Sexual Deception For Pollination? There’s Lots Going On That We Know Nothing About

Criteria for confirming sexual deception

We define pollination by sexual deception as a strategy in which plants lure pollinators with signals that are sexually attractive to the pollinator. While there is considerable diversity among sexually deceptive systems, meeting one or more of the criteria listed below provides confirmation of sexual deception (Table 1). It should be noted that the level of sexual response can vary markedly between individual visitors, meaning that in many cases only a portion of the insects arriving at a flower will show the full potential repertoire of sexual behaviour at the flower (e.g. approach, alight and attempted copulation; Peakall, 1990; Phillips et al., 2013). Further, the level of the sexual response can also vary with the time of day and stage in the season (Paulus and Gack, 1990).

1. Pre-mating behaviour. Any behaviour associated with courtship (e.g. wing fanning of gnats towards Lepanthes; Blanco and Barboza, 2005) or the initiation of mating behaviour towards the flower (e.g. male thynnine wasps attempting to grasp a Drakaea labellum while in flight; Peakall, 1990). The identification of pre-mating behaviour requires an understanding of the sexual behaviour of mating pairs of the pollinator.

2. Attempted copulation. An attempt to initiate copulation with the flower (e.g. Coleman, 1928; Kullenberg, 1961; Peakall, 1989; Paulus and Gack, 1990; Schiestl et al., 1999; Blanco and Barboza, 2005; Phillips et al., 2013).

3. Ejaculation. Sperm release during attempted copulation with the flower. Ejaculation is known for pollinators of two sexually deceptive orchid genera (Blanco and Barboza, 2005; Gaskett et al., 2008), but appears unlikely to occur in most others.

4. Chemical mimicry of sex pheromones. Flowers produce semiochemicals that mimic the chemical composition of the sex pheromone of the pollinator (e.g. Schiestl et al., 1999, 2003; Stokl et al., 2007; Ayasse et al., 2011). Bioassays with synthetic semiochemicals are required to confirm that the specific compounds (or specific blends) elicit sexual attraction and sexual behaviour (e.g. Peakall et al., 2010). Sexual deception may still be involved when a pollinator exhibits a flight pattern indicative of tracking an odour plume, but fails to show any subsequent sexual behaviour at the flower. This distinction may be particularly important in cases where entrapment may prevent the full repertoire of sexual behaviour. Here, sexual attraction could be tested by presenting the scent-producing parts in isolation from the remainder of the flower or by adding chemical extracts from the flower to a dummy female.

In the absence of observations that meet the above criteria, other lines of evidence may be indicative of pollination by sexual deception. However, these traits alone are insufficient to confirm sexual deception (Table 1).

1. Males only. All known cases of sexual deception involve the attraction of male pollinators (Gaskett, 2011), but female-only attraction remains a possibility. It is worth noting that for some insects, such as thynnine wasps, in which the females are wingless, males are the only sex available as potential pollinators (Peakall, 1990). Therefore, when observing pollination by males only, the biology of the insect species needs to be considered before interpreting this as evidence for sexual deception.

2. Food reward absent. There are no confirmed cases of sexually deceptive orchids rewarding pollinators with nectar. However, in Gorteria diffusa (Asteraceae), male and female pollinators take nectar and pollen from flowers, while males are also sexually attracted to the dark spots present in some morphotypes (De Jager and Ellis, 2012).

3. Highly specific. In most species, only one or two pollinator species are attracted via sexual deception (see review in Gaskett, 2011). However, in some sexually deceptive orchids, food-foraging insects can potentially act as pollinators (Steiner et al., 1994).

4. Chemical attractant. All known sexually deceptive orchids attract their pollinators via floral odour. By contrast, in G. diffusa attraction of sexually deceived pollinators is by visual cues (De Jager and Ellis, 2012). Chemical attraction can be confirmed by attracting pollinators to covered flowers (Kullenberg, 1961), an approach flight associated with insects following an odour plume (Stoutamire, 1983) or the rapid attraction of males to floral odours released from artificially presented flowers (Peakall, 1990).

5. Insectiform floral structure. Most species of sexually deceptive orchids have an insectiform floral structure (e.g. Coleman, 1928; Kullenberg, 1961; Stoutamire, 1974; Paulus and Gack, 1990), though there are exceptions (e.g. Phillips et al., 2009). We consider insectiform orchid flowers to have some or all of the following traits: dull coloured, inconspicuous flowers; reduced petals and sepals; a large labellum relative to the remaining petals and sepals; the presence of hairs and/or a pronounced texture. In the case of G. diffusa, dark floral spots on the otherwise brightly coloured ray florets are associated with sexual behaviour (De Jager and Ellis, 2012).

