Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy-to-access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets.

The complete plastid genome sequence of Welwitschia mirabilis: an unusually compact plastome with accelerated divergence rates.

BACKGROUND: Welwitschia mirabilis is the only extant member of the family Welwitschiaceae, one of three lineages of gnetophytes, an enigmatic group of gymnosperms variously allied with flowering plants or conifers. Limited sequence data and rapid divergence rates have precluded consensus on the evolutionary placement of gnetophytes based on molecular characters. Here we report on the first complete gnetophyte chloroplast genome sequence, from Welwitschia mirabilis, as well as analyses on divergence rates of protein-coding genes, comparisons of gene content and order, and phylogenetic implications. RESULTS: The chloroplast genome of Welwitschia mirabilis [GenBank: EU342371] is comprised of 119,726 base pairs and exhibits large and small single copy regions and two copies of the large inverted repeat (IR). Only 101 unique gene species are encoded. The Welwitschia plastome is the most compact photosynthetic land plant plastome sequenced to date; 66% of the sequence codes for product. The genome also exhibits a slightly expanded IR, a minimum of 9 inversions that modify gene order, and 19 genes that are lost or present as pseudogenes. Phylogenetic analyses, including one representative of each extant seed plant lineage and based on 57 concatenated protein-coding sequences, place Welwitschia at the base of all seed plants (distance, maximum parsimony) or as the sister to Pinus (the only conifer representative) in a monophyletic gymnosperm clade (maximum likelihood, bayesian). Relative rate tests on these gene sequences show the Welwitschia sequences to be evolving at faster rates than other seed plants. For these genes individually, a comparison of average pairwise distances indicates that relative divergence in Welwitschia ranges from amounts about equal to other seed plants to amounts almost three times greater than the average for non-gnetophyte seed plants. CONCLUSION: Although the basic organization of the Welwitschia plastome is typical, its compactness, gene content and high nucleotide divergence rates are atypical. The current lack of additional conifer plastome sequences precludes any discrimination between the gnetifer and gnepine hypotheses of seed plant relationships. However, both phylogenetic analyses and shared genome features identified here are consistent with either of the hypotheses that link gnetophytes with conifers, but are inconsistent with the anthophyte hypothesis.

Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns.

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements.

Comparative chloroplast genomics: analyses including new sequences from the angiosperms Nuphar advena and Ranunculus macranthus.

BACKGROUND: The number of completely sequenced plastid genomes available is growing rapidly. This array of sequences presents new opportunities to perform comparative analyses. In comparative studies, it is often useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the genomes reported here: Nuphar advena (from a basal-most lineage) and Ranunculus macranthus (a basal eudicot). We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages) to evaluate features such as the status of ycf15 and ycf68 as protein coding genes, the distribution of simple sequence repeats (SSRs) and longer dispersed repeats (SDR), and patterns of nucleotide composition. RESULTS: The Nuphar [GenBank:NC_008788] and Ranunculus [GenBank:NC_008796] plastid genomes share characteristics of gene content and organization with many other chloroplast genomes. Like other plastid genomes, these genomes are A+T-rich, except for rRNA and tRNA genes. Detailed comparisons of Nuphar with Nymphaea, another Nymphaeaceae, show that more than two-thirds of these genomes exhibit at least 95% sequence identity and that most SSRs are shared. In broader comparisons, SSRs vary among genomes in terms of abundance and length and most contain repeat motifs based on A and T nucleotides. CONCLUSION: SSR and SDR abundance varies by genome and, for SSRs, is proportional to genome size. Long SDRs are rare in the genomes assessed. SSRs occur less frequently than predicted and, although the majority of the repeat motifs do include A and T nucleotides, the A+T bias in SSRs is less than that predicted from the underlying genomic nucleotide composition. In codon usage third positions show an A+T bias, however variation in codon usage does not correlate with differences in A+T-richness. Thus, although plastome nucleotide composition shows "A+T richness", an A+T bias is not apparent upon more in-depth analysis, at least in these aspects. The pattern of evolution in the sequences identified as ycf15 and ycf68 is not consistent with them being protein-coding genes. In fact, these regions show no evidence of sequence conservation beyond what is normal for non-coding regions of the IR.

Distance-based genome rearrangement phylogeny.

Evolution operates on whole genomes through direct rearrangements of genes, such as inversions, transpositions, and inverted transpositions, as well as through operations, such as duplications, losses, and transfers, that also affect the gene content of the genomes. Because these events are rare relative to nucleotide substitutions, gene order data offer the possibility of resolving ancient branches in the tree of life; the combination of gene order data with sequence data also has the potential to provide more robust phylogenetic reconstructions, since each can elucidate evolution at different time scales. Distance corrections greatly improve the accuracy of phylogeny reconstructions from DNA sequences, enabling distance-based methods to approach the accuracy of the more elaborate methods based on parsimony or likelihood at a fraction of the computational cost. This paper focuses on developing distance correction methods for phylogeny reconstruction from whole genomes. The main question we investigate is how to estimate evolutionary histories from whole genomes with equal gene content, and we present a technique, the empirically derived estimator (EDE), that we have developed for this purpose. We study the use of EDE on whole genomes with identical gene content, and we explore the accuracy of phylogenies inferred using EDE with the neighbor joining and minimum evolution methods under a wide range of model conditions. Our study shows that tree reconstruction under these two methods is much more accurate when based on EDE distances than when based on other distances previously suggested for whole genomes.

