Amplification of mitochondrial small subunit ribosomal DNA of ...

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such as classes or orders (Swann and Taylor 1993,. Wilmotte et al 1993). On the other ..... New York: John Wiley & Sons, Inc. 868 p. Bruns TD, Szaro TM. 1992.
Mycologia, 94(5), 2002, pp. 823–833. q 2002 by The Mycological Society of America, Lawrence, KS 66044-8897

Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis Soon Gyu Hong Wonjin Jeong

Ryvarden 1991). However, overlapping and variable morphological characteristics have made the classification of polypores unreliable and unstable, which has been always a nuisance to mycologists (Alexopoulos et al 1996, Hibbett and Donoghue 1995). Molecular systematics has been shown to be a valuable tool in modern fungal taxonomy (Bruns et al 1991, Mitchell et al 1995). Among various molecular methods including DNA-DNA hybridization, RFLP, and sequence analyses, phylogenetic analyses of amino acid or DNA sequences are known to have the highest resolving power (Bruns et al 1991). Due to ubiquitous occurrence and essential functions, DNA sequence data of 18S, 26S, ITS, and mitochondrial rDNAs are most frequently used in recent phylogenetic studies of eukaryotic cells. Sequences of 18S rDNAs are conserved and have been used in phylogenetic analyses of fungi of higher taxonomic ranks such as classes or orders (Swann and Taylor 1993, Wilmotte et al 1993). On the other hand, ITS rDNAs are so variable that they often cannot be aligned accurately between genera and are now commonly used in the systematics of species within a genus (Moncalvo et al 1995a, b, Yan et al 1995). However, mt SSU rDNAs were reported to evolve 16 times faster than 18S rDNAs (Bruns and Szaro 1992), but are less variable than ITS rDNAs. Thus they are believed to have a potential to fill phylogenetic gaps at a family level between those available from 18S and ITS rDNAs. The rDNA found in the nuclear genome of eukaryotes usually consists of tandem repeated units. It is generally considered that the rDNA arrays tend to be homogenized through concerted evolution (Hillis and Dixon 1991). Some examples, however, showed that an organism could have multiple forms of a gene cluster of different sequences (Carranza et al 1996, Tang et al 1996, O’Donnell and Cigelnik 1997, Fatehi and Bridge 1998). Therefore, phylogenies based on 18S or ITS rDNA should be verified by other sources of data. Sequences of mt SSU rDNA serve this purpose. Nearly full-length sequences of 18S and ITS rDNAs for fungi can be amplified by PCR, but only partial sequences of mt SSU rDNAs have been amplified (White et al 1989, Hibbett and Donoghue 1995) so far. For this reason, mt SSU rDNA sequences have not been popular among molecular systematists, and

Korean Collection for Type Cultures, Korean Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, Korea

Hack Sung Jung1 School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151742, Korea

Abstract: There has been a systematic need to seek adequate phylogenetic markers that can be applied in phylogenetic analyses of fungal taxa at various levels. The mitochondrial small subunit ribosomal DNA (mt SSU rDNA) is generally considered to be one of the molecules that are appropriate for phylogenetic analyses at a family level. In order to obtain universal primers for polypores of Hymenomycetes, mt SSU rRNA genes were cloned from Bjerkandera adusta, Ganoderma lucidum, Phlebiopsis gigantea, and Phellinus laevigatus and their sequences were determined. Based on the conserved sequences of cloned genes from polypores and Agrocybe aegerita, PCR primers were designed for amplification and sequencing of mt SSU rDNAs. New primers allowed effective amplification and sequencing of almost full-sized genes from representative species of polypores and related species. Phylogenetic relationships were resolved quite efficiently by mt SSU rDNA sequences, and they proved to be more useful in phylogenetic reconstruction of Ganoderma than nuclear internal transcribed spacer (ITS) rDNA sequences. Key Words: Ganoderma, Hymenomycetes, information content, universal primers

INTRODUCTION

Currently the taxonomy of polypores is primarily based on morphological characteristics, such as the shapes of basidiocarps and hymenophores, hyphal systems, and forms and sizes of basidiospores, and secondarily on mycological features like host relationships and rot types (brown versus white) (Donk 1964, Accepted for publication May 15, 2002. 1 Corresponding author, Email: [email protected]

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TABLE I.

