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Source: Evolution, Vol. 34, No. 4 (Jul., 1980), pp. 712-718 Published by: Society for the Study of Evolution Stable URL: http://www.jstor.org/stable/2408025 . Accessed: 10/03/2014 17:56
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or Xiphosura. Sekiguchi et al. pp. the University of Tsukuba. and the other is Tachypleinae. The American horseshoe crabs. described by Sekiguchi (1973). 34(4). MATERIALS AND METHODS 1 Contributions from the Shimoda Marine Research Center. including Tachypleus tridentatus. gigas. In the present paper. Tachypleus tridentatus. T. ecological. including Limulus polyphemus which is confined to the waters along the east coast of North America. The recent horseshoe crabs are divided into two subfamilies by their distribution and morphological characteristics (Pocock. tridentatus. Thus. Shishikura and Sekiguchi. One is Limulinae. gigas. which was found in the Jurassic lithographic shale of Germany. The fossil Xiphosura is known from all geological systems from the Cambrian onward (St0rmer. 1952). tridentatus except that they developed faster than T. 1980. the systematic relationships of horseshoe crabs are studied by experimental hybridization and are discussed with special reference to the developmental capacity and hemocyanin monomer constituents of interspecific hybrids. The Japanese horseshoe crabs. Smarn Srithunya (Zoological Museum. some embryological. The Asian species were maintained in running seawater at room temperature. which are confined to the waters along the southeast and east coasts of Asia. Massachusetts. 712-718 SYSTEMATICS AND HYBRIDIZATION IN THE FOUR LIVING SPECIES OF HORSESHOE CRABS' KoICHI SEKIGUCHI AND HIROAKI SUGITA Institute of Biological Sciences. rotundicauda. The eggs were artificially inseminated between April and August and kept at 30 C. the appearance of red spots consisting of fine granules around divided nuclei was examined by vital staining using neutral red (Sekiguchi. were obtained from the Supply Department of the Marine Biological Laboratory. 1977. Niihari-gun. morphological. Japan Received October 3. Therefore. 353. 1902). 1978.26. 712 This content downloaded from 58. In order to make clear the relationships of the four species. 1973. These living species are very similar to a fossil specimen. Japan and the Southeast Asian horseshoe crabs. and Carcinoscorpius rotundicauda.. Srinakharinwirot University.Evolution. the University of Tsukuba. so the developmental stage of the embryo was determined according to the normal plate of the Japanese horseshoe crab. hoeveni. Fertilized eggs of T.12 on Mon. are the striking "living fossils" in the present sea. T. and biochemical studies have been carried out in our laboratory (Sekiguchi.189. recorded by Pocock (1902). In order to decide whether they were fertilized or not. 1973). were kindly provided by Dr. Thailand). Woods Hole. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions . 1979 The horseshoe crabs. and the interspecific hybrids developed into swimming larvae through almost the same morphological process as those of T. 1976. As for the Moluccan horseshoe crab. gigas and concluded that only a single species should be recognized. Limulus polyphemus. 1978). and maintained in circulating seawater of 16 C at the Shimoda Marine Research Center. C. 1960. Sakura-mura. were collected from Imari and Kasaoka. T. Mesolimulus walchi. the four species mentioned above are the only members of living Xiphosura. it is considered that the horseshoe crabs have undergone little morphological evolution during their 200-million-year history. Tachypleus tridentatus. Ibaraki 305. No. Waterman (1958) compared it with T. gigas and Carcinoscorpius rotundicauda.
