Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae.

BACKGROUND: The plastid maturase MatK has been implicated as a possible model for the evolutionary "missing link" between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution. RESULTS: Sequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history. CONCLUSION: These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

Canary grasses (Phalaris, Poaceae): biogeography, molecular dating and the role of floret structure in dispersal.

Canary grasses (Phalaris, Poaceae) include 21 species, widely spread throughout the temperate and subtropical regions of the world with two centres of diversity: the Mediterranean Basin and western North America. The genus contains annual and perennial, endemic, cosmopolitan, wild, and invasive species with diploid, tetraploid and hexaploid cytotypes. As such, Phalaris presents an ideal platform to study diversification via historic hybridization and polyploidy events, and geographical dispersal in grasses. We present the first empirical phylogeographic study for Phalaris testing current, intuitive hypotheses on the centres of origin, historic dispersal events and diversification within a geological timeframe. Bayesian methods (beast, version 1.6.2) were used to establish divergence dates, and dispersal-vicariance analyses (rasp, version 2.1b) were implemented for ancestral node reconstructions. Our phylogeographic results indicate that the genus emerged during the Miocene epoch [20.6-8.4 Ma (million years ago)] in the Mediterranean basin followed by dispersal and vicariance events to Africa, Asia and the Americas. We propose that a diploid ancestor of P. arundinacea migrated to western North America via the Bering Strait, where further diversification emerged in the New World. It appears that polyploidy played a major role in the evolution of the genus in the Old World, while diversification in the New World followed a primarily diploid pathway. Dispersal to various parts of the Americas followed different routes. Fertile florets with hairy protruding sterile lemmas showed significant correlation with wider geographical distribution.

Land plant evolutionary timeline: gene effects are secondary to fossil constraints in relaxed clock estimation of age and substitution rates.

PREMISE OF THE STUDY: Land plants play an essential role in the evolution of terrestrial life. Their time of origin and diversification is fundamental to understanding the evolution of life on land. We investigated the timing and the rate of molecular evolution of land plants, evaluating the effects of different types of molecular data, including temporal information from fossils, and using different molecular clock methods. • METHODS: Ages and absolute rates were estimated independently with two substitutionally different data sets: a highly conserved 4-gene data set and matK, a fast-evolving gene. The vascular plant backbone and the crown nodes of all major lineages were calibrated with fossil-derived ages. Dates and absolute rates were estimated while including or excluding the calibrations and using two relaxed clocks that differ in their implementation of temporal autocorrelation. • KEY RESULTS: Land plants diverged from streptophyte alga 912 (870-962) million years ago (Mya) but diversified into living lineages 475 (471-480) Mya. Ages estimated for all major land-plant lineages agree with their fossil record, except for angiosperms. Different genes estimated very similar ages and correlated absolute rates across the tree. Excluding calibrations resulted in the greatest age differences. Different relaxed clocks provided similar ages, but different and uncorrelated absolute rates. • CONCLUSIONS: Whole-genome rate accelerations or decelerations may underlie the similar ages and correlated absolute rates estimated with different genes. We suggest that pronounced substitution rate changes around the angiosperm crown node may represent a challenge for relaxed clocks to model adequately.

Angiosperm phylogeny: 17 genes, 640 taxa.

