Rasamsonia Composticola, a New Thermophilic Species Isolated from Compost in Yunnan, China

Mycol Progress DOI 10.1007/s11557-012-0827-9

ORIGINAL ARTICLE

Rasamsonia composticola, a new thermophilic species isolated from compost in Yunnan, China

Yuan-Ying Su & Lei Cai

Received: 3 March 2012 /Revised: 3 May 2012 /Accepted: 10 May 2012 # German Mycological Society and Springer 2012

Abstract Rasamsonia composticola sp. nov. is described, have a maximum growth temperature at or above 50 °C and illustrated, and compared with similar taxa. This species a minimum temperature of growth at or above 20 °C are produces globose to ellipsoid ascomata, spherical asci borne considered thermophilic, and those that have a thermal in short chains and globose, 1-celled ascospores, typical of maximum near 50 °C and a minimum well below 20 °C Rasamsonia. Anamorph on CYA, MEA, and PDA produces are thermotolerant (Cooney and Emerson 1964). Taxonom- verrucose, rough-walled conidiophores, and hyaline and ically, thermophiles constitute a heterogeneous group cylindrical conidia. This novel species is thermophilic with (Mouchacca 1997, 2007; Salar and Aneja 2007). Thermo- optimal growth temperature of 45–50 °C, and minimum philic fungi are important natural bio-resources capable of growth temperature of 30 °C. Phylogenetic analyses based producing thermo-stable enzymes, which are industrially on combined ITS rDNA, partial calmodulin, and β-tubulin important (Maheshwari et al. 2000). sequences, and combined partial RPB2, Tsr1, and Cct8 gene Houbraken et al. (2012) introduced Rasamsonia, with the sequences were conducted. Both confirmed the generic type species R. emersonii based on a polyphasic study of placement in Rasamsonia and showed its close phylogenetic thermotolerant and thermophilic species in Trichocomaceae. relationships to several species in the genus, such as R. Rasamsonia species are morphologically characterized by emersonii and R. byssochlamydoides. olive-brown ascomata containing spherical, evanescent asci borne in short chains, and penicillate anamorphs with rough- Keywords Calmodulin . Morphology . Phylogeny . walled conidiophores (Houbraken et al. 2012). The com- Talaromyces . β-tubulin bined dataset of partial RNA polymerase II gene (RPB2), putative ribosome biogenesis protein gene (Tsr1) and putative chaperonin complex component TCP-1 gene (Cct8) Introduction sequences showed that Rasamsonia species form a distinct clade within the Trichocomaceae (Houbraken et al. 2012). Temperature is an environmental factor that plays a decisive Currently, the genus Rasamsonia comprises six species, of role in the distribution, diversity, growth and survival of which five were transferred from Talaromyces or Geosmi- microorganisms in ecosystems. Only a few fungi have the thia, and one was newly described (Houbraken et al. 2012). ability to thrive at temperaturew between 45 and 55 °C Several species of the genus have been commercialized; for (Maheshwari et al. 2000). These fungi have been generally example, a thermostable extracellular enzyme from R. emer- categorized as thermophilic and thermotolerant fungi sonii has been used in the wheat-baking process (Waters et (Cooney and Emerson 1964; Mouchacca 2007). Fungi that al. 2010). The objective of this paper is to characterize a novel : species of Rasamsonia isolated from compost in Yunnan, Y.-Y. Su L. Cai (*) China. The combined partial RPB2, Tsr1, and Cct8 gene State Key Laboratory of Mycology, Institute of Microbiology, sequences were adopted, based on the study of Houbraken Chinese Academy of Sciences, Beijing 100101, People’s Republic of China et al. (2012), to confirm the correct generic identification. e-mail: [email protected] ITS-rDNA gene (ITS), partial calmodulin (Cmd), and β- Mycol Progress

Table 1 Strains used in phylogenetic analysis of selected Eurotiales

CBS no. Name Other collections GenBank accession no. (RPB2/Tsr1/Cct8) or reference1

