Evolution of Australian Flora: Hymenopteran Visual Systems

Evolution of Australian Flora Hymenopteran Visual SystemsAbstractVery little spend a penny has been d unrivaled on the organic growing of billed illusion salmagundi, out position of europium and the centralize of attention East. In fact, we know al just about nonhing about the growth of the Australian flora in the context of hymenopterous insect visual bodys. much(prenominal) a study is likely to be important due to the geologic solelyy long isolation of the Australian flora and the spicy proportion of endemic sow species. The aims of this study were to investi approach the color in of Australian aboriginal blossom forths in the context of hymenopteran visual systems, the unlearned discolorise preferences of Australian native bees (Trigona carbonaria), and the interactions amongst native bees and a nourishment cheapjack orchidaceous plant (Caladenia carnea). Firstly, I prime that the difference thresholds of hymenopterans match up with efflorescenceed glossinessizeing diversity and that hymenopterans appear to take a leak been a major contributor to acme semblance evolution in Australia. Secondly, I found that Trigona carbonaria has essential preferences for wavelengths of 422, 437 and 530 nm. Thirdly, I found that bees were able to habituate to orchid extremums based on burnish, thus potentialityly explaining the pabulum garbleizeing polymorphism of Caladenia carnea. Together, my study suggests that the evolution of the Australian flora has been influenced by hymenopterans.1. IntroductionPlant-pollinator interactionsThe interchangeable interactions between pollinators and plants pack been suspected in brainish angiosperm radiation and diversification in the past (Regal 1977 Crepet 1984 McPeek 1996). The obvious mutual benefit is that pollinators depend on the pollen and/or nectar of unfolding plants for viands and, in return, partake in the incidental transfer of pollen necessary for plant reproduction (Faegri and van der Pijl 1978 Harder, Williams et al. 2001). Worldwide, it is estimated that more than 67% of angiosperm plants rely on pollination by insects (Tepedino 1979). Hence, pollinators play a critical aim in the persistence and survival of flowering plants, which be of high value to the piece food chain (Kearns and Inouye 1997 Klein, Vaissiere et al. 2007). boot contort blesss and sensory exploitationgloss is the result of the visible sluttish being absorbed or reflected run into objects and then processed by the eye and brain of an animal (Le golden 1968). Light is part of the electromagnetic spectrum, and can be quantified by the wavelength of antithetic photons of energy (Bueche 1986). The wavelengths reflected off the object be compassd by a visual system as the objects coloring. For font, light that appears blue(a) to a merciful observer can be stack forth by a dominant wavelength of 400nm, whilst light that appears expiration is 700nm. Ultraviolet light m ove between 300-400nm and can be seen by bees, but non gentlemans gentlemans. Flower color have been influenced by the sensory sensory receptors of insects, including their annotateise h everyucination, which is variant to human vision. Humans have a red, blue and light- spirt receptor (Chittka and rise up 2004). In billet insects have a UV, green and blue receptor (Chittka and Wells 2004). As human vision is genuinely assorted to a hymenopterans wring visual system, one cannot discuss a bees colour recognition jibe to human colour terms much(prenominal) as red or blue. at that placefore, this thesis will discuss colors jibe to wavelength. twine is one of the most important patterned foreshadows plants use to communicate study to insect pollinators (Giurfa, Vorobyev et al. 1996 Dyer, Spaethe et al. 2008). Although it is cognize that pollinators select flowers based on morphology, nectar availability, size, and odor (Giurfa, Nez et al. 1994 Kunze and Gumbert 2001 Spaethe, Tautz et al. 2001 Whitney and Gl every distinguish 2007), colour is known to play a critical role in enabling pollinators to detect and discriminate target flowers from a biologically important surpass of up to 50 cm (Giurfa, Vorobyev et al. 1996 Dyer, Spaethe et al. 2008).Our considering of the evolution of colour vision in insects has advanced considerably in youthful years. In the past, studies of colour scholarship were limited due to little selective nurture on the colour visual system of insects (Frisch 1914 Daumer 1956). It is now possible to prize how flower visual