t. Limulus polyphemus.g. L. gigas or C. 0.g. tridentatus sperm.12 on Mon. Therefore. 0. Male T. gigas and C.r. L. We did not consider it reasonable to calculate average fertilizability (fertilized eggs/inseminated eggs) and average hatchability (hatched eggs/fertilized eggs) from all data we obtained because T. as follows: 8.7 mm (T. C. 9').t. Hatchability (hatched/fertilized. Carcinoscorpius rotundicauda. T.8 mm (L.000 eggs of T.000 eggs of C. L.0026 M NaH2PO4 2H2O. Carcinoscorpius rotundicauda. L.000 eggs of T. tridentatus. T. gigas.r. gigas).2 ml of a phosphate buffered saline (0. gigas and C. rotundicauda sometimes contained many bad eggs which could not be distinguished from healthy ones immediately after the insemination.. The fertilizability of T. T. Therefore. %').t.26. the same form as the trilobite larva) was cut with a pair of scissors in about 0. the fertilizability in each experiment was about equal to the values of blastula eggs per inseminated eggs.0 mm (T. C.-. Hemocyanin bands containing copper atoms were detected by the method of Horn and Kerr (1969).. 4. The values shown in Table 2 are representative ones obtained by using eggs and This content downloaded from 58. Fertilizability (fertilizedlinseminated. at rough estimate. This experimental insemination was carried out at least four times. or C. They finally died and gathered mold. gigas.T. In a single experiment of six trials. C.r. tridentatus. and each group was artificially inseminated by sperm from one of the four species. 6. Average hatchability could not be determined by the criteria mentioned above. the appearance of red spots consisting offine granules around divided nuclei was not observed when examined by vital staining using neutral red. The diameters of these eggs were about 3. Tachypleus tridentatus.HYBRIDIZATION IN FOUR HORSESHOE CRABS 1. T. rotundicauda).189. polyphemus eggs..r. 91 59 88 0 0 88 0 0 86 0 84 0 0 0 0 97 99 For examination of the hemocyanin constituent each embryo at Stage 21 (before hatching out.g.0074 M Na2HPO4 12H20.5 mm (C.. T.p. gigas.p. T. pH 7. the data presented in Tables 1 and 2 are some examples carried out in the best conditions. T. 13% of L.. TABLE Male g T.. T. Cr. tridentatus). rotundicauda eggs were fertilized by T.t. T. rotundicauda sperm at almost the same rate as T.t. Most of fertilized eggs shown in Table 1 developed to blastula (Stage 6) irrespective of the species providing sperm. RESULTS Developmental Capacity of the Hybrids The numbers of eggs used were. tridentatus eggs. 3. Sixteen possible combinations of artificial insemination using males and females of four Xiphosura species were carried out at the same time and the representative data of fertilizability is shown in Table 1. gigas sperm and developed -until blastula stage (this result is not included in Table 1). L. and 1... Protein in polyacrylamide gels was fixed and stained overnight according to the method of Waber and Osborn (1969). gigas.p. C. T. rotundicauda.000 eggs of L.g. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions . C. polyphemus. polyphemus sperm could not develop the eggs of Asian species and the sperm from Asian species could not develop L.p. L. polyphemus). In contrast with these results. 89 95 89 - 97 97 99 - 91 80 88 T. and 16. tridentatus eggs by T. rotundicauda sperm was as high as the fertilizability by the T. a > 73 a > T. Limulus polyphemus.g.145 M NaCl. Eggs obtained from one female were divided into four groups. - 713 TABLE 2.p. t.p.1) and this extract was immediately subjected to electrophoresis according to the method of Davis (1964). polyphemus eggs were fertilized by T. T.g. 2.r. Tachypleus tridentatus. L.
