Limitations of Mitochondrial Gene Barcoding in Octocorallia

Molecular Ecology Resources (2011) 11, 19–31 doi: 10.1111/j.1755-0998.2010.02875.x

DNA BARCODING Limitations of mitochondrial gene barcoding in Octocorallia

CATHERINE S. MCFADDEN,* YEHUDA BENAYAHU,† ERIC PANTE,‡ JANA N. THOMA,‡ P. ANDREW NEVAREZ* and SCOTT C. FRANCE‡ *Department of Biology, Harvey Mudd College, Claremont, CA 91711, USA, †Department of Zoology, George S. Wise Faculty of Life Sciences, University of Tel Aviv, Ramat Aviv, Tel Aviv 69978, Israel, ‡Department of Biology, University of Louisiana at Lafayette, PO Box 42451, Lafayette, LA 70504, USA

Abstract The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub-class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral-specific mitochondrial protein-coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra- and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character-based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2· greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy.

Keywords: cnidarians, DNA barcoding, invertebrates, systematics Received 30 December 2009; revision received 15 April 2010; accepted 23 April 2010

ular, current distance-based analytical methods rely on Introduction the existence of a barcoding gap (i.e., a clear distinction DNA barcoding has been envisioned and widely pro- between ranges of genetic distance values typical of intra- moted as a reliable system by which unidentified speci- specific variation vs. interspecific divergence) and a priori mens can be identified to known species by comparison specification of a threshold genetic distance value above to a reference database of molecular exemplars (e.g. which specimens can reliably be assigned to different Hebert et al. 2003a,b). Barcodes based on a fragment of species. The lack of a well-defined barcoding gap in the mitochondrial cytochrome oxidase I gene (COI) have many taxa, and the error rates associated with the use of been demonstrated to work well for species identification threshold values and distance measures for species detec- in many groups of animals, particularly vertebrates tion, dominate discussion in the recent literature (e.g., (Hebert et al. 2004; Ward et al. 2005; Smith et al. 2008; DeSalle et al. 2005; Meyer & Paulay 2005; Hickerson et al. Tavares & Baker 2008; Baker et al. 2009) and arthropods 2006; Meier et al. 2006, 2008; Rach et al. 2008). In addition, (Smith et al. 2005; Hajibabaei et al. 2006). Their effective- it has been recognized from the outset that a lack of mito- ness and appropriateness as tools for species identifica- chondrial gene variation in some invertebrate groups, in tion in many taxa, however, remain controversial on both particular the anthozoan cnidarians, could limit the util- philosophical (DeSalle et al. 2005; Will et al. 2005) and ity of COI as a barcode for species identification (Hebert practical grounds (Meyer & Paulay 2005; Meier et al. et al. 2003b). 2006; Rubinoff et al. 2006; Roe & Sperling 2007). In partic- Anthozoan cnidarians (e.g. scleractinian corals, sea anemones, and octocorals) are unusual among animals in Correspondence: Catherine S. McFadden, Fax: +1 (909) 607 7172; having mitochondrial genomes that evolve relatively E-mail: [email protected] slowly (Shearer et al. 2002). Recent work suggests that the

2010 Blackwell Publishing Ltd 20 DNA BARCODING mitochondrial genome of some scleractinians evolves 5· extended barcode (1845 nt) consisting of the ‘Folmer more slowly than the nuclear genome (Chen et al. 2009), region’ plus an adjacent intergenic region (igr1, Brugler & and 50–100· slower than the mt genomes of most other France 2008) and a 5¢ fragment of msh1 to discriminate oc- animals (Hellberg 2006). No regions of the anthozoan mt tocoral species. The first case study was a comprehensive genome have sufficient variation to distinguish popula- survey of octocoral biodiversity conducted in Eilat (Gulf tions of conspecifics, and few gene regions exhibit of Aqaba, northern Red Sea), Israel, a geographical region enough variability to separate congeneric species (France with a moderate diversity of species that are well known & Hoover 2002; Shearer et al. 2002; Fukami & Knowlton taxonomically relative to other regions of the Indo-Pacific 2005; Concepcion et al. 2006). The low substitution rates (e.g., Grasshoff 2000; Benayahu et al. 2002). The second observed throughout the anthozoan mt genome have case study focused on a group of 10 Mediterranean and consequently led to predictions that COI will be unsuit- North Atlantic species belonging to the soft coral genus able as a species-specific barcode in this group (Hebert Alcyonium. This genus has been the focus of numerous et al. 2003b). Several recent tests of COI sequence data past genetic studies, and species boundaries and clade derived primarily from scleractinian corals show that relationships have been confirmed using a variety of low levels of interspecific divergence result in up to 40% nuclear molecular markers, including allozymes (McFad- of congeners (and occasionally different genera) sharing den 1999) and ribosomal ITS sequences (McFadden et al. identical COI sequences (Huang et al. 2008; Shearer & 2001; McFadden & Hutchinson 2004). In both of these Coffroth 2008). Tests of COI and 16S sequences as spe- case studies we tested the ability of a mitochondrial bar- cies-specific markers for Zoanthinaria (colonial anemo- code (COI alone, msh1, or the extended COI+igr1+msh1) nes) have, however, been more promising, to distinguish known octocoral species using both distinguishing species groups and most morphospecies distance-based and character-based analyses. (Sinniger et al. 2008). Based on scant data, it has been assumed that mem- Methods bers of the diverse anthozoan sub-class Octocorallia share similarly slow rates of mt gene divergence with the other Collection of material anthozoans (Shearer et al. 2002), but no studies have yet systematically examined variation of COI in this group. Case study 1: regional biodiversity survey, Eilat (Gulf of An early study that predated the COI barcoding initiative Aqaba, northern Red Sea). Representatives of as many reported low rates of genetic divergence in octocoral octocoral species as could be found during a 4-day sur- COI, but at the 3¢ region of the gene rather than the 5¢ vey of Eilat reefs down to 30 m were collected using ‘Folmer region’ that was subsequently adopted as a uni- SCUBA. For at least one specimen of each morphospe- versal barcode (France & Hoover 2002). The only study to cies, DNA was extracted immediately from live tissue focus specifically on the ‘Folmer region’ of COI found no using Qiagen’s DNEasy Blood & Tissue Kit following intraspecific variation, but compared only four species the manufacturer’s protocol. Tissue from additional spec- representing three different octocoral families (Calderoń imens was preserved in 95% EtOH for subsequent DNA et al. 2006). Following these initial reports of lack of varia- extraction using the same method. Vouchers of all speci- tion there have been no further published attempts to mens were preserved in 70% EtOH and deposited in the explore the limitations of COI barcoding in octocorals. collections of the Zoological Museum at Tel Aviv Univer- In the present study we sequenced the ‘Folmer region’ sity (ZMTAU) (Table S1). Specimens were identified to of COI across a broad spectrum of octocoral species, morphospecies by YB based on traditional taxonomy, genera, and families, allowing us to estimate average including microscopical examination of sclerites and divergence values across a range of taxonomic levels, other morphological characters. Identifications of mem- and to compare these with values for msh1, an octocoral- bers of the families Acanthogorgiidae, Melithaeidae, specific mitochondrial protein-coding gene that has been Nephtheidae, Plexauridae and Nidaliidae were similarly shown to evolve more rapidly than other coding regions confirmed by L. van Ofwegen, Nationaal Natu- of the octocoral mt genome (France & Hoover 2001; van urhistorisch Museum, Leiden. der Ham et al. 2009). Although previous studies have found that some morphospecies of anthozoans cannot be Case study 2: North Atlantic and Mediterranean species discriminated using COI alone (Calderoń et al. 2006; of Alcyonium. Specimens of 10 Alcyonium species were Shearer & Coffroth 2008), the percentage of correct spe- collected at sites in the Mediterranean Sea (France, Spain) cies identifications that might nonetheless be expected in and North Atlantic coasts of Europe and North America a regional survey of biodiversity has never been esti- from 1990 to 1994 (McFadden 1999; McFadden et al. 2001; mated. We used two case studies to evaluate the ability McFadden & Hutchinson 2004) (Table S2). All specimens of (i) the COI ‘Folmer region’ alone (800 nt), and (ii) an were preserved in liquid N2 immediately following

