Nucleotide structure of the Scytalidium hyalinum and Scytalidium dimidiatum 18S subunit ribosomal RNA gene: evidence for the insertion of a group IE intron in the rDNA gene of S. dimidiatum

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Nucleotide structure of the Scytalidium hyalinum and Scytalidium dimidiatum 18S subunit ribosomal RNA gene: evidence for the insertion of a group IE intron in the rDNA gene of S. dimidiatum Marie Machouart-Dubach a; , Claire Lacroix a;b , Christelle Vaury c , Martine Feuilhade de Chauvin a;b , Christine Bellanne¤ c , Francis Derouin Fre¤de¤ric Lorenzo a a

a;b

,

Laboratoire de Parasitologie-Mycologie, UFR Lariboisie're Saint-Louis-Universite¤ Paris 7, Faculte¤ de Me¤decine, 15 rue de l’e¤cole de me¤decine, 75006 Paris, France b Laboratoire de Parasitologie-Mycologie, Ho“pital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France c Centre d’Etude du Polymorphisme Humain, Ho“pital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France Received 28 August 2001; received in revised form 7 December 2001; accepted 18 December 2001 First published online 1 February 2002

Abstract The molds Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are responsible for dermatomycosis in humans. We sequenced their 18S subunit ribosomal RNA gene to identify these species with molecular biology-based methods. The coding sequences differed by a single polymorphism (A in S. dimidiatum, G in S. hyalinum). Moreover, we found an insert at position 1199 in the 18S rRNA gene sequence of S. dimidiatum. Its potential secondary structure was characteristic of a group IE intron. Bioinformatic and phylogenic group IE intron analyses generated four main homogeneous clusters. The S. dimidiatum intron is original and not related with other known IE group introns. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Group I intron; 18S; Ribosomal subunit; rRNA gene; Scytalidium dimidiatum; Scytalidium hyalinum

1. Introduction Scytalidium dimidiatum (the synanamorph of Nattrassia mangiferae) and Scytalidium hyalinum are ascomycetous fungi mainly causing dermatomycoses in humans. S. dimidiatum, characterized by its black mycelium, was formerly known as a plant pathogen, whereas S. hyalinum, which yields whitish colonies, has only been isolated from humans. Despite these di¡erences in phenotype and ecosystem distribution, several studies have shown similarities in the (G+C) DNA content [1], sterol composition [2] and antigenic cross-reactivity [3] of the two species. Few genetic data on these fungi are available. In order to clarify the taxonomy of S. dimidiatum and S. hyalinum, Roeijmans et al. [4] analyzed the ribosomal genes by using a combina-

* Corresponding author. Tel. : +33 (1) 43 29 65 25; Fax : +33 (1) 43 29 51 92. E-mail address : [email protected] (M. Machouart-Dubach).

tion of molecular typing methods ; polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) pointed to the absence of an intronic structure in the 18S ribosomal gene of S. hyalinum and the presence or absence of one to several introns in the rRNA gene of several S. dimidiatum strains. Among the described intron groups, group I introns are widespread in the fungal kingdom and are characterized by their ability to mediate their own removal from transcripts containing them (self-splicing) [5]. They have been identi¢ed in the genomes of prokaryotes [6], viruses [7], algae [8], fungi [9], lichens [10,11], and plants [12], and in the mitochondrial [13^15] and chloroplast genome [15]. However, many of these introns have not been subjected to secondary structural analysis and remain relegated to the status of unannotated sequences in nucleotide databases. The presence of a group I intron in some organisms has raised several hypotheses on the possible mode of acquisition of this intron from other organisms. Vertical

0378-1097 / 01 / $22.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 4 4 5 - 7