PREMISE OF THE STUDY: Recent analyses employing up to five genes have provided numerous insights into angiosperm phylogeny, but many relationships have remained unresolved or poorly supported. In the hope of improving our understanding of angiosperm phylogeny, we expanded sampling of taxa and genes beyond previous analyses. METHODS: We conducted two primary analyses based on 640 species representing 330 families. The first included 25260 aligned base pairs (bp) from 17 genes (representing all three plant genomes, i.e., nucleus, plastid, and mitochondrion). The second included 19846 aligned bp from 13 genes (representing only the nucleus and plastid). KEY RESULTS: Many important questions of deep-level relationships in the nonmonocot angiosperms have now been resolved with strong support. Amborellaceae, Nymphaeales, and Austrobaileyales are successive sisters to the remaining angiosperms (Mesangiospermae), which are resolved into Chloranthales + Magnoliidae as sister to Monocotyledoneae + [Ceratophyllaceae + Eudicotyledoneae]. Eudicotyledoneae contains a basal grade subtending Gunneridae. Within Gunneridae, Gunnerales are sister to the remainder (Pentapetalae), which comprises (1) Superrosidae, consisting of Rosidae (including Vitaceae) and Saxifragales; and (2) Superasteridae, comprising Berberidopsidales, Santalales, Caryophyllales, Asteridae, and, based on this study, Dilleniaceae (although other recent analyses disagree with this placement). Within the major subclades of Pentapetalae, most deep-level relationships are resolved with strong support. CONCLUSIONS: Our analyses confirm that with large amounts of sequence data, most deep-level relationships within the angiosperms can be resolved. We anticipate that this well-resolved angiosperm tree will be of broad utility for many areas of biology, including physiology, ecology, paleobiology, and genomics.

Inferring phylogenies with incomplete data sets: a 5-gene, 567-taxon analysis of angiosperms.

BACKGROUND: Phylogenetic analyses of angiosperm relationships have used only a small percentage of available sequence data, but phylogenetic data matrices often can be augmented with existing data, especially if one allows missing characters. We explore the effects on phylogenetic analyses of adding 378 matK sequences and 240 26S rDNA sequences to the complete 3-gene, 567-taxon angiosperm phylogenetic matrix of Soltis et al. RESULTS: We performed maximum likelihood bootstrap analyses of the complete, 3-gene 567-taxon data matrix and the incomplete, 5-gene 567-taxon data matrix. Although the 5-gene matrix has more missing data (27.5%) than the 3-gene data matrix (2.9%), the 5-gene analysis resulted in higher levels of bootstrap support. Within the 567-taxon tree, the increase in support is most evident for relationships among the 170 taxa for which both matK and 26S rDNA sequences were added, and there is little gain in support for relationships among the 119 taxa having neither matK nor 26S rDNA sequences. The 5-gene analysis also places the enigmatic Hydrostachys in Lamiales (BS = 97%) rather than in Cornales (BS = 100% in 3-gene analysis). The placement of Hydrostachys in Lamiales is unprecedented in molecular analyses, but it is consistent with embryological and morphological data. CONCLUSION: Adding available, and often incomplete, sets of sequences to existing data sets can be a fast and inexpensive way to increase support for phylogenetic relationships and produce novel and credible new phylogenetic hypotheses.

Molecular evolution of genes encoding allergen proteins in the peanuts genus Arachis: Structural and functional implications.

Food allergies are severe immune responses to plant and animal products mediated by immunoglobulin E (IgE). Peanuts (Arachis hypogaea L.) are among the top 15 crops that feed the world. However, peanuts is among the "big eight food allergens", and allergies induced by peanuts are a significant public health problem and a life-threatening concern. Targeted mutation studies in peanuts demonstrate that single residue alterations in these allergen proteins could result in substantial reduction in allergenicity. Knowledge of peanut allergen proteins is confined to the allotetraploid crop and its two progenitors. We explored frequencies and positions of natural mutations in the hyperallergenic homologues Ara h 2 and Ara h 6 in newly generated sequences for 24 Arachis wild species and the crop species, assessed potential mutational impact on allergenicity using immunoblots and structural modeling, and evaluated whether these mutations follow evolutionary trends. We uncovered a wealth of natural mutations, both substitutions and gaps, including the elimination of immunodominant epitopes in some species. These molecular alterations appear to be associated with substantial reductions in allergenicity. The study demonstrated that Ara h 2 and Ara h 6 follow contrasting modes of natural selection and opposing mutational patterns, particularly in epitope regions. Phylogenetic analysis revealed a progressive trend towards immunodominant epitope evolution in Ara h 2. The findings provide valuable insight into the interactions among mutations, protein structure and immune system response, thus presenting a valuable platform for future manipulation of allergens to minimize, treat or eliminate allergenicity. The study strongly encourages exploration of genepools of economically important plants in allergenicity research.