2010 Blackwell Publishing Ltd DNA BARCODING 21 collection, and stored frozen at )80C. DNA was extracted replicates. Intraspecific variation was estimated as the from frozen tissue using a standard CTAB extraction maximum pairwise distance observed among conspecif- protocol with the addition of Nucleon Phytopure ics, i.e. the coalescent depth; the smallest interspecific dis- (McFadden et al. 2006a). Alcyonium species were identi- tance observed between congeners was used as a fied by CSM using both traditional taxonomic methods measure of interspecific variation (Meier et al. 2008). For and allozyme electrophoresis (e.g. McFadden 1999). the Alcyonium data set, alignment files were viewed in MacClade 4.08 (Maddison & Maddison 2005) to identify Deep-water samples. To estimate mean intra- and inter- nucleotide characters diagnostic for a species (‘pure char- specific divergence values across a wide spectrum of acteristic attributes’, sensu Rach et al. 2008). octocoral taxa we also sequenced gorgonians that had been collected during a series of deep-sea coral expedi- Results tions to Hawaii (1993, 1996, 2006), Alaska (2003-2004), and the North Atlantic (2003–2005), with additional sam- Based on traditional taxonomy, we identified 49 morpho- ples from museum collections or colleagues (Table S3). species of octocorals from Eilat; these represented 23 gen- Whole colonies or portions thereof were sampled using era and nine families, with the majority belonging to the remotely operated vehicles (ROV), human-occupied fleshy octocoral families Alcyoniidae, Nephtheidae, and vehicles or trawls. Fragments for genetic analyses were Xeniidae (Table S1). Sequences for COI and msh1 were preserved in 95–100% EtOH or frozen at )80C. DNA obtained for 1–6 specimens of each morphospecies, was extracted using a modified CTAB protocol (France depending on availability of samples. Sequences for both et al. 1996). Vouchers have been (or will be) deposited at genes were obtained from 7 to 11 individuals for each of the Yale Peabody Museum and USNM (Table S3). Speci- 10 northern hemisphere species of Alcyonium, as well as mens were identified to morphospecies by SCF, EP, JNT, two individuals of the South African A. variabile, and an L. Watling (U. Hawaii), and S. Cairns (USNM). outgroup, the South Atlantic A. haddoni. Among the deep- sea samples, we identified 16 morphospecies representing 10 genera from four families in the Holaxonia-Alcyoniina Amplification and sequencing clade (McFadden et al. 2006b), and 28 morphospecies rep- The ‘universal’ Folmer primers do not amplify COI in resenting at least 12 genera from three families in the Calc- most octocorals (pers. obs.), necessitating the design of axonia clade (Table S3). Sequences for COI and msh1 were octocoral-specific primers for this region (e.g., Calderoń obtained for 1–6 specimens of each morphospecies. A rep- et al. 2006). We amplified a fragment (1.1 kB) that resentative of each unique haplotype from each species encompassed the entire ‘Folmer region’ of COI plus an was deposited in GenBank (Tables S1–S3). adjacent intergenic region (igr1) from all specimens using All edited and aligned sequences included the first primers COII8068F (McFadden et al. 2004) or COII8068xF (5¢) 800 nt of the COI coding region, encompassing the (a degenerate version, Table S4) and COIOCTr (reverse complete ‘Folmer region’ (nucleotide positions 29-736) complement of COIOCTf, France & Hoover 2002). (Folmer et al. 1994). There were no indels in this region, Approximately 760 nt of msh1 was amplified using for- and nucleotide alignments were unambiguous. The igr1 ward primers ND42475F (Brugler & France 2008) or and msh1 regions, however, both exhibited length varia- ND42599F (France & Hoover 2002) and mut3458R (Sań- tion (msh1 typically exhibits amino acid length variation, chez et al. 2003). Alternative primers for each gene region maintaining the correct reading frame despite numerous were used for some problematic deep-sea specimens indels; McFadden et al. 2006b; France 2007). Among (Table S4). Standard PCR protocols were used (e.g. Alcyonium species, igr1 ranged from 107 to 112 nt, with a Sańchez et al. 2003; McFadden et al. 2004; Brugler & final alignment length of 113 nt; msh1 ranged from 726 to France 2008). Amplified products were purified and sent 735 nt due to a 9-nt (3 aa) deletion in one clade. Among to Cogenics (Houston, TX) for sequencing. the other genera, igr1 ranged from 94 nt (Chrysogorgia sp.) to 168 nt (Titanideum frauenfeldii) and msh1 from 723 nt (Radicipes sp.) to 816 nt (Lepidisis sp. B1b) (241–272 aa). Analysis Among closely related taxa (e.g. congeners) nucleotide Nucleotide sequences were aligned using MUSCLE v. 3.6 alignments were unambiguous, but as genetic distance (Edgar 2004) or the LIN-S-I or GIN-S-I algorithm in MA- increased the nucleotide alignment of igr1 and amino FFT (Katoh et al. 2002), and translated protein-coding acid alignment of certain regions within msh1 became regions were adjusted by eye to conform to amino acid increasingly uncertain. We analysed separate alignments alignments. Pairwise distances (uncorrected p) were for the Eilat specimens, deep-sea Holaxonia-Alcyoniina, calculated, and PAUP* (Swofford 2002) was used to con- and deep-sea Calcaxonia; final alignment lengths ranged struct neighbour-joining phylograms with 1000 bootstrap from 164–168 nt for igr1 and 810-888 nt for msh1.

