Barcoding Corals: Limited by Interspecific Divergence, Not Intraspecific Variation

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Molecular Ecology Resources (2008) 8, 247–255 doi: 10.1111/j.1471-8286.2007.01996.x

DNABlackwell Publishing Ltd BARCODING Barcoding corals: limited by interspecific divergence, not intraspecific variation

T. L. SHEARER* and M. A. COFFROTH† *School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332-0230, USA, †Department of Geology, State University of New York at Buffalo, 109 Cooke Hall, Buffalo, NY 14260, USA

Abstract The expanding use of DNA barcoding as a tool to identify species and assess biodiversity has recently attracted much attention. An attractive aspect of a barcoding method to identify scleractinian species is that it can be utilized on any life stage (larva, juvenile or adult) and is not influenced by phenotypic plasticity unlike morphological methods of species identification. It has been unclear whether the standard DNA barcoding system, based on cytochrome c oxidase subunit 1 (COI), is suitable for species identification of scleractinian corals. Levels of intra- and interspecific genetic variation of the scleractinian COI gene were investigated to determine whether threshold values could be implemented to discriminate conspecifics from other taxa. Overlap between intraspecific variation and interspecific divergence due to low genetic divergence among species (0% in many cases), rather than high levels of intraspecific variation, resulted in the inability to establish appropriate threshold values specific for scleractinians; thus, it was impossible to discern most scleractinian species using this gene. Keywords: COI, DNA barcoding, interspecific divergence, intraspecific variation, scleractinian coral, species identification Received 24 January 2007; revision accepted 15 August 2007

DNA barcoding has become an attractive, yet controversial et al. 2003a, b; 2004a; Hajibabaei et al. 2006; Smith et al. 2006; means to identify species over a wide taxonomic range. Witt et al. 2006) and invasive species (e.g. Siddall & Budinoff Such methods utilize a short DNA sequence, rather than 2005), and to assess biodiversity (Janzen et al. 2005; Smith multiple traditional taxonomic characters to characterize et al. 2005). This barcoding system relies on the observation species. Hence, the attraction — rapid and inexpensive gen- that COI sequence divergence between most congeneric eration of sequences with little expertise required, and the species is generally greater than 2% (Hebert et al. 2003b), controversy — species identification determined by only a whereas intraspecific variation is often lower than 1% single complex character rather than multiple taxonomic (Avise 2000). This gap between inter- and intraspecific characters which require expert knowledge of the species variation, or the ‘barcoding gap’ is presumed to be suffi- in question (e.g. Seberg et al. 2003; Will & Rubinoff 2004; ciently wide such that threshold values can be implemented Dasmahapatra & Mallet 2006). to distinguish conspecifics from other taxa (Meyer & Paulay The DNA barcoding system proposed by Hebert et al. 2005). Although this holds true for some taxa (e.g. birds, (2003a, b), based on ~650 bp sequence of the mitochondrial Hebert et al. 2004b; spiders, Barrett & Hebert 2005; butterflies, cytochrome c oxidase subunit 1 (COI) gene, can readily Hajibabaei et al. 2006; fish, Ward et al. 2005), one criticism of discriminate among closely related species across most the barcoding system has been that intraspecific variation animal phyla. In several cases, this system has been utilized may overlap interspecific divergence, resulting in unreliable to distinguish known species (Hebert et al. 2003a, b; Ward taxonomic designations (Meyer & Paulay 2005). Substantial et al. 2005), to identify new or cryptic species (e.g. Hebert overlap has been observed in some taxonomic groups as a result of high levels of intraspecific COI sequence diver- Correspondence: TL Shearer, Fax: (404) 385 4440; E-mail: gence (e.g. cowries, Meyer & Paulay 2005; amphibia, Vences [email protected] et al. 2005). Overlap of inter- and intraspecific variation is

