Molecular Phylogenetics of the Genus Trichosporon Inferred from Mitochondrial Cytochrome b Gene Sequences
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微生物临床杂志 2005年第10期
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan
ABSTRACT
Mitochondrial cytochrome b (cyt b) genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced to determine their molecular phylogenetic relationships. Almost half of the 22 strains investigated (from 11 different species) contained introns in their sequences. Analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. A phylogenetic tree based on the cyt b gene sequences revealed that all species of Trichosporon except Trichosporon domesticum and Trichosporon montevideense had species-specific cyt b genes. Trichosporon sp. strain CBS 5581 was identified as Trichosporon pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale. Analysis of 132-bp deduced amino acid sequences showed a total of 34 (25.75%) variable amino acid sites. T. domesticum and T. montevideense, Trichosporon asahii and Trichosporon asteroides, and Trichosporon gracile and Trichosporon guehoae had identical amino acid sequences. A phylogenetic tree constructed with the ascomycetes Saccharomyces douglasii and Candida glabrata taken as outgroup species and including representative species from closely related genera species of Trichosporon clustered with other basidiomycetous yeasts that contain xylose in their cell wall compositions. These results indicate the effectiveness of mitochondrial cyt b gene sequences for both species identification and the phylogenetic analysis of Trichosporon species.
INTRODUCTION
Trichosporon Behrend is a member of the basidiomycetous yeasts. The species of the genus are causative agents of the superficial to deep-seated mycosis known as trichosporonosis. Generally, it is found that trichosporonosis is caused by six species: Trichosporon asahii, Trichosporon asteroides, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides, and Trichosporon ovoides (16, 19, 36, 37). It is also found that the major causative agents of trichosporonosis differ in causing infection. T. asahii and T. mucoides are involved in deep-seated infection. T. asteroides and T. cutaneum are associated with superficial infection. T. ovoides and T. inkin are involved in white piedra of the head and genital area, respectively. Trichosporon pullulans is not a major causative agent of trichosporonosis and is rarely isolated from fungemia patients (25, 26). Recent studies indicate that the number of patients with illness caused by a Trichosporon species has been increasing (21, 22, 42, 43, 52, 56). Deep-seated trichosporonosis has a high mortality rate, and the prognosis for patients is very poor. Trichosporon species are also responsible for summer-type hypersensitivity pneumonitis (1, 2, 34, 55).
The genus Trichosporon was revised using a combination of physiological and molecular criteria (14, 15). Subsequently, Sugita et al. (35, 36, 38) contributed significantly to the classification and taxonomic position of the genus Trichosporon. Recently, the number of species of the genus Trichosporon has expanded from 19 (17) to 23 (13). All of these classifications are based on the analysis of different regions of the rRNA genes, such as 5S rRNA genes, 26S rRNA genes, 18S rRNA genes, the D1/D2 region of large-subunit rRNA genes, or the internal transcribed spacer region of rRNA genes. There is a need for further evidence based on different DNA sequences to establish phylogenetic relationships (27). It was therefore of interest to find the relationships among various species of Trichosporon by analyzing mitochondrial (mt) DNA rather than rRNA genes.
Mitochondrial genes are an attractive marker for inferring the phylogeny of closely related species because of the rapid evolution of the mitochondrial genome, the lack of recombination, and the strict maternal inheritance (29). Restriction fragment length polymorphism analysis of mt DNA has been shown to be useful for estimating the relationships among fungi (18, 24, 44). The mt cytochrome b (cyt b) gene has been used to study the evolution and phylogenetic relationships of many animals, including birds, mammals, and fish (3, 9, 10, 12, 20, 23), but the sequence of this gene had been determined for only six species of fungi before our study (46). We have reported that the mt cyt b gene is useful for the identification, classification, and phylogenetic analysis of fungi (4-7, 46-49, 53, 54).
We have previously shown that the cyt b gene phylogeny of basidiomycetous yeasts correlates with the cell wall biochemistry and septal ultrastructure (5). However, only two species of Trichosporon, T. aquatile and T. mucoides, were included in that study. In this study, we analyzed the mt cyt b genes of all species of the genus Trichosporon described by Fell et al. (13) and determined their relationships to closely related basidiomycetous yeasts.
