Japanese Society of Grassland Science ISSN1744-6961 Grassland Science 54 (2008) 69–80 © 2008 Blackwell Publishing Ltd 69 Blackwell Publishing Asia ORIGINAL ARTICLE Hybridization between perennial ryegrass and Italian ryegrass in naturalized Japanese populations Hiroyuki Tobina 1 , Masayuki Yamashita 2 , Atsuhiro Koizumi 2 , Masahiro Fujimori 3 , Tadashi Takamizo 4 , Mariko Hirata 5 , Toshihiko Yamada 6 and Hitoshi Sawada 2 1 United Graduate School of Agricultural Science, Gifu University (Shizuoka University), Gifu, Japan 2 Faculty of Agriculture, Shizuoka University, Shizuoka, Japan 3 Yamanashi Prefectural Dairy Experiment Station, Yamanashi, Japan 4 National Institute of Livestock and Grassland Science, Tochigi, Japan 5 Japan Grassland Agriculture & Forage Seed Association, Tochigi, Japan 6 Field Science Center for Northern Biosphere, Hokkaido University, Hokkaido, Japan Keywords DNA markers; interspecific hybrids; Italian ryegrass; naturalization; perennial ryegrass. Correspondence Masayuki Yamashita, Laboratory of Ecology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. Email: abmyama@agr.shizuoka.ac.jp A part of this work was presented at the 60th meeting (March 2005) of the Japanese Society of Grassland Science and the 111th meeting (March 2007) of the Japanese Society of Breeding. Received 30 May 2007; accepted 2 October 2007 doi: 10.1111/j.1744-697X.2008.00108.x Abstract Introduced Lolium species, including perennial ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum), have been widely utilized in Japan for forage, turf and soil conservation. These ryegrasses have escaped from cultivated areas and become naturalized, and this has become a serious issue in recent years. Interspecific hybrids between perennial ryegrass and Italian ryegrass have often been found in naturalized populations. It has also been suggested that hybridization between plant species might serve as a stimulus for the evolution of invasiveness. We surveyed the genetic structure of naturalized ryegrass populations in Japan using genetic markers that distinguished perennial ryegrass and Italian ryegrass. Of the 55 naturalized populations surveyed, 41 exhibited morphological traits of Italian ryegrass. DNA analysis using simple sequence repeat and chloroplast DNA markers characterized 20 of these 41 populations as Italian ryegrass, with the remaining populations as interspecific hybrid derivatives. Approximately half of the naturalized ryegrasses populations in Japan were inferred to include interspecific hybrids. Introduction Recently, the naturalization of exotic plant species in Japan has been recognized as a potentially major threat to biodiversity and local ecosystem integrity (Washitani 2002b; Yamashita 2002). In particular, exotic pasture grasses have become widely naturalized because of their resistance to disturbance and their wide utilization for pasture, slope protection, erosion control and landscape greening (Washitani 2002a; Yamashita 2002). Serious damage to local ecosystem integrity and biodiversity is suspected to have been caused by invasive pasture grasses, and 13 species of exotic pasture grasses have been publicized on an “alien species alert list” by the Ministry of the Environment (2005). To prevent serious damage, it is important to describe the true state and mechanisms of naturalization of exotic pasture grasses and their influence on local ecosystems. Both perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.) are included on the alien species alert list. Ryegrasses (Lolium spp.) have been introduced to Japan since the middle of 19th century (Flora- Kanagawa Association 2001). Sixty-one thousand ha of Italian ryegrass are under cultivation for pasture and forage produc- tion (Ministry of Agriculture, Forestry and Fisheries 2006), while 7000–8000 ha of perennial ryegrass are cultivated mainly in northern Japan (Hokkaido and Tohoku regions), mainly for pasture (Nakamura 2002). Ryegrasses are also used for turf and landscape greening. Over more than 100 years of usage, ryegrass naturalization has occurred. If naturalized populations invade habitats of rare plant species, there is an obvious threat (Ministry of the Environment 2005). Indeed, naturalized ryegrasses have become harmful weeds in some wheat fields and orchards (Asai & Yogo 2005).