polyphemus and the against pure hemocyanins of three Asian other three species. 1975. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions . vation by vital staining. This content downloaded from 58.. Their of Davis (1964). specific hybrids are summarized in Figure 1. T.12 on Mon. rotundicauda.were dissociated into monomers (Sugita mally inseminated larvae (more than and Sekiguchi. T.. 2). tridentatus d hybridswas about 59% dividually from the embryos of T. Hemocyanin monomers in and C. Furthermore. rotundicaudoes not occur in Limulus or Asian Xi. The hybrids of T. Hemocyanin Monomer Patterns in Electrophoresis When Chelicerata hemocyanins were rotundicaudad and the reciprocalcross electrophoresed by using the buffer system were grown into swimming larvae. Judging from the obser. 1. Combinations of artificial insemination and developmental capacity of hybrids. The development protein in individuals was uniform within of interspecific hybrids between T. tridenbut the reciprocal hybrids could not de.t. rotundicauda d and the reciprocal hybrids have colored areas originating from both parents (Fig. T. Stout arrow = fertilized eggs developed into swimming larvae. tridentatus and C.g. g Developmental capacities of these interT. gigas. 80%). The hatchabilityof T.714 K. p. indicating that fertilization did not take place. It was found that good condition. Dashed-line arrow = red spots consisting of fine granules around divided nuclei were not observed. The hybrids of T.189. Carcinoscorpius rotundicauda .the monomers asterisked in Figure 3 of T. tridentatus Y x C.parative studies of hemocyanin monomers though the eggs and sperm were in very have been carried out. and C.same mobility in polyacrylamide-gel eleculus sperm. SEKIGUCHI AND H.tatus. t. L. gigas each species. nuclear division tridentatus. the T. and the velop beyond the blastula stage in spite of hybrids. gigas. tridentatus and C. 1969).. Slender arrow = eggs stopped their development at blastula stage. gigas. Phenotypic Appearance of First-instar Larvae There are obvious phenotypic differences in the appearance of larvae between T. The most striking is the chocolate-brown colored area along the ophthalmic ridge of T. Lamy et al.26. gigas Y x T. C .protein (Horn and Kerr. tridentatus d hybrids have the colored ridge like T. gigas Y x The hemocyanin extract was prepared inT. The electrophoretic pattern of the the high fertilizability. . rotundicauda larvae (data not shown). FIG. rotundicauda. T. rotundicauda was also stopped at these protein bands were identified by its blastula stage while the fertilizability of greenish-gray color developed by copper them was very high. trophoresis.da were immunologically identical (results phosura eggs which are interspecifically will be presented elsewhere) and had the hybridized by using either Asian or Lim. although the first-instar larva of T.g. Antisera bridization between L. Tachypleus tridentatus.. C. tridentatus larva versus the chocolatebrown colored areas along the marginal edges of prosoma and abdomen of C. 2). rotundicauda larva (Fig.r. T. 1977). SUGITA T. Eggs of species at an arrow head were inseminated by sperm of species at an arrow butt. In the case of hy. In order to make clear the relsperm which looked apparently most healthy. tridentatus. Limulus polyphemus. the inseminated eggs horseshoe crabs were prepared and comrdid not show any sign of development al. tridentatus Y x C. hemocyanin molecules hatchabilities were as high as those of nor.. gigas does not bear the chocolate-brown color in comparison with T.
From these electrophoretic patterns and photographs we conclude that hybrids have two complete sets of hemocyanin monomers. derived from both parents.HYBRIDIZATION IN FOUR HORSESHOE CRABS 715 FIG. Tachvpleiustride>ntatus. 1974). Therefore. b.26. c. Wilson et al. hybrid and between T. 2.189. The first-instar larxae of hybrids and the parental species. tridentatus 6. 1973) and Palearctic pond frogs (Kawamura and Nishioka.12 on Mon. ative positions of hemocyanin monomers the lower parts of the gels in Figure 3A are presented as a schema and asterisked monomer bands are lined on the same level. 1975). rotunldicaiuda. tridentatus ? and Carcinoscorpius rotnzdicaiuda 6. d. a. the techniques of backcross and second filial hybridization are not utilizable because it takes more than eight years for the animals to reach maturity (Barthel. DISCUSSION Systematics and Hybridization Natural hybrids and experimental hybrids have been examined in many kinds of animals to study evolutionary relationships. studies using experimental hybridization have clearly revealed the phyletic relationships of European green frogs (Berger. (1974) compared the blood proteins of interspecifically hybridizable pairs of frogs which had undergone little anatomical evolution and mammals which had undergone extensive anatomical evolution. In the case of horseshoe crabs. They discussed the relation between the evolutionary changes in anatomy and in mechanisms of gene expression This content downloaded from 58. the relation between the developmental capacity of hybrids and the phyletic line was examined as the second best way. rotnitdicazda z T. C. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions . For example. hybrid betwveenC.