signals appear to the visual system of hymenopteran pollinators, using spectrophotometer and colorimetry techniques, which allows quantitative evaluations of how complex colour information is perceived by insect pollinators (Chittka 1992) (fig. 1).Previous research has revealed that colour discrimination in hymenopterans is phylogenetically ancient, with different hymenopterans sharing ali ke colour perception (Helversen 1972 Chittka and Menzel 1992). Importantly, colour discrimination in the hymenoptera is known to predate the evolution of flowered colour diversity (Chittka 1996). Here, novel research has revealed remarkable convergence in the evolution and distribution of patterned work in different parts of the world. Specifically, in a seminal paper, Chittka (1996) showed that flowering plants in both Europe and the Middle East have adapted their colour signals to the visual systems of bees, with flower colours in these regions closely matched to the visual receptors of hymenopterans (Chittka 1996). However, outside of Europe and the Middle East, very little work has been done on the evolution of floral colour diversity. In particular, we know almost no turn outg about the evolution of the Australian flora in the context of hymenopteran visual systems. This is an important nous to canvass due to the long isolation of the Australian flora and the high propor tion of endemic plant species. I hypothesise that the Australian floral coloration will closely match the discrimination thresholds of hymenopterans as recent evidence suggests that insect pollinators supported the early spread of flowering plants (Hu, Dilcher et al. 2008). inbred colour preferences of beesCharles Darwin was the first to state that unlearned preferences could allow an inexperienced pollinator to find a food source (Darwin 1877). Pollinators may use certain traits of flowers such as morphology, scent, temperature and colour to locate food (Heinrich 1979 Menzel 1985 Dyer, Whitney et al. 2006 Raine, Ings et al. 2006). Previous studies evaluating innate colour preferences have tended to focus on both species the European honey bee (Apis mellifera) and bumblebee (Bombus terrestris). By occupation, no studies have looked at the innate colour preferences of Australian bees and how this prints their wefts for flowers. We know that European bumblebees and honeybees sh ow strong preferences for violet and blue (400-420nm) throughout their geographic range (Chittka, Ings et al. 2004) ,which interestingly correlates with the most profitable food sources (Lunau and Maier 1995 Chittka and Raine 2006). These preferences be likely to have had an impact on the relative success of different flower colours in regions where these bees atomic reduce 18 dominant pollinators (Chittka and Wells 2004). Consequently, information on the innate preferences of Australian bees will be important to at a lower placestand hymenopteran plant interactions in the Australian context.Pollinator learning and food jerry-built orchidsMost plants reward their pollinators with nectar or pollen. However, some species do not offer floral rewards and, instead, employ a range of deceptive techniques to conjuring trick insects into performing the task of pollination. Deceptive pollination strategies are particularly advantageously known and widespread among orchids (Jerskov, Johnson et al. 2006). For instance, approximately 400 orchid species are known to achieve pollination through sexual deceit, luring unsuspecting male insects to the flower through olfactory, visual and tactile mimicry of potential mates. More green are food deceptive orchids which are believed to number as many a(prenominal) as 6,000 species (one-third of orchids) (Jerskov, Johnson et al. 2009). Food mimicking orchids employ bright colours to falsely propound the presence of a reward to attract naive pollinators (Ackerman 1986 Nilsson 1992 Jerskov, Johnson et al. 2006). The common occurrent of food prank in orchids suggests that this form of pollination by deception is an extremely successful evolutionary strategy (Cozzolino and Widmer 2005).Visits by pollinators to deceptive plants are influenced by pollinator learning. In the case of sexual deception, front research shows that insects promptly learn ungratifying flower decoys and avert them. For example, male insects lear n to avoid areas containing sexually deceptive orchids (Peakall 1990 Wong and Schiestl 2002). However, whether insects can learn to avoid food deceptive orchids re importants to be investigated. In