Translator Disclaimer
1 October 2002 Chromosomes of Japanese Starfishes
Author Affiliations +
Abstract

We developed a method for preparing starfish chromosomes from embryos. Blastulae were treated with colchicine (0.2–4.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7% sodium citrate, and fixed with methanol: acetic acid (3:1). The fixed cells were dropped on a slide and air-dried. We examined the chromosome number in five species of asteroids belonging to 4 families (Luidiidae, Astropectinidae, Asterinidae, and Asteriidae), and all had a diploid number of 44. We analyzed the karyotype in 4 of the species, and all were different. We visualized the nucleolus organizer regions of an Asterina species and an Asterias species and found them to be quite different from each other.

INTRODUCTION

About 1800 species of starfishes have been identified (Hendler et al., 1995; McEdward et al., 2002). While extensive morphological (Fisher, 1911, 1928, 1930; Hayashi, 1940, 1943, 1973; Spencer and Wright, 1966; Fell, 1963; Heddle, 1967; Blake, 1987; Downey, 1973; Clark and Downey, 1992), developmental (Oguro et al., 1976; Komatsu et al., 1979; McEdward, 1992; Chia et al., 1993; Byrne and Cerra, 1996), biochemical (Kubo, 1961; Schopf and Murphy, 1973; Mochizuki and Hori, 1980; Matsuoka, 1981, Matsuoka et al., 1994), and molecular (Lafay et al., 1995; Wada et al., 1996; Knott and Wray, 2000) studies have been systematically carried out on starfishes, cytogenetic studies have not yet been applied to asteroid phylogeny.

Starfish chromosomes have been examined in early embryo sections since the start of the 20th century (Delage, 1901; Tennent and Hogue, 1906; Tennent, 1907; Jordan, 1910). In Japan, Makino and Niiyama (1947), examining testis sections showed that the diploid chromosome number of 4 species ranged from 38 to 50. Later studies with testes showed that the chromosome numbers of 8 species belonging to 4 families were 44 (Delobel, 1971; Colombera, 1974; Colombera et al., 1977; Colombera and Venier, 1980; Colombera and Tagliaferri, 1986). The constancy in chromo-some number necessitates karyotypic comparison, but chromosomes from starfish testes are too small to permit karyo-type analysis. Since I have been able to obtain clear chromosome preparations from sea urchin embryos by the air-drying method (Saotome, 1987), we thought it might be possible to modify the method and apply it to starfish embryos.

In this paper, we describe a method for preparing starfish chromosomes from embryos, and the results of karyo-type and NOR analysis.

MATERIALS AND METHODS

Materials

We used 5 species belonging to 4 families of asteroids and one species of ophiuroid (Table 1). We removed testes and ovaries from adults and immersed them in artificial sea water (Jamarin U, Jamarin Laboratory, Osaka, Japan). We treated the ovaries with 1–2 μM 1-methyladenine for 30 min (Sigma, St Louis, Mo., USA) to induce breakdown of germinal vesicles and obtain mature eggs (Kanatani, 1969). When the motility of sperm released in sea water was poor, we improved it by adding L-histidine (0.1 mM) (Wako Pure Chemical Industries, Osaka, Japan) to the sperm suspension. Eggs were inseminated before extrusion of the second polar body. The embryos (2000 per ml sea water) were cultured at room temperature and only Asterias amurensis embryos at 10–13°C. In Asterina minor and Amphipholis kochii, the several adults cultured at room temperature were transferred to small beakers (200–300 ml), kept at 4–10°C for 1–2 hr, and then returned to room temperature (Yamashita, 1983). We harvested the embryos, which were naturally born during overnight.

