Department of Botany, University of Tennessee
Correspondence: E-mail: elickey{at}utk.edu.
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Abstract |
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Key Words: Artomyces Auriscalpiaceae Auriscalpium Clavicorona group I Intron Lentinellus nr ITS nr small subunit phylogenetic incongruence
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Introduction |
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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 ( 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.
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Methods |
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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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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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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).
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Results |
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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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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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Discussion |
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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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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.
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Acknowledgements |
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Footnotes |
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