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Home , Ancistrolepis, Babyloniidae, Beringius, Buccinoidea, Buccinum, Chrysodomus

VENUS 68 (3–4): 121–137, 2010 ©Malacological Society of Japan

Molecular Phylogeny of (: ) Inferred from Mitochondrial DNA Sequences, with Description of a New

Tomoyuki Nakano1*, Yukito Kurihara1†, Hirofumi Miyoshi2 and Shigeo Higuchi3 1Department of Geology and Palaeontology, National Museum of Nature and Science, Tokyo, 3-23-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan 23-20 Honmachi, Gamagori-shi, Aichi 443-0059, Japan 32-17-13 Kongosawa, Taihaku-ku, Sendai-shi, Miyagi 982-0803, Japan

Abstract: A molecular phylogeny based on sequence data from two mitochondrial genes (COI and 16S) is presented for the Neptunea, including the type species of all the three subgenera. Our results support the previously recognized subdivision of Neptunea into three subgenera (Neptunea s.s., Barbitonia and Golikovia) based on the conchological and radular characters. The fossil record suggests that the divergence of the three subgenera occurred as early as the Late Oligocene to middle Miocene, and that the Atlantic N. antiqua diverged from western Pacific species well before the opening of the Bering Strait in the Pliocene. A new species, Neptunea mikawaensis, is described systematically.

Keywords: Molecular phylogeny, Neptunea, Barbitonia, Golikovia, new species, Neptunea mikawaensis

Introduction

Neptunea is the most species-rich genus in the family Buccinidae. This genus is mainly distributed in the Arctic, Northern Pacific and Northern Atlantic , and inhabits a wide range of depths (e.g. Okutani, 2000; Higuchi, 2006; Fraussen & Terryn, 2007). In 2007, one of the authors (H. M.) obtained specimens of a possibly undescribed species of Neptunea from commercial trawlers operating in the sea area Enshu-nada off Mikawa, Aichi Prefecture, central Japan (Miyoshi et al., 2009). The species is morphologically similar to Neptunea kuroshio Oyama in Kira, 1959, but differs from the latter species in the tall shell shape, the colour of the body whorl and the interior of the shell and the brown filmy periostracum. However, the high degree of intraspecific morphological variation among species of Neptunea (Fraussen & Terryn, 2007) hinders correct identification of these specimens based on the shell morphology alone. On the other hand, modern molecular methodologies have greatly aided resolution of such taxonomic problems in a wide variety of molluscan groups (e.g. Simison & Lindberg, 1999; Nakano & Spencer, 2007). In the confamilial genus , Iguchi et al. (2007b) have identified species reliably by analyzing the mitochondrial 16S gene. However, only a few studies have been made of the genus Neptunea using molecular data (Hayashi, 2005; Iguchi et al., 2007a, b). The systematics and subgeneric classification of Neptunea are still at an early stage. Goryachev (1987) classified Neptunea into three ‘stocks’: N. intersculpta stock, N. lyrata stock and N. polycostata stock, and subdivided each stock into several groups based on shell morphology.

* Corresponding author: [email protected] † Present address: Faculty of Education, Mie University, Tsu, Mie 514-8507, Japan 122 T. Nakano et al.

Japanese workers recognized three subgenera in the genus Neptunea: Neptunea Röding, 1798, Barbitonia Dall, 1916 and Golikovia Habe & Sato, 1973 by combinations of shell and radular morphology (Tiba & Kosuge, 1988). Most recently, Fraussen & Terryn (2007) divided Neptunea into 15 groups. However, there is no congruence among the subdivisions proposed by these authors. We here report the results of morphological, palaeontological and molecular (mitochondrial COI and 16S) analyses of the genus Neptunea. Our aims are to elucidate the status of the subgenera, and to test the status and then describe the new species N. mikawaensis from Japan.

Materials and Methods

Collection of samples Table 1 lists the species and collecting sites of specimens used in this study. We followed Tiba & Kosuge (1988) and Okutani (2000) in the generic assignments of the species. Fifteen individuals of nine Neptunea species, including the type species of all the three subgenera, were newly sequenced, and to these were added the published sequence of N. antiqua (AF373886) (Harasewych & Kantor, 2002). The sequences of Buccinum tsubai and Buccinum striatissimum by Iguchi et al. (2007a, b) were used as outgroups.