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Table 2 Means (±SD) and ranges of genetic distances Mean divergence within taxonomic levels (uncorrected p, expressed as percentage) observed within and Maximum intraspecific genetic distance values (coales- between morphospecies of octocorals collected in Eilat, Israel using different mtDNA barcodes. Within species = maximum cent depth) ranged from 0–1.25% (mean = 0.13%) for COI, intraspecific value (coalescent depth); within genus = minimum 0–1.90% (mean = 0.17%) for msh1, and 0–1.82% (mean = distance between congeners. nt = total length (nucleotides) of 0.16%) for an extended barcode of COI+igr1+msh1, with aligned sequence; n = number of pairwise comparisons a majority of species exhibiting no intraspecific variation in any of the three gene regions (Tables 1–3, Fig. 1). Mini- Within species Within genus mum genetic distances among congeneric species pairs Gene Region(s) nt n =18 n =75 ranged from 0% to 4.75% (mean = 1.2%) for COI,0%to COI coding 800 0.12 (±0.30) 1.11 (±1.05) 9.39% (mean = 3.1%) for msh1, and 0% to 7.12% 0–1.0 0–4.75 (mean = 2.2%) for the extended barcode (Tables 1–3, COI + igr1 1044 0.16 (±0.44) 1.54 (±1.29) Fig. 1). 18.5% of congeneric species pairs shared identical 0–1.75 0–5.28 COI haplotypes, 15.1% shared msh1 haplotypes, and msh1 coding 819 0.16 (±0.46) 3.36 (±2.49) 11.6% shared identical extended barcodes. Specimens 0–1.90 0–9.39 identified to different genera shared the same extended COI + igr1 + msh1 1844 0.16 (±0.45) 2.36 (±1.79) barcode in only one case (Xenia hicksoni and Heteroxenia 0–1.82 0–7.12 ghardaqensis); another pair (Paramuricea placomus and Placogorgia sp.) shared identical msh1 and igr1 sequences, but differed at COI. In both of these cases, genus distinc- old to 0.5%, however, still results in a false-negative rate tions are unclear (see Discussion). of 38% (COI), 23% (msh1) or 25% (extended barcode). A commonly suggested threshold for species detection in barcoding studies is 10· the mean pairwise intra- Case study 1: regional biodiversity survey, Eilat specific genetic distance (Hebert et al. 2004). For octocorals, these values would be 1.3%, 1.7% and 1.6% Among the 49 morphospecies identified from Eilat, we respectively for COI, msh1 and the extended barcode. detected 45 different COI coding sequences. Thirty-four Each of these threshold values results in high rates of taxa (69%) had unique COI haplotypes, but six pairs and false-negative identifications (failure to differentiate spe- one trio of morphospecies shared identical sequences, cies). Including those cases in which different species including one pair in which specimens were identified to shared identical barcode sequences, 62% of pairwise different genera (Xenia, Heteroxenia) (Fig. 2). We detected genetic distances among congeners fell below the 10X intraspecific sequence variation in COI in only five spe- threshold for COI, 36% for msh1, and 45% for the cies (Fig. 2). Addition of igr1 and msh1 sequences to the extended barcode. In only three instances, however, did barcode did not further discriminate any of the species we observe a maximum intraspecific genetic distance that had identical COI coding regions, but did reveal greater than 0.5% (Fig. 1), and in each of those cases fur- intraspecific variation in two additional species (data ther scrutiny suggested the presence of cryptic species not shown). A 10X species detection threshold of 1.3% (see Discussion). Lowering the species detection thresh- for COI would result in 71% false negatives, greatly

Table 1 Means (±SD) and ranges of genetic distances (uncorrected p, expressed as percentage) observed within and between 10 North Atlantic and Mediterranean species of Alcyonium assessed using different mtDNA barcodes. Within species = maximum intraspecific value (coalescent depth); within and between clades = minimum interspecific value. nt = total length (nucleotides) of aligned sequence; # unique = number of species identifiable using pure characteristic attributes (parentheses: number of identifiable Atlantic species). n = number of pairwise comparisons

Within species Within clades Between clades Gene Region(s) nt n =10 n =12 n = 34 # unique

COI coding 800 0.09 (±0.13) 0.08 (±0.14) 2.26 (±0.66) 1 ⁄ 10 (3 ⁄ 7) 0–0.38 0–0.38 1.38–2.88 COI + igr1 913 0.08 (±0.12) 0.15 (±0.27) 2.55 (±0.68) 3 ⁄ 10 (5 ⁄ 7) 0–0.33 0–0.77 1.65–3.31 msh1 coding 735 0.11 (±0.17) 0.31 (±0.38) 4.82 (±1.61) 2 ⁄ 10 (3 ⁄ 7) 0–0.41 0–0.82 2.45–6.62 COI + igr1 + msh1 1648 0.09 (±0.10) 0.22 (±0.28) 3.57 (±1.09) 4 ⁄ 10 (7 ⁄ 7) 0–0.30 0–0.74 2.00–4.78

