Indian J. Fish., 51(4) : 389-399, Oct.-Dec., 2004 389

Genetic relationship between the genus Helice and three grapsid crab species

MD. YOUNUS MIA, REIKO FUSEYA* AND SEIICHI WATANABE* Bangladesh Fisheries Research Institute, Brackishwater Station, Paikgacha, Khulna-9280, Bangladesh *Department of Aquatic Biosciences, Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan

ABSTRACT Horizontal starch gel electrophoresis was used to analyse the genetic variation and relationship between the mud-flat crab of genus Helice and three other grapsid crabs, Chiromantes dehaani, Eriocheir japonica and Hemigrapsus penicillatus. Twenty five allozyme loci of 15 enzymes were investigated. The reaction of phosphoglucomutase (PGM) was found in those crabs except for E. japonica. Gene replacement was found at six loci, ALP*, FH-1*, G3PDH-1*, HK-1*, MPI-1* and MPI-2*. The average proportion of polymorphic loci and heterozygosity were 0.058 and 0.005 in Helice species, 0.091 and 0.023 in C. dehaani, 0.050 and 0.009 in E. japonica and 0.040 and 0.002 in H. penicillatus. Genetic analysis showed that Nei‘s genetic distance was 0.14196 between the genus Helice and H. penicillatus and 1.23512 between C. dehaani and E. japonica.

Introduction Rathbun 1929 are the known species under this genus (Sakai, 1976). The crabs of the genus Helice are the members of the family Grapsidae Electrophoretically detectable (Decapoda). There are many species allozyme genes are useful for analyzing under this family. Within this family genetic divergence among subspecies most of the species inhabit coastal believed to be closely related on the basis waters, though some species like of morphological traits, species and Eriocheir japonica, Chiromantes genera of a variety of organisms. In crab dehaani, C. haematocheir are found in species Takano et al. (1997) reported the estuary to upstream (Sakai, 1976). The genetic divergence between two estuarine crab of genus Helice consists sympatric forms (I and II) of the of four species. Helice tridens latimera estuarine grapsid crab, Hemigrapsus Parisi 1918, H. t. tridens De Haan 1835, penicillatus using isozyme gene markers. H. leachi Hess 1865, and H. t. wuana Fuseya and Watanabe (1996) suggested MD. Younus Mia et al. 390 that the genus Scylla includes at least taken from the legs and stored at –800C. three species based on isozyme analysis The tissues to be analysed for allozymes and Gao and Watanabe (1998) evaluated were homogenized with approximately the level of genetic divergence between equal volumes of the fragments of frozen the Japanese mitten crab, Eriocheir muscle and distilled water. Then, the japonica and the chinese mitten crab, E. homogenates were centrifuged at 12000 sinensis. rpm for 12 minutes at 40C and the supernatant was absorbed onto filter The aim of this study was to paper and used for electrophoresis. estimate the degree of genetic divergence Horizontal starch gel electrophoretic among the former three species of Helice techniques, buffer systems and staining compared to Chiromantes dehaani, procedure followed the method of the Eriocheir japonica and Hemigrapsus Japan Fisheries Resource Conservation penicillatus as out-group species by Association (1989) and Pasteur et al. genetic distance, number of polymorphic (1988). A total of 15 enzymes were loci and number of gene replacements. surveyed; the name, numbers and Materials and methods abbreviations of