Jpn. J. Limnol., 54, 2, 109-116, 1993

Two Types of Semisulcospira reiniana (Brot) (Mesogastropoda: Pleuroceridae) Identified by Electrophoresis

Misako URABE

Abstract

The genetic and morphological variations of a freshwater snail, Semisulcospira reiniana were studied in the Lake Biwa water system and its adjacent two localities . The two types distinguished genetically were distributed sympatrically at Hamaotsu , Uji, and Mino-tsuya. MPI-A type snails had relatively small, and smooth or ribbed embryonic shells. MPI-B type snails had large and ribbed embryonic shells. How- ever, these two types in the same locality could not be distinguished by adult shell morphology. Thus, the convergence of shell morphology with growth was suggested. These results clarified the confusion between these two types in past taxonomic studies of S. reiniana.

Key words: Semisulcospira reiniana, genetic variation, shell morphology, convergence

S. reiniana. 1. Introduction

A common Japanese freshwater snail, Semis- 2. Materials and methods ulcospira reiniana (BROT), was first described Snails were collected at the following local- based solely on adult shell characteristics of ities in 1990 and 1991 (Fig. 1): (1) Hamaotsu, specimens collected at Yokohama (BROT, 1876). Otsu city, on 3 October 1991 (Hamaotsu); (2) However, the exact locality is uncertain today, Shiga Prefectural Fisheries Experimental Sta- and no taxonomical study has been done on the tion, Hikone city, on 5 October 1991 (Hikone); Yokohama specimens. Instead, specimens dis- (3) To-ga-shima, Uji city, on 11 September tributed in the Lake Biwa water system and 1990, and 24 July 1991 (Uji); (4) Takahashi, identified as S. reiniana have been used as Kyoto city, on several days in August 1990, May typifying the species in taxonomic studies and August 1991 (Takahashi); (5) Mino-tsuya, (BOETTGER, 1886; BURCH, 1968; DAVIS, 1969; Gifu prefecture, on 4 June 1991 (Mino-tsuya) : WATANABE, et al., 1989). However, different Mino-tsuya is a spring-fed stream in which karyotypes were reported by BURCH (1968) and water temperature is stable all year round. WATANABE, et al. (1989) and which suggests All snails analyzed in this study had at least that the taxonomic status of this species in the 6 basal cords, and most of them had clear ribs Lake Biwa water system is still unclear. on their shells. Both these features have been In this paper, I report on the genetic varia- regarded as diagnostic characteristics of S. tion, adult and embryonic shell morphologies of reiniana (DAVis, 1969; HABE, 1973). Some indi- S. reiniana to identify characteristics of the viduals at Takahashi and Mino-tsuya were species inhabiting the Lake Biwa water system. without ribs. See URABE (1992) for the reason Further, I disclose new information concerning behind species determination of the former; the

* Contribution from the Laboratory of Ecology, Department of Zoology , Kyoto University, No. 540 110 URABE

Fig. 1. Map of sampling localities. latter anomaly is due to heavy erosion by spring soft body was expelled by crushing the shell water. Thus, all the specimens were identified with pincers. The shell was then weighed with as S. reiniana. a direct reading analytical balance (shell To identify a snail genotype, horizontal weight: SWGT). Then regression equations starch gel electrophoresis was performed. The were calculated between SW and PWL, AL and foot muscle of each snail was kept at -80°C AW, SW and SWGT (both log scales) for the and was homogenized by the same method as purpose of estimating proportion, aperture URABE (1992). Buffer systems and stain shape and relative weight, respectively. In the methods were as follows: case of mature females, the number of embry- MPI (mannosephosphate isomerase) onic shells was counted. The females were buffer system: Tris-citrate, pH 7.0 (SHAW categorized into three types based on the pres- and PRASAD,1970) ence or absence of ribs on embryonic shells: all stain: HARRISand HOPKINSON(1976) smooth embryonic shells (S); mixture (MX); PGMI (phosphoglucomutase 1) and all ribbed(R). The measurements of the buffer system: Amine-citrate, pH 6.1 largest embryonic shells in a brood pouch were (CLAYTONand TRETIAK,1972) conducted under a binocular microscope as stain: HARRISand HOPKINSON(1976) shown in Fig. 2, at which time whorl stages The name of each genotype is given according were also recorded. Individuals infected by to ONIWAand KIMURA(1986a, b). trematodes were excluded from the analysis of For each snail, shell width (SW), penultimate embryonic shells since their reproductive whorl length (PWL), aperture length (AL), and organs were damaged by the infection. aperture width (AW) were measured with a caliper (Fig. 2). After the measurement, the Two Types of Semisulcospira reiniana 111

Fig. 2. Measurements of adult and embryonic shells.

