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Systematics and Hybridization in the Four Living Species of Horseshoe Crabs Author(s): Koichi Sekiguchi and Hiroaki Sugita

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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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 lithographic 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 (Pocock, 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 concluded 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 embryological, morphological, ecological, and biochemical studies have been carried out in our laboratory (Sekiguchi, 1973; Sekiguchi et al., 1976, 1977, 1978; Shishikura and Sekiguchi, 1978). In the present paper, 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.
MATERIALS AND METHODS

1 Contributions from the Shimoda Marine Research Center, No. 353.

The Japanese horseshoe crabs, Tachypleus tridentatus, were collected from Imari and Kasaoka, Japan and the Southeast Asian horseshoe crabs, T. gigas and Carcinoscorpius rotundicauda, were kindly provided by Dr. Smarn Srithunya (Zoological Museum, Srinakharinwirot University, 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 Shimoda Marine Research Center, the University 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).

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HYBRIDIZATION IN FOUR HORSESHOE CRABS


1. Fertilizability (fertilizedlinseminated, 9').-, the appearance of red spots consisting offine 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. TABLE Male g T.t. T.g. C.r. L.p. -

713

TABLE 2. Hatchability (hatched/fertilized, %').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.

a
>

73 a
>

T.t. T.g. Cr. L.p.

89 95 89
-

97 97 99
-

91 80 88

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

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, the same form as the trilobite larva) was cut with a pair of scissors in about 0.2 ml of a phosphate buffered 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). Hemocyanin 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)

from all data we obtained because T. gigas and C. rotundicauda sometimes contained many bad eggs which could not be distinguished from healthy ones immediately after the insemination. They finally died and gathered mold. Therefore, the data presented in Tables 1 and 2 are some examples 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 included in Table 1). Most of fertilized eggs shown in Table 1 developed to blastula (Stage 6) irrespective of the species providing 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

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T. t.

Developmental capacities of these interT.g. specific hybrids are summarized in Figure 1. Phenotypic Appearance of First-instar Larvae There are obvious phenotypic differences 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 chocolatebrown 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).

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 rotundicauda . L. p., Limulus polyphemus.

Hemocyanin Monomer Patterns in Electrophoresis When Chelicerata hemocyanins were rotundicaudad and the reciprocalcross electrophoresed by using the buffer system were grown into swimming larvae. Their of Davis (1964), hemocyanin molecules hatchabilities were as high as those of nor- were dissociated into monomers (Sugita mally inseminated larvae (more than and Sekiguchi, 1975; Lamy et al., 1977). 80%). The hatchabilityof T. gigas Y x The hemocyanin extract was prepared inT. tridentatus d hybridswas about 59% dividually from the embryos of T. tridenbut the reciprocal hybrids could not de- tatus, T. gigas, C. rotundicauda, and the velop beyond the blastula stage in spite of hybrids. The electrophoretic pattern of the the high fertilizability. The development protein in individuals was uniform within of interspecific hybrids between T. gigas each species. Hemocyanin monomers in and C. 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. In the case of hy- protein (Horn and Kerr, 1969). Antisera bridization between L. polyphemus and the against pure hemocyanins of three Asian other three species, the inseminated eggs horseshoe crabs were prepared and comrdid not show any sign of development al- parative studies of hemocyanin monomers though the eggs and sperm were in very have been carried out. It was found that good condition. Judging from the obser- the monomers asterisked in Figure 3 of T. vation by vital staining, nuclear division tridentatus, T. gigas, and C. rotundicaudoes not occur in Limulus or Asian Xi- da were immunologically identical (results phosura eggs which are interspecifically will be presented elsewhere) and had the hybridized by using either Asian or Lim- same mobility in polyacrylamide-gel eleculus sperm. trophoresis. In order to make clear the relsperm which looked apparently most healthy. The hybrids of T. tridentatus Y x C.

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HYBRIDIZATION IN FOUR HORSESHOE CRABS

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FIG. 2. The first-instar larxae of hybrids and the parental species. a, Tachvpleiustride>ntatus. b, hybrid and between T. tridentatus ? and Carcinoscorpius rotnzdicaiuda 6. c, hybrid betwveenC. rotnitdicazda z 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 utilizable 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 anatomy and in mechanisms of gene expression

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fa

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. Furthermore, 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. 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 together in waters along the southeast coast of Asia, are distantly related species among the Asian Xiphosura. The American 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 immunocomparative study of coagulogen of horseshoe crabs (Shishikura and Sekiguchi, 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

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HYBRIDIZATION IN FOUR HORSESHOE CRABS

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American horseshoe crab, L. polyphemus, and compared the gene variation of several 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 belonging to horotelic lines. 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., 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 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.
SUMMARY

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 collecting Tachypleus gigas and Carcinoscorpius rotundicauda in Thailand. We also thank Dr. Tamio Hirabayashi for the revision of the manuscript. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education of Japan.
LITERATURE CITED

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BARTHEL, K. W.

T., AND M. NISHIOKA.

1975. On the

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-

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K., K. NAKAMURA, T. K. SEN, AND H. 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. Comparative study on horseshoe crab coagulogens. J. Exp. Zool. 206:241-246. SHUSTER, C. N., JR. 1962. Serological correspon-

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