Evolution of Australian Flora: Hymenopteran Visual Systems

Evolution of Australian Flora Hymenopteran optical SystemsAbstractVery little work has been d maven on the solicitation of floral influence diversity, outside of Europe and the Middle East. In particular, we dwell virtually nothing about the evolution of the Australian flora in the context of use of hymenopterous louse opthalmic systems. Such a require is apt(predicate) to be important due to the geologically grand isolation of the Australian flora and the extravagantly proportion of turn backemic put species. The aims of this study were to study the work of Australian aborigine peaks in the context of hymenopterous insect visual systems, the innate semblance preferences of Australian native bees (Trigona carbonaria), and the interactions amongst native bees and a forage deceptive orchidaceous implant (Caladenia carnea). Firstly, I found that the discrimination thresholds of hymenopterous insects check up on up with floral people of saturation diversity a nd that hymenopterans bulge out to hurl been a major contributor to skin rash color evolution in Australia. Secondly, I found that Trigona carbonaria has innate preferences for wavelengths of 422, 437 and 530 nm. Thirdly, I found that bees were able to habituate to orchid kicks based on comment, thus po cristaltially explaining the intensity polymorphism of Caladenia carnea. Together, my study suggests that the evolution of the Australian flora has been influenced by hymenopterans.1. IntroductionPlant-pollinator interactionsThe mutual interactions surrounded by pollinators and plants fix been suspected in driving angiosperm radiation and variegation in the past (Regal 1977 Crepet 1984 McPeek 1996). The obvious mutual benefit is that pollinators depend on the pollen and/or ragweed of inflorescence plants for nutrition 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). W orldwide, it is estimated that more(prenominal) than than 67% of angiosperm plants rely on pollination by insects (Tepedino 1979). Hence, pollinators diddle a critical sh atomic round 18 in the persistence and survival of skin rash plants, which ar of high value to the gentlemans gentleman food chain (Kearns and Inouye 1997 Klein, Vaissiere et al. 2007). apex rubric augurs and sensory exploitationColour is the result of the visible un physical objectionable being absorbed or reflected off objects and so processed by the eye and brain of an animal (Le Grand 1968). Light is part of the electromagnetic spectrum, and piece of tail be quantified by the wavelength of distinct photons of energy (Bueche 1986). The wavelengths reflected off the object are perceived by a visual system as the objects colour. For example, clean that appears blue to a human beings percipient can be described by a dominant wavelength of four hundrednm, whilst light that appears red is 700nm. Ultra violet light falls surrounded by 300-400nm and can be seen by bees, but not humans. Flower influence take hold been influenced by the sensory sensory receptors of insects, including their colour resourcefulness, which is different to human vision. Humans have a red, blue and putting green receptor (Chittka and Wells 2004). In business line insects have a UV, green and blue receptor (Chittka and Wells 2004). As human vision is very different to a hymenopterans colour visual system, one cannot discuss a bees colour intelligence according to human colour terms such as red or blue. at that placefore, this thesis depart discuss work according to wavelength.Colour is one of the most important floral forecasts plants use to communicate information to insect pollinators (Giurfa, Vorobyev et al. 1996 Dyer, Spaethe et al. 2008). Although it is know that pollinators appoint meridians based on morphology, nectar avail ability, size, and odour (Giurfa, Nez et al. 1994 Kunze and G umbert 2001 Spaethe, Tautz et al. 2001 Whitney and Glover 2007), colour is cognize to play a critical role in enabling pollinators to find out and discriminate target flowers from a biologically important withdrawnness of up to 50 cm (Giurfa, Vorobyev et al. 1996 Dyer, Spaethe et al. 2008).Our under boothing of the evolution of colour vision in insects has advanced considerably in recent years. In the past, studies of colour science were limited due to little information on the colour visual system of insects (Frisch 1914 Daumer 1956). It is now possible to evaluate how flower visual attach appear to the visual system of hymenopteran pollinators, apply spectrophotometer and colorimetry techniques, which allows quantitative evaluations of how decomposable colour information is perceived by insect pollinators (Chittka 1992) (fig. 1).Previous enquiry has revealed that colour discrimination in hymenopterans is phylogenetically ancient, with different hymenopterans sharing alike (p) colour perception (Helversen 1972 Chittka and Menzel 1992). Importantly, colour discrimination in the hymenoptera is known to predate the evolution of floral colour diversity (Chittka 1996). Here, recent check up on has revealed remarkable convergence in the evolution and distribution of floral color in different parts of the world. Specifically, in a seminal paper, Chittka (1996) fork outed that flowering plants in both Europe and the Middle East have adapt their colour signals to the visual systems of bees, with flower colours in these regions virtually matched to the visual receptors of hymenopterans (Chittka 1996). However, outside of Europe and the Middle East, very little work has been by with(p) on the evolution of floral colour diversity. In particular, we know almost nothing about the evolution of the Australian flora in the context of hymenopteran visual systems. This is an important question to investigate due to the long isolation of the Australian flora and the high proportion of endemic plant species. I hypothesise that the Australian floral colouring will closely match the discrimination thresholds of hymenopterans as recent evidence suggests that insect pollinators support the early spread of flowering plants (Hu, Dilcher et al. 2008).Innate colour preferences of beesCharles Darwin was the first to recite that innate preferences could allow an inexperienced pollinator to find a food character reference (Darwin 1877). Pollinators may use certain traits of flowers such as morphology, odor, temperature and colour to go under 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 cardinal species the European honey bee (Apis mellifera) and bumblebee (Bombus