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Three nonorthologous ITS1 types are present in a polypore fungus Trichaptum abietinum
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 23 (2002) 112–122 www.academicpress.com
Three nonorthologous ITS1 types are present in a polypore fungus Trichaptum abietinum Kwan Soo Ko1 and Hack Sung Jung* School of Biological Sciences, Seoul National University, San 56-1 Shinrim-dong, Kwanag-gu, Seoul 151-742, Republic of Korea Received 25 May 2000; received in revised form 24 August 2001
Abstract To explore phylogenetic relationships of Trichaptum species, internal transcribed spacer (ITS) regions of nuclear ribosomal DNAs were sequenced and analyzed. Gene trees from ITS1 and ITS2 sequences showed striking discrepancy in relationships of eight T. abietinum strains. All strains of T. abietinum had a single orthologous ITS2 type, but there were three paralogous types in the ITS1 region, which were designated Types I, II, and III. PCR amplification tests using type-specific primers showed that Types I and II are present in all strains of T. abietinum. The results suggest that gene duplication of the ancestral ITS1 region might have occurred prior to evolutionary radiation of Trichaptum and both types have been maintained in Trichaptum. However, Type III was amplified only in three T. abietinum strains collected from Korea, indicating that a new local geographic subtype has arisen in Korean strains. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nonorthologous ITS1; Trichaptum abietinum; Concerted evolution
1. Introduction Concerted evolution describes the evolutionary behavior of multigene families, such as ribosomal RNA genes (rDNAs), in which gene copies show a great deal of similarity to each other within an array and within a species but accumulate differences between species (Dover, 1982; Hillis et al., 1991). The two mechanisms most frequently quoted as responsible for concerted evolution are gene conversion (Hillis et al., 1991; Lassner and Dvorak, 1986; Nagylaki, 1984a,b) and unequal crossing-over (Arnheim, 1983; Coen and Dover, 1983; Petes, 1980; Szostak and Wu, 1980). Gene conversion assumes selection or drive for the homogenization of tandem repeats. Unequal crossing-over assumes recombination among tandem repeats either within or between chromosomes, resulting in the stochastic elim-
*
Corresponding author. Fax: +82-2-888-4911. E-mail addresses: [email protected] (Kwan S. Ko), [email protected] (Hack S. Jung). 1 Present address: Department of Microbiology and Cancer Research Center, Seoul National University College of Medicine, Seoul, Korea.
ination of variation in individuals and populations (Odorico and Miller, 1997; Vogler and DeSalle, 1994). Concerted evolution facilitates phylogeny reconstruction in almost completely homogenized multigene families such as nuclear ribosomal loci. For that reason, rDNA is broadly used in phylogenetic studies. The presence of many copies of nuclear rDNAs per genome and their homogenization through concerted evolution greatly reduce intraspecific variation compared to what one can expect based on data of interspecific variation (Carranza et al., 1996). Thus, the rDNA cluster has been assumed to be useful in taxonomic and phylogenetic studies and, in some cases, species delimitation has been made based on sequence variation of the ITS regions (Harlton et al., 1995; Ko et al., 1997; Sreenivasaprasad et al., 1992). However, several examples of nonconcerted evolution have been reported in diverse taxa. Extensive polymorphism within species is not unusual in ITS sequences of angiosperms (Baldwin et al., 1995) and other eukaryotes (Vogler and DeSalle, 1994; Wesson et al., 1992). When the location of nuclear rDNA loci is on nonhomologous chromosomes, concerted evolution is potentially disrupted (Jellen et al., 1994; Suh et al.,
1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 0 0 9 - X
Kwan S. Ko, Hack S. Jung / Molecular Phylogenetics and Evolution 23 (2002) 112–122
1993). When mutation rate exceeds the rate of concerted evolution as in length variants in the multigenic spacer, when pseudogenes evolve (Buckler and Holtsford, 1996), or when interspecific hybridization occurs, nonconcerted evolution also occurs (Odorico and Miller, 1997). In fungi, a nonconcerted pattern of ITS sequences has been reported in Fusarium (O’Donnell and Cigelnik, 1997; O’Donnell et al., 1998; Waalwijk et al., 1996), Scutellospora (Hijri et al., 1999; Hosny et al., 1999), Gigaspora (Zeze et al., 1997), and Ascochyta (Fatehi and Bridge, 1998). Trichaptum is a cosmopolitan wood-rotting genus characterized by a purplish to violet pore surface in actively growing fruitbodies, diagnostic cystidia in the hymenium, and cylindrical spores (Gilbertson and Ryvarden, 1987; Ryvarden and Gilbertson, 1994). It has been classified in the Polyporaceae (Aphyllophorales) and the Hymenomycetes of the Basidiomycota. However, according to recent phylogenetic analyses using molecular sequences, it has been found that Trichaptum is closely related to the members of the Hymenochaetaceae, including Phellinus, Phylloporia, and Inonotus (Hibbett and Donoghue, 1995; Hibbett, 1996; Ko et al., 1997), and ultrastructurally has imperforate parenthosomes in a dolipore apparatus (Langer, 1994; Moore, 1985; Traquair and McKeen, 1978). Species classified in Trichaptum are much alike in general appearance, similar in microscopic characters, and, depending on au-
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thors, problematical as separate species (Gilbertson and Ryvarden, 1987; Ryvarden and Gilbertson, 1994). However, through incompatibility tests and molecular sequences, it was concluded that they are all distinct species (Macrae, 1967; Magasi, 1976; Ko et al., 1997). Trichaptum abietinum, the type species of Trichaptum, is one of the most common polypores with a poroid hymenophore. Trichaptum biforme is similar to T. abietinum in many respects. However, it is found exclusively on hardwoods, while T. abietinum grows mostly on conifers. In addition, basidiocarps of T. biforme tend to be much wider than those of T. abietinum. Trichaptum subchartaceum and T. biforme are very much alike, but T. subchartaceum is restricted to Populus (Gilbertson and Ryvarden, 1987). Trichaptum fusco-violaceum and T. abietinum are similar in having a gelatinous layer between the context and the tubes (Ryvarden and Gilbertson, 1994), but the former is morphologically separated from the latter by its hydnoid hymenophore. Trichaptum laricinum is characterized by a lamellate hymenophore and allantoid spores but is like T. abietinum in other respects. In the present study of nuclear rDNA repeats of T. abietinum, we observed three divergent ITS1 types but only one sequence type for the ITS2 region. As such, the ITS1 gene tree of Trichaptum species including T. abietinum showed a pattern of gene evolution different from that of the ITS2 gene tree and mitochondrial small
Table 1 Trichaptum strains used in this study Species name
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
abietinum abietinum abietinum abietinum abietinum abietinum abietinum abietinum biforme biforme biforme fusco-violaceum fusco-violaceum laricinum laricinum subchartaceum subchartaceum
Strain number
a
CBS 374.68 CCFCb 008387 CBS 375.68 CCFC009931 CCFC010156 SFCc 950815-17 SFC 960608-11 SFC 961028-11 FPd -86522 HHBe -7316 CBS 324.29 FP-133997 HHB-4016 RLGf -4665 RLG-6936 CCFC003928 CCFC003932
Locality
Quebec, Canada Quebec, Canada Ontario, Canada Alberta, Canada Quebec, Canada Ullung Island, Korea Myungsung Mt., Korea Chiak Mt., Korea Maryland, USA Wisconsin, USA Canada Montana, USA Tennessee Montana, USA New York, USA Saskatchewan, Canada Michigan, USA
ITS1 Type
Type Type Type Type Type Type Type Type – – – – – – – – –
I I II II II III III III
GenBank accession ITS1
ITS2
mt-SSU
AF266676 AF266677 AF267648 AF267646 AF267647 AF266680 AF267644 AF267645 U63476 U63473 AF267649 U63472 U63478 U63471 U63477 AF267650 AF266678
AF267651 AF267652 AF266681 AF267653 AF267654 AF267655 AF267656 AF267657
AF408707 AF408708 AF408716 AF036630 AF036629 AF408705 AF036631 AF408706 AF408709 AF036634 AF036635 AF408710 AF408711 – AF408712 AF408713 AF408714
AF267658
AF267659 AF266679
Note. Thirty-three accession numbers typed in boldface are those for the strains sequenced for this study. Accession numbers of U63470s are from Ko et al. (1997) and those of AF03629 and AF036630s are from Ko and Jung (1999). a Centraalbureau voor Schimmelcultures. b Canadian Collection of Fungal Cultures. c Seoul National University Fungus Collection. d USDA Forest Products Laboratory. e H.H. Burdsall, Jr. f R.L. Gilbertson.