addition, high levels of variability in floral traits, particularly flower colour and floral scent, may interrupt the associatory learning of insects by preventing their ability to contract familiar with deceptive flowers (Schiestl 2005). Indeed, disagreement in colour, shape and fragrance is evident in non- feign food-deceptive orchids (Moya and Ackerman 1993 Aragn and Ackerman 2004 Salzmann, Nardella et al. 2007). However, previous studies have only looked at pollinator preference for colour morphs (Koivisto, Vallius et al. 2002), rather than assessing if inconsistent flower colour slows down the ability of naive pollinators to learn unrewarding flower decoys. Furthermore, at that place is a need to incorporate a combining of colour vision science and behavioural ecology to unders tand how a bee perceives the orchid flowers, as bees have a different visual system to humans.Although humans cannot see ultra-violet light, UV sensitivity is common in some animals (Tove 1995). UV sensitivity has been found in insects, birds, fish and reptiles (Marshall, Jones et al. 1996 Neumeyer and Kitschmann 1998 Cuthill, partridge et al. 2000 Briscoe and Chittka 2001). Studies on UV vision in an ecological context have mainly foc apply on species specific signalling and mate excerpt (Bennett, Cuthill et al. 1996 Bennett, Cuthill et al. 1997 Pearn 2001 Cummings, Garc et al. 2006). However, a couple of(prenominal) studies have looked at the role of UV signals in attracting bees to orchids. Previous studies have shown that the presence of UV reflecting direct spiders attracts honeybees to daisies (Heiling, Herberstein et al. 2003). In a similar study, Australian native bees (Austroplebia australis) were attracted but did not land on flowers with UV reflecting crab spiders (H eiling and Herberstein 2004). However, the role of UV signals in orchids is not well studied. In particular, it is not known if the UV signal is important in attracting naive bees to food deceptive orchids. Thus, it will be useful to know if UV signals magnate withal serve to lure naive pollinators to deceptive flowers to understand deceptive pollination.AimsThis project will investigate Australian flower colour diversity in the context of hymenopteran visual systems, the innate colour preferences of Australian native bees (Trigona carbonaria) and their interactions with a food deceptive orchid (Caladenia carnea). This study aims to get by the following questions1. Is there a link between hymenopteran vision and Australian floral coloration?2. Does an Australian native bee (Trigona carbonaria) have innate colour preferences?3. Does a food deceptive orchid (Caladenia carnea) exploit the innate colour preferences of Trigona carbonar2. MethodsPart 1. Is there a link between hymenopt eran vision and Australian floral coloration?Flower collection and spiritual reflectance officiates of Australian native plant flowersAustralian native flowers were unruffled from Maranoa Gardens, Balwyn (melway ref 46 F7). Maranoa Gardens was chosen due to the diverse collection of species from all over Australia. Flowers were collected once a month, from May to January.A colour photograph was taken of the flower for identification. I also took a UV photograph for all flowers, using a digital UV photographic camera Fuji Finepix Pro S3 UVIR modified CCD for UV imaging with correctd UV-vis colourize scales (Dyer, Muir et al. 2004). As UV rays are invisible to the human eye (Menzel and Blakers 1976 Dyer 2001), this photo enabled any UV reflectance areas of the flower to be metrical by the spectrophotometer (Indsto, Weston et al. 2006).The ghostly formula consumptions of flowers were calculated from 300 to 700 nm using a spectrophotometer(S2000) with a PX-2 pulsed atomic nu mber 54 light source attached to a PC running SpectraSuite computer software (Ocean Optics Inc., Dunedin, FL, USA). The spectrophotometer was apply to quantify the colour of the flower as wavelength. The everlasting(a)ness well-worn was a freshly pressed pellet of dry BaSO4, used to calibrate the spectrophotometer. A minimum of iii flowers from from for distributively one one plant were used for severally apparitional synopsis. I evaluated a sample of 111 spectral measurements from Australian flowering plants, encompassing a representative variety of plant families (fig. 2).Correlations between spectral reflectance functions of different plant flowers and trichomatic vision of the honeybeesTo understand if there is a link between hymenopteran vision and Australian native