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inheritance is unlikely to be the only mechanism explaining the distribution of group I introns ; indeed, it has been speculated that horizontal transfer between organisms of evolutionary distinct lineages may occur [16]. Insertions of introns may be explained by the action of a site-speci¢c ‘homing’ endonuclease that recognizes and cleaves the double-stranded insertion (homing) site [17]. Nevertheless, nuclear rDNA open reading frames (ORFs) coding for homing endonucleases are rare, suggesting that other phenomena are responsible for intron acquisition. A reverse splice reaction has been proposed as the mechanism mediating intron insertion into rDNA genes [18]. On the basis of their secondary structural characteristics, group I introns were ¢rst classi¢ed into two subdivisions, IA and IB [5,19], and then reclassi¢ed according to the nucleotide sequences of the conserved core regions. Group I introns are now divided into ¢ve major groups, IA to IE, some of which are further subdivided (IA1, IC3, etc.) [20,21]. In order to develop molecular biology techniques for the identi¢cation of S. dimidiatum and S. hyalinum, we partially sequenced their 18S subunit ribosomal genes and the corresponding cDNAs. We characterized an S. dimidiatum-speci¢c insert as a group IE intron in three di¡erent strains. We performed a phylogenic analysis leading to the division of the group IE introns into four main homogeneous clusters and a ¢fth heterogeneous cluster. Similar results were obtained by the phylogenic tree analysis, the alignment studies of highly conserved structural regions (R and S elements), and localization of the insertion sites into the 18S rRNA gene. These latter results support the fact of an independent evolution of group IE introns prior to their insertion at a preferential site position. After insertion, group IE introns appear to evolve independently of the evolutionary mechanism of the host genes [21].

2. Materials and methods 2.1. Strains This study was performed on S. dimidiatum (IP1278.81, IP2527.99 and IP2528.99) and S. hyalinum (IP1517.83 and IP2526.99) human isolates referenced at the Collection Nationale de Cultures de Microorganismes (Institut Pasteur, France). Each strain is maintained by subculture at 27‡C on Sabouraud-chloramphenicol agar (Biome¤rieux, Marcy l’Etoile, France). 2.2. DNA and RNA extraction DNA was extracted from 3-day cultures, as older colonies of S. dimidiatum contain black pigments that inhibit PCR reactions. DNA was puri¢ed by using a standard phenol^chloroform extraction method [22] followed by RNase treatment for 30 min at 37‡C at a ¢nal concentration of 10 Wg ml31 (Sigma, St. Louis, MO, USA). The absence of contaminating RNA was checked by electrophoresis on ethidium bromide^agarose gel. DNA concentrations were determined with a Hoefer TKO 100 mini£uorometer (Pharmacia Biotech, Orsay, France) using Hoechst 33258 £uorescent dye. The same standard phenol^chloroform extraction method was used for rRNA, with addition of 100 U of RNase inhibitor (Gibco BRL, Life Technologies) to the lysis bu¡er. The pellet was resuspended in 30 Wl of sterile water. 2.3. Selection of primers for PCR ampli¢cation of the 18S gene First, six primer sets were selected to amplify and sequence the 18S gene of the two Scytalidium species (Table 1). These primers were designed from the consensus sequences obtained after alignment of 18S sequences from

Table 1 Primers used for ampli¢cation and sequencing of the 18S gene of S. dimidiatum and S. hyalinum Primer

Sequence

5P-Position

Primer pairs

FaR FaL DH2R DH3L DH3R DH4L DH4R DH5L DH6R DH6L DH8R DH7R

5P-GTATCTGATCGTCTTCGATC-3P 5P-GTAATTCCAGCTCCAATAGCG-3P 5P-CCTCAAACTTCCATCGACTTG-3P 5P-GTTGGTTTCTAGGACCGCCG-3P 5P-CCCGGCCGGACCAGTACA-3P 5P-CTGCGAATGGCTCATTAAATCA-3P 5P-CGCAAGGCCATGCGATTCG-3P 5P-TCCTGCCAGTAGTCATATGCT-3P 5P-CACCTACGGAAACCTTGTTAC-3P 5P-TGCGATAACGAACGAGACCT-3P 5P-CTGATGACATGCGCTTACTAG-3P 5P-GGCATAACAGCAATCGACG-3P

