American Journal of Botany 89(11): 1779±1784. 2002. ECOPHYSIOLOGY OF SEED GERMINATION IN ERYTHRONIUM JAPONICUM (LILIACEAE) WITH UNDERDEVELOPED EMBRYOS1 TETSUYA KONDO,2,4 NORI OKUBO,2 TAKU MIURA,2 KAZUSHIGE HONDA,3 AND YUKIO ISHIKAWA3 2Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan; 3Department of Forestry and Landscape Architecture, Hokkaido College, Senshu University, Bibai 079-0197, Japan Erythronium japonicum (Liliaceae) (Japanese name, katakuri) is indigenous to Japan and adjacent Far East regions. We examined their embryo elongation, germination, and seedling emergence in relationship to the temperature. In incubators, seeds did not germinate at 208/108 (light 12 h/dark 12 h alternating temperature), 208,158,58,or08C with a 12-h light photoperiod for 200 d. They germinated at 158/58 or 108C, starting on day 135. If seeds were kept at 208 or at 258/158C before being exposed to 58C, the seeds germinated, but if kept at 258 or 308C they did not. Embryos at 258/158C grew to half the seed length without germinating; at 08 or 58C, embryos elongated little. Embryos grew and seeds germinated when kept at 258/158C for 90 d and then at 58C. In the ®eld, seeds are dispersed in mid-June in Hokkaido and in Honshu, mid-May to mid-June. Seeds do not germinate immediately after dispersal because the embryo is underdeveloped. Embryos elongated at medium temperatures in autumn after summer heat, and germination ends in No- vember at 88/08C. After germination, seedling emergence was delayed, and most seedlings were observed in early April around the snowmelt when soil cover was 2±3 mm. Key words: ecophysiology; embryo elongation; Erythronium japonicum; seed germination; seedling emergence; temperature. Erythronium japonicum Decne. (Liliaceae) (Japanese name, MATERIALS AND METHODS katakuri) is a typical vernal plant that inhabits the cool-tem- perate mesic deciduous-forest ¯oor. Erythronium japonicum is Seed collectionÐSeeds used in this study were collected from a natural distributed throughout Hokkaido and in broad areas from the population in a deciduous woodland in Asahikawa City, Hokkaido, Japan lowlands to the montane zone in northern and central Honshu, (438529 N, 1428279 E). Light-brown fruits were collected in paper bags on 8 especially in the Japan Sea side of Honshu, Japan. It also oc- June 1998 and 16 June 1999 when some individuals in the population had curs sporadically on the montane zone of southwestern Hon- already dispersed seed. Collected fruits were brought to our laboratory, placed shu, Shikoku, and Kyushu in Japan. It is distributed also in in stainless steel trays, and left to dry for 1 wk. Most of the fruits had dehisced Korea, northeast China, Sakhalin, and the Kurile Islands within this time. The seeds were threshed from the dried fruit by hand, and were then winnowed, put into paper envelopes, and stored in a plastic con- (Ohwi, 1983). In Hokkaido, after the long winter with deep tainer with silica gel at 58C until the start of the experiment. snow, spring is ushered in by many ¯owers immediately after the snow melts. These spring ephemerals include Adonis amu- rensis Regel et Radd, Anemone raddeana Regel, Corydalis Phenology of embryo growth in the outdoor pot experimentsÐSeeds col- lected in 1999 were used. Ten seeds were cut into thin sections with a mi- ambigua Cham. et Schl., Trillium camtschatcense Ker Gawler, crotome, and the maximum embryo length of each seed was measured under and E. japonicum. Their ¯owering and seed setting occur in a dissecting microscope equipped with a micrometer on 25 June 1999. On early spring and mid-June, respectively, and their growth ends the same day, 30 seeds were put into a ®ne-mesh polyester bag, and ®ve such in early summer. There are many popular scenic locations of bags were buried in leaf mold in a nursery ¯at at the depth of 3 cm. The ¯at these indigenous populations, including large-scale vistas of was put in a steel-framed greenhouse without vinyl or other covering at Hok- ¯owers in Japan. kaido University in Sapporo (438049 N, 1418209 E), and the greenhouse was Erythronium japonicum has been studied in terms of its life covered with shade cloth to simulate the light conditions of a forest ¯oor. The cycle, size-class structure, resource allocation (Kawano, Hi- leaf mold was kept moist throughout the experiment. The shade cloth was ratsuka, and Hayashi, 1982), breeding and pollination systems removed, and the ¯at was kept covered with a straw mat from 15 November (Kawano and Nagai, 1982), seed dispersal (Kawano, Hiratsu- 1999 through 18 April 2000. Snow ®rst fell in mid-November and covered ka, and Hayashi, 1982; Ohkawara, Higashi, and Ohara, 1996), the ground from the end of November to the end of March. On 26 July, 25 dry-matter production, environmental