Molecular Phylogenies and Divergence Times of Sea Urchin Species of Strongylocentrotidae, Echinoida

Youn-Ho Lee

Polar Sciences Laboratory, Korea Ocean Research and Development Institute, Ansan, South Korea

Correspondence: E-mail address: ylee{at}kordi.re.kr.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
Sea urchins of the family Strongylocentrotidae have been important model systems in many fields of basic biology, yet knowledge of their evolutionary identities such as the phylogenetic relationships and divergence times remains limited. Here, I inferred molecular phylogenies of seven Strongylocentrotid species (Strongylocentrotus franciscanus, S. nudus, S. purpuratus, S. intermedius, S. droebachiensis, S. pallidus, and Hemicentrotus pulcherrimus) from the analyses of mitochondrial DNA sequences of 12SrDNA (349 nt), 12SrDNA-tRNA(gln) region (862 nt), and a combined sequence of cytochrome oxidase subunit I (COI, 1080 nt) and NADH dehydrogenase subunit I (NDI, 742 nt). The rate of sequence evolution and divergence times for each species were then estimated from the trees with reference to the time of separation between Strongylocentrotidae and Parechinidae, 35 to 50 MYA. The three trees agree well with each other, and the phylogeny is summarized by ((S. franciscanus, S. nudus), (H. pulcherrimus (S. purpuratus, S. intermedius (S. droebachiensis, S. pallidus)))). It is notable that the genus Strongylocentrotus consists of two distinct clades and that H. pulcherrimus branches off within Strongylocentrotus, implying assignment of a separate, monospecific genus to this species inappropriate. The rate of sequence evolution is calibrated to be 0.24%–0.34%/Myr in 12SrDNA, 0.25%–0.36%/Myr in 12SrDNA-tRNA(gln), and 0.65%–0.93%/Myr in COI-NDI combined sequences. S. purpuratus, in particular, shows the significantly higher rate of evolution in the 12SrDNA and 12SrDNA-tRNA(gln) regions compared to other species, suggesting careful use of its sequences in comparative studies. The two clades of Strongylocentrotidae seem to have split 13–19 MYA, and H. pulcherrimus branched off 7.2–14 MYA. In the former clade, S. franciscanus and S. nudus separated 5.7–8.1 MYA. In the latter clade, S. purpuratus, S. intermedius, and the clade of S. droebachiensis and S. pallidus diverged approximately 4.6–12 MYA, and the last two closest species separated 2.1–3.1 MYA.

Key Words: Strongylocentrotidae • sea urchin • phylogeny • evolutionary rate • divergence time • speciation


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
Sea urchins of the family Strongylocentrotidae have been one of the most popular marine organisms for studies of reproductive biology (Vacquier, Swanson, and Hellberg 1995), embryology (Davidson, Cameron, and Ransick 1998; Lee et al. 1999), toxicology (Dinnel et al. 1989), gene regulation (Davidson et al. 2002), as well as evolutionary biology (Peterson, Cameron, and Davidson 2000). A species of this family, S. purpuratus, was recently chosen as a target organism for genome analysis in its entirety by the National Human Genome Research Institute of USA (Pennisi 2002), which will draw more attention to this small family of sea urchins. Species of this family are among the most intensively studied sea urchins by marine ecologists and constitute the majority of the urchin fisheries in North America and east Asia (Lawrence 2001). Yet, the systematics and the phylogenies of the family remain uncertain both at the genus level and at the species level.

According to A. B. Smith (personal communication; the echinoid directory, www.nhm.ac.uk/palaeontology/echinoids), the family Strongylocentrotidae includes five genera: Strongylocentrotus Brandt, Hemicentrotus Mortensen, Allocentrotus Mortensen, Pseudocentrotus Mortensen, and Loxechinus Desor (table 1). Larrain (1995), however, places the genus Loxechinus in the family Echinidae instead of Strongylocentrotidae. The genus Pseudocentrotus only recently became included in this family by Matsuoka (1987), being removed from the family Toxopneustidae after an allozyme study. Based on the molecular tree of bindin, a sperm-borne fertilization protein in the sea urchin, Biermann (1998) showed that two monospecific genera, Allocentrotus and Hemicentrotus, diverged within the genus Strongylocentrotus. Kessing (1991) also found the phylogenetic position of the species H. pulcherrimus among the species of Strongylocentrotus in the molecular phylogeny of cytochrome oxidases I and II (COI-COII)-ATPase8-ATPase6 sequences.


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Table 1 Genera and Species of the Family Strongylocentrotidae.

 
The species taxonomy of Strongylocentrotidae has changed much since Mortensen's monograph on Echinoidea (1943). Jensen (1974) made a thorough revision of the taxonomy and found only 10 species valid in the family (table 1). Recently, Bazhin (1998) reexamined more than 29,000 Russian museum specimens of Strongylocentrotus and made a further revision. He attributed S. pulchellus (A. Agassiz and Clack) to a subordinate synonym of S. intermedius (A. Agassiz) (table 1), and S. polyacanthus apicimagis and S. djakonovi (Baranova) to S. droebachiensis (O. F. Müller) contrary to Jensen's attribution of the two taxa to S. polyacanthus.

