Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses,liverworts, and hornworts) and the lycophytes

The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.     

Exclusive conservation of mitochondrial group II intron nad4i548 among liverworts and its use for phylogenetic studies in this ancient plant clade

Liverworts occupy a pivotal position in land plant (embryophyte) phylogeny as the presumed earliest-branching major clade, sister to all other land plants, including the mosses, hornworts, lycophytes, monilophytes and seed plants. Molecular support for this earliest dichotomy in land plant phylogeny comes from strikingly different occurrences of introns in mitochondrial genes distinguishing liverworts from all other embryophytes. Exceptionally, however, the nad5 gene--the mitochondrial locus hitherto used most widely to elucidate early land plant phylogeny--carries a group I type intron that is shared between liverworts and mosses. We here explored whether a group II intron, the other major type of organellar intron, would similarly be conserved in position across the entire diversity of extant liverworts and could be of use for phylogenetic analyses in this supposedly most ancient embryophyte clade. To this end, we investigated the nad4 gene as a candidate locus possibly featuring different introns in liverworts as opposed to the non-liverwort embryophyte (NLE) lineage. We indeed found group II intron nad4i548 universally conserved in a wide phylogenetic sampling of 55 liverwort taxa, confirming clade specificity and surprising evolutionary stability of plant mitochondrial introns. As expected, intron nad4i548g2 carries phylogenetic information in its variable sequences, which confirms and extends previous cladistic insights on liverwort evolution. We integrate the new nad4 data with those of the previously established mitochondrial nad5 and the chloroplast rbcL and rps4 genes and present a phylogeny based on the fused datasets. Notably, the phylogenetic analyses suggest a reconsideration of previous phylogenetic and taxonomic assignments for the genera Calycularia and Mylia and resolve a sister group relationship of Ptilidiales and Porellales.     

Mosses share mitochondrial group II introns with flowering plants, not with liverworts

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.     

Trans-splicing group II introns in plant mitochondria: the complete set of cis-arranged homologs in ferns, fern allies, and a hornwort.

The fragmentation of group II introns without concomitant loss of splicing competence is illustrated by extraordinary gene arrangements in plant mitochondrial genomes. The mitochondrial genes nad1, nad2, and nad5, all encoding subunits of the NADH dehydrogenase, require trans-splicing for functional assembly of their mRNAs in flowering plants. Tracing the origins of trans-splicing group II introns shows that they have evolved from formerly cis-arranged homologs whose descendants can still be identified in lineages of early branching land plants. In this contribution we present the full set of ancestor introns for all five conserved mitochondrial trans-splicing positions. These introns are strikingly small in the quillwort Isoetes lacustris, the continuous nad2 gene intron in this species representing the smallest (389 nt) land plant group II intron yet identified. cDNA analysis shows correct splicing of the introns in vivo and also identifies frequent RNA editing events in the flanking nad gene exons. Other representatives of the ancestral cis-arranged introns are identified in the fern Osmunda regalis, the horsetail Equisetum telmateia, and the hornwort Anthoceros crispulus. Only the now identified intron in Osmunda carries significant traces of a former maturase reading frame. The identification of a continuous homolog in Anthoceros demonstrates that intron invasion into the affected genes in some cases predated the split of vascular and nonvascular plants more than 400 million years ago. As an alternative to disruption after size increase, the respective introns can get secondarily lost in certain lineages.     

The mitochondrial genomes of the early land plants Treubia lacunosa and Anomodon rugelii: dynamic and conservative evolution

Early land plant mitochondrial genomes captured important changes of mitochondrial genome evolution when plants colonized land. The chondromes of seed plants show several derived characteristics, e.g., large genome size variation, rapid intra-genomic rearrangement, abundant introns, and highly variable levels of RNA editing. On the other hand, the chondromes of charophytic algae are still largely ancestral in these aspects, resembling those of early eukaryotes. When the transition happened has been a long-standing question in studies of mitochondrial genome evolution. Here we report complete mitochondrial genome sequences from an early-diverging liverwort, Treubia lacunosa, and a late-evolving moss, Anomodon rugelii. The two genomes, 151,983 and 104,239 base pairs in size respectively, contain standard sets of protein coding genes for respiration and protein synthesis, as well as nearly full sets of rRNA and tRNA genes found in the chondromes of the liverworts Marchantia polymorpha and Pleurozia purpurea and the moss Physcomitrella patens. The gene orders of these two chondromes are identical to those of the other liverworts and moss. Their intron contents, with all cis-spliced group I or group II introns, are also similar to those in the previously sequenced liverwort and moss chondromes. These five chondromes plus the two from the hornworts Phaeoceros laevis and Megaceros aenigmaticus for the first time allowed comprehensive comparative analyses of structure and organization of mitochondrial genomes both within and across the three major lineages of bryophytes. These analyses led to the conclusion that the mitochondrial genome experienced dynamic evolution in genome size, gene content, intron acquisition, gene order, and RNA editing during the origins of land plants and their major clades. However, evolution of this organellar genome has remained rather conservative since the origin and initial radiation of early land plants, except within vascular plants.     

