A new genus, Limatium gen. n., and two new species, L. pagodula sp. n. and L. aureum sp. n. are described, found on outer slopes of barrier reefs and fringing reefs in the South Pacific. They are rare for cerithiids, which typically occur in large populations. The two new species are represented by 108 specimens sampled over a period of 30 years, only 16 of which were collected alive. Three subadults from the Philippines and Vanuatu likely represent a third species. In addition to their rarity, Limatium species are atypical for cerithiids in their smooth, polished, honey to golden brown shells with distinctive white fascioles extending suture to suture. The radula presents a unique morphology that does not readily suggest an affinity to any of the cerithiid subfamilies. Two live-collected specimens, one of each species and designated as holotypes, were preserved in 95% ethanol and sequenced. Bayesian analysis of partial COI and 16S rDNA sequences demonstrates a placement in the Bittiinae, further extending our morphological concept of the subfamily.

Bittiinae , new genus, new species, marine, DNA

The Cerithiidae is one of 19 families of Cerithioidea currently accepted, and with 219 species considered valid, it is one of the most diverse (Bouchet et al. 2017, MolluscaBase 2018). Members are distributed worldwide from tropical to temperate biotopes, most frequently in shallow waters, with a few species that extend into bathyal depths. As far as is known, most species are microherbivorous grazers and usually occur in large populations (e.g., Houbrick 1992). The family is subdivided into three Recent subfamilies: Cerithiinae, Argyropezinae, and Bittiinae (Bouchet et al. 2017), with the Cerithiinae and Bittiinae containing the majority of the species. As discussed in Strong and Bouchet (2013, and references therein), there is presently no known diagnostic feature of shell morphology or internal anatomy that allows unambiguous placement in Cerithiinae versus Bittiinae. The only possible exception may be internal features of the midgut, but this requires further study to confirm (Strong and Bouchet 2013). The notion that bittiines may be distinguished by small adult size is a frequent misperception.

Unusual polished, golden shells, less than one centimeter in adult length, of an unfamiliar species first became known to us in material collected by the RAPA 2002 expedition to the Austral Islands. Since then, material comprising at least two species has been sorted from residues collected during campaigns led by the MNHN, with particularly rich sources of specimens from the MONTROUZIER expedition to New Caledonia in 1993, and the LIFOU 2000 expedition to the Loyalty Islands. The earliest specimens located thus far were collected during the MUSORSTOM 3 cruise to the Philippines in 1985, from the early years of the Tropical Deep Sea Benthos program (Bouchet et al. 2008). Despite readily identifiable as belonging to the Cerithiidae, their subfamily placement was unclear. Early collections were represented mainly by empty shells, with only a handful of live-collected specimens that had been subsequently dried. This hampered the use of anatomical dissections to explore their systematic placement; the unique radula did not suggest an affinity to any of the cerithiid subfamilies. The ability to assess their affinities more robustly was possible only with the collection of the first live-collected specimen preserved in ethanol during the Moorea Biocode Project in 2008. In 2013 a specimen of a second species was preserved for molecular analysis during the TUHAA PAE 2013 cruise to the Austral Islands. Based on this material, we here describe a new genus, Limatium n. gen., with two new species, L. pagodula sp. n. and L. aureum sp. n.

The only two live-collected, fluid-preserved specimens were those used for sequencing and are the designated holotypes. The head-foot of each was removed through the aperture after drilling a small hole through the abapertural side of the penultimate whorl of the shell. The radulae of the holotypes were extracted and mounted for examination via scanning electron microscopy. However, the radula of Limatium pagodula sp. n. was teratological with thin, flimsy, poorly formed teeth along its length; no other live-collected material exists for this species and the operculum is unknown. In addition to the holotype of Limatium aureum sp. n., a second specimen live collected in Rapa and subsequently dried was used to extract an operculum and a radula for comparative purposes.

The three radulae and single operculum were tissue digested overnight in 100 µl of ATL lysis buffer (Qiagen, Inc.) containing ~50 µg of Proteinase-K, sonicated and rinsed in de-ionized water. Cleaned radulae were mounted directly on glass coverslips; the operculum was attached to the cover slip using a carbon adhesive tab. The cover slips were then attached to aluminum stubs with carbon adhesive tabs, coated with 25-30 nm gold/palladium (60/40), and photographed using an Apreo scanning electron microscope (FEI Company) at the NMNH. Specimens of subadults and juveniles were selected for examination of protoconchs. These shells were mounted on aluminum stubs with Elmer’s glue© and photographed uncoated in charge reduction mode using a Hitachi TM3000 scanning electron microscope (Hitachi High Technologies America, Inc.) also at the NMNH. Shells were photographed using a Canon EOS 50D camera with a Canon MP-E 65 mm f/2.8 1–5× macro lens and Canon MT-24EX macro twin light flash (Canon USA, Inc.).

