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Molecular Ecology (2012) doi: 10.1111/mec.12074 Spatial and temporal patterns of genetic variation in the widespread antitropical deep-sea coral Paragorgia arborea S . H E R R E R A , * † T . M . S H A N K ‡ and J . A . S Á N C H E Z † *Massachusetts Institute of Technology, Woods Hole Oceanographic Institution, Joint Program in Oceanography, 266 Woods Hole Road, Woods Hole, MA 02543, USA, †Laboratorio de Biologia Molecular Marina (BIOMMAR), Departamento Ciencias Biologicas, Universidad de los Andes, Carrera 1E No 18A – 10, Bogota, Colombia, ‡Biology Department, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, MA 02543, USA Abstract Numerous deep-sea species have apparent widespread and discontinuous distributions. Many of these are important foundation species, structuring hard-bottom benthic ecosystems. Theoretically, differences in the genetic composition of their populations vary geographically and with depth. Previous studies have examined the genetic diversity of some of these taxa in a regional context, suggesting that genetic differentiation does not occur at scales of discrete features such as seamounts or canyons, but at larger scales (e.g. ocean basins). However, to date, few studies have evaluated such diversity throughout the known distribution of a putative deep-sea species. We utilized sequences from seven mitochondrial gene regions and nuclear genetic variants of the deep-sea coral Paragorgia arborea in a phylogeographic context to examine the global patterns of genetic variation and their possible correlation with the spatial variables of geographic position and depth. We also examined the compatibility of this morphospecies with the genealogical-phylospecies concept by examining specimens collected worldwide. We show that the morphospecies P. arborea can be defined as a genealogical-phylospecies, in contrast to the hypothesis that P. arborea represents a cryptic species complex. Genetic variation is correlated with geographic location at the basinscale level, but not with depth. Additionally, we present a phylogeographic hypothesis in which P. arborea originates from the North Pacific, followed by colonization of the Southern Hemisphere prior to migration to the North Atlantic. This hypothesis is consistent with the latest ocean circulation model for the Miocene. Keywords: coral, deep sea, DNA barcoding, phylogeography, species, widespread Received 1 June 2012; revision received 28 August 2012; accepted 1 September 2012 Introduction Several marine species, particularly from deep-sea environments, have apparent widespread yet discontinuous distributions (e.g. review by Roberts et al. 2009; Bik et al. 2012). Various mechanisms have been suggested to explain the apparent existence of such species, including recent connectivity among populations mediated by long-distance dispersal (Bucklin et al. 1987; France & Correspondence: Juan A. Sánchez, Fax: +57 1 3394949*2817; E-mail: [email protected] © 2012 Blackwell Publishing Ltd Kocher 1996; Darling et al. 2000; Won et al. 2003; Pawlowski et al. 2007; Lecroq et al. 2009; Etter et al. 2011), large population sizes and similar selective pressures in a stable environment (Bisol et al. 1984; Brinkmeyer et al. 2003; Etter et al. 2011), relatively recent events of colonization mediated by jump dispersal over barriers (Darling et al. 2000; Etter et al. 2011), and cryptic speciation (France & Kocher 1996; Howell et al. 2004). A number of deep-sea coral morphospecies are among these widespread species with discontinuous distributions, for example Lophelia pertusa, Solenosmilia variabilis and Madrepora oculata (Roberts et al. 2009). 2 S . H E R R E R A , T . M . S H A N K and J . A . S Á N C H E Z Deep-sea corals are some of the most conspicuous invertebrate inhabitants of hard-bottom benthic environments worldwide. They are not only more diverse, in terms of number of species, than their shallow counterparts (Cairns 2007), but also they play a fundamental role as foundation species and ecosystem engineers, creating three-dimensional habitats that are occupied by a high diversity of associate species (BuhlMortensen & Mortensen 2005; Costello et al. 2005; Etnoyer & Morgan 2007; Buhl-Mortensen et al. 2010; Shank 2010). Coral ecosystems also support fisheries (D’Onghia et al. 2011; Soeffker et al. 2011) and have been identified as important sources of marine natural products (Leal et al. 2012). Deep-sea corals have evolved in a relatively stable and energy-poor environment; they tend to have slow growth rates (Roberts et al. 2009; Sun et al. 2010), great longevity (Roark et al. 2009) and size-dependent fecundity (Cordes et al. 2001). These characteristics make deep-sea coral ecosystems highly susceptible to disturbance events, especially those generated by human activities, that is, bottom-trawling, deep-sea mining, hydrocarbon extraction, waste disposal, climate change and ocean acidification (reviewed in Ramirez-Llodra et al. 2011). The characterization of spatial distribution patterns of genetic types is of fundamental importance to identify the factors that shape the ranges of deep-sea taxa, and that ultimately drive biodiversity patterns in the ocean (McClain & Mincks 2010). Widespread taxa thus can be used as models to understand how the effects of these factors operate at a global scale. Such information provides critical baseline data with which the potential effects of disturbances on populations inhabiting earth’s largest biome can be assessed. A handful of studies have examined the genetic diversity of deep-sea coral taxa in a regional context (Le Goff-Vitry et al. 2004; Smith et al. 2004; Thoma et al. 2009; Morrison et al. 2011). These have suggested that genetic differentiation does not seem to occur at small geographic scales often associated with discrete features such as individual seamounts or canyons, but presumably at larger scales, that is, broader oceanic regions. However, no studies to date have evaluated such hypotheses throughout the entire known distribution of a putative deep-sea coral species (see Pante & Watling 2012; for a comparison between two distant regions). In this study, we examined the spatial patterns of genetic variation in the widespread bubblegum coral Paragorgia arborea (Linnaeus, 1758) (Octocorallia: Paragorgiidae), which is one of the most prominent coral morphospecies in cold-water sublittoral and bathyal hard-substrate habitats. Paragorgia arborea plays an important ecological role generating microhabitats for numerous species; they are the structural analog of large trees in a rain forest (Buhl-Mortensen & Mortensen 2005; Metaxas & Davis 2005; Watanabe et al. 2009; Buhl-Mortensen et al. 2010). Single colonies of P. arborea can harbour hundreds of individuals from dozens of associated species (e.g. ophiuroids, copepods, shrimp, anemones, polychaetes, ostracods, barnacles, amphipods, hydroids and foraminiferans) (Buhl-Mortensen et al. 2010). The fauna associated