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Varga et al. ARTICLE Table 1. The Aspergillus section Sparsi isolates examined in this study. Species Strain No. Origin A. amazonicus CBS 124228 T = E19D Soil, Makas, Ecuador A. anthodesmis CBS 552.77 T = NRRL 22884 Soil, Ivory Coast A. biplanus CBS 468.65 T = NRRL 5071 Soil, Tilaran, Costa Rica A. biplanus CBS 469.65 = NRRL 5073 Soil, Tilaran, Costa Rica A. biplanus NRRL 5072 Soil, Tilaran, Costa Rica A. conjunctus CBS 476.65 T = NRRL 5080 Forest soil, Palmar, Province of Punteras, Costa Rica A. diversus CBS 480.65 T = NRRL 5074 Soil, Esparta, Costa Rica A. ecuadorensis CBS 124229 T =E19F Soil, Makas, Ecuador A. funiculosus CBS 116.56 T = NRRL 4744 Soil, Ibadan, Nigeria A. haitiensis CBS 464.91 T = NRRL 4569 Soil under sage and cactus, Haiti A. haitiensis CBS 468.91 = NRRL 4568 Desert soil, Haiti A. implicatus CBS 484.95 T Soil, Ivory Coast A. panamensis CBS 120.45 T = NRRL 1785 Soil, Panama A. panamensis NRRL 1786 Soil, Panama A. quitensis CBS 124227 T = E19C Soil, Makas, Ecuador A. sparsus CBS 139.61 T = NRRL 1933 Soil, Costa Rica A. sparsus NRRL 1937 Soil, San Antonio, Texas, USA Isolation and analysis of nucleic acids The cultures used for the molecular studies were grown on malt peptone (MP) broth using 1 % (w/v) of malt extract (Oxoid) and 0.1 % (w/v) bacto peptone (Difco), 2 mL of medium in 15 mL tubes. The cultures were incubated at 25 °C for 7 d. DNA was extracted from the cells using the Masterpure yeast DNA purification kit (Epicentre Biotechnologies) according to the instructions of the manufacturer. Fragments containing the ITS region were amplified using primers ITS1 and ITS4 as described previously (White et al. 1990). Amplification of part of the b-tubulin gene was performed using the primers Bt2a and Bt2b (Glass & Donaldson 1995). Amplifications of the partial calmodulin gene were set up as described previously (Hong et al. 2005). Sequence analysis was performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit for both strands, and the sequences were aligned with the MT Navigator software (Applied Biosystems). All the sequencing reactions were purified by gel filtration through Sephadex G-50 (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in double-distilled water and analyzed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The unique ITS, b-tubulin, and calmodulin sequences were deposited at the GenBank nucleotide sequence database under accession numbers FJ491645–FJ491675, and FJ943936–FJ943941. Data analysis The sequence data was optimised using the software package Seqman from DNAStar Inc. Sequence alignments were performed by MEGA v. 4.0 (Tamura et al. 2007) and improved manually. For parsimony analysis, the PAUP v. 4.0 software was used (Swofford 2002). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed for all data sets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1000 bootstrap replications (Hillis & Bull 1993). An A. ochraceoroseus isolate belonging to section Ochraceorosei of subgenus Nidulantes (Peterson et al. 2008) was used as outgroup in these experiments. The alignments were deposited in TreeBASE () under accession number S11028. Results and discussion Phylogeny We examined the genetic relatedness of section Sparsi isolates using sequence analysis of the ITS region of the ribosomal RNA gene cluster, and parts of the calmodulin and b-tubulin genes. The calmodulin data set included 566 characters, with 288 parsimony informative characters. One of the 56 MP trees is shown in Fig. 1 (tree length: 741, consistency index: 0.7247, retention index: 0.8903). During analysis of a part of the b-tubulin gene, 494 characters were analysed, among which 196 were found to be parsimony informative. The single MP tree based on partial b-tubulin genes sequences is shown in Fig. 2 (length: 507 steps, consistency index: 0.7179, retention index: 0.8938). The ITS data set included 559 characters with 58 parsimony informative characters. One of the 702 MP trees is presented in Fig. 3 (tree length: 180, consistency index: 0.8056, retention index: 0.8763). Phylogenetic analysis of b-tubulin, calmodulin and ITS sequence data indicated that Aspergillus section Sparsi includes 10 species. Aspergillus biplanus and A. diversus are closely related to each other on all trees, while another 188 i m a f U N G U S