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Table 3 Genetic distances (uncorrected p ± SD, expressed as hemisphere Alcyonium species that could be identified percentage) within and between morphospecies of gorgonian unequivocally based on a unique COI haplotype. The octocorals collected on deep-sea cruises using different mtDNA other nine species all shared at least one haplotype ‘barcodes’. For each ‘barcode,’ the first line shows in boldface with another species. means and ranges for all taxa combined, followed by means and ranges for Calcaxonia vs. Holaxonia ⁄ Alcyoniina clades. Addition of the igr1 and msh1 regions to the bar- nt = total length (nucleotides) of alignment of barcode sequence; code sequence resulted in better discrimination of Within species = maximum intraspecific value (coalescent Alcyonium species, separating A. siderium from A. sp. depth); within genus = minimum distance between congeners; A, and A. coralloides from the other three species in n = number of pairwise comparisons for combined that clade (Fig. 3). Not all species were distinguishable, data ⁄ Calcaxonia ⁄ Holaxonia-Alcyoniina, respectively however, even with this extended barcode. The most frequent haplotype found in A. acaule, A. glomeratum Within species Within genus Gene Region(s) nt n =6⁄ 5 ⁄ 1 n =23⁄ 18 ⁄ 5 and A. palmatum was shared by all three species, and A. sp. M2 shared haplotypes with A. bocagei and COI coding 0.21 (±0.51) 0.41 (±0.89) A. hibernicum. Mean minimum genetic distances among 0–1.25 0–4.25 sister taxa belonging to the same clade ranged from Calcaxonia 800 0 (±0) 0.17 (±0.28) 0.08% (COI) to 0.31% (msh1) (Table 1), comparable to 0–0 0–0.88 the mean maximum intraspecific genetic distance val- Holax ⁄ Alcyon 800 1.25 1.28 (±1.68) ues estimated across octocoral taxa. In addition, not all 1.25 0.13–4.25 COI + igr1 0.29 (±0.72) 0.52 (±0.94) sister species were reciprocally monophyletic, and 0–1.75 0–4.39 bootstrap support for species-level clades was gener- Calcaxonia 968 0 (±0) 0.30 (±0.43) ally weak (Fig. 3). 0–0 0–1.19 Although the ‘10X rule’ and reciprocal monophyly Holax ⁄ Alcyon 968 1.75 1.29 (±1.76) both would fail to distinguish sister taxa, a majority of 1.75 0.11–4.39 Alcyonium species nonetheless possessed diagnostic msh1 coding 0.29 (±0.72) 1.25 (±1.58) nucleotide substitutions that allowed character-based 0–1.77 0–7.07 Calcaxonia 888 0 (±0) 0.92 (±0.80) species assignment (Fig. 4). When only the seven species 0–0 0–1.96 that occur in the North Atlantic were included in the Holax ⁄ Alcyon 810 1.77 2.42 (±2.96) analysis, the ability to correctly assign species identity to 1.77 0.14–7.07 a specimen based on diagnostic nucleotide characters COI + igr1 + msh1 0.29 (±0.72) 0.85 (±1.17) was 100%. Species pairs such as A. siderium – A. sp. A 0–1.76 0–5.59 and A. bocagei – A. hibernicum that were not reciprocally Calcaxonia 1856 0 (±0) 0.58 (±0.47) monophyletic and differed by <0.06% (extended barcode) 0–0 0–1.38 Holax ⁄ Alcyon 1778 1.76 1.80 (±2.26) had at least one ‘pure characteristic attribute’ (i.e. a nucle- 1.76 0.12–5.59 otide shared by all members of that species but not by its sister taxon; Rach et al. 2008) that allowed unequivocal species assignment. Among the four Mediterranean spe- underestimating total biodiversity; a 0.5% threshold cies, A. coralloides and A. sp. M2 could be distinguished would still result in 19% false negatives, but would flag from one another and from the other two species using a three potential cryptic species as distinct (see Discussion). character-based approach, but A. acaule and A. palmatum remained inseparable. Case study 2: North Atlantic and Mediterranean Alcyonium species Discussion We detected 11 distinct COI haplotypes among the 10 As anticipated from past studies of other anthozoans species of northern hemisphere Alcyonium. Five species (Shearer et al. 2002; Hellberg 2006; Huang et al. 2008; exhibited no intraspecific variation (Fig. 3). Alcyonium Shearer & Coffroth 2008) and from published estimates glomeratum and A. palmatum each had two haplotypes, of variability in octocoral mitochondrial genes (France & the most common of which they shared with one Hoover 2002; McFadden et al. 2004; Calderoń et al. 2006; another and with A. acaule. The clade comprising van der Ham et al. 2009), COI and msh1 both exhibited lit- A. coralloides, A. hibernicum, A. bocagei and Alcyonium tle intraspecific variation in octocorals (Fig. 1), a potential sp. M2 included 7 COI haplotypes: A. coralloides and advantage for use of either or both gene regions as spe- A. sp. M2 each possessed two unique haplotypes, and cies-specific barcodes. However, both genes also exhib- the remaining three haplotypes were shared by more ited relatively little divergence among congeneric than one species. A. digitatum was the only northern species, with the result that maximum intraspecific and

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COI Coding msh1 Coding COI + igr1 + msh1 (a) 30 30 30

25 25 25

20 20 20 Intra- 15 specific 15 15 10 10 10

5 5 5

0 0 0

(b) 30 30 30

25 25 25

20 20 20 Inter- 15 specific 15 15 10 10 10

5 5 5

0 0 0 0 1.0 2.0 3.0 4.0 5.0 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Uncorrected p (%)

Fig. 1 Frequency histograms of pairwise genetic distance values (uncorrected p) across all octocoral taxa sequenced in this study. Sepa- rate histograms are shown for COI, msh1, and an extended barcode consisting of both of those coding regions plus igr1, an intergenic region adjacent to COI. Pairwise comparisons were made (a) among conspecific individuals (maximum intraspecific distances) and (b) between congeneric species (minimum interspecific distances). Grey bars represent genetic distances of 0, i.e. (a) no intraspecific variation observed or (b) congeners with identical barcode sequences. minimum interspecific genetic distances overlap. In par- cies. The two distinct haplotypes of Sinularia polydactyla ticular, 18.5% of congeneric morphospecies shared identi- collected in Eilat belong to very different clades within cal sequences and were therefore inseparable using the that speciose genus, and morphological characters that standard (COI) molecular barcode. In at least one case, distinguish them at the clade level have subsequently specimens identified to different genera by traditional been identified (McFadden et al. 2009). Likewise, when taxonomy shared identical COI sequences (but see the two divergent msh1 haplotypes of Sarcophyton gemma- below). The percentage of octocoral congeners that share tum found at Eilat are included in a well-sampled phy- COI barcodes is, however, less than the 40% reported for logeny of that genus (McFadden et al. 2006a) they fall scleractinian corals (Shearer & Coffroth 2008). into different clades, suggesting that they, too, represent The lack of a gap between intraspecific and interspe- different species. These examples illustrate cases in cific distance values makes the use of a mitochondrial which a molecular barcode has proven effective for taxon gene barcode problematic for distance-based species discovery in octocorals, alerting taxonomists to previ- identification in octocorals, as specimens whose ously unrecognized cryptic species or misidentifications sequences differ by <1.0% might or might not be conspe- of highly variable, closely related taxa. Our results also cific. Our results do suggest, however, that genetic dis- highlight the need for thorough verification, and in some tances >0.5% are likely to be indicative of species-level cases revision of certain octocoral taxa. As discussed differences. In at least two of the three cases in which in the literature (e.g., DeSalle et al. 2005; Rubinoff et al. specimens identified to the same morphospecies differed 2006), integrated taxonomic approaches utilizing tradi- by >0.5%, subsequent phylogenetic and morphological tional morphological, molecular, and geographical data studies have suggested that they are indeed different spe- will always be necessary to confirm species boundaries,

