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Biol. Bull. 201: 95-103. (August 2001)
© 2001 Marine Biological Laboratory

Biogeography of Asterias: North Atlantic Climate Change and Speciation

John P. Wares

Duke University Zoology, Box 90325, Durham, North Carolina 27708


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Fossil evidence suggests that the seastar genus Asterias arrived in the North Atlantic during the trans-Arctic interchange around 3.5 Ma. Previous genetic and morphological studies of the two species found in the Atlantic today suggested two possible scenarios for the speciation of A. rubens and A. forbesi. Through phylogenetic and population genetic analysis of data from a portion of the cytochrome oxidase I mitochondrial gene and a fragment of the ribosomal internal transcribed spacer region, I show that the formation of the Labrador Current 3.0 Ma was probably responsible for the initial vicariance of North Atlantic Asterias populations. Subsequent adaptive evolution in A. forbesi was then possible in isolation from the European species A. rubens. The contact zone between these two species formed recently, possibly due to a Holocene founding event of A. rubens in New England and the Canadian Maritimes.


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
The North Atlantic Ocean is populated by hundreds of taxa which invaded from the North Pacific following the opening of the Bering Strait about 3.5 million years ago (Ma; Durham and MacNeil, 1967; Vermeij, 1991). Some of these species have maintained genetic contact with source populations in the Pacific until recently (Palumbi and Kessing, 1991; van Oppen et al., 1995), but many of them have subsequently differentiated from the source populations and are now recognized as distinct species (e.g., Gosling, 1992; Reid et al., 1996; Collins et al., 1996). Circumstantial evidence suggests strongly that the seastar genus Asterias (Echinodermata: Asteroidea: Asteriidae: Asteriinae) participated in the trans-Arctic interchange (Worley and Franz, 1983; Vermeij, 1991). Today, two species are recognized in the North Atlantic: A. forbesi on the North American coast, primarily from Cape Hatteras to Cape Cod (Franz et al., 1981), and A. rubens on the American coast primarily from Cape Cod northward (Franz et al., 1981), and on the European coast from Iceland to western France (Clark and Downey, 1992; Hayward and Ryland, 1995). American populations of A. rubens have been previously described as A. vulgaris, a junior synonymy (Clark and Downey, 1992). These species co-occur over a broad range of the North American continental shelf centered on Cape Cod (Gosner, 1978; Menge, 1979; Franz et al., 1981).

Two current hypotheses attempt to explain the recent speciation between A. forbesi and A. rubens. Schopf and Murphy (1973) suggested that they were a germinate species pair formed by a late Pleistocene (0.02–2.5 Ma) vicariance event (i.e., a separation of populations) at Cape Cod, possibly due to lower sea levels during glacial maxima. There is some evidence for hybridization between these seastars, but the separation could be maintained by localized adaptation to the different thermal regimes north and south of Cape Cod (Franz et al., 1981). However, this thermal boundary was latitudinally unstable throughout the Pleistocene (Cronin, 1988) and only in the past 20,000 years (Holocene) has it returned to its current state. If the geographical isolation between these taxa was recent, as proposed in Schopf and Murphy (1973), then strong natural selection within each region has prevented widespread hybridization.

The second hypothesis, based on morphological and paleoceanographic evidence, suggested a late Pliocene (approximately 2.5–5 Ma) separation of Asterias into distinct North American and European species, followed by a Holocene recolonization of North America by the European species A. rubens (Worley and Franz, 1983). This hypothesis would therefore suggest that the differentiation between the two Atlantic species is entirely due to long-term isolation. Thus, subsequent physiological adaptations to warmer water in A. forbesi (Franz et al., 1981) are independent of the speciation event. Essentially, the distinction between these species reflects either primary divergence due to selection or secondary contact following vicariance (Endler, 1977).

