Polar Sciences Laboratory, Korea Ocean Research and Development Institute, Ansan, South Korea
Correspondence: E-mail address: ylee{at}kordi.re.kr.
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Abstract |
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Key Words: Strongylocentrotidae sea urchin phylogeny evolutionary rate divergence time speciation
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Introduction |
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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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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 3.520 MYA. As for the origination of Strongylocentrotidae, he proposed a time frame of 3550 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 1520 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 (3550 MYA; Smith 1988) as a reference time frame, a refined divergence time of each species was then estimated from the phylogenetic trees.
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Materials and Methods |
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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 clockenforced 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 3550 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.
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Results |
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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 1014 MYA for the separation between H. pulcherrimus and the clade of S. purpuratus and S. intermedius (node B in fig. 5), and 8.512 MYA for the divergence between S. purpuratus and S. intermedius (node C; table 2).
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Discussion |
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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. 24). 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 23, 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 speciessuch as S. purpuratus in the 12SrDNA and the 12SrDNA-tRNA(gln) sequences and S. nudus in the COI-NDI sequencesare 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 1.191.70%/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 1318 MYA in the 12SrDNA, or 1319 MYA in the 12SrDNA-tRNA(gln). The two estimates agree well and refine much of the wide range of the previous estimation by Smith (3.520MYA, 1988) (table 3). Hall et al. (1980) once assumed the time as 1520 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 (7.210 MYA and 7.811 MYA, respectively) but the estimate from COI-NDI (1014 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 (8.512 MYA versus 4.66.6 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 23 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 (2.13.1 MYA) than the other estimates, based either on molecular data (Palumbi and Kessing 1991, 35MYA; 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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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).
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Supplementary Materials |
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Acknowledgements |
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Footnotes |
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Literature Cited |
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Bazhin, A. G. 1998. The sea urchin genus Strongylocentrotus in the seas of Russia: taxonomy and ranges. Pp. 563566 in R. Mooi and M. Telford, eds. Echinoderms: San Francisco (Proceedings of the 9th International Echinoderm Conference, San Francisco, August 1996). Balkema, Rotterdam.
Biermann, C. H. 1998. The molecular evolution of sperm bindin in six species of sea urchins (Echinoida: Strongylocentrotidae). Mol. Biol. Evol. 15:1761-1771.
Busslinger, M., S. Rusconi, and M. L. Birnstiel. 1982. An unusual evolutionary behaviour of a sea urchin histone gene cluster. EMBO J. 1:27-33.[ISI]
Cantatore, P., M. Roberti, G. Rainaldi, M. N. Gadaleta, and C. Saccone. 1989. The complete nucleotide sequence, gene organization, and genetic code of the mitochondrial genome of Paracentrotus lividus. J. Biol. Chem. 264:10965-10975.
Davidson, E. H., R. A. Cameron, and A. Ransick. 1998. Specification of cell fate in the sea urchin embryo: summary and some proposed mechanisms. Development 125:3269-3290.
Davidson, E. H., J. P. Rast, and P. Oliveri, et al. (25 co-authors). 2002. A genomic regulatory network for development. Science 295:1669-1678.
Dinnel, P. A., J. M. Link, Q. J. Stober, M. W. Letourneau, and W. E. Roberts. 1989. Comparative sensitivity of sea urchin sperm bioassays to metals and pesticides. Arch. Environ. Contam. Toxicol. 18:748-755.[ISI][Medline]
Durham, J. W. 1966. Classification. Pp. 270295 in R. C. Moore, ed. Treatise on invertebrate paleontology. Geological Society of America and University of Kansas Press, Lawrence, Kansas.
Durham, J. W., and F. S. MacNeil. 1967. Cenozoic migrations of marine invertebrates through the Bering Strait region. Pp. 326349 in D. Hopkins, ed. The Bering Land Bridge. Stanford University Press, Palo Alto, Calif.
Hall, T. J., J. W. Grula, E. H. Davidson, and R. J. Britten. 1980. Evolution of sea urchin non-repetitive DNA. J. Mol. Evol. 16:95-110.[ISI][Medline]
Haq, B. U., J. Hardenbol, and P. R. Vail. 1987. Chronology of fluctuating sea level since the Triassic. Science 235:1156-1166.[ISI]
Hasegawa, M., H. Kishino, and T. Yano. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22:160-174.[ISI][Medline]
Hellberg, M. E. 1998. Sympatric sea shells along the sea's shore: the geography of speciation in the marine gastropod Tegula. Evolution 52:1311-1324.[ISI]
Hellberg, M. E., G. W. Moy, and V. D. Vacquier. 2000. Positive selection and propeptide repeats promoter rapid interspecific divergence of gastropod sperm protein. Mol. Biol. Evol. 17:458-466.
Huelsenbeck, J. P., and F. R. Ronquist. 2001. MrBayes: Bayesian inference of phylogeny. Distributed by the authors. Department of Biology, University of Rochester, N.Y.
Jacobs, H. T., D. J. Elliott, V. B. Math, and A. Farquharson. 1988. Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol. 202:185-217.[ISI][Medline]
Jensen, M. 1974. The Strongylocentrotidae (Echinoidea), a morphologic and systematic study. Sarsia 57:113-148.
Kessing, B. D. 1991. Strongylocentrotid sea urchin mitochondrial DNA: phylogenetic relationships and patterns of molecular evolution. M.Sc. thesis, University of Hawaii, Manoa.
