportion of the total ITS2 type diversity (19 out of 62) wasrepresented by one sample.

The distribution of 28 out of 38 (73%) types fromBarbados and 27 out of 36 (75%) from Belize correspondedto samples from specific host genera (Table 2). S. trenchi(clade D) was detected in a few colonies of Acroporacervicornis, Siderastrea siderea, and the Montastraeaannularis species complex from Belize and was identifiedin some colonies Colpophyllia natans, Diploria sp., M.annularis species complex, S. siderea, and the zoanthidPalythoa caribaeorum from the reefs near Barbados(Table 1). Of the 18 samples comprising eight host generacollected from the nutrient rich and warm (1–2°C aboveambient) site at the rum distillery outfall in Barbados, S.trenchi dominated one colony of Diploria clivosa and co-occurred with C3 in a colony of S. siderea. Twenty-five(out of 38) Symbiodinium types from Barbados and 21types (out of 36) from Belize exhibited restricted depthdistributions (Table 2), with members of clades B and C

a

1 2 3

4 5 6

7 8 9

SSU LSU21

b

5.8

ITS-DGGE- fingerprinting/sequencing

of the Ribosomal arrayc

1

d j

31 7

1 1a

B1d

B1d

B1d

B1j

B1j

B1j

B7

B31

aB

31a

B1

2

B1

2

B1

2

Figure 2 a Symbiodinium spp. identified from cnidarians, including(1) Meandrina meandrites, (2) Favia fragum, (3) Montastraeaannularis, (4) Isophylastea rigida, (5) Montastraea faveolata, (6)Gorgonia sp., (7) Colpophyllia natans, and (8) Acropora palmata,characterized b using ITS-DGGE rDNA fingerprinting, which screensfor numerically dominant intragenomic rDNA sequence variants.Bands from the lower half of each profile (upper half comprisesheteroduplexes) were further characterized by direct sequencing (seetext for details). These sequences were then used in phylogeneticreconstructions


common across all depths, and the few types belonging toclade A occurring predominantly at shallow depths.

To evaluate the relation between depth and geographyand to portray the relative influence of these factors on thedistribution of Symbiodinium diversity, dendrograms basedon presence/absence similarity data were constructed. Theshallow, intermediate, and deep symbiont communitiesfrom Barbados were more similar to each other than toassemblages characterized from analogous depths in thewestern Caribbean (Fig. 3). The diversity data includedfrom the northern Yucatan [9] shows that symbiontdiversities from the Mesoamerican barrier reef are similar,relative to Barbados, and that assemblages from the samerange of depth in these western reefs have the greatestsimilarity (Fig. 3). Differences between shallow and deepenvironments within a location were influenced in part bydifferences in host diversity existing in each habitat.Differences in host diversity surveyed at each locationlikely influenced the overall similarities between symbiont

assemblages. Sites from the western Caribbean are separatedby a distance of about 470 km vs. distances of greater than3,070 km separating them from Barbados, indicating thatdistance corresponds to the level of similarity/dissimilaritybetween symbiont communities.

Phylogenetic Relationships of Symbionts from Easternand Western Caribbean Reefs

Two unrooted phylogenies were reconstructed for clades Band C (Fig. 4a, b) based on sequences screened by DGGEanalyses targeting the dominant intragenomic ITS2 se-quence variant(s) (Fig. 2). While basal sequences corre-sponded to widely distributed symbionts common in two ormore host genera from eastern and western Caribbean reefsystems, most of the sequence diversity “radiating” fromthese “ancestral host generalists” corresponded to hostspecific and/or rare Symbiodinium. The “species” diversityof clade B Symbiodinium based on ITS sequences partitionsinto two subclades, the “B1” and “B19” radiations [sensu6]. However, small sequences differences within the “B1”radiation limited inference of evolutionary relationships.For example, whether types B1d, B31, B32, and B37associated with Millepora, share a recent common ancestorrelative to others in the “B1” radiation cannot be deter-mined with ITS sequence data (Fig. 4a). Similarly, in theclade C radiation, Montastrea cavernosa associated uniquelywith a variety of specific symbiont types (e.g., C3d, C3e, andC3g from Belize and C3o and C3p from Barbados) and asimilar situation was found among those symbionts associatedwith the Mussidae (e.g., C11, C48, C49, and C83). However,the evolutionary relationships between these and most otherclade C Symbiodinium can only be described as an unresolvedradiation (Fig. 4b).

