Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae)

Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PlC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PlC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PlC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PlC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage.     

REEXAMINATION OF PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA): AN ANALYSIS OF atpB AND rbcL GENE SEQUENCES

The chloroplast-encoded atp B gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbc L gene sequence data.1128 base pairs in the coding regions of the atp B gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera— Eudorina, Pleodorina, and Volvox, excluding the section Volvox (= Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbc L gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbc L gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbc L gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atp B gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atp B and rbc L genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.     

Origin and evolution of the colonial volvocales (Chlorophyceae) as inferred from multiple, chloroplast gene sequences

A combined data set of DNA sequences (6021 bp) from five protein-coding genes of the chloroplast genome (rbcL, atpB, psaA, psaB, and psbC genes) were analyzed for 42 strains representing 30 species of the colonial Volvocales (Volvox and its relatives) and 5 related species of green algae to deduce robust phylogenetic relationships within the colonial green flagellates. The 4-celled family Tetrabaenaceae was robustly resolved as the most basal group within the colonial Volvocales. The sequence data also suggested that all five volvocacean genera with 32 or more cells in a vegetative colony (all four of the anisogamous/oogamous genera, Eudorina, Platydorina, Pleodorina, and Volvox, plus the isogamous genus Yamagishiella) constituted a large monophyletic group, in which 2 Pleodorina species were positioned distally to 3 species of Volvox. Therefore, most of the evolution of the colonial Volvocales appears to constitute a gradual progression in colonial complexity and in types of sexual reproduction, as in the traditional volvocine lineage hypothesis, although reverse evolution must be considered for the origin of certain species of Pleodorina. Data presented here also provide robust support for a monophyletic family Goniaceae consisting of two genera: Gonium and Astrephomene.     

Invasion of protein coding genes by green algal ribosomal group I introns

The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.     

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.     

Vertical evolution and intragenic spread of lichen-fungal group I introns

One family within the Euascomycetes (Ascomycota), the lichen-forming Physciaceae, is particularly rich in nuclear ribosomal [r]DNA group I introns. We used phylogenetic analyses of group I introns and lichen-fungal host cells to address four questions about group I intron evolution in lichens, and generally in all eukaryotes: 1) Is intron spread in the lichens associated with the intimate association of the fungal and photosynthetic cells that make up the lichen thallus? 2) Are the multiple group I introns in the lichen-fungi of independent origins, or have existing introns spread into novel sites in the rDNA? 3) If introns have moved to novel sites, then does the exon context of these sites provide insights into the mechanism of intron spread? and 4) What is the pattern of intron loss in the small subunit rDNA gene of lichen-fungi? Our analyses show that group I introns in the lichen-fungi and in the lichen-algae (and lichenized cyanobacteria) do not share a close evolutionary relationship, suggesting that these introns do not move between the symbionts. Many group I introns appear to have originated in the common ancestor of the Lecanorales, whereas others have spread within this lineage (particularly in the Physciaceae) putatively through reverse-splicing into novel rRNA sites. We suggest that the evolutionary history of most lichen-fungal group I introns is characterized by rare gains followed by extensive losses in descendants, resulting in a sporadic intron distribution. Detailed phylogenetic analyses of the introns and host cells are required, therefore, to distinguish this scenario from the alternative hypothesis of widespread and independent intron gains in the different lichen-fungal lineages.     

Evolutionary convergence on highly-conserved 3' intron structures in intron-poor eukaryotes and insights into the ancestral eukaryotic genome

The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3' consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3' splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures.     

Origin and evolution of group I introns in cyanobacterial tRNA genes.