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Fig. 2 Neighbour-joining tree (uncor- Briareum hamrum CO 34187 100 Acabaria erythraea CO 34216 99 rected p) based on COI coding region 81 Acabaria sinaica CO 34200 Acabaria sinaica CO 34197 (800 nt) sequenced for octocoral taxa Clathraria rubrinodis CO 34054 CO 34118 68 Heteroxenia fuscescens collected in a 4-day biodiversity survey at Ovabunda obscuronata CO 34077 100 Heteroxenia ghardaqensis CO 34174 Eilat, Israel. Grey boxes enclose specimens 67 Heteroxenia ghardaqensis CO34176 99 CO 34072 identified to two or more different 85 Xenia hicksoni 79 Xenia hicksoni CO 34073 morphospecies that shared the same COI Ovabunda farauensis CO 34051 Sympodium caeruleum CO 34185 CO 34183 haplotype. Asterisks indicate specimens 95 Anthelia glauca Paralemnalia eburnea CO 34081 identified to the same morphospecies that 100 Paralemnalia eburnea CO 34085 Paralemnalia thyrsoides CO34087 had different COI haplotypes. Numbers 84 Paralemnalia thyrsoides CO 34088 CO 34124 100 74 Rhytisma fulvum on nodes are bootstrap values (% of 1000 Rhytisma fulvum CO 34126 replicates). Rhytisma fulvum CO 34122 * Cladiella pachyclados CO 34084 100 Cladiella pachyclados CO 34129 Cladiella pachyclados CO 34130 100 Cladiella pachyclados CO 34131 Cladiella sphaerophora CO 34132 100 CO 34127 95 Klyxum utinomii Klyxum utinomii CO 34190 Metalcyonium verseveldti CO 33097 Sinularia loyai CO 34154 75 Sinularia leptoclados CO 34095 82 87 Sinularia erecta CO 34144 63 Sinularia gardineri CO 34097 69 Sinularia gardineri CO 34146 Sinularia hirta CO 34100 Sinularia hirta CO 34103 Sinularia hirta CO 34148 85 Sinularia terspilli CO 34156 93 Sinularia querciformis CO 34096 90 Sinularia querciformis CO 34191 Sinularia querciformis CO 34192 99 * 51 Sinularia polydactyla CO 34106 51 Sinularia polydactyla CO 34138 Sinularia polydactyla CO 34181 * 69 68 Sinularia compressa CO 34142 Sinularia compressa CO 34141 Sinularia compressa CO 34143 72 71 91 Sinularia compressa CO 34150 Sinularia compressa CO 34151 Sinularia compressa CO 34140 CO 34152 100 Sinularia cruciata Sarcophyton auritum CO 34060 Sarcophyton pauciplicatum CO 34135 98 Sarcophyton glaucum CO 34063 96 Sarcophyton gemmatum CO 34057 Sarcophyton gemmatum CO 34135 Scleronephthya corymbosa CO 34159 * Dendronephthya klunzingeri CO 34109 Dendronephthya hemprichi CO 34167 95 99 Dendronephthya formosa CO 34179 82 Dendronephthya hemprichi CO 34213 Dendronephthya sp. CO 34227 Dendronephthya sinaiensis CO 34163 93 Dendronephthya savignyi CO 34066 87 Nephthea elatensis CO 34112 Nephthea elatensis CO 34113 Nephthea acuticonica CO 34070 68 55 Nephthea acuticonica CO 34071 Litophyton acutifolium CO 34114 100 Stereonephthya cundabiluensis CO 34208 Stereonephthya cundabiluensis CO 34204 90 Acanthogorgia breviflora CO 34194 Euplexaura sp. CO 34220 Chironephthya sp. 2 CO 34222 100 Chironephthya sp. 1 CO 34203 86 Chironephthya sp. 1 CO 34223 Chironephthya sp. 3 CO 34226 * Tubipora musica CO 34116 0.005 substitutions/site but our results suggest that COI sequence differences of majority of these species lack images of the living animal, >0.5% effectively flag cases worthy of further scrutiny in in situ or otherwise, and also lack sufficient detail to Octocorallia. encompass the morphological variation observed in Evaluation of the effectiveness of molecular barcodes material collected using modern methods such as in octocorals is hindered greatly by the poor state of our SCUBA or ROVs. Identification of species is therefore dif- knowledge of species boundaries in these organisms. ficult to impossible without a thorough re-examination of Cases in which specimens identified to different morpho- type material, and in some cases revision of the relevant species share a barcode can and should motivate addi- genus. In addition, octocoral taxonomy is still based on tional taxonomic work to test species boundaries and an antiquated typological species concept, whereby quantify intraspecific morphological variation. Many specimens that differ from one another in a small set of octocoral species were described in the late 19th and subjectively assessed morphological character states are early 20th centuries based on collections made using deemed different species (McFadden et al. 2006a). trawls and dredges, which typically recover damaged Descriptions of new species are still frequently based on colonies or colony fragments. The descriptions of the vast single specimens (e.g. Watling 2007; van Ofwegen

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A. acaule BAN.AA1 M A. acaule MED.AA4 M A. acaule MAR.9.AA3 M A. glomeratum TRE.AG10 A. palmatum BAN.AP1 M A. palmatum BAN.AP5 M A. palmatum BAN.AP3 M A. palmatum BAN.AP2 M A. acaule MAR.1.AA5 M A. palmatum BAN.AP8 M A. palmatum BAN.AP10 M A. acaule BAN.AA4 M A. glomeratum AZO.AG2 A. glomeratum TRE.AG23 A. glomeratum SAG.AG2 A. acaule MAR.2.AA1 M A. palmatum BAN.AP9 M A. acaule MED.AA1 M A. acaule BAN.AA3 M A. glomeratum GLE.AG5 A. glomeratum GLE.AG23 A. acaule MED.AA8 M A. palmatum BAN.AP7 M A. glomeratum LUZ.AG4 A. palmatum BAN.AP4 M 95 A. glomeratum TRE.AG11 A. acaule MAR.8.AA2 M 100 A. palmatum BAN.AP6 M A. glomeratum LUZ.AG3 A. glomeratum LUZ.AG6 65 A. glomeratum LUZ.AG10 A. bocagei SAG.AC2 A. bocagei SAG.AC4 A. sp. M2 MAR.10.AC4 M A. bocagei SAG.AC23 A. bocagei SAG.AC14 A. bocagei SAG.AC19 66 A. sp. M2 MAR.V.AC30 M A. bocagei SAG.AC5 A. bocagei SAG.AC16 A. sp. M2 MAR.9.AC41 M A. bocagei SAG.AC11 A. sp. M2 MAR.V.AC8 M A. bocagei SAG.AC8 A. bocagei SAG.AC12 A. hibernicum CHA.AH10 A. hibernicum CHA.AH15 A. hibernicum TRE.AC21 A. hibernicum TRE.AH40 A. hibernicum GLE.AH38 A. hibernicum GLE.AH9 A. hibernicum TRE.AH66 A. hibernicum CHA.AH7 A. hibernicum IOM.SU.AH14 A. sp. M2 MAR.9.AC21 M 54 A. hibernicum IOM.GB.AH12 A. hibernicum IOM.BF.AH7 76 A. sp. M2 MAR.4.AC2 M A. sp. M2 MED.AC27 M A. sp. M2 MAR.6.AC2 M A. sp. M2 MAR.10.AC18 M A. coralloides BAN.AC29 M A. coralloides BAN.AC39 M 100 A. coralloides MED.AC34 M A. coralloides MAR.1.AC1 M A. coralloides LUZ.AC33 A. coralloides MAR.7.AC7 M A. coralloides CHA.AC110 95 A. coralloides LUZ.AC23 A. coralloides MAR.8.AC22 M A. coralloides MAR.1.AC2 M 100 A. variabile SAF.18.1 A. variabile SAF.18.2 A. digitatum IOM.BF.AD2 A. digitatum IOM.BF.AD4 A. digitatum CHAAAD12 A. digitatum GLEAAD4 95 A. digitatum IOM.SU.AD3 A. digitatum IOM.SU.AD1 A. digitatum CHA.AD35 A. digitatum IOM.SU.D4 A. digitatum SWE.AD2 A. sp. A IOM.BF.M2 A. siderium NAH.DB2.9 A. siderium NAH.DB4.5 A. siderium NAH.DB5.3 A. siderium NAH.SR.2.4 100 65 A. siderium NAH.SR1.1 A. siderium NAH.SR5.4 A. siderium NAH.DB3.8 A. sp. A IOM.BF.M26 A. sp. A IOM.SU.M11 A. sp. A IOM.BF.M19 A. sp. A IOM.SU.M6 A. sp. A IOM.BF.M20 A. sp. A IOM.BF.M4 100 A. sp. A IOM.SU.M1 A. sp. A IOM.SU.M4 A. sp. A IOM.BF.M25 A. haddoni 0.0005 substitutions/site

