DOI: 10.5061/dryad.gqnk98sn7
title: Opposing effects of Ceanothus velutinus phytochemistry on herbivore communities at multiple scales

description: Identifying the interactions of functional, biotic, and abiotic factors
 that define plant–insect communities has long been a goal of community
 ecologists. Metabolomics approaches facilitate a broader understanding of
 how phytochemistry mediates the functional interactions among ecological
 factors. Ceanothus velutinus communities are a relatively unstudied system
 for investigating chemically mediated interactions. Ceanothus are
 nitrogen-fixing, fire-adapted plants that establish early post-fire, and
 produce antimicrobial cyclic peptides, linear peptides, and flavonoids.
 This study takes a metabolomic approach to understanding how the diversity
 and variation of C. velutinus phytochemistry influences associated
 herbivore and parasitoid communities at multiple spatiotemporal scales.
 Herbivores and foliar samples were collected over three collection times
 at two sites on the east slope of the Sierra Nevada Mountain range. Foliar
 tissue was subjected to LC-MS metabolomic analysis, and several novel
 statistical analyses were applied to summarize, quantify, and annotate
 variation in the C. velutinus metabolome. We found that phytochemistry
 played an important role in plant–insect community structure across an
 elevational gradient. Flavonoids were found to mediate biotic and abiotic
 influences on herbivores and associated parasitoids, while foliar
 oligopeptides played a significant positive role in herbivore abundance,
 even more than abundance of host plants and leaf abundance. The importance
 of nutritional and defense chemistry in mediating ecological interactions
 in C. velutinus plant–herbivore communities was established, justifying
 larger scale studies of this plant system that incorporate other mediators
 of phytochemistry such as genetic and metageomic contributions.
description: 4.1. Study Site <i>Ceanothus
 </i>(Rhamnaceae) is a species-rich North American genus of woody
 perennial that includes about 55 species, with 51 species found in Western
 North America [67] and with the highest diversity (38 species) and
 endemism in the California Floristic Province [68,69].
 <i>Ceanothus</i> have symbiotic relationships with
 nitrogen-fixing bacteria [70] and play an important role in ecosystem
 nitrogen availability [67]. <i>Ceanothus velutinus
 </i>(Tobacco brush) was chosen as the focal species for this study.
 <i>C. velutinus </i>is typically found at elevations between
 1600 m a.s.l. and 2500 m a.s.l. and at times in very dense stands.
 Eighteen plots were established across two sites on the Eastern slope of
 the Sierra Nevada, referred to as the Dog Valley (39°31′39.12″ N, 120°
 1′53.40″ W) and Mt. Rose locations (39°20′33.55″ N, 119°52′11.33″ W). Both
 sites are characterized by eastern facing slopes with Dog Valley having a
 lower minimum elevation (1632 m a.s.l.) and slope (46 m/m) and Mt. Rose
 having a higher maximum elevation (2550 m a.s.l.) and slope (145 m/m). The
 plots were 10 m in diameter and set up in triplicate along an elevational
 gradient between 1600 m a.s.l. and 2600 m a.s.l. at “low”, “medium”, and
 “high” elevation bands (referred to here as subsite). Elevation for each
 band was established relative to the elevational range of each site, ±50
 m. Each plot was at least 10 m away from any trail or road, spaced at
 least 100 m apart from any other plot, and contained at least one
 <i>C. velutinus </i>individual, chosen as the center point of
 the plot. The plots were established in 2018. Ecological and chemical data
 were collected on the same day from each plot monthly from May to October
 of 2018. Due to the extra time involved with locating sites and setting up
 the plots, we repeated data collections in 2019 to ensure we captured a
 complete dataset for all sites over the season. 4.2.
 Ecological Data For each plot, the total plant species
 diversity and the estimated leaf abundance of each plant within the plot
 was determined. Leaf abundance was estimated by counting the leaves on two
 representative branches from a given plant, then estimating the number of
 such branches on the plant. Each <i>C. velutinus</i>
 individual within the plot was numbered and the lepidoptera were collected
 using a beat sheet. Voucher samples were collected for all <i>C.
 velutinus </i>individuals and are available from the herbarium at
 the University of Nevada, Reno Natural History Museum. All collected
 caterpillars were reared individually in plastic cups in the lab at UNR to
 either adult or parasitoid emergence. Host plant foliage was replaced
 every 2 days and all pupae were checked daily. The adult Lepidoptera and
 parasitoids that emerged from the pupae were allowed to fully harden and
 then placed in a freezer for storage before pinning and identification.
 Each caterpillar collected was photographed and assigned a unique voucher
 code, linking the individual to the plot and plant number in the database.