Phylogenetic relationships among Strongylocentrotid species have been studied only for a few species, mostly from North America. Based on the single-copy nuclear DNA sequence differences determined by thermal stability of interspecies DNA duplexes, Hall et al. (1980) showed that S. purpuratus is more closely related with S. droebachiensis than with S. franciscanus. Vawter and Brown (1986) found the same relationship among the three species, based on mtDNA divergence estimated from restriction site maps. Thomas, Maa, and Wilson (1989) analyzed the relationship among S. franciscanus, S. purpuratus, S. intermedius, S. droebachiensis, and S. pallidus with ND5 sequences and found that S. droebachiensis and S. pallidus are the most closely related species and that S. purpuratus comes as a basal taxon to the clade of the two species. Biermann (1998) included more species in her analysis of the sperm bindin sequences and showed that S. purpuratus, S. droebachiensis, S. pallidus, S. polyacanthus, and A. fragilis are closely related, and that H. pulcherrimus and S. franciscanus come close to the five species cluster in order. Her results, however, could not resolve the relationships among the five closely related species any further and retained them in a polytomy. Relationships among the east Asian species such as S. intermedius, S. nudus, and H. pulcherrimus have not been dealt with in all of the previous molecular studies but two: Matsuoka (1987) tried to analyze relationships among S. intermedius, S. nudus, H. pulcherrimus, and P. depressus with an allozyme assay showing that S. intermedius and S. nudus are closely related. Kessing (1991) for the first time made an attempt at reconstructing the molecular phylogeny of the Strongylocentrotid species including both North American and east Asian species. The mtDNA phylogenies he presented gave the first glimpse of the general relationships among the species. However, the phylogenies were not tested statistically for robustness of any clustering of the species, and they also had unresolved polytomies.

Fossil records of Strongylocentrotidae provide only a broad range of time estimates for the divergence of species. Considering that the oldest fossil record of Strongylocentrotus, S antiquus Philip came from the Lower Miocene of Australia (Philip 1965) and that all the reliable fossil records of the extant species of the genus appeared from the Pliocene, Smith (1988) concluded that species of Strongylocentrotus diverged during the time period of 3.5–20 MYA. As for the origination of Strongylocentrotidae, he proposed a time frame of 35–50 MYA for the separation between Strongylocentrotidae and Parechinidae in consideration of Echinoid fossil records. Durham (1966) extended the origination time of this family farther back, to about 65 MYA. Durham also expressed his view that S. purpuratus and S. droebachiensis diverged about 5 MYA (see Busslinger, Rusconi, and Birnstiel 1982). Two molecular studies tried to estimate the divergence times of Strongylocentrotid species. In the study of single-copy nuclear DNA, Hall et al. (1980) estimated the divergence time between S. purpuratus and S. droebachiensis to be about 7 MYA, assuming that divergence between S. purpuratus and S. franciscanus occurred 15–20 MYA. Busslinger, Rusconi, and Birnstiel (1982) came to a similar estimation, about 6 MYA, for the divergence time between S. purpuratus and S. droebachiensis, based on the amount of substitution in silent sites of histone genes.

In the present study, I have attempted to resolve the phylogenetic relationships among the species of Stron-gylocentrotidae by reconstructing molecular phylogenies with mitochondrial DNA sequences, 12SrDNA (349 nt), 12SrDNA-tRNA(gln) region (862 nt), and a combined sequence of cytochrome oxidase subunit I (COI, 1,080 nt) and NADH dehydrogenase subunit I (NDI, 742 nt). Seven species (S. franciscanus, S. nudus, S. purpuratus, S. intermedius, S. droebachiensis, S. pallidus, and H. pulcherrimus) of nine undisputedly recognized species in the family were included. In addition, the rate of sequence evolution was calibrated using the time of separation between Strongylocentrotidae and Parechinidae (35–50 MYA; Smith 1988) as a reference time frame, a refined divergence time of each species was then estimated from the phylogenetic trees.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
Samples and Sequences
Three species of sea urchins, S. intermedius, S. nudus, and H. pulcherrimus were sampled from the east coast of Korea. The voucher specimens have been stored at the Korea Ocean Research and Development Institute. DNA was isolated from the gonad tissue using the Qiagen (Germany) DNA extraction kit according to the manufacturer's instructions. Three mitochondrial genes, 12SrDNA-tRNA(gln) region (862 nt), COI (1,080 nt), and NDI (742 nt), were amplified from the total genomic DNA using the polymerase chain reaction (PCR). The PCR primers were designed from the conserved regions of the genes with reference to the complete mitochondrial DNA sequences of S. purpuratus (Jacobs et al. 1988). For the 12SrDNA-tRNA(gln) region, the forward primer was from the middle of the 12SrDNA gene (L12S, 5'-AAACCAGGATTAGATACCC-3') and the reverse primer from the tRNA-gln gene (HtRNA(gln), 5'-GGAAA-AACGARGARCTTTGA-3'). These primer pairs amplified the region containing the 3'-half of 12SrDNA, the control region, and tRNAs-glu, -thr, -pro, and -gln that correspond to positions 491–1330 of the sequence of S. purpuratus (Jacobs et al. 1988). Primers LNDI (5'-AAGATGCTGGGYTAYAT GCAATT-3') and HNDI (5'-AACATTAACTGATCATASCGRAATCG-3') were used for the NDI gene (positions 2249–3033), and LCOI1490N (5'-TCTACAAACCACAARGA YATT-GG-3') and HCOIN (5'-CCCATTGAAAGAACGTAGTGAAAGTG-3') were used for the COI gene (positions 5809–6935).

Polymerase chain reactions were carried out in the Tgradient thermocycler (Whatman, Germany) using HotStar Taq DNA polymerase (Qiagen, Germany). Three or four reaction mixes of a sample were prepared and different annealing temperatures were applied to each reaction. After 10 min of initial heating at 95°C, amplification was done in 35 repetitions of a three-step cycle (denaturation, 95°C for 1 min; annealing, 48°–55°C for 1 min; extension, 72°C for 1.5 min). Polymerase chain reaction products were then cloned into pCRII-TOPO vector using TOPO TA cloning kit (Invitrogen, Calif.), and at least three clones were sequenced.

