MOLECULAR PHYLOGENY OF JAPANESE GASTROPODS IN THE GENUS

SHIGEAKI KOJIMA,1 NAOTOMO OTA,2 KEISUKE MORI,2 TAIJI KUROZUMI3 AND TOSHIO FUROTA4 1Ocean Research Institute, University of Tokyo, Minamidai 1-15-1, Nakano, Tokyo 164-8639, Japan 2Amakusa Marine Biological Laboratory, Kyushu University, Tomioka 2231, Reihoku, Amakusa, Kumamoto 863-2507, Japan 3Natural History Museum and Institute, Chiba, Aoba-cho 955-2, Chuo, Chiba, Chiba 260-8682, Japan 4Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan (Received 7 September 2000; accepted 20 February 2001)

ABSTRACT

Phylogenetic relationships among four of snails in the genus Batillaria that inhabit intertidal zones of the Japanese Islands were determined from the nucleotide sequences of part of the mitochon- drial gene for cytochrome oxidase I. The analysis indicated that B. multiformis and B. flectosiphonata are closely related to each other and that B. zonalis is the basal of the four species. All snails with a shell that resembled the shell of B. flectosiphonata that were collected from Ariake Bay and the Goto Islands had mitochondrial DNA that corresponded to that of either B. multiformis or B. cumingi. Our results suggest that the morphological characteristics that have been used to discriminate among species in the genus Batillaria need to be revised.

INTRODUCTION logical examination of shells exclusively, because indi- viduals with shells of intermediate morphologies Intertidal snails in the genus Batillaria form a domin- inhabit the same regions. ant group in the muddy tidelands of Japan. With the Taxonomic confusion has been an obstacle to pro- recent exploitation of the coastal areas of Japan, most gress in ecological and zoogeographical studies of of the habitats of such snails have been lost and these fauna on the muddy tidelands of western Kyushu. In snails, in particular B. multiformis (Lischke), are now the present study, we tried to identify Batillaria snails thought to be endangered (Kurozumi 1995; Wada, from the western side of Kyushu on the basis of the Nishihira, Furota, Nojima, Yamahira, Nishikawa, nucleotide sequences of their mitochondrial DNA. Goshima, Suzuki, Kato, Shimamura & Fukuda 1996; Furota 2000). In addition to the three species in the genus Batillaria that have been reported from Japan, namely B. multi- formis, B. cumingi (Crosse) and B. zonalis (Bruguière), MATERIALS AND METHODS Ozawa (1996) described a fourth Japanese species in Sites at which Batillaria snails were sampled are summarized this genus, B. flectosiphonata (Ozawa), that was found in Table 1. Snails that resembled B. flectosiphonata were col- around Iriomote Island, a member of the Ryukyu lected at five sites in Ariake Bay, as well as at a single site on (Nansei) Islands. Ozawa (1996) reported the prelimin- the Goto Islands (Figs. 1 and 2). Morphological characters of ary results of a phylogenetic analysis of the four species Batillaria multiformis, B. cumingi and B. flectosiphonata-like based on the nucleotide sequences of mitochondrial snails collected in Ariake Bay and the Goto Islands are genes for ribosomal RNA. The results suggested that summarized in Table 2. One individual each of two species B. flectosiphonata forms a monophyletic clade with that are closely related to Batillaria spp., namely, Cerithidea B. multiformis and that B. cumingi is a sister species of (C.) rhizophorarum and Cerithideopsilla (Cp.) djadjariensis, the B. flectosiphonata / B. multiformis clade. were collected on the Kiire Coast, Kagoshima Prefecture, for Ozawa (1996) stated that B. flectosiphonata inhabits use as an outgroup for phylogenetic analysis. After extrac- tion of DNA, specimens collected in Ariake Bay and the not only the intertidal regions of the Ryukyu Islands Goto Island were fixed in 100% ethyl alcohol and deposited but is also found along the western coast of Kyushu. at the Natural History Museum and Institute, Chiba. Although snails with shells that resemble those of B. DNA was extracted from the head-foot region of each indi- flectosiphonata are known to inhabit Ariake Bay and vidual by grinding, digestion with sodium dodecyl sulfate the shores of the Goto Islands, west of Kyushu (Fig. 1), (SDS) and extraction with phenol and chloroform. Then part it is difficult to identify such snails from a morpho- (about 700 bp) of the mitochondrial gene for cytochrome

J. Moll. Stud. (2001), 67, 377–384 © The Malacological Society of London 2001 SHIGEAKI KOJIMA ET AL.

Table 1. Sites at which specimens of Batillaria and two related species, Cerithidea rhizophorarum and Cerithideopsilla djadjariensis, were collected.

