INTRODUCTION

The sea urchin Echinometra mathaei was described by Blainville (1825) on the basis of a single rather small sea urchin from Mauritius. Echinometra oblonga was also described in the same paper on the basis of two rather small individuals. The locality of the latter species was unknown. Döderlein (1906) elevated E. oblonga to a separate genus, Mortensia oblonga, because of the morphology of the spicules in the gonads, but Mortensen (1943) claimed these as morphs of E. mathaei, and gave it a trinomial name, E. mathaei oblonga, as if it were a subspecies. Clark and Rowe (1971) followed Mortensen's nomenclature. Kelso (1970) did an extensive study on the ecological distribution, test morphology, spicules in gonads, and gamete compatibility of these two morphs, E. mathaei and E. mathaei oblonga, using Hawaiian individuals. In this study he strongly argued that these two morphs were separate species, E. mathaei and E. oblonga.

E. mathaei occurs in abundance on shallow reefs throughout the tropical to warm Indo-Pacific region (Mortensen, 1943). E. mathaei is well known as a species showing extensive morphological variation in test shape and spine color (Mortensen, 1943). The Okinawan Echinometra species are no exception. Okinawan E. mathaei was divided into two types, A and B, based on spine color. These types showed different distribution patterns, habitat preference, and agonistic behavior (Tsuchiya and Nishihira, 1984, 1985). Uehara and Shingaki reported that these two types were impossible to cross-fertilize and that there were differences in larval morphology and karyotype (Uehara and Shingaki, 1984). Uehara and co-workers (Uehara and Shingaki, 1985; Uehara et al., 1986, 1990; Uehara and Taira, 1987) discovered two additional types, C and D, based on chromosome and spicule characteristics and gamete incompatibility. These studies showed that the four Okinawan types are different species. Recent biochemical studies on enzyme electrophoresis (Matsuoka and Hatanaka, 1991) and on mitochondrial DNA (Palumbi and Metz, 1991) have also suggested that these types are distinct, but very closely related, species. However, the scientific names of these four complex species found in Okinawa are still unclear. This was the reason why the individuals of Echinometra from Mauritius, the type locality of E. mathaei, and the individuals of Echinometra from Okinawa were compared. Okinawan Echinometra are tentatively described in this paper as Echinometra sp. A, B, C, and D.

The distinguishing criteria of Blainville's classification of E. mathaei and E. oblonga were based on the number of porepairs per ambulacral plate, the number of interambulacral tubercle rows, and the color of the specimen (Blainville, 1825). On the other hand, Mortensen included many criteria for his classification: spicules in gonads, tubefeet and the intestinal wall, the apical system, pedicellaria, shape and color of spines, shape and size of test and larval form (Mortensen, 1943). Overlapping criteria included the number of pore-pairs per ambulacral plate. These characteristics were not enough for describing the differences among the four Okinawan species of Echinometra. To distinguish the Okinawan Echinometra species, the characteristics observed on the live specimens, such as chromosome, sperm shape (Arakaki, 1989; Arakaki and Uehara, 1989), and cross-fertilization, were needed. The characteristics comparisons and cross-fertilization experiments between Okinawan Echinometra and Mauritian Echinometra were conducted. Based on these studies, an attempt was made to find counterparts to Okinawan Echinometra among the Mauritian Echinometra species. Since the type locality of E. mathaei is Mauritius, the E. mathaei found in Mauritius must be the same as the original E. mathaei. A comparison of the Echinometra found in Mauritius with those found in Okinawa shows which species of Okinawan Echinometra is E. mathaei.