Other than the above sequences of the three species, 12SrDNA sequences of S. franciscanus, S. pallidus, and S. droebachiensis were obtained from Thomas, Maa, and Wilson (1989; GenBank accession numbers: M27674, M27523, M27672). For S. purpuratus, sequences of 12SrDNA-tRNA(gln), COI, and NDI genes were obtained from the complete mitochondrial DNA sequence (Jacobs et al. 1988; NC_001453). A species of Parechinidae, Paracentrotus lividus, was used as an outgroup to the Strongylocentrotid species in the phylogenetic analyses. The sequence of P. lividus was obtained from Cantatore et al. (1989; the complete mitochondrial DNA sequence: NC_001572).

Phylogenetic Analysis
The sequences were aligned using ClustalW in MacVector (version 6.5.3, Oxford Molecular). Because the lengths of the aligned sequences differed among the species, the 12SrDNA-tRNA(gln) region was analyzed in two ways. First, only the 12SrDNA region (349 nt) was used in reconstructing the tree of all the eight species. Second, the entire region of 12SrDNA-tRNA(gln) (862 nt) was used for the tree of S. intermedius, S. nudus, S. purpuratus, H. pulcherrimus, and the outgroup species, P. lividus. Sequences of COI (1,080 nt) and NDI (742 nt) were also obtained from the five species, and they were concatenated (COI-NDI) for the phylogenetic analysis of the five species. Indel sites in the aligned sequences of the three genes were ignored in measurement of distances between the affected pairwise comparisons in the subsequent phylogenetic analyses.

Phylogenetic trees of the sequences were reconstructed by minimum-evolution (ME), maximum parsimony (MP), maximum likelihood (ML) methods using PAUP* (version 4.0b10; Swofford 1998) and also by the method of Bayesian inference using MrBayes (Huelsenbeck and Ronquist 2001). For the model-based methods (ME and ML) an appropriate model of sequence evolution and its parameters was inferred for each gene using ModelTest (Posada and Crandall 1998). If ModelTest resulted in inapplicable values for the parameters (e.g., gamma shape parameter alpha less than 0.01), MrBayes with the general time-reversible model (GTR) was used for determining such parameters as invariable sites, gamma-distribution shape, and six rate classes of nucleotide changes. Bayesian analyses were run 50,000 generations in four chains, sampling trees every 10 generations. For all three genes, the likelihood scores had reached stationarity by 10,000 generations, and so I discarded the first 1,000 sampled trees ("burnin" = 1,000) and obtained the parameters and a 50%-majority consensus tree from the last 4,000 trees. In analyses of MP and ML, exhaustive searches were performed. For the ME analyses of 12SrDNA and 12SrDNA-tRNA(gln), Log-determinant distances (Lake 1994; Lockhart et al. 1994) were used, because the branch length of S. purpuratus was exceptionally long in these gene trees. Robustness of each branch in the trees was evaluated by the bootstrapping method with 1,000 replicates in the ME and the MP, and 500 replicates in the ML analyses. Heuristic searches were carried out for bootstrapping.

Estimation of the Rate of Sequence Evolution and the Divergence Time
Molecular clock–enforced ML trees were used to estimate the rate of sequence evolution and the divergence time of each species. Heterogeneity of evolutionary rates among the branches was checked by the log-likelihood ratio test between the clock-enforced ML tree and the non-enforced ML tree. If the test had failed, any taxa that showed exceptionally long or short branch lengths were excluded and new tests were carried out. S. purpuratus was excluded in the clock-enforced ML trees of the 12SrDNA and the 12SrDNA-tRNA(gln) sequences because of its long branch, and S. nudus was excluded in the clock-enforced ML tree of the COI-NDI sequences because of its short branch. The rates of sequence evolution were calibrated by dividing the branch length to the midpoint between the Strongylocentrotid species and the outgroup species, P. lividus, by a reference time. The time of 35–50 Myr (Smith 1988) was applied in the calibration as a reference time point for the split between the Strongylocentrotid species and the Parechinid species. Divergence time for each internal node of the trees was then calculated by dividing the branch length of that node by the calibrated rate of sequence evolution.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
DNA Sequences and Phylogenetic Relationships
About 860 bp sequences of the mitochondrial 12SrDNA-tRNA(gln) region were obtained from the four species of Strongylocentrotidae, S. intermedius, S. nudus, H. pulcherrimus, S. purpuratus, and the outgroup species, P. lividus. From the other three species of Strongylocentrotidae, S. franciscanus, S. pallidus, and S. droebachiensis, about 350 bp sequences of the 12SrDNA gene, which align with the above sequences in the first half of the region, were obtained. Alignment of the sequences shows that there are dozens of indels in the 12SrDNA-tRNA(gln) region (fig. 1). The sequence of S. purpuratus is notable in that it has a long deletion in the middle of 12SrDNA gene and also contains several species-specific single nucleotide deletions along the aligned sequences.



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FIG. 1. Aligned DNA sequences of the 12SrDNA-tRNA(gln) regions of seven Strongylocentrotid species and an outgroup species. Dots denote identical nucleotides to the first sequence and dashes are inserted for alignment. Si, S. intermedius; Sp, S. purpuratus; Spa, S. pallidus; Sd, S. droebachiensis; Hp, H. pulcherrimus; Sn, S. nudus; Sf, S. franciscanus; Pl, P. lividus

