DOI: 10.5061/dryad.7wm37pw18
title: Leaf-shelters facilitate the colonization of arthropods and enhance microbial diversity on plants

description: Shelter-building insects are important ecosystem engineers, playing
 critical roles in structuring arthropod communities. Nonetheless, the
 influence of leaf shelters and arthropods on plant-associated microbiota
 remains largely unexplored. Arthropods that visit or inhabit plants can
 contribute to the leaf microbial community, resulting in significant
 changes in plant-microbe interactions. By artificially constructing leaf
 shelters, we provide evidence that shelter-building insects influence not
 only the arthropod community structure but also impact the phyllosphere
 microbiota. Leaf shelters exhibited higher abundance and richness of
 arthropods, changing the associated arthropod community composition. These
 shelters also altered the composition and community structure of
 phyllosphere microbiota, promoting greater richness and diversity of
 bacteria at the phyllosphere. In leaf shelters, microbial diversity
 positively correlated with the richness and diversity of herbivores. These
 findings demonstrate the critical role of leaf shelters in structuring
 both arthropod and microbial communities through altered microhabitats and
 species interactions.
description: <strong>Study area and system</strong>
 We conducted the experiment in the southeastern plains of the
 United States at the J. Nicholene Bishop Biological Field Station
 (32°51’11” N, 87°40’22” W, Akron, AL). The experiment was conducted in
 June 2018 (15 pairs of trees) and in August 2021 (30 pairs of trees), and
 plants were 5-15 meters apart. The average minimum and maximum
 temperatures recorded during the experiment were 19-32°C in 2018 and
 22-32°C in 2021. Throughout the experimental period of the trial conducted
 in June, precipitation averaged 127 mm, whereas in August 2021, it
 averaged 25.4 mm. The study area consisted of an oak-hickory-pine forest.
 We selected the white oak, Quercus alba, as a model system for
 constructing artificial leaf shelters. Q. alba plays an important role in
 providing food and shelter for numerous arthropod species (Lill &
 Marquis 2003; Baer & Marquis 2014). This system is ideal for
 studying the role of ecosystem engineers, considering the rich diversity
 of shelter-building insects on oaks (Quercus spp.), which are dominant
 canopy species in eastern North American forests (Lill & Marquis
 2003; Baer & Marquis 2014). Shelter-building caterpillars occur in
 at least 17 Lepidoptera families including Hesperiidae, Nymphalidae,
 Gelechiidae, Oecophoridae, Lasiocampidae, Pyralidae, Gracillariidae,
 Tortricidae, Geometridae, and Erebidae (Fitzgerald et al. 1991; Fukui
 2001; Lill et al. 2007). Even when present at low densities, the shelters
 constructed by these insects can have disproportionately large effects on
 the diversity and structure of arthropod communities at both local
 (tree-level) and regional (habitat-level) scales due to their high
 densities and broad distributions (Jones et al. 1994; Lill et al. 2007;
 Vieira & Romero 2013; Wetzel et al. 2016). The decline of this
 specialized group could have unprecedented consequences for their host
 plants and the numerous macro- and microorganisms associated with
 them. <strong>Experimental design and data
 collection</strong> We created artificial leaf
 shelters on white oak to investigate whether these structures facilitate
 arthropod colonization and promote phyllosphere microbial diversity. We
 constructed cylindrical leaf rolls with a diameter of 0.6 cm - a common
 size found in natural leaf shelters - using a sterilized pencil. We rolled
 damage-free oak leaves from adaxial to abaxial and secured them with
 sterilized stainless steel hair pins (7cm long and 2mm thick, U shape)
 (Vieira & Romero 2013). We standardized our sampling effort by
 conducting the experiment only on leaves between 1 and 2 meters above the
 ground. Control leaves received a hair pin and were left unrolled. We
 selected 45 oak tree pairs (90 trees in total), with each pair spaced at
 least two meters apart. Trees were randomly assigned to either control or
 leaf shelter treatment. Different pairs (blocks) were separated by at
 least 6 meters. For each tree, we randomly selected and marked five
 undamaged leaves and inspected for the presence of arthropods. To prevent
 cross contamination, we handled leaves and constructed the leaf shelters
 using disposable nitrile gloves. A new pair of gloves was used for each
 individual leaf. After a ten-day period, we collected all experimental
 leaves by encasing each leaf in a sterile plastic bag and cutting the leaf
 at the petiole. Before collecting the control leaves, we carefully
 inspected them, and if necessary, we collected all arthropods present
 before removing the leaves. For leaf shelters, the arthropods were
 consistently found inside the shelters, allowing us to collect the leaf
 with the arthropods inside. The leaves were kept on ice until they could
 be stored in a -20°C freezer. At the laboratory, we recorded the number of
 arthropods, identified them to the lowest taxonomic level possible, and
 categorized them in trophic guilds.
