DOI: 10.5061/dryad.k98sf7mk5
title: Data from: Do sun orchids mimic buzz-pollinated plants? An experimental test of the adaptive significance of false anthers

description: Mimicry implies that an organism gains fitness by resembling a model
 species, and one example is rewardless plants that attract pollinators by
 resembling co-flowering species that provide rewards. While trait matching
 between mimic and model has been characterised in many cases of putative
 floral mimicry, few have demonstrated that resemblance is adaptive and
 dependent on model presence.  Sun orchids (Thelymitra) are
 believed to mimic flowers of buzz-pollinated rewarding plants by
 displaying false anthers. To test the adaptive value of the false anthers
 we examined whether fruit production of T. crinita and T. macrophylla was
 reduced when anthers were experimentally removed or obscured, and whether
 the reduction was stronger when putative model plants were abundant. We
 also assessed visual flower similarity of both orchids and their putative
 model plants according to bee colour perception and identified shared
 pollinators and whether their behaviour on T. crinita was similar to that
 on buzz-pollinated model plants.  Fruit production of both sun
 orchids was strongly reduced (60-71%) by removal or painting of false
 anthers but was not affected by the abundance of model plants. Sun orchid
 flowers closely matched flower colour of co-flowering pollen-rewarding
 species, and T. crinita shared pollinators with the rewarding species.
 Visiting bees attempted to buzz and manipulate the false anther, with a
 behaviour similar to that observed on model plants.  The
 experimental results demonstrate that the false anther is an important
 adaptation to pollination in sun orchids. Striking visual flower
 similarity and shared pollinators between orchids and models suggest that
 sun orchids are pollinated by bees that mistake orchids for
 buzz-pollinated rewarding plants. The adaptive value of the false anther
 did not depend on model plant abundance in the local population,
 indicating that the relevant spatial scale is larger, or that the effects
 of the model species are weak in comparison to effects of other rewarding
 species, i.e., that magnet effects of nectar-rewarding species are
 dominating.  False anthers are widespread in the genus
 Thelymitra, and this “mimicry trait” seems to represent an evolutionary
 novelty that offers unique opportunities to explore adaptations to
 pollination in deceptive plants.
description: <em>2.1 Study system</em>
 <em>Thelymitra </em>is a genus of over 100 nectarless
 orchid species primarily found in Australia. These terrestrial, perennial
 herbs have inconspicuous roots, oval-shaped tubers, and a single leaf near
 the base of the plant (Western Australian Herbarium, 1998). Most species
 within the genus are believed to be bee-pollinated via food deception,
 although some may be autogamous (Beardsell & Bernhardt, 1983;
 Edens-Meier et al., 2014). Unlike typical orchids,
 <em>Thelymitra</em>'s labellum resembles the other
 petals, and flowers have an almost radially symmetric perianth. The
 flower's sexual parts are fused to a short column ending with a
 hood-like structure (mitra) or pseudanthery (Pridgeon et al., 2001; Fig.
 1), hereafter ‘false anther’. Sun orchids, as their common name suggests,
 typically open their flowers in response to daylight, warm temperatures,
 and humidity. Here, we focus on two common species,<em> T.
 macrophylla</em> and <em>T. crinita</em>. The two
 species have similar flower morphology (Fig. 1), but <em>T.
 macrophylla </em>flowers earlier than <em>T.
 crinita</em>. In a previous study, bees of the genera
 <em>Leioproctus</em> and <em>Lasioglossum</em>
 were identified as pollinators of <em>T. macrophylla</em>,
 while only <em>Leioproctus</em> bees pollinated <em>T.
 crinita</em> (Edens-Meier et al., 2013). It is not known whether
 bees attempt to buzz flowers, and whether pollinators vary among
 populations. <em>2.2 Study
 sites</em> We collected data in autumn 2023 and
 2024 in Southwestern Australia. The distribution of <em>T.
 macrophylla</em> and <em>T. crinita</em> ranges from
 Perth to Albany, and from Gingin to Esperance, respectively. Study
 populations of <em>T. macrophylla </em>were in<em>
 Banksia</em> woodland habitats within Warwick and Shanton Park
 bushland reserves, and study populations of <em>T.
 crinita</em> were found in forests dominated by<em> Eucalyptus
 marginata</em> in the Darling Range on the Perth hills (Fig. S1).
