Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae)

Giuseppe  Bonomolo

The northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina aciculata complex are here revised and consist of at least 3 species. The type species, Ocinebrina aciculata (Lamarck, 1822), characterized by a slender shell with rounded whorls and primary and secondary spiral cords of approximately similar size, lives throughout the northeastern Atlantic and Mediterranean Sea at depths usually ranging between 0 and 105 m. Its synonymy is here stabilized by a neotype selection for Murex corallinus Scacchi, 1836. Ocinebrina corallinoides Pallary, 1912 (=Ocinebrina buzzurroi Cecalupo and Mariani, 2008, new synonymy), characterized by a strongly elongate and weakly convex shell and primary and secondary spiral cords of approximately similar size, is endemic to the Gulf of Gabès and is here considered a distinct species, pending genetic studies. Ocinebrina reinai n. sp. is here described from the central Mediterranean Sea (where it is sympatric with O. aciculata) on the basis of morphological diagnostic features of shell (rarest presence of labral tooth, commoner presence of infrasutural apertural denticle, dark spots on the ribs and spiral sculpture with differently sized primary and secondary cords and smaller threads) and radula, confirmed by genetic data. Divergence in COI sequences with sympatric samples of O. aciculata (>7%), confirm their status as a distinct species. A comparative table reporting diagnostic features of the congeneric species of the complex and those with which the new species was previously misidentified is offered.

The genus Muticaria Lindholm, 1925, is currently distributed either in Southeastern Sicily or in the Maltese islands and comprises the species M. syracusana (Philippi, 1836), M. neuteboomi Beckmann, 1990 and M. macrostoma (Cantraine, 1835). For the first time, we report a molecular study on the topotypicous populations of M. syracusana and M. neuteboomi carried out on fragments of the ribosomal 16S rDNA subunit and the cytochrome oxydase I (COI) mitochondrial genes by Neighbour Joining, Maximum Likelihood, Maximum Parsimony and Bayesian Inference algorithms. Our results revealed the existence of nucleotide-sequence divergence (Dxy: 5% for 16S rDNA and 12% for COI sequences) between the two taxa.

bs_bs_banner Zoological Journal of the Linnean Society, 2013. With 4 figures Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae) ANDREA BARCO1, ROLAND HOUART2, GIUSEPPE BONOMOLO3, FABIO CROCETTA4 and MARCO OLIVERIO1* 1 Department of Biology and Biotechnology ‘C. Darwin’, University of Rome ‘La Sapienza’, Viale dell’Università 32, I-00185 Rome, Italy 2 Belgian Royal Institute of Natural Sciences, Rue Vautier, 29, B-1000 Bruxelles, Belgium 3 Via delle Terme 12, I-60035 Jesi, Italy 4 Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Napoli, Italy Received 27 March 2013; revised 2 July 2013; accepted for publication 9 July 2013 We used a molecular phylogenetic approach to investigate species delimitations and diversification in the mussel drills of the Ocinebrina edwardsii complex by means of a combination of nuclear (internal transcribed spacer 2, ITS2) and mitochondrial [cytochrome oxidase subunit I (COI) and 16S] sequences. Our sample included 243 specimens ascribed to seven currently accepted species from 51 sites. Five of the samples were from either the type locality of a nominal species or a close nearby locality (O. edwardsii from Corsica, O. carmelae and O. piantonii from the Kerkennah Islands, O. hispidula from the Gulf of Gabès and O. leukos from the Canary Islands), one from the inferred original locality (O. ingloria from Venice Lagoon), and specimens assigned in the recent literature to O. nicolai. We used a combination of distance- and tree-based species delimitation methods to identify Molecular Operational Taxonomic Units (MOTUs) to compare with the a priori species identifications. The consensus tree obtained by BEAST on the COI alignment allows the recognition of several distinct clades supported by the three species delimitation methods employed. The eight-MOTUs scenario, shared by the Automatic Barcode Gap Discovery (ABGD) and Generalized Mixed Yule-Coalescent (GMYC) methods, comprises the following major clades: clade A contains the south Tunisian species Ocinebrina piantonii Cecalupo, Buzzurro & Mariani from which the sympatric taxon O. carmelae Cecalupo, Buzzurro & Mariani (new synonym) cannot be separated; clades B and C bring together all populations from the Aegean Sea and some from the Ionian Sea, respectively; clade D groups, on the one hand, the south Tunisian samples morphologically assigned to O. hispidula Pallary and, on the other, Atlantic and Alboran Sea samples (including the Canarian taxon O. leukos Houart); clade E includes a sample from the type locality of O. edwardsii and several samples from the Tyrrhenian Sea; clades F and G correspond to a few samples from the Venice Lagoon and the Tyrrhenian Sea, respectively; clade H groups the bulk of samples from the Adriatic Sea, including samples from the Venice Lagoon morphologically identified as Ocinebrina ingloria (Crosse), and some from the Ionian Sea. No final conclusions could be reached to reconcile the currently recognized morphological taxa with the clades suggested by the COI data. The geographical structure proposed by the mitochondrial markers is similar to that found in other marine invertebrates and partially corresponds to the species defined by shell characters. We propose here a framework for the revision of the Ocinebrina edwardsii species complex, suggesting a geographical pattern for the diversification of this group in the studied area. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 doi: 10.1111/zoj.12069 ADDITIONAL KEYWORDS: ABGD – cytochrome oxidase I – DNA-barcoding – GMYC – Mediterranean Sea – species delimitation. *Corresponding author. E-mail: marco.oliverio@uniroma1.it © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 1 2 A. BARCO ET AL. INTRODUCTION The existence of sibling species among marine invertebrates is a widely recognized phenomenon (Knowlton, 1993), emphasized by the increasing use of DNA-based methods for species delimitation (Vogler & Monaghan, 2007). These techniques are contributing to the steady number of new marine species described (Costello, Wilson & Houlding, 2012), allowing the recognition of unknown diversity (Caron et al., 2012) and testing the status of known species (Templeton, 2001). Cryptic diversity, however, does not surface exclusively in poorly known lineages (Richards et al., 2012) or in organisms from relatively unexplored areas (Goetze, 2003). Even within the well-known fauna of the Mediterranean Sea, with its long-lasting tradition of taxonomic studies, many species have turned out to be complexes of sibling lineages (Boisselier-Dubayle & Gofas, 1999; Carreras-Carbonell, Macpherson & Pascual, 2005; Calvo et al., 2009; Sá-Pinto et al., 2010; Xavier et al., 2011). Molluscan taxonomy, in particular, historically has been almost entirely based on shell characters, which are still commonly used even though morphological variation caused by adaptation to environmental pressures has been documented (Vermeij, 1978). Consequently, traditional shell-based taxonomic practice has proved to be problematic for molluscs, and particularly for morphologically diverse groups. Therefore, in several of the most recent studies on marine molluscs, the combination of molecular and traditional approaches has proven to be successful to resolve taxonomically doubtful groups (e.g. Puillandre et al., 2009; Claremont et al., 2011; Zou, Li & Kong, 2012). The genus Ocinebrina Jousseaume, 1880 is widely distributed in the Mediterranean Sea, northeastern Atlantic and northeastern Pacific, with species commonly called small mussel drills. It includes 23 currently accepted living species: 16 in the northeastern Atlantic and Mediterranean and seven in the northeastern Pacific (WoRMS: Appeltans et al., 2012, at http://www.marinespecies.org – accessed on 13 March 2013). The Pacific species are often attributed to other genera, such as Ocenebra Gray, 1847 and Urosalpinx Stimpson, 1865 (two of the c. 29 genera in the subfamily Ocenebrinae Cossmann, 1903: see, for example, Radwin & D’Attilio, 1976; Vermeij & Vokes, 1997; but see WoRMS – accessed on 13 March 2013 – for an updated listing), and their relationships will be dealt with in an ongoing study on the phylogenetics of the Ocenebrinae Cossmann, 1903. The northeastern Atlantic and Mediterranean species are traditionally divided into two groups of morphological affinities: the O. aciculata complex and the O. edwardsii complex. The O. aciculata complex, recently revised by Crocetta et al. (2012), includes O. aciculata (Lamarck, 1822), O. corallinoides Pallary, 1912 and O. reinai Bonomolo and Crocetta, 2012. The O. edwardsii complex includes the following species: O. edwardsii (Payraudeau, 1826), O. carmelae Cecalupo, Buzzurro & Mariani, 2008, O. helleri (Brusina, 1865), O. hispidula Pallary, 1904, O. hybrida (Aradas & Benoît, 1876), O. ingloria (Crosse, 1865), O. inordinata (Houart & Abreu, 1994), O. leukos Houart, 2000, O. miscowichae Pallary, 1920, O. nicolai Monterosato 1884, O. paddeui Bonomolo & Buzzurro, 2006, O. piantonii Cecalupo, Buzzurro & Mariani, 2008 and O. purpuroidea Pallary, 1920 (see Table 2 for a state of the art nomenclature). Ocinebrina edwardsii is traditionally considered to be a common species and one of the most variable European muricids, as witnessed also by the existence of numerous names established for the various morphotypes (see Table 2), whose status has been debated extensively (see, for example, Houart, 2001). Its distribution spans the entire Mediterranean Sea and, in the Atlantic Ocean, it is recorded up to the Bay of Biscay and to the Canary Islands. The shell reaches (and sometimes exceeds) 20 mm in length with up to 5–5.5 teleoconch whorls, has a sealed siphonal canal, five to six small denticles on the internal part of the outer lip and variable coloration. The protoconch is paucispiral (1.25–1.75 whorls) and its morphology is similar to that of O. aciculata. Therefore, a development similar to that described for O. aciculata by Franc (1940) can be assumed, i.e. entirely intracapsular or with a very short pelagic phase. The combination of the high morphological variability of the shell among and within species over a wide geographical range (see Supporting Information Figs S4-S6) has contributed to the confused taxonomy in the O. edwardsii complex. Here, we have applied DNA-based methods to a sample including 243 specimens from the northeastern Atlantic and the Mediterranean Sea (including the area of the type locality of Purpura edwardsii, Corsica) in order to define the limits of this species and check for the presence of cryptic lineages. We also included specimens morphologically ascribed to some of the nominal species of the same complex to investigate their relationships with O. edwardsii: O. ingloria from the Venice Lagoon, O. hispidula from the Gulf of Gabès, O. carmelae and O. piantonii from the Kerkennah Islands, O. leukos from the Canary Islands and O. miscowichae from southern Morocco. In addition, we also processed specimens assigned in the recent literature to O. cf. nicolai and O. nicolai from Spain and Portugal (Rolán, 1983; Afonso et al., 2011; Gofas, 2011), although their morphology was not perfectly matching that of the nominal taxon. Using three © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII markers [mitochondrial cytochrome oxidase subunit I (COI) and 16S, and the nuclear internal transcribed spacer 2 (ITS2)], we applied several methods of species delimitation and used the results to discuss the status of the O. edwardsii complex. We used the ocinebrine Nucella lapillus (Linné, 1758) as an outgroup in the phylogenetic analyses, and also included specimens of Ocinebrina aciculata and O. reinai as recently defined by Crocetta et al. (2012). MATERIAL AND METHODS SAMPLE DATA The specimens used in this work were collected by hand, snorkelling or scuba diving. Sample localities are summarized in Table 1 (see also Figs 1 and 4) with their tissue-processing codes. Each specimen was identified on collection and fixed in 96–100% ethanol. A piece of tissue was later dissected from the foot for DNA extraction. DNA SEQUENCING