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Nationaal Herbarium Nederland, Universiteit Leiden branch, Phanerogams and Cryptogams of the Netherlands and Europe section, P.O. Box 9514, 2300 RA Leiden, The Netherlands
Barbara Gravendeel
Nationaal Herbarium Nederland, Universiteit Leiden branch, Molecular Systematics taskforce, P.O. Box 9514, 2300 RA Leiden, The Netherlands
Thomas W. Kuyper
Wageningen Agricultural University, Department of Environmental Sciences, Subdepartment of Soil Quality, P.O. Box 8005, 6700 EC Wageningen, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Phylogenetic relationships of the European species of Leccinum (Boletales, Boletaceae) were investigated by maximum parsimony, Bayesian and likelihood analyses of nrITS1-5.8S-ITS2 and 28S sequences. The separate gene trees inferred were largely concordant, and their combined analysis indicates that several traditional sectional and species-level taxonomic schemes are artificial. In Leccinum, the nrITS region ranges in size from 694 to 1480 bp. This extreme length heterogeneity is localized to a part of the ITS1 spacer that contains a minisatellite characterized by the repeated presence of CTATTGAAAAG and CTAATAGAAAG core sequences and mutational derivatives thereof. The number of core sequences present in the minisatellite varied from 12 to 36. Intra-individual sequence variation of the minisatellite was always smaller than between different species, indicating that concerted evolution proceeds rapidly enough to retain phylogenetic signal at the infraspecific level. In contrast, the evolutionary pattern exhibited by the major ITS1 repeat types found was homoplastic when mapped onto the species lineages inferred from the combined 5.8S-ITS2 sequences. The minisatellite therefore appears not to be useful for phylogeny reconstruction at or above the species level.
Key words: Boletales, Fungi, ITS, Leccinum, minisatellites, phylogeny, tandem repeats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Den Bakker 2000). Its species are distributed mainly in the northern temperate zone (Singer 1986), and a few species are described from tropical areas. Mycorrhizal partners are mainly deciduous trees of the families Betulaceae and Salicaceae, but some species are associated with Fagaceae, Ericaceae and Pinaceae (Binder and Besl 2000).
Leccinum species have been traditionally sorted into sections based on a pigment causing discoloration of the context on exposure (present in sections Leccinum and Luteoscabra Singer, absent in section Scabra Smith) and cap cuticle (pileipellis) with palisade trichoderm (present in section Luteoscabra; Smith and Thiers 1971, Lannoy and Estades 1995) or cutis-like trichoderm (sections Scabra and Leccinum). Especially section Scabra is defined merely on the absence of characters present in other sections of Leccinum. Absence of characters often indicates symplesiomorphy and generally makes the group defined by such a character paraphyletic (Gravendeel et al 2001). Sequences of the nr 28S gene indicate that section Scabra is indeed paraphyletic and in need of revision (Binder and Besl 2000).
As with many groups of fungi, the phenotypic variation of possible diagnostic characters in Leccinum is poorly understood, as are sexual systems and phylogeographical patterns that can help to facilitate species assessment. As a result, there is considerable variation in the number of species recognized by different authors as different morphological and ecological characters are thought to be of importance for species recognition. In Leccinum, ecological characters are mainly restricted to host preferences. It is a matter of opinion whether a morphologically variable taxon growing on a different host is a new species or just an indication of a less stringent host range. Morphological characters in Leccinum are mainly based on the microscopical structure of the pileipellis and macroscopical characters such as the colour of the cap and discoloration of the context and the stipe scabrosities (Lannoy and Estades 1995).
The extremes for the European Leccinum mycota vary from 13 species according to Watling (1970), who mainly based his species on observations of macroscopical characters in combination with host preference, to 36 species according to Lannoy & Estades (1995). The multiplication of the number of species by the latter authors can be explained by their emphasis on the microscopical characters of the pileipellis. Especially in section Scabra this has lead to considerable taxonomic disagreement (see Table I for the most important views). For example, quite a number of different morphological species and infraspecific taxa are recognized in the L. holopus complex. Lannoy and Estades recognise six species and Watling (1970) and Engel (1978) only one. Within the L. scabrum complex, Lannoy & Estades recognize many more taxa (five species, four varieties and one forma) than Watling (only one species) and Engel (four species). Finally, L. variicolor is divided into two varieties and two formae by Lannoy & Estades, while Engel and Watling recognize only two or one species, respectively.
