AN EVALUATION OF METHODS USED TO ASSESS INTERGENERIC HYBRIDIZATION IN KELP USING PACIFIC LAMINARIALES (PHAEOPHYCEAE) 1 Louis D. Druehl Bamfield Marine Sciences Centre, Bamfield, British Columbia, V0R 1B0, Canada Julie D. Collins Department of Ecology and Evolution, The University of Chicago, 1101 East 57th St., Chicago, Illinois 60637, USA Christopher E. Lane and Gary W. Saunders 2 Centre for Environmental and Molecular Algal Research, Department of Biology, University of New Brunswick, Fredericton, New Brunswick, E3B 6E1, Canada Kelp intergeneric laminarialean hybridizations and hybridization protocol were assessed using sev- en northeast Pacific kelp species: Alaria marginata Postels and Ruprecht, Costaria costata (C. A. Agar- dh) Saunders, Eisenia arborea Areschoug, Lamin- aria saccharina (L) Lamouroux, Lessoniopsis littoralis (Tilden) Reinke, Macrocystis integrifolia Bory, and Nereocystis leutkeana (Mertens) Postels and Ruprecht. Survival and development of sporo- phyte morphologies derived from selfings, separate males and females, and reciprocal crosses were evaluated over 30 weeks of cultivation. All cultures were initiated from cloned gametophytes. Two closely related species, Laminaria angustata Kjell- man and L. japonica Areschoug, demonstrated the efficacy of long-term (up to 30 years) cloned game- tophytes in hybridization studies. Sporophyte morphologies appeared in 34%–69% of control and hybridization trials, and 6%–16% of all trials produced sporophytes in control and hybridization conditions that persisted through 30 weeks of cul- tivation. Sporophytes in control and hybridization conditions could appear normal or abnormal. Usu- ally, the morphology of sporophytes in hybridiza- tions and female controls resembled the female parent, whereas the sporophytes in male controls often had an abbreviated morphology, lacking de- finitive generic features. Species-specific rDNA in- ternal transcribed spacer molecular primers were used to determine the parentage of five putative hy- brids. Only the L. japonica ,/L. angustata < hybrid bore both parental genomes. That negative controls could produce persistent and normal-appearing sporophytes negates their value and emphasizes the importance of molecular confirmation in hy- bridization studies. These findings were applied to critique the only known wild intergeneric hybrid, Pelagophycus/Macrocystis. Key index words: apogamy; inheritance; hybridiza- tion; kelp; Laminariales; parthenogenesis; Protista Abbreviation: ITS, internal transcribed spacer Numerous studies have reported hybridizations be- tween genera and families of the Laminariales (Table 1, Lewis 1996a). Usually, these reports are substanti- ated by the production of normal-appearing sporo- phytes and in some instances chromosome counts. In one instance, molecular evidence confirmed the ge- netic contribution of the parents in an intergeneric hybrid (Liptack and Druehl 2000). The interpretation of hybridization success, based on the morphology of the putative hybrids, is confused by the ability of female kelp gametophytes to give rise directly to sporophyte morphology by way of parthe- nogenesis and the ability of male gametophytes to give rise directly to a sporophyte morphology by apogamy. Schreiber (1930) first reported on parthenogenesis in the Laminariales. Subsequently, numerous authors have reported the occurrence of parthenogenesis in several kelp species (Kemp and Cole 1961, Nakahara 1984, Lewis et al. 1993, Gall et al. 1996). The direct production of sporophytes through male apogamy has been reported by Nakahara and Nakamura (1973) and Nakahara (1984). The studies of Nakahara and Nakamura (1973) and Nakahara (1984) illustrate the ambiguity in assessing hybridization success using morphology and chro- mosome counts. Both studies noted abnormal and normal diploid sporophytes that were derived from fertilized eggs of Alaria crassifolia Kjellman, La- minaria japonica Areschoug, and Agarum clathratum Dumortier (cited as A. cribrosum Bory). These same three species produced normal and abnormal-appear- ing parthenosporophytes that could be either haploid or diploid. Diploidy may also be a poor indicator of hybridization success because parthenosporophytes have been reported to undergo autodiploidization (Fang et al. 1978, Fang and Dai 1980). Lewis et al. 1 Received 11 August 2004. Accepted 15 December 2004. 2 Author for correspondence: e-mail ldruehl@island.net. 250 J. Phycol. 41, 250–262 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.04143.x