the enzymes followed Shaklee et al. (1990) (Table 2). Horizontal starch gel electrophoresis was used in order to estimate the degree The allele frequencies and observed heterozygosities (H ) for each locus were of genetic differentiation. A total of 59 o H. t. latimera, 230 H. t. tridens, 176 H. determined by direct census of the leachi, 25 C. dehaani, 43 H. penicillatus population data. Mean heterozygosity (H ) within population was estimated and 16 E. japonicus were collected (Table e 1). All samples were kept at –200C prior from average values across all samples to electrophoresis analysis. Muscle was (Nei, 1987). A locus was considered to be

TABLE 1: Samples used for electrophoretic analysis in the genus Helice and three grapsid crabs Location Species Number Date Okinawa Helice tridens latimera 57 Mar-97 * H. leachi 40 Mar-97 * Ishigaki H. tridens latimera 2 Nov-98 * H. leachi 26 Nov-98 * Chiba H. t. tridens 76 Jun-97 * Wakayama H. t. tridens 19 Aug-99 Ehime H. t. tridens 33 Nov-98 * Fukuoka H. t. tridens 52 Nov-98 * H. leachi 57 Nov-98 * Kagoshima H. t. tridens 50 Aug-97 * Ogasawara H. leachi 53 Oct-98 * Miyagi Chiromantes dehaani 25 Oct-98 Tokyo Hemigrapsus penicillatus 43 Sep-99 Ogasawara Eriocheir japonica 16 Oct-98 *Samples used in the study of Mia et al. (1999) Genetic relationship between grapsid crabs 391

TABLE 2: Names, numbers and abbreviations of enzymes and buffers used for electrophoresis

Enzyme name (abbreviation) Enzyme number Buffer Aspartate aminotransferase (AAT) 2.6.1.1 CAPM-7 Alkaline phosphatase (ALP) 3.1.3.1 CAPM-7 Esterase (EST) 3.1.1 CAPM-7 Fumarate hydratase (FH) 4.2.1.2 CT-8N Glycerol-3-phosphate dehydrogenase (G3PDH) 1.1.1.8 CAPM-7 Glucose-6-phosphate isomerase (GPI) 5.3.1.9 CT-8N Hexokinase (HK) 2.7.1.1 CT-8N Isocitrate dehydrogenase (IDHP) 1.1.1.42 CT-8 Leucine aminopeptidase (LAP) 3.4.11 CT-7 Lactate dehydrogenase (LDH) 1.1.1.27 CT-8 Malate dehydrogenase (MDH) 1.1.1.37 CAPM-7 Mannose-6-phosphate isomerase (MPI) 5.3.1.8 CT-7 Phosphogluconate dehydrogenase (PGDH) 1.1.1.44 CAPM-7 Phosphoglucomutase (PGM) 2.7.5.1 CAPM-7 Superoxide dismutase (SOD) 1.15.1.1 CT-8N polymorphic when the frequency of the Electrophoretic patterns and most common allele was less than or banding positions of the six species were equal to 0.99 at one or more localities compared on the same gel in order to (Nei, 1987). In order to estimate the analyse the allelic differences among degree of genetic divergence among the them. Electrophoresis revealed 25 loci samples, the genetic distance between encoding the 15 investigated enzymes in samples was calculated using Nei‘s Helice species and Hemigrapsus formulae (Nei, 1972), and dendrogram penicillatus. Twenty-two loci coded by 15 from the matrix of genetic distances were enzymes and twenty loci coded by 14 constructed using the neighbor-joining enzymes were investigated in method (Saitou and Nei, 1987). Chiromantes dehaani and Eriocheir japonica, respectively (Table 3). Bands Results for the enzyme of phosphoglucomutase Isozymic bands were compared on (PGM) was not appeared in E. japonica. the same gel in order to identify common Eight loci, AAT-1*, AAT-2*, GPI*, IDHP*, or different alleles between the genus MDH-1*, MDH-2*, PGDH* and LDH* in Helice and three other grapsid crab Helice species, two loci GPI* and IDHP* species. Bands that appeared at the same in C. dehaani, one locus AAT-2* in E. position on the same gel were assumed