At Hamaotsu, the PGMI alleles of MPI-A type 3. Results were polymorphic, but that of MPI-B type 3-1. Electrophoresis showed no genetic variation. At Uji, the PGMI The allele frequency in each sampling local- alleles completely different between MPI-A ity is shown in Table 1. Two alleles were and MPI-B type. At Mino-tsuya, PGMI was identified in MPI, and no heterozygote was polymorphic in MPI-A type while that of MPI- found. Four alleles were identified in PGMI. B type was fixed on one allele, though this may Two MPI-types were found in the samples be attributable to the small sample size. Only collected at Hamaotsu, Uji, and Mino-tsuya. one MPI allele was observed at Hikone and

Table 1. Allele frequencies of Semisulcospira reiniana at each locality. Specimens from Hamaotsu, Uji, and Mino-tsuya divided into two types according to MPI analysis. 112 URABE

Takahashi. characteristic of adult shells was found by 3-2. Shell morphology which to distinguish two MPI types from the 3-2-1. Adult shell same sampling localities. First, I tested differences in the regression The statistical differences in regression lines lines to compare shell morphology between the between localities are shown in Table 3 (for two MPI-types at the same sampling localities MPI-B type) and Table 4 (for MPI-A type). (Fig. 3 and Table 2). There were no signifi- Although there is no evidence to determine cant differences between MPI-A type and MPI- whether or not the same MPI-type populations B type at either Hamaotsu or Mino-tsuya. At from different localities belong to the same Uji, the slopes of the lines for the SW-PWL biological species, statistical tests were provi- relation were significantly different (P<0.05). sionally conducted within the same MPI-type However, this may be attributable to a small populations. In MPI-B type, the variations of sample size and the narrow range in shell size the lines for SW-PWL relation were small even of MPI-A type (Fig. 3). No morphological when a significant difference was detected. As

Fig. 3. Relationships between shell width (SW) and penultimate whorl length (PWL) (A), aperture length (AL) and aperture width (AW) (B), and shell width (SW) and shell weight (SWGT) (C) at three localities. Open circle: MPI-A type. Solid circle: MPI-B type. Two Types of Semisulcospira reiniana 113

Table 2. Comparisons of regressions of adult shells between two MPI-types at same localities.

*p<0 .05

Table 3. Comparisons of regressions of MPI-B adult shells between localities. •y SW-PWL•z

•y AL-AW•z

•y SW-SWGT•z (log scale)

*p<0 .05 **p<0.01 ***p<0.001 114 URABE

Table 4. Comparisons of regressions of MPI-A adult shells

between localities.

•y SW-PWL•z

•y AL-AW•z

•y SW-SWGT•z(log scale)

*p<0 .05 **p<0.01 ***p<0.001 for AL-AW relations, 5 regression lines could could get no embryo of MPI-A type at Uji. be divided into "Hamaotsu+Hikone" group Almost all the embryonic shells of Hikone, Uji and "Uji+Mino-tsuya+Takahashi" group. MPI-B type, and Takahashi showed pro- As for SW-SWGT relations, a notable signifi- nounced ribs (a few females had MX-type cant difference was detected between speci- embryonic shells in Takahashi) and 0-2 keels. mens at Uji and those at other localities. In There existed common characteristics such MPI-A type, some pairs of the localities as shell size, the presence of ribs and the num- showed significant difference in regression ber of keels through the same MPI-type. The lines. The results did not correspond to those mean shell length of MPI-A type ranged from for MPI-B type. These results show that the 1.35 to 1.47mm, and that of MPI-B type from morphological difference between two MPI- 1.79 to 2.28mm. The embryonic shell sculp- types in the same locality is less than that for ture of the MPI-A type was mixed, while that the same MPI-type between different localities. of MPI-B type was mainly ribbed. 3-2-2. Embryonic shell 4. Discussion The embryonic shell characteristics are shown in Table 5. In Hamaotsu, mean SL, This study shows that at least two genetically mean whorl stage, rib categories and the num- isolated types are distributed sympatrically at ber of keels were different between two MPI- Hamaotsu, Uji, and Mino-tsuya. Although all types. In Mino-tsuya, mean SL-and rib cate- specimens were, in adult shell morphology, typi- gories were different between two MPI-types, cal S. reiniana, two MPI alleles without heter- and the number of keels varied in both types. I ozygote were found as was the case with S. Two Types of Semisulcospira reiniana 115

