Abstract

Sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene delineated seven genotypes within the three varieties of Cryptococcus neoformans via specific combinations of eight nucleotide differences located at positions 10, 11, 15, 19, 108 (ITS1), 221 (5.8S), 298 and 346 (ITS2). The ITS types correlated to polymerase chain reaction fingerprint/random amplification of polymorphic DNA (RAPD) molecular types: with ITS type 1 (ATACTAGC)=C. neoformans var. grubii, molecular types VNI+VNII and the serotype A allele of the AD hybrid, VNIIIA; ITS type 2 (ATATAGGC)=the serotype D allele of the AD hybrid, VNIIIB, and C. neoformans var. neoformans, VNIV; and ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC)=VGI=RAPD type III, ITS type 4 (ACACTGAC)=VGII=RAPD type II, ITS type 5: (ACACTGGG)=VGIII=RAPD type I, ITS type 6 (ACACTGGC)=VGIV=RAPD type IV, all corresponding to C. neoformans var. gattii. Cloned sequences from serotype AD revealed that the hybrid serotype is diploid at the ITS1-5.8S-ITS2 locus carrying the ITS type 1 (ATACTAGC) and the ITS type 2 (ATATAGGC) alleles. ITS sequencing is a useful technique for genotyping the three C. neoformans varieties and for subtyping within C. neoformans var. gattii.

Introduction

Cryptococcus neoformans (C.n.) is a pathogenic fungus with three varieties currently recognized: C.n. var. grubii (serotype A), C.n. var. neoformans (serotype D), and C.n. var. gattii (serotypes B and C) [1–4]. In addition there exists a hybrid of C.n. var. grubii and C.n. var. neoformans, serotype AD. It has recently been proposed that C. neoformans be recognized as two distinct species, C. neoformans (serotypes A, AD, and D) and C. gattii (serotypes B and C) [5]. However, since this proposal was made after this study was finished, we will refer to three varieties of a single species, C. neoformans. C.n. var. grubii has recently been introduced due to genetic characteristics that distinguish it from C.n. var. neoformans[1,6]. It is the most common cause of fungal meningitis in immunocompromised hosts worldwide, and has a global distribution [1,6]. Furthermore, it has been recently reported that the prevalence of C.n. var. neoformans (serotype D) is significantly higher than previously perceived [1]. In contrast, the distribution of human cryptococcosis due to C.n. var. gattii is mainly restricted to tropical and subtropical climates, and typically afflicts immunocompetent hosts [2,7]. However, a recent outbreak of C.n. var. gattii among residents and companion animals of Vancouver Island, British Columbia, on the west coast of Canada, indicates changing patterns of distribution [8].

Several molecular typing methods such as randomly amplified polymorphic DNA (RAPD) analysis [9–11] and polymerase chain reaction (PCR) fingerprinting [12–16] have been used to characterize the genotypes of C. neoformans. Previous studies have sub-classified C. neoformans into eight molecular types, based upon PCR fingerprinting with mini- or microsatellite-specific primers [12,14,16]. Similarly, RAPD analysis has grouped C.n. var. gattii isolates into three subtypes [17]. The comparable genotypes are: C.n. var. grubii (serotype A=molecular types VNI and VNII), the serotype AD hybrid (=molecular type VNIII), C.n. var. neoformans (serotype D=molecular type VNIV), and C.n. var. gattii (serotypes B and C=molecular types VGI=RAPD type III, VGII=RAPD type II, VGIII=RAPD type I, and VGIV) with no specific correlation to serotype. The RAPD types have been correlated to internal transcribed spacer (ITS) sequence types for a limited number of C.n. var. gattii isolates in a previous study [17].

We now extend our analysis of the ITS1-5.8S-ITS2 region to all three varieties of C. neoformans, including 96 isolates representing all eight molecular types previously observed by PCR fingerprinting [14], in order to further characterize these molecular types and to evaluate the usefulness of ITS sequencing as a subtyping method.

