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1 Department of Applied Chemistry and Microbiology, University of Helsinki, 00014 University of Helsinki, Finland
2 Department of Ecology and Systematics, University of Helsinki, 00014 University of Helsinki, Finland
3 Department of Applied Biology, University of Helsinki, 00014 University of Helsinki, Finland
Correspondence
Ilona Oksanen
ilona.oksanen{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 14 November 2003 as DOI 10.1099/ijs.0.02928-0.
Database accession numbers for the new 16S rRNA gene sequences are AY112693, AY112694, AY328896–AY328900 (GenBank) and for the trnL intron AY112695 (GenBank), AJ571702–AJ571720 (EMBL).
Bayesian tree of the 16S and trnL intron sequences, the origin of the strains studied and the list of the strains with no intron PCR products are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Xu et al., 1990; Paquin et al., 1997; Strehl et al., 1999; Besendahl et al., 2000) and type II the eubacterial introns (Rudi & Jakobsen, 2002; Vepritskiy et al., 2002). The type I tRNALeu UAA intron (trnL intron), which is present only as one sequence type in each cyanobacterial strain (Paulsrud & Lindblad, 1998), has been used in taxonomic and ecological studies of cyanobacteria. Within heterocystous cyanobacteria, the host specificity of Nostoc strains (Paulsrud & Lindblad, 1998; Paulsrud et al., 2000, 2001; Costa et al., 1999, 2001; Oksanen et al., 2002; Rikkinen et al., 2002; Summerfield et al., 2002; Linke et al., 2003; Wirtz et al., 2003) and the form species concept of Nostoc commune (Wright et al., 2001) have been studied by comparing nucleotide sequences of the introns and by deriving taxonomic relationships of the similarities. trnL introns are highly variable in their seven loops and are conserved in the stem region. From Nostoc strains, two sequence classes of mostly double-stranded heptamers, 5'-TDNGATT-3' (class I) and 5'-NNTGAGT-3' (class II), have been described in different copy-numbers from the P6b stem–loop of the trnL intron (Costa et al., 2002), causing nucleotide and size variation that is considered useful in strain identification. In previous phylogenetic analyses, the most variable stem–loops, P6b and P9, have been included (Linke et al., 2003; Wirtz et al., 2003) or excluded either partially (Rudi & Jakobsen, 1999) or totally (Paquin et al., 1997; Paulsrud & Lindblad, 1998; Besendahl et al., 2000; Wright et al., 2001) to improve the reliability of the sequence alignment. However, whether the distribution of the two repeat classes follows strain phylogeny has not been tested, nor whether trees based solely on the conserved regions of trnL intron sequences are congruent with those generated from conventional phylogenetic markers, such as the 16S rRNA gene (e.g. Turner et al., 1999).
We constructed the 16S rRNA gene phylogeny of a variety of heterocystous cyanobacteria, including a range of symbiotic Nostoc strains, using maximum-parsimony (MP), maximum-likelihood (ML) and Bayesian inferences. To reveal whether strains with divergent trnL intron P6b stem–loop sequences are more distantly related to each other than strains with similar P6b sequences, we compared the 16S rRNA gene phylogeny with the distribution of repeat classes in the trnL intron sequences. We also evaluated how the results of cluster analyses based on the conserved intron sequences (without loops P6b, P9 and parts of P5) corresponded to the 16S rRNA gene phylogeny that was considered to reflect the species phylogeny of the cyanobacteria studied (e.g. Turner et al., 1999). To reveal the frequency of repeats of the trnL intron, we searched for them on sequence databases.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), Calothrix (Turner et al., 1999; Lyra et al., 2001), Cylindrospermum, Nodularia (Lehtimäki et al., 2000; Lyra et al., 2001) and Nostoc (Miao et al., 1997; Lyra et al., 2001; Rikkinen et al., 2002) from cyanobacterial subsection IV (Castenholtz, 2001) and Tolypothrix from subsection IV.II were included in final analyses (in Supplementary Table A; additional strains studied in Supplementary Table B). Sequences from non-heterocystous Pseudanabaena (Ishida et al., 2001) and Planktothrix (Lyra et al., 2001) from subsection III and Synechococcus (Ishida et al., 2001) from subsection I were used as outgroups. Most of the Nostoc strains originate from lichen symbiosis. There is only one Nostoc strain from each lichen thallus, except for a Lobaria pulmonaria, from the thallus fragments of which two strains were cultured. Jiri Komarek kindly determined Tolypothrix cf. and Nostoc cf. trichormus strains based on morphology. Most of the sequences reported here originate from unialgal and axenic cultures. Axenity was controlled using R2A medium (International Diagnostics Group).
