Complex Evolutionary Patterns of tRNAUAALeu Group I Introns in Cyanobacterial Radiation

Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNAUAALeu genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNAUAALeu genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNAUAALeu group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNAUAALeu genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNAUAALeu group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.     

Nested Evolution of a tRNALeu(UAA) Group I Intron by both Horizontal Intron Transfer and Recombination of the Entire tRNA Locus

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA(Leu)(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA(Ile)(CAU) and tRNA(Arg)(CCU) genes in alpha- and beta-proteobacteria, respectively, than with other cyanobacterial tRNA(Leu)(UAA) group I introns. The divergent tRNA(Leu)(UAA) intron is sporadically distributed both within the Nostoc and the Microcystis radiations. The complete tRNA gene, including flanking regions and intron from Microcystis aeruginosa strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the Microcystis genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA(Leu)(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA(Leu)(UAA) gene which is located within a unstable genome region.     

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.     

Uncultivated cyanobacteria, Chloroflexus-like inhabitants, and spirochete-like inhabitants of a hot spring microbial mat.

Analysis of 16S rRNA sequences retrieved as cDNA (16S rcDNA) from the Octopus Spring cyanobacterial mat has permitted phylogenetic characterization of some uncultivated community members, expanding our knowledge or diversity within this microbial community. Two new cyanobacterial 16S rRNA sequences were discovered, raising to four the number of cyanobacterial sequence types known to occur in the mat. None of the sequences found is that of the cultivated thermophilic cyanobacterium Synechococcus lividus. A new 16S rRNA sequence characteristic of green nonsulfur bacteria and their relatives was discovered, raising to two the number of such sequences known to exist in the mat. Both are unique among the 16S rRNA sequences of cultivated members of this group, including an Octopus Spring isolate of Chloroflexus aurantiacus and Heliothrix oregonensis, whose sequences we report herein. Two spirochete-like 16S rRNA sequences were discovered. One can be placed in the leptospira subdivision of the spirochete group, but the other has such a loose affiliation with the spirochete group that it might actually belong to an as yet unrecognized subdivision or even to a new eubacterial line of descent.     

Uncultivated cyanobacteria, Chloroflexus-like inhabitants, and spirochete-like inhabitants of a hot spring microbial mat.

Analysis of 16S rRNA sequences retrieved as cDNA (16S rcDNA) from the Octopus Spring cyanobacterial mat has permitted phylogenetic characterization of some uncultivated community members, expanding our knowledge or diversity within this microbial community. Two new cyanobacterial 16S rRNA sequences were discovered, raising to four the number of cyanobacterial sequence types known to occur in the mat. None of the sequences found is that of the cultivated thermophilic cyanobacterium Synechococcus lividus. A new 16S rRNA sequence characteristic of green nonsulfur bacteria and their relatives was discovered, raising to two the number of such sequences known to exist in the mat. Both are unique among the 16S rRNA sequences of cultivated members of this group, including an Octopus Spring isolate of Chloroflexus aurantiacus and Heliothrix oregonensis, whose sequences we report herein. Two spirochete-like 16S rRNA sequences were discovered. One can be placed in the leptospira subdivision of the spirochete group, but the other has such a loose affiliation with the spirochete group that it might actually belong to an as yet unrecognized subdivision or even to a new eubacterial line of descent.     

Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments

Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai'i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.     

Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Synechococcus diversity in the California current as seen by RNA polymerase (rpoC1) gene sequences of isolated strains.

Because they are ubiquitous in a range of aquatic environments and culture methods are relatively advanced, cyanobacteria may be useful models for understanding the extent of evolutionary adaptation of prokaryotes in general to environmental gradients. The roles of environmental variables such as light and nutrients in influencing cyanobacterial genetic diversity are still poorly characterized, however. In this study, a total of 15 Synechococcus strains were isolated from the oligotrophic edge of the California Current from two depths (5 and 95 m) with large differences in light intensity, light quality, and nutrient concentrations. RNA polymerase gene (rpoC1) fragment sequences of the strains revealed two major genetic lineages, distinct from other marine or freshwater cyanobacterial isolates or groups seen in shotgun-cloned sequences from the oligotrophic Atlantic Ocean. The California Current low-phycourobilin (CCLPUB) group represented by six isolates in a single lineage was less diverse than the California Current high-phycourobilin (CCHPUB) group with nine isolates in three relatively divergent lineages. The former was found to be the closest known genetic group to Prochlorococcus spp., a chlorophyll b-containing cyanobacterial group. Having an isolate from this group will be valuable for looking at the molecular changes necessary for the transition from the use of phycobiliproteins to chlorophyll b as light-harvesting pigments. Both of the CCHPUB and CCLPUB groups included strains obtained from surface (5 m) and deep (95 m) samples. Thus, contrary to expectations, there was no clear correlation between sampling depth and isolation of genetic groups, despite the large environmental gradients present. To our knowledge, this is the first demonstration with isolates that genetically divergent Synechococcus groups coexist in the same seawater sample.     

Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.     

Novel group I intron in the tRNA(Leu)(UAA) gene of a gamma-proteobacterium isolated from a deep subsurface environment

A group I intron has been found to interrupt the anticodon loop of the tRNA(Leu)(UAA) gene in a bacterium belonging to the gamma-subdivision of Proteobacteria and isolated from a deep subsurface environment. The subsurface isolate SMCC D0715 was identified as belonging to the genus Pseudomonas. The group I intron from this isolate is the first to be reported for gamma-proteobacteria, and the first instance of a tRNA(Leu)(UAA) group I intron to be found in a group of bacteria other than cyanobacteria. The 231-nucleotide (nt) intron's sequence has group I conserved elements and folds into a bona fide group I secondary structure with canonical base-paired segments P1 to P9 and a paired region, P10. The D0715 intron possesses the 11-nt motif CCUACG. UAUGG in its P8 region, a feature not common in bacterial introns. To date, phylogenetic analysis has shown that bacterial introns form two distinct families, and their complex distribution suggests that both lateral transfer and common ancestry have taken part in the evolutionary history of these elements.     