Description of behaviour of the pollinator on the flower

Observations of pollinator behaviour were made in Kings Park, Western Australia, with additional observations at nearby Star Swamp Reserve (Table 2). At both sites, P. sanguinea was present. Flower-visiting gnats were most active between 1000 and 1600 h, though flight times were delayed on cold mornings (e.g. <5 °C). All observations were made between 1000 and 1600 h across 7 days between 21 June and 11 July 2013 (487 min of observation in total). During the observation periods temperature ranged between 17·5 and 22 °C, as measured with a Tinytag data logger (Gemini Data Loggers) suspended 30 cm above the ground (approximating flower height).

We conducted observations of pollinator behaviour based on the baiting method of Stoutamire (1974) and Peakall (1990), in which picked flowers are used to attract pollinators. As observed in sexually deceptive orchids pollinated by thynnine wasps, relocating bait flowers to a new location initiates a renewed response. Picked flowers were maintained in vials of water at all times and stored in a refrigerator at 4 °C between experiments. Three flowering stems were used simultaneously as an attractant, with one to three flowers open per stem. We baited at random positions within the population of fungus gnats, with consecutive positions greater than 3 m apart. Baiting was undertaken for 5-min periods in each position. If no gnats were attracted during this period, orchids were moved to another position. If gnats were attracted, we continued to make observations until visitation ceased. A total of 32 observation periods were conducted.

Pollinator behaviour at the flower was recorded for subsequent detailed evaluation with a Sony Digital HD Video Camera Recorder. Since no courting or mating pairs were observed during the study, the curling of the abdomen below the thorax was taken as evidence of courtship behaviour [as illustrated in Blanco and Barboza (2005) for sciarid gnats], while vigorous probing with the tip of the abdomen was interpreted as attempted copulatory behaviour. The position of the abdomen during copulatory probing was quantified for 29 separate visits. Probes were classed as being made at the labellum apex, along the lower section of the surface of the labellum, towards the upper section of the labellum or immediately below the callus (Fig. 3).

During field observations, pollinator behaviour was quantified across a hierarchy of responses following Peakall (1990) and Phillips et al. (2013). For each floral visitor (Fig. 4) we recorded the presence of pollen on arrival, whether or not the pollinator touched the labellum, the duration of any attempted copulation and the incidence of labellum triggering. If the labellum was triggered, we recorded the duration of pollinator entrapment and whether or not pollination was achieved. For each visitor, the time taken to arrive at an artificially presented bait flower and the total time spent on the flower were also recorded.

Due to the difficulty of observing such small insects in the wild (body length of approximately 3·5 mm), it was only possible to observe their approach to the flower when the sun was low in the sky and so illuminating their wings in flight. Therefore, we could only make general observations of how they approached the flower.

How long does the labellum take to reset?

On 12 July we conducted an experiment in Kings Park bushland to quantify how long it takes the labellum to reset after being trigged. The experiment was conducted between 1430 and 1630 h, coinciding with the period of peak gnat activity. Temperature ranged between 15·7 and 19·1 °C in sunny weather, following rain the previous day. We randomly selected one flower from each of 20 different plants in a population over an area of 15 × 15 m. At 1430 h a wooden skewer was used to trigger all 20 flowers. Each flower was then checked every 2 min to record whether the labellum had returned to the down position as required for pollination.

How many species of fungus gnats are involved?

Thirteen populations of P. sanguinea were visited to collect pollinators (Table 2), using two approaches: (1) baiting for 12 periods of 5 min at each site, during which any visitors to the flower were collected; (2) following observations that gnats are sometimes terminally trapped in Pterostylis flowers (Sargent, 1909; Bernhardt, 1995; Retter, 2009), flowers were dissected to check for the presence of gnats and whether or not they were carrying pollen. The floral dissections were done twice, once early and once late in the flowering season (n = 19–90 flowers per population, 570 flowers in total; Table 2). All gnats collected were placed in 80 % ethanol for subsequent identification and DNA extraction. Any gnats collected carrying pollen were observed under a dissecting microscope (Leica DCF450) to determine the positioning of the pollen on the animal.

