From forgotten taxon to a missing link? The position of the genus Verhuellia (piperaceae) revealed by molecules.

BACKGROUND AND AIMS: The species-poor and little-studied genus Verhuellia has often been treated as a synonym of the genus Peperomia, downplaying its significance in the relationships and evolutionary aspects in Piperaceae and Piperales. The lack of knowledge concerning Verhuellia is largely due to its restricted distribution, poorly known collection localities, limited availability in herbaria and absence in botanical gardens and lack of material suitable for molecular phylogenetic studies until recently. Because Verhuellia has some of the most reduced flowers in Piperales, the reconstruction of floral evolution which shows strong trends towards reduction in all lineages needs to be revised. METHODS: Verhuellia is included in a molecular phylogenetic analysis of Piperales (trnT-trnL-trnF and trnK/matK), based on nearly 6000 aligned characters and more than 1400 potentially parsimony-informative sites which were partly generated for the present study. Character states for stamen and carpel number are mapped on the combined molecular tree to reconstruct the ancestral states. KEY RESULTS: The genus Peperomia is generally considered to have the most reduced flowers in Piperales but this study shows that this is only partially true. Verhuellia, with almost equally reduced flowers, is not part of or sister to Peperomia as expected, but is revealed as sister to all other Piperaceae in all analyses, putting character evolution in this family and in the perianthless Piperales in a different light. A robust phylogenetic analysis including all relevant taxa is presented as a framework for inferring patterns and processes of evolution in Piperales and Piperaceae. CONCLUSIONS: Verhuellia is a further example of how a molecular phylogenetic study can elucidate the relationships of an unplaced taxon. When more material becomes available, it will be possible to investigate character evolution in Piperales more thoroughly and to answer some evolutionary questions concerning Piperaceae.

L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition.

Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2 nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFalpha formation in whole blood (IC50 = 161 nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNgamma and no detectable inhibition of IL-13 formation up to 1 microM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.

The floral scales in Hellmuthia (Cyperaceae, Cyperoideae) and Paramapania (Cyperaceae, Mapanioideae): an ontogenetic study.

BACKGROUND AND AIMS: In 1976 the monotypic genus Hellmuthia was placed in the Hypolytreae s.l., but was subsequently ascribed to the Mapanioideae, tribe Chrysitricheae, mainly because of the presence in Hellmuthia of two lateral, mapanioid-like floral scales with ciliated keels, the anatomy of the nutlet, the embryo and the inflorescence. Recently, based on cladistic analyses and supported by pollen ontogenetic evidence, Hellmuthia was transferred to a Cyperaceae, tribe Cypereae, clade mainly consisting of Ficinia and Isolepis. In this study, the floral ontogeny in Hellmuthia was investigated and compared with the floral ontogeny in Paramapania, with special attention for the floral scales. METHODS: Freshly collected inflorescences of Hellmuthia membranacea and Paramapania parvibractea were investigated using scanning electron and light microscopy. KEY RESULTS: In the conical 'spikelet' in Hellmuthia, proximal bracts occur, each axillating an axis with empty glume-like structures, or a reduced spikelet. Hence, it is a reduced partial inflorescence. In Hellmuthia, the stamen primordia originate before the primordia of the perianth-gynoecium appear. Moreover, a third adaxially positioned 'floral scale' was observed for the first time. The position and relative time of appearance of the floral scales in Hellmuthia are typical for perianth parts in Cyperoideae. The basal position of Hellmuthia within a clade of species with usually perianthless flowers, allows the presence of rudiments of a perianth in Hellmuthia to be interpreted as a primitive state. Development of the lateral 'scales' in Paramapania follows a different pattern. Therefore, it was decided that the lateral 'scales' in Paramapania are different from the lateral perianth parts in Hellmuthia. The pollen grains in Hellmuthia are cyperoid, with one polar and five lateral apertures, of which the membrane is covered with sexinous bodies. The pollen surface is granulate and perforate with microspines. CONCLUSIONS: The floral ontogeny in Hellmuthia occurs according to the general cyperoid pattern. The lateral scales in Hellmuthia are perianth parts, and they are not homologous to the lateral 'scales' in Paramapania.

Evidence of natural hybridization and introgression between Vasconcellea species (Caricaceae) from southern Ecuador revealed by chloroplast, mitochondrial and nuclear DNA markers.