 
In the 12SrDNA gene region, there are 75 variable sites and 29 parsimony informative sites. The ME tree by LogDet distances (fig. 2) using PAUP* reveals that the genus Strongylocentrotus consists of two major separate clades, one of S. franciscanus and S. nudus, and the other of S. purpuratus, S. intermedius, S. droebachiensis, S. pallidus. Differentiation of the two clades is supported by bootstrap values higher than 80%. It is notable that the phylogenetic position of H. pulcherrimus, the only species of the genus Hemicentrotus, is located within the Stron-gylocentrotus species, being clustered with the species in the latter clade. All the internal branches of the tree are well supported by high bootstrap values (the lowest, 76%). In the latter clade, the branch of S. purpuratus is three to four times longer than those of the other species, suggesting its fast rate of sequence evolution. Relative rate tests (Tajima 1993) confirm a significantly higher rate of nucleotide substitution in the sequence of S. purpuratus (P < 0.01). Probably because of this, relationships among S. purpuratus, S. intermedius and the clade of S. pallidus and S. droebachiensis are not resolved. Parsimony analysis of the sequences using PAUP* identifies the ME tree as the maximum parsimony (MP) tree with the same tree topology (RCI = 0.57, ti/tv = 3.8). Each branch of the MP tree is also well supported by relatively high bootstrap values (the lowest, 67%; fig. 2). Bayesian inference of phylogeny for these sequences produces a tree with a couple of polytomies, but it agrees on the monophyly of S. pallidus and S. droebachiensis (probability, 81) and on the monophyletic relationship among the species of the latter major clade in the ME and MP trees (H. pulcherrimus through S. pallidus; probability, 97; fig 2). ModelTest chooses a HKY + G model (Hasegawa, Kishino, and Yano 1985) with alpha = 0.2109 and ti/tv = 3.7916. The likelihood method in PAUP* with the ModelTest parameters identifies the ME tree as one of the highest probability trees (-lnL = 958.46; the maximum likelihood tree, -lnL = 957.47) with similar branch lengths.



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FIG. 2. The minimum-evolution (ME) tree based on LogDet distances among the 12SrDNA sequences (349 nucleotides) from the seven Strongylocentrotid species and an outgroup species, P. lividus. The phylogeny was reconstructed by PAUP* (version 4.0b10; Swofford 1998). Parsimony analysis produces the maximum parsimony (MP) tree (RCI = 0.57; ti/tv = 3.8) with the same topology as the ME tree. Likelihood analysis identifies this ME tree (-lnL = 958.46) as one of the highest probability trees (the maximum likelihood tree, -lnL = 957.47). Bayesian analysis by MrBayes (Huelsenbeck and Ronquist 2001) confirms the monophyly of S. pallidus and S. droebachiensis and that of H. pulcherrimus to S. pallidus with the probabilities, 81 and 97, respectively. Branch support values are from ME bootstrap(1,000 repetitions)/MP bootstrap(1,000)/ (Bayesian) analyses

 
The entire sequences of the 12SrDNA-tRNA(gln) region from S. nudus, S. purpuratus, S. intermedius, H. pulcherrimus, and the outgroup species P. lividus contains 182 variable sites and 37 parsimony informative sites. ModelTest chooses a HKY + G model with alpha = 0.4157 and ti/tv = 3.0023. Maximum likelihood analysis of the sequences with the ModelTest parameters results in a tree that shows the closest relationship between S. intermedius and S. purpuratus, and H. pulcherrimus as a sister species to this clade, leaving S. nudus as the most distantly related species to the other three species (fig. 3). The ME analysis with either the HKY + G model or the LogDet/paralinear model, the MP analysis (ti/tv = 3.0; RCI = 0.53), and the Bayesian method all produce the same topology tree as the ML tree, with similar branch lengths. Robustness of the branching pattern of the tree is strongly supported by bootstrap values higher than 84% in all ML, ME, MP methods and by Bayesian credibility values higher than 98% (fig. 3). The branch length of S. purpuratus in the ML tree is significantly longer than the branch lengths of the other species (P < 0.05).



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FIG. 3. The maximum likelihood (ML) tree among the 12SrDNA-tRNA(gln) sequences (862 nt) from four Strongylocentrotid species and the outgroup species, P. lividus. ME by either HKY + G model distances or LogDet distances, MP (ti/tv = 3.0, RCI = 0.53), and Bayesian analyses produce the same topology tree as the ML tree, with similar branch lengths. Branch support values are from ML bootstrap (500 repetitions)/ME bootstrap(1,000)/MP bootstrap(1,000)/Bayesian analyses

 
The COI and NDI sequences obtained from S. nudus, S. purpuratus, S. intermedius, H. pulcherrimus, and the outgroup species P. lividus aligned well without any stretch of indels (see Supplementary Material online, 1 and 2). The concatenated sequences (COI-NDI sequence) of the two genes comprise 1,822 nucleotides including 540 variable sites and 181 parsimony informative sites. The nucleotide substitution model of a HKY + G with alpha = 0.1912 and ti/tv = 3.7545 was selected by ModelTest for evolution of the sequences. The ML analysis of the sequences with the ModelTest parameters reveals the phylogenetic relationships among the sequences (fig. 4) exactly as depicted by the 12SrDNA-tRNA(gln) sequences, showing that S. intermedius and S. purpuratus are the most closely related among the four species of Stron-gylocentrotidae, and that S. nudus is separated from the other three species by a long internal branch. The ME method with the HKY + G model and the MP method (ti/tv = 3.8; RCI = 0.41) as well as the Bayesian method all result in the same topology tree of the COI-NDI sequence as the ML tree. This topology is strongly supported by bootstrap values greater than 90% in the ML, ME, and MP methods and by 100% Bayesian credibility (fig. 4). The branch length of S. nudus in the ML tree is significantly shorter than the branch lengths of the other species (relative rate test, P < 0.05).



