Evolution of the nad3-rps12 Gene Cluster in Angiosperm Mitochondria: Comparison of Edited and Unedited Sequences

We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms. Received: 24 January 1996 / Accepted: 5 June 1996     

Genes and Processed Paralogs Co-exist in Plant Mitochondria

RNA-mediated gene duplication has been proposed to create processed paralogs in the plant mitochondrial genome. A processed paralog may retain signatures left by the maturation process of its RNA precursor, such as intron removal and no need of RNA editing. Whereas it is well documented that an RNA intermediary is involved in the transfer of mitochondrial genes to the nucleus, no direct evidence exists for insertion of processed paralogs in the mitochondria (i.e., processed and un-processed genes have never been found simultaneously in the mitochondrial genome). In this study, we sequenced a region of the mitochondrial gene nad1, and identified a number of taxa were two different copies of the region co-occur in the mitochondria. The two nad1 paralogs differed in their (a) presence or absence of a group II intron, and (b) number of edited sites. Thus, this work provides the first evidence of co-existence of processed paralogs and their precursors within the plant mitochondrial genome. In addition, mapping the presence/absence of the paralogs provides indirect evidence of RNA-mediated gene duplication as an essential process shaping the mitochondrial genome in plants.     

Phylogeny of the Liliales (Monocotyledons) with special emphasis on data partition congruence and RNA editing

A phylogenetic analysis of the monocot order Liliales was performed using sequence data from three mitochondrial (atp1, cob, nad5) and two plastid genes (rbcL, ndhF). The complete data matrix includes 46 terminals representing all 10 families currently included in Liliales. The two major partitions, mitochondrial and plastid data, were congruent, and parsimony analysis resulted in 50 equally parsimonious trees and a well resolved consensus tree confirming monophyly of all families. Mitochondrial genes are known to include RNA edited sites, and in some cases unprocessed genes are replaced by retro‐processed gene copies, that is processed paralogs. To test the effects on phylogeny reconstruction of predicted edited sites and potentially unintentionally sampled processed paralogs, a number of analyses were performed using subsets of the complete data matrix. In general, predicted edited sites were more homoplasious than the other characters and increased incongruence among most data partitions. The predicted edited sites have a non‐random phylogenetic signal in conflict with the signal of the non‐edited sites. The potentially misleading signal was caused partially by the apparent presence of processed paralogs in Galanthus (Amaryllidaceae), part of the outgroup, but also by a deviating evolutionary pattern of predicted edited sites in Liliaceae compared with the remainder of the Liliales. Despite the problems that processed paralogs may cause, we argue that they should not a priori be excluded from phylogenetic analysis.     

Pentatricopeptide repeat proteins constrain genome evolution in chloroplasts

Higher plants encode hundreds of pentatricopeptide repeat proteins (PPRs) that are involved in several types of RNA processing reactions. Most PPR genes are predicted to be targeted to chloroplasts or mitochondria, and many are known to affect organellar gene expression. In some cases, RNA binding has been directly demonstrated, and the sequences of the cis-elements are known. In this work, we demonstrate that RNA cis-elements recognized by PPRs are constrained in chloroplast genome evolution. Cis-elements for two PPR genes and several RNA editing sites were analyzed for sequence changes by pairwise nucleotide substitution frequency, pairwise indel frequency, and maximum likelihood (ML) phylogenetic distances. All three of these analyses demonstrated that sequences within the cis-element are highly conserved compared with surrounding sequences. In addition, we have compared sequences around chloroplast editing sites and homologous sequences in species that lack an editing site due to the presence of a genomic T. Cis-elements for RNA editing sites are highly conserved in angiosperms; by contrast, comparable sequences around a genomically encoded T exhibit higher rates of nucleotide substitution, higher frequencies of indels, and greater ML distances. The loss in requirement for editing to create the ndhD start codon has resulted in the conversion of the PPR gene responsible for editing that site to a pseudogene. We show that organellar dependence on nuclear-encoded PPR proteins for gene expression has constrained the evolution of cis-elements that are required at the level of RNA processing. Thus, the expansion of the PPR gene family in plants has had a dramatic effect on the evolution of plant organelle genomes.     