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subunit (mt-SSU) rDNA gene trees. From comparison of gene trees and sequence analyses from two ITSs and mt-SSU rDNA, the ITS1 region of T. abietinum was identified as possessing three nonorthologous types. This phenomenon indicates that the ITS1 region of T. abietinum might have escaped from concerted evolution. In addition, it was discovered that a unique ITS1 type was introduced into a certain geographic region as a new subtype and caused an ITS1 polymorphism.
50 ll and the reaction was performed for 30–35 cycles. Each cycle consisted of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, with a final extension step at 72 °C for 10 min. PCR products were purified using Wizard PCR preps (Promega) and directly cycle-sequenced with 35 S-labeled ATP (Hillis et al., 1996), using primers ITS5, ITS2, ITS3, and ITS4 for the ITS region and MS1 and MS2 for the mt-SSU rDNA region (White et al., 1990). 2.3. Phylogenetic analyses
2. Materials and methods 2.1. DNA extraction Fungal strains used in this study are listed in Table 1 along with information of sequence accession numbers and localities. Genomic DNAs were extracted from mycelial cultures or field-collected specimens using the freezing–thawing method of Lecellier and Silar (1994) with some modifications (Ko and Jung, 1997). Cultures were maintained on malt extract agar (malt extract 2%, peptone 0.5%, agar 1.5%) covered with a cellophane disk at 24 °C in complete darkness and cultured mycelium was recovered into an Eppendorf tube using a spatula. For field-collected specimens, dried pieces of fungal specimens from the Seoul National University Fungus Collection were used directly for DNA extraction. A total of 600 ll extraction buffer (50 mM Tris–HCl, 50 mM EDTA, 3% SDS) was added to each sample. The sample was frozen for 1 min in liquid nitrogen and thawed for 1 min in a 75 °C incubator. This process was repeated three to five times until the cells were thoroughly broken. Extracted DNA was purified with a phenol–chloroform extraction and precipitated with 1 volume of isopropanol. The pellet was resuspended in 50 ll of sterile double-distilled water and used as a template for PCR amplification. 2.2. PCR amplification and DNA sequencing The nuclear ITS and partial mt-SSU rDNA regions were amplified using primer pairs ITS5/ITS4 and MS1/ MS2 (White et al., 1990), respectively. To detect other types of the ITS1 region in a strain, three type-specific primers, ITS-Ta1, ITS-Ta2, and ITS-Ta3, were designed and tested individually (Fig. 1) after O’Donnell and Cigelnik (1997). PCR amplifications were performed in a reaction mixture containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2 , 0.1% bovine serum albumin, 0.2 mM of each dATP, dCTP, dGTP, and dTTP, 1 ll of each primer (25 lmol), and 200 ng of template DNA, with 2 Units of Taq DNA polymerase using PTC-100 Programmable Thermal Cycler (MJ Research). Total volume was adjusted to
Sequences of the ITS1 region showed extensive dissimilarity and were manually aligned for maximum optimization, while those of the ITS2 region and the partial mt-SSU rDNA were aligned using the multiple alignment program CLUSTAL X (Thompson et al., 1997) with some manual adjustment. Phylogenetic relationships were estimated from aligned sequences of data sets using PAUP 4.0 (Swofford, 1998) and treating all alignment gaps as missing data. The neighbor-joining option (Saitou and Nei, 1987) of distance analysis and the branch-and-bound option of parsimony analysis were applied for phylogenetic analyses. For the ITS1 region, unambiguously aligned sequences from 1 to 50 and from 343 to 578 in Fig. 1 were used for the reconstruction of phylogenetic trees. Support for phylogenetic groupings was assessed by bootstrap analyses (Hillis and Bull, 1993) of 1000 replications with random addition input order of sequences in the option of the parsimony analysis.
3. Results There were few base substitutions or indels (insertion/ deletion) among sequences of the ITS2 and the partial mt-SSU rDNA regions of T. abietinum. However, extensive sequence variation existed in ITS1 sequences of T. abietinum as shown in the alignment of the ITS1 region of Trichaptum species (Fig. 1). Parsimony trees were inferred from the sequences of ITS1 (Fig. 2A), ITS2 (Fig. 2B), and mt-SSU (Fig. 2C) rDNA. Trees by the neighbor-joining method were very similar to those by the parsimony method. Phylogenetic relationships inferred from the ITS2 and the mt-SSU rDNA sequences showed that eight T. abietinum strains were grouped in a single clade, supported by bootstrap values of 65% (Fig. 2B) and 96% (Fig. 2C). Most of the bootstrap values supporting subgroups within the T. abietinum clade were very low and their resolution was also very weak. In both ITS2 and mt-SSU rDNA trees, the two T. fusco-violaceum isolates formed a monophyletic group as a sister group to the T. abietinum clade (Figs. 2B and C), which is congruent with the previous result (Ko and Jung, 1999). T. biforme and T. subchartaceum were closely related to each other, which was
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Fig. 1. Alignment of ITS1 sequences of Trichaptum abietinum (CBS 374.68 to CBS 375.68), T. fusco-violaceum (FP-133997 and HHB-4016), T. biforme (FP-86522 to CBS 324.29), T. subchartaceum (CCFC003928 and CCFC003932), and T. laricinum (RLG-4665 and RLG-6936). Underlined nucleotides indicate regions used in designing type-specific primers, ITS-Ta1 (50 -TAAATAAGAGATGGTGGCTAG-30 ), ITS-Ta2 (50 -TACCAAAAGCATGTGCTCAG-30 ), and ITS-Ta3 (50 -TAGTAGTACGGCCCAGGTGTG-30 ).