No. Sampling site Species N

1 Shiokawa Tideland, Mikawa Bay Batillaria multiformis 3 2 B. cumingi 3 3 Yone Coast, Okinawa Island B. zonalis 3 4 Miyara Bay, Ishigaki Island B. flectosiphonata 3 5 Myouse, Ariake Bay B. multiformis 3 6 B. cumingi 3 7 B. flectosiphonata-like snail 3 8 Kamitsue River, Ariake Bay B. multiformis 3 9 B. cumingi 3 10 B. flectosiphonata-like snail 4 11 Kurae River, Ariake Bay B. multiformis 3 12 B. cumingi 3 13 B. flectosiphonata-like snail 3 14 Mogine, Ariake Bay B. multiformis 3 15 B. cumingi 3 16 B. flectosiphonata-like snail 3 17 Kurosaki, Ariake Bay B. multiformis 3 18 B. cumingi 6 19 B. flectosiphonata-like snail 3 20 Wakamatsu Island, Goto Islands B. flectosiphonata-like snail 20 21 Kiire Coast, Kagoshima Cerithidea rhizophorarum 1 22 Cerithideopsilla djadjariensis 1

Table 2. Morphological characters of Batillaria multiformis, B. cumingi and B. flec- tosiphonata-like snails collected in Ariake Bay and the Goto Islands.

Species

Character B. multiformis B. cumingi B. flectosiphonata-like snail

Body whorl well inflated less inflated less inflated Axial ribs strong or not strong not strong strong Parietal tooth well developed not developed not developed

oxidase I (COI) was amplified by the polymerase chain and Cp. djadjariensis were amplified with primers COI-B reaction (PCR) using total DNA as template and primers (Hasegawa, Yamaguchi, Kojima & Ohta 1996) and COI-6, COI-6 (Shimayama, Himeno, Sasuga, Yokobori, Ueda & and they were sequenced with primers Gastro-2, Gastro-4 Watanabe 1990) and Gastro-2. The conditions for PCR were and Gastro-3 (Kojima, Segawa, Fujiwara, Hashimoto & as follows: 94°C for 60 s; then 30 to 40 cycles at 92°C for 40 s, Ohta 2000). The nucleotide sequences of all the primers used 50°C for 60 s, and 72°C for 90 s. GenereleaserTM (BioVenture in the present study are given in Table 3. Amino acid Inc., Murfreesboro, TN, USA) was used to sequester pro- sequences of COI were deduced by reference to the modified ducts of cell lysis that might have inhibited the polymerase. genetic code of molluscan mitochondrial DNA (Shimayama Both strands of each amplified fragment were sequenced et al. 1990; Hoffmann Boore & Brown 1992). with an automated sequencer (DSQ-2000L; Shimazu Corp., The genetic distance between sequences was calculated by Kyoto, Japan) with Gastro-4 and UN-1 as internal primers. Kimura’s two-parameter method (Kimura 1980). A phylo- Primers Gastro-2 and Gastro-4 were synthesized on the basis genetic tree was constructed by the neighbour-joining of sequences of genes for COI from several gastropod species. method (Saitoh & Nei 1987) with the program from MEGA These sequences were determined with primers COI-B and package, Version 1.0 (Kumar, Tamura & Nei 1993) and also COI-6 as sequence primers (unpublished data). Primer UN-1 by the maximum-parsimony method with the heuristic was synthesized on the basis of sequences of COI genes search approach of the computer program Parsimony, which of Batillaria snails that had been determined with primer was kindly provided by Dr. K. Tamura of Tokyo Metro- Gastro-4. Fragments of the COI genes of C. rhizophorarum politan University.

378 PHYLOGENY OF BATILLARIA SNAILS

Figure 1. Sites at which samples were collected: 1, Mikawa Bay; 2, Wakamatsu Island, Goto Islands; 3, Kurosaki, Ariake Bay; 4, Myouse, Ariake Bay; 5, Mogine, Ariake Bay; 6, Kamitsue River, Ariake Bay; 7, Kurae River, Ariake Bay; 8, Kiire Coast, Kagoshima; 9, Yone Coast, Okinawa Island; 10, Miyara Bay, Ishigaki Island.

379 SHIGEAKI KOJIMA ET AL.

Figure 2. Shells of Batillaria multiformis from Ariake Bay (a), B. cumingi from Ariake Bay (b), a B. flectosiphonata-like snail from Ariake Bay (c), a B. flectosiphonata-like snail from the Goto Islands (d), B. flectosiphonata from Ishigaki Island (e) and B. zonalis from Okinawa Island (f). Scale bar 1 cm.