japonica and also one locus GPI* in H. to be controlled by the same allele, while penicillatus exhibited polymorphism those that appeared at different positions (Fig. 1). The genotypes of each species were controlled by different alleles. The are shown in Table 3. allele frequencies estimated for the 25 Five enzymes, namely alkaline loci in Helice species and Hemigrapsus phosphatase (ALP), glucose-6-phosphate penicillatus, 22 loci in Chiromantes isomerase (GPI), isocitrate dehaani and 20 loci in Eriocheir japonica dehydrogenase (IDHP), lactate are summarized in Table 3. MD. Younus Mia et al. 392 H. penicillatus Continued... E. japonica C. dehaani and three other grapsid crab species. Ishigaki Ogasawara Miyagi Ogasawara Tokyo Helice Fukuoka Okinawa Kagoshima Fukuoka ayama Ehime Ishigaki Chiba Wak H. t. latimera H. t. tridens H. leachi Okinawa *f 0 0 0 0 0 0 0 0.009 0.025 0 0 0.36 0 0.977 *c 0 0 0 0.105 0*c 0 0.14 0 0 0.01 0*c 0.026 0 0.03 0 0.01 0 0 0.02 0.009 0.01 1 0 0 0 0.063 0 1 0 0.019 0 0.01 0 1 0.988 0 1 0 1 0.98 0 1 *e 0 0 0 0 0 0 0 0.982 0.975 1 1 0.64 0 0.023 *a*b*b 0 000 00 1 0 10 00000 111*d 11 1 01 11111 111*a0 11 1 0 1 11 11111 111*b1 11 1 1 11 11111 111*a1 11 0 1 11 11110 000 0*a1 00 1 0 00 00001 111 1*a1 11 1 01 11110 111*b0 11 0 1 11 11110 000 1 0*a1 00 0 00 00001 111 1*a1 11 1 01 11110 0*b0 0.895 0*a1 1*a 1 0 0.019*b 0.981 0.009 0.981*d 0.851 0 111 0 11 1 11 11110 1 000 00 0 0 1 00 00001 111*a1 11 0 1 1 0 01 11110 0*b0 0 1*a1 0.99 0*a 0 0 0*b 0.974 1 0 0 0.018 0 0*d 0.97 0.982 1 0 0 0 0.962 0 0 0 0 1 0 1 0.991 0.01 0.95 0 0.99 1 0 0 0 0 0.02 0 0 0 1 0.019 0 0 0 0.906 0 0 1 0.01 0 1 0 0 0 0 0 0 0.009 1 0.981 0 0 0 0.031 0 0 0.99 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0.013 0 0 0 0 0 0 0 1 0.02 0 0 0 0 0 Allele frequency, proportion of polymorphic loci and heterozygosity in the genus 3: ABLE LocusAAT-1* AlleleAAT-2* 57 *aALP* 2 0EST-1* EST-2* 76FH-1* 0 19FH-2* G3PDH-1* 0 33G3PDH-2* GPI* 0 52 0 50 0.019HK-1* 57 0HK-2* 40IDHP* 26 1 53 1 25 1 16 0 43 0 0 0 T Species Locality Genetic relationship between grapsid crabs 393 H. penicillatus E. japonica C. dehaani and three other grapsid crab species. Ishigaki Ogasawara Miyagi Ogasawara Tokyo Helice Fukuoka Okinawa Kagoshima Fukuoka yama Ehime Ishigaki Chiba Waka H. t. latimera H. t. tridens H. leachi 0.08 0 0.04 0.08 0.04 0.16 0.04 0.08 0.12 0 0 0.091 0.05 0.04 0.0130.012 0 0 0 0 0.002 0.01 0 0 0.004 0.011 0.005 0.005 0.004 0.011 0.003 0.01 0 0 0.002 0.016 0.001 0.023 0.003 0.002 0.009 0.002 Okinawa *c 0 0 0 0 0 0.01 0 0 0 0 0.009 0 0 0 *c 0 0 0*c 0 1 0 1 0 1 0 1 0.974 1 0.9 0.981 1 1 1 1 0 1 0 1 0 1 0 0 1 a 111 11 1 11111 11 111 11 1 11111 11 *a1 *a1 a 000 00 0 00001 00 *a0 *b 1 1 1 1 1 0.99 1 1 1 1 0.991 0 1 1 a 111 11 1 11111 11 111 11 1 11111 11 *a1 *a1 *a*b*a 0*b 000 00 0 1 00000 10 111 11 1 1 11111 01 *a0 000 00 0 0 0 00000 10 *b1 111 11 1 1 11111 01 *a0 111 11 1 1 11111 01 0*b1 111 11 1 0 11111 01 1*a1 000 00 0 00000 10 1*a1 000 00 0 0 10001 00 0*a0 1*b0 1 0 0 1 0.01 1 0.99 0 0.99 0 0.01 1 1 1 0 0 0.009 1 0.018 0 0 0.1 1 0 0 0 1 0 0 0 1 0 0 1 0 1 1 0 0 1 1 0 *d 0 0 0 0 0 0.019 0 0 0 0 0 0 0 0 Allele frequency, proportion of polymorphic loci and heterozygosity in the genus 3: ABLE SOD-1* SOD-2* P* Ho He LDH* T Species Locality LAP-1* LAP-2* LocusMDH-1* AlleleMDH-2* 57MPI-1* 2MPI-2* 76PGM-1* PGM-2* 19PGDH* 33 52 50 57 40 26 53 25 16 43 MD. Younus Mia et al. 394 ; H. leachi ; H. l., H. tridens tridens . ; H. t. t., Helice tridens latimera Hemigrapsus penicillatus ; H. P., Eriocheir japonica ; E. J., Chiromantes dehaani C. d., Fig .1 Electrophoretic patterns of the eight polymorphic loci: H. t. l., Genetic relationship between grapsid crabs 395 Okinawa Ishigaki Ogasawara and three grapsid crabs Ehime Fukuoka Kagoshima Fukuoka Helice Ishigaki Chiba Wakayama H. leachi H. t. latimera H. t. tridens H. leachi , 8-11: Tokyo Okinawa H. tridens tridens , 3-7: Ogasawara 0 C. dehaani E. japonica H. penicillatus 1.235120.57114 0 0.53678 0 0.73975 0.41317 0.12161 0.0115 0.0053 0.0061 0 Nei‘s genetic distance between the populations in the genus 4: Helice tridens latimera ABLE opulation Miyagi T Species P 11. Ogasawara 0.549701-2: 0.63519 0.17356 0.1687 ##### ##### 0.17996 0.1733 0.1703 0.17169 4E-05 0.0004 1E-05 0 C. dehaani E. japonica H. penicillatus 1. Okinawa2. Ishigaki 0.73972 0.40338 0.745573. Chiba 0.12108 0.406874. Wakayama 0 0.12692 0.74477 0.00085. Ehime 0.40786 0 6. Fukuoka 0.12613 0.74439 0.00087. Kagoshima 0.73980 0.40570 1E-05 0.74323 0.41235 0.125758. Fukuoka 0 0.0005 0.40588 0.127319. Okinawa 0.0006 0.54977 0.12459 4E-05 0.00050 0.0001 3E-05 0.6313010. Ishigaki 0.54790 7E-05 9E-05 0.17169 0.00640 0.62794 5E-05 0.01072 0.55045 0.1664 0.16948 0.00751 0.1722 0.63304 0 8E-05 0.1634 ##### 0.17342 2E-05 0.1693 0.17735 0.1685 0 6E-05 0.1679 0.1744 0.17399 0.171 0.173 0.1681 0 0.168 0.17908 0.165 0.16940 0.1732 0.16645 0.1701 0 0.17154 0.0003 3E-05 0 0.0004 0 MD. Younus Mia et al. 396

Fig. 2. Dendrogram constructed by neighbor-joining method based on Nei‘s genetic distance shows the relationship between population in the genus Helice and three grapsid crabs. H. t. l., Helice tridens latimera; H. t. t., H. tridens tridens; H. l., H. leachi; C. d., Chiromantes dehaani; E. j., Eriocheir japonica; H. P., Hemigrapsus penicillatus. dehydrogenase (LDH), and for both C. dehaani and E. japonica. The phosphogluconate dehydrogenase remaining enzyme phosphoglucomutase (PGDH) appeared in one zone for all (PGM) appeared in two zones for Helice, species. Each of these enzymes was C. dehaani and H. penicillatus indicating controlled by at least one locus. Six that this enzyme was controlled by at enzymes, namely aspartate least two separate loci for these species. aminotransferase (AAT), esterase (EST), Except for the polymorphic loci gene leucine aminopeptidase (LAP), malate replacement was observed at six loci, dehydrogenase (MDH), mannose-6- ALP*, FH-1*, G3PDH-1*, HK-1*, MPI-1* phosphate isomerase (MPI), and and MPI-2*. The genotypes of each superoxide dismutase (SOD) appeared in species for these six loci are shown in two separate zones in all the species, Table 3. indicating that each of the six enzymes At 13 loci, namely AAT-1*, EST-1*, were controlled by at least two separate EST-2*, GPI*, IDHP*, LAP-1*, LAP-2*, loci. Three enzymes, namely fumarate MDH-1*, MDH-2*, PGM-1*, PGM-2*, hydratase (FH), glycerol-3-phosphate SOD-1* and SOD-2* common alleles are dehydrogenase (G3PDH) and hexokinase found. On the contrary, at the remaining (HK) appeared in two zones in Helice 12 loci, namely AAT-2*, ALP*, FH-1*, FH- species and H. penicillatus, but appeared 2*, G3PDH-1*, G3PDH-2*, HK-1*, HK-2*, in one zone in C. dehaani and E. MPI-1*, MPI-2*, PGDH* and LDH* no japonica. It indicates that these enzymes common allele was found. These loci are were controlled by two separate loci for completely divergent. Helice and H. penicillatus and one locus Genetic relationship between grapsid crabs 397

