Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Introns and spacers are a rich and well-appreciated information source for evolutionary studies in plants. Compared to coding sequences, the mutational dynamics of introns and spacers is very different, involving frequent microstructural changes in addition to substitutions of individual nucleotides. An understanding of the biology of sequence change is required for correct application of molecular characters in phylogenetic analyses, including homology assessment, alignment coding, and tree inference. The widely used term “indel” is very general, and different kinds of microstructural mutations, such as simple sequence repeats, short tandem repeats, homonucleotide repeats, inversions, inverted repeats, and deletions, need to be distinguished. Noncoding DNA has been indispensable for analyses at the species level because coding sequences usually do not offer sufficient variability. A variety of introns and spacers has been successfully applied for phylogeny inference at deeper levels (major lineages of angiosperms and land plants) in past years, and phylogenetic structure R in intron and spacer data sets usually outperforms that of coding-sequence data sets. In order to fully utilize their potential, the molecular evolution and applicability of the most important noncoding markers (the trnT–trnF region comprising two spacers and a group I intron; the trnS–G region comprising one spacer and a group II intron in trnG; the group II introns in petD, rpl16, rps16, and trnK; and the atpB–rbcL and psbA–trnG spacers) are reviewed. The study argues for the use of noncoding DNA in a spectrum of applications from deep-level phylogenetics to speciation studies and barcoding, and aims at outlining molecular evolutionary principles needed for effective analysis.     

Evolution of the mitochondrial rps3 intron in perennial and annual angiosperms and homology to nad5 intron 1.

The plant mitochondrial rps3 intron was analyzed for substitution and indel rate variation among 15 monocot and dicot angiosperms from 10 genera, including perennial and annual taxa. Overall, the intron sequence was very conserved among angiosperms. Based on length polymorphism, 10 different alleles were identified among the 10 genera. These allelic differences were mainly attributable to large indels. An insertion of 133 nucleotides, observed in the Alnus intron was partially or completely absent in the other lineages of the family Betulaceae. This insertion was located within domain IV of the secondary-structure model of this group IIA intron. A mobile element of 47 nucleotides that showed homology to sequences located in rice rps3 intron and in intergenic plant mitochondrial genomes was found within this insertion. Both substitution and indel rates were low among the Betulaceae sequences, but substitution rates were increasingly larger than indel rates in comparisons involving more distantly related taxa. From a secondary-structure model, regions involved in helical structures were shown to be well preserved from indels as compared to substitutions, but compensatory changes were not observed among the angiosperm sequences analyzed. Using approximate divergence times based on the fossil record, substitution and indel rate heterogeneity was observed between different pairs of annual and perennial taxa. In particular, the annual petunia and primrose evolved more than 15 and 10 times faster, for substitution and indel rates respectively, than the perennial birch and alder. This is the first demonstration of an evolutionary rate difference between perennial and annual forms in noncoding DNA, lending support to neutral causes such as the generation time, population size, and speciation rate effects to explain such rate heterogeneity. Surprisingly, the sequence from the rps3 intron had a high identity with the sequence of intron 1 from the angiosperm mitochondrial nad5 gene, suggesting a common origin of these two group IIA introns.     

The D4 set: primers that target highly variable intron loops in plant chloroplast genomes

Chloroplast group II introns offer high-quality, rapidly evolving single-copy loci for comparative sequence analysis. These introns feature diagnostic secondary structures with loops that are among the least evolutionarily constrained sequence in plastomes. We exploited these structures to develop universal primers that amplify and sequence the large Domain IV (D4) loop in several angiosperm introns. With a single sequence read, we recover 300-600 nucleotides of highly variable sequence across angiosperms, with rates of change that are equal to or higher than many of the best known intergenic spacers in plant chloroplast genomes.     

Deletion bias in avian introns over evolutionary timescales

The role that introns play in the function and evolution of nuclear genomes is not fully understood. Recent models of intron evolution suggest that selection and drift may interact to maintain introns in multicellular organisms. In addition, deletion mutations are more likely to become fixed than insertion mutations. Examination of indel substitutions over macroevolutionary timescales in pigeons and doves (Aves: Columbiformes) reveals that deletion substitutions outnumber insertion substitutions by over six times. The length of indel events is variable.     