Fig. 3 Neighbour-joining tree based on an extended barcode of COI+igr1+msh1 for 10 northern hemisphere species of Alcyonium. Speci- mens belonging to the same species share the same colour. M: Specimens collected in the Mediterranean Sea. Bootstrap values on nodes are % of 1000 replicates. Tree is rooted using the southern hemisphere A. haddoni as an outgroup.

2008a,b), and therefore intraspecific variation is often not In this study, for example, sequences from the holotype depicted. Furthermore, integrated taxonomic approaches of Iridogorgia fontinalis, described by Watling (2007) from are rarely applied to confirm that morphologically distin- a single specimen, were identical to those from the holo- guishable forms indeed represent different species rather type of I. magnispiralis, calling into question the distinc- than intraspecific or environmentally induced variants. tiveness of the new species. In such cases in which

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uncertainty surrounding the definitions of higher taxa in (a) m541 C276 m009 m040 m493 m150 m581 Octocorallia. For instance, the one case in which speci- A. bocagei SAG.AC2 mens identified to two different genera (Xenia, Heteroxe- A. bocagei SAG.AC8 A. bocagei SAG.AC16 nia) shared identical barcode sequences is more likely A. hibernicum GLE.AH38 because of the lack of a clear distinction between these A. hibernicum CHA.AH10 two genera (Fabricius & Alderslade 2001) than the inabil- A. hibernicum IOM.BF.H7 ity of mtDNA markers to separate well-defined genera. A. coralloides BAN.AC29 Similarly, among the deep-sea taxa an observed lack of A. coralloides MED.AC34 genetic distinction between Paramuricea placomus and A. coralloides MAR.1.AC1 Placogorgia sp. is not surprising, as the sclerites of these A. coralloides MAR.1.AC2 two genera have long been recognized to represent a con- tinuum of morphological variation (Bayer 1959; Grass- (b) hoff 1977). i014 i093 i055 i044 C400 C054 C768 m009 m444 m651 m330 m004 A. digitatum IOM.BF.D2 Large pairwise distances among congeners also reflect A. digitatum CHA.AD12 taxonomic uncertainty. For instance, two species of A. digitatum IOM.SU.D1 Acanthogorgia included here differed from one another by A. digitatum SWE.D2 4–7%, not surprising considering that this genus has pre- A. sp. A IOM.BF.M2 viously been shown to be polyphyletic and to comprise A. siderium NAH.DB2.9 two divergent clades (McFadden et al. 2006b). Among the A. siderium NAH.DB5.3 bamboo corals (Isididae), pairwise genetic distances A. siderium NAH.SR5.4 A. sp. A IOM.SU.M4 between congeners in the nominal genera Isidella, Keratoi- A. sp. A IOM.SU.M1 sis, and Lepidisis range from 0% to 2% for msh1 (all are identical at COI and igr1). Earlier genetic analyses of msh1 and igr4, however, revealed highly divergent clades (c) C180 m591 C204 m279 that do not correspond to currently accepted bamboo A. acaule BAN.AA1 coral taxonomy (France 2007; van der Ham et al. 2009), A. acaule MED.AA4 and a reassessment of morphological characters within A. glomeratum GLE.AG23 the subfamily Keratoisidinae has revealed synapomor- A. glomeratum TRE.AG10 phies sufficiently significant to warrant erecting new gen- A. palmatum BAN.AP4 A. palmatum BAN.AP1 era (France & Watling, unpub. data). In the present A. palmatum BAN.AP8 study, if pairwise genetic distances had been restricted to A. glomeratum AZO.AG2 comparisons within clades (i.e. hypothetical new genera) A. acaule MAR.2.AA1 rather than nominal genera, the maximum interspecific A. glomeratum LUZ.AG3 distance observed among isidids would be only 0.3%. A. glomeratum LUZ.AG6 A. glomeratum LUZ.AG10 Alcyonium as a test case for mtDNA barcoding Fig. 4 Individual nucleotide characters distinguishing species of Alcyonium within each of three distinct clades. For simplicity, Circularities of the type illustrated above are common in only 3–4 specimens of each species are shown (including a repre- groups such as Octocorallia that are poorly known and sentative of each distinct COI + igr1 + msh1 haplotype detected poorly sampled; a lack of sequence divergence among for a species). Species-diagnostic nucleotide positions are morphospecies can be interpreted as either inadequate labelled by gene (C = COI;m=msh1;i=igr1). (a) Alcyonium molecular variation or faulty taxonomy, depending on coralloides clade, excluding A. sp. M2. (b) A. digitatum clade. (c) A. acaule clade. personal bias. For that reason, we used the genus Alcyoni- um as a test of mtDNA barcodes specifically because spe- recognized morphospecies share a barcode sequence(s), cies boundaries in this genus have been confirmed species boundaries should be confirmed using indepen- previously using a variety of independent criteria. A dent criteria. Until such time, a wholesale rejection of molecular phylogenetic study based on the rapidly mitochondrial markers as inadequate for species identifi- evolving nuclear ribosomal internal transcribed spacer cation in octocorals is premature. (ITS) regions separated the northern hemisphere representatives of this genus into three distinct clades among Genetic distances within and among genera which ITS is too divergent (>25%) to align with any cer- tainty (McFadden et al. 2001). Within each clade, how- The distributions of pairwise genetic distance values ever, species exhibit only 1-8% divergence at ITS and are among congeneric species (Fig. 1) reflect additional often difficult to distinguish morphologically (Verseveldt

