DOI: 10.7280/d12h6c
title: Milkweed trait values associated with aridity gradients and drought-induced changes in hostplant quality

description:  The response of herbivorous insects to plant drought stress can
 range from positive to negative, and it has been challenging to understand
 the causes of this variation. We tested whether plant trait values
 associated with aridity gradients might underlie this variation and how
 effects vary among two insect-feeding guilds. Here we propose that plant
 trait values associated with adaptation to arid environments would result
 in positive effects of experimental drought on herbivores, with such plant
 species adaptively shifting resources away from resistance to maintain
 performance under stress. In contrast, plants with trait values associated
 with adaptation to mesic environments would result in negative effects of
 drought because such species lose vigor and thus decline in their
 host-quality. We tested these predictions using experimental manipulations
 in 13 milkweed species (genus Asclepias) adapted to a wide range
 of environmental conditions, and herbivore performance of herbivores. We
 exposed plants to species-specific watering regimes physiologically
 calibrated to maximize (100%) or reduce (50%) stomatal conductance and
 then monitored the performance of the herbivores. Here we present milkweed
 trait values associated with aridity gradient [specific leaf area (SLA),
 relative water content (RWC), water use efficiency (WUE), and maximum
 stomatal conductance (gsmax)] and changes of trait values associated with
 host-plant quality to herbivores [nutrients content (protein, nitrogen,
 and carbohydrates) and plant defensive traits(latex and
 cardenolides)]. 
description: Trait measurements were obtained from 3-5 newly expanded and
 undamaged leaves collected from each experimental plant at the conclusion
 of the greenhouse experiment. Excised leaves were transferred to a paper
 envelope and immediately frozen at -20 °C for later characterization of
 plant traits. For water-use-associated traits, we measured leaves from
 plants in the control treatment as representative of their constitutive
 trait values. To measure SLA, leaf area (cm2)/dry mass (mg), frozen leaves
 were scanned, weighed, dried at 60 °C for 2 days, and reweighed to the
 nearest mg. Leaf water content was calculated as a percentage estimated
 from the difference between leaf fresh weight and leaf dry weight. Dried
 leaves were pulverized using a Mixer Mill (Retsch MM 400) for stable
 isotope and macronutrient analyses. Foliar carbon isotopes ratio (13C and
 12C) was estimated from pulverized foliar tissue as an indicator of WUE
 for a subset of samples (n=46) at the UC-Irvine Mass Spectrometry
 facility. Maximum stomatal conductance (gs max) was the raw stomatal
 conductance values achieved by each species when at its maximum level of
 stomatal conductance. To quantify drought-induced
 changes in hostplant quality, we measured plant defenses and nutritional
 content from plants in the control and drought treatments. Latex exudation
 and cardenolides concentration were measured as the typical plant
 defensive traits in milkweeds. Latex exudation was estimated by excising
 2-3 mm off the tip of a new, undamaged, and fully extended leaf. The latex
 produced within 30 seconds was collected on pre-weighed 1 cm2 filter paper
 discs which were then placed in pre-weighted 2 mL Eppendorf tubes. The
 tubes were immediately reweighed to estimate the mass of wet latex
 collected. Cardenolides concentrations were analyzed from a subsample of
 plants (n=47) from pulverized foliar tissue. We determined cardenolide
 concentration (mg/g dry tissue) from pulverized foliar tissue (n=47) by
 high-performance liquid chromatography following the methods of Züst et
 al. (2019) in the Ecology and Evolutionary Biology department at Cornell
 University. Milkweeds nutritional profiles were
 characterized from pulverized leaf tissue by quantifying nitrogen,
 protein, and total non-structural carbohydrate concentrations for a subset
 of samples (n=46). Nitrogen content (mg/g dry tissue) was estimated during
 stable isotope analysis at the UC-Irvine Mass Spectrometry facility.
 Soluble carbohydrates and carbohydrates from starch were extracted
 separately following protocols described by Chown and Landhässeur (2004)
 and quantified colourimetrically using the phenol-sulfuric acid assay
 (DuBois et al., 1956) optimized for microplate reading (Masuko et al.,
 2005). Protein was extracted by sonication following protocols described
 in Lenhart et al. (2015) and quantified colourimetrically using the
 Bio-Rad Bradford microassay (Bradford, 1976) in a microplate reader. Total
 carbohydrates and protein content were also estimated on a per-mass basis
 (mg/g dry mass). 
description: Excel