In this study, mitochondrial and nuclear sequence data were collected from populations of A. forbesi and A. rubens throughout North America and Europe, as well as from populations of the Pacific sister taxon A. amurensis (Clark and Downey, 1992). Phylogenetic and population genetic assays were used to test the hypotheses described above. It appears that Worley and Franz (1983) were remarkably accurate in suggesting a Pliocene speciation followed by a recent invasion of A. rubens from Europe, even in their prediction of details of timing, mechanisms, and effects. Although selection may have driven some of the divergence, it now seems clear that the initial separation of A. rubens and A. forbesi is due to late Pliocene changes in climate and ocean current flow, whereas North American populations of A. rubens are very recent arrivals.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Asterias specimens were collected from intertidal sites listed in Table 1. Tube feet were immediately placed in 95% ethanol or DMSO buffer (0.25 M EDTA pH 8.0, 20% DMSO, saturated NaCl; Seutin et al., 1991). Species were identified on the basis of key morphological characters described in Clark and Downey (1992) and Hayward and Ryland (1995).


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  		TABLE 1 Collection sites for individuals of each species in this study

 
DNA extraction and amplification
DNA was phenol-extracted from each specimen following the protocol in Hillis et al. (1996). These extractions were stored at -80°C. PCR amplification of an approximately 700-bp portion of the mitochondrial cytochrome c oxidase I (COI) protein-encoding gene was performed using the primers LCO1490 and HCO 2198 from Folmer et al. (1994). Amplification was performed in 50-µl reactions containing 10–100 ng DNA, 0.02 mM each primer, 5 µl Promega 10x polymerase buffer, 0.8 mM dNTPs (Pharmacia Biotech), and 1 unit Taq polymerase (Promega). Reactions took place in a Perkin-Elmer 480 thermal cycler with a cycling profile of 94° (60 s) -40° (90 s) -72° (150 s) for 40 cycles. The internal transcribed spacer (ITS) region was amplified under similar conditions, with an annealing temperature of 50°C and with primers ITS4 and ITS5 (White et al., 1990). For each individual, sequences were obtained for three to four clones, and the consensus sequence was obtained to eliminate Taq error.

PCR products were prepared for sequencing and were cycle-sequenced as in Wares (2001) using both PCR primers. COI sequences representing each individual in this study have been deposited with GenBank (AF240022-240081); ITS sequences were only obtained for 10 individuals, representing each species and region, and are also accessible in GenBank (AF346608-AF346617). Sequences were aligned and edited for ambiguities using complementary fragments in Sequencher 3.0 (Genecodes Corp., Cambridge, MA). No gaps or poorly aligned regions occurred in the COI alignment, but missing characters were trimmed from the ends of the alignment to produce equal sequence lengths for all individuals. In the ITS alignment, all missing or ambiguous characters, including gaps, were removed. Consensus sequences were exported as a NEXUS file for subsequent analysis in PAUP*4.0b4a (Swofford, 1998).

Phylogenetic analysis
A heuristic search for the set of most-parsimonious trees based on the COI data was performed using PAUP*4.0b4a (Swofford, 1998). Trees were rooted using Leptasterias polaris (Asteriinae) and individuals of A. amurensis. Starting trees were obtained via stepwise addition, with simple addition sequence. Tree-bisection-reconnection was used for branch swapping, and branches were collapsed if the maximum branch length was zero.

Maximum-likelihood (ML) phylogenies were also generated in PAUP*. The best-fit model for all likelihood analyses (HKY with {Gamma}-distributed rate variation; Hasegawa et al., 1985; Yang, 1994) was determined by adding parameters until the likelihood description of the neighbor-joining tree did not significantly improve (Goldman, 1993; Cunningham et al., 1998), using the likelihood-ratio test of ModelTest (Posada and Crandall, 1998). A series of bootstrap replicates (100 ML replicates, heuristic search) using PAUP* were performed to determine support for interspecific relationships in the clade. Estimates of the transition-transversion ratio for the HKY model, along with the gamma-distributed parameter for among-site rate heterogeneity, were held constant for bootstrap replicates. A maximum likelihood phylogeny of the ITS sequence data was also generated using the appropriate best-fit model (F81: equal rates among sites, unequal base frequencies).

Estimates of speciation time within the North Atlantic require an estimate of the mutation rate (µ). Because paleontological evidence suggests that Asterias arrived in the North Atlantic during the trans-Arctic interchange about 3.5 Ma (Worley and Franz, 1983; Vermeij, 1991), and because climatic changes shortly thereafter would have prevented additional trans-Arctic migration, this date was used to calibrate the divergence between the Pacific species A. amurensis and the North Atlantic taxa. Other species, including the echinoderm Strongylocentrotus pallidus, have clearly maintained more recent connections across the Arctic (Palumbi and Kessing, 1991). However, S. pallidus appears to be more tolerant of Arctic conditions than Asterias (Worley and Franz, 1983; Palumbi and Kessing, 1991).