MATERIALS AND METHODS

Individual Echinometra specimens were collected at Sesoko Island and at Sunabe along Okinawa's coast in 1995 and 1996. In Mauritius, ten sites were investigated in 1995 (Arakaki and Fagoonee, 1996) and Echinometra specimens were collected from Trou aux Biches, Pte aux Caves, Albion, and Post Lafayette. These ten sites were selected to cover almost all possible environments where Echinometra were expected to live (Fig. 1). These were categorized in three groups: moats protected by coral reefs, rocky shores exposed to strong wave action and a slope beyond the outer-reef. The moats were shallow, 1 m deep, but wide, ranging from 100 m at their narrowest to 1000 m at their broadest. The beach sand consisted of white biogenic calcareous grains. The substratum of the moat was covered with such sand, mid-distant from the coastline to the reef. The outer-half of the moat had coral growth. Reefs sites were as follows: Trou aux Biches, Albion, Flic en Flac, Riambel, Blue Bay, and Ile aux Cerfs. The rocky shores were subjected to wave action and were unprotected by reefs at Pte aux Caves, Gris Gris, and Poste Lafayette. Although Gris Gris had a reef, it was narrow (about 20 m wide). Within the reef no sand subtratum was formed, and wave action was strong both inside the reef and at the reef edge. The outerreef slope, off Pointe aux Cannoniers, was studied using SCUBA at a depth of about 20 m.

Fig. 1

A map of the Indian Ocean and the Pacific Ocean (above) with enlarged maps of Mauritius (left) and Okinawa (right) below. The figures on the maps indicate the sites where the research was conducted in Mauritius, and the sampling was conducted in Okinawa, respectively. 1, Sesoko Island; 2, Sunabe coast; 3, Off Pointe aux Cannoniers; 4, Trou aux Biches; 5, Pte aux Caves; 6, Albion; 7, Flic en Flac; 8, Riambel; 9, Gris Gris; 10, Blue Bay; 11, Ile aux Cerfs; 12, Poste Lafayette.

The characteristics for describing the differences among Okinawan Echinometra spp. were: color of spines, brightness of milled ring and skin around the peristome, spicules in the tubefeet and gonads, the number of pore pairs per ambulacral plate and sperm shape. The Brightness of the milled ring and the skin around the peristome were two important factors, besides the color of the spines, for discriminating the four Echinometra species in the field in Okinawa. Discrimination for Mauritian Echinometra was also done based on these visible characteristics.

The spicules in the gonads were investigated under a microscope (10 × 10). A small portion of gonad was clipped off using a forceps and put on a slide glass and covered by a cover glass and pressed. Several tubefeet were clipped off with a forceps, and the morphology of the spicules was investigated by the same method as above.

The number of pore-pairs per ambulacral plate was counted under the dissecting microscope in every ambulacral plate in a denuded test.

The observation of sperm was carried out with a scanning electron microscope. Sperm samples were obtained as undiluted semen, i.e., “dry sperm” by removing the testes. The samples were prefixed for 30 min at low temperature (about 4°C) with a 1% osmium tetroxide fixative in filtered (0.2 μm) seawater and then postfixed with the same concentration of the osmium tetroxide fixative for one hour at low temperature (about 4°C). After postfixation, the samples were dehydrated in a graded series of ethanol (50% to 80%) for preservation. For the observation, the preserved sperm samples were rehydrated, terminating at 0% ethanol concentration. Several drops of the sample were dropped on a poly-l-lysine coated round cover glass (15 mm in diameter) to spread and adhere the sperm to the cover glass. The glass was put in a balance bottle. The process of dehydration in a graded series of ethanol (50% to 100%) was carried out in the bottle. After dehydration, these samples were treated with isoamyl acetate for 60 min and dried at the critical point of CO₂, and coated with palladium gold using an Ion Coater (Eiko, IB-1). Observations and photography of sperm morphology were carried out with a Hitachi S-530 scanning electron microscope.

Preservation of gametes, which were used for cross-fertilization, was done as follows: Some part of the gonads was removed from live samples and rinsed with filtered (0.2 μm)seawater and wiped with filter paper and put into a stock tube (1.5 cc) and then kept at low temperature (4-5°C) just before fertilization. Since it was possible to preserve sperm longer than eggs, only sperm was preserved for the purpose of cross-fertilization. The sampling of sperm from Mauritian Echinometra spp. for cross-fertilization experiments was carried out with the Albion individuals on the 31st of March, 1996, and the eggs of Okinawan individuals (from Sesoko Island) were fertilized on the 3rd of April, 1996. The sperm from the Mauritian Echinometra spp. were preserved for three days before cross-fertilization. The shedding of eggs was induced by injection of 0.5 M KCl into the coelomic cavity. Eggs were collected by inverting female urchins on a beaker of filtered (0.2 μm) seawater.

