evolutionary relationships of sea-urchins at the molecular level

Comp. Biochem. Physiol. Vol. 97B, No. 1, pp. 31-36, 1990 0305-0491/90 $3.00 + 0.00 Printed in Great Britain © 1990 Pergamon Press plc

MINI-REVIEW

EVOLUTIONARY RELATIONSHIPS OF SEA-URCHINS AT THE MOLECULAR LEVEL

NORIMASA MATSUOKA Department of Biology, Faculty of Science, Hirosaki University, Hirosaki 036, Japan

(Received 20 March 1990)

INTRODUCTION

The taxonomy and phylogeny of the echinoids have been extensively studied by many workers mainly from morphological and/or paleontological stand- points (Clark, 1925; Durham and Melville, 1957; Jackson, 1912; Jensen, 1981; Mortensen, 1928-1952; Nisiyama, 1966, 1968; Philip, 1965; Shigei, 1974, 1986; Smith, 1984, 1988). However, various taxonomic systems have been proposed and there is much disagreement between taxonomists as to the echinoid phylogeny, owing to the different interpretations about the morphological characters used when speculating the phylogenetic relationships among echinoids. Therefore, there are many unresolved problems about the phylogenetic and evolutionary relationships among echinoids (Shigei, 1974).

On the other hand, during the last 10-15 years, the taxonomic, phylogenetic and evolutionary studies have been revitalized by the application of techniques from molecular biology. Protein sequencing, immu- nology, electrophoresis and sequence analysis of ribo- somal RNA or mitochondrial DNA are among the molecular techniques used in evolutionary studies. Such molecular approaches have made it possible for us to estimate the phyiogenetic relationships among taxa and their evolutionary processes quantitatively with common parameters such as enzymes or DNA. In order to clarify many unresolved problems con- cerning echinoid phylogeny, it would be desired to actively introduce the molecular approaches which are more analytic than the traditional and usual morphological methods into echinoid taxonomy. They would provide much valuable, and in some cases critical, information on echinoid taxonomy. I have been investigating the phylogenetic and evolutionary relationships among camarodont sea- urchins of the order Echinoida by using the molecular techniques such as enzyme electrophoresis or immunological method.

In this mini-review, I would like to give an outline of the biochemical systematic studies of the camarodont sea-urchins of the order Echinoida. Firstly, I have introduced the immunological taxonomy by enzyme inhibition method. Then, I have shown the molecular phylogenetic trees of the three families, Toxopneustidae, Strongylocentrotidae and Echino- metridae, of the order Echinoida established by

enzyme electrophoresis. Finally, I have discussed the advantage of molecular methods of measuring the genetic divergence.

31

I. IMMUNOLOGICAL STUDY ON THE PHYLOGENETIC RELATIONSHIPS AMONG CAMARODONT SEA-URCHINS

The phylogenetic relationships among 19 sea- urchin species belonging to four different families of the order Echinoida were examined by the immunological method of enzyme inhibition test with the antibody against glucose-6-phosphate dehydrogenase (G6PD) purified from Strongylocentrotus intermedius of the family Strongylocentrotidae (Matsuoka, 1980, 1986). In this test, the immunological similarity between a reference species (S. intermedius) and a test species was expressed as a relative inhibition potency (RIP) which is the relative value of enzyme activity inhibited by the antibody. Table 1 shows the RIPs of antibody against G6PDs from 19 species of the order Echinoida. The RIP values show that sea-urchins having values close to 1.00 of S. intermedius of the specific antigen are more immunologically closely related to S. intermedius of the family Strongylo- centrotidae. As evident from Table 1, the family Strongylocentrotidae is more closely related to the family Toxopneustidae of the different suborder (Temnopleurina) than to the family Echinometridae of the same suborder (Echinina). The immunological results are not consistent with the generally accepted taxonomic systems proposed by Mortensen (1943), Durham and Melville (1957), Nisiyama (1966) and Shigei (1974, 1986). They strongly argued that the lineage of Strongylocentrotidae-Echinometridae does not have any close relationship to the lineage of Temnopleuridae-Toxopneustidae. In contrast with their taxonomic systems, the immunological results are well consistent with the taxonomic systems by the earlier echinoid taxonomists, Clark (1925), Jackson (1912) and Mortensen (1903) and the recent systems by Jensen (1981) and Smith (1988). They do not divide the two families into two different suborders. Mortensen (1943) described in his monograph that the strongylocentrotids may have been derived from some primitive toxopneustids, though he had no means of proving it. Although his statement seems to be inconsistent with his two suborder systems, his