Fungal strains used for PCR amplification and sequencing mt SSU rDNA

Family Corticiaceae

Ganodermataceae

Species Cytidia salicina Lopharia spadicea Phlebiopsis gigantea

Strain No.a CBS 727.85 (KCTC 6997) CBS 474.48 (KCTC 6710) CBS 429.72 (KCTC 6706)

Pulcherricium caeruleum IFO 4974 (KCTC 6707) Punctularia atropurpurascens IFO 31076 (KCTC 6721) Ganoderma lucidum ATCC 64251 (KCTC 6283)

Ganoderma Ganoderma Ganoderma Ganoderma Ganoderma Ganoderma

lucidum lucidum oerstedii oerstedii oregonense pfeifferi

Ganoderma resinaceum Ganoderma tsugae Ganoderma valesiacum Hericiaceae Hericium coralloides Hymenochaetaceae Phellinus laevigatus Polyporaceae Bjerkandera adusta Junghuhnia nitida Melanoporia nigra Pycnoporus cinnabarinus Trametes versicolor

GenBank accession AF214458 AF214459 AF214479 AF214460 AF214461 AF214475

Restriction enzyme fragment or PCR fragment used PCR product PCR product Hpa I (2.4 kb), 59 end ; base 553 HindIII (1.8 kb), base 499 ; 39 end PCR product

CBS 270.81 (IMSNU 32114) ATCC 46755 (KCTC 6450) ATCC 52411 (KCTC 6286) ATCC 52409 (KCTC 6452) CBS 177.30 (IMSNU 32116) CBS 747.84 (KCTC 6512)

PCR product EcoRI (7.0 kb), 59 end ; base 715 1 (1506 nt intron) 1 716 ; base 1296 HpaI (3.7 kb) base 791 ; 39 end PCR product PCR product PCR product PCR product PCR product PCR product

CBS 842.72 (KCTC 6711) CBS 341.63 (KCTC 6848) IFO 31165 (KCTC 6725) CBS 292.33 (KCTC 6714)

PCR product PCR product PCR product PCR product HindIII (8.4 kb), 59 end ; 39 end HpaI (2.5 kb), 59 end ; base 897 EcoRI (2.0 kb), base 259 ; 39 end PCR product PCR product PCR product PCR product

AF214467 AF214468 AF214469 AF214470 AF214471 AF214477 AF214478 CBS 152.27 (IMSNU 32118) AF214472 ATCC 46754 (KCTC 6455) AF214473 AF214474 CBS 282.33 (KCTC 6513) IFO 7716 (KCTC 6722) AF214462 CFMR 5640 (IMSNU 30079) AF230363 IFO 4983 (KCTC 6717) AF214476 AF214463 AF214464 AF214465 AF042324

a Acronyms for culture collections: ATCC, American Type Culture Collection, Manassas, Virginia; CBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; CFMR, Center for Forest Mycology Research, Madison, Wisconsin; IFO, Institute for Fermentation, Osaka, Japan; IMSNU, Institute of Microbiology, Seoul National University, Seoul, Korea; KCTC, Korean Collection for Type Cultures, Taejon, Korea.

phylogenetic investigation of polypore fungi based on partial sequences of mt SSU rDNA has been found unsatisfactory (Hibbett and Donoghue 1995). In order to design universal primers for polypore fungi of Hymenomycetes, three polypore species, Bjerkandera adusta (Polyporaceae), Ganoderma lucidum (Ganodermataceae), and Phellinus laevigatus (Hymenochaetaceae), and one corticioid fungus, Phlebiopsis gigantea (Corticiaceae), were selected to clone mt SSU rDNAs. Based on conserved sequences of mt SSU rDNAs from B. adusta, G. lucidum, P. gigantea, P. laevigatus, and Agrocybe aegerita (Gonzalez et al 1997), primers were designed for amplification and sequencing of mt SSU rDNAs. In order to assess the phylogenetic informativeness of mt SSU rDNA, similarity value, number of informative sites, skewness, and CI index were used.

MATERIALS AND METHODS

Fungal strains. Strains used for PCR amplification and mt SSU rRNA gene cloning are listed in TABLE I. In order to compare information content of mt SSU rDNA with ITS rDNA, strains of Ganoderma for which ITS sequences are available (Moncalvo et al 1995a) were selected for determining mt SSU rDNA sequences. DNA isolation. For the isolation of mitochondrial DNA which was used in cloning the mt SSU rRNA gene, fungi were grown in 1 L of ME broth (1% malt extract, 0.5% yeast extract, 0.5% peptone) at 24 C for 2 wk with agitation. The mycelia were harvested by filtering, frozen with liquid nitrogen, and then homogenized with a mortar. Mitochondrial DNA was purified from total DNA by bisbenzimideCsCl gradient centrifugation (Raeder and Broda 1985). For the PCR amplification, nucleic acids were extracted from mycelia grown on ME agar covered with a cellophane disc

