Annals of Botany 107: 981 992, 2011 doi:10.1093/aob/mcr048, available online at www.aob.oxfordjournals.

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Carrion mimicry in a South African orchid: owers attract a narrow subset of the y assemblage on animal carcasses
Timotheus van der Niet*, Dennis M. Hansen and Steven D. Johnson
School of Biological and Conservation Sciences, University of KwaZulu-Natal, P. Bag X01, Scottsville, Pietermaritzburg 3209, South Africa * For correspondence. E-mail vdniet@gmail.com
Received: 8 September 2010 Returned for revision: 13 December 2010 Accepted: 3 February 2011 Published electronically: 13 March 2011

Background and Aims Although pollination of plants that attract ies by resembling their carrion brood and food sites has been reported in several angiosperm families, there has been very little work done on the level of specicity in carrion mimicry systems and the importance of plant cues in mediating such specialization. Specicity may be expected, as carrion-frequenting ies often exploit different niches, which has been interpreted as avoidance of interspecic competition. Interactions between the orchid Satyrium pumilum and a local assemblage of carrion ies were investigated, and the functional signicance of oral traits, especially scent, tested. Pollination success and the incidence of pollinator-mediated self-pollination were measured and these were compared with values for orchids with sexual- and food-deceptive pollination systems. Methods and Key Results Observations of insect visitation to animal carcasses and to owers showed that the local assemblage of carrion ies was dominated by blow ies (Calliphoridae), house ies (Muscidae) and esh ies (Sarcophagidae), but owers of the orchid were pollinated exclusively by esh ies, with a strong bias towards females that sometimes deposited live larvae on owers. A trend towards similar partitioning of y taxa was found in an experiment that tested the effect of large versus small carrion quantities on y attraction. GC-MS analysis showed that oral scent is dominated by oligosuldes, 2-heptanone, p-cresol and indole, compounds that also dominate carrion scent. Flesh ies did not distinguish between oral and carrion scent in a choice experiment using olfactory cues only, which also showed that scent alone is responsible for y attraction. Pollination success was relatively high (31.5 % of owers), but tracking of stained pollinia also revealed that a relatively high percentage (46 %) of pollen deposited on stigmas originates from the same plant. Conclusions Satyrium pumilum selectively attracts esh ies, probably because its relatively weak scent resembles that of the small carrion on which these ies predominate. In this way, the plants exploit a specic subset of the insect assemblage associated with carrion. Pollination rates and levels of self-pollination were high compared with those in other deceptive orchids and it is therefore unlikely that this mimicry system evolved to promote outcrossing. Key words: Calliphoridae, deception, oral scent, indole, oligosuldes, osmophore, outcrossing, p-cresol, Sarcophagidae, Satyrium pumilum, specialization.

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IN T RO DU C T IO N Pollination by carrion insects has been documented in a wide range of plant families (Vogel, 1954; Fgri and van der Pijl, 1979; Meve and Liede, 1994; Proctor et al., 1996; Stensmyr et al., 2002; Johnson and Jurgens, 2010; Shuttleworth and Johnson, 2010). Plants attract these pollinators by mimicking the substrate on which the insects rely as a brood or food site (Proctor et al., 1996) through a diverse array of traits such as dark brown or purple ower coloration (Beaman et al., 1988), the frequent presence of bizarre, lliform appendages (Vogel, 1990) and proportionally large owers and inorescences (Davis et al., 2008). Perhaps the most characteristic trait is the putrid oral scent emitted by the owers, which closely resembles that of real carrion (Kite et al., 1998; Stensmyr et al., 2002; Jurgens et al., 2006; Johnson and Jurgens, 2010). Globally, the most important carrion insects include members of the Dipteran families Calliphoridae and

Sarcophagidae (Payne, 1965, Blackith and Blackith, 1990; Hewadikaram and Goff, 1991; Carvalho et al., 2000; Satoshi, 2001; Archer and Elgar, 2003; Shi et al., 2009). The universally observed co-existence of calliphorids and sarcophagids on an ephemeral resource such as carrion, has led to the suggestion that they occupy slightly different niches (Denno and Cothran, 1975). Niche differentiation between these ies occurs along temporal axes such as seasonal variation in carrion abundance (Johnson, 1975; Hanski, 1987; Grassberger and Frank, 2004; Shi et al., 2009), as well as due to specialization according to carrion size (Denno and Cothran, 1975; Kneidel, 1984a), carrion type (Fuller, 1934; Cornaby, 1974; Kneidel, 1984a) and carrion successional stage (Payne, 1965; Cornaby, 1974; Grassberger and Frank, 2004). It is unclear whether carrion-mimicking owers attract a subset of the local assemblage of carrion ies. If so, this could be mediated by differentiation in specic plant traits that are similar to those that determine niche specialization

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van der Niet et al. Carrion mimicry in a South African orchid (Jersakova et al., 2006). The evolution and widespread occurrence of deception in orchids is most commonly explained as a mechanism that reduces geitonogamous self-pollination, and thereby promotes outcrossing (Cozzolino and Widmer, 2005; Jersakova et al., 2006), as oral visitors have a tendency to visit fewer owers on rewardless plants (Johnson and Nilsson, 1999; Jersakova and Johnson, 2006). Apart from elevated levels of outcrossing, deceptive pollination is typically also associated with extremely low levels of pollination success (Tremblay et al., 2005; Scopece et al., 2010). The evolution of mimicry, as a special case of oral deception, may have been driven by selection for increased pollination success to escape high levels of pollen limitation. Johnson (1994), for example, found that fruit set in a Batesian mimic is comparable to that of a rewarding orchid with which it shares habitat and pollinator. Although carrion mimicry is known to occur in orchids (van der Pijl and Dodson, 1966), as far as is known, no studies on carrion mimicry in orchids have documented pollination success and outcrossing rates. It remains to be tested, therefore, whether this form of mimicry also promotes outcrossing and is characterized by relatively high pollination success, or whether it may have evolved for different reasons. These questions were addressed using Satyrium pumilum as the model system. This peculiar member of the large, mainly African orchid genus Satyrium (van der Niet et al., 2005), is characterized by the virtual absence of a owering stalk resulting in the owers being borne at ground level (Fig. 1), which

of ies on real carrion. A comparison among closely related plant species indeed shows variation in traits such as ower size for Rafesia and stapeliads (Beaman et al., 1988; Meve and Liede, 1994) and scent composition among arums and sta peliads (Kite et al., 1998; Jurgens et al., 2006; Johnson and Jurgens, 2010), which may reect mimicry of different sizes and types of carrion. Additionally, carrion mimics are often visited by only one or a few species of carrion y (e.g. Stensmyr et al., 2002), which further suggests that they only attract a subset of the local y assemblage. To ultimately establish the level of specialization of the plant insect interaction for carrion mimics, however, it would be necessary to take the entire assemblage of carrion ies that co-occur with plants into consideration. Apart from one study on carrionmimicking stinkhorn fungi (Sleeman et al., 1997), no one, as far as is known, has studied this for owering plants. Pollination systems that rely on carrion mimicry are often characterized by the absence of a reward and thereby constitute a form of pollination through deception (Dafni, 1984; Schiestl, 2005). Floral deception is particularly widespread among Orchidaceae (Jersakova et al., 2006). The most common types of deception in orchids are food deception, where owers exploit innate preferences of pollinators by a general resemblance to food plants in a community, or conditioned preferences of pollinators by resembling specic food plants (Batesian mimicry), and sexual deception where owers attract male pollinators by mimicking female insects