32 NORIMASA MATSUOKA

Table I. Relative inhibition potencies (RIPs) of anti-S, intermedius G6PD antibody against G6PDs from 19 sea-urchin species of the

order Echinoida

Species RIP

Order Echinoida Suborder Temnopleurina

Family Temnopleuridae Temnopleurus toreumaticus 0.03 Temnopleurus hardwickii 0.02 T. ( Toreumatica ) reevesii 0.02 Mespilia globulus 0.06

Family Toxopneustidae Lytechinus pictus O. 15 Toxopneustes pileolus O. 14 Tripneustex gratilla O. 17 Pseudoboletia maculata O. 18 Pseudocentrotus depressus 0.45

Suborder Echinina Family Strongylocentrotidae

Strongylocentrotus intermedius 1.00 Strongylocentrotus purpuratus 0.58 Strongylocentrotus nudus 0.23 Hemicentrotus pulcherrimus O. 1 I

Family Echinometridae Echinometra mathaei 0.03 Anthocidaris crassispina 0.04 Echinostrephus aciculatus 0.04 Echinostrephus molaris 0.07 Heterocentrotus mammillatus 0.04 Colobocentrotus mertensii 0.05

Nineteen species of sea-urchins of the order Echinoida in this table are classified according to the taxonomic system proposed by Mortensen (1928-1952) and Shigei (1974).

view on the evolutionary origin of the strongylocentrotids is very interesting when considered with the immunological results suggesting a close affinity between the two families. Furthermore, the immunological results are also supported by other compara- tive biochemical studies (Nagaoki and Isono, 1981). Therefore, it is concluded that the generally accepted two suborder systems should be modified.

Of five species of the family Toxopneustidae examined, Pseudocentrotus depressus endemic to Japan showed an outstandingly high RIP value. The results strongly suggest that P. depressus is very closely related to the Strongylocentotidae and the species may be a member of the Strongylocentrotidae. Interestingly, there are two conflicting views on the taxonomic situation of P. depressus from the morphological standpoint. Mortensen (1943) included the species in the family Toxopneustidae. His taxonomic system has been widely accepted by many taxonomists. On the other hand, Clark (1925) referred the species to the family Strongylocentrotidae. The two conflicting views are due to the fact that P. depressus not only has the morphological characters of the Toxopneustidae, but also those of the Strongylo- centrotidae. Such taxonomical controversy may

be settled by the molecular studies which can provide the more objective and quantitative data than the morphological studies. My immunological study is in favor of the view of Clark (1925) which was not supported by other echinoid taxonomists.

2. PHYLOGENETIC RELATIONSHIPS WITHIN THE FAMILY TOXOPNEUSTIDAE AND THE

STRONGYLOCENTROTIDAE ESTIMATED BY ENZYME ELECTROPHORESIS

The immunological study provided much valuable information on the phylogeny of the carmarodont sea-urchins. However, the interrelationships among the members of each family of the order Echinoida remain unclear. In order to clarify the phylogenetic relationships among the members of two related families, Toxopneustidae and Strongylocentrotidae, in addition to the phylogenetic position of P. depressus, I have attempted to establish the molecular phylogenetic tree for the seven members of the two families by the electrophoretic analyses of various enzymes (Matsuoka, 1985, 1987). The sea-urchins examined were the four species of the family Toxopneustidae: Toxopneustes pileolus, Tripneustes gratilla, Pseudoboletia maculata and Pseudocentrotus depressus, and the three species of the family Strongylocentrotidae: Strongylocentrotus intermedius, Strongylocentrotus nudus and Hemicentrotus pulcherrimus. They are commonly found in the shallow water of the seas around Japan and have often been used in developmental, physiological and biochemical studies. The genetic or phylogenetic relationships among them were investigated by polyacrylamide gel electrophoresis of 15 different enzymes, using the supernatant of homogenate of gut and gonad extracted from each individual. The electrophoretic band patterns of 15 enzymes were compared between species, the genotype in each locus of 21 genetic loci scored was inferred and the allele frequencies for all loci in seven species were calculated. Based on these data, the genetic identity (I) and genetic distance (D) between each species were calculated by the method of Nei (1972), in order to quantify the degree of genetic differentiation between species. Table 2 shows the matrices of I and D values between all pairs of seven species examined. Figure 1 shows the molecular phylogenetic tree constructed from the Nei's genetic distance matrix of Table 2 by using the UPGMA clustering method of Sneath and Sokal (1973). According to Nei (1975), genetic distance (D) corre- sponds well with the divergence time (T) from the common ancestor, and T of two taxa can be estimated by T = 5 x 106 D (year). The divergence time estimated from the Nei's equation is also given in the