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according to the freeze-thawing method of Lecellier and Silar (1994). Cloning of mt SSU rDNA. DNA fragments digested with EcoRI, HindIII, or HpaI were ligated with the plasmid pBluescript KS(2) (Stratagene, La Jolla, California, USA). The recombinant plasmids were transformed into competent Escherichia coli DH5a. Dot blotting or PCR screenings were performed to identify recombinant clones containing the mt SSU rRNA genes. For dot blotting, DNA fragments containing the mt SSU rDNA of Trimorphomyces papilionaceus (Hong et al 1993, Jeong et al 1995) were used as probes. For the PCR screening, three sets of primers were used: BAMS1 (59-GTAAAAGCCTACCAAGCCGACG-39) and BAMS2 (59-TTGACAGTGAGGGGTTCATGGG-39) for B. adusta, GLMS1 (59-AACACAATAACCATTCCGCC-39) and GLMS2 (59-TTCCTTCTTCATGCCTCCG-39) for G. lucidum, and PGMS1 (59-TACTAGGGAGATTTCATTCC-39) and PGMS2 (59-TTTAATTTTGGTTCHGATTGAACG-39) for P. gigantea (FIG. 1). SSU rDNA amplification and sequence determination. Mitochondrial SSU rDNA was amplified using BMS05 and BMS173 or BMS173B primers (TABLE II). Amplified mt rDNAs were purified using Wizard PCR prep kit (Promega, Madison, Wisconsin, USA) and cycle-sequenced using internal primers (TABLE II) and Top DNA sequencing kit (Bioneer, Chungju, Korea). Sequences of cloned DNAs were determined by the dideoxy termination method (Sanger et al 1977) using Sequenase kit (Amersham, Buckinghamshire, England). Sequencing reactions were conducted following the supplier’s guide. Evaluation of information contents and phylogenetic potential. Nucleotide sequences of mt SSU rDNA were aligned using the molecular sequence editor PHYDIT 3.0 (formerly called AL16S, Chun 1995) that allows editing, manual, and semi-computer alignment, and comparative analysis of rDNA nucleotide sequence data according to secondary structure model information (available at http://plaza. snu.ac.kr/;jchun/phydit/). Ambiguously aligned sites caused by multiple insertions and deletions found in nine variable domains and stem 15 of secondary structure (FIG. 2) (Neefs et al 1993) were excluded from further analyses. The phylogenetic relationship of mt SSU rDNA sequences of Hymenomycetes were estimated by heuristic search option of PAUP*4.0 beta version (Swofford 1998) with random sequence addition. Skewness values were calculated from 10 000 000 random trees and decay indices by procedures presented by Bruns et al (1992). Sequences of ITS rDNA were aligned using CLUSTALX program (Thompson et al 1997) with a gap open penalty of 5 and a gap extension penalty of 1 and then manually adjusted. Phylogenetic relationship, consistency index, and skewness of tree distribution were estimated by exhaustive search of PAUP* 4.0 beta version (Swofford 1998) for ITS and mt SSU rDNAs of the genus Ganoderma. RESULTS

Cloning and sequencing of SSU rDNA. Screening of a B. adusta mitochondrial DNA library constructed in

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EcoRI site by dot blotting led to the selection of a plasmid carrying a 2.0 kb EcoRI fragment (BAE16, FIG. 1A). It was found that the clone lacked about 300 bp in the 59 region of the gene by the sequence alignment with the A. aegerita gene (Gonzalez et al 1997). By PCR screening of HpaI library using two primers, BAMS1 and BAMS2, a recombinant plasmid carrying 2.5 kb HpaI fragment was selected (BAHP1, FIG. 1A). Cloning of the mt SSU rRNA gene from G. lucidum was performed with the same procedure as the one for B. adusta. Two plasmids carrying mt SSU rRNA gene were selected; a plasmid carrying 7.0 kb EcoRI fragment (GLE78) and a plasmid carrying 3.7 kb HpaI fragment (GLHP1, FIG. 1B). The mt SSU rRNA gene of G. lucidum was found to have a 1506 nt group II intron between the positions 715 and 716 corresponding to the positions 788 and 789 of 16S rDNA of E. coli, which was deduced by comparing with the sequence alignment of E. coli and constructing a secondary structure model of the intron (data not shown). Two clones encoding the SSU rRNA gene of P. gigantea were obtained from HindIII and HpaI libraries. The clones contained a 1.8 kb HindIII fragment (PGH13) and a 2.4 kb HpaI fragment (PGHP1) each (FIG. 1C). The sizes of the mt SSU rRNA of B. adusta, G. lucidum and P. gigantea were deduced as 1831, 1553, and 1892 bp respectively from sequence alignment with the mt SSU rDNA sequence of A. aegerita (Gonzalez et al 1997). Design of primers. The sequences of A. aegerita, B. adusta, G. lucidum, and P. gigantea were aligned with 16S rDNA of E. coli based on the secondary structure model, and it was found that each of the sequences had multiple insertions or deletions in different regions of the gene (FIG. 2). The mt SSU rRNA gene of B. adusta had long insertions in the V2, V4, V8, and V9 regions (Neefs et al 1993). The gene was shorter in the V1, V3, V5, and V7 regions compared to that of E. coli. The V6 region of the gene was slightly longer than that of E. coli. The gene from G. lucidum had a long insertion in the V9 region. It was slightly longer in the V1, V5, V6, and V8 regions and shorter in the V2, V3, V4, and V7 regions than that of E. coli. The gene of P. gigantea had long insertions in the V2, V6, V8, and V9 regions. It was slightly longer in the V1 region and shorter in the V3, V4, V5, and V7 regions than that of E. coli. Although variable domains of the gene had multiple insertions or deletions as described above, the conserved domains were highly conserved among A. aegerita, B. adusta, G. lucidum, and P. gigantea. Primers were designed to amplify and determine the sequence of SSU rRNA genes based on conserved sequences. Requirements for primers were as follows: They were conserved in all compared sequences, no