BACKGROUND AND AIMS: Vasconcellea x heilbornii is believed to be of natural hybrid origin between V. cundinamarcensis and V. stipulata, and is often difficult to discriminate from V. stipulata on morphological grounds. The aim of this paper is to examine individuals of these three taxa and of individuals from the closely related species V. parviflora and V. weberbaueri, which all inhabit a hybrid zone in southern Ecuador. METHODS: Molecular data from mitochondrial, chloroplast and nuclear DNA from 61 individuals were analysed. KEY RESULTS: Molecular analysis confirmed occasional contemporary hybridization between V. stipulata, V. cundinamarcensis and V. x heilbornii and suggested the possible involvement of V. weberbaueri in the origin of V. x heilbornii. In addition, the molecular data indicated unidirectional introgression of the V. cundinamarcensis nuclear genome into that of V. stipulata. Several of the individuals examined with morphology similar to that of V. stipulata had genetic traces of hybridization with V. cundinamarcensis, which only seems to act as pollen donor in interspecific hybridization events. Molecular analyses also strongly suggested that most of the V. x heilbornii individuals are not F(1) hybrids but instead are progeny of repeated backcrosses with V. stipulata. CONCLUSIONS: The results of the present study point to the need for re-evaluation of natural populations of V. stipulata and V. x heilbornii. In general, this analysis demonstrates the complex patterns of genetic and morphological diversity found in natural plant hybrid zones.

Phylogeny of the genus Peperomia (Piperaceae) inferred from the trnK/matK region (cpDNA).

The genus Peperomia is one of the largest genera of basal angiosperms, comprising about 1500-1700 pantropically distributed species. The currently accepted infrageneric classification divides Peperomia into nine subgenera and seven sections. This classification is based on some 200 species, primarily using fruit morphology. The monophyly of these infrageneric taxa has never been tested and molecular phylogenetic studies of a representative sampling within Peperomia do not exist. This paper provides the first molecular phylogeny for the genus Peperomia. Monophyletic clades within Peperomia are identified and previously used morphological characters are critically reviewed. We show that the importance of some morphological characters has been overestimated and that some of these characters presumably have evolved several times independently. Only one previously described subgenus has been confirmed to be monophyletic.

Floral ontogeny in ficinia and isolepis (cyperaceae), with focus on the nature and origin of the gynophore.

BACKGROUND AND AIMS: The generic delimitations of Ficinia and Isolepis, sister genera in the Cypereae, are blurred. Typical Ficinia flowers have a lobed gynophore, which envelops the base of the nutlet, whereas in Isolepis the character is considered to be absent. Some former species of Isolepis, lacking the gynophore, were recently included in Ficinia. The floral ontogeny of representative taxa in Ficinia and Isolepis were investigated with the aim of evaluating the origin and nature of the gynophore in the Cypereae. METHODS: The spikelet and floral ontogeny in inflorescences collected in the field was investigated using scanning electron microscopy (SEM) and light microscopy (LM). KEY RESULTS: SEM images of Isolepis setacea and I. antarctica, Ficinia brevifolia, F. minutiflora, F. zeyheri and F. gracilis, and LM sections of F. radiata, show that the gynoecium in Ficinia is elevated above the flower receptacle by the development of a hypogynous stalk. From its apex, a (often three-)lobed cup is formed, which envelopes the basal part of the later nutlet. In developing flowers of I. antarctica, a rudimentary hypogynous stalk appears. In I. setacea, rudiments of a hypogynous stalk can be observed at maturity. In F. radiata and F. zeyheri, intralocular hairs are present in the micropylar zone. At the surface of developing gynoecia in flowers of F. gracilis, star-shaped cuticular structures appear which disappear again at maturity. CONCLUSIONS: The overall floral ontogeny of all species studied occurs following a typical scirpoid pattern, though no perianth primordia are formed. The gynophore in Ficinia originates as a hypogynous stalk, from which the typical gynophore lobes develop. The gynophore is not homologous with the perianth.

Floral ontogeny in Scirpus, Eriophorum and Dulichium (Cyperaceae), with special reference to the perianth.