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2 1

E
1 2

F I G . 1. Satyrium pumilum and its sarcophagid y pollinators. (A) The habitat of S. pumilum at Leliefontein in the Namaqualand Kamiesberg. (B) Satyrium pumilum in situ (scale bar 1 cm). (C E) Pollination sequence of a S. pumilum ower by a sarcophagid y in an arena (scale bar for all three photos 0.5 cm); (C) the y carrying ve pollinaria from other S. pumilum owers enters an unpollinated ower note the empty stigma (1), and that both its pollinaria are present (2); (D) as the y moves deeper into the ower towards the right-hand spur, it presses an attached pollinium against the stigma, and its thorax against the right-hand viscidium; (E) as it leaves the ower, the y has deposited two massulae on the stigma (1), and removed a pollinarium (2) it now carries six pollinaria.

van der Niet et al. Carrion mimicry in a South African orchid clearly sets it apart from all other satyriums. Johnson (1997) showed that its sister species S. bracteatum (van der Niet et al., 2005) is pollinated by small muscid and sarcophagid ies and suggested, in accordance with earlier speculations by Vogel (1954), that S. pumilum is most likely carrion ypollinated. In this study this prediction is conrmed. Furthermore, this study specically aimed to (a) understand the association between the pollinators and the assemblage of co-occurring carrion ies, (b) test the signicance of oral scent in this mimicry system, particularly with reference to potential pollinator specialization, and (c) quantify levels of pollination success and outcrossing, and compare these with other deceptive systems in orchids. M AT E R I A L S A N D M E T H O D S
Study species and site Carrion y assemblage

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To identify the species composition of the carrion y assemblage at the S. pumilum population, P. capensis carcasses, several days old, were laid out as a bait and samples of visiting ies caught at random using an insect net during several days in September October 2008. Caught ies were killed using ethyl acetate fumes, and identied to family, genus or species level and sexed by specialists (see Acknowledgements).
Arena experiments

Satyrium pumilum Thunb. is distributed throughout the western half of the greater Cape Floristic Region (Born et al., 2007), from Namaqualand in the north, to Riversdale in the east. The study was carried out at a site near Leliefontein, in the Kamiesberg region in Namaqualand, in a large population with several thousand plants scattered over approx. 1 ha. Here, S. pumilum grows in sandy, moist conditions near small streams, in open or shaded habitats under renosterbos bushes (Elytropappus rhinocerotis Less), often forming dense clumps of several dozen plants. The site is part of communal farm land and is grazed by sheep for several weeks in spring. Large numbers of owering plants in this population were observed in four consecutive years (2006 2009), all of which were characterized by relatively high winter and spring rainfall. In 2008, when most of the eldwork was carried out, the population owered for about 8 weeks between early September and late October. Plants have 3.13 + 1.42 open owers (mean + s.d., n 1327) with new owers opening every 3.02 + 1.25 d (mean + s.d., n 17). Individual owers are open for 12.5 + 1.5 d (mean + s.d., n 17). Stigmas become receptive on the day of anthesis, or latest the day thereafter. In contrast to Vogel (1954), no nectar was observed in any of the spurs examined (T. van der Niet, unpublished data). Moreover, the spurs are attened and thus inaccessible to ower visitors.
Pollinator observations

To examine whether different local carrion y species are capable of removing pollinaria and depositing massulae in owers of S. pumilum, 12 arenas made out of transparent empty soft drink bottles were set up. Plants with unvisited owers were put in soil in the bottom of the bottles and ies (up to ve, but generally only a single individual) that had been caught on carrion in the eld released into these arenas. The behaviour of the ies was observed and pollinarium removal and massulae deposition were recorded after 24 h.
Carrion scent quantity and y attraction

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Insect visitors to plants in the eld were observed throughout the duration of the eldwork during two consecutive years (5 d in September 2007 and approx. 5 weeks in September October 2008). If insects were seen close to, or on a plant, their behaviour was observed. Initial observations revealed that visits were very rare. Therefore carrion bait was laid out to attract carrion-visiting insects during several days of the 2008 owering season. Carcasses of the rock hyrax Procavia capensis, a common mammal from the area, which were found along the road, were used. Insects on carrion that carried S. pumilum pollinaria were caught, killed using ethyl acetate fumes, and identied and sexed by specialists (see Acknowledgements). The number of orchid pollinaria on each insect was counted.

To test whether scent quantity has an effect on y attraction, carrion was offered in small versus large quantities to ies in the eld. Part of a leg of P. capensis was used to represent small carrion quantities, and entire P. capensis carcasses represented large quantities. Carrion was placed on the bottom of a bucket (approx. 30 30 cm) that was buried ush with the ground. To exclude visual cues, the bucket was covered with a carton lid that resembled the colour of the surrounding soil. The lid contained a square hole, approx. 10 10 cm in size, over which was mounted a piece of carton, approx. 1 cm above the lid level to let scent escape. An empty bucket with the same cover was also buried to control for y attraction to the bucket. Any ies approaching the buckets were caught, and identied to family level. The experiment was repeated for 3 d during the warmest period of the day (mid-day), as this was found to correspond to the peak of y activity. A 30-min observation period with the small quantity was always followed by a 30-min observation period with the large quantity. The reverse sequence could have potentially resulted in a bias in y attraction during exposure of the small carrion quantity if ies, that would not have been present otherwise, were attracted to the vicinity of the large carrion quantity without being caught. Results (counts for each y family attracted to small versus large carrion quantities) were analysed using a x2 contingency test. The probabilities obtained from separate tests performed on data collected on different days were also combined to create an overall test for signicance (Fisher, 1954). This can be calculated as follows: 2S(ln P), where P is the probability for each separate test. If all the null hypotheses were true, then this quantity would be x2-distributed, with 2k degrees of freedom, where k is the number of separate tests (Sokal and Rohlf, 1995).

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Scent analysis

van der Niet et al. Carrion mimicry in a South African orchid soil. Each lid contained a built-in y trap that allowed ies to enter the pot but not to escape. At the end of each day, the number of ies of each family caught in each pot was recorded. Experiments were run for 6 entire days (approx. 6 h d21) and new plants were used for each day.
Breeding system, pollination success, pollen transfer efciency (PTE) and self-pollination rates

Floral scent was collected in 2007 2009 from plants in the eld and in the laboratory. Samples were taken of entire plants, and of cut owers, to test for spatial variation in scent production. Flower partitioning was based on the results of soaking owers for 24 h in histological 0.01 % neutral red stain to identify osmophores (Vogel, 1990). Two small pieces of P. capensis carcass, of comparable size to that used as small quantity in the experiment that tested for the effect of carrion scent quantity on y attraction were also sampled. Samples obtained using dynamic headspace extraction methods, were analysed by coupled gas chromatography and mass spectrometry (GC-MS). Samples were taken by enclosing plants, ower parts and carrion in polyacetate bags prior to sampling. Air from these bags was then immediately pumped for 20 min through small cartridges lled with 1 mg of Tenaxw and 1 mg of Carbotrapw at a ow rate of 50 mL min21. For every sampling session, controls were taken from an empty polyacetate bag sampled for the same duration. GC-MS analysis of these samples was carried out using a Varian CP-3800 GC (Varian, Palo Alto, CA. USA) equipped with an Alltech EC-WAX column (Alltech Associates, Inc., Deereld, IL, USA) with a 30 m 0.25 mm internal diameter and a 0.25-mm lm thickness, coupled to a Varian 1200 quadrupole mass spectrometer in electron-impact ionization mode. To conrm compound identication, samples were also analysed using a Varian VF-5ms non-polar column (Varian, Palo Alto, CA, USA) with a 30 m 0.25 mm internal diameter and a 0.25-mm lm thickness. Cartridges were placed in a Varian 1079 injector equipped with a Chromatoprobe thermal desorbtion device (Gordin and Amirav, 2000; Dotterl et al., 2005). The ow of helium carrier gas was 1 mL min21. The injector was held at 40 8C for 2 min with a 20 : 1 split ratio and then increased to 200 8C at 200 8C min21 in splitless mode for thermal desorbtion. After a 3-min hold at 40 8C, the temperature of the GC oven was ramped up to 240 8C at 10 8C min21 and held there for 12 min. Compounds were identied using the Varian Workstation software with the NIST05 mass spectral library and compounds were veried using retention times of authentic standards (apart from 2-heptanone and propyl isovalerate) and published Kovats indices. Compounds present at similar abundance in the controls were considered to be contaminants and excluded from the analyses. For quantication of emission rates, standards were injected into cartridges and thermally desorbed under identical conditions to the samples.
Effects of oral scent on y behaviour