Table 3. Nucleotide sequences of primers used in the present study. Y, R, S, W and N denote T or C, A or G, G or C, A or T and G, A, T or C, respectively. Positions refer to the corresponding amino acid residues encoded by the gene for mitochondrial cytochrome oxidase I from Drosophila yakuba.

Name Sequence Position Direction

COI-B 5-GGATGAACNGTNTAYCCNCC-3 123–129 Forward Gastro-3 5-TTAGCTGGTGCTTCNTCNATYYTNGG-3 150–158 Forward Gastro-2 5-GCGTTCTTTGACCCAGCTGGNGGNGGNGAYCC -3 216–226 Forward UN-1 5-TTRATTTTACCRGGATTYGG-3 244–250 Forward Gastro-4 5-ATAATAAARAARTGNTTNGTYCA-3 407–415 Reverse COI-6 5-GGRTARTCNSWRTANCGNCGNGGYAT-3 434–442 Reverse

RESULTS between Batillaria spp. and Cp. djadjariensis, respect- ively (Fig. 3). The nucleotide sequences of parts of the genes for COI The phylogenetic relationships among Batillaria (489 bp) from each of three individuals of four species snails were analyzed by the neighbour-joining (NJ) in the genus Batillaria and from each single specimen of method with C. rhizophorarum and Cp. djadjariensis as two related species are shown in Figure 3. Of the three an outgroup (Fig. 4). Specimens of B. multiformis, B. specimens of B. multiformis, two had an identical cumingi and B. flectosiphonata, respectively, each sequence and there were three nucleotide substitutions formed a monophyletic cluster, and each cluster was in the sequence from the third individual. The nucleo- supported by a high bootstrap probability (89–100%). tide sequences of two specimens of B. cumingi differed On the NJ tree (Fig. 4), B. multiformis and B. flecto- from that of another specimen at a single site only. The siphonata were located close to each other and the sequences from three specimens of B. flectosiphonata monophyly of these two species was supported by a differed at one or two sites from one another. The high bootstrap probability (100%). B. zonalis was the sequences from three specimens of B. zonalis were basal of the four species and the monophyly of the identical. While no amino acid substitutions were other three congeneric species was also supported by a detected among the four species in the genus Batillaria, high bootstrap probability (86%). The topology of a one and three amino acid substitutions were detected 50% majority rule consensus phylogram, obtained by between Batillaria spp. and C. rhizophorarum, and the maximum-parsimony (MP) method, was identical

380 PHYLOGENY OF BATILLARIA SNAILS (Cp). Cerithideopsilla djadjariensis (C) and 54365 (Bc), AB054366 (Bf), AB054367 (Bz), and R denote T or C A G, respectively. The nucleotide Cerithidea rhizophorarum (Bz), B. zonalis (Bf), B. flectosiphonata (Bc), B. cumingi (Bm), Batillaria multiformis Nucleotide sequences from sequences will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases under accession numbers AB054364 (Bm), AB0 AB054368 (C), and AB054369 (Cp). Dots indicate nucleotides that are identical to those in Bm. Underlined codons encode amino acid different from Y Figure 3.

