Morphological and phylogenetic evidence for hybridization and introgression in a sea star secondary contact zone

Glacial cycles and other climatic events have been widely invoked as factors promoting divergence, secondary contact, and hybridization between populations of terrestrial organisms, but the origin and fate of secondary contact in the sea is much less well understood. We studied the distribution of morphological and genetic variation in a northwest Atlantic zone of secondary contact between congeneric sea stars of Asterias that probably separated after the Pliocene as part of the trans-Arctic interchange. These species have similar reproductive biology and can hybridize in the laboratory. However, multivariate analysis of morphological traits scored from sea stars inside and outside the zone of secondary contact clearly indicated two clusters of phenotypes that corresponded to the two taxonomic species. A quantitative analysis of this clustering pattern did not support the hypothesis of a third grouping that might correspond to intermediate hybrid phenotypes. Known F1 hybrids from laboratory matings grouped with one of the two taxonomic species. However, a survey of mtDNA sequence variation clearly indicated that B13% of individuals of one species (Asterias forbesi) are descendants of hybridization events that resulted in introgression of haplotypes of Asterias rubens into populations of A. forbesi. We conclude that morphological phenotypes are inadequate to identify hybrids of Asterias and their descendants, and that hybridization and introgression might be common in this secondary contact zone. Additional key words: phylogeography, Asterias, mtDNA, statistical parsimony The repeated glaciations of the Pleistocene epoch had a significant and well-characterized impact on the phylogeography and genetic differentiation of terrestrial organisms. At the peak of the last glacial maximum 0.02Mya, the Northern Hemisphere ice complex covered most of North America, northern Eurasia, and the polar seas (CLIMAP 1976). Glacial advances caused extirpation or retreat of terrestrial species into ice-free refuges (Pielou 1991; Hewitt 1996, 1999, 2000; Taberlet et al. 1998). Protracted isolation caused many populations to differentiate in allopatry as a result of such vicariance (Endler 1977). Glacial retreat allowed members of these populations to disperse out of isolation and make secondary contact with members of other refugial populations. Depending on the evolution of reproductive barriers in allopatry, the outcomes of secondary contact range from complete reproductive isolation (with possible selection for reinforcement of this barrier) to the formation of stable hybrid zones and introgressive hybridization (Avise et al. 1987; Harrison 1993; Arnold 1997, 2006; Howard & Berlocher 1998). The role of Pleistocene glacial cycles in vicariance, differentiation, and secondary contact in the sea is much less well understood, perhaps in large part because the sources of vicariance in the sea (such as changes in the direction and speed of ocean currents; Benzie 1999) are less easily inferred than the large ice sheets that extirpated or divided terrestrial populations. One likely source of marine vicariance associated with these glacial cycles was sea-level decline and local extirpation associated with glacial advances (Ingolfsson 1992): these effects are expected to have been most severe on near-shore marine species (Palumbi 1994). In the northwest Atlantic, the last glaciation is thought to have been particularly difficult for rocky intertidal animals, perhaps forcing many species from this habitat (see Wares & Cunningham 2001). Shallow-water populations and Invertebrate Biology 126(4): 373–384. r 2007, The Authors Journal compilation r 2007, The American Microscopical Society, Inc. DOI: 10.1111/j.1744-7410.2007.00107.x Current address: Department of Biology, Rollins College, Winter Park, FL 32789-4499, USA. Author for correspondence. Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6. E-mail: [email protected] communities on rocky substrates in New England and Atlantic Canada are expected to be relatively young (of post-glacial age) and descended from refugial populations in Europe, the southeastern United States, or the north Pacific (e.g., Ingolfsson 1992; Wares & Cunningham 2001; Addison & Hart 2005; Vermeij 2005; P. Rawson & F. Harper, unpubl. data). A prominent member of this community is the forcipulate sea star genus Asterias. One species, Asterias rubens LINNAEUS 1758, has an amphi-Atlantic distribution (Tortonese 1963) from Portugal to the United Kingdom, Norway, and Iceland in the northeast Atlantic and from North Carolina to southern Labrador in the northwest Atlantic (Clark & Downey 1992). North American populations of A. rubens were previously described as Asterias vulgaris, a junior synonym (Tortonese 1963; Clark & Downey 1992). Asterias forbesi DESOR 1848 is restricted to the northwest Atlantic and ranges from the eastern shore of Nova Scotia to the Gulf of Mexico (Clark & Downey 1992). Wares (2001) used mtDNA sequences to estimate the initial vicariance of North Atlantic species of Asterias at B3.0Mya, following the formation of the Labrador Current. Low allelic diversity and lack of unique haplotypes in northwest Atlantic populations of A. rubens suggest that this species recently recolonized North America (Wares 2001). Populations of A. forbesi may have survived the Pleistocene glaciations