Group II introns as phylogenetic tools: structure, function, and evolutionary constraints

Group II introns comprise the majority of noncoding DNA in many plant chloroplast genomes and include the commonly sequenced regions trnK/matK, the rps16 intron, and the rpl16 intron. As demand increases for nucleotide characters at lower taxonomic levels, chloroplast introns may come to provide the bulk of plastome sequence data for assessment of evolutionary relationships in infrageneric, intergeneric, and interfamilial studies. Group II introns have many attractive properties for the molecular systematist: they are confined to organellar genomes in eukaryotes and the majority are single-copy; they share a well-defined and empirically tested secondary and tertiary structure; and many are easily amplified due to highly conserved sequence in flanking exons. However, structure-linked mutation patterns in group II intron sequences are more complex than generally supposed and have important implications for aligning nucleotides, assessing mutational biases in the data, and selecting appropriate models of character evolution for phylogenetic analysis. This paper presents a summary of group II intron function and structure, reviews the link between that structure and specific mutational constraints in group II intron sequences, and discusses strategies for accommodating the resulting complex mutational patterns in subsequent phylogenetic analyses.     

Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.     

Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.     

Reevaluation of the cox1 group I intron in Araceae and angiosperms indicates a history dominated by loss rather than horizontal transfer

The origin and modes of transmission of introns remain matters of much debate. Previous studies of the group I intron in the angiosperm cox1 gene inferred frequent angiosperm-to-angiosperm horizontal transmission of the intron from apparent incongruence between intron phylogenies and angiosperm phylogenies, patchy distribution of the intron among angiosperms, and differences between cox1 exonic coconversion tracts (the first 22 nt downstream of where the intron inserted). We analyzed the cox1 gene in 179 angiosperms, 110 of them containing the intron (intron(+)) and 69 lacking it (intron(-)). Our taxon sampling in Araceae is especially dense to test hypotheses about vertical and horizontal intron transmission put forward by Cho and Palmer (1999. Multiple acquisitions via horizontal transfer of a group I intron in the mitochondrial coxl gene during evolution of the Araceae family. Mol Biol Evol. 16:1155-1165). Maximum likelihood trees of Araceae cox1 introns, and also of all angiosperm cox1 introns, are largely congruent with known phylogenetic relationships in these taxa. The exceptions can be explained by low signal in the intron and long-branch attraction among a few taxa with high mitochondrial substitution rates. Analysis of the 179 coconversion tracts reveals 20 types of tracts (11 of them only found in single species, all involving silent substitutions). The distribution of these tracts on the angiosperm phylogeny shows a common ancestral type, characterizing most intron(+) and some intron(-) angiosperms, and several derivative tract types arising from gradual back mutation of the coconverted nucleotides. Molecular clock dating of small intron(+) and intron(-) sister clades suggests that coconversion tracts have persisted for 70 Myr in Araceae, whose cox1 sequences evolve comparatively slowly. Sequence similarity among the 110 introns ranges from 91% to identical, whereas putative homologs from fungi are highly different, but sampling in fungi is still sparse. Together, these results suggest that the cox1 intron entered angiosperms once, has largely or entirely been transmitted vertically, and has been lost numerous times, with coconversion tract footprints providing unreliable signal of former intron presence.     

Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae)

Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the rbcL-462 intron in cases of spontaneous intron loss. On the other hand, the presence of intact to largely degenerate ORFs of the rbcL-462 introns within the three isolates of Gonium viridistellatum and the rare occurrence of the ORF-lacking rbcL-462 intron suggested that the ORFs might degenerate to result in the spontaneous intron loss during a very short evolutionary time following the loss of the ORF function. Thus, the sporadic distribution of the rbcL-462 introns within the colonial Volvocales can be largely explained by an equilibrium between maintenance of the introns by the intron ORF and spontaneous loss of introns when the introns do not have a functional ORF.     

Evidence of the presence of tRNAfMet group I intron in the marine cyanobacterium Synechococcus elongatus

Self-splicing group I introns in tRNA anticodon loops have been found in diverse groups of bacteria. In this work, we identified tRNAfMet group I introns in six strains of marine Synechococcus elongatus. Introns with sizes around 280 bp were consistently obtained in all the strains tested. In a phylogenetic analysis using the nucleotide sequence determined in this study with other cyanobacterial tRNAfMet and tRNALeu intron sequences, the Synechococcus sequence was grouped together with the sequences from other unicellular cyanobacterial strains. Interestingly, the phylogenetic tree inferred from the intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.     

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.     