Genetic variability was estimated by around 0.055. Chow and Fujio (1987) the proportion of polymorphic loci and found the average heterozygosity to be average heterozygosity. The average 0.043 on the basis of studies in 36 proportion of polymorphic loci (P*) was decapod crustacean species. The average 0.058 in Helice species, 0.091 in C. heterozygosities obtained in the present dehaani, 0.050 in E. japonica and 0.040 study were lower than the values, at in H. penicillatus. The mean observed 0.006 in H. t. latimera and H. t. tridens, (H ) and expected heterozygosities (H ) 0.004 in H. leachi, 0.023 in C. dehaani, o e were 0.004 and 0.005 in Helice species, 0.009 in E. japonica and 0.002 in H. 0.016 and 0.023 in C. dehaani, 0.003 and penicillatus. The values of average 0.009 in E. japonica, and both were 0.002 heterozygosities obtained in the present in H. penicillatus (Table 3). study are lower than the values found by other researchers (Irawan et al., 1993; Nei‘s genetic distances between all Irawan and Kijima, 1994; Fuseya and samples based on 25 loci are given in Watanabe, 1996; Gao and Watanabe, Table 4 and Fig. 2. A higher value was 1998). These low values of average observed between different genera than heterozygosities agreed well with the that of between different species. suggestion that relatively low genetic Discussion variability is a general phylogenetic character of crustaceans (Hedgecock et Genetic variability has been al., 1976). Among the six species quantified in many populations and polymorphicity (P*), average observed species of decapod crustacea based on (H ) and expected heterozygosity (H ) of electrophoretically detectable isozyme o e C. dehaani were higher than others, genes (Nelson and Hedgecock, 1980). indicating that this species is genetically Genetic differences between species more variable than other species. include allele that differed after reproductive isolation was established Nei‘s genetic distance calculated (Futuyama, 1986). The degree of genetic from allele frequencies of isozyme genes difference increase according to the term is another useful measurement in of isolation and is considered to estimating the degree of genetic correspond with taxonomic levels. The divergence. Nei (1975) summarized that number of divergent loci, in which no the genetic distance was distributed common allele was observed, is one of the around 0.01 between local races, around indexes for estimating the degree of 0.1 between subspecies and around 1.0 genetic divergence. At the locus AAT-2*, between species for various organisms. ALP*, MPI-1*, MPI-2* and PGDH* In the decapod species, genetic distance alleles *d, *a, *a, *a, *a in E. japonica, was reported as 0.70 between Uca at FH-1*, G3PDH-1*, HK-1* and LDH* speciosa and U. spinicarpa (Salmon et alleles *b, *b, *b and *a in C. dehaani, al., 1979) and 0.979 between respectively were uncommon alleles. Liopetrolisthes mitra and L. patagonicus These uncommon alleles might be a (Weber and Galleguillos, 1991). diagnostic character for respective Hedgecock et al. (1982) summarized the species. This means that we can mean genetic distance was 0.44 ± 0.23 distinguish E. japonica from C. dehaani. among 23 species within 17 genera in decapod crustacea. Genetic distance According to this study, the mean among the grapsid crab species