Variation of Dormancy and Early Flowering Ability in Lilium Longiflorum and L. Formosanum Populations in the Ryukyu Archipelago and Taiwan

Variation of Dormancy and Early Flowering Ability in Lilium longiflorum and L. formosanum Populations in the Ryukyu Archipelago and Taiwan

Narges Mojtahedi1*, Jun-ichiro Masuda1**, Michikazu Hiramatsu2, Nguyen Thi Lam Hai1, Yuki Mizunoe1 and Hiroshi Okubo2

1Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan 2Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

Variations in the dormancy and early flowering ability of seedlings of four populations of Lilium longiflorum, Yaku Shima (LYA), Kikai Jima (LKI), and Ishigaki Jima (LIS) in the Ryukyu Archipelago, Japan and Pitouchiao (LPI), Taiwan, and two populations of L. formosanum, Wulai (FWU), Taiwan and a domesticated Fukuoka population (FFU), Japan were investigated. Seedlings of each population were grown at 15°C for five months. They were then transplanted into an experimental open field for two years or at 15, 20, 25, and 30°C under natural day length for 22 weeks. In the field experiments, FWU, FFU, and LPI populations continued to develop new leaves even at >30°C. Flowering percentage for FFU and LPI was 90% and 19.7%, respectively. Leaf development of LIS, LKI, and LYA was completely arrested from early June in both years, and the flowering percentages were 28, 25, and, 10 in the second year, respectively. Under controlled temperature conditions, LKI and LYA populations produced new leaves only at 15°C. FFU and LPI continued growing at 25 and 30°C, whereas the other populations did not grow. FFU significantly produced the heaviest leaves and bulbs and the highest number of scales per bulb at any temperature. The results showed that high temperature induces bulb dormancy in northern L. longiflorum. Strong correlation with the early flowering ability and bulb dormancy was also found. It seems that the early flowering ability of L. formosanum is largely dependent on the lack or reduction of bulb dormancy after adaptation to the local southern climate. The latitudinal variation of this trait demonstrated the geographic gradient during species habituation in the Ryukyu Archipelago.

Key Words: Dormancy, Lilium formosanum, Lilium longiflorum, Ryukyu Archipelago, temperature.

against the effects of gene flow, local adaptation can still Introduction progress and may result in clinal (Endler, 1977) or The variation in ecophysiological traits of natural ecotypic variation (Turesson, 1922) across a spatially plants often shows more or less a geographically gradient heterogeneous environment. There is often a more pattern along latitude and altitude, such as in Populus gradual change in the environment and clines in a wide tremula (Hall et al., 2007) and Lythrum salicaria (Olsson variety of organisms over greater geographic scales and Agren, 2002). Due to the relatively limited mobile (Endler, 1977). ability of plants, such a gradient has developed as a Geophytes have a special adaptive strategy that allows result of the combination of natural selection under the plants to maintain the ability to survive in seasonally different climates and restricted gene flow among unfavorable climates, such as high or low temperature populations (Judd et al., 1999). Gene flow is normally in summer or winter and summer drought conditions by expected to hinder local adaptation by homogenizing the entering a dormant phase with storage organs that are genetic composition of the local population (Slatkin, ready for subsequent season growth (Dafni et al., 1981; 1985), although, if selection is strong enough to work Okubo, 2000; Rees, 1972; Viémont and Crabbé, 2000). Lilium formosanum is native to the mainland of Taiwan, distributed vertically from coastal areas to Received; December 12, 2012. Accepted; April 5, 2013. * Corresponding author (E-mail: [email protected]). 3000 m high in the mountains (McRae, 1998; Wilson, ** Present address: Graduate School of Life and Environmental 1925). In contrast, L. longiflorum is distributed more Sciences, University of Tsukuba, Tsukuba 305-8572, Japan. widely in the Ryukyu Archipelago between the south-

