Genetic Variability in Two Snail Species Semisulcospira Libertina and Semisulcospira Reiniana

Jpn. J. Genet. (1986) 62, pp. 137-146

Genetic variability in two snail species Semisulcospira libertina and Semisulcospira reiniana

BY Kiyoko ONIWA and Masao KIMURA* Gifu Senior High School, Oonawaba, Gifu 500 and *Faculty of Agriculture , Gifu University, Gifu 501-11

(Received April 18, 1986)

ABSTRACT To evaluate genetic variability within and between populations of two closely related snail species Semisulcospira libertine and Semisulcospira reiniana, enzymes coded for by 18 presumed loci were examined by starch gel electrophoresis. The proportion of polymorphic loci and the expected average heterozygosity per individual for the S. libertine populations were 0.172 and 0.057, respectively. These for the S. reiniana populations were 0.114 and 0.039, respectively. Nei's genetic distance value estimated between the two species was 0.1235. Marked difference in allele frequency between the two species was observed at the MPI and 6PGD loci. Other five loci (ICDH-1, AAT, MDH-1, PHI and Est) were variable in some of the libertine populations, although the five loci were fixed for the wild type allele in all of the reiniana populations. Another locus PGM-1 was variable in both species. These loci seemed to be useful genetical markers for differentiating S, libertina from S. reiniana.

1. INTRODUCTION

Semisulcospira libertina is a black snail and lives in clean rivers through- out Japan. This snail is known as a surveyor of environmental pollutions, because the animal serves as a food for larvae of the firefly Luciola cruciata and as the intermediate host of edema of the lung fluke Paragonimus westermani. Thus the snail is a useful and interesting animal, but no genetical information has been made available. The purpose of this note is to present some genetical data on S. libertina and to compare them with those of another widely distributed snail species Semisulcospira reiniana.

2. MATERIALS AND METHODS Snails were collected from 5 major water systems shown in Fig. 1, (Fig, l and Table 2). Foot muscle was crudely homogenized in deionized water and was centrifuged at 10,000 g for 30 minutes at 0°C. The supernatant was applied to starch gel electrophoresis. Enzymes examined and electrophoretic conditions were listed in Table 1. Enzymes were stained by the methods of 138 K. ONIWA and M. KIMURA

Fig. 1. Map of the central part of Honshu showing collection locations of the samples of Semisulcospira libertina (open circle) and S. reiniana (shaded circle). Samples were collected at 66 sites; Ibigawa River system-Ikeda (ID), Ikeda-II (ID-II), Itonuki (IT), Kami-Ishizu (KI), Kasuga (KS), Motosu (MS), Motosu-Il (MS-II), Motosu-III (MS-III), Nanno (NO), Ogaki (OG), Otani (OT), Otani-II (OT-II), Tado (TD). Kisogawa River system-Akagawa (AK), Akagawa-II (AK-II), Inuyama (IN), Kakami- gahara (KK), Kamiasou (KM), Kani (KN), Kurokawa (KR), Kawaura (KU), Kana- yama (KY), Kanayama-II (KY-II), Mitake (MT), Ogase (OS), Sugibora (SG), Shira- kawa (SK), Wara (WR). Nagaragawa River system-Gifu (GF), Gifu-II (GF-II), Gifu-III (GF-III), Gujo- Hachiman (GH), Ibora (TB), Izira (IJ), Itaya (IY), Kaizu (KZ), Mugegawa (MG), Minami (MN), Miyama (MY), Shiratori (SR), Takatomi (TK), Takatomi-II (TK-II), Tomika (TM), Terao (TO). Machiyagawa River system-Daian (DA), Fujiwara (FJ), Hokusei (HS). Biwako Lake system-Asai (AS), Hikone (HK), Hikone-II (HK-II), Hikone-III (HK-III), Hino (HN), Hannoura (HU), Inae (IE), Ibuki (1K), Kohoku (KH), Katata (KT), Maibara (MB), Makino (MK), Nagahara (NH), Notogawa (NT), Notogawa-II (NT- II), 0 mi (OM), Samegai (SM), Santo (ST), Yoga (YG). S. libertina coexists with S. reiniana at the two sites Itaya and Motosu-III.

Brewer (1970). The genetic variability within population was quantified by measuring the proportion of polymorphic loci (Pp01) and the expected heterozygosity per individual (H). Nei's GST (Nei 1975), a measure of genetic differentiation, was calculated. For the calculation, each population and populations collected from several sampling sites within each water system were tenta- Genetic variability in two snail species 139

Table 1. Enzymes examined and electrophoretic conditions

tively regarded as a subpopulation and as a total population, respectively. To evaluate genetic difference between populations, the genetic distance D of Nei (1975) was also estimated.

3. RESULTS AND DISCUSSION

Electrophoretic pattern and individual variation in 1 8 enzymes AAT, ICDH-1, MDH-1, 6PGD and PHI-Most snails except few had one band. The exceptional few snails had three bands. Est and PGM-1-One-banded and two-banded patterns were observed. In- dividuals showing three-banded or two-banded pattern in these 7 enzymes were assumed to be heterozygote. MPI-All snails of the two species had a pair of bands and inter-species variation in mobility of the enzyme bands was observed. The slower band of S, libertina coincided with that of the faster band of S. reiniana. Other 10 enzymes-No electrophoretic variation was detected either at intra-species level or at inter-species level.

