DOI: 10.5281/zenodo.5667347
title: Disentangling the effects of jasmonate and tissue loss on the sex allocation of an annual plant

description: In this study, we explored norms of reaction in sex expression and sex allocation to herbivory in an experiment designed to uncouple its direct (through tissue loss) and indirect effects (due to defensive jasmonate signalling) in hermaphroditic XX females of the wind-pollinated Mercurialis annua. To uncouple the direct and indirect effects of herbivory on the sex expression and to test the role of jasmonate on conditional sex allocation, we conducted a two-factorial experiment manipulating tissue loss (25% chronic defoliation) and plant anti-herbivore defences via the jasmonate pathway (external application of jasmonate), and measured sexual expression in plants with both a male and a female function. The herbivory treatment applied were: For the control treatment (C), leaves were sprayed with a sham solution containing only water and polysorbate until all leaves were wet (see Supplementary Materials for detailed solution formulae). The herbivory treatment (H) consisted of cutting off half of every second leaf on the plant with scissors and spraying plants with a sham solution until all leaves were wet (defoliation resulted in a 25% reduction of total leaf area over the course of the whole plant’s lifetime). In the jasmonate treatment (JA) plants were sprayed with a solution of methyl-jasmonate and polysorbate until all leaves were wet (polysorbate 20 was used to fix the methyl-jasmonate on the sprayed leaves). Finally, the jasmonate and herbivory treatment (JAH) consisted of cutting off half of every other leaf on the plant with scissors and spraying plants with the methyl-jasmonate solution until all leaves were wet. These treatments were applied repeatedly as plants continued to grow, i.e., they represent chronic stress or manipulation. The first round of treatment was applied one week after repotting the plants (25th of November 2019) and then every two weeks over the next 12 weeks (the last treatment was applied on the 2<sup>nd</sup> of February 2020). On the first round of treatment, when most plants had fewer than six leaves each, we cut off only half a leaf (~10% of the leaf area removed) for plants under the herbivory treatments to avoid seedlings death. Plant sampling consisted of cutting all above-ground plant material of 34 plants per enclosure (<em>N</em> = 272) and recording total height. Plants were then cut in half, lengthwise, creating two distinct segments: top and bottom. The top segment was carefully examined and we counted the number of fruits (immature and mature) and harvested all male flowers using tweezers. Male flowers were stored in paper envelopes, dried and weighed. After phenotyping, plant segments were dried and weighed to obtain plant dry biomass (top + bottom). To estimate seed production, the seeds were isolated from the dried plant materials, stored in paper envelopes and weighed. All materials were dried in an oven at 50°C for at least 14 days and weighed using a digital scale. Variables names and meaning: PlantID: Individual identifier for each plant<br> nb_seeds_estimate.TOP: Number of seeds form the top section of the plant <br> Biomass.BOTTOM: Dry biomass of the bottom plant section (grams) <br> Total_biomass: Dry biomass of the whole aboveground plant materials, except for the male flowers <br> Biomass.TOP: Dry biomass of the bottom plant section (grams) <br> seed_mass_total: Dry biomass of the seeds of the whole plant (top+bottom sections) (grams)<br> seed_mass.BOTTOM: Dry biomass of the seeds from the bottom section (grams)<br> seed_nb_total: Number of seeds from the whole plant (top+bottom sections) <br> Lenght_section.TOP: Length of the top section (cm) <br> Fruit_number.TOP: Number of fruits present on the top sectioon at the time of harvest<br> nb_seeds_estimate.BOTTOM: Number of seeds from the bottom section<br> Height: Plant height (top+bottom sections) (cm) at the time of harvest<br> Fruit_number.BOTTOM: Number of fruits present on the bottom section at the time of harvest <br> DPT: Days-post-treatment = the period elapsed between the last treatment application and the plant sampling date. For logistical reasons, our sampling was spread over 14 days by a team of six assistants.<br> Lenght_section.BOTTOM: Length of the bottom section (cm)<br> Treatment: Herbivory treatments: C=Control; H= 25% chronic tissue loss, JA=exogenous jasmonate application; JAH=tissue loss + jasmonate.<br> Box: Enclosure in which plants were kept. This was a blocking factor with 2 boxes per treatment, each one with 30-32 plants. <br> Date: sampling date <br> seed_mass.TOP: Dry biomass of the seeds on the bottom plant sections (grams)<br> Observer: Identifier for each of the six researchers who sampled plants. We recorder observer identity and included it in our statistical analyses to account for possible biases among assistants.<br> male_fl_mass.TOP: Dry biomass of the male flowers sampled from the top plant section (grams). <br> nb_fl_estimate.TOP: Number of male flowers present on the top plant section at the time of harvest <br> male_fl_mass.BOTTOM: Dry biomass of the male flowers sampled from the bottom plant section (grams). <br> nb_fl_estimate.BOTTOM: Number of male flowers present on the bottom plant section at the time of harvest