Variability and Phylogenetic Incongruence of an SSU nrDNA Group I Intron in Artomyces, Auriscalpium, and Lentinellus (Auriscalpiaceae: Homobasidiomycetes)

Edgar B. Lickey, Karen W. Hughes and Ronald H. Petersen

Department of Botany, University of Tennessee

Correspondence: E-mail: elickey{at}utk.edu.


    Abstract
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Previous research has shown that a group I intron occurs in the SSU ribosomal DNA gene of isolates of Artomyces (Clavicorona, in part) and Lentinellus, but apparently it is absent in an Auriscalpium isolate. However, further investigation revealed that the intron is apparently absent in some species of Artomyces and Lentinellus and is present in at least one species of Auriscalpium. To examine this further, the presence or absence of the group I intron is reported for 13 species of Lentinellus, two species of Auriscalpium, and 16 species of Artomyces. The presence of the intron among the species was variable and is documented for seven species of Lentinellus, one species of Auriscalpium, and 12 species of Artomyces. Furthermore, the presence of the intron was variable among the isolates of several species, and variability of its presence was observed within single isolates, indicating inter-ribosomal repeat heterogeneity. Independent phylogenetic estimations were generated for the intron and nuclear ribosomal internal transcribed spacer regions (ITS). Tests of congruence for the two trees indicated that the data were heterogeneous. Some of the discontinuity between the two phylogenies is due to placement of the Ar. austropiperatus intron within the Lentinellus intron clade. Variability in the length of the intron was observed in populations of the pan-Northern Temperate species Ar. pyxidatus. This was due to the presence of an additional unknown insertional element found only within North American collections of Ar. pyxidatus and absent from European and Asian collections.

Key Words: Artomyces • Auriscalpiaceae • AuriscalpiumClavicorona • group I Intron • Lentinellus • nr ITS • nr small subunit • phylogenetic incongruence


    Introduction
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Group I introns are mobile, intervening sequences that can catalyze their own excision through characteristic secondary and tertiary structure (Cech 1988, 1990; Dujon 1989; Shub, Goodrich-Blair, and Eddy 1994; Westhof and Michel 1996). They have several conserved regions that allow secondary structure to form through base pairing, and these regions have been used to categorize these introns into classes (Cech 1988, 1990; Westhof and Michel 1996). Group I introns have been found in nuclear and organelle genes of eukaryotes, as well as in eubacteria and viruses (Dujon 1989; Shub, Goodrich-Blair, and Eddy 1994). Although apparently homologous introns have been found in related host taxa, consistent with a presumed common evolutionary origin, their occurrence may also be variable among closely related host taxa, suggesting multiple insertional events (Woodson 1996). Some shared occurrences appear to be ancient in origin, such as the intron occurring in the leucine tRNA gene that occurs in both cyanobacteria and chloroplasts, whereas others appear to be much more recent in origin (Turmel et al. 1995). More than 200 different group I introns have been identified, occurring in a wide variety of genes in different hosts (Damberger and Gutell 1994). Particular attention has been paid to the introns found in nuclear, organelle, and bacterial ribosomal RNA genes (Woodson 1996).

Group I introns seem to be especially prevalent in the nuclear small subunit ribosomal RNA genes of fungi. These include the Ascomycete Cryptendoxyla hypophloia (Suh, Jones, and Blackwell 1999), mycorrhizal Cenococcum geophilum (Shinohara, LoBuglio, and Rogers 1996), and lichen-forming Cladonia chlorophaea, Lecanora dispersa, Calicium tricolor, Porpidia crustulata, as well as a non-lichen-forming ally Mycocalicium albonigrum (DePriest and Been 1992; DePriest 1993; Gargas, DePriest, and Taylor 1995). They have also been found in plant parasitic fungi Protomyces, Phialophora gregata, Botrytis, Dumontinia, Encoelia, Grovesinia, Myriosclerotinia, and Sclerotinia (Nishida and Sugiyama 1995; Chen, Gray, and Grau 1998; Holst-Jensen et al. 1999), and the entomothphagus Cordyceps (Nikoh and Fukatsu 2001).

