Journal of The Malacological Society of London Molluscan Studies Journal of Molluscan Studies (2012) 78: 246–255. doi:10.1093/mollus/eys007 A NEW LINEAGE OF CONOIDEA (GASTROPODA: NEOGASTROPODA) REVEALED BY MORPHOLOGICAL AND MOLECULAR DATA YURI I. KANTOR1, ELLEN E. STRONG2 AND NICOLAS PUILLANDRE3,4 1A.N. Severtzov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninski Prosp. 33, Moscow 119071, Russia; 2National Museum of Natural History, PO Box 37012, Washington, DC 20013-7012, USA; 3Museum National d’Histoire Naturelle, Departement Systematique et Evolution, UMR 7138, 43, Rue Cuvier, 75231 Paris, France; and 4Atheris Laboratories, Case Postale 314, CH-1233 Bernex-Geneva, Switzerland Correspondence: Y.I. Kantor; e-mail: [email protected] (Received 26 July 2011; accepted 1 February 2012) ABSTRACT The hyperdiverse group of venomous Conoidea has eluded attempts to construct a robust and stable classification owing to the absence of a robust and stable phylogenetic framework. New molecular data have greatly enhanced our understanding of conoidean evolution, allowing the construction of a new family-level classification. This expanding framework has also allowed the discovery of several in- dependent lineages that merit recognition at familial rank. One of these, based on seven specimens collected over more than 20 years from deep waters off New Caledonia, represents a unique, mono- typic lineage closely related to Mitromorphidae, which we here name as the new family Bouchetispiridae. This new lineage bears a unique combination of teleoconch, protoconch and ana- tomical characters previously unknown within the Conoidea, including a translucent, fusiform shell with sculpture of strong axial ribs crossed by spiral cords, a multispiral protoconch of only 2.5 whorls with punctate sculpture, hypodermic marginal teeth and a multilayered venom bulb with two layers of muscle separated by connective tissue. This lineage may represent the sole survivor of a previously more diverse clade, or is simply one of many unique taxa that have arisen among the isolated sea mounts off New Caledonia. INTRODUCTION The routine use of molecular data in systematic malacology has contributed enormously towards clarifying the phylogeny The Conoidea are a hyperdiverse group of venomous marine and taxonomy of Conoidea, including ‘turrids’. A recently gastropods, representing one of six currently recognized super- published molecular phylogeny of Conoidea (Puillandre et al., families within the Neogastropoda. Owing to their exceptional 2011) and corresponding operational classification (Bouchet levels of species richness and high levels of homoplasy among et al., 2011) were notable breakthroughs. The addition of many features of the shell and anterior alimentary system, the new taxa compared to the previous molecular phylogeny Conoidea have consistently defied attempts to construct a stable (Puillandre et al., 2008) greatly enhanced our knowledge of classification. These efforts have been thwarted primarily by the evolutionary trends within the superfamily. The resulting clas- absence of a robust phylogenetic framework. In addition to the sification includes 15 families, 13 of which were previously clas- well-defined and universally recognized Conidae and sified as ‘turrids’. Most lineages elevated to family rank had Terebridae, the heterogeneous assortment of taxa relegated to already been defined and recognized as families or subfamilies the Turridae s. l. (or ‘turrids’), an acknowledged polyphyletic in previous classifications, such as the one proposed by Taylor taxon (Taylor, Kantor & Sysoev, 1993; Puillandre et al., 2008; et al. (1993) based on anatomical and conchological characters. Tucker & Tenorio, 2009), has posed a particularly daunting However, some of the taxa recognized by Taylor et al. (1993) challenge. The ‘turrids’ include over 360 Recent valid genera were merged with other families (e.g. Pseudomelatomidae sensu and subgenera and 4,000 named living species (Tucker, 2004), Taylor et al., 1993, merged with Crassispirinae) and the while an estimated 80% of species are yet to be described content of many higher taxa was changed by the transfer of (Bouchet, Lozouet & Sysoev, 2009). This assemblage remains numerous genera. One new lineage, the family Horaiclavidae, one of the most intimidating targets in marine malacology, was identified solely with the insight of molecular data. despite their important role in ecosystems and importance in When available, molecular data have been particularly current and future toxicological research (Cabang et al., 2011). useful in resolving the affinities of taxa characterized by # The Author 2012. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved A NEW LINEAGE OF CONOIDEA unusual combinations of morphological characters and/or DNA extraction, amplification and sequencing reduction and loss of diagnostic features of the alimentary system. For example, Toxicochlespira Sysoev & Kantor, 1990, DNA was extracted from foot tissue of the specimen MNHN characterized by an extremely carinated shell similar to IM200735029 using the 6100 Nucleic Acid PrepStation system Cochlespira Conrad, 1865, but possessing hypodermic marginal (Applied Biosystems), following the manufacturer’s recommen- radular teeth very different from the nonhypodermic, duplex dations. Fragments of the mitochondrial genes 12S rRNA, 16S radular teeth of known Cochlespiridae, could not be confi- rRNA and cytochrome oxidase subunit I (COI) were ampli- dently assigned to any suprageneric taxon (Sysoev & Kantor, fied using universal primers 12S1/12SB (Simon, Franke & 1990). Molecular data unequivocally placed it in Mangeliidae. Martin, 1991; Palumbi, 1996), 16Sar/16Sbr (Palumbi, 1996) The latter have very different shell, but similar hypodermic and LCO1490/HCO2198 (Folmer et al., 1994), respectively. m marginal radular teeth. Likewise, the genus Zemacies Finlay, All PCRs were performed in 25 l, containing 3 ng of DNA, 1926 was considered an aberrant genus (lacking radula and 1Â reaction buffer, 2.5 mM MgCl2, 0.26 mM dNTP, 0.3 mM venom apparatus) constituting its own subfamily Zemaciinae each primer, 5% DMSO and 1.5 U of Qbiogene Q-Bio Taq. 8 Sysoev, 2003 within the Turridae (sensu Taylor et al., 1993) Amplification consisted of an initial denaturation step at 94 C 8 that normally possess nonhypodermic radulae. Molecular data for 4 min, followed by 35 cycles of denaturation at 94 C for 8 8 demonstrated that Bathytoma (Borsoniidae), which possesses a 30 s, annealing for 30 s at 54 C, 52 and 50 C for 12S, 16S and 8 hypodermic radula, is its sister group. However, with the COI genes respectively, and extension at 72 C for 1 min. The 8 present state of coverage of the molecular dataset (only 87 of final extension was at 72 C for 5 min. PCR products were 360 genera included), many genera can be only tentatively purified and sequenced by a sequencing facility (Eurofins). All assigned to family, mostly on the basis of radular characters or genes were sequenced in both directions for increased accuracy. sometimes on conchological characters alone (Bouchet et al., Voucher information and sequences have been deposited in 2011). It is clear that among the multitudes of unknown and BOLD (CONO1099-10) and in GenBank (JN640292, inadequately described species the affinities of many are JN662500, JN662502). waiting to be resolved and undoubtedly many hidden inde- pendent lineages remain to be discovered (Bouchet et al., 2009; Phylogenetic analyses Puillandre et al., 2011). Recently, several specimens in the collections of the Muse´um Phylogenetic trees were constructed using a subset of taxa ana- National d’Histoire Naturelle, Paris (MNHN) from deep lysed in Puillandre et al. (2011) (Table 1). As the radula and waters off New Caledonia were recognized as representing an anatomy of MNHN 24504 indicate that it belongs to the clade unusual species of unclear affinity. Conchological characters of Conoidea with hypodermic radulae (as defined in the indicated that it was a neogastropod and examination of the molecular analysis by Puillandre et al., 2011), representatives of radula demonstrated a position among the Conoidea. More this clade were included preferentially, while only a few taxa precisely, owing to the presence of hypodermic marginal from the clade with nonhypodermic radulae were selected as radular teeth, it could be placed among the Conidae sensu closely related outgroups. Taylor et al. (1993) that united all groups of turrids with hypo- Sequences were manually (COI) or automatically (16S and dermic marginal radular teeth. However, the affinity of this 12S) aligned using Muscle online (http://www.ebi.ac.uk/Tools/ unusual species to any one of the currently defined families was msa/muscle/). Phylogenetic analyses were first performed on unclear. Several live-collected specimens have allowed morpho- each gene separately to check for inconsistency between trees. logical and molecular analyses to be carried out, allowing its As trees were mostly congruent, all genes were concatenated in phylogenetic affinities to be assessed. These data indicate that a single dataset. Bayesian Analyses were performed running it represents an independent lineage that cannot be accommo- two parallel analyses in MrBayes (Huelsenbeck, Ronquist & dated in any currently recognized taxon of Conoidea. Hall, 2001), each consisting of eight Markov chains of 20,000,000 generations, with a sampling frequency of one tree from each thousand