The ML estimate of the internal branch length separating the sister taxa (representing net nucleotide divergence d, Nei and Li, 1979) was used to estimate the appropriate mutation rate µ (Edwards and Beerli, 2000), where µ = 0.5 d/(3.5 Ma). Estimates were obtained for the full COI data set (first, second, and third codon positions), as well as third position only. Use of the third-position estimate circumvents problems with branch length estimation when there is strong rate variation (Wares and Cunningham, in press), as well as problems with the potential influences of non-neutral evolution.

Haplotype networks may be more appropriate representations of genealogical relationships within species than are outgroup-rooted phylogenetic trees, because ancestral haplotypes are still present in the population (Crandall and Templeton, 1996). Methods associated with haplotype networks were used to determine the root haplotype for A. rubens. Determination of the root haplotype prevents spurious conclusions about ancestry among populations. Networks were created using a parsimony criterion in the program TCS (alpha version 1.01, Clement et al., 2000); at the same time, a Bayesian analysis of the likelihood that parsimony is violated (Templeton et al., 1992) was performed to ensure that the data set was unlikely to be complicated by homoplasy.

The ML root was determined using GeneTree (Griffiths and Tavaré, 1994); the likelihood of each possible rooted gene tree was determined under an infinite-alleles model. This model assumes that there are no multiply substituted nucleotide sites. The method allows for recoding of characters so that independent substitutions are analyzed separately, but this was not an issue with the A. rubens COI data. The relative likelihood of each tree in comparison with all other possible rooted trees was calculated using 107 simulations in GeneTree.

Tests of rate constancy
Likelihood-ratio tests (Felsenstein, 1988; Goldman, 1993) were used to test the hypothesis that the data collected were consistent with a constant-rate Poisson-distributed process of substitution (molecular clock). This procedure ensures that the data can be used to estimate the time of divergence between A. rubens and A. forbesi. The ML phylogeny was estimated using the best-fit model, and then the likelihood of this phylogeny was recalculated while constraining the estimate to fit the molecular clock model. These likelihood (L) estimates were used to calculate the {chi}2-distributed test statistic {delta} = 2[1n(L0) - 1n(L1)], with (n - 2) degrees of freedom where n is the number of taxa in the tree.

Neutrality tests
Because adaptive selection may have played a role in the divergence between A. rubens and A. forbesi (given a short divergence time; Schopf and Murphy, 1973), polymorphism data for each species were input to DNAsp v.3.5 (Rozas and Rozas, 1999) to test for patterns of non-neutral evolution. Within each species, Tajima’s (1989) test generates a beta-distributed parameter indicating the difference in two estimates (polymorphic sites and number of alleles) of diversity. Significantly low statistics can indicate non-neutral evolution (Tajima, 1989). Additionally, a McDonald-Kreitman test (McDonald and Kreitman, 1991) was performed on each pairwise set of species polymorphism data to determine whether selection has played a role in the divergence between A. rubens and A. forbesi. Also, DNAsp was used to calculate haplotype diversity (H, see eqn. 8.4 in Nei, 1987) and sampling variance for each species or population.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
The COI data set (60 individuals) includes 627 characters, of which 484 are constant, 49 are parsimony-uninformative, and 94 are parsimony-informative. Base frequencies are 33.7% A, 19.6% C, 21.6% G, and 25.1% T for this fragment. Most of the substitutions (92.3%) are at third-position sites; overall, 63% of all third-position characters are polymorphic. These third-position sites are heavily AT-biased (39.0% A, 15.1% C, 11.8% G, and 33.9% T).