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FIG. 4. The maximum likelihood (ML) tree among the COI and NDI combined sequences (1,822 nt) from four Strongylocentrotid species and the outgroup species, P. lividus. ME by HKY + G model distances, MP (ti/tv = 3.8, RCI = 0.41), and Bayesian analyses produce the same topology tree as the ML tree. Branch support values are from ML bootstrap (500 repetitions)/ME bootstrap(1,000)/MP bootstrap(1,000)/Bayesian analyses

 
The Rate of Sequence Evolution and the Divergence Time
The rate of sequence evolution was calibrated from the molecular clock–enforced ML trees, and the divergence time of each species was estimated from the branch length, using the calibrated evolutionary rate. For 12SrDNA, the log likelihood ratio test rejected the assumption of clock-like evolution when all the sequences were included. Without the sequence of S. purpuratus that showed an exceptionally long branch in the phylogenetic tree (fig. 2), the sequences passed the molecular clock assumption: the likelihood of the clock-enforced ML tree (-lnL = 858.4914) was not significantly different from the non-enforced likelihood tree with the same topology (-lnL = 857.1516; the ML tree, -lnL = 856.5948). In this test, a nucleotide substitution model of GTR + G + I with its parameters determined by the Bayesian method (alpha = 1.0221; IP = 0.5383; A-C, 1.3787; A-G, 13.8664; A-T, 3.5755; C-G, 1.1245; C-T, 18.6779, G-T, 1.0000) was applied. The ModelTest method with these sequences resulted in an inapplicable value, 0.0081, for the gamma shape parameter alpha. The molecular clock–enforced ML tree of 12SrDNA but S. purpuratus (fig. 5) shows a congruent tree topology with the ME and MP tree of 12SrDNA (fig. 2) and also with the trees of the 12SrDNA-tRNA(gln) and the COI-NDI genes (figs. 3 and 4). Considering the phylogenetic position of S. purpuratus revealed by the other trees (figs. 2–4 GoGo), the species is added to the clock-enforced ML tree in figure 5 with a broken line at the node of S. intermedius and a clade of S. pallidus and S. droebachiensis as a trichotomous branch.



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FIG. 5. The molecular clock-enforced maximum likelihood (ML) tree among the 12SrDNA sequences from the six Strongylocentrotid species but S. purpuratus and the outgroup species, P. lividus. S. purpuratus was excluded because of its long branch in reconstructing this clock-enforced tree, but it was then added to this tree separately at the position shown by a broken line in figure 2. The log likelihood ratio test shows no significant difference between the clock-enforced tree (-lnL = 858.49) and the clock-non-enforced tree (-lnL = 857.15). The tree is congruent with the trees of 12SrDNA, 12SrDNA-tRNA(gln) and COI-NDI (figs. 2, 3, and 4), and also with the tree of ND5 from S. pallidus, S. droebachiensis, S. intermedius, S. purpuratus, and S. franciscanus (Thomas, Maa, and Wilson 1989) reanalyzed in this study by ML, ME, and MP methods in PAUP* (Swofford 1998) and by the Bayesian method. The scale bar represents the divergence time calibrated by the use of separation between Strongylocentrotidae and Parechinidae 35–50 MYA (Smith 1988) as the reference time point. The internal nodes are labeled (A) through (E)

 
The clock-enforced ML tree calibrates the rate of sequence evolution 0.24%–0.34%/Myr when the time of 35–50 Myr is used in the calibration as a reference time point for the split between the Strongylocentrotid species and the Parechinid species P. lividus. By applying the evolutionary rate to the branches of the tree, divergence time for each node is calculated: 13–18 MYA for the separation between the two major clades of Strongylocentrotidae (node A in fig. 5), 7.2–10 MYA for the separation of H. pulcherrimus (node B), 4.6–6.6 MYA for the divergence among S. intermedius, S. purpuratus, and the clade of S. pallidus and S. droebachiensis (node C), 2.1–3.1 MYA for the split between S. droebachiensis and S. pallidus (node D), and 5.7–8.1 MYA for the split between S. nudus and S. franciscanus (node E; table 2).


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Table 2 The Divergence Time for Each Internal Node in the Mitochondrial Gene Genealogies of Strongylocentrotidae Sea Urchins (Myr).

 
The log likelihood ratio test for 12SrDNA-tRNA(gln) sequences rejected the assumption of clock-like evolution when all the five sequences of S. intermedius, S. purpuratus, S. nudus, H. pulcherrimus, and P. lividus were included. But, the four species without S. purpuratus that showed a long branch in the ML tree (fig. 3) passed the molecular clock assumption: the likelihood of the clock-enforced ML tree (-lnL = 1955.3273) was not significantly different from the non-enforced ML tree (-lnL = 1953.3794). The substitution model of a HKY + G with alpha = 0.1942 and ti/tv = 4.4046 chosen by ModelTest for the four sequences was applied to this test. The molecular clock–enforced ML tree of the 12SrDNA-tRNA(gln) (tree not shown) is consistent with the clock-enforced ML tree of 12SrDNA (fig. 5) and calibrates the rate of sequence evolution 0.25%–0.36%/Myr. Divergence time for each node of the tree then turns out to be 13–19 MYA for the separation between S. nudus and the clade of H. pulcherrimus and S. intermedius (node A in fig. 5), and 7.8–11 MYA for the split between H. pulcherrimus and S. intermedius (node B; table 2).

