DOI: 10.5061/dryad.djh9w0w2p
title: Climatic history, constraints, and the plasticity of phytochemical traits under water stress

description: Environmental stress can induce changes in organismal traits and in
 resulting intraspecific variation. The nature of such effects will depend
 on the plasticity of trait expression and on any ecological constraints to
 such expression. Plants can mitigate abiotic stress, like drought, by
 changing their chemistry, but the ability to induce costly metabolites may
 be under strong local selection and ecologically constrained. Here we
 asked whether climate at the seed source predicts plant chemical
 plasticity in response to water stress and what the consequences are for
 intraspecific variation in phytochemical traits. To this end, we used
 common gardens of two widespread species of western milkweed (Asclepias
 fascicularis and Asclepias speciosa) that had been collected from sites
 across an aridity gradient. Both species produce high concentrations of
 leaf flavonols, which are hypothesized to mitigate water stress by
 functioning as antioxidants. These compounds were found in higher
 constitutive concentrations in plants sourced from drier sites, and both
 species responded to water stress in the common garden by increasing leaf
 flavonol concentrations. Interestingly, flavonol plasticity was higher in
 plants sourced from wetter sites in A. fascicularis, with similar, but
 weaker, patterns in A. speciosa. These opposing patterns in constitutive
 and induced flavonol expression reduced the variation between populations
 in leaf flavonol concentrations under water stress. These results suggest
 that local adaptation in plants can shape phytochemical strategies for
 water limitation but that the cost of metabolite production may ultimately
 limit the range of phytochemical variation.
description: To seed the experiments, we collected seeds
 from six sites spanning 385 km of the Great Basin Desert, USA, in fall
 2016. From highest to lowest climatic water deficit (mm), our seed-source
 sites were: Fallon, NV (FN; 988.98 mm), Auburn, CA (CA; 985.68 mm),
 Pyramid Lake, NV (PL; 919.62 mm), Battle Mountain, NV (BM; 847.74 mm),
 Reno, NV (RN; 587.19 mm),  and Verdi, NV (VE; 460.3
 mm). To
 determine whether intraspecific trait variation could be predicted by
 seed-source CWD and how such variation is affected by water limitation, we
 conducted a drought experiment with <em>A. fascicularis
 </em>and <em>A. speciosa</em> in a glasshouse. We
 germinated 12 <em>A. fascicularis </em>seeds from each of
 three maternal families from all six sites (<em>N </em>= 216),
 and 12 <em>A. speciosa </em>seeds from each of three maternal
 families from four of the sites (FN, PL, BM, and RN; <em>N
 </em>= 144). We randomly assigned six plants from each maternal
 family to the control (well watered) treatment and the other six plants to
 the dry treatment (<em>n </em>= 36 per seed-source site).
 Prior to beginning the dry treatments, mortality was higher for
 <em>A. speciosa</em> than for <em>A.
 fascicularis</em>, such that the final average
 <em>n</em> was ~33 for <em>A. fascicularis
 </em>but ~17 for <em>A. speciosa</em>. We used a
 gravimetric dry-down treatment to expose plants to drought stress for four
 weeks in March–April 2017. Seeds were germinated in Petri dishes under
 lights (L18:D6) at 25° C in November 2016. Plants were grown in 4x9.5-in
 treepots containing 1500 g of a mixture of 2:1:1 parts sand:peat
 moss:composted bark. Pots were completely randomized on tables in the
 glasshouse and fertilized weekly with 24:8:16 N:P:K fertilizer. We
 calculated gravimetric soil water content using 13 treepots with 1500 g of
 the same soil. Saturated mass was measured 2 h after fully saturating the
 pots; dry mass was measured after oven drying for 48 h at 90° C. 100% soil
 saturation was estimated as: <em>saturated mass </em>–
 <em>dry mass</em>. We allowed control plants to dry to 70%
 soil saturation and dry plants to 10% soil
 saturation. To
 verify the efficacy of our drought treatment and to explore differences in
 physiological responses to drought, which may mediate the plant’s
 metabolic allocation, we measured the following plant traits: change in
 plant height; whole-plant dry biomass of roots and shoots; root:shoot
 ratio; leaf mass per area (LMA); and stomatal conductance. Plant height
 was recorded prior to beginning the dry treatment and again prior to
 harvesting the plants. Roots, stems, and leaves were harvested separately,
 washed, and dried at 60° C for 72 h before weighing. Tissues were weighed
 in microcentrifuge tubes, and the weight of the tube was subtracted. Prior
 to weighing, 41 microcentrifuge tubes that had contained stem tissue were
 accidentally discarded; we thus present results on both root:shoot ratios
 and root:leaf ratios. A leaf in position three of the phylotaxis was
 collected for LMA, which was estimated by dividing the dry weight of the
 leaf in mg by the estimated leaf area in mm<sup>2</sup>
 (length x width). Stomatal conductance was measured between 1200–1400 h
 using an SC1 Porometer (Decagon). To
 determine how water stress affected phytochemical trait expression, we
 measured plant UV-absorbent secondary chemistry. Prior to harvest, leaves
 and fine roots were collected and stored at -80° C. These tissues were
 later freeze-dried, ground, and extracted in 100% methanol with a
 cardenolide internal standard (digitoxin). UV-absorbent peaks were
 measured on a high-performance liquid chromatography (HPLC) system with a
 diode array detector recording peaks that absorbed between 200–330 nm. We
 retained peaks for our analysis that could be consistently identified from
 mass fragments using low-resolution mass spectrometry and/or that were
 present in a majority of the samples of a given species (<em>A.
 fascicularis </em>or <em>A. speciosa</em>)<em>
 </em>and respective tissue (leaf or root).
 All
 analyses were conducted in R version 4.0.5 (R Core Team 2021). To
 determine whether trait plasticity under acute water stress depended on
 climatic history at the seed source, we used generalized linear mixed
 models (GLMMs) from the <em>glmmTMB</em> package. Preliminary
 analyses showed strong trait differences and a lack of correspondence in
 phytochemical compounds between species, such that we ran separate models
 for each species. Each saturated model started with the fixed effects of
 water treatment, seed-source CWD, the water x CWD interaction, and the
 random intercept effects of plant maternal family nested within the
 seed-source site. To compare effect sizes among response variables, we
 normalized responses and CWD values using the <em>BBmisc</em>
 package, and we report beta coefficients (<em>β</em>) with
 standard errors. We assessed the residuals of each fitted model, and we
 square-root or log-transformed response variables when these
 transformations provided a better fit to the Gaussian
 distribution.
description: A ReadMe file will be attached with the data.