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.     

Variability of Nuclear SSU-rDNA Group Introns Within Septoria Species: Incongruence with Host Sequence Phylogenies

We report structural features and distribution patterns of 26 different group I introns located at three distinct nucleotide positions in nuclear small subunit ribosomal DNA (SSU-rDNA) of 10 Septoria and 4 other anamorphic species related to the teleomorphic genus Mycosphaerella. Secondary structure and sequence characteristics assigned the introns to the common IC1 and IE groups. Intron distribution patterns and phylogenetic relationships strongly suggested that some horizontal transfer events have occurred among the closely related fungal species sampled. To test this hypothesis, we used a comparative approach of intron- and rDNA-based phylogenies through MP- and ML-based topology tests. Our results showed two statistically well-supported major incongruences between the intron and the equivalent internal transcribed spacer (ITS) tree comparisons made. Such absence of a co-evolutive history between group I introns and host sequences is discussed relatively to the intron structures, the mechanisms of intron movement, and the biology of the Mycosphaerella pathogenic fungi. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Debashish Bhattacharya     

Structural features and evolutionary considerations of group IB introns in SSU rDNA of the lichen fungus Teloschistes

Different species of the lichen-forming ascomycete fungus Teloschistes were found to contain group IB introns at position S1506 in the small subunit ribosomal RNA gene. We have characterized the structural organization and phylogeny of the Teloschistes introns Tco.S1506, Tla.S1506, and Tvi.S1506. Common features to all the introns are a small size, a compact RNA structure, and an atypical catalytic ribozyme core sequence motif. Variations in intron sizes, due to sequence extensions in the P1 and P8 loop segments, were observed in different species and isolates. Phylogenetic analyses based on the ITS1-5.8S-ITS2 region as well as the introns show that the Teloschistes S1506 introns represent a distinct evolutionary isolated cluster among the nuclear group I introns. Furthermore, introns from different lineages of Teloschistes villosus appear not strictly vertically inherited probably due to horizontal transfer in one of the lineages.     

Phylogenetic relationships of the Rhizophoraceae in China based on sequences of the chloroplast gene matK and the internal transcribed spacer regions of nuclear ribosomal DNA and combined data set

The chloroplast gene matK and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA were sequenced from 17 samples of 13 species representing 6 genera of the angiosperm family Rhizophoraceae from China. Phylogenetic analyses were initially conducted based on sequences of the matK gene and the ITS regions, respectively, using Byrsonima crassifolia and Bunchosia armeniaca (Malpighiaceae) as outgroups. The partition–homogeneity test indicated that the two data sets are homogeneous. A combined analysis of the matK and ITS data generated a well supported phylogeny, which is topologically congruent with the two gene trees based on the Templeton test. The combined phylogeny shows that each genus formed a monophyletic group and the monophyletic relationships of the mangrove genera and of the inland genera were strongly supported.     

Genetic variability in Fomes fomentarius reconfirmed by translation elongation factor 1-α DNA sequences and 25S LSU rRNA sequences

The existence of two cryptic species within strains of the wood-decaying fungus Fomes fomentarius was revealed recently based on the internal transcribed spacer (ITS) sequence variability. In this study for the first time the sequences of another molecular markers, partial translation elongation factor 1-α (efa) region and partial 25S large subunit ribosomal RNA gene were obtained and used to evaluate genetic variability of F. fomentarius. Congruent phylogeny was observed for all three markers used confirming the presence of two cryptic species within F. fomentarius. Surprisingly, ITS sequence variability within F. fomentarius was significantly lower compared to the variability of efa sequences (0.023 versus 0.036 nucleotide substitutions per site) questioning the discriminatory power of ITS sequences for fungal species identification.     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

Phylogenetic analysis of LSU and SSU rDNA group I introns of lichen photobionts associated with the genera Xanthoria and Xanthomendoza (Teloschistaceae,lichenized Ascomycetes)