For the COI-NDI gene, four sequences of S. intermedius, S. purpuratus, H. pulcherrimus, and P. lividus, except S. nudus, which has a short branch length in the ML tree of the COI-NDI gene (fig. 4), passed the log likelihood ratio test for the assumption of clock-like evolution. ModelTest provided a substitution model of a HKY + G with alpha = 0.1917 and ti/tv = 3.5129 for the four sequences. The clock-enforced ML tree (-lnL = 5076.2364) was not significantly different from the non-enforced ML tree (-lnL = 5075.4397). The molecular clock-enforced tree of the COI-NDI (tree not shown) agrees well with the tree of the 12SrDNA (fig. 5) and gives the rate of sequence evolution 0.65%–0.93%/Myr. Divergence time for each node of the tree is estimated to be 10–14 MYA for the separation between H. pulcherrimus and the clade of S. purpuratus and S. intermedius (node B in fig. 5), and 8.5–12 MYA for the divergence between S. purpuratus and S. intermedius (node C; table 2).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
Phylogeny of Strongylocentrotidae
Phylogenetic analyses of seven Strongylocentrotid species based on the sequences of 12SrDNA and 12SrDNA-tRNA(gln) region, and the combined sequence of COI and NDI show that relationships among the species are summarized by ((S. franciscanus, S. nudus), (H. pulcherrimus, (S. purpuratus, S. intermedius, (S. droebachiensis, S. pallidus)))). Any trees of the sequences reconstructed by the methods of ML, ME, MP in the PAUP* program (Swofford 1998) and by the method of Bayesian inference of phylogeny in MrBayes (Huelsenbeck and Ronquist 2001) are congruent with the above pattern of species relationships (figs. 2–4GoGo). This phylogeny reveals several intriguing features. First, it suggests that the family Strongylocentrotidae consists of at least two major clades, one of S. franciscanus and S. nudus, and the other of S. purpuratus, S. intermedius, S. droebachiensis, S. pallidus, and H. pulcherrimus. The internal branch connecting the two clades is longer than the other internal branches, and its robustness is strongly supported by bootstrap values higher than 80% and by Bayesian posterior probabilities higher than 97% in all three gene trees (figs. 2–4GoGo). This feature agrees well with some of the previous molecular studies. The molecular evolution study of a nuclear gene, sperm bindin (Biermann 1998), showed that S. franciscanus is clearly separated from the other Strongylocentrotid species including H. pulcherrimus, S. purpuratus, S. droebachiensis, S. pallidus, as well as S. polyacanthus and A. fragilis. The study of single-copy nuclear DNA (Hall et al. 1980) also showed that the relationship between S. franciscanus and S. purpuratus is twice as distant as that between S. purpuratus and S. droebachiensis. The restriction site map study of mtDNA (Vawter and Brown 1986) revealed that S. purpuratus and S. droebachiensis are more closely related to each other than either of them is related to S. franciscanus. Kessing (1991) presented molecular phylogenies of COI-COII-ATPase8-ATPase6 in which S. franciscanus and S. nudus are separated from the other Strongylocentrotid species by a long branch.

A second feature in the present molecular phylogeny of Strongylocentrotidae is that the phylogenetic position of H. pulcherrimus is located within the genus Stron-gylocentrotus. All the trees of 12SrDNA, 12SrDNA-tRNA(gln), and COI-NDI place H. pulcherrimus within Strongylocentrotus (figs. 2–4GoGo). As indicated in the previous paragraph, the phylogenies of the sperm bindin (Biermann 1998) and the COI-COII-ATPase8-ATPase6 sequences (Kessing 1991) also suggested the same phylogenetic position for this species. From the cladistic point of view, this phylogeny raises doubt about the assignment of a separate genus-level classification to the species H. pulcherrimus. In fact, Mortensen (1903) included H. pulcherrimus in Strongylocentrotus in his early publication, but later he attributed it to his new monospecific genus Hemicentrotus (1942, 1943). If H. pulcherrimus retains its genus-level status, the genus Strongylocentrotus should be considered as a paraphyletic group. In Biermann (1998), another monospecific genus, Allocentrotus, was confronted with the same problem of classification as Hemicentrotus because its single species A. fragilis appears within the genus Strongylocentrotus. Considering the deep branching between the two major clades in the phylogeny of Strongylocentrotidae (fig. 2), assignment of a new genus-level classification to the clade of S. nudus and S. franciscanus could be one solution to circumvent the classification problem. H. pulcherrimus constitutes a basal taxon among the Stron-gylocentrotid species in the latter major clade. The branch separating this species from the other species such as S. intermedius, S. purpuratus, S. droebachiensis, and S. pallidus is well supported in the ME tree and the MP tree of the 12SrDNA gene by bootstrap values higher than 70% (fig. 2). All the trees of the 12SrDNA-tRNA(gln) and the COI-NDI manifest such a branching pattern, with bootstrap values above 84% and Bayesian credibility above 98% (figs. 3 and 4). The tree of a nuclear gene, sperm bindin (Biermann 1998), confirms the separation between H. pulcherrimus and the other species by a 100% bootstrap value.

Third, S. pallidus and S. droebachiensis are the most recently diverged species pair among the seven Stron-gylocentrotid species investigated here. These two are the only species in Strongylocentrotidae that occur in the Arctic and north Atlantic coasts (Jensen 1974). Monophyly of the two species is well supported in the ME tree (LogDet distance; bootstrap value 92%), in the MP tree (85%), and in the Bayesian tree (posterior probability, 81%) of the 12SrDNA (fig. 2). Thomas, Maa, and Wilson (1989) also presented the monophyly of the two species in the MP tree of ND5 sequences from S. pallidus, S. droebachiensis, S. intermedius, S. purpuratus, S. franciscanus using only the second base position of codons. I reanalyzed the ND5 sequences using the recently developed algorithms such as ModelTest (Posada and Crandall 1998), ME and ML in PAUP* (Swofford 1998), and Bayesian inference of phylogeny in MrBayes (Huelsenbeck and Ronquist 2001). The results show that the phylogeny is summarized by (S. franciscanus (S. purpuratus, S. intermedius (S. droebachiensis, S. pallidus))), which is congruent with the tree of the 12SrDNA (fig. 2). The monophyletic clade of S. droebachiensis and S. pallidus is again well supported in the new analyses: ME, bootstrap value 93%; ML, 70%; Bayesian inference, probability 85%. On the other hand, Kessing (1991) showed that the two species made an unresolved trichotomy with S. purpuratus. However, when the mtDNA sequence of COI-COII-ATPase8-ATPase6 from S. pallidus, S. droebachiensis, S. purpuratus, and S. franciscanus (Palumbi and Kessing 1991) was reanalyzed, with P. lividus as an outgroup (Cantatore et al. 1989), in the present study, the monophyly of S. pallidus and S. droebachiensis was re-identified in the trees of ME (LogDet distances; bootstrap value, 92%), MP (ti/tv = 3.4; 99%), ML (64%) and Bayesian inference (probability, 74%).

