SUISANZOSHOKU 47(3), 363-368 (1999)

Morphological and Genetic Characteristics in Two Forms of Gibbose Conch, gibberulus gibbosus

Kð¯ji YOKOGAWA *1

(Accepted July 16, 1999)

Abstract: Morphological and genetic features in two forms (white form and purple form) of gibbose conch, gibbosus, were examined. Morphological characters of the two forms showed no significant differences except the shell coloration. Isozymes detected by horizontal starch-gel electrophoresis were used as genetic markers of 22 loci presumed. There were no significant differences between the two forms in the genetic variability and allelic frequencies at any of the loci examined. The genetic distance (D value) between the two forms, calculated from isozymic allele frequencies was 0.0019, being below the inter-subpopulational level. The morphological and genetic results suggested that the white form and the purple form compose the unique Mendelian population, they represented an intra-specific variation.

Key words: Gibberulus gibberulus gibbosus; Morphology; Genetics; Isozyme

Gibbose conch, Gibberulus gibberulus gibbosus Iriomote Island, Taketomi-cho, Okinawa Prefec-

(Roiling) (: Strombiidae), is a com- ture, Japan, on September 13, 1997 by snorkel- mon tropical shell in the Pacific tidal ing. The sampled specimens were immediately waters. It is utilized as food in some tropical transported to the author's laboratory by a cool region where it abundantly occurs1,2). In this delivery service, and they were preserved in a species, it is known that there are two distinct deep refrigerator at -80•Ž. The specimens com- forms; aperture with white coloration (hereafter prised 36 individuals of the white form and 8 in- called white form) and that with purple coloration dividuals of the purple form. Sizes (shell height)

(hereafter called purple form)1-4). In addition, the of the specimens were 32.8-54.6 mm (average purple form has a more contrasty dappled pat- 41.8 mm) in the white form and 34.7-46.2 mm tern on the shell surface than the white form1). (average 41.0 mm) in the purple form.

Since the two forms do not represent sexual Morphologically, 9 sections in the shell shown dimorphism2,4), probabilities that they are intras- in Fig. 1 were measured and each proportion pecific variation or distinct species can be consid- was expressed as a percentage of the shell ered. For this reason, the present study was height (SH, in mm). In addition, total body carried out to examine morphological and genetic weight (shell with soft part, BW, in g) was features in the two forms and assess their tax- weighed and weight index (WI) was calculated onomic status. by the following formula:

WI=BW/SH3•~105 Materials and Methods Immature individuals whose shells were in- Specimens examined in this study were col- complete at the outer lip part were omitted from lected around sandy bank located between the morphological analysis. Number of the omit- Fukapanari (Sotobanare) Island and Uchipanari ted individuals was both one in the two forms. (Uchibanare) Island, which are adjacent to Isozymes detected by horizontal starch-gel

*1 Kagawa Prefectural Fisheries Experimental Station , 75-5 Yashimahigashimachi, Takamatsu, Kagawa 761-0111, Japan. 364 K. Yokogawa

Fig. 1. Measured sections of shell. SH, Shell height; SD, Shell diameter; SW , Shell width; AL, Aperture length; BWL, length; SPH, height; BWD , Body whorl diameter; SWD, Second whorl diameter; TWD, Third whorl diameter. electrophoresis were used as genetic markers. For esterase from the muscle tissue in particular, Each about 0.5g of muscle and visceral mass the thin polyacrylamide-gel electrophoresis after tissue was removed from the specimens in partial Taniguchi and Tashima6) was introduced. For the frozen condition, and it was put into a 1.5 ml micro genetic analysis, all the collected individuals were tube. The sampled tissue was homogenized with examined.

100 ƒÊl and 50 ƒÊl of distilled water, respectively.

The samples were centrifuged at 15, 000 rpm for Results

10 minutes under 0•Ž condition, and the super- natant fluid was used for the electrophoresis. The Morphological Characters following experimental methods and expression of Results of the morphological examinations are loci and alleles were as described by Yokogawa5). shown in Table 1. All the proportions and the

Table 1. Morphometric characteristics of white form and purple form with results of t tests between both average values

*1 Risk percentage for t value . *2 Percentage of shell height . Letters in parenthese represent numbers of individuals examined in each form. Features in Two Forms of Gibbose Conch 365

weight index (WI) of the two forms showed very presumed (Table 2). close values to each other and the t tests be- Fitness of the allelic frequencies in polymor- tween the two forms did not give significance of phic loci, according to Hardy-Weinberg equilib- less than 5% at any of the characters (Table 1). rium, was examined by chi-square test in the This indicated that there were no morphological white form, purple form and both the forms differences between the two forms except for pooled (Table 3). Because no ƒÔ2 values were the shell coloration mentioned earlier. significant at the 5% level in the white and

purple forms, they were both regarded to be Genetic Characters originated from simple Mendelian populations. By electrophoresis, 14 enzymes and 1 non- Also, the pooled population did not show signifi- enzymic protein were detected and 22 loci were cance at any locus, unlike the case of the two

