DOI: 10.5061/dryad.rjdfn2zcw
title: Dataset for: Interactive effects of tree species composition and water availability on growth and direct and indirect defences in Quercus ilex

description: Plant diversity has often been reported to decrease insect herbivory in
 plants. Of the numerous mechanisms that have been proposed to explain this
 phenomenon, how plant diversity influences plant defences via effects on
 growth has received little attention. In addition, plant diversity effects
 may be contingent on abiotic conditions (e.g., resource and water
 availability). Here, we used a long-term experiment to explore the
 interactive effects of tree species composition and water availability on
 growth, direct (i.e. phenolics) and indirect (i.e. Volatile Organic
 Compounds – VOCs) defences and leaf herbivory in Quercus ilex. We
 quantified herbivory by chewing insects, phenolic compounds and VOCs
 in Q. ilex trees growing in stands differing in tree
 species composition (Q. ilex, Q. ilex + Betula
 Pendula, Q. ilex + Pinus
 pinaster and Q. ilex + B.
 pendula + P. pinaster) and water availability (irrigated
 vs control). Both direct and indirect defences were affected by tree
 species composition, but such changes were not mediated by changes in tree
 stem diameter. Q. ilex trees growing in stands
 with P. pinaster had the lowest concentration of both
 direct and indirect defences. Importantly, the effects of tree species
 composition on VOCs were exacerbated on irrigated blocks. Despite
 variation in defences, tree species composition did not affect herbivory
 in Q. ilex. Accordingly, we did not find any association between
 defences and insect herbivory. Our results suggest that changes in the
 micro-environment rather than growth-defence associations may mediate tree
 diversity effects on defences. In addition, reduced defensive investment
 in more diverse stands could negatively impact tree resistance masking the
 beneficial effects of species diversity at reducing insect herbivory.
description: <em><strong>Experimental
 design</strong></em> This study was
 conducted in the ORPHEE experimental trial established in 2008 in
 South-West France (44°440 N, 00°460 W). The experimental design consisted
 of eight blocks and 32 plots within each block. Each plot represented a
 tree species composition treatment, corresponding to 31 possible
 combinations of one to five tree species (<em>Betula
 pendula</em>, <em>Quercus robur</em>, <em>Q.
 pyrenaica</em>, <em>Q. ilex</em>, and <em>Pinus
 pinaster</em>) and an additional plot replicate of the five species
 mixture. Each plot contained 10 rows of 10 trees planted 2 m apart (100
 trees on 400 m²). Tree species mixtures were established according to a
 substitutive design, keeping tree density of tree neighbours equal across
 plots. Within plots, individual trees from different species were planted
 in a regular alternate pattern, such that a tree from a given species had
 at least one neighbour from each of the other species within a 2-m
 radius. From 2015 four out of the eight experimental blocks were allocated
 to an irrigation treatment consisting of sprinkling the equivalent of 3 mm
 precipitation from a 2 m height pole in the centre of each irrigated plot.
 Blocks were irrigated on a daily basis, at night, from May to October. The
 four remaining blocks were kept as controls. This
 datasets collects data for <em>Q. ilex</em>. In particular, we
 focused on <em>Quercus ilex</em> as target species and
 selected six blocks (three irrigated and three control) and four plots
 (tree species composition treatments) in each block corresponding to the
 monoculture of Q. ilex and its combinations with <em>B.
 pendula</em> and <em>P. pinaster</em> (<em>Q.
 ilex</em> + <em>B. pendula</em>, <em>Q.
 ilex</em> + P. pinaster and <em>Q.
 ilex </em>+ <em>B. pendula</em> + <em>P.
 pinaster</em>). Therefore, a total of 24 experimental plots (4 tree
 species composition treatments × 2 irrigation treatments × 3 blocks) were
 included in the study. 
 <em><strong>Sampling and
 measurements</strong></em> At the end of
 the growing season (September 2019), we haphazardly selected
 four <em>Q. ilex</em> trees in each of the 24 plots (N = 96
 trees). Trees in the plot margins were not selected to avoid border
 effects. First, we estimated total height and basal diameter (± 30 cm
 aboveground) in all experimental trees with a tape-measure and a digital
 caliper respectively. After tree growth measurements, we collected VOCs
 for each tree. Briefly, we bagged one branch of each tree with a 1L
 nalophan bag and we trapped the compounds on a charcoal filter by pulling
 air through the filter using an air-sampling pump for 2 h at a rate of 250
 ml min<sup>-1</sup>. Importantly, we sampled air VOCs in empty
 bags (one bag placed in the middle of each plot within each block) as
 controls, in order to identify compounds that may contaminate the blend of
 VOCs taken from the focal trees (e.g., VOCs emitted by neighbour species).
