Research Articles

letters to natureNATURE|VOL 388 | 17 JULY 1997 265Landscape ecology of algalRob Rowan*†, Nancy Knowlton*, Andrew Baker*‡& Javier Jara**Smithsonian Tropical Research Institute, Apartado 2072, Balboa, PanamaUniversity of Guam Marine Laboratory, Mangilao, Guam, 96923, USARosenstiel School of Marine and Atmospheric Science, Unversity of Miami,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Reef-building corals are obligate, mutualistic symbioses ofSymbiodinium spp.)1. Contrary to the earlier, widely acceptedMontastraea annularisandM. faveolata can act as hosts to dynamic, multi-speciesSymbiodinium. Composition of these communities2–4 is not the only mechanism by5,6.The coralsMontastraea annularis and M. faveolata each host three7 of the dinoflagellate genus Symbiodinium,A, B and C, that are identified by restriction-fragment7. A and B are common in shallow-water corals (highirradianceC predominates in deeper corals (lowirradianceA þ C and B þ C, common atNature © Macmillan Publishers Ltd 1997letters to nature266NATURE |VOL 388 | 17 JULY 1997intermediate locations7, suggest that symbionts may actually exist as2.M. faveolata, and all but one colony of M., yielded two or three types of symbionts. As predicted,Symbiodinium Aand B dominated locations with higher, downwellingC dominated locations of lower irradiance (communities 3 andP , 0:001; x2 test). TheseM. annularis, and 6–12m in M. faveolata),Symbiodinium C is predominant overall7. As before7,Symbiodinium Awas more common in M. faveolata than in M..,1 cm) confirmed that symbiontsM. annularis create a localized gradient of low (on the side,Symbiodinium C toB, B þ C, or A (Fig. 2a–d). Analyses of shallower (1–2 m) and7 and intracolony3,8. Bleaching3,8,9. Many bleaching events exhibit intraspecificFigure 1Symbiont communities in M. annularis (a) and M.(b). Each symbol represents one core sample thatSymbiodinium A, B, C or mixtures of taxa summarizedFigure 2Symbiont zonation in columns of M. annularis. a, b, TaqI digests of srRNA genes. a,c) and standards forSymbiodinium A, B and C. b, Standards (see Methods). Lane 1, C alone; 2–10, B þ C mixturesC: B ratios); 11, B alone. c, Column sampled in a; symbols 1–11 (dark,Symbiodinium C; white, Symbiodinium B) represent the data in lanes 1–11, respectively in a.d, Zonation in 14 columns from 14 colonies sampled as in c (differences in shading are forx-axis, location relative to lower side and top centre; y-axis, genotype rangingC to all B or all A (A in one column only); z-axis, depth. e, Zonation in a column8 from vertical for 6 months, presented as in c. f, Zonation in 7 columns (alle,d; zonation in natural columns at a depth of 6m (from d) are plotted forNature © Macmillan Publishers Ltd 1997letters to natureNATURE|VOL 388 | 17 JULY 1997 267that are difficult to explain9–12. Because irradiance and temperature12–14, and the symbionts ofM. annularisand M. faveolata exhibit different associations withM. annularis and M.on 18 September 1995, and bleaching was extensive by the15. At our2. We also observed complex9,10,18 bleachingM. annularis and M. faveolata. Bleaching was,2 m) and deep (.15 m) sites; inM. annularis partitioned as in Fig. 1an ¼ 76 colonies,P , 0:05, x2 test). Some colonies exhibited a ‘ring’ ofM. faveolatacolonies are not easily partitioned into two distinct,6 m) centre. Such12 because theSymbiodinium C’s ‘adaptive9,15,19 (see Fig. 3a, b), if Symbiodinium C were,1 cm from many sites sampled the previous January (Fig. 1). AllSymbiodinium C plus either A or B (or both) werea priori (Fig. 2) thatSymbiodinium C in corals,1 cm awaySymbiodiniumsrRNA RFLPs in these replicate, pre-bleaching pairsSymbiodinium C had decreased in relative abundanceSymbiodinium C were typically close toB underwent a median decrease of 14%, and A moreSymbiodiniumtaxa as symbionts under ‘bleaching conditions’. TheA . BqC.M. annularis and M., which are dominant members of western Atlantic reefs20Figure 3Bleaching in M. annularis (a, c) and M. faveolata (b, d) ata, b) andc, d) patterns. e, Sea surface temperatures (three-week30) at the San Blas Islands,8C in 1983 and 1995 were16 (this study). Records from ourNature © Macmillan Publishers Ltd 1997letters to nature268NATURE |VOL 388 | 17 JULY 1997and are widely used as model systems in reef biology and11,13,18,19,21,22. Each coral ‘species’ encompasses one animal1, in which host taxa alone are adequate units of biodiversity,2–4, and bleaching variability is often12. We conclude that polymorphic symbiont communities20 and bleaching ecology of19. For these corals, and for mutualisms in general, the5,6.M.and M. faveolata in the Bahamas also host Symbiodinium, B and C (data not presented), and published photographs18 and9,10 of bleaching elsewhere strongly resemble our own9.We can attribute thisM. annularis andM. faveolatato symbiont polymorphism and zonation. Moreover,Symbiodinium Aand C (ref. 23; our unpublished data). For otherSymbiondinium taxonomy would be2–and genetic differences among hosts11,14.3,8,21. Alternatively,24–26. Our findingsSymbiodinium to produceM.and M. faveolata might adjust to a warmer AtlanticSymbiodinium A and less Symbiodinium BandC. However, long-term consequences of such replacementsM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .MethodsCoral samples were collected at27 by coring (1.1 cm2 surface area) and,0.12cm2)M. annularis were broken off ,15 cmLaboratory analyses.Symbionts and symbiont DNA were isolated from7 and from fresh28 samples. Nuclear srRNA genes were amplified usingSymbiodinium-biased’ primer ss3Z (all data in Figs 2 and 4), andTaqI and DpnII (data were consistent in every case)7. The biasedSymbiodinium genotypes (discussed in ref. 28), as confirmed by7 and by comparing results from ‘universal’ and ‘biased’ amplificationsA þ C or B þ C) in variousFigure 4Symbiont communities before (January 1995) and during (October 1995)ac, Lanes contain TaqI (a, b) or DpnII (c) digests ofa, Standards for B, C and B:C ratios of 8 : 1 (lane 1) and 1 : 1 (lane 2);M.(lanes 3–6) and M. faveolata (lanes 7–10). b, Standards for A, C and A:Cratios of 2 : 1 (lane 1) and 1 : 8 (lane 2); lane pairs 3 (M. annularis) and 4–7 (M.) compare symbionts as in a. c, Standards for A, B, C and equal amountsA, B and C (lane 1) and A and B (lane 2); lane pair 3 compares symbionts in M., as in a and b. The vertical bracket identifies bands that identify eachd, Densities of A (grey), B (white) and C (black) before and duringa(B þ C, communities 3–10), b (A þ C, communities 3–7) and c (ABC, community 3).e, Chlorophyll contents of the samples reported above, presented as in d.Nature © Macmillan Publishers Ltd 1997letters to natureNATURE|VOL 388 | 17 JULY 1997

