Journal of The Malacological Society of London Molluscan Studies Journal of Molluscan Studies (2019) 00: 1–12. doi:10.1093/mollus/eyz022 Giulia Fassio1 , Valeria Russini1 , Francesco Pusateri2 , Riccardo Giannuzzi-Savelli3 , Tore Høisæter4 , Nicolas Puillandre5 , Maria Vittoria Modica6 , 7 and Marco Oliverio1 , 1 Department of Biology and Biotechnologies “Charles Darwin” Zoology, Sapienza University of Rome, Viale dell’Università 32, I-00185 Roma, Italy; 2 Via Castellana 64, 90135 Palermo; 3 Via Mater Dolorosa 54, 90146 Palermo; 4 Museum of Natural History, University of Bergen, PO Box 7800, Thormøhlens Gate 53A, N-5020 Bergen, Norway; 5 Institut de Systématique Evolution Biodiversité, Muséum National d’Histoire Naturelle, Centre Nationale de la Recherche Scientifique, Sorbonne Université, Ècole Pratique des Hautes Ètudes, Université des Antilles, 57 Rue Cuvier, CP 26, 75005 Paris, France; 6 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121, Naples, Italy; and 7 Institut des Biomolécules Max Mousseron, UMR 5247, University of Montpellier 2, Place Eugène Batallion, F-34095 Montpellier, France Correspondence: M. Oliverio; email: marco.oliverio@uniroma1.it (Received 9 January 2019; editorial decision 9 April 2019 ) ABSTRACT The systematics of several Eastern Atlantic conoidean species, traditionally ascribed to the genus Raphitoma Bellardi, 1847, are revised on the basis of DNA sequence data from three gene regions (cytochrome c oxidase subunit I, 16S rRNA and 12S rRNA). We assign genus ranking to three major lineages (Raphitoma, Cyrillia Kobelt, 1905 and Leufroyia Monterosato, 1884) and suggest that two West African species belong in the subgenus Daphnella (Paradaphne) Laseron, 1954. A new classification, based on molecular systematics and critical study of morphology, is provided for all Eastern Atlantic and Mediterranean species that are currently ascribed to Raphitoma s.l. The genus Clathromangelia Monterosato, 1884 is confirmed as belonging to Raphitomidae. Phylogenetic relationships and genetic distances suggest that Raphitoma maculosa Høisæter, 2016 and R. obesa Høisæter, 2016 may be divergent morphotypes of R. bicolor (Risso, 1826) and Cyrillia aequalis (Jeffreys, 1867), respectively. INTRODUCTION The Raphitomidae are probably the most diverse family of Conoidea, in terms of species richness, ecological range and anatomy (Kantor & Taylor, 2002; Bouchet et al., 2011). The name Raphitomidae Bellardi, 1875 is based on the genus Raphitoma Bellardi, 1847. At the time of its introduction, this genus comprised 34 fossil and Recent species (Bellardi, 1847: 85) that had previously been classified in various genera, such as Pleurotoma and Clathurella. The genus Raphitoma has been particularly well studied in the northeastern Atlantic and Mediterranean, where a recent estimate (Giannuzzi-Savelli et al., 2018) suggested that over 50 extant species occur. These snails, which are usually active at night, live mostly in marine soft-bottom environments at depths ranging from 0–100 m (Raphitoma pseudohystrix has been collected at 700 m). While they inhabit a wide variety of habitats ranging from coastal bioclastic coarse sands to muddy bioclastic coarse sands, they also occur in sandy pockets between rocks and in seagrass meadows, with individuals hiding buried under sand or concealed under stones and in crevices during the day. The limits of the genus are still under debate and Raphitoma s.l., as currently conceived, comprises species with the following shell characters: turreted to biconic-pupoidal shape; small to medium size (5–25 mm) in relation to the family Raphitomidae as whole; protoconch consisting of 3–4.5 whorls when multispiral, with the typical raphitomid diagonally cancellate sculpture; and the last whorl frequently keeled (Giannuzzi-Savelli et al., 2018; Manousis et al., 2018; Fig. 1). While available data on the morphology of the soft parts are scarce, they nonetheless suggest that there is substantial variation in the anatomy of the foregut. Some species, such as R. villaria and R. linearis, have neither a radula nor a venom gland. Others, such as R. purpurea and R. leufroyi, do have a radula, a venom gland or both (Sheridan et al., 1973: 177; Pusateri & Giannuzzi-Savelli, 2008: 124). The arrangement of the foregut has been described for R. purpurea (Sheridan et al., 1973: 177; Miller, 1989: 173), but there is a different arrangement in R. linearis and R. leufroyi, where a rhynchodeal introvert or pseudoproboscis is present (Taylor et al., 1993: 128; Sheridan et al., 1973: 178). The systematic implications of this variability are still unknown, and the problem is further complicated by the lack of a comprehensive phylogenetic framework for the family Raphitomidae. The type species of Raphitoma is R. histrix Bellardi, 1847 [ex Pleurotoma hystrix Cristofori & Jan, 1832, nomen nudum] by subsequent designation (Monterosato, 1872: 54). Raphitoma histrix as almost always conceived is a fossil species (Miocene–Pleistocene) and has a complex nomenclatural history that has been summarized by Giannuzzi-Savelli et al. (2018: 9; see also Dall, 1918: 316; © The Author(s) 2019. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: journals.permissions@oup.com. Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 An assessment of Raphitoma and allied genera (Neogastropoda: Raphitomidae) G. Fassio et al. 2017), using the Q-INS-I algorithm. Ambiguous regions in the 16S rRNA and 12S rRNA alignments were discarded using Gblocks v. 0.91b (Castresana, 2000) with respectively 76% and 64% of the original positions being retained; we used default options. In our phylogenetic analyses we used the three single-gene datasets as well as a combined dataset (COI + 12S rRNA + 16S rRNA). The Bayesian information criterion implemented in jModelTest v. 2.1.7 (Posada, 2008) was used to identify the best substitution models and parameters for each gene partition; the substitution model selected for all datasets was GTR + I + G. Phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian approaches; all analyses were run on the CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010). ML analyses were done using RAxML v. 8 (Stamatakis, 2014). Branch support estimates were based on 1,000 bootstrap replicates. Bayesian analyses were performed using MrBayes v. 