Table 1

Asteroid and ophiuroid species analyzed in this study.

i0289-0003-19-10-1095-t01.gif

Chromosome preparation

Embryos (1×104) at the early blastula stage (250–500 cell stage) or wrinkled blastulae (20–30 embryos) in A. minor were treated with colchicine (0.2–4.0 mg/ml) (Sigma, St Louis, Mo., USA), sampled 4–5 times at 5–30 min intervals before or after a division, collected by centrifugation (300 ×g×5 min), suspended in 1M urea, and dissociated into their component blastomeres by pipetting. The fertilization membranes were simultaneously removed. The colchicine concentration, sampling interval time, and sampling numbers varied with species. A. minor embryos, which have large eggs containing a lot of yolk, were suspended in 0.5 ml sea water in 35 mm dishes, beaten slightly with a bamboo spit (2 mm in diameter), and dissociated into their blastomeres. The dissociated blastomeres were collected by centrifugation, swollen with 7% sodium citrate for 10 min, fixed with methanol: acetic acid (3:1), and washed twice with the fixative. The fixed cells were air-dried (Saotome, 1982a), and the preparations were stained for 10 min with 3% Giemsa solution (Merck, Whitehouse Station, NJ, USA) diluted with phosphate buffer (pH 6.9). We used more than 6 pairs of adults and prepared chromosomes through 2–5 breeding seasons. Since we used only one pair and prepared chromosomes for only one season for Luidia maculata and A. kochii, their data were shown as preliminary ones.

Others

We visualized the nucleolus organizer regions (NORs) by the silver-staining method of Howell and Black (1978), with slight modifications. The preparations stained with Giemsa were destained with methanol after observation, and then stained with 50% silver nitrate solution for 2–3 min at 50–57°C (Saotome, 1991).

We classified chromosomes by arm ratio on the basis of the nomenclature of Levan et al. (1964) into metacentric (m), submetacentric (sm), subtelocentric (st) and telocentric (t) chromosomes. We divided chromosomes into large-sized ones (L) and mediumsized ones (M).

RESULTS

Chromosome preparation

We examined stage of preparation, concentration of colchicine and hypotonic solution, dissociation into single blastomeres, and composition of fixative to establish preparing conditions. Good chromosome preparations could be obtained, when blastulae were treated with colchicine (0.2–4.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7% sodium citrate, and fixed with methanol: acetic acid (3:1).

Chromosomes

Fig. 1a–e show typical metaphase chromosomes from starfish embryos of 5 species. The chromosomes spread over one layer, which made it possible to count them and to analyze the karyotype. The chromosomes were small, ranging in length from 1 μm to 5 μm. Fig. 1f indicates meiotic chromosomes from Asterina minor testes treated with colchicine by air-drying.

Fig. 1

Typical metaphase chromosomes from the embryos of 5 asteroid species. a) Luidia maculata, b) Astropecten scoparius, c) Asterina pectinifera, d) Asterina minor, e) Asterias amurensis, and f) typical meiotic chromosomes from Asterina minor testes. Bar, 5 μm.

i0289-0003-19-10-1095-f01.tif

The modal diploid chromosome numbers in all 4 starfish species were 44 (Fig. 2a–d); the modal haploid number in A. minor testes was 22 (Fig. 2e). Preliminary data in Luidia maculata indicated that 39 (53%) out of 73 cells also had a diploid chromosome number of 44.

Fig. 2

Distribution of chromosome number in embryos of 4 asteroid species, a) Astropecten scoparius (n=72 cells), b) Asterina pectinifera (n=215 cells), c) Asterias amurensis (n=181 cells), d) Asterina minor (n=170 cells), and e) that in Asterina minor testes (n=56 cells).

i0289-0003-19-10-1095-f02.tif

Karyotypes

Asteroids

Astropecten scoparius was characteristic in having 2 pairs of large subtelocentric chromosomes. The karyotype consisted of 9 pairs of metacentric, 7 pairs of submetacentric, and 6 pairs of subtelocentric chromosomes (Fig. 3). Asterina pectinifera had one pair of medium-sized metacentric, 10 pairs of metacentric, and 9 pairs of submetacentric chromosomes, and one pair each of subtelocentric and telocentric chromosomes (Fig. 4a). A. minor showed one pair of medium-sized metacentric, 14 pairs of metacentric, 6 pairs of submetacentric, and one pair of telocentric chromosomes (Fig. 4b). Asterias amurensis had 12 pairs of metacentric, 7 pairs of submetacentric, and 3 pairs of telocentric chromosomes (Fig. 5). Preliminary data for L. maculata showed that it had about 6 pairs of metacentric, two pairs of large submetacentric, about 6 pairs of submetacentric and about 8 pairs of subtelocentric chromosomes.