terrestris). By dividing line, no studies have looked at the innate colour preferences of Australian bees and how this affects their prizes for flowers. We know that Eur opean bumblebees and honeybees show strong preferences for violet and blue (400-420nm) throughout their geographic deviate (Chittka, Ings et al. 2004) ,which interestingly correlates with the most profitable food sources (Lunau and Maier 1995 Chittka and Raine 2006). These preferences are likely to have had an jounce on the recounting success of different flower colours in regions where these bees are dominant pollinators (Chittka and Wells 2004). Consequently, information on the innate preferences of Australian bees will be important to envision hymenopteran plant interactions in the Australian context.Pollinator attending and food deceptive orchidsMost plants give back their pollinators with nectar or pollen. However, some species do not offer floral rewards and, instead, practice a range of deceptive techniques to trick insects into performing the task of pollination. jerry-built pollination strategies are particularly well known and widespread among orchids (Jerskov, Joh nson 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 gross are food deceptive orchids which are believed to number as many as 6,000 species (one-third of orchids) (Jerskov, Johnson et al. 2009). Food mimicking orchids shew use of bright colours to falsely advertise the presence of a reward to attract naive pollinators (Ackerman 1986 Nilsson 1992 Jerskov, Johnson et al. 2006). The habitual occurrence of food deception 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 causa of sexual deception, previous research shows that insects quickly learn un reward flower decoys and avoid them. For example, male insects learn to a void areas containing sexually deceptive orchids (Peakall 1990 Wong and Schiestl 2002). However, whether insects can learn to avoid food deceptive orchids remains to be investigated. In addition, high levels of variability in floral traits, particularly flower colour and floral scent, may interrupt the associative learning of insects by preventing their ability to become familiar with deceptive flowers (Schiestl 2005). Indeed, variation in colour, shape and scent is evident in non- pattern 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 variable flower colour slows de touch the ability of naive pollinators to learn unrewarding flower decoys. Furthermore, there is a need to incorporate a combination of colour vision science and behavioural ecology to understand how a bee perceives the orchid flowers, as bees have a different visual system to humans.Although humans cannot see ultra-violet light, UV esthesia 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 focuse on species specific signalling and mate weft (Bennett, Cuthill et al. 1996 Bennett, Cuthill et al. 1997 Pearn 2001 Cummings, Garc et al. 2006). However, few studies have looked at the role of UV signals in attracting bees to orchids. Previous studies have shown that the presence of UV reflecting holler spiders attracts honeybees to daisies (Heiling, Herberstein et al. 2003). In a exchangeable study, Australian native bees (Austroplebia australis) were attracted but did not land on flowers with UV reflecting crab spiders (Heiling 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 might also serve to enticement 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 address the following questions1. Is there a link surrounded by hymenopteran vision and Australian floral people of colour?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 betwixt hymenopteran vision and Australian floral col oration?Flower collection and apparitional reflectivity maneuvers of Australian native plant flowersAustralian native flowers were collected 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, utilise a digital UV camera Fuji Finepix Pro S3 UVIR modified CCD for UV imaging with calibrated UV-vis grey 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 reflection areas of the flower to be measurable by the spectrophotometer (Indsto, Weston et al. 2006).The ghostlike reflection functions of flowers were mensural from 300 to 700 nm use a spectrophotometer(S2000) with a PX-2 pulsed xenon light source addicted to a PC running SpectraSuite so ftware (Ocean Optics Inc., Dunedin, FL, USA). The spectrophotometer was utilize to quantify the colour of the flower as wavelength. The clear standard was a freshly pressed pellet of dry BaSO4, utilise to calibrate the spectrophotometer. A minimum of three flowers from from to each one one plant were use for each spectral analysis. I evaluated a sample of 111 spectral measurements from Australian flowering plants, encompassing a object lesson 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 utilize the methodology utilize by Chittka and Menzel (1992). In that study, Chittka and Menzel looked for correlations between flower spectra penetrative stairs of different plant flowers and trichomatic vision of the honeybees. Sharp abuses are a rapid change in the spectra wavelength (Ch ittka 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 desktop. Furthermore, a colour signal will be more distinguishable to a pollinator if the sharp steps match up with the crossroad 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 unconquerable by eyesight as described by Chittka and Menzel (1992), as the record of curves varied with each flower. The absolute numbers of sharp steps inwardly each flower spectra were counted. The frequencies are shown in fig. 4b. As hybrid plants are unlifelikely 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 fl ower colour smears in a colour hexagon space. A colour space is a numerical representation of an insects colour perception that is suitable for a wide range of hymenopteran species (Chittka 1992). In a colour space, the distances between reparations of a ii 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 currently unknown, but hymenopteran colorful vision is very similar between species as the colour photoreceptors are phylogenetically ancient (Chittka 1996). Thus, it is possible to model 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 average reflectance from each flower, and calculating the photoreceptor redness (E) va lues, according 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 end as perceived by a bee was calculated by the euclidian distance between ii objects locations in the colour hexagon space (Chittka 1992). exemplar 