Cryptococcus Fildesensis Sp. Nov., a Psychrophilic Basidiomycetous Yeast Isolated from Antarctic Moss

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International Journal of Systematic and Evolutionary Microbiology (2014), 64, 675–679 DOI 10.1099/ijs.0.054981-0

Cryptococcus fildesensis sp. nov., a psychrophilic basidiomycetous yeast isolated from Antarctic moss

Tao Zhang, Yu-Qin Zhang, Hong-Yu Liu, Jing Su, Li-Xun Zhao and Li-Yan Yu

Correspondence Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Li-Yan Yu Medical College, Beijing 100050, PR China [email protected]

Two yeast strains isolated from the moss Chorisodontium aciphyllum from the Fildes Region, King George Island, maritime Antarctica, were classified as members of the genus Cryptococcus based on sequence analyses of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer (ITS) regions. The rRNA gene sequence analyses indicated that the two strains represented a novel species of the genus Cryptococcus, for which the name Cryptococcus fildesensis sp. nov. is proposed (type strain: CPCC 300017T5DSM 26442T5CBS 12705T). The MycoBank number of the novel species is MB 805542.

The genus Cryptococcus Vuillemin (1901) comprises a great gilvescens (Carrasco et al., 2012), Cryptococcus friedmannii variety of asexually reproducing species which differ in (Vishniac, 1985), Cryptococcus laurentii (Vishniac & Hemp- morphology (e.g. cell shape, colony form and colour, forma- fling, 1979; Godinho et al., 2013), Cryptococcus saitoi tion of hyphae or pseudohyphae), physiology (e.g. nutritional (Connell et al., 2008), Cryptococcus victoriae (Carrasco et al., capabilities) and habitat (Fonseca et al., 2011). The genus is 2012; Godinho et al., 2013) and Cryptococcus vishniacii also polyphyletic as species occur in four orders within the (Vishniac & Baharaeen 1979; Vishniac & Hempfling, 1979; Tremellomycetes (Agaricomycotina, Basidiomycota): Tremel- Arenz et al., 2006; Connell et al., 2008) . Species of the genus lales, Trichosporonales, Filobasidiales and Cystofilobasidiales Cryptococcus have also been found in mountainous glacial (Fell et al., 2000; Scorzetti et al., 2002). Therefore, the taxonomy habitats, e.g. Cryptococcus carnescens, Cryptococcus foliicola, of these yeasts should be revised in future. Cryptococcus frias, Cryptococcus fonsecae, Cryptococcus psy- chrotolerans, Cryptococcus aff. tephrensis, Cryptococcus tro- Antarctica is geographically isolated from other continents nadorensis, Cryptococcus victoriae (de Garcia et al., 2012), and one of the harshest ecosystems on earth. Several studies Cryptococcus gilvescens and Cryptococcus terricolus (Turchetti have discussed the yeasts isolated from samples in Antarctica et al., 2008). and indicated that basidiomycetous yeasts (particularly strains of species of the genus Cryptococcus) predominate The flora of Antarctica consists largely of bryophytes in Antarctic habitats. Hitherto, 11 species from the genus (approximately 111 species of mosses and 27 species of Cryptococcus have been found in Antarctica and most liverworts) (Bednarek-Ochyra et al. 2000; Ochyra et al. of them are psychrophilic or psychrotolerant, including 2008) and includes only two native flowering plant species Cryptococcus albidus (Goto et al., 1969), Cryptococcus antarc- (Convey et al., 2011). Given that bryophytes are the ticus (Vishniac & Kurtzman 1992), Cryptococcus adeliensis dominant plant component of the Antarctic terrestrial (Scorzetti et al., 2000), Cryptococcus albidosimilis (Vishniac ecosystem, we investigated the fungal diversity associated & Kurtzman 1992; Connell et al., 2008), Cryptococcus with three bryophyte species in the Fildes Region, i.e. the carnescens (Connell et al., 2008; Loque et al., 2010), liverwort Barbilophozia hatcheri (Evans) Loeske and the Cryptococcus gastricus (Carrasco et al., 2012), Cryptococcus mosses Chorisodontium aciphyllum (Hook. f.) Broth and Sanionia uncinata (Hedw.) Loeske. A total of 29 yeast Abbreviation: ITS, internal transcribed spacer. isolates were isolated from six samples and were recognized by sequence analyses of the D1/D2 domains of the large The GenBank/EMBL/DDBJ accession numbers for the internal subunit rRNA gene and the internal transcribed spacer transcribed spacer sequences of Cryptococcus fildesensis sp. nov. CPCC 30016 and CPCC 30017T are KC894158 and KC894159, (ITS) regions. The majority of these yeasts belonged to respectively, and those for the D1/D2 domains of the large subunit known species including Rhodotorula psychrophenolica, rRNA gene sequences of CPCC 30016 and CPCC 30017T are Mrakia psychrophilia and Mrakia robertii. However, two KC894160 and KC894161, respectively. of them, each isolated from a different sample, were found A supplementary figure is available with the online version of this paper. to belong to a novel species of the genus Cryptococcus,

