The Horticulture Journal 86 (3): 379–388. 2017. e Japanese Society for doi: 10.2503/hortj.OKD-006 JSHS Horticultural Science http://www.jshs.jp/ Genetic Diversity of Lilium auratum var. platyphyllum Endemic to the Izu Archipelago and its Relationship to a Nearby Population of L. auratum var. auratum by Morphological and SSR Analysis Sho Yamamoto1, Tetsuri Kikuchi1, Yutaka Yamagiwa2 and Takashi Handa3* 1Graduate School of Agriculture, Meiji University, Kawasaki 214-8571, Japan 2Shizuoka Research Institute of Agriculture and Forestry, Izu Agricultural Research Center, Higashi-Izu, Shizuoka 413-0411, Japan 3School of Agriculture, Meiji University, Kawasaki 214-8571, Japan Lilium auratum var. auratum Lindl. is distributed in the eastern part of Honshu, the main island of Japan. L. auratum var. platyphyllum Baker is endemic to the Izu archipelago, which consists of nine large islands located in south of Honshu’s Izu peninsula. Both varieties have been used as important parents of Oriental hybrid lily cultivars. They have large white flowers with yellow central stripes and colored spots on their tepals. L. auratum var. platyphyllum has larger flowers and wider leaves than L. auratum var. auratum. L. auratum var. platyphyllum has yellow spots, whereas L. auratum var. auratum has red or brown ones. Natural hybridization between these two taxa has been suggested on the basis of spot colors of populations in the Izu archipelago and the Izu peninsula. However, their genetic diversity and hybridity in nature have not been reported. We performed morphological analysis using 72 individuals of L. auratum var. auratum from seven populations and 72 individuals of L. auratum var. platyphyllum from six populations. We also performed simple sequence repeat (SSR) analysis using 102 individuals of L. auratum var. auratum from seven populations and 134 individuals of L. auratum var. platyphyllum from six populations. Both analyses revealed that L. auratum var. auratum and L. auratum var. platyphyllum are genetically different and that L. auratum var. platyphyllum has genetic diversity among populations in the archipelago. Key Words: geographical type, insular endemic plant, lily, morphological characteristics, population diversity. The Izu archipelago consists of nine large islands: Introduction Izu-Oshima, To-shima, Kouzu-shima, Nii-jima, Lilium auratum has two varieties: L. auratum var. Shikine-jima, Miyake-jima, Mikura-jima, Hachijyo- auratum Lindl. and L. auratum var. platyphyllum Baker jima, and Aoga-shima, from north to south, and several (syn. L. platyphyllum Makino). L. auratum var. auratum small islands (Fig. 1). Izu-Oshima is the largest island is distributed in the eastern part of Honshu (Fig. 1), the and is approximately 20 km from the Izu peninsula. The Japanese mainland (Shimizu, 1987). L. auratum var. altitudes of the highest points of the Izu archipelago platyphyllum is indigenous to the Izu archipelago, lo- range from 109 m (Shikine-jima) to 854 m (Hachjio- cated in south of Honshu’s Izu peninsula. The natural jima). The archipelago was formed as volcanic islands distribution of L. auratum var. platyphyllum in five is- during the Pleistocene era and has never been connect- lands of the archipelago (Izu-Oshima, To-shima, ed to Honshu (Karig, 1975). The islands themselves Kouzu-shima, Miyake-jima, and Mikura-jima) was de- have been separated by ocean since Quaternary glacia- fined by Kikuchi and Kuramoto (2008). tions 20000–80000 years ago when sea levels were 100–120 m lower (Gornitz, 1995). Eighteen plant spe- Received; April 19, 2016. Accepted; September 1, 2016. cies, 21 varieties, one form and two hybrids are endem- First Published Online in J-STAGE on November 3, 2016. ic to the Izu archipelago (Ohba and Akiyama, 2002). This work was supported by by funding from the graduate school of Ohba and Iwatsuki (2006) indicated that the plant spe- Meiji university and from Japanese Society for the Promotion of Science Grants-in-Aid for Scientific Research (No. 15K07301). cies of the Izu archipelago originated in the Honshu. * Corresponding author (E-mail: [email protected]). The relationship of populations in this archipelago and © 2017 The Japanese Society for Horticultural Science (JSHS), All rights reserved. 380 S. Yamamoto, T. Kikuchi, Y. Yamagiwa and T. Handa Fig. 1. Map of the 7 populations of L. auratum var. auratum and 6 populations of L. auratum var. platyphyllum used in this study. 1, Tateyama; 2, Zushi; 3, Miura; 4, Manazuru; 5, Matsuzaki; 6, Higashi-Izu; 7, Shimoda; 8, Izu-Oshima; 9, To-shima; 10, Kouzu-shima; 11, Miyake-jima; 12, Mikura-jima; 13, Aoga-shima. 