Materials and methods

C. neoformans strains used in the study

Ninety-six isolates were studied in this report: 11 strains of C.n. var. grubii, seven strains of C.n. var. neoformans, 10 strains of the serotype AD hybrid and 68 strains of C.n. var. gattii. The strains were obtained either from the culture collection of the Institute of Food Microbiology (IFM) (Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan), or the Australian Medical Fungal Collection (AMFC) (Molecular Mycology Laboratory, Center for Infectious Diseases and Microbiology, ICPMR, Westmead Hospital, Westmead, NSW, Australia). See Table 1 for strain numbers and details of origin. In addition, three different Cryptococcus species were used as outgroups in this study: C. luteolus (CBS943), C. curvatus (CBS570) and C. laurentii (WM601). Fungal strains were inoculated onto potato dextrose agar (Difco, Detroit, MI, USA) slants at 30°C for approximately 48–72 h. Following two subcultures, the serotype of each strain was determined by the slide agglutination test (IATRON Crypto Check, Iatron, Tokyo, Japan) (for results see Table 1).

1

List of C. neoformans strains used in this study and their serotype, origin, RAPD profile, ITS sequence type, PCR fingerprint molecular type and EMBL and DDBJ accession numbers

Isolate Other collection number Serotype Origin RAPD profilea ITS typeb PCR fingerprint molecular typec EMBL and DDBJ accession number 
Cryptococcus neoformans var. grubii 
ATCC90112♦  Human, CSF, Pennsylvania, USA  VNI AJ493550 
WM148♦  Human, CSF, Sydney, Australia  VNI AJ493551 
WM626♦  Human, CSF, Sydney, Australia  VNII AJ493552 
Hamden C3′1♦  Pigeon dropping, Belo Horizonte, Brazil  VNII AJ493553 
RV58146♦  Wood, Zaire  VNII AJ493554 
IFM40216 ATCC 90113 Human, USA   AB034643 
IFM5817 CUH-16 Human, Chiba, Japan   AB087812 
IFM5854 CDC 551 Unknown, CDC, USA   AB087813 
IFM46501 37-518-157 Human, NIH, Thailand   AB087814 
IFM46554 C13 Human, Costa Rica   AB087815 
IFM47273  Human, China   AB087816 
Serotype AD hybrid 
RKI-M1644/90♦  AD Human, Germany  VNIII AJ493555 
RKI-M364/98♦  AD Human, Germany  VNIII AJ493556 
RKI-M3006/94♦  AD Human, Germany  VNIII AJ493557 
WM628♦  AD Human, CSF, Melbourne, Australia  VNIII AJ493558 
IFM5889 UFPR 2536 AD Unknown, Recife, Brazil   AB087818 
IFM45730 I-3a AD Pigeon dropping, China   AB087819 
IFM45751 II-2c AD Pigeon dropping, China   AB087820 
IFM46127 73-4 AD Unknown   AB087824 
IFM46138 CBS132 AD Fermenting fruit juice, Italy  VNIII AB087825 
IFM46140 MCP 9124 AD Unknown, Japan   AB087826 
Cryptococcus neoformans var. neoformans 
B-3501♦  Human, CSF, Richmond, USA  VNIV AJ493559 
RKI-M318/90♦  Human, Germany  VNIV AJ493560 
WM629♦  Human, blood, Melbourne, Australia  VNIV AJ493561 
IFM5881 UFPR 2475 Unknown, Recife, Brazil   AB087817 
IFM46081 AS 128DSL Unknown, Italy   AB087821 
IFM46084 AS 134MD Unknown, Italy   AB087822 
IFM46093 M11 Unknown, Italy   AB087823 
Cryptococcus neoformans var. gattii 
503 2738♦  Human, Papua New Guinea  VGI AJ493562 
WM830♦  Human, Papua New Guinea  VGI AJ493563 
IFM50893 WM 179u Human, CSF, Sydney, Australia  VGI AB087664 
       AJ493564 
IFM5880 UFPR 2262 Human, Recife, Brazil III  AB087629 
IFM45678 856/439 Unknown III  AB087633 