RESULTS

Comparative characteristics

Echinometra from Okinawa and Mauritius are shown in Fig. 2. Echinometra sp. A is characterized by white-tipped spines, a definite bright milled ring and dark skin on the peristome. Echinometra sp. B is characterized by spines with no white tip, with very faded milled rings and dark skin on the peristome. Echinometra sp. C is characterized by spines without a white tip, but with a definite bright milled ring and bright skin on the peristome. In the field, these two Echinometra spp. have exhibit a richly colored variation in spines. Echinometra sp. D is characterized by deep black spines with a faded milled ring and dark skin around the peristome. Only black-spined individuals have been found so far. Mauritian Echinometra individuals could be divided into three groups by appearance. One of the Echinometra which has spines without a white tip, but with a definite bright milled ring and dark skin on the peristome resembles Okinawan Echinometra sp. B described as Echinometra sp. B-like. The spine color of the latter is as abundant as Okinawan Echinometra sp. B and C. The individual in Fig. 2 has a bright milled ring, but individuals which do not have a bright milled ring were also found in the field. The second group of individuals resembles the first group in appearence but has violet spines, described as Violet Echinometra. Violet spine color is very unusual, as it is found neither in Okinawa nor in the other parts of the western Pacific, such as Indonesia, Guam, or Hawaii. The last one has deep black spines with a faded milled ring and dark skin on the peristome, and bears a resemblance to Okinawan Echinometra sp. D described as Black Echinometra.

Fig. 2

The species of Echinometra from Okinawa and Mauritius. The left and the right half are aboral and oral side views, respectively. The letters, A, B, C, and D indicate Echinometra sp. A, B, C, and D from Okinawa, respectively, and VE, EB and BE indicate Violet Echinometra, Echinometra sp. B-like, Black Echinometra from Mauritius, respectively. * The Black Echinometra individual photographed as a specimen preserved in alcohol, but the others were live specimens. The color of the Black Echinometra was faded due to the effects of alcohol preservation.

It is reported that Okinawan Echinometra have three types of spicules, i.e., bihamate spicules (Echinometra sp. A and B) and triradite spicules (Echinometra sp. C and D) in the tubefeet and needle spicules (Echinometra sp. A and B) and triradite spicules (Echinometra sp. C and D) in the gonads (Uehara, 1990). However, in these studies, spicules which are found in the tubefeet and the gonads are divided into four types: bihamete, needle, triradite, and multiple (Fig. 3). “Multiple” means various types of spines. These spicules occur alone or in combination, such as bihamate and triradite, or bihamate-triradiate-needle in the tubefeet, and triradiate and needle in the gonads (Table 1). Spicules in the tubefeet are variable, but it is possible to consider the bihamate spicule as a characteristic of Echinometra sp. A, B, E. Violet Echinometra and Echinometra sp. B-like; and triradiate spicules as a characteristic of Echinometra sp. C, D, and Black Echinometra. Each of these spicule types was found among many individuals of Echinometra.

Fig. 3

The shape of spicules from Echinometra. 1: Bihamate spicules in tubefeet from Okinawan Echinometra. sp. A. 2: Triradiate spicules in tubefeet from Mauritian Black Echinometra. 3: Needle spicules in gonads from Okinawan Echinometra sp. B. 4: Triradiate spicules in gonads from Okinawan Echinometra sp. C. 5: Multiple spicules in gonads from Mauritian Black Echinometra. The longer (1 and 2) and shorter bars (3, 4, and 5) indicate 50 μm and 100 μm, respectively.

Table 1

Appearance and spicules in tubefeet and gonads of Echinometra from Okinawa and Mauritius. A, B, C, D, VE, EB, and BE denote Echinometra sp. A, B, C, D, Violet Echinometra, Echinometra sp. B-like, and Black Echinometra, respectively. The figure in parentheses indicates the number of individuals.

Spicules in the gonads are comparatively stable. The needle spicule is a characteristic of Echinometra sp. A, B, Violet Echinometra, and Echinometra sp. B-like. The triradiate spicule is a characteristc of Echinometra sp. C, D, and Black Echinometra. The multiple spicule is found only in Echinometra sp. D and Black Echinometra. Accordingly, Echinometra from Okinawa and Mauritius can be gathered into two groups by the spicule shape in the tubefeet and the gonads: Echihometra sp. A, B, Violet Echinometra, and Echinometra sp. B-like; and Echinometra sp. C, D, and Black Echinometra.