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Fig. 2. Phylogenetic trees from ITS1 and ITS2 sequences of Trichaptum, inferred by the branch-and-bound option of parsimony analysis in PAUP 4.0 (Swofford, 1998). Sequences of Trichaptum laricinum were used as an outgorup to root trees because T. laricinum was always a good basal taxon to the grouping of Trichaptum in analyses of rDNA sequences along with those of Phellinus (data not shown). Bootstrapping values from 1000 replications are shown at corresponding branches. (A) One of 24 most parsimonious trees inferred from ITS1 sequences (tree length ¼ 92 steps, CI ¼ 0.913, RI ¼ 0.955, RC ¼ 0.872). Groups designated as ITS1 types are boxed. (B) One of 3 most parsimonious trees inferred from ITS2 sequences (tree length ¼ 127 steps, CI ¼ 0.913, RI ¼ 0.952, RC ¼ 0.870). The T. abietinum clade is boxed. (C) One of 2 most parsimonious trees inferred from partial mt-SSU rDNA sequences (tree length ¼ 245 steps, CI ¼ 0.971, RI ¼ 0.956, RC ¼ 0.929). The T. abietinum clade is boxed.
fully or strongly supported by bootstrap analysis (100% and 99% in Figs. 2B and C, respectively). T. laricinum was selected as an outgroup in this study, because it was always placed at a basal position for the grouping of Trichaptum in a preliminary analysis of ITS and mtSSU rDNA sequences along with those of Phellinus species (data not shown) and in a study employing 18S rDNA sequences (Ko et al., 1997). However, in the gene tree inferred from ITS1 sequences (Fig. 2A), eight T. abietinum strains were separated into three distinct groups designated Types. T. abietinum strains CBS 374.68 and CCFC008387 (Type I) were closely related to two T. fusco-violaceum strains. Three T. abietinum strains, SFC 960608-11, SFC 961028-11, and SFC 950815-17 collected in Korea,
formed a distinct clade (Type III). The Type III strains formed a larger group consisting of Type I strains, two strains of T. fusco-violaceum, and Type II strains, which was supported by 100% bootstrap value. The remaining three T. abietinum strains, CCFC009931, CCFC010156, and CBS 375.68, formed another distinct group (Type II). In the phylogenetic tree of ITS1 sequences, T. biforme and T. subchartaceum strains clustered together, which received complete bootstrap support (100%). Comparison of lengths (Table 2) and the alignment of ITS1 sequences (Fig. 1) identified the presence of three distinct ITS1 sequences within T. abietinum. ITS1 sequences of Type I and Type II were 444 and 249–251 bp in length, respectively. Type III ITS1s were 490–491 bp in length. Sequence sites from 51st to 342nd position of
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B
Fig. 2. (continued).
Type I and Type III (Fig. 1) were not properly aligned with any other sequences. The three different ITS1 types of T. abietinum were unambiguously aligned before the 51st nucleotide and after the 342nd nucleotide in the sequence position. However, Type I sequences were aligned quite well with those of two T. fusco-violaceum. In addition to base substitutions, insertions or deletions apparently distinguished three ITS1 types of T. abietinum (Fig. 1). To investigate these divergent ITS1 sequences among three groups of T. abietinum, Type I-, Type II-, and Type III-specific PCR primers were designed (Fig. 1) and used to test whether each strain possessed all three ITS1 type sequences (Fig. 3). Primer pairs ITS-Ta1/ITS4 and ITS-Ta2/ITS4 successfully amplified Type I and Type II sequences from all T. abietinum strains. However, the amplification using the Type III-specific primer ITS-Ta3 and ITS4 was successful only in the three Korean T. abietinum strains (Fig. 3). These results suggest that Type I and Type II ITS1 sequences coexist in
all tested T. abietinum strains and that Type III ITS1 sequences are present only in T. abietinum strains collected from Korea.
4. Discussion A major result of this study is an observed discrepancy among gene trees from nuclear rDNA ITS1, ITS2, and mt-SSU rDNA sequences and a high degree of divergence of ITS1 sequences among T. abietinum strains. The ITS1 gene tree consisted of three clades of T. abietinum due to nonorthologous sequences. T. abietinum strains may possess one of three ITS1 sequence types, but paralogs were not detected within the ITS2 and the mt-SSU rDNA gene trees. From the gene trees of ITS2 sequences (Fig. 2B) and partial mt-SSU rDNA sequences (Fig. 2C) and morphological evidence (Gilbertson and Ryvarden, 1987; Ryvarden and Gilbertson, 1994), T. abietinum has a
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C
Fig. 2. (continued).
Table 2 Nucleotide composition and lengths of the ITS1 sequences Species
A
T
G
C
Length (bp)
T. abietinum (8) Type I (2) Type II (3) Type III (3) T. fusco-violaceum (2) T. biforme (3) T. subchartaceum (2) T. laricinum (2)
27.9–28.3b 25.1–26.1 23.9–24.6 28.4–28.5 23.1–23.5 24.4–24.8 23.7–24.0
29.5–29.7 30.6–31.1 26.9–27.5 29.3–29.7 31.1–31.3 30.3 32.8–34.3
23.2–23.6 22.1–24.0 22.6–22.9 21.9–23.1 24.1–24.2 23.6–24.4. 20.7–21.0
18.7–18.9 20.0–20.7 20.6–21.0 17.4–17.9 21.2–21.6 20.9–21.2 20.6–21.1
444 249–251 490–491 441–443 264–265 254 232–233
Mean
25.5
29.7
23
20.1
a
a
Numbers of studied strains. b Base percentages calculated by PAUP* 4.0 (Swofford, 1998).
closer relationship with T. fusco-violaceum than with any other species. In the gene tree from ITS1 sequences (Fig. 2A), however, only two strains of T. abietinum indicated as Type I, CBS 374.68 and CCFC008387, formed a clade with two strains of T. fusco-violaceum. Strains of
both T. abietinum Type I and T. fusco-violaceum have similar ITS1 lengths (Table 2) and sequence divergence between them was very low in the ITS1 region as shown in Fig. 1. Their relationship was congruent with the gene trees from ITS2 and mt-SSU rDNA sequences and with
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Fig. 3. Examples of PCR-amplified ITS products generated by type- specific primers. The materials are T. abietinum CCFC008387, T. abietinum CBS 375.68, and T. abietinum SFC 950815-17. I lanes represent PCR products generated by a primer pair of ITS-Ta1/ITS4, II lanes by the one of ITS-Ta2/ ITS4, and III lanes by the one of ITS-Ta3/ITS4. M lanes indicate 1-kb DNA ladders.
