Mycoscience (2011) 52:53–58 DOI 10.1007/s10267-010-0066-3 SHORT COMMUNICATION The phylogenetic position of an Armillaria species from Amami-Oshima, a subtropical island of Japan, based on elongation factor and ITS sequences Yuko Ota • Mee-Sook Kim • Hitoshi Neda • Ned B. Klopfenstein • Eri Hasegawa Received: 21 May 2010 / Accepted: 30 July 2010 / Published online: 21 August 2010 Ó The Mycological Society of Japan and Springer 2010 Abstract An undetermined Armillaria species was col- Approximately 40 Armillaria species (Physalacriaceae, lected on Amami-Oshima, a subtropical island of Japan. Agaricales, Basidiomycota) are distributed worldwide The phylogenetic position of the Armillaria sp. was (Volk and Burdsall 1995), and some of these species cause determined using sequences of the elongation factor-1a root disease to plant species (Hood et al. 1991; Dai et al. (EF-1a) gene and the internal transcribed spacer (ITS) 2007). More than 600 species of woody and nonwoody region (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The plants are hosts of Armillaria spp. (Shaw and Kile 1991; phylogenetic analyses based on EF-1a and ITS sequences Fox 2000). In Japan, nine annulate species [A. cepistipes showed that this species differs from known Japanese taxa Velen., A. gallica Marxm. & Romagn., A. jezoensis J.Y. of Armillaria. The sequences of this species and A. novae- Cha & Igarashi, A. mellea subsp. nipponica J.Y. Cha & zelandiae from Southeast Asia were contained in a strongly Igarashi, A. nabsnona T.J. Volk & Burds., A. ostoyae supported clade, which was adjacent to a well-supported (Romagn) Herink (reported to be a synonym of A. solidipes sister clade containing A. novae-zelandiae from Australia Peck by Burdsall and Volk 2008), A. sinapina Be´rube´ & and New Zealand. Dessur., A. singula J.Y. Cha & Igarashi, and one un- described species (Nag. E)] and two exannulate species Keywords Armillaria fuscipes Á Armillaria novae- [A. ectypa (Fr.) Lamoure and A. tabescens (Scop.) Emel] zelandiae have been reported (Nagasawa 1991; Cha et al. 1992, 1994; Cha and Igarashi 1997; Ota et al. 1998, 2009; Kudo and Nagasawa 2003). These 11 species occur in warm-temperate to boreal areas in Japan (Ota et al. 1998, 2009). However, little information is available on Armillaria in subtropical Japan. Amami-Oshima (28°170N 129°220E) is a subtropical Y. Ota (&) Á H. Neda Forestry and Forest Products Research Institute, island in the Ryukyu Archipelago (Nansei Islands) located Matsunosato 1, Tsukuba, Ibaraki 305-8687, Japan approximately 300 km north of Okinawa Island and e-mail: [email protected] 380 km south of Kyushu. The vegetation is typical for evergreen forests of this region, dominated by Castanopsis M.-S. Kim Department of Forestry, Environment, and Systems, sieboldii (Makino) Hatus, Quercus miyagi Koidz, and Kookmin University, Seoul 136-702, Republic of Korea Distylium racemosum Sieb. et Maxim., along with a sec- ondary planted forest composed of Pinus luchuensis Mayr. N. B. Klopfenstein In 1998, an Armillaria sp. was collected on Amami-Oshima. USDA Forest Service, RMRS, 1221 South Main Street, Moscow, ID 83843, USA The morphological characteristics of the basidiocarp were distinct from all previously reported species from the E. Hasegawa warm-temperate to boreal regions of Japan. Recently, Kansai Research Center, sequences of elongation factor-1 alpha (EF-1a) gene and Forestry and Forest Products Research Institute, 68 Nagai-Kyutaro, Momoyama, Fushimi, ribosomal DNA (rDNA regions) [internal transcribed Kyoto 612-0855, Japan spacer (ITS) and intergenic spacer-1 (IGS-1)] have been 123 54 Mycoscience (2011) 52:53–58 used for phylogenetic studies of Armillaria spp. (Maphosa of each primer, 2–2.5 mM of each deoxyribonucleotide et al. 2006; Antonı´n et al. 2009). More recently, Hasegawa triphosphate (dNTP), and 0.5 U Takara Taq or ExTaq et al. (2010) showed that the EF-1a gene was the most (Takara, Tokyo, Japan). The polymerase chain reaction suitable region for identifying eight species of Japanese (PCR) condition was as follows: 94°C for 1 min, 30 cycles Armillaria. Advantages of using EF-1a compared with at 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min. The ITS and IGS-1 regions for phylogenetic analyses of PCR products were purified with MicroSpin Columns and Armillaria are: (1) it is easier to align the sequence data Sephacryl S-300 (GE Healthcare, Piscataway, NJ, USA) because indels are less frequent than those found in the and sequenced with an ABI PRISM 3100 Genetic Analyzer ITS and IGS regions, and (2) EF-1a contains sufficient (Applied Biosystems, Foster City, CA, USA) in both sequence variation to distinguish among closely related directions to ensure accuracy. The sequences are deposited species. in the DNA Data Bank of Japan (accession numbers: The objective of this study was to clarify the phyloge- AB558990–AB559004 