JOURNAL OF BIOLOGY, 25(4): 620–624, 2005

A NATURAL HYBRID MUD (, ) FROM JAPAN

Hideyuki Imai and Masatsune Takeda

(HI, correspondence) Laboratory of Marine Biology and Coral Reef Studies, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan ([email protected]); (MT) Department of Zoology, National Science Museum, Shinjuku, Tokyo 169-0073, Japan ([email protected])

ABSTRACT

A presumed hybrid mud crab of the was examined. The presumed hybrid could not be easily separated morphologically from Downloaded from https://academic.oup.com/jcb/article/25/4/620/2670477 by guest on 23 September 2021 the three Japanese Scylla species, S. serrata, S. paramamosain, and S. olivacea. An analysis of the first internal transcribed spacer (ITS-1) of the nuclear DNA revealed that the hybrid carried genomic DNA from both S. serrata and S. olivacea. Analysis of the maternally inherited 16S rDNA of the mitochondrial DNA demonstrated that the presumed hybrid carried S. olivacea mtDNA. Based on these results, we conclude that the examined is indeed the hybrid offspring of a female S. olivacea and a male S. serrata. The degree of genetic isolation among these species, and the possible causes of hybridization are discussed.

Mud belonging to the genus Scylla are distributed here had the character of S. serrata in the frontal median throughout the West Pacific and Indian Oceans. The spine height. On the other hand, no polygonal patterning four species of mud crabs that have been described to date, on legs indicated S. olivacea. Mitochondrial DNA showed namely S. serrata (Forska˚l, 1775), S. olivacea (Herbst, the character of S. olivacea. Thus, by using Keenan et al. 1796), S. tranquebarica (Fabricius, 1798), and S. para- (1998), we could not discriminate the presumed hybrid. mamosain Estampador, 1949, have very similar morpho- Here, we describe the morphological and genetic character- logical characters: adult Scylla species can be distinguished istics of a hybrid mud crab. using the ratio between the frontal median spine height and width, the presence or absence of obvious cheliped carpal spines, and the presence of a polygonal patterning of the MATERIALS AND METHODS legs (Keenan et al., 1998). Specimens Genetic differences and relatedness among the Scylla spp. have been examined previously using mitochondrial DNA In 1995 the following species and specimens of mud crab were collected at Kino River (348159N, 1358119E ; 348139N, 1358089E) in Wakayama (mtDNA) sequences of cytochrome oxidase I, 16S rDNA Prefecture, Japan: a presumed natural hybrid mud crab (n ¼ 1; National (Keenan et al., 1998; Imai, 1999), 12S rDNA (Imai, 1999), Science Museum Tokyo-Crustacean; NSMT-Cr 14835; Fig. 1); S. serrata and restriction fragment length polymorphism (RFLP) anal- (n ¼ 10); S. paramamosain (n ¼ 10); and S. olivacea (n ¼ 10) (Fig. 2). We ysis of mtDNA (Imai and Numachi, 2002). In addition, also obtained specimens of S. tranquebarica (n ¼ 5) off the coast of Tungkang, Taiwan, in 1999. All were collected during the breeding allozyme analysis has been used to discriminate three season in October. (Fuseya and Watanabe, 1996) or four species (Keenan et al., 1998). However, the results of the allozyme analyses are Morphological Characters controversial, because the data were not definitive. More- We investigated the morphological characteristics of the presumed hybrid over, different researchers have reported different results, specimen according to the method of Keenan et al. (1998) (Table 1). We even though they used the same methodology to examine measured the abdomen width (AW), carapace length (CL), maximum carapace width (CW), carapace width at eighth tooth (8CW), distance alleles at the same locus. More recently, the first internal between frontal lateral spines (DFLS), distance between frontal median transcribed spacer (ITS-1), which separates 18S from 5.8S spines (DFMS), frontal median spine height (FMSH), carapace frontal nuclear ribosomal RNA, was used for species identifica- width (FW), internal carapace width (ICW), ninth laternal spine height tion and for the phylogenetic analysis of and (LSH), posterior width of carapace (PWC), sternum width (SW), DL (dactyl molluscans (Heath et al., 1995; Toro, 1998; Harris and length), inner carpus spine (ICS), inner propodus spine (IPS), merus length (ML), outer carpus spine (OCS), outer propodus spine (OPS), propodus Crandall, 2000; Murphy and Goggin, 2000; Chu et al., depth (PD), propodus length (PL), propodus width (PW), third pereopod 2001; Fernandez et al., 2001; Imai et al., 2004). Imai et al. merus length (3PML), fifth pereopod dactyl length (5PL), and fifth (2004) showed genetic tool for species identification of pereopod dactyl width (5PW), and examined the legs of the specimen to S. serrata (n ¼ 70), S. tranquebarica (n ¼ 10), determine whether there was a polygonal pattern of coloration. S. paramamosain (n ¼ 80), and S. olivacea (n ¼ 61) using Genetic Analysis ITS-1 and 16S rDNA markers. Genomic DNA was extracted from 20 mg of leg tissue using the Get pure To date, there has been no report of natural interspecific DNA kit (Cell, Tissue; Dojindo Molecular Technologies, Japan). We used hybridization within the genus Scylla (Keenan et al., 1998; two pairs of primers, which targeted the first internal transcribed spacer Overton and Macintosh, 2002). Nevertheless, the expansion (ITS-1) of nuclear DNA and 16S rDNA of mtDNA; the primers were of the distributional ranges of several Scylla spp. in Japan identical to those that have been used previously to identify Scylla spp. (Table 2 and Imai et al., 2004). The ITS-1 and 16S rDNA were amplified has increased the frequency of contact among different spe- using polymerase chain reaction (PCR) with the primer sets: SP-1-39 and cies of mud crab, which increases the probability of SP-1-59138 and 16sar-L and 16sbr-H, respectively (Table 2) (Chu et al., interspecific hybridization. The presumed hybrid studied 2001; Palumbi et al., 1991).