CBS 513.88 Aspergillus clavatusa NRRL 1 (0 ATCC 10070CBS 513.650IMI 15949) Houbraken et al. (2012) Aspergillus flavusa NRRL 3357 (0 CBS 1282020ATCC 200026) Houbraken et al. (2012) Aspergillus fumigatusa Af293 Houbraken et al. (2012) Aspergillus nigera Houbraken et al. (2012) Aspergillus terreusa NIH 2624 Houbraken et al. (2012) CBS 100.11NT Byssochlamys nivea ATCC 22260 JF417414; JF417381; JF4174514 CBS 101075HT Byssochlamys spectabilis ATCC 90900 JF417446; JF417412; JF4174546 CBS 295.48IsoT Coccidioides immitisa Strain “RS” Houbraken et al. (2012) Emericella nidulansa FGSC A4 (0 ATCC 381630CBS 112.46) Houbraken et al. (2012) Hamigera avellanea ATCC 104140IMI 0402300NRRL 1938 JF417424; JF417391; JF4174524 Penicillium chrysogenuma Wisconsin 54-1255 Houbraken et al. (2012) CBS 139.45NT Penicillium citrinum ATCC 1109 0 IMI 091961 0 MUCL JF417416; JF417383; JF4174516 29781 0 NRRL 1841 CBS 344.61IsoT Penicillium crustaceum ATCC 18240 0 IMI 086561 0 MUCL JF417428; JF417395; JF4174528 2685 0 NRRL 3332 CBS 325.48 Penicillium expansum ATCC 7861 0 IBT 5101 0 IMI 039761 0 MUCL JF417427; JF417394; JF4174527 29192 0 NRRL 976 CBS 125543NT Penicillium glabrum IBT 22658 0 IMI 91944 JF417447; JF417413; JF4174547 CBS 353.48NT Penicillium namyslowskii ATCC 11127 0 IMI 040033 0 MUCL JF417430; JF417397; JF4174530 29226 0 NRRL 1070 CBS 101.69T Rasamsonia argillacea DTO 97E4 0 IMI 156096 0 IBT 31199 JF417415; JF417382; JF4174515 CBS 128785T Rasamsonia brevistipitata DTO 25H2 0 IBT 31187 JN406530; JN406523; N406520 CBS 413.71T Rasamsonia byssochlamydoides DTO 149D6 0 IBT 11604 JF417437; JF417403; JF4174537 Rasamsonia composticola CGMCC 3.13669T JQ729684; JQ729686; JQ729682 Rasamsonia composticola CGMCC3.14946 JQ729685; JQ729687; JQ729683 CBS 275.58NT Rasamsonia cylindrospora DTO 138F8 0 IBT 31202 0 ATCC JF417423; JF417390; JF4174523 18223 0 IMI 071623 CBS 100538T Rasamsonia eburnea DTO 105D6 0 IBT 17519 JN406532; JN406524; JN406521 CBS 393.64T Rasamsonia emersonii DTO 48I1 0 IBT 21695 0 ATCC JF417434; JF417401; JF4174534 16479 0 IMI 116815 0 IMI 116815ii CBS 398.69 Sagenomella diversispora JF417435; JF417402; JF4174536 CBS 426.67 Sagenomella griseoviridis ATCC 18505 0 IMI 113160 JF417438; JF417404; JF4174538 CBS 427.67IsoT Sagenomella humicola ATCC 18506 0 IMI 113166 JF417439; JF417405; JF4174539 CBS 429.67IsoT Sagenomella striatispora ATCC 18510 0 IMI 113163 JF417440; JF417406; JF4174540 CBS 296.48HT Talaromyces bacillisporus ATCC 10126 0 IMI 040045 0 NRRL 1025 JF417425; JF417392; JF4174525 CBS 100536HT Talaromyces emodensis IBT 14990 JF417445; JF417411; JF4174545 CBS 310.38NT Talaromyces flavus IMI 197477 0 NRRL 2098 JF417426; JF417393; JF4174526 CBS 398.68HT Talaromyces leycettanus ATCC 22469 0 IMI 178525 JF417435; JF417402; JF4174535 CBS 348.51NT Talaromyces luteus CECT 2950 0 IMI 089305 JF417429; JF417396; JF4174529 CBS 371.87 Talaromyces luteus JF417431; JF417398; JF4174531 Talaromyces marneffeia ATCC 18224 (CBS 334.59 0 IMI 68794) Houbraken et al. (2012) CBS 642.68NT Talaromyces mineoluteum IMI 089377 0 MUCL 28666 JF417443; JF417409; JF4174543 Talaromyces stipitatusa ATCC 10500 (0 NRRL 1006 0 CBS 375.48 0 IMI 39805) Houbraken et al. (2012) CBS 252.87HT Talaromyces viridis IMI 288716 JF417422; JF417389; JF4174522 CBS 373.48 Talaromyces trachyspermus ATCC 10497 0 IMI 040043 0 NRRL 1028 JF417432; JF417399; JF4174532 CBS 391.48NT Talaromyces wortmanii ATCC 10517 0 IMI 040047 0 NRRL 1017 JF417433; JF417400; JF4174533 CBS 891.70 Thermoascus aurantiacus IMI 173037 JF417444; JF417410; JF4174544 CBS 181.67T Thermoascus crustaceus ATCC 16462 0 IMI 126333 JF417417; JF417384; JF4174517 CBS 528.71NT Thermoascus thermophilus IMI 123298 0 NRRL 5208 JF417442; JF417408; JF4174542 CBS 218.34 Thermomyces lanuginosus MUCL 8338 JF417418; JF417385; JF4174518 Mycol Progress

