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.     

Origin and evolution of chloroplast group I introns in lichen algae

The history of group I introns is characterized by repeated horizontal transfers, even among phylogenetically distant species. The symbiogenetic thalli of lichens are good candidates for the horizontal transfer of genetic material among distantly related organisms, such as fungi and green algae. The main goal of this study was to determine whether there were different trends in intron distribution and properties among Chlorophyte algae based on their phylogenetic relationships and living conditions. Therefore, we investigated the occurrence, distribution and properties of group I introns within the chloroplast LSU rDNA in 87 Chlorophyte algae including lichen and free‐living Trebouxiophyceae compared to free‐living non‐Trebouxiophyceae species. Overall, our findings showed that there was high diversity of group I introns and homing endonucleases (HEs) between Trebouxiophyceae and non‐Trebouxiophyceae Chlorophyte algae, with divergence in their distribution patterns, frequencies and properties. However, the differences between lichen Trebouxiophyceae and free‐living Trebouxiophyceae were smaller. An exception was the cL2449 intron, which was closely related to ω elements in yeasts. Such introns seem to occur more frequently in lichen Trebouxiophyceae compared to free‐living Trebouxiophyceae. Our data suggest that lichenization and maintenance of lichen symbiosis for millions of years of evolution may have facilitated horizontal transfers of specific introns/HEs between symbionts. The data also suggest that sequencing of more chloroplast genes harboring group I introns in diverse algal groups may help us to understand the group I intron/HE transmission process within these organisms.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus <Emphasis Type="Italic">Tephromela atra</Emphasis> from the Mediterranean region

Recent DNA sequence analyses have revealed the diversity of algal partners in lichen symbioses. Although morphologically similar, different genetic lineages of photobionts are detected in wide geographic ranges of the same lichen fungal species. We studied the photobiont of the genus Trebouxia, which are known as partners of diverse lichen-forming fungal species in the Mediterranean region. We studied the phylogeny of these algae with a multilocus dataset including three loci: ITS, rbcL, and actin type I gene. The two lineages found, informally named Trebouxia sp. 1 and Trebouxia sp. 2, are related to Trebouxia arboricola/decolorans. The cultivation under axenic conditions succeeded only for one of them so far. We used light microscopy, confocal laser scanning microscopy and transmission electron microscopy for phenotypic characterisation. The ultrastructural characters currently used to describe species in the genus do not support the segregation of Trebouxia sp.1 from Trebouxia arboricola. The preferential presence in Mediterranean climates of these strains suggests eco-physiological adaptation. Despite their asexuality in long living lichen symbioses, coccoid algal lichen partners have apparently diversified genetically and physiologically.     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

Fungal specificity and selectivity for algae play a major role in determining lichen partnerships across diverse ecogeographic regions in the lichen‐forming family Parmeliaceae (Ascomycota)

Microbial symbionts are instrumental to the ecological and long‐term evolutionary success of their hosts, and the central role of symbiotic interactions is increasingly recognized across the vast majority of life. Lichens provide an iconic group for investigating patterns in species interactions; however, relationships among lichen symbionts are often masked by uncertain species boundaries or an inability to reliably identify symbionts. The species‐rich lichen‐forming fungal family Parmeliaceae provides a diverse group for assessing patterns of interactions of algal symbionts, and our study addresses patterns of lichen symbiont interactions at the largest geographic and taxonomic scales attempted to date. We analysed a total of 2356 algal internal transcribed spacer (ITS) region sequences collected from lichens representing ten mycobiont genera in Parmeliaceae, two genera in Lecanoraceae and 26 cultured Trebouxia strains. Algal ITS sequences were grouped into operational taxonomic units (OTUs); we attempted to validate the evolutionary independence of a subset of the inferred OTUs using chloroplast and mitochondrial loci. We explored the patterns of symbiont interactions in these lichens based on ecogeographic distributions and mycobiont taxonomy. We found high levels of undescribed diversity in Trebouxia, broad distributions across distinct ecoregions for many photobiont OTUs and varying levels of mycobiont selectivity and specificity towards the photobiont. Based on these results, we conclude that fungal specificity and selectivity for algal partners play a major role in determining lichen partnerships, potentially superseding ecology, at least at the ecogeographic scale investigated here. To facilitate effective communication and consistency across future studies, we propose a provisional naming system for Trebouxia photobionts and provide representative sequences for each OTU circumscribed in this study.     

EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL AND RED ALGAL SMALL SUBUNIT RRNA GENES1

A previous study of the North American biogeography of the red algal genus Hildenbrandia noted the presence of group I introns in the nuclear small subunit (SSU) rRNA gene of the marine species H. rubra (Sommerf.) Menegh. Group IC1 introns have been previously reported at positions 516 and 1506 in the nuclear SSU RNA genes in the Bangiales and Hildenbrandiales. However, the presence of an unclassified intron at position 989 in a collection of H. rubra from British Columbia was noted. This intron is a member of the IE subclass and is the first report of this intron type in the red algae. Phylogenetic analyses of the intron sequences revealed a close relationship between this IE intron inserted at position 989 and similar fungal IE introns in positions 989 and 1199. The 989 IE introns formed a moderately to well‐supported clade, whereas the 1199 IE introns are weakly supported. Unique structural helices in the P13 domain of the 989 and 1199 IE introns also point to a close relationship between these two clades and provide further evidence for the value of secondary structural characteristics in identifying homologous introns in evolutionarily divergent organisms. The absence of the 989 IE intron in all other red algal nuclear SSU rRNA genes suggests that it is unlikely that this intron was vertically inherited from the common ancestor of the red algal and fungal lineages but rather is the result of lateral transfer between fungal and red algal nuclear SSU rRNA genes.     

Combined observational and experimental data provide limited support for facilitation in lichens

It is increasingly recognized that facilitative interactions can shape communities. One of the mechanisms through which facilitation may operate is when one species facilitates the colonization of another through the exchange of shared symbionts. Lichens are symbiotic associations composed of a mycobiont (lichenised‐fungus) and one or two photobionts (algae or cyanobacteria). Different lichen species may have overlapping specificity for photobionts, creating the possibility that facilitation drives lichen community assembly. To investigate whether facilitation occurs in lichens, we combined an observational study (a) with a manipulative field experiment (b). For (a), we quantified the effect of local patch conditions, facilitation and the size of the surrounding metapopulation on colonizations of an epixylic lichen species (Cladonia botrytes) in an area of managed boreal forest. This was done by twice surveying lichens on 293 stumps, located in stands of three age classes. For (b), we treated unoccupied surfaces of 56 cut stumps with algal mixtures of an Asterochloris photobiont and recorded C. botrytes colonizations over three years. In (a), colonization rates of C. botrytes increased with increasing abundance of other lichen species with specificity for Asterochloris photobionts, consistent with an effect of facilitation. However, in the field experiment (b), colonizations of the focal species did not provide support for facilitation. We conclude that our study provides limited support for facilitation in green‐algal lichens, underscoring the importance of combining observational studies with experiments when studying species interactions.     

Heveochlorella (Trebouxiophyceae): a little‐known genus of unicellular green algae outside the Trebouxiales emerges unexpectedly as a major clade of lichen photobionts in foliicolous communities

