Elmar Robbrecht

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.

Aluminum Hyperaccumulation in Angiosperms: A Review of Its Phylogenetic Significance

Aluminum phytotoxicity and genetically based aluminum resistance has been studied intensively during recent decades because aluminum toxicity is often the primary factor limiting crop productivity on acid soils. Plants that grow on soils with high aluminum concentrations employ three basic strategies to deal with aluminum stress. While excluders effectively prevent aluminum from entering their aerial parts over a broad range of aluminum concentration in the soil, hyperaccumulators take up aluminum in their aboveground tissues in quantities above 1000 ppm; that is, far exceeding those present in the soil or in the nonaccumulating species growing nearby. In between these two extremes are indicator species, representing intermediate responses.A list of aluminum hyperaccumulators in angiosperms is compiled on the basis of data in the literature. Aluminum hyperaccumulators include mainly woody, perennial taxa from tropical regions. Recent molecular phylogenies are used to evaluate the systematic and phylogenetic implications of the character. As was hypothesized earlier, our preliminary conclusions support the primitive status of aluminum hyperaccumulation. According to the APG classification system, this phytochemical character is found in approximately 45 families, which belong largely to the eudicots. Aluminum hyperaccumulators are particularly common in basal branches of fairly advanced groups such as rosids (Myrtales, Malpighiales, Oxalidales) and asterids (Cornales, Ericales, Gentianales, Aquifoliales), but the character has probably been lost in the most derived taxa. The feature is suggested to characterize approximately 18 families (e.g., Anisophylleaceae, Cunoniaceae, Diapensiaceae, Memecylaceae, Monimiaceae, Rapateaceae, Siparunaceae, Vochysiaceae, and several monogeneric families). In 27 other families, aluminum hyperaccumulation is restricted to subfamilies, tribes, or genera. Further analyses of a broader range of taxa are needed to examine the origin and taxonomic significance of aluminum hyperaccumulation in several clades. Aluminum hyperaccumulation provides an evolutionary model system for the integration of different biological disciplines, such as systematics, ecology, biogeography, physiology, and biochemistry. Therefore, multidisciplinary approaches are needed to make further progress in understanding the biology of aluminum hyperaccumulators.RésuméLa phytotoxicité et la résistance génétique à l’aluminium ont été étudiées intensivement pendant les dernières décennies en raison du rôle important que joue la toxicité à l’aluminium comme facteur limitant la production des plantes sur les terrains acides. Les végétaux des terres acides ayant une haute concentration d’aluminium, survivent grace à trois stratégies. Les plantes à exclusion d’aluminium empêchent l’élément d’entrer dans les tissus aériens à partir d’un sol à fortes concentrations d’aluminium. Les plantes hyperaccumulatrices d’aluminium cependant contiennent une concentration d’aluminium plus haute que 1000 ppm dans leurs tiges et feuilles, dépassant de beaucoup les concentrations du sol ou des plantes avoisinantes nonaccumulatives. Entre ces deux groupes extrèmes, il y a les plantes indicatrices d’aluminium qui ne font aucun effort pour exclure ou accumuler l’aluminium.Nous présentons une liste d’angiospermes hyperaccumulateurs d’aluminium sur base d’une analyse des données de la littérature. Les plantes hyperaccumulatrices sont surtout des plantes ligneuses et pérennes des régions tropicales. Nous utilisons les nouvelles phylogenèses moléculaires pour évaluer la signification systématique et phylogénétique du signal phytochimique. Comme il avait été supposé préalablement, nos conclusions préliminaires confirment le statut primitif de l’hyperaccumulation d’aluminium. Selon le système de classification APG, cette caractéristique phytochimique a été rapportée dans environs 45 familles, qui appartiennent surtout aux eudicots. Les familles hyperaccumulatrices d’aluminium sont surtout présentes dans les branches basales de groupes généralement évolués comme les rosides (Myrtales, Malpighiales, Oxalidales) et les astendes (Cornales, Ericales, Gentianales, Aquifoliales), mais le caractère a probablement disparu dans les groupes les plus dérivés. La caractéristique semble être constante dans presque 18 familles, comme les Anisophylleacées, Cunoniacées, Diapensiacées, Memecylacées, Monimiacées, Rapateacées, Siparunacées, Vochysiacées et quelques familles monogénériques. Dans 27 autres familles, l’hyperaccumulation d’aluminium est limitée aux sous-familles, tribus ou genres. De nouvelles analyses de divers taxa sont nécessaires pour déterminer l’origine et la signification taxonomique dans certains groupes de plantes. Finalement, l’hyperaccumulation d’aluminium est une excellente donnée permettant d’intégrer différentes disciplines biologiques comme la botanique systématique, l’écologie, la biogéographie, la physiologie et la biochimie. Seulement une approche multidisciplinaire permettra de comprendre tous les secrets des plantes qui accumulent l’aluminium.