MATERIALS AND METHODS
Trichosporon strains and isolation of DNA. The Trichosporon strains used in this study are listed in Table 1. Cultures were grown on yeast extract-peptone-dextrose (1% [wt/vol] yeast extract, 2% [wt/vol] polypeptone, 2% [wt/vol] glucose) slants. One loop of cells from a yeast extract-peptone-dextrose slant was suspended in 1 ml sterile distilled water and used for extraction of total cellular DNA with the Gen Toru Kun kit (Takara Shuzo Co., Ltd., Otsu, Shiga, Japan) as described previously (53).
PCR primers and amplification of the cyt b gene. PCR primers E1M4 (5'-TGRGGWGCWACWGTTATTACTA-3') and E2M4 (5'-GGWATAGMWSKTAAWAYAGCATA-3') were designed as described previously (7), where letters represent bases as follows: K, G or T; M, A or C; R, A or G; S, C or G; W, A or T; and Y, C or T. One microliter of extracted DNA was used as a template for the amplification of the mt cyt b gene with a TaKaRa Ex Taq PCR amplification kit (Takara Shuzo). Reactions were performed in a final reaction volume of 50 μl, containing 10 pmol of each primer, 4 μl of 2.5 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 2.0 U of TaKaRa Ex Taq polymerase, and 5 μl of 10x reaction buffer (Takara Shuzo). Amplification conditions were 94°C for 2 min; followed by 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C, and extension for 1 min at 72°C; and a final extension at 72°C for 10 min.
Sequencing. PCR products were purified with a Qiaquick PCR purification kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Both strands of PCR products were sequenced directly with an ABI Prism model 377 or 310 DNA sequencer with a BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems Japan Co., Ltd., Tokyo, Japan). Amino acid sequences were deduced from the DNA sequences with the yeast mitochondrial genetic code.
Molecular phylogenetic analysis. With the exclusion of the portions of the sequence that included the primers, DNA and amino acid sequences were aligned with GENETYX-MAC genetic information processing software (Software Development Co., Ltd., Tokyo, Japan). This software also generated phylogenetic trees by the unweighted pair-group method with arithmetic mean (UPGMA) and neighbor joining (NJ). The Phylogenetic Analysis Using Parsimony (PAUP) package, version 4.0b4a for Macintosh (41), was used for generating maximum-parsimony (MP) and maximum-likelihood (ML) trees. Standard errors of main branching points in the UPGMA tree were calculated by adopting the method of Nei et al. (33).
Nucleotide sequence accession numbers. Sequences of the cyt b gene of the Trichosporon strains sequenced in this study have been deposited in DDBJ/EMBL/GenBank under the accession numbers listed in Table 1.
RESULTS
The mitochondrial cytochrome b genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced. Among 23 species of the genus Trichosporon studied in this report, 11 species showed an intron in their cyt b gene sequences (Table 1). Interestingly, among four strains of T. asteroides included, two had introns and the other two had no introns. The numbers, sizes, and locations of the introns were variable. The sizes (Table 1) and locations (Fig. 1) of these introns were determined as described previously (5) through comparison of strains with and without introns to maximize amino acid identities.
For each strain, the 396-bp fragment corresponding to nucleotides (nt) 445 to 840 in the Candida glabrata cyt b gene coding sequence (GenBank accession no. X53862) was analyzed. Multiple alignments (not shown) of the coding sequence of each strain of the genus Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. Figure 2 shows the mt cyt b gene UPGMA phylogenetic tree where the ascomycetes Saccharomyces douglasii and C. glabrata were taken as outgroup species. From this phylogenetic tree, it is clear that all other species of the genus Trichosporon except T. domesticum and T. montevideense had a species-specific cyt b gene. Trichosporon sp. strain CBS 5581, whose identification is still unsettled at the species level, was identified as T. pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale.
