DOI: 10.5061/dryad.9w0vt4bc0
title: Species-specific responses to combined water stress and increasing temperatures in two bee-pollinated congeners (Echium, Boraginaceae)

description: 1Water stress and increasing temperatures are two main constraints faced
 by plants in the context of climate change. These constraints affect plant
 physiology and morphology, including phenology, floral traits, and nectar
 rewards, thus altering plant–pollinator interactions. 2. We compared the
 abiotic stress responses of two bee-pollinated Boraginaceae species,
 Echium plantagineum, an annual, and E. vulgare, a biennial. Plants were
 grown for 5 weeks during their flowering period under two watering regimes
 (well-watered and water-stressed) and three temperature regimes (21, 24,
 27°C). 3. We measured physiological traits linked to photosynthesis
 (chlorophyll content, stomatal conductance, and water use efficiency), and
 vegetative (leaf number and growth rate) and floral (e.g., flower number,
 phenology, floral morphology, and nectar production) traits. 4. The
 physiological and morphological traits of both species were affected by
 the water and temperature stresses, although the effects were greater for
 the annual species. Both stresses negatively affected floral traits,
 accelerating flower phenology, decreasing flower size, and, for the annual
 species, decreasing nectar rewards. In both species, the number of flowers
 was reduced by 22–45% under water stress, limiting the total amount of
 floral rewards. 5. Under water stress and increasing temperatures, which
 mimic the effects of climate change, floral traits and resources of
 bee-pollinated species are affected and can lead to disruptions of
 pollination and reproductive success.
description: <b>Growth conditions</b> Seeds
 were provided by Semailles nursery (Faulx-les-Tombes, Belgium). Seeds were
 placed in a germination chamber (Economic Delux model ECD01E; Snijders
 Scientific, Tilburg, The Netherlands) under 20°C/18°C day/night
 temperature and a 16-h light (L):8-h dark (D) photoperiod, for 2 weeks.
 Seedlings were transplanted into pots filled with a 1:1 (v/v) mix of sand
 (0/5, M Pro, The Netherlands) and universal peat compost (DCM, Amsterdam,
 The Netherlands). Plants were grown in the greenhouse at the university
 campus (Louvain-la-Neuve 50°39′58′′N; 4°37′9′′E, Belgium) and were watered
 every 2–3 days with rainwater. Treatments were applied after floral
 transition under controlled conditions in growth chambers (SEFY platform,
 Louvain-la-Neuve) at different temperature and watering regimes.
 To observe the effects of temperature and water stress (and their
 interaction) on vegetative and reproductive development and
 photosynthesis-related parameters, fifteen plants per treatment and
 species were placed under three temperature regimes (21/19°C, 24/22°C, and
 27/25°C day/night) and two watering regimes (well-watered compared to
 water-stressed). The well-watered plants received daily watering (soil
 humidity about 25%, as determined using a Procheck Hand-held Sensor 10 HS
 moisture sensor, Decagon Devises, Inc, Pullman WA, USA), whereas the
 water-stressed plants were watered twice a week (soil humidity of 8–15%).
 The combination of temperature and watering regimes resulted in six
 treatments: 21°C well-watered (21WW), 21°C water-stressed (21WS), 24°C
 well-watered (24WW), 24°C water-stressed (24WS), 27°C well-watered (27WW),
 and 27°C water-stressed (27WS). In total, 90 plants per species were
 monitored in three growth chambers. The photoperiod was set to 16L:8D, and
 relative humidity was maintained at 80 ± 10%. Growth chamber experiments
 lasted for 6 weeks. Water stress was applied after 1 week of acclimation
 to the growth chambers; this initial week was considered week 0.
   <b>Morphological
 traits</b> At week 0, flowering stem height was
 measured. Every week for 6 weeks, the number of axillary stems (for
 <i>E. plantagineum</i>), new leaves (&gt;2 cm),
 inflorescences, and flowers at anthesis were counted per plant. At the end
 of the experiment (week 5), the height of the main flowering stem was
 measured to calculate the growth rate.  
 <b>Physiological traits</b> The
 5th-node leaves of 10 plants per treatment were measured at the beginning
 of the experiment and 2 weeks after inducing stress. The chlorophyll
 content index (CCI) was measured using a chlorophyllometer (Opti-Sciences,
 CCM-200), and three measurements were taken per leaf. An automatic
 porometer (AP4 System, Delta-T Devices) was used to measure the stomatal
 conductance. Gas exchange was measured using an infrared gas analyzer
 (IRGA ADC BioScientific LCI-SD system, serial No.33413, Hoddesdon, UK).
 The instantaneous water use efficiency (WUE<sub>i</sub>) was
 calculated as WUE<sub>i</sub> =
 A<sub>i</sub>/E<sub>i</sub>.
   <b>Floral and nectar
 traits</b> The corolla depth and diameter were
 measured three times, at weeks 1, 3, and 5, on 10 random flowers in each
 treatment. In week 3, flowers were dissected, and floral organs were
 scanned (Ricoh MP C3004 ex PS). The corolla surface area and the length of
 all stamens per flower were calculated using ImageJ software.
 Nectar was extracted with glass capillary tubes (1, 5, or 10 μl,
 depending on the nectar volume; Hirschmann Laborgeräte, Eberstadt,
 Germany) from five flowers per treatment (from five different plants).
 Total sugar concentration (°Brix) was measured with a low-volume hand
 refractometer (Eclipse hand-held refractometer; Bellingham and Stanley,
 Tunbridge Wells, UK). Nectar sugar content per flower (mg) was calculated
 following Prys-Jones and Corbet method (1991).