Wenhu Guo

Complete plastid genomes from Ophioglossum californicum, Psilotum nudum, and Equisetum hyemale reveal an ancestral land plant genome structure and resolve the position of Equisetales among monilophytes

BackgroundPlastid genome structure and content is remarkably conserved in land plants. This widespread conservation has facilitated taxon-rich phylogenetic analyses that have resolved organismal relationships among many land plant groups. However, the relationships among major fern lineages, especially the placement of Equisetales, remain enigmatic.ResultsIn order to understand the evolution of plastid genomes and to establish phylogenetic relationships among ferns, we sequenced the plastid genomes from three early diverging species: Equisetum hyemale (Equisetales), Ophioglossum californicum (Ophioglossales), and Psilotum nudum (Psilotales). A comparison of fern plastid genomes showed that some lineages have retained inverted repeat (IR) boundaries originating from the common ancestor of land plants, while other lineages have experienced multiple IR changes including expansions and inversions. Genome content has remained stable throughout ferns, except for a few lineage-specific losses of genes and introns. Notably, the losses of the rps16 gene and the rps12i346 intron are shared among Psilotales, Ophioglossales, and Equisetales, while the gain of a mitochondrial atp1 intron is shared between Marattiales and Polypodiopsida. These genomic structural changes support the placement of Equisetales as sister to Ophioglossales + Psilotales and Marattiales as sister to Polypodiopsida. This result is augmented by some molecular phylogenetic analyses that recover the same relationships, whereas others suggest a relationship between Equisetales and Polypodiopsida.ConclusionsAlthough molecular analyses were inconsistent with respect to the position of Marattiales and Equisetales, several genomic structural changes have for the first time provided a clear placement of these lineages within the ferns. These results further demonstrate the power of using rare genomic structural changes in cases where molecular data fail to provide strong phylogenetic resolution.

Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates

Rates of nucleotide substitution were previously shown to be several times slower in the plastid inverted repeat (IR) compared with single-copy (SC) regions, suggesting that the IR provides enhanced copy-correction activity. To examine the generality of this synonymous rate dependence on the IR, we compared plastomes from 69 pairs of closely related species representing 52 families of angiosperms, gymnosperms, and ferns. We explored the breadth of IR boundary shifts in land plants and demonstrate that synonymous substitution rates are, on average, 3.7 times slower in IR genes than in SC genes. In addition, genes moved from the SC into the IR exhibit lower synonymous rates consistent with other IR genes, while genes moved from the IR into the SC exhibit higher rates consistent with other SC genes. Surprisingly, however, several plastid genes from Pelargonium, Plantago, and Silene have highly accelerated synonymous rates despite their IR localization. Together, these results provide strong evidence that the duplicative nature of the IR reduces the substitution rate within this region. The anomalously fast-evolving genes in Pelargonium, Plantago, and Silene indicate localized hypermutation, potentially induced by a higher level of error-prone double-strand break repair in these regions, which generates substitutional rate variation.

Unprecedented Heterogeneity in the Synonymous Substitution Rate within a Plant Genome

The synonymous substitution rate varies widely among species, but it is generally quite stable within a genome due to the absence of strong selective pressures. In plants, plastid genes tend to evolve faster than mitochondrial genes, rate variation among species generally correlates between the mitochondrial and plastid genomes, and few examples of intragenomic rate heterogeneity exist. To study the extent of substitution rate variation between and within plant organellar genomes, we sequenced the complete mitochondrial and plastid genomes from the bugleweed, Ajuga reptans, which was previously shown to exhibit rate heterogeneity for several mitochondrial genes. Substitution rates were accelerated specifically in the mitochondrial genome, which contrasts with correlated plastid and mitochondrial rate changes in most other angiosperms. Strikingly, we uncovered a 340-fold range of synonymous substitution rate variation among Ajuga mitochondrial genes. This is by far the largest amount of synonymous rate heterogeneity ever reported for a genome, but the evolutionary forces driving this phenomenon are unclear. Selective effects on synonymous sites in plant mitochondria are generally weak and thus unlikely to generate such unprecedented intragenomic rate heterogeneity. Quickly evolving genes are not clustered in the genome, arguing against localized hypermutation, although it is possible that they were clustered ancestrally given the high rate of genomic rearrangement in plant mitochondria. Mutagenic retroprocessing, involving error-prone reverse transcription and genomic integration of mature transcripts, is hypothesized as another potential explanation.

Predominant and Substoichiometric Isomers of the Plastid Genome Coexist within Juniperus Plants and Have Shifted Multiple Times during Cupressophyte Evolution

Most land plant plastomes contain two copies of a large inverted repeat (IR) that promote high-frequency homologous recombination to generate isomeric genomic forms. Among conifer plastomes, this canonical IR is highly reduced in Pinaceae and completely lost from cupressophytes. However, both lineages have acquired short, novel IRs, some of which also exhibit recombinational activity to generate genomic structural diversity. This diversity has been shown to exist between, and occasionally within, cupressophyte species, but it is not known whether multiple genomic forms coexist within individual plants. To examine the recombinational potential of the novel cupressophyte IRs within individuals and between species, we sequenced the plastomes of four closely related species of Juniperus. The four plastomes have identical gene content and genome organization except for a large 36 kb inversion between approximately 250 bp IR containing trnQ-UUG. Southern blotting showed that different isomeric versions of the plastome predominate among individual junipers, whereas polymerase chain reaction and high-throughput read-pair mapping revealed the substoichiometric presence of the alternative isomeric form within each individual plant. Furthermore, our comparative genomic studies demonstrate that the predominant and substoichiometric arrangements of this IR have changed several times in other cupressophytes as well. These results provide compelling evidence for substoichiometric shifting of plastomic forms during cupressophyte evolution and suggest that substoichiometric shifting activity in plastid genomes may be adaptive.

