Ceropegia species (Apocynaceae) have deceptive pitfall flowers and exploit small flies as pollinators, supposedly by chemical mimicry. Only preliminary data on the composition of flower scents are available for a single species so far, and the mimicry system is not yet understood in any species. We collected data on basic pollination aspects of C. dolichophylla, analyzed floral scent by gas chromatography linked to mass spectrometry, identified electrophysiologically active scent components by gas chromatography coupled with electroantennographic detection, and determined compounds responsible for pollinator attraction in bioassays. We found that flowers of C. dolichophylla are visited by small flies of several taxa. Only Milichiidae and Chloropidae carried pollinaria and are, thus, pollinators. The pollen transfer efficiency at two different sites was 2 % and 4 %, respectively. The floral scent was dominated by spiroacetals, mainly (2S,6R,8S)-8-methyl-2-propyl-1,7-dioxaspiro[5.5]undecane, n-tridecane, and N-(3-methylbutyl)acetamide. This spiroacetal and the acetamide elicited the most intense electrophysiological responses in fly antennae, and bioassays confirmed the capability of the spiroacetal in eliciting behavioral responses in pollinators. Most flies, determined as pollinators of C. dolichophylla, are kleptoparasites. They exploit insect prey of predatory arthropods as food source to which they are attracted by volatiles. 8-Methyl-2-propyl-1,7-dioxaspiro[5.5]undecane and N-(3-methylbutyl)acetamide have not been identified before as volatiles of other plants, however, they are known as insect volatiles. Both compounds occur in the venom glands of paper wasps, a potential food source for the pollinators of C. dolichophylla. We propose that C. dolichophylla shows a kleptomyiophilous pollination strategy. It mimics insect related odors to exploit the food-seeking behavior of its kleptoparasitic pollinators.

The first-branching lineages of the tribe Ceropegieae, Anisotominae, Leptadeniinae, and Heterostemminae, are the focus of the present study. In the ingroup we molecularly analysed 34 samples from southern and (north)eastern Africa and Asia for the nuclear internal transcribed spacer (ITS) region, and five plastid markers (trnT-L, trnL-F and trnH-psbA intergenic spacers, trnL, and rps16 introns). Maximum Parsimony and Bayesian analyses were conducted for phylogenetic reconstruction and Bayesian binary MCMC (BBM) analysis as implemented in RASP for testing biogeographic inference. Sister to the well-known hyperdiverse subtribe Stapeliinae (around 700 species) are the Anisotominae, an African subtribe of c. 30 species, with the tropical Neoschumannia in sister-group position to the remaining genera, Anisotoma, Emplectanthus, Riocreuxia, and Sisyranthus. Emplectanthus is sequenced in full for the first time, and its monophyly as sister to Anisotoma is documented. As in Sisyranthus, new sequences in Riocreuxia, especially in Riocreuxia torulosa s.l., reveal very low genetic variation pointing to an actively radiating and speciating group, possibly in adaptation to pollinator pressure. Molecular and morphological data necessitate the transfer of Brachystelma natalense to Anisotoma, and the new combination Anisotoma natalensis is proposed. Leptadeniinae are sister to the Stapeliinae–Anisotominae clade. The position of the strictly Asian, rheophytic Pentasachme could be resolved as sister to the remaining genera of Leptadeniinae, Conomitra, Leptadenia, and Orthanthera. The position of the S Asian/Australasian wet forest lianas of the genus Heterostemma as sister to the remaining Ceropegieae is confirmed, indicating a humid Asian origin for this tribe known for its arid-adapted African succulents.

Summary: Kleptomyiophily, where flowers imitate wounded insects to attract ‘kleptoparasitic’ flies as pollinators, is one of the most specialized types of floral mimicry and often involves physical trapping devices. However, the diversity of pollinators and functional floral traits involved in this form of mimicry remain poorly understood. We report a novel example of kleptomyiophily in the nontrapping flowers of Ceropegia gerrardii and explore the floral traits responsible for attracting pollinators. The pollinators, reproductive biology and floral traits (including epidermal surfaces, spectral reflectance and the composition of nectariferous petal secretions and scent) were investigated. Attractive volatiles were identified using electrophysiological and behavioural experiments. Ceropegia gerrardii was predominantly pollinated by kleptoparasitic Desmometopa spp. (Milichiidae) flies. The flower corollas extrude a protein‐ and sugar‐containing secretion, similar to the haemolymph of wounded insects, on which the flies feed. Floral scent was chemically similar to that of injured honey bees. Four out of 24 electrophysiologically active compounds, all released by injured honey bees, were identified as key players in pollinator attraction. Our results suggest that C. gerrardii flowers chemically mimic wounded honey bees to attract kleptoparasitic flies and reward them with a secretion similar to the haemolymph on which they would normally feed.