The number of pore-pairs is also a characteristic for discriminating Echinometra. The ratio of 4 pore pairs is dominant in all of the Echinometra specimens from Okinawa and Mauritius (Fig. 4). These Echinometra can be grouped into three according to the ratio of the number of pore-pairs. The first group includes Echinometra sp. A, B, Violet Echinometra, and Echinometra sp. B-like; they have the highest ratio of 4 pore-pairs. Within the group, Echinometra sp. B and Echinometra sp. B-like differ significantly in pore-pair ratio (Table 2). The second group has only one member, Echinometra sp. C. The second group is similar to the first group; however, the number of 4 pore-pairs is less than the first group's while the number of 3 pore-pairs and 5 pore-pairs is more than that of the first group. The ratio of 4 pore-pairs is significantly different from the first group (Table 2). The third group, including Echinometra sp. D and Black Echinometra, has the lowest ratio of 4 pore-pairs and the highest ratio of 5pore pairs (Fig. 4). There is no significant difference in the ratio of 4 pore-pairs between the members of the group (Table 2).

Fig. 4

The ratio of pore pairs of Echinometra from Okinawa (left) and Mauritius (right). The figure in parentheses indicates the number of individuals. The values indicate the mean ± S.D.(%).

Table 2

Comparison of the ratio of four pore pairs of Echinometra from Okinawa and Mauritius. The values indicate a P value which was calculated by ANOVA. The symbols, ***, **, *, and −, indicatesignificant levels, P<0.001, P<0.01, P<0.05, and not significant lev-els, respectively. A, B, C, D, VE, EB, and BE denote Echinometra sp.A, B, C, D, Violet Echinometra, Echinometra sp. B-like, and Black Echinometra, respectively. The figure in parentheses indicates thenumber of individuals.

Sperm size and shape vary among Okinawan Echinometra spp. These differences are also found in Mauritian Echinometra (Fig. 5, Table 3). The sperm head size and shape of Okinawan Echinometra change gradually to longer and slender in accordance with the order of Echinometra sp. A, B, and C. The size changes from 2.91 to 3.45 μm, and the shape (length/width) changes from 2.34 to 3.07. The sperm size and shape of Echinometra sp. D are very different from the others, being very long (5.89 ± 0.13 μm) and slender (6.11 ± 0.48). The sperm size and shape of Mauritian Violet Echinometra and Echinometra sp. B-like are almost the same, resembling those of Echinometra sp. B. The sperm of Mauritian Black Echinometra is a little bit longer and wider than that of Okinawan Echinometra sp. D, but the shape is similar (Fig. 5, Table 3).

Fig. 5

Sperm of Okinawan Echinometra (A, B, C, D) and Mauritian Echinometra (VE, EB, BE). A, B, C, D, VE, EB, and BE denote Echinometra sp. A, B, C, D, Violet Echinometra, Echinometra sp. B-like, and, Black Echinometra, respectively. The bar indicates 2 μm.

Table 3

Sperm head size (length and width) and shape (length/width) of Echinometra from Okinawa and Mauritius. The values indicate the mean ± S.D.. A, B, C, D, VE, EB, and BE denote Echinometra sp. A, B, C, D, Violet Echinometra, Echinometra sp. B-like, and Black Echinometra, respectively. The figure in parentheses indicates the number of sperm. Sperm was obtained from one individual of eachspecies. The data for Okinawan Echinometra are from Arakaki (1989).

Cross-fertilization

The ratio of cross-fertilization between Mauritian Violet Echinometra and Echinometra sp. B-like is very high, with almost 100% success (Table 4). The successful combinations between Black Echinometra sperm and Violet Echinometra, and Echinometra sp. B-like eggs, however, are very low, 0 and 2±0.82%, respectively (Table 4). Sperm from both Mauritian Violet Echinometra and Echinometra sp. B-like easily fertilize the eggs of Okinawan Echinometra sp. B, with 99.67 ± 0.47 and 100% success. Fertilization of eggs from Okinawan Echinometra sp. A and C by the sperm of Mauritian Violet Echinometra is very low, especially the eggs of Echinometra sp. A. The cross-fertilization using Okinawan Echinometra sp. A and B is almost impossible (Uehara et al., 1990). Echinometra sp. B, Mauritian Violet Echinometra and Echinometra sp. B-like are not possible to discriminate in terms of cross-fertilization; they are all easily fertilized. However, Okinawan Echinometra sp. A and C are apparently different from the Mauritian Echinometra, Violet Echinometra and Echinometra sp. B-like.