Foliicolous lichens are formed by diverse, highly specialized fungi that establish themselves and complete their life cycle within the brief duration of their leaf substratum. Over half of these lichen‐forming fungi are members of either the Gomphillaceae or Pilocarpaceae, and associate with Trebouxia‐like green algae whose identities have never been positively determined. We investigated the phylogenetic affinities of these photobionts to better understand their role in lichen establishment on an ephemeral surface. Thallus samples of Gomphillaceae and Pilocarpaceae were collected from foliicolous communities in southwest Florida and processed for sequencing of photobiont marker genes, algal cultivation and/or TEM. Additional specimens from these families and also from Aspidothelium (Thelenellaceae) were collected from a variety of substrates globally. Sequences from rbcL and nuSSU regions were obtained and subjected to Maximum Likelihood and Bayesian analyses. Analysis of 37 rbcL and 7 nuSSU algal sequences placed all photobionts studied within the provisional trebouxiophycean assemblage known as the Watanabea clade. All but three of the sequences showed affinities within Heveochlorella, a genus recently described from tree trunks in East Asia. The photobiont chloroplast showed multiple thylakoid stacks penetrating the pyrenoid centripetally as tubules lined with pyrenoglobuli, similar to the two described species of Heveochlorella. We conclude that Heveochlorella includes algae of potentially major importance as lichen photobionts, particularly within (but not limited to) foliicolous communities in tropical and subtropical regions worldwide. The ease with which they may be cultivated on minimal media suggests their potential to thrive free‐living as well as in lichen symbiosis.     

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.      

Identification of photobionts from the lichen family Physciaceae using algal-specific ITS rDNA sequencing

Abstract:The identity of photobionts from 20 species of the Physciaceae from different habitats and geographical regions has been determined by ITS rDNA sequence comparisons in order to estimate the diversity of photobionts within that lichen group, to detect patterns of specificity of mycobionts towards their photobionts and as a part of an ongoing study to investigate possible parallel cladogenesis of both symbionts. Algal-specific PCR primers have been used to determine the ITS rDNA sequences from DNA extractions of dried lichens that were up to 5 years old. Direct comparisons and phylogenetic analyses allowed the assignment of Physciaceae photobionts to four distinct clades in the photobiont ITS rDNA phylogeny. The results indicate a diversity within the genus Trebouxia Puymaly and Physciaceae photobionts that is higher than expected on the basis of morphology alone. Physciaceae photobionts belonged to 12 different ITS lineages of which nine could unambiguously be assigned to six morphospecies of Trebouxia. The identity of the remaining three sequences was not clarified; they may represent new species. Specificity at the generic level was low as a whole range of photobiont species were found within a genus of Physciaceae and different ranges were detected. The photobionts ofPhyscia (Schreb.) Michaux were closely related and represented one morphospecies of Trebouxia, whereas the algal partners of Buellia De Not and Rinodina (Ach.) S. Gray were in distant lineages of the ITS phylogeny and from several Trebouxia morphospecies. Photobiont variation within a genus of Physciaceae may be due to phylogeny, geographical distance or because photobionts from neighbouring lichens were taken (‘algal sharing’). At the species level Physciaceae mycobionts seem to be rather selective and contained photobionts that were very closely related within one morphospecies of Trebouxia.     

Molecular Characterization of Two Types of rDNA Units in a Single Strain of Candida albicans

ABSTRACT. Strains of the opportunistic fungal pathogen Candida albicans vary in the presence or absence of a self‐splicing group I intron ribozyme (Ca.LSU) in the 25S rRNA gene on chromosome R. Strains of C. albicans typically either lack or contain this ribozyme. However, some strains have both intron‐containing and intronless rRNA genes (rDNA). Pulsed‐field gel electrophoresis analysis of undigested and restricted DNA showed at least six different karyotypes among eight independent colonies of such a heteroallelic strain. In each case, the variation was in chromosome R, and was due to changes in the number of rDNA units. In strains with only one type of rDNA, chromosome R also varied considerably. Polymerase chain reaction amplification spanning the rDNA unit demonstrated that intron‐containing rDNA units are tandemly arrayed, and are immediately adjacent to intronless units in the same cluster. Both types of units were present in the rDNA clusters of both R chromosomes. Possible explanations of these results are loss of Ca.LSU group I intron through purifying selection and/or a relaxation of the commonly accepted concerted evolution of the rDNA units.     