Table 5. Embryonic shell characteristics of Semisulcospira reiniana by sampling locality. Figures in parentheses indicate range of value.

reiniana and S. libertina described by ONIWA converged with growth. and KIMURA (1986a, b). Thus, S. reiniana in Some species or types of Semisulcospira, the Lake Biwa water system and Ibi river sys- which had ribbed adult shells like S. reiniana tem includes two genetically identified popula- and small (and usually unribbed) embryonic tions. shells, have been reported from lakes (PILSBRY, Though the taxonomic status of these types is 1902; DAVIS, 1968; MATSUOKA,et al., 1982) or currently unknown, it is probable that the dif- high altitude locals (FuJITA, 1990). In this ferent MPI types in the same water system study, snails with relatively small embryonic belong to different species. It is also unclear shells (MPI-A type) were found in the Lake whether the same MPI type individuals in dif- Biwa water system, and in Mino-tsuya whose ferent water systems belong to the same water temperature is stable and low due to a species. Further study of local variations or spring-fed stream. These facts prompt consid- genetic distance is needed to clarify the tax- eration of which environmental factors are onomic positions of these types. related to the formation of ribs on adult shells The presence of two types of S. reiniana regardless of embryonic shell types. inhabiting the Lake Biwa water system has never been reported. On the other hand, Acknowledgements Burch's description of the embryonic shell of S. I am very thankful to Prof. Takayoshi reiniana (the specimens at Uji) shows the same SHOTAKE and Dr. Yoshi KAWAMOTO,Primate characteristics as MPI-A type, while that of Research Institute, Kyoto University, for their WATANABE et al. (1989)'s shows MPI-B type help and guidance in the electrophoresis (the specimens at Kita-yamada, Lake Biwa). method. I also wish to thank Prof. Hiroya Therefore, it is clear that these two types have KAWANABEand the members of the laboratory of been confused in past taxonomic studies of S. Animal Ecology, Department of Zoology, Kyoto reiniana. University, for their advice in correcting the A morphological difference between the two manuscript. This research was partially MPI-types at the same locality was recognized supported by Lake Biwa Research Project 91- not in adult shells, but in embryonic shells from 08 from Shiga Prefecture, and the Grant-in-Aid Hamaotsu and Mino-tsuya where specimens of for Scientific Research (B) (No. 02454005), for both types were obtained. These results sug- Scientific Research on Priority Areas (No. gest that the shell morphology of S. reiniana 03269105), and for Co-operative Research (A) 116 URABE

