Steven Dessein

Distribution and evolution of circular miniproteins in flowering plants

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids (∼125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

CARNOY: A new digital measurement tool for palynology

Quantitative data play an important role in palynological research. With the advent of digital imaging in light and electron microscopy, palynologists now have the opportunity to perform measurements faster and more precisely than ever before. Several image analysis software packages already exist for these tasks, but they are often expensive, difficult to use or not adapted to the specific needs of palynologists. After studying the daily workflow of a palynologist, we designed CARNOY, an image analysis application written from the ground up for use in palynology and morphology. CARNOY offers an easy-to-use interface and several features to make measuring easier and faster. The program can export measurements to almost every other software package for further analysis and is available for free on the Internet.

Cyclotide Discovery in Gentianales Revisited—Identification and Characterization of Cyclic Cystine-Knot Peptides and Their Phylogenetic Distribution in Rubiaceae Plants

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.

Phylogeny of the herbaceous tribe Spermacoceae (Rubiaceae) based on plastid DNA data

Abstract In its current circumscription, the herbaceous tribe Spermacoceae s.l. (Rubiaceae, Rubioideae) unites the former tribes Spermacoceae s. str., Manettieae, and the Hedyotis–Oldenlandia group. Within Spermacoceae, and particularly within the Hedyotis–Oldenlandia group, the generic delimitations are problematic. Up until now, molecular studies have focused on specific taxonomic problems within the tribe. This study is the first to address phylogenetic relationships within Spermacoceae from a tribal perspective. Sequences of three plastid markers (atpB-rbcL, rps16, and trnL-trnF) were analyzed separately as well as combined using parsimony and Bayesian approaches. Our results support the expanded tribe Spermacoceae as monophyletic. The former tribe Spermacoceae s. str. forms a monophyletic clade nested within the Hedyotis–Oldenlandia group. Several genera formerly recognized within the Hedyotis–Oldenlandia group are supported as monophyletic (Amphiasma Bremek., Arcytophyllum Willd. ex Schult. & Schult. f., Dentella J. R. Forst. & G. Forst., Kadua Cham. & Schltdl., and Phylohydrax Puff), while others appear to be paraphyletic (e.g., Agathisanthemum Klotzsch), biphyletic (Kohautia Cham. & Schltdl.), or polyphyletic (Hedyotis L. and Oldenlandia L. sensu Bremekamp). Morphological investigations of the taxa are ongoing in order to find support for the many new clades and relationships detected. This study provides a phylogenetic hypothesis with broad sampling across the major lineages of Spermacoceae that can be used to guide future species-level and generic studies.

Towards a new classification of the Arthoniales (Ascomycota) based on a three-gene phylogeny focussing on the genus Opegrapha

A multi-locus phylogenetic study of the order Arthoniales is presented here using the nuclear ribosomal large subunit (nuLSU), the second largest subunit of RNA polymerase II (RPB2) and the mitochondrial ribosomal small subunit (mtSSU). These genes were sequenced from 43 specimens or culture isolates representing 33 species from this order, 16 of which were from the second largest genus, Opegrapha. With the inclusion of sequences from GenBank, ten genera and 35 species are included in this study, representing about 18% of the genera and ca 3% of the species of this order. Our study revealed the homoplastic nature of morphological characters traditionally used to circumscribe genera within the Arthoniales, such as exciple carbonization and ascomatal structure. The genus Opegrapha appears polyphyletic, species of that genus being nested in all the major clades identified within Arthoniales. The transfer of O. atra and O. calcarea to the genus Arthonia will allow this genus and family Arthoniaceae to be recognized as monophyletic. The genus Enterographa was also found to be polyphyletic. Therefore, the following new combinations are needed: Arthonia calcarea (basionym: O. calcarea), and O. anguinella (basionym: Stigmatidium anguinellum); and the use of the names A. atra and Enterographa zonata are proposed here. The simultaneous use of a mitochondrial gene and two nuclear genes led to the detection of what seems to be a case of introgression of a mitochondrion from one species to another (mitochondrion capture; cytoplasmic gene flow) resulting from hybridization.