IFM45699  Human, Taiwan   AB087635 
IFM45713 S-D2d, LY-6 Human, China III  AB087636 
IFM45714 S-D2y, LY-7 Human, China III  AB087637 
IFM50897 WGH-1 Human, Sydney, Australia III VGI AB087668 
IFM50898 W-27 Human, Sydney, Australia III VGI AB087669 
IFM50899 W-35 Human, Sydney, Australia III VGI AB087670 
IFM50900 CBS7523 Eucalyptus camaldulensis debris, Nuriootpa, Australia III VGI AB087671 
IFM50901 CBS7747 Olive seedling underneath Eucalyptus camaldulensis, Barossa Reservoir, Australia III VGI AB087672 
IFM50902 CBS7748♦ Eucalyptus camaldulensis hollow, Balranald, SA, Australia III VGI AB087673 
       AJ493565 
IFM50903 CBS7749 Eucalyptus camaldulensis bark, Balranald, SA, Australia III VGI AB034647 
IFM50904 WM 718♦ Eucalyptus tereticornis, Gold Coast, QLD, Australia III VGI AB087674 
 413/GC2E(2)      AJ493566 
IFM5815  Human, Japan (Patient lived in Singapore) III VGI AB087623 
IFM46640 NACR 3 Unknown, Nagasaki, Japan III VGI AB087639 
IFM48219 A465 Human, Parana, Brazil  VGI AB087651 
CBS6998 NIH 365 Human, CSF, Thailand III  AB087657 
CBS8273 NIH B-3939 Human, brain lesion, Africa III  AB034648 
TH0545  Human, Thailand  VGI AB087675 
TH0886  Human, Thailand  VGI AB087676 
HEC11102♦  Human, urine, Rio de Janeiro, Brazil  VGII AJ493569 
IFM50894 WM 178♦ Human, lung, Sydney, Australia  VGII AB087665 
       AJ493567 
RAM2♦  Eucalyptus camaldulensis, Arnhemland, NT, Australia  VGII AJ493568 
IFM5838  Unknown, Emory University, USA   AB034645 
IFM5851  Human, USA (a subculture of NIH 444) II  AB087624 
IFM5883 UFPR 2527 Human, Recife, Brazil II  AB087631 
IFM46566 C61 Human, Costa Rica II  AB087638 
IFM46914 37-40-40 Human, NIH, Thailand II  AB087640 
IFM46915 37-140-140 Human, NIH, Thailand II  AB087641 
IFM46916 37-362-28 Human, NIH, Thailand II  AB087642 
IFM46917 37-381-47 Human, NIH, Thailand II  AB087643 
IFM46918 37-1852-23 Human, NIH, Thailand II  AB087644 
IFM46919 38-1539-59 Human, NIH, Thailand II  AB087645 
IFM47258 F-1623 Human, Funabashi, Japan (Patient born in Brazil) II  AB087646 
IFM48213 FT 84 Human, Parana, Brazil II  AB087647 
IFM48214 FT 78 Unknown, Parana, Brazil   AB087648 
IFM48216 FT 119 Human, Parana, Brazil II  AB087649 
IFM48218 BV 33 Human, Venezuela II  AB087650 
IFM48221 A 288 Human, Minas Gerais, Brazil II  AB087652 
IFM48222 FT 102 Human, Parana, Brazil   AB087653 
IFM48297 FT 130 Human, Parana, Brazil II  AB087654 
IFM48302 BV 5 Human, Venezuela   AB087655 
CBS6956 NIH 444 Human, sputum, Washington, USA II VGII AB087656 
CBS7750♦  Eucalyptus camaldulensis bark debris, San Francisco, USA II VGII AB087658 
       AJ493570 
CN11  Human, Campinas, Brazil II  AB087659 
CN15  Human, Campinas, Brazil II  AB087660 
CN25  Human, Campinas, Brazil II  AB087661 
CN30  Human, Campinas, Brazil   AB087662 
CN36  Human, Campinas, Brazil II  AB087663 
WM728♦  Eucalyptus sp. debris, San Diego, USA  VGIII AJ493571 
WM726♦  Eucalyptus citriodora, San Diego, USA  VGIII AJ493572 
CN043♦  Human, CSF, Auckland, New Zealand  VGIII AJ493573 
WM161♦  Tree hollow, San Diego, USA  VGIII AJ549318 
CBS6993 NIH 18 Human, CSF, CA, USA  AB034644 
NIH112  Unknown, USA  AB087622 
IFM5873  Unknown, Brazil   AB087625 
IFM5875 UFPR-D1 Unknown, Recife, Brazil  AB087626 
IFM5878 ENCB 1010 G-1 Human, Mexico  AB087627 