Symbiodinium B1 associated with nearly half of the hosttaxa surveyed in both Belize and Barbados and was harboredby hosts across all depths (Table 1; Supplementary Figure 1).Analysis of flanker regions of microsatellite loci Si4.86 (134aligned bases) and Si15 (188 aligned bases) indicated that

Table 2 The proportions (by percent) of ITS2 types exhibiting high host specificity (i.e., found in only one host genus), found at only onelocation (Barbados or Belize), bathymetrically zoned to primarily shallow (<5 m) or deep (>10) dwelling colonies or identified in only one sample(i.e., ecologically rare)

Taxonomic group(no. of ITS2 fingerprint types)

High hostspecificity (%)

Geographicallyrestricted (%)

Exhibiting depth zonation(shallow, % vs. deep, %)

Ecologicallyrare (%)

Barbados A (4) 50 50 25 (25:0) 0

B (17) 94 71 70 (35:35) 53

C (16) 81 69 69 (38:31) 6

Belize A (2) 50 0 50 (50:0) 0

B (14) 86 64 57 (21:36) 36

C (19) 79 79 69 (38:31) 21

Table 2 The proportions (by percent) of ITS2 types exhibiting highhost specificity (i.e., found in only one host genus), found at only onelocation (Barbados or Belize), bathymetrically zoned to primarily

shallow (<5 m) or deep (>10) dwelling colonies or identified in onlyone sample (i.e., ecologically rare)

100

80604020

Barbados ‘shallow’

Barbados ‘Intermediate’

Barbados ‘Deep’

Similarity

south Yucatan ‘deep’

south Yucatan ‘intermediate’

north Yucatan ‘deep’

north Yucatan ‘shallow’

south Yucatan ‘shallow’

eastern Caribbean

western C

aribbean

A

BC

D

Clade Diversity

Barbados

Belize

Figure 3 The relative similarity of Symbiodinium communities fromdifferent locations and habitats was compared based on group-averagelinking using Bray–Curtis coefficients (see text for details). WhileSymbiodinium clades A to D possessed similar proportions of ITS2-type diversity in Belize and Barbados (pie graphs), each regionpossessed distinct compositions likely influenced by their relativegeographic separation


cryptic diversity exists within Symbiodinium B1 that corre-sponds to the hosts they inhabited. These Symbiodiniumpossessed the same dominant ITS2 sequence, type B1, yethad significantly different Si4.86/Si15 sequences (Fig. 5a,Supplementary Table 1). Independent phylogenetic recon-structions based on each locus’ flanker sequence producedcongruent phylogenies (Fig. 5a) and identified three distinctlineages with differing alleles and frequencies (Fig 5b and c).The 4.86 locus did not amplify the symbionts fromDendrogyra cylindrus, but the Si15 flanker sequencesidentified these as representing a fourth lineage, B14

(Fig. 5a). The phylogeny based on the concatenated flankersequences of both loci, to which several additional symbionttypes were included, produced divergent clusters comprisingmostly identical sequences that corresponded to Symbiodi-

nium populations found in specific host taxa, from both eastand west Caribbean locations (Fig. 5d). Host specificityranged from being highly specific, such as those lineagesspecific to Millepora (B11) and Dendrogrya (B14), to onesthat associated with multiple host taxa (e.g., B12, B13;Fig. 5d). With few exceptions, only a single sequence andallele (as determined by the microsatellite motif repeatnumber) was identified, indicating that each sample pos-sessed a single dominant clone–genotype, or strain, com-prising the resident symbiont population.