381 SHIGEAKI KOJIMA ET AL.

Figure 4. Phylogenetic relationships among four species in the genus Batillaria. The phylogenetic tree was constructed by the neighbour- joining method using Cerithidea rhizophorum and Cerithideopsilla djadjariensis as an outgroup. Bootstrap values are shown above branches of clades that are supported by bootstrap values of more than 70%. to that of the NJ tree (data not shown). All conclusions of Kyushu on the basis of the nucleotide sequence of derived by the NJ method were supported by results part of the mitochondrial gene for COI. Preliminary obtained by the MP method with high bootstrap prob- analysis of all four Japanese species in this genus (Figs. abilities (82–99%). 3 and 4) revealed interspecific differences among From 49 Batillaria snails, collected in Ariake Bay, nucleotide sequences within the analyzed region of the and 20 Batillaria snails from the Goto Islands (Table mitochondrial DNA. 1), we found a total of 23 different nucleotide All individuals from Ariake Bay that were identified sequences (data not shown). We analyzed the phylo- as B. multiformis and B. cumingi were found to have genetic relationships among these 69 individuals, B. mitochondrial DNA that corresponded to shell mor- multiformis and B. cumingi from Mikawa Bay, and B. phology both by the neighbour-joining (NJ) method flectosiphonata from Ishigaki Island by the NJ method (Fig. 5) and by the maximum parsimony (MP) method using B. zonalis as an outgroup (Fig. 5). All specimens (data not shown). However, 16 specimens that resem- of B. multiformis collected in Mikawa Bay and Ariake bled B. flectosiphonata, collected in Ariake Bay and the Bay formed a monophyletic cluster with two specimens Goto Islands, formed monophyletic clusters with with B. flectosiphonata-like shells (10a and 10d in either B. multiformis or B. cumingi (Fig. 5). Fig. 5). Other B. flectosiphonata-like snails formed a The three clusters of Batillaria snails indicated in monophyletic cluster with specimens of B. cumingi Figure 5 were distinct from one another, with a smaller from Mikawa Bay and Ariake Bay. The monophyly of range of sequence variation within each cluster than each of the two clusters was supported by a high boot- between clusters on both the NJ tree (Fig. 5) and the strap probability (97% and 99%, respectively). Within MP tree (data not shown). Thus, the clusters appear to the latter cluster, 19 individuals from the Goto Islands be formed by individuals of three different species, and the remainder formed reciprocal monophyletic namely, B. multiformis, B. cumingi and B. flecto- clusters that were supported by high bootstrap pro- siphonata, respectively. The cluster corresponding to babilities (83% and 95%, respectively). All conclusions B. cumingi was formed by two distinct subclusters (Fig. obtained by the NJ method were supported by the MP 5): a cluster composed of almost all individuals from method with high bootstrap probabilities (73–99%; the Goto Islands and a cluster composed of the remain- data not shown). der. Only one individual from the Goto Islands was included in the latter subcluster. Our results suggest DISCUSSION that gene flow between the population of B. cumingi around the Goto Islands and the population in Ariake In the present study, we attempted to identify snails Bay is limited. in the genus Batillaria that inhabit the western coast If the snails that resembled B. flectosiphonata that

382 PHYLOGENY OF BATILLARIA SNAILS

Figure 5. Phylogenetic relationships among Batillaria snails collected from Ariake Bay and the Goto Islands; B. multiformis and B. cumingi from Mikawa Bay; B. flectosiphonata from Ishigaki Island. The phylogenetic tree was constructed by the neighbour-joining method using B. zonalis as an outgroup. Numbers refer to samples indicated in Table 1 and letters after numbers refer to individual specimens. Abbreviations in italics, underlined abbreviations and others denote B. multiformis, B. cumingi and B. flectosiphonata-like snails, respectively. A bootstrap value is shown at each branch of a clade that is supported by a bootstrap value of more than 70%. were found in western Kyushu and had the mitochon- flectosiphonata, namely, a distorted and deeply con- drial DNA of B. cumingi were actually B. cumingi, the cave columella and a siphonal canal which is reflected similarity in terms of shell morphology between them to the left. Both characters are gradational and it is and B. flectosiphonata might be a result of conversion difficult to distinguish B. flectosiphonata from other caused by environmental factors common to their species based on these features alone. No specimens habitats, such as high water temperature. Houbrick from western Kyushu had the latter characteristic, but (1978) stated that convergence of shell characteristics the frequency of individuals with such a feature was is not an uncommon phenomenon in members of the rather low among specimens of B. flectosiphonata family Potamididae, which includes the genus Batil- collected from the Yaeyama Islands by one of the laria. An alternative explanation is that B. flecto- present authors (T. K.). Our results suggest that it may siphonata-like snails of western Kyushu are actually B. be necessary to revise the morphological characteristics flectosiphonata with mitochondria of B. cumingi, which used for discrimination among species in the genus derived from introgression between B. flectosiphonata Batillaria. Moreover, although Fukuda, Yamashita & and B. cumingi at a time when the distribution of the Fujii (1999) reported finding B. flectosiphonata in the two species overlapped. In order to test the latter estuary of the Tagori River, in northern Kyushu, their hypothesis, it will be necessary to examine phylogenetic specimens might have been B. cumingi. relationships among these two species and B. flecto- siphonata-like snails using neutral genetic markers in the nuclear DNA. The present analysis, based on the nucleotide ACKNOWLEDGMENTS sequences of mitochondrial DNA, suggests that B. The authors thank Dr. T. Kimura, Mie University; Dr. Y. flectosiphonata in the Ryukyu Islands is genetically Takada, Ishigaki Tropical Station, Seikai National Fisheries independent from the other three Japanese species in Research Institute; Dr. A. Iijima, Toho University; and Ms. the genus Batillaria (Fig. 4). However, the study failed S. Kamimura, the University of Ryukyus, for samples from to reveal the presence of this species on the western Mikawa Bay, Ishigaki Island and Okinawa Island, respect- coast of Kyushu, as reported by Ozawa (1996). Ozawa ively. Dr. K. Tamura kindly provided the computer program (1996) described two diagnostic characteristics of B. Parsimony. Part of this study was supported by grants from

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