DOI: 10.5061/dryad.cc2fqz6fm
title: Leaf spectroscopy reveals drought response variation in Fagus sylvatica saplings from across the species range

description: The common European beech (F. sylvatica), sensitive to prolonged drought,
 is expected to shift its distribution with climate change. To persist in
 novel environments, young trees rely on the capacity to express diverse
 response phenotypes. Several methods exist to study drought effects on
 trees and their diverse adaptive mechanisms, but these are usually
 destructive and challenging for the large sample numbers needed to
 investigate biological variation. We conducted a common garden experiment
 outdoors, but under controlled watering conditions, with 180 potted
 two-year-old saplings from 16 beech provenances across the species’ range,
 representing three distinct genetic clusters. Drought stress was simulated
 by interrupting irrigation and stomatal conductance and soil moisture were
 used to assess drought severity. We measured leaf reflectance of visible
 to short-wave infrared electromagnetic radiation to determine
 drought-induced changes in biochemical and structural traits derived from
 spectral indices and a model of leaf optical properties.  We
 quantified changes in pigmentation, water balance, nitrogen, lignin,
 epicuticular wax, and leaf mass per area in drought-treated saplings,
 revealing differences in likely adaptive responses to
 drought. Fagus sylvatica saplings from the Iberian
 Peninsula showed signatures of greater drought resistance, i.e., the least
 drought-induced change in spectrally derived traits related to leaf
 pigments and leaf water content. We demonstrate that high-resolution leaf
 spectroscopy is an effective and non-destructive tool to assess individual
 drought responses that can characterize functional intraspecific variation
 among young beech trees. Next, this approach should be scaled up to
 canopy-level or airborne spectroscopy to support drought response
 assessments of forests.
description: <strong>Common garden
 experiment </strong> We
 conducted our experiment with 180 2-year-old saplings from 16 provenances
 across the natural distribution range in June 2023. The saplings were
 transported from the Hauenstein nursery to the experiment site at the
 University of Zurich, Zurich, Switzerland (47° 23’ 44” N, 8° 33’ 05” E;
 elevation 509 m.a.s.l.). We implemented a 2 x 5 randomized block
 arrangement, in which each block formed a 3 x 6 grid. Each block contained
 18 saplings, which were placed in the slots of a steel grid, which were
 spaced approximately 1 m apart to minimize interactions between saplings
 of different blocks. We maximized the diversity of provenances within the
 blocks, so each provenance was represented in every block whenever
 possible. For each provenance, half of the saplings were assigned to the
 drought treatment and half to the control. The placement of the saplings
 within the blocks was randomized. In April 2023, we measured the length of
 the primary shoot using a flexible tape measure from the root collar along
 the shoot to the highest point in its natural position. We then used these
 plant height measurements to select individuals to have a similar
 distribution of heights among the blocks.  
 <strong>Drought treatment </strong>
 We selected a duration of 14 days as a
 target to induce realistic drought stress while avoiding tree mortality in
 the experiment. During the simulated drought period, both groups were
 subjected to a controlled exclusion from rain using cone-shaped rain
 covers which enclosed the entire pot, but the control group was watered
 regularly according to demand and at least every other day. Covers were
 made of waterproof and UV-resistant PVC black pond foil (Heissner GmbH,
 Lauterbach, Hessen, Germany) and fixed on the sapling stem with Parafilm
 (Pechiney Plastic Packaging Inc., Chicago, IL, United States) beneath the
 first stem node. Three thin wooden sticks were used to elevate the cover
 to leave a gap between the foil and the pot to enable air circulation. In
 addition, we inverted the pot trays in the treatment group to avoid the
 retention of water in the soil. Note that we prioritized sapling survival
 by ad hoc irrigation of the drought-treated saplings: when severe wilting
 was observed, we watered wilting saplings with 600 mL water. 
 To monitor soil moisture and air
 temperature, we installed 20 TMS-4 probes (TOMST, Prag, Czech Republic,
 Wild et al., 2019) in pots from block A to F. The probes covered as many
 provenances as possible while capturing the full range of plant heights
 within a single block. Soil moisture and soil temperature were sampled in
 an interval of 15 minutes during the entire experiment. 