the results based on morphological evidence. Thus, it seems that Type I sequences of the ITS1 region reflect the species tree of T. abietinum. Compared to Type I and Type III sequences, Type II sequences that are shorter in length might have been generated by deletion of nucleotides from the 51st to the 342nd sites (Figs. 1 and 3 and Table 2). PCR amplifications using the type-specific primers suggests that both Type I and Type II coexist in individual strains of T. abietinum (Fig. 3). The three Korean strains of T. abietinum possessed an additional ITS1 sequences designated Type III. Three distinct regions were identified within Type III sequences. The first are conserved regions present in all Trichaptum species. The 50 and 30 ends of Type III sequences correspond to this category. The second are regions unambiguously aligned only with sequences of Type I and T. fusco-violaceum. The third are regions unique to Type III sequences. These unique regions consisted of both inserted and unaligned regions. The Type III-specific primer, ITS-Ta3, was designed based upon sequences of the first inserted region (Fig. 1). Results of the PCR assay (Fig. 3) suggest that Type III sequences might exist only in the Korean strains used in the study. Type III sequences of T. abietinum are possibly due to polymorphism endemic to a certain geographic region and could be a subtype of Type I, suggesting that new ITS types can be locally formed and may carry a significance in population genetics. This subtype polymorphism could be placed under homogenization dynamics other than those of Type I and Type II sequences (Vogler and DeSalle, 1994). Ribosomal DNA multigene families are best known for the high degree of intraspecific homogeneity resulting from concerted evolution. For this reason, ribosomal DNA has been used broadly in inferring
phylogenetic relationships. Recently, however, several cases where rDNA sequences have escaped concerted evolution have been stated in various groups such as angiosperms, mosquitoes, tiger beetles, and fungi (Baldwin et al., 1995; Vogler and DeSalle, 1994; Wesson et al., 1992). In fungi, a few examples of nonorthologous ITS sequences have been discovered in ascomycetes (Fatehi and Bridge, 1998; O’Donnell and Cigelnik, 1997; O’Donnell et al., 1998; Waalwijk et al., 1996) and zygomycetes (Hijri et al., 1999; Hosny et al., 1999). Results of the present study indicate that nonorthologous evolution of rDNA sequences also occurs in basidiomycetes. There have been several explanations for intraspecific rDNA polymorphism. First, polymorphic sequences may represent pseudogenes (Buckler et al., 1997). In the processes of divergence of Trichaptum, some types might have lost their ITS functions and possibly became pseudogenes. Pseudogenes have base compositions different from those of functional genes and evolve very rapidly, showing a high AT ratio (Buckler et al., 1997). However, nucleotide compositions among three types did not show significant differences (Table 2). AT ratios of Type II sequences (0.557–0.566) and Type III sequences (0.512–0.519) are lower than those of Type I sequences (0.574–0.581) (Table 2). Conserved sequences of 30 and 50 ends and unambiguously aligned regions also suggest their nonpseudogene state, since pseudogenes usually show random base substitutions (Wang et al., 1997). In addition, the result that they were kept internally homogeneous within the groups of given types suggests that they are not pseudogenes. Second, it can be hypothesized that xenologous ITS1 regions of T. abietinum have been originated from the hybrid of two different species or populations that are in
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the process of speciation (Hugall et al., 1999; Odorico and Miller, 1997). However, none of three types of T. abietinum showed intermediate characteristics of two species, for example, T. abietinum and T. biforme. In addition, Type III sequences seem to be a local subtype of Type I sequences, since they share many unambiguously aligned regions in comparison with those of Type I in T. abietinum and T. fusco-violaceum and the former is restricted to Korea. Thus, it is implausible that type sequences were always derived from hybridization and evolved independently in their original ITS1 regions. Third, rDNA polymorphism within individuals may occur when the pace of concerted evolution is not fast enough to homogenize tandem repeats (Campbell et al., 1997). Polymorphism within the intergenic spacer of the nuclear rDNA of Fusarium oxysporum can be explained by a much slower rate of concerted evolution within a predominately clonal species (Appel and Gordon, 1996). This suggestion is more applicable to incomplete homogenization of rDNA repeats in more complex eukaryotes with longer generation times and larger rDNA loci (Copenhaver and Pikaard, 1996; O’Donnell et al., 1998). However, T. abietinum is an annual mushroom that has a short generation time (Ryvarden and Gilbertson, 1993). Fourth, three types of the ITS1 region of T. abietinum could be located at different chromosomal loci. Location of rDNA loci on nonhomologous chromosomes potentially disrupts concerted evolution (Campbell et al., 1997; Vogler and DeSalle, 1994). Such dispersed location of rDNA repeats on several chromosomes has been reported in Fusarium (Boehm et al., 1994; Fekete et al., 1993), Scilla (Vaughan et al., 1993), and Drosophila (Schl€ otterer and Tautz, 1994). The relatively low heterogeneity within the types of T. abietinum may support this hypothesis. However, this does not seem to readily answer the question why the diversity of ITS2 sequences is extremely low compared to that of ITS1 sequences (Hugall et al., 1999). It is also possible for several types of rDNAs to be located at different loci of the same chromosome (Carranza et al., 1999). Gene duplication might have occurred prior to the divergence of the polypore species T. abietinum, T. fusco-violaceum, T. biforme, and T. subchartaceum. Products of gene duplication, Type I and Type II ITS1 sequences, have been maintained in the same individual of T. abietinum without the process of homogenization or selection. Introduced types might still contain their biological functions (Carranza et al., 1999; Wang et al., 1997). Other additional ITS1 types might have been preserved since they could provide a buffer against deleterious mutations (O’Donnell et al., 1998). For fungi such as Scutellospora castanea, the high levels of nucleotide divergence may be due to impaired genetic exchange among nuclei carrying different ITS
types. Spores of S. castanea are heterokaryotic (Hijri et al., 1999) and may harbor genetically different nuclei and show a nonorthologous ITS pattern (Hosny et al., 1999). Also for strains of T. abietinum, two or three paralogous ITS1 types could be present in heterokaryotic spores. Recently, it was reported that there are more than one genetically different IGS1 regions in dikaryotic individuals of Schizophyllum commune (Basidiomycetes), but not in monokaryon (James et al., 2001). Ribosomal DNA ITS sequences have been most frequently employed in inference of species trees. Direct sequencing of rDNA PCR products provides a rapid and useful tool in phylogenetic studies. However, systemic differences in amplification probabilities between paralogs can result in biased sampling and a false inference of species relationships (Waalwijk et al., 1996). Discrepancy of gene phylogenies inferred from ITS1, ITS2, and mt-SSU rDNA sequences due to nonorthologous sequences shows that caution is needed when inferring species trees only from ITS gene trees. Results of the present study emphasize the importance of survey for gene–gene concordance and thorough in-depth sampling for stable and robust phylogenetic conclusions.
Acknowledgments We sincerely thank Dr. Soon Gyu Hong (Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology) for his helpful advice and technical comments in designing Type-specific primers. K.S. Ko was supported by the BK21 Research Fellowship from the Ministry of Education and Human Resources Development until he graduated from the School of Biological Sciences, Seoul National University.