Downloaded from www.microbiologyresearch.org by 054981 G 2014 IUMS Printed in Great Britain 675 IP: 60.206.66.41 On: Thu, 11 Feb 2016 04:09:55 T. Zhang and others within the order Filobasidiales. The name Cryptococcus BLAST program) and the MyCoID database (using a fildesensis sp. nov. is proposed. pairwise sequence alignment program). Sequence align- ments were done using the MUSCLE algorithm implemented The study area was located in the south-western part in MEGA 5.05 software. Phylogenetic and molecular of King George Island (62u 089 –62u 149 S, 59u 029– evolutionary analyses were also conducted with MEGA 58u 519 W). In the area, bryophytes prevail in wetter 5.05 (Tamura et al., 2011) using the neighbour-joining niches on the ground, whereas lichens predominate in dry method with the maximum composite likelihood distance and rocky places. The native flowering plants are only a measure. Confidence values were estimated from bootstrap minor component of the vegetation (Olech, 2002). A total analysis of 1000 replicates. Pairwise deletion was adopted of 61 species of mosses and 11 species of liverworts have for the treatment of gaps and missing data. been reported from the island (Ochyra, 1998; Ochyra & Vana 1989). From 1982 to 2002, the mean air temperature The D1/D2 sequence indicated that the two novel of the area varied from 26.4 uC in July to 2.3 uCin strains are representatives of a novel species in the order February. Mean annual precipitation is 366.7 mm rainfall Filobasidiales (Fig. 1). Within the order Filobasidiales, equivalent, falling as snow and rain (Setzer et al., 2004). Cryptococcus fildesensis sp. nov. occupied a isolated Sampling of bryophytes was carried out during China’s position with Cryptococcus sp. CBS 12610 (7 nt mis- 28th Antarctic expedition in December 2011. Strain CPCC matches), Cryptococcus sp. CBS 12605 (12 nt mismatches), 300016 was isolated from sample number 451 of the moss which had been isolated from forest soil in Germany, and Chorisodontium aciphyllum (collected at 62u 129 44.770 S, uncultured fungus clone BOTU020, isolated from forest 58u 569 27.470 W, altitude 34 m), whereas strain CPCC leaf litter in the USA (11 nt mismatches) forming a 300017T was isolated from the moss sample number 390 of subclade of nearest relatives, supported by a high boot- Chorisodontium aciphyllum (collected at 62u 139 39.290 S, strap value. Sequence data and phylogenetic analysis of the 58u 569 53.510 W). The samples were cut into segments of ITS region also confirmed the positioning of Cryptococcus 0.1–0.3 cm without surface-sterilization. Between 20 and fildesensis sp. nov. in the order Filobasidiales and 30 tissue segments were then evenly placed in 9 cm- supported the recognition of Cryptococcus fildesensis sp. diameter Petri dishes containing potato dextrose agar nov. as a novel species (a phylogenetic tree based on the (PDA, 1.5 %), tetracycline (50 mg l21) and streptomycin ITS sequences is available as Fig. S1 in the online sulfate (50 mg l21). The plates were incubated at 4 uC, and Supplementary Material). Additionally, the results of all colonies were streaked for purification. Purified yeast mating tests were negative. Therefore, it is proposed that strains were suspended in YM broth supplemented with those two strains represent a novel species of the genus 10 % glycerol (v/v) and maintained at 280 uC. Cryptococcus with the name Cryptococcus fildesensis sp. nov. Morphological characteristics were observed by light microscopy (Axio scope A1; Zeiss) and scanning electron Hitherto, 11 species of the genus Cryptococcus have been microscopy (Quanta 200; FEI). Physiological and bio- found in Antarctica. Among them, eight species are chemical tests were performed by standard methods psychrophilic or psychrotolerant, including Cryptococcus described by Kurtzman et al. (2011). For mating tests, antarcticus, Cryptococcus carnescens, Cryptococcus fried- two strains of 2-day-old cultures were crossed on cornmeal mannii, Cryptococcus gastricus, Cryptococcus gilvescens, agar and incubated at low temperature (12 uC) and at Cryptococcus saitoi, Cryptococcus victoriae and Cryptococcus room temperature (18 uC). The crosses were examined vishniacii (Vishniac & Baharaeen 1979; Vishniac & after 1 month. Hempfling, 1979; Vishniac, 1985; Vishniac & Kurtzman, 1992; Connell et al., 2008; Carrasco et al., 2012). Cryp- Nuclear DNA was extracted using a modified CTAB tococcus albidus, Cryptococcus adeliensis, Cryptococcus albi- method (Cubero et al., 1999). The ITS (ITS1–5.8 S–ITS2) dosimilis and Cryptococcus laurentii are mesophilic as their region of the rRNA gene was amplified with the primers maximum temperatures for growth are ¢30 uC (Vishniac & ITS1F and ITS4 as described by White et al. (1990). Hempfling, 1979; Vishniac & Kurtzman, 1992; Scorzetti Amplification of the ITS region was performed using the et al., 2000). In this study, the two strains of Cryptococcus following conditions: 95 uC for 3 min, followed by 37 fildesensis were isolated from Antarctic moss at 4 uC and cycles of 94 uC for 30 s, 52 uC for 30 s and 72 uC for 30 s; could grow well at 12 uCand18uC, but not at 22 uC and and a final extension at 72 uC for 10 min. The D1/D2 25 uC, indicating that this novel species is psychrophilic. domain was amplified with the primers NL1 and NL4 as described by Kurtzman & Robnett (1991). Amplification of Description of Cryptococcus fildesensis T. Zhang the D1/D2 domain was performed as follows: 94 uC for & L.-Y. Yu sp. nov. 6 min, followed by 40 cycles of 94 uC for 60 s, 50 uC for Cryptococcus fildesensis (fil.des.en9sis. N.L. masc. adj. fildesen- 60 s and 72 uC for 60 s; and a final extension at 72 uC sis referring to the Fildes Region, King George Island, for 5 min. PCR products were purified and sequenced with maritime Antarctica, where the type strain was isolated). the same primers by Sangon Biotech (Beijing, China). The sequences generated in this study were compared with In YM broth, after 7 days at 12 uC, the cells are globose to those of other yeasts in the GenBank database (using the elongate, 1.8–4.662.8–7.7 mm. Polar budding is observed