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, Aanen et al 2000, Peintner et al 2001) and Boletales (Kretzer et al 1996, Binder 1999) and seems to be the ideal marker for studies like this, as it is commonly presented as a straightforward marker for species level phylogenies; Its gene and spacers generally show sufficient variation, are easy to sequence and the results can usually be interpreted without difficulty. However, according to Bruns (2001) ITS has its limitations for phylogenetic analyses, too, due to alignment problems between distantly related species in a genus and occasional lack of variation among closely related species. Moreover, a growing amount of studies indicates considerable length variation in either the ITS1 or ITS2 spacers (Van Herwerden et al 1999, Harris and Crandall 2000, Platas et al 2001, Von der Schulenburg et al 2001, Ko and Jung 2002). In some of these cases (Van Herwerden et al 1999, Platas et al 2001, Von der Schulenburg et al 2001) length polymorphisms are caused by simple repetitive sequence motifs, which prevent unambigous alignment and obscure phylogenetic relationships. Evolution of these repetitive sequences is assumed to be shaped by replication slippage, unequal crossing over and gene conversion, which can lead to the generation of length variation and/or concerted evolution (Dover et al 1993, Lewin 2000). A majority of the species of Leccinum also show extreme length variation of the ITS1 spacer (Binder 1999).
We traced the origin of this length variation back to the presence of an irregular repetitive sequence in this spacer, which can be classified as a minisatellite (tandemly repeated sequences in which the motif ranges in size from 10–100 bp), also known as Variable Number Tandem Repeats (VNTR, Nakamura et al, 1987).
The presence of minisatellites in fungal sequences has been discovered only recently (Andersen & Nillson-Tillgren 1997) and until this date has only been reported for Ascomycetes (Giraud et al 1998, Hamann & Osiewacz, 1998, Platas et al 2001, 2002, Wöstemeyer & Kreibich 2002). The only reports of minisatellite-like repetitive sequences in ITS1 are known from Xylariaceae (Platas et al 2001, 2002). As far as we know this is the first basidiomycete minisatellite in ITS1 documented to date.
The predominant way by which minisatellites mutate is gene conversion and unequal crossing over. DNA slippage or slipped strand mispairing (SSM) is thought to be of minor importance (Goldstein & Schlötterer 1999) in the evolution of minisatellites. Giraud et al (1998) and Platas et al (2002), however, assume SSM to be the most important mutational process for the minisatellites they found.
In this paper we focus on how the presence of a minisatellite in our ITS dataset influences our inferences of the phylogeny of Leccinum. In addition, we try to detect common patterns in the structure of the minisatellite in order to (i) assess its usefulness as a phylogenetic marker and (ii) elucidate the evolution of this repetitive sequence from a phylogentic perspective.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Korhonen (1995). If mentioned, fresh material was fixed on CTAB in the field, otherwise herbarium material was used. Of the most common species of section Scabra several collections representing populations separated by a considerable geographic distance (mainly Norway/Sweden vs. The Netherlands) were included. Voucher specimens are deposited in L (Nationaal Herbarium Nederland, University Leiden branch, The Netherlands), GENT (Herbarium of the University of Ghent, Belgium), H (Botanical Museum of the University of Helsinki, Finland) and O (Botanical Museum, University of Oslo, Norway). The 28S sequence data of Binder & Besl (2000) were obtained from GenBank. Most material from which the sequences were derived was provided by either the authors of the species or before mentioned authorities on the genus (Korhonen, Lannoy & Estades), which make the identifications of the material, to our opinion, reliable.