974 543 1386 801 654 55 247 12* 1780* 1283 1570 1407

FaL DH3L DH4L DH5L DH6L DH7R; DH8R

The 5P-position is relative to the 18S rDNA sequence of S. hyalinum (AF258606) except for those tagged with * which are relative to the 18S rDNA sequence (Z75578) of S. cerevisiae. Nucleotide 1 of the 18S rDNA consensus sequences of S. hyalinum corresponds to nucleotide 33 of the 18S rDNA sequence (Z75578) of S. cerevisiae.

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various ¢lamentous fungi and yeasts [23], including the international Saccharomyces cerevisiae reference Z75578. Primers were selected to obtain overlapping PCR products throughout the 18S sequence. 2.4. PCR PCR was performed in a reaction mixture of 25 Wl containing 25 ng DNA matrix, 1.5 mM MgCl2 , 100 WM each dNTP, 25 pmol of each primer and 1 U Taq polymerase (Eurogentec, Seraing, Belgium). Ampli¢cation conditions varied according to the primer set (Table 1). The PCR conditions consisted of denaturation for 3 min at 94‡C, followed by 35 ampli¢cation cycles at 94‡C for 1 min, 1 min at 60‡C, 1 min at 72‡C and then one cycle at 72‡C for 5 min. Five microliters of PCR product was electrophoresed in 2% agarose gel in the presence of ethidium bromide, and visualized under UV light. 2.5. Reverse transcription (RT) studies RT was performed in a total volume of 20 Wl by 50-min incubation at 42‡C followed by 15-min incubation at 70‡C to inactivate reverse transcriptase. The reaction mixture contained a sixth of the volume of the RNA extraction products, and the following ¢nal reagent concentrations: 1U hexanucleotide mix (Boehringer Mannheim, Mannheim, Germany), 500 WM dNTP mix, 40 U RNAse inhibitor, 1U ¢rst strand bu¡er, 100 mM dithiothreitol, and 1 U Superscript II (Gibco BRL, Life Technologies). Two microliters of RT products were PCR-ampli¢ed (RT-PCR) with primers DH6L and DH8R (Table 1). 2.6. Sequencing of PCR products The PCR and RT-PCR products of S. dimidiatum and S. hyalinum were puri¢ed in a reaction mixture of 20 Wl containing 4 Wl PCR product (at a minimal concentration of 5 ng Wl31 ), 2 U Exonuclease I (Amersham) and 2 U shrimp alkaline phosphatase (Amersham). After 1 h at 37‡C, the reaction was stopped by heating for 15 min at 72‡C. The two strands of ampli¢ed DNA were sequenced using the PCR primers and the BigDye Terminator kit (Applied Biosystems) on an automated sequencer (Applied Biosystems 377XL).

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www.doubletwist.com/) were used for alignments [25]. The Restriction Enzyme Database (REBASE) web site http://rebase.neb.com/rebase/rebase.homing.html was consulted for the nucleotide structure of digestion sites of homing endonucleases. A secondary structural model of the intron sequence was predicted using the aligned sequences of selected intron I types obtained by BLAST analysis (see Results and Discussion) in order to localize and match the P, Q, R and S elements. The 2D structure was estimated using the RNAStructure 3.5 software, and the Comparative RNA Web Site http://www.rna.icmb.utexas.edu was also consulted to help to predict stem structures [26]. The model was manually optimized according to structural conventions for group I introns [20,27]. Brie£y, a group I intron is composed of nine base-paired segments (P1^P9) and a speci¢c intron core structure (P3, P8) including the four elements P, Q, R and S, typically conserved among group I introns, and for which consensus sequences have been established [21]. The insertion site of this intron within S. dimidiatum 18S DNA was numbered according to the Escherichia coli 16S nucleotide sequence J01859 [28,29]. 2.8. Phylogenic analysis A set of 21 fungal small-subunit rRNA sequences, corresponding to 22 IE introns (Xylaria polymorpha contains two IE group introns), were recovered from GenBank. These sequences were aligned using CLUSTAL W (http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw _in.pl) [30]. Group IE intron sequences with or without S. dimidiatum were studied. The resulting alignments with a total length of 8937 or 8544 nucleotides corresponding to 17 612 or 16 687 characters were analyzed using the PHYLIP package (http://www.genebee.msu.su/services/ phtree_full.html) or (http://www.infobiogen.fr/services/ analyseq/cgi-bin/phylo_in.pl) with the following parameters: homological fragment only, random scale, throw o¡ columns with N unknown nucleotide and the cluster algorithm (arithmetic mean of pairwise distances between elements of two groups) [31]. One-hundred bootstrap replicates were performed to assess the con¢dence limits of the trees.