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MYCOLOGIA Primers used in PCR amplification and sequencing

Primer

Direction

BMS05 BMS35 BMS55 BMS65 BMS105a BMS105B BMS115 BMS145a BMS155 BMS33 BMS53 BMS63a BMS63B BMS103a BMS103B BMS113 BMS133a BMS133B BMS153a BMS153B BMS173a BMS173B

Forward Forward Forward Forward Forward Forward Forward Forward Forward Reverse Reverse Reverse Reverse Reverse Reverse Reverse Reverse Reverse Reverse Reverse Reverse Reverse

59 to 39 Sequence TTAATTTTGGTTCNGATTGAACG GGGTAAAGGCCTACCAAGCC GAGTGAACTAGCTAACTGAAATC CTGGTGCCAGAAGACTCGGTAA ATTAGTCGGTCTCGAAGCAAACG CCATTCCGCCTTGTGAGTAC TACAGGTGTTGCATGGCTGTC AGCTAATCATTAAAAAAAGT TGAACTAACTGTCTGTCGCTGG GGCTTGGTAGGCCTTTACCC GATTTCAGTTAGCTAGTTCACTC TTACCGAGTCTTCTGGCACCAG CTTACCGAGTCTTCTGGCAC CACTTCGTTTGCTTCGAGACCGAC GTACTCACAAGGCGGAATGG GACAGCCATGCAACACCTGTA GGGCCATAACGACTTGTCTTAGC CATNAGGGCCATAACGACTTGTC CGACAGACAGTTAGTTCATCACG AGCGACAGACAGTTAGTTCATC TGCTATGACTTTTGAGATGTTAC CTACCNTGCTATGACTTTTG

Position in E. coli 13–15 25–277 384–406 512–533 919–939 874–893 1043–1063 1275–1294 1391–1412 Compl. 258–277 Compl. 384–406 Compl. 512–533 Compl. 515–534 Compl. 874–893 Compl. 922–994 Compl. 1043–1063 Compl. 1187–1210 Compl. 1193–1215 Compl. 1386–1408 Compl. 1389–1410 Compl. 1480–1502 Compl. 1489–1508

a Primers did not work or were not conserved for some species and can be replaced by those labeled B after the name. However, BMS145 can be replaced by BMS155.

significant hairpin loops were formed in secondary structure prediction, primer-dimers were not formed by each pair of forward and reverse primers, so that any DNA regions targeted could be amplified, and DNA sequences could be read bidirectionally assuming that 300 bps were read by one sequencing reaction (BMS05, BMS35, BMS55, BMS65, BMS105, BMS115, BMS145, BMS33, BMS53, BMS63, BMS103, BMS113, BMS133, BMS153, BMS173, TABLE II). PCR reactions with primers BMS05 and BMS173 produced DNAs from 1.5 to 1.9 kb long for species of TABLE I from various families of Aphyllophorales except for Cytidia salicina, Phellinus laevigatus, and Pulcherricium caeruleum of the Corticiaceae and Hymenochaetaceae. In order to know which primer was fit for these template DNAs, we tested the fidelity of primers to template DNA by PCR reactions using four other primer sets, BMS05 and BMS113, BMS05 and BMS133, BMS55 and BMS173, and BMS65 and BMS173. Of the four sets of primers tested, two PCR reactions using BMS173 primer did not produce any DNA fragments, suggesting that the BMS173 primer did not work for some DNA templates from Aphyllophorales. In order to find DNA regions conserved in these species and to improve fidelity of primers, mt SSU rDNA from Phellinus laevigatus was cloned. The selected clone contained an 8.4 kb HindIII frag-