An important observation was finding some Symbiodi-nium possessing an unusual ITS2 fingerprint instead sharedidentical flanker sequences with particular B1 types. Theseinvariably were characterized from samples of the samehost species. For example, the Si4.86 and Si15 flankersequences of B24 found in one sample of Diploria strigosafrom Belize matched with Symbiodinium B12 found inDiploria spp. samples from both Belize and Barbados. In asimilar example, the flanker sequences of B20 harbored bya colony of Dichocoenia stokesii from Belize, matched withSymbiodinium B13 found in D. stokesii from both regions(Fig. 5d). Finally, B1k harbored by D. cylindrus inBarbados matched with flanker sequences of the B14 foundin the D. cylindrus from Belize (Fig. 5d). While “B1” wasalso detected in other hosts, such as gorgonians andanemones, complete analysis of this previously unrealizeddiversity across all potential hosts and geographic locationswas beyond the scope of this study

Discussion

Toward Delimiting Species of SymbiodiniumUsing a Combination of Approaches

The combination of ITS2-DGGE analysis and microsatel-lite flanker sequencing provided independent and comple-mentary data on the genetic diversity and phylogeneticrelationships of ecologically differentiated Symbiodinium.The clear discontinuity between sequence clusters anddifferences in motif repeats indicates that the phylogeneticgroupings illustrated in Fig. 5d do not share alleles and aretherefore genetically (i.e., reproductively) isolated. Thisevidence, when combined with ecological data, provides astrong case that speciation of Symbiodinium is influencedprimarily by ecological specialization (i.e., host–symbiontspecificity, high- and low-light adaptation) and geographicisolation [39].

While DGGE is commonly employed in the analysis ofbacterial community assemblages, it is mainly used here toscreen eukaryotic ribosomal DNA for the most commonintragenomic sequence variants [31]. The analysis of aparticular genome generates a reproducible fingerprint, with

C53a(53)

C48

C83C7

C7a

C3LC3b

C3a

C16

C80C81

(1a)

C1a-i

C1p

C52

C47

C4

C82a

C82b

C46a

(46)

C1C3

C3e

C3g

C3d

C3p

C3q

C11

C49

C3o

Mussidae

Porites

M. cavernosa

b

a West Caribbean (Belize)

East Caribbean (Barbados)

1 change

B1d

B32

B31

B37

B1jB1g

B1LB1e

B24

B1k

B5

B34

B1i

B7B1m

B38

B2

B33

B6

B20B17

B23

B30

B19

MilleporaMillepora

B1*

Greater Caribbean

Figure 4 Phylogenetic reconstructions using maximum parsimony(unrooted) of ITS2 sequences of clade B (a) and clade C Symbiodi-nium (b) characterized from zooxanthellate reef cnidarians in western(Belize, black circles) or eastern Caribbean locations (Barbados, graytriangles), including some found in both regions (open squares).Sequences used in these analyses were of dominant intragenomicvariants screened by ITS2-DGGE fingerprinting (see [31])


the brightest band or bands, apparently corresponding thosesequences that have been become numerically dominantthrough concerted evolution (Fig. 2c; [40, 41]). Thisprocess filters out rare functional and nonfunctionalvariants that, when added together, represent a majorcomponent of the ribosomal array, but do not providedependable genetic markers [30, 31]. The analysis ofeukaryotic rDNA in this manner should be limited tosamples usually dominated by a single species. Becauseresident Symbiodinium populations in a host colony oftenappear to be clonal ([7, 21, 36, 42–45]; see results ofmicrosatellite sequencing), a single repeatable profilecharacteristic of the dominant resident symbiont is pro-

duced (Fig. 2c). In cases where a second or third symbiontco-occurs, the individual fingerprints of each symbiont aretypically discernable in the sample profile [9].

Recent comparisons of microsatellite flanker sequencesand allelic variation indicated that Symbiodinium B1 [14;B184 sensu 44] may actually comprise multiple crypticspecies [21]. Subtle yet repeatable differences amongbanding patterns in the ITS-DGGE fingerprints of Symbio-dinium B1 from different host genera indicated that thegenomes of these particular symbionts had undergonedifferentiation and divergence, yet the dominant rDNArepeat was still the “B1” sequence. In the present study,phylogenetic analyses of flanker sequences from the