In a phylogenetic study of homobasidiomycetes using the nuclear small subunit ribosomal RNA gene, Hibbett (1996) found that Auriscalpiaceae species Artomyces pyxidatus ({equiv} Clavicorona pyxidata, Lickey et al. 2003), Lentinellus montanus, L. ursinus and L. omphalodes and Tricholomataceae species Panellus stypticus all contained a group I intron in the same position corresponding to base 943 of the small subunit ribosomal gene of Escherichia coli (see Gargas, DePriest, and Taylor 1995). The intron was apparently absent in another member of the Auriscalpiaceae, Auriscalpium vulgare. Because the intron occurred in two separate evolutionary lineages, Panellus stypticus and Artomyces-Lentinellus, Hibbett concluded that the intron was probably acquired in two separate events, adding that the introns in Artomyces and Lentinellus are more similar to each other than either is to the intron in Panellus stypticus. Once acquired in the Auriscalpiaceae lineage, Hibbett suggested that the intron was probably lost in Auriscalpium (Hibbett 1996). Variation in the presence/absence of the intron in one isolate of L. montanus and an isolate of L. ursinus was also noted and may represent inter-ribosomal repeat heterogeneity (Hibbett 1996). For his survey, Hibbett (1996) sampled only one specimen each of Ar. pyxidatus, Au. vulgare, L. omphalodes (L. micheneri in this study), andL. ursinus (probably L. omphalodes in this study) and two specimens of L. montanus.

In this study, the range and extent of the occurrence of the group I intron in the SSU nrDNA gene was examined among and within species of Artomyces, Auriscalpium, and Lentinellus. Phylogenetic analyses were undertaken to elucidate the evolutionary history of the intron and to determine if it is congruent with that of the ribosomal ITS 1 – 5.8S – ITS 2 sequences.


    Methods
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Cultures and DNA Extraction
The treatment of Artomyces species ({equiv}Clavicorona subg. Ramosa) follows the recent revision by Lickey, Hughes, and Petersen (2003), which includes previously undescribed species. The taxonomy and nomenclature of Lentinellus is presently in a state of flux and reorganization, but taxonomic groups have tentatively been delimited. The following taxonomic groupings and names are sensu R.H.P. (work in progress). Monokaryon and dikaryon cultures used in this study (see Supplementary Material online) were established from basidiomata collected in nature and are maintained in the culture collection at the University of Tennessee as described by Hughes et al. (1999). These include 84 isolates of seven species of Lentinellus, 15 isolates of two species of Auriscalpium, and 325 isolates of 15 species of Artomyces. Each collection is represented by a voucher specimen and, for most cases, dikaryon and monokaryon cultures. The DNA extraction protocol followed that used by Hughes et al. (1999).

Polymerase Chain Reaction Amplification
The region of the nuclear ribosomal SSU gene in which the intron was located was amplified using the forward primer SR1c and reverse primer NS6 (Hibbett 1996; table 1). For collections in which only a ca. 800-bp fragment was obtained, additional amplification reactions were performed using primers 943a-sel and 943b-sel (table 1; modified from Hibbett 1996), which are internal to the intron, and in paired combinations of SR1c with 943b-sel and 943a-sel with NS6 as described in Hibbett (1996). Cycle parameters were 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes, with a final polymerase chain reaction (PCR) extension at 72°C for 1 minute (modified from Hibbett 1996). The PCR products were visualized by gel electrophoresis in 1.5% TBE agarose gels. A few representative isolates were electrophoresed in a 1.5% agarose gel with a 1x alkaline buffer according to FMC Bioproducts (1997).


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Table 1 List of Primers Used For PCR Assay and Sequencing of SSU Nuclear Ribosomal DNA and the Associated Group I Intron.