 After collecting the VOCs, we stored the filters at -80ºC until chemical
 analyses. Right after VOCs collection, we haphazardly collected 20 fully
 expanded and developed leaves throughout the tree’s canopy. Importantly,
 because <em>Q. Ilex</em> is an evergreen species, sampled
 leaves may have consisted of one to three cohorts of leaves (i.e. produced
 between 2017 and 2019; up to two-years old). For each leaf, we visually
 estimated the percentage of leaf area removed by insect herbivores (mostly
 leaf chewers) using the following scale: 0 = no damage; 1 = 1–5% damaged;
 2 = 6–10% damaged; 3 = 11–25% damaged; 4 = 26–50% damaged; 5 = 51–75%
 damaged; 6 = &gt;75% damaged (“leaf herbivory” hereafter). We averaged
 class values across all leaves to obtain a mean value per tree for
 statistical analyses. We selected a subset of 4-5 leaves with little or no
 evidence of herbivory for further chemical analyses of phenolic compounds.
 Leaves were oven-dried for 48 h at 40ºC.
 <strong><em>Chemical
 analyses</em></strong> Quantification of volatile
 organic compounds (VOCs) To analyse VOCs,
 we performed gas chromatography and mass spectrometry analyses. To extract
 the compounds from the charcoal traps, we first added 5 μl of naphthalene
 (20 ng ml<sup>−1</sup>) as an internal standard to the
 traps (Pellissier et al., 2016), and then eluted their contents with 400
 μl of dichloromethane. We then injected 2 μl of the extract for each
 sample into a gas chromatograph (GC) coupled with a mass selective
 detector (MSD) fitted with a 30 m × 0.25 mm × 0.25 mm film thickness
 HP-5MS fused silica column. We operated the GC in splitless mode with
 helium as the carrier gas (constant flow rate 0.9 ml
 min<sup>−1</sup>). The GC oven temperature program was: 1 min
 hold at 40°C, and then 10°C min<sup>−1</sup> ramp to 240°C. We
 identified individual volatile compounds (i.e., terpenes) using Kovats
 retention index from published work, the NIST Standard Reference Database
 1A v17, and by comparison with commercial standards when available.
 Volatile emissions are reported as nanograms naphthalene equivalents. For
 subsequent analyses, we selected VOCs identified as either monoterpenes or
 sesquiterpenes. We quantified individual monoterpenes and sesquiterpenes
 relative to the internal standard and used for statistical analyses those
 exhibiting a relative abundance higher than 1%. Importantly, for those
 compounds present in both the samples and the corresponding control, we
 only consider those which intensity in the sample was at least double than
 in the control. Finally, we quantified the total concentration of VOCs as
 the sum of concentrations of all individual compounds.
 Quantification of phenolic
 compounds We extracted phenolic compounds
 from 20 mg of dry leaf tissue with 1 ml of 70% methanol in an ultrasonic
 bath for 15 min, followed by centrifugation (Moreira et al., 2020) and
 transferred the extracts to chromatographic vials. To analyse the phenolic
 compounds, we performed chromatographic analyses using ultra-high
 performance liquid chromatography equipped with a Nexera SIL-30AC injector
 and one SPD-M20A UV/VIS photodiode array detector. The compound separation
 was carried out on a Kinetex 2.6 μm C18 82–102 Å, LC Column 100 × 4.6 mm,
 protected with a C18 guard cartridge. The flow rate was 0.4 ml
 min<sup>−1</sup> and the oven temperature was set at 25°C. The
 mobile phase consisted of two solvents: water–formic acid (0.05%) (A) and
 acetonitrile–formic acid (0.05%) (B), starting with 5% B and using a
 gradient to obtain 30% B at 4 min, 60% B at 10 min, 80% B at 13 min and
 100% B at 15 min. The injection volume was 15 μl. For phenolic compound
 identification, we used an ultra-performance liquid chromatography coupled
 with electrospray ionization quadrupole time-of-flight mass spectrometry.
 We identified four groups of phenolic compounds: flavonoids, ellagitannins
 and gallic acid derivates (‘hydrolysable tannins’ hereafter),
 proanthocyanidins (‘condensed tannins’ hereafter) and hydroxycinnamic acid
 precursors to lignins (‘lignins’ hereafter). We quantified flavonoids as
 rutin equivalents, condensed tannins as catechin equivalents, hydrolysable
 tannins as gallic acid equivalents, and lignins as ferulic acid
 equivalents . The quantification of these was conducted by external
 calibration using the corresponding calibration curve at 0.25, 0.5, 1, 2
 and 5 μg ml<sup>−1</sup> for each of the four standards used
 (rutin, catechin, gallic acid and ferulic acid). We expressed phenolic
 compound concentrations in mg g<sup>−1</sup> tissue on a dry
 weight basis.