DOI: 10.5061/dryad.4mw6m90h6
title: Vertical stratification of leaf physical traits exerts bottom-up pressures on insect herbivory in a sugar maple temperate forest

description: Do vertical gradients in temperate forest structure insect herbivore
 communities?  We tested the hypothesis that the increase in light
 intensity from understory to forest canopy level drives differences in
 leaf physical traits and budburst phenology that impact insect herbivores
 and thus play a role in structuring both herbivore communities and the
 leaf damages they cause. Twelve sugar maple (Acer saccharum) sites were
 monitored in southern Quebec, examining insect herbivore patterns from
 understory to the shaded and sun canopy over the summers of 2020, 2021,
 and 2022. Additionally, we recorded leaf physical traits, temperature,
 humidity, and sun exposure. Our findings revealed that leaf thickness
 increased along the vertical gradient in 2021, making leaves less
 favorable to herbivores in the canopy level. Accordingly, we recorded a
 consistent decrease in insect herbivory damage rates from the understory
 to the shaded canopy and sun canopy in 2020 and 2021, driven by leaf
 cutters, skeletonizers, stipplers, and leaf miners. These results support
 our hypothesis that variation in plant physical traits due to sun exposure
 contributes to the vertical stratification of insect damage. In 2022, the
 gradient of insect herbivore abundance corroborated the observed damage
 trends from the previous years. Moreover, we calculated an average annual
 herbivory rate of 9.1% of the leaf surface in our study site, suggesting
 limited evidence supporting a significant contribution of background
 herbivory to the decline of sugar maple forests. Overall, our study
 highlights the importance of vertical gradients in structuring insect
 herbivore communities and emphasizes the role of leaf traits in mediating
 these interactions.
description: Study site and plant species We conducted
 our sampling at the Kenauk Nature property, located in southwestern
 Quebec, Canada (45°42'N; 74°53'W). The property spans two
 regions from the Outaouais river valley to the Laurentian Mountains. Based
 on the provided domains map by the Quebec Ministry of Forests it is in a
 maple/bitternut hickory domain of temperate deciduous forest (Quebec
 Ministry of Forests 2023). To consider the variation in insect herbivory
 throughout the growing season, three sampling sessions were conducted at
 the end of June, end of July, and middle of August in 2020, 2021, and
 2022. During each session, we sampled the same 12 sites, each spaced more
 than 15 meters apart and tree species composition was relatively uniform
 in all sites. These sites were selected based on the similarity in tree
 size and safety considerations for climbing. Each site comprised one
 mature sugar maple tree and one sapling. We collected samples from both
 the sunlit and shaded canopy levels of the mature trees, as well as from
 the branches of saplings in the understory. Canopy branches were sampled
 at a height of 10-13 meters above ground on mature trees averaging around
 16 meters in height, and the understory branches were defined as those on
 surrounding sugar maple saplings ranging from 1-3 meters in height. For
 visual clarity and to present all sampling events and their respective
 timings in one place, an infographic has been included as Figure
 1. Assessment of insect herbivore damages
 Leaf herbivory damage is recognized as a good measure of the
 structure of insect herbivore communities and their interactions with host
 plants (Schowalter et al. 1986b; Schowalter 2006). Among the methods of
 assessing leaf herbivory damage, visual assessments are accurate enough
 for comparative analysis (Landsberg 1989; Johnson et al. 2016). In this
 study, herbivory damages were visually quantified as the percentage of
 leaf area damaged by different insect feeding guilds (Wint, G. R. W 1983;
 Houston et al. 1990; Thomas et al. 2010; Johnson et al. 2016). We averaged
 the damage scores for 15 fully expanded leaves per stratum per site in
 both 2020 and 2021. Nine insect herbivory damage types identified on sugar
 maples (Thomas et al. 2010) were assessed, including leaf rollers, leaf
 skeletonisers, leaf cutters, leaf stippling, spindle galls, erineum gall,
 ocellate gall, leaf miner maple leaf-cutter and maple leaf-blotch miner
 (Fig 1). Trained interns conducted assessments using the guidebook by
 Houston et al. (1990), which features clear images of insect herbivores
 induced damages on sugar maple leaves (Houston et al. 1990). During
 assessments, each leaf was visually divided into four parts, and the
 percentage of observed damage per part was estimated. The sum of these
 percentages yielded the total percentage of a particular damage type on
 the leaf. To ensure consistency in assessment methods, subsequent interns
 received supervision from the previous year's intern, and the first
 20-30 leaves were assessed together. To capture both spatial and temporal
 effects on herbivory damage, we randomly collected 15 leaves per stratum
 per site at three sampling sessions (June, July, and August). In total
 1620 leaves were collected (12 trees*3 strata*15 leaves*3 sampling
 dates)(Schowalter et al. 1986a; Zehnder et al. 2009; Turcotte et al.