Table 1 (continued)

CBS no. Name Other collections GenBank accession no. (RPB2/Tsr1/Cct8) or reference1

CBS 224.63 Thermomyces lanuginosus MUCL 8337 JF417419; JF417386; JF4174519 CBS 236.58HT Thermomyces thermophilus ATCC 10518 0 IMI 048593 0 NRRL 2155 JF417420; JF417387; JF4174520 CBS 247.57 Trichocoma paradoxa MUCL 39666 0 IBT 31159 JF417421; JF417388; JF4174521 CBS 512.65NT Warcupiella spinulosa ATCC 16919 0 IMI 075885 0 NRRL 4376 JF417441; JF417407; JF4174541

CBS culture collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands (WDCM 133); DTO internal culture collection of CBS-KNAW Fungal Biodiversity Centre; IMI CABI Genetic Resources Collection, Surrey, UK (WDCM 214); IBT culture collection of the Center for Microbial Biotechnology (CMB) at Department of Systems Biology, Technical University of Denmark (WDCM 758); NRRL ARS Culture Collection, U.S. Department of Agriculture, Peoria, Illinois, USA (WDCM 97); ATCC American Type Culture Collection, Manassas, VA, USA (WDCM 1); MUCL Mycotheque de l'Universite catholique de Louvain, Louvain-la-Neuve, Belgium (WDCM 308). WDCM WFCC-World Data Centre for Microorganisms a Sequences derived from published genome sequences tubulin (TUB2) regions were also sequenced and analyzed lock plastic bags and returned to the laboratory, where 10 g of to infer its phylogenetic relationships. each sample were added into a shake flask with 90 mL sterile water, and shaken at 120 rpm for 30 min at 45 °C. The extract was diluted to a series of concentrations, i.e. 10−2,10−3,10−4, − − − − Materials and methods 10 5,106,107,and108, and a 0.2-mL extract from each concentration was spread onto potato-dextrose agar (PDA) Isolation, cultural and morphological characterization containing ampicillin (100 μg/mL) and streptomycin (100 μg/mL) (3 replicates). All plates were incubated at Composts made from rice straw and cow dung were collected 45 °C for 3–10 days with daily examination. Individual colo- from Yunnan, China, by Lei Cai. Samples were placed in zip- nies were picked and inoculated to a new PDA plate. Isolates

Table 2 Rasamsonia isolates used in this study

Name CBS no. Other collections GenBank accession numbers (ITS/BenA/Cmd)

R. argillacea CBS 101.69T DTO 97E4 0 IMI JF417491; JF417456; JF417501 156096 0 IBT 31199 R. argillacea CBS 102.69 DTO 97E5 0 IBT 31200 JF417490; JF417457; JF417502 R. brevistipitata CBS 128785 T DTO 25H2 0 IBT 31187 JF417488; JF417454; JF417499 R. brevistipitata CBS 128786 DTO 26B1 0 IBT 31188 JF417489; JF417455; JF417500 R. byssochlamydoides CBS 413.71 T DTO 149D6 0 IBT 11604 JF417476; JF417460; JF417512 R. byssochlamydoides CBS 533.71 DTO 138G6 0 IBT 11601 0 IBT 31184 JF417477; JF417461; JF417513 R. cylindrospora CBS 275.58NT DTO 138F8 0 IBT 31202 0 ATCC 18223 0 IMI 071623 JF417470; JF417448; JF417493 R. cylindrospora CBS 432.62 DTO 138F7 0 IBT 31201 JF417471; JF417449; JF417492 R. composticola CGMCC 3.13669T JF970184; JF970183; JQ729688 R. composticola CGMCC3.14946 JQ178360; JQ060951; JQ729689 R. eburnea CBS 100538T DTO 105D6 0 IBT 17519 JF417483; JF417462; JF417494 R. eburnea CBS 124445 DTO 49D7 0 IBT 31193 JF417472; JF417450; JF417495 R. emersonii CBS 393.64T DTO 48I1 0 ATCC 16479 0 IMI 116815 0 IMI JF417478; JF417463; JF417510 116815ii 0 IBT 31218 0 IBT 21695 R. emersonii CBS 355.92 DTO 138G4 JF417482; JF417465; JF417508 R. emersonii CBS 549.92 DTO 138G5 0 CBS 814.70 0 IMI 154228 0 IBT JF417481; JF417464; JF417507 31203 0 IBT 31181 0 IBT 24759 Trichocoma paradoxa CBS 103.73 IBT 31160 JF417486; JF417469; JF417506