with this coral can be two to three times richer than the fauna associated with equivalent shallow-water tropical gorgonians (Buhl-Mortensen & Mortensen 2004, 2005). Paragorgia arborea has been reported from polar, subpolar and subtropical regions of all of the world’s oceans. This conspicuous and locally abundant species can grow massive colonies, which can reach up to 8 m in height (Sánchez 2005). Paragorgia arborea lives in regions of high productivity (Sarmiento & Gruber 2006 depth-integrated primary production > 10 mol/C/m2/ year) and high export fluxes (Sarmiento & Gruber 2006 particle export at 100 m > 2 mol/C/m2/year), water temperatures lower than 12 °C and relatively high local current velocities of 5–30 cm/s (Mortensen & Buhl-Mortensen 2004; Bryan & Metaxas 2006; Etnoyer & Morgan 2007; Roberts et al. 2009; Watanabe et al. 2009). The known distribution of P. arborea in the Northern Hemisphere includes numerous observations in both eastern and western North Atlantic waters and also in the eastern and western North Pacific (WNP), from Japan to the Aleutian Islands and to the Californian seamounts. In the Southern Hemisphere, it has been reported around the Crozet Islands, the Patagonian Shelf and the western South Pacific off New Zealand (Grasshoff 1979; Tendal 1992). Since the publication of these records, both fishing pressure and scientific research in the deep sea have increased significantly, and the number of new records for this species has increased in tandem. Some of these records can now be found in biodiversity databases such as the Ocean Biogeographic Information System (OBIS, http://www. iobis.org) and Global Biodiversity Information Facility (GBIF, http://data.gbif.org); however, many others remain unconsolidated in scattered publications and local databases. Thus, an updated picture of the global distribution of this species is in order. In this study, we provide an up-to-date summary of the global distribution of P. arborea and genetic insights into the global phylogeography of this species. By examining the genealogy of mitochondrial and nuclear genetic variants from specimens collected over nearly its entire known distribution, we tested the compatibility of the morphospecies P. arborea with the genealogical-phylospecies concept. We evaluated the hypothesis that the morphospecies P. arborea is a complex of © 2012 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF DEEP-SEA CORAL PARAGORGIA ARBOREA 3 cryptic species in a barcoding framework. We also examined the global patterns of genetic variation and their possible correlation with the spatial variables of geographic position and depth. We propose a scenario that could explain the observed evolutionary and present-day patterns in this and other species. Methods Global distribution To illustrate the currently known global distribution of Paragorgia arborea, we plotted on a gridded geographic map all the unique records available to date from the databases of the OBIS, the GBIF, the Smithsonian Institution National Museum of Natural History (http://www.mnh.si.edu/rc/), the Yale University Peabody Museum of Natural History (www.peabody. yale.edu), the Harvard University Museum of Comparative Zoology (www.mcz.harvard.edu), the Muséum National d’Histoire Naturelle France (www.mnhn.fr), the National Institute of Water & Atmospheric Research (www.niwa.cri.nz) and several local databases and publications (Bruntse & Tendal 2000; Wareham & Edinger 2007; Mortensen et al. 2008; Roberts et al. 2008; Hibberd & Moore 2009; Laptikhovsky 2011). Geographic coordinates were reconstructed using Google Earth for records with known collection locality, but no latitude and longitude information. Similarly, missing depth data were reconstructed using data from the global GEBCO_08 30 arc-second grid. Molecular methods We analysed a total of 130 specimens of P. arborea available from various museum and laboratory collections (see Table S1, Supporting information). The examined material, collected since 1878, covers most to the known geographic distribution as well as the entire depth distribution of P. arborea (see Table S1, and Figs S1–S4, Supporting information for comparison). For more details on the sequencing of old specimens, see the Appendix S1 (Supporting information). Additional material from other paragorgiid morphospecies was included for comparisons. Total DNA was extracted from dry or ethanol-preserved (70–96%) samples using a CTAB–proteinase K–PCI protocol (Coffroth et al. 1992) or using an automated extraction system (AutoGenprep 965; AutoGen Inc.) as described in the study by Herrera et al. (2010). DNA was eluted in TE buffer and stored at !70 °C. Mutation rates in octocoral mitochondria are significantly lower than in most other organisms (Bilewitch & Degnan 2011). The implication of this lower mutation © 2012 Blackwell Publishing Ltd rate is that mitochondrial markers in octocorals are useful to infer phylogeographic patterns and connectivity at broader spatial and temporal scales. Thus, to maximize the amount of variability captured from the genome of this organelle, we obtained sequences from seven gene regions, amplified by five primer pairs (Herrera et al. 2010), adding up to approximately 3000 base pairs (bp). These regions include the 3′-end of the NADH dehydrogenase subunit 6 (nad6), the nad6-nad3 intergenic spacer (int), the 5′-end of the NADH dehydrogenase subunit 3 (nad3), the 3′-end of the cytochrome c oxidase subunit I (cox1), the 5′-end of the DNA mismatch repair protein – mutS – homolog (mtMutS), two different regions of the large subunit ribosomal RNA (16S) and the 5′-end of the NADH dehydrogenase subunit 2 (nad2). We also sequenced the nuclear ribosomal internal transcribed spacer 2 (ITS2). In octocorals, ITS has been assessed in a number of groups providing enough resolution for diverse phylogenetic inferences (Alcyoniidae McFadden et al. 2001; McFadden & Hutchinson 2004; Nephtheidae van Ofwegen & Groenenberg 2007). ITS2 has also provided enough resolution for intraspecific and phylogeographic studies in Caribbean shallowwater octocorals (Sánchez et al. 2007; Gutiérrez-Rodriguez et al. 2009). Furthermore, ITS2 has also provided valuable information for the analysis of genetic structure of deep-sea corals (Le Goff-Vitry et al. 2004; Miller et al. 2011). Polymerase chain reactions (PCR) and sequencing reactions for mitochondrial gene regions were performed following the protocols used by Herrera et al. (2010). ITS2 PCR amplicons, from a subset of 19 geographically representative individuals, were examined to assess the possibility of intragenomic variants through denaturing gradient gel electrophoresis (DGGE). Gels contained 8% polyacrylamide, 19 TAE buffer and a linear urea–formamide denaturing gradient from 45% to 80%. The gels were pre-ran at 60 °C and 90 V for 30 min, followed by 13 h at 60 °C and 90 V. Gels were stained with ethidium bromide for 15 min and visualized using a Bio-Rad Chemidoc system. PCR products