DOI: 10.5061/dryad.2ngf1vj15
title: Adaptive evolution can mitigate the negative effects of temperature stress on plant-pollinator interactions

description: Temperature stress negatively affects various aspects of plant fitness,
 including plant-pollinator interactions, but whether plants can overcome
 these adverse effects through adaptive evolution is largely unknown. Here,
 we conducted a six-generation evolution experiment using fast cycling
 Brassica rapa plants at ambient and elevated temperatures, with
 bumblebee-pollination. At the end of the experiment, we re-grew the
 evolved genotypes at different temperatures. We phenotyped the plants and
 conducted pollinator bioassays to assess adaptive evolution, evolutionary
 trait divergence, and the evolution of heat-mediated phenotypic
 plasticity. We found that plants that had evolved with
 bumblebee-pollination in both temperature regimes had higher seed set than
 control plants, which suffered lower seed set when evolved under elevated
 temperatures. We also showed that the number of flowers, a trait that
 largely determined plant attractiveness to bumblebees and seed set, was
 increased in bumblebee-pollinated plants, and so was heat-induced
 phenotypic plasticity in flower number. Plants that evolved with high
 temperature showed increased UV-reflection, and a stronger association
 between flower size and nectar content (honest signalling), and reduced
 scent emission. Our results show that plants that evolve under
 pollinator-mediated selection can mitigate at least some of the negative
 effects of temperature stress through adaptive evolution.
description: # Adaptive evolution can mitigate the negative effects of temperature
 stress on plant-pollinator interactions ## Description of data ####
 data_local_adapt_traits.csv This dataset incorporates all plant traits
 measured in this experiment, as well as data on bumblebee visitation on
 plants, and on the reproductive success of the plants. The dataset
 includes plants from genotypes which were grown for 6 generations at 23°C
 or at 27°C plus 6 “hot” days of 30°C and visited by *Bombus terrestris*
 bumblebees or hand pollinated (control). It also includes Generation 1
 (G1) plants. On the last generation, all plants were grown at 23°C or 27°C
 plus 6 “hot” days of 30°C. Among the phenotypic measurements there are
 visual, morphological, reward traits and the composition of flower scent,
 obtained through volatile collection and GC-MS. To acquire bumblebee
 behavioral data and plant reproductive success, plants were set in groups
 of 24, in a 5 x 5 matrix, where we allowed up to 6 bees to visit plants
 sequentially (until 50-70% of plants were visited). Each matrix contained
 plants from the same pollination genotype (bumblebee genotype or control)
 and compared the attractiveness and reproductive success of 12
 hot-genotype plants versus 12 ambient-genotype plants. There are a few
 mismatches between the phenotypic data and the behavioral/fitness data.
 Not all plants we phenotyped could be used in behavioral assays and vice
 versa. This is the main source of NAs in this document. Description of
 variables * *temp_environment*: The temperature at which we grew all
 plants on the last generation, during which we made all measurements. H
 means hot environment (27°C plus 6 “hot” days of 30°C) and C means ambient
 environment (23°C). * *temp_genotype*: The temperature at which plant
 genotypes were kept during 6 generations. C means ambient-genotype (kept
 at 23°C) and H means hot-genotype (kept at 27°C plus 6 “hot” days of
 30°C). * *poll_genotype*: The pollinator visitation regime under which
 plant genotypes were kept during 6 generations. B means bumblebee-genotype
 (plants visited by *Bombus terrestris* bumblebees) and H means control
 (plants that were hand pollinated). G means G1 plants. * *rep*: All
 treatments were divided into replicates A and B. A and B were composed of
 49 plants belonging to different full-sibling families. At the start of
 the experimental evolution, plants from the same replicates belonged to
 the same 49 families across treatments. * *cohort*: planting week. Plants
 from all treatments were grown and tested over a six-week period. *
 *plant*: Individual ID for each plant. * *matrix*: code indicating whether
 plants were tested in the same matrix during bumblebee visitation
 bioassays. For instance, HB1 means that bumblebee-genotype plants were
 tested in the hot environment on week one, and CH4 that control plants
 were tested in the ambient environment on week 4. * *height*: plant height
 measured in cm, to the nearest 0.5 cm. Plants were measured using a ruler,
 from the base to the top of the highest inflorescence. * *leaves*: total
 number of leaves on the week that the plants were tested. * *flowers*:
 total number of open flowers, counted on the week when plants were tested,
 and before bioassays. * *nectar_per_flower*: estimation of nectar amount
 per flower, in microliters (µl). Nectar was collected from three flowers
 using microcapillaries, and the resulting amount was then divided by 3. *
 *nectar_per_plant*: estimation of nectar amount per plant, calculated by
 multiplying the nectar amount per flower by the number of open flowers. As
 we only could measure nectar amounts on three flowers per plant, when
 nectar per flower was 0, we added n/a on nectar per plant, as we cannot
 know for sure whether the plant had absolutely no nectar. *
 *flowering_time*: days from sowing until opening of the first flower. *
 *petal_area*: area per flower petal. We measured the petal area of all