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) was added and mixed with the material. Samples were incubated at 65 °C for 60 minutes in a water bath and occasionally mixed. An equal volume (500 µL) of chloroform-isoamyl alcohol (24:1) was added to the samples and briefly mixed by vortexing. Tubes were centrifuged at 13 000 rpm for 10 minutes. The upper aqueous layer was removed to a clean and labeled 2-mL reaction tube. Another 450 µL chloroform-isoamyl alcohol was added to the reaction tube and mixed by energetically shaking the solution for 5 to 10 minutes. The samples were again centrifuged for 10 minutes at 13 000 rpm. The 500 µL of the upper phase was then transferred to a new 1.5 mL reaction tube. Precipitation took place by adding 500 µL cold isopropanol to the samples. The samples were then shaken for 5 to 10 minutes at room temperature and subsequently centrifuged for 10 minutes at 5000 rpm. The supernatant was poured off and removed as much as possible by putting the samples upside down on a paper towel. The last isopropanol was then evaporated by drying the samples in a speedvac for 20 minutes on a low temperature. The remaining pellets were dissolved in 300 µL TE. Traces of RNA were removed by adding 3 µL RNAse A (concentration 50–100 units/mL, Sigma) to each sample and incubation for 1 hour at 37 °C. The DNA was precipitated for a second time by adding 150 µL 7.5 M NH4 acetate and 1125 µL 100% ethanol. The samples were then left to precipitate for 30 minutes at -20 °C and subsequently centrifuged for 10 minutes at 13 000 rpm at 4 °C. The ethanol solution was poured off and the remaining pellets were washed twice by addition of 500 µL 76% ethanol-10 mM NH4 acetate, storage for 20 minutes at -20 °C and centrifugation of the samples at 13 000 rpm at 4 °C. The ethanol mix was then poured off and the pellets were dried in a speedvac for 20 minutes. The pellets were dissolved in 50 µL TE and stored at -20 °C. The DNA extraction of the CTAB-fixed material differed only in the grinding step. Sterilized sea sand was added to the samples fixed in a 1.5-mL tube filled with 500 µL CTAB. The samples were ground with a micropestle until the tissue seemed to be homogenized enough for DNA extraction. The rest of the procedure was the same as for the dried material from the 65 °C water bath step onwards. If DNA samples appeared to be contaminated with black or yellow pigments and hampered PCR amplifications, the samples were cleaned using columns of the Dneasy DNA isolation kit (Qiagen, USA).
PCR, cloning and DNA sequencing –
The nr ITS1-5.8S-ITS2 region was amplified with primers ITS5 and ITS4 (White et al 1990) using 1 µL of total genomic DNA in a 75 µL reaction mixture. PCR parameters were 3 min at 94 °C followed by 35 cycles of 1 min at 94 °C, 1 min at 52 °C and 1 min at 72 °C. The final extension was 3 min at 72 °C. The PCR products were electrophoresed in a 1.25% agarose gel in 1x TBE (pH 8.3) buffer, stained with ethidium bromide to confirm a single product and cleaned following the Qiagen Qiaquick PCR Cleanup protocol (Qiagen, USA). In cases where cloning was necessary, PCR products were cut out and subsequently cleaned following the QIAquick Gel Extraction Kit's manual (Qiagen, USA).
For several taxa direct sequencing was not possible. We therefore cloned the PCR products following the pGEM-T Easy Vector protocol (Promega, USA). Per sample, ten transformed bacterial colonies were screened for possible intra-individual length polymorphisms by touching them with a sterile pipette tip and using that as template for PCR. If PCR products of aberrant length were found, two or more transformed colonies representing the length polymorphisms found, were sequenced. When the PCR products appeared to be of the same length, we generally sequenced only one colony. In the case of L. versipelle, we sequenced two clones of the same length, to see if these were identical.
The cleaned PCR products or plasmids were sequenced using the ITS2, ITS3, ITS4 and ITS5 primers (White et al 1990) and in some cases standard universal forward and reverse plasmid primers. Samples were sequenced on an ABI 377 automated sequencer (Perkin-Elmer Applied Biosystems, USA) using standard dye-terminator chemistry following the manufacturer's protocols.