 Morphotypes were later condensed and validated using reared adult
 specimens and larval photographs as references. 4.3.
 Sample Preparation Immediately after caterpillars were
 collected using a beat sheet, leaf samples were collected from each
 <i>C. velutinus </i>individual for phytochemical profiling.
 Young leaves were cut from the terminal end of the branches, placed in
 paper bags, and stored in a cooler with dry ice until the end of the
 collection day, and subsequently stored at −80 °C. Leaves were then
 transferred to plastic centrifuge tubes with a tungsten steel bead,
 lyophilized, and ground to a fine powder at 30 Hz for two minutes using a
 tissue lyser (Qiagen Tissuelyser II; Hilden, Germany). The ground plant
 material (~20 mg) was transferred to screw-cap scintillation vials with 1
 mL of 70% aq solution of HPLC-grade, denatured ethanol (Fisher,
 Pittsburgh, PA, USA) in 18 MΩ water. The samples were vortexed, sonicated
 for 10 min and incubated overnight on a shaker at room temperature and
 randomized before filtration through a 96 well filter plate with 1.0 mL
 capacity and 1.0 μM glass fiber filters (Acroprep 96, Pall Corporation,
 Port Washington, NY, USA) into a 96 well plate with 1 mL glass inserts,
 sealed with a silicone cap mat, and stored at −20 ℃ for less than one week
 until analysis. 4.4. LC-TOF analysis of foliar plant
 tissue Chromatography was performed on an Agilent 1200
 analytical HPLC equipped with a binary pump, autosampler, column
 compartment and diode array UV detector, coupled to an Agilent 6230
 Time-of-Flight mass spectrometer via an electrospray ionization source
 (ESI-TOF; gas temperature: 350 °C, flow: 8 L/m; nebulizer pressure: 35
 psig; VCap: 3500 V; fragmentor: 175 V; skimmer: 65 V; octopole: 750 V).
 Internal standards were selected to represent the major metabolite classes
 found in <i>Ceanothus</i>: flavonoid glycosides (narignin),
 flavonoid aglycones (naringenin), and peptides (cholecystokinin fragment),
 but were not found in plants. Multiple internal standards were used to
 account for differences in ionization efficiency among phytochemical
 classes. Extracts (1.00 μL) were co-injected with 1.00 μL of internal
 standard mix (naringenin, 100. μM; naringin, 101 μM, Cholecystokinin
 Fragment 30–33 Amide, (52 μM) Sigma-Aldrich) and eluted at 0.400 mL/min
 through a Kinetex EVO C18 column (Phenomenex, 2.1 × 100 mm, 2.6 μ, 100 Å)
 at 40 °C. The linear binary gradient was comprised of buffers A (water
 containing 10 mM ammonium acetate) and B (acetonitrile) changing over 25
 min accordingly: 0 min 5% B, ramp to 40% B at 8 min, ramp to 100% B at 14
 min, 14–20 min 100% B ramping to 0.70 mL/min before re-equilibrating the
 column from 21–25 min at 5% B, 0.4 mL/min. The
 agilent-formatted raw data were converted to mzML format in ProteoWizard
 MSConvert 3.0 [71] before processing using the Bioconductor package XCMS
 [72] in R. Peaks and were extracted using the centwave function before
 retention time correction using obiwarp, peak density correspondence
 analysis, and gap-filling to yield 1308 aligned retention time and
 <i>m</i>/<i>z</i> bins (chromatographic features).
 Features were visually inspected to remove 260 peaks representing areas of
 baseline noise above peak threshold. CAMERA [73], an XCMS wrapper, was
 then used to identify groups of features (pseudospectra) having similar
 retention time and were correlated across samples (r &gt; 0.7), with
 similar (r &gt; 0.5) peak shape and by isotopic pattern. The CAMERA
 command <i>annotate</i> was used to identify and remove
 features resulting from ammonium adducts that might lead to the
 misidentification of nitrogenous compounds. The feature within a
 pseudospectrum group with the highest abundance was then chosen to
 represent the peak area of each of the 300 resulting pseudospectra. To
 minimize between-run batch effects, each peak bin was subjected to ANOVA
 analysis grouped by batch and 133 peaks exhibiting significant batch
 effects (<i>p </i>&lt; 0.10) were excised.
description: These data include the LC-TOF peak abundances of
 <em>m/z</em>-rt peak bins generated from analysis of
 <em>Ceanothus velutinus </em>leaf extracts and the ecological
 data with which they are associated. ReadMe files will be uploaded with
 keys describing column names in tables.