Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome.

Aristolochia, a genus in the magnoliid order Piperales, has been famous for centuries for its highly specialized flowers and wide medicinal applications. Here, we present a new, high-quality genome sequence of Aristolochia fimbriata, a species that, similar to Amborella trichopoda, lacks further whole-genome duplications since the origin of extant angiosperms. As such, the A. fimbriata genome is an excellent reference for inferences of angiosperm genome evolution, enabling detection of two novel whole-genome duplications in Piperales and dating of previously reported whole-genome duplications in other magnoliids. Genomic comparisons between A. fimbriata and other angiosperms facilitated the identification of ancient genomic rearrangements suggesting the placement of magnoliids as sister to monocots, whereas phylogenetic inferences based on sequence data we compiled yielded ambiguous relationships. By identifying associated homologues and investigating their evolutionary histories and expression patterns, we revealed highly conserved floral developmental genes and their distinct downstream regulatory network that may contribute to the complex flower morphology in A. fimbriata. Finally, we elucidated the genetic basis underlying the biosynthesis of terpenoids and aristolochic acids in A. fimbriata.

Genetic relationships and polyploid origins in the Lippia alba complex.

PREMISE: Plant genomes vary in size and complexity due in part to polyploidization. Latitudinal analyses of polyploidy are biased toward floras of temperate regions, with much less research done in the tropics. Lippia alba has been described as a tropical polyploid complex with diploid, triploid, tetraploid, and hexaploid accessions. However, no data regarding relationships among the ploidal levels and their origins have been reported. Our goals are to clarify the relationships among accessions of Lippia alba and the origins of each ploidal level. METHODS: We investigated 98 samples representing all five geographical regions of Brazil and all ploidal levels using microsatellite (SSR) allelic variation and DNA sequences of ITS and trnL-F. Nine morphological structures were analyzed from 33 herbarium samples, and the chemical compounds of 78 accessions were analyzed by GC-MS. RESULTS: Genetic distance analysis, the alignment block pattern, as well as RAxML and Bayesian trees showed that accessions grouped by ploidal level. The triploids form a well-defined group that originated from a single group of diploids. The tetraploids and hexaploid grouped together in SSR and trnL-F analyses. The recovered groups agree with chemical data and morphology. CONCLUSIONS: The accessions grouped by ploidal level. Only one origin of triploids from a single group of diploids was observed. The tetraploid origin is uncertain; however, it appears to have contributed to the origin of the hexaploid. This framework reveals linkages among the ploidal levels, providing new insights into the evolution of a polyploid complex of tropical plants.

Divergent gene expression levels between diploid and autotetraploid Tolmiea relative to the total transcriptome, the cell, and biomass.

PREMISE OF THE STUDY: Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea, we present an integrated approach for cross-ploidy comparisons that account for differences in transcriptome size and cell density and make multiple comparisons of transcript abundance. METHODS: We use RNA spike-in standards in concert with cell size and density to identify and correct for differences in transcriptome size and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass. KEY RESULTS: In total, ~17% of all loci were identified as differentially expressed (DEGs) between the diploid and autopolyploid species. The per-transcriptome normalization, the method researchers typically use, captured the fewest DEGs (58% of total DEGs) and failed to detect any DEGs not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEGs were recovered, respectively. The discrepancy between per-transcriptome and per-cell recovery of DEGs occurs because per-transcriptome normalizations are concentration-based and therefore blind to differences in transcriptome size. CONCLUSIONS: While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid-like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis-related functions.

Terrestrial species adapted to sea dispersal: Differences in propagule dispersal of two Caribbean mangroves.

A central goal of comparative phylogeography is to understand how species-specific traits interact with geomorphological history to govern the geographic distribution of genetic variation within species. One key biotic trait with an immense impact on the spatial patterns of intraspecific genetic differentiation is dispersal. Here, we quantify how species-specific traits directly related to dispersal affect genetic variation in terrestrial organisms with adaptations for dispersal by sea, not land-the mangroves of the Caribbean. We investigate the phylogeography of white mangroves (Laguncularia racemosa, Combretaceae) and red mangroves (Rhizophora mangle, Rhizophoraceae) using chloroplast genomes and nuclear markers (thousands of RAD-Seq loci) from individuals throughout the Caribbean. Both coastal tree species have viviparous propagules that can float in salt water for months, meaning they are capable of dispersing long distances. Spatially explicit tests of the role of ocean currents on patterning genetic diversity revealed that ocean currents act as a mechanism for facilitating dispersal, but other means of moving genetic material are also important. We measured pollen- vs. propagule-mediated gene flow and discovered that in white mangroves, seeds were more important for promoting genetic connectivity between populations, but in red mangroves, the opposite was true: pollen contributed more. This result challenges our concept of the importance of both proximity to ocean currents for moving mangrove seeds and the extent of long-distance pollen dispersal. This study also highlights the importance of spatially explicit quantification of both abiotic (ocean currents) and biotic (dispersal) factors contributing to gene flow to understand fully the phylogeographic histories of species.

Karyotypic variation and pollen stainability in resynthesized allopolyploids Tragopogon miscellus and T. mirus.