3. Results and discussion 2.7. Similarity searches and secondary structure modeling The publicly available sequences analyzed here were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/). The BLASTN 2.2.1 (http://www.ncbi.nlm.nih.gov/blast/) and BLAST2 (http://blast.genome.ad.jp/) programs were used to identify similar sequences to those of the S. dimidiatum intron [24]. The Multiple Alignment tool of GeneBee (http://www.genebee.msu.su/) and the Multi-Align GeneTool Editor programs of DoubleTwist (http://

3.1. Nucleotide structure of S. dimidiatum and S. hyalinum 18S sequences We sequenced the two strands of the 18S gene of three S. dimidiatum strains and two S. hyalinum strains, using the primers described in Table 1 for PCR and sequencing. These genomic sequences are deposited at GenBank, under the following accession numbers: S. dimidiatum IP1278.81: AF258603 (18S rRNA gene), AF204293 (V4

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domain), AF349941 (cDNA), S. dimidiatum IP2527.99 and IP2528.99: AF258604 and AF258605, respectively (18S rRNA gene), S. hyalinum IP1517.83: AF258606 (18S rRNA gene), AF204294 (V4 domain), and AF349942 (cDNA), S. hyalinum IP2526.99: AF258607 (18S rRNA gene). The 18S sequences of the three S. dimidiatum strains resulted in 2136/2137 nucleotides and those of the two S. hyalinum strains resulted in 1743 nucleotides. In S. dimidiatum, sequencing of PCR products revealed the presence of a 393-bp insertion in two strains (IP1278.81 and IP2527.99, AF258603 and AF258604, respectively) and a 394-bp insertion in one strain (IP2528.99, AF208605). The latter contained an additional T at position 56 in a poly-T stretch. This 11T (IP1278.81 and IP2527.99) or 12T (IP2528.99) polymorphism was con¢rmed in several double-strand sequencing runs. The 18S sequences were identical between the two Scytalidium species except for a G (S. hyalinum) to A (S. dimidiatum) polymorphism at position 144 in the V2 domain. This polymorphism was con¢rmed in the ¢ve strains of Scytalidium. The location of the polymorphic domains (V1 to V9, excluding the prokaryotic domain V6) was in agreement with previously described eukaryotic models [32] (Fig. 1). As reported by Roeijmans et al [4] for ¢ve human source strains, no intronic insertion in the 18S rRNA gene of S. hyalinum was observed. 3.2. Characterization of the S. dimidiatum intronic insertion To con¢rm the presence of a putative intronic structure in the genomic 18S rRNA gene, we reverse-transcribed S. dimidiatum and S. hyalinum total RNA then PCR-ampli¢ed the corresponding cDNA. Electrophoresis of the PCR products revealed 288-bp amplicons with the

Fig. 1. Schematic representation of the small ribosomal subunit 18S gene. The numbering is relative to the 18S rDNA sequence of S. hyalinum (AF258606). The hatched boxes correspond to the polymorphic region V1^V9. The black box corresponds to the group I intron of S. dimidiatum inserted at position 1397. The sites of the PCR and sequencing primers are indicated with arrows. +: note the G (S. hyalinum) to A (S. dimidiatum) polymorphism at position 144 of the V2 domain.