620 IMAI AND TAKEDA: A NATURAL HYBRID MUD CRAB 621 Downloaded from https://academic.oup.com/jcb/article/25/4/620/2670477 by guest on 23 September 2021

Fig. 1. Dorsal view of a hybrid mud crab. Carapace width ¼ 155 mm.

A two-temperature PCR that was used to amplify ITS-1 was performed We conducted double digestion of the amplified 16S rDNA fragment with in a total volume of 25 lL reaction mixture. It contained 2.5 lL of 2.5 mM two endonucleases Dra I and Hind III (Takara Shuzo, Japan), which have dNTPs, 1 lL each of 25 pM primers, 1 lL of template DNA, 2.5 lLof been reported to be effective for identifying Scylla species (Imai et al., recombinant Z-Taq buffer, and 0.5 units of Z-TaqÔ (Takara Shuzo, Japan) 2004). The digested PCR product was electrophoresed in 1.5% agarose in a GeneAmpÒ 9700 thermal cycler (Perkin-Elmer, USA). We used gel (TreviGelÔ500, Trevigen, U.S.A.) and stained with ethidium bromide the following PCR parameters: 60 s of denaturation at 948C, followed by for imaging. 25 cycles of 2 s at 988C, and 20 s at 728C. The 16S rDNA was amplified in a 25-lL reaction mixture that contained Histology 2.5 lL of 2.5 mM dNTPs, 1 lL each of 25 pM primers, 1 lL of template The testes of sexually mature S. serrata and the presumed hybrid were DNA, 2.5 lL of recombinant Ex buffer, and 0.5 units of ExTaqÔ DNA dissected and fixed in Bouin’s solution. Pieces of fixed testis were polymerase (Takara Shuzo, Japan). The PCR amplification profile was dehydrated in ethanol, embedded in paraffin, sectioned at 6 lm thick, and 2 min denaturation at 948C, 30 cycles of 60 s denaturation at 948C, 30 s finally stained in Mayer’s hematoxylin plus eosin (HE). annealing at 458C, 90 s extension at 728C; and 10-min extension at 728C.

RESULTS Morphological Characters The presumed hybrid specimen is a male (see Materials and Methods). The width and length of the carapace is 155.0 mm and 100.2 mm, respectively. The general shape of the carapace, chelipeds, and ambulatory legs are consistent with the genus Scylla. The color of the legs of the presumed hybrid (Fig. 1) was uniformly dark moss-green, without any polygonal patterning. The cutting and prehensile edges of the pincers were of a rusty brown color. Further morphological classification of the presumed hybrid specimen was carried out using morphometric char- acters and ratios presented by Keenan et al. (1998). The results were as shown in Table 3. Based on these measure- ments, we calculated five ratios (Table 4) and combined these data with those of Keenan et al. (1998) (who examined only Scylla spp. specimens with an ICW . 95 mm). In the Fig. 2. Dorsal aspect of the upper part of the carapace of four species of Scylla and the hybrid. (A) S. serrata. (B) S. paramamosain. (C) S. olivacea. presumed hybrid specimen, the size and ratios of the carapace (D) S. tranquebarica. (E) Hybrid. (F, G) legs and left cheliped of the fell within the range of S. serrata, but were distinct from hybrid, showing the diagnostic features. Scale bars ¼ 10 mm. S. tranquebarica (FW/ICW, FMSH/FW, and FMSH/DFMS 622 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 25, NO. 4, 2005