DNA extraction and PCR.
Total DNA was either extracted by the DNeasy tissue kit (Qiagen) with previous modifications (Lohtander et al., 2000) or obtained directly from crushed cells. DNA polymerases used were Taq (Sigma), Platinum Taq (GibcoBRL) or Fast Start Taq (Roche). The trnL intron sequence was amplified in a nested PCR with two sets of primers: cyanobacterial specific primers A and C (Paulsrud & Lindblad, 1998) and TL25 and TL23 (Biniszkiewicz et al., 1994). First amplification with primers A and C was carried out in 20 µl as described in Paulsrud & Lindblad (1998) with the addition of 1 M betaine (Sigma). DNA was re-amplified with primers TL25 and TL23 in 50 µl with 0·5 or 1·0 µl PCR product and 1 M betaine at an annealing temperature of 60 °C for 1 min in 35 cycles following the instructions from the DNA polymerase manufacturer. Cyanobacterial 16S rRNA was amplified with primers PCR 18 and PCR1 (Wilmotte et al., 1993) in 25 or 50 µl at an annealing temperature of 56 °C for 1 min for 30 cycles following the instructions from the DNA polymerase manufacturer.
DNA sequencing.
PCR products were purified with Microcon-PCR filters (Millipore). The PCR product of the trnL intron was sequenced bidirectionally with primers TL25 and TL23 (Biniszkiewicz et al., 1994). The 16S rRNA gene was sequenced with primers from Wilmotte et al. (1993). Cycle sequencing of the trnL intron and the 16S rRNA gene was performed using the ABI Prism dye terminator cycle sequencing ready reaction kit (Perkin-Elmer) and PCR products were purified on MicroSpin G-50 columns (Amersham Pharmacia Biotech). Sequence reactions were analysed with an ABI Prism 377 automated sequencer or with an ABI Prism 310 Genetic Analyzer (PE Biosystems).
Sequence alignment.
The 16S rRNA gene and trnL intron sequences were aligned using the CLUSTAL W program (Thompson et al., 1994). Alignments were refined manually and pairwise sequence identities were obtained with BioEdit 5.0.0 (available at http://www.mbio.ncsu.edu/BioEdit/). The GenBank database was queried with parts of the trnL intron P6b sequence using BLAST (Altschul et al., 1997) and Cyanobase, with their BLAST search (http://www.kazusa.or.jp/cyano/blast.html). A secondary structure of a trnL intron from a Nostoc (Fig. 1) is based on the model of Cech et al. (1994).
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). The dataset with 41 strains was used in all other analyses except hypothesis testing. In the first trnL intron dataset, sequences of Pseudanabaena and Synechococcus were excluded from the MP, ML and Bayesian analyses, since their inclusion in these analyses resulted in unresolved trees. From the second trnL intron dataset, Calothrix and Planktothrix strains, these being most distantly related to Nostoc, were removed to test whether this would improve the resolution of the tree. In all intron alignments, the loop regions P6b, P9 and 15 nucleotides from the P5 loop (Supplementary Figure) were removed. In addition, only one representative was included from strains that shared identical intron sequence in phylogenetic analyses of the intron (Table 1; datasets 1 and 2). To ensure stability of resulting groupings, three different phylogenetic approaches with varying algorithms and a test of alternative hypotheses were applied to the sequence datasets. The support values, bootstrap proportion and the Bayesian posterior probabilities applied for phylogenetic groups measure different parameters from the models. Bayesian posterior probabilities indicate the probability of the model (phylogenetic tree) on estimated priors, whereas the bootstrap analysis indicates the relative probability of the simulated data (sequences) on a given model (Alfaro et al., 2003). Thus, the two support values do not necessarily always correlate (Suzuki et al., 2002; Douady et al., 2003). Substitution models can create additional biases in the analyses as different models are used: unweighted parsimony versus the best-fitting, weighted substitution models in the likelihood-based ML and Bayesian inference (Posada & Crandall, 2001).