CBS culture collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands; DTO internal culture collection of CBS-KNAW Fungal Biodiversity Centre; IMI CABI Genetic Resources Collection, Surrey, UK; IBT culture collection of Center for Microbial Biotechnology (CMB) at Department of Systems Biology, Technical University of Denmark; ATCC American Type Culture Collection, Manassas, VA, USA Mycol Progress

Fig. 1 Phylogram of tree 66/100 CBS 353.48NT Penicillium namyslowskii generated from maximum 64/- CBS 125543NT Penicillium glabrum parsimony analysis based on 100/100 CBS 139.45NT Penicillium citrinum combined dataset of partial 99/100 CBS 344.61IsoT Penicillium crustaceum RPB2, Cct8, and Tsr1 -/- 100/100 Wisconsin 54-1255 Penicillium chrysogenum sequences. Values above the CBS 325.48 Penicillium expansum branches are parsimony -/- FGSC A4 Aspergillus nidulans bootstrap (≥ 50 %) and 93/100 CBS 295.48IsoT Hamigera avellanea significant Bayesian posterior CBS 398.68HT Talaromyces leycettanus probability (≥ 90 %). The tree is -/- 92/100 CBS 512.65NTWarcupiella spinulosa rooted with Coccidioides NRRL1 Aspergillus clavatus immitis 99/100 92/100 52/- Af293 Aspergillus fumigatus NRRL 3357 Aspergillus flavus CBS 513.88 Aspergillus niger 95/- -/99 NIH 2624 Aspergillus terreus 86/- CBS 181.67T Thermoascus crustaceus 100/100 CBS 528.71NT Thermoascus thermophilus -/- CBS 891.70 Thermoascus aurantiacus CBS 100.11NT Byssochlamys nivea 100/100 CBS 101075HT Byssochlamys spectabilis 100/100 CGMCC 3.13669T Rasamsonia composticola 100/100 CGMCC 3.14946 Rasamsonia composticola 82/- 84/98 CBS 393.64T Rasamsonia emersonii T Rasamsonia 94/100 CBS 101.69 Rasamsonia argillacea -/- CBS 413.71T Rasamsonia byssochlamydoides CBS 275.58NT Rasamsonia cylindrospora 61/93 94/94 CBS 398.69 Sagenomella diversispora 100/100 CBS 426.67 Sagenomella griseoviridis 100/100 CBS 429.67IsoT Sagenomella striatispora 52/- CBS 427.67IsoT Sagenomella humicola 56/- CBS 247.57 Trichocoma paradoxa 100/- 100/100 CBS 218.34 Thermomyces lanuginosus 100/100 CBS 224.63 Thermomyces lanuginosus CBS 236.58HT Thermomyces thermophilus 74/100 100/100 CBS 348.51NT Talaromyces luteus CBS 371.87 Talaromyces luteus 97/100 CBS 310.38NT Talaromyces flavus 100/100 ATCC 18224 Talaromyces marneffei 100/100 CBS 252.87HT Talaromyces viridis 100/100 ATCC 10500 Talaromyces stipitatus CBS 373.48 Talaromyces trachyspermus 100/100 100/100 CBS 642.68NT Talaromyces mineoluteum 96/100 CBS 100536HT Talaromyces emodensis CBS 296.48HT Talaromyces bacillisporus 91/100 CBS 391.48NT Talaromyces wortmanii Strain “RS” Coccidioides immitis 100 were subsequently identified and stored at 4 °C. Isolates of DNA exaction and PCR reaction Rasamsonia composticola, described below, were also grown on Czapek yeast autolysate agar (CYA; Pitt 1979), Malt Isolates were grown on PDA and incubated at 45 °C for extract agar (MEA; Pitt 1979), and PDA for cultural charac- 5 days. Genomic DNA was extracted by using a Biospin terization. Colony color (surface and reverse) were assessed Fungus Genomic DNA Extraction Kit (BioFlux®) accord- after 5 days’ incubation, using the color charts of Rayner ing to the manufacturer’s protocol. Quality and quantity of (1970). Morphological characters were studied based on the DNA were estimated visually by staining with GelRed on colony grown on MEA at 45 °C. In order to determine the 1 % agarose gel electrophoresis. Parts of the RPB2, Tsr1, optimal temperature for growth, mycelia discs (0.5 cm diam.) and Cct8 genes were amplification and sequenced following were placed on CYA, MEA, and PDA, and incubated at 25– the procedure of Houbraken et al. (2012). TUB2, Cmd, and 55 °C with at 5 °C intervals in triplicate. Colony size was ITS rDNA regions from the strain were amplified by PCR recorded after 5 days. reactions. Primer pairs and PCR amplification conditions Mycol Progress