from DGGE-excised bands were subsequently cleaned and sequenced. Complementary chromatograms were assembled and edited using the SEQUENTM CHER 4.8 software (Gene Codes Corp.). Sequences of each region were aligned independently using MAFFT 6.8 (Katoh et al. 2002). The G-INS-i and Q-INS-i algorithms (gap opening penalty = 1.53, offset value = 0.07) were employed for the protein coding and ribosomal regions, respectively. Secondary structures of ribosomal regions were inferred to improve the alignments, following the protocols used in the study by Herrera et al. (2010). To correct possible mistakes, all 4 S . H E R R E R A , T . M . S H A N K and J . A . S Á N C H E Z alignments of protein coding sequences were visually inspected and translated to amino acids in GENEIOUS 5.3 (Drummond et al. 2010), using the genetic code of Hydra attenuata (Pont-Kindon et al. 2000). No unusual stop codons or suspicious substitutions were identified, suggesting that no nuclear pseudogenes were sequenced (Lopez et al. 1994; Bensasson et al. 2001). Mitochondrial sequences were concatenated for each individual and treated as one single locus in most subsequent analyses, given that the mitochondrial genome is assumed to be nonrecombining. Mitochondrial and ITS2 genetic variants, with alignment gaps included as an informative state (Giribet & Wheeler 1999), were identified using DNASP 5.0 (Librado & Rozas 2009) and will be referred hereafter as haplotypes. Gene trees and molecular clock To evaluate the compatibility of P. arborea with the functional definition of genealogical-phylospecies sensu De Queiroz (2007), that is, all alleles of a given locus in individuals of P. arborea being ‘descended from a common ancestral allele not shared with those of other species’ (Avise & Ball 1990; Baum & Shaw 1995), we performed independent phylogenetic analyses of the mitochondrial and ITS2 haplotypes. Homologous sequences from eight other paragorgiid morphospecies were included as outgroups (see Table S1, Supporting information). Phylogenetic estimation was performed using Bayesian inference (BI) in MRBAYES 3.12 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) as implemented in the CIPRES portal (http://www.phylo. org). Most likely nucleotide substitution models were selected for each region based on the Akaike Information Criteria (AIC) as implemented in JMODELTEST 2.0. Models for the mitochondrial regions are shown in Table S2 (Supporting information). The general time reversible model with a gamma-distributed rate variation across sites (GTR+G) was selected for the ITS2. Default prior distribution settings were assumed for all parameters. Four independent analyses of 10 000 000 Monte Carlo Markov chain (MCMC) generations (94 chains) were run with a sampling frequency of 1000 generations (burn-in = 25%). Combined BI analysis of the mitochondrial locus was performed with explicit character partitions for each concatenated region, along with their independently selected models of evolution. To account for the rate variation among partitions (Marshall et al. 2006), we allowed the rates to vary under a flat Dirichlet prior distribution (ratepr = variable). The parameters of nucleotide frequencies, substitution rates, gamma shape and invariant site proportion were unlinked across partitions. MCMC runs were analysed in the program TRACER 1.5 (Rambaut & Drummond 2007). Convergence was indicated by the ‘straight hairy caterpillar’ (Drummond et al. 2007) shape of the stationary posterior-distribution trace (generations vs. log-likelihood) of each parameter. Other examined convergence and mixing diagnostics included the standard deviation of partition frequencies (<0.01), the potential scale reduction factor (PSRF) (ca. 1.00), the effective sample sizes (EES) (>200) and the similitude of posterior probabilities of specific nodes between different runs in the program AWTY (http://ceb.csit.fsu.edu/awty) (Nylander et al. 2008). High correlations between runs and no obvious trends in the split frequency plots were observed. Tree files for each run were combined, after burn-in, using the program LOGCOMBINER v1.7.1 (Drummond et al. 2012). The most probable trees were summarized into a maximum clade credibility tree using TREEANNOTATOR v1.7.1 (Drummond et al. 2012). A Bayesian-MCMC joint estimation of gene genealogy and divergence times was performed in BEAST 1.7.1 (Drummond et al. 2012) for the mitochondrial marker assuming the same substitution model mentioned above. We assumed an uncorrelated relaxed lognormal molecular clock model, which allows for the variation in mutation rates among branches, with the Yule model of constant speciation rate (Yule 1925; Gernhard 2008) and the coalescent model of constant population size (Kingman 1982), as the tree priors. Additional sequences from specimens of the sister family, Coralliidae, were added to estimate divergence time within the phylogeny as this family contains some of the few fossils available for Octocorallia. The coralliid node was calibrated implementing a normal prior distribution for the time to the most recent common ancestor (TMRCA) with a mean of 83.5 million years before present (Myr BP) and a standard deviation of 0.7, corresponding to Campanian age stratum, in which the oldest known fossil in this family has been found (Schlagintweit & Gawlick 2009). Three MCMC independent analyses were run for 30 000 000 generations with a sampling frequency of 3000 (burn-in = 25%). Convergence diagnostics (generations plot and EES) were also examined for the combined runs in TRACER 1.5 (Rambaut & Drummond 2007) as mentioned above. The most probable trees were summarized into a maximum clade credibility tree with median node heights using TREEANNOTATOR v1.7.1 (Drummond et al. 2012). To infer the historical patterns of dispersal in P. arborea, we used the Bayesian phylogeography framework proposed by Lemey et al. (2009), as implemented in BEAST 1.7.1 (Drummond et al. 2012). We mapped the geographic ocean region where each haplotype was sampled to the time-scaled mitochondrial gene genealogy, which was inferred with the assumption of an uncorrelated relaxed lognormal molecular © 2012 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF DEEP-SEA CORAL PARAGORGIA ARBOREA 5 clock and the coalescent constant-population-size tree prior, as explained above. This framework allows the reconstruction of discrete states of geographic location for ancestral nodes by posterior probability estimation. Barcoding and species delimitation To test for the possibility of cryptic species in the morphospecies P. arborea, we calculated pairwise uncorrected distances among individuals of P. arborea and other paragorgiid morphospecies for the mtMutS and cox1 sequences, as proposed by McFadden et al. (2011), in PAUP* 4.0b10 (Swofford 2002). Neighbour-joining trees were built using the calculated distances. We also examined the ITS2 secondary structures for the presence of compensatory base changes (CBCs) using the visualization