Using the mt yeast genetic code, conversion of the 396-bp cyt b gene sequences deduced the 132-bp amino acid sequences. Multiple alignments (Fig. 1) of the deduced sequences of each strain of the genus Trichosporon under investigation showed a total of 34 (25.75%) variable amino acid sites. A phylogenetic tree constructed based on amino acid sequences is shown in Fig. 3. T. domesticum and T. montevideense, T. asahii and T. asteroides, and Trichosporon gracile and Trichosporon guehoae contained identical amino acid sequences. However, all other species of Trichosporon had species-specific amino acid sequences.
With the ascomycetes S. douglasii and C. glabrata taken as outgroup species, the basidiomycetous yeasts were divided into two major clusters correlated with cell wall biochemistry, the finding which concerns the presence or absence of xylose and of septal ultrastructure (dolipore or "simple" pore). For either the cyt b gene DNA or the amino acid sequence-based phylogenetic tree, the outcomes were similar (Fig. 2 through 4). The species of Trichosporon clustered in the section of basidiomycetous yeasts contain xylose in their cell wall compositions and have dolipore septa. All the species of Trichosporon were grouped in such a manner that the genus Trichosporon should be monophyletic if Cryptococcus curvatus and Cryptococcus humicolus were not included in this cluster.
DISCUSSION
In this study, analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. To ensure that these variations were not due to polymerase errors, we used the TaKaRa Ex Taq polymerase, which has an approximately fourfold-lower error rate than standard Taq DNA polymerase. Moreover, we sequenced both strands of each PCR product and repeated each PCR and sequencing reaction.
The presence of an intron in the cyt b gene is not uncommon. Basically, most fungi, e.g., Aspergillus nidulans, Neurospora crassa, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, contain an intron(s) in their cyt b gene sequences (28). cyt b gene sequences of Pneumocystis carinii and C. glabrata do not contain introns (11, 45). Among the 23 species of Trichosporon studied, 11 species had an intron(s) in the region sequenced. Strains with or without introns occurred for T. asteroides. The presence or absence of introns in T. asteroides strains is not the first occurrence, and we found in our previous study that all strains except the type strain of Cryptococcus neoformans have introns in this region of the cyt b gene (7). T. asahii, T. asteroides, Trichosporon coremiiforme, Trichosporon dulcitum, T. faecale, Trichosporon jirovecii, T. ovoides, and T. pullulans had introns in the cyt b gene that started at nt 47 (Fig. 1 and Table 1), which is the same position as that of intron II of the Neurospora crassa cyt b gene. Other basidiomycetous yeasts, such as Rhodotorula acheniorum, Rhodotorula ferulica, and C. neoformans (except the type strain), have introns in the same location (5, 7). The second introns of T. asteroides and T. pullulans and introns of Trichosporon brassicae and T. inkin began at nt 63 (Table 1), which is similar to the location of intron I of the Aspergillus nidulans and intron III of the Saccharomyces cerevisiae cyt b genes. Trichosporon moniliiforme had an intron that started at nt 377 of the cyt b gene coding sequence. Rhodotorula acheniorum, Rhodotorula hinnulea, and Rhodotorula phylloplana have introns in the same location (5). These findings suggest two possible evolutionary events. The first possibility is that these introns appeared in these locations prior to the separation of these species and that some species are losing introns over time. Alternatively, these introns appeared in these locations after the separation of these species.
Beside UPGMA trees, we generated trees using MP, ML, and NJ (Fig. 4). For either DNA or amino acid sequences, the trees were almost similar in topology irrespective of the generation method and correlated with those determined by cell wall biochemistry (the finding concerning the presence or absence of xylose) (50, 51) and septal ultrastructure (dolipore or "simple" pore) (8, 31). However, use of the UPGMA method results in the correct topology when the distance measure used is exactly linear with evolutionary time and the substitution rate of an amino acid in cytochrome b is presumably in good proportion to evolutionary time (32). Wang et al. (46-49) and Yokoyama et al. (54) compared different methods and have shown that UPGMA is the best method to construct phylogenetic trees based on the amino acid sequences of cytochrome b in fungi. Analyses of the cyt b gene of Rhodotorula and related basidiomycetous yeasts had similar outcomes (5). The negligible differences between phylogenetic trees constructed from nucleotide and amino acid sequences using any of the methods are due to the fact that different nucleotide codons may give rise to the same amino acid, which may produce differences in topologies of the nucleotide- and amino acid-based phylogenetic trees.