100/100 CGMCC 3.13669T R. composticola Model of evolution was estimated by using Mrmodeltest 100/100 CGMCC 3.14946 R. composticola 2.3 (Nylander 2004). Posterior probabilities (PP) (Rannala 100/100 CBS 549.92 R. emersonii and Yang 1996; Zhaxybayeva and Gogarten 2002)were CBS 393.64T R. emersonii determined by Markov Chain Monte Carlo sampling 83/- 100/100 CBS 355.92 R. emersonii (BMCMC) in MrBayes 3.0b4 (Huelsenbeck and CBS 413.71T R. byssochlamydoides Ronquist 2001), using the estimated model of evolution. 100/100 CBS 533.71 R. byssochlamydoides Six simultaneous Markov chains were run for 1,000,000 69/- T 100/100 CBS 101.69 R. argillacea generations and trees were sampled every 100th gener- 100/100 CBS 102.69 R. argillacea ation (resulting in 10,000 total trees). The first 2,000 CBS 100538T R. eburnea trees, which represented the burn-in phase of the anal- 100/- 100/100 CBS 124445 R. eburnea 75/100 yses, were discarded and the remaining 8,000 trees were 100/100 CBS 128785T R. brevistipitata used for calculating posterior probabilities (PP) in the CBS 128786 R. brevistipitata majority rule consensus tree. NT 100/100 CBS 275.58 R. cylindrospora CBS 432.62 R. cylindrospora

CBS 103.73 Trichocoma paradoxa 10 Results

Fig. 2 Phylogram of tree generated from maximum parsimony analy- The combined partial RPB2, Tsr1, and Cct8 gene dataset β sis based on combined dataset of ITS, and partial -tubulin sequences included sequences from 46 fungal strains (Table 1). The and calmodulin sequences. Values above the branches are parsimony bootstrap (≥50 %) and significant Bayesian posterior probability final dataset comprised 2,393 characters after alignment; of (≥90 %). The tree is rooted with Trichocoma paradoxa these, 1,178 characters were parsimony informative, and 1,079 were constant. Parsimony analysis generated only were followed as previously described (Hong et al. 2006; one tree (TL099,68, CI00.246, RI00.530, RC00.130, Houbraken et al. 2012). DNA sequencing was performed at HI00.754) shown in Fig. 1. For the Bayesian analyses, the the SinoGenoMax Company, Beijing. best-fit model (GTR + I + G) was selected by MrModeltest 2.3. The branches with significant Bayesian posterior Sequence alignment and phylogenetic analyses probability (≥ 90 %) were shown in the phylogenetic tree (Fig. 1). Sequences from forward and reverse primers were aligned The combined dataset of ITS, Cmd, and TUB2 included to obtain a consensus sequence. Sequences of one isolate sequences from 17 fungal strains (Table 2). The final dataset from compost, along with reference sequences obtained comprised 1,784 characters after alignment; of these, 455 from GenBank (Tables 1, 2), were aligned by Clustal X characters were parsimony informative, and 1,206 were (Thompson et al. 1997). Alignments were optimized manu- constant. Parsimony analysis generated only one tree ally in Bioedit for maximum alignment and to minimize (TL0950, CI00.805, RI00.861, RC00.693, HI00.195) gaps (Hall 1999). shown in Fig. 2. For the Bayesian analyses, the best-fit Phylogenetic analyses were performed by using PAUP* model (HKY+ G) was selected by MrModeltest 2.3. The 4.0b10 (Swofford 2002). Ambiguously aligned regions isolates from compost, described below as Rasamsonia were excluded from all analyses. Unweighted parsimony composticola, were most closely related to R. emersonii (UP) analysis was performed. Trees were inferred using and R. byssochlamydioides. the heuristic search option with TBR branch swapping and The phenotypic differences between R. composticola 1,000 random sequence additions. Maxtrees were unlimited, and other known Rasamsonia species are provided in branches of zero length were collapsed and all multiple Table 3. parsimonious trees were saved. Descriptive tree statistics such as tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy Taxonomy index (HI), were calculated for trees generated under different optimality criteria. Clade stability was assessed in a Rasamsonia composticola Y.Y. Su & L. Cai, sp. nov. bootstrap analysis with 1,000 replicates, each with 10 repli- Figs. 3 and 4 cates of random stepwise addition of taxa. The Shimodaira- MycoBank no.: MB 800249 Hasegawa test (SH test) (Shimodaira and Hasegawa 1999) Etymology: composticola, referring to the habitat of the was performed in order to determine whether trees were species. significantly different. Trees were visualized in Treeview Diagnostic features: Thermophilic with optimal growth (Page 1996). temperature of 45–50 °C, and minimum growth temperature Table 3 Synopsis of characters of Rasamsonia species