Table 2. Enzymes, protein and tissues examined

Table 3. Chi-square tests of fitness for Hardy-Weinberg equilibrium in groups of white form, purple form and both forms pooled

* 1 Number of individuals in which genotypes were revealed at each locus . * 2 Risk percentage to reject the equilibrium . 366 K. Yokogawa

forms of a pen shell, Atrina pectinata reported within a species. The probability can be by Yokogawa7), in which the two forms were supported by the facts that the two forms do not clarified to be the distinct species. represent sexual dimorphism2,4) and they are not Values to indicate the genetic features in the caused by environmental factors because the white form, purple form and both the forms two forms inhabit together anywhere1,2,4), furth- pooled, are shown in Table 4. These values er the two forms do not represent distinct spe- were fairly similar in the two forms and the cies. pooled population. The heterozygosity ratio Ueno4) mentioned that there were no morpho-

(Ho/He) indicated approximately 1 in both the logical differences except the aperture coloration forms (Table 4), suggesting no prominent excess and regarded the two forms as the same species of homozygotes or heterozygotes. although he showed no data on that. While Allelic frequencies at the 22 loci with ƒÔ2 Tsuchida and Shimura1) examined a regression heterogeneities between the two forms are between shell height (SH) and shell diameter shown in Table 5. In general, the allelic frequen- (SD) in the two forms of this species from cies were very similar between the two forms. Fiji by using many specimens, detecting a statis- The ƒÔ2 heterogeneities between the two forms tically significant difference between the two resulted in too small values to reach the signifi- forms at the inclination of the linear regression cant level of 5% at any of the loci examined formulae. Namely, the SD of the purple form is

(Table 5). relatively greater than that of the white form. In The genetic distance (D value)8) between the this study, however, the difference of the two forms, calculated from isozymic allele fre- SD/SH between the two forms was not signifi- quencies resulted in 0.0019, being a figure below cant (Table 1) the inter-populational level9,10) . Supposing the phenomenon detected by Tsuchida and Shimural) were universal, no sig- Discussion nificance recognized in this study could be caused by the small numbers of specimens, in The results given in this study showed that particular by that of the purple form. If so, the there were no morphological and genetic differ- proportional difference detected by them can ences except for the shell coloration between reflect the genetic variation as same as the shell the two forms of Gibberulus gibberulus gibbosus. coloration. This suggests that the two forms are not distinct Yokogawa11-14)examined total biological fea- species, they may reflect the genetic variation tures in two forms of Hirase's scallop, Volachla- mys hirasei, concluding that the two forms rep- Table 4. Genetic features in groups of white form, purple resent genetic dimorphism, and both the forms form and both forms pooled including much variation in the shell coloration and morphology. This suggests that there can be genetically coded morphological polymorphism also in the shell proportion. Thus in G. gibberu- lus gibbosus, the allele(s) for the morphological characteristic(s) can link with that(those) for the coloration. Tsuchida and Shimura1) purchased an amount of this species, which comprised 266 individuals P* : Polymorphism less than 0.95. (41.2%) of the white form and 380 individuals P : Polimorphism over 0.95. Ho : Observed heterozygosity. (58.8%) of the purple form at Suva Market, Fiji. He : Expected heterozygosity. Similarly, Cernohorsky (1965) reported that the Letters in parenthese represent numbers of individuals Fijian G. gibberulus gibbosus populations compris- examined in each group. ed almost equal numbers of the two forms. Features in Two Forms of Gibbose Conch 367

Table 5. Allelic frequencies of white and purple forms with ƒÔ2 heterogeneities between both frequencies

* 1 Degree of freedom . * 2 Risk percentage for chi -square value .

In this study, however, the specimens consist the shell coloration. The Japanese archipelago is of 36 individuals (81.8%) of the white form and 8 located at the northern margin of distribution of individuals (18.2%) of the purple form, consider- this species15), where genetically independent ably differing from the Fijian populations. This local populations of the tropical shell species may reflect the differences in genetic composi- occasionally can be established by the Black tion between the Fijian and the Japanese popula- Current * 2. This may be a reason why the Fijian tions, especially in the frequencies of alleles for and the Japanese populations genetically differ