DNA extraction, amplification and sequencing Living specimens were preserved in 95% ethanol. Total DNA was extracted from a fragment of the foot-muscle tissue. Extraction was performed using High Pure PCR Template Preparation Kit (Roche). The mitochondrial large-subunit ribosomal RNA (16S rRNA) and cytochrome-c oxidase I (COI) genes were amplified and sequenced. The universal 16S primers, 16Sar (5’-CGC CTG TTT ATC AAA AAC AT-3’) and 16Sbr (5’-CCG GTC TGA ACT CAG ATC ACG T-3’) (Palumbi et al., 1991) were used to amplify 16S. The universal invertebrate COI primer LCO1490 (5’-GGT CAA CAA ATC ATA AGA TAT TGG-3’) and HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) (Folmer et al., 1994) were used to amplify COI. PCR amplification was performed in 25 μl of reaction volume containing 10 mM Tris-HCl ph 8.3, 50 mM KCL, 1.5 mM

MgCl2, 200 μM dNTPs, 0.2 μM of a forward and reverse PCR primer, 0.5 mg/ml BSA (Sigma), 2 units of Taq polymerase (Takara), and 1 μl of template DNA solution. The cycling parameters for amplification consisted of an initial denaturation for 3 min at 94°C; followed by 30 cycles of denaturation for 45 s at 94°C, annealing for 90 s at a gene-specific annealing temperature (50°C for 16S, 48–50°C for COI), and extension for 120 s at 72°C; and ending with a 5 min extension at 72°C. Successful PCR products were purified using High Pure PCR Product Purification Kit (Roche). Direct double-stranded cycle sequencing of 25 to 30 ng of each PCR product was performed in both directions using the Applied Biosystems BigDye v 3 dye terminator cycle sequencing kit. Cycle sequencing was performed using an Applied Biosystems GeneAmp PCR System 9700. The cycling parameters were 25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C. Sequencing reaction products were purified using ethanol precipitation and analysed on an ABI PRISM 3130 DNA sequencer. Sequences were verified by forward and reverse comparisons. All sequences have been deposited in GenBank under accession numbers AB498781–AB498795 (16S rRNA) and AB498766–AB498780 (COI).

Phylogenetic analyses Sequences of 16S were aligned using the ClustalX alignment program, run at default parameters (Thompson et al., 1997). Further manual adjustments to improve alignments were made by eye. Sequences of COI were aligned using MacClade v.4.03 (Maddison & Maddison, 2002) with Molecular Phylogeny of Neptunea 123 COI 16S ID AB498779 AB498794 N2 AB498774 AB498789 N8 AB498775 AB498790 N11 AB498778 AB498793 N6 AF373886 – Off Hirajisone,Nagasaki Pref.,Japan, 470–487m AB498780 AB498795 N15 Off Soma, FukushimaPref., Japan, 300–400m AB498767 AB498782 N7 Off Samani, Hokkaido, Japan, 100–130m Japan, Hokkaido, Samani, Off Off Shichigahama, Miyagi Pref.,Japan, 20–40m Off Soma, FukushimaPref., Japan, 20–40m AB498777 AB498792 N5 Off Soma, FukushimaPref., Japan, 250–350mOff Soma, FukushimaPref., Japan, 250–350mOff Soma, FukushimaPref., Japan, 300–400m AB498768 AB498769 AB498783 N13 AB498784 N14 AB498771 AB498786 N12 Enshu-nada,off Mikawa, Aichi Pref., Japan, 300–400m AB498773 AB498788 N3 Locality n. sp. (Sowerby III, 1899) Off Soma, Fukushima Pref., Japan, 350–400m AB498770 AB498785 N10 Scarlato, 1952Scarlato, 110–120m Japan, Hokkaido, Samani, Off Dall, 1916

Habe & Sato, 1972 Sato, & Habe (Dall, 1907) Off Soma, Fukushima Pref., Japan, 300–400m AB498766 AB498781 N9 s.s . (Bernardi, 1857)* Off Shichigahama, Miyagi Pref.,Japan, 20–40m AB498776 AB498791 N4

Oyama in Kira, 1959 Enshu-nada,off Mikawa, Aichi Pref., Japan, 300–400m AB498772 AB498787 N1 Kira, 1959* (Linneaus, 1758)* (Linneaus, Sakurai & Tiba, 1969 Tiba, & Sakurai 80–100m Japan, Pref., Fukushima Soma, Off (Pilsbry, 1901) Röding, 1798 Golikovia Barbitonia Neptunea Specimens used inthis study.