Sequence similarity, alignment and phylogenetic analysis – Putatively homologous parts of Leccinum nrITS1-5.8S-ITS2 sequences were assessed using Dotplot analyses as implemented in MegAlign version 4.03 (DNASTAR, Inc. 1999) with a windowsize of 10 and a stringency of 90 percent.
Sequences were aligned using the Clustal option of MegAlign. First the whole dataset was aligned with the gap length penalty lowered to 5 instead of 10 to allow for considerable length variation of the ITS1 spacer. Heterobasidion annosum (Fr.) Bref. (Kasuga et al 1993, GenBank X70021) and Boletus mirabilis Murrill (Berbee et al unpubl, GenBank AF335451) were used to identify borderlines between 18S nrDNA, ITS1, 5.8S, ITS2 and 28S in Leccinum. Alignment and trees are made available on TreeBASE (SN1169).
Because the ITS1 spacer in Leccinum appeared to consist of up to 70 to 80 percent of an array of tandem repeats (see also Binder 1999), it was necessary to make two separate data sets. One data set consisted of the ITS1 spacer (including an alignable but hardly variable region of 220 bp and a very variable region of a tandemly repeated sequence, differing in length from 130 to 848 bp), the complete 5.8S-ITS2 region and the first 40 bp of 28S gene (hereafter called ITS). In the other data set the tandem repeat was left out of the sequences (this data set is called hereafter ITS without the minisatellite region). Both alignments were fine scaled in MegAlign using the clustal option, with the gap length penalty lowered to 3. Further manual adjustments were made in MacClade version 3.04 (Maddison and Maddison 1992). Sequences are deposited in GenBank (AF454560 until AF454591).
Both data sets were analyzed separately and together and with nr28S sequences collected by Binder and Besl (2000). Results of the separate and combined analyses were compared by eye and Partition Homogeneity tests (Farris et al 1995).
Phylogenetic reconstruction was done using PAUP* version 4.0b10 (Swofford 1999) for the maximum parsimony (MP) and maximum likelihood (ML) analyses and MrBayes 2.01 (Huelsenbeck and Ronquist 2001) for the Baseyian analysis. In all analyses Leccinum crocipodium (Letellier) Watling and L. carpini (R. Schulz) Moser ex Reid were specified as outgroup as suggested by Binder and Besl (2000), who identified these species to belong to the sistergroup of the sections Scabra and Leccinum in their phylogeny of Boletales based on nr28S sequences. In the MP analyses 1000 heuristic searches were performed, each with a random addition sequence, MAXTREES set to 10 000, and TBR branch swapping. Bootstrapping in these analyses was performed using 1000 replicates, each with one heuristic search with the addition sequence as given, MAXTREES set to 100, and TBR branch swapping. All characters were assessed as independent, unordered and equally weighted. Gaps were treated as missing characters. For the ML analysis, the appropriate nucleotide substitution model was first determined using Modeltest version 3.06 (Posada and Crandall 1998). A ML analysis was then performed using the substitution model suggested by Modeltest (see results). In the ML analysis 1000 heuristic searches were performed, each with a random addition sequence, MAXTREES set to autoincrease, and TBR branch swapping. For the Bayesian analysis default settings were changed to estimated base frequencies and gamma shape and six substitution types to meet the requirements for the model as determined by Modeltest. The number of generations was set to five million and one tree was saved per 500 generations. The cladogram and posterior credibility values for the clades found are based on the outcome of the last four million generations.
Characterisation of minisatellite –
The boundaries of the irregular tandem repeat array in ITS1 were designated by means of a dotplot analyses as implemented in the alignment program Megalign of the DNAstar package. To visualize the presence of (irregular) tandemly repeats, the dotplot parameters were set to a windowsize of 10 bp (the minimum size of repeats) and a stringency (percent match) of 90 percent. By plotting the sequence against itself under these settings, the repetitive region will become easily visible as a square region composed of short lines parallel to the main diagonal (see Fig. 5 and Fig. 6). To see to what extent the repetition within this region could be explained by direct, regular tandem repetition, we used the program Tandem Repeats Finder Version: 2.02 (Benson 1999), using the default settings for a subset of the sequences.