ment. Alignment of P. laevigatus sequence with four previously determined mt SSU rDNA sequences revealed that second and third base positions (A and T) from the 39 end of BMS173 primer were not conserved in P. laevigatus. Thirteen internal primers were generally good for determining sixteen mt SSU rDNA sequences from the strains listed in TABLE I except for C. salicina, P. laevigatus and P. caeruleum. However, sequencing reactions using two primers, BMS63 and BMS145, were not satisfactory for J. nitida, L. spadicea, M. nigra, and P. atropurpurascens. In the cases of BMS105, BMS103, BMS133, and BMS153, aligned sequences had non-conserved sites in the primer regions even though sequencing with these primers was successful. To improve fidelity of primers to template DNAs, primers were modified based on additional 17 mt SSU rDNA sequences (BMS105B, BMS155, BMS63B, BMS103B, BMS133B, BMS153B, and BMS173B, TABLE II). These primers were good for PCR amplification and sequencing of mt SSU rDNA from C. salicina, J. nitida, L. spadicea, M. nigra, P. laevigatus, P. caeruleum, and P. atropurpurascens. Two external primers, BMS05 and BMS173B, were designed based on conserved sequences of five species of distant evolutionary relationships (Hibbett and Donoghue 1995, Hibbett et al 1997). PCR amplifica-

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FIG.1. Organization and restriction map of the mt DNA region encoding SSU rRNA. Clones BAE16, GLE78, and PGH13 were selected by dot-blotting and BAHP1, GLHP1, and PGHP1 by PCR screening using primers indicated by arrows. Primers BAMS1, BAMS2, GLMS1, GLMS2, and PGMS1 were designed using flanking sequences between EcoRI and HpaI sites. PGMS2 was designed based on the conserved sequences between Bjerkandera adusta and Ganoderma lucidum. Sequences of primers were presented in the Materials and Methods. Restriction sites abbreviations: D, HindIII; E, EcoRI; H, HpaI.

tion tests using these two primers on Amanita verna (Amanitaceae, Agaricales), Pleurotus ostreatus (Pleurotaceae, Agaricales), Coniophora puteana (Coniophoraceae, Aphyllophorales) and Hymenochaete tabacina (Hymenochaetaceae, Aphyllophorales) were satisfactory (data not shown), suggesting a possibility that they work well for a wide range of fungi from Hymenomycetes, including Agaricales and Aphyllophorales. DISCUSSION

By aligning mt SSU rDNA sequences from fourteen fungal species of Hymenomycetes and 16S rDNA of E. coli based on the secondary structure model, it was found that the sequences had multiple insertions and deletions in the variable domains (FIG. 2). Large in-

sertions or deletions were found in the V2, V4, V6, V8, and V9 domains. The other variable domains also had multiple insertions or deletions, so that sequence alignment was not possible except among the closely related taxa. Stem 15 of the secondary structure model was previously known to be a very conserved domain (Neefs et al 1993). However, J. nitida and M. nigra had multiple insertions in this region. The two species did not form a monophyletic group in the phylogenetic analysis (FIG. 3A) and the two insertion sequences could not be aligned. Therefore, it is speculated that such large insertions in stem 15 of those two species could have been obtained by independent insertion events of two different origins. Similarity values among fourteen species of Hymenomycetes for the 1085 aligned sites are presented

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FIG. 2. Map of mt SSU rDNA from fourteen Hymenomycetes species and 16S rRNA of E. coli. Nine variable domains and stem 15 region were indicated as open boxes.

in TABLE III. Similarity values ranged from 83.93% between A. aegerita and P. laevigatus to 99.22% between B. adusta and L. spadicea. High similarity values from 96.8% to 98.55% were found in four closely related species, C. salicina, G. lucidum, P. cinnabarinus, and T. versicolor (also see FIG. 3). Among these species, 435 additional sites were alignable in variable domains. Informative sites were increased three times, from seven to twenty-one. Phylogenetic analysis of 1085 aligned sites of fourteen mt SSU rDNAs yielded one most parsimonious tree of 764 steps, a skewness value of g1 5 20.8512

(p , 0.01) and a consistency index of 0.664. The number of informative sites was 221 (FIG. 3A). The deeply left-skewed data structure of the mt SSU rDNA indicates that the data have non-random structures that likely reflect phylogenetic signal (Hillis and Huelsenbeck 1992). The high similarity values also indicated that mt SSU rDNA sequences of Hymenomycetes are not saturated with substitutions. Two inferred monophyletic groups, which were supported by high bootstrap values and decay indices, were recognized by the phylogenetic analysis. One group includes C. salicina, G. lucidum, P. cin-

TABLE III. Similarity values (lower left) and different/total nucleotides (upper right) of conserved domains among 14 mt SSU rDNA sequences

A. aeger C. salic L. spadi P. gigan P. caeru P. atrop G. lucid H. coral P. laevi B. adust J. nitid M. nigra P. cinna T. versi

A. aeger

C. salic

L. spadi

P. gigan

P. caeru

P. atrop

G. lucid

— 84.68 86.83 83.98 85.91 84.7 84.09 85.74 83.93 86.88 85.74 84.76 84.37 84.84

158/1031 — 88.72 85.55 85.74 85.41 98.55 87.62 85.02 88.76 86.05 86.82 97.29 97

135/1025 116/1028 — 92.03 93.59 87.32 88.52 89.5 87.88 99.22 88.04 88.73 89.01 89.01