Fig. 2. Ampli¢cation pattern of one S. hyalinum strain (IP1517.83) and one S. dimidiatum strain (IP2528.99) using the DH6L^DH8R primer set £anking the intron insertion site. Lane 1: 100-bp ladder, lane 2: PCR without DNA (negative control), lane 3: DNA from S. dimidiatum, lane 4: cDNA from S. dimidiatum, lane 5: DNA from S. hyalinum, lane 6: cDNA from S. hyalinum.

cDNA of S. dimidiatum and both the genomic DNA and cDNA of S. hyalinum. The size of the PCR products obtained from S. dimidiatum genomic DNA was 681/682 bp, which con¢rmed the presence of an intronic insertion in the 18S gene of S. dimidiatum (Fig. 2). Sequencing of RT-PCR and PCR products con¢rmed the intron size of 393/394 bp and the splice site at nucleotide 1397 relative to the S. hyalinum 18S sequence (position 1199 in the E. coli J01859 sequence). We did not test the capacity for self-splicing in vitro. Although we found a single intron in three di¡erent S. dimidiatum strains of human origin, Roeijmans et al. [4] reported 18S RNA amplicons of di¡erent sizes in S. dimidiatum strains of various origins. In two strains of human origin, the amplicons were of 1800 bp in size, indicating the absence of an intron in their 18S rRNA gene. The 18S rRNA amplicons of three strains of plant and human origin and four strains of plant, human and bovine origin were of 2100 or 3000 bp, indicating the presence of one or several introns in the 18S rRNA gene of these S. dimidiatum strains. This genetic heterogeneity of SSU is poorly documented but has been observed in other species as in di¡erent strains of Exophiala dermatitidis [33]. The allelic heterogeneity is also rarely observed, but has also been found in a Candida albicans strain with some intron-containing and intronless 25S rRNA genes [34]. In our study, the corresponding sequencing graph of each PCR ampli¢cation product (DH6L^DH8R amplicons) was totally pure, pointing to the absence of genetic

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Fig. 3. Putative structural model of the S. dimidiatum group IE intron. Intronic structure sequences are given in uppercase letters, and 5P- and 3P-exons in lowercase letters. Boldface letters indicate conserved core sequence elements P, Q, R and S. R: splice site. ‡:guanosine cofactor-binding site. Conserved helical regions are designated P1 to P13. Insert: 5P^3P base-pairing of exon^intron junctions with IGS.

heterogeneity of the 18S rRNA genes per haploid genome of S. hyalinum and S. dimidiatum strains. This pro¢le is the most frequently described in the literature [35]. 3.3. Modeling of the S. dimidiatum insert as a group IE intron BLAST analysis of the total intronic sequence (intronic structure from sequence AF258604: 393 nucleotides) with available database sequences yielded 73 hits corresponding mainly to fungi, algae, lichens and yeast. The scores were very low, with E-values from 0.021 to 5.2 corresponding to matches of 23/393 or 19/393 nucleotides. The structure of the sequences matching with the query corresponded principally to the S element of group I introns (consensus sequence described by Suh et al. [21]). We performed a second BLAST analysis with the putative S element of the S. dimidiatum intron (ggcttAAGGTACGTGCTaa: 19 nucleotides, S element is underlined). We obtained 142 hits with E-values from 3U1034 (19/19 nucleotides) to 0.26 (14/19 nucleotides). The high hits correspond to SSU group IE introns described by Suh et al. [21], Grube et al. [11] and Bhattacharya et al. [36]. Consequently, using these BLAST analysis results (localization