program 4SALE (Seibel et al. 2006); CBCs are altered pairings in a helix of the secondary structure of the ITS2 RNA transcript, and empirical work has suggested that they could be used as indicators of species boundaries in most metazoans (Muller et al. 2007; Coleman 2009). We also used the coalescent-based species delimitation method described by Pons et al. (2006) and Monaghan et al. (2009), as implemented in the SPLITS R-package (available from http://r-forge.r-project.org/ projects/splits/). This likelihood method is based on a general mixed Yule-coalescent (GMYC) model, which estimates phylospecies boundaries in a clock-constrained calibrated tree by identifying increases in branching rates (looking forward in time). Such increases are assumed to be characteristic of transition points between interspecific speciation–extinction processes and intraspecific coalescent processes, that is, populations (Pons et al. 2006; Monaghan et al. 2009). Single- and multiple-threshold models with explicit and upper and lower limits for the estimation of scaling parameters (0 and 10, respectively) were used in the analysis of the time-calibrated trees obtained with the Yule and coalescent models tree priors. Genetic variability Genetic variability among individuals and populations was measured for each locus according to the haplotype diversity (h) and genetic diversity (average number of pairwise differences hp) indices (Tajima 1983) using ARLEQUIN 3.5 (Excoffier & Lischer 2010). Fu’s Fs statistic was calculated to determine whether the observed pattern of polymorphism was consistent with a neutral model of evolution (Tajima 1989; Fu 1997). Global FST statistics were calculated to evaluate for possible differentiation in the genetic composition among populations worldwide. Pairwise comparisons of population differ© 2012 Blackwell Publishing Ltd entiation were made in Arlequin and significance values estimated after 1000 permutations. To visualize the spatial patterns of genetic variation for each marker, the specimens were colour-coded according to haplotype, their geographic collection coordinates were plotted using IMAP v3.5 (Biovolution), and their collection depth was plotted on an X-Y scatter plot. To assess the amount of variability in the populations of P. arborea that was represented in our samples, we generated haplotype accumulation curves (Gotelli & Colwell 2001) by calculating estimates of the mean and variance for the number of accumulated haplotypes through 1000 random permutations, using the program R-package SPIDER v1.1 (Brown et al. 2012). Results Global distribution A total of 341 high-confidence geographic location records of Paragorgia arborea were gathered (see Fig. S3, Supporting information). Paragorgia arborea is an antitropical taxon, occupying a band between 30° and 70° degrees of latitude in both hemispheres. These bands are, in general, areas of high surface primary productivity and export (Sarmiento & Gruber 2006). Most records of P. arborea are from depths shallower than 1000 m, indicating a preference for upper-bathyal environments. Despite the fact that other coral species that share part of their ranges with P. arborea have been commonly observed in tropical and subtropical regions (e.g. Lophelia pertusa and Madrepora oculata), P. arborea has never been found in these areas. This suggests that the currently known distribution of P. arborea is not a result of undersampling at lower latitudes. Molecular data A total of 92 specimens were positively screened for the mitochondrial marker. The concatenated mitochondrial alignment for the morphospecies P. arborea had a length of 2922 bp, of which 2881 were invariable sites (pairwise identity of 99.7%); eight sites were parsimonyinformative. The mean ungapped sequence length was 2917.9 bp (SD = 2.8 bp), with a range of 2910 and 2921 bp. The G-C content was 39.5%. The only noncoding region in the mitochondrial locus data set, the nad6nad3 intergenic spacer (int), contained one indel, but no nucleotide substitutions. The ITS2 was successfully sequenced for 48 specimens, of which 83% overlapped with the mitochondrial set. The ITS2 alignment had a length of 312 bp, of which 301 were invariable (pairwise identity of 99.2%); 35 sites were parsimony-informative. The mean ungapped sequence length for ITS2 was 6 S . H E R R E R A , T . M . S H A N K and J . A . S Á N C H E Z 282 bp (SD 0.9 bp), ranging between 280 and 284 bp. The G-C content was 41.1%. No intragenomic variability was revealed, using DGGE, in the ITS2. The predicted secondary structure of ITS2 showed the characteristic shape of a helicoidal ring with four helixes (Coleman 2007); stems III and IV were particularly long in this species (Fig. S5, Supporting information). The number of haplotypes and genetic diversity estimates for each population with both loci are shown in Figs 1 and 2. The nad6-int-nad3 region (hereafter referred as nad6 for simplicity) contained most of the variable sites (21) and the greatest number of haplotypes (11) found in the individuals with complete mitochondrial data sets. Haplotype differences were also located in the ITS2: one at helix I, three at helix III and five at helix IV; the remaining two were free nucleotides at the structure main ring (Fig. S5, Supporting information). Gene genealogies and phylogeographic history Individual mitochondrial gene trees were largely congruent, although resolution was generally low (Fig. S6, Supporting information). The inferred phylogeny based on each independent loci (i.e. concatenated mitochondrial and ITS2) highly supported the monophyly of P. arborea (Fig. 3) and had much greater clade resolution. Both Bayesian and neighbour-joining analyses inferred the same evolutionary relationships. Branch lengths were appreciably shorter within the clade of P. arborea, when compared to the ones among morphospecies. Relationships within Paragorgia were not fully resolved, particularly among Paragorgia wahine, Paragor- gia yutlinux and Paragorgia sp. 1. The systematic relationships of Paragorgia spp. are outside of the scope of this study and will not be further discussed. The time-scaled trees estimated assuming the coalescent model of constant population size had, in general, shorter shallower and longer deeper branches than the tree estimated assuming the Yule model of constant speciation rate (Figs S7 and S8, Supporting information). Consequently, the time to the TMRCA of the genus Paragorgia was estimated to be 61 Myr BP (95% CI: 31–101) under the coalescent model and 54 Myr BP (95% CI: 41–94) under the Yule model. The TMRCA of P. arborea based on the coalescent model and the Yule model was 10.1 Myr BP (95% CI: 4.4–18.8) and 14.1 Myr BP (95% CI: 6.7–26.3), respectively. The Bayesian phylogeographic analysis indicates that the lineage of P. arborea likely originated in the North Pacific (posterior probability 0.38, see Fig. 4). Dispersal to the South Pacific and subsequent colonization of the North Atlantic likely occurred