symbionts creates variation

in episodes of coral bleaching

Miami, Florida 33149, USA

heterotrophic animals and phototrophic dinoflagellates


belief that corals harbour only one symbiont, we found that the

ecologically dominant Caribbean corals

communities of

follows gradients of environmental irradiance, implying

that physiological acclimatization

which corals cope with environmental heterogeneity. The importance

of this diversity was underlined by analysis of a natural

episode of coral bleaching. Patterns of bleaching could be

explained by the preferential elimination of a symbiont associated

with low irradiance from the brightest parts of its distribution.

Comparative analyses of symbionts before and after bleaching

from the same corals supported this interpretation, and suggested

that some corals were protected from bleaching by hosting an

additional symbiont that is more tolerant of high irradiance and

temperature. This ‘natural experiment’ suggests that temporal

and spatial variability can favour the coexistence of diverse

symbionts within a host, despite the potential for destabilizing

competition among them

distantly related taxa


length polymorphisms (RFLPs) in genes encoding small ribosomal


habitats), whereas

habitats). Mixed samples

complex communities that track differences in irradiance within a


To test this hypothesis we sampled four locations in each of 46

colonies (Fig. 1). All


irradiance (communities 1 and 2, unshaded colony tops),


4, colony sides and shaded colony tops) (

patterns of intra-colony zonation largely disappear at slightly

greater depths (8–11m in



Analyses at a finer scale (

occupy distinct but overlapping habitats (Fig. 2). Unshaded columns


no downwelling) to high (on the top, full downwelling) irradiance,

which we sampled along transects. At intermediate depths (3–7 m)