 2014). For understory, leaves were collected by hand from accessible
 branches on the ground.  Canopy leaves were accessed using single rope
 technique and climbing gear and were cut with a two-meter pole pruner;
 shaded leaves were collected from mid-center canopy branches under
 near-complete canopy cover receiving less light, while sunlit leaves were
 sampled from higher and more marginal canopy branches with less than 50%
 canopy cover to ensure greater sun exposure. Due to the limitations of
 single rope techniques, sampling was often confined to the inner section
 of the tree crown (Basset 1991; Thomas et al. 2010; Maguire et al. 2014).
 Environmental factors such as light intensity, temperature, and humidity
 were recorded at each sampled branch during the three sampling sessions to
 account for microhabitats differences between branches in sun-exposed
 canopies, shaded canopies, and understory. Light intensity was measured
 using a Reed light meter model R1930, while temperature and humidity were
 recorded using a Reed temperature and humidity monitor model
 R6000. Measurement of leaf physical traits and
 phenology of leaf bud burst In 2021, we evaluated the
 effect of microhabitat heterogeneity on leaf quality by collecting 36
 undamaged, fully expanded leaves per stratum per sampling date (total of
 108 leaves per stratum=3 leaves* 12 site* 3 sampling dates). Sampling size
 was determined based on previous studies that measured leaf traits in a
 vertical gradient of deciduous forests (Corff and Marquis 1999; Fortin and
 Mauffette 2002; Murakami et al. 2005; Zvereva et al. 2020). The leaf
 sampling was synchronized with the herbivore damage sampling. Fresh leaves
 were weighed on-site, and their thickness was measured using a digital
 caliper. Subsequently, the leaves were pressed, labeled, transported to
 the lab, and dried in an oven for 72 hours. Dried leaves were then weighed
 again, and all leaves were scanned to measure leaf area using ImageJ
 software. All efforts were made to ensure that dried leaves remain intact
 and free from any folding or breaking to maintain measurement accuracy.
 This allowed us to determine water content and specific leaf area (SLA)
 for all sampled leaves, facilitating the comparison of leaf traits
 variation across the three sugar maple strata. In 2022,
 we conducted a comparative study on the phenology of leaf-flush between
 sugar maple saplings and the canopy of mature sugar maples trees. On May
 10, 2022, we recorded the bud burst ranking by visually inspecting 10 buds
 on a single sugar maple sapling with the naked eye, and 10 buds on the
 canopy of mature trees adjacent to the selected sapling using binoculars.
 The bud burst ranking was assessed on a scale of 0 to 30; with 0
 representing small buds, 10 indicating swollen buds, 20 indicating small
 leaves, and 30 indicating fully expanded leaves (Hannerz 1999). This
 recording process was repeated at 20 sites which were distinct from the 12
 sites used for herbivory damages and leaf trait measurements but were
 located in close proximity to those sampled sites and exhibit similar tree
 species composition. Each sites spaced at least 10 meters apart and
 included one mature sugar maple with a height of 10-15 meters to assess
 canopy level bud burst timing and one sugar maple sapling with a height of
 1-3 meters representing the understory. Insect
 herbivore species identification To establish a link
 between the observed herbivory damage types to the actual insect herbivore
 community on sugar maple trees, we employed the beat sheeting method to
 collect insects from all three strata at the same 12 sugar maple sites
 used for herbivory damage assessments. Although sampling was conducted
 during the summer of 2020, 2021 and 2022, technical issues resulted in the
 collection of fewer than 20 individual insect herbivores in the first two
 years, which were subsequently excluded from the analysis. Therefore, only
 data from 2022 was considered for analysis. Using the beat sheeting
 method, three branches from each stratum were struck with a one-meter
 length stick, while an 85 cm* 85 cm sheet with a detachable jar at the
 center was positioned beneath the branch. All organisms that dislodged
 onto the sheet were carefully collected in the jar. Subsequently, the jars
 containing samples from the canopy strata were lowered to the ground and
 filled with 70% ethanol to prevent predation on the herbivores within the
 container. Upon retrieval, the samples were sorted into morphotypes and
 identified to the lowest possible taxonomic level in the lab. Each sample
 was meticulously labeled, pinned, and deposited at the Lyman Entomological
 Museum in St-Anne-de-Bellevue, QC, Canada.