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were found by comparison of primary and secondary structures with yeast sequences using secondary structure predictions performed in RNAdraw version 1.1 (Matzura and Wennberg 1996). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Using Modeltest, the general time-reversible model was chosen, with variable sites assumed to follow a gamma distribution (shape set to 0.6920), nucleotide frequencies set to A:0.2823, C:0.2040, G: 0.2236, T:0.2900 and substitution rates set to 2.1753 (A
C), 4.0993 (AG), 2.0767 (AT), 1.9933 (CG), and 5.9494 (CT). ML analyses of the ITS sequences resulted in a single tree with log Likelihood score -2975.37353 (see Fig. 3). Topology and resolution are generally congruent with the MP analysis of the ITS sequences without the minisatellite (see above) and indicate that section Scabra is nested within section Leccinum. However, the position of L. variicolor differs in both types of analysis. While L. variicolor (clade D) is a sistergroup to clade E in the parsimony analyses, it is placed within clade E in the ML analysis.
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Position and characterization of minisatellite –
PCR products of the ITS region in Leccinum ranged from 694 to1480 bp (Fig. 4). ITS1 varied in length from 150 to 868 bp, 5.8S measured ca 158 bp and ITS2 ranged from 270 to 355 bp, with the exception of L. carpini, which showed an ITS2 sequence of 574 bp. The length variation observed in the ITS1 spacer in most species of Leccinum examined by us is caused by an insert varying in length from 240 to 749 bp. According to Van Nues et al (1994) the ITS1 spacer in yeasts is organised in two functionally and structurally distinct halves with highly conserved nucleotide regions at three different positions. In relation to the position of the A2 processing site, we assume the insert in the ITS1 spacer in Leccinum is situated in domain III (see Fig. 5). The ITS1 spacer in Boletaceae inspected by us in general is very short (ca 210 bp) and sequence variation is very low. Variation, consisting of mono- or dinucleotide tandem repeats, is mainly found in two regions associated with processing sites A2 and A3. This variation, that seems to be associated with the processing sites, is virtually absent in those species of Leccinum that contain a minisatellite. For most Leccinum species several accessions were sequenced. The length of the ITS1 spacer could vary considerably within one species. When comparing all sequences of one species, indels appeared to be interspersed throughout the whole ITS1 spacer and were not restricted to certain positions (see L. duriusculum, L. piceinum and L. variicolor in Fig. 6).
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Although minisatellites usually are so variable that they can even be used in forensic research (Meyer and Mitchell 1995), the minisatellite found in Leccinum displays considerably less allelic variation. For instance, all species of clade A in Fig. 3 show only two alleles (a long and a short variant), irrelevant of their geographical origin. The same holds for L. piceinum and L. vulpinum. Although the accessions of the latter were collected at a geographical distance of at least 1600 kilometers, the long length variant of the minisatellite of both species is identical. In contrast, the minisatellites found in the species of clade B in Fig. 3 are less homogeneous. Although we have sampled only five specimens, we can discriminate between a long allele present in Scandinavian accessions of L. scabrum and L. rotundifoliae and a shorter allele present in Dutch accessions of L. scabrum and L. rigidipes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Taxonomic implications –
Separate and combined analyses of sequences of the nr28S gene (Binder and Besl 2000) and the ITS sequences analyzed here indicate that there seems to be no evolutionary based reason to differentiate between the sections Leccinum and Scabra as suggested by Smith and Thiers (1971) and Lannoy and Estades (1995). Most of the species belonging to section Scabra form a monophyletic clade. This clade is nested within section Leccinum, thereby making the latter section paraphyletic. Moreover, L. variicolor seems not related to the other members of section Scabra, making this section polyphyletic. Our results indicate that L. variicolor represents a lineage that has diverged from section Leccinum and subsequently lost the pigments causing blackish discoloration.