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 248 DNA BARCODING less frequently due to low levels of interspecific sequence and 0.2 mm of each primer. The forward primer was a divergence. This variability in the rate of evolution among shortened version of the Folmer et al. (1994) universal taxa indicates that no standard level of sequence diver- primer, LCOI1490: 5′-CAAATCATAAAGATATTGG-3′. The gence can be implemented to discern species (Will & reverse primer was the Folmer et al. (1994) universal primer, Rubinoff 2004) and the utility of this barcoding system HCOI2198: 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′. should be evaluated on a taxon-by-taxon basis. These primers amplified 705 base pairs (bp) of the COI gene A genetic identification system would be particularly in the coral mitochondrial genome. Thermal cycling protocol useful for organisms such as scleractinian corals, whose conditions consisted of a denaturing step of 2 min at 95 °C identification may be uncertain due to phenotypic plastic- followed by 40 cycles of 95 °C for 30 s, 47 °C for 90 s and ity, morphologically cryptic species or lack of identification 72 °C for 90 s. The amplified products were purified through tools at various life stages (Hebert et al. 2003a). This system a 2.0% agarose gel in 1× TAE buffer. Reactions for cycle would be an extremely useful tool to characterize morpho- sequencing were performed using the SequiTherm EXCEL logical variants of a species, identify cryptic or endemic II DNA Sequencing Kit-LC (Epicentre Technologies) accord- species and rapidly assess and compare biodiversity of coral ing to modifications specified by LI-COR Biotechnology reefs. A genetic method to identify newly settled Caribbean Division. COI sequences were read in both directions on a scleractinian recruits has recently been described, in part, LI-COR Gene ReadIR 4200 automatic DNA sequencer. COI to overcome the difficulty in morphological identification sequences were submitted to GenBank (Table 1, Reference 1). of this early life stage (Shearer & Coffroth 2006). In that In addition to the sequences obtained in this study, study, the COI gene was useful in resolving species for 97 COI sequences from Caribbean, western Atlantic and Indo- some coral genera with high recruitment rates on artificial Pacific scleractinians were downloaded from GenBank substrate (agaricids and poritids). This was possible due to (Table 1). All sequences were aligned manually using seqapp the relatively small number of Caribbean scleractinian spe- 1.9a (Gilbert 1992) and trimmed to the minimum number of cies and unusually low levels of intraspecifc mitochondrial corresponding nucleotides. Pairwise Kimura’s 2-parameter sequence variation observed within corals often resulting (K2P) genetic distances among all species, within families, in a species-specific genetic signal (Shearer et al. 2002). Low within genera and within species were calculated using levels of COI sequence differentiation within coral species paup* (version 4.0b10 Swofford 2002). K2P is the best DNA should improve the capacity of this gene to distinguish substitution model for low genetic distances (Nei & Kumar species by minimizing the ‘noise’ created by overlapping 2000). To determine levels of intraspecific nucleotide vari- intra- and interspecific genetic distances. Limited taxonomic ation, sequences from multiple colonies of 27 species were comparisons within the phylum Cnidaria, within which analysed (Table 2). scleractinians are classified, indicate that nucleotide poly- morphism in COI is insufficient to discern some closely Results related species (Shearer et al. 2002; Hebert et al. 2003b); however, several species can be discerned using this gene COI sequences generated from this study were obtained from (Shearer & Coffroth 2006). Levels of interspecific diver- 49 samples, comprising 30 Caribbean species representing gence must be more thoroughly examined to assess the 17 genera in 11 families (Table 1, Reference 1). The 705 bp potential for DNA barcoding in corals. This study evaluates COI sequences were aligned with scleractinian sequences whether intra- and interspecific variation in COI of Carib- downloaded from GenBank (Table 1) and trimmed to a bean, western Atlantic and Indo-Pacific scleractinian corals 615 bp sequence containing no indels. In total (newly gener- is amenable to establishing threshold values critical to ated sequences plus those downloaded from GenBank), distinguish species using this barcoding gene. 40 Caribbean, four western Atlantic and 46 Indo-Pacific scleractinian coral species, representing 44 genera in 14 families (Table 1) were analysed. To ensure COI sequences Materials and methods were cnidarian in origin and not from symbiotic dino- Tissue from 30 Caribbean coral species (Table 1, Reference 1) flagellates associated with corals, homology of nucleotide was collected and preserved in high salt solution (250 mm sequences with azooxanthellate cnidarian COI (Metridium EDTA, 20% DMSO, saturated NaCl, pH 7.5), 95% ethanol senile GenBank Accession no. U36783) was confirmed (data or frozen in liquid nitrogen. DNA was isolated following not shown). the protocol described in Shearer et al. (2005) or Coffroth Intraspecific nucleotide variation was low (mean 0.048%, et al. (1992) and frozen at –20 °C until amplified. SE ± 0.017) even at third codon positions, with sequence Amplifications were performed in 10 μL volume solutions differences observed in 10 of the 27 congeners analysed with 10–50 ng genomic DNA, 1 U Taq DNA polymerase (Table 2). Variation was attributed to substitution at one and a final concentration of 0.2 mm of each dNTP, 10 mm out of 615 bp (0.163%) in most cases, which is equivalent