Polyphasic taxonomy of Aspergillus section Sparsi A. conjunctus NRRL 5080 A. conjunctus CBS 476.65 A. anthodesmis CBS 552.77 A. panamensis NRRL 1785 A. panamensis CBS 120.45 A. amazonicus CBS 124228 A. ecuadorensis CBS 124229 A. quitensis CBS 124227 A. diversus NRRL 5074 A. diversus CBS 480.65 A. biplanus NRRL 5072 A. biplanus NRRL 5071 A. biplanus NRRL 5073 A. biplanus CBS 468.65 A. biplanus CBS 469.65 A. implicatus CBS 484.95 A. funiculosus NRRL 4744 A. sparsus NRRL 1933 A. sparsus CBS 139.61 A. sparsus NRRL 1937 A. hai6ensis NRRL 4569 A. hai6ensis CBS 464.91 Fig. 1. One of the MP trees obtained based on A. hai6ensis NRRL 4568 phylogenetic analysis of calmodulin sequence data of A. hai6ensis CBS 468.91 Aspergillus section Sparsi. Numbers above branches A. ochraceoroseus CBS 550.77 are bootstrap values. Only values above 70 % are indicated. ARTICLE A. funiculosus NRRL 4744 A. diversus NRRL 5074 A. diversus CBS 480.65 A. panamensis NRRL 1785 A. panamensis CBS 120.45 A. panamensis NRRL 1786 A. anthodesmis CBS 552.77 A. conjunctus CBS 476.65 A. amazonicus CBS 124228 A. ecuadorensis CBS 124229 A. quitensis CBS 124227 A. implicatus CBS 484.95 A. sparsus NRRL 1933 A. sparsus CBS 139.61 A. sparsus NRRL 1937 A. hai6ensis NRRL 4569 A. hai6ensis CBS 464.91 A. hai6ensis NRRL 4568 A. hai6ensis CBS 468.91 A. ochraceoroseus CBS 550.77 A. biplanus NRRL 5073 A. biplanus CBS 469.65 A. biplanus NRRL 5071 A. biplanus NRRL 5072 A. biplanus CBS 468.65 Fig. 2. The single MP tree obtained based on phylogenetic analysis of b-tubulin sequence data of Aspergillus section Sparsi. Numbers above branches are bootstrap values. Only values above 70 % are indicated. v o l u m e 1 · n o . 2 189
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T h e G l o b a l M y c o l o g i c
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IMA vision My first opportunity to
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undescribed fungal diversity will r
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Fungi on German TV: Focus on black
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REPORTS IMC9 Edinburgh: a selection
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vier’s favour was that they publi
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Welcome to the pressure dome: inves
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REPORTS IMC9: Moments to reminisce
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IMA Medals awarded at IMC9 The high
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Society of America, in the form of
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ecognition based on several criteri
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8. Preparing agreed responses to In
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Physiological differences between w
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Trizodia acrobia, hyphae enveloping
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International Society for Fungal Co
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steering committee mentioned earlie
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Iranian Mycological Society It give
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The Forayer, and courses was missed
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accounts of studies of particular g
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62 nd Annual Applied Microscopy Con
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IMA Fungus · volume 1 · no 2: 101
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Johansonia al. 1990) and LSU1Fd (Cr
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Mycology in Egypt ACKNOWLEDGEMENTS
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Mycology in Egypt Aleem AA (1952b)
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