Removal of Noisy Characters from Chloroplast Genome-Scale Data Suggests Revision of Phylogenetic Placements of <Emphasis Type="Italic">Amborella</Emphasis> and <Emphasis Type="Italic">Ceratophyllum</Emphasis>

It is widely appreciated that noisy, highly variable data can impede phylogeney reconstruction. Researchers have for a long time omitted problematic data from phylogenetic analyses, such as the third-codon positions and variable regions. In the analyses of the phylogenetic relations of the angiosperms; however, inclusion of complete gene sequences into genomic-scale alignments has become a common practice. Here we demonstrate that this practice can be misleading. We show that support of the basal-most position of Amborella trichopoda among the angiosperms in the chloroplast genomic data is based only on a tiny subset (< 1% of the total alignment length) of the most variable positions in alignment, exhibiting mean maximum likelihood (ML) distance among the angiosperm operational taxonomic units (OTUs) approximately 36 substitutions/site. Exclusion of these positions leads to disappearance of the basal Amborella branch. Likewise, the recently reported sister-group relationship of Ceratophyllum to the eudicots is based on the presence of 2% of the most variable positions in the genomic alignment, exhibiting, on average, 20 substitutions/site in comparison among the angiosperm OTUs. These observations highlight a need for excluding a certain proportion of saturated positions in alignment from phylogenomic analyses.     

Chloroplast genome phylogenetics: why we need independent approaches to plant molecular evolution

The traditional approach to plant molecular phylogenetics involves amplifying, sequencing and analyzing one or a few genes from many species and is conducive to broad taxon sampling. An independent approach involves chloroplast genome sequencing, providing much larger amounts of data per taxon but for a smaller number of species. In principle, the two strategies can inform each other but in practice their results sometimes conflict for reasons that are currently debated. An Opinion article published in the October 2004 issue of Trends in Plant Science cautioned against the pursuit of genome-based phylogenies. Here, we provide a different perspective on issues at the heart of the current debate and defend the use of chloroplast genome phylogenetics for crucial species because it provides an independent test of hypotheses generated by the traditional approach.     

Evolutionary analysis of 58 proteins encoded in six completely sequenced chloroplast genomes: Revised molecular estimates of two seed plant divergence times

Fifty-eight homologous protein sequences from the completely sequenced chloroplast genomes ofZea mays, Oryza sativa, Nicotiana tabacum, Pinus thunbergii, Marchantia polymorpha andPoryphyra purpurea were investigated. Analyzed individually, only 40 of the 58 proteins gave the true, known topology for these species. Trees constructed from the concatenated 14295 amino acid alignment and from automatically generated subsets of the data containing successively fewer polymorphisms were used to estimate theNicotiana-Zea andPinus-angiosperm divergence times as 160 and 348 million years, respectively, with an uncertainty of about 10%. These estimates based upon phylogenetic analysis of protein data from complete chloroplast genomes are in much better accordance with current interpretations of fossil evidence than previous molecular estimates.This paper is dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday.     

The mitochondrial genome of Malus domestica and the import-driven hypothesis of mitochondrial genome expansion in seed plants

Mitochondrial genomes of spermatophytes are the largest of all organellar genomes. Their large size has been attributed to various factors; however, the relative contribution of these factors to mitochondrial DNA (mtDNA) expansion remains undetermined. We estimated their relative contribution in Malus domestica (apple). The mitochondrial genome of apple has a size of 396 947 bp and a one to nine ratio of coding to non-coding DNA, close to the corresponding average values for angiosperms. We determined that 71.5% of the apple mtDNA sequence was highly similar to sequences of its nuclear DNA. Using nuclear gene exons, nuclear transposable elements and chloroplast DNA as markers of promiscuous DNA content in mtDNA, we estimated that approximately 20% of the apple mtDNA consisted of DNA sequences imported from other cell compartments, mostly from the nucleus. Similar marker-based estimates of promiscuous DNA content in the mitochondrial genomes of other species ranged between 21.2 and 25.3% of the total mtDNA length for grape, between 23.1 and 38.6% for rice, and between 47.1 and 78.4% for maize. All these estimates are conservative, because they underestimate the import of non-functional DNA. We propose that the import of promiscuous DNA is a core mechanism for mtDNA size expansion in seed plants. In apple, maize and grape this mechanism contributed far more to genome expansion than did homologous recombination. In rice the estimated contribution of both mechanisms was found to be similar.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

Automated Removal of Noisy Data in Phylogenomic Analyses

Noisy data, especially in combination with misalignment and model misspecification can have an adverse effect on phylogeny reconstruction; however, effective methods to identify such data are few. One particularly important class of noisy data is saturated positions. To avoid potential errors related to saturation in phylogenomic analyses, we present an automated procedure involving the step-wise removal of the most variable positions in a given data set coupled with a stopping criterion derived from correlation analyses of pairwise ML distances calculated from the deleted (saturated) and the remaining (conserved) subsets of the alignment. Through a comparison with existing methods, we demonstrate both the effectiveness of our proposed procedure for identifying noisy data and the effect of the removal of such data using a well-publicized case study involving placental mammals. At the least, our procedure will identify data sets requiring greater data exploration, and we recommend its use to investigate the effect on phylogenetic analyses of removing subsets of variable positions exhibiting weak or no correlation to the rest of the alignment. However, we would argue that this procedure, by identifying and removing noisy data, facilitates the construction of more accurate phylogenies by, for example, ameliorating potential long-branch attraction artefacts.     