Growth on CYA Growth on CYA Optimum and maximum Shape and size conidia Ascomata Ascospores (7 days, 25 °C) (7 days, 37 °C) growth temperature on MEA

R. argillacea 15–25 mm 30–40 mm Optimum 36 °C; maximum Cylindrical or ovoid, (3–) 3.5–4.5 Absent above 50 °C (–5.0)×1.5–2.0 (–2.3) μm R. brevistiptata 7–13 mm 11–17 mm Optimum 33 °C; maximum Ellipsoidal or ovoid, (2.0–) 2.5–3.0 Absent slightly above 45 °C (3.5)×1.7–2.1 μm R. byssochlamydoides No growth 19–27 mm Optimum around 45 °C, maximum Cylindrical, 4–8×1–2.5 μm Present, 9–12.5×6.5–7.5 μm Globose to subglobose, between 50 and 55 °C 3.7–5.5×3.5–4.5 μm R. cylindrospora 3–8mm 5–10 mm Optimum 36 °C; maximum at Cylindrical, 4.0–5.0×1.6–2.1 μm Absent or slightly above 50 °C R. eburnea 14–20 mm 30–40 mm Optimum 36 °C; maximum slightly Cylindrical at first, becoming Occasionally present, 10.5–13 Pale yellow, subglobose above 50 °C ellipsoidal or ovoid, 2.5–3.5 (15)×8–9.5 (–11) μm to more or less ovoid, (–4)×1.8–2.5 μm 4–5×4–4.5 μm R. emersonii No growth 18–30 mm Optimum between 45 and 50 °C, Cylindrical, pale brown, 3.5–4.5 Present, 8–10.5×6.5–8 μm Yellow, subglobose maximum around 55 °C (–5.0)×1.5–3.0 μm to ovoid, 3.5–4× 2.5–3.5 μm R. composticola No growth 25–40 mm Optimum between 45 and 50 °C, Cylindrical, hyaline, slightly larger Present, 6.7–11×6–8 μm Hyaline to yellow, globose, maximum around 55 °C at one end, 3–8.75×1.5–3.75 μm 2–3.8 μm diam ðx ¼ 4:7 1:1 2 0:6μm

References: Salar and Aneja (2007); Houbraken et al. (2012) yo Progress Mycol Mycol Progress

Fig. 3 Rasamsonia composticola. a, b Ascocarps forming on MEA. c Ascocarps. d, e Asci and globose ascospores. f Scanning electron micrographs of ascocarp. g, h Scanning electron micrographs of asci and ascospores. Scale bars (c) 50 μm; (d–g)10μm; (h)2μm