AND ALIGNMENT DNA extraction was performed after tissue digestion in proteinase K using a phenol–chloroform protocol (Hillis, Moritz & Mable, 1996) with slight modifications as described by Oliverio & Mariottini (2001b). A fragment of the mitochondrial COI was amplified using the universal primers LCO1490 and HCO2198 (Folmer et al., 1994), part of the mitochondrial 16S was obtained with the primers 16SA (Palumbi et al., 2002) and CGLeuUURR (Hayashi, 2005), and the entire nuclear ITS2 rRNA gene was amplified using the primers ITS-3d and ITS-4r (Oliverio & Mariottini, 2001a). Polymerase chain reaction (PCR) amplifications were performed in 25 μL containing 2.5 μL of BIOLINE 10× buffer, 0.5 μL of 10 mM deoxynucleoside triphosphates (dNTPs) mix, 0.4 μL of each primer (10 mM), 2.5–3 μL of 50 mM MgCl2, 1 U of BIOLINE TaqPolymerase and 0.5–1 μL of genomic DNA. The DNA fragments were amplified with an initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C (COI and 16S) and 62 °C (ITS2) for 40 s and extension at 72 °C for 50 s. These cycles were followed by an extension at 72 °C for 10 min. PCR products were purified with the ExoSAP protocol. A mix of 2 μL containing 20 U μL−1 Exonuclease I (New England Biolabs, Ipswich, MA, USA) and 1 U μL−1 Shrimp Alkaline Phosphatase (Roche, Basel, Switzerland) was used to purify 5 μL of PCR product. Fragments were sequenced by Macrogen Inc. (Seoul, South Korea) using the same PCR primers in BigDye terminator cycling conditions (Applied Biosystems, Carlsbad, CA, USA). Reacted products were purified 3 using ethanol precipitation and run using an automatic sequencer AB3730XL (Applied Biosystems). Forward and reverse sequences were assembled and reciprocally edited with Sequencher (v. 4.1.4; Gene Codes Corporation, Ann Arbor, MI, USA). The COI dataset was aligned manually and translated into amino acids to check for stop codons. The 16S and ITS2 dataset was aligned with MAFFT (Katoh et al., 2002) using the Q-INS-i algorithm (Katoh & Toh, 2008), which accounts for secondary structures in the sequences. The nucleotide substitution models were selected by jModelTest (Posada, 2008) using the Bayesian Information Criterion (Schwarz, 1978). The likelihood scores for 88 possible substitution models were calculated with PhyML (Guindon & Gascuel, 2003) using a tree obtained separately for each model with BIONJ (Gascuel, 1997) employing a JC model (Jukes & Cantor, 1969). SPECIES DELIMITATION Three methods of species delimitation were applied to identify Molecular Operational Taxonomic Units (MOTUs) (Blaxter, 2004) in the COI dataset: the Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al., 2012a), the Generalized Mixed Yule-Coalescent (GMYC) technique (Pons et al., 2006) and statistical parsimony network analysis (Posada & Crandall, 2001; Templeton, 2001). ABGD is a distance-based method designed to detect the so-called ‘barcode gap’ in the distribution of pairwise distances calculated in a COI alignment (Puillandre et al., 2012a, b). A distance value corresponding to the most reliable gap was used to group the sequences in MOTUs. The web-based ABGD program (available at http://wwwabi.snv.jussieu.fr/ public/abgd/) was employed to generate a preliminary partition of sequences, using a distance matrix calculated from the COI dataset. The COI alignment was submitted and processed in ABGD (excluding the outgroup O. miscowichae) using the Kimura twoparameter (K2P) model (Kimura, 1980) and the following settings: a prior for the maximum value of intraspecific divergence between 0.001 and 0.1, 25 recursive steps within the primary partitions defined by the first estimated gap, and a gap width of 0.1. The GMYC method identifies the species– population boundary in a sample of multiple species and populations using a time-calibrated (ultrametric) tree and a likelihood statistic (Pons et al., 2006). The boundary represents the shift of the tree branching rates from a Yule (interspecific) to a coalescent (intraspecific) model, and is estimated as the likelihood peak of the transition along the branches. An ultrametric tree based on the COI alignment (excluding the outgroup O. miscowichae) was generated with © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 N Sampling locality, environmental data Coordinates 902 903 904 905, 923 907 909 920, 1299 921 922 924 927 936 965, 1058, 1059, 1061, 1062 959 960 961 962 963, 1033 5 5 5 3, 4 11 16 3, 7 2 7 4 6 3 1, 1, 10, 3, 2 Italy, Venice Lagoon, low tide on/under stones Tunisia, Djerba Island, south-east of Adjim, 0.5 m depth, amidst Posidonia rhizomes Italy, Augusta, Megara harbour, 0–1 m depth, on/under stones Italy, Marzamemi, harbour, 0–1 m depth, on/under stones Italy, Siracusa, Punta Asparano, 1 m depth, cave, amidst calcareous algae Italy, Lampedusa Island, Cala Francese, low depth, in cave Italy, Pozzuoli, ‘o’ valjone’ harbour, 0.5 m depth, on/under rocks amidst algae Italy, Bacoli, Punta Pennata, 0.5 m depth, amidst mussels Italy, Marina di Schiavonea, 0–1 m depth, on/under rocks and amidst mussels Italy, Marsala, 0–1 m depth, under stones Greece, Limnos Island, 0–1 m depth, on/under stones Greece, Heraklion, Lygaria, Crete, 0–1 m depth, on/under stones Spain, Bayona, Cabo Silleiro, low tide, on rocks 45°17′18″N, 33°41′49″N, 37°13′50″N, 36°44′30″N, 36°59′32″N, 35°29′43″N, 40°49′18″N, 40°47′35″N, 39°38′29″N, 37°47′50″N, 39°49′58″N, 35°23′56″N, 42°06′40″N, 012°16′18″E 010°47′25″E 015°13′27″E 015°07′03″E 015°15′41″E 012°37′26″E 014°07′08″E 014°05′11″E 016°34′00″E 012°25′45″E 025°15′55″E 025°01′41″E 008°53′57″W 4 6 4 3 1, 5 Croatia, Črvar, 0–1 m depth, on/under stones Croatia, Katoro, 0–1 m depth, on/under stones Italy, Gallipoli, Punta Pizzo, 0–1 m depth, on/under stones Italy, Muggia, Punta Sottile, 0–1 m depth, on/under stones Spain, Vigo, low tide, on rocks 45°27′14″N, 45°27′56″N, 40°00′01″N, 45°36′20″N, 42°13′52″N, 013°31′03″E 013°30′31″E 017°59′37″E 013°43′10″E 008°42′44″W 969 1026 1029 1044 1045 1046, 1293 2 4 2 5 5 5, 7 Italy, Marettimo Island, Cammello shoal, 10 m depth, under stones Italy, Campomarino, 0–1 m depth, on/under stones Croatia, Umag, Sol Stella Maris, 0–1 m depth, on/under stones Tunisia, Sfax, fishing nets residuals, from Posidonia meadows Italy, Pugnochiuso, 0–1 m depth, on/under stones Italy, Pellestrina, Venice Lagoon, low tide under stones 37°59′21″N, 012°13′55″E 40°18′03″N, 017°30′13″E 45°27′02″N, 013°30′45″E 34°42′N, 010°42′E 41°47′14″N, 016°11′22″E 45°15′13″N, 012°17′57″E 1048 1049 1050 1051 1052 1053 1054 1055 1063 5 6 5 1 1 1 1 7 4 Tunisia, Djerba Island, Borj Kastil, 0.5 m depth, amidst Posidonia rhizomes Italy, Siracusa, harbour, 0.5 m depth, amidst mussels Croatia, Murter Island, 1–10 m depth, on/under rocks Tunisia, Kerkennah Islands, Ra’s Bu Numah, 0.5–0.8 m depth, amidst Posidonia rhizomes Tunisia, Kerkennah Islands, Borj el-Hissar, −0.5 m, under stones Tunisia, Kerkennah Islands, Ra’s ‘Āmir, −0.5 m, near a small stone Tunisia, Kerkennah Islands, near Kraten, low tide in very quiet waters, sandy beach Greece, Milos Island, low depth, on/under rocks Spain, O Grove, low depth, on/under rocks 33°41′00″N, 010°51′27″E 33°04′N, 015°17′E 43°47′48″N, 015°36′34″E 34°47′00″N, 011°12′56″E 34°42′42″N, 011°09′06″E 34°40′60″N, 011°07′00″E 34°43′N, 011°11′E 36°39′43″N, 024°25′41″E 42°30′N, 008°52′W 1064 1065 1106 1107 1109 1280 1281 1282 1290 1291 1294 1295 1296 1298 1300 1308 1133 1060 1037, 1042 1038 1039 187 1 5 4 3 3 5 1 2 2 3 1 6 3 6 4 3 5 6 1, 1 1 1 1 Croatia, Cres Island, Punta Križa, low depth, on/under rocks Portugal, Peniche, Peniche de Cima beach, very low tide, rocky bottom, under and on rocks Italy, Sardina, Santa Maria Navarrese, 3 m depth, on rocks Italy, Zannone Island, 10 m depth, on rocks Italy, Marettimo Island, Punta Mugnone, 18 m depth, under stones Spain, Benalmádena, 1–5 m depth, on rocks Spain, Calahonda, low tide, amidst rocks and algae Spain, Barbate, low tide, amidst rocks Spain, Tenerife Islands, La Caleta, low depth, on/under rocks, Spain, Tenerife Islands, Punta de Teno, −15 m Portugal, off Ponta da Piedade, low depth, on/under rocks Italy, Cefalù, low tide on rocks France, Corsica, Porto Vecchio, Saint-Cyprien beach, 0-0.5 m depth, on granitic rocks Greece, Kavala, −0.5 m, under rocks Italy, Palinuro, tide level amidst algae Italy, Sardinia, Cagliari, Marina Piccola, harbour, −2 m, on rocks Morocco, Dakhla, low water, beach, on rocks Spain, Bayona, Cabo Silleiro, low tide, on rocks Italy, Tor Paterno MPA, −25 m Italy, Tor Paterno MPA, −25 m Italy, Tor Paterno MPA, −20 m UK, Portobello, intertidal 44°38′29″N, 014°30′20″E 39°22′15″N, 009°22′18″W 39°59′15″N, 009°41′26″E 40°57′54″N, 013°03′26″E 37°59′15″N, 012°01′28″E 36°35′03″N, 004°31′07″W 36°29′04″N, 004°41′08″W 36°11′00″N, 005°56′09″W 22°28′00″N, 016°47′00″W 28°20′31″N, 016°55′23″W 37°03′32″N, 008°38′54″W 38°00′57″N, 014°06′55″E 41°36′N, 009°17′E 40°54′49″N, 024°22′42″E 40°02′07″N, 015°17′07″E 39°11′28″N, 009°09′53″E 23°41′N, 015°55′W 42°06′40″N, 008°53′ 57″W 41°36′18″N, 012°20′30″E 41°36′21″N, 012°20′28″E 41°36′13″N, 012°20′30″E 55°57′N, 030°6′W A priori morphological identification MOTU O. ingloria O. hispidula O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii (partim) O. nicolai (partim) sensu Rolán (1983) O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii (partim) O. nicolai (partim) sensu Rolán (1983) O. edwardsii O. edwardsii O. edwardsii O. hispidula O. edwardsii O. edwardsii IV H III D II C II C II C II C III E, G III E IV H III E II B II B III D O. hispidula O. edwardsii O. edwardsii O. piantonii O. carmelae O. hispidula O. hispidula O. edwardsii O. edwardsii (partim) O. nicolai (partim) sensu Rolán (1983) O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. nicolai sensu Gofas (2011) O. leukos O. edwardsii O. nicolai sensu Afonso et al. (2011) O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. edwardsii O. miscowichae O. aciculata O. aciculata O. reinai O. reinai Nucella lapillus IV IV IV IV III H H H H D III E II C IV H III D IV H III F IV H III D II C IV H IA IA III D III D III D III D IV H III D III E III E III E III D III D III D III D III D III D III E III E II B III G III E – – – – – – A. BARCO ET AL. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 BAU voucher code 4 Table 1. Collection sites with relevant BAU voucher codes, number of assayed specimens (N), their preliminary identification on collection and the Molecular Operational Taxonomic Unit (MOTU) in which they were included, according to the four-MOTUs (I–IV) and eight-MOTUs (A–H) hypotheses (see text). Vouchers from each sampling site are figured in Supporting Information Figs S4–S6. CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII 5 Figure 1. Location map of the sampling sites. Numbers of the sites as in Table 1. BEAST (Drummond & Rambaut, 2007), using the substitution model proposed by jModelTest. A relaxed lognormal clock, a substitution rate fixed to one (no calibrations were used as our aim was to estimate the branching rates only) and a constant coalescent prior (which is thought to be more conservative than a Yule prior for species delimitation: see Monaghan et al., 2009) were used. Four BEAST runs were performed, each with 108 generations sampling every 104. Two samples of 104 trees were obtained, and convergence was evaluated, reading the log files with Tracer (Rambaut & Drummond, 2003), to verify that the effective sample size (ESS) values were > 200. Trees from all the runs were combined with LogCombiner and then summarized in a maximum credibility tree with TreeAnnotator (both included in the BEAST package). The consensus tree was analysed, applying the single-threshold GMYC function from the SPLITS package (Ezard, Fujisawa & Barraclough, 2009) in R (http://www.R-project.org). Having found no incongruence between ITS2 and COI single-gene analyses (see below), we also analysed with the same settings the combined COI + ITS2 dataset, where the clock and substitution model were unlinked for each gene, and the substitution models suggested in jModelTest were used. The statistical parsimony network analysis calculates the maximum number of mutational steps constituting a parsimonious connection between two haplotypes (Posada & Crandall, 2001; Templeton, 2001). The haplotypes are then joined into networks following the algorithms proposed by Templeton, Crandall & Sing (1992), and those separated by more mutational steps (i.e. with probability of secondary mutations exceeding 5%) remain