 
The nuclear ribosomal ITS 1–5.8S-ITS 2 (ITS) region was amplified using forward primer ITS 5 and reverse primer ITS 4 (White et al. 1990). Cycle parameters were an initial melting cycle of 94°C for 4 minutes, 35 repetitions of a three step amplification cycle of 94°C for 1 minute, 52°C for 1 minute, and 72°C for 1 minute, ending with an extension cycle of 72°C for 1 minute.

Southern Blots
Southern Blots of intron-containing SSU PCR products were performed according to Amersham (1987) using the gel in figure 1. DNA was fixed to Amersham Hybond N+ nylon membranes by exposure to ultraviolet light. A 148-bp probe for the SSU region adjacent to the intron insertion site was obtained from a PCR amplicon of Ar. adrienneae (isolate 7387), which does not contain the intron. A portion of the SSU region of this isolate was amplified using primers SR1C and NS6, and the product was digested with restriction enzyme HaeIII. The148-bp fragment, which corresponds to bases 1265 to 1403 in the small subunit gene of Saccharomyces cerevisiae (Rubstov et al. 1980), was then excised from a 1.5%TAE low-melting-temperature agarose gel (NuSieve GTG agarose, FMC Bioproducts). The excised fragment was purified using a Promega Wizard PCR Purification Kit following manufacturer's directions, labeled with 32P and used to probe the blots (Sambrook, Fritsch, and Maniatis 1989).



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FIG. 1. Variability in PCR product length among Artomyces pyxidatus isolates. Lanes are: A, Phi x HaeIII size marker; B, GB 1702 without 2° insert (Sweden); C, 9255 (Canada); D, 9256 (Canada); E, 8396 (Mexico); F, Ar. adrienneae 7388 (without intron); G, 8896 without 2° insert (Russia); H, 4567 (Georgia); I, 4568 (Georgia); J, 4573 (Georgia); K, Phi x HaeIII; L, Lambda HindIII size marker; M, 56667 (Georgia); N, 1508 (Wisconsin); O, 1513 (Wisconsin); P, H7524 (Arizona); Q, 4990 (Maine); R, HBB810 (Maryland); S, 9229 (Tennessee); T, 9230 (Tennessee); U, 9232 (Tennessee); and V = Phi x HaeIII size marker

 
DNA Sequencing, Alignment, and Comparison
Five isolates of Ar. pyxidatus, two isolates of L. castoreus, and one isolate each of Ar. austropiperatus, Au. villipes, L. cochleatus, L. montanus, L. micheneri, L. flabelliformis, and L. "subaustralis" (nom. prov.) were chosen for sequencing of both the SSU group I intron and the ITS region (see table in Supplementary Material online). The PCR products were electrophoresed and then excised from a 1.5% TAE low-melting-temperature agarose gel and purified as described above. Purified products were sequenced with an automated ABI 373 DNA sequencer (ABIPrism Dye Terminator cycle sequencing, PerkinElmer) with primers NS41 and NS51 (table 1) for the intron and primers ITS 5 and ITS 4 for ITS (Whiteet al. 1990). Two additional internal primers (table 1) were needed to accommodate the longer intron length of some Ar. pyxidatus isolates. Sequences of each gene were manually corrected and aligned using the LineUp and SeqLab programs in the Genetics Computer Group package (2001) and deposited in GenBank (table 2). Sequences of the small subunit gene of Saccharomyces cerevisiae (GenBank J01353; Rubstov et al. 1980) and sequences for Ar. pyxidatus and L. omphalodes (GenBank U59066 and U59078, respectively; Hibbett 1996) were compared to the intron alignment for reference purposes.


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Table 2 GenBank Accession Numbers of Group I Intron and ITS Sequences of Isolates Used in This Study.

