DOI: 10.7280/d1rt3j
title: Haplopappus floral volatiles, flower visitors' preferences, and floral-visitors interaction outcomes along an Andean elevational gradient

description: The diversity of specialized molecules produced by plants radiating along
 ecological gradients is thought to arise from plants’ adaptations to local
 conditions. Therefore, closely related species growing in similar habitats
 should phylogenetically converge, or diverge, in response to similar
 climates, or similar interacting animal communities. We here asked whether
 closely related species in the genus Haplopappus (Asteraceae) growing
 within the same elevation bands in the Andes, converged to produce similar
 floral odours. To do so, we combine untargeted analysis of floral volatile
 organic compounds (VOCs) with insect olfactory bioassay in congeneric
 Haplopappus  (Asteraceae) species growing within the same
 elevation bands along the Andean elevational gradient. We then asked
 whether the outcome of biotic interactions (i.e., pollination versus seed
 predation) would also converge across species within the same elevation.
 We found that flower odours grouped according to their elevational band,
 and that the main floral visitor preferred floral heads from low-elevation
 band species. Furthermore, the cost-benefit ratio of predated vs.
 fertilized seeds was consistent within elevation bands, but increased with
 elevation, from 6:1 at low to 8:1 at high elevations. In the light of our
 findings, we propose that climate and insect community changes along
 elevation moulded a common floral odour blend, best adapted for the local
 conditions. Moreover, we suggest that at low elevation where floral
 resources are abundant, the per capita cost of attracting seed predators
 is diluted, while at high elevation, sparse plants incur a higher
 herbivory cost per capita. Together, our results suggest that
 phytochemical convergence may be an important factor driving plant-insect
 interactions and their ecological outcomes along ecological gradients.
description: <strong><em>Floral
 volatile organic compounds sampling and
 analysis:</em></strong> To analyse differences in
 flower VOC production across elevational bands, we sampled the headspace
 of single floral heads (or capitula) of natural populations of Haplopappus
 species occurring at three distinct elevational zones (2-3 species per
 elevational band, n=5-10), using polydimethylsiloxane (PDMS)-coated
 Twisters (Gerstel, 10mm length, 0.5mm film thickness). Sampling occurred
 during January 2018, between 11 AM and 2 PM, during sunny days. For each
 Haplopappus species, we randomly selected one floral head per plant with
 florets in the floral disk in full bloom and bagged it using 1L oven bags
 (Tangan No34, Turkey). Next, the headspace samples were pumped on the
 Twisters with a handpump set to 200 mL per minute. The pump was connected
 to a glass tube within which we inserted the adsorptive Twister, which was
 inserted into the bag and allowed to collect for two hrs. Additionally, we
 collected control samples (VOCs collected from the vegetative part of the
 plant but without the flowers inside the bag) using the same methodology
 as the floral samples. The hermetically sealed Twisters tubes were stored
 in a cooler in the field and then stored in a freezer at -20 °C within the
 same day, where they remained frozen until gas chromatography (GC)
 analysis. After including 1μL internal standard (5μg mL-1 naphthalene in
 dichloromethane) directly to each Volatile compounds were thermally
 desorbed using a Multipurpose Sampler MPS (Gerstel, Mülheim an der Ruhr,
 Germany), and injected onto a HP-5MS column, 30 m x 0.25mm x 0.25um at 40°
 C for 30 s. For VOCs separation, the GC oven temperature was increased at
 5 °C per minute until 160 °C, which was held for 0.01 min before
 increasing the temperature again at 3 °C per minute until 200 °C, which
 was held for four minutes before a final temperature ramp at 100 °C per
 minute until reaching 250 °C for three minutes. VOCs were then detected by
 within an Agilent 5975C (Agilent, Santa Clara, CA, USA) mass spectrometer
 (MS). The mass detector in EI mode at 70 eV was used to scan over the mass
 range from m/z 33 to 350. To identify the molecules, we compared each peak
 found in the chromatograms with the NIST05 database. Compounds present in
 control samples were removed from our final analysis. Tentative compound
 identification was done using AMDIS and NIST libraries, as well as with
 comparisons with in- house libraries of mono- and sesquiterpenes
 mixtures. <strong><em>Insect preference
 bioassay:</em></strong> To investigate whether floral heads from
 different elevation bands were differentially attractive to floral
 visitors, we measure individual preferences from the most common insect
 visitors, <em>Dioxyna chilensis</em> and from
 <em>Trupanea </em>sp. seed predators, towards
 <em>Haplopappus </em>floral heads growing at the two extremes
 of the elevation gradient (i.e., low- and high-elevation species). Both
 fly species have been described in (Soto Andrades, n.d.). We constructed seven arenas by taping a
 BugDorm mesh lid to the bottom of an inverted a plastic pot (Figure S2).
 We inserted a pair of inflorescences in each arena with <em>H.
 foliosus</em> from low elevation and <em>H.
 scrobiculatus</em> from high elevation. <em>D.
 chilensis</em> and <em>Trupanea </em>sp. flies emerging
 from inflorescences collected from all <em>Haplopappus
 </em>species along the elevation gradient were used for the choice
 bioassay. Each bioassay consisted of placing a pair of flies, a male and a
 female of the same species, in an arena for ten minutes and counting the
 number of landings on each inflorescence (n = 30 pairs for <em>D.
 chilensis</em>, and 20 pairs for <em>Trupanea </em>sp.).
 Flies were introduced in pairs to incentivize them to make a choice as
 both fly species mate in the flowers. Females were identified based on the
 presence of an ovipositor. In a few instances, one or both flies landed on
 a flower once for the entire duration of the bioassay. We converted
 landing counts to proportions so that a fly that could not decide between
 flowers would not outweigh a fly who made a clear choice from the start.
 Proportions of landing was estimated by dividing the number of landings on
 each floral head by the total number of landings. 
 <strong><em>Predation vs. fertilized seed counts across
 species:</em></strong> To assess the potential positive or negative
 effects of floral visitors, we randomly sampled ten capitula that were at
 the mature stage across approximately 20 plants for each
 <em>Haplopappus </em>species (n = 18-34 plants per species,
 with a total of ~1500 capitula). Capitula were placed individually in
 plastic tubes with pierced lids, so to let them fully mature under
 controlled conditions in the laboratory. After a minimum of two weeks,
 each capitulum, was analysed under a stereo microscope for quantifying
 fertile (fully swollen) seed production, as well as the number of seeds
 that were visibly damaged by an herbivory event. In most cases, a small
 exit hole can be observed in the seed, meaning that herbivory by an insect
 larva had happened inside the seed. We opted to not quantify unfertile
 seeds since we could not assess the fertility of half-swollen
 seeds.
description: Microsoft excel or any other data processing software