ab

c

d

Figure 5 Phylogenetic analysisof microsatellite flanker regionssequenced from “B1” and otherrelated ITS2 types harbored byscleractinians (stony corals) andMillepora spp. (fire corals). aThe correspondence of phylog-enies based on loci Si4.86 [44]and Si15 [36]. Variation andfrequency of motif copy numberfor Si4.86 (b) and Si15 (c) foreach of three lineages (desig-nated by black, gray, and whitebars). d A partial clade Bphylogeny based on concatenat-ed Si4.86 and Si15 flankerregions. Symbiodinium B19from the gorgonian Plexaurellasp. was designated as the out-group. The numbers of samplessequenced are indicated in pa-rentheses next to the ITS2 typedesignation. Bootstrap valuesbased on 1,000 replicates areprovided for each branch. Blackcircles, gray triangles, and opensquares correspond to samplesfrom Belize, Barbados, and thegreater Caribbean, respectively.The star symbol indicates thatSi4.86 did not amplify the B14

and B1k from Dendrogyracylindrus


microsatellite loci 4.86 [21] and Si15 [36] cleanly delin-eated several constituent lineages of “B1” that differedmarkedly in their host–habitat range (Fig. 5a, b). Thephylogenetic congruence between each microsatellite flank-er sequence as well as significant differences in motifrepeats indicate that these are genetically isolated, indepen-dently evolving, sympatric lineages (Fig. 5a, b). Symbiodi-nium B1 was initially viewed as a host-generalist because itassociated with a majority of reef cnidarians living in theCaribbean [9]. It is now clear that it comprises numerouscryptic species some of which are highly specific whileothers are still somewhat generalized (Fig. 5d). Finally,these data indicate that two or more organisms whosegenomes are dominated by the same “ancestral” ITSsequences may comprise separate species.

The data provided by flanker sequences also suggeststhat certain rare ITS2-DGGE fingerprint types may repre-sent geographic and/or host-specific variants within aSymbiodinium species. The four distinctive “B1” lineagesshown in Fig. 5a contained representatives whose ribosom-al arrays were characterized by distinct ITS2 sequences thatdiffered by one to several base changes from the ancestralB1sequence (Fig. 4a). For example, the flanker sequencesof rare types characterized as B20 from D. stokesii and B24from Diploria sp. possessed Si4.86 and Si15 flankersequences identical to the “B1” types found in these coralsfrom both Belize and Barbados, respectively (Fig. 5d).These rare ITS2-DGGE fingerprint types may indicatenewly diverged lineages or may instead be uncommongenetic variants in the population. While B20 and B24 eachpossessed unusual allele sizes for one or both micro-satellites, their sample number (n=1) precluded a compar-ison of allele frequencies to further assess the possibilitythat these unique types represent genetically isolatedsubpopulations.

While ITS-DGGE fingerprinting, in many cases, offersa consistent and often sufficient “first-pass” at delimitingoperational taxonomic units, additional sequence datasuch as microsatellite flanker sequences [21] and/orplastid genes [30] can provide a clearer and more detailedphylogenetic reconstruction of evolutionary relationshipsamong closely related Symbiodinium spp. (Fig. 5d). Futurestudies of Symbiodinium diversity would benefit fromemploying a combination of genetic markers, if they areavailable. Presently microsatellites with phylogeneticallyinformative flanker sequences are only available for cladeB Symbiodinium.

The present lack of accurate, unified, species taxonomyfor Symbiodinium has unquestionably inhibited the abilityof investigators to compare and discuss their findings in thecontext of previously published research. The establishmentof a standardized nomenclature wherein species delimita-tion is based on detailed genetic and ecological data is

therefore essential for addressing fundamental questionsabout coral–algal symbioses. Knowledge of symbiontspecies diversity and distribution is especially importantwhen selecting hosts to carryout experimental manipulationswhen testing hypotheses. For example, hosts associatingwith clade C Symbiodinium may harbor different specieswithin the clade and therefore, proper interpretation mustaccount for the possibility that significant physiologicaldifferences exist between closely related taxa (see below).Furthermore, “species-level” characterizations are necessarybefore appropriately analyzing population genetic data todescribe how geographic isolation, dispersal, and/or ecolog-ical specialization limit gene flow among Symbiodiniumpopulations [21].