disconnected. This method has been used previously to differentiate species in a mixed sample (Pons et al., 2006; Hart & Sunday, 2007). It was applied to our COI dataset (excluding the outgroup O. miscowichae) using TCS (Clement, Posada & Crandall, 2000) with a 95% limit of tolerance. We also analysed the ITS sequences for fixed sites between MOTUs defined in the species delimitation tests using DNAsp (Librado & Rozas, 2009) PHYLOGENETIC ANALYSIS Bayesian phylogenetic trees for each single gene and for the combined COI + ITS2 and COI + ITS2 + 16S datasets were estimated using MrBayes (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). Sequences of Nucella lapillus (Linnaeus, 1758) were used to root the trees and, where available, sequences of O. aciculata, O. reinai and O. miscowichae were included to test their relationships with the species included in this study. Two Metropolis-coupled Markov chain Monte Carlo (MC3) algorithms, with four chains each, were run for 8 × 107 generations using the substitution models selected by jModelTest, and sampling every 8 × 103. Convergences of the chains were evaluated by plotting values of the standard deviation of average split frequencies, and with the Potential Scale of Reduction Factor (PSRF) (Gelman & Rubin, 1992). All the analyses in MrBayes were computationally intensive, and thus run on the University of Oslo Bioportal (Kumar et al., 2009). © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 6 A. BARCO ET AL. To test for incongruence among trees obtained with single-gene analyses, we compared clades recovered with high support in each topology. Conflict among clades with high support can be seen as evidence for divergent evolutionary histories, and can be used as an argument against multiple-gene phylogenetic analyses. However, we found no conflict among single-gene trees, and combined the datasets to estimate Bayesian topologies using COI + ITS2 and COI + ITS2 + 16S. RESULTS SEQUENCE ANALYSIS We obtained 208 original sequences of COI, 58 of 16S and 155 of ITS2, and included in our analyses 38 COI sequences of O. cf. edwardsii and O. hispidula from a previous study (Barco, Corso & Oliverio, 2013). Ten COI sequences of O. aciculata and O. reinai from Crocetta et al. (2012) were included in the phylogenetic analyses, together with six new COI sequences of O. aciluata from Spain (BAU 1060). The COI alignment spanned 658 bp (no stop codon detected), 167 of which were variable and 133 were parsimony informative; the 16S alignment comprised 763 bp, 83 of which were variable and 58 were parsimony informative; the ITS2 alignment comprised 559 sites, 157 of which were variable and 66 were parsimony informative. Accession numbers for the sequences are provided in Supporting Information Table S1. The substitution models selected by jModelTest were the TPM2uf + I + Γ (Kimura, 1981) for COI, HKY + Γ (Hasegawa, Kishino & Yano, 1985) for ITS2 and TIM3 + I + Γ (Posada, 2003) for 16S. The distances for the ABGD analysis on the COI dataset were obtained using the K2P model (Kimura, 1980) implemented in the online software. SPECIES DELIMITATION The ABGD method on the 236 COI sequences proposed nine distinct groupings ranging from 67 MOTUs to a single one depending on the value of the maximum intraspecific divergence used as a boundary between inter- and intraspecific distance values. The results obtained for K2P values lower than 0.012 were discarded for the unrealistic assumption of more than 10 species. The histogram of the pairwise distances (Fig. 2) shows a multimodal distribution for the frequencies, with a relevant minimum for values of K2P distance between 0.020 and 0.025, corresponding approximately to the partitions 15–17 (K2P distance = 0.014–0.021, eight MOTUs) and 18 (K2P distance = 0.026, four MOTUs). All of the four BEAST runs reached ESS values of > 200, and the ‘burnin’ was set for one-half of the Figure 2. Histogram of the frequency of Kimura twoparameter (K2P) genetic divergence in all pairwise comparisons of cytochrome oxidase subunit I (COI) sequences. generations. A final sample of 104 trees was used to obtain the maximum clade credibility tree shown in Figure 3. The single-threshold model in GMYC was significantly better than the null model of no shift in branching rates (P < 0.001). The GMYC method proposed eight MOTUs (confidence interval, 2–18). In addition, in the combined dataset (COI + ITS2), the GMYC method performed better than the null model, but the dataset was divided into only three MOTUs (confidence interval, 1–36; Supporting Information Fig. S1). The 238 COI sequences comprised 70 distinct haplotypes, 37 of which were unique. Using a 95% cut-off, the haplotypes were grouped by statistical parsimony analysis into nine independent networks, considered here as equivalent to nine MOTUs (Fig. 3). The MOTUs defined by the three species delimitation methods in the eight-MOTUs scenario, based on the COI alignment, are named I–IV (four-MOTUs scenario: ABGD) and A–H (eight-MOTUs scenario: ABGD, GMYC, TCS) in Figure 3. Concordance was found among the three analyses, with only a single difference: group D was recovered as a single MOTU in the ABGD and GMYC analyses, but split into two by TCS (D1 and D2). In the two-genes (COI + ITS2) GMYC test, the threshold time for the coalescent/Yule boundary was lower (–0.044) than in the single-gene test (–0.01), resulting in the groups A + B + C and D + E + F + G, respectively, clustered into two MOTUs (Fig. S1). Conserved sites in ITS2 were calculated for the MOTUs suggested in the species delimitation analysis (data not shown). Fixed differences were found only between more distantly related groups (e.g. B–D1, B–D2 and B–F), whereas most sister groups © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII 7 Figure 3. Consensus tree of a final sample of 104 trees obtained by BEAST on the cytochrome oxidase subunit I (COI) alignment. The black bars on the right delimit the Molecular Operational Taxonolllmic Units (MOTUs) as defined by the three species delimitation methods employed [Automatic Barcode Gap Discovery (ABGD), Generalized Mixed YuleCoalescent (GMYC) and TCS]. Posterior supports > 0.95 are reported, and only for the nodes subtending the MOTUs. Voucher shells for each MOTU are figured (shells not to scale). 3A, MOTUs A–C; 3B, MOTUs D1–H. showed no fixed differences, usable as diagnostic for each MOTU. PHYLOGENETIC ANALYSIS In the MrBayes analysis, convergence was reached by the sampling chains in almost all analyses, as shown by the average standard deviation of split frequencies lower than 0.01 and by PSRF values of 1.00 for each parameter estimated by the MC3 algorithm. However, for the single-gene ITS2 analysis, the convergence was difficult to reach even after setting a starting tree, increasing the number of generations, the number of MCMC chains or the λ value. In all the analyses, we employed a ‘burnin’ of about 25% of the sampled trees, whereas, in the ITS2 analysis, we used a 50% ‘burnin’. The single-gene Bayesian analyses produced three topologies (Supporting Information Fig. S2). In the COI and ITS2 analyses, using Nucela lapillus as an outgroup, the O. aciculate complex (O. aciculata and O. reinai) and the O. edwardsii complex were reciprocally monophyletic. In particular, in the COI analysis, O. miscowichae was defined as the sister taxon to the remaining lineages of the O. edwardsii complex. We obtained a high congruence between the COI and 16S trees (Fig. S2A,C), whereas the topology of ITS2 (Fig. S2B) was divergent and less resolved. Groups A–H of the BEAST-GMYC test were also obtained in the COI and 16S trees with high posterior probability (> 0.96), except for group G, which was unresolved in the 16S tree. In the ITS2 tree, none of the clades obtained with the other alignments was recovered. The limited amount of sequence variability observed in ITS2 probably prevented clear resolution of the phylogenetic signal in this alignment. We found no incongruence among highly supported clades in the single-gene trees, and thus also computed the combined-gene trees. In the combined-gene analyses (COI + ITS2, Supporting Information Fig. S3A; COI + ITS2 + 16S, Fig. S3B), we obtained two topologies similar to those of the COI and 16S single-gene analyses. In both cases, groups E and G of the BEAST-GMYC test had a moderate support (between 0.90 and 0.95), whereas all the others had high support values (> 0.96). © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 8 A. BARCO ET AL. Figure 3. Continued. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII DISCUSSION As a starting point to define the threshold between intraspecific and interspecific pairwise comparisons, we searched those values (or range of values) that separated sympatric samples falling in distinct clades. The minima in the multimodal distribution for the frequencies of the pairwise distances (Fig. 2) corresponding to values higher than 0.031, defined in ABGD, partition with a single species for all of our samples. This is an unrealistic scenario which would classify the Tunisian specimens identified as O. piantoni and O. carmelae as conspecific with the sympatric specimens of O. hispidula, despite a K2P distance of > 0.09 in sympatry. A single-species hypothesis would also produce a complete lack of geographical pattern in the phylogenetic trees, with northern Sicily and Tyrrhenian samples more closely related to Adriatic and (some) Tunisian ones than to southern Sicily and Aegean samples. Conversely, the values of the K2P distance between 0.020 and 0.026 corresponded approximately to the partitions 15–17 (K2P distance = 0.014–0.021, eight MOTUs) and 18 (K2P distance = 0.026, four MOTUs). The eightMOTUs scenario was the same as that depicted by the GMYC method, and the four-MOTUs scenario was within the confidence limits (2–18 MOTUs) of the GMYC results. For COI, the K2P distance range representing the intra-/interspecific boundary in the eight-MOTUs/ four-MOTUs scenarios (0.014–0.026) is similar to that obtained from the analysis of the COI sequences in other studies on marine gastropods (Castelin et al., 2010; Reid, Dyal & Williams, 2010; Williams et al., 2011; Nuñez et al., 2012; Puillandre et al., 2012b). This suggests that our COI distance values represent a reliable boundary between congeneric Ocinebrina species. However, deep mitochondrial divergences within other mollusc species have been repeatedly observed (e.g. Quattro et al., 2001; Wilson, Schrödl & Halanych, 2009), raising the need for support with nuclear data before taking taxonomic decisions. Several previous studies have provided evidence of a general concordance between results from COI and ITS2 sequence analyses and their usefulness in the species delimitation of molluscs (Calvo et al., 2009; Puillandre et al., 2010; 2011). However, it is also known that too large or too small an intraspecific variation of ITS2 may confound the detection of cryptic species (Vilas, Criscione & Blouin, 2005). According to the species delimitation methods based on the COI dataset, a set of lineages that possibly represent cryptic species emerged within the O. edwardsii complex. A final taxonomic revision of the complex, also including the description of new species, will depend on the detection of a genetic 9 distinction also at the nuclear level, and, eventually, on the identification of diagnostic morphological characters. Unfortunately, the lack of objectively distinguishable shell characters (at least at this point of our study), as well as the absence of a defined phylogenetic pattern in the selected nuclear sequences, prevented us from providing such a taxonomic revision. Therefore, we discuss the pattern emerging from the mitochondrial data, and its congruence with the preliminary shell-based identifications, under the assumption that, in our case, the mitochondrial phylogenetic signal is more reliable than the ITS2 one. The small mussel drills of the O. edwardsii complex are common northeastern Atlantic and Mediterranean gastropods displaying striking morphological variability and, consequently, a complicated taxonomic history. As a probable consequence of their variability, numerous nominal taxa have been erected based exclusively on shell morphology for these gastropods (Houart, 2001; Cecalupo et al., 2008). Thirteen species based on a purely morphological approach are accepted currently in the northeastern