Table 1. Morphological characters of adult mud crab and genetical methods useful in species identification. Morphological characters and genetical methods of species identification described by Keenan et al. (1998) and Imai et al. (2004), respectively.

Frontal lobe spines Cheliped Polygonal patterning ITS-1 16S rDNA Species Shape Height Carpus spines Propodus spines last legs genotype haplotype S. serrata blunt point height both obvious obvious presence B 2 S. tranquebarica blunted moderate both obvious obvious presence D 1 S. paramamosain triangular moderatly high inner absent obvious weak presence E 4, 5 outer reduced S. olivacea rounded low inner absent reduced absence C 3 outer reduced

in Table 4), S. paramamosain (FW/ICW and FMSH/DFMS mud crab exhibited a single PCR product, which is char- Downloaded from https://academic.oup.com/jcb/article/25/4/620/2670477 by guest on 23 September 2021 in Table 4), and S. olivacea. acteristic of homozygotic individuals. By contrast, the PCR Although this might be explained by normal morpholog- product of the presumed hybrid was a double fragment, ical variation in chelipeds (the thickness of the propodi and which would be characteristic of a heterozygote with alleles the armature of the fingers differs among individuals ac- from S. serrata and S. olivacea, i.e., the PCR product from cording to their age and sex), the ratio based on the carpus the presumed hybrid suggested that this individual carried spines (specifically, ICS/OCS) of the presumed hybrid fell genomic DNA that had been inherited from S. serrata within the range of S. serrata. Regarding the palm of the and S. olivacea. cheliped (which is armed with a pair of distinct spines on the The restriction profiles of 16S rDNA are shown in Fig. 4. dorsal margin posterior to the insertion of the movable Intraspecific polymorphism was not observed, and all of the finger), the spines of the presumed hybrid specimen were specimens could be discriminated. Because the restriction smaller than those of S. serrata and S. tranquebarica, but pattern of the presumed hybrid was identical to that of were similar in size to those of S. paramamosain. The frontal S. olivacea, the maternal parent of the presumed hybrid was lobe spines of the presumed hybrid specimen were blunt and an S. olivacea female. These results demonstrate that the high, unlike those of S. paramamosain (see Keenan et al., presumed hybrid that was examined in the present study was 1998). Although the weighting of the characters might be the offspring of a female S. olivacea and a male S. serrata. somewhat different, the most useful morphological ratios that can be used to discriminate between the different species Histology of the Testis of mud crab are ICS/OCS, FMSH/FW, and FW/ICW, ac- The structure and hematoxylin–eosin pattern of staining of cording to Keenan et al. (1998). The presumed hybrid the testes of normal S. serrata males revealed the existence specimen was distinct from all species of mud crab with of spermatophores, and the histological appearance of the respect to the ratio of FW/ICW, distinct from S. tranque- testes of S. serrata was identical to those of the blue crab, barica and S. olivacea with respect to FMSH/FW, and Callinectes sapidus (see Johnson, 1980). By contrast, the distinct from S. paramamosain and S. olivacea with respect testis of the presumed hybrid did not contain spermato- to ICS/OCS. Despite an overall apparent similarity to phores (Fig. 5), even though the specimen had been cap- S. serrata, the presumed hybrid specimen differed from tured at the same point in the breeding season. S. serrata in the lack of the polygonal color patterning that characterizes the chelipeds and legs of the latter species. In DISCUSSION addition, the hybrid could be distinguished from S. serrata Keenan et al. (1998) analyzed allozymes and mtDNA by the armature of chelipedal carpus, which bore two blunt in four species of mud crabs of the genus Scylla, and spines that were neither obvious nor reduced. concluded that there is no evidence for hybridization among these species. This conclusion has been supported by other Genetic Analysis studies. For example, Imai and Numachi (2002) used The results of the PCR amplification of ITS-1 are presented RFLP of whole mtDNA to analyze 338 individuals of the in Fig. 3. Intraspecific polymorphism was not detected. The genus Scylla (119, 107, and 112 specimens of S. serrata, PCR data could be used to distinguish S. serrata and S. paramamosain, and S. olivacea, respectively) that were S. olivacea, whereas the length of the PCR product was the obtained from locations throughout the range of distribution same for S. tranquebarica and S. paramamosain, which, of this genus. In addition, Imai et al. (2002) analyzed the consequently, could not be distinguished. Each species of