The Primary Influence of Host Species Identityon the Ecology and Evolution of Symbiodinium

The specificity exhibited by most Symbiodinium spp. for aparticular host species, and/or genus, emphasizes theimportance of host specialization in the ecology andevolution of these symbionts. “ecological specialists”represent a majority of the Symbiodinium communitydiversity while “ecological generalists,” those symbiontsassociated with numerous host species representing multi-ple genera, are few [14, 28]. Plainly stated, most species ofcoral are incompatible with most species of Symbiodinium.From an evolutionary point of view, hosts provide thedefinitive habitat where symbiont proliferation is probablygreatest. The natural selection of particular genotypes as aconsequence of ecological specialization would over timelimit genetic exchange, ultimately leading to populationsubdivision, reproductive isolation, and speciation [46].

Geographic Partitioning

Faunal community differences, confirmed by populationgenetic studies and the modeling of sea surface currents,indicate that the tropical Western Atlantic comprises severaldistinct biological subprovinces [29, 47, 48]. While relativeecological dominance of Symbiodinium clades A, B, C, andD appears similar throughout much of the Caribbean[Table 1; Fig. 3; 9, 27], major differences in the presenceand absence of ITS2 types were evident between easternand western regions (Figs. 3 and 4a, b). Indeed approxi-mately 80% (49 of 61) of Symbiodinium observed in oneregion did not exist in the other (Fig. 4a, b). Pastevolutionary responses to major environmental changes inthe north-western Atlantic may have facilitated the uniqueecological success of clade B relative to clade C [6]. Themost recent of these events was the Pliocene/Pleistoceneboundary where the onset of North American glaciationscorrelates with increased marine extinctions in the western


tropical Atlantic [49]. Indeed, much of the present diversityof Symbiodinium in the region is differentiated by minimalITS sequence divergence (Fig. 4a, b) and is likely the resultof adaptive radiations originating during or subsequent tothe Pliocene/Pleistocene transition [6].

In several cases, Symbiodinium types found in Belizeshared close phylogenetic affinities to counterparts possess-ing similar ecological traits in Barbados (e.g., hostdistribution). For example, C1a-j occurred in deep Poritesastreoides in Belize while the closely related C80 occurredin P. astreoides from low-light environments in Barbados(Fig. 4b, Table 1). This same phylogeographic pattern wasalso observed for the symbionts of Porites porites (e.g., C4and C82a) as well as deep and/or shaded colonies of the M.annularis complex (C7 in Belize compared with C7a inBarbados). East–west differences in symbiont were alsoevident among Agaricia spp. (C3a in Belize compared withC3b and C3q in Barbados, Fig. 4b). It is likely thatgenetically similar symbionts associated with the same hosttaxa are geographic subpopulations of a broadly distributedspecies of symbiont. However, these slight yet fixeddifferences among intragenomically dominant ITS se-quence indicate the existence of an effective barrier todispersal between eastern and western provinces. Based onprevious rate estimates of ITS divergence [one baseconversion in the dominant ITS2 per 0.75–1.3 Myr; 6],the observed sequence differences correspond to effectiveperiods of genetic isolation ranging tens to hundreds ofthousands of years.

Sequence data that provides increased phylogeneticresolution is now needed to discern the evolutionaryrelatedness among many of the closely related membersof the clade C radiation. For example, it is not clear whetherthe C3 variants harbored by M. cavernosa share a morerecent common ancestor than with genetically similar typesspecific to other host species (Fig. 4b). Phylogeneticallyinformative flanker sequences, such as those employed herefor clade B, are not yet available; however, recentcomparative analyses of the noncoding region of the plastidpsbA minicircle DNA indicate that this region may providethe required resolution [50, 51].