Fig. 3

Karyotype of Astropecten scoparius. m=metacentric, sm=submetacentric, st=subtelocentric chromosome Bar, 5 μm

i0289-0003-19-10-1095-f03.tif

Fig. 4

Karyotypes of two Asterinidae species. a) Asterina pectinifera and b) Asterina minor. t=telocentric chromosome Bar, 5 μm

i0289-0003-19-10-1095-f04.tif

Fig. 5

Karyotype of Asterias amurensis. Bar, 5 μm

i0289-0003-19-10-1095-f05.tif

Ophiuroids

We tried to prepare chromosomes from several ophiuroid species but succeeded only with Amphipholis kochii (Fig. 6). We made only three preparations because of the difficulty in getting mature adults and harvesting well-developed embryos. The preliminary results indicated that 42 (72%) out of 58 cells had a diploid chromosome number of 42, which had three pairs of metacentric, three pairs of submetacentric, 7 pairs of subtelocentric, and 8 pairs of telocentric chromosomes (Fig. 7).

Fig. 6

Preliminary metaphase chromosomes from Amphipholis kochii embryos. Bar, 5 μm

i0289-0003-19-10-1095-f06.tif

Fig. 7

Preliminary karyotype from Amphipholis kochii embryos. Bar, 5 μm

i0289-0003-19-10-1095-f07.tif

NORs

The NORs of A. pectinifera occurred on two regions. One was large (long arrow) and located on the long arm of a metacentric chromosome pair, while the other was small (short arrow) and located on a terminal position of a telocentric chromosome pair (Fig. 8a). The NORs of A. amurensis were located on a terminal position of three telocentric chromosome pairs (arrows in Fig. 8b).

Fig. 8

NORs in a) Asterina pectinifera and b) Asterias amurensis. Arrows indicate Ag-NORs.

i0289-0003-19-10-1095-f08.tif

DISCUSSION

In this paper, we reported a method for preparing starfish chromosomes from embryos. The timing of the preparation was critical because colchicine inhibited cytoplasmic division but did not stop nuclear division. Indeed, we observed many polyploid cells after long colchicine treatment, and were not able to obtain metaphase plates in those shortly after the stoppage of cleavage. Many metaphase plates could be prepared during the M phase at the early blastula stage (250 or 500 blastomeres). In Asterina minor eggs, which are large and contain a lot of yolk, wrinkled blastulae were good for preparation. Though we do not understand why colchicine did not stop nuclear division, we were not able to obtain good metaphase plates without it. Colchicine may affect chromosome morphology. We could prepare well-spread chromosomes from A. minor testes (Fig. 1f) and count clearly chromosome number of 22 as a haploid number (Fig. 2e), but not analyze karyotype owing to chromosome smallness. Though karyotype analysis might be also difficult in the metaphase plates from testes of other species (Colombera, 1974; Colombera et al., 1977; Colombera and Venier, 1980; Colombera and Tagliaferri, 1986), we found that chromosome preparations from embryos were good to analyze karyotype.