The partial COI sequence for the holotype of Limatium pagodula sp. n., was produced under the Moorea Biocode Project (see Geller et al. 2013). For the other newly generated sequences, genomic DNA was extracted from roughly one cubic millimeter of 95% ethanol-preserved foot tissue using an automated phenol:chloroform extraction on the Autogenprep965 (Autogen, Holliston, MA) using the mouse tail tissue protocol with a final elution volume of 50 µl. A 658 base pair (bp) fragment of the cytochrome c oxidase I (COI) gene was amplified using degenerate Folmer primers (dgLCO/dgHCO) (Meyer et al. 2005) with M13 tails, and using JGLCO (Geller et al. 2013) in combination with C1-N-2191R (aka “Nancy”) (Simon et al. 1994). A ~510 bp fragment of the 16S rRNA gene was amplified using the universal primers 16SAR/BR (Palumbi et al. 1991). PCR reactions were performed with 1 μl of undiluted DNA template in 20 μl volumes. Reaction volumes for COI contained 10 µl of Promega Go-Taq® Hotstart Master Mix (1 unit Promega Go-Taq®, 400 μM dNTPs, 4 mM MgCl₂), 0.3 µl 10 µM of each primer, 0.25 µg/μl of BSA, and 1.25% DMSO. Amplification consisted of an initial denaturation step at 95 °C for 7 min, followed by 45 cycles of denaturation at 95 °C for 45 s, annealing at 42 °C for 45 s, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. Reaction volumes for 16S, also in 20 μl volumes, contained 1 μl of undiluted template DNA, 1 unit Biolase DNA Polymerase (Bioline), 2 µl 10X reaction buffer, 500 μM dNTPs, 2 mM MgCl2, 0.25 μg/μl of BSA, and 0.3 µl 10 µM of each primer. Amplification consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 30 s, extension at 72 °C for 45 s and a final extension at 72 °C for 3 min. PCR products were purified prior to sequencing using the Exo-SAP-IT protocol (Amersham Biosciences, Piscataway, NY). Sequencing reactions were performed with 1 µl of purified PCR product, 1.75 µl BigDye buffer, and 0.5 µl BigDye (ABI, Foster City, CA), and run in the thermal cycler for 30 cycles of 30 s at 95 °C, 30 s at 50 °C, 4 min at 60 °C, and then held at 10 °C. Sequencing reactions were purified using Millipore Sephadex plates (MAHVN-4550, Millipore, Billerica, MA) per manufacturer’s instructions and analyzed on an ABI 3730XL DNA Analyzer Capillary Array.

Chromatograms were trimmed, assembled, and edited as necessary in Geneious 11.0.2. Sequences were aligned separately for each gene with ClustalX (Thompson et al. 1997) using default parameters as implemented in Geneious. COI was translated into amino acids to check for stop codons and frameshift mutations. All newly generated sequences have been deposited in GenBank (Table 1).

The best fit partitions and models for phylogenetic analyses were determined with PartitionFinder 1.1.1 (Lanfear et al. 2012) which favored the following scheme: COI: SYM+I+G, F81+I, HKY+G, for the first, second and third codon position, respectively; and GTR+I+G for 16S. A subset of the cerithiid dataset of Strong and Bouchet (2013) was used, augmented with newly generated COI and 16S sequences for Ittibittium houbricki (Ponder, 1993) and Bittium reticulatum (da Costa, 1778) (see Table 1). In addition to Limatium, the Bittiinae was represented by five of the 12 Recent genera currently accepted, four by their type species or their subjective synonyms (see Table 1). Bayesian analysis of the concatenated dataset for 29 cerithiids and one outgroup (Litiopidae) was inferred with MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003) on the CIPRES Science Gateway, using the schemes and models indicated by PartitionFinder. Bayesian analyses, consisting of two independent replicates with four heated chains each (0.02), and three swaps per swapping cycle, were run for 50,000,000 Markov chain Monte Carlo (MCMC) generations with a sampling frequency of one tree every 1000 generations. The first 25% were discarded as burn-in. Tracer 1.6 (Rambaut et al. 2014) was used to assess MCMC convergence and to ensure that all ESS values exceeded 200. A majority rule consensus tree was constructed with the sumt command. Nodal support was assessed with posterior probability of each node.

Houbrick’s (1993) generic review of the Bittiinae published 25 years ago remains the authoritative resource for comparative anatomy and systematics of bittiines. At that time, Houbrick (1993) recognized nine genera in the Bittium-group: Bittium, Argyropeza, Bittiolum, Cacozeliana, Ittibittium, Lirobittium, Neostylidium [then as Stylidium], Varicopeza, and Zebittium; Cassiella was identified as a possible member but its placement uncertain given the paucity of anatomical data. In 2006, Bandel established a separate subfamily for Argyropeza. Since then, the Bittiinae has been expanded (MolluscaBase 2018) to include the Recent genera Cerithidium (provisionally excluded by Houbrick 1993), and Pictorium.