3.2.3 (Huelsenbeck & Ronquist, 2001); analyses were run for 10,000,000 generations, with trees sampled every 1,000 generations and 25% burn-in (for all other parameters we used default settings). Convergence of MCMC was assumed to have occurred when the effective sample size was >200 and the potential scale reduction factor was approximately 1, as calculated with Tracer v. 1.7 (Rambaut et al., 2018) Branches with bootstrap values (BS) ≥70% and posterior probabilities (PP) ≥0.95 were considered to be strongly supported. MATERIAL AND METHODS The dataset is composed of 62 specimens representing 14 raphitomid genera from the Mediterranean Sea, North Sea and Indo-Pacific region. DNA sequence data were generated by us for 28 of these specimens; sequence data for the remaining individuals were obtained from GenBank (Table 1). The specimens sampled included 17 species ascribed to the genus Raphitoma s.l.: Raphitoma aequalis, R. bicolor, R. concinna, R. cordieri, R. corimbensis, R. densa, R. horrida, R. laviae, R. leufroyi, R. linearis, R. maculosa, R. obesa, R. philberti, R. pseudohystrix, R. purpurea, R. rubroapicata and an unidentified Raphitoma sp. The dataset also included 13 other raphitomid or putative raphitomid genera: Clathromangelia Monterosato, 1884; Hemilienardia Boettger, 1895; Eucyclotoma Boettger, 1895; Rimosodaphnella Cossmann, 1916; Veprecula Melvill, 1917; Pleurotomella Verrill, 1872; Phymorhynchus Dall, 1908; Pseudodaphnella Boettger, 1895; Spergo Dall, 1895; Taranis Jeffreys, 1870; Thatcheria Angas, 1877; Daphnella Hinds, 1844; and Teretiopsis Kantor & Sysoev, 1989. Specimens from two other conoidean families were also included. These groups are the Clathurellidae (the putative sister group of the raphitomids) and the Mangeliidae (considered to be sister to the clade comprising the Raphitomidae and Clathurellidae) (Abdelkrim et al., 2018). The outgroup comprised three species of Conidae DNA was isolated from a piece of foot tissue following a standard proteinase K/phenol–chloroform extraction protocol (Oliverio & Mariottini, 2001). Three mitochondrial gene fragments were amplified: the 658-bp barcode region of cytochrome c oxidase subunit I (COI), with universal primers LCO1490 and HC02198 (Folmer et al., 1994); an approximately 500-bp region of the 16S rRNA gene, with primers 16SA (Palumbi, 1996) and CGLeuR (Hayashi, 2003) or 16SH (Espiritu et al., 2001); and an approximately 600-bp region of the 12S rRNA, with primers 12SI and 12SIII (Oliverio & Mariottini, 2001). The following PCR conditions were used: initial denaturation (94 ◦ C for 4 min), 35 cycles of denaturation (94 ◦ C for 30 s), annealing (48–51 ◦ C for COI, 52 ◦ C for 16S rRNA and 58–60 ◦ C for 12S rRNA for 40 s) and extension (94 ◦ C for 60s) and final extension (72 ◦ C for 10 min). Amplicons were purified using Exosap-IT (USB Corporation) and sequenced by Macrogen Inc. (The Netherlands). COI sequences were aligned using Geneious v. 11 (Kearse et al., 2012). Sequences for 16S rRNA and 12S rRNA were aligned with the online version of MAFFT v. 7 (Kuraku et al., 2013; Katoh et al., RESULTS The final datasets consisted of 62 COI sequences, 47 16S rRNA sequences and 34 12S rRNA sequences. Single-gene and combined analyses yielded topologically similar trees. The trees obtained from the concatenated dataset tended to show higher branch support values and this was especially so in the case of the Bayesian analysis (Fig. 2; Supplementary Material Figs S1–S7). The three families Raphitomidae, Clathurellidae and Mangeliidae together formed a strongly supported monophyletic group. Our Bayesian analyses recovered the Clathurellidae as sister to the raphitomid clade, but this relationship was not strongly supported (e.g. PP = 0.71 for combined dataset, Fig. 2). We found consistently strong support for the monophyly of the Raphitomidae. Within the Raphitomidae, specimens of the genus Raphitoma s.l. were distributed across five clades. Raphitoma leufroyi and R. concinna were strongly supported as sister species (BS = 99%, PP = 1); these two species together with R. rubroapicata and the genus Hemilienardia formed a clade that was strongly supported in the ML analysis (BS = 85%), but not in the Bayesian analysis (PP = 0.94). The Bayesian analysis showed strong support for the clade comprising R. corimbensis, Rimosodaphnella and Veprecula (PP = 0.95) and the clade comprising the ‘Raphitoma’ sp. from the Philippines (MNHN-IM2007-17882) and Eucyclotoma cymatodes (PP = 0.99). Relationships between these two clades and other raphitomids were unresolved. The two species of Clathromangelia, which were strongly supported as sister taxa (BS = 99%, PP = 1), formed a clade with Pseudodaphnella, Eucyclotoma and a ‘Raphitoma’ sp. (MNHN-IM-2007-17882) in the Bayesian analysis (PP = 1). This clade was nested within the raphitomid clade. Most of the specimens ascribed to Raphitoma s.l. formed a strongly supported clade only in the Bayesian analyses of the 12S rRNA, 16S rRNA and combined datasets (PP = 1 in Fig. 2; see also Supplementary Material Figs S1, S3, S5); this large clade was not strongly supported in most of the remaining analyses (Supplementary Material Figs S2, S4, S6, S7). However, we consistently found strong support for two sublineages within this clade. The first sublineage comprised R. linearis, R. aequalis and R. obesa (BS = 100%, PP = 1). The second sublineage consisted of R. pseudohystrix, R. bicolor, R. cordieri, R. densa, R. horrida, R. laviae, R. maculosa, R. philberti and R. purpurea (BS = 100%, PP = 1); in this sublineage, R. pseudohystrix 2 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 van Aartsen et al., 1984: 89–90; Rolán et al., 1998: 105). Raphitoma pseudohystrix (Sykes, 1906) appears to be the closest extant relative of R. histrix; while the teleoconch of the former is almost identical to that of the latter, the protoconch in the extant species is paucispiral whereas in R. histrix it is multispiral. According to current taxonomy, at least eight nominal genera are included in the synonymy of Raphitoma s.l. (see Systematic Descriptions below). Høisæter (2016) argued that DNA sequencebased phylogenetic studies would most likely show that Raphitoma s.l. consists of several genus-level taxa, for which available names could be employed. By carrying out a molecular