The diploid chromosome number was 44 in all 5 asteroid species we studied. Since all 8 species (Astropecten bispinosus, Astropecten arantiacus, Ophidiaster ophidianus, Hacelia attenuata, Asterina gibbosa, Asterina pectinifera, Asterina coronata japonica, and Echinaster sepositus) belonging to 4 families reported until now also had a diploid chromosome number of 44, (Delobel, 1971; Colombera, 1974; Colombera et al., 1977; Colombera and Venier, 1980; Colombera and Tagliaferri, 1986), the basic diploid chromo-some number for asteroids may be 44. The high aneuploidy frequency (17%–22%) we observed may have been caused by contamination of embryos showing polyspermy, which induces irregular cleavage and results in abnormal chromo-some separation (Saotome, 1982b). When adults matured fully, good chromosome preparations could be obtained owing to the high rate of germinal vesicle breakdown and fertilization, and synchronous cleavage. When they did not mature fully, however, the fertilization rate was low and cleavage was not synchronous. In those cases, the addition of more concentrated sperm solution was apt to induce polyspermy. The cases showing polyspermy, of course, K. Saotome and M. Komatsu were not used for chromosome preparations.

The karyotypes from 5 species were compared on the basis of chromosome size and arm ratio. The karyotype of Astropecten scoparius (chromosome formula; (9 m+7 sm+(2 L+4) st) belonging to Astropectinidae, differed from the karyotypes of Asterina pectinifera ((1M+10) m+9 sm+1 st+1 t) and Asterina minor ((1M+14) m+6 sm+1 t) (both Asterinidae) as well as Asterias amurensis (12 m+7 sm+3 t) (Asteriidae) (Fig. 3, 4, 5). Astropectinidae is distinct from Asterinidae and Asteriidae morphologically (Hayashi, 1974) and immunologically (Mochizuki and Hori, 1980), which is consistent with the karyological difference. The A. scoparius karyotype may be close to the karyotype of L. maculata (about 6 m+(2L+about 6) sm+about 8 st), belonging to Luidiidae. Paleontological evidence suggests that the order Platyasterida (Luidiidae) comprises the more primitive starfish from which the Paxillosida (Astropectinidae) diverged (Fell, 1963; Spencer and Wright, 1966). Moreover, A. scoparius resembles L. maculata in its nonbrachiolarian mode of development (Oguro et al., 1976). These relationships may be consistent with the similarity in their karyotypes, but clear karyotype analysis is needed to clarify them. The karyotypes of A. pectinifera and A. minor were similar but differed in number of meta- and submetacentric chromosomes. Though A. pectinifera and A. minor belong to the same genus, they differ in the size and yolk content of eggs and in type of larval development–indirect (A. pectinifera; Komatsu, 1984) VS. direct (A. minor ; Komatsu et al. 1979). Immunological data indicate that A. pectinifera differs considerably from A. minor compared with other species belonging to genus Asterina (Mochizuki and Hori, 1980; Matsuoka, 1981). Those developmental and immunological differences may be correlated with karyological differences. Structural changes, such as inversion may have occurred between meta- and submetacentric chromosomes in the course of evolution. The karyotype of A. amurensis was quite different from that of other three species we examined. Asteriidae is a well- characterized family, and well-defined by the possession of both straight and crossed pedicellariae and compressed ambulacrals, by which it is distinguished from all other asteroids (Fisher, 1911; Clark and Downey, 1922; Hyman, 1955; Spencer and Wright, 1966; Blake, 1987; Gale, 1987). Asteriidae is also well differentiated from the other families immunologically (Mochizuki and Hori, 1980). These morphological and immunological differences are consistent with the karyological one. A. pectinifera and A. amurensis are similar in larval development (indirect type), but differ morphologically. We were also able to distinguish them by karyotype and NORs (Fig. 8). We could visualize NORs in only 2 species out of 5 asteroid species. There was variation in number of NORs. 10 cells (45.5%) out of 22 cells had 4 Ag-NOR sites in A. pectinifera and cells of 57% (4/7 cells) had 6 Ag-NOR sites in A. amurensis. The NORs on one pair of telocentric chromosomes were common to A. pectinifera and A. amurensis, but NORs on one pair of metacentric chromosomes was specific for A. pectinifera. Further data about NORs in other species may make it possible to discuss asteroid phylogeny. We, furthermore, have to compare karyotypes in many species belonging to each order to discuss phylogeny at the order level.