description: We have submitted our raw data including Insect herbivory damages on
 leaves, recorded environmental factors, and measured leaf traits in 2020
 and 2021(**Leaf damage.trait.2020.2021.csv**), insect herbivore specimen
 information collected in 2022 (**herbivore.guilds.22.csv**), recorded
 budburst phenology raw data for all sampled trees in 2022
 (**sugar.maple.budburst.22.csv**), data needed for leaf trait correlation
 with leaf herbivory damages in RDA analysis (**rda.dam.trait.21.csv**),
 four data sets created out of *Leaf damage.trait.2020.2021.csv* for
 illustrating line graphs for insect herbivory damages variation thorough
 time and between tree strata for 2020 and 2021 (**Av.dam.sampling.20.csv,
 Av.dam.sampling.21.csv, Av.dam.20.strata.csv, Av.dam.21.strata.csv**) and
 R script (**Damage.trait.20.21.R**). ### Descriptions #### **Leaf
 damage.trait.2020.2021.csv** * *Month*: Sampling performed in 1 = June, 2=
 July and 3=August * *Site*: each selected 12 sites includes one mature
 sugar maple tree for collecting data from shaded and sun canopy levels and
 one sugar maple sapling not further than one meter apart from the mature
 tree to collect data from the understory. * *Strata*: In each site, three
 strata were sampled including sun canopy level, shaded canopy level, and
 understory * *Av.leaf.roller*: Average percentage of leaf area damaged by
 insect leaf rollers for 15 leaves per stratum (%) * *Av.leaf.cutters*:
 Average percentage of leaf area damaged by insect leaf cutters for 15
 leaves per stratum (%) * *Av.leaf.skeletonisers*: Average percentage of
 leaf area damaged by insect leaf skeletonisers for 15 leaves per stratum
 (%) * *Av.spindle.gall*: Average percentage of leaf area damaged by insect
 spindle gall makers for 15 leaves per stratum (%) * *Av.erineum.gall*:
 Average percentage of leaf area damaged by insect erineum gall makers for
 15 leaves per stratum (%) * *Av.ocellate.gall*: Average percentage of leaf
 area damaged by insect ocellate gall makers for 15 leaves per stratum (%)
 * *Av.leaf.miner.maple.cutter*: Average percentage of leaf area damaged by
 insect leaf miner maple cutter for 15 leaves per stratum (%) *
 *Av.leaf.blotch.miner*: Average percentage of leaf area damaged by insect
 leaf blotch miner for 15 leaves per stratum (%) * *Av.total*: sum of
 percentage of leaf area damaged by any insect herbivores per15 leaves
 which was then averaged for 15 leaves per stratum (%) * *Av.exposed*: sum
 of percentage of leaf area damaged by insect herbivore exposed feeders
 including; cutters, skeletonisers and stippling, per15 leaves which was
 then averaged for 15 leaves per stratum (%) * *Av.sheltered*: sum of
 percentage of leaf area damaged by shelter builder insect herbivores
 including; all three gall makers, two miners and rollers, per15 leaves
 which was then averaged for 15 leaves per stratum (%) *
 *Av.leaf.thickness*: Average leaf thickness measured for 3 leaves per
 stratum only in 2021(mm) * *Av.water.content*: Average leaf water content
 measured for 3 leaves per stratum only in 2021(%) * *Av.SLA*: Average
 specific lead area ( leaf area/dried leaf mass) measured for 3 leaves per
 stratum only in 2021(mm2g-1) * *Av.leaf.area*: Average leaf area measured
 for 3 leaves per stratum only in 2021(mm2) * *av.hum*: Average humidity
 measured 3 times per stratum (%) * *av.temp*: Average temperature measured
 3 times per stratum ('C) * *av.light*: Average light intensity
 measured 3 times per stratum (L) #### **herbivore.guilds.22.csv** *
 *Month*: sampling repeated three times thorough the season including in