(No. 02304002) from the Ministry of Education, late Tamiji Kawamura Freshwater Biology in Science and Culture, Japan. Japan 307-341. Hokuryukan (in Japanese). HARRIS,H., and D. A. HOPKINSON(1976): Handbook of enzyme electrophoresis in human genetics. 摘 要 Elsevier, Amsterdam. MATSUOKA,K., and Fossil Mollusc Research Group 電 気 泳 動 法 に よ っ て 区分 さ れ た チ リメ ン カ ワ ニ ナ for Nojiri-ko Excavation (1982): On the fossil Semisulcospira reiniana(Brot)の2型 embryonic shell of 琵 琶 湖 水 系 及 び そ の 周 辺 の2地 点 よ り得 られ た (Gould) (Mesogastropoda: Pleuroceridae) チ リ メ ンカ ワニ ナSemisulcospira reinianaを,遺 from the latest Pleistocene Nojiri-ko Forma- 伝 的 変 異 と成 殻 ・胎 殻 形 態 の 面 か ら調 べ た 。 浜 大 tion, Nagano Prefecture, Central Japan; A comparative study of recent and fossil Semisul- 津 ・宇 治 ・美 濃 津 屋 の3ケ 所 で は,遺 伝 的 に 区 別 cospira. Chikyu-kagaku, 36: 175-184 (in され る2型 が 生 息 して いた 。MPI-A型 は,比 較 的 Japanese, with English abstract). 小 さ く平 滑 ま た は 縦 助 の あ る胎 貝 を持 っ て い た 。 ONIWA,K., and M. KIMURA(1986a): Genetic varia- MPI-B型 は大 き く縦 肋 の あ る胎 貝 を持 っ て い た 。 bility in two snail species Semisulcospira liber- し か し,同 じ地 点 か ら得 られ た これ ら2型 は,成 tina and Semisulcospira reiniana. Jpn. J. Genet., 殻 形 態 で は 区 別 で きな か っ た 。 これ らの 結 果 か ら, 61: 137-146. 過 去 の分 類 学 的 研 究 に お い て は2型 が 混 同 され て ONIWA,K., and M. KIMURA(1986b): Genetic varia- きた こ と を指 摘 し,さ ら に成 殻 の 収 斂 現 象 に つ い bility in six snail species of the genus Semisul- て 示 唆 し た 。 cospira. Jpn. J. Genet., 62: 503-514. PILSBRY, H. A. (1902): Revision of Japanese References Viviparidae, with Notes on Melania and Bith- ynia. Proc. Acad. Natur. Sci. Philadelphia. 54: BOETTEBER, O.(1886): Zur Kenntniss der Melanian 115-121, pl. 9. Chinas and Japans. Jahrb. Deut. Malakozool. SHAW, C. R., and R. PRASAD (1970): Starch gel Ges., 13: 1-16. electrophoresis of enzymes-A compilation of BROT, A. (1876): Die Melaniaceen (Melanidae). recipes. Biological Genetics, 4: 297-320. Abbild. Natur. Syst. Conchy. Cab. Martini & URABE, M. (1992): A discrimination and mor- Chemnitz. Nurnberg., p. 337, pl. 34. phological comparison of two snail species of BURCH, J. B. (1968): Cytotaxonomy of some the genus Semisulcospira in a single river. Japanese Semisulcospira (Streptoneura: Pleur- Venus, 50: 270-286 (in Japanese, with English oceridae). J. Conchyliol., 107: 3-51. abstract). CLAYTON,J. W., and D. N. TRETIAK(1972): Amine- WATANABE,C. N., K. H. NAKAMURA,and M. NISHINO citrate buffers for pH control in starch gel (1989): Syuyou teisei doubutsu no Bunruiga- electrophoresis. J. Fish. Res. Bd. Canada, 29: kuteki saikentou ni kansuru kenkyu. (Tax- 1169-1172. onomical reinvestigation of the freshwater DAVIS, G. M. (1968): Biosystematic analysis of snails, genus Semisulcospira in Lake Biwa.) Semisulcospira trachea (: Pleurocer- Report of the project research: Landscape ecol- idae). Proc. Sym. . 1: 16-35. Marine ogy on the shore of Lake Biwa: Lake Biwa Biological Association of India, Bangalore Research Institute (in Japanese, unpublished). Press. DAVIS,G. M. (1969): A taxonomic study of some (著者:浦 部 美 佐 子,京 都 大 学 理 学 部 動 物 学 教 室, species of Semisulcospira in Japan (Mesogas- 〒606-01京 都 市 左 京 区 北 白 川 追 分 町;Misako tropoda: Pleuroceridae). Malacologia, 7: 211- URABE, Department of Zoology, Faculty of Science, 294. Kyoto University. Oiwakecho, Kitashirakawa, Sa- FUJITA, T. (1990): Some species of Semisulcospira kyoku, Kyoto 606-01) from the Matsumoto Basin and neighboring mountains, Central Japan. Journal of the Japan Received: 12 June 1992 Volklore Museum and Matsumoto City Accepted: 27 November 1992 Museum, 1: 33-40 (in Japanese). HABE, T. (1973): Mollusca. In M. Ueno (ed.), The