Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae.

Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues.

Monilophyte mitochondrial rps1 genes carry a unique group II intron that likely originated from an ancient paralog in rpl2.

Intron patterns in plant mitochondrial genomes differ significantly between the major land plant clades. We here report on a new, clade-specific group II intron in the rps1 gene of monilophytes (ferns). This intron, rps1i25g2, is strikingly similar to rpl2i846g2 previously identified in the mitochondrial rpl2 gene of seed plants, ferns, and the lycophyte Phlegmariurus squarrosus Although mitochondrial ribosomal protein genes are frequently subject to endosymbiotic gene transfer among plants, we could retrieve the mitochondrial rps1 gene in a taxonomically wide sampling of 44 monilophyte taxa including basal lineages such as the Ophioglossales, Psilotales, and Marattiales with the only exception being the Equisetales (horsetails). Introns rps1i25g2 and rpl2i846g2 were likewise consistently present with only two exceptions: Intron rps1i25g2 is lost in the genus Ophioglossum and intron rpl2i846g2 is lost in Equisetum bogotense Both intron sequences are moderately affected by RNA editing. The unprecedented primary and secondary structure similarity of rps1i25g2 and rpl2i846g2 suggests an ancient retrotransposition event copying rpl2i846g2 into rps1, for which we suggest a model. Our phylogenetic analysis adding the new rps1 locus to a previous data set is fully congruent with recent insights on monilophyte phylogeny and further supports a sister relationship of Gleicheniales and Hymenophyllales.

Reverse U-to-C editing exceeds C-to-U RNA editing in some ferns - a monilophyte-wide comparison of chloroplast and mitochondrial RNA editing suggests independent evolution of the two processes in both organelles.

BACKGROUND: RNA editing by C-to-U conversions is nearly omnipresent in land plant chloroplasts and mitochondria, where it mainly serves to reconstitute conserved codon identities in the organelle mRNAs. Reverse U-to-C RNA editing in contrast appears to be restricted to hornworts, some lycophytes, and ferns (monilophytes). A well-resolved monilophyte phylogeny has recently emerged and now allows to trace the side-by-side evolution of both types of pyrimidine exchange editing in the two endosymbiotic organelles. RESULTS: Our study of RNA editing in four selected mitochondrial genes show a wide spectrum of divergent RNA editing frequencies including a dominance of U-to-C over the canonical C-to-U editing in some taxa like the order Schizaeales. We find that silent RNA editing leaving encoded amino acids unchanged is highly biased with more than ten-fold amounts of silent C-to-U over U-to-C edits. In full contrast to flowering plants, RNA editing frequencies are low in early-branching monilophyte lineages but increase in later emerging clades. Moreover, while editing rates in the two organelles are usually correlated, we observe uncoupled evolution of editing frequencies in fern mitochondria and chloroplasts. Most mitochondrial RNA editing sites are shared between the recently emerging fern orders whereas chloroplast editing sites are mostly clade-specific. Finally, we observe that chloroplast RNA editing appears to be completely absent in horsetails (Equisetales), the sister clade of all other monilophytes. CONCLUSIONS: C-to-U and U-to-C RNA editing in fern chloroplasts and mitochondria follow disinct evolutionary pathways that are surprisingly different from what has previously been found in flowering plants. The results call for careful differentiation of the two types of RNA editing in the two endosymbiotic organelles in comparative evolutionary studies.

Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns.

The "Monilophyte" clade comprising ferns, horsetails and whisk ferns receives unequivocal support from molecular data as the sister clade to seed plants. However, the branching order of its earliest emerging lineages, the Equisetales (horsetails), the Marattiales, the Ophioglossales/Psilotales and the large group of leptosporangiate ferns has remained dubious. We investigated the mitochondrial nad2 and rpl2 genes as two new, intron-containing loci for a wide sampling of taxa. We found that both group II introns - nad2i542g2 and rpl2i846g2 - are universally present among monilophytes. Both introns have orthologues in seed plants where nad2i542g2 has evolved into a trans-arrangement. In contrast and despite substantial size extensions to more than 5kb in Psilotum, nad2i542g2 remains cis-arranged in the monilophytes. For phylogenetic analyses, we filled taxonomic gaps in previously investigated mitochondrial (atp1, nad5) and chloroplast (atpA, atpB, matK, rbcL, rps4) loci and created a 9-gene matrix that also included the new mitochondrial nad2 and rpl2 loci. We extended the taxon sampling with two taxa each for all land plant outgroups (liverworts, mosses, hornworts, lycophytes and seed plants) to minimize the risk of phylogenetic artefacts. We ultimately obtained a well-supported molecular phylogeny placing Marattiales as sister to leptosporangiate ferns and horsetails as sister to all remaining monilophytes. In addition, an indel in an exon of the here introduced rpl2 locus independently supports the placement of horsetails. We conclude that under dense taxon sampling, phylogenetic information from a prudent choice of loci is currently superior to character-rich phylogenomic approaches at low taxon sampling. As here shown the selective choice of loci and taxa enabled us to resolve the long-enigmatic diversifications of the earliest monilophyte lineages.

Horizontal gene transfer of chlamydial-like tRNA genes into early vascular plant mitochondria.

Mitochondrial genomes of lycophytes are surprisingly diverse, including strikingly different transfer RNA (tRNA) gene complements: No mitochondrial tRNA genes are present in the spikemoss Selaginella moellendorffii, whereas 26 tRNAs are encoded in the chondrome of the clubmoss Huperzia squarrosa. Reinvestigating the latter we found that trnL(gag) and trnS(gga) had never before been identified in any other land plant mitochondrial DNA. Sensitive sequence comparisons showed these two tRNAs as well as trnN(guu) and trnS(gcu) to be very similar to their respective counterparts in chlamydial bacteria. We identified homologs of these chlamydial-type tRNAs also in other lycophyte, fern, and gymnosperm DNAs, suggesting horizontal gene transfer (HGT) into mitochondria in the early vascular plant stem lineages. These findings extend plant mitochondrial HGT to affect individual tRNA genes, to include bacterial donors, and suggest that Chlamydiae on top of their recently proposed key role in primary chloroplast establishment may also have participated in early tracheophyte genome evolution.

Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata.

RNA editing in chloroplasts and mitochondria of land plants differs significantly in abundance. For example, some 200-500 sites of cytidine-to-uridine RNA editing exist in flowering plant mitochondria as opposed to only 30-50 such C-to-U editing events in their chloroplasts. In contrast, we predicted significantly more chloroplast RNA editing for the protein-coding genes in the available complete plastome sequences of two species of the spike moss genus Selaginella (Lycopodiophyta). To evaluate these predictions we investigated the Selaginella uncinata chloroplast transcriptome. Our exhaustive cDNA studies identified the extraordinary number of 3415 RNA-editing events, exclusively of the C-to-U type, in the 74 mRNAs encoding intact reading frames in the S. uncinata chloroplast. We find the overwhelming majority (61%) of the 428 silent editing events leaving codon meanings unaltered directly neighboring other editing events, possibly suggesting a sterically more flexible RNA-editing deaminase activity in Selaginella. No evidence of RNA editing was found for tRNAs or rRNAs but we identified a total of 74 editing sites in cDNA sequences of four group II introns (petBi6g2, petDi8g2, ycf3i124g2, and ycf3i354g2) retained in partially matured transcripts, which strongly contribute to improved base-pairing in the intron secondary structures as a likely prerequisite for their splicing.