MADRON˜ O, Vol. 55, No. 2, pp. 132–142, 2008 GENETIC EVIDENCE OF HYBRIDIZATION BETWEEN OENOTHERA WOLFII (WOLF’S EVENING PRIMROSE) AND O. GLAZIOVIANA, A GARDEN ESCAPE JENNIFER DEWOODY*1,LEONEL ARGUELLO2,DAVID IMPER3,ROBERT D. WESTFALL4 AND VALERIE D. HIPKINS1 1 USDA Forest Service, Pacific Southwest Research Station, National ForestGeneticsLab, 2480 Carson Road, Placerville, CA 95667 2 U.S. Department of the Interior, National Park Service, Redwood National and State Parks, P.O. Box 7, Orick, CA 95555 3 U.S. Fish and Wildlife Service, 1655 Heindon Road, Arcata, CA 95521 4 USDA Forest Service, Pacific Southwest Research Station, P.O. Box 245, Berkeley, CA 94701-0245 ABSTRACT Isozyme analysis of the rare Oenothera wolfii (Wolf’s evening primrose) and the garden escape, O. glazioviana, indicates that hybridization between these species may be more widespread than morphological evidence indicates. Although both species contained low amounts of genetic variation, unique alleles were identified in both taxa. Analysis of 22 populations, including pure populations of each species, identified eight populations as containing putative hybrid individuals. Four of these putative hybrid populations were considered pure O. wolfii based on morphological analysis. This study confirms that the native O. wolfii may be at risk not only from habitat destruction, but potentially from genetic swamping where it co-occurs with O. glazioviana. These results can be used as baseline information for future genetic monitoring efforts. Key Words: complex heterozygote, genetic swamping, hybridization, isozymes, Oenothera. Although habitat loss usually poses the great- completely describe hybrid swarms of individuals, est threat to a rare species’ survival, there is particularly if second-generation hybrids or back- evidence that hybridization with widespread cross individuals occur frequently. Genetic infor- related taxa poses an immediate threat to some mation may provide greater power to identify species (Rhymer and Simberloff 1996). A rare hybrids if unique alleles occur in either or both species may become functionally extinct through pure species. Even in the absence of unique alleles, genetic swamping after repeated hybridization given sufficient variation in neutral, bi-parentally and backcrossing with a more common species inherited, genetic markers (for example, iso- (Levin et al. 1996). Management efforts to zymes), statistical methods exist to identify not minimize hybridization in order to protect a rare only first-generation hybrid individuals, but also species may not be justified if hybridization second-generation hybrids and introgressed indi- results from natural processes. By contrast, viduals resulting from backcrosses with either artificial hybrid zones arising from human- parental species (Rannala and Mountain 1997; mediated habitat modification or species intro- Rieseberg et al. 1998; Nason et al. 2002). duction may require management action to Oenothera wolfii [Munz] Raven, W. Dietr. minimize the potential loss of a rare species Stubbe (Onagraceae) (Wolf’s evening primrose) (Rhymer and Simberloff 1996; Allendorf et al. is a biennial to short-lived perennial native to the 2001). In order to minimize the effects of artificial coastal areas of northern California and southern hybrid zones, managers must be able to distin- Oregon. Populations of this species are rare and guish between pure populations of a rare species patchy in distribution, found on moderately and hybrid swarms where the two species coexist. disturbed sites, including the upper margin of Frequently, hybrids display phenotypes interme- beach strand and coastal bluffs (Imper 1997). diate to either parent species, although hybrid While disturbance resulting from continued morphology may be extreme to either parent development and recreation along the coast have (Schwarzback et al. 2001). Due to these varia- created new habitat for O. wolfii in some tions, morphology alone may be insufficient to instances, the net effect of human encroachment has been negative for existing populations (Imper 1997). As a result, O. wolfii is listed as threatened * Author for correspondence. Current address: Uni- versity of Southampton, School of Biological Sciences, by the state of Oregon, and both the California Southampton, SO16 7PX, United Kingdom. +44-(0)79- Native Plant Society and the Oregon Natural 8363-0570 (voice), +44-23-8059-4459 (fax), j.dewoody@ Heritage Program list this species as endangered soton.ac.uk. throughout its range (Imper 1997). 