Finally, the evolution of S. purpuratus is particular among the Strongylocentrotid species in that the branch of S. purpuratus is significantly longer than those of the other species in two of the three gene trees investigated (figs. 2 and 3). Relative rate tests (Tajima 1993) show that the rate of sequence evolution has been significantly higher in S. purpuratus both for the 12SrDNA (P < 0.01) and for the 12SrDNA-tRNA(gln) (P < 0.05). In the alignment of the 12SrDNA-tRNA(gln) sequences among the Strongylocentrotid species (fig. 1), the sequence of S. purpuratus has several specific indels, indicating its fast evolution. Biermann (1998) also showed that in the ME tree (LogDet distance) of the sperm bindin the branch of S. purpuratus is almost twice as long as those of other closely related species. Probably because of the high rate of mutation, the phylogenetic position of S. purpuratus in the trees of the 12SrDNA and of the bindin gene has not been resolved, being retained in a polytomy with S. intermedius, S. droebachiensis, and S. pallidus (fig. 2; Biermann 1998). In contrast, the trees of COI-NDI and ND5 (reanalyzed in the present study) do not show any significantly longer branch for S. purpuratus than for the other species. A wide range of variation in the rate of sequence evolution for S. purpuratus compared to other species of Strongylocentrotidae could invoke caution when sequences of S. purpuratus are being used in studies of comparative biology and evolution.

The Rate of Sequence Evolution and Divergence Time of the Strongylocentrotid Species
Species-specific difference in the rate of sequence evolution is manifested in S. purpuratus, as mentioned in the previous section. The present study also shows that the evolutionary rate differs by a factor of 2–3, depending on the genes of mtDNA in Strongylocentrotidae. In the 12SrDNA and the 12SrDNA-tRNA(gln), the rates are calibrated to be 0.24%–0.34%/Myr and 0.25%–0.36%/Myr, but in the combined sequence of COI and NDI, it turns out to be 0.65%–0.93%/Myr, although the same reference time point is applied and any aberrantly evolving species—such as S. purpuratus in the 12SrDNA and the 12SrDNA-tRNA(gln) sequences and S. nudus in the COI-NDI sequences—are excluded in the calibration. Reanalysis of the other mtDNA sequences of the Stron-gylocentrotid species such as the COI-COII-ATPase8-ATPase6 (Palumbi and Kessing 1991) and the ND5 (Thomas, Maa, and Wilson 1989) results in the evolutionary rates 0.68%–0.97%/Myr and 1.19–1.70%/Myr, respectively. The higher rates in the protein-coding genes compared to the structural genes such as the 12SrDNA and tRNAs could be due in part to the selection pressure-free changes in the synonymous sites and in part to the saturation effect of nucleotide substitution, especially of transitional changes in the deep branch (Vigilante et al. 1991).

With the rate of sequence evolution calibrated in Strongylocentrotidae, new or refined estimates of divergence time for each Strongylocentrotid species are made. The time of separation between the two major clades of Strongylocentrotidae, S. franciscanus and S. nudus versus H. pulcherrimus, S. purpuratus, S. intermedius, S. droebachiensis, and S. pallidus is estimated to be 13–18 MYA in the 12SrDNA, or 13–19 MYA in the 12SrDNA-tRNA(gln). The two estimates agree well and refine much of the wide range of the previous estimation by Smith (3.5–20MYA, 1988) (table 3). Hall et al. (1980) once assumed the time as 15–20 MYA in calibration of the rate of single-copy DNA evolution. For the time of origination of H. pulcherrimus, two estimates from 12SrDNA and 12SrDNA-tRNA(gln) are almost identical (7.2–10 MYA and 7.8–11 MYA, respectively) but the estimate from COI-NDI (10–14 MYA) is older than the other two estimates (table 2). Such difference between estimates is even higher for the diversification event among S. purpuratus, S. intermedius, and the clade of S. pallidus and S. droebachiensis: the estimate from COI-NDI is almost twice as old as that from 12SrDNA (8.5–12 MYA versus 4.6–6.6 MYA). Although both estimates provide refinements to the proposal of Smith (1988), the younger estimate agrees better with other estimations for this trichotomy (Hall et al. 1980; Busslinger, Rusconi, and Birnstiel 1982; Palumbi and Wilson 1990; Palumbi and Kessing 1991; table 3). The older estimates from the COI-NDI might be attributable to the higher effect of substitution saturation on the deepest calibration time point in the protein-coding gene compared to the structural gene. Moreover, the saturation effect would arise much earlier in the COI-NDI than in the 12SrDNA, because the sequence evolves 2–3 times faster in the former gene. The older estimates from the COI-NDI could be considered as the upper bounds of the time for the cladogenic events. Then, the time estimates for the separation of H. pulcherrimus and for the diversification event at the trichotomy would range from 7.2 to 14 MYA and from 4.6 to 12 MYA, respectively (table 3). For the time of split between S. droebachiensis and S. pallidus, the present study gives a younger estimate (2.1–3.1 MYA) than the other estimates, based either on molecular data (Palumbi and Kessing 1991, 3–5MYA; table 3) or on the fossil records (Durham and MacNeil 1967; older than 3.5MYA). Durham and MacNeil (1967) interpreted the fossil records of the two species from Pliocene deposits in Japan, Greenland, and northern Europe as indications of their presence in the north Pacific before the Bering Sea-way was first opened about 3.5 MYA, through which the species migrated to the Arctic and the Atlantic. To resolve the contradiction between the present estimate from the 12SrDNA and that from the fossil records, another independent molecular estimation such as the one based on a nuclear gene and/or a finer dating the fossil records would be required.