this gradient coincides with the transition from

deeper (9–12 m) corals (Fig. 2d) show that depth

zonation of the symbionts occur in parallel. These consistent

patterns strongly argue that zonation is controlled by ambient

irradiance. Furthermore, experimentally toppled columns, which

experienced immediate and severe changes in irradiance gradients,

largely re-established expected patterns of symbiont zonation

during a six-month period (Fig. 2e,f). This response shows that

the patterns are maintained dynamically.

Symbioses between corals and dinoflagellates are stable mutualisms,

with the notable exception of coral bleaching, which involves

the loss of symbionts and/or photosynthetic pigments

is an ecologically important but poorly understood response to

environmental stress

variation distributed within and among habitats in ways



according to the code shown below. Columns in the data

matrices represent individual coral colonies (depth increases

from left to right), and rows represent locations of higher (rows 1

and 2) and lower (rows 3 and 4) irradiance, as defined in the

diagrams to the left. Samples were collected in January 1995.

Transect from column side to top (lanes 1–11; positions shown in

(numbers below are

clarity only);

from all

transplanted to 90

at a depth of 6 m) transplanted and sampled along two transects (white and black) as in

presented as in

comparison (grey).

act synergistically to induce bleaching

irradiance (Figs 1 and 2), we hypothesized that symbiont polymorphism

underlies this variation.

We observed ‘paling’ in several colonies of


second week in October, both in Panama and elsewhere

study site, this event was ‘typical’: like a similar event there in 1983

(ref. 16), it followed a prolonged excursion above the mean summer

maximum of temperature (Fig. 3e); it also coincided with atypically

high water clarity (data in ref. 17), which implies increased

irradiance at depth

patterns in both

rare or slight at both shallow (

between, however, both species displayed a curious pattern, with

shallower colonies bleached preferentially in shaded places

(Fig. 3a,b) and deeper colonies bleached preferentially in unshaded

places (Fig. 3c,d). Among

(communities 1 and 2 versus 3 and 4) and by depth (above 8m

versus below 8 m), this difference was significant (

64 bleached;

bleaching at the boundary between column top and side (Fig. 3a).

irradiance microhabitats, but they clearly showed the same reciprocal

pattern (Fig. 3b,d), with a shallower (

observations have previously been hard to explain

environment is isothermal, and the associations with irradiance and

colony morphology are inconsistent.

Symbiont zonation provides a simple hypothesis to explain these

bleaching patterns. Bleaching was disproportionately common in

what seems to be the upper limit of

zone’: low-irradiance parts of corals in shallower water, and highirradiance

parts of corals in deeper water. Slight increases in

temperature and irradiance might push these symbioses, but not

others, beyond some physiological limit, resulting in bleaching. This

hypothesis accounts for our bleaching observations, including areas

of slight bleaching

expelled selectively from mixed symbiont communities.

An analysis of symbionts collected in late October supported this

interpretation of events. Post-bleaching samples were obtained

available samples from communities that had previously contained

mixtures of

identified (Fig. 1) and analysed. We reasoned

these sites accurately defined the limit of

under non-bleaching conditions. Such mixtures also allow the fates

of different symbionts to be compared directly. We also tested

archived samples taken at the same time as, and

from, the original (pre-bleaching event) samples. In every case,

were equivalent (data not shown), indicating that the small distance

between pre- and post-bleaching samples was unlikely to be


As predicted,

in all 18 communities tested (see Fig. 4a–c). Absolute

responses of different symbionts within a mixed community were

compared by using estimates of relative abundances (from RFLP

data; see Fig. 4a–c) to partition direct counts of symbionts into each

genotype (Fig. 4d). Losses of

100%, whereas

than doubled in 3 of 5 instances. The single sample that contained

all three symbionts exhibited these same trends (Fig. 4c, d). Changes

in colony chlorophyll contents and subjective assessments of

bleaching paralleled changes in symbiont numbers (Fig. 4e).