* 2 Yokogawa , unpublished data. 368 K. Yokogawa from each other. 5) Yokogawa, K. (1997): Morphological and genetic dif- Masuda and Shinomiya16) examined genetic ferences between Japanese and Chinese red ark shell characteristics of a nautilus, Nautilus pompilius, Scapharca broughtonii. Fisheries Sci., 63(3), 332-337. 6) Taniguchi, N. and K. Tashima (1978): Genetic varia populations from Fiji and the Philippines, detect- tion of liver esterase in red sea bream. Bull. Japan. ing considerable differences between the two Soc. Sci. Fish., 44(6), 619-622 (in Japanese). populations. This information suggests that there 7) Yokogawa, K. (1996): Genetic divergence in two forms of pen shell Atrina pectinata. Venus, 55(1), 25- can be genetic isolation between distant popula- 39 (in Japanese). tions, and add weight to the probability that the 8) Nei, M. (1972): Genetic distance between populations. Fijian and the Japanese G. gibberulus gibbosus Amer. Nat., 106, 283-292. populations are genetically different. 9) Nei, M. (1990): Molecular evolutionary genetics (Trans -lated from English by T. Goj•¬bori and N. Sait•¬). Baifu

kan, Tokyo, vii+433 p. (in Japanese).

Acknowledgements 10) Japanese Society to Protect Aquatic Resources (Nihon-

Suisanshigen-Hogokyokai) eds. (1989): Population The author is grateful to Miss Ikuko Makino, analyses of fishes and shellfishes by isozyme. Nihon- Suisanshigen-Hogokyokai, Tokyo, 555 p. (in Japanese). Nihon Veterinary and Science University, 11) Yokogawa, K. (1997): Characteristics in two forms of who assisted in transporting the specimens used Hirase's scallop Volachlamys hirasei-1, shell morpholo- in this study. Also, I wish to thank Mr. Thomas gy. Venus, 56(4), 319-329 (in Japanese). Verghese, University of Malaya, Malaysia, for 12) Yokogawa, K. (1997): Characteristics in two forms of Hirase's scallop Volachlamys hirasei-2, soft part mor grammatically checking the manuscript. phology and its seasonal changes. Venus, 56(4), 331- 339 (in Japanese). References 13) Yokogawa, K. (1998): Characteristics in two forms of Hirase's scallop Volachlamys hirasei-3, age and growth. 1) Tsuchida, E. and S. Shimura (1996): Some commercial Venus, 57(1), 49-56. (in Japanese). mollusks collected at Suva market, Fiji (2). Chiribotan, 14) Yokogawa, K. (1998): Characteristics in two forms of 27(1), 16-20 (in Japanese). Hirase's scallop Volachlamys hirasei-4, genetic charac 2) Cernohorsky, W. O. (1965): The Strombiidae in Fiji. - ters. Venus, 57(1), 57-71 (in Japanese). Rec. Fiji Mus., 1(1), 1-18, 6 pls. 15) Arakawa, Y. (1986): Gibbose conch Gibberulus gibberu 3) Wang, R., Q. Zhang, X. Qu, Y. Cai, and Y. Zhang lus gibbosus (Rð±ding), in •gThe Great Animal Illustrated Encyclopedia, •h(ed. by T. Okutani), Sekai- (1988): Coloured illustrations of aquatic mollusks in China. Zhejiang Publ. House Sci. Tech., Hangzhou, iii bunkasha, Tokyo, pp. 94 (in Japanese). +v+x+255 p. (in Chinese). 16) Masuda, Y. and A. Shinomiya (1986): Genetic variabil 4) Ueno, S. (1994): Why does gibboue conch, which in ity in a nautilus Nautilus pompilius. Monthly Ocean Sci -habits sandy beach of Iriomote Island gather? Mar. ence, 18(10), 661-665 (in Japanese). Sci. Mus. Tokai Univ., 24(5), 2-3, 1 pl. (in Japanese).

ネ ジマ ガキ ガ イ2型 の形 態 的お よび遺伝 的特 徴

横川浩治

ネ ジマ ガキ ガイ2型(殻 口の 白色型 と紫 色型)の 形態 的 お よび遺伝 的差異 につい て調べ た。殻 の外 部形 態 につ いて,型 の標 徴 で ある殻 の色彩 を除 き,調 べ たす べ ての形 質 にお いて両型 の平 均値 には全 く有 意差 は認 め られ なか った。遺 伝形 質 と して アイ ソザ イム を調 べ22遺 伝 子座 を推定 したが,両 型 の 遺伝 的諸 特性 値 は非常 に類似 した値 を示 し,さ らに遺伝 子頻 度 につ いて も両型 問 で有 意差 が認 め られ た遺 伝 子座 は全 くなか った。 アイ ソザ イム系遺 伝子 に よる両集 団 問の遺 伝 的距離(D値)は0.0019と な り,地 域集 団 問以下 の水 準 であ った。 以上 の形態 的 お よび遺伝 的解析 結 果 か ら,ネ ジマ ガキ ガ イの 白色 型 と紫色 型 は同一 種 内の遺伝 的多型現 象 で ある可能性 が強 く示唆 された。