Many tRNA(Leu)UAA genes from plastids contain a group I intron. An intron is also inserted in the same gene at the same position in cyanobacteria, the bacterial progenitors of plastids, suggesting an ancient bacterial origin for this intron. A group I intron has also been found in the tRNA(fMet) gene of some cyanobacteria but not in plastids, suggesting a more recent origin for this intron. In this study, we investigate the phylogenetic distributions of the two introns among cyanobacteria, from the earliest branching to the more derived species. The phylogenetic distribution of the tRNA(Leu)UAA intron follows the clustering of rRNA sequences, being either absent or present in clades of closely related species, with only one exception in the Pseudanabaena group. Our data support the notion that the tRNA(Leu)UAA intron was inherited by cyanobacteria and plastids through a common ancestor. Conversely, the tRNA(fMet) intron has a sporadic distribution, implying that many gains and losses occurred during cyanobacterial evolution. Interestingly, a phylogenetic tree inferred from intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.     

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.     

PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA) INFERRED FROM rbcL GENE SEQUENCE DATA

The chloroplast-encoded large subunit of the ribulose-1, 5-bisphosphate carboxylase / oxygenase (rbcL) gene was sequenced from 20 species of the colonial Volvocales (the Volvacaceae, Goniaceae, and Tetrabaenaceae) in order to elucidate phylogenetic relationships within the colonial Volvocales. Eleven hundred twenty-eight base pairs in the coding regions of the (rbcL) gene were analyzed by the neighbor-joining (NJ) method using three kinds of distance estimations, as well as by the maximum parsimony (MP) method. A large group comprising all the anisogamous and oogamous volvocacean species was resolved in the MP tree as well as in the NJ trees based on overall and synonymous substitutions. In all the trees constructed, Basichlamys and Tetrabaena (Tetrabaenaceae) constituted a very robust phylogenetic group. Although not supported by high bootstrap values, the MP tree and the NJ tree based on nonsynonymous substitutions indicated that the Tetrabaenaceae is the sister group to the large group comprising the Volvocaceae and the Goniaceae. In addition, the present analysis strongly suggested that Pandorina and Astrephomene are monophyletic genera whereas Eudorina is nonmonophyletic. These results are essentially consistent with the results of the recent cladistic analyses of morphological data. However, the monophyly of the Volvocaceae previously supported by four morphological synapomorphies is found only in the NJ tree based on nonsynonymous substitutions (with very low bootstrap values). The genus Volvox was clearly resolved as a polyphyletic group with V. rousseletii Pocock separated from other species of Volvox in the rbcL gene comparisons, although this genus represents a monophyletic group in the previous morphological analyses. Furthermore, none of the rbcL gene trees supported the monophyly of the Goniaceae; Astrephomene was placed in various phylogenetic positions .     

Spliceosomal introns as tools for genomic and evolutionary analysis

Over the past 5 years, the availability of dozens of whole genomic sequences from a wide variety of eukaryotic lineages has revealed a very large amount of information about the dynamics of intron loss and gain through eukaryotic history, as well as the evolution of intron sequences. Implicit in these advances is a great deal of information about the structure and evolution of surrounding sequences. Here, we review the wealth of ways in which structures of spliceosomal introns as well as their conservation and change through evolution may be harnessed for evolutionary and genomic analysis. First, we discuss uses of intron length distributions and positions in sequence assembly and annotation, and for improving alignment of homologous regions. Second, we review uses of introns in evolutionary studies, including the utility of introns as indicators of rates of sequence evolution, for inferences about molecular evolution, as signatures of orthology and paralogy, and for estimating rates of nucleotide substitution. We conclude with a discussion of phylogenetic methods utilizing intron sequences and positions.     