phylogenetic study of the raphitomids, we seek to explore this issue. Our dataset consists of representatives of at least 13 recognized genera of Raphitomidae (18% of the approximately 70 genera known for this family; MolluscaBase, 2018), as well as two species of Clathromangelia, a genus that has been considered to be a raphitomid (Oliverio, 1995) or a clathurellid (Bouchet et al., 2011). The dataset also includes 14 species, which, on the basis of morphology, have been ascribed to Raphitoma s.l.; these include the type species of Cenodagreutes, Cyrillia, Leufroyia, Lineotoma and Philbertia, the apparent closest relatives of the type species of Cordieria and Cyrtoides, and the closest extant relative of the (fossil) type species of Raphitoma. Table 1. List of material used in the study along with voucher registration numbers, collection localities, GenBank accession numbers for sequences and relevant references. GenBank accession numbers Taxon Voucher ID Locality COI 16S rRNA 12S rRNA References Cyrillia aequalis (Jeffreys, 1867) ZMBN-020209-O Norway, 60◦ 13′ 48′′ N, 5◦ 12′ E JF834219 JF834214 Cyrillia aequalis (Jeffreys, 1867) ZMBN-E-345-66a Norway, 60◦ 18′ N, 5◦ 10′ 48′′ E JF834221 Høisæter (2016) Cyrillia aequalis (Jeffreys, 1867) ZMBN-E-345-66b Norway, 60◦ 18′ N, 5◦ 10′ 48′′ E JF834225 Høisæter (2016) Cyrillia aequalis (Jeffreys, 1867) MT09383 North Sea, 57◦ 53′ 56.4′′ N, 0◦ 54′ 57.6′′ W KR084567 Barco et al. (2016) Cyrillia aequalis (Jeffreys, 1867) MT09222 North Sea, 55◦ 22′ 15.6′′ N, 0◦ 12′ 25.2′′ W KR084390 Cyrillia linearis (Montagu, 1803) BAU-2234 Italy, Giannutri Is., loc. Le Cerniette, MK410632 MK410605 MK410623 MK410599 This study Raphitomidae Høisæter (2016) Barco et al. (2016) MK410585 This study 42◦ 15′ 10′′ N, 011◦ 05′ 32′′ E Cyrillia linearis (Montagu, 1803) BAU-2912.1 Italy, Giglio Is., Cala Cupa, 42◦ 22′ 06′′ N, 10◦ 55′ 12′′ E, 10–20 m Cyrillia obesa (Høisæter, 2016) ZMBN-E-37-68 Norway, 60◦ 18′ N, 5◦ 07′ 48′′ E JF834220 MK410610 Høisæter (2016); this study Clathromangelia granum (Philippi, BAU-3082.1 Italy, Scilla, 38◦ 15′ 23′′ N, 15◦ 42′ 45′′ E, MK410624 MK410600 This study MK410627 MK410601 This study EU015740 HQ401674 HQ401607 Puillandre et al. (2008) MK410635 MK410608 MK410587 This study EU015678 HQ401676 HQ401610 Puillandre et al. (2008) 1844) 35–37 m BAU-1545 Greece, Astypalea Is., VYLLAS, 36◦ 35′ 02′′ N, 026◦ 25′ 24′′ E, 1–7 m, under rocks MNHN-IM-2007-17927 Salomon Is., Vella Gulf, SALOMON 2, 8◦ 3′ 32.4′ S, 156◦ 54′ 32.4′′ E Daphnella (Paradaphne) corimbensis BAU-2989 Canary Islands, Tenerife, Radazul, 28◦ 24′ 08′′ N, 16◦ 19′ 5′′ W, 20 m Rolán, Otero-Schmitt & Fernandes, 1998 Eucyclotoma cymatodes (Hervier, MNHN-IM-2007-17903 Philippines, Pamilacan Is., PANGLAO 2004, 9◦ 29′ 24′′ N, 123◦ 56′ 0′′ E 1897) Hemilienardia acinonyx Fedosov, MNHN-IM-2009-33593 Philippines, Panglao Is., Momo beach KX233238 KX233249 MNHN-IM-2007-17861 Philippines, Panglao Is., Sungcolan Bay, EU015683 HQ401684 Fedosov et al. (2017) Stahlschmidt, Puillandre, Aznar-Cormano & Bouchet, 2017 Hemilienardia calcicincta (Melvill & HQ401618 Puillandre et al. (2008) PANGLAO 2004, 9◦ 38′ 30′′ N, Standen, 1895) 123◦ 49′ 12′′ E Leufroyia concinna (Scacchi, 1836) ZMBN-H-3-69a Norway, 60◦ 33′ N, 4◦ 52′ 12′′ E JF834222 Leufroyia concinna (Scacchi, 1836) ZMBN-E-23-67 Norway, 60◦ 18′ N, 5◦ 10′ 48′′ E JF834223 Leufroyia concinna (Scacchi, 1836) ZMBN-020209-F Norway, 60◦ 13′ 48′′ N, 5◦ 12′ E JF834224 JF834218 Leufroyia concinna (Scacchi, 1836) BAU-2254.1 Croatia, Biograd, 43◦ 55′ 51′′ N, MK410616 MK410593 MK410633 MK410606 Høisæter (2016) Høisæter (2016) Høisæter (2016) MK410580 This study 15◦ 26′ 42′′ E Leufroyia concinna (Scacchi, 1836) BAU-2237 France, La Ciotat, Figuerolles, This study 43◦ 09′ 53′′ N, 5◦ 35′ 45′′ E, 15 m Leufroyia leufroyi (Michaud, 1828) BAU-2240.1 Croatia, Sevid, 43◦ 28′ 46′′ N, 16◦ 02′ 08′′ E, MK410613 This study 2–4 m (Continued) Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 3 Daphnella sp. Systematics of Raphitoma Clathromangelia loiselieri Oberling, 1970 Table 1. Continued GenBank accession numbers Taxon Voucher ID Leufroyia leufroyi (Michaud, 1828) BAU-1742 Locality COI Sardinia, Villasimius, 39◦ 07′ 43′′ N, MK410628 16S rRNA 12S rRNA References MK410584 This study 9◦ 32′ 17′′ E ‘Phymorhynchus’ sp. MCR-1256 Mid-Cayman Spreading Centre, Beebe Plouviez et al. (2015) KJ566952 KM979537 EU015657 HQ401701 HQ401640 Puillandre et al. (2008) EU015700 HQ401688 HQ401624 Puillandre et al. (2008) MK410630 MK410603 MK410619 MK410595 vent chimneys Pleurotomella sp. MNHN-IM-2007-17848 New Caledonia, Lansdowne, EBISCO, 20◦ 4′ 52.32′′ S, 160◦ 20′ 2.34′′ E Pseudodaphnella aureotincta (Hervier, MNHN-IM-2007-17878 Philippines, Pamilacan Is., PANGLAO 2004, 9◦ 29′ 24′′ N, 123◦ 56′ 6′′ E 1897) Raphitoma bicolor (Risso, 1826) BAU-1897 France, St. Maxime, 43◦ 18′ 49′′ N, This study 6◦ 40′ 22′′ E, intertidal Raphitoma cordieri (Payraudeau, 1826) BAU-2262.1 Croatia, Sukosan, 44◦ 02′ 04′′ N, MK410582 This study 15◦ 18′ 57′′ E Raphitoma cordieri (Payraudeau, 1826) BAU-2262.2 Croatia, Sukosan, 44◦ 02′ 04′′ N, MK410625 This study 15◦ 18′ 57′′ E BAU-2257.1 Croatia, Sukosan, 44◦ 02′ 10′′ N, MK410617 MK410594 MK410629 MK410602 MK410581 This study G. Fassio et al. Raphitoma densa (Monterosato, 1884) 15◦ 18′ 55′′ E BAU-1895 Italy, Torre Colimena, 40◦ 17′ 39′′ N, This study 17◦ 45′ 17′′ E, 3 m Raphitoma horrida (Monterosato, 1884) BAU-2264.1 Croatia, Dugi Otok, 43◦ 59′ N, 15◦ 05′ 34′′ E MK410620 MK410596 Raphitoma horrida (Monterosato, 1884) BAU-1900 Corsica, Tour d′ Ancone, 42◦ 02′ 36′′ N, MK410631 MK410604 MK410612 MK410590 MK410577 This study MK410615 MK410592 MK410579 This study MK410591 MK410578 This study MK410583 This study This study 8◦ 43′ 20′′ E, 10 m Raphitoma horrida (Monterosato, 1884) BAU-1906.1 France, St. Maxime, 43◦ 18′ 49′′ N, 6◦ 40′ 22′′ E, intertidal Raphitoma laviae (Philippi, 1844) BAU-2253.1 Croatia, Telascjca, 43◦ 53′ 30′′ N, 15◦ 09′ 33′′ E Raphitoma laviae (Philippi, 1844) BAU-2246.1 Croatia, Zaton, 44◦ 13′ 07′′ N 15◦ 09′ 41′′ E MK410614 Raphitoma maculosa (Høisæter, 2016) ZMBN-040809_X Norway, 60◦ 18′ N, 5◦ 07′ 48′′ E MK410638 Raphitoma philberti (Michaud, 1829) BAU-2365.1 Croatia, Biograd, 43◦ 55′ 51′′ N, MK410622 Høisæter (2016); this study MK410598 This study 15◦ 26′ 42′′ E Raphitoma philberti (Michaud, 1829) BAU-2258.1 