Palynological characters and their phylogenetic signal in Rubiaceae

In the 1990s Rubiaceae became a hot spot for systematists, mainly due to the comprehensive treatment of the family by Robbrecht in 1988. Next to the exploration of macromolecular characters to infer the phylogeny, the palynology of Rubiaceae finally received the attention it deserves. This article aims to present a state-of-the-art analysis of the systematic palynology of the family. The range of varíation in pollen morphology is wide, and some of the pollen features are not known from other angiosperm taxa; e.g., a looplike or spiral pattern for the position of apertures in pantoaperturate grains. We compiled an online database at the generic level for the major pollen characters and orbicule presence in Rubiaceae. An overview of the variation is presented here and illustrated per character: dispersal unit, pollen size and shape, aperture number, position and type, sexine ornamentation, nexine pattern, and stratification of the sporoderm. The presence/absence and morphological variation of orbicules at the generic level is provided as well. The systematic usefulness of pollen morphology in Rubiaceae is discussed at the (sub)family, tribal, generic, and infraspecific levels, using up-to-date evolutionary hypotheses for the different lineages in the family. The problems and opportunities of coding pollen characters for cladistic analyses are also treated.

Pollen morphology of NW European representatives confirms monophyly of Rubieae (Rubiaceae)

Abstract This study focuses on the six genera of Rubieae that occur in NW Europe: Asperula, Crucianella, Cruciata, Galium, Rubia, and Sherardia. The pollen morphology of 29 species was studied using light microscope and scanning electron microscope observations. Several features demonstrate the advanced nature of the tribe: simple apertures, up to 13 ectocolpi, supratectal microspines, a characteristic and unique endopattern, and absence of orbicules. Asperula, Cruciata, Galium, and Rubia cannot be distinguished by pollen morphology only. The monospecific genus Sherardia can be recognized easily by the high numbers of apertures (10–13) that are slit-like and the very fine perforations in the tectum. Some Crucianella species have relatively larger perforations in the tectum. None of the species investigated produces orbicules, which makes Rubieae, next to Gardenieae, the second rubiaceous tribe entirely without orbicules. Our palynological data are interpreted in the broader perspective of all herbaceous Rubiaceae. The Rubieae are unique among Rubiaceae in the combination of the following pollen features: several colpate apertures, a perforate and microechinate tectum, a relatively small size, the absence of endoapertures, a coarse nexine area beneath the ectocolpi, and the absence of orbicules. The predictive value of pollen morphology is therefore extremely high at the tribal level and supports the monophyly of Rubieae.

Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspects

Pollen morphology of 43 African species of the genus Spermacoce has been investigated by scanning electron and light microscopy. The genus is eurypalynous, which is reflected in the remarkable variation of almost all pollen characters. The average equatorial diameter (E) ranges from 15.8 w m to 115.5 w m. Grains are colporate or pororate. The number of apertures varies from 3 up to more than 25. The majority of species has apertures situated only at the equator (being zonoaperturate), but a few species have pantoaperturate grains. The endoaperture is generally an endocingulum, often with a secondary lolongate or lalongate thinning at the ectocolpus; endocolpi and endopores are also observed. The sexine is usually perforate, but eutectate, foveolate, and (micro)reticulate tecta were also found. Supratectal elements are present as granules, microspines or spines. The inner nexine surface is granular, often with irregular grooves (endocracks). Among native African species, nine pollen types are recognized mainly on the basis of pollen size, aperture morphology and tectum peculiarities. In two of the pantoaperturate types, apertures are in a configuration not yet recorded for the angiosperms in general. Some evolutionary trends are proposed that await verification by further systematic study. Pollen morphological characters have a high taxonomic value in the genus Spermacoce . They provide almost unique identification marks for the species, which enables sharpening of species boundaries. Small groups of related species often share the same pollen type. The genus Borreria , previously separated from Spermacoce on the basis of its fruit morphology only, is not supported by pollen data.

Endophytic Bacteria in Toxic South African Plants: Identification, Phylogeny and Possible Involvement in Gousiekte

