Warming reduces mid-summer flowering plant reproductive success through advancing fruiting phenology in an alpine meadow

Description

Notes

Methods

2.3 Soil temperature and moisture measurements

We monitored the soil temperature and moisture of each plot during the growing season from April to October in 2021 and 2022. EM50 Data collection System (Decagon Devices, Inc., USA) was used to monitor soil temperature and moisture at 5 cm soil depth [36]. Data were sampled at 1-minute intervals, and then the 15-minute average was automatically stored in the logger. We chose soil temperature instead of air temperature because previous studies showed that soil temperature was a better predictor of alpine plant phenology than air temperature [38].

2.4 Phenology monitoring

Dominance and flowering frequency of common plants were used as criteria to select for phenological and reproduction monitoring [36, 39]. In late August (when the cumulative biomass of plant communities reached the maximum) from 2019 to 2020, one 50 × 50 cm quadrat was randomly selected at least 0.5 m from the edge of each plot. We harvested all the stems and leaves in each quadrat, sorted them into species, dried them to a constant weight at 70℃, and then weighed them to 0.001g to estimate the aboveground biomass of individual species between treatments. We counted the relative biomass and abundance of individual species. In the fifth and sixth years of the warming and precipitation change treatments (2021 and 2022), we selected the six common species with a high frequency of sexual reproduction in communities for phenological and reproduction observation to enable enough plant material for trait collection and ensure that these species represent well the studied communities (Table 1). The selected pool of six species made up 60-70% of the relative cover, 70% of the total biomass of the plant community, and represented common flowering functional types found in the alpine meadow on the eastern Tibetan Plateau [29, 38, 39].

The monitored species were classified into two flowering functional groups: early-spring flowering species (K. graminifolia and A. rivularis) that flowered before the beginning of June and mid-summer flowering species (A. obtusiloba, Pleurospermum camtschaticum, E. nutans and P. pratensis) that flowered between the beginning of June and July [30, 38]. As soon as each of the six focus species produces obvious flower buds or flowering stems (for grasses), five individuals of each species in each plot will be marked with color-coded tags (five individuals that lost tags during the study were replaced by similar-sized individuals). The marked individuals were monitored during the growing season at intervals of 5-7 days [4, 57]. We recorded the first flower budding day (hereafter referred to as "FFBD"); the first flowering day (hereafter referred to as "FFD"); and the first fruit setting day (hereafter referred to as "FFSD"). The phenological duration was calculated as the number of days between the onset and conclusion of each phenological event [23, 47].

2.5 Reproductive output and success

At each phenological census through the growing season, we also counted the total number of open flowers and fruits on all marked individuals of each species. For grasses, we recorded the number of racemes or inflorescences [58]. Individual, graminoid species, were defined as genets of visibly integrated ramets [57], and where possible rhizomatous species were partially exposed to determine genet size [59]. For clarity, we hereafter use 'flower' and 'fruit' for all species. With these variables, reproductive output was estimated as the number of flowers and fruits produced by each marked individual, and reproductive success was estimated as the proportion of flowers on each plant that successfully produced fruits/seeds [7, 43]. Notably, P. pratensis is a typical single-inflorescence plant, only 0 and 1 were obtained according to the reproductive success calculation method in this study. Thus, only five of the six focus species were counted for reproductive output and success. All reproductive phenological events and reproductive indicators were averaged for the five individuals of each species within each plot for further analysis [57, 60].