C. in contrast with the frogs. d.12 on Mon. the relation between T. rotundicauda 9 and T. rotundicauda. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions . Thus. gigas. tridentatus. had experienced rapid evolutionary change in the systems regulating the expression of genes. SUGITA A B a fa b c d e f FIG. rotundicauda are immunologically identical with each other. Polyacrylamide-gel electrophoretic patterns of hemocyanin extracts (A) and of their hemocyanin monomers (B). SEKIGUCHI AND H. T. Tachypleus gigas and C. hybrid between T. gigas Q and T. tridentatus 9 and Carcinoscorpius rotundicauda d. the further the development of their hybrids advances.26. The American species. T. This leads one to expect that hybrids between very distantly related species could not develop because the breakdown in gene regulation would be so extensive. and proposed that the marked restriction of interspecific hybridization among mammals occurred because the mammals. f. Furthermore. Tachypleus gigas. 1962). polyphemus.716 K. is considered to be isolated from any species of Asia in almost equal degree. tridentatus and T. (1970) detected protein polymorphism using many enzymes of This content downloaded from 58. they suggested that evolutionary loss of ability for two species to hybridize probably resulted from the accumulation of incompatibilities between the two systems to regulate the expression of genes during embryonic development. tridentatus d. the closer the taxonomic relationship between the parental species is. Asterisked hemocyanin monomers of T. hybrid between C. L.189. a. 3. 1978) and by serological comparison among horseshoe crabs (Shuster. c. tridentatus. which are living together in waters along the southeast coast of Asia. e. tridentatus and C. When the systematic relationships of two species are discussed from a viewpoint of developmental ability of their hybrids in the case of the horseshoe crabs. b. rotundicauda is closer than the relation between T. This conclusion is supported by results obtained from immunocomparative study of coagulogen of horseshoe crabs (Shishikura and Sekiguchi. rotundicauda. are distantly related species among the Asian Xiphosura. Phenotypic Appearance and Gene Regulatory System Selander et al. tridentatus d. hybrid between T. gigas because the hybrid between the former species developed further than that between the latter ones. and C.
1973. Tachypleus tridentatus. 9:256266. V. NISHIOKA. C. I. Limulus: a living fossil Horseshoe crabs aid interpretation of an Upper Jurassic environment (Solnhofen). AND M. Bannister (ed. R. These results mean that the maternal and paternal genes of hemocyanin monomers and. 1974) may have remained almost unchanged in the horseshoe crabs. St. The taxonomy of recent species POCOCK.).HYBRIDIZATION IN FOUR HORSESHOE CRABS 717 American horseshoe crab. horseshoe crabs. This similarity in the mechanisms of the gene regulation may be one of the reasons why the horseshoe crab has undergone little morphological evolution during its 200million-year history. 121:404-427.. Biol. Scorpion hemocyanin subunits: Properties. polyphemus. 1964. Disc electrophoresis-I1. that is. Berlin. Soc. Mag. SEKIGUCHI. 1969. In J. J. 7. Springer-Verlag.Y. AND J. J. Ser. Naturwissenschaften 61:428-433. Asamushi 10:161-164. They reported L. Herpetol. 1973. which is a "phylogenetic relic. J. 1902.189. Comp. We also thank Dr. Hemocyanins and certain other major protein constituents. fruit flies. the genes participating in the color development were expressed equally in any hybrid which developed to a swimming larva. BAGLIN. Bull. Limulus polyphemus. Callinectes sapidus-1. 7:1-10. mice. .26. This content downloaded from 58.. S. W. Nat. K. The electrophoretic data of hemocyanin monomers and color development of hybrids suggests that the three species of Asian horseshoe crabs have very similar mechanisms for regulating gene expression during embryonic development. Embryonic development of the horse-shoe crab studied by vital staining. 37-49. Rep. 1975. K. T. frogs. KAWAMURA. Tachypleus tridentatus. 1970). 15:153-162. Systematics and hybridization in European green frogs of Rana esculenta complex. and man. Syst. The hemolymph proteins of the blue crab. association. Zool. Tokyo Kyoiku Daigaku Sec. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education of Japan. LITERATURE CITED 1974. 