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100 Bullera taiwanensis JCM11143 (AB079065) 0.01 52 Cryptococcus sp. CBS 9089 (AF444720) 72 Cryptococcus sp. PYCC 4949 (AF444689) Cryptococcus filicatus CBS 10875 (EU433984) Cryptococcus sp. PYCC 5266 (AF444691) 100 Cryptococcus silvicoIa CBS 10099T (AY898955) Cryptococcus cylindricus CBS 8680T (AF181534) 63 Syzygospora sorana voucher UBC F16310 (EU541305) Uncultured fungus clone B05A10 (KC701830) 74 Cryptococcus sp. CBS 12605 (HG324303) 57 Uncultured fungus clone BOTU020 (KC588721) 94 Cryptococcus sp. CBS 12610 (HG324302) 80 Cryptococcus fildesensis CPCC 300016 (KC894160) 97 Cryptococcus fildesensis CPCC 300017T (KC894161) Cryptococcus gastricus CBS 2288T (AF137600) 100 Cryptococcus gilvescens CBS 7525T (AF181547) 85 Cryptococcus agrionensis CBS 10799T (EU627786) CBS 10871T (AY731791) 96 Cryptococcus ibericus T 99 Cryptococcus aciditolerans CBS 10872 (AY731790) 100 Cryptococcus metallitolerans CBS 10873T (AY731789) 98 Cryptococcus keelungensis CBS 10876T (EF621562) 100 Cryptococcus sp. CBS 11770 (FN824499) Filobasidium sp. CBS 10188 (EU002812) 92 Cryptococcus terricolus CBS 4517T (AF181520) Cryptococcus aerius CBS 155T (AF075486) 61 81 Cryptococcus fuscescens CBS 7189T (AF075472) 69 Cryptococcus phenolicus CBS 8682T (AF181523) 98 Cryptococcus terreus CBS 1895T (AF075479) 90 Cryptococcus chernovii CBS 8679T (AF181530) 96 Cryptococcus stepposus CBS 10265T (DQ222456) 76 Cryptococcus magnus CBS 140T (AF181851) T 87 75 Cryptococcus oeirensis CBS 8681 (AF181519) Cryptococcus wieringae CBS 1937T (AF181541) Filobasidium uniguttulatum CBS 1730T (AF075468) 100 Cryptococcus friedmannii CBS 7160T (AF075478) 72 Cryptococcus saitoi CBS 1975T (AF181540) 94 Cryptococcus cerealis CBS 10505T (FJ473376) Cryptococcus randhawii CBS 10160T (AJ876599) 100 Cryptococcus antarcticus CBS 7687T (AF075488) 99 Cryptococcus bhutanensis CBS 6294T (AF137599) 85 Cryptococcus adeliensis CBS 8351T (AF137603) Cryptococcus albidus CBS 142T (AF075474) 98 Cryptococcus uzbekistanensis CBS 8683T (AF181508) T 58 Cryptococcus albidosimilis CBS 7711 (AF137601) Cryptococcus diffluens CBS 160T (AF075502) 96 Cryptococcus liquefaciens CBS 968T (AF181515) 83 Cryptococcus arrabidensis CBS 8678T (AF181535) Syzygospora bachmannii isolate AM72 (JN043613) 100 Syzygospora alba isolate AM147 (JN043616) Syzygospora pallida isolate AM26 (JN043615) Holtermanniella wattica CBS 9496 (AY138478) CBS 10162T (AY462119) 100 Holtermanniella festucosa 99 Holtermanniella mycelialis CBS 7712T (AJ311450) 92 Holtermanniella nyarrowii CBS 8804T (AY006480)