 Due to an unusual heatwave that caused flowering of<em> T. crinita
 </em>in the Perth region to end early, we also included a population
 in the Margaret River region, at Flat Rock bushland, Augusta (Fig. S1).
 Both <em>Thelymitra</em> species are abundant in the study
 regions, with typical population sizes ranging from 20 to 100 plants. The
 flowering time ranges from September to October for <em>T.
 marcrophylla</em> and from October to November for <em>T.
 crinita </em>(Western Australian Herbarium, 1998). Both orchids
 co-occur with a range of rewarding species with similar flower colour,
 including both pollen-rewarding buzz-pollinated species and
 nectar-rewarding species that are not buzz-pollinated (Edens-Meier et al.,
 2014; Brundrett et al, 2024). In the <em>T. macrophylla</em>
 study populations,<em> </em>main co-flowering species were
 <em>Orthrosanthus laxus </em>(Iridaceae) and<em>
 Sowerbaea laxiflora </em>(Asparagaceae) (Fig. 1 C,D that both are
 pollen-rewarding and buzz-pollinated. In the <em>T.
 crinita</em> populations, the main co-flowering species were
 pollen-rewarding <em>Thysanotus manglesianus</em> and
 <em>Agrostocrinum hirsutum</em> (Asphodelaceae) (Fig. 1 G,H),
 and nectar-rewarding <em>Lechenaultia biloba</em>,
 <em>Dampiera linearis</em>, and <em>Scaveola
 calliptera</em> (Goodeniaceae). <em>2.3
 Field observations and experiments</em> To
 determine flower colour similarity, we measured flower colour in one
 population of each orchid species, including all main co-flowering
 rewarding species (Table 1). To identify orchid pollinators and their
 behaviour on orchids and model plants, we conducted pollinator
 observations in three populations of <em>T. crinita
 </em>(Table 1). We initially included both orchid species in our
 observations, but due to very low insect activity in <em>T.
 macrophylla</em> populations, we focused on <em>T.
 crinita</em>. To test the adaptive significance of false anthers and
 whether it depends on rewarding context, we used two experimental
 approaches, anther removal and anther painting. With anther removal,
 physical damage may influence bee behaviour on flowers. With anther
 obscuration by paint, false anthers remained intact, and only the visual
 signal was removed. First, we examined the effect of removing the false
 anther in two populations of<em> T. macrophylla</em>, one with
 putative model plants (<em>O. laxus, S. laxiflora</em>) and
 one without (Table 1). Second, we examined the effect of obscuring the
 false anther using paint in two populations of each of the two species
 (Table 1). In the anther-painting experiment on <em>T.
 macrophylla</em>, we could not identify any workable sites with
 variation in model abundance, and the experiment was conducted in two
 population that both had abundant model plants. In <em>T.
 crinita</em>, we conducted the anther-painting experiment in two
 populations that differed quantitatively in local abundance of putative
 model plants. In both populations, we quantified abundance of model and
 magnet plants by estimating the number of flowers per species in five
 randomly distributed 5 × 5 m<sup> </sup>plots per population.
 We counted all flowering stems and all flowers on ten stems per plant, and
 estimated total number of flowers by multiplying number of stems by mean
 number of flowers per stem. We included all rewarding species with
 blue-violet flower colour as potential model (<em>A.
 hirsutum,</em> <em>T. manglesianus </em>which are
 pollen-rewarding) and magnet plants (<em>D. linearis, L.
