systematics and hybridization in the four hsc

Systematics and Hybridization in the Four Living Species of Horseshoe CrabsAuthor(s): Koichi Sekiguchi and Hiroaki SugitaSource: Evolution, Vol. 34, No. 4 (Jul., 1980), pp. 712-718Published by: Society for the Study of EvolutionStable URL: http://www.jstor.org/stable/2408025 .Accessed: 10/03/2014 17:56

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Evolution, 34(4), 1980, pp. 712-718

SYSTEMATICS AND HYBRIDIZATION IN THE FOUR LIVING SPECIES OF HORSESHOE CRABS'

KoICHI SEKIGUCHI AND HIROAKI SUGITA Institute of Biological Sciences, the University of Tsukuba,

Sakura-mura, Niihari-gun, Ibaraki 305, Japan

Received October 3, 1979

The horseshoe crabs, or Xiphosura, are the striking "living fossils" in the present sea. These living species are very similar to a fossil specimen, Mesolimulus walchi, which was found in the Jurassic litho- graphic shale of Germany. Therefore, it is considered that the horseshoe crabs have undergone little morphological evolution during their 200-million-year history. The fossil Xiphosura is known from all geological systems from the Cambrian onward (St0rmer, 1952).

The recent horseshoe crabs are divided into two subfamilies by their distribution and morphological characteristics (Po- cock, 1902). One is Limulinae, including Limulus polyphemus which is confined to the waters along the east coast of North America, and the other is Tachypleinae, including Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda, which are confined to the waters along the southeast and east coasts of Asia. As for the Moluccan horseshoe crab, T. hoeveni, recorded by Pocock (1902), Waterman (1958) compared it with T. gigas and con- cluded that only a single species should be recognized. Thus, the four species mentioned above are the only members of living Xiphosura. In order to make clear the relationships of the four species, some em- bryological, morphological, ecological, and biochemical studies have been carried out in our laboratory (Sekiguchi, 1973; Sekiguchi et al., 1976, 1977, 1978; Shi- shikura and Sekiguchi, 1978).

In the present paper, the systematic relationships of horseshoe crabs are studied

1 Contributions from the Shimoda Marine Re- search Center, No. 353.

by experimental hybridization and are discussed with special reference to the developmental capacity and hemocyanin monomer constituents of interspecific hybrids.

MATERIALS AND METHODS

The Japanese horseshoe crabs, Tachy- pleus tridentatus, were collected from Im- ari and Kasaoka, Japan and the Southeast Asian horseshoe crabs, T. gigas and Car- cinoscorpius rotundicauda, were kindly provided by Dr. Smarn Srithunya (Zoo- logical Museum, Srinakharinwirot Uni- versity, Thailand). The American horseshoe crabs, Limulus polyphemus, were obtained from the Supply Department of the Marine Biological Laboratory, Woods Hole, Massachusetts, and maintained in circulating seawater of 16 C at the Shi- moda Marine Research Center, the Uni- versity of Tsukuba. The Asian species were maintained in running seawater at room temperature.

The eggs were artificially inseminated between April and August and kept at 30 C. In order to decide whether they were fertilized or not, the appearance of red spots consisting of fine granules around divided nuclei was examined by vital staining using neutral red (Sekiguchi, 1960, 1973). Fertilized eggs of T. gigas, C. rotundicauda, and the interspecific hybrids developed into swimming larvae through almost the same morphological process as those of T. tridentatus except that they developed faster than T. tridentatus, so the developmental stage of the embryo was determined according to the normal plate of the Japanese horseshoe crab, Tachypleus tridentatus, described by Sekiguchi (1973).

712


HYBRIDIZATION IN FOUR HORSESHOE CRABS 713

TABLE 1. Fertilizability (fertilizedlinseminated, 9').-, the appearance of red spots consisting of fine granules around divided nuclei was not observed when examined by vital staining using neutral red. T.t., Tachypleus tridentatus. T.g., T. gigas, C.r., Carcinoscorpius rotundicauda. L.p., Limulus polyphemus.

Male

T.t. T.g. C.r. L.p.