The congruence of the ITS data with the 28S data (and not the minisatellite data found in ITS1) makes it clear that the minisatellite is unsuitable for phylogenetic analysis of relations above the species level in Leccinum. However, processes like concerted evolution make it an ideal marker to reveal relations at and below the species level, as homogenization seems to take place in its tandem repeat arrays.
Leccinum rigidipes and L. rotundifoliae share almost identical ITS sequences with L. scabrum populations which were collected on locations nearby. Leccinum rigidipes is distinguished from L. scabrum by the presence of cylindrical elements in the pileipellis and a rigid, clavate stipe. However, L. scabrum often has a clavate stipe, too, and cylindrical elements in the pileipellis are difficult to distinguish, as this character seems to be not discrete. Transitions between cylindrical and other elongate elements appear to be rather common. Leccinum rotundifoliae was considered ‘a truly distinctive species' according to Smith and Thiers (1971). We think that it might just be an ecotype associated with the dwarf shrub Betula nana and that it owes its distinctive smaller size to the mountainous and tundra environments it grows in. A similar case was found for Hebeloma alpinum by Aanen et al (2000).
The second interesting group consists of the L. holopus complex (clade A in Figs. 2 and 3). This clade of diverse, morphologically clearly circumscribed species is very homogenous in its ITS sequences if we do not take into account the length variations of the ITS1 spacer. Many species recognized by Lannoy & Estades (1995) within this group are distinguished by the presence or absence of cylindrical elements in the pileipellis and the absence or presence of pigment in the pileipellis and the stipe. As already mentioned, the presence or absence of cylindrical elements is not discrete. Nr ITS1-5.8S-ITS2 sequences indicate that recognition of L. brunneogriseolum, L. holopus, L. nucatum, and L. pulchrum as separate species may not be warranted. However, we think it would be premature to consider this entire group conspecific as lack of ITS variation not necessarily provides evidence of conspecifity (Bruns 2001). Even if we do consider albinism and pileipellis characters invalid for species delimitation, there is still enough morphological variation to distinguish at least two species or species groups. More accessions of additional localities need to be studied and the outcomes of additional molecular markers have to be obtained, before decisions about definitive species delimitations can be made. The current ITS perspective agrees reasonably well with Watling's (1970) conservative view of species, but whether additional molecules or increased sampling will distinguish more taxa remains to be investigated.
Functionality of ITS1 spacer –
The presence of a large and heterogeneous insert in the ITS1 spacer could indicate a loss of function for this region in Leccinum. Muir et al (2001) have shown that multiple ITS variants in one genome can indicate some of these variants have actually lost their function. They also show that these non-functional ITS regions are characterized by the presence of more mutations, especially in the more conserved regions. According to Van Nues et al (1994) deletions in domain III severely reduce the formation of 17S rRNA. Given the fact that, in those samples in which we found the insert, no other sequences lacking the insert were present, we have to conclude the ITS1 region is likely to be still functional. It appears that insertions within this domain are tolerated. Moreover, sequence divergence between Leccinum and several Boletus species in regions immediately adjacent to the insert (see Fig. 5) is virtually absent, indicating that the adjacent regions are relatively conserved, also in the samples containing the minisatellite.
Characterization of minisatellite –
The minisatellite found in the ITS1 spacer of Leccinum is AT-rich (59–64%). Other minisatellites reported in fungi are either GC-rich (Anderson & Torsten 1996) or AT-rich (Giraud et al. 1998). AT-richness is a character shared with the ITS-region in which the minisatellite is positioned.
Both the dotplot analysis and the mapping of the core sequence variants show that the minisatellite region containss a high level of repetition. A search for tandem repeats however shows some minisatellite regions to contain no such repeats, while others can only partly be explained by direct repetition. An explanation for this result is almost certainly the fact that repetition within a sequence is only detected with the Tandem Repeats Finder software when repeats are rather perfect (>80% match) copies of each other. Repeats therefore have to be relatively unaffected by mutations to be detected with this kind of software. However, the patterns found with the tandem repeat software are, in addition to the patterns found when mapping the core sequences, not completely meaningless. One of the common patterns found with the tandem repeat software is the presence of a region consisting of three tandem repeats of ca 116 bp (see Fig. 6). Comparison of these regions with the patterns found when mapping the core sequences show that this 116 bp tandem repeat region is also (almost) identical in its core sequence pattern. We conclude that both methods to analyze the patterns of repetition point towards the same homologous regions in several species.