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) for each dataset in PAUP 4.0b10 (Swofford, 2002). The model was chosen with the MODELTEST 3.06 program using the Akaike Information Criterion (Posada & Crandall, 1998; Table 1
).
MP analysis.
MP analyses were conducted in equally weighted heuristic searches in PAUP 4.0b10a using the TBR branch-swapping algorithm and a random addition sequence with 1000 replicates (Swofford, 2002). Strict consensus trees were constructed from the most parsimonious trees. Non-parametric MP bootstrap analysis in PAUP incorporated random addition of sequences with replicates, and TBR branch-swapping. Random addition sequences of 100 replicates were used for the 16S rRNA, and combined sequence data and 1000 replicates for the trnL intron data.
ML analysis.
ML analyses were conducted in PAUP 4.0b10 (Swofford, 2002) using best-fitting substitution models with their associated parameters (Table 1) in heuristic searches with options TBR and random addition sequence with 30 replicates. ML non-parametric bootstrap analysis in PAUP incorporated TBR branch-swapping, starting trees by neighbour-joining analysis and 10 000 bootstrap replicates.
Bayesian inference.
Bayesian inference was applied with MrBayes 3.0b3 and 3.0b4 (Huelsenbeck & Ronquist, 2001; Huelsenbeck et al., 2001). MrBayes implements a Markov chain Monte Carlo method that runs many chains simultaneously. With the best-fitting nucleotide substitution model, two or three separate runs incorporating four chains were performed for a million generations, of which every 100th was sampled, generating a total of 10 000 trees each run. Runs started from a random tree. To ensure that the data were from stationary phase, likelihood scores were plotted on a diagram and unstable initial cycles were removed. The proportion of discarded initial cycles was 50 % in analyses of 16S rRNA gene sequences and in combined datasets of 16S rRNA gene sequences and trnL intron sequences, and 10 % in analyses of the trnL intron sequences. Majority rule (50 %) consensus trees were viewed with TreeView 1.6.6 (Page, 1996). The confidence of groupings, such as the paraphyly of Nostoc strains with the trnL intron repeat class I in the 16S rRNA gene tree, was studied from the taxon partition, which gave the number of trees in which each grouping showed and the posterior probabilities for these trees.
Shimodaira–Hasegawa test.
An alternative hypothesis for the constructed 16S rRNA gene tree was tested with the one-tailed Shimodaira–Hasegawa log-likelihood test (Shimodaira & Hasegawa, 1999; Goldman et al., 2000). The null hypothesis was a monophyletic origin of Nostoc strains with the class I repeat in the P6b stem–loop of the trnL intron. Thus, a constrained tree from the 16S rRNA gene dataset, in which all the Nostoc strains with class I repeats were forced to be monophyletic, was constructed with TreeView 1.6.6 (Page, 1996). Constrained and non-constrained sequence data (n=40, excluding strain Calothrix PCC 7102) were analysed with ML methods. The statistical difference between the constrained and non-constrained trees (–lnL0=6668·6208, –lnL1=6590·1152) was tested as implemented in PAUP with re-sampling estimated log-likelihood option (RELL) and 1000 bootstrap replicates.