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1964, 1973; Feldman 1973; Weinberg 1977; Manuel 1981; ising as barcodes for Octocorallia than our extended Groot & Weinberg 1982). Alcyonium therefore provides a mitochondrial barcode. Other mitochondrial protein- rigorous test of the applicability of mitochondrial barcoding and rDNA regions typically exhibit levels of codes to octocorals, because it includes sister species interspecific variation similar to or less than that of known to be extremely similar both genetically and mor- COI, and the utility of mt intergenic regions is limited phologically, but demonstrated via allozymes or repro- by extreme length variation among taxa (van der Ham ductive differences to be reproductively isolated in et al. 2009). The internal transcribed spacers (ITS-1, sympatry (McFadden 1999; McFadden et al. 2001). ITS-2) frequently lack sufficient variation to discrimi- In this taxonomically challenging genus, the extended nate sister taxa (McFadden et al. 2001; Aguilar & Sań- COI+igr1+msh1 barcode correctly identified 7 of 10 species chez 2007) and, in addition, often exhibit when a character-based analysis was applied. Within the intraindividual sequence polymorphisms (McFadden A. coralloides clade (Fig. 3), the extended barcode failed to et al. 2001; McFadden & Hutchinson 2004; Duenãs & distinguish the Mediterranean species A. sp. M2 from two Sańchez 2009). Few nuclear markers have yet been Atlantic species, A. bocagei and A. hibernicum. Based on a identified that exhibit adequate levels of variation to study of shared ITS polymorphisms, however, it has been distinguish species of octocorals, and those that have proposed that A. bocagei and A. hibernicum both arose (e.g. SRP54; Concepcion et al. 2008) have proven diffi- from hybrid speciation events, and that A. sp. M2 may cult to amplify reliably across diverse taxa (pers. obs.). have been one of the progenitors (McFadden & Hutchin- For now, we suggest continued use of an extended son 2004), a scenario that could explain the sharing of mitochondrial barcode for octocorals: msh1 + igr1 mitochondrial haplotypes. The failure of mitochondrial encompasses the most variable regions of most octo- barcodes to distinguish taxa with histories of hybridiza- coral mt genomes, while COI allows comparison to tion or introgression is well known (Rubinoff et al. 2006). other taxa for which an extensive database already The other cases in which a character-based analysis of the exists. extended barcode failed to distinguish species occurred In addition to an apparent two-fold difference in rates among the three species in the A. acaule clade. This is the of evolution between the COI and msh1 coding regions, one clade of Alcyonium for which we lack confirmation of our data also suggest some rate heterogeneity among the species boundaries using allozyme data. different taxa included in our study. In particular, deep- sea Calcaxonia exhibited significantly less genetic variation at both COI and msh1 than other taxa (Table 3); we COI or msh1 as an octocoral barcode? observed no intraspecific variation in any of the five calc- Screening of variation across the octocoral mitochon- axonian species for which multiple individuals were drial genome suggests that the msh1 gene, unique to sampled, and genetic distances among congeners of Calc- this cnidarian subclass, is the most rapidly evolving axonia were an order of magnitude lower for COI and protein-coding region in an otherwise slowly evolving 2–4· lower for msh1. Whether these observed differences genome (van der Ham et al. 2009), and therefore the reflect actual rate differences, or, alternatively, are arte- most promising candidate for a mitochondrial barcode. facts of the smaller sample size for intraspecific compari- For pairwise comparisons among congeners, genetic sons or more narrowly defined genera in this group distances at msh1 were typically 2–3· greater than remain to be tested. those at COI (Tables 1–3). Overlap between intra- and interspecific genetic distances were, however, compara- Conclusions ble for the two gene regions (Fig. 1), and in character- based analyses msh1 was often no more effective than As has been reported previously for other anthozoans COI at distinguishing species. All of the species pairs (Huang et al. 2008; Shearer & Coffroth 2008), the absence that shared identical COI sequences in the Eilat survey of a significant gap between intra- and interspecific were also identical at msh1; among the deep-sea calc- genetic distance values imposes a limit on the use of axonian taxa, however, some morphospecies that mitochondrial barcodes (COI, msh1, or extended shared COI haplotypes did differ at msh1. Among the COI + igr1 + msh1) for species identification in Octocor- North Atlantic Alcyonium species, each gene alone dis- allia. Overlap between intra- vs. interspecific genetic distinguished just three of seven species, whereas com- tances will give rise to false negatives, i.e. an bining the two coding regions plus igr1 revealed underestimate of biodiversity as a result of different spe- enough pure characteristic attributes to distinguish all cies being lumped together because of lack of sequence seven species reliably. divergence. Our data suggest, however, that intraspecific Alternative molecular markers that have been variation (coalescent depth) rarely if ever exceeds 0.5% at explored to date do not appear to be any more prom- either COI or msh1, making it generally safe to conclude