Phylogenetic signal in matK vs. trnK: a case study in early diverging eudicots (angiosperms).

The matK gene has been among the most useful loci for resolving plant phylogenetic relationships at different evolutionary time-scales, but much less is known about the phylogenetic utility of the flanking trnK intron, especially for deep level phylogenetics. We compared the relative performance of matK and trnK intron regions for resolving the relationships of the early diverging eudicots (angiosperms). The two regions display similar nucleotide compositions and distributions of rate variation among sites. The trnK intron sequences also provide similar levels of phylogenetic information per-site as matK. Combining the trnK intron sequences with matK increases overall bootstrap support for the early diverging eudicots compared to analyses of matK alone. MP, ML and Bayesian analyses provide strong support for eudicots, the sister group relationship of Ranunculales to remaining eudicots, and a Buxales+Trochodendraceae+core eudicots clade. matK and the trnK intron support conflicting positions for Buxales and Trochodendrales in relation to the core eudicots.

Amino acid variation in the 10 kDa Oryza prolamin seed storage protein.

The deduced amino acid variability for the 10 kDa prolamin was determined for 16 Oryza species, both cultivated (rice) and wild. Prolamin, a seed storage protein and site of nitrogen and sulfur accumulation, is sequestered in the subaleurone layer of the starchy endosperm for use during seedling germination. The 10 kDa prolamin amino acid distribution for the cultivated species (O. sativa and O. glaberrima) was determined and compared to those of wild and, hitherto unknown, noncultivated Oryza species. Four wild species (O. granulata, O. australiensis, O. brachyantha, and O. meyeriana) exhibited the greatest residue heterogeneity in both the signal and mature peptide regions. A breakdown of the essential amino acid variance among three Central/South American and one African endemic wild species is also presented and compared with those of rice.

Impact of gene molecular evolution on phylogenetic reconstruction: a case study in the rosids (Superorder Rosanae, Angiosperms).

Rate of substitution of genomic regions is among the most debated intrinsic features that impact phylogenetic informativeness. However, this variable is also coupled with rates of nonsynonymous substitutions that underscore the nature and degree of selection on the selected genes. To empirically address these variables, we constructed four completely overlapping data sets of plastid matK, atpB, rbcL, and mitochondrial matR genes and used the rosid lineage (angiosperms) as a working platform. The genes differ in combinations of overall rates of nucleotide and amino acid substitutions. Tree robustness, homoplasy, accuracy in contrast to a reference tree, and phylogenetic informativeness are evaluated. The rapidly evolving/unconstrained matK faired best, whereas remaining genes varied in degrees of contribution to rosid phylogenetics across the lineage's 108 million years evolutionary history. Phylogenetic accuracy was low with the slowly evolving/unconstrained matR despite least amount of homoplasy. Third codon positions contributed the highest amount of parsimony informative sites, resolution and informativeness, but magnitude varied with gene mode of evolution. These findings are in clear contrast with the views that rapidly evolving regions and the 3rd codon position have inevitable negative impact on phylogenetic reconstruction at deep historic level due to accumulation of multiple hits and subsequent elevation in homoplasy and saturation. Relaxed evolutionary constraint in rapidly evolving genes distributes substitutions across codon positions, an evolutionary mode expected to reduce the frequency of multiple hits. These findings should be tested at deeper evolutionary histories.

Evaluating evolutionary constraint on the rapidly evolving gene matK using protein composition.