2008] DEWOODY ET AL.: HYBRIDIZATION IN OENOTHERA 133 While habitat loss is affecting O. wolfii, of two distinct genomes. Multiple reciprocal hybridization with a common congener, O. translocations across the genome have produced glazioviana Micheli, may prove the more imme- a single linkage group consisting of both sets of diate threat (Imper 1997). As a garden escape chromosomes at meiosis. As a result, the 14 (i.e., a horticultural species of hybrid origin that chromosomes in a diploid individual form a has become established in natural areas), O. single ring instead of seven bivalents. This glazioviana may be interfertile with O. wolfii, phenomenon persists through self-pollination producing artificial hybrid zones where the coupled with balanced lethals, with gametophytic species coexist. Several factors support this and sporophytic lethals persisting in the hetero- hypothesis. First, introgression is common be- zygous state. Although self-fertilization may tween many members of this genus. Greenhouse produce embryos homozygous for either genome, experiments have shown that hybridization be- only heterozygous embryos survive to produce tween O. wolfii and other members of the genus viable seed, since those embryos homozygous for readily occurs (Wasmund and Stubbe 1986). one genome will also be homozygous for either Second, individuals of hybrid origin have been the sporophytic or gametophytic lethal allele. As identified at the California-Oregon border area a result, alleles are not independently assorted, based on morphological traits (Carlson et al. and populations are not randomly mating. This 2001). Hybrids are fertile, vigorous, and display a mechanism explains the lack of genotypic diver- greater fitness than either parent species (Imper sity and recombination observed in the data set 1997). Although genetic typing of hybrid individ- (see Results), and prevents the use of statistical uals indicates that hybrids tend to breed true, analyses typical of co-dominant genetic data (e.g., there is evidence of hybrids back-crossing with O. admixture analyses or population assignment wolfii (Imper 1997). Third, O. wolfii is potentially tests). susceptible to genetic swamping by O. glazioviana Samples were collected from populations at 22 based on the mating systems of each species. O. sites whose taxonomy was determined by mor- wolfii is self-compatible and produces the major- phological traits (Tables 1 and 3, Fig. 1). Typical ity of its seed via self-pollination (Carlson et al. O. wolfii plants produce small (,5 cm) pale- 2001). This breeding system is a consequence of yellow corollas having petals that do not overlap, O. wolfii’s complex heterozygous genome, which with sepals covered in dense long-spreading is maintained through self-fertilization and bal- pubescence, both villous and glandular pubes- anced lethals, and results in approximately half of cence occur on fruits, and plants have a reddish the mature pollen grains being sterile (Wasmund upper stem (Imper 1997). By contrast, typical O. and Stubbe 1986). In contrast, O. glazioviana is glazioviana plants produce larger (.8 cm) bright an outcrossing species (Imper 1997). Given the yellow flowers having substantial overlap in the asymmetry of available pollen between these petals, minimal pubescence on either sepals or parent species asymmetric gene flow might occur fruit, green upper stems, and more wrinkled and as O. glazioviana pollen swamps O. wolfii stigmas lighter green foliage than observed on O. wolfii at sympatric sites. Together, this evidence sug- (Imper 1997). Field observations identified four gests that hybridization likely occurs between this populations as O. glazioviana (nos. 1 to 4; the rare endemic and the widespread garden escape. populations of the garden escape most proximate This study reports an investigation into the to O. wolfii), 14 populations as O. wolfii (nos. 6 to extent and structure of hybrid zones between O. 19), and three populations as intermediates or wolfii and O. glazioviana using isozymes, which putative hybrids (nos. 20 to 22). Field observa- are putatively neutral, bi-parentally inherited, tions could not distinguish between O. glaziovi- molecular markers. Three questions were ad- ana and O. elata, a common congener at one site dressed: First, does sufficient genetic variation (no. 5), and one population appeared to be O. exist to discriminate between pure O. wolfii and wolfii, but occurred in a novel location (no. 15). O. glazioviana populations? Second, can hybrid A single leaf was collected from between four and populations be identified using these molecular 25 individuals in each population for subsequent markers? Third, what is the frequency of hybrid genetic analyses. individuals in natural populations of O. wolfii? Tissue was prepared for isozyme analysis Ultimately, these genetic findings provide greater following the liquid nitrogen procedure using insight and guidelines for management plans and Gottleib (1981) extraction buffer,