The karyotype figures in this paper show paired chromosomes but we also observed a pair of heterotypic chromosomes, which might represent sex chromosomes. In A. pectinifera, about half of karyotypes examined had heterotypic chromosomes (data are not shown). More detailed examination by several banding and FISH methods is needed to determine whether the asteroids indeed have sex chromosomes.

Some palentological evidence suggests that the ophiuroids are more closely related to the asteroids than to the echinoids (Fell, 1963; Spencer and Wright, 1966). Our preliminary data showed that the ophiuroid A. kochii (Amphiuridae) had a diploid chromosome number of 42 (Fig. 6). Since diploid chromosome number of Ophiomyxa pentagona (Ophiomyxidae) and Macrophiothrix longipeda (Ophiotrichidae) has been reported to be 42, and that of Ophiothrix fragilis (Ophiotrichidae), Ophioderma longicauda (Ophiodermatidae), and Ophiocomina nigra (Ophiocomidae) is 44 (Colombera, 1974; Colombera and Tagliaferri, 1986), chromosome numbers of 42 and 44 may exist in ophiuroids. A. kochii had many subtelocentric and telocentric chromosomes (Fig. 7), which contrasted with the karyotypes of asteroids. Since we found considerable difference in karyo-type between asteroids and ophiuroids, karyotypic comparison may be an important phylogenetic tool.

To elucidate chromosome evolution in asteroids and ophiuroids, we need chromosomal data from many species. This method described here is applicable to species from which we can consistently obtain mature eggs and sperm, fertilized eggs, and well-developed embryos.

Acknowledgments

We are grateful to the staff of the Misaki Marine Biological Station, University of Tokyo, Kanagawa Prefecture, the Tateyama Marine Biological Station, Ochanomizu University, Chiba Prefecture, and the Amakusa Marine Biological Laboratory, Kyushu University, Kumamoto Prefecture, for kindly supplying materials.

REFERENCES

1.

D. B. Blake 1987. A classification and phylogeny of post-Palaeozoic sea stars (Asteroidea: Echinodermata). J Nat Hist 21:481–528. Google Scholar

2.

M. Byrne and A. Cerra . 1996. Evolution of intragonadal development in the diminutive asterinid sea stars Patiriella vivipara and P. parvivipara with an overview of development in the Asterinidae. Biol Bull 191:17–26. Google Scholar

3.

F. S. Chia, C. Oguro, and M. Komatsu . 1993. Sea-star (Asteroid) development. Oceanogr Mar Biol Ann Rev 31:223–257. Google Scholar

4.

A. M. Clark and M. E. Downey . 1992. Starfishes of the Atlantic. Chapman and Hall. London. Google Scholar

5.

D. Colombera 1974. Chromosome evolution in the phylum Echinodermata. Z zool Syst Evolutforsch 12:299–308. Google Scholar

6.

D. Colombera and F. Tagliaferri . 1986. The male chromosomes of five species of echinoderms together with some technical hints. Caryologia 39:347–352. Google Scholar

7.

D. Colombera and G. Venier . 1980. Il numero dei cromosomi di cinque specie di echinodermi. Caryologia 33:503–507. Google Scholar

8.

D. Colombera, G. Venier, and R. Vitturi . 1977. Chromosome and DNA in the evolution of echinoderms. Biol Zbl 96:43–49. Google Scholar

9.

Y. Delage 1901. Études expérimentales sur la maturation cytoplasmique et sur la parthénogénèse artificielle chez les échinodermes. Arch Zool Exp 9:285–326. Google Scholar

10.

N. Delobel 1971. Determination du nombre chromosomique chez une asteride: Echinaster sepositus. Caryologia 24:247–250. Google Scholar

11.