A choice experiment was set up to test whether scent, in the absence of visual cues, triggers y attraction and whether ies discriminate between oral versus carrion scent. A small quantity of carrion ( piece of a leg of a P. capensis carcass) and approx. ten S. pumilum plants were placed in separate plant pots, with an additional empty pot used as a control. The three pots were dug into the soil in a triangle with approx. 1 m distance between all pots and with the top of the pot at ground level. To exclude visual cues, the pots were covered with carton lids that resembled the colour of the surrounding

To test whether S. pumilum relies on pollinators for fruit set, a breeding system experiment was set up with four treatments per plant: (1) cross-pollination (using three pollinia as pollen donors, removed from plants at least several metres away from a focal plant); (2) self-pollination (using a pollinium from the focal plant); (3) unmanipulated owers with the pollinaria intact, to test for autogamy; (4) removal of both pollinaria and leaving a ower unpollinated to test for apomixis. All owers used for the experiment had unpollinated stigmas. Treated plants were covered with mesh bags after the treatment to exclude all insects for the duration of the experiment. All four treatments were applied to a total of 20 plants. Fruit set was recorded after approx. 6 weeks. Pollination rates of S. pumilum were assessed in four surveys during the owering season in 2008 to account for intraseasonal variation. At each survey, 50 plants at least 1 m away from each other were randomly selected. One recently wilted ower from each plant was inspected for pollination by recording removal of pollinaria and/or massulae deposition. To assess natural fruit set, all ovaries of 21 randomly sampled plants from the eld were inspected for swelling at the end of the owering season in October 2008. PTE, the proportion of removed massulae deposited on conspecic stigmas (e.g. Johnson et al., 2005) was calculated, using the sample of 200 owers from the surveys of pollination success. A proxy for the total number of massulae deposited was calculated as the product of the number of owers with massulae deposition, multiplied by the average number of massulae deposited on pollinated stigmas of 32 randomly selected owers (eight owers collected during each of four surveys). The total number of massulae removed was calculated as the product of the number of pollinia removed multiplied by the average number of massulae per pollinium of 27 randomly selected owers (one pollinium counted per ower sampled from different plants, collected during the 2007 and 2008 owering seasons). To assess rates of pollinator-mediated self-pollination, the PTE of massulae transported to stigmas of the same plant was quantied using pollinia labelled with histological stains throughout the 2008 owering season (Peakall, 1989). Groups of stained plants planted within a 1 m radius, only contained one plant of each stain colour (1 % fast green, 0.2 % rhodamine pink, 1 % methyl blue, 1 % gentian violet, all stock solutions mixed with a drop of tween soap) and different groups were separated by at least 10 m. This distance was deemed adequate for avoiding between-plot stain movement in this dense population. Plants were monitored in the eld on a daily basis and once a plant was visited it was taken to the laboratory for counting of the removed pollinaria and stained massulae deposition. The self-pollination rate is calculated by dividing the PTE of self-pollination by the overall

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van der Niet et al. Carrion mimicry in a South African orchid PTE (which includes self-pollination + outcrossing). To calculate the PTE of self-pollination, the total number of stained selfed massulae deposited was divided by the number of stained massulae removed (see above) (Johnson and Brown, 2004). R E S U LT S
Pollinator observations

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Although insects were rarely seen visiting plants, a large number of ies carrying S. pumilum pollinaria were observed and caught. In all cases these were Sarcophagidae (Table 1 and Figs 1 and 2). Fifteen ies with pollinaria (1 male, 14 females) were caught in association with plants and 60 ies with pollinaria (12 males, 48 females) were caught in association with carrion. The male : female ratio did not differ signicantly between pollinators caught in association with plants or carrion (x2 1.49, P 0.22). Male ies carried sig1 nicantly fewer pollinaria than females [males 2.8 + 2.2 (mean + s.d., n 13), females 8.7 + 7.8 (mean + s.d., n 62); Mann Whitney, U 176.5, P 0.0015].
Carrion y assemblage

F I G . 2. Members of the carrion y assemblage at Leliefontein on a carcass. The majority of ies are Chrysomya chloropygia (blue), but also several Muscidae (Orthellia sp.; green) are present. Note the single sarcophagid y in the middle, carrying the yellow orchid pollinaria (see insert).

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the arena. Several calliphorid ies [Calliphora croceipalpus 4 out of 4 (including 1 male), Chrysomya chloropygia 3 out of 6 (all females)] also successfully removed S. pumilum pollinaria.
Small versus large quantity carrion

On carrion laid out as bait, a total of 314 ies were caught (Table 2). The main visitors were callophorid ies (90.8 %) of the genera Chrysomya (C. chloropyga represented 82.5 % of the ies caught), Lucilia, and Calliphora (Table 2 and Figs 2 and 3). Muscid ies made up 5.4 %, mainly of the genera Orthellia and Ophyra. Sarcophagid ies made up 3.5 % of the assemblage. A single individual belonging to the Anthomyiidae was caught. In contrast, of all ies caught in association with plants (on plants, on experimental set-ups including plants, or carrying pollinaria which by denition means they visited plants) 98.5 % were Sarcophagidae, mostly of the genus Sarcophaga (Table 2 and Figs 2 and 3).
Arena experiments

Sarcophagids visited small carrion in signicantly higher proportions than members of other y families when compared with large carrion (Fig. 3) (test results for separate days: x2 1 2.82, P 0.093, x2 3.99, P 0.046, x2 5.82, P 0.016; 1 1 test for overall signicance: x2 19.208, P , 0.01). 6
Scent analysis

Sarcophagidae released in arenas with plants visited and pollinated S. pumilum owers in 44 % of the cases (n 16; Fig. 1; video in Supplementary Data, available online). In one case, sarcophagid larvae were found in owers inside

Neutral red staining resulted in highly localized absorption of stain (Fig. 4). On the outside surface of the ower, absorption mainly occurred in the spur region and at the side of the labellum towards the landing platform. Inside the ower, stain absorption was restricted to the oor of the spur apex, which is densely carpeted with papillae. The scent of S. pumilum owers contains only six compounds (Table 3). This was conrmed using two different

TA B L E 1. Satyrium pumilum pollinators (as identied by the presence of orchid pollinaria on the sarcophagid ies) caught in association with plants and carrion during 2007 and 2008
Plant Male Sarcophaga (Bercaea) sp. (arno and/or inaequalis)* Sarcophaga (Liosarcophaga) redux Sarcophaga (Nesbittia) guillarmodi Sarcophagidae indet. spp. Total 0 1 (1) 0 0 1 (1) Female 0 9 (10.6 + 6.1) 0 5 (7.0 + 4.5) 14 (9.3 + 5.7) Male 0 12 (3.0 + 2.2) 0 0 12 (3.0 + 2.2) Carrion Female 6 (2.7 + 1.9) 30 (8.1 + 6.9) 1 (11) 11 (12.4 + 12.1) 48 (8.5 + 8.3)

Fly species names are given according to Pape (1996). The numbers represent the total number of male and female ies caught of each species, with the mean number of pollinaria/y + s.d. in parenthesis. * Females of these species are currently inseparable (T. Pape, Natural History Museum, Denmark, pers. comm.)

