DOI: 10.5061/dryad.kprr4xh29
title: Data from: Floral trait differentiation in Anacamptis coriophora: phenotypic selection on scents, but not on colour

description: Current divergent selection may promote floral trait differentiation among
 conspecific populations in flowering plants. However, whether this applies
 to complex traits such as colour or scents has been little studied, even
 though these traits often vary within species. In this study, we compared
 floral colour and odour as well as selective pressures imposed upon these
 traits among seven populations belonging to three subspecies of the
 widespread, generalist orchid Anacamptis coriophora. Colour was
 characterised using calibrated photographs and scents were sampled using
 dynamic headspace extraction and analysed using gas chromatography-mass
 spectrometry. We then quantified phenotypic selection exerted on these
 traits by regressing fruit set values on floral trait values. We showed
 that the three studied subspecies were characterised by different floral
 colour and odour, with one of the two predominant floral volatiles emitted
 by each subspecies being taxon-specific. Plant size was positively
 correlated with fruit set in most populations, while we found no apparent
 link between floral colour and female reproductive success. We detected
 positive selection on several taxon-specific compounds in A. coriophora
 subsp. fragrans, whereas no selection was found on floral volatiles of A.
 coriophora subsp. coriophora and A. coriophora subsp. martrinii. This
 study is one of the first to document variation in phenotypic selection
 exerted on floral scents among conspecific populations. Our results
 suggest that selection could contribute to ongoing chemical divergence
 among A. coriophora subspecies.
description: Three populations of <i>A. c. coriophora</i>, three
 populations of <i>A. c. fragrans</i> and one population of
 <i>A. c. martrinii</i> – each comprising between 50 and 100
 individuals – were sampled in southern France during spring/summer
 2016. In each of the seven populations, 50 to 65
 individuals were randomly selected and marked. For each individual, plant
 height and inflorescence length (distance between uppermost and lowermost
 flowers) were measured to the nearest 0.5 mm with a tape measure and
 flowers were counted. In six populations out of seven
 (three of <i>A. c. coriophora</i>, two of <i>A. c.
 fragrans</i> and one of <i>A. c. martrinii</i>), floral
 colour was characterised using calibrated photographs. For each
 individual, one of the lowermost flowers was photographed using a
 standardised procedure (front view and planar position, indirect natural
 sunlight) with a Nikon D70 digital camera equipped with a Novoflex 35 mm
 lens. A ruler and two reference greys (23 % and 35 % of average
 reflectance) were included in each photograph for further scaling and
 colour calibration, respectively. Each photograph was calibrated using the
 Image Calibration and Analysis Toolbox plugin (Troscianko and Stevens,
 2015) in the software ImageJ (Schneider <i>et al.</i>, 2012).
 After this calibration step, on each photograph, the labellum was manually
 selected and its colour characterised using (i) RGB values, to provide a
 measure of floral colour as we humans perceive it, and (ii) the excitation
 values of the SW (short wavelength) and MW (medium wavelength)
 photoreceptors of the honeybee (<em>Apis mellifera</em>). For
 each labellum, we measured one mean and one standard deviation values for
 each of the R, G, B, SW and MW channels. In each of the
 seven populations, floral odour was sampled <i>in situ</i>
 using headspace dynamic extraction. For each individual, the inflorescence
 was enclosed in a ~10*5 cm Nalophan® bag for 30 min. ChromatoProbe® quartz
 microvials filled with 3 mg of a 1:1 mix of Tenax-TA and Carbotrap® were
 used as adsorbent traps, and 1 µl of internal standard (n-tetradecane, 100
 ng µl<sup>-1</sup>) was injected in these traps. Two traps –
 one for mass spectrometry analyses, used for compound identification, and
 one for flame ionisation detection analyses, used for compound
 quantification – connected to a pump by silicon tubes were inserted into
 the bag after 20 min. The air inside the bag was then pumped through the
 two traps for 10 min at a rate of 40 ml min<sup>-1</sup>.
 After floral odour extraction, each trap was put into a vial and stored at
 -20°C until further analyses. Floral odour was analysed by gas
 chromatography (GC)-mass spectrometry (MS) and flame ionization detection
 (FID) using a Trace 1310 gas chromatograph coupled with a flame ionization
 detector and an ISQ mass spectrometer (Thermo-Electron, Milan, Italy) and
 equipped with an OPTIMA® 5-MS-HT capillary column (30 m × 0.25 mm × 0.25
 µm, Macherey-Nagel, Düren, Germany). Traps were handled with a
 Multi-Purpose Sampler (Gerstel, Mülheim an der Ruhr, Germany) and desorbed
 with a double stage desorption system composed of a Thermal Desorption
 Unity (TDU) and a Cold Injection System (CIS; Gerstel, Mülheim an der
 Ruhr, Germany). First, traps were desorbed splitless with a temperature of
 250°C on the CIS trap cooled at -80°C using liquid nitrogen. Then, the CIS
 trap was heated to 250°C with a 1:4 split ratio to inject the compounds
 into the column. Oven temperature was held at 40°C for 3 min, increased
 from 40°C to 210°C at a rate of 5°C/min and from 220 to 250°C at a rate of
 10°C/min, and held at 250°C for 2 min. The temperatures of transfer line
 and ion source of the mass spectrometer were 250°C and 200°C,
 respectively, and acquisition was set from 38m/z to 350m/z, at a 70eV
 ionisation energy. The FID was heated to 250°C. Retention times of a
 series of <i>n</i>-alkanes (Alkanes standard solution, 04070,
 Sigma Aldrich®) were used to convert retention times into retention
 indices. Compounds were identified on the GC-MS samples based on their
 retention indices and mass spectra, which were compared to those recorded
 in databases (Adams, 2007). Peak areas were measured on the GC-FID samples
 using the software Xcalibur (Thermo-Electron, Milan, Italy) and were
 converted to absolute (<i>i.e.</i> in ng) and relative
 (<i>i.e.</i> in %) amounts using the peak area of the internal
 standard (n-tetradecane). One month after the
 measurement of floral traits, the fruit set of each individual was
 measured and used as a proxy of female reproductive success.
description: In the floral colour dataset, two measurements were made for
 three individuals; in this case, the average value was used in subsequent
 analyses.