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Table 3 Comparison of the Divergence Time Estimates for Each Internal Node of Strongylocentrotid sea Urchins Among the Studies.

 
Phylogeny, Geographical Distribution, and Cladogenic Events
Information on the phylogeny and the geographical distribution of the Strongylocentrotid species provides clues to the possible cause of speciation. The sister species, S. franciscanus and S. nudus, show disjunct distribution, inhabiting the northeast Pacific and the northwest Pacific, respectively (Jensen 1974; Bazhin 1998). So do the sister species, S. purpuratus and S. intermedius. Such distribution patterns between the sister species pairs imply that speciation could have been invoked by the trans-Pacific vicariant event (Vermeij 1989). During the cold climate period, the lowstand of sea level together with glaciation would push southward the amphi-Pacific species, resulting in disjunct sister species on either side of the Pacific coast, as shown in marine snails (Vermeij 1989) and abalones (Lee and Vacquier 1995). Chronology of sea level fluctuation (Haq, Hardenbol, and Vail 1987) indicates correlation between the lowstand of sea level and the cladogenesis of the sister species pairs. The sea level dropped drastically, over 80–100 m, at 10.5 MYA, and since then it has fluctuated frequently until recently. This period of frequent sea level change overlaps with the divergence times between S. purpuratus and S. intermedius (4.6–12 MYA), and between S. franciscanus and S. nudus (5.7–8.1 MYA; table 3).

Geographical distribution of H. pulcherrimus, which is restricted to the East/Japan Sea and the regions only proximal to it (Jensen 1974), implies that its speciation is also due to the sea level change. The East/Japan Sea is a semi-enclosed neritic sea, having only narrow, shallow straits connected to the north Pacific. Therefore, during the lowstands of sea level since the Meiocene, water flow in and out of the East/Japan Sea has been very limited (Tada 1994; Park et al. 2000). At this condition of the sea, gene flow between populations inside and outside of the sea could have been restricted and speciation followed. Divergence between Tegula rusticus and T. nigerrima was attributed to such paleoceanographic condition of the East/Japan Sea (Hellberg 1998). Genetic differentiation among populations of Turbo cornutus inside and outside of the sea was also ascribed to more recent events of glacio-eustatic sea level change and the divergent current pattern (Kojima, Segawa, and Hayashi 1997). For the two Caribbean sea urchins E. viridis and E. lucunter, which have diverged into separate species within the Caribbean Sea in the last 1.5 Myr, allopatry caused by glaciation-induced sea level drops was elicited to explain the speciation (McCartney, Keller, and Lessios 2000).

A problem, however, lies in explaining speciation by sea level change alone. The chronology of sea level fluctuation reveals that most of the lowstands of sea level lasted less than one million years (Haq, Hardenbol, and Vail 1987), rendering the allopatry of populations resulting from sea level drop only transient. Unless the separations among populations during the period of low sea level are retained even after a rise in the sea level, it would be implausible for those populations to go into speciation. Transient allopatry might allow too little time for the isolated populations to accumulate genetic differentiation sufficient to confer reproductive isolation between the populations upon subsequent contact (Hellberg 1998). However, if only a few gamete recognition proteins become diversified during the transient allopatry, it would provide the necessary means of reproductive isolation and endow the once-isolated populations to differentiate further, even after the subsequent contact, as suggested for speciation of Tegula (Hellberg 1998). In the sea urchins of Strongylocentrotidae, Biermann (1998) showed that the sperm bindin, a key molecule for the sperm to bind and fertilize the egg, evolves by strong selection, indicating its possible involvement in speciation. Such a strong positive selection phenomenon in the evolution of sperm-borne proteins has been observed in several marine organisms such as Echinometra sea urchins (Metz and Palumbi 1996), abalones (Lee, Ota, and Vacquier 1995; Swanson and Vacquier 1995), and Tegula (Hellberg, Moy, and Vacquier 2000). Although McCartney, Keller, and Lessios (2000) did not incline to such an explanation, speciation of E. viridis and E. lucunter in the Caribbean Sea, without any present land barriers between them, could be also explained by transient allopatry linked with differentiation of any gamete recognition proteins. In fact, gametic incompatibility, although unidirectional, is observed between the two sympatric species, yet the allopatric species E. viridis and E. vanbrunti remain completely interfertile (Lessios and Cunningham 1990).


    Supplementary Materials
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
The sequences newly obtained in the present study have been deposited in GenBank under accession numbers AF525450-5 and AF525767-9. Alignments of the COI and the NDI sequences of the Strongylocentrotid species are available at the journal's Web site.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
 Literature Cited
 
I thank Drs. J. Y. Lee and Y. J. Park for collecting sea urchin samples and Mrs. J. I. Kwak, W. S. Chung, S. E. Yoon, and Ms. H. Park for their technical help. I also thank Drs. E. H. Davidson, V. D. Vacquier, M. E. Hellberg, and two anonymous reviewers for helpful advice and detailed comments on the manuscript. This work was supported by Korea Ocean Research and Development Institute grants PE98745 and PE83500 and Korea Ministry of Science and Technology grant PN46700.


    Footnotes
 
William Jeffery, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Materials
 Acknowledgements
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Accepted for publication March 24, 2003.