DOI: 10.5061/dryad.fn2z34v2c
title: Ancient insect vision tuned for flight amongst rocks and plants underpins natural flower colour diversity - rock, mineral, stick, bark, leaf, bird- and insect-flower petal reflectance spectra

description: Understanding the origins of flower colour signalling to pollinators is
 fundamental to evolutionary biology and ecology. Flower colour evolves
 under pressure from visual systems of pollinators, like birds and insects,
 to establish global signatures among flowers with similar pollinators.
 However, an understanding of the ancient origins of this relationship
 remains elusive. Here, we employ computer simulations to generate
 artificial flower backgrounds assembled from real material sample spectra
 of rocks, leaves, and dead plant materials, against which to test flowers’
 visibility to birds and bees. Our results indicate how flower colours
 differ from their backgrounds in strength, and the distributions of
 salient reflectance features when perceived by these key pollinators, to
 reveal the possible origins of their colours. Since Hymenopteran visual
 perception evolved before flowers, the terrestrial chromatic context for
 its evolution to facilitate flight and orientation consisted of rocks,
 leaves, sticks, and bark. Flowers exploited these pre-evolved visual
 capacities of their visitors, and in response evolved chromatic features
 to signal to bees, and differently to birds, against a backdrop of other
 natural materials. Consequently, it appears that today’s flower colours
 may be an evolutionary response to the vision of diurnal pollinators
 navigating their world millennia prior to the first flowers.
description: <strong>Material samples, data
 collection</strong> <strong>Background
 materials data (non-floral surfaces): rocks, minerals, sand, shells, dry
 leaves, dry bark, wood, dry seeds, and green
 leaves.</strong> Background surface
 data samples were collected across a 2400 km range from the tropical north
 (16.9°, 145.7°) to the temperate southern tip (39.1°, 146.2°) of mainland
 Australia. N = 507 total natural background surface
 data samples were collected: n = 65 green leaves; n = 96 dry leaves, bark,
 wood etc. samples; n = 346 rocks/mineral samples. 
 <strong>Flower data (petal surfaces): insect- and
 bird-pollinated flowers.</strong> 
 Flower data has previously been described and analysed
 <sup>9,29</sup> and is made available here.
 <strong>Spectral reflectance
 measurement</strong> Reflectance curves were
 measured for background material and floral samples from 300-700 nm (see
 below – <strong>Marker point calculations</strong>) using a
 spectrophotometer with quartz optics and a PX-2 pulsed xenon light source
 (USB2000+, Ocean Optics Inc., Dunedin, FL, USA) attached to a computer
 running SPECTRA SUITE software. Reflectance profiles were measured
 relative to a Lambertian PTF WS-1 reflectance standard (Ocean Optics,
 USA). Multiple readings, usually three, were taken from within a square
 region of each surface (see associated article, <strong>Fig.
 1B</strong> for examples). Where sample surfaces were conspicuously
 heterogeneous to human visual systems several sections were sampled
 individually and used in this study. In the case of fresh green leaves
 specifically, one measurement was taken near the base, one in the middle,
 and one near the tip of each. These three spectra were then used to
 calculate an average reflectance spectrum for the leaf.
 To assist us in making accurate spectrophotometer readings, we
 built a black, curved-wall sample enclosure and covered this with a black
 cardboard lid. The lid was perforated with a tiny hole through which a
 fibre-optic light was channelled which prevented ambient light from
 hitting the sample during measurement. Room lighting was turned off. The
 optical fibre was held in an aluminium block that also helped eliminate
 stray illumination. The fibre was held approximately 6 mm above each
 sample, illuminating a circular patch 3-4 mm in diameter. Sample data
 points recorded between 300 and 700 nm in wavelength that represent
 reflectance spectra for three background surfaces are illustrated (see
 associated article, <strong>Fig. 1B</strong>).
 <strong>Marker point
 calculations</strong> Marker points are
 locations on surface spectral reflectance curves centrally located within
 sudden changes in spectral reflectance (see associated article,
 <strong>Fig. 1B</strong>) <sup>14,
 SuppRef-1</sup>. The severity of the jump is measured as a threshold
 over which it occurs from its base reflectance to its peak.  For this
 study, marker points were identified as the midpoint of any change in
 reflectance of at least a pre-specified percentage threshold value,
 occurring within a wavelength range of &lt; 50 nm. We calculated
 thresholds for each background sample at 5% reflectance jump and for each
 flower sample at 20% reflectance jump, in order to compare the properties
 of different materials. The 20% threshold for flower spectra was taken
 from the literature as being suited to such evolutionarily enhanced
 signals<sup>16</sup>. The 5% threshold for natural backgrounds
 that do not constitute evolutionarily enhanced reflectance curves was
 determined empirically to provide a similar mean number of marker points
 per sample as the 20% threshold had done for the flowers (see associated
 article, Supp <strong>Table 1</strong>).
 Marker point calculations were performed using the
 Open Source Spectral-MP software<sup>SuppRef-1</sup> with
 parameters: Threshold 5% (backgrounds), 20% (flowers); Range 50 nm;
 Smoothing window 21 points; Lookahead 5 points; Interval 300-700 nm. The
 study interval of 300-700 nm was selected to ensure we analysed regions in
 which the spectrophotometer readings were reliable, noting that the
 extremities of these regions are outside the range of sensitivity of avian
 and hymenopteran pollinator vision (see associated article,
 <strong>Fig. 1B</strong>).
 <strong>References</strong>
 <em>(Citation numbering refers to original article
 bibliographic data, reproduced here without change for
 clarity)</em> <strong>9.</strong>
 Shrestha, M., Dyer, A. G., Boyd-Gerny, S., Wong, B. B. M. & Burd,
 M. Shades of red: bird pollinated flowers target the specific colour
 discrimination abilities of avian vision. <em>New
 Phytologist</em> <strong>198</strong>, 301–310
 (2013). <strong>14.</strong> Chittka, L.
