Cryptococcus antarcticus var. circumpolaris var. nov., a basidiomycetous yeast from Antarctica

Antonie van Leeuwenhoek 83: 231–233, 2002.  2002 Kluwer Academic Publishers. Printed in the Netherlands.

231

Cryptococcus antarcticus var. circumpolaris var. nov., a basidiomycetous yeast from Antarctica 1, 2 Helen S. Vishniac * and Silvano Onofri 1

Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA; 2 Dipartimento di Scienze Ambientali, Universita della Tuscia, 01 100 Viterbo, Italy; * Author for correspondence (e-mail: hsvishi@ okstate.edu; phone: (11) 405 744 5564; fax: (11) 405 744 6790) Received 18 December 2001; accepted in revised form 13 June 2002

Key words: Antarctica, Cryptococcus, Filobasidiales, Soil yeast

Abstract Cryptococcus antarcticus Vishniac & Kurtzman var. circumpolaris Vishniac and Onofri var. nov. (Filobasidiales, Tremellomycetidae, Hymenomycetes), an anamorphic yeast with ca. 73% nDNA relatedness to Cryptococcus antarcticus var. antarcticus, differs in failure to assimilate raffinose, a lower maximum temperature for growth, fatty acid profile, and in a single nucleotide change in the D2 region of LSU rDNA.

Introduction

Materials and methods

When Cryptococcus antarcticus Vishniac & Kurtzman (Vishniac and Kurtzman 1992) was described it was deemed inappropriate to segregate the two biotype 52 isolates as a new variety, even though the nDNA relatedness determined for a representative of this biotype, MYSW A834-66Y604 / 52 (ATCC 76661), to the type of the species was in the range which allows such a designation. Given the precision of spectrophotometric reassociation, in which ‘‘replicate determinations often differ by 5%’’ (Kurtzman 1998), a relatedness of 73% is very near the 70% limit mentioned by Kurtzman. However, the two biotype 52 isolates studied both came from University Valley, Dry Valleys, Victoria Land, Antarctica. Although collected in different years, they could be considered derivatives of the same population, even, given the small size of yeast populations in Antarctic soils, descendants of the same cell. Examination of possible varietal status appeared appropriate after the isolation of an additional Antarctic representative and additional characterization of this biotype. The formal description of varieties should provide clues to the mechanisms of speciation in this largely anamorphic group of yeasts.

Data regarding the isolation and characterization of biotype 52 can be found in the original description of Cryptococcus antarcticus (Vishniac and Kurtzman 1992). The additional Antarctic isolate CCFEE 5107 (5 ATCC MYA 1214), isolated by plating soil on CMC (carboxymethylcellulose) agar and Czapek agar at 10 8C, came from the surface of coarsely sandy soil collected on 27 December 96 at the base of a vertical slope of Beacon sandstone, north-exposed without visible colonization, at Battleship Promontory, Dry Valleys (Ross Desert), 768 54.9719 S, 1618 01.7399 E, 1200 m above sea level. Battleship Promontory, overlooking Atalatna Valley, is composed of extremely weathered Beacon Sandstone. Superficial colonization by lichens or other organisms is very rare or lacking, while, because of the relatively milder climate (compared with other parts of the Dry Valleys), cryptoendolithic colonization is particularly abundant, mainly of the lichen-dominated endolithic community. As determined by the Soil Water and Forage Analytical Laboratory of Oklahoma State University, sample water content was 1.00%, pH 6.0, electrical conductivity (EC in mmhos / cm) 640, total soluble salts (TSS) 422 ppm. Hitherto unpublished data for

234 Antarctic soil sample A834-66 indicate that var. circumpolaris also occurs at pH 7.4, EC 1,530, and TSS 1,009. Conventional methods of characterization (Kurtzman and Fell 1998; Barnett et al. 2000), were employed to identify and compare the biotype 52 isolates with representatives of other biotypes in Cryptococcus antarcticus, with the additional use of N-acetyl-glucosamine, L-malate, and saccharate (glucarate) as substrates. Fatty acid analysis was performed by Microbial Identifications, Inc. (Newark, Delaware). MIDI Labs (Newark, Delaware) sequenced a portion of of the D2 LSU rDNA, using the proprietary method of PE Biosystems.

Results and discussion Isolate CCFEE 5107 conformed to the description of biotype 52 in exibiting a maximum temperature of growth .15 but ,20 8C, growing without added vitamins, utilizing L-arabinose, cellobiose, Dglucitol, gluconate, glucuronate, myo-inositol, 2ketogluconate, maltose, mannitol, melezitose, amethyl glucoside, L-rhamnose, salicin, soluble starch, succinate, sucrose, trehalose, and D-xylose as sole substrates, although failing to utilize citrate, and in developing a yellow color on nitrate or glucuronate containing media. Cadaverine, L-lysine, and nitrate were similarly utilized as sole N-sources. As to the assimilation of N-acetyl-glucosamine, L-malate, and saccharate (glucarate), which were not included in published descriptions of the species (Vishniac and Kurtzman 1992; Kurtzman and Fell 1998; Barnett et al. 2000), L-malate was assimilated by all tested strains of C. antarcticus; N-acetyl-glucosamine and saccharate were not. Minor points of difference with the description of Barnett et al. (2000) were noted – C. antarcticus is typically urease positive, though our isolates are sometimes very weakly so. The reported negative outcome is therefore not surprising. No assimilation of lactate has been observed in this