Neptunea Table 1. 1. Table Nepunea fukueae fukueae Nepunea Neptunea ennae Subgenus Subgenus Neptunea arthritica Neptunea constricta constricta Neptunea Neptunea frater intersculpta Neptunea Neptunea kuroshio Neptunea mikawaensis Neptunea polycostata Subgenus Genus Species Asterisks(*) indicate thespecies type ofsubgenera. 124 T. Nakano et al. reference to translated amino acid sequence. Two genes were tested for congruence of phylogenetic signal using ILD test (Farris et al., 1995), as implemented by the partition homogeneity test in PAUP 4.0 version b10 (Swofford, 2002). Pairwise molecular distances of the partial 16S and COI genes were calculated by Kimura’s two-parameter method (Kimura, 1980). Subsequent phylogenetic analyses were performed with PAUP* v. 4b10 (Swofford, 2002) for Neighbor-joining (NJ) (Saito & Nei, 1987) (Kimura’s two-parameter method; Kimura, 1980), equally-weighted maximum parsimony (MP) and bootstrap values (Felsenstein, 1985, 1988). MrBayes v.3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) was used to estimate Markov-chain Monte-Carlo Bayesian posterior probabilities. The NJ bootstrap analyses consisted of 10,000 replicates. The weighted MP bootstrap analyses consisted of 1,000 replicates using a heuristic search (with 10 random addition sequence replicates and TBR branch-swapping). The models of nucleotide substitution for the Bayesian analysis were selected using Modeltest (Posada & Crandall, 1998). The model selected for each gene region was HKY + G for both genes. Bayesian analysis was performed using MrBayes v.3.1.2 with the following settings for the two partitions (i.e. genes). Rate variation across sites was modeled using a gamma distribution, with a proportion of the sites being invariant (rate = gamma). The shape, proportion of invariable sites, state frequency, and substitution rate parameters were estimated for each partition separately. The Markov-chain Monte-Carlo Search was run with four chains for 3,000,000 generations, with trees being sampled every 100 generations (the first 5,000 trees, i.e.

Fig. 1. NJ phylograms generated from (A) 16S and (B) COI sequences. Numbers on branches are NJ bootstrap values / equally weighted MP bootstrap values / Bayesian posterior probabilities. Molecular Phylogeny of Neptunea 125

500,000 generations, were discarded as burn-in).

Estimating divergence times A likelihood ratio test (Felsenstein, 1981) testing for departure from a clock-like rate of evolution was not significant for our molecular data (P > 0.05). Therefore, our molecular data are consistent with clocklike behavior. We transformed the Bayesian tree of COI data set using the nonparametric rate smoothing (Sanderson, 1997) as implemented in TreeEdit ver. 1.0a10 (http:// evolve.zoo.ox.ac.uk).

Calibration points In order to calibrate the divergence times within Neptunea, we used two alternative fossil records as calibration points: (C1) oldest record of Neptunea s.s. and (C2) oldest record of N. (Barbitonia) (see Appendix).

Fig. 2. Bayesian phylograms, estimated using Markov-chain Monte-Carlo (MCMC) Bayesian posterior probabilities, generated from 1,169 bp of combined 16S and COI data. Numbers on branches are NJ bootstrap values / equally weighted MP bootstrap values / Bayesian posterior probabilities. 126 T. Nakano et al. – – – – 0.00 0.20 – – – – – – s) and interspecific pairwise comparisons. Figures are genetic 0.00 0.00 0.00 0.15–0.91 – – – – 1.99 2.19 1.58 2.39 2.99 1.99 3.43–3.64 1.19 – 0.59 1.19 1.39 0.99 1.99 – 2.21–2.42 2.61–2.82 2.40–2.61 2.82–3.03 3.02–3.23 2.62–2.83 – 1.99 2.19 2.39 2.39 3.00 2.00 2.62–2.82 –– 1.19 1.19 1.78 1.96 1.58 –1.39 12345678910 – – 6.92 8.668.65 7.497.68 7.82 7.00–7.51 1.38–1.69 3.25 6.70 7.48–7.81 6.83 7.49 6.84 8.447.897.84 7.64 9.01 10.0–10.2 7.30 10.0–10.2 8.52 9.34–9.68 8.72 8.18 10.2 10.4 9.70 9.38 9.58 8.85 7.65 9.02 6.97 8.37 8.33 6.33 8.64–8.82 8.49–8.81 the models selected by Modeltest: HKY + G. the modelsby selectedModeltest: HKY

n. sp. n. 8.28 7.33 4.55–4.87 8.19 4.06 n. sp. n. – 1.79 0.79 2.18 0.99 Genetic distances Genetic (%): (in onintraspecific italic diagonal N. fukueae N. fukueae N. antiqua N. N. frater N. kuroshio N. mikawaensis N. polycostata N. constricta intersculpta N. N. arthritica N. ennae N. polycostata N. mikawaensis N. arthritica N. ennae N. intersculpta N. N. kuroshio N. frater N. antiqua N. N. constricta COI 1. 3. 5. 6. 7. 2. 2. 4. 10. 10. 8. 9. 10. 7. 7. 6. 6. 8. 8. 9. 9. 4. 4. 5. 3. 3. 16S 1. 2. distances calculatedusing Table 2.Table Molecular Phylogeny of Neptunea 127

Radula morphology The of N. mikawaensis n. sp. was removed from the , placed in 20% KOH at room temperature overnight, and rinsed in distilled water, and observed under a scanning electron microscope.