between the midMiocene and early Pliocene. Genetic distances, CBCs and GMYC Maximum uncorrected genetic distances among conspecifics and minimum uncorrected genetic distances among congeners were used to measure the intraspecific and interspecific variation, respectively, of mtMutS and cox1 sequences as in the study by McFadden et al. (2011). The maximum distances within P. arborea, 0.3% for mtMutS and 0.9% for cox1, were in general smaller than the distances among morphospecies of Paragorgia, Fig. 1 The global geographic distribution of mitochondrial haplotypes in Paragorgia arborea. The gene tree in the centre of the figure shows the inferred relationships among haplotypes. Each haplotype is indicated by a different colour. Framed circles represent individuals. Pie charts indicate the frequency of haplotypes in each population (global region): North Atlantic (NA), South Atlantic (SA), South Pacific (SP), western North Pacific (WNP) and eastern North Pacific (ENP). The size of each pie is proportional to the number of samples from each population (n). The number of haplotypes (H), haplotype diversity (h), genetic diversity (hp) and the Fu’s Fs statistic are also indicated. © 2012 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF DEEP-SEA CORAL PARAGORGIA ARBOREA 7 Fig. 2 The global geographic distribution of nuclear internal transcribed spacer 2 haplotypes in Paragorgia arborea. The gene tree in the centre of the figure shows the inferred relationships among haplotypes. Each haplotype is indicated by a different colour. Framed circles represent individuals. Pie charts indicate the frequency of haplotypes in each population: North Atlantic (NA), South Indian Ocean (SI), South Pacific (SP), western North Pacific (WNP) and eastern North Pacific (ENP). The size of each pie is proportional to the number of samples from each population (n). The number of haplotypes (H), haplotype diversity (h), genetic diversity (hp) and the Fu’s Fs statistic are also indicated. NP NP NP NP NP NP NP NP SP SP NP SP SA NA SP SP NA NA NA Time (million years) Fig. 3 Unrooted gene tree hypotheses for the mitochondrial (top) and nuclear internal transcribed spacer 2 (bottom) markers in Paragorgia. which ranged between 0.5–6.1% and 0–2.3% for mtMutS and for cox1, respectively (Table S3, Supporting information). The minimum distances between P. arborea © 2012 Blackwell Publishing Ltd Fig. 4 Maximum clade credibility ultrametric time-scaled mitochondrial gene tree for Paragorgia arborea. Branch colours show the most probable location states: North Atlantic (NA) in blue, South Pacific (SP) in green, South Atlantic (SA) in violet and North Pacific (NP) in orange. Pie charts show the posterior probabilities of location states for each ancestral node (total pie area = 1). The most probable location state of each node is also indicated. and the other morphospecies of Paragorgia ranged between 1.8–5.1% for mtMutS and 0.6–1.8% for cox1. A similar pattern was observed for ITS2 distances, 0.6% in 8 S . H E R R E R A , T . M . S H A N K and J . A . S Á N C H E Z intraspecific comparisons within P. arborea (maximum distances) and 3.3–7.4% in interspecific comparisons among morphospecies of Paragorgia. The minimum distances between P. arborea and the other morphospecies ranged between 3.3 and 5.6%. The predicted ITS2 secondary structure was essentially the same for all haplotypes of P. arborea and no CBCs or hemi-CBCs were observed. A transition point between species- and populationlevel branching patterns was identified, by the singlethreshold GMYC method, at ca. 21 Myr BP for the time-scaled mitochondrial gene genealogy estimated with the Yule model tree prior (Fig. S7, Supporting information). For the time-scaled gene genealogy estimated with the coalescent model tree prior, this transition point was inferred to be at ca. 16 Myr BP (Fig. S8, Supporting information). In both cases, the GMYC model showed a marginally significant (i.e. a = 0.05) better fit to the data than the null model of uniform coalescent branching rates (LR = 4.84, d.f. = 3, P = 0.18, compared to LR = 5.84, d.f. = 3, P = 0.12, respectively). The implementation of a multiple-threshold GMYC model did not yield a significantly (i.e. a = 0.05) better fit than the single-threshold GMYC for either case (v2 = 2.55, d.f. = 3, P = 0.47 and v2 = 0.83, d.f. = 3, P = 0.84, respectively). Spatial patterns of genetic variation Overall, in P. arborea, 16 haplotypes were defined based on the mitochondrial locus and 11 based on the ITS2. The genealogical relationships among haplotypes inferred by the Bayesian inference did not reveal reciprocal monophyly of the specimens from Northern and Southern Hemispheres, or from different oceans, for example Pacific vs. Atlantic (Figs 1 and 2). In fact, a number of haplotypes were shared across large geographic spans. Genetic diversity, as measured by the haplotype diversity (h, the probability that two randomly chosen haplotypes are different in a population sample) and the average number of nucleotide differences between all pairs of haplotypes in the sample (hp), was highest in the western North Pacific Ocean (mitochondrial h = 0.74, SD = 0.11, and hp = 7.44, SD = 0.11; nuclear h = 0.75, SD = 0.14, and hp = 2.68, SD = 1.81) and South Pacific Ocean (SP; mitochondrial h = 0.79, SD = 0.03, and hp = 6.37, SD = 3.10; nuclear h = 0.73, SD = 0.09, and hp = 1.64, SD = 1.15) regions, intermediate in the North Atlantic Ocean (NA; mitochondrial h = 0.26, SD = 0.12, and hp = 0.46, SD = 0.42; nuclear h = 0.71, SD = 0.11, and hp = 1.02, SD = 0.82) and lowest in the eastern North Pacific Ocean (ENP; mitochondrial hp = 0.00; nuclear h = 0.49, SD = 0.18, and hp = 1.16, SD = 0.91) (Figs 1 and 2). Overall, the relative levels of genetic diversity for both loci were highly similar (see Figs 1 and 2). No significant deviations from neutrality were found in either locus (i.e. a = 0.05). Mitochondrial haplotypes m15 and m12 were shared between the NA and SP regions, although their frequencies were dissimilar (Fig. 1). Haplotype m15 was the dominant form in the NA, with a frequency of 0.86, whereas only one specimen was found having the m12 variant. In the SP, these haplotypes represented two of the three most common ones, with frequencies of 0.26 for m15 and 0.31 for m12. The rest of the haplotypes in these regions represented private alleles, that is, variants exclusive to a particular area. The haplotype from the single specimen from the South Atlantic Ocean (SA) region was a private allele. No haplotypes from the North Pacific Ocean (NP) were shared with other regions. Within the NP region, there was a clear break between western and eastern subregions, separated by the Alaska Peninsula. All haplotypes within these subregions represented private alleles. Two dominant haplotypes were found in the WNP, m2 and m7, with frequencies of 0.5 and 0.21, respectively. In the ENP, there was a single haplotype. In the ITS2 data set, regional differences were less pronounced than