Evolution, 42(2), 1988, pp. 227-238 A MOLECULAR REEXAMINATION OF INTROGRESSION BETWEEN HELIANTHUS ANNUUS AND H. BOLANDERI (COMPOSITAE) loREN H. RIESEBERG, I DOUGLAS E. SOLTIS, Department ofBotany, Washington State University, Pullman, WA 99164 AND JEFFREY D. PALMER Department ofBiology, University ofMichigan, Ann Arbor, MI48109 Abstract. -Heiser (1949) hypothesized that a weedy race of Helianthus bolanderi had originated by the introgression of genes from H. annuus into a serpentine race of H. bolanderi. Although Heiser's investigation of these species is frequently cited as one ofthe best examples ofintrogression in plants, definitive evidence of gene exchange is lacking (Heiser, 1973). To determine whether the weedy race of H. bolanderi actually originated via introgression, we analyzed allozyme, chlo­ roplast-DNA (cpDNA), and nuclear-ribosomal-DNA (rDNA) variation. Evidence from enzyme electrophoresisdid not support the proposed introgressive origin ofweedy H. bolanderi. We detected a total of 37 low-frequency alleles distinguishing the serpentine race of H. bolanderi from H. annuus. Weedy H. bolanderi possessed only four of the 37 marker alleles. Further analysis demonstrated that serpentine H. bolanderi combined seven of the 35 alleles distinguishing H. annuus from weedy H. bolanderi, indicating that serpentine H. bolanderi shares three more marker alleles with H. annuus than does weedy H. bolanderi. These results are similar to expectations for race divergence from a single common ancestor and suggest that, ifintrogression occurred, the majority of marker alleles were rapidly lost following the initial hybridization event. Even more compelling evidence opposing Heiser's (1949) hypothesis, however, was from re­ striction-fragment analysis of cpDNA and nuclear rDNA. We detected a total of 17 cpDNA and five rDNA restriction-site mutations among the 19 populations examined. No parallel or back mutations were observed in phylogenetic trees constructed using eithercpDNAor rDNA mutations, and both phylogenies were completely congruent regarding the alignment of all three taxa. In addition, the weedy race ofH. bolanderi possessed a unique cpDNA, which was outside the range of variation observed among populations of either ofthe presumed parental species. Mean sequence divergences between the cpDNAs of weedy H. bolanderi and those ofserpentine H. bolanderi and H. annuus were 0.30% and 0.35%, respectively. These estimates are comparable to sequence­ divergence values observed between closely related species in other plant groups. Given the lack of parallel or convergent mutations in the cpDNA and rDNA phylogenetic trees, the complete congruence of these trees with flavonoid- and allozyme-variation patterns, and the presence of a unique and divergent chloroplast genome in the weedy race of H. bolanderi, we suggest that the weedy race of H. bolanderi was not derived recently through introgression, as hypothesized, but is relatively ancient in origin. Received June 9,1987. Accepted October 27,1987 For the past 50 years, interspecific gene permanent addition of genes from one flow via hybridization and subsequent in­ species into another, resulting in the cre­ trogression has been implicated as one of ation and establishment ofa new type, has the primary factors bringing about evolu­ rarely been demonstrated (Heiser, 1973). tionary change in many plant groups (An­ Nevertheless, numerous studies have im­ derson, 1949; Stebbins, 1959, 1969; Grant, plicated introgression in the origin and es­ 1981). However, the frequency of occur­ tablishment ofnew races, varieties, subspe­ rence and importance ofintrogression in the cies, and species (e.g., Heiser, 1949, 1951b; evolution of plants is unknown. Although Grant, 1950; Levin, 1963; Ornduff, 1967; the occurrence of backcrossed individuals Bloom, 1976; Davis, 1985). Although in­ in hybrid swarms is well documented (e.g., trogression may have occurred in these ex­ Heiser, 1949; Alston and Turner, 1963, amples, they are based primarily on mor­ Levin, 1975; Soltis and Soltis, 1986), the phological data and historical inference, making conclusive documentation of in­ trogression difficult. Other equally plausible I Present address: Rancho Santa Ana Botanic Gar­ hypotheses are convergent evolution, reten­ den, 1500 N. College, Claremont, CA 91711. tion ofancestral characters, and phenotypic 227 228 L. H. RIESEBERG ET AL. plasticity. However, the use of precise ge­ sive hybrid swarms in areas ofcontact. Us­ netic markers provided by allozyme varia­ ing morphological and cytological data, tion and restriction-site mutations in spe­ Heiser (1949) hypothesized that the weedy cific DNA sequences may resolve ambiguous race ofH. bolanderi developed in California questions of introgression. through