 <strong>Phyllosphere microbiome
 analysis</strong> To investigate whether leaf
 shelters promote microbial diversity in the phyllosphere, we swabbed all
 leaves with sterile cotton swabs to sample the bacterial community
 (Medical Wire MW113). We swabbed the surface of each leaf 30 times. In
 2018 (trial 1), we used one cotton swab for all five leaves per plant. In
 2021 (trial 2), we used a different cotton swab for each of the five
 leaves per plant. After swabbing the leaves, each swab was placed inside a
 2 mL screw cap tube and dry stored at -80 °C. We extracted DNA from the
 swabs using the DNeasy kit (Qiagen) with slight modifications to the
 manufacture’s protocol to increase DNA yield (Kueneman et al. 2014). We
 then PCR-amplified the V4 region of the bacterial 16S rRNA gene using
 dual-indexed 515F and 806R barcoded-primers (Kozich et al. 2013). Pooled
 PCR amplicons of all the samples were purified using a QlAquick Gel
 Extraction Kit (Qiagen) and sent for sequencing on an Illumina MiSeq
 sequencer at TUFTs Genomic core facility in Boston, Massachusetts, United
 States. We received bacterial sequences demultiplexed by sample.
 Subsequently, we imported forward reads into Quantitative Insights into
 Microbial Ecology (QIIME2) to extract alpha and beta diversity metrics for
 bacterial microbiomes. Prior to analyzing sequence data, we trimmed
 sequences to 150 bp, filtered by quality scores, and used the Deblur
 pipeline to cluster sequences into operational taxonomic units (OTUs)
 based on 97% similarity. We assigned taxonomy to OTUs using the Greengenes
 13.8 reference sequence database. We then removed chloroplast and
 mitochondrial sequences and removed reads from any OTU contributing less
 than 0.005% of total reads (Bokulich et al. 2013). We rarefied the OTU
 table to 1000 reads for 2018, and 1580 reads for 2021 based on rarefaction
 curves. The bacterial community extracted from each of the five swabs per
 tree in 2021 were summed to provide a single data point, except for the
 phylogenetic diversity, which was averaged across the swabs. The raw DNA
 sequences can be found on NCBI under the ID project:
 PRJNA1116723. <strong>Statistical
 analyses</strong> We used multivariate analysis
 of variance (MANOVA, Roy’s greatest root) to assess the effects of leaf
 shelters and year on arthropod community diversity metrics (abundance,
 richness, and Shannon diversity) and the main feeding guilds recorded
 (herbivores and predators), followed by univariate analyses. To test
 whether leaf shelters influence arthropod and microbial communities, we
 conducted a generalized linear mixed models (GLMM) with a Gaussian
 distribution for continuous and Poisson for count data. We modelled
 treatment as a fixed effect, year as a random effect, and the following
 variables as response variables: abundance, richness, and Shannon
 diversity of arthropods and richness, diversity, evenness (Pielou), and
 phylogenetic diversity of bacteria. To test whether leaf shelters alter
 arthropod and bacterial community composition, we conducted permutational
 multivariate analysis of variance (PERMANOVA) using the “adonis2” function
 and Bray-Curtis dissimilarity coefficient; we used Monte Carlo permutation
 (999) to test the significance of these results (Oksanen 2008). The
 PERMANOVA was performed using quantitative (relative abundance) data. We
 visualized the community composition similarity with nonmetric
 multidimensional scaling (NMDS). We used the “envfit” function to identify
 the most important species contributing to the composition of bacterial
 and arthropod community structures. Additionally, to investigate the
 relationship between bacterial diversity and arthropod abundance,
 richness, and diversity, we used Pearson correlation analysis using the
 base R function “cor.test”. This analysis was performed separately for
 each year due to variations in the arthropod community composition between
 years. We then investigated the contribution of specific arthropod guilds
 (herbivores and predators) to bacterial diversity. In 2021, the abundance
 of herbivores, omnivores and detritivores was low. Therefore, we only
 analyzed the relationship between predators and bacterial diversity for
 that year. We also conducted a Pearson correlation analysis to examine the
 relationship between arthropod body size and bacterial richness. To
 identify specific association between bacterial taxa and either of the two
 treatments we conducted a Linear Discriminant Analyzes Effect Size (LEfSe)
 to estimate the effect size of each treatment and characterize the
 differences between the two microbial communities. All analyses, except
 LefSe, were conducted in R version 4.4.1 (2024-06-14), using the “vegan”,
 “glmmTMB”, and “metaMDS” packages. LefSe was performed on the Huttenhower
 Lab Galaxy Server 2.0 website for LefSe.