in southern refugia off the southeastern coast of North America, and has subsequently expanded its range northward (Harris et al. 1998). Populations of the two species are now sympatric in a broad zone of secondary contact from about Cape Cod to northeastern Nova Scotia. What is the typical fate of such secondary contact zones in the sea? Many shallow temperate marine communities include pairs or groups of closely related congeners that appear to be reproductively isolated as good biological species (Miner 1950; Mayr 1954; Palumbi 1994; Kozloff & Price 1996). However, Gardner (1997) suggested that hybridization in the marine environment might be as common as in other environments. Many of Gardner’s examples of hybridization in marine invertebrates were based on the observation of morphologically intermediate specimens. Additional examples may be difficult to detect using morphology alone; some heterospecific crosses produce offspring that more closely resemble the phenotype of one parental species (Lamb & Avise 1987; Byrne & Anderson 1994). Analysis of genetic markers independent of morphological traits may often reveal allele or haplotype sharing that is consistent with hybridization and introgression (DePamphilis & Wyatt 1990; Paige & Capman 1993; Bert et al. 1996; Addison & Hart 2005). Previous morphological, reproductive, and genetic evidence for hybridization between North Atlantic species of Asterias is mixed. The morphological similarities between the sibling species have been well described (Verrill 1866; Coe 1912; Aldrich 1956; Downey 1973). One important consequence of these similarities has been disagreement over the identification and frequency of putative hybrids. Some studies included anecdotal accounts of morphological intermediates that were considered to be hybrids (Clark 1904; Perlmutter & Nigrelli 1960; Ernst 1967; Walker 1973). Menge (1986) estimated that 1.4% of 295 Asterias species from Boston Harbor, MA, were morphological intermediates, but did not report which characters were variable. Clark & Downey (1992) suggested that hybrids were common from Cape Cod to Maine but that these hybrids did not reach sexual maturity. In contrast, in an extensive survey of skeletal characters, Worley & Franz (1983) concluded that hybrids were not present and assigned thousands of specimens to one or the other species. They suggested that coastal populations of forbesilike animals from Maine were morphological variants of A. rubens or relict populations of A. forbesi that had been isolated from southern populations by periodic climate changes. The latter hypothesis is plausible in light of the expected oscillations in the latitudinal distribution of shallow-water marine organisms in the North Atlantic associated with cyclical Pleistocene climate change (Wares & Cunningham 2001). Laboratory studies suggest that these two species are potentially reproductively compatible. They have overlapping spawning seasons in the zone of secondary contact (Smith 1940; Boolootian 1966; Menge 1986), and share similar natural histories (feeding activity and diet, Menge 1979) and habitats (Menge 1986). Natural hybridization and introgression are likely to occur because adults of the two species release their gametes in close proximity to each other, hybrid fertilization rates in the laboratory are often high (Ernst 1967; Harper & Hart 2005), and hybrid zygotes can mature into viable and fertile F1 adults (Harper & Hart 2005). However, despite this mixed morphological and reproductive evidence for hybridization potential, there is no genetic evidence that hybridization in Asterias leads to introgression in natural populations. A recent phylogenetic analysis of speciation in North Atlantic Asterias species using mtDNA (COI) and nuclear ITS sequences (Wares 2001) did not find any 374 Harper & Hart Invertebrate Biology vol. 126, no. 4, fall 2007 evidence of shared haplotypes between A. forbesi and A. rubens (a potential indicator of hybridization and introgression). Although Wares’ study was not specifically designed to detect introgression, its results (combined with the morphological interpretation of Worley & Franz 1983) suggest that the Asterias zone of secondary contact may not be a hybrid zone. Here, we use morphological and genetic analyses of sympatric and allopatric Asterias populations to reconsider the potential for hybridization and introgression in this zone of secondary contact. In the morphological analysis, specimens were examined and scored for the five qualitative characters (Clark & Downey 1992) and three morphometric characters (Worley & Franz 1983) analyzed in previous studies. We used principal components analysis (PCA) and clustering algorithms to determine whether a significant cluster of morphological intermediates was quantitatively supported. This approach provides an objective method for identifying morphological intermediates between the two parental phenotypes that are possible hybrids. We then obtained mtDNA sequences of the highly variable control region from a subset of animals identified in the morphological survey to look for introgression in sympatric populations. Our sampling design emphasized specimens within the contact zone, including some with morphological characters intermediate between the parental species. The results suggest that introgression between Asterias species may be frequent and asymmetrical without leaving an easily detected signature of morphological intermediates.