 
Phylogenetic Reconstruction
Phylogenetic relationships of the intron and the ITS 1–5.8S–ITS 2 region were estimated separately using PAUP* 4.0 (Swofford 1998). Ambiguously aligned regions were excluded from the analysis, and gaps were treated as missing data. A branch-and-bound search was used to find the most parsimonious trees, and bootstrap analyses of 100 replicates were conducted with a branch-and-bound search using equally weighted maximum parsimony.

Phylogenetic Comparisons
Several comparisons were made between the phylogenetic estimations based on the intron and ITS sequences with a direct focus on levels of character incongruence. Incongruence metrics of Mickevich and Farris (1981) and Miyamoto (in Kluge 1989; Swofford 1991) (IMF and IM, respectively), and the homogeneity test (HTF) of Farriset al. (1994) were calculated using PAUP and have been described by Johnson and Soltis (1998). The parameters for HTF were 1,000 replicates using a branch-and-bound search. Templeton's (1983) significantly least parsimonious test (SLPT) was also used to test for congruence. This test has recently been criticized for its inability to test accurately for congruence between data sets of different size and evolutionary rate, often resulting in an overestimation of congruence (Shimodaira and Hasegawa 1999; Goldman, Anderson, and Rodrigo 2000; Dowton and Austin 2002).


    Results
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
PCR Assay for Group I Intron
Based on Saccharomyces cerevisiae sequence (Rubstov et al. 1980), the expected length of the SSU region amplified using PCR primers SR1c and NS6 should be approximately 800 bp without the intron. All isolates of Auriscalpium vulgare exhibited a PCR product of this size using these primers. No larger fragments were recovered for these isolates using intron-specific primers 943a-sel and 943b-sel, or with combinations of these primers with SSU primers SR1c and NS6. Four isolates of Au. villipes, two from Mexico, one from Costa Rica, and one from Paraguay, all showed the presence of an approximately 1,300-bp fragment, which was shown by sequencing to contain the group I intron identified by Hibbett (1996). The two collections from Mexico, however, exhibited two fragments, one of each size, indicating an apparent polymorphism.

The 1,300-bp intron-containing fragment was present in members of a North American clade consisting of three closely related species, L. flabelliformis, L. montanus, and L. occidentalis nom. prov., but one collection from Australia, L. novae-zelandiae, with similar ITS sequences lacked the 1,300-bp fragment, exhibiting only the ca. 800-bp fragment lacking the intron. Lentinellus subaustralis from the southeastern United States and L. micheneri (Europe, western United States, and Greenland) were well-supported clades based on ITS sequences, and both produced a 1,300-bp fragment. Presence of the intron was variable in L. castoreus, a global species, with the intron present in southeastern United States, Costa Rican, and Argentinean collections, but it is apparently absent in European collections and collections from California. Amplifications of L. ursinus using SR1c and NS6 produced only the ca. 800-bp fragment expected when the intron is lacking, but when primers internal to the intron (943a-sel and 943b-sel) were used, an amplification product was seen in five of 13 collections tested. With amplifications using one primer internal to the intron and an external primer (SR1c with 943b-sel and 943a-sel with NS6), the larger 1,300-bp product containing the intron was obtained for the five collections but not for the remaining eight. This is consistent with the findings reported by Hibbett (1996), who suggested that the intron may be present in low copy number and that PCR bias was responsible for the failure of the 1,300-bp intron-containing fragment to amplify when primers SR1cand NS6 were used. Intron presence or absence in L. ursinus did not have an apparent geographical pattern. Detectable intron presence varied in both Europe and North America; it was variable in L. pulvinulus, a southern hemisphere species, in L. cochleatus, a northern hemisphere species, and in L. tridentinus, a European species. The intron was not present in L. vulpinus and L. sublineolatus, both north temperate species. Occasionally, some isolates were observed with both fragments, indicating a presence/absence polymorphism.