Molecular evolutionary processes and conflicting gene trees: The hominoid case

Molecular evolutionary processes modify DNA over time, creating both newly derived substitutions shared by related descendant lineages (phylogenetic signal) and “false” similarities which confound phylogenetic reconstruction (homoplasy). However, some types of DNA regions, for example those containing tandem duplicate repeats, are preferentially subject to homoplasy-inducing processes such as sporadically occurring concerted evolution and DNA insertion/deletion. This added level of homoplasic “noise” can make DNA regions with repeats less reliable in phylogenetic reconstruction than those without repeats. Most molecular datasets which distinguish among African hominoids support a human-chimpanzee clade; the most notable exception is from the involucrin gene. However, phylogenetic resolution supporting a chimpanzee-gorilla clade is based entirely on involucrin DNA repeat regions. This is problematic because (1) involucrin repeats are difficult to align, and published alignments are contradictory; (2) involucrin repeats are subject to DNA insertion/deletion; (3) gorillas are polymorphic in that some do not have repeats reported to be synapomorphies linking chimpanzees and gorillas. Gene tree/species tree conflicts can occur due to the sorting of ancestrally polymorphic alleles during speciation. Because hominoid females transfer between groups, mitochondrial and nuclear gene flow occur to the same extent, and the probability of conflict between mitochondrial and nuclear gene trees is theoretically low. When hominoid intraspecific mitochondrial variability is taken into account [based on cytochrome oxidase subunit II (COII) gene sequences], humans and chimpanzees are most closely related, showing the same relative degree of separation from gorillas as when single individuals representing species are analyzed. Conflicting molecular phylogenies can be explained in terms of molecular evolutionary processes and sorting of ancient polymorphisms. This perspective can enhance our understanding of hominoid molecular phylogenies. © 1994 Wiley-Liss, Inc.     

Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein

Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.     

Evolutionary dynamics of wheat mitochondrial gene structure with special remarks on the origin and effects of RNA editing in cereals

We investigated the evolutionary dynamics of wheat mitochondrial genes with respect to their structural differentiation during organellar evolution, and to mutations that occurred during cereal evolution. First, we compared the nucleotide sequences of three wheat mitochondrial genes to those of wheat chloroplast, alpha-proteobacterium and cyanobacterium orthologs. As a result, we were able to (1) differentiate the conserved and variable segments of the orthologs, (2) reveal the functional importance of the conserved segments, and (3) provide a corroborative support for the alpha-proteobacterial and cyanobacterial origins of those mitochondrial and chloroplast genes, respectively. Second, we compared the nucleotide sequences of wheat mitochondrial genes to those of rice and maize to determine the types and frequencies of base changes and indels occurred in cereal evolution. Our analyses showed that both the evolutionary speed, in terms of number of base substitutions per site, and the transition/transversion ratio of the cereal mitochondrial genes were less than two-fifths of those of the chloroplast genes. Eight mitochondrial gene groups differed in their evolutionary variability, RNA and Complex I (nad) genes being most stable whereas Complex V (atp) and ribosomal protein genes most variable. C-to-T transition was the most frequent type of base change; C-to-G and G-to-C transversions occurred at lower rates than all other changes. The excess of C-to-T transitions was attributed to C-to-U RNA editing that developed in early stage of vascular plant evolution. On the contrary, the editing of C residues at cereal T-to-C transition sites developed mostly during cereal divergence. Most indels were associated with short direct repeats, suggesting intra- and intermolecular recombination as an important mechanism for their origin. Most of the repeats associated with indels were di- or trinucleotides, although no preference was noticed for their sequences. The maize mt genome was characterized by a high incidence of indels, comparing to the wheat and rice mt genomes.     

The problems of molecular phylogenetics with the example of squamate reptiles: Mitochondrial DNA markers

The review considers the current problems of molecular phylogenetics based on mitochondrial and chromosomal DNA sequences. The emphasis is placed on mtDNA markers, which are widely employed in reconstructing molecular evolution, but often without a critical analysis of the physiological and biochemical features of mitochondria that affect the adequacy and reliability of the results. In addition to the factors that make mtDNA-based phylogenies difficult to interpret (unrecognized hybridization and introgression events, ancestral polymorphism, and nuclear paralogs of mtDNA sequences), attention is paid to the nonneutrality and unequal mutation rates of mtDNA genes and their fragments, violations of uniparental inheritance of mitochondria, recombination events, natural heteroplasmy, and mtDNA haplotypic diversity. These factors may influence the congruence of phylogenetic inferences and trees constructed for the same organisms with different mtDNA markers or with mitochondrial and nuclear markers. The review supports the viewpoint that mitochondrial genes and their fragments fail to provide reliable evolutionary markers when considered without a thorough study of the environmental conditions and life of the taxa. The influence of external conditions on the metabolism and physiology of mitochondria cannot be taken into account in full nor modeled well enough for phylogenetic applications. It is assumed that mtDNA is valuable as a phylogenetic marker primarily because its complete sequence may be analyzed to identify the apomorphic and synmorphic properties of a taxon and to search for informative nuclear paralogs of mtDNA for phylogeographical studies and estimations of relative evolution times.     