A remarkable feature of the minisatellite here is the fact that we found the number of core sequences to be even in all but one sample (L. variicolor SW). According to Platas et al (2001) inverted repeats are associated with stable folding positions of a tandem repeat array. Although we could not find inverted repeats of considerable size within the minisatellites examined, a secondary structure prediction (data not shown) of the minisatellite region with the Mfold 3.0 software (Mathews et al 1999, Zucker et al 1999) shows this region to fold in such a way that most of the core sequences form a stem-like structure. These stem-like structure consist either of an AA, AB or BB pair of core sequences. This property of the core sequences might enable the minisatellite region to fold into a stable secondary structure within the ITS spacer, which may explain why it can be situated in a rather conservative part of this spacer. It is probably also putting a strong constraint on the minisatellite variants that can persist within the organism, since badly folding mutants can have a deleterious effect on the organism.
Mechanisms causing length polymorphisms – Mapping of the core sequences shows the minisatellites found can be divided into two groups. One group consists of L. rotundifoliae, L. piceinum and L. duriusculum, three phylogentically rather unrelated species, with a minisatellite size of 550 to 575 bp, and a rather identical core sequence pattern. The other group comprises L. variicolor, L. versipelle, L. holopus and L. palustre, in which the minisatellite is considerably shorter or longer in size. Although some of these species (L. versipelle and L. holopus) show pieces of their minisatellite to be homologous to patterns found in the other group, they are all characterized by patterns that indicate a rather drastic expansion or contraction of the region. Assuming the longest minisatellite is to be the primitive state of the sequence, length polymorphisms found in closely related accessions (L. rotundifoliae and L. scabrum, L. variicolor NL and L. variicolor SW) or within one individual (L. piceinum, L. duriusculum) can best be explained by simple deletion processes.
The mechanism causing length polymorphisms in fungal minisatelitte-like sequences mentioned in literature is slipped strand mispairing (SSM) (Giraud et al 1998, Platas et al 2002). This process is mostly considered to be responsible for repetitive sequences consisting of repeats of 10 bp or less (microsatellites). The main reasons for Giraud et al (1998) to assume SSM as the main mutational mechanism are the particular structures of the minisatellites and the absence of evidence for recombination between alleles of the flanking regions. We did not find any evidence to assume allelic recombination in the flanking region. However, since the flanking regions seem to be relatively conserved, there are not much variable sites found in these regions anyway. Moreover, unequal crossing over does not necessarily imply recombination in the flanking regions (McAllister and Werren 1999). Neither the patterns found by mapping the core sequences, nor the Tandem Repeats Finder analyses, revealed patterns that lead to the conclusion that SSM is not the most probable process responsible for the length polymorphisms found. Gene conversion and unequal crossing over therefore seem to be better candidates to explain the processes involved in expansion of the minisatellite region.
Phylogenetic and systematic utility of minisatellite – The structure and length of the minisatellite region appear not to be phylogenetically informative, as they do not correlate well with main monophyletic clades in the molecular phylogenies reconstructed. This is probably because the mutational processes responsible for the structure and size of the minisatellites act under strong structural constraints. Although these characteristics make the ITS1 spacer unsuitable as a phylogenetic marker at or above the species level in Leccinum, it does not mean that large parts of ITS1 are completely useless from a systematic point of view. Combined with morphological and ecological data ITS1 sheds new light on the status of many morphologically defined species. The minisatellite in ITS1 seems to unique for Leccinum and is specific in its length and composition for most clades which coincide with morphologically circumscribed species or species-groups, making it an ideal marker for PCR based further ecological and evolutionary investigations of traditionally controversial species complexes in Leccinum.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication June 24, 2004.
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