Croatia, Vrsi, 44◦ 16′ 56′′ N, 15◦ 12′ 35′′ E MK410618 This study Raphitoma philberti (Michaud, 1829) BAU-1893.1 Greece, Limnos, Koukonisi Bay, MK410611 This study Raphitoma philiberti (Michaud, 1829) BAU-3046 39◦ 53′ 07′′ N, 25◦ 16′ 16′′ E Greece: Astypalea Is., Vai, VYLLAS MK410636 MK410588 This study MK410589 This study 2017, 36◦ 35′ 13′′ N, 026◦ 24′ 10′′ E, 1–6 m, under rocks Raphitoma pseudohystrix (Sykes, 1906) BAU-3205 Malta, SW, off Gnejna Bay, MK410637 MK410609 35◦ 49′ 54.3′′ N, 14◦ 17′ 15.2′′ E, 220 m, fine sand and mud (Continued) Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 4 Raphitoma densa (Monterosato, 1884) Table 1. Continued GenBank accession numbers Taxon Voucher ID Raphitoma purpurea (Montagu, 1803) BAU-2337.1 Locality COI 16S rRNA 48◦ 48′ 5′′ N, MK410621 MK410597 France, Ploubazlanec, 48◦ 48′ 5′′ N, MK410626 France, Ploubazlanec, 12S rRNA References This study 3◦ 00′ 10′′ W, intertidal Raphitoma purpurea (Montagu, 1803) BAU-2337.3 This study 3◦ 00′ 10′′ W, intertidal Raphitoma purpurea (Montagu, 1803) BAU-2338 France, Ploubazlanec, 48◦ 48′ 5′′ N, MK410634 MK410607 MK410586 This study EU015713 HQ401703 HQ401642 Puillandre et al. (2008) 3◦ 00′ 10′′ W, intertidal Raphitoma rubroapicata (E. A. Smith, MNHN-IM-2007-17890 Philippines, Panglao Is., off Momo beach, PANGLAO 2004, 9◦ 36′ 30′′ N, 1885) 123◦ 45′ 18′′ E ‘Raphitoma’ sp. MNHN-IM-2007-17882 Philippines, Balicasag Is., PANGLAO Puillandre et al. (2008) EU015704 2004, 9◦ 30′ 54′′ N, 123◦ 41′ 12′′ E Rimosodaphnella sp. MNHN-IM-2007-17836 New Caledonia, Koumac Sector, around Puillandre et al. (2008) EU015645 HQ401704 EU015650 HQ401682 HQ401616 Puillandre et al. (2008) HQ401584 HQ401707 HQ401645 Puillandre et al. (2011) KR087296 KR088045 KR087382 ] Fedosov et al. (2015) EU015654 HQ401708 HQ401646 Puillandre et al. (2008) EU015736 FJ868138 FJ868124 Puillandre et al. (2008) EU015705 HQ401717 HQ401654 Puillandre et al. (2008) Ouaco, BOA1, 20◦ 48′ 42′′ S, MNHN-IM-2007-17841 New Caledonia, SE Fairway, EBISCO, 21◦ 32′ 36′′ S, 162◦ 28′ 36′′ E MNHN-IM-2007-42296 Philippines, AURORA 2007, 15◦ 56′ 34.2′′ N, 121◦ 50′ 11.4′′ E Taranis sp. MNHN-IM-2013-52046 Papua New Guinea, Bismarck Archipelago, W Kairiru I., 3◦ 19′ 26.4′′ S, 143◦ 27′ 14.4′′ E Teretiopsis cf. hyalina (Sysoev & MNHN-IM-2007-17845 New Caledonia, SE Fairway, EBISCO, 21◦ 28′ 8′′ S, 162◦ 33′ 54′′ E Bouchet, 2001) Thatcheria mirabilis (Angas, 1877) MNHN-IM-2007-17924 Salomon Is., SE Isabel, SALOMON 2, 8◦ 16′ 54′′ S, 159◦ 59′ 42′′ E Veprecula cf. spanionema (Melvill, MNHN-IM-2007-17883 Philippines, Balicasag Is., PANGLAO 2004, 9◦ 30′ 54′′ N, 123◦ 41′ 12′′ E 1917) Clathurellidae Lienardia crassicostata (Pease, 1860) NA NA JF823629 JF823611 JF823590 Cabang et al. (2011) Lienardia nigrotincta (Montrouzier in MNHN-IM-2007-42607 Vanuatu, E Luganville, Segond Channel, HQ401575 HQ401666 HQ401599 Puillandre et al. (2011) EU015679 HQ401698 HQ401634 Puillandre et al. (2008) HQ401572 HQ401660 HQ401590 Puillandre et al. (2011) HQ401582 HQ401699 HQ401635 Puillandre et al. (2011) SANTO 2006, 15◦ 30′ 58′′ S, 167◦ 11′ 52′′ E Souverbie & Montrouzier, 1873) Nannodiella ravella (Hedley, 1922) MNHN-IM-2007-17904 Philippines, Panglao Is., off San Isidro, PANGLAO 2004, 9◦ 33′ 54′′ N, 123◦ 50′ 30′′ E Mangeliidae Anticlinura sp. (Thiele, 1934) MNHN-IM-2007-42513 Salomon Is., Sta Isabel, SALOMON 2, 8◦ 47′ 0′′ S, 159◦ 37′ 54′′ E Propebela cf. scalaris (Møller, 1842) MNHN-IM-2007-42325 Norway, Hornsund, Svalbard (Continued) Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 5 Taranis sp. Systematics of Raphitoma 164◦ 24′ 12′′ E Spergo sp. Table 1. Continued GenBank accession numbers Taxon Locality COI 16S rRNA 12S rRNA References MNHN-IM-2007-17925 Salomon Is., Choiseul, SALOMON 2, EU015738 HQ401711 HQ401649 Puillandre et al. (2008) KJ550437 KJ550900 KJ551133 Puillandre et al. (2014) KJ550497 KJ550930 KJ551134 Puillandre et al. (2014) KJ550006 KJ550745 KJ551370 Puillandre et al. (2014) 6◦ 37′ 12.6′′ S, 156◦ 12′ 44.4′ E Kantor, 1990) Conidae MNHN-IM-2007-30883 Philippines, Bohol Is., Ubajan, PANGLAO 2004, 9◦ 41′ 30′′ N, 12350′ 60′′ E Conus textile (Linnaeus, 1758) MNHN-IM-2007-30900 Vanuatu, NW Aésé Is., SANTO 2006, 15◦ 25′ 7′′ S, 167◦ 14′ 10′′ E Conus ventricosus (Gmelin, 1791) NA Djerba, Tunisia Institutional abbreviations are as follows: BAU, Department of Biology and Biotechnologies, ‘Sapienza’ University, Rome; MNHN, Muséum national d’Histoire naturelle, Paris; MT, German Centre for Marine Biodiversity Research, Senckenberg Institute, Wilhelmshaven; ZMBN, University Museum of Bergen Natural History Collections. NA indicates that specimen registration data were not available. Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 6 Conus radiatus (Gmelin, 1791) G. Fassio et al. Toxicochlespira pagoda (Sysoev & Voucher ID Systematics of Raphitoma was sometimes strongly supported as sister to the clade containing the other members of the sublineage. explore the relationship between this species and lineages currently placed in the genus Daphnella Hinds, 1844 (which may prove to be polyphyletic). We suggest a provisional classification of R. corimbensis and R. bedoyai in Paradaphne Laseron, 1954 (type species: Daphnella botanica Hedley, 1922 by original designation), which is currently ranked as a subgenus of Daphnella Hinds, 1844. The rationale for this classification is that the shell features of the type species of Paradaphne are strikingly similar to R. bedoyai and R. corimbensis. Our findings suggest that R. rubroapicata (E.A. Smith, 1885) and the ‘Raphitoma’ sp. (IM-2007-17882) do not belong in the genus Raphitoma, but further work involving broader taxon sampling is needed to clarify their relationships. On the basis of the phylogenetic results presented here and shell morphological data, we propose the following new classification for the bulk of Mediterranean/East Atlantic species currently ascribed to Raphitoma s.l., as previously conceived (Tables 2–4). DISCUSSION The Bayesian analyses showed a sister-group relationship between the Raphitomidae and Clathurellidae; this agrees with the most recent phylogenetic hypotheses on the Conoidea. These hypotheses were based on the most extensive taxon sampling achieved to date (Puillandre et al., 2011; Abdelkrim et al., 2018), but were not strongly supported. The genus Clathromangelia was