Of the 16 species of Artomyces surveyed with primers SR1c and NS6, increased fragment sizes were observed in Ar. pyxidatus isolates and Argentinean isolates of Ar. austropiperatus. The large fragment in all Eurasian Ar. pyxidatus isolates and in the Argentinean Ar. austropiperatus isolates was approximately 1,300 bp, indicating presence of the intron. Considerable variation, however, was found in fragment sizes among North American Ar. pyxidatus isolates. Comparisons of fragment lengths of several North American Ar. pyxidatus isolates and Ar. adrienneae isolate 7387, which lacks the intron, are shown in figure 1. The size of these PCR fragments ranged from approximately 1,300 bp to a little more than 2,100 bp. As with Au. villipes, a fragment of 800 bp was sometimes observed in Ar. pyxidatus isolates, but it was always in a polymorphic condition.

Approximately two-thirds of the North American Ar. pyxidatus isolates surveyed exhibited multiple primary and secondary fragments. The high annealing temperature (60°C) in the PCR cycle should have been sufficiently stringent to avoid non-target amplification. Southern blots probed with a 148-bp SSU fragment showed that the variable primary bands and the multiple secondary bands were likely of small subunit origin and not the result of non-target priming and amplification. Alkaline gels, which should prevent secondary structure formation, indicated that the multiple bands were probably not the result of heteroduplexing or other secondary structure.

Artomyces isolates, which initially appeared to lack the intron using the SSU primers, were surveyed further with the intron-specific primers 943a-sel and 943b-sel. Of these 69 isolates, 32 exhibited fragments of the expected size of the intron. All isolates of Ar. cristatus, Ar. stephenii, and Ar. Taxon "B" exhibited positive results for the intron, whereas all isolates of Ar. carolinensis, Ar. colensoi, Ar. dichotomus, and Ar. piperatus exhibited no amplified fragments. Amplification of the intron was variable among isolates of Ar. adrienneae (intron amplified in seven of eight isolates), Australasian Ar. austropiperatus (two of four), Ar. candelabrus (three of 11), Ar. costaricensis (two of three), Ar. microsporus (one of two), Ar. novae-zelandiae (one of eight), Ar. tasmaniensis (two of three), and Ar. turgidus (three of eight). Additionally, fragments larger than expected for these two primers were amplified from two Ar. adrienneae isolates, and isolates of Ar. cristatus and Ar. Taxon "B." Further surveys with combinations of SSU and intron-specific primers indicated that the intron is present in the 943 position in the SSU.

Sequence Comparisons
The aligned sequences of the SSU group I intronwere compared to those reported by Hibbett (1996) and Rubstov et al. (1980; Saccharomycyes cerevisiae SSU rRNA, GenBank J01353). The alignment verified that the increased PCR fragment size in isolates of Lentinellus, Artomyces, and Auriscalpium were due to what Hibbett (1996) identified as a group I intron in a position corresponding to base 943 of Escherichia coli (see Gargas, DePriest, and Taylor 1995), or base 1156 of the S. cerevisiae sequence.

Sequence comparisons also verified that the larger, highly variable fragments amplified from North American Ar. pyxidatus isolates, as well as the Ar. adrienneae, Ar. cristatus, and Ar. Taxon "B" isolates were due to an additional insertion located between conserved regions R and S of the group I intron (fig. 2). These secondary insertions ranged in size from about 100 to over 1,000 bp, with differences in length apparently caused by indels of variable size. All secondary insertions were alignable on the 3' and 5' ends of the longest insertion sequenced from Ar. pyxidatus isolate 56667, with significant sections missing from the middle (fig. 2). A basic Blast search in GenBank revealed no matches for these insertions, but a search for potential secondary structure using RNAdraw (Matzura and Wennborg 1996) indicated that secondary structure was strongly probable. A search of the unknown sequences using the Genetics Computer Group program PepData indicated that several putative open reading frames (ORFs) were present.