laboratory, but delayed (D) characters are unreliable as they can easily fail to be manifested. Only isolates of C. antarcticus identifiable as biotype 52 failed to assimilate raffinose; this character therefore becomes a positive means of identification for C. antarcticus var. antarcticus, as does the difference in maximum temperatures for growth. C.antarcticus var. antarcticus isolates had maximum temperatures for growth of .20 but ,25 8C (Vishniac and Kurtzman 1992). Fatty acid profiles often fail to be defining or identifying for eukaryote microbes (Osterhout and Merz 1996; Vishniac and Kurtzman 1992) but since the fatty acid profile of MYSW A834-66Y604 / 52 was reportedly less similar to that of the type of C. antarcticus than were those of other C. antarcticus isolates (Vishniac and Kurtzman 1992), it appeared worthwhile to reexamine the fatty acid profiles of A834-66Y604 / 52, CCFEE 5107, and the type of C. antarcticus. The results, shown in Table 1, indicate a greater resemblance between CCFEE 5107 and A83466Y604 than between these strains and the type of C. antarcticus. The Euclidian distance (calculated by Microbial ID software as the distance in two-dimensional – principal component – space between two strains) between A834-66Y604 and CCFEE 5107 was approximately 2, but these did not connect with the type of C. antarcticus (i.e. C. antarcticus var. antarcticus) until ca. 16. The D1 / D2 region of large subunit ribosomal DNA has proved to be useful in constructing a phylogenetic tree of the Cryptococcus albidus clade (inter alia), to which C. antarcticus belongs, and of its parent order, the Filobasidiales (Tremellomycetidae, Hymenomycetes) (Fonseca et al. 2000; Fell et al. 2000). Although only the D2 region was sequenced by MIDI labs, this region proved informative. Both C. antarcticus var. circumpolaris strains exhibited the same sequence; that of the type was deposited in GenBank as AF324933. These sequences were compared, using BLAST (www.ncbi.nlm.nih.gov / BLAST), with sequence AF075488 for the type of Cryptococcus antarcticus (ATCC 76663 5 CBS 7687) submitted by

Table 1. Fatty acid content of Cryptococcus antarcticus var. antarcticus and C. antarcticus var. circumpolaris. Fatty acid, % C. ant. var. antarcticus (type) A812-20bY693 / 64 C. ant. var.circumpolaris A834-66Y604 (type) CCFEE 5107

16:00

16:0 2OH

18:00

18:1 CIS 9

18:2 CIS 9,12

9.63

0.6

0.81

29.53

57.49

8.74

0.3

4.09

38.54

45.95

3.56

37.61

47.11

9.79

235 Fell et al. As expected from the recorded 102% relatedness for nDNA similarity to the type (Vishniac and Kurtzman 1992), the 342 base sequence of MYSW A834-66Y699 / 61 (C. antarcticus var. antarcticus) was identical with that of the type. The sequences of CCFEE 5107 and MYSW A834-66Y604 / 52 were 99% similar to that of the type of C. antarcticus, differing in the substitution of c for the t of position 489 of the GenBank sequence of C. antarcticus. A single base difference might be dismissed as erroneous, had it not been found in both of these isolates, linked by their similarity in other respects but not in either of the two sequenced isolates of C. antarcticus var. antarcticus. We therefore consider the result consonant with varietal status (Fell et al. 2000) for the biotype 52 strains.

by failure to assimilate raffinose as sole carbon source, failure to grow at 20 8C, and a single base substitution (c for t) in a base sequence in the D2 region of LSU rDNA.

Acknowledgements The senior author wishes to thank Paul Epstein for assistance with Latin, John Bartell of MIDI Labs for discussions of the sequence data, and Karen Dohrman of Microbial ID, Inc. for her efforts in fatty acid analysis. S. O. wishes to thank the Italian National Program for Research in Antarctica (PNRA), in the framework of which this work is carried out, and the National Science Foundation (USA) for logistic support in Battleship Promontory.

Conclusions Since the biotype 52 isolates constitute a discrete group, identifiable by both physiological and molecular methods, we concluded that they should be described as a new variety of Cryptococcus antarcticus. Description of Cryptococcus antarcticus var. circumpolaris Vishniac & Onofri var. nov. Cryptococcus antarcticus var. circumpolaris var. nov. A typo differt raffinosum non assimilans, plus quam 20 8C non crescens et ad sequentiam basis in D2-regione LSU DNA singulatim (c pro t) substitutus. Holotypus: MYSW A834-66Y604 / 52 (5 NRRL Y-17459 5 ATCC 76661 5 CBS 7689), e solo in lapide arenario, Battleship Promontory, Antarcticus, 28 December 1983, a H. S. Vishniac isolatus est. á Etymology: Latin ‘‘cir-cum-pol-ár-is’’ (adjective 5 around a pole) Cryptococcus antarcticus var. circumpolaris var. nov. is typified by MYSW A834-66Y604 / 52 (5 NRRL Y-17459 5 ATCC 76661 5 CBS 7689). This new variety, Cryptococcus antarcticus var. circumpolaris, is distinguished from var. antarcticus

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