From these data we can tentatively rank the ‘fitness’ of the different

ranking obtained in this manner is:

Our study provides a fuller understanding of


the study site on 28 October 1995 showing ‘shallow’ (

‘deep’ (

running means, from satellite data

Panama. Temperatures above 29

associated with coral bleaching

study site (at 7-m depth) since 1993 (Marine Environmental

Sciences Program, Smithsonian Tropical Research Institute)

corroborate satellite data.


and dynamic, multi-species communities of symbiotic dinoflagellates.

This strongly contradicts the ‘one host, one symbiont’ view of

reef corals

environmental variability is accommodated largely by

physiological acclimatization

not understood

underlie the broad distributions

these corals. Directed shifts in symbiont populations following

extreme environmental change (Figs 2e, f and 4) suggest that similar

shifts may also occur over annual cycles of environmental


ability to cope with environmental change through changes in

symbiont community composition reflects the selective advantage

of hosting several distinct symbionts, despite the potential for

destabilizing competition among them

How typical and important are the patterns documented here?




(Fig. 3a–d). With respect to Caribbean corals in general, bleaching

is often predominant at intermediate depths

pattern (and its within-colony correlate) in

at least three other species of Caribbean corals host (at least) both

species, which might host multiple but not so distantly related

symbionts, refinements of

essential. However, symbiont polymorphism does not exclude the

significance of other attributes that are important features of coral

biology, such as physiological acclimatization of hosts and symbionts


It has been hypothesized that a global warming trend, with

increased frequencies of world wide coral bleaching induced by

increasing temperature or ultraviolet irradiation, could have catastrophic

consequences for many living coral reefs

coral communities may adjust to climate change by recombining

their existing host and symbiont genetic diversities

supply a precedent for this idea: that one species of coral can

flexibly host more than one taxon of

symbioses with distinct ecological properties. For example,


ocean by hosting more

would depend on how they affect rates of coral growth and


Field collections and manipulations.

Aguadargana reef in San Blas, Panama

freezing immediately in liquid nitrogen (data in Figs 1 and 4). Other colonies

(data in Fig. 2) were sampled by removing a defined circular area (

of living tissue from freshly collected colonies with an airbrush. In transplant

experiments (Fig. 2e, f), columns of

below the living tissue, turned on their sides, and cemented (at the non-living

base) back to the colony at a comparable location; this increased (new top),

decreased (new side), or did not change (side) local irradiance. All 28

transplants at a depth of 6m seemed to be normal after 6 months. Analyses

of non-transplanted (control) columns showed that natural zonation patterns

were stable over this period (data not shown).


the ‘universal’ PCR primers ss5 and ss3 (all data in Fig. 1) or a combination of

ss5 and the ‘

analysed with

primer (ss3Z) does not discriminate (within this study) against unknown,



of 30 samples that contained two genotypes (

an episode of coral bleaching.

srRNA genes.

lane pairs compare symbionts before (left) and during (right) bleaching in






bleaching (left and right bars of each pair, respectively) in samples reported in

Samples were scored as ‘normal’ (not marked) or ‘slightly pale’, ‘pale’, or

‘bleached’ (marked by asterisks) when collected.

