Cyclotides are a unique class of ribosomally synthesized cysteine‐rich miniproteins characterized by a head‐to‐tail cyclized backbone and three conserved disulfide‐bonds in a knotted arrangement. Originally they were discovered in the coffee‐family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide‐containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome‐mining, we identified nine novel cyclotide‐containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 438–452, 2013.

Summary: Flowers have been hypothesized to contain either modules of attraction and reproduction, functional modules (pollination‐effecting parts) or developmental modules (organ‐specific). Do pollination specialization and syndromes influence floral modularity? In order to test these hypotheses and answer this question, we focused on the genusErica: we gathered 3D data from flowers of 19 species with diverse syndromes via computed tomography, and for the first time tested the above‐mentioned hypotheses via 3D geometric morphometrics. To provide an evolutionary framework for our results, we tested the evolutionary mode of floral shape, size and integration under the syndromes regime, and – for the first time – reconstructed the high‐dimensional floral shape of their most recent common ancestor. We demonstrate that the modularity of the 3D shape of generalist flowers depends on development and that of specialists is linked to function: modules of pollen deposition and receipt in bird syndrome, and access‐restriction to the floral reward in long‐proboscid fly syndrome. Only size and shape principal component 1 showed multiple‐optima selection, suggesting that they were co‐opted during evolution to adapt flowers to novel pollinators. Whole floral shape followed an Ornstein–Uhlenbeck (selection‐driven) evolutionary model, and differentiated relatively late. Flower shape modularity thus crucially depends on pollinator specialization and syndrome.

Inflorescences of Araceae pollinated by cyclocephaline scarab beetles are visited frequently by a wide array of other arthropods that exploit floral resources without taking part in pollination, including earwigs, flies, and true bugs. To date, nothing is known about the cues these insect visitors use to locate the inflorescences and whether or to what extent floral scents play a role. An aroid visited by large numbers of plant bugs (Miridae) in addition to cyclocephaline scarab beetle pollinators is the Neotropical species Dieffenbachia aurantiaca. We identified the plant bug species and investigated their behavior and arrival time on the inflorescences. To test the importance of olfactory cues in locating their host we conducted experiments with open and gauze-bagged inflorescences as well as natural scent samples of D. aurantiaca. Inflorescence scents were analyzed by gas chromatography linked to mass spectrometry (GC/MS), and the attractive potential of the main scent compound was determined by behavioral assays. Three species of Neella, the most common one being N. floridula, visited the inflorescences at nightfall, shortly after the beginning of scent emission, and showed feeding and copulation activity. Bagged inflorescences as well as natural scent samples attracted similar numbers of plant bugs as the non-bagged inflorescences, showing that olfactory cues are sufficient for them to locate their host. Cis-jasmone was the major component within the inflorescence scent bouquet. In two-choice field bioassays, this compound proved to be highly attractive to Neella, and thus obviously plays a key role in finding host plants.