Andrew J. Alverson

Rapid Evolution of Enormous, Multichromosomal Genomes in Flowering Plant Mitochondria with Exceptionally High Mutation Rates

A pair of species within the genus Silene have evolved the largest known mitochondrial genomes, coinciding with extreme changes in mutation rate, recombination activity, and genome structure.

Methods for Obtaining and Analyzing Whole Chloroplast Genome Sequences

During the past decade, there has been a rapid increase in our understanding of plastid genome organization and evolution due to the availability of many new completely sequenced genomes. There are 45 complete genomes published and ongoing projects are likely to increase this sampling to nearly 200 genomes during the next 5 years. Several groups of researchers including ours have been developing new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this chapter. The most important developments that enhance our ability to generate whole chloroplast genome sequences involve the generation of pure fractions of chloroplast genomes by whole genome amplification using rolling circle amplification, cloning genomes into Fosmid or bacterial artificial chromosome (BAC) vectors, and the development of an organellar annotation program (Dual Organellar GenoMe Annotator [DOGMA]). In addition to providing details of these methods, we provide an overview of methods for analyzing complete plastid genome sequences for repeats and gene content, as well as approaches for using gene order and sequence data for phylogeny reconstruction. This explosive increase in the number of sequenced plastid genomes and improved computational tools will provide many insights into the evolution of these genomes and much new data for assessing relationships at deep nodes in plants and other photosynthetic organisms.

Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids

BackgroundThe Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids.ResultsThe Vitis vinifera chloroplast genome is 160,928 bp in length, including a pair of inverted repeats of 26,358 bp that are separated by small and large single copy regions of 19,065 bp and 89,147 bp, respectively. The gene content and order of Vitis is identical to many other unrearranged angiosperm chloroplast genomes, including tobacco. Phylogenetic analyses using maximum parsimony and maximum likelihood were performed on DNA sequences of 61 protein-coding genes for two datasets with 28 or 29 taxa, including eight or nine taxa from four of the seven currently recognized major clades of rosids. Parsimony and likelihood phylogenies of both data sets provide strong support for the placement of Vitaceae as sister to the remaining rosids. However, the position of the Myrtales and support for the monophyly of the eurosid I clade differs between the two data sets and the two methods of analysis. In parsimony analyses, the inclusion of Gossypium is necessary to obtain trees that support the monophyly of the eurosid I clade. However, maximum likelihood analyses place Cucumis as sister to the Myrtales and therefore do not support the monophyly of the eurosid I clade.ConclusionPhylogenies based on DNA sequences from complete chloroplast genome sequences provide strong support for the position of the Vitaceae as the earliest diverging lineage of rosids. Our phylogenetic analyses support recent assertions that inadequate taxon sampling and incorrect model specification for concatenated multi-gene data sets can mislead phylogenetic inferences when using whole chloroplast genomes for phylogeny reconstruction.

Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella

Shaping Plant Evolution Amborella trichopoda is understood to be the most basal extant flowering plant and its genome is anticipated to provide insights into the evolution of plant life on Earth (see the Perspective by Adams). To validate and assemble the sequence, Chamala et al. (p. 1516) combined fluorescent in situ hybridization (FISH), genomic mapping, and next-generation sequencing. The Amborella Genome Project (p. 10.1126/science.1241089) was able to infer that a whole-genome duplication event preceded the evolution of this ancestral angiosperm, and Rice et al. (p. 1468) found that numerous genes in the mitochondrion were acquired by horizontal gene transfer from other plants, including almost four entire mitochondrial genomes from mosses and algae. Much of the mitochondrial DNA genome of the flowering plant Amborella trichopoda originated from other organisms. We report the complete mitochondrial genome sequence of the flowering plant Amborella trichopoda. This enormous, 3.9-megabase genome contains six genome equivalents of foreign mitochondrial DNA, acquired from green algae, mosses, and other angiosperms. Many of these horizontal transfers were large, including acquisition of entire mitochondrial genomes from three green algae and one moss. We propose a fusion-compatibility model to explain these findings, with Amborella capturing whole mitochondria from diverse eukaryotes, followed by mitochondrial fusion (limited mechanistically to green plant mitochondria) and then genome recombination. Amborella’s epiphyte load, propensity to produce suckers from wounds, and low rate of mitochondrial DNA loss probably all contribute to the high level of foreign DNA in its mitochondrial genome.