of the S element) we determined the core and then the helices and stems of the putative secondary nucleotide structure of the insert. It was typical of group I introns [20,37] (Fig. 3). It was composed of nine base-paired segments (noted P1^P9) comprising the structures P3, P8 forming the intron core. In these segments, the four elements P, Q, R and S were in keeping with the consensus sequences of group I [5,21]. According to the splice model of Burke et al. [27], the internal guide sequence (IGS) and the requisite U and G residues at the 5P- and 3P-splice sites are present in the S. dimidiatum intron. In addition, ¢ve consensus nucleotides speci¢c for this group I [37] are conserved, namely the G and C of the second base-pair of P7 (binding site of the guanosine-cofactor involved in a self-splicing process [20]), the 2A in the J4/5 segments, and the A at position J6/7. The presence of an additional basepaired segment, P13, involved in stabilizing the auto-catalytic core, suggests that the S. dimidiatum intron belongs to group IE1

1

The putative secondary structure was con¢rmed by Franc°ois Michel (Centre de Ge¤ne¤tique Mole¤culaire, CNRS de Gif-sur-Yvette, France) [38].

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Table 2 Conserved group IE intronic R and S regions of 18S rRNA genes

The clusters I^V were determined from the phylogenic tree (Fig. 4). *The position of these introns in the rRNA gene (R.R. Gutell, personal communication) were : C. capitata: 1481^1887; X. polymorpha intron 1: 1562^ 2006 ; X. polymorpha intron 2: 2222^2666; R. necatrix: 1602^2011. i1: intron 1; i2: intron 2. **The reference sequences of the R and S elements are from C. hypophloia. The resulting polymorphisms are bold and underlined.

3.4. Phylogenetic analysis of the group IE intron A phylogenetic analysis was performed using three data entries: (i) the phylogram (Fig. 4) resulting from the cluster algorithm analysis of 18S rRNA group IE introns [11,21,36], (ii) the alignment studies (Table 2) of the corresponding R and S elements and, (iii) the intronic insertion site position (Table 2) into the 18S rRNA gene. 3.4.1. Phylogram analysis The analysis of the phylogram principal ancestry nodes showed four unlinked homogeneous clusters and a ¢fth heterogeneous cluster that contains four congruent ascomycetes (Cryphonectria parasitica, E. dermatitidis, Cladonia merochlorophaea intron 2 and Stegobium paniceum intron 2) (Fig. 4). The bootstrap values (I: 64^100%, II: 50^ 100%, III: 78^100% and IV: 50^88%) provide a good support for the monophyly of the homogeneous IE intron clusters. S. dimidiatum belongs to the cluster IV, along with the black yeast Nadsoniella (Exophiala) nigra. When we compared this phylogram to that obtained without the S. dimidiatum intron sequence, we found that cluster I was retained whereas the clusters II and III were grouped together and unlinked to cluster I (data not shown). These two phylograms were consistent with the

common eukaryotic 18S rRNA gene introns studied by Bhattacharya et al [36] and Suh et al. [21]. The splitting of clusters when the S. dimidiatum sequence was added to the alignment strongly suggests the originality of the S. dimidiatum intron and the polyphyly of the group IE introns. 3.4.2. Alignment studies of the R and S elements The highly conserved R and S regions including the P7 and P7P paired segments of previously studied 18S rRNA gene introns are described in Table 2. To analyze these nucleotide alignments, the Cryptendoxyla hypophloia (AF015912, partial 18S rRNA gene sequence; 664 nucleotides; intron 87^519) intronic sequence was used as reference, because it has been extensively studied as a member of the IE intron group [21,36]. Cluster analyses were performed taking into account the main polymorphisms observed in the R and S elements. The aligned nucleotide sequences of the S regions identi¢ed three groups with a good correlation with the phylogram clusters. The ¢rst group (polymorphism in position 5: G, noted G5) corresponds to the phylogenic cluster I, except for Mycocalicium albonigrum intron 1 (polymorphism T5) and with the addition of E. dermatitidis (cluster V). The second group is represented by the clusters II^IV

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Fig. 4. Phylogram of IE intron of the 18S rRNA gene. Scale changes are shown on the abscisse’s axis. The insertion site positions, the organism accession numbers and the abbreviations are given in Table 2.