To assess the degree of pollinator specificity, we sequenced the mitochondrial CO1 region, a region commonly used for DNA barcoding in Diptera (Meier et al., 2008). A total of 24 gnats were sequenced from the three populations where live gnats were collected (Kings Park, Shenton Park and Star Swamp). DNA was extracted from whole gnats using a modified salt extraction method (Bruford et al., 1998). Briefly, the gnats were added to 250 μl TNES buffer with 30 μl proteinase K (10 mg ml⁻¹), mixed gently and incubated at 55 °C for a minimum of 18 h. The buffer was then transferred to a tube containing 95 μl of 4 m ammonium acetate, mixed, incubated on ice for 20 min, then centrifuged for 20 min at 16 000 × g. The intact pollinator was returned to storage in 80 % ethanol. Following centrifugation, the supernatant (400 μl) was transferred to a clean tube and 1 mL of cold 100 % ethanol was added and the tube contents were thoroughly mixed before incubation for at least 18 h to precipitate the DNA. A pellet of DNA was formed by spinning at 16 000 × g for 20 min. The supernatant was discarded and the DNA pellet was washed with 500 μl of cold 70 % ethanol and allowed to dry before resuspending in 80 μl TE.

Amplification and sequencing followed the methods of Griffith et al. (2011). We used the primers of Folmer et al. (1994) as recommended by the Consortium for the Barcoding of Life (http://www.barcoding.si.edu/protocols.html). Sequences were edited and aligned using Geneious v6·1·6 created by Biomatters Ltd. A phylogenetic analysis was undertaken using the Phyml plugin in Geneious, using a substitution model of GTR + G. To quantify the genetic divergence within morphologically defined gnat species, we calculated the average Kimura 2 parameter (K2P) distances (Kimura, 1980) and the average percentage of varying base pairs using MEGA 5·05 (Tamura et al., 2011).

Are all populations of Pterostylis sanguinea attractive to the same pollinator species?

To test whether all study populations of P. sanguinea attracted the same pollinator, two flowering stems from each population were picked for baiting in the Kings Park gnat population. When possible we collected individuals representing both green and brown colour forms. Picked orchids were artificially presented within the gnat population and left exposed until a gnat arrived at the flower and was observed to attempt copulation. These gnats were then captured and included in the DNA sequencing dataset.

Is pollinator attraction by floral odour or visual cues?

The ability of gnats to locate hidden flowers was investigated by obscuring flowers in a black plastic screen made from a washed 1·5 L PET bottle. The screen was 15 cm high but open at the top and raised 5 cm off the ground, allowing access to the concealed flowers. Each trial consisted of a 2-min presentation without flowers within the screen, followed by a 2-min presentation with the flowers. The number of gnats approaching the screen and locating the hidden flowers was recorded for 30 trials in bushland in Kings Park.

Site of production of the sexual attractant

Following the observation that copulatory behaviour was only ever observed on the labellum, we tested the hypothesis that the labellum is the sole source of the chemical attractant by dissecting flowers. Each trial consisted of the sequential presentation of the flower without the labellum for the first 5 min, followed by simultaneous presentation of both the flower (minus labellum) and the labellum only (each presented pinned on a separate wooden skewer). The number of gnats responding was recorded along with evidence of sexual behaviour. This trial was repeated on five occasions per flower, for a total of nine flowers.

Does the morphology of the labellum determine the orientation of the pollinator?

Since gnats typically alighted on the labellum facing upwards, we tested whether the orientation of the labellum influenced their behaviour. We removed the labella and affixed them to wooden skewers using UHU All Purpose Adhesive. Labella were either positioned in the natural vertical position or rotated 180 °. Trials were undertaken by presenting a labellum to the fungus gnats until a response was achieved. For every response we alternated between the treatments used. We recorded whether each gnat faced upwards, downwards or sideways and whether they exhibited sexual behaviour (bending and/or probing of the abdomen). Each subsequent trial was conducted at least 1 m from the previous location. If no gnats were attracted within 5 min the trial was terminated. This experiment was conducted using labella from six different plants, for a total of 32 observations per treatment. We employed G-tests calculated in GenAlEx 6·5 (Peakall and Smouse, 2006, 2012) to compare both the proportion of gnats copulating with the flower and the proportion showing abdomen bending between treatments.

The influence of population size and density on fruit set

Fruit set (percentage of flowers in a population setting fruit) was recorded for 19 populations in 2008 and 2012. Population size ranged from five to 127 plants. The position of all individuals, as measured with a GPS, was uploaded to ArcGIS 9·3·1 and the minimum convex polygon was calculated to determine the area and density of the population. Regression analysis was conducted in PASW statistics 18 (SPSS Inc., 2009) to determine whether there is a relationship between fruit set and plant population size or density. The 2008 and 2012 data sets were analysed separately. As a test for resource-limited fruit set, in 2008 all flowers on six plants were cross-pollinated by hand in 13 populations. Pollen was sourced from plants more than 5 m from the pollen recipient.