Table 2. Genetic identities (above diagonal) and genetic distances (below diagonal) between seven sea-urchin species from the two families, Toxopneustidae and Strongylocentrotidae, from the order Echinoida

Species 1 2 3 4 5 6 7

(I) Toxopneustes pileolus - - 0.471 0.542 0.335 0.395 0.314 0.287 (2) Tripneustes gratilla 0.753 - - 0.474 0.314 0.327 0.222 0.172 (3) Pseudoboletia maculata 0.612 0.747 - - 0.288 0.348 0.345 0.240 (4) Pseudocentrotus depressus 1.094 1.158 1.245 - - 0.472 0.418 0.381 (5) Strongylocentrotus intermedius 0.929 1.118 1.056 0.751 - - 0.629 0.562 (6) Strongylocentrotus nudus 1.158 1.505 1.064 0.872 0.464 - - 0.570 (7) Hemicentrotus pulcherrimus 1.248 1.760 1.427 0.965 0.576 0.562 - -

Evolutionary relationships of sea-urchins 33

DIVERGENCE T I M E ( M Y ) ~ e 5 4 s ,z I ?

I I I i I

TOXOPNEUST£$ PILEOLU$

P$EUDOBOLETIA MACULATA

TRIPNEUSTE$ GRATILLA

PSEUDOCENTROTU$ DEPRE$$U$

HEMICENTROTU$ PULCHERRIMUS

$TRONGYLOCENTROTUS INTERMEDIUS

STRONGYLOCENTROTUS NUDU$

£ 4 1~o o~5 6 GENETIC DISTANCE

Fig. I. A molecular phylogenetic tree of seven sea- urchin species from the two families, Toxopneustidae and Strongylocentrotidae, from the order Echinoida. This dendrogram was constructed from the Nei's genetic distances by using the UPGMA clustering method. The divergence time estimated from the Nei's equation is also

given.

to those of Toxopneustidae. This result is consistent with the previous immunological data. These biochemical results are strongly supported by the DNA hybridization test (Poltaraus and Antonov, 1984), cross-fertilization test (Noguchi, 1982), karyological study (Saotome, pers. commn) and zoogeographical evidence. Putting this various evidence together, it is concluded that P. depressus should be included taxonomically into the family Strongylocentrotidae. Recently, Shigei (1986), who is one of the echinoid taxonomists in Japan, modified his previous taxonomic system and transferred the taxonomic position of P. depressus from the family Toxopneustidae to the family Strongylocentrotidae.

(5) From the phylogenetic tree shown in Fig. 1, it is strongly suggested that the three strongylocentrotids endemic to Japan might have been evolved from the P. depressus-like sea-urchin which diverged from the primitive toxopneustids. Their divergence times estimated from the molecular data are roughly consistent with the paleontological data reported by Nisiyama (1966).

molecular phylogenetic tree. The phylogenetic tree demonstrates the following:

(1) Seven species are divided into two large clusters. One consists of three species of the Toxopneustidae which are widely distributed in the Indo-West Pacific region. The other consists of three species of the Strongylocentrotidae and P. depressus which was included taxonomically in the Toxopneustidae. The genetic distance between these two clusters is smaller than the D values observed between different families of other animals. It suggests the close affinity between these two families of the order Echinoida.

(2) Among three species of the Toxopneustidae, T. pileolus is more closely related to P. maculata than to T. gratilla. This relationship is consistent with the karyological study by Shingaki and Uehara (1984). Mortensen (1943) and Clark (1925) proposed the opposite views as to the taxonomic position of the genus Pseudoboletia: the Toxopneustidae and the Strongylocentrotidae, respectively. The biochemical results are consistent with the view of Mortensen (1943).

(3) Among three species of the Strongylocentro- tidae, the two species of the genus Strongylocentrotus (S. intermedius and S. nudus) are closely related to each other than they are to H. pulcherrimus. How- ever, the genetic distances among them are relatively small and they may be included in the same genus, Strongylocentrotus. Their close relationships are also suggested by the DNA hybridization test by Poltaraus and Antonov (1984).