We studied group I introns in sterile cultures of selected groups of lichen photobionts, focusing on Trebouxia species associated with Xanthoria s. lat. (including Xanthomendoza spp.; lichen‐forming ascomycetes). Group I introns were found inserted after position 798 (Escherichia coli numbering) in the large subunit (LSU) rRNA in representatives of the green algal genera Trebouxia and Asterochloris. The 798 intron was found in about 25% of Xanthoria photobionts including several reference strains obtained from algal culture collections. An alignment of LSU‐encoded rDNA intron sequences revealed high similarity of these sequences allowing their phylogenetic analysis. The 798 group I intron phylogeny was largely congruent with a phylogeny of the internal transcribed spacer region, indicating that the insertion of the intron most likely occurred in the common ancestor of the genera Trebouxia and Asterochloris. The intron was vertically inherited in some taxa, but lost in others. The high‐sequence similarity of this intron to one found in Chlorella angustoellipsoidea suggests that the 798 intron was either present in the common ancestor of Trebouxiophyceae, or that its present distribution results from more recent horizontal transfers, followed by vertical inheritance and loss. Analysis of another group I intron shared by these photobionts at small subunit position 1512 supports the hypothesis of repeated lateral transfers of this intron among some taxa, but loss among others. Our data confirm that the history of group I introns is characterized by repeated horizontal transfers, and suggests that some of these introns have ancient origins within Chlorophyta.     

A Group I Intron in the Nuclear Small Subunit rRNA Gene of Cryptendoxyla hypophloia, an Ascomycetous Fungus: Evidence for a New Major Class of Group I Introns

The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns. Received: 11 January 1998 / Accepted: 12 October 1998     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

Use of Nuclear and Mitochondrial Single Nucleotide Polymorphisms to Characterize English Walnut (Juglans regia L.) Genotypes

English walnut (Juglans regia L.) is the most economically important species, for both food and timber, of the 21 species belonging to the genus Juglans. This study was undertaken to analyze and compare DNA sequences of the mitochondrial cytochrome oxidase subunit II (COX2) and ribosomal DNA (rDNA) genes in the molecular characterization of 30 English walnut genotypes. rDNA sequences revealed the presence of 402 variations, including 101 in 3′ ends of 18S, 21 in internal transcribed spacer 1(ITS1), 170 in ITS2, 30 in 5.8S, and 80 in 5′ ends of 28S regions. Cox2 intron I sequences showed 769 variable positions and GG insertion/deletion at 3′ end regions. Based on single nucleotide polymorphism markers of rDNA and cox2 intron I sequences, an amplification refractory mutation system was used to fingerprint 18 out of 30 walnut genotypes. The findings revealed that the cox2 intron I region, either alone or in conjunction with rDNA, could be used effectively in identifying these walnut genotypes.     

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.     

Re-evaluation of Japanese Phytophthora isolates based on molecular phylogenetic analyses

Over the past 40 years in Japan, Phytophthora isolates have been collected from various diseased host tissues and infested soils and identified using morphological characters. In order to develop a molecular method for the characterization of Japanese Phytophthora species, we obtained nuclear ribosomal ITS and LSU and mitochondrial coxI DNA sequences from 151 isolates representing 21 known species and 10 unidentified isolates. These were compared with similar sequences from representative isolates of known species. Of these, 124 isolates were found to have been correctly identified. Among the remaining 37 isolates, 19 showed high homology with other described species. The remaining 18 isolates showed only low levels of homology with any known species, and generated monophyletic sub-clades in a phylogenetic tree based on the ITS and nLSU regions and the coxI gene. Therefore, these isolates are candidates for new species, falling into six groups. Together, the Japanese isolates were found to represent phylogenetically diverse groups of species. In a sequence variation analysis, the ITS regions and the coxI genes were found to be more variable than the nLSU sequences, suggesting that they will be more useful for Phytophthora identification.     

Characterization of Variability in Some Pathogenic Species of Alternaria Based on the Nucleotide Sequence of Ribosomal DNA

The internal transcribed spacer (ITS) sequences within the ribosomal DNA (rDNA) region were targeted to delineate genetic variability among eight Alternaria species that cause economically important diseases in crops. The rDNA regions of Alternaria species comprising of rRNA genes and the ITS regions were cloned and sequenced. Phylogenetic relationship based on the rDNA sequences and PCR-RFLP of amplified rDNA sequences clustered eight species of Alternaria into three major groups. A. macrospora and A. helianthi accumulated wide genetic variations and are distantly related to rest of the six species which formed two major groups. Group I comprised of three species viz., A. dianthicola, A. brassicae and A. citri, while group II had A. longipes, A. porri and A. alternata. Incorporation of unique stretches of nucleotides and single nucleotide substitutions within relatively conserved ITS1 and ITS2 regions led to clustering of the members of Alternaria species in each group. The divergent sequences within the ITS regions can be employed to design species-specific PCR primer for use in molecular diagnostics.     