In specialized pollination systems, floral scents are crucial for flower–pollinator communication, but key volatiles that attract pollinators are unknown for most systems. Deceptive Ceropegia trap flowers are famous for their elaborate mechanisms to trap flies. Recent studies revealed species-specific floral chemistry suggesting highly specialized mimicry strategies. However, volatiles involved in fly attraction were until now identified in C. dolichophylla and C. sandersonii, only. We here present data on C. stenantha for which flower scent and pollinators were recently described, but volatiles involved in flower–fly communication stayed unknown. We performed electrophysiological measurements with scatopsid fly pollinators (Coboldia fuscipes) and identified 12 out of 13 biologically active floral components. Among these volatiles some were never described from any organism but C. stenantha. We synthesized these components, tested them on antennae of male and female flies, and confirmed their biological activity. Overall, our data show that half of the volatiles emitted from C. stenantha flowers are perceived by male and female fly pollinators and are potentially important for flower–fly communication in this pollination system. Further studies are needed to clarify the role of the electrophysiologically active components in the life of scatopsid fly pollinators, and to fully understand the pollination strategy of C. stenantha.

Deceptive Ceropegia pitfall flowers are an outstanding example of synorganized morphological complexity. Floral organs functionally synergise to trap fly-pollinators inside the fused corolla. Successful pollination requires precise positioning of flies headfirst into cavities at the gynostegium. These cavities are formed by the corona, a specialized organ of corolline and/or staminal origin. The interplay of floral organs to achieve pollination is well studied but their evolutionary origin is still unclear. We aimed to obtain more insight in the homology of the corona and therefore investigated floral anatomy, ontogeny, vascularization, and differential MADS-box gene expression in Ceropegia sandersonii using X-ray microtomography, Light and Scanning Electronic Microscopy, and RT-PCR. During 10 defined developmental phases, the corona appears in phase 7 at the base of the stamens and was not found to be vascularized. A floral reference transcriptome was generated and 14 MADS-box gene homologs, representing all major MADS-box gene classes, were identified. B- and C-class gene expression was found in mature coronas. Our results indicate staminal origin of the corona, and we propose a first ABCDE-model for floral organ identity in Ceropegia to lay the foundation for a better understanding of the molecular background of pitfall flower evolution in Apocynaceae.

Elaborated kettle trap flowers to temporarily detain pollinators evolved independently in several angiosperm lineages. Intensive research on species of Aristolochia and Ceropegia recently illuminated how these specialized trap flowers attract particular pollinators through chemical deception. Morphologically similar trap flowers evolved in Riocreuxia; however, no data about floral rewards, pollinators, and chemical ecology were available for this plant group. Here we provide data on pollination ecology and floral chemistry of R. torulosa. Specifically, we determined flower visitors and pollinators, assessed pollen transfer efficiency, and analysed floral scent chemistry. R. torulosa flowers are myiophilous and predominantly pollinated by Nematocera. Pollinating Diptera included, in order of decreasing abundance, male and female Sciaridae, Ceratopogonidae, Scatopsidae, Chloropidae, and Phoridae. Approximately 16% of pollen removed from flowers was successfully exported to conspecific stigmas. The flowers emitted mainly ubiquitous terpenoids, most abundantly linalool, furanoid (Z)-linalool oxide, and (E)-β-ocimene—compounds typical of rewarding flowers and fruits. R. torulosa can be considered to use generalized food (and possibly also brood-site) deception to lure small nematocerous Diptera into their flowers. These results suggest that R. torulosa has a less specific pollination system than previously reported for other kettle trap flowers but is nevertheless specialized at the level of Diptera suborder Nematocera.