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van der Niet et al. Carrion mimicry in a South African orchid owers have a quantitatively different scent composition compared with whole plants (e.g. elevated rates of p-cresol), this cutting effect would affect both ower parts equally, as the cut surface on each part has by denition the same surface area. Apart from propyl isovalerate, all ower compounds [DMDS and dimethyl trisulde (DMTS), 2-heptanone, p-cresol and indole] were also found in carrion scent, where they comprise a large proportion of the entire blend (Table 3). Emission rates of small pieces of carrion were much higher than of individual owers.

GC columns, but only results using the EC-WAX column which gave the best separations are reported here. The dominant compound is dimethyl disulde (DMDS) which comprises .90 % of the blend. There is clear spatial quantitative and qualititative intraoral variation in scent production, with the oligosuldes and 2-heptanone and propyl isovalerate produced in the spur region (Fig. 4), and not (or only in trace amounts) by the rest of the ower, whereas p-cresol and indole are produced by all ower parts. The spur region also has higher emission rates than the rest of the ower (Fig. 4). Although cut

TA B L E 2. Members of the 2008 carrion y assemblage at Leliefontein, South Africa caught in association with S. pumilum plants and carrion, respectively
Plant (%) Male Anthmyiidae Calliphoridae Anthomyia tempestatum Calliphora croceipalpis Chrysomyia chloropyga Chrysomyia marginata Lucilia cuprina Lucilia sericata Morellia sp. 1 Musca sp. 1 Muscina sp. 1 Ophyra capensis Orthellia sp. 1 Orthellia sp. 2 Orthellia sp. 3 Sarcophaga (Bercaea) africa Sarcophaga (Nesbittia) guillarmodi Sarcophaga (Liopygia) nodosa Sarcophaga (Liosarcophaga) redux Sarcophaga (Bercaea) sp. (arno and/or inequalis)* Indet. spp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12 (17.9) 0 0 12 Female 0 0 0 0 0 0 0 0 1 (1.5) 0 0 0 0 0 1 (1.5) 0 37 (55.2) 4 (6.0) 12 (17.9) 55 Male 0 5 (1.6) 41 (13.1) 1 (0.3) 0 1 (0.3) 1 (0.3) 0 0 1 (0.3) 0 0 0 0 0 0 5 (1.6) 0 0 55 Carrion (%) Female 1 (0.3) 9 (2.9) 218 (69.4) 3 (1.0) 5 (1.6) 2 (0.6) 1 (0.3) 5 (1.6) 0 3 (1.0) 2 (0.6) 1 (0.3) 3 (1.0) 1 (0.3) 2 (0.6) 1 (0.3) 1 (0.3) 1 (0.3) 0 259

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Muscidae

Sarcophagidae

Total

Sarcophagid species names are given according to Pape (1996). * Females of these species are currently inseparable (T. Pape, Natural History Museum, Denmark, pers. comm.).

100 90 80 70 Percentage (%) 60 50 40 30 20 10 0

66 285 Calliphoridae Muscidae Sarcophagidae

100 90 80 70 60 50 40 30 20

Other fly families Sarcophagidae 104

23 19

34

17 11 0 1 S. pumilum Carrion Source

10 0 Small Large Carrion scent quantity

F I G . 3. Fly families caught on different sources: (A) ies caught in association with plants versus carrion; (B) ies caught in association with large versus small quantities of carrion scent. Numbers of caught ies are given above the bars.

van der Niet et al. Carrion mimicry in a South African orchid

A
DMDS

987

Propyl isovalerate

B
2-Heptanone

Flower without spur Spur

DMTS

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Indole

00 05 10 15 20 25 Scent emission (ng flower1 min1)

F I G . 4. Local variation in scent emission of S. pumilum owers. (AD) Patterns of neutral red staining (scale bar 5 mm): (A) unstained ower lateral view; (B) stained ower lateral view the grey arrow highlights the spur region with high levels of neutral red stain absorption; (C) cut unstained ower, (D) cut stained ower the grey arrow highlights the oor of the spur region with high levels of neutral red stain absorption. (E) Scent emission rates of individual compounds in the spur region versus the rest of the ower. Total emission rates were 3.70 ng ower21 min21 in the spur and 0.92 ng ower21 min21, respectively.

Effects of oral scent on y behaviour

Only Sarcophagidae were attracted in the choice experiment. These ies were equally attracted to carrion and plant scent [number of ies d21: 3.00 + 1.84 in plant trap versus 3.33 + 0.49 in carrion trap (mean + s.e., n 6 pairs); Wilcoxon signed-rank test, Z 0.95, P 0.34]. Empty control buckets did not attract any ies at all. Flies therefore signicantly prefer carrion scent and plant scent to no scent, respectively (carrion versus empty: Wilcoxon signed-rank test, Z 2.21, P 0.027; plants versus empty: Wilcoxon signed-rank test, Z 2.03, P 0.042).
Breeding system, pollination success, PTE and self-pollination rates

TA B L E 3. Mean percentages (+ s.e.) of individual scent compounds in the blend of whole plants and small carrion, arranged according to retention times of the EC-WAX column, and mean emission rates (+ s.e.)
Whole plant (n 3) DMDS Propyl isovalerate 2-Heptanone DMTS p-Cresol Indole Other compounds Emission rates (ng ower21 min21) 93.80 + 2.92 0.14 + 0.14 1.91 + 0.57 1.54 + 1.19 0.63 + 0.63 1.97 + 1.47 0 0.80 + 0.33 Small carrion (n 2) 17.49 0.0 Trace 34.40 13.82 0.82 33.46* 31.41

There were no fruits set in bagged owers with the pollinaria removed. Outcrossing resulted in 94 %, and selfpollination in 100 % fruit set, respectively. Fruit set in bagged intact owers was 11 %. Evidence of visitation was found in 31.5 % of wilted owers. Natural fruit set was 38.0 + 34.8 % (mean + s.d., n 21 plants). Overall, 10.8 % of removed massulae were deposited on stigmas. Of removed stained massulae, 5.0 % were deposited on stigmas of the same plant (n 22).

* Twenty-eight additional compounds were produced in carrion, with an average proportion of 1.19 %. Calculated including only the compounds DMDS + DMTS + p-cresol + indole.

Combining these results leads to a self-pollination rate of 45.8 %. There was no correlation between the proportion of self-pollination and the number of owers per plant (Spearmans r 0.21, P 0.34).