Variable Frequency of Plastid RNA Editing among Ferns and Repeated Loss of Uridine- to-Cytidine Editing from Vascular Plants

The distinct distribution and abundance of C-to-U and U-to-C RNA editing among land plants suggest that these two processes originated and evolve independently, but the paucity of information from several key lineages limits our understanding of their evolution. To examine the evolutionary diversity of RNA editing among ferns, we sequenced the plastid transcriptomes from two early diverging species, Ophioglossum californicum and Psilotum nudum. Using a relaxed automated approach to minimize false negatives combined with manual inspection to eliminate false positives, we identified 297 C-to-U and three U-to-C edit sites in the O. californicum plastid transcriptome but only 27 C-to-U and no U-to-C edit sites in the P. nudum plastid transcriptome. A broader comparison of editing content with the leptosporangiate fern Adiantum capillus-veneris and the hornwort Anthoceros formosae uncovered large variance in the abundance of plastid editing, indicating that the frequency and type of RNA editing is highly labile in ferns. Edit sites that increase protein conservation among species are more abundant and more efficiently edited than silent and non-conservative sites, suggesting that selection maintains functionally important editing. The absence of U-to-C editing from P. nudum plastid transcripts and other vascular plants demonstrates that U-to-C editing loss is a recurrent phenomenon in vascular plant evolution.

Evolution of Plant Mitochondrial Intron-Encoded Maturases: Frequent Lineage-Specific Loss and Recurrent Intracellular Transfer to the Nucleus

Among land plants, mitochondrial and plastid group II introns occasionally encode proteins called maturases that are important for splicing. Angiosperm nuclear genomes also encode maturases that are targeted to the organelles, but it is not known whether nucleus-encoded maturases exist in other land plant lineages. To examine the evolutionary diversity and history of this essential gene family, we searched for maturase homologs in recently sequenced nuclear and mitochondrial genomes from diverse land plants. We found that maturase content in mitochondrial genomes is highly lineage specific, such that orthologous maturases are rarely shared among major land plant groups. The presence of numerous mitochondrial pseudogenes in the mitochondrial genomes of several species implies that the sporadic maturase distribution is due to frequent inactivation and eventual loss over time. We also identified multiple maturase paralogs in the nuclear genomes of the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens, and the representative angiosperm Vitis vinifera. Phylogenetic analyses of organelle- and nucleus-encoded maturases revealed that the nuclear maturase genes in angiosperms, lycophytes, and mosses arose by multiple shared and independent transfers of mitochondrial paralogs to the nuclear genome during land plant evolution. These findings indicate that plant mitochondrial maturases have experienced a surprisingly dynamic history due to a complex interaction of multiple evolutionary forces that affect the rates of maturase gain, retention, and loss.

Multiple origins of endosymbionts in Chlorellaceae with no reductive effects on the plastid or mitochondrial genomes

Ancient endosymbiotic relationships have led to extreme genomic reduction in many bacterial and eukaryotic algal endosymbionts. Endosymbionts in more recent and/or facultative relationships can also experience genomic reduction to a lesser extent, but little is known about the effects of the endosymbiotic transition on the organellar genomes of eukaryotes. To understand how the endosymbiotic lifestyle has affected the organellar genomes of photosynthetic green algae, we generated the complete plastid genome (plastome) and mitochondrial genome (mitogenome) sequences from three green algal endosymbionts (Chlorella heliozoae, Chlorella variabilis and Micractinium conductrix). The mitogenomes and plastomes of the three newly sequenced endosymbionts have a standard set of genes compared with free-living trebouxiophytes, providing no evidence for functional genomic reduction. Instead, their organellar genomes are generally larger and more intron rich. Intron content is highly variable among the members of Chlorella, suggesting very high rates of gain and/or loss of introns during evolution. Phylogenetic analysis of plastid and mitochondrial genes demonstrated that the three endosymbionts do not form a monophyletic group, indicating that the endosymbiotic lifestyle has evolved multiple times in Chlorellaceae. In addition, M. conductrix is deeply nested within the Chlorella clade, suggesting that taxonomic revision is needed for one or both genera.

High and Variable Rates of Repeat-Mediated Mitochondrial Genome Rearrangement in a Genus of Plants

For 30 years, it has been clear that angiosperm mitochondrial genomes evolve rapidly in sequence arrangement (i.e., synteny), yet absolute rates of rearrangement have not been measured in any plant group, nor is it known how much these rates vary. To investigate these issues, we sequenced and reconstructed the rearrangement history of seven mitochondrial genomes in Monsonia (Geraniaceae). We show that rearrangements (occurring mostly as inversions) not only take place at generally high rates in these genomes but also uncover significant variation in rearrangement rates. For example, the hyperactive mitochondrial genome of Monsonia ciliata has accumulated at least 30 rearrangements over the last million years, whereas the branch leading to M. ciliata and its sister species has sustained rearrangement at a rate that is at least ten times lower. Furthermore, our analysis of published data shows that rates of mitochondrial genome rearrangement in seed plants vary by at least 600-fold. We find that sites of rearrangement are highly preferentially located in very close proximity to repeated sequences in Monsonia. This provides strong support for the hypothesis that rearrangement in angiosperm mitochondrial genomes occurs largely through repeat-mediated recombination. Because there is little variation in the amount of repeat sequence among Monsonia genomes, the variable rates of rearrangement in Monsonia probably reflect variable rates of mitochondrial recombination itself. Finally, we show that mitochondrial synonymous substitutions occur in a clock-like manner in Monsonia; rates of mitochondrial substitutions and rearrangements are therefore highly uncoupled in this group.