Variation in sequence and RNA editing within core domains of mitochondrial group II introns among plants

The 3' regions of several group II introns within the mitochondrial genes nad1 and nad7 show unexpected sequence divergence among flowering plants, and the core domains 5 and 6 are predicted to have weaker helical structure than those in self-splicing group II introns. To assess whether RNA editing improves helical stability by the conversion of A-C mispairs to A-U pairs, we sequenced RT-PCR amplification products derived from excised intron RNAs or partially spliced precursors. Only in some cases was editing observed to strengthen the predicted helices. Moreover, the editing status within nad1 intron 1 and nad7 intron 4 was seen to differ among plant species, so that homologous intron sequences shared lower similarity at the RNA level than at the DNA level. Plant-specific variation was also seen in the length of the linker joining domains 5 and 6 of nad7 intron 3; it ranged from 4 nt in wheat to 11 nt in soybean, in contrast to the 2-4 nt length typical of classical group II introns. However, this intron is excised as a lariat structure with a domain 6 branchpoint adenosine. Our observations suggest that the core structures and sequences of these plant mitochondrial introns are subject to less stringent evolutionary constraints than conventional group II introns.     

Did the ancestral globin gene of plants and animals contain only two introns?

All vertebrate globin genes contain two introns, while plant globin genes contain three. It is widely thought that the plant gene structure reflects the structure of the primordial globin gene and that a common ancestor of all animals lost the central intron shortly after the divergence of plants and animals more than one billion years ago. The recent discovery of a discordant central intron in some animal globin genes suggests that this model is incorrect. We propose that the typical vertebrate two-intron gene structure is the primordial eukaryotic form, and that following the distructure is the primordial eukaryotic form, and that following the divergence of plants and animals, a common ancestor of plants gained a central intron in the globin gene.     

Plant Mitochondrial RNA Editing

RNA editing affects messenger RNAs and transfer RNAs in plant mitochondria by site-specific exchange of cytidine and uridine bases in both seed and nonseed plants. Distribution of the phenomenon among bryophytes has been unclear since RNA editing has been detected in some but not all liverworts and mosses. A more detailed understanding of RNA editing in plants required extended data sets for taxa and sequences investigated. Toward this aim an internal region of the mitochondrial nad5 gene (1104 nt) was analyzed in a large collection of bryophytes and green algae (Charales). The genomic nad5 sequences predict editing in 30 mosses, 2 hornworts, and 7 simple thalloid and leafy liverworts (Jungermanniidae). No editing is, however, required in seven species of the complex thalloid liverworts (Marchantiidae) and the algae. RNA editing among the Jungermanniidae, on the other hand, reaches frequencies of up to 6% of codons being modified. Predictability of RNA editing from the genomic sequences was confirmed by cDNA analysis in the mosses Schistostega pennata and Rhodobryum roseum, the hornworts Anthoceros husnotii and A. punctatus, and the liverworts Metzgeria conjugata and Moerckia flotoviana. All C-to-U nucleotide exchanges predicted to reestablish conserved codons were confirmed. Editing in the hornworts includes the removal of genomic stop codons by frequent reverse U-to-C edits. Expectedly, no RNA editing events were identified by cDNA analysis in the marchantiid liverworts Ricciocarpos natans, Corsinia coriandra, and Lunularia cruciata. The findings are discussed in relation to models on the phylogeny of land plants. Received: 2 April 1998 / Accepted: 4 August 1998     

Intron phylogeny: a new hypothesis

The three major classes of intron are clearly of unequal antiquity. Structured (often self-splicing and sometimes mobile) introns are the most ancient, probably dating (at least for group I) from the ancestral (eubacterial) cell 3500 million years ago, and were originally restricted to tRNA. Protein-spliced introns (usually in tRNA) probably evolved from them by a radical change in splicing mechanism in the common ancestor of eukaryotes and archaebacteria, perhaps only about 1700 million years ago. Spliceosomal introns probably evolved from group-II-like self-splicing introns after the origin of the nucleus between 1700 and 1000 million years ago, and were probably mostly inserted into previously unsplit protein-coding genes after the origin of mitochondria 1000 million years ago.     