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van der Niet et al. Carrion mimicry in a South African orchid DISCUSSION of the local carrion y assemblage, which was dominated by C. chloropyga (Calliphoridae). Specic pollination by sarcophagid ies, with a similar bias towards females, has been documented previously in Rafesiaceae by Pape and Banziger (2000), and Banziger and Pape (2004). While Pape and Banziger (2000) did note y family-specic attraction to owers by carrion ies, they did not quantify the abundance of local carrion ies that were not attracted to owers, nor did they suggest potential causes for selective attraction. The present results raise the question of why the large numbers of non-sarcophagid carrion ies observed in the vicinity of the orchid never carried pollinaria. Calliphorids are known pollinators of plants with similar traits as S. pumilum, such as the dead horse arums (Stensmyr et al., 2002). Shuttleworth and Johnson (2010) showed that members of Calliphoridae, Muscidae and Sarcophagidae were all pollinators of two species of Eucomis (Hyacinthaceae). The present arena experiments showed that two calliphorid species (including C. chloropyga) were capable of removing pollinaria. We therefore argue that calliphorids and muscids are potential pollinators of S. pumilum and that a mechanical mismatch between plant and insect cannot explain the absence of non-sarcophagids with pollinaria in the eld. A second explanation may be that S. pumilum mimics a specic brood site which only attracts sarcophagids, but not members of other carrion y families. On carrion laid out as bait, however, sarcophagids with orchid pollinaria were found frequently co-occurring with calliphorids and muscids (Fig. 2). We therefore conclude that brood site specialization is not likely to explain the absence of non-sarcophagids with pollinaria in the eld, either. Instead, we argue that the specialized interaction between S. pumilum and sarcophagid ies, with a bias towards females, can be explained by an aspect of carrion y niche differentiation which is different from brood site specialization. The initial observation that only Sarcophagidae carried pollinaria of S. pumilum, despite the presence of a much larger carrion y assemblage including Calliphoridae, led us to attempt to replicate a situation in the eld where only sarcophagids were attracted to a particular, real carrion source. Another preliminary observation, that only sarcophagids were attracted to trace scent of carrion, led us to hypothesize that the quantity of carrion scent may determine which ies are attracted to a particular source. Indeed, the experiment using small versus large quantities of carrion resulted in the attraction of signicantly fewer Calliphoridae and Muscidae to small carrion quantities, much resembling the pollinator community (Figs 2 and 3). Even though the small quantity of carrion in the experiment still attracted some nonsarcophagid ies, as opposed to S. pumilum, we suspect that a further reduction of carrion scent quantity would have ultimately led to a complete elimination of the non-sarcophagid element from the y assemblage attracted to the carrion. The approx. 10-fold smaller scent emission rates of typical S. pumilum plants compared with that of the small carrion used in the experiment, may thus explain the complete absence of non-sarcophagids among the orchids pollinators. Although the possibility cannot be excluded that the small carrion used in the experiment differed not only quantitatively (smaller absolute amount of volatiles emitted), but also

Results from this study show that S. pumilum is pollinated exclusively by sarcophagid carrion ies, mainly females, despite the presence of large numbers of carrion ies from other families in the same area. The experiment testing for the effect of scent quantity on y attraction revealed that the ies attracted to small quantities of carrion showed a resemblance to those attracted to S. pumilum. Given the key role of S. pumilum scent, which is similar to that of real carrion, and which was equally attractive to sarcophagid ies, we speculate that the basis for the selective attraction of sarcophagid ies by S. pumilum owers and small items of carrion is that the quantity of scent emitted in both cases is much lower than that for whole animal carcasses. Finally, it was shown that pollination rates are comparatively high in this deceptive orchid, although this is associated with high rates of pollinator-mediated self-pollination.
Plant pollinators and the carrion y assemblage

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The results, which demonstrate pollination of S. pumilum by carrion ies, conrm the predictions made by Vogel (1954) and Johnson (1997) based on plant traits such as the putrid scent and dull brown coloration. Bolus (1893 1896) had already noted that S. pumilum resembles a Stapelia in both its colour and scent, a genus well-known for its carrion ypollinated owers (Meve and Liede, 1994). Apocynaceae pollinaria (in one case identied as Stapelia hirsuta var. hirsuta; P. Bruyns, University of Cape Town, South Africa, pers. comm.) were found attached to several ies which also carried S. pumilum pollinaria. Interestingly, S. pumilum and many Stapelia owers share a tendency to present their owers at ground-level, a trait which we interpret as facilitating pollination by carrion ies which tend to alight on the ground before walking or ying further towards owers. The fact that among all other satyriums this character state is only found in S. pumilum, along with the single occurrence of carrion mimicry, strongly suggests that it is an adaptation in the context of this pollination system (cf. Coddington, 1988). Finally, inorescence stalks of S. pumilum have been repeatedly observed to elongate after owering (T. van der Niet, unpubl. res.; H. Starker, Vienna, Austria, pers. comm.). This reinforces the notion that ground-borne owers are an adaptation that is only benecial during the time of owering. Despite very rarely seeing ies visiting S. pumilum plants in the eld, many ies carrying the orchids pollinaria were caught, mainly in association with carrion. We interpret this as evidence that the ies have visited the plants and are effective pollinators. This is further supported by the arena experiments, which showed that ies pollinate S. pumilum. Finally, the pollinaria carried by the ies caught in the eld match those of S. pumilum, and not of any other of the few co-owering orchid species. One of the most notable ndings of this study was that, in comparison to the full assemblage of ies attracted to real carrion, there was a strong bias in taxonomic composition of carrion ies that pollinated the orchid. All caught ies that carried orchid pollinaria were Sarcophagidae. In contrast, members of this family represented only a small proportion

van der Niet et al. Carrion mimicry in a South African orchid qualitatively (fewer volatiles emitted) compared with the large carrion, we argue that a quantitative explanation of the results is more likely. If the small carrion lacked certain compounds that are essential to attract Calliphoridae and Muscidae, a complete absence of these families during the time that small carrion was exposed would be expected. Instead, only a reduction in the number of these ies attracted to the carrion scent was found. The differential visitation of carrion and plants between sarcophagids and other y families may be related to fundamental differences in life-history traits. Sarcophagidae lay few, live, larvae whereas Calliphoridae lay many eggs (Kamal, 1958; Denno and Cothran, 1976). This allows Sarcophagidae to exploit smaller pieces of carrion as it reduces the amount of intraspecic competition (e.g. Denno and Cothran, 1975; Braack, 1987). Some researchers argue that specialization along carrion size gradients is contentious (e.g. Kuusela and Hanski, 1982; Kneidel, 1984b) and that co-existence between different carrion y families is rather a function of variation in seasonal carrion abundance (Hanski and Kuusela, 1980; Hanski 1987), carrion succession stage (Payne, 1965; Cornaby, 1974; Shi et al., 2009) or resource patchiness (e.g. Hanski, 1987). Several studies including the present one, however, support the notion that Sarcophagidae are proportionally more common on small carrion (e.g. Monnig and Cilliers, 1944; Denno and Cothran, 1975). A nal line of indirect evidence in favour of carrion size as a major determinant of y attraction is a morphological difference between Sarcophagidae and Calliphoridae: Sarcophagidae have more sensory pits in their antennae than Calliphoridae (e.g. Chapman, 1982; Sukonstason et al., 2004). With olfactory reception being the most likely function of these sensory pits (e.g. Sukonstason et al., 2004 and references therein), this difference could explain how Sarcophagidae are better able to detect small quantities of carrion scent (Sukontason et al., 2004). This particularly applies to the females, who have more sensory pits than males (Chapman, 1982; Sukontason et al., 2004). This, in turn, may explain the bias towards females that was found among sarcophagid ies carrying pollinaria, which was also found in other carrion mimicking plants (e.g. Pape and Banziger, 2000; Stensmyr et al., 2002). Ecological niche differentiation among carrion ies may then explain why only Sarcophagidae, and not Calliphoridae, visit S. pumilum. The scent quantity of S. pumilum probably mimics that of small carrion, on which Sarcophagidae proportionally outnumber Calliphoridae while the reverse is found on large carrion. This differentiation may result from the scent quantity of small carrion being too weak to be detected by Calliphoridae under eld conditions. Alternatively, these ies are simply preferentially attracted to stronger carrion scent cues (Spivak et al., 1991). The interaction between S. pumilum and sarcophagid ies can be regarded as highly specialized, especially in the light of the local carrion y assemblage. We do not fully understand, however, why this specialized interaction has evolved. The morphology of S. pumilum requires ies of a certain size for effective contact between the y body and the orchid viscidia (i.e. pollination). A size difference between Sarcophagidae and Calliphoridae, could have driven selective attraction of the