165/1030 149/1031 82/1029 — 92.44 85 85.06 86.92 84.91 92.06 85.74 86.23 85.84 85.74

145/1029 147/1031 66/1030 78/1032 — 84.92 85.16 87.11 85.87 93.42 85.17 86.72 86.23 85.65

157/1026 150/1028 130/1025 154/1027 155/1028 — 85.12 89.03 86.65 87.56 86.31 85.42 85.41 85.6

164/1031 15/1034 118/1028 154/1031 153/1031 153/1028 — 87.04 84.34 88.57 85.08 86.53 96.9 96.8

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FIG. 3. Phylogenetic analysis of full-length (A) and partial (B) mt SSU rDNA sequences of Hymenomycetes and distribution of tree lengths for 10 000 000 random trees. The consensus tree of three equally most parsimonious trees was presented for MS1/MS2 region of mt SSU rDNA. The phylogenetic tree was obtained with heuristic search of PAUP* 4.0 beta. Bootstrap values from a sample of 1000 replicates and decay values are shown at internal branches (bootstrap value/decay value).

nabarinus, and T. versicolor. The species, M. nigra, was found to be related to this group by relatively high bootstrap value and decay index data. Two genera, Pycnoporus and Trametes, previously were recogTABLE III.

nized as closely related taxa based on the morphological characteristics such as a di-trimitic hyphal system, generative hyphae with clamps, hyaline, thinwalled, cylindric, smooth, and non-amyloid basid-

Extended

H. coral

P. laevi

B. adust

J. nitid

M. nigra

P. cinna

T. versi

147/1031 128/1034 108/1029 135/1032 133/1032 113/1030 134/1034 — 88.64 89.55 88.2 86.06 87.8 87.61

165/1027 154/1028 124/1023 155/1027 145/1026 137/1026 161/1028 117/1030 — 87.83 86.5 83.95 85.39 85.59

135/1029 116/1032 8/1031 82/1033 68/1034 128/1029 118/1032 108/1033 125/1027 — 88.28 89.06 89.05 89.15

147/1031 144/1032 123/1028 147/1031 153/1032 141/1030 154/1032 122/1034 139/1030 121/1032 — 85.87 86.42 86.61

157/1030 136/1032 116/1029 142/1031 137/1032 150/1029 139/1032 144/1033 165/1028 113/1033 146/1033 — 87.31 87.6

161/1030 28/1033 113/1028 146/1031 142/1031 150/1028 32/1033 126/1033 150/1027 113/1032 140/1031 131/1032 — 97.29

156/1029 31/1032 113/1028 147/1031 148/1031 148/1028 33/1032 128/1033 148/1027 112/1032 138/1031 128/1032 28/1032 —

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FIG. 4. Phylogenetic analyses of ITS (A) and mt SSU rDNAs (B) from Ganoderma and distribution of tree lengths for all possible trees. The consensus tree of 4 equally most parsimonious trees was presented for ITS sequences. The phylogenetic trees were obtained with exhaustive search of PAUP* 4.0 beta. Bootstrap values from a sample of 1000 replicates and decay indices are shown at internal branches (bootstrap value/decay index).

iospores, absence of cystidia, formation of white rot, tetrapolarity, and growth almost exclusively on angiosperms (Ryvarden 1991). The genus Ganoderma also has the same morphological characteristics except that it forms double-wall basidiospores (Gilbertson and Ryvarden 1987). The close phylogenetic relationship of C. salicina with three poroid fungi, G. lucidum, P. cinnabarinus, and T. versicolor, was not supported by morphological characteristics because it is a resupinate fungus with smooth hymenophores, monomitic hyphal system, numerous dendrohyphidia, and allantoid basidiospores. Concrete conclusions await analysis of other strains and related species. The genus Melanoporia has similar morphological characteristics except that it causes brown rot (Gilbertson and Ryvarden 1987). However, it is premature to include M. nigra in this group because the similarity values among M. nigra and other species of this group were not high (TABLE III). The other monophyletic group includes B. adusta, L. spadicea, P. caeruleum and P. gigantea. The genus Bjerkandera has been classified in the Polyporaceae by Donk (Donk 1964) based on the poroid hymenop-

hores. However, a close relationship of B. adusta to corticioid fungi has been presented in subsequent phylogenetic studies (Hibbett and Donoghue 1995, Hibbett et al 1997). It is evident that B. adusta, a poroid fungus, is closely related to three corticioid fungi, and C. salicina, a corticioid fungus, is closely related to three poroid fungi based on phylogenetic relationships (FIG. 3A) and similarity values (TABLE III). Although it was not possible to reach concrete taxonomic conclusions with such a small number of taxa, it is obvious that mt SSU rDNA sequences contain considerable information, enough to resolve phylogenetic relationships among fungal species of Hymenomycetes. In terms of sequence lengths, partial mt SSU rDNA sequences amplified by MS1 and MS2 primers (White et al 1989) for the set of same taxa had 68 informative sites out of total 319 unambiguously aligned sites. Heuristic search of the sequences produced three equally parsimonious trees of 257 steps (FIG. 3B). The consensus tree had polytomous unresolved branches. Bootstrap values and decay indices were much lower in a number of branches than those from the tree of full-length mt