(4) Pseudocentrotus depressus is more closely related to the members of Strongylocentrotidae than

3. PHYLOGENETIC RELATIONSHIPS WITHIN THE FAMILY ECH1NOMETRIDAE ESTIMATED

BY ENZYME ELECTROPHORESIS

Another large family of the order Echinoida is the Echinometridae. The phylogenetic relationships within the family have not yet been examined and still remain unclear. The family Echinometridae found in Japanese waters is represented by six species of five different genera. They are Echinometra mathaei, Anthocidaris crassispina, Echinostrephus aciculatus, Echinostrephus molaris, Heterocentrotus mammillatus and Colobocentrotrus mertensff. Most of them are widely distributed over the tropical and subtropical regions of the Indo-West Pacific, and commonly found in shallow water. Each of these five genera is characterized by the highly specialized external morphology and therefore the family has been regarded as a heterogeneous group. Thus, it seems to be difficult to speculate their phylogenetic relationships by only morphological studies. Furthermore, there is little quantitative information available con- cerning the genetic relationships among them. In order to clarify the phylogenetic relationships among six members of the family Echinometridae, I attempted to establish the molecular phylogenetic tree of the six members of the family by electrophoretic study (Matsuoka and Suzuki, 1987, 1989). Following the comparison of the electrophoretic band patterns of 18 different enzymes, the genetic identity (I) and genetic distance (D) between each species were calculated from allele frequencies at 26 genetic loci by the method of Nei (1972). Table 3

Table 3. Genetic identities (above diagonal) and genetic distances (below diagonal) between six sea-urchin species of the family Echinometridae from the order Echinoida

Species I 2 3 4 5 6 (I) Anthocidaris crassispina -- 0.631 0.587 0.579 0.380 0.487 (2) Echinometra mathaei 0.460 -- 0.585 0.581 0.434 0.509 (3) Echinostrephus aciculatus 0.533 0.536 -- 0.963 0.390 0.426 (4) Echinostrephus molaris 0.546 0.543 0.038 -- 0.369 0.442 (5) Heterocentrotus mammillatus 0.968 0.835 0.942 0.997 - - 0.476 (6) Colobocentrotus mertensii 0.719 0.675 0.853 0.816 0.742 --


D I V E R G E N C E T I M E ( M Y ) 5 4 8 2 I o

i i t ANTHOCIDARIS I CRASSISPINA ECHINOM£TRA MATHAEI l ECHINOSTREPHUS 'E ACICULATUS ECHINOSTREPHUS MOLARIS HETEROCENTROTUS MAMMILLATUS COLOBOC£NTROTUS MERTEN$11

1:o o'.s b GENETIC DISTANCE

Fig. 2. A molecular phylogenetic tree of six sea-urchin species from the family, Echinometridae, from the order Echinoida. This dendrogram was constructed from the Ners genetic distances by using the UPGMA clustering method. The divergence time estimated from the Ners equation is

also given.

shows the matrices of 1 and D values between all pairs of Echinometridae. Based on the matrix of D values, the molecular phylogenetic tree of the six echino- metrids was constructed by the UPGMA clustering method (Fig. 2). The divergence time calculated from the Nei's equation described above is also given in the dendrogram. The phylogenetic tree demonstrates the following:

(1) Six species of the Echinometridae are divided into two large clusters. One consists of Anthocidaris crassispina, Echinometra mathaei and two Echino- strephus species, and the other Heterocentrotus rnammillatus and Colobocentrotus mertensii. The results suggest that the family Echinometridae is constituted by two different lineages and therefore the family might be classified into two subfamilies.

(2) Heterocentrotus rnammillatus and C. mertensii are related to each other, but they are largely genetically differentiated from the other four species. The results are roughly consistent with those inferred from morphological characters such as the shape of spines. From the molecular estimation of their divergence time, they are considered to be the more primitive species among the family Echinometridae.

(3) The two morphologically very similar species of the genus Echinostrephus (E. aciculatus and E. molaris) are little differentiated genetically from each other. Their genetic similarity verified by the electrophoretic study and their morphological and ecological similarity strongly suggest that these two sea-urchins may belong to one and the same species.

(4) Anthocidaris crassispina endemic to Japan is more closely related to E. mathaei than to Echino- strephus. Although A. crassispina has been considered to be one of the relic endemic species having old evolutionary origin from the paleontological study (Nisiyama, 1966; Nishimura, 1985), the divergence time estimated from the molecular data suggested that A. crassispina had speciated in the relatively recent geological age of late Pliocene. Since E. mathaei is the most predominant species of the family Echinometridae and widely distributed in the Indo- West Pacific region, it may be speculated that A. crassispina has derived from Echinometra species and

become a Japanese endemic species after going up north from the Indo-West Pacific to Japanese waters.