Hybridization and the EvolutionSteven V. Vollmer* and Stephen R. PalumbiHundreds of coral species coexist sympatrically on reefs, reproducing in massspawningAcropora corals show that mass spawningA. cervicornis and A. palmata areAcropora prolifera are entirely F1 hybrids of these two species,1 individuals can reproduce asexually and form long-lived, potentially immortalDiverse reef-building coral assemblages have1), thereby providing2). Laboratory2, 3). Interspecific4) that some species-rich genera3).Acropora, the world’s most speciose coral5), exemplify this view (24). Most ofAcropora arose over the6, 7), and many2, 8). One3),Department of Organismic and Evolutionary Biology,Fig. 1.The Caribbean Acropora species: (A) A. cervicornis and (B) A. palmata, and (C) the bushy andD) palmate F1 hybrid A. prolifera morphs from Puerto Rico.RE P O R T SCIENCE VOL 296 14 JUNE 20022023Downloaded on April 7, 2010hypothesis is lacking. Polyphyletic sequence811) withoutAcropora (Fig. 1).Acropora cervicornisand A. palmata are sister12, 13). Both have distinct morphologiesA. cervicornis occurs throughoutA. palmata occurs primarily14, 15). Both species spawn synchronously16) and canAcropora, occurs Caribbean-wide, where it7, 14, 15). It is morphologicallyA. cervicornis and A. palmata,7, 15). Pax-C intron data showing10). Morphological variation in A. prolifera isA.morphs—a thin, highly branchedAcroporaspecies at introns of the nuclear17). TheA. cervicornisandA. palmata are genetically distinctA. proliferais actually a first-generation1) hybrid. Acropora cervicornis and A. palmatawere reciprocally monophyletic at minicollagenA. prolifera (n 22) were heterozygous at minicollagen, containingA. cervicornisandA. palmata formed three distinct alleles: A,(Fig. 2B). Allele A was exclusive toA. cervicornis. B alleles were exclusive to A., but the variant Bwas shared betweenA. prolifera (n 28) were heterozygous26; B/B‘ ” 2). TheA. prolifera at these1 hybrid.A. cervicornis and hybrid A. prolifera.A. palmata, A. cervicornis, and hybridA. prolifera. All three haplotypes were foundA. prolifera, indicating that hybrid crossesA. palmata (B haplotype) or A. cervicornis(A haplotype) “mothers.”A. cervicorniscolonies possess all three haplotypeA. palmata colonies do not. The datapalmata” (B) haplotypes areA. cervicornis through backcrossingA. cervicornis with hybrid A. prolifera. IntrogressedA. cervicornis were(20%) and sampled at every site.A. cervicornisindicates the mtDNA introgression has18, 19), polyphyletic patterns inA. prolifera, i.e., the bushy and1 hybrids, they differ in which speciespalmatamaternal and mitochondrial background,cervicornis background. This suggestsFig. 2.Maximum likelihood (ML) trees for (A) minicollagen, (B) calmodulin, and (C) mitochondrial31) with estimated model32) inMODELTEST 3.06 (33). Major allele/haplotype clades are labeled. Tick marksn). Site abbreviations: Yucatan ( Y); Panama (Pa); Jamaica ( Ja); Puerto Rico (PR); St. Croix)50%) from 300 replicates are labeled on relevant nodes. The Pacific congenerAcropora nasutawas used as the outgroup. Sequences are available in GenBank (accession numbers654.81).592.86). (C)$*model (ln score 2014.96).A. prolifera hybrids are shown in blue; bushy hybrids are in red.RE P O R T S202414 JUNE 2002 VOL 296 SCIENCE www.sciencemag.orgDownloaded on April 7, 2010that maternal and/or cytoplasmic effects account20); however, a rarely explored alternative is21) to our data and the publishedPax-C data (10) to estimate the rate ofM) in units 2 + theNe) andm)] and test null hypotheses of noM 0) using likelihood ratio22). Results [Table 1 and supplemental23)] indicate that the mitochondrialM 0.20), roughly equivalent toNf (i.e., mtDNA effective populationPax-C data were also consistent with low levelsM 0.30), whereas the minicollagenallele at calmodulin is a retained24), and/or the25).A. proliferashows that complete barriers to hybridizationA. palmata and A.. However, the observation that A.hybrid populations are composed1 individuals suggests thatA. proliferais severely limited or that hybrid breakdownA. proliferaare reproductive, produceA.. Yet, the limited introgression1 hybrids are26). Where F1 hybrids dominate,27). Such F1 hybrids should be common1 offspring are long-lived. Like many corals28), hybrid A. prolifera can propagate29), allowing forAcropora morphotypesAcropora show that reefbuilding30), but has never been postulated5), suggests that morphologically3) without genetic isolation.References and Notes1. P. L. Harrisonet al., Science 223, 1186 (1984).Coral Reefs16, s53 (1997).Corals in Space and Time: The Biogeography(CornellAnnu. Rev. Ecol. Syst. 25,Corals of the World (Australian Institute!!!!,R. Kelley, Assoc. Australas. Palaeontol. Mem.6, 1 (1988).Staghorn Corals of the World: A Revision(Scleractinia; Astrocoeniina;) Worldwide, with Emphasis on(Commonwealthet al., Mol. Biol. Evol. 16, 1607 (1999).Mol. Biol. Evol. 14, 465Mol. Ecol. 9, 1363 (2000).Mol. Biol. Evol. 18, 1315 (2001).J. Paleontol.68, 951 (1994).Proc. 8th Int. Coral Reef1, 423 (1997).Ecology 40, 67 (1959).Stony Corals (Cnidaria: Anthozoa:) of Carrie Bow Cay, Belize (SmithsonianBull. Mar. Sci.64, 189 (1999).A. cervicornis,, and A. prolifera, respectively) were3, 3, 3; Panama 7, 5, 0;3, 4, 0; Puerto Rico 41, 22, 19 (10 bushy,0, 12, 6. DNA extractions used,g), and standard phenol-chloroform-GAGGTTGATGCTGATGGTGAG-3)-CAGGGAAGTCTATTGTGCC-3). The$ bp) plus-GCTTAGACAGGTTGGTTGATTGCCC-) and CO3r (5-CTCCCAAATACATAATTTGAACTAA-), and two internal sequencing primers, CRseqf (5) and CRseqr-ATAACCCAACAAAGTCTAATTCCC-3). AmplificationsMol. Biol. Evol. 5, 568 (1988).Evolution 55, 859Molecular Markers, Natural History and(Chapman & Hall, New York, 1994).Genetics 158, 885 (2001).Acropora cervicornis and A. palmata were treated asM in units of 2Nem) was used to estimate the rate ofM versusM0 to test the null hypotheses of ancestralP values were divided by 2 after (21)23)]. The model searched forM and T within the bounds of 0 toScience OnlineTheor. Popul. Biol. 38, 331Evolution 47, 1814 (1990).Natural Hybridization and Evolution(Oxford Univ. Press, Oxford, 1997).Am. J. Bot.79, 101 (1992).Mar. Ecol. Prog. Ser. 7, 207 (1982).Proc. 8th Int. Coral Reef Symp. 2,Population Biology and EvolutionK. Wohrmann, V. Loeschecke, Eds. (Springer-Verlag,PAUP*: Phylogenetic Analysis Using(and Other Methods), version 4.0. (SinauerJ. Mol. Evol. 36 (1993).Bioinformatics 14, 817Table 1.Estimated genetic introgression. ResultsM inNem units) and the results of the likelihood ratioP 0.05; **P 0.01.Nem LRT PMinicollagen 0.00 0.00 1.000 (NS)Pax-C 0.30 6.02 0.007**RE P O R T SCIENCE VOL 296 14 JUNE 20022025