234 J. Japan. Soc. Hort. Sci. 82 (3): 234–241. 2013. 235 ern islands of Kyushu in Japan and the mainland of population were cultured in an incubator (16 h light/8 h Taiwan across 1300 km distance. Based on genetic dark illumination at about 5000 lux provided by cool and morphological analyses, L. formosanum and white light, 70% humidity, 15°C) for 5 months in a L. longiflorum populations have a close relationship mixture of sphagnum peat moss: vermiculite (Redi-Earth (Nishikawa et al., 2001), and it has been confirmed by Sun Gro Horticulture Distribution Inc., Bellevue, WA, allozyme analysis that L. formosanum is a derivative of USA) and transplanted into plastic pots (15 cm diameter, L. longiflorum. L. formosanum shows early onset of 12 cm height) containing red loam : commercial soil (4 flowering among Lilium species (Hiramatsu et al., : 1) to grow in an open field or in temperature-controlled 2001a). It is considered that this trait was obtained by rooms in the phytotron of the Biotron Institute, Kyushu shifting the anthesis time to late summer to ensure first- University. About 250 mg ammonium nitrate (JCAM year biomass accumulation (Saruwatari, 2009). Varia- AGRI. Co., Ltd, Tokyo, Japan) was used as the fertilizer tions in flowering rates of L. longiflorum seedlings in each pot and leaf mold was used after transplantation (Hiramatsu et al., 2002) and the degrees of self- to prevent soil evaporation. Daily maximum and compatibility and -incompatibility with flower morphol- minimum temperatures were recorded at both ends of ogy were also shown in association with their geographic the field. distribution (Sakazono, 2009; Sakazono et al., 2006, 2008). Seasonal growth patterns in an open field In this study, the growth of L. longiflorum and Five individuals of each FWU, LPI, LIS, and LKI L. formosanum seedlings was investigated in association population in 2009, and twenty each of FFU, LPI, LIS, with their bulb dormancy to clarify the ecological and LKI, and LYA were used in 2010. All the plants were evolutional background of the species in their natural placed randomly in the field after transplantation and habitats. leaf number was measured every week for two years. Number of scales and total leaves and flowering rates Materials and Methods (%) in the first and second years were measured for all Plant materials and culture conditions populations planted in 2010 in the open field. Four populations of L. longiflorum, Yakushima (30°3'N, LYA), Kikai Jima (28°4'N, LKI), and Ishigaki Effect of temperature on seedling growth Jima (24°4'N, LIS) in the Ryukyu Archipelago, Japan Ten plants of each FFU, LPI, LIS, LKI, and LYA and Pitouchiao (25°1'N, LPI), Taiwan and two population were transplanted at the end of May 2010, populations of L. formosanum, Wulai (FWU), Taiwan and cultured at 15, 20, 25, or 30°C under natural day and Fukuoka (FFU), Japan were used (Fig. 1). The length in the phytotron. Total leaf number was measured Fukuoka population (FFU) is not an originally natural every week after transplantation for 22 weeks. At harvest, habitat, but has been naturalized for a long time. As fresh and dry weights of bulbs and leaves and number FWU and FFU showed similar growth characteristics, of scales per bulb were measured. The leaves and bulbs FFU was also used for the experiments. Seeds of each were dried in an oven for 3–4 hours at 90°C followed by 3–4 days at 70°C for dry weight measurement.

Statistical analysis Factorial experiments based on a Completely Randomized Design (CRD) were used for both experiments. Data were statistically analyzed using the General Linear Models (GLM) Procedure of SPSS (Ver. 16). The means were compared using Tukey’s multiple range tests. Correlation between leaf and bulb dry weight was analyzed statistically. Results Seasonal growth patterns in an open field FWU and FFU continued leaf development even in the high temperatures of July and August, continued from October to April (Fig. 2). FWU plants flowered from late August untill the late October, and the FFU plants flowered from late September till the end of October in the first year. The leaf development of FWU and FFU plants in the first year was similar to the second Fig. 1. Geographic distribution of Lilium formosanum Wulai (□) and Fukuoka (●) populations and L. longiflorum (○) populations year, although the starting time of flowering changed to studied. late June and July in the second year, respectively 236 N. Mojtahedi, J. Masuda, M. Hiramatsu, N. T. L. Hai, Y. Mizunoe and H. Okubo

(Fig. 2). LPI also developed new leaves until the end of temperature reached 26°C in both years to the middle October, and slow leaf development continued until of November. Low temperatures in winter suppressed February of the next year. This growth pattern was their growth until the middle of February in the following repeated in the second year (Fig. 2). Flowering times of year and nearly all populations started to develop new LPI also shifted from July–August in the first year to leaves when the maximum temperature reached 15°C. May–June in the second year (Fig. 2). Leaf development continued until the end of May, and The growth of LIS and LKI was slow, and completely 28% of LIS, 25% of LKI, and 10% of LYA plants first arrested from early May when maximum daily flowered in the second year (Fig. 2, Table 1).

Fig. 2. Leaf development of four populations of Lilium longiflorum (LPI, LIS, LKI, LYA) and two populations of L. formosanum (FWU, FFU) during 24 months after transplantation in the field. A: May 2009–March 2011, B: May 2010–March 2012. The line between two arrows shows the flowering period in each population. FWU: L. formosanum-Wulai; FFU: L. formosanum-Fukuoka; LPI: L. longiflorum-Pitouchiao; LKI: L. longiflorum-Kikai Jima; LYA: L. longiflorum-Yakushima; LIS: L. longiflorum-Ishigaki Jima.

Table 1. Total and scaly leaf number in the first year and frequency of flowered plants in the first and second years in five populations of Lilium.