Tris-HCl (pH 8.3), 50 mm KCl, 0.001% gelatin, 2.5 mm MgCl2, to rates of sequencing error (e.g. Clark & Whittam 1992;

Table 1 Taxonomic classification, Gen- Taxon Location Accession no. Reference Bank Accession numbers and references for Caribbean, western Atlantic and Indo- Order Scleractinia Pacific scleractinian coral species included Suborder Astrocoeniina in this study. References: 1, this study; Family Acroporidae 2, van Oppen et al. (2002); 3, Fukami et al. Acropora cervicornis Caribbean AY451340 1 (2004); 4, Snell et al. (1998); 5, Medina et al. Acropora palmata Caribbean AY451341–42 1 (1999) Acropora tenuis Indo-Pacific AF338425 2 Family Astrocoeniidae Stephanocoenia michellini Caribbean AY451343 1 Family Pocilloporidae Madracis mirabilis Caribbean AY451344 1 Suborder Caryophyliina Family Caryophyliidae Eusmilia fastigiata Caribbean AY451345 1 AB117294 3 Suborder Faviina Family Faviidae Caulastraea furcata Indo-Pacific AB117274 3 Cladocora arbuscula Caribbean AB117292 3 Colpophyllia natans Caribbean AY451346–47 1 AB117228 3 Cyphastrea chalcidicum Indo-Pacific AB117259 3 Cyphastrea serailia Indo-Pacific AB117257–58 3 Diploastrea heliopora Indo-Pacific AB117290 3 Diploria clivosa Caribbean AB117226 3 Diploria labyrinthiformis Caribbean AY451348 1 AB117224 3 Diploria strigosa Caribbean AY451349 1 AF108716–17 4 AB117225 3 Echinopora gemmacea Indo-Pacific AB117263 3 Echinopora pacificus Indo-Pacific AB117261–62 3 Favia favus Indo-Pacific AB117267 3 Favia fragum Caribbean AY451350–51 1 AB117222–23 3 Favia leptophylla Caribbean AB117229–30 3 Favia pallida Indo-Pacific AB117265–66 3 Favia stelligera Indo-Pacific AB117264 3 Favites chinensis Indo-Pacific AB117269 3 Favites halicora Indo-Pacific AB117268 3 Goniastrea aspera Indo-Pacific AB117271 3 Goniastrea pectinata Indo-Pacific AB117270 3 Leptoria irregularis Indo-Pacific AB117272 3 Leptoria phrygia Indo-Pacific AB117273 3 Manicina areolata Caribbean AB117227 3 Montastraea annularis Caribbean AY451352 1 AF013737 5 AB117260 3 Montastraea cavernosa Caribbean AY451353–56 1 AF108710–15 4 AF051094 4 AF013736 5 AB117288–89 3 Montastraea curta Indo-Pacific AB117278 3 Montastraea faveolata Caribbean AY451357 1 AF013738 5 Montastraea franksi Caribbean AY451358 1 Montastraea magnistellata Indo-Pacific AB117279 3 Montastraea valenciennesi Indo-Pacific AB117280 3 Oulophyllia bennettae Indo-Pacific AB117277 3 Oulophyllia crispa Indo-Pacific AB117275–76 3 Platygyra daedalea Indo-Pacific AB117281 3 Platygyra lamellina Indo-Pacific AB117282 3 Solenastrea bournoni Caribbean AY451359 1 AB117291 3