of Reef Coral Diversity

events where hybridization appears common. In the Caribbean, DNA

sequence data from all three sympatric

does not erode species barriers. Species

distinct at two nuclear loci or share ancestral alleles. Morphotypes historically

given the name

showing morphologies that depend on which species provides the egg for

hybridization. Although selection limits the evolutionary potential of hybrids,


hybrids with unique morphologies.

served as the foundation for complex reef

ecosystems with exceptional biodiversity and

productivity. Yet, the evolutionary genesis of

coral diversity remains mired in a paradox.

As many as 105 coral species from 36 genera

and 11 families reproduce in yearly, synchronous

mass-spawning events (

overwhelming opportunities for

hybridization among congenerics (

crosses from a number of mass-spawning

genera demonstrate that viable hybrids

occur among congenerics (

hybridization should blur coral species

boundaries and stifle species diversification,

yet many mass-spawning coral groups have

rapidly diversified. The juxtaposition of high

hybridization potential and high species diversity

in mass-spawning corals has confused

the picture of coral evolution and cast such

doubt on the cohesiveness of coral species

boundaries (

have been considered hybrid swarms (

group (

the 115 species of

past 5 million years (My) (

are capable of hybridizing with sympatric

congenerics in laboratory crosses (

prominent hypothesis proposes that interspecific

hybridization promotes reticulate evolution

and morphological diversification in the

absence of genetically distinct species (

even though a genetic mechanism for this

Harvard University, 16 Divinity Avenue, Cambridge,

MA 02138, USA.

*To whom correspondence should be addressed. Email:


data for corals continue to be taken as direct

evidence of reticulate evolution (

due consideration to alternatives such as

incomplete lineage sorting.

To examine the potential role of hybridization

in coral speciation, we analyzed DNA sequence

variation at three loci in the three sympatric

species of Caribbean

species with fossil records dating back at least 3

to 3.6 My (

and habitat preferences. The arborescent

“staghorn” coral

forereef and backreef habitats, whereas the

robust “elkhorn” coral

in high–wave energy reef-crest habitats


over a few nights each summer (

potentially hybridize. The third species,


varies from being locally rare to occurring in

large patches (

intermediate between

causing many to consider it a species of

hybrid origin (

high heterozygosity support this possibility


high and yet surprisingly discrete. In Puerto

Rico, for example, there are two discrete


form we term the “bushy” morph (Fig. 1C), and

a thicker formwith palmate, flattened branches

we call the “palmate” morph (Fig. 1D).