Evolution of Oleosin in Land Plants

Oleosins form a steric barrier surface on lipid droplets in cytoplasm, preventing them from contacting and coalescing with adjacent droplets. Oleosin genes have been detected in numerous plant species. However, the presence of oleosin genes in the most basally diverging lineage of land plants, liverworts, has not been reported previously. Thus we explored whether liverworts have an oleosin gene. In Marchantia polymorpha L., a thalloid liverwort, one predicted sequence was found that could encode oleosin, possessing the hallmark of oleosin, a proline knot (-PX₅SPX₃P-) motif. The phylogeny of the oleosin gene family in land plants was reconstructed based on both nucleotide and amino acid sequences of oleosins, from 31 representative species covering almost all the main lineages of land plants. Based on our phylogenetic trees, oleosin genes were classified into three groups: M-oleosins (defined here as a novel group distinct from the two previously known groups), low molecular weight isoform (L-oleosin), and high molecular weight isoform (H-oleosin), according to their amino-acid organization, phylogenetic relationships, expression tissues, and immunological characteristics. In liverworts, mosses, lycophytes, and gymnosperms, only M-oleosins have been described. In angiosperms, however, while this isoform remains and is highly expressed in the gametophyte pollen tube, two other isoforms also occur, L-oleosins and H-oleosins. Phylogenetic analyses suggest that the M-oleosin isoform is the precursor to the ancestor of L-oleosins and H-oleosins. The later two isoforms evolved by successive gene duplications in ancestral angiosperms. At the genomic level, most oleosins possess no introns. If introns are present, in both the L-isoform and the M-isoform a single intron inserts behind the central region, while in the H-isoform, a single intron is located at the 5′-terminus. This study fills a major gap in understanding functional gene evolution of oleosin in land plants, shedding new light on evolutionary transitions of lipid storage strategies.     

Slow molecular evolution in 18S rDNA, rbcL and nad5 genes of mosses compared with higher plants

The evolutionary potential of bryophytes (mosses, liverworts and hornworts) has been debated for decades. Fossil record and biogeographical distribution patterns suggest very slow morphological evolution and the retainment of several ancient traits since the split with vascular plants some 450 million years ago. Many have argued that bryophytes may evolve as rapidly as higher plants on the molecular level, but this hypothesis has not been tested so far. Here, it is shown that mosses have experienced significantly lower rates of molecular evolution than higher plants within 18S rDNA (nuclear), rbcL (chloroplast) and nad5 (mitochondrial) genes. Mosses are on an average evolving 2-3 times slower than ferns, gymnosperms and angiosperms; and also green algae seem to be evolving faster than nonvascular plants. These results support the observation of a general correlation between morphological and molecular evolutionary rates in plants and also show that mosses are 'evolutionary sphinxes' regarding both morphological and molecular evolutionary potential.     

Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae

Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid liverworts.     

Introducing Intron Locus cox1i624 for Phylogenetic Analyses in Bryophytes: On the Issue of Takakia as Sister Genus to All Other Extant Mosses

Liverworts are well supported as the sister group to all other land plants (embryophytes) by molecular data. Observations strongly supporting this earliest dichotomy in embryophyte evolution are the strikingly different introns occurring in the mitochondrial DNAs of liverworts versus non-liverwort embryophytes (NLE), including the mosses. A final conclusion on the most basal lineages of mosses, for which genera such as Sphagnum and Takakia are the most likely candidates, is lacking. We have now investigated cox1i624, a mitochondrial group I intron conserved between the moss Physcomitrella patens and the liverwort Marchantia polymorpha. Focusing on a sampling of liverwort and moss genera, which had previously been identified as early branching taxa in their respective clades, we find that group I intron cox1i624 is universally conserved in all 33 mosses and 11 liverworts investigated. The group I intron core secondary structure is well conserved between the two ancient land plant clades. However, whereas dramatic size reductions are seen in the moss phylogeny, exactly the opposite is observed for liverworts. The cox1i624g1 locus was used for phylogenetic tree reconstruction also in combination with data sets of nad5i753g1 as well as chloroplast loci rbcL and rps4. The phylogenetic analyses revealed (i) very good support for the Treubiopsida as sister clade to all other liverworts, (ii) a sister group relationship of the nematodontous Tetraphidopsida and Polytrichopsida and (iii) two rivalling hypotheses about the basal-most moss genus with mitochondrial loci suggesting an isolated Takakia as sister to all other mosses and chloroplast loci indicating a Takakia–Sphagnum clade.     