 & Menzel, R. The evolutionary adaptation of flower colors and the
 insect pollinators' color vision systems. <em>Journal of
 Comparative Physiology A</em> <strong>171</strong>,
 171-181 (1992). <strong>16.</strong> Dyer,
 A. G.<em> et al.</em> Parallel evolution of angiosperm colour
 signals: common evolutionary pressures linked to hymenopteran vision.
 <em>Proc. Royal Soc. London B</em>
 <strong>279</strong>, 3605-3615 (2012).
 <strong>29.</strong> Burd, M., Stayton, C. T.,
 Shrestha, M. & Dyer, A. G. Distinctive convergence in Australian
 floral colours seen through the eyes of Australian birds. <em>Proc.
 R. Soc. B</em> <strong>281</strong> (2014).
 <strong>SuppRef-1</strong>. Dorin, A., Shrestha, M.,
 Herrmann, M., Burd, M. & Dyer, A. G. Automated calculation of
 spectral-reflectance marker-points to enable analysis of plant
 colour-signalling to pollinators. <em>MethodsX</em>
 <strong>7</strong>, 1-9, https://doi.org/10.1016/j.mex.2020.100827 (2020).
description: # Ancient insect vision tuned for flight amongst rocks and plants
 underpins natural flower colour diversity - rock, mineral, stick, bark,
 leaf, bird- and insect-flower petal reflectance spectra
 The dataset contains
 spectral reflectance data collected from: * Australian **background
 materials**, i.e. non-floral surfaces, likely to appear behind flowers
 when viewed by pollinating insects and birds : rocks, minerals, sand,
 shells, dry leaves, dry bark, wood, dry seeds, green leaves. * Australian
 **flowers** (petal surfaces): insect- and bird-pollinated flowers. The
 data was used to test theories of flower colour evolution targeting visual
 systems of insect and bird pollinators. It supports the hypothesis that
 flowers have evolved colours to allow their specific pollinators to detect
 them against natural backgrounds that form the visual context for
 pollinator foraging. Reflectance curves were measured for background
 material and floral samples from 300-700 nm using a spectrophotometer with
 quartz optics and a PX-2 pulsed xenon light source (USB2000+, Ocean Optics
 Inc., Dunedin, FL, USA) attached to a computer running SPECTRA SUITE
 software. Reflectance profiles were measured relative to a Lambertian PTF
 WS-1 reflectance standard (Ocean Optics, USA). ## Description of the data
 and file structure Spectral reflectance data are collected in sets: 1.
 **Backgrounds** 1. Bark, dry leaves, sticks (96 samples) 2. Green leaves
 (65 samples) 3. Rocks and Minerals (346 samples) 2. **Insect-only
 pollinated flower** petals (153 samples) 3. **Bird-only pollinated
 flower** petals (68 samples) Although file names all have a numerical
 prefix, these are not indicative of any relationship between samples –
 they are simply the order in which the samples were measured over several
 years of data collection. Many file prefixes are missing as they refer to
 samples that are not relevant to the present dataset. File labels
 "insect-only" and "bird-only" refer to the fact that,
 based on the literature, these species have a clearly dominant pollinator
 (either insect or bird). **Backgrounds** spectral reflectance data are
 stored in CSV files generated by Open Access software Spectral-MP** when
 fed with the raw spectral reflectance data. These data are formatted as
 follows (refer to Spectral-MP publication and software manual for more
 detail): * column 1 = wavelength * column 2 = spectral reflectance
 (0-100%), mean value of 3 raw spectrophotometer readings of a material
 taken in the same area of the surface on all materials except green leaves
 where samples were taken at leaf base, middle and tip. * column 3 =
 spectral reflectance (0-100%) smoothed values of column 2 with smoothing
 applied by Spectral-MP with input parameters: Threshold 20%; Range 50 nm;
 Smoothing window 21 points (i.e. +/- 10 points either side of central
 point); Lookahead 5 points; Interval 300-700 nm. * column 4 = spectral
 reflectance (0-100%) sections of curve (if any) identified by Spectral-MP
 as being monotonically increasing or decreasing (parameters as for column
 3) * column 5 = spectral reflectance (0-100%) marker points (if any)
 identified by Spectral-MP at 20% threshold (parameters as for column 3). *
 Notes: Image.PNG image files in the directories are produced by SpectralMP
 and show the plotted spectral reflectance graph for each material with
 (Threshold=20%) marker points (if any). Material name for each sample
 appears in the file name, e.g., 1223_EucalyptusDryBark_Bark_1.csv. Files
 Histogram_H/V.png and histogram.csv are produced automatically by the
 SpectralMP software and depict histograms (_H/V.png indicates
 horizontal/vertical formatting, .csv = data file) of the marker points for
 the encapsulating folder placed in 10 nm wide bins. **Insect and
 Bird-pollinated flower petal** spectral reflectance data are stored in CSV
 files formatted as follows: * column 1 = wavelength * column 2 = spectral
 reflectance (0-100%) * Notes: Files \_FlowerData FileList 2023 Bird
 Pollinated Only.csv and \_FlowerData FileList 2023 Insect Pollinated
 Only.csv contain the species lists for the spectral data files which are
 simply numbered file names, e.g., 112.csv ## Code/Software \**
 **Spectral-MP software** is detailed/linked in: Dorin, A., Shrestha, M.,
 Herrmann, M., Burd, M. & Dyer, A. G. Automated calculation of
 spectral-reflectance marker-points to enable analysis of plant
 colour-signalling to pollinators. *MethodsX* **7**, 1-9,
 (2020)