DOI: 10.5061/dryad.xksn02vs0
title: Striking variation of pollinator attracting scent within a highly specialized pollination system

description: Although attractive scents play a crucial role in reproduction in
 insect-pollinated plants, the degree of variation of this signal within
 and among populations remains understudied. Depending on the specifics of
 the reproductive system of the plant under scrutiny, it is possible to
 formulate predictions regarding this variation. In plants with separate
 sexes (dioecious species) and with highly specific pollination, one would
 predict (i) males to emit more scent than females, owing to sexual
 selection, (ii) scent bouquets to have a strictly similar composition
 between sexes, to guarantee efficient pollen transfer and (iii) variation
 of scent bouquet among populations that should mirror neutral genetic
 divergence. These hypotheses were tested in the European fan palm,
 Chamaerops humilis, by collecting scent in eight populations from three
 regions, Spain, Sardinia and Sicily, quantifying densities of pollinators
 - Derelomus sp. and Meligethinus pallidulus - and genotyping the plants on
 a set of neutral markers. Males emitted more scent than females. We
 detected some differences in bouquet composition between sexes, showing an
 imperfect inter-sex mimicry in some populations. We also found a strong
 geographical effect, with individuals sampled in Sicily emitting a
 strikingly different scent bouquet, that contained high proportions of a
 volatile compound that was never detected in the other two regions.
 Geographical variation of scent composition did not mirror neutral genetic
 structure: Sardinian and Spanish populations emitted similar scent
 bouquets but displayed very high levels of genetic differentiation. On the
 reverse, Sicilian populations showed both strong scent differences and
 appeared clearly genetically differentiated from populations found
 elsewhere, without any depletion in neutral genetic diversity. Synthesis:
 Our study confirmed higher scent emission rates in males compared to
 females, consistent with expectations of sexual selection. However, we
 also discovered significant variation in the composition of the floral
 bouquet, which was unexpected given the highly specific pollination
 context. Together with observations of spatial genetic structure, these
 findings suggest a shift in plant-pollinator interactions within the
 species across different regions. 
description: Three types of data, described below, were collected for our
 study: microsatellite genotypes, pollinator observations, and odors
 emitted by the leaves of the dwarf palm Chamaerops humilis. Data was
 collected from two Spanish (SPA1 and SPA2), three Sicilian (SIC1, 2
 & 3), and three Sardinian natural populations (SAR1, 2 &
 3) of <em>Chamaerops humilis. </em>Data was obtained as
 follows: <strong>MICROSATELLITE
 DATA</strong> : 377 plants from the eight populations were genotyped
 using thirteen neutral microsatellite markers (see 'Genotype'
 tab, missing allles are coded as "000"). Six of these markers
 were developed by Guarino et al. (Molecular Ecology Resources Primer
 Development, 2013, marker names : GRD72UV01A0I0S, GRD72UV01BCJA2,
 GRD72UV01BR6MX, GRD72UV01AW9C6, cons470, GRD72UV01BS052), and seven were
 developed for the needs of this study and have not yet been published
 (Ch09, Ch14, Ch15, Ch18, Ch21, Ch30, Ch52). The primers for the newly
 developped markers are available in the 'Primers' tab. Three
 single-step multiplex PCR assays were used to genotype the 377 individuals
 (Multiplex 1 : Ch09, Ch15, Ch18, Ch30, Ch52; Multiplex 2 : GRD72UV01A0I0S,
 Ch14, GRD72UV01BCJA2, Ch21; Multiplex 3 : GRD72UV01BR6MX, GRD72UV01AW9C6,
 cons470, GRD72UV01BS052). PCR reactions were performed in a 10 µL volume
 containing 1x multiplex PCR master mix (Qiagen Hilden, Germany), 5-20 ng
 of genomic DNA, and 0.2 µM of labeled forward and unlabeled reverse
 primers. The PCR cycling program was as follows: (i) an initial
 denaturation phase of 95°C for 15 min (ii) an amplification phase of 30
 cycles of 94°C for 30 s, annealing temperature of 55°C for 1 min 30 s and
 72°C for 1 min and (iii) a final extension phase at 60°C for 30 min.