The rapidly evolving chloroplast matK gene has nucleotide and amino acid substitution rates suggestive of progression toward a pseudogene state. However, molecular evidence has demonstrated that matK is expressed and functional. We explore in this paper the underlying factors behind the mode and tempo of matK evolution that allow this protein coding gene to accommodate such elevated rates of substitution and yet maintain functionality. Conservative amino acid replacement may reconcile the fast evolutionary rate in matK with conservation in protein function. Based on this premise, we have examined putative amino acid sequences for MATK from across green plants to determine constraint on this protein as indicated by variation in composition of amino acid side chain category. Amino acids in the MATK ORF were divided into six categories based on chemical properties of their side chains: nonpolar, uncharged (pH 7), basic, acidic, aromatic, and "special" (amino acids that specifically affect protein structure, i.e., proline, glycine, and cysteine). The amount of standard deviation (SD) in side chain composition was used as a measure of variation and constraint, where a low SD implied high evolutionary constraint and a high SD implied low constraint. Further, we used secondary structure prediction to evaluate if conservation observed in side chain composition was reflected in stable predicted structure. The results of this study demonstrate evolutionary constraint on MATK, identify three regions of functional importance, show highly conserved secondary structure, and support the putative function of MATK as a group II intron maturase.

Expression of matK: functional and evolutionary implications.

Strong phylogenetic signal from matK has rendered it an invaluable gene in plant systematic and evolutionary studies at various evolutionary depths. Further, matK is proposed as the only chloroplast-encoded group II intron maturase, thus implicating MATK in chloroplast posttranscriptional processing. For a protein-coding gene, matK has an unusual evolutionary mode and tempo, including relatively high substitution rates at both the nucleotide and amino acids levels. These evolutionary features have raised questions about matK function. In the current study, we examined matK RNA and protein from representative land plant species to provide insight into functional aspects of this unusual gene. We report the first evidence of a transcript for matK separate from the trnK precursor and demonstrate that a full-length MATK protein exists in five angiosperm species. We also show that matK RNA and protein levels are regulated by light and developmental stage, suggesting functional roles for this putative maturase. Specifically, matK expression increased after etiolation and decreased at 4 weeks after germination. This work provides evidence for the expression of the only putative chloroplast-encoded group II intron maturase and insight into regulation mechanisms relating to plant development and, indirectly, to photosynthesis.

Phylogenetic utility of rapidly evolving DNA at high taxonomical levels: contrasting matK, trnT-F, and rbcL in basal angiosperms.

The prevailing view in molecular systematics is that relationships among distantly related taxa should be inferred using DNA segments with low rates of evolution. However, recent analyses of sequences from the rapidly evolving matK and trnT-trnF regions yielded well resolved and highly supported trees for early diverging angiosperms. We compare here the phylogenetic structure in matK, trnT-F, and rbcL datasets for the same 42, primarily basal angiosperm taxa. Phylogenetic trees based on matK or trnT-F are far more robust than those based on rbcL. Combined analysis of the rapidly evolving regions provides support for higher-level relationships stronger than that derived from analyses of multi-gene datasets of up to several fold the number of characters analyzed here. In addition to displaying a higher percentage of parsimony-informative characters, the average phylogenetic signal per informative character is significantly higher in the datasets from rapidly evolving DNA than in the more slowly evolving rbcL, as detected using resampling of identical numbers of parsimony-informative characters from the data matrices and subjecting different statistics for overall tree robustness and phylogenetic signal to significance tests. Automated via a set of scripts, the method used here should be easily extendable to comparisons of a broader range of genomic regions for varying taxon samplings. The relative performance of markers correlates not only with a lower mean homoplasy in matK and trnT-trnF compared to rbcL, but in particular correlates negatively with the percentage of sites exhibiting maximum or close to maximum homoplasy. A likelihood ratio test confirms that the rapidly evolving gene matK evolves significantly closer to neutrality, which may be one of the underlying factors for lower levels of overall homoplasy. Our results are in line with evidence from simulation studies suggesting that the deleterious effect of multiple hits in using rapidly evolving DNA at rather deep phylogenetic levels may have been overestimated, and thus promote extending the use of rapidly evolving DNA to deeper phylogenetic levels.