Origins and Recombination of the Bacterial-Sized Multichromosomal Mitochondrial Genome of Cucumber

This work presents the cucumber mitochondrial genome sequence. Its extremely large size reflects the proliferation of dispersed repeats, large introns, and an abundance of chloroplast and nuclear DNA. The genome has an unusual multichromosomal structure, and computational analyses reveal a large number of recombinationally active repeats. Members of the flowering plant family Cucurbitaceae harbor the largest known mitochondrial genomes. Here, we report the 1685-kb mitochondrial genome of cucumber (Cucumis sativus). We help solve a 30-year mystery about the origins of its large size by showing that it mainly reflects the proliferation of dispersed repeats, expansions of existing introns, and the acquisition of sequences from diverse sources, including the cucumber nuclear and chloroplast genomes, viruses, and bacteria. The cucumber genome has a novel structure for plant mitochondria, mapping as three entirely or largely autonomous circular chromosomes (lengths 1556, 84, and 45 kb) that vary in relative abundance over a twofold range. These properties suggest that the three chromosomes replicate independently of one another. The two smaller chromosomes are devoid of known functional genes but nonetheless contain diagnostic mitochondrial features. Paired-end sequencing conflicts reveal differences in recombination dynamics among chromosomes, for which an explanatory model is developed, as well as a large pool of low-frequency genome conformations, many of which may result from asymmetric recombination across intermediate-sized and sometimes highly divergent repeats. These findings highlight the promise of genome sequencing for elucidating the recombinational dynamics of plant mitochondrial genomes.

COMPARATIVE SEQUENCE ANALYSIS OF DIATOM SILICON TRANSPORTERS: TOWARD A MECHANISTIC MODEL OF SILICON TRANSPORT†

Silicon is an important element in biology, for organisms ranging from unicellular algae to humans. It acts as a structural material for both plants and animals, but can also function as a metabolite or regulator of gene expression, affecting a wide range of cellular processes. Molecular details of biological interaction with silicon are poorly understood. Diatoms, the largest group of silicifying organisms, are a good model system for studying this interaction. The first proteins shown to directly interact with silicon were diatom silicon transporters (SITs). Because the basis for substrate recognition lies within the primary sequence of a protein, identification of conserved amino acid residues would provide insight into the mechanism of SIT function. Lack of SIT sequences from a diversity of diatoms and high sequence conservation in known SITs has precluded identification of such residues. In this study, PCR was used to amplify partial SIT sequences from eight diverse diatom species. Multiple gene copies were prevalent in each species, and phylogenetic analysis showed that SITs generally group according to species. In addition to partial SIT sequences, full‐length SIT genes were identified from the pennate diatom, Nitzschia alba (Lewin and Lewin), and the centric diatom Skeletonema costatum (Greville) Cleve. Comparing these SITs with previously identified SITs showed structural differences between SITs of centrics and pennates, suggesting differences in transport mechanism or regulation. Comparative amino acid analysis identified conserved regions that may be important for silicon transport, including repeats of the motif GXQ. A model for silicon uptake and efflux is presented that is consistent with known aspects of transport.

The “fossilized” mitochondrial genome of Liriodendron tulipifera: ancestral gene content and order, ancestral editing sites, and extraordinarily low mutation rate