IFM5879 ENCB 1010 G-2 Human, Mexico  AB087628 
IFM5882 UFPR 2526 Unknown, Recife, Brazil  AB087630 
IFM5884 UFPR 2528-1 Human, Recife, Brazil  AB034646 
IFM5885 UFPR 2528-2 Human, Recife, Brazil  AB087632 
IFM50895  Human, Melbourne, Australia VGIII AB087666 
B-5742♦  Human, CSF, Punjab, India IV VGIV AJ493574 
IFM50896 WM 779♦ Cheetah, Johannesburg, South Africa IV VGIV AB087667 
       AJ493575 
Outgroups 
Cryptococcus luteolus 
CBS943♦ WM775  Air, Tokyo, Japan    AJ493576 
Cryptococcus curvatus 
CBS570♦ WM56  Human, sputum, The Netherlands    AJ493577 
Cryptococcus laurentii 
ICPMR156-0720♦ WM601  Human, Sydney, Australia    AJ493578 
Isolate Other collection number Serotype Origin RAPD profilea ITS typeb PCR fingerprint molecular typec EMBL and DDBJ accession number 
Cryptococcus neoformans var. grubii 
ATCC90112♦  Human, CSF, Pennsylvania, USA  VNI AJ493550 
WM148♦  Human, CSF, Sydney, Australia  VNI AJ493551 
WM626♦  Human, CSF, Sydney, Australia  VNII AJ493552 
Hamden C3′1♦  Pigeon dropping, Belo Horizonte, Brazil  VNII AJ493553 
RV58146♦  Wood, Zaire  VNII AJ493554 
IFM40216 ATCC 90113 Human, USA   AB034643 
IFM5817 CUH-16 Human, Chiba, Japan   AB087812 
IFM5854 CDC 551 Unknown, CDC, USA   AB087813 
IFM46501 37-518-157 Human, NIH, Thailand   AB087814 
IFM46554 C13 Human, Costa Rica   AB087815 
IFM47273  Human, China   AB087816 
Serotype AD hybrid 
RKI-M1644/90♦  AD Human, Germany  VNIII AJ493555 
RKI-M364/98♦  AD Human, Germany  VNIII AJ493556 
RKI-M3006/94♦  AD Human, Germany  VNIII AJ493557 
WM628♦  AD Human, CSF, Melbourne, Australia  VNIII AJ493558 
IFM5889 UFPR 2536 AD Unknown, Recife, Brazil   AB087818 
IFM45730 I-3a AD Pigeon dropping, China   AB087819 
IFM45751 II-2c AD Pigeon dropping, China   AB087820 
IFM46127 73-4 AD Unknown   AB087824 
IFM46138 CBS132 AD Fermenting fruit juice, Italy  VNIII AB087825 
IFM46140 MCP 9124 AD Unknown, Japan   AB087826 
Cryptococcus neoformans var. neoformans 
B-3501♦  Human, CSF, Richmond, USA  VNIV AJ493559 
RKI-M318/90♦  Human, Germany  VNIV AJ493560 
WM629♦  Human, blood, Melbourne, Australia  VNIV AJ493561 
IFM5881 UFPR 2475 Unknown, Recife, Brazil   AB087817 
IFM46081 AS 128DSL Unknown, Italy   AB087821 
IFM46084 AS 134MD Unknown, Italy   AB087822 
IFM46093 M11 Unknown, Italy   AB087823 
Cryptococcus neoformans var. gattii 
503 2738♦  Human, Papua New Guinea  VGI AJ493562 
WM830♦  Human, Papua New Guinea  VGI AJ493563 
IFM50893 WM 179u Human, CSF, Sydney, Australia  VGI AB087664 
       AJ493564 
IFM5880 UFPR 2262 Human, Recife, Brazil III  AB087629 
IFM45678 856/439 Unknown III  AB087633 
IFM45699  Human, Taiwan   AB087635 
IFM45713 S-D2d, LY-6 Human, China III  AB087636 
IFM45714 S-D2y, LY-7 Human, China III  AB087637 
IFM50897 WGH-1 Human, Sydney, Australia III VGI AB087668 
IFM50898 W-27 Human, Sydney, Australia III VGI AB087669 
IFM50899 W-35 Human, Sydney, Australia III VGI AB087670 
IFM50900 CBS7523 Eucalyptus camaldulensis debris, Nuriootpa, Australia III VGI AB087671 
IFM50901 CBS7747 Olive seedling underneath Eucalyptus camaldulensis, Barossa Reservoir, Australia III VGI AB087672 
IFM50902 CBS7748♦ Eucalyptus camaldulensis hollow, Balranald, SA, Australia III VGI AB087673 
       AJ493565 