 <strong>Leaf
 spectroscopy </strong> 
 Leaf spectroscopy measurements were taken during the second half
 of the drought (days 9 to 14, corresponding to June 19th to 24th, 2023).
 We constrained the measurement time to ±3 hours from solar noon.
 Therefore, within one day, we measured two blocks (one control, one
 treatment block) totalling 36 individuals and 72 leaves (2/individual),
 except for one day when 72 individuals corresponding to 144 leaves were
 measured. We used the ASD FieldSpec 4 Standard-Res spectrometer (ASD Inc.,
 Boulder, USA), which measures electromagnetic radiation across a total of
 2151 bands covering the spectral range from 350 nm to 2500 nm. We used a
 plant probe with a leaf clip (model A122317, serial No 455, ASD Inc.,
 Boulder, USA) with a calibrated low-intensity halogen light source. The
 leaf clip allows the non-destructive handling of the leaves and the
 isolation of external illumination. We used the spectral acquisition
 software RS3 (ASD Inc., Boulder, CO, USA). For each sapling, we measured
 two random sun-exposed top-of-canopy leaves. We placed the leaf clip on
 the leaf adaxial surface, filling the measurement window of the plant
 probe and avoiding the midrib. Spectral measurements consisted of four
 successive measurements for each leaf: the white reference (Rw), the white
 reference with target leaf (Tw), the black reference (Rb), and the black
 background reference with target leaf (Tb). We gathered 5 readings per
 measurement, totalling 20 measurements per leaf. We recalibrated
 (optimized) the sensor gain with the RS3 software against the white
 reference background of the leaf clip after measuring 10 saplings to
 correct for differences in temperature sensitivity of the three detectors
 resulting in deviations of the white reference reflectance value from one.
 The leaf clip and plant probe were regularly checked for dirt or other
 contamination and cleaned, or white and black reference stickers were
 replaced as needed.  <strong>Stomatal
 conductance </strong> 
 During the drought, we measured stomatal conductance (gs) of a
 subset of the saplings (n=141) with an SC-1 leaf porometer (METER Group,
 Pullman, WA United States) according to the manufacturer’s protocol. Along
 with the spectral measurements, we constrained the measurement time to ±3
 hours from solar noon due to high variation during the day. Given this
 constraint and the significant time required for each measurement, it was
 not feasible to measure all the beech saplings in a single day. Therefore,
 we measured two to three blocks each day. To standardize the strong and
 immediate effects of changing conditions on stomatal conductance during
 the 6-hour measurement window, we always measured a sapling from the
 control directly after measuring one from the treatment. With this
 ”paired” approach, the solar radiation and air temperature were on average
 similar for treatment and control, which allows comparison between them.
 We calibrated the device according to the manufacturer’s manual on each
 measurement day. When measuring, we took the values as soon as they
 stabilized. Per sapling, we measured two representative sun-exposed leaves
 on the adaxial side.
description: # Leaf spectroscopy reveals drought response variation in *Fagus
 sylvatica* saplings from across the species range
 [https://doi.org/10.5061/dryad.cc2fqz6fm](https://doi.org/10.5061/dryad.cc2fqz6fm) In this experiment, we collected four types of data:  1\) Leaf spectral data for n=180 plants (reflectances). Converted from raw .asd files  to .csv using the R package spectrolab and added metadata.  2\) Plant stomatal conductance for n=141 plants (in [mmol/m2s]). Provided as .xlsx file including metadata. 3\) Plant height for n=180 plants (in [cm]). Provided as .xlsx file including metadata. 3\) Soil moisture data (3936 time points for n=20 plants in uncalibrated Time Domain Transmission (TDT) units). Provided as .xlsx file including metadata. ## Description of the data and file structure Each data file (.xlsx) contains the necessary metadata describing the data collected, units, and how sampling and collection occurred (one single file with a tab for data and another for metadata per dataset). The variable "site" (values BES – SLK) appearing in the data files is described in the file metadata_trees.xlsx. This file also describes the location of the sites and the date of seed collection. The variable "collection" (values "1 - WNW") refers to either the mother tree (integers) or exposition (E: East, N: North, W: West, S: South). ## Code/Software To process the data, we used R (v4.3.1, R core team 2023). Spectral data were processed with the spectrolab package (v0.0.18, Meireles et al 2020).