Striking variation of pollinator attracting scent within a highly specialized pollination system
Data files
Jan 16, 2025 version files 204.45 KB
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Chumilis_Data.xlsx
202.22 KB
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README.md
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Abstract
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.
README: Striking variation of pollinator attracting scent within a highly specialized pollination system
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)
Methods
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 Chamaerops humilis. Data was obtained as follows:
MICROSATELLITE DATA : 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).
POLLINATOR OBSERVATION DATA - In Sardinia and Sicily, a mouth aspirator was used to capture pollinators (Derelomus sp. and Meligethinus pallidulus) 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.
SCENT DATA -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.