Table 1 Continued Taxon Location Accession no. Reference

Family Meandrinidae Dendogyra cylindrus Caribbean AB117299 3 Dichocoenia stokesi Caribbean AY451360 1 AB117298 3 Meandrina braziliensis Western Atlantic AB117297 3 Meandrina meandrites Caribbean AY451361–62 1 AB117295–96 3 Family Merulinidae Hydnophora exesa Indo-Pacific AB117285 3 Hydnophora grandis Indo-Pacific AB117286 3 Merulina ampliata Indo-Pacific AB117283 3 Merulina scabricula Indo-Pacific AB117284 3 Family Mussidae Acanthastrea echinata Indo-Pacific AB117249–50 3 Acanthastrea rotundata Indo-Pacific AB117251 3 Cynarina lacrymalis Indo-Pacific AB117246 3 Isophyllia sinuosa Caribbean AB117238 3 Lobophyllia corymbosa Indo-Pacific AB117241 3 Lobophyllia hemprichii Indo-Pacific AB117240 3 Lobophyllia pachysepta Indo-Pacific AB117242 3 Mussa angulosa Caribbean AB117239 3 Mussismilia braziliensis Western Atlantic AB117231 3 Mussismilia harttii Western Atlantic AB117232 3 Mussismilia hispida Western Atlantic AB117233 3 Mycetophyllia aliciae Caribbean AY451364 1 AB117235 3 Mycetophyllia danaana Caribbean AB117234 3 Scolymia cubensis Caribbean AB117236–37 3 Scolymia vitiensis Indo-Pacific AB117247 3 Scolymia sp. Indo-Pacific AB117248 3 Symphyllia agaricia Indo-Pacific AB117243 3 Symphyllia radians Indo-Pacific AB117245 3 Symphyllia recta Indo-Pacific AB117244 3 Family Oculinidae Oculina diffusa Caribbean AB117293 3 Oculina sp. Caribbean AY451365 1 Family Pectiniidae Echinophyllia aspera Indo-Pacific AB117252 3 Echinophyllia echinoporoides Indo-Pacific AB117254 3 Echinophyllia orpheensis Indo-Pacific AB117253 3 Mycedium elephantotus Indo-Pacific AB117387–88 3 Oxypora lacera Indo-Pacific AB117255–56 3 Pectinia alcicornis Indo-Pacific AB117385 3 Pectinia paeonia Indo-Pacific AB117386 3 Family Trachyphyllidae Trachyphyllia geoffroyi Indo-Pacific AB117287 3 Suborder Fungiina Family Agariciidae Agaricia agaricites Caribbean AY451366–67 1 AF112120–21 4 Agaricia fragilis Caribbean AY451368 1 Agaricia lamarcki Caribbean AY451369 1 Agaricia tenuifolia Caribbean AY451370–72 1 Leptoseris sp. Caribbean AY451373 1 Family Poritidae Porites astreoides Caribbean AY451374–79 1 Porites branneri Caribbean AY451380 1 Porites divaricata Caribbean AY451381 1 Porites furcata Caribbean AY451382 1 Porites porites Caribbean AY451383–84 1 Family Siderastreidae Siderastrea radians Caribbean AY451385 1 Siderastrea siderea Caribbean AY451386–87 1

Table 2 Percentage and range of intraspecific sequence variation (Kimura’s 2-parameter genetic distance), including total number of base-pair (bp) differences for 27 scleractinian coral species based on 615 bp COI sequence. Mean % genetic distance is reported for species with multiple comparisons. NA, range not quantified due to single comparison. GenBank Accession numbers are given in Table 1