PREMISE OF THE STUDY: Polyploidy has extensively shaped the evolution of plants, but the early stages of polyploidy are still poorly understood. The neoallopolyploid species Tragopogon mirus and T. miscellus are both characterized by widespread karyotypic variation, including frequent aneuploidy and intergenomic translocations. Our study illuminates the origins and early impacts of this variation by addressing two questions: How quickly does karyotypic variation accumulate in Tragopogon allopolyploids following whole-genome duplication (WGD), and how does the fertility of resynthesized Tragopogon allopolyploids evolve shortly after WGD? METHODS: We used genomic in situ hybridization and lactophenol-cotton blue staining to estimate the karyotypic variation and pollen stainability, respectively, of resynthesized T. mirus and T. miscellus during the first five generations after WGD. KEY RESULTS: Widespread karyotypic variation developed quickly in synthetics and resembled that of naturally occurring T. mirus and T. miscellus by generation S4 . Pollen stainability in resynthesized allopolyploids was consistently lower than that of natural T. mirus and T. miscellus, as well as their respective diploid progenitor species. Logistic regression showed that mean pollen stainability increased slightly over four generations in resynthesized T. mirus but remained at equivalent levels in T. miscellus. CONCLUSIONS: Our results clarify some of the changes that occur in T. mirus and T. miscellus immediately following their origin, most notably the rapid onset of karyotypic variation within these species immediately following WGD.

Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining.

Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.

Phylogenetic signal detection from an ancient rapid radiation: Effects of noise reduction, long-branch attraction, and model selection in crown clade Apocynaceae.

Crown clade Apocynaceae comprise seven primary lineages of lianas, shrubs, and herbs with a diversity of pollen aggregation morphologies including monads, tetrads, and pollinia, making them an ideal group for investigating the evolution and function of pollen packaging. Traditional molecular systematic approaches utilizing small amounts of sequence data have failed to resolve relationships along the spine of the crown clade, a likely ancient rapid radiation. The previous best estimate of the phylogeny was a five-way polytomy, leaving ambiguous the homology of aggregated pollen in two major lineages, the Periplocoideae, which possess pollen tetrads, and the milkweeds (Secamonoideae plus Asclepiadoideae), which possess pollinia. To assess whether greatly increased character sampling would resolve these relationships, a plastome sequence data matrix was assembled for 13 taxa of Apocynaceae, including nine newly generated complete plastomes, one partial new plastome, and three previously reported plastomes, collectively representing all primary crown clade lineages and outgroups. The effects of phylogenetic noise, long-branch attraction, and model selection (linked versus unlinked branch lengths among data partitions) were evaluated in a hypothesis-testing framework based on Shimodaira-Hasegawa tests. Discrimination among alternative crown clade resolutions was affected by all three factors. Exclusion of the noisiest alignment positions and topologies influenced by long-branch attraction resulted in a trichotomy along the spine of the crown clade consisting of Rhabdadenia+the Asian clade, Baisseeae+milkweeds, and Periplocoideae+the New World clade. Parsimony reconstruction on all optimal topologies after noise exclusion unambiguously supports parallel evolution of aggregated pollen in Periplocoideae (tetrads) and milkweeds (pollinia). Our phylogenomic approach has greatly advanced the resolution of one of the most perplexing radiations in Apocynaceae, providing the basis for study of convergent floral morphologies and their adaptive value.

Phylogeny of extant and fossil Juglandaceae inferred from the integration of molecular and morphological data sets.

It is widely acknowledged that integrating fossils into data sets of extant taxa is imperative for proper placement of fossils, resolution of relationships, and a better understanding of character evolution. The importance of this process has been further magnified because of the crucial role of fossils in dating divergence times. Outstanding issues remain, including appropriate methods to place fossils in phylogenetic trees, the importance of molecules versus morphology in these analyses, as well as the impact of potentially large amounts of missing data for fossil taxa. In this study we used the angiosperm clade Juglandaceae as a model for investigating methods of integrating fossils into a phylogenetic framework of extant taxa. The clade has a rich fossil record relative to low extant diversity, as well as a robust molecular phylogeny and morphological database for extant taxa. After combining fossil organ genera into composite and terminal taxa, our objectives were to (1) compare multiple methods for the integration of the fossils and extant taxa (including total evidence, molecular scaffolds, and molecular matrix representation with parsimony [MRP]); (2) explore the impact of missing data (incomplete taxa and characters) and the evidence for placing fossils on the topology; (3) simulate the phylogenetic effect of missing data by creating "artificial fossils"; and (4) place fossils and compare the impact of single and multiple fossil constraints in estimating the age of clades. Despite large and variable amounts of missing data, each of the methods provided reasonable placement of both fossils and simulated "artificial fossils" in the phylogeny previously inferred only from extant taxa. Our results clearly show that the amount of missing data in any given taxon is not by itself an operational guideline for excluding fossils from analysis. Three fossil taxa (Cruciptera simsonii, Paleoplatycarya wingii, and Platycarya americana) were placed within crown clades containing living taxa for which relationships previously had been suggested based on morphology, whereas Polyptera manningii, a mosaic taxon with equivocal affinities, was placed firmly as sister to two modern crown clades. The position of Paleooreomunnea stoneana was ambiguous with total evidence but conclusive with DNA scaffolds and MRP. There was less disturbance of relationships among extant taxa using a total evidence approach, and the DNA scaffold approach did not provide improved resolution or internal support for clades compared to total evidence, whereas weighted MRP retained comparable levels of support but lost crown clade resolution. Multiple internal minimum age constraints generally provided reasonable age estimates, but the use of single constraints provided by extinct genera tended to underestimate clade ages.