1–7, L. auratum var. auratum; 8–13, L. auratum var. platyphyllum. Honshu has been investigated by the morphology in genetic differences among populations (Haruki et al., some plant species: for example, Campanula punctata 1998). However, the genome size of lilies is large and and C. punctata var. microdantia (Inoue and Kawahara, the C values of Lilium species are variable, ranging 1990; Oiki et al., 2001), Hosta longipes (Yamada and from 22 to 104 pg and averaging 56.3 pg (Peruzzi et al., Maki, 2014), Ligustrum obtusifolium, and 2009). Varshney et al. (2007) suggested that individual L. ovalifolium (Yamada et al., 2014). These studies re- amplified fragment length polymorphism (AFLP) or vealed morphological differentiation and diversity be- RAPD fragments are complex in a large genome tem- tween the Izu archipelago and Honshu. plate, because the band number is large. Microsatellites L. auratum var. auratum and L. auratum var. or simple sequence repeat (SSR) markers are useful platyphyllum have racemose inflorescences and large tools for genetic analysis because of their biparental in- white flowers with yellow central stripes, and colored heritance and hypervariability. Some studies of Lilium spots on recurved tepals. L. auratum var. platyphyllum employed SSR markers. Horning et al. (2003) devel- has larger flowers and wider leaves than those of oped six SSR markers in L. philadelphicum. Arzate- L. auratum var. auratum. L. auratum var. auratum Fernández et al. (2005) applied inter simple sequence shows red or brown spots on tepals. In contrast, repeat (ISSR) markers in L. maculatum var. L. auratum var. platyphyllum has yellow spots on the bukosaense. Kawase et al. (2010) developed three SSR tepals, although some individuals of this variety on Izu- markers in L. japonicum. Lee et al. (2011) developed 19 Oshima have been reported to have red spots (Shimizu, EST-SSR markers in L. regale using an expressed se- 1971, 1987). Our preliminary investigation also found quence tag (EST) database. Yuan et al. (2013) devel- that some L. auratum var. platyphyllum individuals had oped 118 EST-SSR markers in L. regale and related red spots on their tepals (Yamamoto et al., 2014a). It species, and applied them to Lilium cultivars. These has been proposed that L. auratum var. auratum and EST-SSR markers were adopted for a wild population L. auratum var. platyphyllum were hybridized in Izu- of L. auratum in our previous study (Yamamoto et al., Oshima by pollinators flying from the Izu peninsula 2014b). (Shimizu, 1971), although Izu-Oshima is 20 km from Although L. auratum var. platyphyllum is an impor- the Izu peninsula by sea. tant genetic resource of Oriental hybrid lilies, there is Several molecular approaches have been applied to little information about its genetic diversity. The wild the genus Lilium. Randomly amplified polymorphic population of L. auratum var. platyphyllum in the Izu DNA (RAPD) markers applied to L. japonicum showed archipelago has been decreasing and is listed in the red Hort. J. 86 (3): 379–388. 2017. 381 list of threatened species in Tokyo as a vulnerable plant Morphological analysis (Ohba, 2011). The Aoga-shima population was once Twenty-four characteristics were measured at flower- diminished by a volcanic eruption. The populations on ing time: (1) flower diameter; (2) outer tepal length; (3) Hachijyo-jima and Miyake-jima were once destroyed outer tepal width; (4) number of red spots on outer by excessive harvesting by humans for ornamental and tepal; (5) number of yellow spots on outer tepal; (6) edible use, and the Miyake-jima population was re- margin undulation of outer tepal; (7) recurving degree planted from the Mikura-jima population. of outer tepal; (8) shape of outer tepal; (9) inner tepal Our preliminary studies using sequence related am- length; (10) inner tepal width; (11) number of red spots plified polymorphism (SRAP) marker analysis and on inner tepal; (12) number of yellow spots on inner morphological observation suggested that L. auratum tepal; (13) spot size of inner tepal; (14) margin undula- var. platyphyllum has genetic diversity in some popula- tion of inner tepal; (15) recurving degree of inner tepal; tions of the Izu archipelago (Yamamoto et al., 2012). (16) shape of inner tepal; (17) stamen length; (18) stig- However, the precise genetic differences among popu- ma length; (19) stigma color; (20) coloration of stem lations in the Izu archipelago are still unclear, and the anthocyanin; (21) distribution of stem anthocyanin; (22) genetic relationship between L. auratum var. auratum leaf arrangement; (23) leaf length; and (24) leaf width. and L. auratum var. platyphyllum have not yet been an- At first these morphological data were measured in four alyzed. L. auratum var. auratum population (Tateyama, 15; In the present study, we aimed to assess the genetic Zushi, 11; Miura, 12; Manazuru, 8) and six L. auratum diversity of a wild population of L. auratum var. var. platyphyllum population (Izu-Oshima, 26; To- platyphyllum by morphological and SSR analysis. We shima, 12; Kouzu-shima, 12; Miyake-jima, 13; Mikura- also clarified the genetic relationship between jima, 3; Aoga-shima, 6). To avoid misleading L. auratum var. auratum and L. auratum var. correlations, which can be affected by climate or annual platyphyllum. changes, we selected 10 characteristics for further sta- tistical analysis: (a) number of red spots on outer tepal; Materials and Methods (b) number of red spots on inner tepal; (c) number of Plant materials for morphological analysis yellow spots on outer tepal; (d) number of yellow spots Seventy-two individuals of L.