The best-fit model (HKY + {Gamma}) was used to estimate distances among individuals to determine whether there is any evidence for saturation at third-position characters in the COI coding region. A plot of pairwise genetic distances versus number of third-position substitutions does not indicate any pattern of saturation (data not shown); in fact, all of the information within each species is based on third-position substitutions. Additionally, the best-fit model was reestimated for this character partition; likelihood-ratio tests indicate that the HKY model with invariant sites (I = 0.213) and no rate variation describes the third-position data effectively. The Asterias data sets do not reject the molecular clock model, whether all positions are considered (P = 0.163), or only third positions (P = 0.231).

Maximum-likelihood analysis was used to determine the interspecific gene tree, using all codon positions and the HKY + {Gamma} model (Tr:Tv 8.256, {alpha} = 0.0608, four rate classes). The ML tree (L = 1472.87) is presented in Figure 1A, including all individuals sampled within A. amurensis, A. rubens, and A. forbesi. Bootstrap support is indicated on the tree, with each species being fully resolved in 100% of replicates. The Pacific species A. amurensis is basal to a strongly supported clade of Atlantic species in this phylogeny.



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  		Figure 1. Phylogenetic trees for Asterias generated using the best-fit maximum likelihood model in each data set (COI: HKY + {Gamma}; ITS: F81). (A) Cytochrome c oxidase I phylogeny of inter- and intraspecific Asterias relationships. Here all characters (first, second, and third position) are included; an identical topology is found using parsimony or distance methods, or looking at third-position characters alone. Bootstrap support for each species is indicated by the numbers below each branch. These data do not reject a molecular clock model. The divergence across the Arctic (between A. amurensis and the Atlantic species) is considered to be 3.5 Ma; this generates an estimate of about 3.0 Ma for the divergence between A. rubens and A. amurensis (see Table 2 and Discussion). Haplotypes A–D of A. rubens are found on both the North American and European coasts (A: Maine (n = 8), Nova Scotia (n = 2), Newfoundland (n = 2), Iceland (n = 1), Norway (n = 2), Ireland (n = 1); B: Maine (n = 2), Nova Scotia (n = 4), Newfoundland (n = 3), Iceland (n = 1), Ireland (n = 2), France (n = 2); C: Maine (n = 1), Norway (n = 3), Ireland (n = 2), and France (n = 1); D: Ireland (n = 1), and Maine (n = 1)). Amphi-Atlantic haplotype B is the maximum likelihood root (index = 0.857). (B) Internal transcribed spacer (ITS) phylogeny of inter- and intraspecific Asterias relationships. Likelihood ratio tests do not reject a hypothesis of proportional branch lengths (P > 0.10) suggesting that, aside from substantial differences in substitution rate, the two phylogenies are equivalent representations of interspecific differentiation. A nearly identical phylogeny is reconstructed when indels are included in the ITS data.

 
Following exclusion of missing and ambiguous characters in the ITS data set (length of fragment varies from 413 to 482 bases when gaps included), these data include 368 characters of which 343 are constant, 1 is parsimony-uninformative, and 24 are parsimony-informative. Indels did not vary within species and were removed (analysis with gapped characters included produced nearly identical results). Parsimony analysis produced a single most-parsimonious tree of 25 steps, and the ML phylogeny (best-fit model F81, no rate variation) is shown in Figure 1B. Under a variety of mutational models, this phylogeny is statistically indistinct from the COI phylogeny in Figure 1A. Likelihood-ratio tests indicate that, in addition to a similar interspecific topology, branch lengths on the COI and ITS phylogenies are proportional (P > 0.10), though the substitution rate is significantly different (P < 0.05). Bootstrap replicates of the ITS data also indicate strong support for differentiation among these species. The ITS data do not reject a molecular clock model.

Divergence among these species is indicated in Table 2. HKY + {Gamma} distances in the COI fragment indicate that A. amurensis, A. forbesi, and A. rubens have been isolated from each other for a similar amount of time; assuming trans-Arctic isolation around 3.5 Ma, A. rubens and A. forbesi have been separated for at least 3.0 Ma. Although the estimated divergence date is higher when all codon positions are included (Table 2), and these data do not reject a molecular clock, neutrality tests (see below) suggest that some second-position substitutions may be under selection. Therefore, third-position sites may be more appropriate for the divergence estimate. The estimated divergence time is also higher when the ITS data are used; however, there is no reason to believe that speciation predated the appearance of Asterias in the North Atlantic, and the long branch leading to A. forbesi is not easily explained since it appears in both phylogenies (one using a protein-coding gene, one using untranslated spacer region data). This longer branch appears to influence the age estimates of the COI (all positions) and ITS data sets strongly.