 Electrophoresis was conducted on a capillary sequencer (Applied
 Biosystems® 3130 Genetic Analyzer) after mixing 1.5 µL of the PCR reaction
 with 0.25 µL of the GeneScan 500 LIZ size standard (Applied Biosystems)
 and 9.75 µL of deionized formamide. Alleles were then called using
 GeneMapper® (Thermo Fisher Scientific, Waltham, USA).
 <strong>POLLINATOR OBSERVATION DATA</strong> - In
 Sardinia and Sicily, a mouth aspirator was used to capture pollinators
 (<em>Derelomus </em>sp. and <em>Meligethinus
 pallidulus</em>) found in the prophyll (large bract surrounding the
 inflorescence) of one to four receptive inflorescences for each of the
 plants for which scent was characterized. The data set includes, for each
 plant, the number of pollinators of each taxa, the plant sex (male or
 female) and the number of inflorescences that were
 investigated.  <strong>SCENT DATA
 </strong>-The 'Scent' tab contains the relative
 proportions of the 92 volatile organic compounds detected in the studied
 natural populations (each column is a molecule), as well as information on
 the population of origin, sex of the plant, work session (plants that
 share the same letter were extracted at the same date at the same time)
 and total scent production (ng.h-1). The scent extraction process involved
 collecting volatiles from 212 individuals of Chamaerops humilis using the
 headspace technique. A full leaf from each plant was sealed inside a
 polyethylene terephthalate bag (Nalophane®, Kalle Nalo GmbH) for one hour,
 allowing scent accumulation between 10 am and 4 pm. Control samples,
 consisting of empty bags, were used to account for ambient VOCs. After
 accumulation, air was pumped out at a rate of 200 mL min⁻¹ through
 adsorbent traps containing a mix of Tenax® and Carbotrap®, with internal
 standards (n-Nonane and n-Dodecane). Traps were stored at -20 °C before
 analysis. The volatile extracts were analyzed at the “Platform for
 Chemical Analyses in Ecology” (PACE) in Montpellier, France, using gas
 chromatography-mass spectrometry (GC-MS) for compound identification and
 on FID for quantity estimation. Molecular Ecology
 Resources Primer Development C, Arranz SE, Avarre J-C, Balasundaram C et
 al (2013). Permanent genetic resources added to Molecular Ecology
 Resources database 1 December 2012–31 January 2013. Molecular Ecology
 Resources 13(3): 546-549.
description: # Striking variation of pollinator attracting scent within a highly
 specialized pollination system
 [https://doi.org/10.5061/dryad.xksn02vs0](https://doi.org/10.5061/dryad.xksn02vs0) ## Description of the data and file structure Three types of data, described below, were collected for our study: microsatellite genotypes, pollinator observations, and odors emitted by the leaves of the dwarf palm *Chamaerops humilis*. Data was collected from two Spanish populations (SPA1 and SPA2), three Sicilian populations (SIC1, 2 & 3), and three Sardinian populations (SAR1, 2 & 3). ### Files and variables #### File: Chumilis\_Data.xlsx **"Primers" tab : Microsatellite primers designed for the study.** Variables : * Marker : Name of microsatellite marker * Dye : Fluorescent dye used for electrophoresis * Repeat motif : Type of repeat motif * Forward sequence * Reverse sequence * Size range (bp) : Allele size in base pairs **"Genotypes" tab : Genotypes for the 377 *Chamaerops humilis* individuals included in the study (thirteen neutral microsatellite markers, missing alleles are coded as "000")** Variables : * Population : Population of origin (8 natural populations) * Following columns : Individual genotypes (one column per allele) for each of the 13 microsatellite markers **"Pollinators" tab : Pollinator counts** Variables * Population : Population of origin (6 natural populations) * Plant sex : Plant sex (male : M, female : F) * Number of Meligethinus sp. : total number of *Meligethinus* sp. observed on the plant * Number of Derelomus sp. : total number of *Derelomus* sp. observed on the plant * Number of inflorescences : number of inflorescences that were screened on the plant (one to four) **"Scent" tab :** **Leaf scent extracted using the head-space technique and analyzed using GC-MS** Variables * Population : (8 natural populations) * Plant sex : Plant sex (male : M, female : F) * Work session : plants that share the same letter were extracted at the same date at the same time * Total scent production : total scent production (ng.h-1) * Following columns : relative proportions of the 92 volatile organic compounds detected in the studied natural populations (each column is a molecule)