Atlantic–Mediterranean area (Table 2). The specimens used in our analysis were classified morphologically on collection into seven of these nominal species (O. carmelae, O. edwardsii, O. hispidula, O. ingloria, O. leukos, O. miscowichae, O. piantonii). Of these, O. miscowichae proved to be the sister species to the O. edwardsii complex (when Nucella lapillus was used as an outgroup, and O. aciculata was sister to the O. miscowichae– O. edwardsii pair). In our dataset, specimens morphologically ascribed to O. helleri, O. nicolai and O. paddeui (Table 2) were not available for DNA extraction. Their inclusion in a future dataset will probably affect the taxonomic and nomenclatural patterns. Within the O. edwardsii complex, our analyses proposed a number of MOTUs ranging from four to eight, and a preliminary hypothesis of species delimitation only partially concordant with our a priori identifications. THE OCINEBRINA EDWARDSII COMPLEX In the four-MOTUs scenario, supported by the ABGD method only (yet within the confidence limit of the GMYC analysis), the four major lineages (I–IV) comprised the following samples: MOTU-I: specimens morphologically identified as O. piantonii and O. carmelae from the Gulf of Gabès. MOTU-II: specimens morphologically identified as O. edwardsii from the Aegean Sea, Ionian Sea, southwestern Sicily and Lampedusa Island. MOTU-III: specimens morphologically identified as O. edwardsii from the Tyrrhenian Sea (including © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Type locality [listed localities] Type material Currently accepted as Notes Murex aciculatus Lamarck, 1822: 176 Vannes area (France) Not known O. aciculata Murex acrisius Nardo, 1847: 59–60 Gulf of Venice (Italy) 14 syntypes MSNVE 21980 (Crocetta et al., 2012) O. aciculata Type species of Ocinebrina Jousseaume, 1880 and Corallinia Bucquoy & Dautzenberg, 1882 (in Bucquoy, Dautzenberg & Dollfus, 1882: 24) Originally referring to: Chiereghin (unpublished*): figs. 713–714. Subjective junior synonym (Crocetta et al., 2012) Ocinebrina edwardsi [sic!] var. apiculata Pallary, 1902a: 314 Murex baeticus Reeve, 1845: pl. 32 (fig. 162) Tanger (Morocco) Not known O. edwardsii Not known 3 syntypes NHMUK 1972024 – one in Houart (2001) as BMNH 1621845 Holotype MNHM 33489 (Cecalupo et al., 2008) Holotype MNHM 33491 (Cecalupo et al., 2008) Holotype nr./1, Senckenberg Mus. (Houart, 2001) Not known O. edwardsii Probable subjective senior synonym of Murex hybridus O. corallinoides Subjective junior synonym (Crocetta et al., 2012) O. carmelae New subjective junior synonym of O. piantonii (? = Murex hybridus) Ocinebrina buzzurroi Cecalupo & Mariani, 2008: in Cecalupo et al., 2008: 90, pl. 43 (figs. 1–7) Ocinebrina carmelae Cecalupo, Buzzurro & Mariani, 2008: 98, pl. 48 (figs. 1–8) Amyclina compacta Nordsieck, 1968: 140, pl. 23 (fig. 80.45) Ocinebrina corallinoides Pallary, 1912: 221, plate (fig. 48) Murex corallinus Scacchi, 1836: 12 (fig. 15) Portovenere (Italy) Murex costulatus Nardo, 1847: 55–56 (sp. 8) Cres Island area (Croatia) Neotype MZN Z7010 (Crocetta et al., 2012) Not known Ocinebrina edwardsi [sic!] var. crassata Pallary, 1902b: 12, pl. 1 (figs. 10–11) Ocinebrina cyclopus Monterosato, 1884: 112 Purpura edwardsii Payraudeau, 1826: 155, pl. 7 (figs. 19–20) Ocinebrina erronea Cecalupo, Buzzurro & Mariani, 2008: 92, pls. 42 (figs. 5–6), 44 (figs. 7–16) and 45 (figs. 1–10) Tanger (Morocco) Not known Palermo (Italy) Corsica (France) Syntypes in MCZR (Settepassi, 1977) Not known O. edwardsii O. edwardsii Sfax and Kerkennah Islands (Tunisia) Not designated O. hispidula NW of Bou Grara Sea (Tunisia) Holotype MNHN 0362 (Crocetta et al., 2012) O. corallinoides [Zadar, Šibenik, Hvar, Dubrovnik, Budva] (Croatia and Montenegro) Gulf of Gabès (Tunisia) Not known O. helleri 3 syntypes MNHN 1001 (Houart, 2001; Giannuzzi-Savelli et al., 2003; present paper) Not known O. hispidula O. hybrida Probable subjective junior synonym of Murex baeticus Not known O. aciculata Subjective junior synonym (Houart, 2001; Crocetta et al., 2012) Holotype MNHN 0993 (Fair, 1976; Houart, 2001; Giannuzzi-Savelli et al., 2003; present paper) Holotype MMF 25429 (Houart & Abreu, 1994) O. ingloria Ocinebrina aciculata exilis Houart, 2001: 19 (figs. 8–9), 51 (fig. 62), 62, 143 (figs. 167–168), 176 (figs. 324–325) Fusus helleri Brusina, 1865: 8 Ocinebrina edwardsi [sic!] var. hispidula Pallary, 1904: 231, pl. 7 (fig. 18) Murex hybridus Aradas & Benoît, 1876: 272, pl. 5 (fig. 9) Murex inconspicuus Sowerby G.B. II, 1841: 5 (67), plates (figs. 81) Murex inglorius Crosse, 1865: 213, pl.6 (fig.4) Ocenebra inordinata Houart & Abreu, 1994: 123, 129 (figs. 11–13) Borj el Hissar (Tunisia) Borj el Hissar (Tunisia) Sfax, Gulf of Gabès (Tunisia) Baia (Italy) Palermo (Italy) Jersey Island (Bailiwick of Jersey) Not known Madeira Island O. edwardsii O. corallinoides O. aciculata O. inordinata Subjective junior synonym (Houart, 2001; Crocetta et al., 2012) Originally referring to: Chiereghin (unpublished*): figs. 693–694. Hereby first considered subjective junior synonym of Fusus helleri. Primary junior homonym of Murex costulatus Schröter 1805 and Murex costulatus Risso, 1826 Hereby considered as belonging to the Ocinebrina edwardsii complex Type species of Dentocenebra Monterosato, 1917 (= Ocinebrina Jousseaume, 1880) Subjective junior synonym (Cossignani & Ardovini, 2011), hereby assigned to Cecalupo et al. (2008) since Muricopsis erroneus Settepassi, 1977 is unavailable (ICZN, 1999: Art. 11.4) Subjective junior synonym (Bonomolo & Buzzurro, 2006; Cecalupo et al., 2008; Crocetta et al., 2012) = Murex hellerianus Brusina, 1866: 63 (unnecessary spelling emendation: Houart, 2001) A. BARCO ET AL. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Nominal taxon 10 Table 2. List of available nominal taxa from the northeastern Atlantic and the Mediterranean currently included in the genus Ocinebrina. Names currently considered as valid are given in bold. The type locality or, in substitution (in square brackets), the list of localities reported in the original description and the location of the type material are given when known. We have excluded unavailable names, such as infrasubspecific names (ICZN, 1999: Art. 45.5, 45.6) or nomina nuda (ICZN, 1999: Art. 12 and Glossary), but also all names introduced by Settepassi (1977), which has sometimes been considered as a consistently non-binomial work, under ICZN, 1999: Art. 11.4 [see, for example, WoRMS (2012) for Ocinebrina edwardsi var. fasciatus Settepassi, 1977. Accessed through: World Register of Marine Species at http://www.marinespecies.org/aphia.php?p=taxdetails&id=403955 on 13 March 2013] [Chioggia and Venice] (Italy) Presumably not existing Merex [sic!] labiosus Nardo, 1847: 55–56 (sp. 9) Kvarner Gulf (Croatia) Presumably not existing Ocenebra erinaceus Ocinebrina leukos Houart, 2000: 464 (figs. 7–10) La Isleta, Lanzarote (Spain) O. leukos Ocinebrina miscowichi Pallary, 1920: 40, plate (figs. 5–6) Ocinebrina nicolai Monterosato, 1884: 112 Essaouira (Morocco) Holotype MNHN 0966 (Houart, 2000; present paper) 32 syntypes MNHN 0176 and 0177 (Houart, 2001; present paper) Syntypes in MCZR (Settepassi, 1977) O. miscowichae Originally referring to: Chiereghin (unpublished*): figs. 695–696. New subjective junior synonym of Murex inglorius Originally referring to: Chiereghin (unpublished*): figs. 695–696. Currently accepted as Ocenebra erinaceus, and here considered as a member of the O. edwardsii complex. Primary junior homonym of Murex labiosus Gray, 1828 and Murex labiosus Wood, 1828 Spelling emended by Houart (1997) Murex orcomenus Nardo, 1847: 57–58 (sp. 11) [Corsica, Sardegna, Lipari] (Italy and France) Northern Adriatic Sea Ocinebrina paddeui Bonomolo & Buzzurro, 2006: 1 (figs. 1–6) Murex pereger Brugnone, 1873: 10, plate (fig. 17) Holotype MNHN 29909 (Bonomolo & Buzzurro, 2006) Not known O. paddeui African shores Mediterranean Not known O. edwardsii Mondello (Italy) Not known O. edwardsii Borj el Hissar (Tunisia) Holotype MNHM 33490 (Cecalupo et al., 2008) O. piantonii Probable subjective junior synonym of Murex baeticus Not known O. aciculata Not known 3 syntypes NHMUK 1972021 (Crocetta et al., 2012) Not known O. edwardsii Subjective junior synonym (Houart, 2001; Crocetta et al., 2012) Primary junior homonym of Murex pumilus Broderip, 1833 and Murex pumilus A. Adams, 1853 Rabat (Morocco) 8 syntypes MNHN 0931 (Houart, 2001) O. purpuroidea Procida Island (Italy) Holotype MNHN 24566 (Crocetta et al., 2012) O. reinai Murex edwardsi [sic!] f. perigmus De Gregorio, 1885: 253 Murex edwardsi [sic!] f. perilus De Gregorio, 1885: 253 Ocinebrina piantonii Cecalupo, Buzzurro & Mariani, 2008: 96, pl. 49 (figs. 1–10), pl. 50 (figs. 1–6) Murex pistacia Reeve, 1845: pl. 34, fig. 174 Off Capo Caccia, Alghero (Italy) O. nicolai Not known Originally referring to: Chiereghin (unpublished*): figs. 699–700.It is probably a senior synonym of Murex inglorius, but its status as nomen oblitum (ICZN, 1999: Art. 23.9.1.2) should be verified O. hybrida Murex pumilus Küster, 1869 (in Küster & Kobelt, 1839–1878: 118, pl. 35: figs. 8–10) Ocinebrina purpuroidea Pallary, 1920: 39, plate (figs. 16–17, 24) Ocinebrina reinai Bonomolo & Crocetta, 2012: (in Crocetta et al., 2012: 180, figs. 1 K-1 L, 2A-2F, 2I, 2 L, 3) Murex semiclausus Küster, 1869: 111, pl. 34 (figs. 6–7) Murex subaciculatus Locard, 1886: 164 Not known Not known O. edwardsii Toulon (France) Not known O. aciculata Fusus titii Stossich, 1865: 31 Northern Adriatic Sea Not known O. aciculata Ocinebra [sic!] wardiana Baker, 1891: 134, pl. 11 (fig. 5) Murex weinkauffianus Crosse, 1866: 274, pl. 8 (fig. 4) ‘Australia’ (see discussion in Vokes, 1994) Zadar (Croatia) Holotype CAS 20698 (Vokes, 1994) O. aciculata Not known O. helleri Subjective junior synonym (Houart, 2001). Primary junior homonym of Murex pereger Beyrich, 1854 Originally referring to: Bucquoy et al. (1882): pl. 2 (fig. 3). Subjective junior synonym (Houart, 2001) Originally referring to: Murex aciculatus in Hidalgo (1870): pl. 13 (figs. 7–8); Murex aciculatus var. curta Bucquoy et al. (1882): page 25. Subjective junior synonym (Houart, 2001; Crocetta et al., 2012) Subjective junior synonym (Houart, 2001; Crocetta et al., 2012) Subjective junior synonym (Houart, 2001; Crocetta et al., 2012) Subjective junior synonym (Houart, 2001) *Chiereghin S. (unpublished). Descrizione de′ crostacei, de′ testacei e de′ pesci che abitano le lagune e golfo veneto. [An unpublished manuscript referred to by Nardo, (1847) who then made available several otherwise unavailable names.] CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Ocinebrina labiosa Monterosato, 1884: 112 11 12 A. BARCO ET AL. Corsica, type locality of O. edwardsii); specimens morphologically identified as O. hispidula (from the Gulf of Gabès); specimens from the Mediterranean and Atlantic coasts of Spain and Portugal, including the Canary Islands; part of the specimens from the Venice Lagoon. MOTU-IV: specimens from the Adriatic Sea and a few localities in the Ionian Sea. In the eight-MOTUs scenario, supported by all three methods (ABGD, GMYC, TCS), the eight major lineages maintained a geographical coherence, and only MOTU-A corresponded exactly to MOTU-I. We cannot decide with the present data between the two scenarios (four-MOTUs or eight-MOTUs). Pending the availability of more reliable and congruent data from the nuclear genes to support such a decision, we discuss the geographical patterns in the O. edwardsii complex focusing on the more detailed (albeit admittedly less conservative) eight-MOTUs scenario. In the eight-MOTUs scenario, all the specimens attributed preliminarily to O. edwardsii were distributed among seven MOTUs: clade B, including specimens from localities in the Aegean Sea; clade C from the Ionian Sea, southwestern Sicily and Lampedusa Island; clade D2 from the Atlantic coast of Spain and Portugal, the Canary Islands and Málaga Province; clade E from several localities across the Tyrrhenian Sea; clade F from Pellestrina, a locality within the Venice Lagoon; clade G from Campania (Pozzuoli and Palinuro); and clade H from the Adriatic Sea and a few localities in the Ionian Sea. Of these groups, we provisionally identify MOTU-E as representing the ‘true’ O. edwardsii, because it included the specimens sampled in Corsica, its type locality (Payraudeau, 1826). Among our samples, this group is apparently restricted to the Tyrrhenian