confirmed as belonging to the Raphitomidae, as has previously been suggested on the basis of anatomical and protoconch data (Oliverio, 1995). Our finding that Clathromangelia is a member of a clade containing Pseudodaphnella and Eucyclotoma is not surprising given the similarity in shell morphology between these three taxa and particularly between Clathromangelia and Pseudodaphnella. This study shows that most of the species ascribed to Raphitoma s.l. fall into three clades, and we propose that these distinct lineages should be ranked as genera. We propose to use the name Raphitoma for the clade containing Raphitoma pseudohystrix (believed to be the closest extant relative of the type species of Raphitoma), R. bicolor, R. cordieri, R. densa, R. horrida, R. laviae, R. maculosa, R. philberti and R. purpurea. We note that a strongly supported sister-group relationship of R. pseudohystrix to the other species in the sublineage was recovered in some analyses. We also note that R. pseudohystrix never formed a clade with other morphologically similar spiny shelled raphitomids, such as R. cordieri and R. horrida. The clade comprising R. linearis, R. aequalis and R. obesa may be the sister group of Raphitoma s.s., but this relationship was strongly supported in only three of the eight analyses we carried out. We propose, therefore, to treat the clade of R. linearis, R. aequalis and R. obesa as a distinct genus for which the name Cyrillia Kobelt, 1905 is available (see Systematic Descriptions below). Our results show that the R. leufroyi + R. concinna lineage is not nested within the clade that contains most of the Raphitoma species or the clade of R. linearis + R. aequalis + R. obesa. We use the generic name Leufroyia Monterosato, 1884 for the R. leufroyi + R. concinna lineage. Raphitoma corimbensis was not related to the lineages Raphitoma, Cyrillia or Leufroyia and, as suggested by its shell morphology (and by that of its likely close relative, R. bedoyai Rolán, Otero-Schmitt & Fernandes, 1998), further studies of its systematic position should SYSTEMATIC DESCRIPTIONS Family RAPHITOMIDAE Bellardi, 1875 Genus Raphitoma Bellardi, 1847 (Table 2; Fig. 1) Raphitoma Bellardi, 1847: 612. [type species Raphitoma histrix Bellardi, 1847 (ex Pleurotoma hystrix Cristofori & Jan, 1832, nomen nudum) SD, Monterosato, 1872: 54]. Homotoma Bellardi, 1875: 22 (type species Murex reticulatus Renier, 1804; SD, Powell, 1966). Cordieria Monterosato, 1884: 131 (type species Murex reticulatus Renier, 1804.; SD, Crosse, 1885; erroneously credited to Brocchi, 1814, ICZN, 1999, Art. 67.7; not Rouault, 1848). Philbertia Monterosato, 1884: 132 (type species Pleurotoma bicolor Risso, 1826; SD, Crosse, 1885). Peratotoma Harris & Burrows, 1891: 113 (replacement name for Homotoma Bellardi, 1875, not Guérin-Ménéville, 1844). Cyrtoides F. Nordsieck, 1968: 176 [type species Pleurotoma rudis Scacchi, 1836 (not G.B. Sowerby I, 1834; renamed Cordieria pupoides Monterosato, 1884 and R. neapolitana F. Nordsieck, 1977) OD]. Diagnosis: Shell small to medium size for family, ranging in height from 5 mm (R. laviae) to 25 mm (R. cordieri, R. bourguignati); shape turreted to biconic-pupoidal; suture impressed. 7 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 Figure 1. Raphitoma hystrix Bellardi, 1847, the type species of the genus Raphitoma Bellardi, 1847. A–C. Neotype (MRSN n. cat. 011.16.008) from Colli Astesi (Italy: Pliocene, Piacentian); shell height 17.6 mm. D. Protoconch of a shell from Stirone River (Italy: Pliocene, Piacentian); scale bar = 100 μm. G. Fassio et al. Figure 3. Cyrillia linearis (Jeffreys, 1867), the type species of the genus Cyrillia Kobelt, 1905. A–E. Shell from a depth of 1 m, Lastovo (Croatia); shell height 7 mm. Scale bar = 100 μm. Protoconch: If multispiral, then 3–4.5 whorls, with protoconch I (embryonic shell) of 0.5–0.7 whorls, with reticulate sculpture of spirals and orthocline axial striae, and protoconch II (larval shell) of 2.3–3.5 whorls, with diagonally cancellate sculpture and often keeled last whorl; if paucispiral, then 2 whorls, with large nucleus and reticulate sculpture. Teleoconch with slender spire of 5 8 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 Figure 2. Phylogenetic relationships among conoideans, as illustrated by the Bayesian majority consensus tree of the combined dataset (COI + 12S rRNA + 16S rRNA). The tree is rooted on a composite outgroup comprising three species of Conus. Support values are given as posterior probabilities for the Bayesian analysis (only values ≥0.95 are shown) and as bootstrap percentages for the ML analysis (only values ≥70% are shown). Closed circles indicate branches with bootstrap support >95% and posterior probabilities >0.98. Shells of vouchers are indicated by asterisks and are not to scale. Scale bar indicates substitutions per site. Systematics of Raphitoma Table 2. List of Recent species of the genus Raphitoma with their geographic range and the type of protoconch. Species NEA WA Mac Med P + p R. alleryana (Sulliotti, 1889) + p R. alternans (Monterosato, 1884) + p R. arnoldi (Pallary, 1906) + p + m R. atropurpurea (Locard & Caziot, 1900) + + R. bartolinorum Pusateri & Giannuzzi-Savelli, 2018 + R. bernardoi Rolán, Otero-Schmitt & Fernandes, 1998 ∗ R. bicolor (Risso, 1826) [= ?R. maculosa] R. bourguignati (Locard, 1891) + + + R. bracteata (Pallary, 1904) R. brunneofasciata Pusateri, Giannuzzi-Savelli & Oliverio, 2013 R. christfriedi Rolán, Otero-Schmitt & Fernandes, 1998 R. contigua (Monterosato, 1884) + m + m + p + m + m + m m + + R. corbis (Potiez & Michaud, 1838) ∗ R. cordieri (Payraudeau, 1826) ∗ R. densa (Monterosato, 1884) p m + + + m + + m R. digiulioi Pusateri & Giannuzzi-Savelli, 2017 + m R. ebreorum Pusateri & Giannuzzi-Savelli, 2018 + m + m + p R. echinata (Brocchi, 1814) sensu Auctores + + + + R. farolita F. Nordsieck, 1977 R. formosa (Jeffreys, 1867) m + R. griseomaculata Pusateri & Giannuzzi-Savelli, 2018 R. hispidella Pusateri & Giannuzzi-Savelli, 2019 + ∗ R. horrida (Monterosato, 1884) R. kharybdis Pusateri & Giannuzzi-Savelli, 2018 ∗ R. laviae (Philippi, 1844) R. lineolata (Bucquoy, Dautzenberg & Dollfus, 1883) + R. locardi Pusateri, Giannuzzi-Savelli & Oliverio, 2013 ∗ R. maculosa Høisæter, 2016 [=? R. bicolor] + p + m + p + p + m + m + m m + R. melitis Kontadakis & Mbazios, 