T.t. 89 97 91 - 73 T.g. 95 97 80 - a Cr. 89 99 88

> L.p. - - 99

For examination of the hemocyanin constituent each embryo at Stage 21 (be- fore hatching out, the same form as the trilobite larva) was cut with a pair of scis- sors in about 0.2 ml of a phosphate buff- ered saline (0.0074 M Na2HPO4 12H20, 0.0026 M NaH2PO4 2H2O, 0.145 M NaCl, pH 7.1) and this extract was immediately subjected to electrophoresis according to the method of Davis (1964). Protein in polyacrylamide gels was fixed and stained overnight according to the method of Waber and Osborn (1969). He- mocyanin bands containing copper atoms were detected by the method of Horn and Kerr (1969).

RESULTS Developmental Capacity of the Hybrids

The numbers of eggs used were, at rough estimate, as follows: 8,000 eggs of T. tridentatus, 6,000 eggs of T. gigas, 4,000 eggs of C. rotundicauda, and 16,000 eggs of L. polyphemus. The diameters of these eggs were about 3.0 mm (T. tridentatus), 3.7 mm (T. gigas), 2.5 mm (C. rotundicauda), and 1.8 mm (L. polyphemus). Eggs obtained from one female were divided into four groups, and each group was artificially inseminated by sperm from one of the four species. This experimental insemination was carried out at least four times. We did not consider it reasonable to calculate average fertilizability (fertilized eggs/inseminated eggs) and average hatchability (hatched eggs/fertilized eggs)

TABLE 2. Hatchability (hatched/fertilized, %'). T. t., Tachypleus tridentatus. T.g., T. gigas. C.r., Car- cinoscorpius rotundicauda. L.p., Limulus polyphemus.

Male

T.t. T.g. C.r. L.p.

T.t. 91 0 86 0 g T.g. 59 88 0 0 a C.r. 88 0 84 0

> L.p. 0 0 0 97

from all data we obtained because T. gigas and C. rotundicauda sometimes contained many bad eggs which could not be distin- guished from healthy ones immediately after the insemination. They finally died and gathered mold. Therefore, the data presented in Tables 1 and 2 are some ex- amples carried out in the best conditions.

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. The fertilizability of T. tridentatus eggs by T. gigas or C. rotundicauda sperm was as high as the fertilizability by the T. tridentatus sperm. T. gigas and C. rotundicauda eggs were fertilized by T. tridentatus, T. gigas, or C. rotundicauda sperm at almost the same rate as T. tridentatus eggs. In contrast with these results, L. polyphemus sperm could not develop the eggs of Asian species and the sperm from Asian species could not develop L. polyphemus eggs. In a single experiment of six trials, 13% of L. polyphemus eggs were fertilized by T. gigas sperm and developed

-until blastula stage (this result is not in- cluded in Table 1). Most of fertilized eggs shown in Table 1 developed to blastula (Stage 6) irrespective of the species pro- viding sperm. Therefore, the fertilizability in each experiment was about equal to the values of blastula eggs per inseminated eggs.

Average hatchability could not be determined by the criteria mentioned above. The values shown in Table 2 are representative ones obtained by using eggs and


714 K. SEKIGUCHI AND H. SUGITA

T. t. . g T.g.

FIG. 1. Combinations of artificial insemination and developmental capacity of hybrids. Eggs of species at an arrow head were inseminated by sperm of species at an arrow butt. Stout arrow = fertilized eggs developed into swimming larvae. Slender arrow = eggs stopped their development at blastula stage. Dashed-line arrow = red spots consisting of fine granules around divided nuclei were not observed, indicating that fertilization did not take place. T.t., Tachypleus tridentatus. T.g., T. gigas. C .r., Carcinoscorpius rotund icauda . L. p., Limulus polyphemus.

sperm which looked apparently most healthy.

The hybrids of T. tridentatus Y x C. rotundicauda d and the reciprocal cross were grown into swimming larvae. Their hatchabilities were as high as those of nor- mally inseminated larvae (more than 80%). The hatchability of T. gigas Y x T. tridentatus d hybrids was about 59% but the reciprocal hybrids could not develop beyond the blastula stage in spite of the high fertilizability. The development of interspecific hybrids between T. gigas and C. rotundicauda was also stopped at blastula stage while the fertilizability of them was very high. In the case of hybridization between L. polyphemus and the other three species, the inseminated eggs did not show any sign of development although the eggs and sperm were in very good condition. Judging from the obser- vation by vital staining, nuclear division does not occur in Limulus or Asian Xi- phosura eggs which are interspecifically hybridized by using either Asian or Lim- ulus sperm.