A structural and phylogenetic analysis of the group IC1 introns in the order Bangiales (Rhodophyta)

Our previous study of the North American biogeography of Bangia revealed the presence of two introns inserted at positions 516 and 1506 in the nuclear-encoded SSU rRNA gene. We subsequently sequenced nuclear SSU rRNA in additional representatives of this genus and the sister genus Porphyra in order to examine the distribution, phylogeny, and structural characteristics of these group I introns. The lengths of these introns varied considerably, ranging from 467 to 997 nt for intron 516 and from 509 to 1,082 nt for intron 1506. The larger introns contained large insertions in the P2 domain of intron 516 and the P1 domain of intron 1506 that correspond to open reading frames (ORFs) with His-Cys box homing endonuclease motifs. These ORFs were found on the complementary strand of the 1506 intron in Porphyra fucicola and P. umbilicalis (HG), unlike the 516 intron in P. abbottae, P. kanakaensis, P. tenera (SK), Bangia fuscopurpurea (Helgoland), and B. fuscopurpurea (MA). Frameshifts were noted in the ORFs of the 516 introns in P. kanakaensis and B. fuscopurpurea (HL), and all ORFs terminated prematurely relative to the amino acid sequence for the homing endonuclease I-Ppo I. This raises the possibility that these sequences are pseudogenes. Phylogenies generated using sequences of both introns and the 18S rRNA gene were congruent, which indicated long-term immobility and vertical inheritance of the introns followed by subsequent loss in more derived lineages. The introns within the florideophyte species Hildenbrandia rubra (position 1506) were included to determine relationships with those in the Bangiales. The two sequences of intron 1506 analyzed in Hildenbrandia were positioned on a well-supported branch associated with members of the Bangiales, indicating possible common ancestry. Structural analysis of the intron sequences revealed a signature structural feature in the P5b domain of intron 516 that is unique to all Bangialean introns in this position and not seen in intron 1506 or other group IC1 introns.     

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5′-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5′-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3′-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5′-SS and BPS sequences. Intriguingly, several deletion mutants between the 5′-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5′-SS and BPS sequences and 3′-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5′-SS and BPS play an essential role in IME.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

The self-splicing intron in the Neurospora apocytochrome b gene contains a long reading frame in frame with the upstream exon.

We have determined the DNA sequence of intron 1 and flanking exons in the mitochondrial apocytochrome b gene of the Neurospora laboratory strain 74A and the natural isolate North Africa. In contrast to a previous report, we find that this intron contains an open reading frame (ORF) of 951 bases in frame with the upstream exon. The putative intron-encoded protein resembles those of other intron ORFs with respect to length, calculated isoelectric point, and proportion of basic, acidic, polar, and non-polar amino acids; however, no amino acid sequences resembling the "decapeptides" characteristic of maturase-like ORFs were found. Coupled with the previous finding that this intron is capable of self-splicing in vitro in the absence of proteins, the observations discussed here raise the possibility that other introns with long, in-frame ORFs may also be capable of RNA-catalyzed splicing in vitro.     

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.     

Discovery of a group I intron in the SSU rDNA of Ploeotia costata (Euglenozoa)

The gene coding for the small ribosomal subunit RNA of Ploeotia costata contains an actively splicing group I intron (Pco.S516) which is unique among euglenozoans. Secondary structure predictions indicate that paired segments P1-P10 as well as several conserved elements typical of group I introns and of subclass IC1 in particular are present. Phylogenetic analyses of SSU rDNA sequences demonstrate a well-supported placement of Ploeotia costata within the Euglenozoa; whereas, analyses of intron data sets uncover a close phylogenetic relation of Pco.S516 to S-516 introns from Acanthamoeba, Aureoumbra lagunensis (Stramenopila) and red algae of the order Bangiales. Discrepancies between SSU rDNA and intron phylogenies suggest horizontal spread of the group I intron. Monophyly of IC1 516 introns from Ploeotia costata, A. lagunensis and rhodophytes is supported by a unique secondary structure element: helix P5b possesses an insertion of 19 nt length with a highly conserved tetraloop which is supposed to take part in tertiary interactions. Neither functional nor degenerated ORFs coding for homing endonucleases can be identified in Pco.S516. Nevertheless, degenerated ORFs with His-Cys box motifs in closely related intron sequences indicate that homing may have occurred during evolution of the investigated intron group.