Results

Molecular data PCR amplification of 16S gave a product approximately 550 bp, and subsequent sequencing of this product yielded approximately 510 bp readable sequence (509 bp for N. arthritica and 507 bp for the other species), which, after alignment, gave a total of 511 bp. The COI product was approximately 700 bp long, and sequencing routinely gave an exactly 658 bp read. The partition homogeneity test detected no significant incongruity between gene partitions (P = 0.80). Sequences of 16S and COI were therefore combined for analysis. The combined aligned dataset of 1,169 bp characters (511 bp for 16S and 658 bp for COI), including the outgroup taxa (Buccinum tsubai and Buccinum striatissimum), had 212 variable and 166 parsimony-informative characters. Three datasets were used for constructing trees. Two independent gene datasets included 9 species (16S) and 10 species (COI), respectively (Fig. 1). The third, combined dataset included 15 individuals of the 9 species for which both genes had been sequenced (Fig. 2).

Fig. 3. Phylogram representing the Bayesian tree of COI transformed using nonparametric rate smoothing (Sanderson, 1997). The time scale was calibrated using three alternative fossil records. (A) C2, oldest record of N. (Barbitonia) (B) C1, oldest record of Neptunea s.s. Star marks denote the oldest reliable fossil record of each subgenus. The outgroup is not shown in the phylograms. 128 T. Nakano et al.

Molecular phylogeny All the phylogenetic trees, whether constructed using NJ, MP or Bayesian analysis, recovered three clades which correspond to the three subgenera, Neptunea s.s., N. (Golikovia) and N. (Barbitonia): bootstrap support for these clades ranged from 58% to 100% for the NJ and MP analyses, and the Bayesian analysis gave posterior probabilities of between 0.42 and 1.00 (Figs. 1, 2). The Neptunea s.s. clade consisted of four or five species: N. kuroshio, N. frater, N. intersculpta, N. antiqua (only COI sequence; Fig. 2B) and N. mikawaensis n. sp. The N. (Golikovia) clade included N. ennae and N. fukueae. The only representative of N. (Barbitonia) was N. arthritica. Topologies estimated from the 16S and COI data were identical except the placement of N. intersculpta. Neptunea intersculpta was clustered with N. constricta + N. polycostata in the 16S tree, whereas it was sister to N. kuroshio + N. frater + N. mikawaensis n. sp. in the COI tree. However, these clades were supported by lower bootstrap values (<= 64%). Interspecific genetic distances among the ten species ranged from 0.99 to 3.64% (for 16S), 1.38 to 10.4% (for COI), whereas the intraspecific distances ranged from 0.00 to 0.20% (for 16S), 0.00 to 0.91% (for COI) (Table 2).

Divergence times Estimation of divergence times within Neptunea is shown in Fig. 3, based on the two alternative fossil records (see Appendix). The divergence times of Neptunea calibrated by C1 better fits the fossil records than that of C2. However, we could not reject the divergence times based on C2, due to the uncertainty of the ancestral morphology of three subgenera. In both cases, the Atlantic N. antiqua diverged from the western Pacific species well before the opening of the Bering Strait in the Pliocene.

Discussion

Molecular phylogeny of Neptunea Only a few molecular studies including Neptunea species have been done until now. Hayashi (2005) used only N. intersculpta as a representative to elucidate the phylogenetic relationship of the Buccinidae at the intra- and supra-familial level using complete mitochondrial 16S. Neptunea intersculpta has also been used as an outgroup to examine the species boundaries of Japanese Buccinum species based on the partial mitochondrial 16S (Iguchi et al., 2007b). Iguchi et al. (2007a) analyzed COI sequences to examine the intraspecific variation within Buccinum tsubai and N. polycostata. Most recently, phylogenetic analysis has been used to examine the generic relationships within the family Buccinidae based on a fragment of nuclear 28S sequences including four species of Neptunea (N. cumingi, N. lyrata decemcostata, N. eulimata and an uncertain species) (Dong et al., 2008). Due to the limited sampling of species, relationships among Neptunea have been unclear. The present study is the first attempt to reconstruct the molecular phylogeny of the genus Neptunea. Our molecular phylogenetic analyses demonstrate three clades within Neptunea s.l. : the Neptunea s.s. clade, N. (Golikovia) clade, and N. (Barbitonia) clade. This supports the subgeneric division of Neptunea based on combination of shell and radular characters proposed by Tiba & Kosuge (1988) (Table 3). Neptunea s.s. is characterized by a large shell size, convex whorls, oval and radular configuration that consists of a central tooth with three cusps and two lateral teeth with three to five cusps. The shell shape and radular configuration of N. (Barbitonia) are similar to those of Neptunea s.s., but the former is distinguished from the latter by the presence of spiral cords within the outer lip (Tiba & Kosuge, 1988). According to our observation, however, the spiral cords within the outer lip in N. (Barbitonia) commonly disappear in mature specimens. Shells of N. (Golikovia) differ from those of other two subgenera in the absence of external sculpture Molecular Phylogeny of Neptunea 129 except on the early whorls, and in the presence of two cusps on the marginal tooth. Goryachev (1987) divided Neptunea into three stocks; N. intersculpta, N. lyrata and N. polycostata, based on Golikov’s (1963) work on the shape of the penis, radula and egg capsule. He further subdivided the three stocks into several groups based on conchological characters (Table 4). Neptunea (Barbitonia) and N. (Golikovia) are treated as the same stock in his classification. However, our data do not support the Goryachev’s classification, because his N. polycostata stock is polyphyletic, and N. polycostata and N. constricta, which he classified as different stocks, are in a close relationship in our phylogenetic trees. Probably, the convergence