flowers, I used the methodology used by Chittka and Menzel (1992). In that study, Chittka and Menzel looked for correlations between flower spectra sharp step of different plant flowers and trichomatic v ision of the honeybees. Sharp move are a rapid change in the spectra wavelength (Chittka and Menzel 1992) (see fig. 3 for an example of a sharp step). These steps cross over different receptors, thereby producing vivid colours that stand out from the background. Furthermore, a colour signal will be more distinguishable to a pollinator if the sharp steps match up with the overlap of receptors in a visual system. Thus, the main feature of a flower wavelength is a sharp step. For this study, I defined a sharp step as a change of greater than 20 % reflectance in less than 50 nm of the bee visual spectrum. The midpoint of the slope was determined by eyesight as exposit by Chittka and Menzel (1992), as the nature of curves varied with each flower. The absolute numbers game of sharp steps within each flower spectra were counted. The frequencies are shown in fig. 4b. As hybrid plants are bionicly selected by humans, hybrid flowers were not included in the analyses.Generating a Hexagon colour spaceTo evaluate how flower colours are seen by bees, I plotted the flower colour positions in a colour hexagon space. A colour space is a numerical representation of an insects colour perception that is adapted for a wide range of hymenopteran species (Chittka 1992). In a colour space, the distances between locations of a two colour objects link with the insects capacity to differentiate those colours. To make the colour space, the spectral reflectance of the colour objects were required, as well as the receptor sensitivities of the insect. For Trigona carbonaria, the exact photoreceptors are presently unknown, but hymenopteran trichromatic vision is very similar between species as the colour photoreceptors are phylogenetically ancient (Chittka 1996). Thus, it is possible to manakin hymenopteran vision with a vitamin A1 visual template (Stavenga, Smits et al. 1993) as described by Dyer (1999). I then predicted how the brain processed these colour signals by using the aver age reflectance from each flower, and calculating the photoreceptor upheaval (E) values, fit in to the UV, blue and green receptor sensitivities (Briscoe and Chittka 2001) using the methods explained by Chittka (1992). The UV, blue and green E-values of flower spectra were used as coordinates and plotted in a colour space (Chittka 1992). The colour difference as perceived by a bee was calculated by the Euclidean distance between two objects locations in the colour hexagon space (Chittka 1992).Modelling the distributions of Australian flower colours according to bees perceptionI analysed the most frequent flower colour according to a bees colour perception using the methods of Chittka, Shmida et al. (1994). I plotted the Australian flower colours in a colour space (Fig 5a). A colour space is a graphical representation of a bees colour perception. A radial grid of 10 degree sectors was determined over the distribution of colour loci and the number of floral colour loci within each sector was counted(fig. 5b).Part 2. Does an Australian native bee (Trigona carbonaria) have innate colour preferences?Insect model and housingTrigona carbonaria is an Australian native stingless bee that lives in colonies of 4000-10000 psyches (Heard 1988). In the wild, stingless bees live in hollows inside trees (Dollin, Dollin et al. 1997). Trigona carbonaria has a similar social structure to the honeybee (Wille 1983). They are common to North easterly Australia and are a potentially important pollinator for several major technical crops (Heard 1999). A research colony (ca. 4000 adults and 800 hunt down individuals) of T. carbonaria was propagated for the try outs by Dr Tim Heard (CSIRO Entomology, great hundred Meiers Rd, Indooroopilly 4068, Australia) as described in the paper by Heard (1988). Bees were maintained in laboratory conditions so that no previous impinging with flowers had been do. For this study, a colony was placed in a pine nest incase (27.5 x 20 x 31 cm LWH) and connected to the foraging sports stadium by a 16 cm plexiglass tube, containing individual shutters to control bee movements. All laboratory experiments were conducted in a Controlled Temperature Laboratory (CTL) at Monash University, Clayton, School of Biological Sciences (CTL room G12C dimensions 3 x 5m), during the months of July 2009- January 2010. Relative humidity (RH) was set to 30%, and the temperature was set to 27 C (SPER-Scientific Hygrometer, Arizona, USA), as this set up approximately matches conditions in Queensland for insect pollinators (Heard and Hendrikz 1993). Illumination (10/14 hr day/night) was provided by four Phillips Master TLS HE slimline 28W/865 UV+ mean solar day fluorescent tubes (Holland) with specially fitted high oftenness (1200Hz) ATEC Jupiter EGF PMD2x14-35 electronic dimmable ballasts which closely matches twenty-four hours conditions for trichromatic hymenoptera (Dyer and Chittka 2004). The flight arena (1.2 x 0.6 x 0.5m LWH) was mad e of a coated steel frame with laminated white wooden side panels. The arena floor was painted foliage green, and the arena lid was cover with UV transparent plexiglass. Experiments were conducted from 1pm-3pm to control for time of day, as this is when bees are most active (Heard and Hendrikz 1993).Pre-trainingBees were habituated to the flight arena for seven days. Naive foragers (i.e. bees that had never encountered real or artificial flowers) were initially pre-trained to forage in the flight arena on three rewarding aluminium sanded disks (25 mm in diameter), with a 10-l droplet of 15% (w/w) sucrose solution placed in the centre. The disks were placed on vertical plastic cylinders (diameter = 25 mm, height = 100 mm), to put together them to a higher place the floor of the flight arena so that bees learnt to fly to the disks. Pre-training allows bees to become habituated to visiting artificial flowers for further experiments. The aluminium sanded disks were chosen as unbiased stimuli because they have an even spectral reflectance curve in the spectral visual range of the bees, fig. 6. The sucrose solution reward on these training disks was refilled using a pipette after it was consumed by foraging bees. The spatial positions of these training disks were pseudo randomized, so that bees would not learn to associate particular locations with reward. Bees were allowed a minimum of two hours to forage on the pre-training disks before info collectionInnate colour preference testingTo test the innate colour preferences of naive bees, I performed simultaneous natural selection experiments with flower-naive bees using artificial flowers that simulated the floral colours of natural flowers. The aluminum rewarding disks were replaced by the ten unrewarding, aslant artificial disks in the original flight arena. imitative flower stimuli were cut in a circle (70 mm diameter) from order colour papers of the HKS-N-series (Hostmann-Steinberg K+E Druckfarben, H. Sch mincke Co., Germany). In each experiment the same set of ten test colours (1N pale yellow, 3N arrant(a) yellow, 21N light pink, 32N pink, 33N purple, 50N blue, 68N green, 82N brown, 92N grey, back of 92N white) were used. These colours were chosen as they have been used in innate colour experiments with other hymenopterans (Giurfa, Nez et al. 1995 Kelber 1997 Gumbert 2000), and the colours are also widely used in other bee colour experiments (Giurfa, Vorobyev et al. 1996). The coloured paper disks were placed on vertical plastic cylinders (diameter = 15 mm height = 50 mm), to raise them above the floor of the flight arena. The gate was shut in the arena to turn back the bees used in each trial were separated from the next trial. The number of landings and approaches to the stimuli were save for one hour. nigh 200 bees were used for each trial. The spatial positions of the artificial flowers were pseudo randomised in a counter balance fashion every 15 minutes. After ea ch trial, the colour disks were aired and wiped with a paper create from raw material to remove possible scent marks, which are known to affect experiments with honeybees (Schmitt and Bertsch 1990 Giurfa and Nez 1992). I conducted each subsequent trial after removing the used bees from the system, to ensure that the bees in the next trial were replaced with naive foragers.It is known that perception of colour can be influenced by background colour (Lunau, Wacht et al. 1996). Therefore, I also tested colour choices on other background colours of grey and black. The results are qualitatively similar (fig. 8b), so only data from the biologically relevant green background was used for subsequent compendium.Analysis of colour stimuliAs bees see colours differently to humans, I quantified stimuli according to louvre parameters wavelength, brightness, purity (saturation), chromatic contrast to the background and green receptor contrast. preponderant wavelength was