Ultrastructural and molecular analyses have shown that basidiomycetous yeasts are distributed among the three main phylogenetic lines of the Basidiomycota, namely, the Hymenomycetes, Urediniomycetes, and Ustilaginomycetes (30, 39, 40). Analysis of the D1/D2 region categorized the hymenomycetous yeasts into four major clades: the Tremellales, the Trichosporonales, the Filobasidiales, and the Cystofilobasidiales. With the exception of T. pullulans, which occurred in the Cystofilobasidiales, all other species of Trichosporon grouped in the Trichosporonales (13). We were unable to amplify the cyt b gene of the type strain of T. pullulans (CBS 2532) using the primers in this study but did perform amplification and sequencing of the cyt b gene of T. pullulans (CBS 2535). Phylogenetic analysis of the cyt b gene revealed that T. pullulans (CBS 2535) was closely related to T. ovoides; however, CBS 2535 was not included in the study of Fell et al. (13). Further study is required to confirm the phylogenetic position of T. pullulans, for example, to sequence and analyze both D1/D2 of the rRNA genes and the mt cyt b gene of several strains of T. pullulans.
In addition to the species of the genus Trichosporon, the order Trichosporonales included Apiotrichum porosum, Cryptococcus curvatus, and C. humicolus (13). We did not include A. porosum in this study; however, the cyt b gene of C. curvatus (90% bootstrap support with T. sporotrichoides) and C. humicolus (97% bootstrap support with T. mucoides) showed close relationships with species of Trichosporon. C. curvatus and C. humicolus do not produce arthroconidia (14-16); why are they connected to the arthroconidium-producing genus Trichosporon This question has yet to be answered.
Physiologically, T. asahii cannot be distinguished from T. asteroides (17). Analysis of the D1/D2 region of rRNA genes demonstrated close relationships among T. asahii, T. faecale, and T. asteroides (13), which is similar to the outcome of the cyt b gene phylogeny. T. asahii differed from T. asteroides by 16 bases (4.04%), but none of these substitutions was nonsynonymous, producing identical protein sequences. It is interesting to mention here that although these organisms show a very close relationship based on their DNA or amino acid sequences, they differ in causing infection: T. asahii is involved in deep-seated infection, whereas T. asteroides is involved with superficial infection. Similarly, T. domesticum and T. montevideense are closely related species (13). However, their cyt b gene sequences (396 bp) were identical. Therefore, further studies are necessary to confirm the relationships of T. domesticum and T. montevideense, for example, by sequencing the complete cyt b gene.
Trichosporon laibachii IFM 48580 is equivalent to strain IFM 48562. Strains IFM 48579 and IFM 48623 had identical cyt b gene sequences and differed from the type strain IFM 48580 (CBS 5790) by 11 bases, of which one was nonsynonymous. Trichosporon multisporum is considered a specific species closely related to T. laibachii (13), which is similar to what was found in this study. cyt b genes of T. multisporum and T. laibachii differed by 8 nt (2.02%), of which one was nonsynonymous. Further study is required to confirm whether strains IFM 48579 and IFM 48623 are varieties of T. laibachii or are distinct species.
In conclusion, we have shown that species of Trichosporon clustered in the cyt b gene phylogenetic analysis with the set of basidiomycetous yeasts that contain xylose in their cell wall compositions and have dolipore septa. This observation correlates with cell wall biochemistry and septal ultrastructure findings. The results of this study also indicate that mt cyt b gene sequences are effective for both species identification and determining phylogenetic relationships of Trichosporon species.
ACKNOWLEDGMENTS
This study was performed as part of the program "Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)" through the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (2001). We also thank the Institute for Fermentation, Osaka, Japan, for a grant for this study.
Present address: Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430.
Present address: Department of Pathogenobiology, School of Basic Medical Science, Norman Bethune Medical Division of Jilin University, 2 Xinmin Street, Changchun, Jilin, China.