Table 3. Shell characters of Neptunea s.s., N. (Barbitonia) and N. (Golikovia). Neptunea s.s. N. (Barbitonia) N. (Golikovia) Shell thickness Typically thin Thin Thick Spiral sculpture Very strong carinae to Evenly rounded ribs, Evenly rounded ribs, subobsolete threads. Often commonly only on early commonly only on early not readily distinguishable whorls whorls into primary, secondary and tertiary spirals Axial sculpture Typically absent, or as Peripheral knobs on later Absent rather narrow folds or whorls lamellae when present Inner side of outer lip Smooth With spiral lirae in juve- Smooth niles Protoconch Small (ca. 3mm: N. Large (ca. 4.1 mm: N. Small (ca. 2.2 mm: N. mikawaensis n. sp., arthritica, NSMT-Mo ennae), 2 whorls NSMT-Mo 76946, 3mm: 71363; 4.5 mm: N. N. frater*) to large (3.9– arthritica, NSMT-Mo 4.1 mm: N. inter- 71362), 2 whorls sculpta*), 2 whorls Radula Typically 3 (sometimes 4) 3–6 cusps on central teeth 3 cusps on central teeth cusps on lateral and and 3 or 4 cusps on lateral and 2 cusps on lateral central tooth. Central teeth teeth teeth wide *The protoconch data of N. frater and N. intersculpta are from Hasegawa (2009).

Table 4. Taxonomic subdivision of Neptunea by Goryachev (1987). Neptunea intersculpta stock (1) N. intersculpta group: *N. intersculpta (Sowerby) (2) N. constricta group: *N. constricta (Dall), N. varicifera (Dall), N. lamellosa Golikov stock (1) N. lyrata group: N. lyrata (Gmelin), N. stielesi Smith (2) N. beringiana group: N. beringiana (Middendorf), N. ventricosa (Gmelin) (3) N. communis group: N. communis (Middendorf), N. denselirata Broegger (4) N. despecta group: N. despecta (Linnaeus) (5) N. antiqua group: *N. antiqua (Linnaeus), N. contraria (Linnaeus) Neptunea polycostata stock (1) N. polycostata group: *N. polycostata Scarlato, N. laticostata Golikov, N. vinosa (Dall), N. amianta (Dall) (syn. N. insularis (Dall), N. pribiloffensis (Dall)) (2) N. bulbacea group: N. bulbacea (Bernardi), N. rugosa Golikov (3) Golikovia group: N. smirnia (Dall), *N. fukueae Kuroda (4) N. arthritica group: *N. arthritica (Bernardi), N. cumingi (Crosse) Asterisks (*) indicate the species included in our phylogenetic analysis. 130 T. Nakano et al. was due to the shape of the egg capsule or penis, since the distinctions in of radula morphology are consistent with molecular phylogenetic trees. Due to the limited sampling of species, our molecular data are not sufficient to evaluate the subdivision of Neptunea s.s. by other workers.