calculated by tracing a line from the centre of the colour hexagon through the stimulant drug location to the corresponding spectrum locus wavelength (Wyszecki and Stiles 1982). Brightness was measured as the sum of excitation values of the UV, blue and green receptors (Spaethe, Tautz et al. 2001). spectral purity of the arousal was calculated by the percentage distance of the stimulus in relation to the end of the spectrum locus (Chittka and Wells 2004). Chromatic contrast was calculated as the distance of a colour stimulus from the centre of the colour hexagon relative to the background. Chromatic contrast is important as perception can be affected by background colour (Lunau, Wacht et al. 1996). jet receptor contrast was measured as the green receptor excitation from a stimulus relative to the background (Giurfa, Nez et al. 1995). This contrast is relevant as green receptors and green contrast are known to affect motion in bees (Srinivasan, Lehrer et al. 1987).statistical analysesThe impact of wa velength on number of landings by Trigona carbonaria was investigated using a single factor analysis of variance (ANOVA) and a post hoc Tukeys HSD test (=0.05) (Quinn and Keough 2002) using the number of landings as the dependent covariant and wavelength of stimuli as the mugwump multivariate. Brightness, purity (saturation), chromatic contrast to the background and green receptor contrast of stimuli were analysed using the Spearmans rank correlation test against choices. Statistical analyses were conducted using R statistical and graphical environment (R Development nubble Team, 2007). Statistical significance was set to P0.05.Part 3. Does a food deceptive orchid (Caladenia carnea) exploit the innate colour preferences of Trigona carbonaria?Plant modelCaladenia carnea is a widespread species, common to eastern Australia. The orchid is highly variable in colour, ranging from pink to white. It is pollinated by Australian native bees of the Trigona species (Adams and Lawson 1993).W ith bright colours and fragrance, this orchid achieves pollination by food mimicry (Adams and Lawson 1993). Thus, due to the colour variation of the orchid, C. carnea is an excellent model with which to examine floral exploitation of potential pollinators. Caladenia carnea flowers were supplied by private growers from the Australasian Native Orchid Society.Can Trigona carbonaria perceive a difference between pink and white flowers of Caladenia carnea?Colorimetric analysis of the pink and white Caladenia carnea flowers were used to investigate whether different colours of the orchid would be perceived as similar or different to a bees visual system. A spectrophotometer was used to take four measurements of each flower colour (pink versus white). The actual measurements used in the analysis were an average of each colour (Dyer, Whitney et al. 2007). To predict the probability with which insect pollinators would discriminate between different flowers, these spectra were plotted as loci in a hexagon colour space (Chittka 1992) (see hexagon colour space methods).Choice experimentsI conducted trials testing the preferences of bees when offered a dichotomous choice between a white versus pink Caladenia carnea flower. Each trial took place inside a flight arena. Each white and pink flower used in a trial were matched for size, placed into indiviual plastic containers (diameter= 5 cm, height=5 cm) and placed in the arena with a distance of 10 cm between flower centres. Each container was covered with successful WrapTM (The Clorox Company, Oaklands, CA, USA) to remove olfactory cues as they are known to inuence the choice behaviour of honeybees (e.g. Pelz, Gerber et al. 1997 Laska, Galizia et al. 1999). Approximately 50 bees were let into the arena for each trial. The rst contact made by a bee with the buoyant WrapTM within a distance of 4 cm, was recorded as a choice of that ower (Dyer, Whitney et al. 2007). The number of landings were recorded to the flowers for fiv e minutes. After each trial, the blithesome WrapTM was changed to prevent scent marks. In addition, individual flowers and spatial positions were randomised. Individual bees were sacrificed after each trial to avoid pseudo replication.Does the UV signal affect the attraction of bees to orchid flowers?To investigate whether the UV reectance of the dorsal sepal affected the response of bees, I offered bees the choice between two white orchids, one with a UV signal and the other without (N=16). The UV signal was removed by applying a thin layer