Scaly leaf number in Total leaf number Frequency of first year Frequency of second year the first year ± SE the first year ± SE flowered plants (%) flowered plants (%) FFU 15.1a ± 1.4 54.1a ± 2.2 90 97 LPI 7.3b ± 0.8 24.5b ± 3.3 19.7 55 LIS 6.7b ± 1.1 6.7c ± 1.1 0 28.7 LKI 5.5b ± 0.3 5.5c ± 0.3 0 25 LYA 4.1 b ± 0.1 4.1c ± 0.1 0 10

Different letters in each column indicate significant differences at the 5% level by Tukey’s multiple-range test. FFU, LPI, LIS, LKI, LYA (see Fig. 2). J. Japan. Soc. Hort. Sci. 82 (3): 234–241. 2013. 237

Ninety percent of FFU plants flowered in the first growing. FFU and LPI continued growing at 25 and year. Among L. longiflorum populations, LPI had a 30°C, whereas the others did not. higher frequency of first year flowering (19.7%), while FFU significantly produced the heaviest leaves and LIS, LKI, and LYA did not flower at all in the first year bulbs (fresh and dry) and the highest number of scales (Table 1). FFU population developed the highest number per bulb at any temperature (Fig. 4). LPI also showed of scaly leaves and total leaves, while the average scaly the highest values among L. longiflorum populations leaf number of L. longiflorum populations was less than (Fig. 4, Table 2). The lightest bulbs and leaves were eight. The total leaf number in LPI was higher than those obtained at 15°C in all populations. Analysis of variance of other L. longiflorum populations. for each parameter showed significant differences among populations and temperatures, although F values for Effect of temperature on seedling growth population in all parameters were higher than All populations continued to develop new leaves at temperature (Table 2). High positive correlations of 15°C, although LYA stopped growing one month after growth between aerial and underground organs were also transplantation (Fig. 3). LYA stopped developing new found (Fig. 5). leaves after three weeks at 20°C while others kept

Fig. 3. Leaf number of four populations of Lilium longiflorum and one population of L. formosanum during 22 weeks after transplantation at different temperatures. FFU, LPI, LIS, LKI, LYA (see Fig. 2). 238 N. Mojtahedi, J. Masuda, M. Hiramatsu, N. T. L. Hai, Y. Mizunoe and H. Okubo

Fig. 4. Fresh and dry weights of leaves and bulbs, and number of scales per bulb of Lilium longiflorum and one population of L. formosanum at different temperatures after 22 weeks of transplantation. Different letters in each population indicate significant differences at the 5% level by Tukey’s multiple range test. FFU, LPI, LIS, LKI, LYA. (see Fig. 2).

and Roberts, 1970; Wilkins, 2005) and on separate scales Discussion (Okubo et al., 1988). In the present experiment, the one- Contradictory responses among L. longiflorum popu- year-old seedlings of L. longiflorum populations also lations and previous studies on these populations showed summer dormancy, which was repeated in the (Hiramatsu et al., 2001a, b) clearly showed that second year too. temperature plays an important role. It was previously It is concluded that L. formosanum populations have shown that bulb dormancy in L. longiflorum populations no or weak dormancy in summer, whereas L. longiflorum in the northern Ryukyu Archipelago can be induced at showed a wide range of dormancy. LYA showed the high temperatures (Mojtahedi et al., 2013). deepest dormancy while LPI had the lowest. Thus, the The expression of dormancy traits of the seedlings of further north population, the deeper the dormancy is L. longiflorum populations in the northern Ryukyu within L. longiflorum populations. The evidence of Archipelago that finally controls the total growth amount such local adaptation has been recently demonstrated during a specific period seems to be controlled by in Arabidopsis lyrata (Brassicaceae) populations temperature. Summer dormancy of L. longiflorum has (Riihimäki and Savolanien, 2004), Lythrum salicaria L. already been recognized in large flowering bulbs (Wang (Olsson and Agren, 2002), and Beta vulgaris L. (Boudry J. Japan. Soc. Hort. Sci. 82 (3): 234–241. 2013. 239

Table 2. Analysis of variance of different temperatures on growth parameters of Lilium populations after 22 weeks.

Parameter Source d.f. Sum of square Mean of square F Value Fresh weight of leaves Population 4 947.53 236.88 55.78** Temperature 3 147.41 49.13 11.57** Population × Temperature 158.79 13.23 3.12** Dry weight of leaves Population 4 18.7 4.67 66.29** Temperature 3 2.18 0.72 10.33** Population × Temperature 2.31 0.19 2.73** Fresh weight of bulb Population 4 645.39 163.09 83.72** Temperature 3 52.95 17.065 9.06** Population × Temperature 42.21 3.51 1.81NS Dry weight of bulb Population 4 56.32 14.08 78.17** Temperature 3 3.63 1.21 6.73** Population × Temperature 4.33 0.36 2.01** Number of scales per each bulb Population 4 3044.38 761.09 66.63** Temperature 3 292.13 97.37 8.52** Population × Temperature 501.94 41.82 3.66** Cumulative leaf number Population 4 24888.49 6222.12 678.34** Temperature 3 1324.79 441.59 48.14** Population × Temperature 2899.19 241.6 26.34**

** P < 0.001; NS: not significant.

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