Historical biogeography of Neptunea Historical biogeography can be discussed from a combination of the species-level phylogeny, recent distributions, divergence times and fossil record. Recent molecular biogeographic works on gastropods have focused on taxa mainly inhabiting the intertidal rocky shores (e.g. Reid et al., 1996; Koufopanou et al., 1999; Williams & Reid, 2004; Nakano & Ozawa, 2004, 2007). In these works, divergence times estimated from molecular data and fossil records are sometimes incongruent, probably because gastropods in such habitats have a much lower fossilization potential than those in shallow marine, soft-bottom environments. In the case of Neptunea, however, many fossil species have been reported from Japan, the Russian Far East and Alaska (e.g. Nelson, 1978; Amano, 1997, and references therein). Our molecular data suggest that the diversification of Neptunea chiefly occurred in the Late Oligocene to middle Miocene and agree well with the fossil records documented by Nelson (1978). The oldest fossil record of Neptunea is unclear because the oldest records described by many authors are based on poorly preserved materials (see Appendix). Some of the buccinid gastropods documented from the Paleogene of Hokkaido appear to represent a stem group that diverged from an ancestral form before the divergence of the Neptunea s.s. clade. Our results suggest that the divergence of N. (Barbitonia) + N. (Golikovia) from Neptunea s.s. occurred in the Late Oligocene and Middle Miocene (Fig. 3). However, the oldest reliable fossil record of N. (Barbitonia) is from the early Late Miocene (Fig. 3B). The time gaps between the molecular clock estimates and the available fossil records is likely due to the uncertainty of the generic and subgeneric assignment of poorly preserved Paleogene and Miocene materials, and/or the time gap between divergence and morphological evolution. The oldest fossil records of Neptunea s.s. are older than the estimated time of divergence (Fig. 3A). If this estimation is true, this fossil record may belong to a stem group of Neptunea. In both cases, further taxonomic studies based on better preserved materials may provide better divergence times. Neptunea is one of the genera that supposedly invaded the North Atlantic from the North Pacific after the opening of the Bering Strait (MacNeil, 1965; Strauch, 1972). The Aleutian- Commander Island Arc between the northwestern and northeastern Pacific is a plausible migration root for marine molluscs (Vermeij et al., 1990). Littorinid gastropods (Reid, 1996) and patellogastropod limpets (Nakano & Ozawa, 2004, 2007) are also thought to have migrated via this route. After the opening of the Bering Strait during the Pliocene, 295 molluscan species extended their distribution from the northern Pacific to Arctic-Atlantic basins (Vermeij, 1991). Neptunea is likely to have spread to the Atlantic during this period, since the first stratigraphic occurrence of Neptunea s.s. in the Atlantic is known from the Oorderen Member of the Lillo Formation in Antwerp, which is dated as 2.7–3.2 Ma (F.P. Wesselingh, personal communication, 2008). Nevertheless, our estimation of divergence times suggests that N. antiqua had diverged from the Japanese species examined well before the opening of the Bering Strait (latest Miocene to Early Pliocene; Marincovich & Gladenkov, 1999). More comprehensive sampling of Pacific Neptunea species may reveal taxa that are more closely related to the Atlantic species.

Systematics

Family Buccinidae Rafinesque, 1815 Genus Neptunea Röding, 1798 Type species, by subsequent designation (Sandberger, 1861): antiquus Linnaeus, 1758. Molecular Phylogeny of Neptunea 131

Neptunea mikawaensis n. sp. (Figs. 4A–D, 5)

Neptunea sp.: Miyoshi et al., 2009, p. 86, fig. 1A, B.

Diagnosis: Shell thick, obese fusiform; whorls sculptured by sharp ribs with many interstitial secondary riblets; brown filmy periostracum. Descriptions (Fig. 4): Shell thick, whitish brown, obese-fusiform. Protoconch of 1.5 whorls, small, white or pinkish white, no clear distinction from teleoconch. Teleoconch consisting of about six convex whorls with rounded periphery and distinct suture. Body whorl well inflated, strongly constricted at base. Surface sculptured by sharp spiral ribs with many interstitial secondary riblets. Whorls covered with brown filmy periostracum. Outer lip simple and sharp. Aperture

Fig. 4. Shell morphology of Neptunea species. A–B. Neptunea mikawaensis n. sp., holotype, NSMT-Mo 76946, 9.1 × 4.6 mm, Sea area Enshu-nada, central Japan. C–D. Neptunea mikawaensis n. sp., paratype #1, NSMT-Mo 76947, 5.8 × 3.1 mm, Sea area Enshu-nada, central Japan. E–F. Neptunea yokoyamai, NSMT- PM12140, 4.3 × 2.1 mm, Koshiba, Kanazawa District, Yokohama City, Kanagawa Pref., Japan. G–H. Neptunea kuroshio, NSMT-Mo 76948, 7.5 × 4.2 mm, Sea area Enshu-nada, central Japan. 132 T. Nakano et al. broadly ovate, white within. Anterior canal long, hemitubular and slightly oblique. corneous, thick, dark brown and semi-oval in shape, pointed towards siphonal canal with a terminal nucleus. Radula (Fig. 5) stenoglossate; narrow rachidian tooth with four cusps and massive, strong laterals with four cusps. Cusps of rachidian tooth similar in shape and size. Outermost cusp of lateral largest and separated from next one by deep trough. Second, third and innermost cusps of lateral similar in shape and close to each other. Innermost cusp of lateral slightly bigger than second and third ones. Type locality: Sea area Enshu-nada, off Mikawa, Aichi Prefecture, central Japan, 300–400 m depth. Type materials: Holotype, NSMT-Mo 76946; paratype #1, NSMT-Mo 76947; paratype #2, Miyoshi collection; paratypes #3–7, Higuchi collection (Table 5). Distribution: Known only from type locality. Etymology: Named after Mikawa (an old district name for the western part of Shizuoka Prefecture), the type locality of this new species. Remarks: Neptunea mikawaensis n. sp. is morphologically similar to the sympatric species N. kuroshio, but the former has a more inflated body whorl and a thicker shell than N. kuroshio. The anterior canal is slightly oblique and the shell is whitish brown in N. mikawaensis n. sp., whereas