IFM50903 CBS7749 Eucalyptus camaldulensis bark, Balranald, SA, Australia III VGI AB034647 
IFM50904 WM 718♦ Eucalyptus tereticornis, Gold Coast, QLD, Australia III VGI AB087674 
 413/GC2E(2)      AJ493566 
IFM5815  Human, Japan (Patient lived in Singapore) III VGI AB087623 
IFM46640 NACR 3 Unknown, Nagasaki, Japan III VGI AB087639 
IFM48219 A465 Human, Parana, Brazil  VGI AB087651 
CBS6998 NIH 365 Human, CSF, Thailand III  AB087657 
CBS8273 NIH B-3939 Human, brain lesion, Africa III  AB034648 
TH0545  Human, Thailand  VGI AB087675 
TH0886  Human, Thailand  VGI AB087676 
HEC11102♦  Human, urine, Rio de Janeiro, Brazil  VGII AJ493569 
IFM50894 WM 178♦ Human, lung, Sydney, Australia  VGII AB087665 
       AJ493567 
RAM2♦  Eucalyptus camaldulensis, Arnhemland, NT, Australia  VGII AJ493568 
IFM5838  Unknown, Emory University, USA   AB034645 
IFM5851  Human, USA (a subculture of NIH 444) II  AB087624 
IFM5883 UFPR 2527 Human, Recife, Brazil II  AB087631 
IFM46566 C61 Human, Costa Rica II  AB087638 
IFM46914 37-40-40 Human, NIH, Thailand II  AB087640 
IFM46915 37-140-140 Human, NIH, Thailand II  AB087641 
IFM46916 37-362-28 Human, NIH, Thailand II  AB087642 
IFM46917 37-381-47 Human, NIH, Thailand II  AB087643 
IFM46918 37-1852-23 Human, NIH, Thailand II  AB087644 
IFM46919 38-1539-59 Human, NIH, Thailand II  AB087645 
IFM47258 F-1623 Human, Funabashi, Japan (Patient born in Brazil) II  AB087646 
IFM48213 FT 84 Human, Parana, Brazil II  AB087647 
IFM48214 FT 78 Unknown, Parana, Brazil   AB087648 
IFM48216 FT 119 Human, Parana, Brazil II  AB087649 
IFM48218 BV 33 Human, Venezuela II  AB087650 
IFM48221 A 288 Human, Minas Gerais, Brazil II  AB087652 
IFM48222 FT 102 Human, Parana, Brazil   AB087653 
IFM48297 FT 130 Human, Parana, Brazil II  AB087654 
IFM48302 BV 5 Human, Venezuela   AB087655 
CBS6956 NIH 444 Human, sputum, Washington, USA II VGII AB087656 
CBS7750♦  Eucalyptus camaldulensis bark debris, San Francisco, USA II VGII AB087658 
       AJ493570 
CN11  Human, Campinas, Brazil II  AB087659 
CN15  Human, Campinas, Brazil II  AB087660 
CN25  Human, Campinas, Brazil II  AB087661 
CN30  Human, Campinas, Brazil   AB087662 
CN36  Human, Campinas, Brazil II  AB087663 
WM728♦  Eucalyptus sp. debris, San Diego, USA  VGIII AJ493571 
WM726♦  Eucalyptus citriodora, San Diego, USA  VGIII AJ493572 
CN043♦  Human, CSF, Auckland, New Zealand  VGIII AJ493573 
WM161♦  Tree hollow, San Diego, USA  VGIII AJ549318 
CBS6993 NIH 18 Human, CSF, CA, USA  AB034644 
NIH112  Unknown, USA  AB087622 
IFM5873  Unknown, Brazil   AB087625 
IFM5875 UFPR-D1 Unknown, Recife, Brazil  AB087626 
IFM5878 ENCB 1010 G-1 Human, Mexico  AB087627 
IFM5879 ENCB 1010 G-2 Human, Mexico  AB087628 
IFM5882 UFPR 2526 Unknown, Recife, Brazil  AB087630 
IFM5884 UFPR 2528-1 Human, Recife, Brazil  AB034646 
IFM5885 UFPR 2528-2 Human, Recife, Brazil  AB087632 
IFM50895  Human, Melbourne, Australia VGIII AB087666 
B-5742♦  Human, CSF, Punjab, India IV VGIV AJ493574 
IFM50896 WM 779♦ Cheetah, Johannesburg, South Africa IV VGIV AB087667 
       AJ493575 
Outgroups 
Cryptococcus luteolus 
CBS943♦ WM775  Air, Tokyo, Japan    AJ493576 
Cryptococcus curvatus 
CBS570♦ WM56  Human, sputum, The Netherlands    AJ493577 
Cryptococcus laurentii 
ICPMR156-0720♦ WM601  Human, Sydney, Australia    AJ493578 