Sample % genetic Species size distance (bp) Range

Acanthastrea echinata 20 NA Acropora palmata 20 NA Agaricia agaricites 40 0 Agaricia tenuifolia 30 0 Colpophyllia natans 30 0 Cyphastrea serailia 2 0.163 (1) NA Dichocoenia stokesi 2 0.163 (1) NA Diploria labyrinthiformis 2 0.163 (1) NA Fig. 1 Histogram of intra- and interspecific (within and among Diploria strigosa 40 0 families) genetic divergence (Kimura 2-parameter model; K2P) Echinopora pacificus 2 0.326 (2) NA among 90 scleractinian species (pairwise comparisons; N = 4005). Eusmilia fastigiata 20 NA Due to the relatively small number of congeneric comparisons Favia fragum 40 0 (N = 83), these comparisons were included with the confamilial Favia leptophylla 2 0.163 (1) NA sequences (also see Table 4). Favia pallida 2 0.163 (1) NA Meandrina meandrites 4 1.07 (0–9) 0–1.98 Montastraea annularis 30 0 Montastraea cavernosa 14 0.023 (0–1) 0–0.163 Discussion Montastraea faveolata 20 NA Mycedium elephantotus 2 0.163 (1) NA Occasionally in metazoans, high rates of nucleotide sub- Mycetophyllia aliciae 20 NA stitution may lead to an overlap of intra- and interspecific Oulophyllia crispa 20 NA variation which results in unreliable taxonomic identification Oxypora lacera 2 1.150 (7) NA when attempting to identify species based on the COI Porites astreoides 60 0 barcoding system described by Hebert et al. (2003a, b) (e.g. Porites porites 40 0 Meyer & Paulay 2005; Vences et al. 2005). In scleractinian Scolymia cubensis 20 NA corals, low levels of intraspecific COI sequence variation, Siderastrea siderea 20 NA Solenastrea bournoni 20 NA even at third codon positions, should enhance the utility of this barcoding method. However, this barcoding system has limited use in identification of scleractinian corals due to very low levels of interspecific divergence among seem- ingly distantly related taxa (i.e. among different genera and Ewing & Green 1998; Ewing et al. 1998). Although in most families). This pattern of slow evolution in the COI gene is cases sample sizes were low, low intraspecific divergence a characteristic of anthozoan mitochondrial DNA in general is a common pattern in anthozoan mitochondrial genes (e.g. France et al. 1996; van Oppen et al. 1999; Shearer et al. (reviewed in Shearer et al. 2002; Hellberg 2006). Larger intra- 2002; Tseng et al. 2005; Hellberg 2006). Threshold values specific differences (> 1% as in Meandrina meandrites and typically implemented to discern species (2–3%: Hebert Oxypora lacera) suggest population differentiation, specimen et al. 2003b) could not be utilized with scleractinians due to misidentification or presence of cryptic species. low interspecific divergence among many species. In fact, Mean sequence divergence between confamilial species genetic distances between 84.3% of congeners were < 2% (mean 1.90%, SE ± 0.045; calculated using only unique haplo- and nearly 50% of all 4005 pairwise species comparisons types for each species, i.e. eliminating the intraspecific com- resulted in < 3% sequence divergence (Table 5). Implementing ponent) was significantly greater than the mean intraspecific the standard sequence threshold proposed by Hebert et al. distance (t-test, t = 38.93, P < 0.0001); however, the range of (2004b), 10 times the mean intraspecific variation for the confamilial divergences (0–5.799%, including congeneric group under study (0.048% mean intraspecific variation; comparisons) overlapped intraspecific differences (0–1.98%; 0.48% threshold), was also not appropriate since almost 40% Fig. 1). Of 21 COI haplotypes shared by multiple species, of scleractinian congeners in this analysis exhibited genetic 10 were shared by different genera and three were shared distances of 0%. An appropriate threshold value specific by different families (Table 3). for scleractinians could not be defined due to the overlap