BackgroundThe mitochondrial genomes of flowering plants vary greatly in size, gene content, gene order, mutation rate and level of RNA editing. However, the narrow phylogenetic breadth of available genomic data has limited our ability to reconstruct these traits in the ancestral flowering plant and, therefore, to infer subsequent patterns of evolution across angiosperms.ResultsWe sequenced the mitochondrial genome of Liriodendron tulipifera, the first from outside the monocots or eudicots. This 553,721 bp mitochondrial genome has evolved remarkably slowly in virtually all respects, with an extraordinarily low genome-wide silent substitution rate, retention of genes frequently lost in other angiosperm lineages, and conservation of ancestral gene clusters. The mitochondrial protein genes in Liriodendron are the most heavily edited of any angiosperm characterized to date. Most of these sites are also edited in various other lineages, which allowed us to polarize losses of editing sites in other parts of the angiosperm phylogeny. Finally, we added comprehensive gene sequence data for two other magnoliids, Magnolia stellata and the more distantly related Calycanthus floridus, to measure rates of sequence evolution in Liriodendron with greater accuracy. The Magnolia genome has evolved at an even lower rate, revealing a roughly 5,000-fold range of synonymous-site divergence among angiosperms whose mitochondrial gene space has been comprehensively sequenced.ConclusionsUsing Liriodendron as a guide, we estimate that the ancestral flowering plant mitochondrial genome contained 41 protein genes, 14 tRNA genes of mitochondrial origin, as many as 7 tRNA genes of chloroplast origin, >700 sites of RNA editing, and some 14 colinear gene clusters. Many of these gene clusters, genes and RNA editing sites have been variously lost in different lineages over the course of the ensuing ∽200 million years of angiosperm evolution.

Intragenomic nucleotide polymorphism among small subunit (18S) rdna paralogs in the diatom genus Skeletonema (Bacillariophyta)

Morphological features of the siliceous cell wall traditionally have been used to diagnose and classify species of diatoms, though an increasing number of studies distinguish new species, in part, by phylogenetic analysis of rDNA sequences. Intragenomic sequence variation is common among the hundreds to thousands of rDNA cistrons present within a genome, and this variation has strong potential to obscure species boundaries based on rDNA sequences. We screened six Skeletonema culture strains for intragenomic nucleotide polymorphisms in the small subunit (SSU) rDNA gene and found that all strains had polymorphic sites, with proportions ranging from 0.57% to 1.81%. In all cases, transitions accounted for more than 70% of nucleotide differences at polymorphic sites. Polymorphic sites were split nearly evenly in the SSU rRNA molecule between the base‐paired regions of helices (52%) and the unpaired regions of loops and bulges (48%). Phylogenetic analysis showed that SSU rDNA genotypes were monophyletic for two of the six culture strains examined. Genotypes from the other four culture strains either showed little or no phylogenetic structure compared with genotypes of other conspecific culture strains or had phylogenetic structure that was incongruent with existing species boundaries. Moderate to strong support for monophyly was recovered for four of the seven species included in the analysis. Phylogenetic results combined with the low sequence divergence of SSU rDNA genotypes within species suggest that concerted evolution has not proceeded to completion in these species and/or that the rate at which variation is being generated exceeds the rate at which concerted evolution is expunging variation.

Extensive Loss of RNA Editing Sites in Rapidly Evolving Silene Mitochondrial Genomes: Selection vs . Retroprocessing as the Driving Force

Theoretical arguments suggest that mutation rates influence the proliferation and maintenance of RNA editing. We identified RNA editing sites in five species within the angiosperm genus Silene that exhibit highly divergent mitochondrial mutation rates. We found that mutational acceleration has been associated with rapid loss of mitochondrial editing sites. In contrast, we did not find a significant difference in the frequency of editing in chloroplast genes, which lack the mutation rate variation observed in the mitochondrial genome. As found in other angiosperms, the rate of substitution at RNA editing sites in Silene greatly exceeds the rate at synonymous sites, a pattern that has previously been interpreted as evidence for selection against RNA editing. Alternatively, we suggest that editing sites may experience higher rates of C-to-T mutation than other portions of the genome. Such a pattern could be caused by gene conversion with reverse-transcribed mRNA (i.e., retroprocessing). If so, the genomic distribution of RNA editing site losses in Silene suggests that such conversions must be occurring at a local scale such that only one or two editing sites are affected at a time. Because preferential substitution at editing sites appears to occur in angiosperms regardless of the mutation rate, we conclude that mitochondrial rate accelerations within Silene have “fast-forwarded” a preexisting pattern but have not fundamentally changed the evolutionary forces acting on RNA editing sites.