Developmental capacities of these interspecific hybrids are summarized in Fig- ure 1.

Phenotypic Appearance of First-instar Larvae

There are obvious phenotypic differ- ences in the appearance of larvae between T. tridentatus and C. rotundicauda. The most striking is the chocolate-brown colored area along the ophthalmic ridge of T. tridentatus larva versus the chocolate- brown colored areas along the marginal edges of prosoma and abdomen of C. rotundicauda larva (Fig. 2). The hybrids of T. tridentatus Y x C. rotundicauda d and the reciprocal hybrids have colored areas originating from both parents (Fig. 2). Furthermore, the T. gigas Y x T. tridentatus d hybrids have the colored ridge like T. tridentatus, although the first-instar larva of T. gigas does not bear the chocolate-brown color in comparison with T. tridentatus and C. rotundicauda larvae (data not shown).

Hemocyanin Monomer Patterns in Electrophoresis

When Chelicerata hemocyanins were electrophoresed by using the buffer system of Davis (1964), hemocyanin molecules were dissociated into monomers (Sugita and Sekiguchi, 1975; Lamy et al., 1977). The hemocyanin extract was prepared in- dividually from the embryos of T. tridentatus, T. gigas, C. rotundicauda, and the hybrids. The electrophoretic pattern of the protein in individuals was uniform within each species. Hemocyanin monomers in these protein bands were identified by its greenish-gray color developed by copper protein (Horn and Kerr, 1969). Antisera against pure hemocyanins of three Asian horseshoe crabs were prepared and comr- parative studies of hemocyanin monomers have been carried out. It was found that the monomers asterisked in Figure 3 of T. tridentatus, T. gigas, and C. rotundicauda were immunologically identical (results will be presented elsewhere) and had the same mobility in polyacrylamide-gel electrophoresis. In order to make clear the rel-



FIG. 2. The first-instar larxae of hybrids and the parental species. a, Tachvpleius tride>ntatus. b, hybrid between T. tridentatus ? and Carcinoscorpius rotnzdicaiuda 6. c, hybrid betwveen C. rotnitdicazda z and T. tridentatus 6. d, C. rotunldicaiuda.

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. From these electrophoretic patterns and photographs we conclude that hybrids have two complete sets of hemocyanin monomers, derived from both parents.

DISCUSSION Systematics and Hybridization

Natural hybrids and experimental hybrids have been examined in many kinds of animals to study evolutionary relationships. For example, studies using experimental hybridization have clearly revealed the phyletic relationships of European green frogs (Berger, 1973) and

Palearctic pond frogs (Kawamura and Nishioka, 1975). In the case of horseshoe crabs, the techniques of backcross and second filial hybridization are not utiliza- ble because it takes more than eight years for the animals to reach maturity (Barthel, 1974). Therefore, the relation between the developmental capacity of hybrids and the phyletic line was examined as the second best way.

Wilson et al. (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. They discussed the relation between the evolutionary changes in anato- my and in mechanisms of gene expression



A B

a fa b c d e f

FIG. 3. Polyacrylamide-gel electrophoretic patterns of hemocyanin extracts (A) and of their hemocyanin monomers (B). a, Tachypleus gigas. b, hybrid between T. gigas Q and T. tridentatus d. c, T. tridentatus. d, hybrid between T. tridentatus 9 and Carcinoscorpius rotundicauda d. e, hybrid between C. rotundicauda 9 and T. tridentatus d. f, C. rotundicauda. Asterisked hemocyanin monomers of T. gigas, T. tridentatus, and C. rotundicauda are immunologically identical with each other.

and proposed that the marked restriction of interspecific hybridization among mammals occurred because the mammals, in contrast with the frogs, had experienced rapid evolutionary change in the systems regulating the expression of genes. Fur- thermore, they suggested that evolutionary loss of ability for two species to hy- bridize probably resulted from the accumulation of incompatibilities between the two systems to regulate the expression of genes during embryonic development. 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. Thus, the closer the taxonomic relationship between the parental species is, the further the development of their hybrids advances. 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, the relation between T. tridentatus and C. rotundicauda is closer than the relation between T. tridentatus and T. gigas because the hybrid between the former species developed further than that between the latter ones. Tachypleus gigas and C. rotundicauda, which are living to- gether in waters along the southeast coast of Asia, are distantly related species among the Asian Xiphosura. The Ameri- can species, L. polyphemus, is considered to be isolated from any species of Asia in almost equal degree. This conclusion is supported by results obtained from im- munocomparative study of coagulogen of horseshoe crabs (Shishikura and Sekigu- chi, 1978) and by serological comparison among horseshoe crabs (Shuster, 1962).