We obtained sequence data for the Caribbean

minicollagen and calmodulin genes, and at the

mitochondrial putative control region (

nuclear data indicate that the species

and that the morphologically intermediate species


(Fig. 2A). All of the

one allele from each of the two species’

clades. The calmodulin data for

B, and B


species, making it either a shared ancestral allele

or an introgressed allele from recent or

historical hybridization. As with minicollagen,

all of the

at calmodulin (A/B

complete heterozygosity of

two nuclear loci strongly suggests that every

individual sampled was a F

Mitochondrial data show that the 45

unique haplotypes form a polytomy with

three clades (Fig. 2C), labeled as haplotypes

A, B, and C. The A and C haplotypes contained


The B haplotypes contained all three



occur in both directions. Hybrids receive maternally

inherited mitochondrial DNAs from


Although hybrid crosses occur in either direction,

mitochondrial DNA (mtDNA) introgression

appears unidirectional because

clades, but

indicate that “

passed to


B haplotypes in

common (

The presence of multiple B variants in

occurred more than once. Because nuclear loci

should sort more slowly than maternally inherited

mtDNAs (

the mitochondrial data but not the minicollagen

data are consistent with recent introgression

rather than incomplete lineage sorting.

In Puerto Rico, we sampled two distinct

morphs of

palmate morphs (Fig. 1, C and D). Although

all individuals, irrespective of morphology,

are F

donated its egg and mitochondrion to the

hybridization event. All bushy hybrids had a

whereas all of the palmate hybrids

had a

putative control region. Likelihood searches were conducted in PAUP* 4.0b8 (

parameters and 25 random-addition heuristic searches with tree-bisection-reconnection branch swapping.

Models of sequence evolution were evaluated on distance-based topologies with hierarchical

likelihood ratio tests (

along major branches indicate substitutions. Sample sizes (alleles or haplotypes) are labeled in

parentheses (

(SC). Bootstrap values (

AF507116 to AF507373). (A) Minicollagen ML tree constructed with a K80 model (ln score

(B) Calmodulin ML tree constructed with a HKY model (1 of 4 trees; ln score

Mitochondrial putative control region ML tree constructed with a F81


for the marked differences in these two

hybrid morphotypes. Thus, coral morphology

appears sensitive to not only nuclear genetic

effects, but also to nuclear-cytoplasmic interactions

within a hybrid nuclear genome.

Differential introgression of loci characterizes

many terrestrial hybridization systems


that the pattern is due to ancestral polymorphism.

We applied a two-population Bayesian

coalescent model (

introgression [as migration (

product of effective population size (

migration (

introgression (

tests (LRTs) (

material (

data are consistent with low levels of

introgression (

one haplotype crossing the species boundary

every 5

size) generations. For the nuclear loci, the

of introgression (

and calmodulin data were both consistent

with no introgression, suggesting that the

shared B

ancestral allele. Such differential cytoplasmic

and nuclear introgression is consistent with selection

against hybrid genotypes that is thought

to result from selection against nuclear genes in

foreign genetic backgrounds (

breakup of coadapted gene complexes in backcrossed

individuals (

The existence of hybrid

have not evolved between



almost entirely of F

the reproductive potential of hybrid

occurs in later generations. Some hybrid

viable gametes, and are interfertile with


suggests that they are essentially sterile

“mules,” which have little genetic impact on

either parent species. Strict F

often ecologically rare in natural hybridization

systems (

selection manifest as hybrid infertility

or hybrid breakdown has been inferred, as

here (

only when hybridization is frequent or



clonally by fragmentation (

long-lived, potentially immortal hybrid genotypes.

These “immortal mules” may accumulate

over time, providing the opportunity for

rare backcrosses, and for the ecological persistence

of a diverse suite of

that is greater than the number of

species on reefs.