requirements (Sawada et August, 23 September, 22 October, and 21 November 1999, a bag was taken al., 1997), growth and reproduction as examined by a mathe- from the mold and embryos of ten seeds were measured as above. All but matical model (Yokoi, 1976), and population structures and three of the seeds had germinated by 21 November, so on this day, only three dynamics (Yokoi, 1976; Sawada et al., 1997; Takada, Nakay- embryos were measured. The temperature of the soil surface above the bag ama, and Kawano, 1998). However, germination information was measured every 15 min with an electric thermograph throughout the needed for propagation and maintenance of population num- experiment. The daily mean, maximum, and minimum temperatures were cal- bers has not been reported. We examined the germination ecol- culated. ogy and temperature requirements for germination under both outdoors and laboratory conditions. Phenology of germination in the outdoor pot experimentsÐSeeds col- lected in 1999 were used. One hundred seeds were put in a ®ne-mesh poly- 1 Manuscript received 25 January 2002; revision accepted 16 May 2002. ester bag and three such bags were buried in leaf mold in a nursery ¯at at 4 Author for reprint requests (FAX: 181-11-667-8837; e-mail: kondo@ the depth of 3 cm on 25 June 1999. The ¯at was placed in the steel-framed res.agr.hokudai.ac.jp). greenhouse and treated as in the experiment on embryo growth. 1779 1780 AMERICAN JOURNAL OF BOTANY [Vol. 89 The three bags were lifted, and seeds were examined for germination every month from June 1999 to January 2000. Here, germination is distinguished from emergence of seedlings. Germination was said to have occurred when the radicle emerged from the seed, and seedlings were said to have emerged when a cotyledon appeared above the ground. Germinated seeds were re- moved from the bags, which were buried again. In winter, the ¯at was dug out from snow for examination of germination. The maximum snow cover was about 1.5 m. The temperature of the soil surface was measured as above. Phenology of seedling emergence in the outdoor pot experimentsÐSeeds collected in 1998 were used. Three pots 19 cm in diameter were ®lled with a 1 : 1 mixture of peat moss and vermiculite. One hundred seeds were sown in each pot and covered with 2±3 mm of sieved soil on 16 June 1998. The pots were covered with shade cloth to prevent dryness and protect against insects and placed in the greenhouse. The soil was kept moist throughout the experiment. The shade cloth was removed and the pots were covered with a straw mat from 16 October 1998 until 19 April 1999. Pots were examined monthly for seedlings that had emerged, and emerged seedlings were removed from the pots. The maximum snow cover was about 1.5 m. The temperature of the soil surface was measured as above. Fig. 1. Temperatures in the ®eld and mean lengths (61 SD) of embryos of Erythronium japonicum. Lengths of ten embryos were measured on 25 June, and on the same day, 150 seeds were buried in leaf mold. On 26 July, Effects of constant or alternating temperatures on germinationÐSeeds 25 August, 23 September, and 22 October, the seeds were unburied, ten were collected in 1999 were treated with 500 ppm (parts per million) of benomyl chosen at random, and lengths of embryos were measured. On 21 November, for 24 h for bacterial control before being used in this experiment. On 24 only three embryos could be measured; all other seeds had germinated. June 1999, the experiment was started in a temperature- and light-controlled incubator with a 12-h photoperiod with seeds in 9-cm glass petri dishes on a double layer of ®lter paper moistened with distilled water. Five constant tem- perature treatment was effective for the germination. Seeds were placed at peratures (08,58,108,158, and 208C) and two regimes of alternating temper- 258/158C for 30 d, 60 d, 90 d, or 120 d and then moved to 58C. atures with 12 h at each temperature (158/58 and 208/108C, light 12 h/dark 12 h) were used. At constant temperatures, seeds were exposed to light for 12 h Effects of temperature on embryo growthÐIn an examination of the ef- each day; and at 12/12 h alternating temperatures, seeds were in light during fects of temperature on embryo growth, seeds were kept at 08,58,or258/158C the high-temperature period and in the dark during the low-temperature pe- throughout the experiment, or else at 258/158C for 90 d before being moved riod. The light source was 40-W white ¯uorescent tubes, and the irradiance to 58C. The lengths of ten embryos were measured on 25 June, 25 July, 25 at the level of the seeds was about 20 mmol´m22 ´s21 on a double layer of August, 23 September, 22 October, 21 November, and 21 December as in the ®lter paper moistened with distilled water.