T=type culture.

aRAPD pattern obtained with primer A-1 in Japan [16].

bITS types 5′→3′: 1=ATACTAGC; 2=ATATAGGC; 3=GCGCTGGC; 4=ACACTGAC; 5=ACACTGGG; 6=ACACTGGC; 7=ACGCTGGC (sequence position 10, 11, 15, 19, 108 (ITS1), 221 (5.8S rRNA gene), 298 and 346 (ITS2); ♦ ITS sequences are determined in Australia using primers SR6R and LR1 [20], all other ITS sequences are determined in Japan using primers ITS4 and ITS5 [18].

cMolecular types obtained by PCR fingerprinting with primer M13 (5′-GAGGGTGGCGGTTCT-3′) in Australia [12,13].

Extraction of DNA

DNA extractions were carried out independently in Japan or Australia using different extraction protocols. DNA extractions in Japan were carried out according to the procedures described by Tamura et al. [18,19]: 250 μl of GPT reagent (6 M guanidine thiocyanate, dissolved in 50 mM Tris pH 8.3) and 450 μl of Tris (pH 8.0)-buffered phenol were added to a suspension of washed yeast cells in a microfuge tube and the mixture was boiled for 15 min; 250 μl of chloroform–isoamyl alcohol was then added, and the aqueous phase separated by centrifugation at 12 000×g, mixed with an equal amount of 100% isopropanol and 1/10 volume of 3 M sodium acetate, and placed at −20°C for 1 h. Samples were centrifuged at 12 000×g for 20 min. The nucleic acid pellet was then washed with ice-cold 70% ethanol, dried and resuspended in sterile TE buffer at a concentration of 5 μg ml−1. DNA extractions undertaken in Australia were carried out as previously described [13].

RAPD analysis

RAPD analysis was undertaken in Japan. The RAPD reactions were performed in a 25-μl volume containing 12.5 ng of genomic DNA, 20 pmol of primer A-1 (5′-ATT GCG TCC A-3′) [17–19], and one PCR bead (Ready-to-Go PCR bead, Amersham Pharmacia Biotech, Piscataway, NJ, USA). An initial denaturation of 94°C for 4 min was followed by 35 cycles of 94°C for 2 min, 32°C for 2 min, and 72°C for 2 min, followed by a final extension at 72°C for 10 min. All reaction products were characterized by electrophoresis on 1.5% agarose gels in 1×TBE buffer at 80 V for 90 min, and then stained in a 0.5 μg ml−1 ethidium bromide solution, and visualized by UV transillumination.

PCR fingerprinting

PCR fingerprinting analysis was undertaken in Australia. Molecular types for many of the strains included in this study have been established in a global molecular epidemiology study [12,14,16]. These data were generated according to a previously described protocol [14] and molecular types were assigned based upon comparisons to an established set of standard molecular type strains: WM148 (serotype A, VNI), WM626 (serotype A, VNII), WM628 (serotype AD, VNIII), WM629 (serotype D, VNIV), WM179 (serotype B, VGI), WM178 (serotype B, VGII), WM161 (serotype B, VGIII), and WM779 (serotype C, VGIV) [12,14,16].

ITS1-5.8S-ITS2 sequencing

Amplification of the ITS1-5.8S-ITS2 fragment was performed either in Japan or in Australia, using slightly different protocols. For the Japanese protocol, amplification reactions were performed in 30-μl volumes containing 12.5 ng of genomic DNA, 20 pmol each of primers ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) and ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) [20] and one PCR bead (Ready-to-Go PCR bead, Amersham). An initial denaturation at 94°C for 4 min was followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a final extension at 72°C for 10 min [18]. The PCR products were purified using a PCR product pre-sequencing kit (USB, Cleveland, OH, USA), and then sequenced directly using a Big Dye terminator reagent kit, including Taq DNA polymerase, according to the protocol recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). Sequencing was performed on a 310 automated DNA sequencer (Applied Biosystems).