Structural and functional properties of the cyanobacterial photosystem I complex

Photosystem I (PSI) complexes have been isolated from two cyanobacterial strains, Synechococcus sp. PCC 7002 and 6301. These complexes contain six to seven low molecular mass subunits in addition to the two high molecular mass subunits previously shown to bind the primary reaction center components. Chemical cross-linking of ferredoxin to the complex identified a 17.5-kDa subunit as the ferredoxin-binding protein in the Synechococcus sp. PCC 6301-PSI complex. The amino acid sequence of this subunit, deduced from the DNA sequence of the gene, confirmed its identity as the psaD gene product. A 17-kDa subunit cross-links to the electron donor, cytochrome c-553, in a manner analogous to the cross-linking of plastocyanin to the higher plant PSI complex. Using antibodies raised against the spinach psaC gene product (a 9-kDa subunit which binds Fe-S centers A and B), we identified an analogous protein in the cyanobacterial PSI complex.     

Molecular evolution and phylogenetic utility of the petD group II intron: a case study in basal angiosperms

Sequences of spacers and group I introns in plant chloroplast genomes have recently been shown to be very effective in phylogenetic reconstruction at higher taxonomic levels and not only for inferring relationships among species. Group II introns, being more frequent in those genomes than group I introns, may be further promising markers. Because group II introns are structurally constrained, we assumed that sequences of a group II intron should be alignable across seed plants. We designed universal amplification primers for the petD intron and sequenced this intron in a representative selection of 47 angiosperms and three gymnosperms. Our sampling of taxa is the most representative of major seed plant lineages to date for group II introns. Through differential analysis of structural partitions, we studied patterns of molecular evolution and their contribution to phylogenetic signal. Nonpairing stretches (loops, bulges, and interhelical nucleotides) were considerably more variable in both substitutions and indels than in helical elements. Differences among the domains are basically a function of their structural composition. After the exclusion of four mutational hotspots accounting for less than 18% of sequence length, which are located in loops of domains I and IV, all sequences could be aligned unambiguously across seed plants. Microstructural changes predominantly occurred in loop regions and are mostly simple sequence repeats. An indel matrix comprising 241 characters revealed microstructural changes to be of lower homoplasy than are substitutions. In showing Amborella first branching and providing support for a magnoliid clade through a synapomorphic indel, the petD data set proved effective in testing between alternative hypotheses on the basal nodes of the angiosperm tree. Within angiosperms, group II introns offer phylogenetic signal that is intermediate in information content between that of spacers and group I introns on the one hand and coding sequences on the other.     

Molecular characterization of cyanobacterial diversity in a shallow eutrophic lake

We have studied the diversity of pelagic cyanobacteria in Lake Loosdrecht, The Netherlands, through recovery and analysis of small subunit ribosomal RNA gene sequences from lake samples and cyanobacterial isolates. We used an adapted protocol for specific amplification of cyanobacterial rDNA for denaturing gradient gel electrophoresis (DGGE) analysis. This protocol enabled direct comparison of cyanobacterial community profiles with overall bacterial profiles. The theoretical amplification specificity of the primers was supported by sequence analysis of DNA from excised DGGE bands. Sequences recovered from these bands, in addition to sequences obtained by polymerase chain reaction (PCR) and cloning from lake DNA as well as from cyanobacterial isolates from the lake, revealed a diverse consortium of cyanobacteria, among which are representatives of the genera Aphanizomenon, Planktothrix, Microcystis and Synechococcus. One numerically important and persistent cyanobacterium in the lake, Prochlorothrix hollandica, appeared to co-occur with an unknown but related species. However, the lake is dominated by filamentous species that originally have been termed 'Oscillatoria limnetica-like'. We show that this is a group of several related cyanobacteria, co-occurring in the lake, which belong to the Limnothrix/Pseudanabaena group. The available variation among the coexisting strains of this group can explain the persistent dominance of the group under severe viral pressure.     

Identification of two cyanobacterial strains isolated from the Kotel'nikovskii hot spring of the Baikal rift

Two cyanobacterial strains, Pseudanabaena sp. 0411 and Synechococcus sp. 0431, were isolated from a sample collected in the Kotel'nikovskii hot spring of the Baikal rift. According to the results of light and transmission electron microscopy, as well as of the phylogenetic analysis of the 16S rRNA gene, these cyanobacteria were classified as Pseudanabaena sp. nov. and Synechococcus bigranulatus Skuja. The constructed phylogenetic tree shows that the studied strains are positioned in the clades of cyanobacteria isolated from hydrothermal vents of Asia and New Zealand, separately from marine and freshwater members of these genera, including those isolated from Lake Baikal.     

Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

Rare group I intron with insertion sequence element in a bacterial ribonucleotide reductase gene

A rare group I intron in a cyanobacterial ribonucleotide reductase gene has been characterized. It contains a mobile insertion sequence element not required for RNA splicing. Ribonucleotide reductase genes were found to be hot spots for all three types of self-splicing intervening sequences, including group I and II introns and inteins.     

Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat.

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.     

Nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8.

The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.     

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.