 four petals from three flowers by scanning the flat, excised petals and
 calculating the area on ImageJ. * *uv_absorbance*: The relative area of UV
 absorbance per petal. Calculated by dividing the area of UV absorbance per
 petal (in cm2, obtained by photographing flattened petals using a camera
 with a UV tranmission filter) by the petal area. * *uva*: Relative diffuse
 reflectance of UVA color, measured 0.5 mm below the top edge of the petal
 with a spectrometer. * *uvb*: Relative diffuse reflectance of UVB color,
 measured 0.5 mm below the top edge of the petal with a spectrometer. *
 *yellow*: Relative diffuse reflectance of yellow color, measured 0.5 mm
 below the top edge of the petal with a spectrometer. * *chosen*: Indicates
 whether plants were visited (1) or not (0) by bumblebees in the
 pollination bioassays. * *seed_number*: Total number of seeds per plant,
 collected and counted after the bioassays, and once fruits were mature. *
 *fruit_number*: Total number of siliques per plant, collected and counted
 after the bioassays, and once fruits were mature. * *seeds_per_fruit*:
 number of seeds per plant divided by the number of fruits per plant. *
 *fruit_set*: number of fruits divided by the number of open flowers at the
 time of pollination. * *bee_flower_visits*: number of flower visits by
 bumblebees received by each plant. * *bee_plant_visits*: number of overall
 plant visits by bumblebees received by each plant. *
 *bee_seconds_per_flower*: average seconds spent per flower by a bumblebee,
 when visiting a plant. Measurement obtained by dividing the time spent per
 plant by the number of flower visits. * *bee_seconds_per_plant*:
 cumulative time spent by all bumblebee visitors on a plant. Calculated by
 measuring the time elapsed between arrival on a plant and departure from
 it for each bumblebee visitor, and then adding the times together for an
 individual plant. * *run*: code indicating when the scent samples from the
 plant were measured in the GC-MS. Plants sharing the code were measured on
 the same run. * *Benzaldehyde - ZZ.alpha.Farnesene_D*: Individual flower
 scent compounds identified and quantified by the GC-MS. Scent amounts
 expressed as pg / flower / l / h, transformed by ln(1+x). * *scent_sum*:
 Sum of the amounts from all identified flower scent compounds, expressed
 as pg / flower / l / h, transformed by ln(1+x). * *sum_aromatics*: Sum of
 the amounts from all identified aromatic flower scent compounds, expressed
 as pg / flower / l / h. Aromatic scent compounds detected in this study
 are: benzaldehyde, methyl Benzoate, phenylethyl alcohol, 2-amino
 benzaldehyde, p-anisaldehyde, methyl anthranilate, phenylacetaldehyde,
 benzyl Nitrile, methyl salicilate and indole. * *lnBenz - lntotal*: ln(x +
 1)-transformed values of scent emission for the individual scent compunds
 and their sum. Before transformation, scent amounts expressed as pg /
 flower / l / h. #### reaction_norms.csv This dataset includes the
 temperature reaction norms of all plant phenotypic traits measured, and
 from plants of all treatments. For every treatment, we grew pairs of
 half-sibling plants, one at an ambient (23°C) and one at hot (27°C plus 6
 “hot” days of 30°C) temperature, which allowed us to obtain temperature
 reaction norms by calculating the difference between the trait values of
 sibling plant pairs grown at ambient and hot temperature environments.
 Description of variables As we display the temperature reaction norms from
 the plant phenotypic traits present in the dataset above
 (**data_local_adapt_traits.xlsx**), all the column headings have the same
 meaning as in that dataset. Except for the fact that here, instead of
 showing individual measurements, we are showing temperature reaction norms
 of traits, obtained from half-sibling plant pairs grown at different
 temperature environments. N/a are present if data was missing from one (or
 both) plants in the half-sib pair. #### haldanes.csv This dataset contains
 the evolutionary rates, measured in Haldanes, of each trait per replicate,
 for all treatments. Haldanes were obtained by calculating the absolute
 difference between the replicate trait means at Generation 8 vs. 1,
 dividing that by the pooled SD per trait per replicate, and dividing the
 result by the number of generations under selection (6). A more detailed
 description and the pertinent formulas are available in the methods
 section of the main manuscript. Description of variables *
 *temp_genotype*: The temperature at which plant genotypes were kept during
 6 generations. C means ambient-genotype (kept at 23°C) and H means
 hot-genotype (kept at 27°C plus 6 “hot” days of 30°C). * *poll_genotype*:
 The pollinator visitation regime under which plant genotypes were kept
 during 6 generations. B means bumblebee-genotype (plants visited by
 *Bombus terrestris* bumblebees) and H means control (plants that were hand
 pollinated). * *rep*: All treatments were divided into replicates A and B.
 A and B were composed of 49 plants belonging to different full-sibling
 families. At the start of the experimental evolution, plants from the same
 replicates belonged to the same 49 families across treatments. * *traits*:
 All of the phenotypic traits which were measured from plants of all
 treatments. As mentioned above, haldanes were calculated per traits per
 treatment replicate. * *haldane*: Evolutionary rates measured in
 Haldane's index for synchronic comparisons. * *trait_cat*: column
 indicating whether the traits used to calculate haldanes consist of flower
 scent compounds (scent) or all other phenotypic traits measured
 (non-scent).