of 30 °C, conidiophores regularly branched, conidia hyaline, 5 2:9 0:8μm, n030), verrucose, hyaline, strait. Penicilli 3–8.75×1.5–3.75 μm, ascomata present, ascospores hya- biverticillate, terverticillate, often asymmetric; metulae 6– line, globose, 2–3.8 μm diam. 15×1.5–4 μm(x ¼ 9:9 2:6 2:3 0:9μm, n030), often Growth temperature 30–55 °C, with an optimal with enlarged apices, 2–4 per branch, usually with verrucose growth temperature between 45–50 °C on CYA, MEA walls. Phialides 3–6 per metulae, 5.5–9×1.5–3 μm(x ¼ 6: and PDA. Colonies on MEA 70–80 mm diam after 8 1 2 0:5μm, n030), cylindrical with long collula, 1– 5 days at 45 °C, surface texture velutinous, white, 3 μm, verruculose. Conidia smooth, hyaline, cylindrical, sparse, with floccose buff aerial mycelia in centre slightly larger at one end, 1-celled, 3–8.75×1.5–3.75 μm (Fig. 4). Ascomata often confluent, yellow-brown to ( x ¼ 4:7 1:1 2 0:6μm, n040), formed in chains orange-brown, globose to ellipsoid, 100–250 μmin (Fig. 4). diameter. Coverings scanty, consisting of inconspicuous Specimen examined: China, Yunnan, Kunming, composts networks of hyphae, surrounded by twisted and yellow- made from rice straw and cow dung, January 2006, L. Cai, ish hyphae, about 1 μmindiameter.Asci evanescent, HMAS 242447 (holotype, dried culture); ex-type living globose to subglobose, 8-spored, 6.7–11×6–8 μm(x ¼ 7 culture CGMCC 3.13669; ibid SUYY1088, living culture :8 1 7 1μm, n030), formed in short chains. Asco- CGMCC3.14945; ibid SUYY1089, living culture spores hyaline, 1-celled, smooth, globose, 2–3.8 μmdiam CGMCC3.14946. Duplicate strains have been deposited in (x ¼ 3 0:6μm, n040) (Fig. 3). CBS. Conidiophores arising from the subsurface, surface or Habitat: Composts made from rice straw and cow dung. aerial mycelium on MEA, 30–200×2–5 μm(x ¼ 80 20: Distribution: China. Mycol Progress

Fig. 4 The anamorphic state of Rasamsonia composticola. a–c Upper and reverse view of colony respectively on CYA, MEA, PDA 4 days after inoculation at 45°C. d–f Penicilli. g–j Conidia. k–n Scanning electron micrographs of penicilli and conidia. Scale bars (d–j)10μm; (k)20μm; (l)10μm; (m, n)5μm

Discussion by remnants of gelatinous covering materials, which is different from that of R. composticola (Salar and Aneja Morphological characters and molecular phylogenetic analy- 2007). Rasamsonia composticola is also comparable to R. ses confirmed the generic placement of this novel species in emersonii. However, two species are different in the color, Rasamsonia. Rasamsonia was erected for a distinct lineage in shape, and size of the ascospores (hyaline to yellow, glo- Trichocomaceae comprising thermotolerant or thermophilic bose, diameter 2–3.8 μminR. composticola vs. yellow, species with olive-brown ascomata, spherical, evanescent asci subglobose to ovoid, 3.5–4.0×2.5–3.5 μminR. emersonii) borne in short chains, and penicillate anamorphs with distinct- and conidia (hyaline, cylindrical, often clavate, and 3–8.75× ly rough-walled conidiophores. Currently, the genus Rasam- 1.5–3.75 μminR. composticola vs. often pale brown, sonia comprises six species. Except for R. brevistipitata,other cylindrical or truncated and 3.5–4.0×1.5–3 μminR. emer- species were previously accommodated in Talaromyces or sonii) (Salar and Aneja 2007; Houbraken et al. 2012). In the Geosmithia (Houbraken et al. 2012). Many species in Talar- phylogenetic tree, R. composticola formed a distinct lineage omyces and Geosmithia have recently been reclassified based (Fig. 1) from R. byssochlamydioides and R. emersonii. Ge- on their phylogenetic affinities. For example, eight species of netically, R. composticola shares 93 and 92 % identity to R. Geosmithia have been transferred to Talaromyces s. str., Pen- emersonii type strain (CBS 393.640NRRL 3321) in ITS icillium s. str., and Rasamsonia (Houbraken and Samson and TUB2 sequences, respectively, while it shares 90 and 2011; Samson et al. 2011). 92 % to R. byssochlamydioides type strain (CBS 413.71) in Rasamsonia composticola is most closely related to R. ITS and TUB2 sequences. Several strains identified as R. emersonii and R. byssochlamydioides. Rasamsonia compos- emersonii (CBS 549.920CBS 814.70) in Houbraken et al. ticola differs from R. byssochlamydioides in producing (2012) share 99 % similarity in ITS and TUB2 sequen- smaller ascospores (2–3.8 μm in diameter vs. 3.7–5.5× ces to R. composticola. The morphological characters 3.5–4.5 μm) (Salar and Aneja 2007; Houbraken et al. should be examined to confirm their relationships with 2012). Ascospores of R. byssochlamydioides are covered R. composticola. Mycol Progress