Suitability of chloroplast LSU rDNA and its diverse group I introns for species recognition and phylogenetic analyses of lichen-forming Trebouxia algae

To date, species identification of lichen photobionts has been performed principally on the basis of microscopic examinations and molecular data from nuclear-encoded genes. In plants, the chloroplast genome has been more readily exploited than the nuclear genome for systematic investigations. At the present time, very little information is available about the chloroplast genome of lichen-forming algae. For this reason, we have sequenced a portion of the gene encoding for the chloroplast large sub-unit rRNA (LSU rDNA) as a new molecular marker. Sequencing of the chloroplast LSU rDNAs revealed the existence of an unusual diversity of group I introns (a total of 31) within 15 analyzed Trebouxia species. The number, sequence and insertion site of these introns were very different among species, contributing to their recognition. A relatively large intron-free portion of the chloroplast LSU rDNA and part of the nuclear ribosomal cistron (18S–5.8S–26S) between the nuclear internal transcribed spacers (nrITS) were subjected to phylogenetic analyses. The obtained results indicate that data combination from both nuclear and chloroplast sequences can improve phylogenetic accuracy. Herein, we propose the suitability of both intronic and exonic sequences of the chloroplast LSU rDNA for species recognition, and an exonic sequence spanning from position 879 to 1837 in the Escherichia coli 23S rDNA for phylogenetic analyses of Trebouxia phycobionts.     

Evolutionary inferences based on ITS rDNA and actin sequences reveal extensive diversity of the common lichen alga Asterochloris (Trebouxiophyceae,Chlorophyta)

The genus Asterochloris is one of the most common lichen photobionts. We present a molecular investigation of 41 cultured strains, for which nuclear-encoded ITS rDNA and partial actin I sequences were determined. The loci studied revealed considerable differences in their evolutionary dynamics as well as sequence variation. As compared to ITS data, the actin sequences show much greater variation, and the phylogenies yield strong resolution and support of many internal branches. The partitioning of ITS dataset into several regions yielded better node resolution. We recognized 16 well-supported monophyletic lineages, of which one represents the type species of the genus (Asterochloris phycobiontica), and six correspond to species previously classified to the genus Trebouxia (T. erici, T. excentrica, T. glomerata, T. irregularis, T. italiana and T. magna). Only 15% of isolated photobionts considered in our study could be assigned with certainty to previously described species, emphasizing amazing cryptic variability in Asterochloris. Concurrently with the formal delimitation of the genus Asterochloris, we propose new combinations for the former Trebouxia species; furthermore, T. pyriformis is reduced to a synonym of A. glomerata. The present knowledge of global diversity of Asterochloris algae is discussed.     

The dynamic loss and gain of introns during the evolution of the Brassicaceae

Sequence comparison allows the detailed analysis of evolution at the nucleotide and amino acid levels, but much less information is known about the structural evolution of genes, i.e. how the number, length and distribution of introns change over time. We constructed a parsimonious model for the evolutionary rate of intron loss (IL) and intron gain (IG) within the Brassicaceae and found that IL/IG has been highly dynamic, with substantial differences between and even within lineages. The divergence of the Brassicaceae lineages I and II marked a dramatic change in the IL rate, with the common ancestor of lineage I losing introns three times more rapidly than the common ancestor of lineage II. Our data also indicate a subsequent declining trend in the rate of IL, although in Arabidopsis thaliana introns continue to be lost at approximately the ancestral rate. Variations in the rate of IL/IG within lineage II have been even more remarkable. Brassica rapa appears to have lost introns approximately 15 times more rapidly than the common ancestor of B. rapa and Schenkiella parvula, and approximately 25 times more rapidly than its sister species Eutrema salsugineum. Microhomology was detected at the splice sites of several dynamic introns suggesting that the non‐homologous end‐joining and double‐strand break repair is a common pathway underlying IL/IG in these species. We also detected molecular signatures typical of mRNA‐mediated IL, but only in B. rapa.     