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  		TABLE 2 Internal branch lengths (based on best-fit likelihood model) separating Asterias species (lower triangle*, all 3 matrices)

 
A McDonald-Kreitman test (McDonald and Kreitman, 1991) rejects a pattern of neutral substitution between A. rubens and A. forbesi (P < 0.01, Table 3). Despite branch lengths that do not reject the molecular clock model, there is an excess of amino acid replacement substitutions between the Atlantic species. The replacement substitutions between A. rubens and A. forbesi do not include any first-position substitutions. Half (8/16) of the amino acid substitutions do not involve a change in charge or polarity, whereas almost half (7/16) of the changes substitute a basic residue for an uncharged or nonpolar residue. However, there does not seem to be an obvious pattern to these changes between A. rubens and A. forbesi. Other species comparisons do not reject the neutral model of substitution (Table 3). Within each species, Tajima’s (1989) test is nonsignificant (A. amurensis, D = 0.837, P > 0.10; A. forbesi, D = -0.705, P > 0.10; A. rubens, D = -1.482, P > 0.10), indicating that there is no reason to suspect non-neutral evolution in the intraspecific comparisons.


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  		TABLE 3 McDonald-Kreitman tests on each Asterias species pair using cytochrome c oxidase I (COI) translated data

 
Additionally, Bayesian analysis (Templeton et al., 1992; Clement et al., 2000) of the COI data within A. rubens indicates greater than 95% confidence that the intraspecific gene tree is parsimonious. The ML root haplotype is found on both coasts of the Atlantic (Fig. 1A, Haplotype B), and this haplotype is at least an order of magnitude more likely to be the ancestral haplotype than any other haplotype of A. rubens (likelihood index = 0.857). All North American haplotypes are also found in Europe; the unique haplotypes found in Europe contribute to a significantly higher allelic diversity (P < 0.01, Table 4). The ITS data are consistent with the COI data in that there is no allelic diversity among North American and European individuals of A. rubens (n = 6).


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  		TABLE 4 Comparisons of haplotype diversity (H, see eqn. 8.4 in Nei 1987, calculated in DNAsp 3.50, Rozas and Rozas 1999) for the cytochrome c oxidase I fragment in each species and population of A. rubens

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
Understanding the mechanisms that are responsible for the divergence of Asterias rubens and A. forbesi first requires that the timing of their divergence be estimated. Estimates based on the molecular calibrations reported here suggest that these species last shared a common ancestor at least 3.0 Ma (Table 2), not long after the genus first arrived in the North Atlantic (around 3.5 Ma; Worley and Franz, 1983; Vermeij, 1991). Note, however, that asterozoan skeletons are rarely preserved in the fossil record, because they lack rigidly articulated skeletons and rapidly disintegrate (Barker and Zullo, 1980); indeed, fossils of A. forbesi have been reported only twice, each time in Pleistocene interglacial sediments. Thus, little direct evidence points to the first appearance of Asterias in the North Atlantic (Durham and MacNeil, 1967; Worley and Franz, 1983), and the biogeographic data used in this paper is therefore based on consistent fossil evidence from other cold temperate species that participated in the trans-Arctic exchange. Nevertheless, there is reason to believe that Asterias also spread from the Pacific to the Atlantic at about 3.5 Ma (Worley and Franz, 1983). Miocene and early Pliocene temperatures were around 5°–6°C warmer in the North Atlantic and Arctic, permitting the initial trans-Arctic passage of temperate species (Berggren and Hollister, 1974; Vermeij, 1991), but then two dramatic changes were initiated around 3.0 Ma that appear to play a role in speciation within the North Atlantic.