and AB36893, Table 1). netic position of an Armillaria sp. collected on Amami- The sequence data reported by Hasegawa et al. (2010) Oshima, a subtropical island of Japan, using sequences of was used for eight known Japanese species (A. cepistipes, EF-1a and ITS region. Two isolates of Armillaria sp. were A. gallica, A. mellea, A. nabsnona, A. ostoyae, A. sinapina, collected from Amami-Oshima, Kagoshima Prefecture, A. tabescens) and Nagasawa’s E (Nag. E). Nag. E is a from basidiocarp tissue and multiple spores and established Japanese Armillaria species reported by Nagasawa (1991) in culture (Table 1). For comparisons, A. ectypa from without valid description. Armillaria jezoensis and A. sin- Japan was also sequenced (Table 1). Two isolates of gula were not used for DNA analysis because cultured Tricholoma matsutake and T. magnivelare (Table 1) were isolates could not be found. Some sequences from Gen- analyzed as outgroups in the preliminary analysis. The Bank, which showed the highest similarities with the examined isolates are deposited in the culture banks of the examined isolates according to a BLAST search, were also Forestry and Forest Products Research Institute (FFPRI) in used for the analyses. Sequence alignments were examined Tsukuba, Ibaraki, Japan, and the Microbial Ecology Lab of using Clustal X (Jeanmougin et al. 1998) followed by FFPRI; Japanese voucher specimens have been deposited visual editing and alignment using BioEdit (Hall 1999). in the Mycological Herbarium of FFPRI (TFM). All gaps and ambiguously aligned regions were removed Isolates were grown on liquid MYG medium [2% (w/v) for analyses. The sequence alignments were deposited in malt extract, 0.2% (w/v) yeast extract, 2% (w/v) glucose] at TreeBase as submission no. S10511. Phylogenetic analyses 25°C in the dark, and mycelia were harvested after 10 days were conducted using PAUP* ver. 4.0b10 (Swofford 2002) in culture. DNA was extracted from cultured mycelia using for the neighbor-joining (NJ) method, and PhyML (Guin- a DNeasy extraction kit (Qiagen, Valencia, CA, USA) fol- don and Gascuel 2003) was used for the maximum likeli- lowing the manufacturer’s protocol. Primers EF595F/ hood (ML) method. NJ trees were generated using p, K2P, EF1160R (Maphosa et al. 2006) and ITS1-F/ITS4-B (Gardes and HKY85 distances. The confidence for the internal and Bruns 1993) were used to amplify a portion of the EF- branches of the resulting trees were tested statistically 1a gene and the ITS region, respectively. Each 20-ll using bootstrap analysis (Felsenstein 1985), with 1,000 reaction mixture contained 10 ng of template DNA (or no bootstrap replications through a FastStep search using the DNA template for a negative control), 10 mM Tris– default settings. For ML trees, the initial tree was con- hydrochloride (HCl) (pH 8.3), 50 mM potassium chloride structed by BioNJ; the HKY85 substitution model was used (KCl), 1.5 mM magnesium chloride (MgCl2), 0.1–0.2 mM with an estimated proportion of invariant sites (0.515 for Table 1 Isolates used in this study Species Isolate Original Location Host Specimen EF accession ITS accession no. no. no. (TFM) no. no. Armillaria sp. 00-13 S841 Amami, Kagoshima, Japan Decayed wood 26911 AB558990 AB558998 Armillaria sp. 00-14 S842 Amami, Kagoshima, Japan Decayed wood AB558991 AB558999 A. ectypa Je-2 Aomori, Japan Phragmites australis 27105 AB558992 AB559001 Je-4 Aomori, Japan Phragmites australis 27109 AB558993 AB559002 Je-7 Aomori, Japan Phragmites australis 27212 AB558994 AB559003 Je-9 Aomori, Japan Phragmites australis 27213 AB558995 AB559004 Tricholoma matsutake Tmm4 Tm029 Shiga, Japan Unknown AB558996 AB559005 T. magnivelare Tmm27 Tp-C3 Canada Unknown AB558997 AB036893 123 Mycoscience (2011) 52:53–58 55 100/100 2006-20-01 (AB510807) 2002-06-03 (AB510806) 96-1-8 (AB510804) A. tabescens 96- 3- 3 (AB510805) 100/100 Je-2 Je-4 Je-7 A. ectypa Je-9 100/100 CMW4980 (DQ435648) Australia A. hinnulea 83/86 CMW4981 (DQ435649) Australia 99/97 CMW4967(DQ425651) Australia 100/99 CMW4722 (DQ435652) New Zealand A. novae-zelandiae 89/84 PNZ87.54(FJ618650) PNG CMW4143 (DQ435654) Indonesia 99/80 00-14 99/97 00-13 96-37-1 (AB510769) Armillaria sp. from Amami-Oshima 2003-71-13 (AB510767) 100/100 94-2-1 (AB510768) 94-35-01 (AB510770) 2000-23-02 (AB510773) Nag.E NE4 (AB510771) 94-43-08(AB510772) -/81 NA4 (AB510761) NA17 (AB510759) 94/91 NA13 (AB510760) A. gallica 100/100 00-4-4 (AB510762) NB3(AB510763) 75/93 00-3-1 (AB510766) NB4 (AB510764) A. nabsnona 00-16-4 (AB510765) A-12 (AB510801) 100/100 A-10 (AB510800) 94-7 (AB510799) 94-5 (AB510802) 89-07 (AB510796) A. mellea var. nipponica 97-6 (AB510797) 94-10-1 (AB510798) 94-68 (AB510803) 91-01-10 (AB510785) 100/100 NC8 (AB510782) 2002-66-3 (AB510781) 89-03B-09 (AB510780) 94-75-07 (AB510779) A. ostoyae 94-8-2 (AB510783) -/84 88-01-19 (AB510784) 96-7-1 (AB510774) 97/95 05-13-2 (AB510776) 96-8-1 (AB510777) A.