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Three nonorthologous ITS1 types are present in a polypore fungus Trichaptum abietinum Kwan Soo Ko1 and Hack Sung Jung* School of Biological Sciences, Seoul National University, San 56-1 Shinrim-dong, Kwanag-gu, Seoul 151-742, Republic of Korea Received 25 May 2000; received in revised form 24 August 2001
Abstract To explore phylogenetic relationships of Trichaptum species, internal transcribed spacer (ITS) regions of nuclear ribosomal DNAs were sequenced and analyzed. Gene trees from ITS1 and ITS2 sequences showed striking discrepancy in relationships of eight T. abietinum strains. All strains of T. abietinum had a single orthologous ITS2 type, but there were three paralogous types in the ITS1 region, which were designated Types I, II, and III. PCR amplification tests using type-specific primers showed that Types I and II are present in all strains of T. abietinum. The results suggest that gene duplication of the ancestral ITS1 region might have occurred prior to evolutionary radiation of Trichaptum and both types have been maintained in Trichaptum. However, Type III was amplified only in three T. abietinum strains collected from Korea, indicating that a new local geographic subtype has arisen in Korean strains. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nonorthologous ITS1; Trichaptum abietinum; Concerted evolution
1. Introduction Concerted evolution describes the evolutionary behavior of multigene families, such as ribosomal RNA genes (rDNAs), in which gene copies show a great deal of similarity to each other within an array and within a species but accumulate differences between species (Dover, 1982; Hillis et al., 1991). The two mechanisms most frequently quoted as responsible for concerted evolution are gene conversion (Hillis et al., 1991; Lassner and Dvorak, 1986; Nagylaki, 1984a,b) and unequal crossing-over (Arnheim, 1983; Coen and Dover, 1983; Petes, 1980; Szostak and Wu, 1980). Gene conversion assumes selection or drive for the homogenization of tandem repeats. Unequal crossing-over assumes recombination among tandem repeats either within or between chromosomes, resulting in the stochastic elim-
*
Corresponding author. Fax: +82-2-888-4911. E-mail addresses: [email protected] (Kwan S. Ko), [email protected] (Hack S. Jung). 1 Present address: Department of Microbiology and Cancer Research Center, Seoul National University College of Medicine, Seoul, Korea.
ination of variation in individuals and populations (Odorico and Miller, 1997; Vogler and DeSalle, 1994). Concerted evolution facilitates phylogeny reconstruction in almost completely homogenized multigene families such as nuclear ribosomal loci. For that reason, rDNA is broadly used in phylogenetic studies. The presence of many copies of nuclear rDNAs per genome and their homogenization through concerted evolution greatly reduce intraspecific variation compared to what one can expect based on data of interspecific variation (Carranza et al., 1996). Thus, the rDNA cluster has been assumed to be useful in taxonomic and phylogenetic studies and, in some cases, species delimitation has been made based on sequence variation of the ITS regions (Harlton et al., 1995; Ko et al., 1997; Sreenivasaprasad et al., 1992). However, several examples of nonconcerted evolution have been reported in diverse taxa. Extensive polymorphism within species is not unusual in ITS sequences of angiosperms (Baldwin et al., 1995) and other eukaryotes (Vogler and DeSalle, 1994; Wesson et al., 1992). When the location of nuclear rDNA loci is on nonhomologous chromosomes, concerted evolution is potentially disrupted (Jellen et al., 1994; Suh et al.,
1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 0 0 9 - X
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1993). When mutation rate exceeds the rate of concerted evolution as in length variants in the multigenic spacer, when pseudogenes evolve (Buckler and Holtsford, 1996), or when interspecific hybridization occurs, nonconcerted evolution also occurs (Odorico and Miller, 1997). In fungi, a nonconcerted pattern of ITS sequences has been reported in Fusarium (O’Donnell and Cigelnik, 1997; O’Donnell et al., 1998; Waalwijk et al., 1996), Scutellospora (Hijri et al., 1999; Hosny et al., 1999), Gigaspora (Zeze et al., 1997), and Ascochyta (Fatehi and Bridge, 1998). Trichaptum is a cosmopolitan wood-rotting genus characterized by a purplish to violet pore surface in actively growing fruitbodies, diagnostic cystidia in the hymenium, and cylindrical spores (Gilbertson and Ryvarden, 1987; Ryvarden and Gilbertson, 1994). It has been classified in the Polyporaceae (Aphyllophorales) and the Hymenomycetes of the Basidiomycota. However, according to recent phylogenetic analyses using molecular sequences, it has been found that Trichaptum is closely related to the members of the Hymenochaetaceae, including Phellinus, Phylloporia, and Inonotus (Hibbett and Donoghue, 1995; Hibbett, 1996; Ko et al., 1997), and ultrastructurally has imperforate parenthosomes in a dolipore apparatus (Langer, 1994; Moore, 1985; Traquair and McKeen, 1978). Species classified in Trichaptum are much alike in general appearance, similar in microscopic characters, and, depending on au-
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thors, problematical as separate species (Gilbertson and Ryvarden, 1987; Ryvarden and Gilbertson, 1994). However, through incompatibility tests and molecular sequences, it was concluded that they are all distinct species (Macrae, 1967; Magasi, 1976; Ko et al., 1997). Trichaptum abietinum, the type species of Trichaptum, is one of the most common polypores with a poroid hymenophore. Trichaptum biforme is similar to T. abietinum in many respects. However, it is found exclusively on hardwoods, while T. abietinum grows mostly on conifers. In addition, basidiocarps of T. biforme tend to be much wider than those of T. abietinum. Trichaptum subchartaceum and T. biforme are very much alike, but T. subchartaceum is restricted to Populus (Gilbertson and Ryvarden, 1987). Trichaptum fusco-violaceum and T. abietinum are similar in having a gelatinous layer between the context and the tubes (Ryvarden and Gilbertson, 1994), but the former is morphologically separated from the latter by its hydnoid hymenophore. Trichaptum laricinum is characterized by a lamellate hymenophore and allantoid spores but is like T. abietinum in other respects. In the present study of nuclear rDNA repeats of T. abietinum, we observed three divergent ITS1 types but only one sequence type for the ITS2 region. As such, the ITS1 gene tree of Trichaptum species including T. abietinum showed a pattern of gene evolution different from that of the ITS2 gene tree and mitochondrial small
Table 1 Trichaptum strains used in this study Species name
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
abietinum abietinum abietinum abietinum abietinum abietinum abietinum abietinum biforme biforme biforme fusco-violaceum fusco-violaceum laricinum laricinum subchartaceum subchartaceum
Strain number
a
CBS 374.68 CCFCb 008387 CBS 375.68 CCFC009931 CCFC010156 SFCc 950815-17 SFC 960608-11 SFC 961028-11 FPd -86522 HHBe -7316 CBS 324.29 FP-133997 HHB-4016 RLGf -4665 RLG-6936 CCFC003928 CCFC003932
Locality
Quebec, Canada Quebec, Canada Ontario, Canada Alberta, Canada Quebec, Canada Ullung Island, Korea Myungsung Mt., Korea Chiak Mt., Korea Maryland, USA Wisconsin, USA Canada Montana, USA Tennessee Montana, USA New York, USA Saskatchewan, Canada Michigan, USA
ITS1 Type
Type Type Type Type Type Type Type Type – – – – – – – – –
I I II II II III III III
GenBank accession ITS1
ITS2
mt-SSU
AF266676 AF266677 AF267648 AF267646 AF267647 AF266680 AF267644 AF267645 U63476 U63473 AF267649 U63472 U63478 U63471 U63477 AF267650 AF266678
AF267651 AF267652 AF266681 AF267653 AF267654 AF267655 AF267656 AF267657
AF408707 AF408708 AF408716 AF036630 AF036629 AF408705 AF036631 AF408706 AF408709 AF036634 AF036635 AF408710 AF408711 – AF408712 AF408713 AF408714
AF267658
AF267659 AF266679
Note. Thirty-three accession numbers typed in boldface are those for the strains sequenced for this study. Accession numbers of U63470s are from Ko et al. (1997) and those of AF03629 and AF036630s are from Ko and Jung (1999). a Centraalbureau voor Schimmelcultures. b Canadian Collection of Fungal Cultures. c Seoul National University Fungus Collection. d USDA Forest Products Laboratory. e H.H. Burdsall, Jr. f R.L. Gilbertson.
