Phenotyping
We focused on ten traits related to differential water availability
across the entire plant life-cycle.
Germination fraction was censused per pot after germination had
ceased at the beginning of the experiment. It typically decreases
towards arid populations to hedge against more frequent unfavorable
years (Tielbörger et al . 2012; Lampei et al. 2017; ten
Brink et al. 2020). As climate manipulations increased (dry
plots) and decreased (wet plots) the occurrence of unfavorable years, we
expected reduced germination fractions being favored in dry plots andvice-versa in wet plots.
Days to flowering (since first irrigation) were determined by
inspecting plants daily for the first open flower. Accelerated phenology
is expected by theory and repeatedly found in annual plants from drier
sites (reviewed in Kigel et al. 2011), and we expected earlier
phenology in plants descending from dry manipulated plots and more arid
sites. Moreover, the number of leaves at the day of first flowering
(leaf number at flowering ) provided an ontogenetic phenological
measure and a non-destructive measure of plant size. It disentangled
whether phenology changed via accelerated development (days to
flowering) or shifted ontogeny (leaves at flowering) (Kigel et
al . 2011).
Stomata density and carbon isotopes(δ 13C) assessed gas exchange and water use
efficiency. Stomata density was quantified by automated high-throughput
microscopy (Dittberner et al. 2018; see Supplementary Methods).
As lower stomata density may decrease maximum transpiration (Liuet al ., 2012) we expected lower stomata density in plants
descending from drier conditions. Due to high costs, carbon isotopic
ratios (δ 13C, see supplementary Methods), were
analyzed only for a subset (14 genotypes per site, rainfall treatment,
and four water levels: 15ml – 50ml). We expected that plants from drier
sites and plots exhibit higher water use efficiency, i.e. higherδ 13C (Li 1999; Hartman & Danin 2010).
Plant height was measured on a fixed day (12thApril) before the onset of senescence. Moreover, abovegroundvegetative biomass was determined at the end of the experiment
(May 15th 2014) as the dry weight (24 h, 70°C) of all
stems and leaves. We expected greater height and vegetative biomass in
plants from wetter conditions as adaptation to intensified aboveground
competition (Westoby 1998; Schiffers & Tielbörger 2006).
Total seed number per plant quantified fitness. Moreover, we
estimated the selfing rate per plant visually as percent of flowers that
developed into viable seeds; it served as covariate in some analyses
because B. didyma populations may differ in self-compatibility
(Gibson-Forty 2018).
Reproductive allocation quantified the biomass allocation to
reproductions (i.e. weight of all diaspores and flower remains) relative
to the vegetative biomass. Reproductive allocation should be higher in
plants from drier conditions as they require less investment in
vegetative tissue for outgrowing neighbors (Aronson et al. 1993).
Diaspore weight (maternal investment per single offspring) was
measured across 30 randomly picked diaspores per plant. Diaspore weight
consists of c. 50% of seed mass in B. didyma and both are
strongly correlated (r²=0.88, p<0.001; determined for 15 seeds
in 32 randomly picked individuals across sites). Although investment per
offspring is a crucial feature of plant life cycles (Westoby 1998),
predicting its evolutionary response to aridity is controversial (Kurzeet al. 2017).