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former family. We found no evidence for this, however, among y families at the study site. Instead of viewing the selective attraction of Sarcophagidae, using oral scent quantity as a lter, as an extreme example of specialization, with a tness benet over a generalized pollination system in which calliphorids and muscids would also be attracted, our observation may rather be the result of a constraint of scent emission rates. As the size of oral parts, scent emission rates and insect attraction can be correlated (Miyake and Yafuso, 2003), owers of S. pumilum (already among the largest in the genus) would have to increase further in size to elevate scent emission, which could lead to a conict with other oral functions such as plant pollinator t.
Attractant cues

The observation that a female sarcophagid deposited live larvae on a S. pumilum ower suggests successful carrion mimicry by the plant. Carrion ies use olfactory and visual cues to nd carrion (Wall and Fisher, 2001), so the expectation is that mimics resemble their models in these aspects. Although visual cues were not explicitly tested in this study, S. pumilum, with its dull maroon-brown coloration (Fig. 1), has the typical appearance of a carrion mimic (e.g. Fgri and van der Pijl, 1979; Beaman et al., 1988; Proctor et al., 1996). Flowers of S. pumilum clearly mimic carrion scent, although the oral scent compounds only represent a subset of the compounds found in carrion. This result is consistent with scent analyses in some carrion-mimicking stapeliads (Jurgens et al., 2006). Stensmyr et al. (2002) found that mainly oligosulde compounds from both carcasses and carrion-mimicking arums elicited strong antennal responses of blowies, even though the blend of both carcass and plants contained several additional compounds. This suggests that for a plant to be a successful carrion mimic, the scent of its owers may need only to contain a few key compounds of the model to attract the operator. Sarcophagid ies were equally attracted to carrion and plant scent. Additionally, a comparison between sarcophagid y species that visit carrion and plants shows strong overlap at the species level (Table 2). Although, theoretically, esh ies may be attracted by different scent compounds in plants and carrion, a phenomenon that has been reported for calliphorid ies (Kaib, 1974), we consider this unlikely. All the compounds in the oral bouquet of S. pumilum (apart from propyl isovalerate, which comprises ,0.14 % of the entire bouquet) are found among two-thirds of the volatiles emitted by the carcass of a rock hyrax, a common mammal across the distribution range of S. pumilum, and whose carcasses are visited by sarcophagid ies. In addition, the most important scent compound in S. pumilum, dimethyl disulde, has been shown to elicit strong antennal responses in carrion frequenting insects (Stensmyr et al., 2002; Kalinova et al., 2009) and, recently, Shuttleworth and Johnson (2010) showed that adding a mix of oligosuldes to a wasp-pollinated plant is sufcient to attract sarcophagid ies. The co-occurrence of oligosuldes and p-cresol + indole in the scent of S. pumilum, respectively, seems at odds with comparative scent patterns found in Araceae (Kite et al., 1998) and stapeliads (Jurgens et al., 2006). In both cases two distinct

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van der Niet et al. Carrion mimicry in a South African orchid relatively simple scent bouquet which only contains compounds that largely overlap with compounds from real carrion. As these compounds are unusual for owers that do not mimic carrion, we presume that they therefore have a functional role in attracting carrion insects. Similarly compoundpoor bouquets were found for carrion-mimicking stapeliads (Jurgens et al., 2006; Johnson and Jurgens, 2010). The absence of non-active compounds may limit the opportunity for insects to learn to associate certain scent cues with the deceptive nature of the orchid, which could prevent avoidance behaviour towards the orchid by experienced individuals (e.g. Kunze and Gumbert, 2001), although this mechanism remains to be tested for ies. Compared with the few studies available that measure levels of outcrossing for deceptive versus rewarding orchids, the proportion of massulae deposited as a result of self-pollination in S. pumilum is more typical for rewarding species than for deceptive species (Jersakova et al., 2006). This means that both pollination success and self-pollination rates of this carrion-mimicking deceptive orchid are closer to the ranges found for rewarding orchids. Johnson (1994) already showed that fruit set in a Batesian orchid mimic was comparable to that of a rewarding species that shared the same pollinator. There are unfortunately very few comparative data on fruit set and pollinator-mediated self-pollination rates available across deceptive pollination systems to arrive at broad-scale generalizations about the evolution of certain mimicry systems with regards to pollination success and outcrossing. The results seem, however, to indicate that deception through carrion mimicry may have evolved for different reasons than to promote outcrossing, which to date is considered one of the major drivers of the evolution of deceit pollination in orchids (Cozzolino and Widmer, 2005; Jersakova et al., 2006). This is further supported by the absence of protandry in S. pumilum, which also promotes outcrossing in orchids (Jersakova and Johnson, 2007). Stigmas are receptive from the rst day after anthesis on the multi-owered inorescences which last for at least 2 weeks. An alternative explanation for the evolution of carrion mimicry, may be Stebbins most effective pollinator principle (Stebbins, 1970) which emphasizes the role of a geographically variable pollinator mosaic in oral evolution (Johnson, 2010). Plants adapt to pollinators which are locally most effective. The arid Karoo region of South Africa, the centre of diversity of many carrion mimics such as the Stapelieae (White and Sloane, 1933), is characterized by low diversity of owering plants and of important pollinator groups such as bees (Kuhlmann, 2009). On the other hand, herbivorous mammals and their carcasses have likely been present historically (Milton et al., 1990), and calliphorid and sarcophagid carrion ies are recorded as being common in the area (Hepburn, 1943). The locally large proportion of ies, compared with other major pollinator groups, may have rendered them relatively effective pollinators, and may have driven the evolution of carrion mimicry in several unrelated plant groups.
Conclusions

scent proles were recognized: scent of plant species dominated by oligosuldes was associated with carrion mimicry, whereas scent of plant species dominated by p-cresol, indole and 2heptanone was associated with dung mimicry. Neither of these studies, however, included the scent of carrion and dung in their analyses. In a recent analysis, Johnson and Jurgens (2010) indeed showed that carrion samples were dominated by oligosuldes, whereas dung samples contained p-cresol and indole. Several plant and stinkhorn fungus samples in their analysis, however, contained a combination of oligosuldes as well as p-cresol and indole, similar to the present ndings. Dekeirsschieter et al. (2009) showed that volatiles emitted by a pig carcass, at several decomposition stages, also included these compounds together, in agreement with the present nding of compounds emitted by rock hyrax carcasses. A partitioning of plant mimicry systems into carrion versus dung mimicry according to scent proles is therefore not yet supported by conclusive evidence and a broader sampling of particularly various kinds of carrion and dung is required. For rewarding plant species, the location of the nectaries often facilitates positioning of insect visitors so as to assure contact with anthers and stigma. Deceptive plant species require alternative mechanisms for insect positioning to effect pollination. Due to the proportionally large size of carrion-mimicking owers (Davis et al., 2008), contacting anthers and stigma is further complicated. Some species have evolved complex trap owers, where insects are guided by olfactory and visual cues towards a trap chamber where pollination takes place while insects are held captive, such as in Aristolochia and Ceropegia (Vogel, 1990; Proctor et al., 1996; Burgess et al., 2004). Carrion-mimicking orchids represent perhaps the most complicated case, as the possession of a gynostemium requires even more precise positioning and renders trap mechanisms often impossible. The present results from the neutral red staining and the scent analysis suggest that correct positioning of the insect with regard to the viscidia and stigma in S. pumilum is achieved by spatial variation in scent production. Flies are thus initially attracted to the entire inorescence as a unit, as it perceives the whole scent bouquet from a distance. Once a y has reached an inorescence, it is strongly attracted to the particular scent that is emitted from the spur region, which could be considered an osmophore (Vogel, 1990). As a y explores a ower and approaches this spur region, it is forced to enter the owers hood, thereby touching the viscidia and/or depositing massulae from previously attached pollinaria on its abdomen, and so effecting pollination (Fig. 1).
Pollination and self-pollination rates