Fig. 5. Radula of Neptunea mikawaensis n. sp., paratype #1, NSMT-Mo 76947. Scale bar = 50 μm.

Table 5. The measurements of N. mikawaensis n. sp. (mm). Depositry SL SW SL/SW Holotype NSMT-Mo 76946 91.5 48.9 1.87 Paratype #1 NSMT-Mo 76947 57.5 33.8 1.7 Paratype #2 H. Miyoshi collection 83.2 43.3 1.92 Paratype #3 S. Higuchi collection 97.1 41.5 2.33 Paratype #4 S. Higuchi collection 88.4 44 2.00 Paratype #5 S. Higuchi collection 96.0 50.5 1.90 Paratype #6 S. Higuchi collection 86.4 49.1 1.76 Paratype #7 S. Higuchi collection 87.9 47.8 1.84 Molecular Phylogeny of Neptunea 133 the canal is almost straight and the shell white to yellowish white in N. kuroshio. Additionally, the thin periostracum is brown in color in N. mikawaensis n. sp., whereas it is yellowish brown in N. kuroshio. Miyoshi et al. (2009) have already recognized the similarity between two fossil species of Neptunea from Japan and N. mikawaensis n. sp. These are Neptunea noboriensis Ozaki, 1954 from the Pliocene deep-water deposits of Shikoku and Okinawa Island, southwestern Japan and Neptunea yokoyamai Kuroda, 1954 from Plio-Pleistocene deposits from the Pacific side of central Honshu, central Japan. Neptunea mikawaensis n. sp. and N. noboriensis exhibit almost identical whorl profile and surface sculpture in younger growth stages, but the former differs distinctly from the latter in having a thicker shell wall and higher and more alternating spiral cords in fully grown shells. Neptunea mikawaensis n. sp. is easily distinguished from N. yokoyamai in having a more deeply incised suture and more rounded primary spiral cords.

Acknowledgements

We are grateful to Dr. J. Hashimoto, the crews of the F/V Tokai-maru no. 7 and Dr. Y. Kano for providing the material. We are also grateful to Dr. T. Kase and Dr. D. G. Reid for comments on the manuscript. Dr. F. P. Wesselingh provided the biostratigraphic information of Neptunea in the North Sea Basin. Mr. Y. Kuwahara and Mr. T. Haga shared their knowledge on Neptunea. This study was supported by a Grant-in- Aid for JSPS fellows no. 207024 to TN from the Japan Society for the Promotion Science.

References

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(Received May 14, 2009 / Accepted December 23, 2009)

Appendix

Critical evaluation of the fossil record of three subgenera within Neptunea Although there are a number of buccinid gastropods previously assigned to the genus Neptunea in the Paleogene of Japan and the Russian Far East, their , relationships and generic assignments remain matters of controversy and uncertainty. In general, the generic assignments of these Paleogene species are difficult due to their poor preservation. Strauch (1972), Titova (1994) and Oleinik (2001) regarded altispiratus Nagao, 1928 from the Eocene of Kyushu and Kamchatka as the oldest species of Neptunea, but without critical evaluation. On the other hand, Nelson (1978) assigned C. altispiratus to the genus as it has the distinctly angulated base of the body whorl that is characteristic of that genus. In our opinion, the generic assignment of C. altispiratus is still uncertain, because the details of the siphonal and columellar characters are inaccessible due to the incomplete preservation of the holotype. Similarly, species previously described as Neptunea from the Oligocene of Hokkaido cannot be certainly assigned to the genus as they have a less inflated body whorl, a very short siphonal canal and/or a less twisted , and later workers placed them mostly in Ancistrolepis, or Trominina (e.g. Oyama et al., 1960; Titova, 1994). Nelson (1978) recognized Chrysodomus modestus Kuroda, 1931 as the oldest species of Neptunea s.s. We adopt here Nelson’s conservative view, but the current chronostratigraphic framework of the Japanese 136 T. Nakano et al.