Identifying the basal angiosperm node in chloroplast genome phylogenies: sampling one's way out of the Felsenstein zone.

While there has been strong support for Amborella and Nymphaeales (water lilies) as branching from basal-most nodes in the angiosperm phylogeny, this hypothesis has recently been challenged by phylogenetic analyses of 61 protein-coding genes extracted from the chloroplast genome sequences of Amborella, Nymphaea, and 12 other available land plant chloroplast genomes. These character-rich analyses placed the monocots, represented by three grasses (Poaceae), as sister to all other extant angiosperm lineages. We have extracted protein-coding regions from draft sequences for six additional chloroplast genomes to test whether this surprising result could be an artifact of long-branch attraction due to limited taxon sampling. The added taxa include three monocots (Acorus, Yucca, and Typha), a water lily (Nuphar), a ranunculid (Ranunculus), and a gymnosperm (Ginkgo). Phylogenetic analyses of the expanded DNA and protein data sets together with microstructural characters (indels) provided unambiguous support for Amborella and the Nymphaeales as branching from the basal-most nodes in the angiosperm phylogeny. However, their relative positions proved to be dependent on the method of analysis, with parsimony favoring Amborella as sister to all other angiosperms and maximum likelihood (ML) and neighbor-joining methods favoring an Amborella + Nymphaeales clade as sister. The ML phylogeny supported the later hypothesis, but the likelihood for the former hypothesis was not significantly different. Parametric bootstrap analysis, single-gene phylogenies, estimated divergence dates, and conflicting indel characters all help to illuminate the nature of the conflict in resolution of the most basal nodes in the angiosperm phylogeny. Molecular dating analyses provided median age estimates of 161 MYA for the most recent common ancestor (MRCA) of all extant angiosperms and 145 MYA for the MRCA of monocots, magnoliids, and eudicots. Whereas long sequences reduce variance in branch lengths and molecular dating estimates, the impact of improved taxon sampling on the rooting of the angiosperm phylogeny together with the results of parametric bootstrap analyses demonstrate how long-branch attraction might mislead genome-scale phylogenetic analyses.

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.

Chloroplast DNA rearrangements in Campanulaceae: phylogenetic utility of highly rearranged genomes.

BACKGROUND: The Campanulaceae (the "hare bell" or "bellflower" family) is a derived angiosperm family comprised of about 600 species treated in 35 to 55 genera. Taxonomic treatments vary widely and little phylogenetic work has been done in the family. Gene order in the chloroplast genome usually varies little among vascular plants. However, chloroplast genomes of Campanulaceae represent an exception and phylogenetic analyses solely based on chloroplast rearrangement characters support a reasonably well-resolved tree. RESULTS: Chloroplast DNA physical maps were constructed for eighteen representatives of the family. So many gene order changes have occurred among the genomes that characterizing individual mutational events was not always possible. Therefore, we examined different, novel scoring methods to prepare data matrices for cladistic analysis. These approaches yielded largely congruent results but varied in amounts of resolution and homoplasy. The strongly supported nodes were common to all gene order analyses as well as to parallel analyses based on ITS and rbcL sequence data. The results suggest some interesting and unexpected intrafamilial relationships. For example fifteen of the taxa form a derived clade; whereas the remaining three taxa--Platycodon, Codonopsis, and Cyananthus--form the basal clade. This major subdivision of the family corresponds to the distribution of pollen morphology characteristics but is not compatible with previous taxonomic treatments. CONCLUSIONS: Our use of gene order data in the Campanulaceae provides the most highly resolved phylogeny as yet developed for a plant family using only cpDNA rearrangements. The gene order data showed markedly less homoplasy than sequence data for the same taxa but did not resolve quite as many nodes. The rearrangement characters, though relatively few in number, support robust and meaningful phylogenetic hypotheses and provide new insights into evolutionary relationships within the Campanulaceae.

Fast phylogenetic methods for the analysis of genome rearrangement data: an empirical study.

Evolution operates on whole genomes through mutations that change the order and strandedness of genes within the genomes. Thus analyses of gene-order data present new opportunities for discoveries about deep evolutionary events, provided that sufficiently accurate methods can be developed to reconstruct evolutionary trees. In this paper we present two new methods of character coding for parsimony-based analysis of genomic rearrangements: one called MPBE-2, and a new parsimony-based method which we call MPME (based on an encoding of Bryant), both variants of the MPBE method. We then conduct computer simulations to compare this class of methods to distance-based methods (NJ under various distance measures). Our empirical results show that two of our new methods return highly accurate estimates of the true tree, outperforming the other methods significantly, especially when close to saturation.