Phylogeny and Self-Splicing Ability of the Plastid tRNA-Leu Group I Intron

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7–3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis. The first two authors (Dawn Simon and David Fewer) have contributed equally to this work. Present address (David Fewer): Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Helsinki, Finland     

High photobiont diversity in the common European soil crust lichen Psora decipiens

The genetic diversity of green algal photobionts (chlorobionts) in soil crust forming lichens was studied as part of the SCIN-project (Soil Crust InterNational). A total of 64 lichen samples were collected from four different sites along latitudinal and altitudinal gradients in Europe (Tabernas/Spain; Hochtor-Großglockner/Austria; Gynge Alvar/Sweden; Ruine Homburg/Germany). The dominant lichen species at all four sites was Psora decipiens, often occurring with Buellia elegans, Fulgensia bracteata, F. fulgens and Peltigera rufescens. Genetic identification of chlorobionts was carried out using the nuclear marker (nrITS) and a chloroplast marker (psbL-J). We found P. decipiens to be associated with several different species of Trebouxia and Asterochloris, although previously described to only have Asterochloris sp. The phylogenetic analyses revealed a high chlorobiont diversity with 12 well supported clades, including Trebouxia asymmetrica, T. jamesii, T. impressa and other, as yet taxonomically unidentified clades (Trebouxia sp. URa1-4, T. sp. URa6, T. sp. URa7-13). Additionally, five clades of Asterochloris were identified (A. magna, A. sp. URa14 -17). Most of the chlorobiont species appeared to be cosmopolitan, but five clades were unevenly distributed between the sampling sites with only Trebouxia being found in the warm and dry Spanish habitats and combinations of Trebouxia and Asterochloris in the cooler and more humid habitats. The wide range of chlorobiont species might contribute to the observed domination of P. decipiens at all four research sites of the SCIN project which range from a desert in Spain to an alpine site in the Alps of Austria.     

Novel Group I Introns Encoding a Putative Homing Endonuclease in the Mitochondrial <Emphasis Type="Italic">cox1</Emphasis> Gene of Scleractinian Corals

Analyses of mitochondrial sequences revealed the existence of a group I intron in the cytochrome oxidase subunit 1 (cox1) gene in 13 of 41 genera (20 out of 73 species) of corals conventionally assigned to the suborder Faviina. With one exception, phylogenies of the coral cox1 gene and its intron were concordant, suggesting at most two insertions and many subsequent losses. The coral introns were inferred to encode a putative homing endonuclease with a LAGLI-DADG motif as reported for the cox1 group I intron in the sea anemone Metridium senile. However, the coral and sea anemone cox1 group I introns differed in several aspects, such as the intron insertion site and sequence length. The coral cox1 introns most closely resemble the mitochondrial cox1 group I introns of a sponge species, which also has the same insertion site. The coral introns are also more similar to the introns of several fungal species than to that of the sea anemone (although the insertion site differs in the fungi). This suggests either a horizontal transfer between a sponge and a coral or independent transfers from a similar fungal donor (perhaps one with an identical insertion site that has not yet been discovered). The common occurrence of this intron in corals strengthens the evidence for an elevated abundance of group I introns in the mitochondria of anthozoans. [Reviewing Editor: Dr. Niles Lehman]     

Characterization of a group I intron in the nuclera rDNA differentiatingPhialophora gregata f. sp.adzukicola fromP. gregata f. sp.sojae

An insertion sequence was detected near the 3′ end of the nuclear small subunit rDNA in isolates ofPhialophora gregata f. sp.adzukicola, the causal agent of the brown stem rot disease of adzuki bean. This insertion sequence was absent in isolates ofP. gregata, f. sp.sojae which causes brown stem rot of soybean. The insertion sequence is 304 bp long and contains all the characteristics of group I introns. These characteristics include, the four conserved sequence elements (P, Q, R, and S), a U at the 5′ splice site of the exon, a G at the 3′ splice site of the intron, a putative internal guiding sequences; the sequence also fits a secondary structure model for group I introns. Similar to most group I introns found in nuclear small subunit rDNA, the intron was located in a highly conserved region and is devoid of long open reading frames. This intron provides a convenient marker for use in conventional PCR to separateP. gregata f. sp.adzukicola fromP. gregata f. sp.sojae.     

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.