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ABSTRACT
Mitochondrial cytochrome b (cyt b) genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced to determine their molecular phylogenetic relationships. Almost half of the 22 strains investigated (from 11 different species) contained introns in their sequences. Analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. A phylogenetic tree based on the cyt b gene sequences revealed that all species of Trichosporon except Trichosporon domesticum and Trichosporon montevideense had species-specific cyt b genes. Trichosporon sp. strain CBS 5581 was identified as Trichosporon pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale. Analysis of 132-bp deduced amino acid sequences showed a total of 34 (25.75%) variable amino acid sites. T. domesticum and T. montevideense, Trichosporon asahii and Trichosporon asteroides, and Trichosporon gracile and Trichosporon guehoae had identical amino acid sequences. A phylogenetic tree constructed with the ascomycetes Saccharomyces douglasii and Candida glabrata taken as outgroup species and including representative species from closely related genera species of Trichosporon clustered with other basidiomycetous yeasts that contain xylose in their cell wall compositions. These results indicate the effectiveness of mitochondrial cyt b gene sequences for both species identification and the phylogenetic analysis of Trichosporon species.
INTRODUCTION
Trichosporon Behrend is a member of the basidiomycetous yeasts. The species of the genus are causative agents of the superficial to deep-seated mycosis known as trichosporonosis. Generally, it is found that trichosporonosis is caused by six species: Trichosporon asahii, Trichosporon asteroides, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides, and Trichosporon ovoides (16, 19, 36, 37). It is also found that the major causative agents of trichosporonosis differ in causing infection. T. asahii and T. mucoides are involved in deep-seated infection. T. asteroides and T. cutaneum are associated with superficial infection. T. ovoides and T. inkin are involved in white piedra of the head and genital area, respectively. Trichosporon pullulans is not a major causative agent of trichosporonosis and is rarely isolated from fungemia patients (25, 26). Recent studies indicate that the number of patients with illness caused by a Trichosporon species has been increasing (21, 22, 42, 43, 52, 56). Deep-seated trichosporonosis has a high mortality rate, and the prognosis for patients is very poor. Trichosporon species are also responsible for summer-type hypersensitivity pneumonitis (1, 2, 34, 55).
The genus Trichosporon was revised using a combination of physiological and molecular criteria (14, 15). Subsequently, Sugita et al. (35, 36, 38) contributed significantly to the classification and taxonomic position of the genus Trichosporon. Recently, the number of species of the genus Trichosporon has expanded from 19 (17) to 23 (13). All of these classifications are based on the analysis of different regions of the rRNA genes, such as 5S rRNA genes, 26S rRNA genes, 18S rRNA genes, the D1/D2 region of large-subunit rRNA genes, or the internal transcribed spacer region of rRNA genes. There is a need for further evidence based on different DNA sequences to establish phylogenetic relationships (27). It was therefore of interest to find the relationships among various species of Trichosporon by analyzing mitochondrial (mt) DNA rather than rRNA genes.
Mitochondrial genes are an attractive marker for inferring the phylogeny of closely related species because of the rapid evolution of the mitochondrial genome, the lack of recombination, and the strict maternal inheritance (29). Restriction fragment length polymorphism analysis of mt DNA has been shown to be useful for estimating the relationships among fungi (18, 24, 44). The mt cytochrome b (cyt b) gene has been used to study the evolution and phylogenetic relationships of many animals, including birds, mammals, and fish (3, 9, 10, 12, 20, 23), but the sequence of this gene had been determined for only six species of fungi before our study (46). We have reported that the mt cyt b gene is useful for the identification, classification, and phylogenetic analysis of fungi (4-7, 46-49, 53, 54).
We have previously shown that the cyt b gene phylogeny of basidiomycetous yeasts correlates with the cell wall biochemistry and septal ultrastructure (5). However, only two species of Trichosporon, T. aquatile and T. mucoides, were included in that study. In this study, we analyzed the mt cyt b genes of all species of the genus Trichosporon described by Fell et al. (13) and determined their relationships to closely related basidiomycetous yeasts.