introgression ofgenes from the re­ The annual species of Helianthus (sun­ cently introduced H. annuus into the ser­ flowers) are particularly favorable for study­ pentine race of H. bolanderi. Although H. ing introgression (Stebbins and Daly, 1961; annuus and H. bolanderi do hybridize, and Heiser et aI., 1969; Levin, 1975). They are although the weedy race ofH. bolanderi ap­ widespread, self-incompatible, and weedy, proaches H. annuus in a number of mor­ often colonizing disturbed habitats. Heiser phological characters, no evidence of gene (1947,1949, 1951a, 1951b, 1954, 1961)has exchange away from hybrid swarms has been shown that hybridization occurs frequently documented. In fact, even Heiser (1973) and that hybrids, although nearly sterile, can considered his earlier morphological find­ produce some offspring by backcrossing with ings to be less than definitive and envi­ the parental species. Heiser (1965) suggest­ sioned that new biochemical and genetic ed that ecological amplitude and genetic tools might help solve ambiguous questions variation in some of these species has been of introgression. increased by this process. Finally, this group Although allozyme variation has been has been the subject ofextensive cytological used successfully to investigate introgres­ and morphological studies (Heiser, 1947, sion in animal species (e.g., Selander et aI., 1949, 1951a, 1951b, 1954,1961), and 1969; Hunt and Selander, 1973; Avise and members of this group are often cited as Smith, 1974; Avise and Saunders, 1984), outstanding examples of introgression. the use of electrophoretic markers to study The most frequently cited example ofin­ introgression in plants has been limited trogression in Helianthus, indeed one ofthe (Levin, 1975; Bell and Lester, 1978; Soltis classic cases of introgression in plants, in­ and Soltis, 1986; Doebley et aI., 1984; Sol­ volves the two annual sunflowers of Cali­ tis, 1985). fornia, Helianthus annuus and H. bolanderi Comparison of cytoplasmic and nuclear (Heiser, 1949). Helianthus annuus is a com­ genetic markers has produced perhaps the mon roadside weed in California, occurring most suggestive evidence for introgression frequently in the central valley and in south­ in nature, in both plants and animals. Palm­ ern California. Helianthus annuus was al­ er et al. (1983) demonstrated a discrepancy ready in the state when the first botanical between biparental nuclear and maternal­ collections were made and was used by the cpDNA phylogenies in Brassica and native Americans for various purposes suggested the occurrence of introgression. (Heiser, 1949). Since it does not now occur Similarly, comparison of karyotypes and in natural sites in California, it was very cpDNAs in Pisum (Palmer et aI., 1985) sug­ likely introduced quite recently by native gested one possible instance in which cy­ Americans (Heiser, 1949; Stebbins and toplasms may have been exchanged be­ Daly, 1961). Helianthus bolanderi, in con­ tween related species. Studies of mitochon­ trast, is native to California and was shown drial-DNA variation in deer (Carr et aI., by Heiser(1949) to consist oftwo races. One 1986), Drosophila (Powell 1983; Solignac of these races is restricted to the foothill and Monnerot, 1986), mice (Ferris et aI., regions of California and grows chiefly on 1983), sunfish (Avise and Saunders, 1984), serpentine-derived soils. This race is re­ tree frogs (Lamb and Avise, 1986), trout ferred to as the serpentine race ofH. bolan­ (Gyllensten et aI., 1985), and water frogs den (Heiser, 1949). The other race is weedy (Spolsky and Uzzell, 1984) have also un­ and invades fields, ditches, and roadsides covered possible cases of introgression. in the central valley of California; it is re­ Here we examine allozyme, chloroplast­ ferred to as the weedy race of H. bolanderi DNA, and nuclear-ribosomal-DNA varia­ (Heiser, 1949). The weedy race of H. bo­ tion among populations of H. annuus and landeri is sympatric with H. annuus in the H. bolanderi. As part of an exhaustive ex­ central valley, and the two taxa form exten- amination of introgression in these taxa INTROGRESSION IN HELIANTHUS 229 (Rieseberg, 1987), some of the populations (I.dh-l, 2), leucine aminopeptidase (Lap-I,2), employed in this study have also been ex­ malate dehydrogenase (Mdh-2,3), malic en­ amined using flavonoid chemistry and mor­ zyme (Me-I), phosphoglucoisomerase (Pgi­ phology. Two specific questions are ad­ 1,2), phosphoglucomutase (Pgm-I,3), dressed in this study: 1) are allozyme, 6-phosphogluconate dehydrogenase (Pgd-3), cpDNA, and rDNA data consistent with the and triosephosphate isomerase (Tpi-I,2,3). hypothesized origin ofthe weedy race ofH. All enzymes were resolved on 12.5% starch bolanderi through introgression? 2) are DNA