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FIG. 2. Relative locations of the group I intron and secondary insert. The arrow indicates the position homologous to 943 of Escherichia coli (Gargas, DePriest, and Taylor 1995) and 1156 of Saccharomyces cerevisiae (Rubstov et al. 1980). Also illustrated are the regions of homology in secondary insertion sequences of four North American Ar. pyxidatus isolates. The European isolate (GB 1702) does not contain the insertion. Solid lines represent completely aligned areas, and dotted lines represent "missing" sections

 
Phylogenetic Analyses
The resulting alignment of the group I intron sequences after excluding the ambiguously aligned regions was 387 characters, of which 58 were parsimony informative. A branch-and-bound search of these sequences for representative isolates (see table in Supplementary Material online) yielded a single most parsimonious tree of 117 steps (fig. 3). Bootstrap analysis indicated strong support for Ar. pyxidatus (bootstrap = 95%) and a clade composed of L. montanus, L. "subaustralis," L. micheneri, L. flabelliformis, and L. castoreus ("core" Lentinellus group; bootstrap = 100%). However, Ar. austropiperatus and L. cochleatus were paired on a moderately well-supported branch sister to the Lentinellus clade (bootstrap = 87%). The single Au. villipes isolate composed a branch between Ar. pyxidatus and the Ar. austropiperatusLentinellus clade.



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FIG. 3. Comparison of phylogenetic estimations for group I intron and ITS sequences of isolates of Artomyces, Auriscalpium, and Lentinellus. Both trees are the result of branch-and-bound searches with maximum parsimony and are mid-point rooted. Numbers above branches represent branch length, and numbers below branches represent bootstrap values from 100 replicates. The intron tree is the single most parsimonious tree, 117 characters long, CI = 0.78, and RI = 0.86. The ITS tree is one of three most parsimonious trees, 150 characters long,CI = 0.81, and RI = 0.87

 
The resulting alignment of the ITS sequences after excluding the ambiguously aligned regions was 544 characters, of which 62 were parsimony informative. A branch-and-bound search using sequences of the same isolates as above yielded three most parsimonious trees of 150 steps (fig. 3). Bootstrap analysis indicated strong support for Ar. pyxidatus (bootstrap = 99%) and moderate support for the "core" Lentinellus group (bootstrap = 73%). Unlike the intron phylogeny, Ar. austropiperatus grouped strongly with Ar. pyxidatus, and Au. villipes allied with the Lentinellus clade (bootstrap = 100%). However, the relationship among Au. villipes, L. cochleatus, and the core Lentinellus group was unresolved.

The intron and ITS phylogenies appeared topologically different (fig. 3). High incongruence metrics indicated that there was heterogeneity between the data sets with IMF = 0.17 and IM = 0.65. An analysis of the data sets using the partition homogeneity test described by Farris et al. (1994) revealed that the heterogeneity was substructured among taxa (table 3). When all groups were analyzed together, there was significant heterogeneity between the data sets of intron and ITS sequences, but when subsets of Artomyces, exclusively Ar. pyxidatus, Lentinellus with A. villipes, and exclusively Lentinellus were analyzed separately, the data sets were not significantly heterogeneous. However, significant heterogeneity was observed when the Lentinellus data sets were analyzed without L. cochleatus (P = 0.038).


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Table 3 Results of the Homogeneity Test (HTF) of Farris et al. (1994).

 
Templeton's (1983) "significantly least parsimonious" (SLPT) test suggests that the intron and ITS data sets are significantly incongruent (P < 0.001, table 4). The intron data set was not congruent with any of the three most parsimonious ITS phylogenies, and the ITS data set was not congruent with the most parsimonious intron phylogeny. In this particular case the results of the SLPT test, which may overestimate congruence, should be considered valid.


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Table 4 Results of the Significantly Least Parsimonious Test (SLPT) of Templeton (1983).