Despite the absence of a diagnostic feature that allows unambiguous placement in the subfamily (Strong and Bouchet 2013), the common perception of bittiines is that they are small in adult size, turreted, with a predominating beaded or spiral sculpture, and cream, gray, tan to dull brown in color. This concept of bittiine teleoconch morphology was expanded by the description of the genus Pictorium, based on a small type species formerly placed in Cerithium, with a pupoid, brilliantly colored reddish-purple shell (Strong and Bouchet 2013). Limatium species differ from all other bittiines in their smooth, glossy, polished shells with a rich honey to golden brown background color. This unique shell morphology is unknown in the Cerithiidae and further expands the conchological concept of the subfamily. The distinctive white fascioles also may be a diagnostic feature of the genus, but further comparative material is required to be certain, particularly of the suspected additional species from Vanuatu and the Philippines. Clearly, more material, and more sequencable material, is needed to understand intraspecific variation within Limatium.

In addition to shell morphology, Limatium differs from known bittiines in several unique features of the radula. Most possess a rachidian with an attached basal portion and a freely projecting face. The face of the tooth has a central to basal constriction that can be quite strongly developed in some species. Thickenings at the lower, outer corners of the tooth extend onto the radular membrane beyond the sides of the tooth. The upper projecting margin of the tooth often forms a prominent crest from below which the teeth project. The cutting edge spans the entire anterior edge of the tooth, or the majority of it, and bears a single, strong central cusp and two to three denticles on each side (e.g., Houbrick 1980b, 1993, Gofas 1987, Ponder 1993, Hasegawa 1998). The rachidian of Limatium has a basal plate that is hexagonal to septagonal in shape, with a central elevated portion that has a U-shaped, rounded lower edge roughly midway down the face of the tooth. The cutting edge is restricted to this elevated portion, and bears only three dagger-like cusps. This configuration is unknown among the Bittiinae, and even more broadly among the Cerithiidae. In terms of operculum morphology, most bittiines are characterized by a multispiral operculum (Houbrick 1980b, 1993, Gofas 1987, Hasegawa 1998); Bittiolum and Cassiella differ in possessing an ovate, paucispiral operculum with a small nucleus (Houbrick 1993, Ponder 1993). In contrast, Limatium possesses a subcircular, paucispiral operculum of only ~3 whorls and a large nucleus. Argyropezinae conform to the bittiine configuration of rachidian morphology and possess a circular, multispiral operculum (Houbrick 1980a).

The phylogenetic analysis supported Limatium and Cacozeliana as sister taxa, although more comprehensive taxon sampling is required to assess the affinities of bittiine genera. The analysis confirmed that Bittium, as presently conceived is polyphyletic (Strong and Bouchet 2013). Inclusion of the type species, Bittium reticulatum, for the first time allows us to tie the genus-group name to a clade including B. latreillei and B. simplex. As in the analysis of Strong and Bouchet (2013), Bittium impendens, only cautiously retained in Bittium by Houbrick (1993), is robustly supported as the sister group to Pictorium; Bittium glareosum is the sister to them but its placement is not supported. Additional sampling is required to resolve the affinities of Indo-Pacific species currently placed in Bittium, with Bittiums.s. possibly retained only for species from the Atlantic.

Limatium is exceptionally rare, represented in museum and personal collections by a scant 108 specimens known to us as enumerated herein, only 16 of them collected alive. As described in Strong and Bouchet (2013), Pictorium also had been rare in museum collections and had never been collected alive prior to the 1980's. Like Limatium, the latter genus is also small and found in steep, hard-bottom habitats that are too deep for diving and too steep for dredging. The use of lumun-lumun and tangle nets in the commercial shell trade in the Philippines, and the adoption of new collecting techniques as brushing baskets and suction sampling in biodiversity surveys (Albano et al. 2011), have revolutionized access to these challenging habitats. While the number of specimens of Pictorium rose dramatically particularly since 2004, the number of Limatium specimens also has increased but not so dramatically, and live collected specimens remain elusive. The unique radular morphology of Limatium suggests a life habit different from that of most other bittiines, which may explain why they remain so tantalizingly rare.

Figure 5. 

Phylogeny of Cerithiidae based on Bayesian analysis of partial COI and 16S sequences. Catalogue numbers for vouchers indicated after species name. Posterior probabilities greater than .50 are shown at the nodes. See Table 1 for details.

We thank Jean Tröndlé (La Force, France) who sorted the gastropods of the RAPA 2002 expedition and recognized Limatium aureum as a potentially new species. We are grateful to Jean Letourneux (Tahiti) for his generosity in sharing and donating specimens of the two new species from his personal collections, including several designated paratypes. We thank Alexander Fedosov (Russian Academy of Sciences) for collecting the sequenced holotype of L. aureum during the TUHAA PAE research cruise at the invitation of Cecile Debitus (IRD). We are also indebted to Chris Meyer (NMNH), Gustav Paulay (UF) and the Moorea Biocode Project for making the sequenced holotype of L. pagodula available to us for study and for allowing us to use the image of the live animal and the COI sequence. We thank Serge Gofas (University of Malaga) for the material of Bittium reticulatum from Spain. We are grateful to Freya Goetz and Yolanda Villacampa (both NMNH) for producing the scanning electron micrographs of the protoconchs, operculum and radulae, and to Gilberto Marani (MNHN) for producing the map figure. We also thank Kazunori Hasegawa (National Museum of Nature and Science, Tokyo) for his very thorough and knowledgeable review.