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subunit (mt-SSU) rDNA gene trees. From comparison of gene trees and sequence analyses from two ITSs and mt-SSU rDNA, the ITS1 region of T. abietinum was identified as possessing three nonorthologous types. This phenomenon indicates that the ITS1 region of T. abietinum might have escaped from concerted evolution. In addition, it was discovered that a unique ITS1 type was introduced into a certain geographic region as a new subtype and caused an ITS1 polymorphism.
50 ll and the reaction was performed for 30–35 cycles. Each cycle consisted of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, with a final extension step at 72 °C for 10 min. PCR products were purified using Wizard PCR preps (Promega) and directly cycle-sequenced with 35 S-labeled ATP (Hillis et al., 1996), using primers ITS5, ITS2, ITS3, and ITS4 for the ITS region and MS1 and MS2 for the mt-SSU rDNA region (White et al., 1990). 2.3. Phylogenetic analyses
2. Materials and methods 2.1. DNA extraction Fungal strains used in this study are listed in Table 1 along with information of sequence accession numbers and localities. Genomic DNAs were extracted from mycelial cultures or field-collected specimens using the freezing–thawing method of Lecellier and Silar (1994) with some modifications (Ko and Jung, 1997). Cultures were maintained on malt extract agar (malt extract 2%, peptone 0.5%, agar 1.5%) covered with a cellophane disk at 24 °C in complete darkness and cultured mycelium was recovered into an Eppendorf tube using a spatula. For field-collected specimens, dried pieces of fungal specimens from the Seoul National University Fungus Collection were used directly for DNA extraction. A total of 600 ll extraction buffer (50 mM Tris–HCl, 50 mM EDTA, 3% SDS) was added to each sample. The sample was frozen for 1 min in liquid nitrogen and thawed for 1 min in a 75 °C incubator. This process was repeated three to five times until the cells were thoroughly broken. Extracted DNA was purified with a phenol–chloroform extraction and precipitated with 1 volume of isopropanol. The pellet was resuspended in 50 ll of sterile double-distilled water and used as a template for PCR amplification. 2.2. PCR amplification and DNA sequencing The nuclear ITS and partial mt-SSU rDNA regions were amplified using primer pairs ITS5/ITS4 and MS1/ MS2 (White et al., 1990), respectively. To detect other types of the ITS1 region in a strain, three type-specific primers, ITS-Ta1, ITS-Ta2, and ITS-Ta3, were designed and tested individually (Fig. 1) after O’Donnell and Cigelnik (1997). PCR amplifications were performed in a reaction mixture containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2 , 0.1% bovine serum albumin, 0.2 mM of each dATP, dCTP, dGTP, and dTTP, 1 ll of each primer (25 lmol), and 200 ng of template DNA, with 2 Units of Taq DNA polymerase using PTC-100 Programmable Thermal Cycler (MJ Research). Total volume was adjusted to
Sequences of the ITS1 region showed extensive dissimilarity and were manually aligned for maximum optimization, while those of the ITS2 region and the partial mt-SSU rDNA were aligned using the multiple alignment program CLUSTAL X (Thompson et al., 1997) with some manual adjustment. Phylogenetic relationships were estimated from aligned sequences of data sets using PAUP 4.0 (Swofford, 1998) and treating all alignment gaps as missing data. The neighbor-joining option (Saitou and Nei, 1987) of distance analysis and the branch-and-bound option of parsimony analysis were applied for phylogenetic analyses. For the ITS1 region, unambiguously aligned sequences from 1 to 50 and from 343 to 578 in Fig. 1 were used for the reconstruction of phylogenetic trees. Support for phylogenetic groupings was assessed by bootstrap analyses (Hillis and Bull, 1993) of 1000 replications with random addition input order of sequences in the option of the parsimony analysis.
3. Results There were few base substitutions or indels (insertion/ deletion) among sequences of the ITS2 and the partial mt-SSU rDNA regions of T. abietinum. However, extensive sequence variation existed in ITS1 sequences of T. abietinum as shown in the alignment of the ITS1 region of Trichaptum species (Fig. 1). Parsimony trees were inferred from the sequences of ITS1 (Fig. 2A), ITS2 (Fig. 2B), and mt-SSU (Fig. 2C) rDNA. Trees by the neighbor-joining method were very similar to those by the parsimony method. Phylogenetic relationships inferred from the ITS2 and the mt-SSU rDNA sequences showed that eight T. abietinum strains were grouped in a single clade, supported by bootstrap values of 65% (Fig. 2B) and 96% (Fig. 2C). Most of the bootstrap values supporting subgroups within the T. abietinum clade were very low and their resolution was also very weak. In both ITS2 and mt-SSU rDNA trees, the two T. fusco-violaceum isolates formed a monophyletic group as a sister group to the T. abietinum clade (Figs. 2B and C), which is congruent with the previous result (Ko and Jung, 1999). T. biforme and T. subchartaceum were closely related to each other, which was
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Fig. 1. Alignment of ITS1 sequences of Trichaptum abietinum (CBS 374.68 to CBS 375.68), T. fusco-violaceum (FP-133997 and HHB-4016), T. biforme (FP-86522 to CBS 324.29), T. subchartaceum (CCFC003928 and CCFC003932), and T. laricinum (RLG-4665 and RLG-6936). Underlined nucleotides indicate regions used in designing type-specific primers, ITS-Ta1 (50 -TAAATAAGAGATGGTGGCTAG-30 ), ITS-Ta2 (50 -TACCAAAAGCATGTGCTCAG-30 ), and ITS-Ta3 (50 -TAGTAGTACGGCCCAGGTGTG-30 ).