 biloba</em>, <em>S. calliptera </em>which are both
 nectar and pollen-rewarding) regardless of the reward type, because buzz
 pollinators forage on both nectar- and pollen-rewarding flowers. The mean
 number of rewarding flowers per plot was 3.9 times higher in the
 population with high model abundance (mean ± SD = 87.6 ± 15.0) compared to
 the population with low model abundance (mean ± SD = 22.6 ± 12.4), and the
 ratio of mimics to models were approximately half in the former (0.45 and
 0.89, respectively). <em>2.4 Flower
 colour</em> To characterise flower colour in the
 two deceptive orchids and the co-flowering pollen- and nectar-rewarding
 species, we collected inflorescences in the field and brought them to the
 laboratory, where we measured six flowers per species using a field
 spectrometer (JAZ, Ocean Insight, Orlando, FL). On each flower, we took
 one measurement on the surface of the tepals in the orchids and of the
 petals in the co-flowering plants. To quantify colour similarity according
 to bee visual perception, we used the conventional bee vision model
 (colour hexagon space) based on photoreceptor spectral sensitivity of
 honeybees (Chittka, 1992). We quantified colour similarity by calculating
 the mean Euclidian distance between hexagon colour loci of
 <em>Thelymitra</em> and putative models and magnets. In
 addition, we used false colour photography as per Lunau et al.<sup>
 </sup>(2021), to identify colour patterns and details in the central
 area of the orchid flowers. This technique combines colour and UV
 photographs and is a robust method for obtaining detailed information on
 flower colour patterns under natural light conditions in the field
 (Verhoeven et al., 2018; Lunau et al., 2020). The technique splits colour
 photos into the three colour channels blue, green, and red, and by
 combining the green- and blue-channel photos with the UV photos, the
 resulting assemblage illustrates how bees perceive flower colour, i.e.,
 including UV and excluding red. <em>2.5
 Pollinator observations</em> To identify
 pollinators of <em>T. crinita</em>, we assessed the approach
 of all visitors to the flowers (attraction to the flower without
 necessarily landing), their behaviour and potential landing, and the
 capability to remove and deposit orchid pollinia, combining direct
 pollinator observations with camera recordings. Because natural visitation
 rate was very low, we created experimental arrays of <em>T.
 crinita</em> inflorescences close to rewarding species, to increase
 the likelihood of observing visitors (Scaccabarozzi et al., 2020). Arrays
 were formed by three cut orchid inflorescences placed in glass vials (one
 inflorescences per vial, each with 4–6 flowers) spaced 10 cm apart, and
 located ca 1 m from flowering individuals of the main rewarding species,
 <em>T. manglesianus</em> and <em>A. hirsutum</em>.
 In Perth hills, we conducted 80 and 20 ten-minute trials in the first and
 second population, respectively, and in the Augusta population, we
 conducted 200 ten-minute trials. This resulted in 50 hours pollinator
 observations in total. In each period, we also recorded insect behaviour
 using an EOS M video camera (Canon, Tokyo, Japan) for subsequent viewing
 in slow motion. Observations were done between 9 am and 3 pm (typical
 flower anthesis) during sunny or partly sunny days, when temperatures were
 above 22°C (measured 20 cm above the ground with a Smartsensor AR827,
 Sinosource Scientific Company, China). To determine if
 <em>T. crinita</em> shares pollinators with putative model
 species, we also conducted pollinator observations and camera recordings
 of <em>T. manglesianus </em>and <em>A.
 hirsutum,</em> during some of the orchid arrays. For both species,
 we performed 21 ten-minute observation periods, yielding a total of 3
 hours and 30 min observations per species. When possible, pollinators of
 both <em>T. crinita</em> and the putative model plants were
 identified in the field. In other cases, specimens were netted and later
 identified at the Museum of Western Australia.
 <em>2.6 False anther manipulations</em>
 <em>a)     </em><em>Anther removal
 experiment</em> On 24 September 2023, we randomly
 tagged 50 plants in each of two <em>T. macrophylla</em>
 populations separated by approximately 1 km within the Warwik Bushland
 Reserve, Perth. Putative model plants were present in one of the
 populations. In each population, we randomly assigned half of the plants
 to an anther removal treatment, while the other half were used as controls
 (n=25 per treatment). In the anther removal treatment, we carefully
 excised the false anthers (i.e., the upper part of the column, Fig. S2)
 from all open flowers using scissors. Most flowers were already open at
 the time of the experiment, and any remaining buds were removed. Prior to
 conducting manipulations, we recorded number of flowers per individual and
 examined if flowers had been visited. All flowers with pollinia removal or
 deposition were marked and excluded from the treatments (no difference
 between treatments; <em>P </em>&gt; 0.05). In both
 populations, we recorded fruit production of all plants two weeks after
 removing false anthers. To test if anther removal
 affected the ability to produce fruits, we supplementally hand-pollinated
 all flowers on 16 additional plants in another population in the Warwick
 bushland reserve, where eight had their false anther removed as described
 above, and eight were unmanipulated controls. We recorded fruit production
 two weeks later. Anther removal in <em>T. macrophylla</em> did
 not affect the ability to produce fruits (one-way ANOVA; F<sub>1,12
 </sub>= 0.35, <em>P </em>= 0.56).