MATERIALS AND METHODS
Trichosporon strains and isolation of DNA. The Trichosporon strains used in this study are listed in Table 1. Cultures were grown on yeast extract-peptone-dextrose (1% [wt/vol] yeast extract, 2% [wt/vol] polypeptone, 2% [wt/vol] glucose) slants. One loop of cells from a yeast extract-peptone-dextrose slant was suspended in 1 ml sterile distilled water and used for extraction of total cellular DNA with the Gen Toru Kun kit (Takara Shuzo Co., Ltd., Otsu, Shiga, Japan) as described previously (53).
PCR primers and amplification of the cyt b gene. PCR primers E1M4 (5'-TGRGGWGCWACWGTTATTACTA-3') and E2M4 (5'-GGWATAGMWSKTAAWAYAGCATA-3') were designed as described previously (7), where letters represent bases as follows: K, G or T; M, A or C; R, A or G; S, C or G; W, A or T; and Y, C or T. One microliter of extracted DNA was used as a template for the amplification of the mt cyt b gene with a TaKaRa Ex Taq PCR amplification kit (Takara Shuzo). Reactions were performed in a final reaction volume of 50 μl, containing 10 pmol of each primer, 4 μl of 2.5 mM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 2.0 U of TaKaRa Ex Taq polymerase, and 5 μl of 10x reaction buffer (Takara Shuzo). Amplification conditions were 94°C for 2 min; followed by 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C, and extension for 1 min at 72°C; and a final extension at 72°C for 10 min.
Sequencing. PCR products were purified with a Qiaquick PCR purification kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Both strands of PCR products were sequenced directly with an ABI Prism model 377 or 310 DNA sequencer with a BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems Japan Co., Ltd., Tokyo, Japan). Amino acid sequences were deduced from the DNA sequences with the yeast mitochondrial genetic code.
Molecular phylogenetic analysis. With the exclusion of the portions of the sequence that included the primers, DNA and amino acid sequences were aligned with GENETYX-MAC genetic information processing software (Software Development Co., Ltd., Tokyo, Japan). This software also generated phylogenetic trees by the unweighted pair-group method with arithmetic mean (UPGMA) and neighbor joining (NJ). The Phylogenetic Analysis Using Parsimony (PAUP) package, version 4.0b4a for Macintosh (41), was used for generating maximum-parsimony (MP) and maximum-likelihood (ML) trees. Standard errors of main branching points in the UPGMA tree were calculated by adopting the method of Nei et al. (33).
Nucleotide sequence accession numbers. Sequences of the cyt b gene of the Trichosporon strains sequenced in this study have been deposited in DDBJ/EMBL/GenBank under the accession numbers listed in Table 1.
RESULTS
The mitochondrial cytochrome b genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced. Among 23 species of the genus Trichosporon studied in this report, 11 species showed an intron in their cyt b gene sequences (Table 1). Interestingly, among four strains of T. asteroides included, two had introns and the other two had no introns. The numbers, sizes, and locations of the introns were variable. The sizes (Table 1) and locations (Fig. 1) of these introns were determined as described previously (5) through comparison of strains with and without introns to maximize amino acid identities.
For each strain, the 396-bp fragment corresponding to nucleotides (nt) 445 to 840 in the Candida glabrata cyt b gene coding sequence (GenBank accession no. X53862) was analyzed. Multiple alignments (not shown) of the coding sequence of each strain of the genus Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. Figure 2 shows the mt cyt b gene UPGMA phylogenetic tree where the ascomycetes Saccharomyces douglasii and C. glabrata were taken as outgroup species. From this phylogenetic tree, it is clear that all other species of the genus Trichosporon except T. domesticum and T. montevideense had a species-specific cyt b gene. Trichosporon sp. strain CBS 5581, whose identification is still unsettled at the species level, was identified as T. pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale.