 

    Discussion
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Data from PCR assay and SSU DNA sequences confirmed Hibbett's (1996) observation of the presence of a group I intron in Lentinellus and Artomyces for the species that he surveyed. However, the group I intron was not found in all species of these two genera, and it was found in at least one Auriscalpium species. Additionally, in a few species, some isolates apparently contain the intron, and other isolates of the same species do not (see table in Supplementary Material online). There seems to be no underlying geographic pattern because intron-present and intron-absent isolates were derived from collections with little or no obvious geographic separation, and the intron was found in isolates from around the world. In L. ursinus, Hibbett (1996) did not detect the intron by PCR amplification with primers NS6 and SR1c, but he was able to determine its presence by using a combination of intron-specific primers. The apparent absence of the intron was attributed to a PCR bias where, in an isolate that exhibits inter-ribosomal repeat heterogeneity, intron-less regions amplified at a higher frequency than regions containing the intron (Hibbett 1996). In the present study, the intron was not found in any of our L. ursinus isolates when we used primers SR1c and NS6. This putative heterogeneous inter-ribosomal repeat pattern was observed in isolates of some species of the three genera where two distinct fragments in the primary PCR products amplified with SR1c and NS6. Often, one or more of the fragments would appear less concentrated (fig. 1), possibly reflecting frequency differences of intron-present and intron-absent ribosomal repeats, or simply indicating that some regions amplify better than others because of primer annealing properties.

An additional secondary insertion was discovered within the group I intron between conserved regions R and S. Such secondary insertions have been found primarily in the North American populations of Ar. pyxidatus but have also been found in a few isolates of Ar. adrienneae, Ar. cristatus, and Ar. Taxon "B." With the exception of the Ar. cristatus isolate (GB1701) of a collection from Sweden, the occurrence of this insertion has only been found in the New World. Considerable length heterogeneities of PCR fragments amplified with the SSU primers were observed both among and within individual isolates (fig. 1). A comparison of sequences of gel-isolated fragments indicates they are due to significant and variable degradation in the middle of the secondary insert (fig. 2). Such variability within single isolates further suggests heterogeneous inter-ribosomal repeats. Based on putative secondary RNA-like structure and ORFs, this insertion may either be another group I intron or some other type of transposable element. They appeared to have no sequence similarity with anything in the GenBank database, and they may represent a new element. Literature searches have not yielded any reports of similar secondary insertions. DePriest and Been (1992) and DePriest (1993) noted significant size variations of the small subunit nuclear ribosomal DNA gene in the lichen-forming fungus Cladonia chlorophaea. Sequence and restriction site analysis revealed that the cause of the variation was due to several different group I introns inserted at five different sites (DePriest and Been 1992, DePriest 1993), but little variation in the sizes of the introns was observed.

Phylogenetic estimations based on intron sequences indicate that strong phylogenetic signal exists within Artomyces and Lentinellus (fig. 3), and the close similarity of intron sequences within the Auriscalpiaceae may indicate that the intron had been acquired before these three genera diverged. When the intron phylogeny was compared to phylogenies based on ITS sequences, however, significant heterogeneity existed between the two data sets (tables 3 and 4). It was evident that some of the difference between data sets was due in part to the discordance within the Lentinellus group (table 3). Within Lentinellus, the intron was present in four closely related species with little ITS sequence divergence. Many factors could be responsible for the observed incongruence between the data sets, including lineage sorting and variable rates of evolution between the ITS regions and introns. There was also conflict in the placement of the Ar. austropiperatus intron within the Lentinellus clade (fig. 3).