Table 4

Cross-fertilization among Echinometra from Okinawa and Mauritius. The values indicate the mean fertilization ratio ±S.D.(%). These values were calculated by three replicates of 100 inseminated eggs. A, B, C, VE, EB, and BE denote Echinometra sp. A, B, C, Violet Echinometra, Echinometra sp. B-like, and Black Echinometra, respectively. The sperm used for the insemination of Okinawan Echinometra was collected from different individuals which had inseminated Mauritian Echinometra.

Distribution of the Echinometra spp. in Mauritius

The ten sites surveyed in Mauritius cover habitats ranging from calm water (eg. off Pointe aux Cannoniers) to shallow (Trou aux Biches, Albion, Flic en Flac, Riambel, Blue Bay, Ile aux Cerfs) and very rough water (Pte aux Caves, Gris Gris, Poste Lafayette) conditions (Table 5). The substratum, varies from sand, sandy bottom with seaweeds, shingle, coral rubble, coral, to rock. These sites cover almost all the possible environments where Echinometra live.

Table 5

Abundance and distribution of Echinometra in Mauritius. VE, EB, and BE denote Violet Echinometra, Echinometra sp. B-like, and Black Echinometra, respectively. Symbols, ++, +, and −, denote abundance of species: abundant, not many, and not found, respectively.

Violet Echinometra and Echinometra sp. B-like tended to be distributed in calm water, and Black Echinometra in strong wave areas, such as Pte aux Caves, Gris Gris, and Poste Lafayette (Table 5). These distribution patterns on the reef are similar to those of Okinawan Echinometra sp. B and D, respectively (Arakaki, 1989; Nishihira et al., 1991; Tsuchiya and Nishihira, 1984).

DISCUSSION

The white-tipped spines of Echinometra sp. A are its most prominent characteristc. Therefore discrimination of this species can be done by this characteristic in the field. No Mauritian Echinometra individuals have this type of spine. In regard to spicules in the tubefeet and the gonads, pore-pair number, sperm shape, and distribution on a reef, it is rather difficult to discriminate Echinometra sp. A from Echinometra sp. B-like and Violet Echinometra, but cross-fertilization between them is almost impossible. From the results of cross-fertilization it should be understood that Echinometra sp. A and those of Mauritian Echinometra, Violet Echinometra and Echinometra sp. B-like are different species. Cross-fertilization between Echinometra sp. A and Black Echinometra was not carried out because viable sperm of the species was not sampled. However, certain differences (the length and the shape of sperm head and 4 pore-pairs ratio) between them are apparent. It is understood that if the sperm is different, the species should be different (Koike, 1985). Okinawan Echinometra sp. C is characterized by a definite milled ring, bright skin on the peristome, triradiate spicule in the tubefeet and the gonads, and a somewhat longer and slender sperm head than Echinometra sp. A and B. Among these characteristics, the brightness of the milled ring and the skin on the peristome, and the shape of spicules in the tubefeet are not so reliable because these characteristics vary. However, the shape of spicules in the gonads is stable, with Echinometra sp. C having triradiate spicules only. In Mauritian Echinometra, Black Echinometra have triradiate spicules, but Violet Echinometra and Echinometra sp. B-like do not have this type of spicule. Cross-fertilization between Echinometra sp. C and Violet Echinometra/Echinometra sp. B-like has a very low success rate. The sperm head size and shape, and the 4 pore-pairs ratio of Echinometra sp. C are very different from those of Black Echinometra. Thus, it could be said that the counterparts of Okinawan Echinometra sp. A and C are not distributed in Mauritius.

Almost all of the characteristics of Violet Echinometra and Echinometra sp. B-like are similar, namely, the brightness of the milled ring and the skin on the peristome, spicules in tubefeet/gonads, ratio of pore-pairs, and the sperm shape. In addition, they easily fertilize each other. It is not possible to distinguish Violet Echinometra from Echinometra sp. B-like by these characteristics. However, it is possible to recognize Violet Echinometra, and distinguish them from Echinometra sp. B-like, solely on the basis of their violet-colored spines as those are only found in the Violet Echinometra.