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Fig. 2. Phylogenetic trees from ITS1 and ITS2 sequences of Trichaptum, inferred by the branch-and-bound option of parsimony analysis in PAUP 4.0 (Swofford, 1998). Sequences of Trichaptum laricinum were used as an outgorup to root trees because T. laricinum was always a good basal taxon to the grouping of Trichaptum in analyses of rDNA sequences along with those of Phellinus (data not shown). Bootstrapping values from 1000 replications are shown at corresponding branches. (A) One of 24 most parsimonious trees inferred from ITS1 sequences (tree length ¼ 92 steps, CI ¼ 0.913, RI ¼ 0.955, RC ¼ 0.872). Groups designated as ITS1 types are boxed. (B) One of 3 most parsimonious trees inferred from ITS2 sequences (tree length ¼ 127 steps, CI ¼ 0.913, RI ¼ 0.952, RC ¼ 0.870). The T. abietinum clade is boxed. (C) One of 2 most parsimonious trees inferred from partial mt-SSU rDNA sequences (tree length ¼ 245 steps, CI ¼ 0.971, RI ¼ 0.956, RC ¼ 0.929). The T. abietinum clade is boxed.
fully or strongly supported by bootstrap analysis (100% and 99% in Figs. 2B and C, respectively). T. laricinum was selected as an outgroup in this study, because it was always placed at a basal position for the grouping of Trichaptum in a preliminary analysis of ITS and mtSSU rDNA sequences along with those of Phellinus species (data not shown) and in a study employing 18S rDNA sequences (Ko et al., 1997). However, in the gene tree inferred from ITS1 sequences (Fig. 2A), eight T. abietinum strains were separated into three distinct groups designated Types. T. abietinum strains CBS 374.68 and CCFC008387 (Type I) were closely related to two T. fusco-violaceum strains. Three T. abietinum strains, SFC 960608-11, SFC 961028-11, and SFC 950815-17 collected in Korea,
formed a distinct clade (Type III). The Type III strains formed a larger group consisting of Type I strains, two strains of T. fusco-violaceum, and Type II strains, which was supported by 100% bootstrap value. The remaining three T. abietinum strains, CCFC009931, CCFC010156, and CBS 375.68, formed another distinct group (Type II). In the phylogenetic tree of ITS1 sequences, T. biforme and T. subchartaceum strains clustered together, which received complete bootstrap support (100%). Comparison of lengths (Table 2) and the alignment of ITS1 sequences (Fig. 1) identified the presence of three distinct ITS1 sequences within T. abietinum. ITS1 sequences of Type I and Type II were 444 and 249–251 bp in length, respectively. Type III ITS1s were 490–491 bp in length. Sequence sites from 51st to 342nd position of
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B
Fig. 2. (continued).
Type I and Type III (Fig. 1) were not properly aligned with any other sequences. The three different ITS1 types of T. abietinum were unambiguously aligned before the 51st nucleotide and after the 342nd nucleotide in the sequence position. However, Type I sequences were aligned quite well with those of two T. fusco-violaceum. In addition to base substitutions, insertions or deletions apparently distinguished three ITS1 types of T. abietinum (Fig. 1). To investigate these divergent ITS1 sequences among three groups of T. abietinum, Type I-, Type II-, and Type III-specific PCR primers were designed (Fig. 1) and used to test whether each strain possessed all three ITS1 type sequences (Fig. 3). Primer pairs ITS-Ta1/ITS4 and ITS-Ta2/ITS4 successfully amplified Type I and Type II sequences from all T. abietinum strains. However, the amplification using the Type III-specific primer ITS-Ta3 and ITS4 was successful only in the three Korean T. abietinum strains (Fig. 3). These results suggest that Type I and Type II ITS1 sequences coexist in
all tested T. abietinum strains and that Type III ITS1 sequences are present only in T. abietinum strains collected from Korea.
4. Discussion A major result of this study is an observed discrepancy among gene trees from nuclear rDNA ITS1, ITS2, and mt-SSU rDNA sequences and a high degree of divergence of ITS1 sequences among T. abietinum strains. The ITS1 gene tree consisted of three clades of T. abietinum due to nonorthologous sequences. T. abietinum strains may possess one of three ITS1 sequence types, but paralogs were not detected within the ITS2 and the mt-SSU rDNA gene trees. From the gene trees of ITS2 sequences (Fig. 2B) and partial mt-SSU rDNA sequences (Fig. 2C) and morphological evidence (Gilbertson and Ryvarden, 1987; Ryvarden and Gilbertson, 1994), T. abietinum has a
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C
Fig. 2. (continued).
Table 2 Nucleotide composition and lengths of the ITS1 sequences Species
A
T
G
C
Length (bp)
T. abietinum (8) Type I (2) Type II (3) Type III (3) T. fusco-violaceum (2) T. biforme (3) T. subchartaceum (2) T. laricinum (2)
27.9–28.3b 25.1–26.1 23.9–24.6 28.4–28.5 23.1–23.5 24.4–24.8 23.7–24.0
29.5–29.7 30.6–31.1 26.9–27.5 29.3–29.7 31.1–31.3 30.3 32.8–34.3
23.2–23.6 22.1–24.0 22.6–22.9 21.9–23.1 24.1–24.2 23.6–24.4. 20.7–21.0
18.7–18.9 20.0–20.7 20.6–21.0 17.4–17.9 21.2–21.6 20.9–21.2 20.6–21.1
444 249–251 490–491 441–443 264–265 254 232–233
Mean
25.5
29.7
23
20.1
a
a
Numbers of studied strains. b Base percentages calculated by PAUP* 4.0 (Swofford, 1998).
closer relationship with T. fusco-violaceum than with any other species. In the gene tree from ITS1 sequences (Fig. 2A), however, only two strains of T. abietinum indicated as Type I, CBS 374.68 and CCFC008387, formed a clade with two strains of T. fusco-violaceum. Strains of
both T. abietinum Type I and T. fusco-violaceum have similar ITS1 lengths (Table 2) and sequence divergence between them was very low in the ITS1 region as shown in Fig. 1. Their relationship was congruent with the gene trees from ITS2 and mt-SSU rDNA sequences and with
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Fig. 3. Examples of PCR-amplified ITS products generated by type- specific primers. The materials are T. abietinum CCFC008387, T. abietinum CBS 375.68, and T. abietinum SFC 950815-17. I lanes represent PCR products generated by a primer pair of ITS-Ta1/ITS4, II lanes by the one of ITS-Ta2/ ITS4, and III lanes by the one of ITS-Ta3/ITS4. M lanes indicate 1-kb DNA ladders.