At that time, warm North Atlantic currents were displaced by the formation of the cold-water Labrador Current. This event created a significant thermal gradient in the North Atlantic, and tropical-temperate faunas were abruptly replaced with polar and subpolar faunas on the continental shelf off Nova Scotia and the rest of New England (Berggren and Hollister, 1974; Worley and Franz, 1983; Cronin, 1988). As Northern Hemisphere glaciation began, the present-day latitudinally controlled faunal provincialization was established as well (Berggren and Hollister, 1974). This dramatic cooling of the northwestern North Atlantic probably initiated the separation of North Atlantic Asterias into European and North American populations with very little genetic contact (Worley and Franz, 1983). Subsequent Pleistocene glaciation would have prevented the long-term establishment of populations in New England, as most of the North American coast from Long Island Sound northward was covered by a kilometer of ice during glacial maxima (Kelley et al., 1995).

Pacific and Atlantic populations of other species appear to have had more recent trans-Arctic genetic contact than the estimates above would suggest for Asterias (Palumbi and Kessing, 1991; van Oppen et al., 1995). Moreover, rapid climatic fluctuations (Cronin, 1988; Roy et al., 1996) during the Pleistocene could have permitted large-scale changes in the geographic range of cold temperate species. However, both the sea urchin Strongylocentrotus pallidus (Palumbi and Kessing, 1991) and the red alga Phycodrys rubens (van Oppen et al., 1995) appear to have greater tolerance for Arctic waters than Asterias does. Worley and Franz (1983) report that expansion of Asterias populations into habitats as far north as Greenland only occurs periodically, and that these populations cannot tolerate colder waters (Franz et al., 1981). However, the indirect morphological and paleontological evidence is bolstered by the molecular evidence, which strongly suggests that A. rubens and A. forbesi diverged shortly after their ancestral lineage separated from the Pacific A. amurensis. The estimates of mutation rate presented here are very similar to other estimates for both the COI fragment (Knowlton and Weigt, 1998; Schubart et al., 1998; Wares, 2001; Wares and Cunningham, in press) and the ITS fragment (Schlötterer et al., 1994; van Oppen et al., 1995). Thus these data strongly support earlier inferences of a late Pliocene trans-Arctic passage and subsequent speciation within the Atlantic.

An analysis of genealogical patterns within A. rubens confirms that the North American populations of this species are descendants of a recent colonization from Europe that probably followed the most recent glacial maximum (about 20,000 BP, Holder et al., 1999). The genealogical data presented here fit several important patterns that suggest a recent range expansion (Wares, 2000). All North American haplotypes are identical to the most-common European haplotypes (Fig. 1A). Generally, invading haplotypes are the most deeply nested haplotype in the European (putative source) population. This is to be expected, because deeply nested ancestral haplotypes are often the most common (Castelloe and Templeton, 1994), and therefore have a higher probability of participating in long-distance dispersal events. Haplotype B (Fig. 1A) is a good illustration of this expectation—it is closely related to each other haplotype and has a high copy number in both European and American populations. These observations contribute to the high likelihood (85.7%, more than an order of magnitude greater likelihood than any other haplotype) that this is the ancestral allele in A. rubens.

Additionally, allelic diversity is significantly lower in North American A. rubens than in Europe (Table 4), a signal of recent range expansion (Hewitt, 1996; Austerlitz et al., 1997). However, the North American colonization is difficult to date because there are no unique haplotypes in North America; ancestral allelic polymorphism tends to inflate indirect estimates of population size and age (Kuhner et al., 1998; Edwards and Beerli, 2000). The lack of unique diversity in North America also prevents the meaningful use of other phylogeographic methods; for instance, statistics of the geographic dispersion of haplotypes (for review see Templeton, 1998) are uninformative (Wares, unpubl. data). This is primarily because even closely related individuals (identical haplotypes) are distributed across the entire geographic range of A. rubens. It is possible that the multiple shared alleles between Europe and North America represent a multiple-invasion history; Asterias larvae are planktotrophic and disperse in the water column for 6 or more weeks (Clark and Downey, 1992).