in the mitochondrial data set but, similarly, the haplotype frequencies varied greatly among regions (Fig. 2). Haplotypes i1 and i6 had near-cosmopolitan distributions. Haplotype i1 was found within all regions with frequencies of 0.23 (NA), 0.67 [South Indian Ocean (SI)], 0.07 (SP), 0.25 (WNP) and 0.09 (ENP). Haplotype i6 was found in specimens from the NA, SP and ENP, with frequencies of 0.15, 0.07 and 0.73, respectively. Haplotype i2 was shared between NA and SP, being dominant in both regions, with frequencies of 0.54 and 0.43, respectively. Lastly, haplotype i5 was found in both the NA (frequency = 0.09) and the SI (frequency = 0.33). Private alleles were found in the Pacific, with high frequencies in the SP (i7, frequency = 0.36) and the NWP (i8, frequency = 0.5), but none were found in the NA or SI. No detectable differentiation in the haplotype distributions could be explained by depth differences (Fig. 5). Haplotype accumulation curves revealed that the global mitochondrial and nuclear ITS2 diversities have not been fully sampled, as indicated by the steep slopes of both lines (Fig. S9, Supporting information). Both markers showed similar levels of diversity at given sampling efforts, ITS2 being slightly lower than the mitochondrial. When the individual genes in the mitochondrial data set were examined individually, it was clear that there are significant differences in their contributions to overall diversity estimates and that a single one does not capture the diversity found in the combined mitochondrial marker. By far, the gene that © 2012 Blackwell Publishing Ltd 0 0 500 500 Depth (m) Depth (m) PHYLOGEOGRAPHY OF DEEP-SEA CORAL PARAGORGIA ARBOREA 9 1000 1500 1000 1500 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 Mitochondrial haplotype 1 2 3 4 5 6 7 8 ITS2 haplotype 9 10 11 Fig. 5 The distribution of mitochondrial (left) and nuclear internal transcribed spacer 2 ITS2 (right) haplotypes of Paragorgia arborea with depth. Individuals are represented by dots. Each haplotype is indicated by a different colour, as in Figs 1 and 2. The prefix m denotes mitochondrial haplotypes, and the prefix i denotes nuclear ITS2 haplotypes (haplotype numbers are equivalent to the ones in Table S1, Supporting information). captures the largest mitochondrial diversity in terms of haplotypes in P. arborea is nad6 (11), followed by nad2 (6), mtMutS (5), 16S (4) and cox1 (3). The combination of nad6 and 16S captures 14 haplotypes, and the addition of nad2 to these two regions captures all 16 haplotypes found in the combined mitochondrial marker. Discussion The data and analyses generated in this study showed that the morphospecies Paragorgia arborea can be defined as a genealogical-phylospecies, in contrast to the hypothesis that P. arborea represents a cryptic species complex. Genetic variation in this lineage is correlated with geographic location at the basin-scale level, but not with depth. We present a phylogeographic hypothesis for P. arborea in which this independently evolving lineage originates from the North Pacific, followed by colonization of the Southern Hemisphere prior to migration to the North Atlantic. We argue that this hypothesis is consistent with the latest ocean circulation model for the Miocene. A globally distinct evolving lineage? The distinction among species, incipient species and structured populations in many deep-sea invertebrates remains contentious due to the difficulties in defining the species boundaries for certain groups and to the paucity of the genetic, ecological and taxonomic data available to date (Vrijenhoek 2009; McFadden et al. 2011). Commonly used species concepts (e.g. biological species concept) have been traditionally developed in terrestrial models. However, the biological and ecological information required to apply such concepts to deepsea organisms (e.g. reproductive success, behaviour) is, at this time, impracticable to obtain. It is now recognized that a combination of morphologic and phylogenetic criterions is most practical to discern among deep-sea coral species (e.g. Herrera et al. 2010; Pante & Watling 2012). © 2012 Blackwell Publishing Ltd Here, we examined, for the first time, the compatibility of traditional taxonomical identifications and molecular information in a putative deep-sea coral species at a global scale. The mitochondrial and nuclear gene trees have congruent topologies, showing that alleles of P. arborea have a common ancestor not shared with other paragorgiid morphospecies. Thus, the morphospecies P. arborea is compatible with the genealogicalphylospecies concept. Furthermore, the branch lengths among haplotypes of P. arborea are much shorter than the branches among other putative species, which is to be expected for genetic variability within a phylospecies. The only consistent morphological variant corresponds to the populations found in the NP, which were previously referred as Paragorgia pacifica Verrill, 1922 but synonymized with P. arborea by Grasshoff (1979). Individuals from these populations seem to have reduced sclerite size and ornamentation when compared to the characteristics that defined P. arborea prior to Grasshoff synonymizing the two (Sánchez 2005). Our data do not support P. pacifica as a valid species (see discussion below); we rather suggest that it may represent a subspecies. Taken together, this evidence indicates that P. arborea is a globally distinct lineage, implying that identifications based on morphology can accurately distinguish this taxon. A complex of cryptic species? The presence of cryptic species complexes has been detected in various presumed widespread marine morphospecies (e.g. clams Goffredi et al. 2003; isopods Raupach et al. 2007; limpets Johnson et al. 2008; gastropods Duda et al. 2009; Vrijenhoek 2009). Despite being morphological indistinguishable, cryptic species have been detected through molecular data, on the basis of genetic dissimilarity. Here, we tested for the possibility of cryptic species within the specimens of P. arborea, by analysing the pairwise uncorrected genetic distances among haplotypes using a DNA barcoding framework 10 S . H E R R E R A , T . M . S H A N K and J . A . S Á N C H E Z based on the cox1 and mtMutS gene regions (see McFadden et al. 2011). Under this framework, pairwise uncorrected distances greater than 1% for mtMutS or cox1 genes can be confidently used to indicate cryptic species (McFadden et al. 2011). Based on this threshold, the maximum intraclade distances (0.3% for mtMutS and 0.9% for cox1) among specimens of P. arborea do not suggest the presence of cryptic species. These distances are consistent with the intraspecific distances found within other paragorgiid species, for example 0.8% for mtMutS in Sibogagorgia cauliflora (Herrera et al. 2010). However, the suggested threshold is unidirectional, meaning that distances smaller than 1% do not imply the absence of species boundaries. Additional genetic and biological data are needed to test for this