Sea (eastern Corsica, eastern Sardinia, northern Sicily and western Italian coast: Fig. 4). The sister lineage to clade E is composed of the D1 and D2 lineages (clade D). The D2 lineage included specimens morphologically ascribed to O. edwardsii sampled along the Atlantic coasts of Spain and Portugal, from the Canary Islands and from Málaga Province; the specimens ascribed to O. leukos from the Canary Islands; and specimens from populations referred in the recent literature to O. [cf.] nicolai from Galicia (Rolán, 1983) (samples 963, 965, 1033, 1058, 1059, 1061–1063), from Barbate (sample 1282) (Gofas, 2011) and from off Ponta da Piedade–Algarve (sample 1294) (Afonso et al., 2011) (Fig. 4). The specimens of O. edwardsii sampled along the Atlantic coast of Spain and Portugal displayed a rather distinctive shell morphology with respect to the most common forms found in the Mediterranean. Ocinebrina leukos was described from Lanzarote (Canary Islands) as living sympatrically with, yet morphologically easily distinguished from, local O. edwardsii (Houart, 2000). The latter, however, has been recorded as highly variable morphologically, even in the same locality (Houart, 2000), and, according to our molecular data, O. leukos may fall within the variability of this lineage (D2). Ocinebrina nicolai was originally described from Corsica, Sardinia and Lipari Islands (Monterosato, 1884). Specimens from the Monterosato collection (see Settepassi, 1977: 38–44), however, are morphologically different from those figured recently under this name (e.g. Rolán, 1983; Houart, 2001; Afonso et al., 2011; Gofas, 2011). Therefore, the association of the name O. nicolai with one of our MOTUs is postponed pending the genetic analysis of topotypical specimens. According to the ABGD and GMYC analyses, clade D also included the specimens from Gabès ascribed to O. hispidula, although a distinct well-supported clade (D1) for this lineage has been recovered in all the topologies. The large genetic distance between specimens identified as O. hispidula from Gabès and specimens classified preliminarily as O. edwardsii from Sicily (clade C) has been reported recently by Barco et al. (2013). Ocinebrina hispidula has also been reported from other Atlantic and Mediterranean localities, such as Mogador (Ardovini & Cossignani, 2004), Sicily (Settepassi, 1977; Giannuzzi-Savelli et al., 2003), Cyprus (Houart, 2001; Öztürk, Buzzurro & Benli, 2004) and the Baleares (Pons-Moyà & Pons, 2002). Although these records may have been based on extremely spiny morphotypes of other taxa of the O. edwardsii complex, or on mislabelled specimens, according to our analyses (ABGD and BEASTGMYC), O. hispidula is included in a more widely distributed MOTU (clade D). Even in the less conservative eight-MOTUs scenario, MOTU-D probably represents a single species ranging from the northeastern Atlantic to the western and central Mediterranean Sea, which has recently colonized the Gulf of Gabès with a population displaying a strikingly deviating morphology (Fig. 4), a phenomenon well known in the Gabès molluscan fauna (Cecalupo et al., 2008), and with a distribution compatible with that of other intertidal molluscs (Calvo et al., 2009). Furthermore, the young age of the Gulf of Gabès (Burollet, Clairefond & Winnock, 1979; Stocchi, Colleoni & Spada, 2009) would not be compatible with the recent evolution of an endemic species. Ocinebrina erronea Cecalupo, Buzzurro & Mariani, 2008 (see Table 2 for nomenclatural details) has been synonymized recently with O. hispidula by Cossignani & Ardovini (2011). A single specimen of the O. erronea morphotype (sample 1053) was included in our dataset and, in all the analyses, it fell © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII 13 Figure 4. Same tree as in Figure 3 obtained by BEAST on the cytochrome oxidase subunit I (COI) alignment, and distribution maps of the vouchers, grouped in the clades resulting from the COI analysis. within the O. hispidula clade (group D1), indicating that it is not a distinct species. Ocinebrina piantonii and O. carmelae were described as new species from the Kerkennah Islands by Cecalupo et al. (2008), living sympatrically (and syntopically) amidst the rhizomes of Posidonia oceanica (L.) Delile, 1813. The morphological differences between the two nominal taxa are indeed very subtle, possibly ending up as just two colour forms: a white phenotype (O. piantonii) and a dark phenotype (O. carmelae). Furthermore, the latter is indistinguishable from O. hybrida (Aradas & Benoît, 1876) (which is possibly a synonym of O. baetica (Reeve, 1845): see Table 2), a rare species for which we had no specimens for DNA extraction. We assayed two specimens from the Gulf of Gabès, representing the two morphotypes, which proved clearly to belong to a single species (clade A), not related phylogenetically to O. hispidula. They fall into an eastern Mediterranean group including clades B (Aegean Sea) and C (Ionian Sea). Specimens collected at four localities across the Aegean Sea (clade B) were indistinguishable morphologically from those classified preliminarily as O. edwardsii from other Mediterranean localities. The COI and 16S sequences of these specimens were particularly divergent from all the others, allowing the identification of a well-distinct mitochondrial lineage. Furthermore, the mitochondrial data also suggest a well-defined population structure within the group, with three deeply separated clusters corresponding to distinct sampling localities (northern, central and southern Aegean Sea) (Fig. 4). Another clade (C) is represented by the specimens collected in the Ionian Sea, southwestern Sicily and Lampedusa Island. Similar to group B, clade C © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 14 A. BARCO ET AL. is well defined geographically (Fig. 4), but the shells are rather indistinguishable from specimens ascribed morphologically to O. edwardsii from other Mediterranean localities. The sister group relationship with the Aegean clade suggests an allopatric origin of both lineages from an ancestral eastern Mediterranean clade (see below). Ocinebrina ingloria was described originally by Crosse (1865) without specifying any locality. Other names may apply to this entity (e.g. Murex orcomenus Nardo, 1847, see Table 2), but the assessment of the status was beyond the scope of this study. Neglected for over a century, it was recorded as O. ingloria recently from the Adriatic Sea, from Grado and the Venice Lagoon (which was interpreted as the most likely locality of the type material) by Houart (2001) and from Palermo by Giannuzzi-Savelli et al. (2003). The population at Grado seems to be extinct, together with other intertidal molluscan species, as a result of the intense human activity in the area (see Crocetta, 2011), whereas the record from Palermo seems likely to be incorrect because such a morphotype has never been found there (G. Bonomolo, unpublished). We have assayed specimens from the Venice Lagoon (morphologically and geographically representing the nominal taxon O. ingloria) which, despite evident morphological differences, were grouped in clade H with other specimens classified morphologically as O. edwardsii from the Adriatic Sea and a few localities in the Ionian Sea (Fig. 4). Apparently all specimens of the O. edwardsii complex from the Adriatic Sea belong in the same lineage as O. ingloria, again suggesting that shell variation, as currently approached within this group, is rather misleading, and that a single species is involved in this lineage. The sole geographical exception to clade H is represented by some specimens sampled at Pellestrina (clade F), within the area of the Venice Lagoon. All specimens from this site were indistinguishable from each other morphologically (see, for example, vouchers 1046_1 and 1046_3: Fig 3B, Supporting Information Fig. S4). After the first analyses split the first lot of specimens from this locality (sample 1046) into groups F and H, we collected other specimens from the same locality (sample 1293) in order to rule out the possibility of contamination, and obtained the same results. Thus, according to this evidence, two morphologically indistinguishable MOTUs live sympatrically at this site in the Venice Lagoon. The sister clade to MOTU-F from Pellestrina is clade G from Pozzuoli and Palinuro (central Tyrrhenian Sea). Clades F and G were clearly distinct MOTUs in all of our species delimitation analyses, and both lineages possibly represent relict populations isolated on the two sides of the Italian peninsula. BIOGEOGRAPHICAL PATTERNS Comparing our molecular data with the fossil record of Ocinebrina would allow the drawing of a timecalibrated tree and a comparison between the estimated origin of clades and the history of the Mediterranean. Unfortunately, although fossil Ocinebrina shells are relatively abundant, their species classification based on shell morphology would be rather misleading. What has emerged from the present study is that shell characters used in the taxonomy of O. edwardsii and related species do not always define monophyletic lineages, and we have therefore no basis for using fossils to calibrate node ages in the phylogeny. Such an analysis may become possible only after a thorough revision of shell characters used in this group. A comparison with previous studies, however, might be helpful in understanding the observed geographical distribution of molecular lineages. The present-day Mediterranean marine fauna is the result of a long and troubled geological history (Taviani, 2002) peaking in the Messinian Salinity Crisis (MSC) (Krijgsman et al., 1999) and the following Pliocene and Quaternary glacial/interglacial cycles. These events contributed to the shaping of the current fauna into defined biogeographical categories (Bianchi & Morri, 2000), and the geographical structure among populations of Mediterranean organisms can generally be traced back to these major events (Patarnello, Volckaert & Castilho, 2007). Species with short or no planktonic larval stage (such as the northeastern Atlantic Ocinebrina species) are supposed to show a more structured distribution across a reduced geographical range (Cunha et al., 2005, 2008; Meyer et al., 2005; Paulay & Meyer, 2006) than those with planktonic larvae. Thus, in the Mediterranean Sea, the phylogeography of marine organisms, and especially of those with reduced planktonic stages, is expected to reflect these events. This is the case for reef-building gastropods of the Dendropoma petraeum (Monterosato, 1884) complex, which has been shown to include four cryptic species with a clear east–west Mediterranean subdivision (Calvo et al., 2009). This geographical structure has been attributed to vicariant cladogenesis favoured by the reduced planktonic larval stage of the species. We observed a high similarity between the geographical distributions of the lineages of Ocinebrina and that found by Calvo et al. (2009) in the D. petraeum complex. Our group D has a distribution similar to that of the western Mediterranean Dendropoma clade. The continuous range across the Atlanto-Mediterranean border found in many marine species is generally explained as a re-colonization process completed by propagules from extant Atlantic populations © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII establishing new populations in the Mediterranean. However, restricted gene flow through the Straits of Gibraltar has also been detected in species with high dispersal potential (Sá-Pinto et al., 2012). In species with a short-lived planktonic stage, the dispersal across long distances possibly has been accomplished by rafting or with floating debris (Martel & Chia, 1991; Cunningham & Collins, 1998). The combination of these factors and the effect of the Algerian current flowing along the northern coast of Africa could explain the presence of this lineage in the Gulf of Gabès and its genetic distinctiveness. The repeated instances of separation of the western and eastern Mediterranean basins by the Sicilian sill during the Quaternary glacial cycles are often correlated with the present-day distinction between corresponding lineages (e.g. Oliverio, 1994, 1996; Calvo et al., 2009; Sá-Pinto et al., 2010; Mejri et al., 2011). We found that three of our groups (MOTU-A, MOTU-B and MOTU-C) formed a wellsupported clade (MOTU-I + MOTU-II) in both the BEAST and MrBayes