The Caribbean

corals diversify not only through conventional

species formation, but also through

the unprecedented formation of long-lived coral

hybrid morphotypes. In effect, hybridization,

through the formation of asexual coral hybrid

lines, generates new morphologies and potentially

new ecotypes without speciation. Similar

clonal niche partitioning is known for rare parthenogenetic

taxa (

for an ecosystem-defining group like

reef-building corals. Although it remains to be

seen how pervasive coral hybrid “mules” are,

the variety of intermediate morphologies in corals,

especially in regional endemics and putative

subspecies (

unique hybrids may be common. Because

of the potential for natural hybridization in

mass-spawning corals, the coral reticulate evolution

hypothesis suggested that genetic exchange

between “species” generates discrete

coral morphologies (

Instead, we suggest that reef-building coral

diversity is enhanced by hybridization through

the production of long-lived asexual hybrid

morphotypes, which have little evolutionary


2. B. L. Willis, R. C. Babcock, P. L. Harrison, C. C. Wallace,

3. J. E. N. Veron,

and Evolution of the Scleractinia

Univ. Press, Ithaca, NY, 1995).

4. C. C. Wallace, B. L. Willis,

237 (1994).

5. J. E. N. Veron,

of Marine Science, Townsville, Australia, 2000).


7. C. C. Wallace,

of the Coral Genus Acropora


Morphology, Phylogeny and Biogeography

Scientific and Industrial Research Organisation,

Melbourne, Australia, 2000).

8. M. Hatta

9. D. M. Odorico, D. J. Miller,


10. M. J. H. van Oppen, B. L. Willis, H. W. J. A. van Vugt,

D. J. Miller,

11. M. J. H. van Oppen, B. J. McDonald, B. Willis, D. J.


12. A. F. Budd, T. A. Stemann, K. G. Johnson,

13. A. F. Budd, K. G. Johnson,


14. T. F. Goreau,

15. S. D. Cairns, Ed.,


Institution Press, Washington, DC, 1982).

16. M. de Graaf, G. J. Geertjes, J. J. Videler,

17. A total of 131 individuals were sampled across five sites

in the Caribbean; samples per site and species (

A. palmata

as follows: Yucatan


9 palmate); St. Croix

a CTAB (hexadecyltrimethylammonium bromide) buffer,

proteinase K (100

extraction methods. Amplifications were obtained

with GeneAmp XL PCR kits under normal polymerase

chain reaction (PCR) conditions, 30 to 35 cycles, and

annealing temperatures of 51° to 54°C. A 374–base

pair (bp) fragment of minicollagen, including the second

intron, was amplified with published primers. A calmodulin

intron (343 bp) was amplified with coral-specific

primers CalMf (5

and CalMr2 (5

mitochondrial putative control region (933

83 bp of cytochrome oxidase III was amplified with

primers CRf (5





were sequenced directly; heterozygous nuclear

alleles were observed as double peaks confirmed in

samples sequenced in both directions.

18. P. Pamilo, M. Nei,

19. S. R. Palumbi, F. Cipriano, M. Hare,


20. J. C. Avise,


21. R. Nielson, J. Wakeley,


separate populations. Models were run independently

for each gene. The mode of the integrated posterior

probability distribution for the migration parameter


introgression. LRTs compared probabilities of


Multiple simulations confirm model convergence

[supplemental material (

the best values of

10 for both parameters.

23. Supplementary material is available on



24. N. Takahata, M. Slatkin,


25. R. S. Burton,

26. M. L. Arnold,

27. J. D. Nason, N. C. Ellstrand, M. L. Arnold,

28. R. C. Highsmith,

29. A. Bowden-Kerby,

2063 (1997).

30. R. C. Vrijenhoek, in

Berlin, 1984), pp. 175–197.

31. D. L. Swofford,


Associates, Sunderland, MA, 1996).

32. N. Goldman,

33. D. Posada, K. A. Crandall,


34. Thanks to those who assisted with field collections;

to R. Nielson and J. Wakeley for assistance with the

coalescent modeling; and to P. Barber, S. Belliveau, E.

Weil, and four anonymous reviewers for valuable

comments. Photographs were provided by H. Ruiz.

Supported by National Science Foundation grants

(S.R.P.) and a NIH Genetics Training Grant Fellowship


3 January 2002; accepted 3 May 2002

of the Bayesian coalescent modeling for each gene

showing the estimated rates of introgression (


tests (LRTs). NS, not significant;*

Gene 2

Calmodulin 0.08 2.17 0.071 (NS)

MtDNA control region 0.20 4.31 0.019*

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