Table 3. Measurements (mm) of meristic characteristics of a male Table 2. Sequences of the primers used. presumed hybrid. See Materials and Methods for abbreviations.

L primer Sequences H primer Sequences Carapace AW CL CW 8CW DFLS DFMS FMSH FW ICW LSH PWC SW 48.4 100.2 155.0 152.6 7.2 7.0 3.6 52.0 143.6 5.7 48.0 76.4 SP-1-39 59-ATTTAGCTGCG- SP-1-59138 59-CACACCGCC- Chelipeds DL ICS IPS ML OCS OPS PD PL PW GTCTTCATC-39 CGTCGCTACTA-39 58.0 1.0 1.0 61.4 1.0 2.0 52.6 121.0 35.6 16sar-L 59-CGCCTGTTTAT- 16sbr-H 59-GGTTTGAAC- Pereopods 3PML 5PL 5PW CAAAAACAT-39 TCAGATCATGT-39 47.0 47.0 24.0 IMAI AND TAKEDA: A NATURAL HYBRID MUD CRAB 623

Table 4. Ranges and means (in parentheses) of two morphometric characters, and ratios for four species of mud crab and a male presumed hybrid (data for the four species are from Keenan et al., 1998). See Materials and Methods for abbreviations.

Ratio (mm) Male (NSMT-Cr 14835) S. serrata n S. tranquebarica n S. paramamosain n S. olivacea n CW 155.0 102.4–204.4 (147.3) 60 102.5–143.8 (120.8) 24 111.1–145.7 (122.5) 9 98.8–138.1 (112.2) 63 ICW 143.6 95.5–191.7 (138.4) 68 97.1–137.8 (113.7) 25 104.8–134.1 (114.7) 9 95.0–133.9 (107.5) 66 ICS/OCS 1.000 0.500–1.583 (0.940) 55 0.467–1.429 (0.980) 24 0.188–0.941 (0.352) 9 0.000–0.250 (0.006) 63 FMSH/FW 0.069 0.041–0.095 (0.061) 67 0.031–0.053 (0.043) 23 0.040–0.081 (0.058) 9 0.018–0.037 (0.029) 64 FW/ICW 0.362 0.335–0.406 (0.371) 68 0.383–0.443 (0.412) 25 0.364–0.386 (0.377) 9 0.371–0.451 (0.415) 66 LSH/ICW 0.040 0.018–0.046 (0.031) 60 0.022–0.044 (0.031) 24 0.022–0.049 (0.034) 9 0.015–0.037 (0.022) 63 FMSH/DFMS 0.500 0.254–0.544 (0.418) 63 0.216–0.371 (0.310) 23 0.283–0.483 (0.369) 8 0.143–0.316 (0.221) 64