M. E. Downey 1973. Starfishes from the Caribbean and the Gulf of Mexico. Smithsonian Contr Zool 126:1–158. Google Scholar

12.

H. B. Fell 1963. The phylogeny of sea-stars. Phil Trans Roy Soc London, Ser B 246:381–435. Google Scholar

13.

W. K. Fisher 1911. Asteroidea of the North Pacific and adjacent waters. Part 1. Phanerozonia and Spinulosa. US Nat Mus Bull 76. Google Scholar

14.

W. K. Fisher 1928. Asteroidea of the North Pacific and adjacent waters. Part 2. Forcipulata (part). US Nat Mus Bull 76. Google Scholar

15.

W. K. Fisher 1930. Asteroidea of the North Pacific and adjacent waters. Part 3. Forcipulata (concluded). US Nat Mus Bull 76. Google Scholar

16.

A. S. Gale 1987. Phylogeny and classification of the Asteroidea (Echinodermata). Zool Soc Linnean Soc 89:107–132. Google Scholar

17.

R. Hayashi 1940. Contributions to the classification of the sea-stars of Japan. I. Spinulosa. J Fac Sci Hokkaido Imp Univ Ser VI Zool 7:107–204. Google Scholar

18.

R. Hayashi 1943. Contributions to the classification of the sea-stars of Japan. II. Forcipulata, with the note on the relationships between the skeletal structure and respiratory organ of the sea-stars. J Fac Sci Hokkaido Imp Univ Ser VI Zool 8:133–281. Google Scholar

19.

R. Hayashi 1973. The Sea-Stars of Sagami Bay. Ed by. Biological Laboratory Imperial Household. Tokyo. Google Scholar

20.

R. Hayashi 1974. Asteroidea. In “Systematic Zoology Vol 8b”. Ed by T. Uchida Nakayama Book Company. Tokyo. pp. 84–141. in Japanese. Google Scholar

21.

D. Heddle 1967. Versatility of movement and the origin of the asteroids. In “Echinoderm Biology”. Symp zool Soc LondEd by N. Millott Academic Press. New York. pp. 125–141. Google Scholar

22.

G. L. Hendler, J. E. Miller, D. L. Pawson, and P. M. Kier . 1995. Sea Stars, Sea Urchins and Allies. Smithsonian Institution. Washington DC. Google Scholar

23.

W. M. Howell and D. A. Black . 1978. A rapid technique for producing silver-stained nucleolus organizer regions and trypsin-Giemsa bands on human chromosomes. Hum Genet 43:53–56. Google Scholar

24.

L. H. Hyman 1955. The Invertebrates: Echinodermata. McGraw-Hill. New York. Google Scholar

25.

H. E. Jordan 1910. The relation of nucleoli to chromosomes in the egg of Cribrella sanguineolenta Lütken. Arch f Zellforsch 5:394–405. Google Scholar

26.

H. Kanatani 1969. Induction of spawning and oocyte maturation by L-methyladenine in starfishes. Exp Cell Res 57:333–337. Google Scholar

27.

K. E. Knott and G. A. Wray . 2000. Controversy and consensus in asteroid systematics: New insights to ordinal and familial relationships. Amer Zool 40:382–392. Google Scholar

28.

M. Komatsu, Y. T. Kano, H. Yoshizawa, S. Akabane, and C. Oguro . 1979. Reproduction and development of the hermaphroditic sea-star, Asterina minor Hayashi. Biol Bull 157:258–274. Google Scholar

29.

M. Komatsu 1984. Late development of Asterina pectinifera: Scanning electron microscopic observations of the skeleton and larva. Zool Sci 1:942. Google Scholar

30.

K. Kubo 1961. Studies on the systematic serology of sea-stars. V. Jpn J Zool 13:15–37. Google Scholar

31.

B. Lafay, A. B. Smith, and R. Christen . 1995. A combined morphological and molecular approach to the phylogeny of asteroids (Asteroidea: Echinodermata). Syst Biol 44:190–208. Google Scholar

32.