Phylogenetic analysis of cpDNA restriction sites andrps16 intron sequences reveals relationships among Apiaceae tribes Caucalideae, Scandiceae and related taxa

Evolutionary relationships among members of Apiaceae (Umbelliferae) tribe Caucalideae Spreng. and related taxa were inferred from maximum parsimony analyses of chloroplast DNA restriction sites andrps16 intron sequences and the results compared to an existing phylogeny for the group based on nuclear ribosomal DNA internal transcribed spacer sequences. While these three data sets were not similar in size or composition, the relationships among the shared taxa, with few exceptions, were concordant. Three major lineages are recognized, coinciding with the previously delimited Scandiceae subtribes Daucinae Dumort. (Agrocharis, Ammodaucus, Cuminum, Daucus, Orlaya, Pachyctenium, Pseudorlaya), Torilidinae Dumort. (Astrodaucus, Caucalis, Glochidotheca, Lisaea, Szovitsia, Torilis, Turgenia, Yabea), and Scandicinae Tausch (Anthriscus, Kozlovia, Myrrhis, Osmorhiza, Scandix). Included in Daucinae is representation from tribe Laserpitieae (Laser, Laserpitium, Melanoselinum, Monizia, Polylophium). Daucinae and Torilidinae arise as sister taxa in the chloroplast DNA-based phylogenies, whereas in the ITS trees relationships among the three major lineages are unresolved. Unexpectedly, three species ofFerula ally with Daucinae and Torilidinae. The position ofArtedia is equivocal, occurring either sister to Daucinae in the ITS trees, within Torilidinae in the intron trees, or sister to Torilidinae upon analysis of combined ITS and intron data.Chaetosciadium trichospermum emerges withinTorilis, and is recognized asTorilis trichosperma (L.) Spreng.     

Evidence for the Ancestral Origin of Group I Introns in the SSUrDNA of Naegleria spp.

ABSTRACT The sequence variation within the group I intron in five Naegleria spp. was studied and compared with the sequence variation within the flanking small subunit ribosomal DNA. Considerable sequence divergence was observed in the introns as well as in the rDNA. In the intron deletions and insertions are only detected in the sequence contributing to the secondary structure, not in the open reading frame. Most of the sequence variation is detected in the unpaired loops. In the case of nucleotide substitution in helices, compensating base pair changes were observed. The sequence variation does not induce variation in the secondary structure model. The phylogenetic tree based on the intron sequences is similar to the tree based on the flanking rDNA sequences. This observation indicates that the intron might have been acquired at an early stage in evolution, and lost in the majority of Naegleria spp.     

ASSESSING PHYLOGENETIC AFFINITIES AND SPECIES DELIMITATIONS IN KLEBSORMIDIALES (STREPTOPHYTA): NUCLEAR‐ENCODED rDNA PHYLOGENIES AND ITS SECONDARY STRUCTURE MODELS IN KLEBSORMIDIUM,HORMIDIELLA, AND ENTRANSIA1

Nuclear‐encoded SSU, group I intron, and internal transcribed spacer (ITS) rDNA sequences were obtained for 16 strains of green algae representing species of Klebsormidium, Hormidiella attenuata, and Entransia fimbriata (for taxonomic authorities, see Table S1 in the supplementary material). The SSU phylogeny resolved a well‐supported clade Klebsormidiales in the Streptophyta that comprised authentic Klebsormidium isolates described recently in a monograph by G. M. Lokhorst and various strains from culture collections. The H. attenuata and En. fimbriata pair was the sister group of Klebsormidium. Certain isolates from culture collections previously identified as “Klebsormidium” emerged as Trebouxiophyceae. Strains assigned to Koliella, Gloeotila, and Stichococcus previously allied with Klebsormidium because of shared morphological and ultrastructural characteristics also belonged to Trebouxiophyceae. Group I introns inserted at Escherichia coli position 516 were found in K. nitens and SAG strain 384‐1, and at position 1506 in H. attenuata and En. fimbriata. Introns were not observed in other Klebsormidiales. Unambiguous alignment of ITS regions of Klebsormidiales was only possible after thermodynamic folding had predicted eight conserved helical domains. The ITS phylogeny provided support for five of the morphospecies recognized by Lokhorst (K. flaccidum, K. elegans, K. bilatum, K. crenulatum, K. mucosum), but the sequences of K. dissectum, K. fluitans, and K. nitens formed an unresolved clade. The species with the earliest origin in the Klebsormidium phylogeny was K. flaccidum. The incongruence between Lokhorst’s morphology‐based cladograms and the ITS phylogenies demonstrated the need for a critical reappraisal of the taxonomy and the morphological and molecular species concept in Klebsormidium on the basis of a more extensive taxonomic and geographic sampling strategy.     