Alternatively, episodes of horizontal transmission could be responsible for the conflicting phylogenies. Some group I introns have been shown to be self-excising elements, often exhibiting protein-coding sequences which are thought to be mobile and perhaps transmissible (Cech 1988, Turmel et al. 1995). Nishida and Sugiyama (1995), Hibbett (1996), Holst-Jensen et al. (1999) have presented potential examples of horizontal transmission by group I introns, but the mechanism of transmission from one host to another or possible vectors remain unknown. It might be assumed that for transmission to occur, an organism infected with the intron must be in close proximity to an uninfected one. With limited fossil records, ancient distributions of fungi remain largely unknown. However, present-day distributions of the taxa with the group I intron are wide-ranging; Ar. pyxidatus (1541, China; GB1702, Sweden; 1513, Wisconsin; 56667, Georgia; H7524, Arizona), Ar. austropiperatus (8335, Argentina), Ar. candelabrus (2637, New Zealand), Ar. tasmaniensis (3905, Tasmania), Ar. turgidus (New Guinea), L. castoreus (8685, Louisiana; 4101, North Carolina), L. "subaustralis" (9159, Tennessee), L. micheneri (6701, Alaska), L. montanus (OKM, Montana), L. flabelliformis (9981, Austria), L. cochleatus (9985, Austria), and Au. villipes (4396, Mexico), for example. Because of the long geographic distances separating these taxa and the presence of strong phylogenetic signal in the intron sequences, it appears unlikely that there was recent horizontal transmission of the intron among these taxa.

Based on his higher taxonomic level phylogenetic estimation using SSU ribosomal DNA sequences, Hibbett (1996) hypothesized either that the Auriscalpiaceae clade gained the intron in a single event with a subsequent loss in Auriscalpium or that it was gained once in each of the Lentinellus and Clavicorona (Artomyces) clades. The expanded data set of the present study shows that the intron is present in Auriscalpium, but that many Lentinellus and Artomyces species do not contain this intron. A phylogenetic estimation of Artomyces based on ITS sequences (modified from Lickey, Hughes, and Petersen 2003; fig. 4) illustrates the distribution of introns among the species. On this strict consensus tree, it is apparent that the presence of the intron is widely distributed across several branches. Neither many losses nor many gains would seem to provide the most parsimonious explanation. If the intron presence in the Auriscalpiaceae is due to a single transmission event, it is likely that there were repeated losses of the intron along several evolutionary branches.



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FIG. 4. Phylogenetic estimation of Artomyces based on nrDNA ITS sequences (modified from Lickey et al. 2003). This is a strict consensus tree with bootstrap values indicated on the major groups. Lentinellus "subaustralis" (9159), Auriscalpium villipes (4396), and Au. vulgare (4242) were used as the outgroup. Black lines indicate presence of the group I intron

 
Several mechanisms have been proposed which could result in the loss of an intron, and in some cases, losing an intron may be easier than gaining one, especially if there is a mutation in the homing recognition sequence (Dujon 1989, Lambowitz and Belfort 1993). Alternatively, the introns may never really be lost. They instead may be present, lying latent in some other location in the genome. Potential harboring places for an intron would include the mitochondria where it may be transposed into the nuclear genome (Turmel et al. 1995). However, there was no evidence, when intron-specific primers were used, that this intron exists in some other location (this study; Hibbett 1996).

Group I introns have been identified in the ribosomal genes of a number of fungi, but their origin is unknown. The existence of genetically similar intron sequences in several members of the Auriscalpiaceae may provide evidence that their origin is due to a single infection event that occurred before the divergence of current genera and species, with consequent losses of the intron along several lineages. However, intron similarity could also be due to repeated horizontal events from a common source or even a combination of horizontal and vertical transmission. The presence of a second insertional element in North and Central American Ar. pyxidatus isolates suggests that these elements were present before the radiation of Ar. pyxidatus in the New World and lends credence to a hypothesis of a single infection followed by radiation.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Many thanks to Laura McGhee and Jennifer Mace for their help in the laboratory. We are indebted to David Hibbett for sharing his primers and advice. We also thank D. Hibbett, T. Eickbush, and an anonymous reviewer for their very helpful reviews, which greatly improved this manuscript. This research was supported by a Partnership for Enhancing Expertise in Taxonomy grant from the National Science Foundation DEB 95–21526 to R.H.P.


    Footnotes
 
Thomas Eickbush, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication June 19, 2003.





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