Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

Natural selection acting on integrated phenotypes: covariance among functional leaf traits increases plant fitness.

Plant functional strategies are usually accomplished through the simultaneous expression of different traits, and hence their correlations should be promoted by natural selection. The adaptive value of correlations among leaf functional traits, however, has not been assessed in natural populations. We estimated intraspecific variation in leaf functional traits related to the primary metabolism and anti-herbivore defence in a population of Turnera velutina. We analysed whether natural selection favoured the expression of individual traits, particular combinations of traits or leaf phenotypic integration. Patterns of covariation among traits were related to water and nitrogen economy, and were similar among genotypes, but the magnitude of their phenotypic integration differed by 10-fold. Although families did not differ in the mean values of leaf functional traits, directional selection favoured low nitrogen content and low chemical defence, high content of chlorophyll, sugar in extrafloral nectar and trichome density. Families with higher phenotypic integration among leaf traits grew faster and produced more flowers. We suggest that the coordinated expression of leaf traits has an adaptive value, probably related to optimisation in the expression of traits related to water conservation and nitrogen acquisition.

Self-discrimination in vine tendrils of different plant families.

Previous study reported a novel type of self-discrimination in the tendrils of the vine Cayratia japonica (Vitaceae). However, whether self-discrimination in tendrils is common in vine plant species has not been elucidated. Here, we investigated whether tendrils of Momordica charantia var. pavel (Cucurbitaceae), Cucumis sativus (Cucurbitaceae) and Passiflora caerulea (Passifloraceae) can discriminate self and non-self plants. We also investigated whether the tendrils of M. charantia and C. sativus can discriminate differences in cultivars to determine the discrimination ability for genetic similarity. We found that tendrils of the M. charantia and P. caerulea were more likely to coil around non-self plant than self plants, but not in C. sativus. Our findings support the common occurrence of self-discrimination in tendrils in different plant taxa, although some species lacked it. Furthermore, tendrils of M. charantia more rapidly coil around different cultivars than around same cultivars. The tendrils of M. charantia may can discriminate differences in cultivars.

Could refuge theory and rivers acting as barriers explain the genetic variability distribution in the Atlantic Forest?

The Atlantic Forest is one of the most species-rich ecoregions in the world. The historical origins of this richness and the evolutionary processes that produced diversification and promoted speciation in this ecosystem remain poorly understood. In this context, focusing on Passiflora contracta, an endemic species from the Atlantic Forest distributed exclusively at sea level along forest edges, this study aimed to characterize the patterns of genetic variability and explore two hypotheses that attempt to explain the possible causes of the genetic diversity in this region: the refuge and riverine barrier theories. We employed Bayesian methods combined with niche modeling to identify genetically homogeneous groups, to determine the diversification age, and identify long-term climate stability areas to species survival. The analyses were performed using molecular markers from nuclear and plastid genomes, with samples collected throughout the entire geographic distribution of the species, and comparisons with congeners species. The results indicated that populations were genetically structured and provided evidence of demographic stability. The molecular markers indicated the existence of a clear structure and the presence of five homogeneous groups. Interestingly, the separation of the groups coincides with the geographical locations of local rivers, corroborating the hypothesis of rivers acting as barriers to gene flow in this species. The highest levels of genetic diversity and the areas identified as having long-term climate stability were found in the same region reported for other species as a possible refuge area during the climatic changes of the Quaternary.

Multivariate analysis of backcross progeny of Passiflora L. (Passifloraceae) for pre-breeding genotype selection.

The Ward-MLM procedure was used to evaluate genetic variation in four backcross progenies and in their parents, hybrid F1 HD13 and donor parent Passiflora sublanceolata. Sixteen quantitative descriptors and five qualitative characteristics of relevance to ornamental flower production were assessed. Using the pseudo-F and pseudo-T² criteria, we identified four groups among these plants in two evaluation periods. In both evaluations, the BC1 plants showed greater dissimilarity to their recurrent parent, but showed high genetic similarity with the P. sublanceolata parent. The first two canonical variables produced by the Ward-MLM procedure accounted for over 90% of the variation in both evaluation periods, enabling the representation of diversity through two-dimensional graphics. Groups II and IV were formed in the first assessment period. Groups I and IV formed in the second period and showed plants with selection potential. We found that it was essential to use both qualitative and quantitative variables for this analysis. Assessments of quantitative descriptors indicate that the selection of BC1 plants can be performed in any of the four progenies. Because of the similarities observed for some floral descriptors between BC1 and the P. sublanceolata parent, a second generation backcross was not recommended. However, the selection of BC1 plants for evaluation and direct use as an ornamental cultivar, or as a resource in other breeding programs, can be recommended.

Dissection of evolutionary networks to assess their role in the evolution of robustness, function, and diversification.