possibility. The uniformity of predicted ITS2 secondary structures and the absence of CBCs or hemi-CBCs are also consistent with intraspecific levels of variation (Muller et al. 2007; Coleman 2009; Ruhl et al. 2010). However, similar to the mitochondrial barcoding threshold discussed above, this criterion is also unidirectional; thus, the absence of cryptic species is not implied (Coleman 2009). Lastly, the branching transition points inferred by the GMYC likelihood method indicate that the lineage of P. arborea is independently evolving with a branching pattern characteristic of a population-level coalescent process (Figs S7 and S8, Supporting information). In summary, the levels and patterns of genetic variability in mitochondrial and ITS2 loci do not provide actual evidence for cryptic species boundaries within P. arborea. Global patterns of genetic variation Genetic diversity in P. arborea is not randomly distributed, as it would be expected under a scenario of global panmixia. It is highest at the ENP and SP populations and lowest at WNP and NA populations, as indicated by the haplotype diversity and the average pairwise differences among alleles (Figs 1 and 2). The significantly high global FST value (0.61 for the mitochondrial locus and 0.39 for the ITS2, see Table 1) indicates that there are significant differences in the genetic composition among worldwide populations, when defined at the basin/regional scale. This is consistent with the results from other studies of deep-sea corals (e.g. Smith et al. 2004; Thoma et al. 2009; Miller et al. 2011; Morrison et al. 2011). However, to test over scales smaller than regional for genetic structuring, it will be necessary to examine larger numbers of independent, highly variable markers (e.g. Le Goff-Vitry et al. 2004 and Morrison et al. 2011). Regional geographic differences were sorted out by comparing the FST values of genetic differentiation among populations (Table 1). The pairwise differences Table 1 Global and pairwise FST values for the mitochondrial (top) and nuclear ITS2 (bottom) markers among populations of Paragorgia arborea Mitochondrial FST global NA SP WNP ENP Nuclear ITS2 FST global NA SP WNP ENP SI 0.61 NA SP WNP 0.27 0.67 0.98 0.39 0.67 0.74 ENP 0.39 NA SP WNP ENP 0.16 0.47 0.27 0.13 0.51 0.43 0.31 0.48 0.24 0.26 SI NA, North Atlantic; SA, South Atlantic; SP, South Pacific; WNP, western North Pacific; ENP, eastern North Pacific; SI, South Indian Ocean; ITS2, internal transcribed spacer 2. All values are significant (i.e. a = 0.05). among populations for both markers suggest strong differentiation between the EPN population and all the other populations, including the neighbouring WNP. There is also a significant break between North and South Pacific populations. South Pacific and North Atlantic populations are the less dissimilar, which suggests a more recent connection between them. Gene genealogies of P. arborea showed no reciprocal monophyly of alleles among populations. Two nonexclusive and equally plausible mechanisms could have lead to this observed pattern: (i) gene flow between populations for which recent connectivity could be conceived given a temporal continuity of favourable environmental conditions, and (ii) incomplete lineage sorting caused by a rapid succession of divergence events among populations, combined with large ancestral effective population sizes (Maddison 1997; Edwards 2009). The earliest, divergent lineage of alleles as well as the highest genetic diversity was found in the WNP, which lends support to the idea that P. arborea originated in this region. The nuclear ITS2 showed signs of lower genetic differentiation among populations than the mitochondrial locus. The effects of differing effective population sizes on processes such as genetic drift and genetic sweeps could explain this difference given that the mitochondrial genome has one quarter the effective population size of the nuclear genome (given that it is haploid and assuming maternal inheritance only). Similarly, the nuclear gene tree had much lower resolution compared © 2012 Blackwell Publishing Ltd P H Y L O G E O G R A P H Y O F D E E P - S E A C O R A L P A R A G O R G I A A R B O R E A 11 to the mitochondrial one, which is likely due to the smaller number of phylogenetically informative sites present in the short ITS2 sequence. In contrast to patterns observed in other deep-sea organisms (Cho & Shank 2010; Etter et al. 2011; Miller et al. 2011), depth does not appear to be an important large-scale structuring factor in populations of P. arborea. This is perhaps not surprising given the widespread distribution of this organism, which suggests that it is capable of living under a relatively broad range of conditions. Alternatively, as mentioned above, small-scale genetic structuring related to depth could be revealed with higher-resolution markers. The mitochondrial nad6 gene contained the greatest amount diversity in terms of haplotypes in this data set, that is, was the most variable mitochondrial marker. This result contrasts with previous studies, in which the mtMutS gene has been found to be significantly more variable than any other mitochondrial gene region (France & Hoover 2001; McFadden et al. 2004, 2010; Herrera et al. 2010). We suggest that the levels of variation among different mitochondrial gene regions in octocorals vary among taxa (see McFadden et al. 2010, 2011; Bilewitch & Degnan 2011), and thus, there is not a single universal region that provides the largest amount of variability. For the samples of P. arborea examined here, we found that the combination of nad6 + 16S +nad2 is the most informative. The nuclear ITS2 still seems to be a good cost-effective alternative to detect genetic variation among individuals, in the absence of intragenomic variants. Phylogeographic hypothesis Here, we suggest a phylogeographic scenario in which P. arborea originated in the North Pacific, possibly in the WNP followed by colonization of the South Pacific and spreading eastward around the Southern Hemisphere in a stepping stone fashion (possibly via the Antarctic Circumpolar Current). The colonization of the North Atlantic seems to have occurred through a more recent dispersal event from the South Pacific, via the Central American Seaway, or from the SA. Similarities between other deep-sea coral taxa from the South Pacific and the North Atlantic have been independently observed (Thoma et al. 2009; Pante & Watling 2012), which gives support to the idea of a more recent connection between South Pacific and North Atlantic deep-sea communities. This scenario is an alternative to the trans-Arctic interchange hypothesis (Vermeij 1991), which suggests a recent North Pacific and North Atlantic connection as indicated by the distributions of several shallow-water taxa (e.g. red algae Vanoppen et al. 1995; asteroids, bivalves, gastropods, barnacles © 2012 Blackwell Publishing Ltd Wares & Cunningham 2001; seagrass Olsen et al. 2004; cnidarians Govindarajan et al. 