BACKGROUND AND AIMS: Based on molecular phylogenetic analysis, it has been suggested recently that the Cyperaceae comprises only two subfamilies: the Mapanioideae and the Cyperoideae. In most flowers of the Cyperoideae, the whorl of inner stamens is reduced, resulting in tetracyclic flowers. In the more primitive (scirpoid) genera within the Cyperoideae, the perianth consists of two polysymmetric whorls, whereas the perianth parts in the more derived genera have been subject to modifications and/or reduction. Comparative studies of the many silky hairs of Eriophorum and of the eight bristles of Dulichium have given rise to much discussion about their homology. METHODS: The spikelet and floral ontogeny in freshly collected inflorescences was investigated using scanning electron microscopy. KEY RESULTS: Complete floral ontogenies are presented for Scirpus sylvaticus L., Eriophorum latifolium Hoppe and Dulichium arundinaceum (L.) Britton, with special reference to the perianth. The results in S. sylvaticus confirm the trimerous monocot-like organization of the flower. It is used as a model for floral development in Cyperoideae. In the early developmental stages, the androecium of E. latifolium is surrounded by a massive perigonial primordium, from which the many hair-like bristles originate. Consequently, the stamens develop among the hair primordia, more or less simultaneously. The hairs are arranged in whorls, which develop centripetally. The development of the perianth in D. arundinaceum starts with the formation of three initial perianth primordia opposite the stamens. Subsequently, two more abaxial bristle primordia, alternating with the stamens, originate simultaneously with the appearance of three adaxial bristle primordia in the zone where an adaxial inner perianth primordium is expected. CONCLUSIONS: The floral development in E. latifolium and D. arundinaceum can be considered as variations upon the scirpoid floral ontogenetic theme.

Phylogenetic analysis of the highland papayas ( Vasconcellea) and allied genera (Caricaceae) using PCR-RFLP.

The chloroplast and mitochondrial DNA diversity of 61 genotypes belonging to 18 Vasconcellea species, the so-called highland papayas, was studied by PCR-RFLP analysis of two non-coding cpDNA regions ( trnM- rbcL and trnK1- trnK2) and one non-coding mtDNA region ( nad4/1- nad4/2). This sample set was supplemented with six genotypes belonging to three other Caricaceae genera: the monotypic genus Carica, including only the cultivated papaya, and the genera Jacaratia and Cylicomorpha. Moringa ovalifolia was added as an outgroup species. The PCR-amplified cpDNA regions were digested with 18 restriction endonucleases, the mtDNA region with 11. A total of 22 point mutations and four insertion/deletions were scored in the sample. A higher level of interspecific variation was detected in the two cpDNA regions in comparison to the analysis of the mtDNA. Wagner parsimony and Neighbor-Joining analysis resulted in dendrograms with similar topologies. PCR-RFLP analysis supported the monophyly of Caricaceae, but among the 26 mutations scored, an insufficient number of markers discriminated between the different Caricaceae genera included in this study. Hence the inference of the intergeneric relationships within Caricaceae was impossible. However, some conclusions can be noted at a lower taxonomic level. The Caricaceae species were divided into two lineages. One group included only Vasconcellea spp., whereas the second included the remaining Vasconcellea spp., together with the papaya genotypes and those from the other Caricaceae genera. This may indicate a higher level of inter-fertility for the Vasconcellea species from the latter clade in interspecific crossings with papaya. The putative progenitors of the natural sterile hybrid V. x heilbornii, i.e. V. stipulata and V. cundinamarcensis, were only distantly related to V. x heilbornii. This indicates that probably none of these species was involved as the maternal progenitor in the origin of V. x heilbornii. Surprisingly, V. x heilbornii had organellar genome patterns identical with V. weberbaueri, suggesting a possible involvement of this species in the origin of V. x heilbornii. On the basis of discrepancy between morphological traits and the cpDNA profiles of some pairs of Vasconcellea species, we believe that besides V. x heilbornii, some other species have originated through interspecific hybridization. A reticulate evolution for Vasconcellea has therefore been suggested. Finally, intraspecific cpDNA variation was detected in V. microcarpa, thus providing molecular evidence for the high diversity previously indicated by morphological observations.

AFLP analysis of genetic relationships among papaya and its wild relatives (Caricaceae) from Ecuador.

The AFLP technique was used to assess the genetic relationships among the cultivated papaya ( Carica papaya L.) and related species native to Ecuador. Genetic distances based on AFLP data were estimated for 95 accessions belonging to three genera including C. papaya, at least eight Vasconcella species and two Jacaratia species. Cluster analysis using different methods and principal co-ordinate analysis (PCO), based on the AFLP data from 496 polymorphic bands generated with five primer combinations, was performed. The resulted grouping of accessions of each species corresponds largely with their taxonomic classifications and were found to be consistent with other studies based on RAPD, isozyme and cpDNA data. The AFLP analysis supports the recent rehabilitation of the Vasconcella group as a genus; until recently Vasconcella was considered as a section within the genus Carica. Both cluster and PCO analysis clearly separated the species of the three genera and illustrated the large genetic distance between C. papaya accessions and the Vasconcella group. The specific clustering of the highly diverse group of Vasconcella x heilbornii accessions also suggests that these genotypes may be the result of bi-directional introgression events between Vasconcella stipulata and Vasconcella cundinamarcensis.