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 storage-battery grid of 10 degree sectors was lay 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 car bonaria) have innate colour preferences?Insect model and holdTrigona carbonaria is an Australian native stingless bee that resides in colonies of 4000-10000 individuals (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 Eastern Australia and are a potentially important pollinator for several major commercial crops (Heard 1999). A research colonisation (ca. 4000 adults and 800 foraging individuals) of T. carbonaria was propagated for the experiments by Dr Tim Heard (CSIRO Entomology, 120 Meiers Rd, Indooroopilly 4068, Australia) as described in the paper by Heard (1988). Bees were maintained in lab conditions so that no previous contact with flowers had been made. For this study, a colony was placed in a pine nest box (27.5 x 20 x 31 cm LWH) and connected to the foraging eye socket by a 16 cm plexiglass tube, containing indivi dual shutters to mesh bee movements. All laboratory experiments were conducted in a Controlled Temperature Laboratory (CTL) at Monash University, Clayton, School of Biological Sciences (CTL way G12C dimensions 3 x 5m), during the months of July 2009- January 2010. Relative humidity (RH) was solidifying to 30%, and the temperature was stiff 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 iv Phillips Master TLS HE slimline 28W/865 UV+ daylight fluorescent tubes (Holland) with specially fitted high frequency (1200Hz) ATEC Jupiter EGF PMD2x14-35 electronic dimmable ballasts which closely matches daylight conditions for trichromatic hymenoptera (Dyer and Chittka 2004). The escapism bailiwick (1.2 x 0.6 x 0.5m LWH) was made of a coated make frame with laminated fair wooden side panels. The arena floor was motley foli age green, and the arena lid was covered 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 fledge arena for seven days. Naive foragers (i.e. bees that had never en riposteed real or artificial flowers) were ab initio pre-trained to forage in the escape valve arena on three rewarding aluminium sanded disks (25 mm in diameter), with a 10-l droplet of 15% (w/w) saccharose solution placed in the centre. The disks were placed on vertical formative cylinders (diameter = 25 mm, height = 100 mm), to drum them above 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 however experiments. The aluminium sanded disks were chosen as neutral stimuli because they have an even spectral reflectance curve in the spectral visual range of the bees, fig. 6. The saccharose 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 randomised, 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 data collectionInnate colour preference testingTo test the innate colour preferences of naive bees, I performed simultaneous choice 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, coloured artificial disks in the original flight arena. Artificial flower stimuli were cut in a circle (70 mm diameter) from standardized colour papers of the HKS-N-series (Hostmann-Steinberg K+E Druckfarben, H. Schmincke Co., Germany). In each experiment the same set of ten test colours (1N pale yellow, 3N saturate d yellow, 21N light pink, 32N pink, 33N purple, 50N blue, 68N green, 82N brown, 92N grey, back of 92N sporting) 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 ensure the bees used in each running play were separated from the neighboring trial. The number of landings and approaches to the stimuli were recorded for one hour. Approximately 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 each trial, the colour disks were aired and wiped with a paper tissue to remove p ossible scent marks, which are known to affect experiments with honeybees (Schmitt and Bertsch 1990 Giurfa and Nez 1992). I conducted each posterior 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 analysis.Analysis of colour stimuliAs bees see colours differently to humans, I quantified stimuli according to five parameters wavelength, brightness, uprightness (saturation), chromatic contrast to the background and green receptor contrast. Dominant wavelength was calculated by canvass a line from the centre of the colour hexagon through the stimulus location to the corresponding spect rum 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 stimulus 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 impact by background colour (Lunau, Wacht et al. 1996). Green 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 wavelength on number of landings by Trigona carbonaria was investigated using a hit 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 reliant variable and wavelength of stimuli as the independent variable. 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 Core 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).With bright colours and fragrance, this orchid achieves pollination by food mimicry (Adams and Lawson 1993). Thus, due t o 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 prospect 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 Glad 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 Glad 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 five minutes. After each trial, the Glad WrapTM was changed to prevent scent marks. In addition, individual flowers and spatial positio ns were randomised. undivided 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 hydroplane was permeable to all wavelengths of light above 300 nm and did not obscure the reflectance of flowers.Do bees display 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 stage experiment to investigate if bees could learn to habituate to a non-rewarding flower colour over time and whether bees familiarised 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 minutes. 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 affectionate to spectral differences (fig. 4b). There are three clear peaks in sharp steps (fig. 4b). It is known that hymenopteran trichomats are all sensitive to spectral differences at approximately 400 and 500 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 powerfully 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? resolution of brightness, spectral purity, chromatic contrast and green rece ptor contrast on colour choicesThere was no significant final result 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 kind 7b). There was no significant correlation effect of chromatic contrast on choice frequency (rs = 0.042, n=10, p= 0.907 trope 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