 <em>b)     </em><em>Paint
 experiment</em> On 4 October 2023, we randomly
 tagged 40 plants at flowering peak in each of two <em>T. macrophylla
 </em>populations (both with abundant model plants) in Shenton
 Bushland Reserve, Perth. In each population, we randomly assigned half of
 the plants to an anther paint treatment, while the other half were used as
 controls (n=20 per treatment). In the paint treatment, we obscured false
 anthers in all open flowers by applying a colour that matched the colour
 of the column (Eraldo Di Paolo, Acrylic Paint, Warm Blue; colour match
 examined by spectrometer measurements and false colour photography; Fig.
 2A). To control for potential side-effects of painting, we also applied
 paint to the backside of the corolla of all flowers in the control
 treatment. On 17 October 2023, we randomly tagged 50 plants at flowering
 peak in each of two <em>T. crinita </em>populations (high vs.
 low abundance of model plants) on the Darling Scarp, Perth Hills. In each
 population, we randomly assigned half of the plants to an anther paint
 treatment, while the other half were used as controls (n=25 per
 treatment). Treatments were conducted as described for <em>T.
 macrophylla</em>. Prior to manipulations, we recorded number of
 flowers per individual and marked and excluded all flowers with pollinia
 removal or deposition for both orchids (no difference between treatments;
 both <em>P </em>&gt; 0.05). In both experiments, we
 recorded fruit production of all plants two weeks after the anther
 manipulations. To test the ability for autogamous
 pollination in both orchids, we bagged the inflorescences on ten
 individuals of each species<em> </em>at the bud stage, and
 recorded fruit production four weeks later. None of the bagged
 inflorescences in any of the two species developed fruits.
 <em>2.7 Statistical analysis</em>
 We initially checked if number of flowers per plant differed
 between treatment groups (anther removal or paint vs. control) or
 populations at the onset of the experiments, by using two-way ANOVAs
 including the interaction between population and treatment. Number of
 flowers did not differ between treatments or populations in any of the
 experiments (all<em> P </em>&gt; 0.31).
 To examine the effects of treatment (anther removal or paint vs.
 control), population, and their interaction on number of fruits produced
 in each of the three experiments, we used Generalised Linear Models with
 Poisson distribution and log link. Data were in some cases zero-inflated,
 and we used a negative binomial distribution when we detected
 overdispersion of the residuals. We used the package glmm TMB in R Studio
 (version 4.2.3).
description: # Do sun orchids mimic buzz-pollinated plants? An experimental test of the
 adaptive significance of false anthers Dataset DOI:
 [10.5061/dryad.k98sf7mk5](10.5061/dryad.k98sf7mk5) ## Description of the
 data and file structure Datasets are named in the file description and
 accompany the result section. ### Files and variables #### File:
 Datafile_Thelymitra_crinita_anther_paint.xls **Description:** Dataset of
 anther paint experiment on T. crinita. ##### Variables * plant: number of
 plant ID * flower: number of flowers per plant * fruit: number of fruits
 per plant * anther_painted: 0 for control and 1 for anther painted ####
 File: Datafile_Thelymitra_macrophylla_anther_paint.xls
 **Description:** Dataset of anther paint experiment on T. macrophylla.
 ##### Variables * population: 1 for population 1 and 2 for population 2 *
 plant: number of plant ID * flower: number of flowers per plant * fruit:
 number of fruits per plant * anther_painted: 0 for control and 1 for
 anther painted #### File: Datafile_Thelymitra_macrophylla_crossing.xls
 **Description:** Dataset of crossing experiments. ##### Variables *
 Flower: number of flowers per plant * Fruit: number of fruits per plant *
 Treatment: 0 for control, 1 for hand pollination #### File:
 Datafile_Thelymitra_macrophylla_anther_removal.xls
 **Description:**  Dataset of anther removal experiment on T. macrophylla.
 ##### Variables * population: 0 for population with no models and 1 for
 population with models * plant: number of plant ID * flower: number of
 flowers per plant * fruit: number of fruits per plant * anther_removed: 0
 for control and 1 for anther removed #### File: Insects_caught.xlsx
 **Description:** List of insects caught during observations. #####
 Variables * Code: specimen code * Date: date of specimen collection *
 Site: site of collection * Plant species: species where insect caught *
 Insect species: identified insect species * Sex: gender of specimen *
 Length (mm): insect body length