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FIG. 5. Sequence alignment of V4 domain (stem P23-1) from nine Ganoderma strains and three sister taxa. The boxed regions (1 and 2) base pair with those of opposite sites (19 and 29).

SSU rDNA. Distribution of random trees was less leftskewed than the one of full-length mt SSU rDNA sequences. From these results, it is apparent that sequences of full-length mt SSU rDNA are increasing phylogenetic information and resolving power considerably more than partial sequences. Analysis of 476 aligned sites of ITS sequences from 9 strains of Ganoderma yielded 4 most parsimonious trees of 47 steps, a skewness value of g1 5 22.1366 (p , 0.01), a consistency index of 1.000 and 18 informative sites. The strict consensus tree was presented in FIG. 4A. Analysis of 1469 aligned sites of mt SSU rDNA for the same suite of taxa yielded one parsimonious tree of 111 steps, a skewness value of g1 5 21.5850 (p , 0.01) and a consistency index of 0.865. The number of informative sites was 62 (FIG. 4B). The number of most parsimonious trees itself was regarded as a poor indicator of data quality in phylogenetic analyses because a single solution can be found from random data with high probability (Hillis and Huelsenbeck 1992). However, ITS rDNA sequences of Ganoderma have only 19 informative sites and the poor resolution of the phylogenetic relationship is likely to have originated from the poverty of phylogenetic information. On the other hand, parsimony analysis of mt SSU rDNAs produced one most parsimonious tree, which had a relatively high value of CI, bootstrap values, and decay indices. The number of informative sites was 3.3 times as many as that of ITS rDNA sequences. Eighty-four percent of informative sites (52/62) were distributed in nine variable domains. Average similarities among nine sequences of conserved domains and variable domains of mt SSU rDNA and ITS rDNA were 99.43, 94.51, and 96.67% each. The rate of evolutionary change of variable domains of mt SSU rDNA was found to be faster than ITS rDNA.

Multiple insertions or deletions were found in the V4 domain of mt SSU rDNA. The sequence alignment of these domains was presented in FIG. 5. The sister taxa of the genus Ganoderma, namely C. salicina, P. cinnabarinus, and T. versicolor, had homologous sequences with G. lucidum ATCC 46755 and 5 other strains with long sequences. Therefore, it is concluded that multiple deletion events caused the short V4 domain of G. oerstedii ATCC 52411, G. pfeifferi CBS 747.84 and G. resinaceum CBS 152.27. It is not certain whether additional deletion events occurred in G. oerstedii ATCC 52411 or multiple insertion events occurred in a common ancestor of G. pfeifferi CBS 747.84 and G. resinaceum CBS 152.27 after large deletions in a common ancestor of the three species. Multiple insertions or deletions of mt SSU rDNA were observed also within the genus Agrocybe (Gonzalez and Labarere 1998) and Suillus (Bruns and Szaro 1992). By comparative analysis of the secondary structures of variable domains from the genus Agrocybe, it was proposed that molecular studies of variable domains could be a good alternative method for determining relationships between species (Gonzalez and Labarere 1998). In the preliminary study of the genus Ganoderma, we also conclude that the insertion or deletion events are congruent with the phylogenetic analysis of primary sequences, and that analysis of multiple insertion and deletion events could be a good phylogenetic marker. From phylogenetic analyses of several genera of Hymenomycetes and strains of the genus Ganoderma, it is clear that mt SSU rDNA sequences can be used as molecular markers to resolve phylogenetic relationships of both higher and lower rank of taxa, because the region is composed of conserved and variable domains. In addition, insertion and deletion events as well as the primary sequence can be used

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as important molecular markers to resolve phylogenetic relationships. ACKNOWLEDGMENTS

The authors are grateful to Dr. K.S. Bae of KCTC (Korean Collection for Type Cultures), KRIBB (Korea Research Institute for Bioscience and Biotechnology), who kindly provided fungal strains for research collaboration. This work was supported by the Brain Korea 21 Project of the Ministry of Education and Human Resources Development and partly by the Young Scientist Research Grant of the Korea Research Foundation.