mtDNA D-loop region of 843 S. paramamosain specimens mtDNA, which suggests the possibility of a founder effect, Downloaded from https://academic.oup.com/jcb/article/25/4/620/2670477 by guest on 23 September 2021 by RFLP. The haplotypes that were revealed by these whereas the other two species are polymorphic. Before 1980, analyses were not shared among the species and were in S. olivacea was not found in the Kino River (S. Hamano, agreement with the morphological characteristics of each personal communication), Lake Hamana (Ito, 1994), or species. In the present report, we demonstrate the existence Orido Bay (S. Kishiyama and I. Hori, personal communi- of a natural hybrid mud crab of the genus Scylla that appears cation), but sufficient numbers of this species to support to have been produced by cross-breeding between a female commercial catches have been reported recently in each of S. olivacea and a male S. serrata. In addition, we believe these areas. The explanation of how S. olivacea has extended that the hybrid specimen that we examined was sterile, its distribution is not clear. The microhabitats of S. serrata, because there were no gametes within its testes, even though S. paramamosain, and S. olivacea are somewhat different, the size of the individual suggested that it had reached but do overlap. is usually encountered in maturity. Moreover, the presumed hybrid male was captured a limited area in the vicinity of river mouths or the innermost during the breeding season at a time at which male crabs (of parts of bays. The distribution of S. paramamosain is wider the genus Scylla) exhibit gametogenesis. Thus, this crab was than that of S. olivacea, but far smaller than that of S. serrata, probably a F1 representative of a sterile generation. This is which is encountered most frequently in estuaries and in agreement with the observation that laboratory-produced bays (Oshiro, 1988). Our description of a hybrid between hybrid fresh water prawns and lobsters are sterile, although S. olivacea and S. serrata suggests that hybridization be- it is worth noting that hybrids of Penaeus spp. can be tween these two species occurred after the habitat of reproductively active (Shokita, 1978; Talbot et al., 1983; S. serrata was invaded by S. olivacea. Bray et al., 1990). In Japan, three species of mud crab (S. serrata, S. paramamosain, and S. olivacea) are widely distributed ACKNOWLEDGEMENTS throughout various habitats in the west of the country We are grateful to Dr. J.-H. Cheng (Tungkang Marine Laboratory, Taiwan (Oshiro, 1988). Scylla olivacea is the least abundant of the Fisheries Research Institute) for kindly providing S. tranquebarica three species, although its distribution has extended recently specimens. We are also grateful to Dr. T. Kitano (Tokai University) for within Japan. In Urado Bay, Lake Hamana, and the Kino assistance with the histology. We thank Mr. S. Hamano (Kino River Fisheries Cooperative Association) and Mr. S. Kishiyama and Mr. I. Hori River, S. paramamosain is the numerically dominant species (Shimizu City Fisheries Cooperative Association) for their useful of mud crab (Yamakawa and Suzuki, 1985; Oshiro, 1988), suggestions. We are grateful to Dr. K. Numachi for discussion and critical whereas S. olivacea and S. serrata are less abundant. In the reading of the manuscript. This study was supported by Grant-in-Aid for Yaeyama Islands, S. serrata is dominant (Oshiro, 1988). Young Scientists (B15780136) and the 21st Century COE program of the There are three species of mud crab in Orido Bay, Japan: S. serrata and S. paramamosain are co-dominant, while S. olivacea is less abundant (Oshiro and Imai, 2003). Imai and Numachi, (2002) showed S. olivacea is genetically monomorphic in Orido Bay using RFLP analysis of whole

Fig. 4. Electrophoresis fragments of 16S rDNA produced by double digestion with DraI and HindIII. Lanes marked with M are the molecular Fig. 3. ITS-1 PCR products from four species of Scylla and the hybrid. marker (100 bp DNA ladder). Fragment sizes are shown on the right. 1, S. paramamosain;2,S. serrata;3,S. olivacea; 4, hybrid; 5, Species of Scylla in the lanes (from left to right) are S. tranquebarica, S. tranquebarica;M,k-EcoT14I digest molecular marker. S. serrata, S. paramamosain, S. olivacea, and the hybrid. 624 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 25, NO. 4, 2005