1960. which are "phylogenetic relics. L. E. LAMY. 1977. T. Acad. A normal plate of the development of the Japanese horse-shoe crab. J. Proceedings in Life Sciences: Structure and Function of Haemocyanin.-C. B. Mar. dissociation. 11:61-78.. Biochem. and consequently the gene regulatory systems whose changes may provide the basis for anatomical evolution (Wilson et al. Proc. Hist. p. and that the two genera of Asian horseshoe crabs are closely related to each other. BERGER. N.12 on Mon. Ann. Ann. with special reference to the isolating mechanisms between different species. and Carcinoscorpius rotundicauda. L. M. 29:493-508. Tamio Hirabayashi for the revision of the manuscript. BARTHEL. SUMMARY shoe crabs. and compared the gene variation of several animal species. DAVIS. On the To examine evolutionary relationships of horseshoe crabs. The polyacrylamide-gel electrophoresis of hemocyanin monomers obtained from interspecific hybrids revealed that any hybrid has two complete sets of hemocyanin monomers derived from the parents.. probably. polyphemus. HORN. The impressive stability of external morphological features manifested by the fossil since 200 million years ago may be explained in the terms of temporal uniformity of the organismenvironment relationship and/or genetic homeostasis (Selander et al. of Limulus. Smarn Srithunya for collecting Tachypleus gigas and Carcinoscorpius rotundicauda in Thailand. WEILL. Sci. gigas. that is. Jap. From the developmental capacity of hybrids we conclude that the American horseshoe crab." is not less genetically variable than some other animals belonging to horotelic lines. These data suggest that the parental species of hybrids have very similar mechanisms for regulating gene expression during embryonic development. This is also the case with morphological characteristics of the hybrids. Sci." experimental hybridization among all living species was carried out. KERR. is distantly isolated from the Asian horse- pond frogs in the Palearctic region.. B. AND M. LAMY. Physiol. ACKNOWLEDGMENTS We thank Dr. they developed all colored areas originating from both parents. Method and application to human serum proteins. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions .
Jap. Biochem. SHISHIKURA. AND V. H. Evidence from studies of interspecific hybridization. from the Bay of Bengal and the Gulf of Siam. K.Y. Morphological variation and distribution of a horseshoe crab.. Proc. SHUSTER.). SEKIGUCHI AND H. Syst. MACHJAJIB. ST0RMER. Serological correspon- This content downloaded from 58. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. AND H. K. F. 206:241-246. Proc. 1978. LEWONTIN. K. Acad. AND K. WEBER. AND T.. T.. T. AND W. MAXSON. USA 71:28432847. J. Biol. N. E. NAKAMURA. A. MAKIOKA. AND H. Two types of molecular evoluSARICH. Comparative study on horseshoe crab coagulogens. Milkman K. Sci. 1977. Carcinoscorpius rotundicauda. a phylogenetic "relic.189. Corresponding Editor: A. Jap. S. Proc. R. SRITHUNYA. NAKAMURA. S. Tachypleus gigas. 1962. M. Tachypleus gigas and Carcinoscorpius rotundicauda. Zool.718 SEKIGUCHI. WILSON. Zool. YAMASAKI. Soc. Jap. HeterogeSUGITA. On the doubtful validity of Tachypleus hoeveni Pocock. from the Bay of Bengal and the Gulf of Siam. 1975. Proc. Soc. Chem. A study on the egg-laying habits of the horseshoe crabs. and the horseshoe crab (Tachypleus tridentatus).. SUGITA. SEKIGUCHI. 1970. L. C. L.26. Zool. 12:13-20. C.. Soc. Syst. SEKIGUCHI. R. S. 36:1-17. 1976. Y. neity of the minimum functional unit of hemocyanins from the spider (Argiope bruennichii). K. Zool. 1969. R. 26:630-639. H. K. 10 Mar 2014 17:56:09 PM All use subject to JSTOR Terms and Conditions . Yale Peabody Mus. N. Postilla. AND K. Zoologica. Phylogeny and taxonomy of fossil horseshoe crabs.. S. 1958. NAKAMURA. Soc. SEKIGUCHI. Genetic variation in the horseshoe crab (Limulus polyphemus). 15:24-30. K." Evolution 24:402-414. AND M. SEN. K. SELANDER. 1952. T. Nat.. 47:1-8. C. 244:4406-4412. Syst. K. 1974. 78:713-718. YANG. OSBORN. SESHIMO.. Paleontol. J. the scorpion (Heterometrus sp.. Morphological variation of a horseshoe crab. in Chonburi area of Thailand. JR. SUGITA dence among horseshoe "crabs" (Limulidae). NISHIWAKI.12 on Mon. J. 13:39-45. Zool. Exp. WATERMAN. JOHNSON. E. an Indonesian horseshoe crab (Xiphosura). tion. 1978. J. K. SEKIGUCHI.
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