New molecular marker for phylogenetic reconstruction of black yeast-like fungi (Chaetothyriales) with hypothetical EIF2AK2 kinase gene

In eukaryotes, phosphorylation of the α-subunit of eIF2 is a mechanism to adjust cellular gene expression profiles in response to specific signals. The eIF2α kinases are a group of serine–threonine kinases that perform important functions in response to infection, proteotoxicity, and nutrient scavenging. The conserved nature of eIF2α kinases among fungi makes them potential evolutionary markers, which may contribute to deeper understanding of taxonomy and evolution. To date, only few studies are available of eIF2α kinases in black yeasts, which are members of Chaetothyriales containing potential agents of a gamut of major human diseases, such as chromoblastomycosis, phaeohyphomycosis and mycetoma. To establish the phylogenetic validity of sequences of eIF2α kinases hypothetical genes, we compared these genes between members of different classes of fungi, including black yeasts and allies, aiming at evaluation of the phylogeny of this group using an alternative molecular marker, compared to standard ribosomal genes. Trees generated with eIF2α kinase sequences of fungi were compared with those generated by ribosomal internal transcribed spacers (ITS rDNA) sequences from the same species. Sequences used were obtained from the protein Non-redundant database of NCBI, were aligned using CLUSTALX v1.8 and alignments were analyzed with RAxML v8.2.9 on the CIPRES Science Gateway portal. The trees generated had similar topologies, demonstrating that eIF2α kinases hypothetical gene sequences present a coherent reflection of evolution among fungi, compared to trees reconstructed by the use of ribosomal sequences. Our preliminary findings with a limited dataset strongly suggest that the evolution of kinases among black yeasts follows a similar path as revealed by ribosomal data, which underlines the validity of current taxonomy of black yeasts and relatives.     

Phylogenetic evidence for horizontal transmission of group I introns in the nuclear ribosomal DNA of mushroom-forming fungi

Group I introns were discovered inserted at the same position in the nuclear small-subunit ribosomal DNA (nuc-ssu-rDNA) in several species of homobasidiomycetes (mushroom-forming fungi). Based on conserved intron sequences, a pair of intron-specific primers was designed for PCR amplification and sequencing of intron-containing rDNA repeats. Using the intron-specific primers together with flanking rDNA primers, a PCR assay was conducted to determine presence or absence of introns in 39 species of homobasidiomycetes. Introns were confined to the genera Panellus, Clavicorona, and Lentinellus. Phylogenetic analyses of nuc-ssu-rDNA and mitochondrial ssu-rDNA sequences suggest that Clavicorona and Lentinellus are closely related, but that Panellus is not closely related to these. The simplest explanation for the distribution of the introns is that they have been twice independently gained via horizontal transmission, once on the lineage leading to Panellus, and once on the lineage leading to Lentinellus and Clavicorona. BLAST searches using the introns from Panellus and Lentinellus as query sequences retrieved 16 other similar group I introns of nuc-ssu-rDNA and nuclear large-subunit rDNA (nuc-lsu-rDNA) from fungal and green algal hosts. Phylogenetic analyses of intron sequences suggest that the mushroom introns are monophyletic, and are nested within a clade that contains four other introns that insert at the same position as the mushroom introns, two from different groups of fungi and two from green algae. The distribution of host lineages and insertion sites among the introns suggests that horizontal and vertical transmission, homing, and transposition have been factors in intron evolution. As distinctive, heritable features of nuclear rDNAs in certain lineages, group I introns have promise as phylogenetic markers. Nevertheless, the possibility of horizontal transmission and homing also suggest that their use poses certain pitfalls.      

Multiple Group I Introns in the Small-Subunit rDNA of Botryosphaeria dothidea: Implication for Intraspecific Genetic Diversity

Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.