Using the mt yeast genetic code, conversion of the 396-bp cyt b gene sequences deduced the 132-bp amino acid sequences. Multiple alignments (Fig. 1) of the deduced sequences of each strain of the genus Trichosporon under investigation showed a total of 34 (25.75%) variable amino acid sites. A phylogenetic tree constructed based on amino acid sequences is shown in Fig. 3. T. domesticum and T. montevideense, T. asahii and T. asteroides, and Trichosporon gracile and Trichosporon guehoae contained identical amino acid sequences. However, all other species of Trichosporon had species-specific amino acid sequences.
With the ascomycetes S. douglasii and C. glabrata taken as outgroup species, the basidiomycetous yeasts were divided into two major clusters correlated with cell wall biochemistry, the finding which concerns the presence or absence of xylose and of septal ultrastructure (dolipore or "simple" pore). For either the cyt b gene DNA or the amino acid sequence-based phylogenetic tree, the outcomes were similar (Fig. 2 through 4). The species of Trichosporon clustered in the section of basidiomycetous yeasts contain xylose in their cell wall compositions and have dolipore septa. All the species of Trichosporon were grouped in such a manner that the genus Trichosporon should be monophyletic if Cryptococcus curvatus and Cryptococcus humicolus were not included in this cluster.
DISCUSSION
In this study, analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. To ensure that these variations were not due to polymerase errors, we used the TaKaRa Ex Taq polymerase, which has an approximately fourfold-lower error rate than standard Taq DNA polymerase. Moreover, we sequenced both strands of each PCR product and repeated each PCR and sequencing reaction.
The presence of an intron in the cyt b gene is not uncommon. Basically, most fungi, e.g., Aspergillus nidulans, Neurospora crassa, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, contain an intron(s) in their cyt b gene sequences (28). cyt b gene sequences of Pneumocystis carinii and C. glabrata do not contain introns (11, 45). Among the 23 species of Trichosporon studied, 11 species had an intron(s) in the region sequenced. Strains with or without introns occurred for T. asteroides. The presence or absence of introns in T. asteroides strains is not the first occurrence, and we found in our previous study that all strains except the type strain of Cryptococcus neoformans have introns in this region of the cyt b gene (7). T. asahii, T. asteroides, Trichosporon coremiiforme, Trichosporon dulcitum, T. faecale, Trichosporon jirovecii, T. ovoides, and T. pullulans had introns in the cyt b gene that started at nt 47 (Fig. 1 and Table 1), which is the same position as that of intron II of the Neurospora crassa cyt b gene. Other basidiomycetous yeasts, such as Rhodotorula acheniorum, Rhodotorula ferulica, and C. neoformans (except the type strain), have introns in the same location (5, 7). The second introns of T. asteroides and T. pullulans and introns of Trichosporon brassicae and T. inkin began at nt 63 (Table 1), which is similar to the location of intron I of the Aspergillus nidulans and intron III of the Saccharomyces cerevisiae cyt b genes. Trichosporon moniliiforme had an intron that started at nt 377 of the cyt b gene coding sequence. Rhodotorula acheniorum, Rhodotorula hinnulea, and Rhodotorula phylloplana have introns in the same location (5). These findings suggest two possible evolutionary events. The first possibility is that these introns appeared in these locations prior to the separation of these species and that some species are losing introns over time. Alternatively, these introns appeared in these locations after the separation of these species.
Beside UPGMA trees, we generated trees using MP, ML, and NJ (Fig. 4). For either DNA or amino acid sequences, the trees were almost similar in topology irrespective of the generation method and correlated with those determined by cell wall biochemistry (the finding concerning the presence or absence of xylose) (50, 51) and septal ultrastructure (dolipore or "simple" pore) (8, 31). However, use of the UPGMA method results in the correct topology when the distance measure used is exactly linear with evolutionary time and the substitution rate of an amino acid in cytochrome b is presumably in good proportion to evolutionary time (32). Wang et al. (46-49) and Yokoyama et al. (54) compared different methods and have shown that UPGMA is the best method to construct phylogenetic trees based on the amino acid sequences of cytochrome b in fungi. Analyses of the cyt b gene of Rhodotorula and related basidiomycetous yeasts had similar outcomes (5). The negligible differences between phylogenetic trees constructed from nucleotide and amino acid sequences using any of the methods are due to the fact that different nucleotide codons may give rise to the same amino acid, which may produce differences in topologies of the nucleotide- and amino acid-based phylogenetic trees.