of sunscreen (Hamilton SPF 30+, Adelaide, SA, Australia) over the dorsal sepal. Spectral reflectance measurements were taken to ensure that the sunscreen prevented any reflection of UV light (below 395 nm) from the sepals and did not change the reflectance properties of the orchid. In addition, spectral measurements of orchid sepals under Glad WrapTM confirmed that the foil was permeable to all wavelengths of light above 300 nm and did not obsc ure the reflectance of flowers.Do bees show preferences when choosing between pink versus white orchid flowers?To assess whether bees show a preference for pink or white variants of the orchid Caladenia carnea, I offered bees a simultaneous choice between a pink or white flower (N=16). See procedures for choice testing.Do bees habituate to non-rewarding orchids based on differences in floral coloration?I conducted a two deliver experiment to investigate if bees could learn to habituate to a non-rewarding flower colour over time and whether bees adjusted their subsequent flower choice depending on the flower colour encountered previously. At stage 1 of the experiment, native bees were presented with one flower, either white or pink. Flowers were placed in a container with Glad WrapTM. Landings to the flower were recorded at the start and again at the 30 min mark. At stage 2, the flower from stage 1 was swapped with a new flower colour and the number of landings were scored for 5 mi nutes. Flowers were randomised and Glad WrapTM changed to prevent scent marks after each trial. Once again, bees were used only once per experiment.Statistical analysesFor experiments 2, 3 4, numbers of landings by naive bees to flower pairs were compared using two tailed paired t-tests. A two factor ANOVA was used to analyse whether bees habituate to non-rewarding orchids based on differences in floral coloration. The dependent variable was the number of landings and the two independent variables were previous flower colour and new flower colour.3. ResultsPart 1. Is there a link between hymenopteran vision and Australian floral coloration?Correlations between the inflection curves of different plant flowers and trichomatic vision of hymenopteransThe analysis of 111 spectral reflection curves of Australian flowers reveals that sharp steps occur at those wavelengths where hymenoterans are most sensitive to spectral differences (fig. 4b). There are three clear peaks in sharp steps (f ig. 4b). It is known that hymenopteran trichomats are all sensitive to spectral differences at approximately 400 and calciferol nm (Menzel and Backhaus 1991 Peitsch, Fietz et al. 1992). Hence, the peaks at 400 and 500 nm can be discriminated well by hymenopteran trichomats, as illustrated by the inverse / function (solid curve shown in fig. 4a) of the honeybee (Helversen 1972), which is an empirically determined threshold function which shows the region of the electromagnetic function that a bees visual system discriminates colours best. In summary, the spectral position of receptors of trichomatic hymenopterans are correlates with steps in the floral spectra of Australian flowers.The distributions of Australian flower colours according to bees perceptionThe floral colour loci are strongly clustered in the colour hexagon (fig. 5a). blue green flowers are the most common in the perception of bees, while pure UV flowers were the rarest (fig. 5b).Part 2. Does an Australian native bee (Trigona carbonaria) have innate colour preferences?Effect of brightness, spectral purity, chromatic contrast and green receptor contrast on colour choicesThere was no material incumbrance of stimulus brightness on choice frequency (rs= 0.333, n=10, p= 0.347 fig. 7a). There was no significant effect of spectral purity on choice frequency (rs = 0.224, n=10, p= 0.533 figure 7b). There was no significant correlation effect of chromatic contrast on choice frequency (rs = 0.042, n=10, p= 0.907 figure 7c). There was no significant effect of green receptor contrast on choice frequency (rs = 0. 0.552, n=10, p= 0.098 figure 7d).Effect of wavelength on colour choicesStimuli colours are plotted in figure 8a, as they appear to a human viewer to enable readers to understand the correlation between colour choices. However, all statistical analyses were conducted with stimuli plotted as wavelength due to the different visual perception of bees and humans (Kevan, Chittka et al. 2001). There is a significant effect of wavelength on the number of landings by Trigona carbonaria (Single factor ANOVA, F9,110 = 5.60, P