Allele frequencies at 8 variable loci Assuming that each of the 8 enzymes is controlled by a separate autosomal locus with codominant alleles, allele frequency at each locus was calculated and shown in Table 2. Pronounced difference in allele frequency between the two snail species was observed in two enzyme loci MPI and 6PGD (Table 2). Populations of 140 K. ONIWA and M. KIMURA

O O d N

C) O N

41 41 N N

G\l

Cd H Genetic variability in two snail species 141

O U

H 142 K. ONIWA and M. KIMURA

U Genetic variability in two snail species 143

S. libertina and S. reiniana appear to be fixed for the alleles MPIA and MPIB, respectively, although one snail typed MPI B was detected in the libertina population Samegai and one individual typed MPI A in each of the two reiniana populations Takatomi and Tomika. These three exceptional snails may be invaders from the neighbouring snail population of different species. An- other interesting fact was an abscence of the allele 6PGDA from the libertina population and of the allele 6PGDD from the reiniana population. Appear- ance of the two exceptional snails, one with the genotype A/B in the libertina population Nanno and another with the genotype A/B in the libertina population Omi, may be explained by supposing a possible invasion by snails from the neighbouring reiniana populations. So far no heterozygote snail for the 6PGD alleles A and D has been collected. Other 5 loci were variable in some of the libertina populations. The ICDH-1 locus was fixed for the allele A in all but two libertina populations. Rare allele D was detected in the Kami-Ishizu (n=132, qD= 0.015) and Kasuga (n=32, qD=0.016) populations. The AAT locus was fixed for the allele A in all except 4 libertina populations. Exceptional populations in which allele B was segregating were the Makino (n =129, qB= 0.016), Hannoura (n = 34, qB= 0.074), Ibuki (n=24, qB= 0.292) and Asai (n=48, qB= 0.042) where n is the sample size and qB is the frequency of the allele B. As to the MDH-1 locus, the wild type allele was B in both species. A mutant allele C was observed in the following five libertina populations; Kami-Ishizu (n=132, qC=0.008), Kurokawa (n=7, qC= 0.143), Akagawa (n=29, qC= 0.172), Shirakawa (n=60, qC= 0.508) and W ara (n=11, qC= 0.091) . No variant allele was found at the PHI and Est loci in the reiniana populations. In some libertina populations, frequency of the mutant allele Estc was higher than that of the wild allele EstB (Table 2).

Variability within and between species Genetic variability within each snail population was estimated by calculat- ing the proportion of polymorphic loci (Pp01) and the average heterozygosity per individual (H). These two estimates were averaged within each water system and shown in Table 3. Mean Ppoly± S.D. estimated for the libertina and reiniana species were 0.172 ± 0.059 and 0.114 ± 0.036, respectively. Mean H ± S.D, for these two species were 0.057±0.023 and 0.039±0.008, respectively. Thus the genetic variability within each libertina population seemed to be somewhat higher than that within each reiniana population. Selander and Kauf man (1973) found that, in small immobile animals (inver- tebrates), the level of genetic heterozygosity was higher than that in large mobile animals (most vertebrates). Nevo (1978) reported that Pp01 and H for the 27 species of invertebrata (except insecta) were 0.399 ± 0.275 (mean ± S.D.) and 0.1001±0.074, respectively. Furthermore Fujio et al. (1985) sur- 144 K. ONIwA and M. KIMURA

O O.N

`IJ

H Genetic variability in two snail species 145 veyed genetic variation in 25 species of marine molluscs, and estimated Pp01 and H to be 0.412±0.30 (mean ± S.E.) and 0.147±0.11, respectively. The level of genetic variability estimated for the two snail species appears to be somewhat lower than the level expected from these references. To evaluate the relative magnitude of genetic differentiation among populations within each snail species, Nei's GST (Nei 1975) was estimated. GST5 calculated for several populations in each species were presented in Table 3. The level of GST within S. libertina species was about 6 times higher than that in S. reiniana. The mean GST value of 0.174 for S. libertina was somewhat higher than the mean FST value of 0.116 for the European land snail Helix aspersa reported by Selander and Kaufman (1975). Nei's (1975) genetic distances among the 68 populations of S. libertina and S. reiniana were estimated. Several comparisons can be made. Of which the average genetic distance in the within-species comparisons was 0.0396 ± 0.036 (mean ± S.D.) for the S, libertina and 0.0021 ± 0.0026 for the S. reiniana. In comparisons between populations drawn from different species, the mean genetic distance was 0.1235 ± 0.0287. The average genetic distance between the two species was apparently larger than the distance within species. S. libertina was shown to be genetically more variable than S, reiniana. The reason for this fact is not known. S. libertina lives in clean mountainous streams and is sensitive to water pollution, whereas S. reiniana inhabits in rivers and paddy fields and can live in muddy waters. The difference in genetic variability may reflect difference in their tolerances to environmental changes. Davis (1969) published papers to establish basic taxonomic concepts for 10 species of the fresh water snail genus Semisulcospira and discussed several morphological and cytological characters of basic importance in defining the species. He relegated the taxa to 2 species complexes, the S. libertina and the S, niponica groups. S. libertina and S. reiniana are the main species in the former complex and sometimes may be confused with each other, as the populations of S. libertina which have nodulate ribs appear very much like S. reiniana. Precise difference between the taxa is that the former has a chromosome number of n =18 while the latter has n = 20. Results of the present study shows that electrophoresis of mannose phosphate isomerase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, aspartate amino- transf erase, phosphoglucomutase and esterase presents a very useful tool for separating closely related these two snail species.

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