Fig. 1. Phylogenetic placement of Cryptococcus fildesensis sp. nov. within the order Filobasidiales. Neighbour-joining analysis of the D1/D2 domain of the large subunit rRNA gene sequences. Numbers on the branches are bootstrap values (1000 replicates; values ,50 % are not shown). Species of the genus Holtermanniella in the order Holtermanniales were used as the outgroup in the analysis. Numbers in parentheses are GenBank accession numbers. Bar, 0.01 substitutions per nucleotide position.

Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 677 IP: 60.206.66.41 On: Thu, 11 Feb 2016 04:09:55 T. Zhang and others and cells occur singly or in pairs (Fig. 2). After 1 month at Germany, as DSM 26442T and in the Centraalbureau voor 12 uC, sediment is present. After 1 month at 12 uC, the Schimmelcultures (CBS), Utrecht, The Netherlands, as CBS streak culture is butyrous and cream-coloured. The surface 12705T. Strain CPCC 300016 is an additional strain of the is smooth and the margin entire. Pseudohyphae are not species and has been deposited in the above two observed in cultures grown on cornmeal agar. No collections, as DSM 26441 and CBS 12704, respectively. fermentation is observed. D-Arabinose, cellobiose, D- The MycoBank number of the novel species is MB 805542. galactose, D-glucose, lactose (weakly), maltose, D-mannose, melezitose, melibiose (weakly), sucrose, trehalose, D-xylose, L-arabinose, L-rhamnose (weakly), L-sorbose (weakly), N- Acknowledgements acetyl-D-glucosamine, methyl a-D-glucoside, D-glucuronic This research was supported by the National Infrastructure of acid, succinic acid, salicin (weakly), D-mannitol, ethanol, Microbial Resources (no. NIMR-2013-3), the National Natural glycerol (weakly), inositol and D-sorbitol are assimilated. Science Foundation of China (NSFC) (nos: 30970038 and Citric acid, raffinose, D-ribose, D-galactitol, DL-lactic acid, 31170041), the National Science and Technology Major Special erythritol, methanol, ribitol, hexadecane, inulin and Project on Major New Drug Innovation (nos: 2012ZX09301002-001 and 2012ZX09301002-003) and the Special Fund for Health-scientific soluble starch are not assimilated. Ammonium sulfate, Research in the Public Interest (no. 201002021). We thank the potassium nitrate, sodium nitrite, cadaverine and L-lysine Chinese Arctic and Antarctic Administration (CAA) and Shunan Cao are assimilated; creatinine, creatine, ethylamine and of China’s 28th Antarctic expedition team for sample collection. imidazole are not assimilated. No extracellular starch-like compounds are produced. Reaction with Diazonium Blue B and urease production are positive. Vitamins are not References required for growth. No growth is observed in the presence Arenz, B. E., Held, B. W., Jurgens, J. A., Farrell, R. L. & Blanchette, of 0.1 % (w/v) cycloheximide, 10 % sodium chloride/5 % R. A. (2006). Fungal diversity in soils and historic wood from the Ross glucose or 50 % glucose. Weak growth is observed in the Sea Region of Antarctica. Soil Biol Biochem 38, 3057–3064. presence of 0.01 % (w/v) cycloheximide. 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