Ultrastructural and molecular analyses have shown that basidiomycetous yeasts are distributed among the three main phylogenetic lines of the Basidiomycota, namely, the Hymenomycetes, Urediniomycetes, and Ustilaginomycetes (30, 39, 40). Analysis of the D1/D2 region categorized the hymenomycetous yeasts into four major clades: the Tremellales, the Trichosporonales, the Filobasidiales, and the Cystofilobasidiales. With the exception of T. pullulans, which occurred in the Cystofilobasidiales, all other species of Trichosporon grouped in the Trichosporonales (13). We were unable to amplify the cyt b gene of the type strain of T. pullulans (CBS 2532) using the primers in this study but did perform amplification and sequencing of the cyt b gene of T. pullulans (CBS 2535). Phylogenetic analysis of the cyt b gene revealed that T. pullulans (CBS 2535) was closely related to T. ovoides; however, CBS 2535 was not included in the study of Fell et al. (13). Further study is required to confirm the phylogenetic position of T. pullulans, for example, to sequence and analyze both D1/D2 of the rRNA genes and the mt cyt b gene of several strains of T. pullulans.
In addition to the species of the genus Trichosporon, the order Trichosporonales included Apiotrichum porosum, Cryptococcus curvatus, and C. humicolus (13). We did not include A. porosum in this study; however, the cyt b gene of C. curvatus (90% bootstrap support with T. sporotrichoides) and C. humicolus (97% bootstrap support with T. mucoides) showed close relationships with species of Trichosporon. C. curvatus and C. humicolus do not produce arthroconidia (14-16); why are they connected to the arthroconidium-producing genus Trichosporon This question has yet to be answered.
Physiologically, T. asahii cannot be distinguished from T. asteroides (17). Analysis of the D1/D2 region of rRNA genes demonstrated close relationships among T. asahii, T. faecale, and T. asteroides (13), which is similar to the outcome of the cyt b gene phylogeny. T. asahii differed from T. asteroides by 16 bases (4.04%), but none of these substitutions was nonsynonymous, producing identical protein sequences. It is interesting to mention here that although these organisms show a very close relationship based on their DNA or amino acid sequences, they differ in causing infection: T. asahii is involved in deep-seated infection, whereas T. asteroides is involved with superficial infection. Similarly, T. domesticum and T. montevideense are closely related species (13). However, their cyt b gene sequences (396 bp) were identical. Therefore, further studies are necessary to confirm the relationships of T. domesticum and T. montevideense, for example, by sequencing the complete cyt b gene.
Trichosporon laibachii IFM 48580 is equivalent to strain IFM 48562. Strains IFM 48579 and IFM 48623 had identical cyt b gene sequences and differed from the type strain IFM 48580 (CBS 5790) by 11 bases, of which one was nonsynonymous. Trichosporon multisporum is considered a specific species closely related to T. laibachii (13), which is similar to what was found in this study. cyt b genes of T. multisporum and T. laibachii differed by 8 nt (2.02%), of which one was nonsynonymous. Further study is required to confirm whether strains IFM 48579 and IFM 48623 are varieties of T. laibachii or are distinct species.
In conclusion, we have shown that species of Trichosporon clustered in the cyt b gene phylogenetic analysis with the set of basidiomycetous yeasts that contain xylose in their cell wall compositions and have dolipore septa. This observation correlates with cell wall biochemistry and septal ultrastructure findings. The results of this study also indicate that mt cyt b gene sequences are effective for both species identification and determining phylogenetic relationships of Trichosporon species.
ACKNOWLEDGMENTS
This study was performed as part of the program "Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)" through the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (2001). We also thank the Institute for Fermentation, Osaka, Japan, for a grant for this study.
Present address: Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430.
Present address: Department of Pathogenobiology, School of Basic Medical Science, Norman Bethune Medical Division of Jilin University, 2 Xinmin Street, Changchun, Jilin, China.
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