There is evidence that natural selection has played some role in the overall divergence between these species. A significant number of amino acid replacement substitutions distinguish A. rubens from A. forbesi (Table 3), all of them reflecting second- or third-position nucleotide substitutions. There is no obvious pattern to the amino acid replacements, as most of them involve substitutions among uncharged or nonpolar amino acids. Two of the three species in the genus Asterias are found in cold-temperate waters, while A. forbesi is found in the warmer mid-Atlantic region (Schopf and Murphy, 1973; Franz et al., 1981). Many of the physiological differences between A. rubens and A. forbesi (Franz et al., 1981) reflect this latitudinal distribution. However, the possibility that these amino acid substitutions are related to physiological differences in the warm-temperate A. forbesi has never been tested. The difference in temperature between the habitats of A. rubens and A. forbesi is unlikely to contribute to differences in metabolic rate that could accelerate the mutation rate (for review see Rand, 1994). Nevertheless, this hypothesis is worth examination, because A. forbesi is supported by relatively long branches in both the COI and the non-coding ITS region (Table 2, Fig. 1B). If natural selection is playing a role in the amino acid divergences of the mitochondrial COI gene between A. rubens and A. forbesi, there is no reason why a noncoding nuclear sequence should reflect the same increase in divergence rate.

In conclusion, the biogeographic response of Asterias to late Pliocene climatic and oceanographic change fits a pattern predicted by Worley and Franz (1983). Following the arrival of Asterias in the North Atlantic around 3.5 Ma (Worley and Franz, 1983; Vermeij, 1991), populations were established on both the European and North American coasts during a period when the North Atlantic was as much as 5–6°C warmer (Berggren and Hollister, 1974). The formation of the Labrador Current 3.0 Ma rapidly changed the faunal composition of the intertidal Canadian Maritimes and New England coast, and Asterias populations in this region probably went extinct. An American population survived under the conditions of the mid-Atlantic coast and Gulf Stream waters (A. forbesi), and the European population (A. rubens) has recently recolonized the cold-temperate shores of New England and the Canadian Maritimes. Thus, the zone of sympatry between these two species appears to be a zone of secondary contact. Hybridization is considered rare between these species (Schopf and Murphy, 1973; Worley and Franz, 1983), but whether behavioral mechanisms (Franz et al., 1981) or gametic recognition mechanisms (Hellberg and Vacquier, 1999; Pernet, 1999) are responsible is unclear.

The genetic data presented here illustrate a strong concordance between paleoceanographic changes and indirect estimates of speciation between the North Atlantic Asterias species. The species boundaries are phylogenetically quite distinct, and the divergence estimates based on these genetic data appear to support a late Pliocene, rather than late Pleistocene or Holocene, separation. A better understanding of the balance between oceanographic and climatic changes in the late Pliocene and Pleistocene, and of the response of species based on varying life-history characters to these changes, will enable us to predict the responses of other taxa (Cunningham and Collins, 1998; Wares and Cunningham, in press).


   Acknowledgments
 
I thank G. Manchenko, A. Ingólfsson, J. Maunder, B. O’Connor, D. Garbary, D. M. Rand, and C. Damiani for assistance in the field collecting seastars. The manuscript was greatly improved thanks to discussions with T. Turner and the suggestions of two anonymous reviewers. These analyses were done in the laboratory of C. W. Cunningham, whose aid during this and other projects was invaluable. A National Science Foundation Dissertation Improvement Grant (NSF DEB-99-72707) to J. P. W. funded this study.


   Footnotes
 
Current address: Dept. of Biology, University of New Mexico, Castetter Hall, Albuquerque, NM 87131. E-mail: jpwares{at}unm.edu


   Literature Cited
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

    Austerlitz, F., B. Jung-Muller, B. Godelle, and P.-H. Gouyon. 1997. Evolution of coalescence times, genetic diversity and structure during colonization. Theor. Popul. Biol.51:148–164.
    Barker, L., and V. A. Zullo. 1980. Asterias forbesi, (Desor) (Asterozoa, Asteroidea) from the Pleistocene "Coquina" at Fort Fisher, New Hanover County, North Carolina. J. Elisha Mitchell Sci. Soc.96:39–44.
    Berggren, W. A., and C. D. Hollister. 1974. Paleogeography, paleobiogeography, and the history of circulation in the Atlantic Ocean. Soc. Econ. Paleontol. Mineral.20:126–186.
    Castelloe, J., and A. R. Templeton. 1994. Root probabilities for intraspecific gene trees under neutral coalescent theory. Mol. Phylogenet. Evol.3:102–113.[Medline]
    Clark, A. M., and M. E. Downey. 1992. Starfishes of the Atlantic., Chapman & Hall, London.
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