description: # Leaf-shelters facilitate the colonization of arthropods and enhance
 microbial diversity on plants Journal: Ecology Letters Authors: M.S.
 Danilo F. B. dos Santos M.S. Jacob E. Herschberger M.S. Bijay Subedi Dr.
 Victoria M. PociusWesley Dr. J. Neely Dr. Sasha E. Greenspan Dr. C.
 Guilherme Becker Dr. Gustavo Q. Romero Dr. Mônica F. Kersch-Becker Data
 collection: Trial 1 - June 2018 Trial 2 - August 2021 Contact information:
 M.S. Danilo F. B. dos Santos: [dxf5343@psu.edu](mailto:dxf5343@psu.edu)
 Dr. Dr. Mônica F. Kersch-Becker: [mfkb@psu.edu](mailto:mfkb@psu.edu)
 Keywords: ecosystem engineering, plant-insect interactions, plant
 microbiome, leaf shelters, phyllosphere Funding: * Department of
 Entomology at the Pennsylvania State University and the USDA National
 Institute of Food and Agriculture and Hatch Appropriations under Project
 #PEN04923 and Accession #7006440. * FAPESP (Grants
 2019/08474-8,2022/10765-3 and 2023/01589-0), by a CNPq-Brazil productivity
 grant, and funding from the Royal Society, Newton Advanced Fellowship
 (Grant No. NAF/R2/180791). * Distinguished Huck Fellowship from the Huck
 Institute of Life Sciences at Pennsylvania State University. Dataset
 description This dataset contains records of plant-insect-bacteria
 interactions through artificial leaf shelters in oak trees in North
 America. The study aimed to investigate the impacts of leaf shelters on
 the arthropod community and on the bacterial community inside the
 shelters. The study also aimed to investigate correlations between
 arthropod visits and the bacterial community in shelters. File information
 Dataset files: 1. “Arthropods_Microbiome_GLMM_PERMANOVA.csv” - This is the
 main dataset containing records of arthropods visits and the bacterial
 community present in the treatments. For each treatment, control or leaf
 shelter, we recorded the number of arthropods and identified the
 arthropods in laboratory. Additionally, all leaves were swabbed for
 bacterial analyses. From the number of arthropods visits and the number of
 observed features of bacteria, community metrics such as richness, Shannon
 diversity, evenness, and Phylogenetic diversity were calculated. These
 records and metrics were use to compare the effects of shelters in
 arthropods and bacterial taxa. This dataset also provides the data used
 for the correlation analyses. 2. “Arthropod_T1_Permanova.csv” and
 “Arthropod_T2_Permanova.csv” - These files contain the records of
 arthropod frequency per samples, which provide the matrix to perform the
 PERMANOVA test for both trials. This file provides the comparison between
 the community composition for arthropods between control leaves and leaf
 shelters. 3. “Microbiome_T1.csv” and Microbiome_T2.csv” - These files
 contain the records of observed features of the Operational Taxonomic
 Units (OTUs) per samples, which provide the matrix to performa the
 PERMANOVA test for both trials. This file provides the comparison between
 the community composition for bacteria taxa between control leaves and
 leaf shelters. 4. “insect.body.lenght.csv” - This dataset contains the
 measurements taken from the insects recorded in Trial 1 and the bacterial
 community metrics also from trial 1 to make the correlation between body
 size and bacterial richness. 5. “metadata.T1.csv” and “metadata.T2.csv” -
 These are the dataset that contains the samples identity and the OTUs that
 were recorded in each leaf sample. These metadata will help to create the