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RESULTS AND DISCUSSION |
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; Paulsrud, 2001; Rikkinen et al., 2002). Recently, the variable P6b region has also been used for constructing phylogenies (Linke et al., 2003; Wirtz et al., 2003). However, in our phylogenetic analyses of the 16S rRNA gene and of 16S rRNA gene and trnL intron combined, some cyanobacteria sharing identical repeat classes in their trnL intron P6b loop fell into paraphyletic groups consistently (Fig. 2a). Thus, the analyses of the 16S rRNA gene sequences show that cyanobacterial strains with the same repeat class in their trnL intron are not always more closely related to strains with a different repeat class or without these repeats. For example, three Nostoc strains that harboured the class I repeat in their P6b intron loop were intermixed with strains that harboured the class II repeat (Fig. 2a). Nostoc 9.4.19 with class I repeat and Nostoc 9.4.22 with class II repeat were monophyletic in phylogenetic analyses of the conserved trnL intron sequence but highly different in their P6b sequence (Fig. 2). While, in the P6b sequence, they had 39 nt (49 %) differences, in all other parts of the intron, the difference was only 3 nt, and sequence similarity of the 16S rRNA gene was 98 %. Similarly, strains Calothrix PCC 7102 with class I repeat and Calothrix PCC 7714 with class II repeat had 31 nt differences (including 10 gaps) in their P6b sequence but they were monophyletic in the 16S rRNA gene tree, indicating that nucleotide differences in the P6b loop do not predict strain phylogeny, and therefore it is essential to exclude the stem–loop in phylogenetic analyses. The Bayesian taxon partition on a much larger number of trees (hypotheses) than in the ML analysis also supported this finding. The group of Nostoc strains with both repeat classes (Nostoc B in Fig. 2a) was monophyletic with full Bayesian posterior probability in every tree. Finally, the alternative hypothesis of a monophyletic origin of the Nostoc strains containing class I repeats in the P6b loop of the trnL intron was rejected by a statistically highly significant (P=0·001) difference in the Shimodaira–Hasegawa test (Shimodaira & Hasegawa, 1999). Hence, it is highly unlikely that the 16S rRNA gene tree topology in Fig. 2(a) would have been obtained by chance. However, although the 16S rRNA gene is a common phylogenetic tool (Woese, 2000) and the test supported our 16S rRNA tree, we cannot explicitly trust the 16S rRNA gene phylogeny alone without knowledge of possible variation among 16S rRNA gene copies. Nevertheless, phylogenetic analyses of the conserved intron sequences without the most variable loops resulted in a tree topology similar to that of the 16S rRNA gene tree, thus providing additional support for the 16S rRNA gene phylogeny.
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). Another explanation could be that the 485-nt long alignment of 16S rRNA gene sequences with too few informative characters can result in erroneous or poorly resolved phylogeny (parsimony analysis; data not shown). Wirtz et al. (2003) used another approach by excluding sequence fragments from the beginning and end of the trnL intron stem region and including in analyses the total P6b loop sequence that contained both nucleotide and length variation due to repeats. A repeat, however, is seen as a unit of one mutational event (e.g. Costa et al., 2002). Therefore, if the vertical inheritance and homology of the sequences has been confirmed, coding of single events and removing nucleotide sequence of repeats from the alignment would be a more preferable method. Nevertheless, as we show here that repeats in the trnL intron can lead to erroneous phylogenies, they should not be used at all in phylogenetic analyses.
In the case of the trnL intron sequence, a small number of informative characters (Table 1) clearly restricts phylogenetic group formation. This relates to the short length of the sequences and to the stable secondary and tertiary structure required by the self-splicing property of the group I intron (Xu et al., 1990), which restricts the number of random mutations in the stem regions. In the study of Paquin et al. (1997), a subtree based mainly on the intron stem was highly unresolved. In our analyses, the inclusion of cyanobacteria that were distantly related to Nostoc also resulted in unresolved trees (not shown). Only after the distant relatives were removed, did some hierarchy become evident (Fig. 2b, c), although only with weak support values. Hence, the conserved parts of the trnL intron alone do not provide enough sequence variation for hierarchical analyses. As the conserved intron sequence was combined with the 16S rRNA gene data, the resulting tree topology in main clades (Supplementary Figure) was nearly identical to the 16S rRNA gene tree without the intron, indicating that phylogenetic information of the conserved parts of the intron sequence was not in strong conflict with that of the 16S rRNA gene. We did not obtain trnL intron PCR products from some strains of the genera Anabaena, Aphanizomenon, Calothrix, Cyanothece, Fisherella, Microcystis, Oscillatoria and Tolypothrix (Supplementary Table B). The missing introns resulted either from technical problems or from sporadic distribution of the intron. It is obvious that sporadic distribution of the intron will be problematic if it is used as a molecular marker among strains that potentially lack it.
Congruence of phylogenetic methods
In accordance with Suzuki et al. (2002) and Douady et al. (2003), the bootstrap support values and Bayesian posterior probabilities were not always in the same category (Fig. 2). However, the phylogenetic trees obtained by different phylogenetic methods were generally very similar, with only some minor differences in tree topology. Nostoc group A (Fig. 2a) was well supported in all analyses, but Nostoc strain VI.2.2 fell outside this well-resolved group in the Bayesian analyses of 16S rRNA gene sequences and the combined dataset. Moreover, the weak posterior probability of this branch in Bayesian analyses indicated that the position of the strain was unresolved. In a Bayesian taxon partition with the 16S rRNA gene data, the monophyly of Nostoc A (without strain VI.2.2) was found in separate runs in nearly all trees.