A. Levan, K. Fredga, and A. A. Sandberg . 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52:201–220. Google Scholar

33.

L. R. McEdward 1992. Morphology and development of a unique type of pelagic larva in the starfish Pteraster tesselatus (Echinodermata: Asteroidea). Biol Bull 182:177–187. Google Scholar

34.

L. R. McEdward, W. B. Jaekle, and M. Komatsu . 2002. Chapter 26 Phylum Echinodermata: Asteroidea. In “Atlas of Marine Invertebrate Larvae”. Ed by C. M. Young Academic Press. London. pp. 449–512. Google Scholar

35.

S. Makino and H. Niiyama . 1947. A study of chromosomes in echinoderms. J Fac Sci Hokkaido Univ Ser VI Zool 9:225–232. Google Scholar

36.

N. Matsuoka 1981. Phylogenetic relationships among five species of starfish of the genus, Asterina: an electrophoretic study. Comp Biochem Physiol 70B:739–743. Google Scholar

37.

N. Matsuoka, K. Fukuda, K. Yoshida, M. Sugawara, and M. Inamori . 1994. Biochemical systematics of five asteroids of the family asteriidae based on allozyme variation. Zool Sci 11:343–349. Google Scholar

38.

Y. Mochizuki and S. H. Hori . 1980. Immunological relationships of starfish hexokinases: Phylogenetic implication. Comp Biochem Physiol 65B:119–125. Google Scholar

39.

C. Oguro, M. Komatsu, and Y. T. Kano . 1976. Development and metamorphosis of the sea-star, Astropecten scoparius Valenciennes. Biol Bull 151:560–573. Google Scholar

40.

K. Saotome 1982a. A method for chromosome preparation of sea urchin embryos. Stain Technol 57:103–105. Google Scholar

41.

K. Saotome 1982b. Abnormality of cleavage and separation of chromosome in sea urchin embryos. The 15th annual meeting of developmental biologyAbstract, in Japanese. 77. Google Scholar

42.

K. Saotome 1987. Chromosome numbers in 8 Japanese species of sea urchins. Zool Sci 4:483–487. Google Scholar

43.

K. Saotome 1991. Chromosome number and nucleolus organizer regions of the irregular sea urchin Peronella japonica. Chromo-some Info Serv 50:32–34. Google Scholar

44.

T. J. M. Schopf and L. S. Murphy . 1973. Protein polymorphism of the hybridizing seastars Asterias forbesi and Asterias vulgaris and implications for their evolution. Biol Bull 145:589–597. Google Scholar

45.

W. K. Spencer and C. W. Wright . 1966. Asterozoans. In “Treatise on Invertebrate Paleontology Part U” Ed by R. C. Moore Geol Soc Am, Univ Kansas Press. pp. 4–107. Google Scholar

46.

D. H. Tennent 1907. Further studies on the parthenogenetic development of the starfish egg. Biol Bull 13:309–316. Google Scholar

47.

D. H. Tennent and M. J. Hogue . 1906. Studies on the development of the starfish egg. J Exp Zool 3:519–541. Google Scholar

48.

H. Wada, M. Komatsu, and N. Satoh . 1996. Mitochondrial rDNA phylogeny of the asteroidea suggests the primitiveness of the Paxillosida. Mol Phylogenet Evol 6:97–106. Google Scholar

49.

M. Yamashita 1983. Electron microscopic observations during monospermic fertilization process of the brittle-star Amphipholis kochii Lütken. J Exp Zool 228:109–120. Google Scholar
Kyoko Saotome and Mieko Komatsu "Chromosomes of Japanese Starfishes," Zoological Science 19(10), 1095-1103, (1 October 2002). https://doi.org/10.2108/zsj.19.1095
Received: 12 April 2002; Accepted: 1 July 2002; Published: 1 October 2002
JOURNAL ARTICLE
9 PAGES


SHARE
ARTICLE IMPACT
Back to Top