Imai, H. 1999. Studies on molecular variability of the three species in mud crabs, genus Scylla and swimming crab Portunus trituberculatus, and its use for stocking as the genetic marker.—Ph.D. thesis, Tokai University, Shizuoka. 120 pp. ———, J.-H. Cheng, K. Hamasaki, and K. Numachi. 2004. Identification of mud crabs (genus Scylla) using ITS-1 and 16S rDNA markers.— Aquatic Living Resources 17: 31–34. ———, and K. Numachi. 2002. Intra- and interspecific genetic variability and relationships among mud crabs, Scylla spp. (Decapoda: Portunidae), demonstrated by RFLP analysis of mitochondrial DNA.—Journal of Animal Genetics 29: 3–11. ———, Y. Obata, S. Sekiya, T. Shimizu, and K. Numachi. 2002. Mitochondrial DNA markers confirm successful stocking of mud crab juveniles, () into a natural population.—Suisan- zoshoku 50: 149–156. Ito, M. 1994. Mud crab in Lake Hamana.—Hamana 404: 7–8. Johnson, P. T. 1980. The reproductive system, histology of the blue crab, Downloaded from https://academic.oup.com/jcb/article/25/4/620/2670477 by guest on 23 September 2021 Callinectes sapidus. Pp. 327–346, Praeger Publishers, New York. Keenan, C. T., P. J. F. Davie, and D. L. Mann. 1998. A revision of the genus Scylla de Hann, 1833 (Crustacea: Decapoda: Brachyura: Fig. 5. Histological appearance of the testis of mud crabs (Scylla spp.) that Portunidae).—The Raffles Bulletin of Zoology 46: 217–245. was captured during the breeding season. (A) Cross-section of the testis of Murphy, N. E., and C. L. Goggin. 2000. Genetic discrimination of S. serrata. (B) Cross-section of the testis of the hybrid mud crab. Tissue in sacculinid parasites (Cirripedia, Rhizocephala): implication for control of A and B was stained with hematoxylin and eosin. introduced green crabs (Carcinus maenas).—Journal of Crustacean Biology 20: 153–157. Oshiro, N. 1988. Mud crabs (Scylla spp.). Pp. 218–229 in S. Shokita, ed., in Tropical Areas. Midorishobo Co Ltd., Tokyo. University of the Ryukyus from the Ministry of Education, Culture, Sports, ———, and H. Imai. 2003. Portunidae. Pp. 262–265 in M. Nishida, Science and Technology of Japan. N. Shikatani, and S. Shokita, eds. The Flora and Fauna of Inland Waters in the Ryukyu Islands. Tokai University Press, Tokyo. Overton, J. L., and D. J. Macintosh. 2002. Estimated size at sexual maturity for female mud crabs (genus Scylla) from two sympatric species within LITERATURE CITED Ban Don Bay, Thailand.—Journal of Crustacean Biology 22: 790–797. Bray, W. A., A. L. Lawrence, L. J. Lester, and L. L. Smith. 1990. Hy- Palumbi, S., A. Martin, S. Romano, W. O. MacMillan, L. Stice, and bridization of Penaeus setiferus (Linnaeus, 1767) and Penaeus schmitti G. Grabowski. 1991. The Simple Fool’s Guide to PCR, version 2.0. Burkenroad, 1936 (Decapoda).—Journal of Crustacean Biology 10: Department of Zoology and Kewalo Marine Laboratory, University of 278–283. Hawaii, Honolulu. Chu, K. H., C. P. Li, and H. Y. Ho. 2001. The first internal transcribed Shokita, S. l978. Laval development of interspecific hybrid between spacer (ITS-1) of ribosomal DNA as a molecular marker for phylogenetic Macrobrachium asperulum from Taiwan and Macrobrachium shokitai and population analyses in Crustacea.—Marine Biotechnology 3: from the Ryukyus.—Bulletin of the Japanese Society of Scientific 355–361. Fisheries 44: 1187–1195. Ferna´ndez, A., T. Garcı´a, L. Asensio, M. A´ . Rodriguez, I. Gonza´lez, P. E. Talbot, P., D. Hedgecock, W. Borgeson, P. Wilson, and C. Thaler. 1983. Harna´ndez, and R. Martı´n. 2001. PCR-RFLP analysis of the internal Examination of spermatophore production by laboratory-maintained transcribed spacer (ITS) region for identification of 3 clam species.— lobsters (Homarus).—Journal of the World Mariculture Society 14: Journal of Food Science 66: 657–661. 271–278. Fuseya, R., and S. Watanabe. 1996. Genetic variability in the mud crab Toro, J. E. 1998. PCR-based nuclear and mtDNA markers and shell genus Scylla (Decapoda: Portunidae).—Fisheries Science 78: 95–109. morphology as an approach to study the taxonomic status of the Chilean Harris, D. J., and K. A. Crandall. 2000. Intragenomic variation within ITS1 blue mussel, Mytilus chilensis (Bivalvia).—Aquatic Living Resources and ITS2 of freshwater crayfishes (Decapoda: Cambaridae): implications 11: 347–353. for phylogenetic and microsatellite studies.—Molecular Biological Yamakawa, H., and K. Suzuki. 1985. Mud crab, mystery of three types. Pp. Evolution 17: 284–291. 110–117 in M. Okiyama and K. Suzuki, eds. Marine Organisms in Japan, Heath, D. D., P. D. Rawson, and T. J. Hilbish. 1995. PCR-based nuclear Ecology of Invasion and Disturbance. Tokai University Press, Tokyo. markers identify alien blue mussel (Mytilus spp.) genotypes on the west coast of Canada.—Canadian Journal of Fisheries Aquatic Sciences 52: RECEIVED: 2 February 2004. 2621–2627. ACCEPTED: 5 May 2005.