(polymorphism T5), with the addition of M. albonigrum intron 1 (cluster I) and C. parasitica (cluster V). The third group (polymorphisms C1, T5 and A8) included two members of the heterogeneous cluster V, without C. parasitica and E. dermatitidis. The analysis of the sequence alignments corresponding to the nucleotide R regions is more complex, but a good correlation with the phylogram and the S-groups was observed. The polymorphisms T4 and A11 are principally associated with the phylogenic cluster I, in addition of E. dermatitidis (cluster V) and except for Rhodosporidium dacryoidum and M. albonigrum intron 1 (polymorphisms T4 and G11). The polymorphisms C4 and G11 are associated with the clusters II^IV, excepted for C. hypophloia (polymorphisms C4 and A11) and S. dimidiatum (T4 and G11). 3.4.3. Intronic insertion site positions Insertion of group I introns has been reported in at least 17 nucleotide positions in the eukaryotic SSU rDNA, 12 of which are represented in fungal taxa [29], sites 989 and 1199 (according to the E. coli SSU rRNA gene sequence) being the most prevalent. Published data and the results of this study show only four insertion site positions corresponding to the 18S rRNA group IE intron (217, 516, 989 and 1199) [21,36]. We analyzed the correlation between the insertion site positions and the di¡erent phylogenic clusters and groups that we identi¢ed by the phylogenic tree analysis and the R and S sequence alignments, respectively.

In our study the introns inserted at 516 insertion site position are recovered in the cluster I, except for E. dermatitidis (cluster V). The intron inserted at positions 989 and 1199 are grouped together in the clusters II^IV, except for C. parasitica and C. merochlorophaea intron 2 (cluster V). Cluster V is heterogeneous with a rare position at 287 (S. paniceum intron 2). This is in agreement with previous phylogenetic analyses suggesting that the introns inserted at the same site of the 18S rRNA gene are monophyletic [8,11,39], e.g. the ICI introns inserted at position 516 [36]. We also examined the correlation between the insertion site positions and the polymorphisms of the groups determined by the R and S alignments. Every intron inserted at the 989 and 1199 sites has the T5 polymorphism of the S element. This polymorphism is G5 for the introns inserted at site 516, except for M. albonigrum intron 1 (polymorphism T5). In addition, every intron inserted at the 516 site has the T4 polymorphism of the R element. Finally, we observed the following trends of the IE intron characteristics (R and S polymorphisms, insertion site) and the phylogram: (i) the phylogenic clusters II^IV correspond preferentially to the T5 polymorphism of the S element with insertion sites at position 989 or 1199, (ii) the phylogenic cluster I corresponds preferentially to the T4 polymorphism of the R element and the G5 polymorphism of the S element with the insertion site position 516. We have described some exceptions above. These characteristic elements could be used in order to classify intronic structures (IE group, phylogeny). The monophylies of the IE intron at position 516, 989