Molecular evolution of insertions and deletion in the chloroplast genome of silene

Insertions, deletions, and inversions in the chloroplast genome of higher plants have been shown to be extremely useful for resolving phylogenetic relationships both between closely related taxa and among more basal lineages. Introns and intergenic spacers from the chloroplast genome are now increasingly used for phylogenetic and population genetic studies of populations from a single species, and it is therefore interesting to know whether indels can provide useful data and hence increase the power of intraspecific studies. Here, we show that indels in three cpDNA intergenic spacers and one cpDNA intron for two species of Silene evolve at slightly higher rates than base pair substitutions. Repeat indels appear to have the highest rate of evolution and are thus more prone to homoplasy. We show that coded indel data have high information content for phylogenetic analysis, and indels thus provide useful information to infer phylogenetic relationships at the intraspecific level.     

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.     

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.     

Nuclear-encoded rDNA group I introns: origin and phylogenetic relationships of insertion site lineages in the green algae

Group I introns are widespread in eukaryotic organelles and nuclear- encoded ribosomal DNAs (rDNAs). The green algae are particularly rich in rDNA group I introns. To better understand the origins and phylogenetic relationships of green algal nuclear-encoded small subunit rDNA group I introns, a secondary structure-based alignment was constructed with available intron sequences and 11 new subgroup ICI and three new subgroup IB3 intron sequences determined from members of the Trebouxiophyceae (common phycobiont components of lichen) and the Ulvophyceae. Phylogenetic analyses using a weighted maximum-parsimony method showed that most group I introns form distinct lineages defined by insertion sites within the SSU rDNA. The comparison of topologies defining the phylogenetic relationships of 12 members of the 1512 group I intron insertion site lineage (position relative to the E. coli SSU rDNA coding region) with that of the host cells (i.e., SSU rDNAs) that contain these introns provided insights into the possible origin, stability, loss, and lateral transfer of ICI group I introns. The phylogenetic data were consistent with a viral origin of the 1512 group I intron in the green algae. This intron appears to have originated, minimally, within the SSU rDNA of the common ancestor of the trebouxiophytes and has subsequently been vertically inherited within this algal lineage with loss of the intron in some taxa. The phylogenetic analyses also suggested that the 1512 intron was laterally transferred among later-diverging trebouxiophytes; these algal taxa may have coexisted in a developing lichen thallus, thus facilitating cell- to-cell contact and the lateral transfer. Comparison of available group I intron sequences from the nuclear-encoded SSU rDNA of phycobiont and mycobiont components of lichens demonstrated that these sequences have independent origins and are not the result of lateral transfer from one component to the other.      

Eu-Chlorella large subunit rDNA sequences and group I introns in ribosomal DNA of the paramecian symbiotic alga NC64A

Although the examination of large subunit ribosomal RNA genes (LSU rDNA) is advanced in phylogenetic studies, no corresponding sequence data from trebouxiophytes have been published, with the exception of ‘Chlorella’ellipsoidea Gerneck. We determined the LSU rDNA sequence of Chlorella vulgaris Beijerinck and of the symbiotic alga of green paramecium, Chlorella sp. NC64A. A total of 59 nucleotide substitutions were found in the LSU rDNA of the two species, which are disproportionately distributed. Primarily, 65% of the substitutions were encountered in the first 800 bp of the alignment. This segment apparently has evolved eight times faster than the complete SSU rDNA sequence, making it a good candidate for a phylogenetic marker and giving a resolution level intermediate between small subunit (SSU) rDNA and internal transcribed spacers. Green algae are known as a group I intron‐rich group along with rhodophytes and fungi. NC64A is particularly rich in the introns; five introns were newly identified from the LSU rDNA sequence, which we named Cnc.L200, Cnc.L1688, Cnc.L1926, Cnc.L2184 and Cnc.L2437, following the insertion positions. In the present study we analyzed these introns with three others (Cnc.S943, Cnc.S1367 and Cnc.S1512) that had already been found in NC64A SSU rDNA. Secondary structure modeling placed these introns in the group I intron family, with four introns belonging to subgroup C1 and the other four introns belonging to subgroup E. Five of the intron insertion positions are unique to the paramecian symbiont, which may indicate relatively recent events of intron infections that includes transpositions. Intron phylogeny showed unprecedented relationships; four Cnc. IC1 introns made a clade with some green algal introns with insertions at nine different positions, whereas four Cnc. IE introns made a clade with the S651 intron (Chlorella sp. AN 1–3), which lay as a sister to the S516 insertion position subfamily.