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.

A survey of the systematic wood anatomy of the Rubiaceae

Recent insight in the phylogeny of the Rubiaceae, mainly based on macromolecular data, agrees better with wood anatomical diversity patterns than previous subdivisions of the family. The two main types of secondary xylem that occur in Rubiaceae show general consistency in their distribution within clades. Wood anatomical characters, especially the fibre type and axial parenchyma distribution, have indeed good taxonomic value in the family. Nevertheless, the application of wood anatomical data in Rubiaceae is more useful in confirming or negating already proposed relationships rather than postulating new affinities for problematic taxa. The wood characterised by fibre-tracheids (type I) is most common, while type II with septate libriform fibres is restricted to some tribes in all three subfamilies. Mineral inclusions in wood also provide valuable information with respect to systematic relationships.

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.

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.

Pollen morphological support for the Catesbaeeae-Chiococceae-Exostema-complex (Rubiaceae)

The pollen morphology of the Catesbaeeae-Chiococceae-Exostema complex as recently treated by Delprete (1966) was examined with LM and SEM. The group is remarkably stenopalynous; typical representatives have medium sized, 3-colpate pollen with a perforate tectum covered with microspines. The inner nexine ornamentation is pronounced and offers more variation than the sexine pattern. A typology of the inside structures is presented based on LM observations and SEM observations of sectioned grains. Orbicules are common and numerous in the Catesbaeeae and Exostema-group; for most genera of the Chiococceae confirmation is needed of orbicule presence. All orbicules observed are relatively large (1-4 μm) and spiny. Pollen and orbicule morphology proved to be a powerful tool to delimit the Catesbaeeae-Chiococceae-Exostema complex. The overall delimitation of the complex is corroborated with our pollen data. The genera Mastixiodendron and Placocarpa, however, can be excluded from the complex based on their pollen m...

Identification, origin, and evolution of leaf nodulating symbionts of Sericanthe (Rubiaceae)

Bacterial leaf symbiosis is an intimate association between bacteria and plants in which endosymbionts are housed within leaf nodules. This phenomenon has been reported in three genera of Rubiaceae (Pavetta, Psychotria, and Sericanthe), but the bacterial partner has only been identified in Psychotria and Pavetta. Here we report the identification of symbiotic bacteria in two leaf nodulating Sericanthe species. Using 16S rRNA data and common housekeeping genetic markers (recA and gyrB) we studied the phylogenetic relationships of bacterial endosymbionts in Rubiaceae. Endosymbionts of leaf nodulating Rubiaceae were found to be closely related and were placed as a monophyletic group within the genus Burkholderia (β-Proteobacteria). The phylogenetic analyses revealed a pattern of strict host specificity and placed the two investigated endosymbionts at two distinct positions in the topology of the tree, suggesting at least two different evolutionary origins. The degree of sequence divergence between the Sericanthe endosymbionts and their relatives was large enough to propose the Sericanthe endosymbionts as new species (‘Candidatus Burkholderia andongensis’ and ‘Candidatus Burkholderia petitii’). In a second part of this study, the pylogenetic relationships among nodulating and non-nodulating Sericanthe species were investigated using sequence data from six chloroplast regions (rps16, trnG, trnL-trnF, petD, petA-psbJ, and atpI-atpH). Overall, genetic variation among the plastid markers was insufficient to enable phylogenetic estimation. However, our results could not rule out the possibility that bacterial leaf symbiosis originated once in a common ancestor of the Sericanthe species.

Wood Anatomy of the Predominantly African Representatives of the Tribe Psychotrieae (Rubiaceae-Rubioideae)

Wood samples of representatives of Chassalia, Chazaliella, Gaertnera, Hymenocoleus, Pagamea and Psychotria are examined. The generic delimitation of these predominantly African Psychotrieae, which is mainly based on fruit morphology, is compared with wood anatomical variation patterns. Part of the variation observed is related to habit, e. g. wide vessels in the tree species Psychotria dermatophylla. Other features do have systematic significance, as shown by a cluster analysis of the data obtained. The genus pair Gaertnera/Pagamea differs obviously from the other genera and is wood anatomically clearly distinguished by the presence of fibre-tracheids and parenchyma bands. Chassalia, Chazaliella, Hymenocoleus and Psychotria have rather similar wood structure, although variation in vessel diameter, vessel arrangement, ray composition and axial parenchyma occurs. Several uncommon features are recorded: the presence of few to numerous openings in one oblique perforation plate, irregular reticulate perforation plates and multiple vessel-ray perforations with marked irregularity.