2019 + m R. mirabilis (Pallary, 1904) + p R. nivea (J. T. Marshall in Sykes, 1906) + p + p R. oblonga (Jeffreys, 1867) m + R. papillosa (Pallary, 1904) ∗ R. philberti (Michaud, 1829) + R. pruinosa (Pallary, 1906) ∗ R. pseudohystrix (Sykes, 1906) + R. pumila (Monterosato, 1890) R. pupoides (Monterosato, 1884) ∗ R. purpurea (Montagu, 1803) + + + + p + p + p + m + m + m R. radula (Monterosato, 1884) + m R. skylla Pusateri & Giannuzzi-Savelli, 2018 + m R. smriglioi Pusateri & Giannuzzi-Savelli, 2013 + p R. sophiae Kontadakis & Mbazios, 2019 + m R. spadiana Pusateri & Giannuzzi-Savelli, 2012 + R. strucki (Maltzan, 1883) R. syrtensis F. Nordsieck, 1977 + R. zelotypa Rolán, Otero-Schmitt & Fernandes, 1998 + p ? + p m Species included in our molecular systematic analyses are indicated by an asterisk. Abbreviations are as follows: NEA, North East Atlantic; WA, West Africa; Mac, Macaronesia; Med, Mediterranean; m, multispiral; P, protoconch; p, paucispiral. (R. brunneofasciata) to 9 (R. cordieri) uniformly convex whorls; reticulate-cancellate sculpture, axials broader than spirals. Fine granular microsculpture occasionally present on whole teleoconch (R. papillosa) or on first whorl only (R. philberti). Outer lip thickened, with 7–13 inner denticles. Columella simple, slightly sinuous anteriorly. Siphonal canal very short (R. contigua) to moderately long (R. cordieri). Siphonal notch wide, plain or intorted. Remarks: As type species of Cordieria, Crosse (1885) designated ‘Murex reticulatus Brocchi, 1814’ (following the indication by Monterosato (1884: 131) “C. reticulata, (Ren.) Brocc. / = Murex reticulatus ed echinatus, Brocc. - 1814, p. 423, t. 8, f. 3”). However, Murex reticulatus Brocchi (1814: 435, pl. 9, f ig. 12) is not a raphitomid, but a species of Genota Gray, 1847 (Borsoniidae). It is clear that Monterosato, (1884: 131) confused M. reticulatus 9 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 Raphitoma alida Pusateri & Giannuzzi-Savelli, 2016 G. Fassio et al. Table 3. List of Recent species of the genus Cyrillia with their geographic range and the type of protoconch. Species NEA ∗ Cyrillia aequalis (Jeffreys, 1867) + WA Mac Med + + m + m + m C. ephesina (Pusateri, Giannuzzi-Savelli & Stahlschmidt, 2017) C. kabuli (Rolán, Otero-Schmitt & Fernandes, 1998) m + + + m + C. zamponorum (Horro, Gori & Rolán, 2019) m + Species included in our molecular systematic analyses are indicated by an asterisk. Abbreviations are as follows: NEA, North East Atlantic; WA, West Africa; Mac, Macaronesia; Med, Mediterranean; P, protoconch; m, multispiral. Brocchi with Murex reticulatus Renier (which is also invalid: ICZN, 1999: Op. 316); the latter is probably the same as Murex echinatus Brocchi, 1814 (=Raphitoma echinata) and it was this species that Monterosato (1884) was indicating. Therefore, we retain Crosse’s (1885) designation, but as an incorrect citation (ICZN, 1999: Art. 67.7), and use Renier’s name which, even if unavailable, can be designated as the type species for Cordieria and Homotoma; see ICZN, 1999: Art 67.1.2). The phylogenetic results presented here do not support any further splitting of this genus. In this respect it is important to note that the species traditionally ascribed to the ‘genera’ Philbertia and Cordieria (=Peratotoma) are distributed across the tree. Similarly, the grouping of species in the phylogeny does not correspond to differences in larval development (as indicated by their multispiral or paucispiral protoconch) and this is consistent with the currently accepted view that larval development is not a reliable taxonomic character at the genus level (Bouchet, 1990). The genetic distance between R. maculosa and R. bicolor is small (<1%); this level of variation could well fall within the variation of the latter species when a denser sampling of R. bicolor is carried out. In contrast, our phylogenetic data indicate that a DNA-barcode-based approach could potentially be used to discriminate between closely related species of Raphitoma (e.g. R. philberti and R. densa in the COI phylogeny; see Supplementary Material Figs S1, S2). DNA barcodes should be used in combination with shell morphology to define species limits in this difficult group of neogastropods. slightly sinuous anteriorly. Siphonal canal short; siphonal notch plain. Remarks: Cirillia Monterosato, 1884 is preoccupied by Cirillia Rondani, 1856, but the emended name Cyrillia Kobelt, 1905 is available and has already been used (e.g. Ceulemans et al., 2018). This is a clear case of a demonstrably intentional emendation (ICZN, 1999: Art. 33.2), since the prescriptions of the Code are met: “there is an explicit statement of intention” and “both the original and the changed spelling are cited and the latter is adopted in place of the former” (ICZN, 1999: Art. 33.2.1). As an intentional, yet unjustified emendation, the name that should be used is Cyrillia Kobelt, 1905 (ICZN, 1999: Art. 33.2.3). Genus Cyrillia Kobelt, 1905 Diagnosis: Shell medium to large size for family, from 15 mm (L. concinna) to 24 mm (L. villaria); shape suboval (L. erronea) to fusiform (L. villaria). Protoconch of 3–3.5 whorls with protoconch I (embryonic shell) of 0.5–0.7 whorls, with reticulate sculpture of spirals and orthocline axial striae, and protoconch II (larval shell) of 2.5–3 whorls, with diagonally cancellate sculpture, sometimes lightly keeled last whorl. Teleoconch with slender spire of 5 (L. concinna) to 7 (L. villaria) uniformly convex whorls; sculpture of thin, numerous low spiral cords and broader, wavy axial ribs. Microsculpture of dense, rather conspicuous growth lines, or rugae; no granules or pustules. Inner lip smooth with no denticles. Columella simple, slightly sinuous anteriorly. Siphonal canal short (L. erronea) to moderately long (L. villaria); siphonal notch wide, plain. Cirillia aequalis and C. linearis lack radula and venom gland. Our phylogenetic results suggest that denser sampling may show C. obesa to be simply a colour variant of C. aequalis. Cyrillia zamponorum from São Tomé Island and another probably undescribed species from Madagascar (N. Puillandre & M. Oliverio, unpubl.) indicate that this lineage has a wide geographical distribution. Genus Leufroyia Monterosato, 1884 (Table 4; Fig. 4) Leufroyia Monterosato, 1884: 134 (type species Pleurotoma