2010 Blackwell Publishing Ltd DNA BARCODING 29 that specimens differing by >1% are indeed different spe- well-sampled phylogenies. Only through such collabo- cies. Given the poor state of octocoral taxonomy and our rative, integrated approaches will our ability to confi- lack of understanding of morphological characters in dently identify species of octocorals using molecular many genera, the potential for identification of cryptic barcodes continue to improve. species and genera using mtDNA barcodes is still high, as evidenced by the outcome of our Eilat biodiversity Acknowledgements study as well as some other recent studies (McFadden et al. 2006a, 2009; van der Ham et al. 2009). For specimens We thank Vanessa Brisson for assistance in the laboratory, Leen that differ by <1% at COI or msh1, however, it will not be van Ofwegen for identification of specimens, the Interuniversity possible to conclude without additional biological, mor- Institute at Eilat for assistance and use of facilities, and Alex Shlagman for professional curatorial skills. We also thank the phological or molecular study whether or not species Israel Nature and Parks Authority for providing collection per- boundaries are present. mits, and the Eilat-Ashkelon Pipeline Company (EAPC) for Interspecific divergence values <1% do not, however, allowing research on their premises. This research was sup- preclude use of a mitochondrial barcode to assign species ported by the Assembling the Cnidarian Tree of Life project (NSF identity in cases in which clades have been well sampled grants EF-0531570 to C.S. McFadden and EF-0531779 to P. Cart- both intra- and interspecifically (e.g., Meyer & Paulay wright), and by grants #NA05OAR4601061 from NOAA’s Office 2005). Nucleotide character-based analysis successfully of Ocean Exploration and OCE-0624601 from NSF’s Ocean Sci- ences Division–Biological Oceanography Program to S.C. France. identified specimens of Alcyonium in cases where sister P.A. Nevarez was supported by Howard Hughes Medical Insti- species differed by <0.1%; specimens could reliably be tute award 52005123 to Harvey Mudd College. Grants from - assigned to species that differed from one another by Sigma Xi (GIAR G20061021830514629) and the American only a single nucleotide when that difference represented Museum of Natural History (Lerner Gray Fund) allowed E. Pante a pure characteristic attribute (sensu Rach et al. 2008). In to sample specimens from the MNHN collection (Paris, France). the regional biodiversity survey at Eilat, our ability to detect cryptic species of Sinularia and Sarcophyton was further enhanced by the existence of well-sampled msh1 References phylogenies for those genera (McFadden et al. 2006a, Aguilar C, Sańchez JA (2007) Phylogenetic hypotheses of gorgoniid 2009). As other octocoral genera are sampled exhaus- octocorals according to ITS2 and their predicted RNA secondary struc- tively and a database of exemplar sequences grows, our tures. Molecular Phylogenetics and Evolution, 43, 774–786. ability to assign species identifications confidently using Baker AJ, Tavares ES, Elbourne RF (2009) Countering criticisms of single mitochondrial DNA gene barcoding in birds. Molecular Ecology character-based approaches will improve, despite the rel- Resources, 9(Suppl. 1), 257–268. ative lack of interspecific divergence observed in Octo- Bayer FM (1959) A review of the gorgonacean genus Placogorgia Studer, corallia. with a description of Placogorgia tribuloides, a new species from the Neither COI nor an extended barcode encompass- Straits of Florida. Journal of the Washington Academy of Sciences, 49, 54– 61. ing additional, more variable regions of the octocoral Benayahu Y, Yosief T, Schleyer MH (2002) Soft corals (Octocorallia, Alcy- mitochondrial genome (igr1 + msh1) meets all of the onacea) of the southern Red Sea. Israel Journal of Zoology, 48, 273–283. criteria required of an ideal, species-specific genetic Brugler MR, France SC (2008) The mitochondrial genome of a deep-sea marker. Given the poor state of octocoral taxonomy, bamboo coral (Cnidaria, Anthozoa, Octocorallia, Isididae): genome structure and putative origins of replication are not conserved among however, and our current lack of understanding of octocorals. Journal of Molecular Evolution, 67, 125–136. morphological variation in this group, these imperfect Calderoń I, Garrabou J, Aurelle D (2006) Evaluation of COI and ITS mark- molecular markers can and will nonetheless provide ers as tools for population genetic studies of temperate gorgonians. Journal of Experimental Marine Biology and Ecology, 336, 184–197. valuable guidance and assistance, as we search for a Chen I-P, Tang C-Y, Chiou C-Y et al. (2009) Comparative analyses of cod- stable genus- and species-level taxonomy in this ing and noncoding DNA regions indicate that Acropora (Anthozoa: diverse, neglected and ecologically important group of Scleractinia) possesses a similar evolutionary tempo of nuclear vs. organisms. Studies that combine molecular character- mitochondrial genomes as in plants. Marine Biotechnology, 11, 141–152. Concepcion GT, Medina M, Toonen RJ (2006) Noncoding mitochondrial state data (i.e. barcodes) with traditional morphological loci for corals. Molecular Ecology Notes, 6, 1208–1211. taxonomic approaches have already greatly increased Concepcion GT, Crepeau MW, Wagner D, Kahng SE, Toonen RJ (2008) our understanding of species boundaries and intrac- An alternative to ITS, a hypervariable, single-copy nuclear intron in lade relationships and have identified new, diagnostic corals, and its use in detecting cryptic species within the octocoral genus Carijoa. Coral Reefs, 27, 323–336. morphological character states useful for field identifi- DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, spe- cation in several speciose and ecologically important cies delimitation and DNA barcoding. Philosophical Transactions of the genera of octocorals (McFadden et al. 2006a, 2009; Royal Society B, 360, 1905–1916. France 2007). We urge traditional taxonomists and Duenãs LF, Sańchez JA (2009) Character lability in deep-sea bamboo corals (Octocorallia, Isididae, Keratoisidinae). Marine Ecology Progress molecular systematists to continue to work together to Series, 397, 11–23. better understand species boundaries and to construct

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Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Supporting Information Other Methods). Version 4. Sinauer Associates, Sunderland, Massachu- setts. Additional supporting information may be found in the Tavares ES, Baker AJ (2008) Single mitochondrial gene barcodes reliably online version of this article. identify sister-species in diverse clades of birds. BMC Evolutionary Biol- ogy, 8, 81–95. Table S1 Morphospecies of octocorals identified from Thoma JN, Pante E, Brugler MR, France SC (2009) Deep-sea octocorals collections made during a 4-day biodiversity survey at and antipatharians show no evidence of seamount-scale endemism in the NW Atlantic. Marine Ecology Progress Series, 397, 25–35. Eilat, Israel Verseveldt J (1964) Notes on Mediterranean Alcyonium species (Coelenter- ata: Octocorallia). Zoologische Mededelingen Leiden, 39, 155–167, pls. XI– Table S2 Species of Alcyonium included in the compari- XII. son of barcode sequences. Each unique haplotype Verseveldt J (1973) On the validity of Alcyonium siderium Verrill (Coelent- sequenced for a species was deposited in GenBank erata: Octocorallia). Zoologische Mededelingen Leiden, 46, 209–216, pls. 1– 2. Table S3 Deep-sea octocorals analyzed, including loca- Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005) DNA bar- tion and depth of collection coding Australia’s fish species. Philosophical Transactions of the Royal Society B, 360, 1847–1857. Table S4 Primers used to amplify the regions of COI, Watling L (2007) A review of the genus Iridogorgia (Octocorallia: Chrysog- igr1, and msh1 used as molecular ‘barcodes’ orgiidae) and its relatives, chiefly from the North Atlantic Ocean. Journal of the Marine Biological Association of the United Kingdom, 87, 393– Please note: Wiley-Blackwell are not responsible for the 402. Weinberg S (1977) Revision of the common Octocorallia of Mediterranean content or functionality of any supporting information circalittoral. II. Alcyonacea. Beaufortia, 25, 131–166. supplied by the authors. Any queries (other than missing Will KW, Mishler BD, Wheeler QD (2005) The perils of DNA barcod- material) should be directed to the corresponding author ing and the need for integrative taxonomy. Systematic Biology, 54, 844– for the article. 851.

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