2005). Paragorgia arborea shares a similar phylogeographic history and genetic diversity patterns with the spiny dogfish Squalus acanthias (Verissimo et al. 2010) and the bryozoan Membranipora membranacea (Schwaninger 2008), both of which have modern antitropical distributions. The time of divergence between the WNP and South Pacific populations of the spiny dogfish has been estimated to be around 7.8 Myr BP and approximately 13.3 Myr BP (9.9–21.9) for the bryozoan, which is comparable to our estimates of 4.5 Myr BP (95% CI = 2.0– 8.3 using the coalescent model) and 8.1 Myr BP (95% CI = 3.6–15.3 using the Yule model) for P. arborea (Fig. 4, Figs S7 and S8, Supporting information). The timing of colonization of the North Atlantic has been estimated to be between 3.6 and 5.3 Myr BP for the dogfish, 6.2 Myr BP (95% CI = 4.6–10.2) for the bryozoan and between 1.7 Myr BP (95% CI = 0.4–3.6) and 4.0 Myr BP (95% CI = 1.3–8.3) for P. arborea. The similar and independently estimated times for these events give support to the idea that a common set of oceanographic conditions in the Miocene and early Pliocene lead to the current distributions of these species. The latest Miocene ocean circulation models (Butzin et al. 2011) indicate that there was a dominant southward horizontal flow that carried deep waters from the WNP to the South Pacific, passing along the eastern side of the New Zealand landmass, during the mid- to late Miocene (~5–15 Myr BP). This flow decreased during the late Miocene. The Antarctic Circumpolar Current started to develop during the mid- to late Eocene (ca. 37 –40 Myr BP) (Scher 2006) and thus was already well established as the dominant feature of ocean circulation during the Miocene, transporting massive amounts of water eastward. At the same time, during the mid-Miocene, the deep-water formation in the North Atlantic and its southward flow were absent or weak, likely due to the dominant barotropic water flux from the Pacific to the Atlantic. The formation of deep water in this time period mainly took place in the Southern Ocean. Deepwater formation in the North Atlantic and the dominant southward flow, as we know them today, were later established during the late Miocene as the Central American Seaway closed (Butzin et al. 2011). Evolutionary migrations inferred from genetic diversity patterns presented here for P. arborea are consistent with this history of ocean circulation. Historical changes in the global patterns of ocean circulation and climate may have caused shifts in the habitat and thus the distribution of P. arborea. Widespread ocean cooling during glacial periods in the late Miocene–early Pliocene (Mercer & Sutter 1982) and throughout the Quaternary (Ehlers et al. 2011) could have aided the trans-equatorial 12 S . H E R R E R A , T . M . S H A N K and J . A . S Á N C H E Z exchange by increasing the area of suitable habitat for stepping stone populations towards the tropics (McIntyre et al. 1989). Isolated relict low-latitude populations might still exist. We hypothesize that the described set of conditions could explain the current distribution patterns of many other marine taxa (e.g. deep-sea coral associates, such as ophiuroids and chirostylid crabs) and thus might have played an important role shaping extant deep-sea faunal diversity. Acknowledgements Support for this study was generously provided by a mini-grant from the Global Census of Marine Life on Seamounts Project (CenSeam) to J.A.S. and S.H., a grant from the Facultad de Ciencias, Department of Biological Sciences of the Universidad de los Andes to J.A.S, the National Systematics Laboratory of NOAA’s National Marine Fisheries Service, a Smithsonian Graduate Student Fellowship to S.H., an award from the Systematics Research Fund of the Systematics Association and the Linnean Society of London to S.H., and a Grantin-Aid of Research from the Sigma Xi Research Society to S.H. We are especially thankful to S.D. Cairns, A.G. Collins, C.L. Agudelo, N. Ardila, L. Dueñas, A. Ormos, J. Hunt, L. Weigt, L. Monroy, M. Herrera and M. Sangrey for their generous support, assistance and advise. Laboratory work was performed at the Laboratories of Analytical Biology NMNH, Smithsonian Institution and BIOMMAR, Universidad de los Andes. Samples were generously provided by P. Alderslade (CSIRO), A. Andouche (MNHN), A. Andrews (MLML), A. Baco (FSU), A. Baldinger (MCZ), J. A. Boutillier (DFO), S.D. Cairns (USNM), S. Davies (DFO), M. Eriksson (UUZM), Y. Imahara (WPMNH), D. Janussen (SMF), E. Lazo-Wasem (YPM), P. Lozouet (MNHN), L. Lundsten (MBARI), S. Mills (NIWA), K. Schnabel (NIWA), and B. Stone (NOAA), D. Tracey (NIWA), and R. Weber (Te Papa Tongarewa). We also thank J. McDermott, N. Roterman and C. Munro for their comments on earlier versions of the manuscript. We are grateful for the helpful input from the editor and two anonymous reviewers. References Avise JC, Ball RM (1990) Principles of genealogical concordance in species concepts and biological taxonomy. 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Yule GA (1925) A mathematical theory of evolution, based on the conclusions of Dr. J.C. Willis. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 213, 21–87. S.H. and J.A.S. conceived and designed the study. S.H. performed the research, analysed the data and wrote the article with contributions from T.M.S. and J.A.S. © 2012 Blackwell Publishing Ltd Data accessibility Sample information and locations are provided as supporting information in Table S1 (Supporting information). DNA sequences are available in GenBank and accession numbers appear in Table S1 (Supporting information). DNA sequence alignments are available in DRYAD doi:10.5061/dryad.ns23j. Supporting information Additional Supporting Information may be found in the online version of this article. Appendix S1 Sequencing of old samples. Table S1 Collection and sequence information for the specimens used in this study. Table S2 Nucleotide substitution models for mitochondrial gene partitions, as selected by the AIC criterion in JMODELTEST. Table S3 Interspecific and intraspecific (i.e. coalescent depths) uncorrected pairwise distances (%) among haplotypes of species of Paragorgia and Sibogagorgia. Fig. S1 Sampling location of specimens of Paragorgia arborea examined in this study. Fig. S2 Depth distribution of samples shown in Fig. S1 (Supporting information). Fig. S3 The revised geographic distribution of Paragorgia arborea. Fig. S4 Depth distribution of records shown in Fig. S3 (Supporting information). Fig. S5 Predicted ITS2 secondary structure of Paragorgia arborea. Fig. S6 Individual mitochondrial gene tree hypotheses in Paragorgia. Fig. S7 Fit of the GMYC single-threshold model to the mitochondrial time-calibrated gene tree generated with the Yule model tree prior. Fig. S8 Fit of the GMYC single-threshold model to the mitochondrial time-calibrated gene tree generated with the coalescent model tree prior. Fig. S9 Haplotype accumulation curves in Paragorgia arborea.