Clade D Symbiodinium in the Caribbean apparentlycomprises a single species, S. trenchi (D1a). There was noclear ecological nor perceivable genetic differentiation ofthis symbiont. ITS2 DGGE fingerprints appeared identicalamong samples from different hosts in both regions (datanot shown). This contrasts with the Indo-Pacific wheremembers of clade D are genetically diverse with manyexhibiting regional endemism [7]. While seemingly hap-hazard in its host and depth distribution, S. trenchi wasmost frequently detected in colonies of the M. “annularis”complex [52] and S. siderea (Table 1). This symbiontbecame much more prevalent and was found in many new

hosts species during the mass coral bleaching event thatoccurred in Barbados just months after completion of the2005 summer survey [52; see “Materials and Methods”section]. The unusual ecology of this enigmatic host-generalist symbiont certainly deserves further investigation.

Partitioning to High- and Low-Irradiance Habitats

The existence of “shallow high-light” and “deep low-light”Symbiodinium in the cnidarian communities from Belizeand Barbados suggests that depth (i.e., irradiance) alsocontributes substantially to the niche partitioning of thesesymbionts [9, 12, 13, 53]; not surprising given that thesedinoflagellates are sedentary photoautotrophs. Clade ASymbiodinium constitutively express UV adsorbingmycrosporine-like amino acids and appear to have theability to upregulate cyclic electron transport aroundPhotosystem I [54, 55]. These physiological traits explainwhy members of clade A are predominantly found incompatible hosts from shallow habitats [9, 12]. However,Symbiodinium A3 was observed in some colonies from lowphotic environments indicating that it may also possessadaptations for utilizing a wide range of irradiance [Table 1;Supplementary Fig. 1; 56].

Although comparative studies are still few, differences inphotophysiology (and thermal tolerance) clearly existbetween sibling species of Symbiodinium [4, 34, 53, 56–60]. Numerous members of clade B and C were found incolonies from either “shallow” (1–5 m) or “deep” (>10 m)habitats [Table 2; Supplementary Fig. 1]. This apparentdepth zonation is confounded by the habitat and depthpreferences of the host. Nonetheless, there is sufficientevidence to indicate that certain related Symbiodiniumappear adapted to high and/or low irradiances. For example,B1j was found exclusively in shallow colonies of M.annularis and M. faveolata, as opposed to B7 harbored bycolonies of Madracis at all depths [25].

Physiological adaptation probably occurs in conjunctionwith host–habitat specialization. Symbiodinium C11 asso-ciates exclusively with Caribbean Mussidae. As broodingcorals [61], mussid larvae probably acquire their sym-bionts through maternal inheritance (i.e., vertical trans-mission), a mode of transmission that facilitates theevolution of host-specialized Symbiodinium [6]. Thesefleshy solitary, corymbose, or plating species occurtypically at depths below 10 m [62]. Presumably, asymbiont that is specific to a particular host species orgenus must also have physiological adaptations necessaryto function optimally in the environments where the hostis most abundant [63]. This and other examples highlightthe important interplay of external (abiotic) and internal(biotic) factors that influence ecological specialization andSymbiodinium diversification.


The Influence of High Specificity on Coral–AlgalSymbioses Subjected to Rapid Climate Change

The evidence presented here contributes substantively to agrowing body of work demonstrating that specificity amongcoral–algal symbioses, while influenced in part by externalenvironmental conditions (e.g., irradiance, temperature) andgeographic location, depends primarily on molecular–cellu-lar interactions between host and symbiont entities. Thisspecificity will seriously limit the extent to which host–symbiont partners recombine to form associations tolerant ofwarming sea surface temperatures [5, 15]. Reconstructions ofpast evolutionary events suggest that the development ofhost–symbiont partnerships requires time scales beyond thecurrent pace of climate change [6]. The biological responseof these symbioses to rapid climate warming may thereforedepend on existing stress tolerant combinations and/or therapid spread of thermally tolerant host-generalist symbionts[7, 52, 64].

Acknowledgments Funding for this research was provided byPennsylvania State University, Florida International University, andthe National Science Foundation (IOB 0544854) to T. C. LaJeunesse,a UWI Research Grant to H. A. Oxenford, and a UWI postgraduateaward to J. C. Finney. A Coral Reef Research Permit to WF/TCL/HAO was obtained in June 2005 through the CZMU, Government ofBarbados. Funding for work in Belize was provided by the CaribbeanCoral Reef Ecosystems (CCRE) Program, Smithsonian Institution.This is contribution no. 879 to the CCRE program.

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