LITERATURE CITED

Alexopoulos CJ, Mims CW, Blackwell M. 1996. Introductory mycology. 4th ed. New York: John Wiley & Sons, Inc. 868 p. Bruns TD, Szaro TM. 1992. Rate and mode differences between nuclear and mitochondrial small-subunit rRNA genes in mushrooms. Mol Biol Evol 9:836–855. ———, Vilgalys R, Barns SM, Gonzalez D, Hibbett DS, Lane DJ, Simon L, Stickel S, Szaro TM, Weisburg WG, Sogin ML. 1992. Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences. Mol Phylogenet Evol 1:231–241. ———, White TJ, Taylor JW. 1991. Fungal molecular systematics. Annu Rev Myco Syst 22:525–564. Carranza S, Giribet G, Ribera C, Baguna J, Riutort M. 1996. Evidence that two types of 18S rDNA coexist in the genome of Dugesia (Schmidtea) mediterranea (Platyhelminthes, Turbellaria, Tricladida). Mol Biol Evol 13: 824–832. Chun J. 1995. Computer-assisted classification and identification of actinomycetes [PhD Thesis]. Newcastle, UK: University of Newcastle upon Tyne. 416 p. Donk MA. 1964. A conspectus of the families of Aphyllophorales. Persoonia 3:199–324. Fatehi J, Bridge P. 1998. Detection of multiple rRNA-ITS regions in isolates of Ascochyta. Mycol Res 102:762–766. Gilbertson RL, Ryvarden L. 1987. North American polypores. Vol. 2. Megasporoporia–Wrightoporia. Oslo, Norway: Fungiflora. 451 p. Gonzalez P, Barroso G, Labarere J. 1997. DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita. Gene 184:55–63. ———, Labarere J. 1998. Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe. Appl Environ Microbiol 64: 4149–4160. Hibbett DS, Donoghue MJ. 1995. Progress toward a phylogenetic classification of the Polyporaceae through parsimony analysis of mitochondrial ribosomal DNA sequences. Can J Bot 73(Suppl 1):S853–S861. ———, Pine EM, Langer E, Langer G, Donoghue MJ. 1997.

Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences. Proc Natl Acad Sci USA 94:12002–12006. Hillis DM, Dixon MT. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Q Rev Biol 64:411– 453. ———, Huelsenbeck JP. 1992. Signal, noise, and reliability in molecular phylogenetic analyses. J Hered 83:189– 195. Hong SG, Kang YW, Jung HS. 1993. Sequence analysis of the small subunit ribosomal RNA gene of Trimorphomyces papilionaceus mitochondria. Kor J Microbiol 31: 471–477. Jeong WJ, Hong SG, Kang YW, Jung HS. 1995. Restriction and transcription maps of mitochondrial DNA of Trimorphomyces papilionaceus. J Microbiol 33:149–153. Lecellier G, Silar P. 1994. Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Curr Genet 25:122–123. Mitchell JI, Roberts PJ, Moss ST. 1995. Sequence or structure?: a short review on the application of nucleic acid sequence information to fungal taxonomy. Mycologist 9:67–75. Moncalvo JM, Wang HF, Hseu RS. 1995a. Gene phylogeny of the Ganoderma lucidum complex based on ribosomal DNA sequences. Comparison with traditional taxonomic characters. Mycol Res 99:1489–1499. ———, ———, ———. 1995b. Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87:223–238. Neefs JM, Van de Peer Y, De Rijk P, Chapelle S, De Wachter R. 1993. Compilation of small ribosomal subunit RNA structures. Nucl Acids Res 21:3025–3049. O’Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116. Raeder U, Broda P. 1985. Rapid preparation of DNA from filamentous fungi. Letters Appl Microbiol 1:17–20. Ryvarden L. 1991. Genera of polypores: nomenclature and taxonomy. Syn Fung 5:1–363. Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467. Swann EC, Taylor JW. 1993. Higher taxa of basidiomycetes: an 18S rRNA gene perspective. Mycologia 85:923–936. Swofford DL. 1998. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland, Massachusetts: Sinauer Associates. Tang J, Toe` L, Back C, Unnasch TR. 1996. Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex. Mol Biol Evol 13:244–252. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882. White TJ, Bruns T, Lee S, Taylor J. 1989. Amplification and direct sequencing of fungal ribosomal RNA genes for

HONG

ET AL:

AMPLIFICATION

OF MT

phylogenetics. In: Innis M, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press Inc. p 315–322. Wilmotte A, van de Peer Y, Goris A, Chapelle S, de Baere R, Nelissen B, Neefs JM, Hennebert GL, de Wachter R.

SSU RDNA

FROM POLYPORES

833

1993. Evolutionary relationships among higher fungi inferred from small ribosomal subunit RNA sequence analysis. System Appl Microbiol 16:436–444. Yan ZH, Rogers SO, Wang CJK. 1995. Assessment of Phialophora species based on ribosomal DNA internal transcribed spacers and morphology. Mycologia 87:72–83.