Phylogeny of Eleusine (Poaceae: Chloridoideae) based on nuclear ITS and plastid trnT-trnF sequences.

Phylogenetic relationships in the genus Eleusine (Poaceae: Chloridoideae) were investigated using nuclear ITS and plastid trnT-trnF sequences. Separate and combined data sets were analyzed using parsimony, distance, and likelihood based methods, including Bayesian. Data congruence was examined using character and topological measures. Significant data heterogeneity was detected, but there was little conflict in the topological substructure measures for triplets and quartets, and resolution and clade support increased in the combined analysis. Data incongruence may be a result of noise and insufficient information in the slower evolving trnT-trnF. Monophyly of Eleusine is strongly supported in all analyses, but basal relationships in the genus remain uncertain. There is good support for a CAIK clade (E. coracana subsp. coracana and africana, E. indica, and E. kigeziensis), with E. tristachya as its sister group. Two putative ITS homeologues (A and B loci) were identified in the allotetraploid E. coracana; the 'B' locus sequence type was not found in the remaining species. Eleusine coracana and its putative 'A' genome donor, the diploid E. indica, are confirmed close allies, but sequence data contradicts the hypothesis that E. floccifolia is its second genome donor. The 'B' genome donor remains unidentified and may be extinct.

Evolutionary implications of substitution patterns in prolamin genes of Oryza glaberrima (African rice, Poaceae) and related species.

Patterns of sequence variation of nuclear genes encoding 10-kDa and 16-kDa prolamin seed storage proteins were examined in Oryza glaberrima (African rice, Poaceae) and O. barthii and compared to available sequences for the genus to assess potential application of these gene families in evolutionary studies. Sequence variation among species in 10-kDa genes was very low. In contrast, the 16-kDa genes have undergone rapid evolution, displaying a larger number of length and point mutations that in some cases result in frame shift or produce truncated protein or pseudogenes. The proportion of nonsynonymous substitution is high in both genes. Although nonsynonymous mutations did not alter the overall profile of the protein, pronounced shifts in proportions of some amino acids were evident and could have systematic application. The data provide support for a proposed direct evolution of the Asian (O. sativa) and African rice from O. rufipogon and O. barthii, respectively. Patterns of amino acid frequencies of the 10-kDa genes show the distinctness of O. rufipogon and O. longistaminata from the other species. The study underscores the potential application of the prolamin genes as markers from the nuclear genome for evolutionary studies in grasses at different taxonomic levels.

Angiosperm phylogeny based on matK sequence information.

Plastid matK gene sequences for 374 genera representing all angiosperm orders and 12 genera of gymnosperms were analyzed using parsimony (MP) and Bayesian inference (BI) approaches. Traditionally, slowly evolving genomic regions have been preferred for deep-level phylogenetic inference in angiosperms. The matK gene evolves approximately three times faster than the widely used plastid genes rbcL and atpB. The MP and BI trees are highly congruent. The robustness of the strict consensus tree supercedes all individual gene analyses and is comparable only to multigene-based phylogenies. Of the 385 nodes resolved, 79% are supported by high jackknife values, averaging 88%. Amborella is sister to the remaining angiosperms, followed by a grade of Nymphaeaceae and Austrobaileyales. Bayesian inference resolves Amborella + Nymphaeaceae as sister to the rest, but with weak (0.42) posterior probability. The MP analysis shows a trichotomy sister to the Austrobaileyales representing eumagnoliids, monocots + Chloranthales, and Ceratophyllum + eudicots. The matK gene produces the highest internal support yet for basal eudicots and, within core eudicots, resolves a crown group comprising Berberidopsidaceae/Aextoxicaceae, Santalales, and Caryophyllales + asterids. Moreover, matK sequences provide good resolution within many angiosperm orders. Combined analyses of matK and other rapidly evolving DNA regions with available multigene data sets have strong potential to enhance resolution and internal support in deep level angiosperm phylogenetics and provide additional insights into angiosperm evolution.