the results based on morphological evidence. Thus, it seems that Type I sequences of the ITS1 region reflect the species tree of T. abietinum. Compared to Type I and Type III sequences, Type II sequences that are shorter in length might have been generated by deletion of nucleotides from the 51st to the 342nd sites (Figs. 1 and 3 and Table 2). PCR amplifications using the type-specific primers suggests that both Type I and Type II coexist in individual strains of T. abietinum (Fig. 3). The three Korean strains of T. abietinum possessed an additional ITS1 sequences designated Type III. Three distinct regions were identified within Type III sequences. The first are conserved regions present in all Trichaptum species. The 50 and 30 ends of Type III sequences correspond to this category. The second are regions unambiguously aligned only with sequences of Type I and T. fusco-violaceum. The third are regions unique to Type III sequences. These unique regions consisted of both inserted and unaligned regions. The Type III-specific primer, ITS-Ta3, was designed based upon sequences of the first inserted region (Fig. 1). Results of the PCR assay (Fig. 3) suggest that Type III sequences might exist only in the Korean strains used in the study. Type III sequences of T. abietinum are possibly due to polymorphism endemic to a certain geographic region and could be a subtype of Type I, suggesting that new ITS types can be locally formed and may carry a significance in population genetics. This subtype polymorphism could be placed under homogenization dynamics other than those of Type I and Type II sequences (Vogler and DeSalle, 1994). Ribosomal DNA multigene families are best known for the high degree of intraspecific homogeneity resulting from concerted evolution. For this reason, ribosomal DNA has been used broadly in inferring
phylogenetic relationships. Recently, however, several cases where rDNA sequences have escaped concerted evolution have been stated in various groups such as angiosperms, mosquitoes, tiger beetles, and fungi (Baldwin et al., 1995; Vogler and DeSalle, 1994; Wesson et al., 1992). In fungi, a few examples of nonorthologous ITS sequences have been discovered in ascomycetes (Fatehi and Bridge, 1998; O’Donnell and Cigelnik, 1997; O’Donnell et al., 1998; Waalwijk et al., 1996) and zygomycetes (Hijri et al., 1999; Hosny et al., 1999). Results of the present study indicate that nonorthologous evolution of rDNA sequences also occurs in basidiomycetes. There have been several explanations for intraspecific rDNA polymorphism. First, polymorphic sequences may represent pseudogenes (Buckler et al., 1997). In the processes of divergence of Trichaptum, some types might have lost their ITS functions and possibly became pseudogenes. Pseudogenes have base compositions different from those of functional genes and evolve very rapidly, showing a high AT ratio (Buckler et al., 1997). However, nucleotide compositions among three types did not show significant differences (Table 2). AT ratios of Type II sequences (0.557–0.566) and Type III sequences (0.512–0.519) are lower than those of Type I sequences (0.574–0.581) (Table 2). Conserved sequences of 30 and 50 ends and unambiguously aligned regions also suggest their nonpseudogene state, since pseudogenes usually show random base substitutions (Wang et al., 1997). In addition, the result that they were kept internally homogeneous within the groups of given types suggests that they are not pseudogenes. Second, it can be hypothesized that xenologous ITS1 regions of T. abietinum have been originated from the hybrid of two different species or populations that are in
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the process of speciation (Hugall et al., 1999; Odorico and Miller, 1997). However, none of three types of T. abietinum showed intermediate characteristics of two species, for example, T. abietinum and T. biforme. In addition, Type III sequences seem to be a local subtype of Type I sequences, since they share many unambiguously aligned regions in comparison with those of Type I in T. abietinum and T. fusco-violaceum and the former is restricted to Korea. Thus, it is implausible that type sequences were always derived from hybridization and evolved independently in their original ITS1 regions. Third, rDNA polymorphism within individuals may occur when the pace of concerted evolution is not fast enough to homogenize tandem repeats (Campbell et al., 1997). Polymorphism within the intergenic spacer of the nuclear rDNA of Fusarium oxysporum can be explained by a much slower rate of concerted evolution within a predominately clonal species (Appel and Gordon, 1996). This suggestion is more applicable to incomplete homogenization of rDNA repeats in more complex eukaryotes with longer generation times and larger rDNA loci (Copenhaver and Pikaard, 1996; O’Donnell et al., 1998). However, T. abietinum is an annual mushroom that has a short generation time (Ryvarden and Gilbertson, 1993). Fourth, three types of the ITS1 region of T. abietinum could be located at different chromosomal loci. Location of rDNA loci on nonhomologous chromosomes potentially disrupts concerted evolution (Campbell et al., 1997; Vogler and DeSalle, 1994). Such dispersed location of rDNA repeats on several chromosomes has been reported in Fusarium (Boehm et al., 1994; Fekete et al., 1993), Scilla (Vaughan et al., 1993), and Drosophila (Schl€ otterer and Tautz, 1994). The relatively low heterogeneity within the types of T. abietinum may support this hypothesis. However, this does not seem to readily answer the question why the diversity of ITS2 sequences is extremely low compared to that of ITS1 sequences (Hugall et al., 1999). It is also possible for several types of rDNAs to be located at different loci of the same chromosome (Carranza et al., 1999). Gene duplication might have occurred prior to the divergence of the polypore species T. abietinum, T. fusco-violaceum, T. biforme, and T. subchartaceum. Products of gene duplication, Type I and Type II ITS1 sequences, have been maintained in the same individual of T. abietinum without the process of homogenization or selection. Introduced types might still contain their biological functions (Carranza et al., 1999; Wang et al., 1997). Other additional ITS1 types might have been preserved since they could provide a buffer against deleterious mutations (O’Donnell et al., 1998). For fungi such as Scutellospora castanea, the high levels of nucleotide divergence may be due to impaired genetic exchange among nuclei carrying different ITS
types. Spores of S. castanea are heterokaryotic (Hijri et al., 1999) and may harbor genetically different nuclei and show a nonorthologous ITS pattern (Hosny et al., 1999). Also for strains of T. abietinum, two or three paralogous ITS1 types could be present in heterokaryotic spores. Recently, it was reported that there are more than one genetically different IGS1 regions in dikaryotic individuals of Schizophyllum commune (Basidiomycetes), but not in monokaryon (James et al., 2001). Ribosomal DNA ITS sequences have been most frequently employed in inference of species trees. Direct sequencing of rDNA PCR products provides a rapid and useful tool in phylogenetic studies. However, systemic differences in amplification probabilities between paralogs can result in biased sampling and a false inference of species relationships (Waalwijk et al., 1996). Discrepancy of gene phylogenies inferred from ITS1, ITS2, and mt-SSU rDNA sequences due to nonorthologous sequences shows that caution is needed when inferring species trees only from ITS gene trees. Results of the present study emphasize the importance of survey for gene–gene concordance and thorough in-depth sampling for stable and robust phylogenetic conclusions.
Acknowledgments We sincerely thank Dr. Soon Gyu Hong (Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology) for his helpful advice and technical comments in designing Type-specific primers. K.S. Ko was supported by the BK21 Research Fellowship from the Ministry of Education and Human Resources Development until he graduated from the School of Biological Sciences, Seoul National University.
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