Distribution and hypothetical origin of the repeats
The length of trnL intron sequences varied greatly, ranging from 257 to 346 nt (Fig. 2b; Supplementary Table A) due to the loops, mainly P6b and P9. Generally, Nostoc strains had long introns (272–346 nt), usually with four repeat copies and sometimes with additional insertions in their P6b loops. The trnL introns from Pseudanabaena, Synechococcus and Aphanizomenon strains were the shortest and only had a 17-nt P6b. While only the Nostoc and the Calothrix strains had recognizable repeat classes in their P6b loops, Nodularia also had one class II heptanucleotide motif and Cylindrospermum two motifs close to the class I repeat. The P5 loops of the two Anabaena strains were six nucleotides longer than any other strain, while Pseudanabaena and Planktothrix had the shortest, these being only three and four nucleotides long. Large length variation in loops demonstrates how difficult it is to find evolutionary homologies between single nucleotides in intron loops. The class II repeat sequence GCTGAGT of the trnL intron of some Nostoc strains, a Nodularia strain and a Tolypothrix strain was found as two copies in an internal transcribed spacer between tRNAAla and 23S genes (rRNA operon) from a heterocystous Gloeotrichia species (AF105135). The sequence TGCTGAGT AATGAGT GCTGAGT GTTGAGT in the P6b loop of the trnL intron of Nostoc 152 (PCC 9237/1) was found with two mismatches and an E-value of 0·01 from the mcyB gene of Nostoc PCC 7120 (total genome available at http://www.kazusa.or.jp/cyano/blast.html) and with one mismatch in the internal transcribed spacer of the Gloeotrichia species (underlined nucleotides). The 26-nt AT-rich repeat site in the P6b loops of the introns of Nostoc strains, AAAATTCAAAATCT AAAATCCAAAAT was found with one mismatch from other heterocystous cyanobacteria. This sequence was found in the repeat family STRR6 between the nifS and nifU genes of Anabaena azollae (Jackman & Mulligan, 1995), between the genes rbcL and rbcX in Anabaena 133 (Gugger et al., 2002) and in the repeat region of the apcEA1B1C operon of Calothrix PCC 7601 (Houmard et al., 1990). As the class II repeat of the trnL intron and other tandemly repeated heptanucleotide sequences (Meeks et al., 2001) are found at multiple sites in many heterocystous cyanobacteria, they are not trnL intron specific and seem to lack the functional constancy and genetic stability required from phylogenetic markers (Ludwig & Schleifer, 1999).
The tandemly repeated heptamers in the P6b loop of the trnL intron correspond to a common definition of minisatellites by size (6- to 100-nt motif spanning 0·5 kb to several kilobases) and by being hypervariable in the loci (Vergnaud & Denoeud, 2000). The evolutionary origin of the heptanucleotide repeat classes is unclear. Possible mechanisms, such as slipped-strand mispairing and errors of DNA polymerase in DNA duplication, have been suggested (Costa et al., 2002). Repeats may also originate from intra- or intergenomic recombination, from transposase or they may have been transferred by a phage (Lewin, 2000). Theories of lateral intron transfer and recombination of the entire tRNA locus suggested for the trnL intron type II (Rudi & Jakobsen, 1997; Rudi et al., 2002) may not explain the evolution of intron type I, since cyanobacteria with different repeat classes in their introns nonetheless shared the most closely related introns as indicated by the conserved stem sequences of the introns. Thus, the entire locus may not have been transferred laterally, but only the P6b region of the intron may have undergone recombination between strains. Among Nostoc strains, various evolutionary events have occurred in the P6b region. Although most Nostoc strains here have long P6b sequences, with repeats and other insertions, some Nostoc commune strains lack the P6b loop (Wright et al., 2001). It would be necessary to confirm their genus identity and phylogeny to reveal the most probable evolutionary explanation for P6b in those strains.
To conclude, sequence differences in the hypervariable regions of the trnL intron cannot be used as a measure of taxonomic relationships. In contrast to eukaryotic micro- and minisatellites, repeats in the trnL intron are not applicable in population genetics, since their evolution seems to involve not only verticality but also laterality.
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ACKNOWLEDGEMENTS |
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