Background South African plant species of the genera Fadogia, Pavetta and Vangueria (all belonging to Rubiaceae) are known to cause gousiekte (literally ‘quick disease’), a fatal cardiotoxicosis of ruminants characterised by acute heart failure four to eight weeks after ingestion. Noteworthy is that all these plants harbour endophytes in their leaves: nodulating bacteria in specialized nodules in Pavetta and non-nodulating bacteria in the intercellular spaces between mesophyll cells in Fadogia and Vangueria. Principal Findings Isolation and analyses of these endophytes reveal the presence of Burkholderia bacteria in all the plant species implicated in gousiekte. Although the nodulating and non-nodulating bacteria belong to the same genus, they are phylogenetically not closely related and even fall in different bacterial clades. Pavetta harborii and Pavetta schumanniana have their own specific endophyte – Candidatus Burkholderia harborii and Candidatus Burkholderia schumanniana – while the non-nodulating bacteria found in the other gousiekte-inducing plants show high similarity to Burkholderia caledonica. In this group, the bacteria are host specific at population level. Investigation of gousiekte-inducing plants from other African countries resulted in the discovery of the same endophytes. Several other plants of the genera Afrocanthium, Canthium, Keetia, Psydrax, Pygmaeothamnus and Pyrostria were tested and were found to lack bacterial endophytes. Conclusions The discovery and identification of Burkholderia bacteria in gousiekte-inducing plants open new perspectives and opportunities for research not only into the cause of this economically important disease, but also into the evolution and functional significance of bacterial endosymbiosis in Rubiaceae. Other South African Rubiaceae that grow in the same area as the gousiekte-inducing plants were found to lack bacterial endophytes which suggests a link between bacteria and gousiekte. The same bacteria are consistently found in gousiekte-inducing plants from different regions indicating that these plants will also be toxic to ruminants in other African countries.

Phylogeny of tricalysia (rubiaceae) and its relationships with allied genera based on plastid dna data: resurrection of the genus empogona

Abstract Recent studies on the circumscription of the tribe Coffeeae (Rubiaceae) revealed a weakly supported clade containing Tricalysia A. Rich. and the allied genera Argocoffeopsis Lebrun, Calycosiphonia Pierre ex Robbr., Belonophora Hook. f., Diplospora DC., Discospermum Dalzell, Nostolachma T. Durand, and Xantonnea Pierre ex Pit. The phylogenetic relationships of Tricalysia and these allied taxa are investigated further using sequence data from four plastid regions (trnL-F intron and intergenic spacer, rpL16 intron, accD-psa1 intergenic spacer, and PetD). Our results demonstrate that Tricalysia sensu Robbrecht is not monophyletic. The genus name Tricalysia should be restricted to taxa from subgenus Tricalysia; subgenus Empogona (Hook. f.) Robbr. is sister to the genus Diplospora and is recognized at the generic level. The 34 necessary new combinations for Empogona Hook. f. are provided: E. acidophylla (Robbr.) J. Tosh & Robbr., E. aequatoria (Robbr.) J. Tosh & Robbr., E. africana (Sim) J. Tosh & Robbr., E. aulacosperma (Robbr.) J. Tosh & Robbr., E. bequaertii (De Wild.) J. Tosh & Robbr., E. bracteata (Hiern) J. Tosh & Robbr., E. breteleri (Robbr.) J. Tosh & Robbr., E. buxifolia (Hiern) J. Tosh & Robbr. subsp. buxifolia, E. buxifolia subsp. australis (Robbr.) J. Tosh & Robbr., E. cacondensis (Hiern) J. Tosh & Robbr., E. concolor (N. Hallé) J. Tosh & Robbr., E. coriacea (Sond.) J. Tosh & Robbr., E. crepiniana (De Wild. & T. Durand) J. Tosh & Robbr., E. deightonii (Brenan) J. Tosh & Robbr., E. discolor (Brenan) J. Tosh & Robbr., E. filiformistipulata (De Wild.) Bremek. subsp. filiformistipulata, E. filiformistipulata subsp. epipsila (Robbr.) J. Tosh & Robbr., E. glabra (K. Schum.) J. Tosh & Robbr., E. gossweileri (S. Moore) J. Tosh & Robbr., E. kirkii Hook. f. subsp. junodii (Schinz) J. Tosh & Robbr., E. lanceolata (Sond.) J. Tosh & Robbr., E. macrophylla (K. Schum.) J. Tosh & Robbr., E. maputenis (Bridson & A. E. van Wyk) J. Tosh & Robbr., E. ngalaensis (Robbr.) J. Tosh & Robbr., E. nogueirae (Robbr.) J. Tosh & Robbr., E. ovalifolia (Hiern) J. Tosh & Robbr. var. ovalifolia, E. ovalifolia var. glabrata (Oliv.) J. Tosh & Robbr., E. ovalifolia var. taylorii (S. Moore) J. Tosh & Robbr., E. reflexa (Hutch.) J. Tosh & Robbr. var. reflexa, E. reflexa var. ivorensis (Robbr.) J. Tosh & Robbr., E. ruandensis (Bremek.) J. Tosh & Robbr., E. somaliensis (Robbr.) J. Tosh & Robbr., E. talbotii (Wernham) J. Tosh & Robbr., and E. welwitschii (K. Schum.) J. Tosh & Robbr.