4, CONCLUSION

At present, there are various molecular methods of measuring protein or DNA divergence between different taxa. As expected, there exist high correlations among different measures of genetic divergence. The genetic distance (D) obtained by electrophoresis is also highly correlated with other measures of genetic divergence (Sarich, 1977; Highton and Larson, 1979). For example, O'Brien et al. (1985) examined the genetic relationship of the giant panda with various species of bears and raccoons by using immunological distance of albumin, DNA hybridization data, electrophoretic distance, and chromosomal banding pattern. As a result, high correlations among all these data were observed, and they have indicated that the panda is a bear rather than a raccoon, closing a long-standing controversy among zoologists. The biochemical systematic studies of sea-urchins also showed the high correlation between immunological data obtained by enzyme inhibition method and electrophoretic data. Namely, these two data demonstrated the close relationship between Pseudocentrotus depressus and the family Strongylocentrotidae.

Protein electrophoresis is one of the most powerful techniques among various methods of measuring genetic divergence in evolutionary studies. Further- more, this method is more simple and less expensive than other molecular methods. Since the introduction by O'Farrell (1975) of two dimensional electrophoresis, a number of authors (Aquadro and Avise, 1981; Goldman et al., 1987; Ohnishi et al., 1983) have used this method to study the evolutionary relationship of related species instead of standard electrophoresis. Although this method permits an examination of a large number of polypeptides on one gel, it does not detect as much variation as ordinary electrophoresis does. The proportion of polymorphic loci detectable by this method is lower than that detectable by ordinary electrophoresis (Aquadro and Avise, 1981; Ohnishi et al., 1983). The reason for this seems to be that the proteins studied by this technique are mainly structural proteins such as actin, tubulin, etc. and these proteins are less variable because of stronger functional constraints (Kimura, 1983).

In recent years, many authors have used mitochondrial DNA to study evolutionary relationships of organisms. However, Nei (1987) suggested that the resolving power of mitochondrial DNA is not neces- sarily higher than that of protein electrophoresis. This is particularly so when the restriction enzyme technique is used. According to the estimation of Nei (1987), electrophoresis is expected to survey about 100 nucleotides/locus. If we examine 60 loci by electrophoresis, it is equivalent to studying 6000 nucleotides. This is much larger than the number of nucleotides (895) sequenced by Brown et al. (1982) for human and ape mitochondrial DNAs. In their study of the evolution of human and ape

Evolutionary relationships of sea-urchins 35

mitochondrial DNAs, Ferris et al. (1981) used 18 6-base enzymes and one 4-base enzyme. The average number of restriction sites per sequence for all 6-base enzymes was 42, whereas the number for the 4-base enzyme was seven. Therefore, the total number of nucleotides assayed is 42 × 6 + 7 × 4 = 280. This number is even smaller than the number of nucleotides sequenced by Brown et al. (1982).

Generally, the echinoids or sea-urchins have been regarded as an animal group having old evolutionary origins. However, the molecular phylogenetic trees of the three families clearly demonstrated that most of the sea-urchin species of the order Echinoida have recent evolutionary origins and had speciated in relatively recent geological times. The biochemical systematic studies showed high correlation between the genetic distances and the taxonomic ranks in sea-urchins. Furthermore, the genetic distances obtained between different species or genera in sea- urchins were comparable to those between taxa of the corresponding ranks in many other animal groups reported hitherto (Ayala, 1982; Ferguson, 1980). The results strongly suggest that the morphological evolutionary rate of sea-urchins is not slower than those of other animal groups. On the other hand, almost all of members of the order Echinoida from Japanese waters have the same diploid chromosome number (2n = 42), as reported by Saotome (1987). It suggests that the chromosomal evolutionary rate of sea- urchins is much slower than the molecular evolutionary rate.

When considering the two conflicting views of the taxonomic position of Pseudocentrotus depressus proposed by Mortensen (1943) and Clark (1925), it may be apparent that the speculation of the phylogenetic relationships among echinoids by only morphological characters is considerably difficult. It is mainly due to the difference among taxonomists in the choice and interpretation of the morphological characters available for speculating the phylogenetic relationships.

On the other hand, molecular approaches make it possible for us to estimate the phylogenetic relationships quantitatively with the common parameters such as enzymes or DNA. They can provide more objective, quantitative and analytic data than the morphological studies. There is no doubt that the molecular or biochemical approaches are significant and useful in the phylogenetic and evolutionary studies.

In future, the immunological study on the phylogeny of camarodont sea-urchins and the molecular phylogenetic trees within the three families, Toxopneustidae, Strongylocentrotidae and Echino- metridae, of the order Echinoida established by the electrophoretic studies would provide principal and valuable information for further research of their evolution and for the reconstruction of new taxonomic systems which sufficiently reflects their phylogenetic and evolutionary relationships.

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evolutionary relationships of sea-urchins at the molecular level

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