Biological systems are remarkably robust in the face of environmental, mutational, and developmental perturbations. Analyses of molecular networks reveal recurrent features, such as modularity, that have been implicated in robustness and evolvability. Multiple theoretical models account for these features, yet few empirical tests of these models exist. Here I develop a set of broadly applicable methodologies to enable expanded empirical evaluation of model predictions. The methodologies focus on the inference and analysis of networks that depict evolutionary correlations among characters. I apply these methodologies to analyze an evolutionary network at a larger scale of organization among 42 stem anatomical and morphological characters of 52 species in the genus Adenia (Passifloraceae). I evaluate a model predicting that modular evolutionary networks will evolve in response to environmental change. The evolutionary network of Adenia is modular and "small-world," and the three diagnosed modules correspond roughly to functions of transport, storage, and mechanical support. The phylogenetically informed analyses suggest that the storage module is more impacted by environmental change than expected by chance. These results corroborate the hypothesis that modularity reduces the impact of environmental change, but this result requires further empirical evaluation that can be aided by the proposed methods in additional study systems.

Pollinator-mediated selection on floral morphology: evidence for transgressive evolution in a derived hybrid lineage.

Hybridization between closely related lineages is a mechanism that might promote substantive changes in phenotypic traits of descendants, resulting in transgressive evolution. Interbreeding between divergent but morphologically similar lineages can produce exceptional phenotypes, but the potential for transgressive variation to facilitate long-term trait changes in derived hybrid lineages has received little attention. We compare pollinator-mediated selection on transgressive floral traits in both early-generation and derived hybrid lineages of the Piriqueta cistoides ssp. caroliniana complex. The bowl-shaped flowers of morphotypes in this complex have similar gross morphologies and attract a common suite of small insect pollinators. However, they are defined by significant differences in characters that generate pollinator interest and visitation, including floral area and petal separation. In common garden experiments, patterns of pollen deposition in early-generation recombinant hybrids indicate that Piriqueta's pollinators favour flowers with greater area and reduced petal separation. Changes in floral morphology in derived hybrid lineages are consistent with predictions from selection gradients, but the magnitude of change is limited relative to the range of transgressive variation. These results suggest that hybridization provides variation for evolution of divergent floral traits. However, the potential for extreme transgressive variants to contribute to phenotypic shifts may be limited due to reduced heritability, evolutionary constraints or fitness trade-offs.

Multi-locus phylogenies of the genus Barteria (Passifloraceae) portray complex patterns in the evolution of myrmecophytism.

The four species of the central African genus Barteria show variation in habitat and in degree of association with ants. Whereas B. solida, restricted to submontane forests, attracts opportunistic ants to extrafloral nectar, the three other species, found in lowland rainforests (B. fistulosa, B. dewevrei) and in littoral scrub (B. nigritana), possess stem domatia of varying shapes and degrees of specialisation, hosting either non-specific arboreal ants (B. nigritana, some B. dewevrei) or two large species of ants of the genus Tetraponera Smith, 1852 that are specific to some species of Barteria (B. fistulosa, some B. dewevrei). We aimed to investigate whether this variation represents an evolutionary trend toward increasing specialisation of mutualism or the reduction or loss of myrmecophytic traits. For this, we determined phylogenetic relationships within the genus using DNA sequences (primarily nuclear ITS) and microsatellite genotypes (11 loci) on a large sample of individuals, mostly from Cameroon and Gabon. The two types of markers support an initial dichotomy that groups B. dewevrei with B. nigritana and B. fistulosa with B. solida respectively. Within these pairs, species do not appear reciprocally monophyletic. At microsatellite loci, B. nigritana forms a clade embedded within B. dewevrei; and within both B. solida and B. fistulosa, geographical populations show levels of differentiation similar to that observed between populations of B. solida and B. fistulosa. Geographic distance alone does not account for genetic differentiation between species, which indicates reproductive isolation. Divergence in each of the two pairs implies evolutionary transitions in habitat and in myrmecophytism. Specialised mutualism with specific ant species of the genus Tetraponera has been lost in species found in more marginal habitats.

Molecular phylogenetic analysis of Passifloraceae sensu lato (Malpighiales) based on plastid and nuclear DNA sequences.

A phylogenetic analysis of Passifloraceae sensu lato was performed using rbcL, atpB, matK, and 18S rDNA sequences from 25 genera and 42 species. Parsimony analyses of combined data sets resulted in a single most parsimonious tree, which was very similar to the 50% majority consensus tree from the Bayesian analysis. All nodes except three were supported by more than 50% bootstrap. The monophyly of Passifloraceae s.l. as well as the former families, Malesherbiaceae, Passifloraceae sensu stricto, and Turneraceae were strongly supported. Passifloraceae s.s. and the Turneraceae are sisters, and form a strongly supported clade. Within Passifloraceae s.s., the tribes Passifloreae and Paropsieae are both monophyletic. The intergeneric relationships within Passifloraceae s.s. and Turneraceae are roughly correlated with previous classification systems. The morphological character of an androgynophore/gynophore is better used for characterizing genera grouping within Passifloraceae s.s. Other morphological characters such as the corona and aril are discussed.