Kojima, S., R. Segawa, and I. Hayashi. 1997. Genetic differentiation among populations of the Japanese turban snail Turbo (Batillus) cornutus corresponding to warm currents. Mar. Ecol. Prog. Ser. 150:149-155.[ISI]
Lake, J. A. 1994. Reconstructing evolutionary trees from DNA and protein sequences: Paralinear distances. Proc. Natl. Acad. Sci. USA 91:1455-1459.[Abstract]
Larrain, A. P. 1995. Biodiversity in Chilean echinoderms: state of the art and biosystematic synopsis. Gayana Zool. 59:73-96.
Lawrence, J. M. 2001. Edible sea urchins: biology and ecology. Elsevier, Amsterdam. 419 pp.
Lee, Y.-H., G. M. Huang, R. A. Cameron, G. Graham, E. H. Davidson, L. Hood, and R. J. Britten. 1999. EST analysis of gene expression in early cleavage-stage sea urchin embryos. Development 126:3857-3867.
Lee, Y.-H., T. Ota, and V. D. Vacquier. 1995. Positive selection is a general phenomenon in the evolution of abalone sperm lysin. Mol. Biol. Evol. 12:231-238.[Abstract]
Lee, Y.-H., and V. D. Vacquier. 1995. Evolution and systematics in Haliotidae (Mollusca: Gastropoda): inferences from DNA sequence of sperm lysin. Mar. Biol. 124:267-278.[CrossRef][ISI]
Lessios, H. A., and C. W. Cunningham. 1990. Gametic incompatibility between species of the sea urchin Echinometra on the two sides of the Isthmus of Panama. Evolution 44:933-941.[ISI]
Lockhart, P. J., M. A. Steel, M. D. Hendy, and D. Penny. 1994. Recovering evolutionary trees under a more realistic model of sequence evolution. Mol. Biol. Evol. 11:605-612.
Matsuoka, N. 1987. Biochemical study on the taxonomic situation of the sea-urchin, Pseudocentrotus depressus. Zool. Sci. 4:339-347.[ISI]
McCartney, M. A., G. Keller, and H. A. Lessios. 2000. Dispersal barriers in tropical oceans and speciation in Atlantic and eastern Pacific sea urchins of the genus Echinometra. Mol. Ecol. 9:1391-1400.[CrossRef][ISI][Medline]
Metz, E. C., and S. R. Palumbi. 1996. Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin. Mol. Biol. Evol. 13:397-406.[Abstract]
Mortensen, T. 1903. The Danish Ingolf Expedition. IV(1), Copenhagen. 193pp.
Mortensen, T. 1942. New Echinoidea (Camarodonta). Vidensk. Meddr Dansk Naturh. Foren. 106:225-231.
Mortensen, T. 1943. A monograph of the Echinoidea III(3) Camarodonta. C. A. Reitzel, Copenhagen. 446pp.
Palumbi, S. R., and B. D. Kessing. 1991. Population biology of the trans-Arctic exchange: mtDNA sequence similarity between Pacific and Atlantic sea urchins. Evolution 45:1790-1805.[ISI]
Palumbi, S. R., and A. C. Wilson. 1990. Mitochondrial DNA diversity in the sea urchins Strongylocentrotus purpuratus and S. droebachiensis. Evolution 44:403-425.[ISI]
Park, S.-C., D.-G. Yoo, C.-W. Lee, and E.-I. Lee. 2000. Last glacial sea-level changes and paleogeography of the Korea (Tsushima) Strait. Geo-Marine Letters 20:64-71.
Pennisi, E. 2002. Chimps and fungi make genome "Top Six.". Science 296:1589-1591.
Peterson, K. J., R. A. Cameron, and E. H. Davidson. 2000. Bilaterian origins: significance of new experimental observations. Dev. Biol. 219:1-17.[CrossRef][ISI][Medline]
Philip, G. M. 1965. Classification of echinoids. J. Paleontol. 39:45-62.
Posada, D., and K. A. Crandall. 1998. ModelTest: Testing the model of DNA substitution. Bioinformatics 14:817-818.[Abstract]
Smith, A. B. 1988. Phylogenetic relationship, divergence times, and rates of molecular evolution for Camarodont sea urchin. Mol. Biol. Evol. 5:345-365.
Swanson, W. J., and V. D. Vacquier. 1995. Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa. Proc. Natl. Acad. Sci. USA 92:4957-4961.[Abstract]
Swofford, D. L. 1998. PAUP*: phylogenetic analysis using parsimony(*and other methods). Version 4. Sinauer Associates, Sunderland, Mass.
Tada, R. 1994. Paleoceanographic evolution of the Japan Sea. Palaeogeogr. Palaeoclimatol. Palaeoecol. 108:487-508.[CrossRef][ISI]
Tajima, F. 1993. Simple method for testing molecular clock hypothesis. Genetics 135:599-607.
Thomas, W. K., J. Maa, and A. C. Wilson. 1989. Shifting constraints on tRNA genes during mitochondrial DNA evolution in animals. New Biologist 1:93-100.[Medline]
Vacquier, V. D., W. J. Swanson, and M. E. Hellberg. 1995. What have we learned about sea urchin sperm bindin? Dev. Growth Differ. 37:1-10.[ISI]
Vawter, L., and W. M. Brown. 1986. Nuclear and mitochondrial DNA comparisons reveal extreme rate variation in the molecular clock. Science 234:194-196.[ISI][Medline]
Vermeij, G. J. 1989. Geographical restriction as a guide to the causes of extinction: the case of the cold northern oceans during the Neogene. Paleobiol. 15:335-356.[ISI]
Vigilante, L., M. Stoneking, H. Herpending, K. Hawkes, and A. C. Wilson. 1991. African populations and the evolution of human mitochondrial DNA. Science 253:1503-1507.[ISI][Medline]