Phenotypic Appearance and Gene Regulatory System

Selander et al. (1970) detected protein polymorphism using many enzymes of



American horseshoe crab, L. polyphemus, and compared the gene variation of sev- eral animal species, that is, horseshoe crabs, mice, frogs, fruit flies, and man. They reported L. polyphemus, which is a "phylogenetic relic," is not less genetically variable than some other animals belong- ing to horotelic lines. The impressive sta- bility of external morphological features manifested by the fossil since 200 million years ago may be explained in the terms of temporal uniformity of the organism- environment relationship and/or genetic homeostasis (Selander et al., 1970), and consequently the gene regulatory systems whose changes may provide the basis for anatomical evolution (Wilson et al., 1974) may have remained almost unchanged in the 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. This is also the case with morphological characteristics of the hybrids, that is, they developed all colored areas originating from both parents. These results mean that the maternal and paternal genes of hemocyanin monomers and, probably, the genes participating in the color development were expressed equally in any hybrid which developed to a swimming larva. These data suggest that the parental species of hybrids have very similar mechanisms for regulating gene expression during embryonic development. This similar- ity in the mechanisms of the gene regulation may be one of the reasons why the horseshoe crab has undergone little morphological evolution during its 200- million-year history.

SUMMARY To examine evolutionary relationships

of horseshoe crabs, which are "phylogenetic relics," experimental hybridization among all living species was carried out. From the developmental capacity of hybrids we conclude that the American horseshoe crab, Limulus polyphemus, is distantly isolated from the Asian horse-

shoe crabs, Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda, and that the two genera of Asian horseshoe crabs are closely related to each other. 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.

ACKNOWLEDGMENTS We thank Dr. Smarn Srithunya for col-

lecting Tachypleus gigas and Carcinoscor- pius rotundicauda in Thailand. We also thank Dr. Tamio Hirabayashi for the re- vision of the manuscript. This work was supported in part by a grant-in-aid for sci- entific research from the Ministry of Ed- ucation of Japan.

LITERATURE CITED

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BERGER, L. 1973. Systematics and hybridization in European green frogs of Rana esculenta com- plex. J. Herpetol. 7:1-10.

DAVIS, B. J. 1964. Disc electrophoresis-I1. Meth- od and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.

HORN, E. C., AND M. S. KERR. 1969. The hemo- lymph proteins of the blue crab, Callinectes sapi- dus-1. Hemocyanins and certain other major protein constituents. Comp. Biochem. Physiol. 29:493-508.

KAWAMURA, T., AND M. NISHIOKA. 1975. On the pond frogs in the Palearctic region, with special reference to the isolating mechanisms between different species. Proc. Jap. Soc. Syst. Zool. 11:61-78.

LAMY, J., J. LAMY, M.-C. BAGLIN, AND J. WEILL. 1977. Scorpion hemocyanin subunits: Properties, dissociation, association, p. 37-49. In J. V. Bannister (ed.), Proceedings in Life Sci- ences: Structure and Function of Haemocyanin. Springer-Verlag, Berlin.

POCOCK, R. I. 1902. The taxonomy of recent species of Limulus. Ann. Mag. Nat. Hist. Ser. 7, 9:256- 266.

SEKIGUCHI, K. 1960. Embryonic development of the horse-shoe crab studied by vital staining. Bull. Mar. Biol. St. Asamushi 10:161-164.

. 1973. A normal plate of the development of the Japanese horse-shoe crab, Tachypleus tridentatus. Sci. Rep. Tokyo Kyoiku Daigaku Sec. B. 15:153-162.



SEKIGUCHI, K., K. NAKAMURA, T. K. SEN, AND H. SUGITA. 1976. Morphological variation and distribution of a horseshoe crab, Tachypleus gigas, from the Bay of Bengal and the Gulf of Siam. Proc. Jap. Soc. Syst. Zool. 12:13-20.