leufroyi Michaud, 1828; SD Crosse, 1885). (Table 3; Fig. 3) Cirillia Monterosato, 1884: 133 [type species Murex linearis Montagu, 1803, SD Crosse, 1885; not Rondani, 1856 (Diptera)]. Cyrillia Kobelt, 1905: 367 (unjustified emendation of Cirillia Monterosato, 1884). Cenodagreutes E. H. Smith, 1967: 1 (type species Cenodagreutes aethus E. H. Smith, 1967 = Defrancia aequalis Jeffreys, 1867; OD). Lineotoma F. Nordsieck, 1977 (replacement name for Cirillia Monterosato, 1884, not Rondani, 1856). Diagnosis: Shell small in size for family, from 5 mm (C. linearis) to 10 mm (C. ephesina); biconic, suture impressed. Protoconch 3.5–4 whorls, multispiral, with protoconch I (embryonic shell) of 0.5– 0.7 whorls, with reticulate sculpture of spirals and orthocline axial striae, and protoconch II (larval shell) of 3.3–3.5 whorls, with diagonally cancellate sculpture and weakly keeled last whorl. Teleoconch with slender spire of 5 (C. linearis) to 7 (C. ephesina) convex whorls, with reticulate-cancellate sculpture; axials broader than spirals. Microsculpture of granules or pustules; growth lines seldom obvious. Outer lip thickened, with 7–13 inner denticles, the 2 anterior-most stronger. Columella simple, Remarks: The protoconch is wider (diameter = c. 220–250 μm) and lower than in the ‘multispiral’ propoconch of species of Raphitoma and Cyrillia. ACKNOWLEDGEMENTS We thank Jean Louis Delemarre, Michel Le Quement, Constantin Mifsud and Jakov Prkić for providing critical specimens for our molecular work. Extralimital material was collected as part of the MNHN Tropical Deep-Sea Benthos (Aurora, BOA1, EBISCO 10 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 ∗ C. linearis (Montagu, 1803) ∗ C. obesa (Høisæter, 2016) [=? C. aequalis] P Systematics of Raphitoma Table 4. List of Recent species of the genus Leufroyia with their geographic range and the type of protoconch. Species NEA ∗ Leufroyia concinna (Scacchi, 1836) + WA Mac + L. erronea Monterosato, 1884 ∗ L. leufroyi (Michaud, 1828) + + L. villaria (Pusateri & Giannuzzi-Savelli, 2008) + + Med P + m + m + m + m Species included in our molecular systematic analyses are indicated by an asterisk. Abbreviations are as follows: NEA, North East Atlantic; WA, West Africa; Mac, Macaronesia; Med, Mediterranean; P, protoconch; m, multispiral. and Salomon 2) and Our Planet Reviewed (Papua Niugini, Santo 2006) programmes, or stand-alone expeditions (Panglao 2004); Bouchet (2009) and Bouchet et al. (2008, 2016) provide details on the context of the expeditions and the partnerships involved. All expeditions operated under permits provided by the host countries and satisfy the conditions set by the Convention on Biological Diversity for access to genetic resources. Stefano Bartolini, Vittorio Garilli, Andrea Nappo and Bruno Sabelli provided help with photography. Part of the molecular work was conducted by Louise Lindblom in the DNA Lab at the University of Bergen. SEM photos were taken at the Laboratory of Technological and Functional Analyses of Prehistoric Artifacts of Sapienza University of Rome, with the kind help of Cristina Lemorini (Department of Classics). The work was funded partly by Sapienza University of Rome (grant AR11715C7E17226C/2017 to VR and RM11715C818F7955/2017 to MO). Virginie Héros and Philippe Bouchet (MNHN) commented on initial drafts of the manuscript, two anonymous reviewers provided constructive feedback and David Reid suggested a number of editorial improvements. BARCO, A., RAUPACH, M.J., LAAKMANN, S., NEUMANN, H. & KNEBELSBERGER, T. 2016. Identification of North Sea molluscs with DNA barcoding. Molecular Ecology Resources, 16: 288–297. BELLARDI, L. 1847 [1848]. Monografia delle Pleurotome fossili del Piemonte. Memorie della Reale Accademia delle Scienze di Torino, serie 2, 9: 531–650, pls 1–4. [Journal issue published in 1848, but a separate distributed in 1847 as: Monografia delle Pleurotome Fossili del Piemonte. Torino]. BOUCHET, P. 1990. Turrid genera and mode of development: the use and abuse of protoconch morphology. Malacologia, 32: 69–77. BOUCHET, P. 2009. From specimens to data, and from seashells to molluscs: the Panglao Marine Biodiversity Project. Vita Malacologica, 8: 1–8. BOUCHET, P., HEROS, V., LOZOUET, P. & MAESTRATI, P. 2008. A quarter-century of deep-sea malacological exploration in the South and West Pacific: where do we stand? How far to go. Tropical deep-sea benthos, Vol. 25, Paris. Mémoires du Muséum National d’Histoire Naturelle, 196: 9–40. BOUCHET, P., KANTOR, Y.I., SYSOEV, A. & PUILLANDRE, N. 2011. A new operational classification of the Conoidea. Journal of Molluscan Studies, 77: 273–308. CABANG, A.B., IMPERIAL, J.S., GAJEWIAK, J., WATKINS, M., CORNELI, P.S., OLIVERA, B.M. & CONCEPCION, G.P. 2011. Characterization of a venom peptide from a crassispirid gastropod. Toxicon, 58: 672–680. CASTRESANA, J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution, 17: 540–552. CEULEMANS, L., VAN DINGENEN, F. & LANDAU, B.M. 2018. The lower Pliocene gastropods of Le Pigeon Blanc (Loire-Atlantique, northwest France). Part 5—Neogastropoda (Conoidea) and Heterobranchia (fine). Cainozoic Research, 18: 89–176. REFERENCES VAN AARTSEN, J.J., MENKHORST, H.P.M.G. & GITTENBERGER, E. 1984. The marine Mollusca of the Bay of Algeciras, Spain, with general notes on Mitrella, Marginellidae and Turridae. Basteria, Supplement 2: 1–135. ABDELKRIM, J., AZNAR-CORMANO, L., FEDOSOV, A.E., KANTOR, Y.I., LOZOUET, P., PHUONG, M.A., ZAHARIAS, P. & PUILLANDRE, N. 2018. Exon-capture-based phylogeny and diversification of the venomous gastropods (Neogastropoda, Conoidea). Molecular Biology and Evolution, 35: 2355–2374. 11 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 Figure 4. Leufroyia leufroyi (Michaud, 1828), the type species of the genus Leufroyia Monterosato, 1884. A–E. Shell from a depth of 40 m, Ile Rousse (Corsica); shell height 11 mm. Scale bar = 100 μm. G. Fassio et al. PUILLANDRE, N., SAMADI, S., BOISSELIER, M.C., SYSOEV, A.V., KANTOR, Y.I., CRUAUD, C., COULOUX, A. & BOUCHET, P. 2008. Starting to unravel the toxoglossan knot: molecular phylogeny of the ’turrids’ (Neogastropoda: Conoidea). Molecular Phylogenetics and Evolution, 47: 1122–1134. RAMBAUT, A., DRUMMOND, A.J., XIE, D., BAELE, G. & SUCHARD, M.A. 2018. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67: 901–904. ROLÁN, E., OTERO SCHMITT, J. & FERNANDES, F. 1998. The family Turridae s.l. (Mollusca, Gastropoda) in Angola (West Africa), 1. Subfamily Daphnellinae. Iberus, 16: 95–118. SHERIDAN, R., VAN MOL, J.-J. & BOUILLON, J. 1973. Étude morphologique du tube digestif de quelques Turridae (Moll.-Gastr.Prosobr.-Toxogl.) de la région de Roscoff. Cahiers de Biologie Marine, 14: 159–188. SMITH, E.H. 1967. Two new species of British turrids. Veliger, 10: 1–4. STAMATAKIS, A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30: 1312–1313. TAYLOR, J.D., KANTOR, Y.I. & SYSOEV, A.V. 1993. Foregut anatomy, feeding mechanisms, relationships and classification of Conoidea (Toxoglossa) (Gastropoda). Bulletin of the Natural History Museum, London (Zoology), 59: 125–170. 12 Downloaded from https://academic.oup.com/mollus/advance-article-abstract/doi/10.1093/mollus/eyz022/5610862 by guest on 02 November 2019 MANOUSIS, T., KONTADAKIS, C., MBAZIOS, G. & POLYZOULIS, G. 2018. The family Raphitomidae (Mollusca: Gastropoda: Conoidea) in the Greek Seas with the description of two new species. Journal of Biological Research-Thessaloniki, 25: 14. MILLER, J.A. 1989. The toxoglossan proboscis: structure and function. Journal of Molluscan Studies, 55: 167–182. MILLER, M.A., PFEIFFER, W. & SCHWARTZ, T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 November 2010, New Orleans, pp. 1–8. IEEE, Piscataway. MONTEROSATO, T.A. 1872. Notizie intorno alle conchiglie Mediterranee. Michele Amenta, Palermo. MONTEROSATO, T.A. 1884. Nomenclatura generica e specifica di alcune conchiglie mediterranee. Virzi, Palermo. MONTEROSATO, T.A. 1980. Conchiglie della profondità del mare di Palermo. Naturalista Siciliano, 9: 181–191. NORDSIECK, F. 1968. Die europäischen Meeres-Gehäuseschnecken (Prosobranchia). Vom Eismeer bis Kapverden und Mittelmeer. Gustav Fischer, Stuttgart. OLIVERIO, M. 1995. The systematics of the radula-less gastropod Clathromangelia (Caenogastropoda, Conoidea). Zoologica Scripta, 24: 193–201. OLIVERIO, M. & MARIOTTINI, P. 2001. A molecular framework for the phylogeny of Coralliophila and related muricoids. Journal of Molluscan Studies, 67: 215–224. PALUMBI, S.R. 1996. Nucleic acid II: the polymerase chain reaction. In: Molecular systematics, (D.M. Hillis, G. Moritz & B.K. Mable, eds) pp. 205–247. Sinauer Associates, Sunderland, MA. PLOUVIEZ, S., JACOBSON, A., WU, M. & VAN DOVER, C.L. 2015. Characterization of vent fauna at the Mid-Cayman Spreading Center. Deep Sea Research Part I: Oceanographic Research Papers, 97: 124–133. POSADA, D. 2008. JModelTest: phylogenetic model averaging. Molecular Biology and Evolution, 25: 1253–1256. POWELL, A.W.B. 1966. The molluscan families Speightiidae and Turridae. An evaluation of the valid taxa both recent and fossil, with lists of characteristic species. Bulletin of the Auckland Institute and Museum, 5: 1–184. PUILLANDRE, N., BOUCHET, P., DUDA, T.F., KAUFERSTEIN, S., KOHN, A.J., OLIVERA, B.M., WATKINS, M. & MEYER, C. 2014. Molecular phylogeny and evolution of the cone snails (Gastropoda, Conoidea). Molecular Phylogenetics and Evolution, 78C: 290–303. PUILLANDRE, N., KANTOR, Y.I., SYSOEV, A., COULOUX, A., MEYER, C., RAWLINGS, T., TODD, J.A. & BOUCHET, P. 2011. The dragon tamed? A molecular phylogeny of the Conoidea (Gastropoda). Journal of Molluscan Studies, 77: 259–272. CROSSE, H. 1885. Nomenclature generica e specifica di alcune Conchiglie Mediterranee, pel marchese di Monterosato. Journal de Conchyliologie, 33: 139–142. DALL, W.H. 1918. Notes on the nomenclature of the mollusks of the family Turritidae. Proceedings of the United States National Museum, 54: 313–333. ESPIRITU, D.J.D., WATKINS, M., DIA-MONJE, V., CARTIER, G.E., CRUZ, L.J. & OLIVERA, B.M. 2001. Venomous cone snails: molecular phylogeny and the generation of toxin diversity. Toxicon, 39: 1899–1916. FEDOSOV, A.E., PUILLANDRE, N., KANTOR, Y.I. & BOUCHET, P. 2015. Phylogeny and systematics of mitriform gastropods (Mollusca: Gastropoda: Neogastropoda). Zoological Journal of the Linnean Society, 175: 336–359. FEDOSOV, A.E., STAHLSCHMIDT, P., PUILLANDRE, N., AZNARCORMANO, L. & BOUCHET, P. 2017. Not all spotted cats are leopards: evidence for a Hemilienardia ocellata species complex (Gastropoda: Conoidea: Raphitomidae). European Journal of Taxonomy, 268: 1–20. 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. GIANNUZZI-SAVELLI, R., PUSATERI, F. & BARTOLINI, S. 2018. A revision of the Mediterranean Raphitomidae (Gastropoda: Conoidea) 5: loss of planktotrophy and pairs of species, with the description of four new species. Bollettino Malacologico, 54, Supplement 11: 1–77. HARRIS, G.F. & BURROWS, H.W. 1891. The Eocene and Oligocene beds of the Paris Basin. University College, London. HAYASHI, S. 2003. 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. HØISÆTER, T. 2016. A taxonomic review of the Norwegian species of Raphitoma (Gastropoda: Conoidea: Raphitomidae). Fauna Norvegica, 36: 9–32. HORRO, J., GORI, S. & ROLÁN, E. 2019. Raphitoma zamponorum a new species from São Tomé Island (Gastropoda: Raphitomidae). Iberus, 37: 1–6. HUELSENBECK, J.P. & RONQUIST, F. 2001. MrBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17: 754–755. INTERNATIONAL COMMISSION ON ZOOLOGICAL NOMENCLATURE (ICZN). 1999. International code of zoological nomenclature. Edn 4. International Trust for Zoological Nomenclature, London. KANTOR, Y.I. & TAYLOR, J.D. 2002. Foregut anatomy and relationships of raphitomine gastropods (Gastropoda: Conoidea: Raphitominae). In: Systematics, phylogeny and biology of the Neogastropoda (M. Oliverio & R. Chemello, eds), Bollettino Malacologico, Supplement 5: 161–174. KATOH, K., ROZEWICKI, J. & YAMADA, K.D. 2017. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics, 30: 3059. KEARSE, M., MOIR, R., WILSON, A., STONES-HAVAS, S., CHEUNG, M., STURROCK, S., BUXTON, S., COOPER, A., MARKOWITZ, S., DURAN, C., THIERER, T., ASHTON, B., MEINTJES, P. & DRUMMOND, A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28: 1647–1649. KOBELT, W. 1905; 1887–1908. Iconographie der schalentragenden europäischen Meeresconchylien. Part 3: 1-24, pls 59–62 [1902]; 25–200, pls 63–78 [1903]; 201–272, pls 79–84, 86–87 [1904]; 273–406 [1905]. T. Fischer, Cassel. KURAKU, S., ZMASEK, C.M., NISHIMURA, O. & KATOH, K. 2013. aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acids Research, 41: W22–W28.