 June, July and August * *Site*: Each 12 sampled site includes one mature
 sugar maple tree for collecting data from shaded and sun canopy levels and
 one sugar maple sapling not further than one meter apart from the mature
 tree to collect data from the understory. * *Strata*: In each site, three
 strata were sampled including sun canopy level, shaded canopy level, and
 understory * *cutter, roller, skeletoniser, stippling *: the number of
 insect leaf cutters, insect leaf rollers, insect leaf skeletoniser, insect
 leaf stippling individuals collected from three strata in 12 sites *
 *total*: the total number of insect herbivore individuals collected from
 three strata in 12 sites #### **sugar.maple.budburst.22.csv** * *Site*:
 each selected 20 sites includes one mature sugar maple tree for collecting
 data from canopy levels and one sugar maple sapling not further than one
 meter apart from the mature tree to collect data from the understory. *
 *Tree.type*: two sugar maple trees, including one mature tree and one
 sapling, per site were observed to recordedbudburst ranking *
 *Rank.budburst*: The bud burst ranking was assessed on a scale of 0 to 30;
 with 0 representing small buds, 10 indicating swollen buds, 20 indicating
 small leaves, and 30 indicating fully expanded leaves * *Av.temp*: Average
 temperature was measured 3 times close to per tree ('C). ####
 **rda.dam.trait.21.csv** * *Tree*: equal to Site number in previous data.
 each selected 12 sites in 2021includes one mature sugar maple tree for
 collecting data from shaded and sun canopy levels and one sugar maple
 sapling not further than one meter apart from the mature tree to collect
 data from the understory. * *leaf.type*: In each site, leaves were
 collected from three strata including sun canopy level, shaded canopy
 level, and understory * *Thickness*: Average leaf thickness measured 3
 times per stratum (mm) * *Water*: Average leaf water content measured for
 3 leaves per stratum only in 2021(%) * *SLA*: Average specific lead area (
 leaf area/dried leaf mass) measured for 3 leaves per stratum only in
 2021(mm2g-1) * rol, cut, sk,st: percentage of leaf area damaged by insect
 leaf rollers, leaf cutters, leaf skeletonisers and leaf stippling
 respectively each averaged for 15 leaves per stratum (%) * sum.g, sum.m:
 percentage of leaf area damaged by leaf gall makers ( spindle gall,
 erineum gall and ocellate gall) averaged for for 15 leaves per stratum and
 sum of percentage of leaf area damaged by leaf miners ( leaf miner maple
 cutter and leaf blotch miner) averaged for for 15 leaves per stratum (%) *
 *total*: sum of percentage of leaf area damaged by any insect herbivores
 per15 leaves which was then averaged for 15 leaves per stratum (%) ####
 **Av.dam.sampling.20.csv, Av.dam.sampling.21.csv, Av.dam.20.strata.csv,
 Av.dam.21.strata.csv** * In all for data columns C to L have the same
 description as lines 4 to 12 in *Leaf damage.trait.2020.2021.csv.* * *Tree
 *in all four data* *: equal to Site number in previous data. each selected
 12 sites includes one mature sugar maple tree for collecting data from
 shaded and sun canopy levels and one sugar maple sapling not further than
 one meter apart from the mature tree to collect data from the understory.
 * *Sampling* in the first two data: Includes three sampling months
 including; June, July and August * *leaf.type* in the last two data: In
 each site, leaves were collected from three strata including sun canopy
 level, shaded canopy level, and understory ### Code/Software R is required
 to run *Damage.trait.20.21.R*; the script was created using version
 2023.12.1+402.