Comparative analysis of a translocated copy of the trnK intron in carnivorous family Nepenthaceae

During phylogenetic analysis of the Nepenthaceae cpDNA trnK intron, it became apparent that a second non-functional copy of the locus was present in most of the investigated taxa. The translocation event was older than the radiation of all recent Nepenthaceae, and the translocated pseudogenized copy was conserved in nearly all members of the plant family. Using single chloroplast PCR and inverse PCR, we could exclude a plastom location for the second copy. Although translocation into the nucleus is possible, mitochondrial localization seems more likely based on these data. In total, the translocated sequence contained at least 3525 base pairs (bp) that were homologous to the Spinacia oleracea chloroplast genome. Comparative phylogenetic analysis of the non-functional copy revealed a high amount of homoplasies compared to topologies from the cpDNA trnK intron phylogenetic reconstruction. Therefore, this copy proved to be insufficient for phylogenetic reconstruction of the family. Since two different paralogs of the non-functional copy were found in one species, it is feasible that different paralogs were conserved in different groups and that paralogous sequences were included in the data matrix. These data demonstrate that phylogenetic analyses of pseudogenized copies of phylogenetically relevant loci should be performed with great caution. In addition, pseudogenized copies can exist in nearly every member of a plant family, and can be PCR-amplified at levels comparable to the specific copy. In this case, the inclusion of such copies can easily remain unnoticed, thus leading to faulty hypotheses.     

Molecular phylogeny and character evolution in terete-stemmed Andean opuntias (Cactaceae-Opuntioideae)

The cacti of tribe Tephrocacteae (Cactaceae-Opuntioideae) are adapted to diverse climatic conditions over a wide area of the southern Andes and adjacent lowlands. They exhibit a range of life forms from geophytes and cushion-plants to dwarf shrubs, shrubs or small trees. To confirm or challenge previous morphology-based classifications and molecular phylogenies, we sampled DNA sequences from the chloroplast trnK/matK region and the nuclear low copy gene phyC and compared the resulting phylogenies with previous data gathered from nuclear ribosomal DNA sequences. The here presented chloroplast and nuclear low copy gene phylogenies were mutually congruent and broadly coincident with the classification based on gross morphology and seed micro-morphology and anatomy. Reconstruction of hypothetical ancestral character states suggested that geophytes and cushion-forming species probably evolved several times from dwarf shrubby precursors. We also traced an increase of embryo size at the expense of the nucellus-derived storage tissue during the evolution of the Tephrocacteae, which is thought to be an evolutionary advantage because nutrients are then more rapidly accessible for the germinating embryo. In contrast to these highly concordant phylogenies, nuclear ribosomal DNA data sampled by a previous study yielded conflicting phylogenetic signals. Secondary structure predictions of ribosomal transcribed spacers suggested that this phylogeny is strongly influenced by the inclusion of paralogous sequence probably arisen by genome duplication during the evolution of this plant group.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

小麦叶绿体蛋白质编码基因RNA编辑位点的测定及与返白现象的关系

RNA编辑是一种转录后基因加工修饰现象,广泛存在于高等植物细胞器中。已有研究表明,RNA编辑与植物发生白化或者黄化有关。通过PCR、RT-PCR及测序的方法,对具有阶段性白化特性的小麦（Triticum aestivum）返白系FA85及其野生型矮变一号（Aibian 1）的叶绿体蛋白质编码基因RNA编辑位点进行了测定,在14个基因上发现了26个编辑位点。有5个编辑位点在2个株系之间存在编辑效率的差异,且这些差异的位点均位于编码叶绿体RNA聚合酶的基因上,其中3个位点编辑前后对应的蛋白质二级结构可能有差异。对2个株系叶绿体中PEP、NEP及PEP、NEP共同依赖基因转录水平的检测显示,除psbA和clpP外,其它基因在小麦返白系中的转录水平均有不同程度的下降。这种转录水平的显著下降及叶绿体RNA聚合酶基因上RNA编辑位点编辑效率的改变,可能与小麦返白系叶片的返白有关。