analyses, supporting the existence of an eastern Mediterranean lineage. In the three-gene Bayesian analysis (Fig. S3B), the Adriatic clade (all the Adriatic specimens with the exception of group F) is closely related to groups A, B and C, suggesting an early separation of the ancestor of these four groups from a western Mediterranean clade ancestor. The phylogenetic relationships of groups F and G with the others are still unclear, making it difficult to draw their position in this scenario. According to BEAST analysis (Fig. 2), both lineages are closely related to the western Mediterranean group (clades D and E), but, in other analyses, support for such a position is lower. The long branches of these groups and their sympatry with other clades (F with H in the Adriatic Sea and G with E in the Tyrrhenian Sea) would suggest early allopatric isolation followed by secondary contact. The actual distribution of these two groups, however, is still unknown, and further studies with denser sampling are required to understand their status. ACKNOWLEDGEMENTS Carlos Afonso (Faro, Portugal), Giuseppe Colamonaco (Altamura, Italy), Fabio Daga (Carbonia, Italy), Carlos H. Durais de Carvalo (Alvaiazere, Portugal), Jean-Louis Delemarre (St. Nazaire, France), Francisco Déniz (Las Palmas, Gran Canaria, Spain), Serge Gofas (Málaga, Spain), Didier Marcellesi (Marseille, France), Loris Perini (Chioggia, Italy), Walter Renda (Amantea, Italy), Emilio Rolán (Vigo, Spain), Paolo Russo (Venice, Italy), Mirco Vianello (Chioggia, Italy) and Diego Viola (Trieste, Italy) pro- 15 vided samples. Amelia MacLellan (NHMUK, London, UK) provided the photographs of syntypes of Murex baeticus. Ermanno Quaggiotto (Longare, Italy) and Michele Reina (Palermo, Italy) helped with bibliography. Three anonymous reviewers significantly helped to improve this article with precious hints and critiques on a previous version of the manuscript. REFERENCES Afonso CML, Bonomolo G, Monteiro P, Bentes L, Oliveira F, Veiga P, Rangel MO, Sousa I, Leite L, Gonçalves JMS. 2011. First record of Ocinebrina nicolai (Mollusca: Gastropoda: Muricidae: Ocenebrinae) in northeastern Atlantic waters. Marine Biodiversity Records 3: 1–4. Appeltans W, Bouchet P, Boxshall GA, De Broyer C, de Voogd, Gordon, NJ, Hoeksema DP, Horton BW, Kennedy T, Mees M, Poore J, Read GCB, Stöhr G, Walter S, Costello TC, MJ, eds. 2012. World register of marine species. Available at: http://www.marinespecies.org [accessed 13 March 2013]. Aradas A, Benoît L. 1876. Conchigliologia vivente marina della Sicilia e delle isole che la circondano. Parte terza. Atti dell’Accademia Gioenia di Scienze Naturali in Catania 6: 1–329. Ardovini R, Cossignani T. 2004. West African seashells. Ancona: L’Informatore Piceno. Baker FC. 1891. Description of new species of Muricidæ, with remarks on the apices of certain forms. Proceedings of the Rochester Academy of Science 1: 129–137. Barco A, Corso A, Oliverio M. 2013. Endemicity in the Gulf of Gabès: the small mussel drill Ocinebrina hispidula is a distinct species in the Ocinebrina edwardsii complex (Muricidae: Ocenebrinae). Journal of Molluscan Studies 79: 273–276. Bianchi CN, Morri C. 2000. Marine biodiversity of the Mediterranean Sea: situation, problems and prospects for future research. Marine Pollution Bulletin 40: 367–376. Blaxter ML. 2004. The promise of a DNA taxonomy. Philosophical Transactions of the Royal Society B: Biological Sciences 359: 669–679. Boisselier-Dubayle MC, Gofas S. 1999. Genetic relationships between marine and marginal-marine populations of Cerithium species from the Mediterranean Sea. Marine Biology 135: 671–682. Bonomolo G, Buzzurro G. 2006. Description of a new Muricid for the Mediterranean sea: Ocinebrina paddeui (Mollusca, Gastropoda, Muricidae, Ocenebrinae). Triton 13: 1–4. Brugnone GA. 1873. Miscellanea Malachologica. Pars prima. Palermo: Michele Amenta. Brusina S. 1865. Conchiglie dalmate inedite. Verhandlungen der Kaiserlich-Königlichen Zoologisch-Botanischen Gesellschaft in Wien 15: 3–42. Brusina S. 1866. Contribuzione pella fauna dei molluschi dalmati. Verhandlungen der Kaiserlich-Königlichen Zoologisch-Botanischen Gesellschaft in Wien 16: 1–134. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 16 A. BARCO ET AL. Bucquoy EJ, Dautzenberg P, Dollfus GF. 1882. Les Mollusques marins du Roussillon. Tome I (Gastropodes). Paris: Baillière J.B. and Fils. Burollet PF, Clairefond P, Winnock E. 1979. La mer pélagienne. Etude sédimentologique et écologique du plateau tunisien et du golfe de Gabès. Annales de l’Université de Provence 5: 1–345. Calvo M, Templado J, Oliverio M, MacHordom A. 2009. Hidden Mediterranean biodiversity: molecular evidence for a cryptic species complex within the reef building vermetid gastropod Dendropoma petraeum (Mollusca: Caenogastropoda). Biological Journal of the Linnean Society 96: 898–912. Caron DA, Countway PD, Jones AC, Kim DY, Schnetzer A. 2012. Marine protistan diversity. Annual Review of Marine Science 4: 467–493. Carreras-Carbonell J, Macpherson E, Pascual M. 2005. Rapid radiation and cryptic speciation in Mediterranean triplefin blennies (Pisces: Tripterygiidae) combining multiple genes. Molecular Phylogenetics and Evolution 37: 751– 761. Castelin M, Lambourdiere J, Boisselier MC, Lozouet P, Couloux A, Cruaud C, Samadi S. 2010. Hidden diversity and endemism on seamounts: focus on poorly dispersive neogastropods. Biological Journal of the Linnean Society 100: 420–438. Cecalupo A, Buzzurro G, Mariani M. 2008. Contributo alla conoscenza della malacofauna del Golfo di Gabès (Tunisia). Quaderni della Civica Stazione Idrobiologica di Milano 31: 1–267. Claremont M, Williams ST, Barraclough TG, Reid DG. 2011. The geographic scale of speciation in a marine snail with high dispersal potential. Journal of Biogeography 38: 1016–1032. Clement M, Posada D, Crandall KA. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657–1659. Cossignani T, Ardovini R. 2011. Malacologia Mediterranea. Atlante delle conchiglie del Mediterraneo – 7.500 foto a colori. Ancona: L’Informatore Piceno. Costello MJ, Wilson S, Houlding B. 2012. Predicting total global species richness using rates of species description and estimates of taxonomic effort. Systematic Biology 61: 871– 883. Crocetta F. 2011. Marine alien Mollusca in the Gulf of Trieste and neighbouring areas: a critical review and state of knowledge (updated in 2011). Acta Adriatica 52: 247–260. Crocetta F, Bonomolo G, Albano PG, Barco A, Houart R, Oliverio M. 2012. The status of the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina aciculata complex (Mollusca: Gastropoda: Muricidae), with the description of a new species. Scientia Marina 76: 177– 189. Crosse H. 1865. Description d’espèces nouvelles. Journal de Conchyliologie 13: 213–215. Crosse H. 1866. Description d’un Murex nouveau de l’Adriatique. Journal de Conchyliologie 14: 274–276. Cunha RL, Castilho R, Rüber L, Zardoya R. 2005. Patterns of cladogenesis in the venomous marine gastropod genus Conus from the Cape Verde Islands. Systematic Biology 54: 634–650. Cunha RL, Tenorio MJ, Afonso C, Castilho R, Zardoya R. 2008. Replaying the tape: recurring biogeographical patterns in Cape Verde Conus after 12 million years. Molecular Ecology 17: 885–901. Cunningham CW, Collins TM. 1998. Beyond area relationships: extinction and recolonization in molecular marine biogeography. In: De Salle R, Schierwater B, eds. Molecular approaches to ecology and evolution. Basel: Birkhaeuser Verlag, 297–321. De Gregorio A. 1885. Studi su talune conchiglie Mediterranee viventi e fossili. Siena: Tipografia all’insegna dell’ancora. Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. Ezard T, Fujisawa T, Barraclough TG. 2009. Splits: species’ limits by threshold statistics. Available at: http:// R-Forge.R-project.org/projects/splits/ Fair RH. 1976. The murex book: an illustrated catalogue of recent Muricidae (Muricinae, Muricopsinae, Ocenebrinae). Honolulu, HI: Sturgis Printing Co. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299. Franc A. 1940. Recherches sur le développement d’Ocinebra aciculata, Lamarck (Mollusque Gastéropode). Bulletin biologique de la France et de la Belgique 74: 327–345. Gascuel O. 1997. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Molecular Biology and Evolution 14: 685–695. Gelman A, Rubin DB. 1992. Inference from iterative simulation using multiple sequences. Statistical Science 7: 457–472. Giannuzzi-Savelli R, Pusateri F, Palmeri A, Ebreo C. 2003. Atlante delle conchiglie marine del Mediterraneo. Vol. 4 (Neogastropoda: Muricoidea). Rome: Evolver. Goetze E. 2003. Cryptic speciation on the high seas; global phylogenetics of the copepod family Eucalanidae. Proceedings of the Royal Society B: Biological Sciences 270: 2321–2331. Gofas S. 2011. Familia muricidae. In: Gofas S, Moreno D, Salas C, eds. Moluscos marinos de Andalucía – I. Málaga: Servicio de Publicaciones e Intercambio Científico, Universidad de Málaga, 278–286. Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696–704. Hart MW, Sunday J. 2007. Things fall apart: biological species form unconnected parsimony networks. Biology Letters 3: 509–512. Hasegawa M, Kishino H, Yano T. 1985. Dating of the human–ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22: 160–174. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Hayashi S. 2005. The molecular phylogeny of the Buccinidae (Caenogastropoda: Neogastropoda) as inferred from the complete mitochondrial 16S rRNA gene sequences of selected representatives. Molluscan Research 25: 85–98. Hidalgo JG. 1870. Moluscos marinos de España, Portugal y las Baleares. Madrid: Impr. de M. Ginesta. Hillis DM, Moritz C, Mable BK. 1996. Molecular systematics. Sunderland, MA: Sinauer Associates. Houart R. 1997. The west African Muricidae. II. Ocenebrinae, Ergalataxinae, Tripterotyphinae, Typhinae, Trophoninae & Rapaninae. Apex 12: 49–91. Houart R. 2000. New species of Muricidae (Gastropoda) from the northeastern Atlantic and the Mediterranean Sea. Zoosystema 22: 459–469. Houart R. 2001. A review of the recent Mediterranean and Northeastern Atlantic species of Muricidae. Rome: Evolver. Houart R, Abreu AD. 1994. The Muricidae (Gastropoda) from Madeira with the description of a new species of Ocenebrinae. Apex 9: 119–130. Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755. ICZN [International Commission on Zoological Nomenclature]. 1999. International code of zoological nomenclature, Fourth edn. London: International Trust for Zoological Nomenclature. Jousseaume FP. 1880. Division méthodique de la famille des Purpuridae. Le Naturaliste: Journal des échanges et des nouvelles 2: 335–336. Jukes TH, Cantor CR. 1969. Evolution of protein molecules. In: Munro HM, ed. Mammalian protein metabolism. New York: Academic Press, 21–132. Katoh K, Misawa K, Kuma KI, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059– 3066. Katoh K, Toh H. 2008. Recent developments in the MAFFT multiple sequence alignment program. Briefings in Bioinformatics 9: 286–298. Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120. Kimura M. 1981. Estimation of evolutionary distances between homologous nucleotide sequences. Proceedings of the National Academy of Sciences 78: 454–458. Knowlton N. 1993. Sibling species in the sea. Annual Review of Ecology and Systematics 24: 189–216. Krijgsman W, Hilgen FJ, Raffi I, Sierro FJ, Wilson DS. 1999. Chronology, causes and progression of the Messinian salinity crisis. Nature 400: 652–655. Kumar S, Skjæveland T, Orr RJS, Enger P, Ruden T, Mevik B, Burki F, Botnen A, Shalchian-Tabrizi K. 2009. AIR: a batch-oriented web program package for construction of supermatrices ready for phylogenomic analyses. BMC Bioinformatics 10: 357. Küster HC, Kobelt W. 1839–1878. Die geschwänzten und bewehrten Purpurschnecken (Murex, Ranella, Tritonium, Trophon, Hindsia) in abbildungen nach der Natur mit 17 Beschreibungen. Systematisches Conchylien-Cabinet von Martini und Chemnitz 3: 1–99, 110–336, pls A, B, 1–77, 4b, 37a, 38a, 39a. Lamarck JBM. 1822. Histoire naturelle des animaux sans vertèbres, présentant les caractères généraux et particuliers de ces animaux, leur distribution, leurs classes, leurs familles, leurs genres, et la citation des principales espèces qui s’y rapportent; précédée d’une introduction offrant la détermination des caractères essentiels de l’animal, sa distinction du végétal et des autres corps naturels; enfin, l’exposition des principes fondamentaux de la zoologie. Vol. 7. Paris. Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451–1452. Locard A. 1886. Prodrome de malacologie française. Vol. 2. Catalogue général des Mollusques vivants de France. Mollusques marins. Lyon: Librairie Henri Georg. Martel A, Chia FS. 1991. Drifting and dispersal of small bivalves and gastropods with direct development. Journal of Experimental Marine Biology and Ecology 150: 131–147. Mejri R, Arculeo M, Ben Hassine OK, Lo Brutto S. 2011. Genetic architecture of the marbled goby Pomatoschistus marmoratus (Perciformes, Gobiidae) in the Mediterranean Sea. Molecular Phylogenetics and Evolution 58: 395–403. Meyer CP, Geller JB, Paulay G. 2005. Fine scale endemism on coral reefs: archipelagic differentiation in Turbinid gastropods. Evolution 59: 113–125. Monaghan MT, Wild R, Elliot M, Fujisawa T, Balke M, Inward DJG, Lees DC, Ranaivosolo R, Eggleton P, Barraclough TG, Vogler AP. 2009. Accelerated species inventory on Madagascar using coalescent-based models of species delineation. Systematic Biology 58: 298–311. di Monterosato TA. 1884. Nomenclatura generica e specifica di alcune conchiglie mediterranee. Palermo: Virzi. di Monterosato TA. 1917. Molluschi viventi e quaternari raccolti lungo le coste della Tripolitania dall’ing Camillo Crema. Bollettino della Società Zoologica Italiana 3: 1–28. Nardo GD. 1847. Sinonimia moderna delle specie registrate nell′ opera intitolata: Descrizione de′ crostacei, de′ testacei e de′ pesci che abitano le lagune e golfo veneto, rappresentati in figure, a chiaro-scuro ed a colori dall′ Abate Stefano Chiereghin Ven. Clodiense. Venezia. Nordsieck F. 1968. Die europäischen MeeresGehäuseschnecken (Prosobranchia). Stuttgart: G. Fischer. Nuñez JJ, Vejar-Pardo A, Guzmán BE, Barriga EH, Gallardo CS. 2012. Phylogenetic and mixed Yulecoalescent analyses reveal cryptic lineages within two South American marine snails of the genus Crepipatella (Gastropoda: Calyptraeidae). Invertebrate Biology 131: 301– 311. Oliverio M. 1994. Developmental vs genetic variation in two Mediterranean rissoid gastropod complexes. Journal of Molluscan Studies 60: 461–465. Oliverio M. 1996. Life-histories, speciation and biodiversity in Mediterranean prosobranch gastropods. Vie et Mileu 46: 163–169. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 18 A. BARCO ET AL. Oliverio M, Mariottini P. 2001a. Contrasting morphological and molecular variation in Coralliophila meyendorffii (Muricidae, Coralliophilinae). Journal of Molluscan Studies 67: 243–246. Oliverio M, Mariottini P. 2001b. A molecular framework for the phylogeny of Coralliophila and related muricoids. Journal of Molluscan Studies 67: 215–224. Öztürk B, Buzzurro G, Benli HA. 2004. Marine molluscs from Cyprus: new data and checklist. Bollettino Malacologico 39: 49–78. Pallary P. 1902a. Diagnoses de quelques coquilles nouvelles provenant du Maroc. Journal de Conchyliologie 49: 226– 228. Pallary P. 1902b. Liste des mollusques testacés de la Baie de Tanger. Journal de Conchyliologie 50: 1–39. Pallary P. 1904. Addition à la faune malacologique du Golfe de Gabès. Journal de Conchyliologie 52: 212–248. Pallary P. 1912. Sur la faune de l’ancienne lagune de Tunis. Bulletin de la Société d’histoire naturelle d’Afrique du Nord 3: 215–228. Pallary P. 1920. Mission zoologique. Malacologie. In: Exploration scientifique du Maroc organisée par la Société de Géographie de Paris et continuée par la Société des Sciences Naturelles du Maroc. Deuxième fascicule. Rabat/Paris: Institut Scientifique Chérifien. Palumbi S, Martin A, Romano S, McMillan WO, Stice L, Grabowski G. 2002. The simple fool’s guide to PCR Version 2.0. Honolulu, HI: Department of Zoology and Kewalo Marine Laboratory, University of Hawaii. Patarnello T, Volckaert FAMJ, Castilho R. 2007. Pillars of Hercules: is the Atlantic–Mediterranean transition a phylogeographical break? Molecular Ecology 16: 4426–4444. Paulay G, Meyer C. 2006. Dispersal and divergence across the greatest ocean region: do larvae matter? Integrative Comparative Biology 46: 269–281. Payraudeau BC. 1826. Catalogue descriptif et méthodique des Annélides et des Mollusques de l’île de Corse. Paris. Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S, Kamoun S, Sumlin WD, Vogler AP. 2006. Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology 55: 595–609. Pons-Moyà J, Pons GX. 2002. Primera cita d’Ocinebrina hispidula (Pallary, 1904) (Mollusca: Gastropoda: Muricidae) per a les aigües Ibero-Balears. Bolletí de la Societat d’Història Natural de les Balears 45: 81–85. Posada D. 2003. Using MODELTEST and PAUP* to select a model of nucleotide substitution. In: Current Protocols in Bioinformatics. New York: John Wiley & Sons, Inc. Posada D. 2008. jModelTest: phylogenetic model averaging. Molecular Biology and Evolution 25: 1253–1256. Posada D, Crandall KA. 2001. Intraspecific gene genealogies: trees grafting into networks. Trends in Ecology and Evolution 16: 37–45. Puillandre N, Baylac M, Boisselier MC, Cruaud C, Samadi S. 2009. An integrative approach to species delimitation in Benthomangelia (Mollusca: Conoidea). Biological Journal of the Linnean Society 96: 696–708. Puillandre N, Lambert A, Brouillet S, Achaz G. 2012a. ABGD, automatic barcode gap discovery for primary species delimitation. Molecular Ecology 21: 1864–1877. Puillandre N, Meyer CP, Bouchet P, Olivera BM. 2011. Genetic divergence and geographical variation in the deep-water Conus orbignyi complex (Mollusca: Conoidea). Zoologica Scripta 40: 350–363. Puillandre N, Modica MV, Zhang Y, Sirovich L, Boisselier MC, Cruaud C, Holford M, Samadi S. 2012b. Large-scale species delimitation method for hyperdiverse groups. Molecular Ecology 21: 2671–2691. Puillandre N, Sysoev AV, Olivera BM, Couloux A, Bouchet P. 2010. Loss of planktotrophy and speciation: geographical fragmentation in the deep-water gastropod genus Bathytoma (Gastropoda, Conoidea) in the western Pacific. Systematics and Biodiversity 8: 371–394. Quattro J, Chase M, Rex M, Greig T, Etter R. 2001. Extreme mitochondrial DNA divergence within populations of the deep-sea gastropod Frigidoalvania brychia. Marine Biology 139: 1107–1113. Radwin GE, D’Attilio A. 1976. Murex shells of the world. Stanford, CA: Stanford University Press. Rambaut A, Drummond AJ. 2003. Tracer v. 1.5.0. Available at: http://evolve.zoo.ox.ac.uk/software/ Reeve LA. 1845. Conchologia Iconica, or, illustrations of the shells of Molluscous animals. Vol. 3. London. Reid DG, Dyal P, Williams ST. 2010. Global diversification of mangrove fauna: a molecular phylogeny of Littoraria (Gastropoda: Littorinidae). Molecular Phylogenetics and Evolution 55: 185–201. Richards TA, Jones MDM, Leonard G, Bass D. 2012. Marine fungi: their ecology and molecular diversity. Annual Review of Marine Science 4: 495–522. Rolán E. 1983. Moluscos de la Ria de Vigo – I. Gasteropodos. Thalassas 1: 1–383. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Sá-Pinto A, Baird SJE, Pinho C, Alexandrino P, Branco M. 2010. A three-way contact zone between forms of Patella rustica (Mollusca: Patellidae) in the central Mediterranean Sea. Biological Journal of the Linnean Society 100: 154–169. Sá-Pinto A, Branco MS, Alexandrino PB, Fontaine MC, Baird SJE. 2012. Barriers to gene flow in the marine environment: insights from two common intertidal limpet species of the Atlantic and Mediterranean. PLoS ONE 7: e50330. Scacchi A. 1836. Catalogus Conchyliorum Regni Neapolitani quae usque adhuc reperit A. Scacchi. Napoli: Typis FiliatreSebetii. Schwarz G. 1978. Estimating the dimension of a model. Annals of Statistics 6: 461–464. Settepassi F. 1977. Atlante Malacologico. I molluschi marini viventi nel Mediterraneo volume II. Roma: Museo di Zoologia. Sowerby GB II. 1832–1841. The conchological illustrations, or coloured figures of all the hitherto unfigured Recent shells. London. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Stocchi P, Colleoni F, Spada G. 2009. Bounds on the time–history and Holocene mass budget of Antarctica from sea-level records in SE Tunisia. Pure and Applied Geophysics 166: 1319–1341. Stossich A. 1865. Enumerazione dei Molluschi del Golfo di Trieste. Programma della Civica Scuola Reale Superiore di Trieste. 1865: 21–58. Taviani M. 2002. The Mediterranean benthos from late Miocene up to present: ten million years of dramatic climatic and geologic vicissitudes. Biologia Marina Mediterranea 9: 445–463. Templeton AR. 2001. Using phylogeographic analyses of gene trees to test species status and processes. Molecular Ecology 10: 779–791. Templeton AR, Crandall KA, Sing CF. 1992. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132: 619–633. Vermeij GJ. 1978. Biogeography and adaptation: patterns of marine life. Cambridge: Harvard University Press. Vermeij GJ, Vokes EH. 1997. Cenozoic Muricidae of the Western Atlantic region. Part XII–the subfamily Ocenebrinae (in part). Tulane Studies in Geology and Paleontology 29: 68–118. Vilas R, Criscione CD, Blouin MS. 2005. A comparison between mitochondrial DNA and the ribosomal internal 19 transcribed regions in prospecting for cryptic species of platyhelminth parasites. Parasitology 131: 839–846. Vogler AP, Monaghan MT. 2007. Recent advances in DNA taxonomy. Journal of Zoological Systematics and Evolutionary Research 45: 1–10. Vokes EH. 1994. The muricid types of Frank Collins Baker. The Nautilus 107: 118–123. Williams S, Apte D, Ozawa T, Kaligis F, Nakano T. 2011. Speciation and dispersal along continental coastlines and Island arcs in the Indo-West Pacific turbinid gastropod genus Lunella. Evolution 65: 1752–1771. Wilson NG, Schrödl M, Halanych KM. 2009. Ocean barriers and glaciation: evidence for explosive radiation of mitochondrial lineages in the Antarctic sea slug Doris kerguelenensis (Mollusca, Nudibranchia). Molecular Ecology 18: 965–984. Xavier R, Zenboudji S, Lima FP, Harris DJ, Santos AM, Branco M. 2011. Phylogeography of the marine isopod Stenosoma nadejda (Rezig, 1989) in North African Atlantic and western Mediterranean coasts reveals complex differentiation patterns and a new species. Biological Journal of the Linnean Society 104: 419–431. Zou S, Li Q, Kong L. 2012. Monophyly, distance and character-based multigene barcoding reveal extraordinary cryptic diversity in Nassarius: a complex and dangerous community. PLoS ONE 7: e47276. doi:10.1371/journal. pone.0047276. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Result of the Generalized Mixed Yule-Coalescent (GMYC) analysis for the combined COI + ITS2 dataset. Figure S2. Results of the single-gene Bayesian analyses (by MrBayes). From left to right: COI, ITS2, 16S. Figure S3. Results of the multiple-gene Bayesian analyses (by MrBayes). From left to right: COI + ITS2 and COI + ITS2 + 16S. Figure S4. Shells of representative voucher specimens from each sampling site. ID numbers as in Table 1. Scale bar, 10 mm. Figure S5. Shells of representative voucher specimens from each sampling site. ID numbers as in Table 1. Scale bar, 10 mm. Figure S6. Shells of representative voucher specimens from each sampling site. ID numbers as in Table 1. Scale bar, 10 mm. Table S1. Sequence Accession Numbers of the sequences of Ocinebrina spp. used in this work, within the framework of the BOLD Project ‘BOCI’ (Barcoding of Ocinebrina). © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII SUPPORTING INFORMATION Additional Supporting Information in the online version of this article at the publisher’s web-site Figure S1. Result of the Generalized Mixed Yule-Coalescent (GMYC) analysis for the combined COI + ITS2 dataset. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Figure S2. Results of the single-gene Bayesian analyses (by MrBayes). From left to right: COI, ITS2, 16S. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Figure S3. Results of the multiple-gene Bayesian analyses (by MrBayes). From left to right: COI + ITS2 and COI + ITS2 + 16S. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Figure S4. Shells of representative voucher specimens from each sampling site. ID numbers as in Table 1. Scale bar, 10 mm. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Figure S5. Shells of representative voucher specimens from each sampling site. ID numbers as in Table 1. Scale bar, 10 mm. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Figure S6. Shells of representative voucher specimens from each sampling site. ID numbers as in Table 1. Scale bar, 10 mm. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 A. BARCO ET AL. SUPPORTING INFORMATION CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII Table S1. Sequence Accession Numbers of the sequences of Ocinebrina spp. used in this work, in the framework of the BOLD Project “BOCI” (Barcoding of Ocinebrina). BOLD Process ID BOCI001-12 BOCI002-12 BOCI003-12 BOCI004-12 BOCI005-12 BOCI006-12 BOCI007-12 BOCI008-12 BOCI009-12 BOCI010-12 BOCI011-12 BOCI012-12 BOCI013-12 BOCI014-12 BOCI015-12 BOCI016-12 BOCI017-13 BOCI259-13 BOCI018-13 BOCI019-13 BOCI020-13 BOCI021-13 BOCI022-13 BOCI023-13 BOCI024-13 BOCI025-13 BOCI026-13 BOCI027-13 BOCI028-13 BOCI029-13 BOCI030-13 BOCI031-13 BOCI032-13 BOCI033-13 BOCI034-13 BOCI035-13 BOCI036-13 BOCI037-13 BOCI038-13 BOCI039-13 BOCI040-13 BOCI041-13 BOCI042-13 BOCI043-13 BOCI044-13 BOCI045-13 BOCI046-13 BOCI047-13 BOCI048-13 BOCI049-13 BOCI050-13 BOCI051-13 BOCI052-13 BOCI053-13 BOCI054-13 BOCI055-13 BOCI056-13 Sample ID SP5_03 R2_01 R2_02 R2_03 SP2_04 SP3_02 SP3_04 SP4_13 SP4_15 SP4_17 SP4_18 SP4_19 BAU1037 BAU1038 BAU1039 BAU1042.1 BAU903.1 BAU903.2 BAU903.3 BAU903.4 BAU903.5 BAU903.6 BAU904.1 BAU904.2 BAU905.1 BAU905.2 BAU905.3 BAU905.4 BAU907.1 BAU907.2 BAU907.3 BAU907.4 BAU907.5 BAU907.6 BAU907.7 BAU907.8 BAU907.9 BAU907.10 BAU907.11 BAU909.1 BAU909.2 BAU909.3 BAU909.4 BAU909.5 BAU909.6 BAU909.7 BAU909.8 BAU909.9 BAU909.10 BAU909.11 BAU909.12 BAU909.13 BAU909.14 BAU909.15 BAU909.16 BAU902.1 BAU902.2 GenBank 16S GenBank ITS2 KF153517 KF153543 KF153507 KF153502 KF153582 KF153496 KF367631 KF153484 KF153530 KF153545 KF153572 KF367620 KF153568 KF367622 KF367629 KF367628 KF367621 KF367614 KF367616 KF367611 KF367602 KF367601 KF367612 KF367605 KF367607 KF367627 KF367613 KF367599 KF367632 KF153617 KF367617 KF367624 KF367630 KF367625 KF367615 KF367609 KF367626 KF367610 KF367619 KF367603 KF367606 KF367600 KF367608 KF367623 KF367618 KF367604 KF153486 KF153596 GenBank COI-5P FR851904 FR851905 FR851908 FR851906 FR851909 FR851913 FR851907 FR851903 FR851902 FR851910 FR851911 FR851900 FR851899 FR851912 FR851914 FR851901 KC883664 KC883666 KC883674 KC883673 KC883665 KC883658 KC883660 KC883642 KC883677 KC883654 KC883645 KC883644 KC883656 KC883648 KC883650 KC883678 KC883672 KC883655 KC883668 KC883657 KC883641 KC883676 KC883661 KC883669 KC883675 KC883670 KC883659 KC883652 KC883671 KC883653 KC883663 KC883646 KC883649 KC883643 KC883651 KC883667 KC883662 KC883647 KF153270 KF153455 © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Identification Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina reinai Ocinebrina cf. aciculata Ocinebrina reinai Ocinebrina reinai Ocinebrina reinai Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina reinai Ocinebrina reinai Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina reinai Ocinebrina reinai Ocinebrina cf. aciculata Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina ingloria Ocinebrina ingloria A. BARCO ET AL. BOCI057-13 BOCI058-13 BOCI059-13 BOCI060-13 BOCI260-13 BOCI061-13 BOCI062-13 BOCI063-13 BOCI064-13 BOCI065-13 BOCI066-13 BOCI067-13 BOCI068-13 BOCI069-13 BOCI070-13 BOCI071-13 BOCI072-13 BOCI073-13 BOCI074-13 BOCI075-13 BOCI076-13 BOCI077-13 BOCI078-13 BOCI079-13 BOCI080-13 BOCI265-13 BOCI081-13 BOCI082-13 BOCI083-13 BOCI084-13 BOCI085-13 BOCI086-13 BOCI087-13 BOCI088-13 BOCI089-13 BOCI090-13 BOCI092-13 BOCI093-13 BOCI266-13 BOCI091-13 BOCI094-13 BOCI095-13 BOCI096-13 BOCI097-13 BOCI098-13 BOCI099-13 BOCI100-13 BOCI101-13 BOCI102-13 BOCI103-13 BOCI104-13 BOCI105-13 BOCI261-13 BOCI262-13 BOCI106-13 BOCI107-13 BOCI108-13 BOCI109-13 BOCI110-13 BOCI111-13 BOCI112-13 BOCI113-13 BOCI114-13 SUPPORTING INFORMATION BAU902.3 BAU902.4 BAU902.5 BAU920.1 BAU920.2 BAU920.3 BAU920.4 BAU921.1 BAU921.2 BAU922.1 BAU922.2 BAU922.3 BAU922.4 BAU922.5 BAU922.6 BAU922.7 BAU922.8 BAU923.1 BAU923.2 BAU923.3 BAU923.4 BAU924.1 BAU924.2 BAU924.3 BAU924.5 BAU927.1 BAU927.4 BAU927.5 BAU927.6 BAU927.7 BAU927.8 BAU927.9 BAU936.1 BAU936.2 BAU936.3 BAU959.1 BAU959.2 BAU959.3 BAU959.4 BAU959.11 BAU960.1 BAU960.2 BAU960.3 BAU960.4 BAU960.5 BAU960.6 BAU961.1 BAU961.2 BAU961.3 BAU961.4 BAU962.1 BAU962.2 BAU962.3 BAU962.4 BAU962.5 BAU963 BAU965.2 BAU968.1 BAU968.2 BAU969.1 BAU969.2 BAU1026.1 BAU1026.2 KF153510 KF153658 KF153665 KF153654 KF153650 KF153595 KF153488 KF153609 KF153585 KF153563 KF153601 KF153574 KF153630 KF153566 KF153559 KF153506 KF153602 KF153540 KF153614 KF153482 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII KF153316 KF153394 KF153437 KF153452 KF153475 KF153435 KF153328 KF153431 KF153409 KF153465 KF153384 KF153295 KF153420 KF153430 KF153376 KF153413 KF153403 KF153310 KF153354 KF153466 KF153369 KF153261 KF153265 KF153438 KF153629 KF153610 KF153532 KF153520 KF153528 KF153580 KF153640 KF153534 KF153578 KF153590 KF153575 KF153477 KF153358 KF153333 KF153348 KF153427 KF153349 KF153360 KF153424 KF153451 KF153322 KF153447 KF153421 KF153646 KF153639 KF153664 KF153628 KF153632 KF153533 KF153544 KF153538 KF153556 KF153542 KF153564 KF153604 KF153606 KF153518 KF153505 KF153512 KF153552 KF153607 KF153641 KF153620 KF153661 KF153616 KF153494 KF153599 KF153483 KF153633 KF153516 KF153262 KF153359 KF153375 KF153365 KF153398 KF153371 KF153410 KF153468 KF153472 KF153330 KF153401 KF153308 KF153319 KF153361 KF153281 KF153461 KF153266 KF153442 KF153326 KF153408 KF153471 KF153441 © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Ocinebrina ingloria Ocinebrina ingloria Ocinebrina ingloria Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina nicolai Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii A. BARCO ET AL. BOCI115-13 BOCI116-13 BOCI117-13 BOCI118-13 BOCI119-13 BOCI120-13 BOCI121-13 BOCI122-13 BOCI123-13 BOCI124-13 BOCI125-13 BOCI126-13 BOCI127-13 BOCI128-13 BOCI129-13 BOCI130-13 BOCI131-13 BOCI132-13 BOCI133-13 BOCI134-13 BOCI135-13 BOCI136-13 BOCI137-13 BOCI138-13 BOCI139-13 BOCI140-13 BOCI141-13 BOCI142-13 BOCI143-13 BOCI144-13 BOCI145-13 BOCI146-13 BOCI147-13 BOCI148-13 BOCI149-13 BOCI150-13 BOCI151-13 BOCI152-13 BOCI153-13 BOCI154-13 BOCI155-13 BOCI156-13 BOCI157-13 BOCI158-13 BOCI159-13 BOCI160-13 BOCI161-13 BOCI162-13 BOCI163-13 BOCI164-13 BOCI165-13 BOCI166-13 BOCI167-13 BOCI168-13 BOCI169-13 BOCI170-13 BOCI171-13 BOCI172-13 BOCI173-13 BOCI174-13 BOCI175-13 BOCI176-13 BOCI267-13 SUPPORTING INFORMATION BAU1026.4 BAU1026.5 BAU1029.1 BAU1029.3 BAU1033.1 BAU1033.2 BAU1033.4 BAU1033.5 BAU1033.6 BAU1044.1 BAU1044.2 BAU1044.3 BAU1044.4 BAU1044.5 BAU1045.1 BAU1045.2 BAU1045.3 BAU1045.4 BAU1045.5 BAU1046.1 BAU1046.2 BAU1046.3 BAU1046.4 BAU1046.5 BAU1048.1 BAU1048.2 BAU1048.4 BAU1048.5 BAU1048.6 BAU1049.1 BAU1049.2 BAU1049.3 BAU1049.4 BAU1049.5 BAU1049.6 BAU1050.1 BAU1050.2 BAU1050.3 BAU1050.4 BAU1050.5 BAU1051 BAU1052 BAU1053 BAU1054 BAU1055.1 BAU1055.2 BAU1055.3 BAU1055.4 BAU1055.5 BAU1055.6 BAU1055.7 BAU1058 BAU1059.1 BAU1059.10 BAU1059.2 BAU1059.3 BAU1059.4 BAU1059.5 BAU1059.6 BAU1059.7 BAU1059.8 BAU1059.9 BAU1060.1 KF153636 KF153546 KF153522 KF153619 KF153489 KF153523 KF153663 KF153648 KF153603 KF153493 KF153498 KF153490 KF153605 KF153561 KF153560 KF153579 KF153547 KF153583 KF153642 KF153511 KF153659 KF153662 KF153645 KF153600 KF153554 KF153651 KF153567 KF153666 KF153643 KF153550 KF153594 KF153491 KF153553 KF153618 KF153647 KF153621 KF153627 KF153487 KF153558 KF153509 KF153655 KF153492 KF153611 KF153514 KF153635 KF153521 KF153500 KF153597 KF153504 KF153501 KF153513 KF199906 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII KF153327 KF153334 KF153380 KF153336 KF153453 KF153273 KF153473 KF153339 KF153263 KF153467 KF153279 KF153293 KF153274 KF153470 KF153406 KF153404 KF153425 KF153382 KF153433 KF153378 KF153318 KF153457 KF153463 KF153392 KF153289 KF153391 KF153300 KF153414 KF153454 KF153479 KF153303 KF153309 KF153412 KF153272 KF153464 KF153386 KF153450 KF153275 KF153389 KF153282 KF153271 KF153402 KF153283 KF153302 KF153314 KF153440 KF153377 KF153277 KF153478 KF153284 KF153321 KF153351 KF153335 KF153331 KF153297 KF153458 KF153307 KF153301 KF153320 KF153426 KF153368 KF153280 KF199915 © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina hispidula Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina piantonii Ocinebrina carmelae Ocinebrina hispidula Ocinebrina hispidula Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. aciculata A. BARCO ET AL. BOCI268-13 BOCI269-13 BOCI270-13 BOCI271-13 BOCI272-13 BOCI273-13 BOCI177-13 BOCI178-13 BOCI179-13 BOCI274-13 BOCI180-13 BOCI181-13 BOCI263-13 BOCI182-13 BOCI183-13 BOCI184-13 BOCI185-13 BOCI186-13 BOCI187-13 BOCI188-13 BOCI189-13 BOCI190-13 BOCI191-13 BOCI192-13 BOCI193-13 BOCI194-13 BOCI195-13 BOCI196-13 BOCI197-13 BOCI198-13 BOCI199-13 BOCI200-13 BOCI201-13 BOCI202-13 BOCI203-13 BOCI204-13 BOCI205-13 BOCI206-13 BOCI207-13 BOCI208-13 BOCI209-13 BOCI210-13 BOCI211-13 BOCI212-13 BOCI213-13 BOCI214-13 BOCI215-13 BOCI216-13 BOCI217-13 BOCI218-13 BOCI219-13 BOCI220-13 BOCI221-13 BOCI222-13 BOCI223-13 BOCI224-13 BOCI225-13 BOCI226-13 BOCI227-13 BOCI228-13 BOCI229-13 BOCI230-13 BOCI231-13 SUPPORTING INFORMATION BAU1060.2 BAU1060.3 BAU1060.4 BAU1060.5 BAU1060.6 BAU1060.7 BAU1061.1 BAU1061.2 BAU1061.3 BAU1061.4 BAU1062.1 BAU1062.2 BAU1063.1 BAU1063.2 BAU1063.3 BAU1063.4 BAU1063.5 BAU1064.1 BAU1065.1 BAU1065.2 BAU1065.3 BAU1065.4 BAU1065.6 BAU1106.1 BAU1106.2 BAU1106.3 BAU1107.1 BAU1107.2 BAU1107.3 BAU1109.1 BAU1109.2 BAU1109.3 BAU1133.1 BAU1133.2 BAU1133.3 BAU1133.4 BAU1133.6 BAU1280.1 BAU1280.2 BAU1280.3 BAU1280.4 BAU1280.5 BAU1281 BAU1282.1 BAU1282.2 BAU1290.1 BAU1290.3 BAU1291.2 BAU1291.3 BAU1291.4 BAU1293.1 BAU1293.2 BAU1293.3 BAU1293.4 BAU1293.5 BAU1293.6 BAU1293.7 BAU1294 BAU1295.1 BAU1295.2 BAU1295.3 BAU1295.4 BAU1295.5 KF153615 KF153634 KF153649 KF199907 KF199905 KF199902 KF199903 KF199904 KF199908 KF153481 KF153537 KF153519 KF153562 KF153551 KF153612 KF153581 KF153625 KF153497 KF153531 KF153588 KF153503 KF153586 KF153637 KF153524 KF153549 KF153587 KF153644 KF153589 KF153527 KF153485 KF153571 KF153529 KF153624 KF153656 KF153622 KF153631 KF153626 KF153499 KF153536 KF153652 KF153653 KF153623 KF153576 KF153584 KF153573 KF153598 KF153541 KF153557 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII KF199916 KF199914 KF199911 KF199912 KF199913 KF199917 KF153264 KF153364 KF153317 KF153388 KF153332 KF153387 KF153480 KF153278 KF153429 KF153299 KF153291 KF153344 KF153356 KF153443 KF153395 KF153305 KF153436 KF153381 KF153340 KF153385 KF153439 KF153390 KF153276 KF153337 KF153444 KF153346 KF153268 KF153417 KF153350 KF153290 KF153353 KF153374 KF153294 KF153393 KF153355 KF153446 KF153366 KF153286 KF153306 KF153312 KF153456 KF153269 KF153296 KF153342 KF153407 KF153363 KF153343 KF153445 KF153422 KF153434 KF153287 KF153419 KF153459 KF153370 KF153399 © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina cf. aciculata Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina miscowichae Ocinebrina miscowichae Ocinebrina miscowichae Ocinebrina miscowichae Ocinebrina miscowichae Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina nicolai Ocinebrina nicolai Ocinebrina leukos Ocinebrina leukos Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina nicolai Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii A. BARCO ET AL. BOCI232-13 BOCI233-13 BOCI234-13 BOCI264-13 BOCI235-13 BOCI236-13 BOCI237-13 BOCI238-13 BOCI239-13 BOCI240-13 BOCI241-13 BOCI242-13 BOCI243-13 BOCI244-13 BOCI245-13 BOCI246-13 BOCI247-13 BOCI248-13 BOCI249-13 BOCI250-13 BOCI251-13 BOCI252-13 BOCI253-13 BOCI254-13 BOCI255-13 BOCI256-13 BOCI257-13 BOCI258-13 SUPPORTING INFORMATION BAU1295.6 BAU1296.2 BAU1296.3 BAU1296.4 BAU1296.5 BAU1296.6 BAU1296.7 BAU1296.8 BAU1298.1 BAU1298.2 BAU1298.3 BAU1298.4 BAU1298.5 BAU1298.6 BAU1299.1 BAU1299.2 BAU1299.3 BAU1299.4 BAU1299.5 BAU1299.6 BAU1299.7 BAU1300.1 BAU1300.3 BAU1300.4 BAU1300.5 BAU1308.1 BAU1308.2 BAU1308.3 KF153535 KF153593 KF153508 KF153592 KF153569 KF153565 KF153515 KF153608 KF153660 KF153548 KF153657 KF153638 KF153570 KF153591 KF153526 KF153495 KF153555 KF153577 KF153539 KF153525 CRYPTIC DIVERSITY IN OCINEBRINA EDWARDSII KF153362 KF153449 KF153313 KF153415 KF153411 KF153324 KF153474 KF153460 KF153315 KF153383 KF153405 KF153462 KF153416 KF153448 KF153345 KF153285 KF153396 KF153373 KF153423 KF153367 KF153341 KF153338 KF153469 KF153428 KF153347 KF153397 KF153323 © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013 Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii Ocinebrina cf. edwardsii

RELATED PAPERS

RELATED TOPICS

Log In

Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae)

Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae)

Related Papers

RELATED PAPERS

RELATED TOPICS