Table 3 COI nucleotide sequence haplo- Nucleotide haplotype Species Scleractinian family types shared among scleractinian species. GenBank Accession numbers are given in Caribbean Acropora spp. Acropora cervicornis Acroporidae Table 1. Where multiple haplotypes exist Acropora palmata Acroporidae among conspecifics, the Accession number Agaricia spp. 1 Agaricia agaricites Agariciidae of the shared sequence is given in paren- Agaricia tenuifolia Agariciidae theses. Single-species haplotypes (N = 39) Agaricia spp. 2 Agaricia fragilis Agariciidae are not included in table Agaricia lamarcki Agariciidae Eusmilia sp./ Eusmilia fastigiata Caryophyliidae Dichocoenia sp. Dichocoenia stokesi (AY451360) Meandrinidae Caulastraea sp./ Caulastraea furcata Faviidae Oulophyllia spp. Oulophyllia bennettae Faviidae Oulphyllia crispa Faviidae Cyphastrea spp. Cyphastrea chalcidicum Faviidae Cyphastrea serailia (AB117257) Faviidae Diploria spp. Diploria clivosa Faviidae Diploria strigosa Faviidae Caribbean Faviidae spp./ Diploria labyrinthiformis (AY451348) Faviidae Mussismilia spp. Favia fragum Faviidae Favia leptophylla (AB117230) Faviidae Manicina areolata Faviidae Mussismilia braziliensis Mussidae Mussismilia harttii Mussidae Mussismilia hispida Mussidae Echinopora spp. Echinopora gemmacea Faviidae Echinopora pacificus (AB117262) Faviidae Echinopora sp./ Echinopora pacificus (AB117261) Faviidae Leptoria sp. Leptoria irregularis Faviidae Favites spp./ Favites chinensis Faviidae Montastraea spp. Favites halicora Faviidae Montastraea magnistellata Faviidae Montastraea valenciennesi Faviidae Goniastrea sp./ Goniastrea pectinata Faviidae Merulina spp. Merulina ampliata Merulinidae Merulina scabricula Merulinidae Montastraea annularis Montastraea annularis Faviidae species complex Montastraea faveolata Faviidae Montastraea franksi Faviidae Platygyra spp. Platygyra daedalea Faviidae Platygyra lamellina Faviidae Meandrina spp. Meandrina braziliensis Meandrinidae Meandrina meandrites (AB117295) Meandrinidae Hydrophora spp. Hydnophora exesa Merulinidae Hydnophora grandis Merulinidae Lobophyllia spp./ Lobophyllia hemprichii Mussidae Symphyllia spp. Lobophyllia pachysepta Mussidae Symphyllia agaricia Mussidae Symphyllia radians Mussidae Symphyllia recta Mussidae Caribbean Mussidae spp. Mycetophyllia aliciae Mussidae Mycetophyllia danaana Mussidae Scolymia cubensis Mussidae Echinophyllia spp./ Echinophyllia aspera Pectiniidae Oxypora sp. Echinophyllia echinoporoides Pectiniidae Echinophyllia orpheensis Pectiniidae Oxypora lacera (AB117256) Pectiniidae Mycedium sp./ Mycedium elephantotus (AB117388) Pectiniidae Pectinia sp. Pectinia paeonia Pectiniidae Caribbean Porites spp. Porites branneri Poritiidae Porites divaricata Poritiidae Porites furcata Poritiidae Porites porites Poritiidae

Table 4 COI amino acid sequence haplo- Amino acid haplotype Species Family types shared among scleractinian species. GenBank Accession numbers are given in Caribbean Acroporidae Acropora cervicornis Acroporidae Table 1. Where multiple haplotypes exist Acropora palmata Acroporidae among conspecifics, the Accession number Caribbean Caryophyliidae/ Eusmilia fastigiata Caryophyliidae of the shared sequence is given in paren- Caribbean Meandrinidae Dendogyra cylindrus Meandrinidae theses. Single-species haplotypes (N =13) Dichocoenia stokesi Meandrinidae are not included in table Meandrina meandrites Meandrinidae (AB117296) Indo-Pacific Faviidae/ Caulastraea furcata Faviidae Merulinidae/Mussidae/ Echinopora gemmacea Faviidae Pectiniidae/Trachyphyllidae Echinopora pacificus Faviidae Favia favus Faviidae Favia pallida Faviidae Favia stelligera Faviidae Favites chinensis Faviidae Favites halicora Faviidae Goniastrea pectinata Faviidae Leptoria irregularis Faviidae Leptoria phrygia Faviidae Montastraea curta Faviidae Montastraea magnistellata Faviidae Montastraea valenciennesi Faviidae Oulophyllia bennettae Faviidae Oulophyllia crispa Faviidae Platygyra daedalea Faviidae Platygyra lamellina Faviidae Hydnophora exesa Merulinidae Hydnophora grandis Merulinidae Merulina ampliata Merulinidae Merulina scabricula Merulinidae Acanthastrea echinata Mussidae Cynarina lacrymalis Mussidae Lobophyllia hemprichii Mussidae Lobophyllia pachysepta Mussidae Scolymia sp. Mussidae Scolymia vitiensis Mussidae Symphyllia agaricia Mussidae Symphyllia radians Mussidae Symphyllia recta Mussidae Mycedium elephantotus Pectiniidae Oxypora lacera (AB117255) Pectiniidae Pectinia alcicornis Pectiniidae Pectinia paeonia Pectiniidae Trachyphyllia geoffroyi Trachyphyllidae Caribbean Faviidae/ Cladocora arbuscula Faviidae Caribbean Oculinidae Montastraea cavernosa Faviidae Solenastrea bournoni Faviidae Oculina sp. Oculinidae Caribbean Faviidae/ Colpophyllia natans Faviidae Caribbean Mussidae Diploria clivosa Faviidae Diploria labyrinthiformis Faviidae Diploria strigosa Faviidae Favia fragum Faviidae Favia leptophylla Faviidae Manicina areolata Faviidae Mussismilia braziliensis Mussidae Mussismilia harttii Mussidae Mussismilia hispida Mussidae Indo-Pacific Faviidae Cyphastrea chalcidicum Faviidae Cyphastrea serailia Faviidae

