Itayguara Ribeiro da Costa

Myrteae phylogeny, calibration, biogeography and diversification patterns: Increased understanding in the most species rich tribe of Myrtaceae.

Myrteae (c. 2500 species; 51 genera) is the largest tribe of Myrtaceae and an ecologically important groups of angiosperms in the Neotropics. Systematic relationships in Myrteae are complex, hindering conservation initiatives and jeopardizing evolutionary modelling. A well-supported and robust phylogenetic hypothesis was here targeted towards a comprehensive understanding of the relationships within the tribe. The resultant topology was used as a base for key evolutionary analyses such as age estimation, historical biogeography and diversification rate patterns. One nuclear (ITS) and seven chloroplast (psbA-trnH, matK, ndhF, trnl-trnF, trnQ-rps16, rpl16 and rpl32-trnL) DNA regions for 115 taxa representing 46 out of the 51 genera in the tribe were accessed and analysed using maximum likelihood and Bayesian inference tools for phylogenetic reconstruction. Dates of diversification events were estimated and contrasted using two distinct fossil sets (macro and pollen) in BEAST. The subsequent dated phylogenies were compared and analysed for biogeographical patterns using BioGeoBEARS and diversification rates using BAMM. Myrteae phylogeny presents strong statistical support for three major clades within the tribe: Australasian group, Myrtus group and Main Neotropical Lineage. Dating results from calibration using macrofossil are an average of 20 million years older and show an early Paleocene origin of Myrteae, against a mid-Eocene one from the pollen fossil calibration. Biogeographic analysis shows the origin of Myrteae in Zealandia in both calibration approaches, followed by a widespread distribution throughout the still-linked Gondwana continents and diversification of Neotropical endemic lineages by later vicariance. Best configuration shift indicates three points of acceleration in diversification rates, all of them occurring in the Main Neotropical Lineage. Based on the reconstructed topology, several new taxonomic placements were recovered, including: the relative position of Myrtus communis, the placement of the Blepharocalyx group, the absence of generic endemism in the Caribbean, and the paraphyletism of the former Pimenta group. Distinct calibration approaches affect biogeography interpretation, increasing the number of necessary long distance dispersal events in the topology with older nodes. It is hypothesised that biological intrinsic factors such as modifications of embryo type and polyploidy might have played a role in accelerating shifts of diversification rates in Neotropical lineages. Future perspectives include formal subtribal classification, standardization of fossil calibration approaches and better links between diversification shifts and trait evolution.

Polyploidy and polyembryony in Anemopaegma (Bignonieae, Bignoniaceae)

Polyploidy is a key process in plant evolution, with the asexual formation of embryos representing a way through which polyploids can escape sterility. The association between polyploidy and polyembryony is known to occur in Bignoniaceae. In this study, we investigate polyembryony in four polyploid species of Anemopaegma: A. acutifolium, A. arvense, A. glaucum and A. scabriusculum as well as in one diploid species, A. album. Polyembryony was observed only in polyploid species. We used seed dissection and germination tests to compare the number of polyembryonic seeds. We tested how the pollen source influences the number of polyembryonic seeds and the number of embryos per seed and tested the correlation between the number of viable seeds per fruit and mean number of embryos per seed. The number of polyembryonic seeds observed by seed dissection was higher than the number of polyembryonic seeds determined by the germination test, with the number of embryos produced per seed being higher than the number of seedlings. The dissection of seeds of A. glaucum indicated that a higher number of polyembryonic seeds and a higher number of embryos were present in seeds from cross-pollination than in seeds from self-pollination. On the other hand, germination tests indicated that a higher number of polyembryonic seeds were present in fruits from self-pollination than from cross-pollination. The mean number of embryos per seed was not influenced by the number of viable seeds per fruit in fruits from open pollination. These results indicate a positive relationship between polyembryony and polyploidy in Anemopaegma.

DNA methylation patterns and karyotype analysis of off-type and normal phenotype somatic embryos of feijoa

Acca sellowiana is a native species to the southern region of Brazil, with secondary dispersion in Uruguay northeast. The potentially economic interest of this species motivated many fundamental and applied researches, such as micropropagation protocols development, including somatic embryogenesis (SE). Previous studies have reported the high frequency of off-type somatic embryos in A. sellowiana SE and low conversion efficiency, possibly due to this high off-type somatic embryos formation. In this sense, this study was aimed to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) in the induction and development of somatic embryos in direct and indirect SE through cytogenetic and DNA methylation analysis. The results of the present work reinforces the previous findings that 2,4-D plays a key role in the SE induction in A. sellowiana. However, the continuous presence of this auxin in the culture medium enhances the rate of off-type somatic embryos. This auxin also apparently affects the cells and chromosomes size in normal and off-type somatic embryos and DNA methylation patterns of the analyzed samples. These findings may indicate that the off-type somatic embryos formation may be related to genetic and/or epigenetic changes caused by long-term cultivation in A. sellowiana. The intermediate phase of callus also indicated increased formation of off-types, corroborating other studies that showed that indirect SE could cause increased formation of anomalies in regenerated plantlets.

Characterization of a methionine-rich protein from the seeds of Cereus jamacaru Mill. (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Cereus jamacaru (Cactaceae), to which we gave the trivial name of cactin. This protein has a molecular mass of 11.3 kDa and is formed by a light chain (3.67 kDa) and a heavy chain (7.63 kDa). This protein was isolated using a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of cactin was determined and found to resemble that of the 2S seed reserve protein from the Brazil nut, a protein remarkable for its high methionine content. The usefulness of cactin as a molecular marker in the taxonomy of the Cactaceae is discussed.

Isolation and Characterisation of a Reserve Protein from the Seeds of Cereus jamacaru (Cactaceae)

We describe here the isolation and characterisation of a major reserve protein from the seeds of Cereus jamacaru. (Cactaceae). This protein has a molecular mass of 5319 kDa and was isolated by a combination of gel filtration chromatography and reverse phase HPLC. The amino acid composition of the protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The usefulness of this protein as a molecular marker in the Cactaceae is also discussed.

Investigating the floral and reproductive biology of the endangered microendemic cactus Uebelmannia buiningii Donald (Minas Gerais, Brazil)

The Cactaceae are known to be amongst the most endangered plant families of the world due to reduction of their habitats and activities of collectors. As the species of the family are dependent on animals to perform cross pollination, and hence seed production, their population performance may be further negatively affected by interrupted biotic interactions. For efficient conservation of rare species, knowledge on reproductive biology and pollinators is of prime importance. In our study we focused on Uebelmannia buiningii Donald, a microendemic cactus from the Serra Negra State Park, Minas Gerais state, Brazil. During four field expeditions to three localities of the species between September 2012 and September 2013, we measured flowers, detected nectar-guides and osmophores and performed pollen viability tests. We studied the reproductive system of the species using manual self- and cross-pollination tests and observed pollinators. Our results revealed that the flowering period takes place during the dry season, between April and October, and that the diurnal flowers open between 7:00 a.m. and 5 p.m. The flowers are shortly tubular with yellow perianth-segments. We found neither nectar nor nectar-guides, and osmophores appeared as glands within the flower tube. Whereas pollen viability was 90.25%, manual cross-pollination tests have shown cross-pollination with gametophytic incompatibility. We observed two bee species visiting the flowers and acting as effective pollinators: Dialictus opacus and Plebeia sp. The combination of low reproductive activity with gametophytic incompatibility, together with the reduced number of individuals in a population and low number of populations, makes the endemic cactus U. buiningii a critically endangered species.