was also heterozygosity per individual was reported as 1.431 between Helice tridens MD. Younus Mia et al. 398 and H. japonica, 2.775 between H. this result raise confusion about the tridens and Chiromantes dehaani, and taxonomical position of H. penicillatus 2.111 between H. japonica and C. and calls for further research to be done dehaani (Irawan et al., 1993); 0.0004 on Helice species and H. penicillatus. To between E. japonica and E. sinensis (Gao solve this problem further study like and Watanabe, 1998), within the mud DNA and mtDNA analyses are needed crab, genus Scylla it was 0.059 between to define the taxonomical relationship S. serrata and S. tranquebarica, 0.126 between Helice species and H. between S. tranquebarica and S. penicillatus clearly. oceanica and 0.187 between S. serrata Acknowledgement and S. oceanica (Fuseya and Watanabe, 1996). The genetic distance (1.431) Financial support from Hirose between Helice species (Irawan et al., International Scholarship Foundation, 1993) was higher than the values Japan is greatly acknowledged. (0.00033, 0.16966 and 0.17017) obtained References among the three Helice species (Mia et al., 1999). The genetic distance between Chow, S. and Y. Fujio 1987. Comparison of C. dehaani and E. japonica, C. dehaani intraspecific genetic diversity levels and H. penicillatus, E. japonica and H. among local populations in decapod penicillatus, H. t. latimera and C. crustacean species with some references dehaani, H. t. tridens and C. dehaani, of phenotypic diversity. Nippo. Sui. Gak., 53(5): 691-693. H. leachi and C. dehaani, H. lecahi and E. japonica obtained in the present study Fuseya, R. and S. Watanabe 1996. Genetic was found higher than an average value variability in the mud crab genus Scylla between crustacean species (Hedgecock (Brachyura: Portunidae). Fish. Sci., et al., 1982). The value is considered to 62(5): 705-709. be enough not only for different species Futuyama, D.J. 1986. Evolutionary Biolgy. but also for separate subgenera Sinauer Associate, Inc., Sundeland, according to the values summarized by Mass. Nei (1975). Gao, T. and S. Watanabe 1998. Genetic The study reveals that all the three variation among local populations of the Japanese mitten crab, Eriocheir grapsid crab species viz. C. dehaani, E. japonica De Haan. Fish. Sci., 64(2): 198- japonica and H. penicillatus are 205. genetically different as is evident from the divergent loci. This finding is to be Hedgecock, D., R. A. Shleser and K. Nelson 1976. Applications of biochemical used for the management of these three genetics to aquaculture. J. Fish. Res. Bd. species separately. Chiromantes dehaani Canada, 33: 1108-1119. is more different from other species investigated here due to its higher Hedgecock, D., M.L. Tracey and K. Nelson genetic distances. 1982. The Biology of Crustacea, vol. 2, Embryology, Morphology and Genetic L. The genetic analysis also shows that G. Abele (Ed.), Academic Press, New Hemigrapsus penicillatus is genetically York, 283pp. close with H. t. latimera and H. t. tridens Irawan, B., A. Kijima and Y. Fujio 1993. than H. leachi (Fig. 2). In fact, on the Genetic divergence among the three basis of the existing classification of species of estuarine crab, Helice tridens, Helice it should not have happened. So, H. japonica and Chiromantes dehaani Genetic relationship between grapsid crabs 399

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