Tertiary indicates that the oldest occurrence of this species is not in the Upper Oligocene as recognized by Nelson (1978) but in the upper Lower Miocene (e.g. Gladenkov, 2008). Well preserved specimens of this species in the upper Lower Miocene were illustrated by Itoigawa et al. (1981) from the Yamanouchi Formation of the Mizunami Group, central Japan. Therefore, we calculated the divergence time as 18 Ma for the node of Neptunea s.s. (C1). The fossil record of the subgenus N. (Barbitonia) is restricted to the Neogene and Quaternary of Japan and its adjacent areas. Although this subgenus is quite common in Upper Pleistocene shallow-water marine deposits in central and northern Japan, its Miocene and Pliocene fossil record is very limited. A specimen described by Noda (1992) as Neptunea sp. aff. arthritica (Bernardi) from the Lower Miocene Sankebetsu Formation of Hokkaido is too poorly preserved to allow subgeneric assignment. The oldest reliable record of this subgenus is represented by an unnamed species from the lower Upper Miocene Itahana Formation, central Honshu, Japan (Y. Kurihara, unpublished). This unnamed species bears distinct spiral lirae on the inner side of the outer lip, which is the diagnostic character of the subgenus. The geologic age of the Itahana Formation is estimated as ca. 11 Ma by microfossil biostratigraphy and radiometric dating (Takahashi & Hayashi, 2004). Therefore, the second calibration point (C2) is 11 Ma for the first node of the N. (Barbitonia) clade. (C2). Nelson (1978) reviewed the distribution of the subgenus N. (Golikovia) in space and time. He doubted the assignment of some Oligocene and early Miocene species of this subgenus from the Russian Far East because of their poor state of preservation. According to him, the oldest reliable species of this subgenus include N. (G.) nikkoensis Nomura, 1937 in the western Pacific and N. (G.) plafkeri Kanno, 1971 in the eastern Pacific. Neptunea (G.) nikkoensis ranges from the Miocene to the Upper Pliocene (see Amano, 1997 for synonymy). The sole Miocene record of this species is from the Kubiki Formation of Niigata Prefecture, central Japan (Noda, 1962), which is currently mapped as the Upper Miocene Sugawa Formation (Takeuchi et al., 1990). Kanno (1971) recorded N. (G.) plafkersi from the lower part of the Yakataga Formation in the Yakataga District and its correlated Topsy Formation in the Lituya district of Alaska (see also Nelson, 1978). These formations had been considered as the early Middle Miocene, but Lagoe et al. (1993) later suggested that the lower part of the Yakataga Formation is of latest Miocene in age. Molecular Phylogeny of Neptunea 137

ミトコンドリア DNAに基づくエゾボラ属の分子系統

中野智之・栗原行人・三好博文・樋口滋雄

要 約

エゾボラ属 Neptuneaは，エゾバイ科 Buccinidaeの中で最も種数の多いグループであり，北極海から北 太平洋，北大西洋の幅広い水深に分布している。これまでにエゾボラ属の系統分類は，形態学的にいく つかのグループに細分されているものの（例えば， Goryachev, 1987; Tiba & Kosuge, 1988; Fraussen & Terryn, 2007），それぞれの分類は整合的ではなかった。そこで本研究では，エゾボラ属の 3亜属全ての 模式種を含む 10種を解析の対象とし，ミトコンドリア DNAの 16S rRNAと COI遺伝子の部分配列に基 づき，分子系統解析を行った。その結果，エゾボラ属は Neptunea s.s., N.（ Barbitonia）， N.（ Golikovia） の 3つのグループに区分でき， Tiba & Kosuge（ 1988） の分類を支持する結果となった。また，三好・他 （2009）で報告された遠州灘産の Neptunea sp.は，同所的に産出するヒメエゾボラモドキと遺伝的に異な り，さらには化石種ヨコヤマエゾボラ N. yokoyamai Kuroda, 1961とノボリエゾボラ N. noboriensis Ozaki, 1956とも形態学的に区別できる事から，下記のように新種 として記載した。

Neptunea mikawaensis n. sp. ミカワエゾボラ（新種・新称） 殻長は 7 cm前後。殻は紡錘型で丸みは弱く，なで肩である。殻表には数本の強い螺肋があり，螺肋の 間には細い間肋がある。殻は茶褐色で，殻表には薄いフィルム状の殻皮がある。体層はやや膨れ，殻底 はくびれる。縫合は不顕著。殻口内壁は紫褐色で，前管溝はやや長く突きでる。