Echinometra sp. B share common characteristics with Violet Echinometra and Echinometra sp. B-like, and fertilization between them is very successful. However, individuals with violet spines, the spine color of Violet Echinometra, were never found in Okinawa. Some Echinometra sp. B-like individuals have bright milled rings, but this characteristic is variable. Many individuals, presumed to be the same species as Okinawan Echinometra sp. B, with a bright milled ring, are found in Indonesia (Arakaki and Uehara, 1995). Among these three Echinometra, Mauritian Violet Echinometra are distinguished by violet-colored spines, but it is not possible to distinguish Echinometra sp. B from Mauritian Echinometra sp. B-like. Thus, it is appropriate to infer that Okinawan Echinometra sp. B is the same as Mauritian Echinometra sp. B-like.

Black Echinometra are distinguished by uniform black spines, 4 pore-pairs ratio, sperm head shape, cross-fertilization, and distribution on a reef from Violet Echinometra and Echinometra sp. B-like. Black Echinometra are also distinguished from Okinawan Echinometra sp. A, B, C by these characteristics. Cross-fertilization between Mauritian Black Echinometra and Okinawan Echinometra was not performed because viable sperm of Black Echinometra was not brought back to Okinawa. But the data are sufficient to show Black Echinometra are a different species from Okinawan Echinometra sp. A, B, C. Cross-fertilization between Okinawan Echinometra sp. D and Mauritian Echinometra was not conducted, but Echinometra sp. D can be distinguished from Violet Echinometra and Echinometra sp. B-like by the characteristics mentioned above. Resemblances between Okinawan Echinometra sp. D and Mauritian Black Echinometra are noted in the uniform back spines, dark milled ring and skin on the peristome, 4 pore-pairs ratio, sperm head shape, and distribution on a reef. Therefore, it could be said that Okinawan Echinometra sp. D is the same as Mauritian Black Echinometra.

The above mentioned Mauritian Echinometra were described as E. mathaei (Echinometra sp. B-like), and E. mathaei violacea (Violet Echinometra) by Mortensen (1943). E. mathaei violacea was described as follows: the test of E. mathaei violacea agrees with a typical E. mathaei. But it is not possible to apply the naked tests to the E. mathaei violacea because when these are naked their distinguishing characteristic is no longer visible. In addition to their highly distinct purplish violet spines, E. mathaei violacea abundantly developed tridentate pedicellariae and two dense series of triradiate spicules in the aboral tubefeet. The distribution of this variety is restricted to the western Indian Ocean. Research was carried out by Arakaki and Fagoonee (1996) on this variety which was collected in Mauritius and Mahé (Seychelles). This variety is not found in Okinawa, Indonesia and other parts of the Pacific Ocean. It is found in the west Indian Ocean only. It is meaningful to distinguish this variety from E. mathaei.

Mortensen (1943) described Echinometra, which has black spines, as E. mathaei oblonga. While Kelso (1970) described this sea urchin as E. oblonga based on his studies done in Hawaii. Among Hawaiian Echinometra, it was shown that E. mathaei and E. oblonga were impossible to cross-fertilize (Kelso, 1970; Metz et al., 1994). Therefore, it is approprite to describe Echinometra, which has black spines, as E. oblonga or as a species separate from E. mathaei.

Among the four species of Okinawan Echinometra, it could be said that Echinometra sp. B is the same as E. mathaei while the other species are different. Because the type locality of E. mathaei is Mauritius (Blainville, 1825), Echinometra sp. B, which has the most similar characteristics with Mauritian E. mathaei, would be E. mathaei. However, it is indispensable to consult the holotype of E. mathaei in the future. For Okinawan Echinometra sp. A and C, a new species name would be needed. Okinawan Echinometra sp. D is thought to be the same species as Mauritian E. oblonga, but whether Okinawan Echinometra sp. D is E. oblonga remains as a ploblem for future research. Because the type locality of E. oblonga is not known, it is suggested that the E. oblonga describe in the Indo-West Pacific is a complex species (Uehara, personal communication). To decide on a species name for Okinawan Echinometra sp. D, thorough research is needed in the future.