 LEfSE plots. 6. “otu.count.T1” and “otu.count.T2” - These dataset provide
 a matrix of the number of observed OTUs for each single sample. This
 matrix is used to calculate the relative abundance that is depicted in the
 LEfSE plots. 7. “taxonomy.lefse.T1” and “taxonomy.lefse.T2 - These dataset
 contains the taxonomic information for the OTUs that were significant
 according to the LEfSE analyses. File Formats: • CSV (Comma-Separated
 Values) for data files. • .R for the scripts used in R - version 4.4.1
 (2024-06-14) Variables descriptions 1. File:
 “Arthropods_Microbiome_GLMM_PERMANOVA.csv” “plant_id” - the unique plant
 identity “block” - the subset of experimental unit “treatment” - the
 groups of leaves assigned as control or leaf shelter “trial” - the data
 point collection, in this case we had two datapoint collections
 “Omnivore_count” and all other variables that ends in “_count” represents
 the guild name and the frequency of arthropods for that specific guild
 “Omnivore_rich” and all other variables that ends in “_rich” represents
 the guild name and the richness of arthropods for that specific guild
 “Omnivore_shann” and all other variables that ends in “_shann” represents
 the guild name and the Shannon diversity index of arthropods for that
 specific guild “micro_rich” - represents the richness for the bacterial
 taxa “micro_phylo_div” - represents the Phylogenetic diversity index
 (Faith) for the bacterial taxa “micro_pielou” - represents the evenness
 for the bacterial taxa “micro_shann” - represents the Shannon diversity
 index for the bacterial taxa 2. File: “Arthropod_T1_Permanova.csv” and
 “Arthropod_T2_Permanova.csv” “Plant-id” - the unique plant identity
 “treatment” - the groups of leaves assigned as control or leaf shelter
 “trial” - the data point collection, in this case we had two datapoint
 collections All other columns are arthropods identification to the lower
 taxonomic level possible 3. File: “Microbiome_T1.csv” “Sample_Type” -
 represents the treatments “lf” for leaf shelter and “c” for control “ID” -
 represents the plant number All other columns are bacterial taxons 3.1
 File: Microbiome_T2.csv” “plant_number” - the number assigned for each
 plant “treatment” - the groups of leaves assigned as control or leaf
 shelter All other columns are bacterial taxons 1. File:
 “insect.body.lenght.csv” “plant_id” - the number assigned for each plant
 “treatment” - the groups of leaves assigned as control or leaf shelter
 “mean_body_length_mm” - is the insect body length measured in millimiters
 “otu_richness” - represents the richness for the bacterial taxa
 “otu_diversity” - represents the Shannon diversity index for the bacterial
 taxa 2. File: “metadata.T1.csv” OBS: only the two first columns are
 important for this file. “plant_id” - plant identity “treatment” - the
 groups of leaves assigned as control or leaf shelter 5.1 File:
 “metadata.T2.csv” Same as in item (5) 1. File: “otu.count.T1” “otu” -
 represents the OTUs coded as OTU1….OTUx for all OTUs found in trial 1 All
 the remaining columns represents the leaf treatment “lf1a…lf10a” for
 control and “lf1b…lf15b” for leaf shelter 6.1 File: “otu.count.T2” Same as
 in item (6) 1. File: “taxonomy.lefse.T1” “otu” - represents the OTUs
 codename “taxon” - represents the name for the bacterial species 7.1 File:
 “taxonomy.lefse.T2 Same as in item (7) In all datasets “NA” represents
 data not available. \#LEfSE analyses on Galaxy website
 ([http://galaxy.biobakery.org/](http://galaxy.biobakery.org/))