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or 1199 were strongly supported by these latter analysis. These results are identical to those of the phylogram without the S. dimidiatum intron sequence (data not shown). Its addition splits the cluster corresponding to insertion sites 989 and 1199 into two parts (clusters II and III of this study). This signi¢es that the S. dimidiatum intron would be one of potential ancestors of the organisms corresponding to clusters II and III with bootstrap values of 50 and 33%. The heterogeneity of the cluster V indicates that other factors could also exist. Several papers have provided evidence that group I introns were transferred horizontally to a distinct insertion site prior to their vertical transmission and divergence [8,21,39^41]. Our phylogenic results support the fact of an independent evolution of group IE introns prior to their insertion at a preferential site position. The mechanism of site-speci¢c intron insertion is unknown. The polymorphic nucleotide structures of the R and S elements described here (Table 2) are only indicative tags of other active structures involved in the insertion mechanism and in site speci¢city. After insertion, the evolution of the group IE intron appears to occur independently (such as the T2 polymorphism in the R element of X. polymorpha introns 1 and 2, Rosellinia necatrix intron 1) of the evolutionary mechanism of the host genes [8,21,40]. This ordered evolution mechanism is illustrated by the ascomycete X. polymorpha in which the SSU rRNA gene contains two group IE introns (Fig. 4, Table 2) inserted at 989 (cluster III) and 1199 (cluster II) positions. These introns have 66.7% nucleotidic identity and identical R and S polymorphic elements (Table 2). Moreover, the 989 intron has never been described as being inserted at position 1199 and vice versa. We suspect the existence of a unknown intron ancestor(s) with an intronic structure speci¢c to horizontal transfer to position 989 or the 1199. This speci¢city could correspond in part to the nucleotide structures of paired segments P1, P1P and P10, P10P at the exon^ intron junction. We analyzed the totality of the BLAST hits, despite the low E-values obtained and not only the top BLAST results [42]. Most of the intron hits corresponded to the 18S rRNA gene of ascomycetous fungi that shared a common ecosystem and are parasites of fruit trees or timber trees, or soil dwellers. In the BLAST list, no match corresponded to an intronic or non-intronic structure (invertebrate eukaryotes or bacterial species) describing sister species or ancestor (18S or another gene). This is probably due to the small number of sequenced introns IE and the numerous annotationless sequences in the databases. Indeed, the intron sequences and, in some cases, the corresponding insertion sites are not always properly annotated in the GenBank entry. Some introns are probably set in an artifactual position in the phylogenetic tree. Unlike S. dimidiatum, a plant pathogen mainly recovered from fruit trees, S. hyalinum has only been recovered from humans. Few nucleotide data are available for Scy-

talidium sp. We cannot conclude whether the group IE intron was inserted in an ancestor of S. dimidiatum and S. hyalinum and was subsequently lost in S. hyalinum, or if this insertion occurred after the phylogenic separation of these two species, as S. dimidiatum has been described with intron(s) (this study and [4]) and without intron [4]. Although morphogenetic characteristics di¡erentiate these two Scytalidium species (such as mycelium pigmentation, intronic insertion(s) in the 18S gene, and the A/G polymorphism), Roeijmans et al. [4] considered S. hyalinum as a synonym of S. dimidiatum. The presence of this group IE intron in the 18S rRNA of S. dimidiatum is compatible with this view. In addition to its phylogenic relevance, the presence of a type I intron(s) in some S. dimidiatum strains could indicate a particular drug susceptibility pro¢le, S. dimidiatum and S. hyalinum being resistant to most available antifungal drugs [43]. Mercure et al. reported the identi¢cation of a group I intron in the 25S ribosomal RNA of some C. albicans strains [44] with high susceptibility to 5-£uorocytosine, 5-£uorouracil [45] and pentamidine [35]. It was suggested that the in vivo self-splicing of the intron or the adoption of its secondary structure could be blocked by antifungal drugs [46^48]. The presence of group I introns in essential genes of the fungus may have potential value in the search for new drug targets in S. dimidiatum.

Acknowledgements The authors thank Franc°ois Michel (Centre de Ge¤ne¤tique Mole¤culaire, CNRS de Gif-sur-Yvette, France) for his help in the construction of the putative intron secondary model, Debashish Bhattacharya (Department of Biological Sciences, University of Iowa, IA, USA) and Robin R. Gutell (Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, USA) for personal communication on group IE introns, Joe«lle Dupont (Laboratoire de Cryptogamie, Muse¤um d’Histoires Naturelles, Paris, France) for helpful discussions on the phylogeny of ascomycetous fungi, and David Young for checking the English. This study was supported by a grant from the Ministe're de l’Enseignement de la Recherche et de la Technologie to M.M.-D.

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