Respuesta in vitro de diferentes biotipos y explantos de Passiflora caerulea L / In vitro response of different biotypes and explants of Passiflora caerulea L

Passiflora caerulea L., al igual que otras especies de la familia Passifloraceae, es utilizada en la medicina popular por sus propiedades antiespasmódicas y para el tratamiento de la ansiedad, el insomnio y el nerviosismo. La belleza de sus flores les otorga valor ornamental, mientras que sus frutos son apreciados por su importancia alimenticia. Se evaluó la respuesta in vitro de diferentes explantos y tres biotipos de P. caerulea: Corral de Bustos (provincia de Córdoba), Zavalla (provincia de Santa Fe) y Neuquén (provincia de Neuquén). Se utilizaron dos tipos de explantos: entrenudos y segmentos nodales, y como medio de cultivo Murashige y Skoog (1962) (MS), suplementado con vitaminas de Gamborg (1976) y 1 mg/L-1 de benciladenina (BA). Las respuestas fueron diferentes según el genotipo y el explanto. Los entrenudos ubicados tanto horizontal como verticalmente en medio de cultivo generaron callos como única respuesta. El biotipo de Neuquén mostró los mayores porcentajes de segmentos nodales con brotes. A través de estudios histológicos se determinó que en medio de cultivo MS con 1 mg/L-1 de BA, los segmentos nodales de P. caerulea originan brotes a partir de las yemas axilares preformadas y raíces que parten de callos en la base de los mismos. En iguales condiciones, los entrenudos originan callo como única respuesta.

As other species of the Passifloraceae family, Passiflora caerulea L. is used in popular medicine for its antispasmodic properties and as a remedy for anxiety, insomnia and nervousness. It is also highly prized for the ornamental value of its beautiful flowers, as well as for the nutritional importance of its fruits. The in vitro response of different explants and three biotypes of P. caerulea: the Corral de Bustos (Province of Córdoba), the Zavalla (Province of Santa Fe) and the Neuquén (Province of Neuquén) genotypes, was evaluated using two types of explants: internodes and nodal segments on Murashige and Skoog (1962) (MS) culture medium supplemented with Gamborg’s vitamins (1976) and 1 mg.L-1 of benzyladenine (BA). There were different responses depending on the genotype and the explant. The internodes placed both horizontally and vertically in the culture medium produced callus as sole response. The Neuquén biotype showed the highest percentages of nodal segments with shoots. Histological tests allowed to establish that in MS culture medium with 1 mg.L-1 of BA, the nodal segments of P. caerulea produce shoots from the preformed axillary buds and roots that develop from the callus situated on its base. Under similar conditions, the internodes produce callus as sole response.

Phylogeographic inferences concerning evolution of Brazilian Passiflora actinia and P. elegans (Passifloraceae) based on ITS (nrDNA) variation.

BACKGROUND AND AIMS: Passiflora actinia and P. elegans, two markedly parapatric species, have their southern and northern distribution limits, respectively, in the most southern part of the Brazilian Atlantic Rain Forest. Despite the fact that they are classified in different taxonomic series, previous phylogenetic studies of this genus revealed a high genetic similarity between them. The aim of the present work was to analyse in more detail their geographical range in this region of overlap, to investigate intraspecific genetic variability and phylogeographic structure, and to search for possible hybrids. METHODS: Eighty-two localities were searched for these species, and nuclear internal transcribed spacer (ITS) sequences were investigated for 32 individuals of P. actinia, 20 of P. elegans and one putative interspecific hybrid. Plastid trnL-trnF and psbA-trnH were examined for 12 plants of each species and the putative hybrid. KEY RESULTS: Both species showed a high level of intraspecific and intra-individual ITS variability. Network analysis revealed a north-south geographic gradient in their intra and interspecific relationships. Mismatch analyses suggested a recent population expansion of P. elegans. The plastid markers showed restricted variability but, together with the nuclear data, they contributed to the identification of an interspecific hybrid of intermediate morphology at the border of the distribution of these two species. Both genetic and morphological data indicate the absence of an extensive hybridization zone between these species. CONCLUSIONS: Gene flow between lineages is the possible cause for the presence of different ITS sequences within a given plant, the absence of homogenization being due to the high degree of vegetative reproduction in the two species. Differentiation of P. actinia into geographic groups and the origin of P. elegans may have been influenced by the Atlantic Forest migration towards southern Brazil. The genetic pattern of the interspecific hybrid indicates that plastid inheritance in these species is at least sometimes paternal.