Table 4 Continued Amino acid haplotype Species Family

Caribbean Faviidae Montastraea annularis Faviidae Montastraea faveolata Faviidae Montastraea franksi Faviidae Caribbean Meandrinidae/ Meandrina meandrites Meandrinidae Western Atlantic Meandrinidae/ (AY451361–62, AB117295) Meandrinidae Caribbean Mussidae/ Meandrinia braziliensis Mussidae Indo-Pacific Mussidae Mussa angulosa Mycetophyllia aliciae Mussidae Mycetophyllia danaana Mussidae Scolymia cubensis Mussidae Lobophyllia corymbosa Mussidae Indo-Pacific Pectiniidae Echinophyllia aspera Pectiniidae Echinophyllia echinoporoides Pectiniidae Echinophyllia orpheensis Pectiniidae Oxypora lacera (AB117256) Pectiniidae Caribbean Agariciidae Agaricia agaricites Agariciidae Agaricia tenuifolia Agariciidae Agaricia fragilis Agariciidae Agaricia lamarcki Agariciidaes Caribbean Poritiidae Porites branneri Poritiidae Porites divaricata Poritiidae Porites furcata Poritiidae Porites porites Poritiidae

Table 5 Percentage of pairwise comparisons with mean genetic lies, share identical sequences, and (ii) congeneric species distances (Kimura’s 2-parameter genetic distance) of 0%, < 2% are often less similar than noncongeners. The lack of diver- and < 3% across all species, within families, within genera and gence of this mitochondrial gene among many species reduces within species. N indicates total number of pairwise comparisons. the utility of this COI barcoding method in scleractinians Number in parentheses denotes observed number of pairwise and points to the need to generate alternate markers for comparisons the use of distinguishing coral species. This gene might be Within Within Within useful if a suite of mitochondrial and nuclear genes, rather Genetic All species family genus species than a single gene, were used to genetically identify species distance N = 4005 N = 824 N =83 N = 157 (Vences et al. 2005).

0% 1.8% (73) 6.9% (57) 39.8% (33) 82.8% (130) < 2% 30.0% (1203) 60.2% (496) 84.3% (70) 100% (157) Acknowledgements < 3% 49.2% (1971) 86.3% (711) 98.8% (82) 100% (157) We thank the Florida Keys National Marine Sanctuary and the Flower Garden Banks National Marine Sanctuary for permission to collect scleractinian coral samples. Special thanks to D. Brazeau M. Hellberg, H. Lasker, M. Medina, S. Santos, D. Taylor. This research was between intraspecific and interspecific genetic distances, supported by NOAA’s National Undersea Research Program making it impossible to discern most scleractinian species (M.A.C. 2000–15; M.A.C. & J. Ault 2002–12, the National Science using the COI gene. Foundation (M.A.C. OCE-95–30057 and OCE-99–07319). In addition to inherently low nucleotide substitution rates, the inability to use this barcoding system in scleractinians also results from difficulties in taxonomic classification and References an unclear understanding of patterns of evolution within Avise JC (2000) Phylogeography. The History and Formation of Species. this order. It has been indicated that this barcoding system Harvard University Press, Cambridge, Massachusetts. will be most useful in taxonomically well-understood groups Barrett RDH, Hebert PDN (2005) Identifying spiders through (e.g. Barrett & Hebert 2005; Meyer & Paulay 2005), which DNA barcodes. Canadian Journal of Zoology-Revue Canadienne de Zoologie, 83, 481–491. is not the situation for scleractinian corals. Relationships Clark AG, Whittam TS (1992) Sequencing errors and molecular among COI nucleotide sequences are not consistent with evolutionary analysis. Molecular Biology and Evolution, 9, 744–752. traditional taxonomic classification of the Scleractinia at two Coffroth MA, Lasker HR, Diamond ME, Bruenn JA, Bermingham E levels: (i) multiple species, sometimes from different fami- (1992) DNA fingerprints of a gorgonian coral: a method for

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