Mitochondrial DNA of Vitis vinifera and the issue of rampant horizontal gene transfer

The mitochondrial genome of grape (Vitis vinifera), the largestorganelle genome sequenced so far, is presented. The genomeis 773,279 nt long and has the highest coding capacity amongknown angiosperm mitochondrial DNAs (mtDNAs). The proportionof promiscuous DNA of plastid origin in the genome is also thelargest ever reported for an angiosperm mtDNA, both in absoluteand relative terms. In all, 42.4% of chloroplast genome of Vitishas been incorporated into its mitochondrial genome. In orderto test if horizontal gene transfer (HGT) has also contributedto the gene content of the grape mtDNA, we built phylogenetictrees with the coding sequences of mitochondrial genes of grapeand their homologs from plant mitochondrial genomes. Many incongruentgene tree topologies were obtained. However, the extent of incongruencebetween these gene trees is not significantly greater than thatobserved among optimal trees for chloroplast genes, the commonancestry of which has never been in doubt. In both cases, weattribute this incongruence to artifacts of tree reconstruction,insufficient numbers of characters, and gene paralogy. Thisfinding leads us to question the recent phylogenetic interpretationof Bergthorsson et al. (2003, 2004) and Richardson and Palmer(2007) that rampant HGT into the mtDNA of Amborella best explainsphylogenetic incongruence between mitochondrial gene trees forangiosperms. The only evidence for HGT into the Vitis mtDNAfound involves fragments of two coding sequences stemming fromtwo closteroviruses that cause the leaf roll disease of thisplant. We also report that analysis of sequences shared by bothchloroplast and mitochondrial genomes provides evidence fora previously unknown gene transfer route from the mitochondrionto the chloroplast.     

Analysis of Acorus calamus chloroplast genome and its phylogenetic implications

Determining the phylogenetic relationships among the major lines of angiosperms is a long-standing problem, yet the uncertainty as to the phylogenetic affinity of these lines persists. While a number of studies have suggested that the ANITA (Amborella-Nymphaeales-Illiciales-Trimeniales-Aristolochiales) grade is basal within angiosperms, studies of complete chloroplast genome sequences also suggested an alternative tree, wherein the line leading to the grasses branches first among the angiosperms. To improve taxon sampling in the existing chloroplast genome data, we sequenced the chloroplast genome of the monocot Acorus calamus. We generated a concatenated alignment (89,436 positions for 15 taxa), encompassing almost all sequences usable for phylogeny reconstruction within spermatophytes. The data still contain support for both the ANITA-basal and grasses-basal hypotheses. Using simulations we can show that were the ANITA-basal hypothesis true, parsimony (and distance-based methods with many models) would be expected to fail to recover it. The self-evident explanation for this failure appears to be a long-branch attraction (LBA) between the clade of grasses and the out-group. However, this LBA cannot explain the discrepancies observed between tree topology recovered using the maximum likelihood (ML) method and the topologies recovered using the parsimony and distance-based methods when grasses are deleted. Furthermore, the fact that neither maximum parsimony nor distance methods consistently recover the ML tree, when according to the simulations they would be expected to, when the out-group (Pinus) is deleted, suggests that either the generating tree is not correct or the best symmetric model is misspecified (or both). We demonstrate that the tree recovered under ML is extremely sensitive to model specification and that the best symmetric model is misspecified. Hence, we remain agnostic regarding phylogenetic relationships among basal angiosperm lineages.     

Noncoding sequences from the slowly evolving chloroplast inverted repeat in addition to rbcL data do not support gnetalean affinities of angiosperms

We developed PCR primers against highly conserved regions of the rRNA operon located within the inverted repeat of the chloroplast genome and used these to amplify the region spanning from the 3' terminus of the 23S rRNA gene to the 5' terminus of the 5S rRNA gene. The sequence of this roughly 500-bp region, which includes the 4.5S rRNA gene and two chloroplast intergenic transcribed spacer regions (cpITS2 and cpITS3), was determined from 20 angiosperms, 7 gymnosperms, and 16 ferns (21,700 bp). Sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) from the same or confamilial genera were analyzed in both separate and combined data sets. Due to the low substitution rate in the inverted repeat region, noncoding sequences in the cpITS region are not saturated with substitutions, in contrast to synonymous sites in rbcL, which are shown to evolve roughly six times faster than noncoding cpITS sequences. Several length polymorphisms with very clear phylogenetic distributions were detected in the data set. Results of phylogenetic analyses provide very strong bootstrap support for monophyly of both spermatophytes and angiosperms. No support for a sister group relationship between Gnetales and angiosperms in either cpITS or rbcL data was found. Rather, weak bootstrap support for monophyly of gymnosperms studied and for a basal position for the aquatic angiosperm Nymphaea among angiosperms studied was observed. Noncoding sequences from the inverted repeat region of chloroplast DNA appear suitable for study of land plant evolution.