DOI: 10.5061/dryad.v15dv41w7
title: Spatial and temporal heterogeneity in pollinator communities maintains within-species floral odour variation

description: Flowering plants emit complex bouquets of volatile organic compounds
 (VOCs) to mediate interactions with their pollinators. These bouquets are
 undoubtedly influenced by pollinator-mediated selection, particularly in
 deceptively-pollinated species that rely on chemical mimicry. However,
 many uncertainties remain regarding how spatially and temporally
 heterogeneous pollinators affect the diversity and distribution of floral
 odour variation. Here, we characterized and compared the floral odours of
 ten populations of deceptively-pollinated Arum maculatum (Araceae), and
 inter-annual and decadal variation in pollinator attraction within these
 populations. Additionally, we transplanted individuals from all sampled
 populations to two common garden sites dominated by different pollinator
 species (Psychoda phalaenoides or Psycha grisescens), and compared
 pollinator attraction rates to investigate whether populations maintained
 odour blends adapted to a specific pollinator. We identified high within-
 and among-population variation in a common blend of VOCs found across the
 range of A. maculatum. We also observed shifts in pollinator community
 composition within several populations over 1-2 years, as well as over the
 past decade. Common garden experiments further revealed that transplanted
 inflorescences generally attracted the dominant local pollinator species
 in both transplant sites. However, one population (Forêt du Gâvre, France)
 appears to exclusively attract P. grisescens, even when transplanted to a
 P. phalaenoides-dominated site. Together, our results suggest that
 maintaining diverse floral odour bouquets within populations may be
 advantageous when pollinator communities vary over short timescales. We
 propose that temporally-replicated ecological data are one potential key
 to understanding variation in complex traits such as floral odour, and in
 some cases may reveal resiliency to shifting pollinator communities.
description: Floral odour collection and
 identification <em>Arum
 maculatum</em> inflorescences open for an anthesis cycle of roughly
 24h, with VOC emissions peaking in the late afternoon / early evening of
 the first flowering day. We therefore carried out all floral odour
 sampling between 18:00 at the earliest and 20:30 at the latest. We sampled
 dynamic headspace volatile organic compounds (VOCs) using
 polydimethylsiloxane (PDMS) coated Gerstel Twister® (Mülheim an der Ruhr,
 Germany) stir bar sorptive extraction. Inflorescences were wrapped in
 inert oven bags (Tangan No34 distributed by Migros, Zurich, Switzerland)
 cut open at least 8cm above the tip of the spathe to prevent condensation,
 due to the strong thermogenesis of the appendix. Twisters® were inserted
 in a glass tube through the oven bag at a height even with, but not
 contacting, the tip of the spadix. 6L of air was pumped over Twisters® at
 a standard rate of 200mL per minute for 30 minutes – except for five
 samples from Conteville, France, where sampling was carried out at the
 same rate over only 15 minutes. At every sampling site, at least one empty
 oven bag was placed approximately 5 meters away from any inflorescences,
 and ambient air was passed over Twisters® identically
 to <em>A. maculatum</em> inflorescences; these samples were
 used as controls to filter out ambient air VOCs. All samples were
 transported in glass vials on ice, and stored at -21°C until
 analysis. Gas
 Chromatography We applied 1µL of internal
 standard (5µg mg/mL naphthalene in dichloromethane) directly to each
 Twister® immediately before processing. Using a Multipurpose Sampler
 (Gerstel, Mülheim an der Ruhr, Germany), VOCs were thermally desorbed and
 separated on a HP-5MS column, 30 m x 0.25mm x 0.25µm at 40°C for 30 sec,
 increasing temperature by 5°C per min to 160°C, which was held for 0.01
 min before increasing 3°C per min to reach 200°C, which was held for 4
 min, finally increasing at 10°C per min. until reaching 250°C for 3
 min. Volatile Data
 Processing We aligned peaks by retention time
 within each population. Major ions were recorded for each integrated peak
 using Agilent Chemstation software. Putative compound identifications were
 then derived from NIST 2.3 (library version 17) hits confirmed for the
 same peak in several spectra; all names used in the final analysis should
 be considered hypotheses. Compounds present in blank samples with a mean
 quantity anywhere near those
 within <em>A. maculatum</em> samples were removed prior to
 further analyses. Quantitative values were obtained by dividing compound
 peak areas by the internal standard, then multiplying by the internal
 standard concentration, and finally scaling based on sampling time (for
 the few samples run for less than 30 minutes).
 Pollinator identification
 On the morning after floral odour collection, all trapped
 pollinators were collected from within each inflorescence and preserved in
 70% ethanol. All pollinators were identified to at least the suborder
 level. Psychodidae were further identified to species level using
 taxonomic information and illustrations (Ježek 1990). First, the number of
 antennal segments were counted. 15 segments indicated specimens were
 likely <em>P. phalaenoides</em> or <em>P. crassipennis</em>. 16 segments indicated either <em>P. grisescens</em> or <em>P. trinodulosa</em> - wing venation patterns were then examined for 16-segmented specimens, as P. trinodulosa has a characteristic disconnection in one branched vein. To confirm the final species identity (particularly when intact antennae were not available) and sex of all psychodids, the reproductive anatomy of specimens were also examined: Psychodid abdomens were separated, flattened, cleared in a diluted solution of potash, and mounted on a slide in glycerol beside their decapitated head, and wings laid out flat. 
description: The data presented here include individual-level data on 1)
 floral VOC emissions, in proportional format, 2) pollinator attraction at
 the species level for Psychodidae, and family level for other taxa, and 3)
 associated metadata, namely population of origin, transplant location (if
 applicable), and date of VOC sampling. The VOC data have been pre-filtered
 to remove compounds identified in blank (ambient air) samples, and
 compounds that consistently represented less than 1% of floral odour
 blends. These data may be used to reproduce all analyses and figures in
 our article. Analysis scripts are avalable from the corresponding author
 on reasonable request.