A total of six WR populations occurring in the early (code as WRE) and late (WRL) rice-cultivation seasons were collected from three rice fields in Leizhou, Guangdong Province of China (Table S1). The geographic distance between the sampled rice fields was >30 km. WR populations collected from the same field were treated as a population pair (e.g., CDE/L, DCE/L, or HJE/L pair). About 60 randomly selected samples were collected from each WR population of either the early- or late-season rice field at the spatial distance intervals of >10 m to avoid similar sampling genotypes. Matured panicles from a WR plant were collected as an independent sample. The average duration of the early rice-cultivation seasons (ES) was from March 1st to June 25th, whereas late rice-cultivation seasons (LS) were from July 20th to November 5th.
Historical climate data collection and analysis
The 10-year air temperature data (2011~2020) in Leizhou was collected from the Tianqi Database (http://lishi.tianqi.com/leizhou/index.html, in Chinese). The air temperature data included the daily minimum and maximum temperatures (Table S2). The averages of the minimum and maximum temperatures were defined as the daily average air temperature. Also, the 10-year day length data (2006~2015) was collected from an open website (https://richurimo.51240.com/leigaozhen__richurimo/, in Chinese). The day length data included the daily time of sunrise and sunset (Table S3). The differences between the time of sunrise and sunset were defined as the day lengths.
In addition, the WR growth period was artificially divided into four stages, corresponding to different rice growth phases: seedling (S), tillering (T), flowering (F), and ripening (R). The average values of each year in the ten years were used to estimate differences in air temperatures and day lengths between the early and late rice-cultivation seasons at different stages.
Design of common garden experiments
Common garden experiments were conducted in a rice field in Leizhou, Guangdong Province of China (Figure S1) in the early and late rice-cultivation seasons, respectively. In the experiments, 60 WR samples were included from each population, and 10 well-developed seeds from each sample were used to determine the ratios of seed germination. Seeds were germinated in black boxes placed in the experimental rice field (Figure S1b). Thirty-day-old seedlings (≥3 seedlings from the same sample) were transplanted to the experimental rice field. In the experiment, each plot containing 36 seedlings was arranged with a 6 ´ 6 grid (Figure S1c) and 30 cm between the hills and rows. Six plots (replicates) were included for the same treatments of each WR population. All plots were arranged in the experimental field with a completely randomized design with 60 cm spacing between the plots. About 80 days after seed germination, panicles were enclosed in mesh nylon bags to avoid accidental shattering of seeds (Figure S1d).
Measurements of fitness related traits
Vegetative growth traits, including plant height, number of tillers per plant, and leaf length/width, were measured in the common garden experiments. Plant height was defined as the distance from the ground to the tip of the longest leaf, and the number of tillers per plant referred to the total of branches that emerged from the main stem culm of a plant. Leaf length and width of the main stem culm were also involved, including the top-first leaf (the first leaf on the top of the main stem culm) and top-second leaf (the second leaf on the top of the main stem culm). The leaf length was defined as the distance from the leaf tip to the base, and the leaf width was measured at the middle of the leaf.
To detect the differences in vegetative growth between the early- and late-season WR populations at different stages, we measured these traits every 20 days after seed germination (DAG), including the 20, 40, 60, and 80 DAG. At the 20 DAG, considering the elder seedlings are too weak to avoid harm during the measurement, the leaf length and width measurements were abandoned. Therefore, only plant height and number of tillers per plant were measured. After transplanting, the measurements of plant height, number of tillers, and leaf length/width for each plant individual were conducted in all planting plots at 40, 60, and 80 DAG. Experimental data obtained from the measurements and recording was mainly used to estimate differences in growth and development between the early- and late-season WR populations in the same rice-cultivation season and to compare the performance of the same population in different rice-cultivation seasons.
The flowering time of each plant from beginning to end was recorded, and a dynamic pattern of flowering time was constructed in each population. The beginning of flowering was defined as the date of the first flower emerging, and the rise of heavy panicles indicated the end of flowering. The proportion of flowering plants per day was marked and recorded in each population, which was used to construct the flowering time patterns of weedy rice populations. To estimate more vast differences in flowering time between the early- and late-season WR populations, the average flowering time in different phases, including 1%, 30%, 50%, and 80% plants flowered, were also calculated to make further comparisons between the two-season WR populations.
The reproductive traits were closely associated with the flowering time in rice. Therefore, the number of seeds per plant, seed setting rate, and 100-seeds weight were measured for each plot. After harvesting and threshing, the seed air cleaning instrument (CFY-2, Top Cloud-agri Technology Company, Zhejiang, China) was used to separate the full seeds. Seeds counting was conducted in an electronic seed counter (PME, Shanco Instruments, Shanghai, China), and 100-seeds weighting used an analytical balance.
Estimate magnitude of local adaptation
Fitness-related traits, such as plant height, number of tillers, and reproductive traits, were used to quantify the local adaptation (LA) of WR populations in the native rice-cultivation environment. In addition, the flowering time was also involved in this analysis based on the data of days to flowering, and early flowering was regarded as an adaptive trait. The quantitative measure of local adaptation was the relative fitness of the native population at a field site in a given year minus the relative fitness of a nonnative population at that site, following the equation from Hereford :
Where W represents the mean fitness of native and nonnative populations, and avg (W) represents the mean fitness of all populations. In this study, the early-season WR populations were native in the early rice-cultivation season, and the late-season WR populations were native in the late rice-cultivation season. Generally, positive LA values indicate local adaptation in the native populations (Hereford, 2009). However, the negative LA values of flowering time also indicate local adaptation for early flowering in WR.
Principal component analysis (PCA)
Nine phenotypical data, including the number of tillers per plant at 40 (A), 60 (B), and 80 (C) DAG, plant height at 40 (D), 60 (E), and 80 (F) DAG, flowering time (G), seed setting rate (H) and 100-seeds weight (I), were used in the principal component analysis. The PCAs were conducted in R v.4.1.2 (https://cran.r-project.org/bin/windows/base/) with package “ggbiplot”, and the confidence intervals of the ellipse was set as 95%. Additionally, the principal component analysis for variables, including contributions, coordinates, and correlation, was conducted using the R package “FactoMineR”. The top three principal components (PC1, PC2, and PC3) were selected in the analyses according to their explained variance.
Statistical analyses of data
Two-way ANOVA (analysis of variance) was used to determine the factors, including the population (the corresponding early- and late-season WR populations), population pair (CDE/L, DCE/L, and HJE/L from different collecting sites), and transplant season (the EARLY and LATE rice-cultivation season), affecting the plant growth and development significantly. Four groups of ANOVAs conducted: ⅰ) estimation of the effects of population and population pair in the EARLY rice-cultivation season (Table 1), ⅱ) estimation of the effects of population and population pair in the LATE rice-cultivation season (Table S4), ⅲ) estimation of the effect of transplant season and population pair in the early-season weedy rice populations (Table S5) and ⅳ) estimation of the effect of transplant season and population pair in the late-season weedy rice populations (Table S6). The first two groups of ANOVAs aimed to estimate the differences in growth and development traits, which were measured in common garden experiments, between the early- and late-season WR populations in the early and late rice-cultivation environments, respectively. The latter two groups of ANOVAs aimed to estimate the differences in growth and development of the same population in different rice-cultivation seasons.
In addition, difference analyses included in this study were tested based on the student t-test , and the method of two-tails and equal variance test of two samples were adopted. Two-way ANOVAs and student t-test were both performed using the software IBM SPSS Statistics ver. 22.0 for Windows (SPSS Inc., IBM Company Chicago, IL, USA, 2010).