Rasamsonia composticola grows faster on MEA and PDA Houbraken J, Spierenburg H, Frisvad J (2012) Rasamsonia, a new than on CYA, and the colony characteristics varied among the genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species. Antonie van Leeuwenhoek 101:403–421 three media, particularly in producing darker pigmentation on Huelsenbeck JP, Ronquist FR (2001) MRBAYES: bayesian inference PDA. The species is exclusively thermophilic, and did not of phylogenetic trees. Biometrics 17:754–755 grow at room temperature. The optimum temperature for Maheshwari R, Kamalam PT, Balasubramanyam DV (1987) The bio- – growth is 45–50 °C. Thermophilic species often occur in soils geography of thermophilic fungi. Curr Sci 56:151 155 Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their or other habitats where decomposition of plant material takes physiology and enzymes. Microbiol Mol Biol Rev 64:461–488 place. Rasamsonia composticola was isolated from compost Mouchacca J (1997) Thermophilic fungi: biodiversity and taxonomic made from rice straw and cow dung, wherein the warm, status. Cryptogam Mycol 18:19–69 humid, and aerobic environment may provide the basic phys- Mouchacca J (2007) Heat tolerant fungi and applied research: addition to the previously treated group of strictly thermotolerant species. iological conditions for its development, and its occurrence World J Microb Biotech 23:1755–1770 was probably due to the propagule dissemination from self- Nylander JAA (2004) MrModeltest v2. Program distributed by the heating masses of organic material (Maheshwari et al. 1987; author. Evolutionary Biology Centre, Uppsala University Salar and Aneja 2007). Only 42 out of approximately 100,000 Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Applic Biosci 12:357–358 recorded fungal species can thrive at temperatures between 45 Pitt JI (1979) Geosmithia gen. nov. for Penicillium lavendulum and and 55 °C (Mouchacca 1997,Maheshwarietal.2000,Salar related species. Can J Bot 57:2021–2030 and Aneja 2007). Various studies on thermophilic fungi have Rannala B, Yang Z (1996) Probability distribution of molecular evo- been stimulated by the prospect of their capabilities of secret- lutionary trees: a new method of phylogenetic inference. J Mol Evol 43:304–311 ing high levels of commercially important enzymes with Rayner RW (1970) A Mycological Colour Chart. Commonwealth higher temperature optima (Salar and Aneja 2007). The dis- Mycological Institute. Kew covery of this new thermophilic species demonstrates that Salar RK, Aneja KR (2007) Thermophilic fungi: taxonomy and bio- – there might be more unknown diversity in this economically geography. J Agric Technol 3:77 107 Samson R, Yilmaz N, Houbraken J, Spierenburg H, Seifert K, Peterson important fungal group. S, Varga J, Frisvad J (2011) Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium sub- Acknowledgements This work was financially supported by CAS genus Biverticillium. Stud Mycol 70:159–183 (KSCX2-YW-Z-1026) and NSFC (NSFC 31110103906). Shimodaira H, Hasegawa M (1999) Multiple comparisons of log- likelihoods with applications to phylogenetic inference. Mol Biol Evol 16:1114–1116 References Swofford DL (2002) PAUP*. Phylogenetic analysis using parsimony (*and other methods), 4th edn. Sinauer, Sunderland Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG Cooney DG, Samson RA (1964) Thermophilic fungi – an account of (1997) The clustal X windows interface: flexible strategies for their biology, activities and classification. Freeman, San Francisco multiple sequence alignment aided by quality analysis tools. Nucl Hall TA (1999) Bioedit: a user-friendly biological sequence alignment Acids Res 24:4876–4882 editor and analysis program for windows 95/98/NT. Nucl Acids Waters DM, Murray PG, Ryan LAM, Arendt EK, Touhy MG (2010) Symp Ser 41:95–98 Talaromyces emersonii thermostable enzyme systems and their Hong SB, Cho HS, Shin HD, Frisvad JC, Samson RA (2006) Novel applications in wheat baking systems. J Agric Food Chem Neosartorya species isolated from soil in Korea. Int J Syst Evol 58:7415–7422 Microbiol 56:477–486 Zhaxybayeva O, Gogarten JP (2002) Bootstrap, Bayesian probability Houbraken J, Samson R (2011) Phylogeny of Penicillium and the segre- and maximum likelihood mapping: exploring new tools for com- gation of Trichocomaceae into three families. Stud Mycol 70:1–51 parative genome analyses. Genomics 3:1–15