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Pollination rates and fruit set in S. pumilum are high compared with those of other deceptive orchids (Neiland and Wilcock, 1998; Tremblay et al., 2005; Scopece et al., 2010). This is particularly so in comparison with sexually deceptive orchids (Neiland and Wilcock, 1998; Scopece et al., 2010), another system which relies mainly on scent cues for insect attraction (Schiestl, 2005). A key difference between sexually deceptive orchids and S. pumilum may be the presence of a large number of non-active scent compounds in the former group (e.g. Mant et al., 2005). In contrast, S. pumilum has a

We have conclusively shown that, of all insect species visiting carrion, only a very minor proportion pollinated the

van der Niet et al. Carrion mimicry in a South African orchid carrion-mimicking plant species S. pumilum. We argued that this most likely reects niche differentation among carrion insects, with a critical role for scent quantity in brood- and food-site location. One implication of this study is that it may be necessary to reinterpret the sapromyiophilous pollination syndrome from one considered to be generalized for carrion ies to one that may reect several specialized interactions involving different groups of ies. Our study underlines the need to dene specialization in terms of the proportion of animal species in local assemblages that could potentially pollinate owers, and to understand this specicity in terms of the ecology and life history of the insects involved. S U P P L E M E N TARY D ATA Supplementary data are available online at www.aob.oxfordjournals.org and consist of a video showing a sarcophagid y that carries several S. pumilum pollinaria repeatedly entering a S. pumilum ower. AC KN OW L E DG MEN T S We thank Ruth Cozien for extensive help during eld work, including unpleasant experiments involving carrion. We thank Annelise le Roux for providing excellent research facilities at the Succulent Karoo Knowledge Center, and Sarah and Joseph Grootman for facilitating eldwork on communal land. Thomas Pape (Natural History Museum of Denmark) identied Sarcophagidae, Mervyn Mansell (University of Pretoria) identied Calliphoridae, Ray Miller (University of KwaZulu-Natal) identied Calliphoridae and Muscidae. Adam Shuttleworth and Ruth Cozien provided useful comments on an earlier version of this manuscript. L I T E R AT U R E C I T E D
Archer MS, Elgar MA. 2003. Effects of decomposition on carcass attendance in a assemblage of carrion-breeding ies. Medical and Veterinary Entomology 17: 263271. Banziger H, Pape T. 2004. Flowers, faeces and cadavers: natural feeding and laying habits of esh ies in Thailand (Diptera: Sarcophagidae, Sarcophaga spp.). Journal of Natural History 38: 16771694. Beaman RS, Decker PJ, Beaman JH. 1988. Pollination of Rafesia (Rafesiaceae). American Journal of Botany 75: 1148 1162. Blackith RE, Blackith RM. 1990. Insect manifestations of small corpses. Journal of Natural History 24: 699709. Bolus H. 1893 1896. Icones orchidearum austro-africanarum extratropicarum, Vol. 1 (in two parts). London: William Wesley & Son. Born J, Linder HP, Desmet P. 2007. The greater Cape Floristic Region. Journal of Biogeography 34: 147 162. Braack LEO. 1987. Community dynamics of carrion-attendant arthropods in tropical African woodland. Oecologia 72: 402409. Burgess KS, Singeld J, Melendez V, Kevan PG. 2004. Pollination biology of Aristolochia grandiora (Aristolochiaceae) in Veracruz, Mexico. Annals of the Missouri Botanical Garden 91: 346 356. Carvalho LML, Thyssen PJ, Linhares AX, Palhares FAB. 2000. A checklist of arthropods associated with pig carrion and human corpses in southeastern Brazil. Memorias do Instituto Oswaldo Cruz 95: 135 138. Chapman RF. 1982. The insects: structure and function. Cambridge, MA: Harvard University Press. Coddington JA. 1988. Cladistic tests of adaptational hypotheses. Cladistics 4: 3 22. Cornaby BW. 1974. Carrion reduction by animals in contrasting tropical habitats. Biotropica 6: 51 63.

991

Cozzolino S, Widmer A. 2005. Orchid diversity: an evolutionary consequence of deception? Trends in Ecology and Evolution 20: 487494. Dafni A. 1984. Mimicry and deception in pollination. Annual Review of Ecology and Systematics 15: 259 278. Davis CC, Endress PK, Baum DA. 2008. The evolution of oral gigantism. Current Opinion in Plant Biology 11: 4957. Dekeirsschieter J, Verheggen FJ, Gohy M, et al. 2009. Cadaveric volatile organic compounds released by decaying pig carcasses (Sus domesticus L.) in different biotopes. Forensic Science International 189: 46 53. Denno RF, Cothran WR. 1975. Niche relationships of a assemblage of necrophagous ies. Annals of the Entomological Society of America 68: 741754. Denno FR, Cothran WR. 1976. Competitive interactions and ecological strategies of sarcophagid and calliphord ies inhabiting rabbit carrion. Annals of the Entomological Society 69: 109113. rgens A. 2005. Qualitative and quantitative analyses Dotterl S, Wolfe LM, Ju of ower scent in Silene latifolia. Phytochemistry 66: 203213. Fgri K, van der Pijl L. 1979. The principles of pollination ecology. New York, NY: Pergamon. Fisher RA. 1954. Statistical methods for research workers. Edinburgh: Oliver & Boyd. Fuller ME. 1934. The insect inhabitants of carrion: a study in animal ecology. Bulletin of the Council for Scientic and Industrial Research 82: 5 62. Gordin A, Amirav A. 2000. SnifProbe: new method and device for vapor and gas sampling. Journal of Chromatography 903: 155 172. Grassberger M, Frank C. 2004. Initial study of Arthropod succession on pig carrion in a central European urban habitat. Journal of Medical Entomology 41: 511523. Hanski I. 1987. Carrion y community dynamics: patchiness, seasonality, and coexistence. Ecological Entomology 12: 257266. Hanski I, Kuusela S. 1980. The structure of carrion y communities: differences in breeding seasons. Annales Zoologici Fennici 17: 185 190. Hepburn GA. 1943. Sheep blowy research. V. Carcasses as sources of blowies. Onderstepoort Journal of Veterinary Science and Animal Industry 18: 5972. Hewadikaram KA, Goff ML. 1991. Effect of carcass size on rate of decomposition and arthropod succession patterns. The American Journal of Forensic Medicine and Pathology 12: 235 240. Jersakova J, Johnson SD. 2006. Lack of oral nectar reduces self-pollination in a y-pollinated orchid. Oecologia 147: 60 68. Jersakova J, Johnson SD. 2007. Protandry promotes male pollination success in a moth-pollinated orchid. Functional Ecology 21: 496504. Jersakova J, Johnson SD, Kindlmann P. 2006. Mechanisms and evolution of deceptive pollination in orchids. Biological Reviews 81: 219 235. Johnson MD. 1975. Seasonal and microseral variations in the insect populations on carrion. American Midland Naturalist 93: 7990. Johnson SD. 1994. Evidence for Batesian mimicry in a buttery-pollinated orchid. Biological Journal of the Linnean Society 53: 91 104. Johnson SD. 1997. Insect pollination and oral mechanisms in south African species of Satyrium (Orchidaceae). Plant Systematics and Evolution 204: 195206. Johnson SD. 2010. The pollination niche and its role in the diversication and maintenance of the southern African ora. Philosophical Transactions of the Royal Society B Biological Sciences 365: 499516. Johnson SD, Brown M. 2004. Transfer of pollinaria on birds feet: a new pollination system in orchids. Plant Systematics and Evolution 244: 181188. rgens A. 2010. Convergent evolution of carrion and faecal Johnson SD, Ju scent mimicry in y-pollinated owers and a stinkhorn fungus. South African Journal of Botany 76: 796807. Johnson SD, Nilsson LA. 1999. Pollen carryover, geitonogamy, and the evolution of deceptive pollination systems in orchids. Ecology 80: 2607 2619. Johnson SD, Neal PR, Harder LD. 2005. Pollen fates and the limits on male reproductive success in an orchid population. Biological Journal of the Linnean Society 86: 175190. Jurgens A, Dotterl S, Meve U. 2006. The chemical nature of fetid oral scents in stapeliads (Apocynaceae-Asclepoadoideae-Ceropegieae). New Phytologist 172: 452468. Kaib M. 1974. Die Fleisch- und Blumenduftrezeptoren auf der Antenne der Schmeissiege Calliphora vicina. Journal of Comparative Physiology 95: 105 121.

