Plant regeneration strategy plays a critical role in species survival and can be used as a proxy for the evolutionary response of species to climate change. However, information on the effects of key plant traits and phylogenetic relatedness on seed germination is limited at large regional scales that vary in climate. To test the hypotheses that phylogenetic niche conservatism plays a critical force in shaping seed ecophysiological traits across species, and also drives their response to climatic fluctuation, we conducted a controlled experiment on seed germination and determined the percentage and rate of germination for 249 species in subtropical China under two temperature regimes (i.e., daily 25ºC; daily alternating 25/15ºC for each 12 h). Germination was low with a skewed distribution (mean = 38.9% at 25ºC, and 43.3% at 25/15ºC). One fifth of the species had low (<10%) and slow (4–30d) germination, and only a few (8%) species had a high (>80%) and rapid (1.2–6.6d) germination. All studied plant traits (including germination responses) showed a significant phylogenetic signal, with an exception of seed germination percentage under the alternating temperature scenario. Generalized linear models (GLMs) and phylogenetic generalized estimation equations (GEEs) demonstrated that growth form and seed dispersal mode were strong drivers of germination. Our experimental study highlights that integrating plant key traits and phylogeny is critical to predicting seed germination response to future climate change.

Seed collection

Mature seeds of the 249 common species were collected between August and November in 2015 and 2016. Seeds were collected from one to three populations for each species with at least 20–30 randomly selected individuals. All seeds of each species were pooled, air-dried at room temperature for 1–2 days, and then stored in dry conditions at 4ºC (mean = 15 days, to avoid effects of low temperature and dry storage on the physiology of the seeds) until the onset of germination tests. Seed mass of each species was determined by weighing three replicates of 100 seeds. Seed vigor was assessed with the tetrazolium test before initiation of experiments (Hendry & Grime, 1993). Three replicates of 50 seeds of each species were placed on moist filter paper at room temperature for 24 h and then sliced along the longitudinal axis with a scalpel. Both seed sections were incubated in a 0.1% aqueous solution of tetrazolium chloride for 24 h at 25ºC in darkness. Seeds with a strong red-stained embryo were considered to be viable.

Germination test

The final germination percentage and rate (speed) of each species were determined under laboratory conditions. For each species, three replicates of 50 seeds were placed in 9-cm-diameter Petri dishes on two layers of filter paper moistened with distilled water. Seeds of each species were incubated under two temperature regimes: either a constant temperature of daily 25ºC (the mean high temperature during the spring germination period), or alternating temperatures of 25ºC and 15ºC for each 12h (the high and low mean annual temperatures). Seeds were in the same daily light-dark cycle in each temperature regime. Light was provided by fluorescent tubes (PPFD, 25–30μmol/m-2s-1 at seed level) for 12 h each day (i.e., during the high-temperature phase of the alternating temperature regime). Seed germination was monitored every 24 h for 30 days, and a seed was considered to be germinated when the radical was visible to the naked eye. Germinated seeds were counted and then discarded. At the end of the germination tests, viability of non-germinated seeds was determined by opening each seed with a needle to check if the embryo was firm and white (viable) or soft and gray (non-viable) .

Two germination indices were used in this study. Germination percentage (GP) was calculated as GP = GN*100/SN, where GP is the final germination percentage (%), GN is the total number of germinated seeds, and SN is the total number of seeds tested. The mean value of the three replicates was calculated. Germination rate (GR) was calculated as GR = ∑(Gi×i) / ∑Gi, where i is number of days between seed sowing (day 0) and seed germination, and Gi is the number of seeds germinated on day i. GR corresponds to the mean germination time of the fraction of seeds that germinated.

Four traits were selected to capture information on various aspects of plant ecological strategy. Seed mass and seed dispersal syndrome provide information about a species’ reproductive strategy. Maximum plant height and growth form provide crucial information about resource acquisition, competitive ability, and life-history strategy. Plant seed dispersal mode (unassisted, vertebrate, wind), growth form (annual or biennial herb, perennial herb, shrub, or tree), and height were extracted from the Flora of China (http://www.iplant.cn/foc/).