SEKIGUCHI, K., K. NAKAMURA, AND H. SESHIMO. 1978. Morphological variation of a horseshoe crab, Carcinoscorpius rotundicauda, from the Bay of Bengal and the Gulf of Siam. Proc. Jap. Soc. Syst. Zool. 15:24-30.

SEKIGUCHI, K., S. NISHIWAKI, T. MAKIOKA, S. SRITHUNYA, S. MACHJAJIB, K. NAKAMURA, AND T. YAMASAKI. 1977. A study on the egg-laying habits of the horseshoe crabs, Tachypleus gigas and Carcinoscorpius rotundicauda, in Chonburi area of Thailand. Proc. Jap. Soc. Syst. Zool. 13:39-45.

SELANDER, R. K., S. Y. YANG, R. C. LEWONTIN, AND W. E. JOHNSON. 1970. Genetic variation in the horseshoe crab (Limulus polyphemus), a phylogenetic "relic." Evolution 24:402-414.

SHISHIKURA, F., AND K. SEKIGUCHI. 1978. Com- parative study on horseshoe crab coagulogens. J. Exp. Zool. 206:241-246.

SHUSTER, C. N., JR. 1962. Serological correspon-

dence among horseshoe "crabs" (Limulidae). Zoologica, N.Y. Zool. Soc. 47:1-8.

ST0RMER, L. 1952. Phylogeny and taxonomy of fossil horseshoe crabs. J. Paleontol. 26:630-639.

SUGITA, H., AND K. SEKIGUCHI. 1975. Heteroge- neity of the minimum functional unit of hemocyanins from the spider (Argiope bruennichii), the scorpion (Heterometrus sp.), and the horseshoe crab (Tachypleus tridentatus). J. Biochem. 78:713-718.

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Corresponding Editor: A. E. Milkman


Article Contentsp. 712p. 713p. 714p. 715p. 716p. 717p. 718

Issue Table of ContentsEvolution, Vol. 34, No. 4 (Jul., 1980), pp. 617-824Front MatterDensity-Dependent and Density-Independent Causes of Extinction of a Salamander Population [pp. 617-621]The Evolutionary Significance of Bumble Bee Color Patterns: A Mimetic Interpretation [pp. 622-637]Morphometric Differentiation in New Zealand Populations of the House Sparrow (Passer domesticus) [pp. 638-653]Chromosomal and Morphological Correlates in Two New World Sparrows (Emberizidae) [pp. 654-662]Maintenance of the Three Sex Chromosome Polymorphism in the Platyfish, Xiphophorus maculatus [pp. 663-672]Some Characteristics of Hybrids Derived from the Sulfur Butterflies, Colias eurytheme and C. philodice: Phenotypic Effects of the X-Chromosome [pp. 673-687]The Effect of X-Chromosome Inheritance on Mate-Selection Behavior in the Sulfur Butterflies, Colias eurytheme and C. philodice [pp. 688-695]Population Genetics of Chironomus stigmaterus Say (Diptera: Chironomidae): II. Protein Variability in Populations of the Southwestern United States [pp. 696-704]Genetic Divergence Among Fishes of the Eastern Pacific and the Caribbean: Support for the Molecular Clock [pp. 705-711]Systematics and Hybridization in the Four Living Species of Horseshoe Crabs [pp. 712-718]Modes of Speciation and Inferences Based on Genetic Distances [pp. 719-729]A Multifactorial Genetic Investigation of Speciation Theory Using Drosophila melanogaster [pp. 730-737]Reproductive Isolation in Asclepias: Lock and Key Hypothesis Reconsidered [pp. 738-746]Environmental Determinants of Outcrossing in Impatiens capensis (Balsaminaceae) [pp. 747-761]Sexual Dimorphism in the Strawberry Fragaria chiloensis [pp. 762-768]Components of Fitness and Frequency-Dependent Overdominance in Hermaphrodite Plants [pp. 769-778]Heterozygosity of a Neutral Locus Linked to a Self-Incompatibility Locus or a Balanced Lethal [pp. 779-788]Gene Flow, Effective Population Sizes, and Genetic Variance Components in Birds [pp. 789-798]Group Selection: The Phenotypic and Genotypic Differentiation of Small Populations [pp. 799-812]Group Selection: The Genetic and Demographic Basis for the Phenotypic Differentiation of Small Populations of Tribolium castaneum [pp. 813-821]Book ReviewReview: untitled [pp. 822-824]

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systematics and hybridization in the four hsc

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