Group II introns: structure, folding and splicing mechanism

Group II introns are large autocatalytic RNAs found in organellar genomes of plants and lower eukaryotes, as well as in some bacterial genomes. Interestingly, these ribozymes share characteristic traits with both spliceosomal introns and non-LTR retrotransposons and may have a common evolutionary ancestor. Furthermore, group II intron features such as structure, folding and catalytic mechanism differ considerably from those of other large ribozymes, making group II introns an attractive model system to gain novel insights into RNA biology and biochemistry. This review explores recent advances in the structural and mechanistic characterization of group II intron architecture and self-splicing.     

Intron distribution in Plantae: 500 million years of stasis during land plant evolution

Little is known about the evolution of the intron-exon organization in the more primitive groups of land plants, and the intron distribution among Plantae (glauco-, rhodo-, chloro- and streptophytes) has not been investigated so far. The present study is focused on some key species such as the liverwort Marchantia polymorpha, representing the most ancient lineage of land plants, and the streptophycean green alga Mesostigma viride, branching prior to charophycean green algae and terrestrial plants. The intron distribution of six genes for sugar phosphate metabolism was analyzed including four different glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the sedoheptulose-1,7-bisphosphatase (SBP) and the glucose-6-phosphate isomerase (GPI). We established 15 new sequences including three cDNA and twelve genomic clones with up to 24 introns per gene, which were identified in the GPI of Marchantia. The intron patterns of all six genes are completely conserved among seed plants, lycopods, mosses and even liverworts. This intron stasis without any gain of novel introns seem to last for nearly 500 million years and may be characteristic for land plants in general. Some unique intron positions in Mesostigma document that a uniform distribution is no common trait of all streptophytes, but it may correlate with the transition to terrestrial habitats. However, the respective genes of chlorophycean green algae display largely different patterns, thus indicating at least one phase of massive intron rearrangement in the green lineage. We moreover included rhodophyte and glaucophyte reference sequences in our analyses and, even if the well documented monophyly of Plantae is not reflected by a uniform intron distribution, at least one GPI intron is strictly conserved for 1.5 billion years.     

The phylogeny of mosses – Addressing open issues with a new mitochondrial locus: Group I intron cobi420

Mosses are an ancient clade of land plants, set apart from the two other monophyletic groups of bryophytes, the liverworts and the hornworts. Different molecular data sets seem to converge towards a consensus backbone phylogeny of mosses. Nevertheless, for some crucial nodes open questions remain, which obviously require additional phylogenetic information. We here report that a group I intron in the mitochondrial cob gene (cobi420) is universally conserved in the mosses, including basal genera such as Sphagnum and Takakia. Well resolved phylogenetic trees were obtained for 56 mosses of wide phylogenetic sampling, e.g. supporting a placement of Drummondia branching after the genera Timmiella and Catoscopium basal in the Dicranidae. Taxon gaps have been filled in the previously established data sets nad5i753, nad2i156, the nad5-nad4 intergenic region and chloroplast rbcL and rps4 genes. A concatenated 6-loci analysis suggests reconsideration of a Funariidae concept which includes Gigaspermaceae but leaves the isolated genus Timmia at the radiation of arthrodontous mosses unresolved.     

The mitochondrial genome of Chara vulgaris: insights into the mitochondrial DNA architecture of the last common ancestor of green algae and land plants

Mitochondrial DNA (mtDNA) has undergone radical changes during the evolution of green plants, yet little is known about the dynamics of mtDNA evolution in this phylum. Land plant mtDNAs differ from the few green algal mtDNAs that have been analyzed to date by their expanded size, long spacers, and diversity of introns. We have determined the mtDNA sequence of Chara vulgaris (Charophyceae), a green alga belonging to the charophycean order (Charales) that is thought to be the most closely related alga to land plants. This 67,737-bp mtDNA sequence, displaying 68 conserved genes and 27 introns, was compared with those of three angiosperms, the bryophyte Marchantia polymorpha, the charophycean alga Chaetosphaeridium globosum (Coleochaetales), and the green alga Mesostigma viride. Despite important differences in size and intron composition, Chara mtDNA strikingly resembles Marchantia mtDNA; for instance, all except 9 of 68 conserved genes lie within blocks of colinear sequences. Overall, our genome comparisons and phylogenetic analyses provide unequivocal support for a sister-group relationship between the Charales and the land plants. Only four introns in land plant mtDNAs appear to have been inherited vertically from a charalean algar ancestor. We infer that the common ancestor of green algae and land plants harbored a tightly packed, gene-rich, and relatively intron-poor mitochondrial genome. The group II introns in this ancestral genome appear to have spread to new mtDNA sites during the evolution of bryophytes and charalean green algae, accounting for part of the intron diversity found in Chara and land plant mitochondria.     

nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.