Downloaded from http://aob.oxfordjournals.org/ by guest on May 18, 2012

992

van der Niet et al. Carrion mimicry in a South African orchid

van der Pijl L, Dodson CH. 1966. Orchid owers: their pollination and evolution. Coral Gables, FL: University of Miami Press. Proctor M, Yeo P, Lack A. 1996. The natural history of pollination. Portland, OR: Timber Press. Satoshi S. 2001. Filth ies collected by the late Dr. T. Ohse in Ethiopia and thirteen African countries with record of Dr. Kanos collection in Africa. 2. Calliphoridae and Sarcophagidae. Medical Entomology and Zoology 52: 209 217. Schiestl FP. 2005. On the success of a swindle: pollination by deception in orchids. Naturwissenschaften 92: 255 264. Scopece G, Cozzolino S, Johnson SD, Schiestl FP. 2010. Pollination efciency and the evolution of specialized deceptive pollination systems. American Naturalist 175: 98 105. Shi Y-W, Liu X-S, Wang H-Y, Zhang R-J. 2009. Seasonality of insect succession on exposed rabbit carrion in Guangzhou, China. Insect Science 16: 425 439. Shuttleworth A, Johnson SD. 2010. The missing stink: sulphur compounds can mediate a shift between y and wasp pollination systems. Proceedings of the Royal Society B Biological Sciences 277: 28112819. Sleeman DP, Jones P, Cronin JN. 1997. Investigations of an association between the stinkhorn fungus and badger setts. Journal of Natural History 31: 983992. Sokal RR, Rohlf FJ. 1995. Biometry: the principles and practice of statistics in biological research. New York, NY: W.H. Freeman & Co. Spivak M, Conlon D, Bell WJ. 1991. Wind-guided landing and search behavior in eshies and blowies exploiting a resource patch (Diptera: Sarcophagidae, Calliphoridae). Annals of the Entomological Society of America 84: 447 452. Stebbins GL. 1970. Adaptive radiation of reproductive characteristics in angiosperms. I. Pollination mechanisms. Annual Review of Ecology and Systematics 1: 307 326. Stensmyr MC, Urru I, Collu I, Celander M, Hansson BS, Angioy AM. 2002. Rotting smell of dead-horse arum orets. Nature 420: 625 626. Sukontason K, Sukontason KL, Piangjai S, et al. 2004. Antennal sensilla of some forensically important ies in families Calliphoridae, Sarcophagidae and Muscidae. Micron 35: 671 679. Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN. 2005. Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversication. Biological Journal of the Linnean Society 84: 1 54. Vogel S. 1954. Blutenbiologische Typen als Elemente der Sippengliederung. Jena: Gustav Fischer Verlag. Vogel S. 1990. The role of scent glands in pollination: on the structure and function of osmophores. Washington, DC: Smithsonian Institution Libraries and National Science Foundation. Wall R, Fisher P. 2001. Visual and olfactory cue interaction in resourcelocation by the blowy, Lucilla sericata. Physiological Entomology 26: 212 218. White A, Sloane BL. 1933. The Stapelieae: an introduction to the study of this tribe of Asclepiadaceae. Pasadena, CA: Abbey San Encino Press.

Kalinova B, Podskalska H, Ruzicka J, Hoskovec M. 2009. Irresistible bouquet of death how are burying beetles (Coleoptera: Silphidae: Nicrophorus) attracted by carcasses. Naturwissenschaften 96: 889 899. Kamal AS. 1958. Comparative study of thirteen species of sarcophagous Calliphoridae and Sarcophagidae (Diptera). I. Bionomics. Annals of the Entomological Society 51: 261 271. Kite GC, Hetterscheid WLA, Lewis MJ, et al. 1998. Inorescence scents and pollinators of Arum and Amorphophallus (Araceae). In: Owens SJ, Rudall PJ, eds. Reproductive biology. Kew, Richmond: Royal Botanic Gardens, 295315. Kneidel KA. 1984a. Inuence of carcass taxon and size on species composition of carrion-breeding Diptera. American Midland Naturalist 111: 57 63. Kneidel KA. 1984b. Competition and disturbance in communities of carrionbreeding diptera. Journal of Animal Ecology 53: 849 865. Kuhlmann M. 2009. Patterns of diversity, endemism and distribution of bees (Insecta: Hymenoptera: Anthophila) in southern Africa. South African Journal of Botany 75: 726 738. Kunze J, Gumbert A. 2001. The combined effect of color and odor on ower choice behavior of bumble bees in ower mimicry systems. Behavioral Ecology 12: 447456. Kuusela S, Hanski I. 1982. The structure of carrion y communities: the size and the type of carrion. Holarctic Ecology 5: 337348. Mant J, Peakall R, Schiestl FP. 2005. Does selection on oral odor promote differentiation among populations and species of the sexually deceptive orchid genus Ophrys? Evolution 59: 14491463. Meve U, Liede S. 1994. Floral biology and pollination in stapeliads new results and a literature review. Plant Systematics and Evolution 192: 99 116. Milton SJ, Siegfried WR, Dean WRJ. 1990. The distribution of Epizoochoric plant species: a clue to the prehistoric use of arid Karoo rangelands by large herbivores. Journal of Biogeography 17: 2434. Miyake T, Yafuso M. 2003. Floral scents affect reproductive success in ypollinated Alocasia odora (Araceae). American Journal of Botany 90: 370376. Monnig HO, Cilliers PA. 1944. Sheep blowy research. VII. Investigations in the cape winter-rainfall areas. Onderstepoort Journal of Veterinary Science and Animal Industry 19: 71 77. Neiland MRM, Wilcock CC. 1998. Fruit set, nectar reward, and rarity in the Orchidaceae. American Journal of Botany 85: 16571671. van der Niet T, Linder HP, Bytebier B, Bellstedt DU. 2005. Molecular markers reject monophyly of the subgenera of Satyrium (Orchidaceae). Systematic Botany 30: 263274. Pape T. 1996. Catalogue of the Sarcophagidae of the world (Insecta: Diptera). Memoirs of Entomology International 8: 1 558. Pape T, Banziger H. 2000. Two new species of Sarcophaga (Diptera: Sarcophagidae) among pollinators of newly discovered Sapria ram (Rafesiaceae). The Rafes Bulletin of Zoology 48: 201 208. Payne JA. 1965. A summer carrion study of the baby pig Sus scrofa Linnaeus. Ecology 46: 592602. Peakall R. 1989. A new technique for monitoring pollen ow in orchids. Oecologia 79: 361 365.

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