For the Australian protocol, amplification reactions were performed in a 50-μl volume, containing 30 ng of genomic DNA, 1×PCR buffer (Applied Biosystems, Branchburg, NJ, USA), 0.2 mM each of dATP, dCTP, dGTP and dTTP, 3 mM magnesium acetate, 25 ng each of primers SR6R (5′-AAG TAAG AAG TCG TAA CAA GG-3′) and LR1 (5′-GGT TGG TTT CTT TTC CT-3′) [21] and 2.5 U Taq DNA polymerase (Applied Biosystems). An initial denaturation at 97°C for 3 min was followed by 20 cycles of 97°C for 35 s, 50°C for 55 s, and 72°C for 45 s+4 s/cycle. This was followed by 10 cycles of 97°C for 45 s, 50°C for 55 s, and 72°C for 2 min+4 s/cycle, and finally 72°C for 6 min. PCR products were purified using the GeneClean Spin kit (Bio101, Quantum Technologies, Carlsbad, CA, USA) and 100 ng of the purified DNA was used to sequence both directions of the amplified fragment on an ABI 373 automated DNA sequencer (Applied Biosystems) or on a LiCor automated DNA sequencer (LiCor, Lincoln, NE, USA).

Cloning of ITS1-5.8S-ITS2 sequences from serotype AD

In light of the observations of multiple alleles at the URA5 locus in serotype AD [21], the serotype AD ITS1-5.8S-ITS2 regions were cloned and sequenced to determine whether multiple alleles also existed at this locus. A 10-μl ligation reaction containing 1×Rapid ligation buffer (Promega, Madison, WI, USA), 50 ng pGEM cloning vector (Promega), 40 ng of purified PCR product, 3 U T4 DNA ligase was incubated at room temperature for 3 h and then stored at 4°C overnight. 6 μl of ligation reaction was combined with 40 μl of competent Escherichia coli JM109 cells, incubated on ice for 20 min, followed by 42°C for 2 min, and again on ice for 5 min. 950 μl SOC medium (20 g l−1 Bacto-tryptone (Oxoid, Basingstoke, UK), 5 g l−1 Bacto-yeast extract (Oxoid), 10 mM NaCl, 2.5 mM KCl, 20 mM MgSO4, and 20 mM glucose) was added to the transformation mixture and incubated at 37°C for 1 h with shaking. 200 μl of transformed cells were spread on to LB agar containing 50 mg l−1 ampicillin that had been coated with 40 mg ml−1 of X-gal (Promega) and 100 mg IPTG (Promega). The plates were incubated at 37°C overnight. White colonies were picked from the agar with a sterile toothpick and used as the template in a PCR to re-amplify the ITS region. Amplified ITS1-5.8S-ITS2 fragments were purified and sequenced as described above.

Sequence editing and phylogeny

Sequence alignment was performed in Japan using the CLUSTALW software [23], while in Australia sequences were manually aligned using the program SeqPup v.0.6 (http://iubio.bio.Indiana.edu/soft/molbio/seqpup). Phylogenetic relationships between representative sequences of each ITS type were estimated by maximum-parsimony analysis using the software PAUP* 4.0b10 (PPC/Altivec) for Macintosh (Swofford 1998, Florida State University, Miami, FL, USA). The search for the most parsimonious trees was performed using the heuristic search option of PAUP* 4.0b10. Bootstrap analysis with 100 bootstrap replications using the heuristic search option of the program PAUP* 4.0b10 was carried out to test the robustness of the internal branches.

Results

ITS sequence signatures to subtype the three varieties of C. neoformans

ITS sequences, including the 5.8S gene, were determined for 11 C.n. var. grubii strains, seven C.n. var. neoformans strains, 10 serotype AD hybrid strains and 68 C.n. var. gattii strains. EMBL and DDBJ accession numbers for these sequences are given in Table 1. All C. neoformans strains revealed an identical ITS1-5.8S-ITS2 region length of 463 bp. The outgroup cryptococcal species investigated possessed different ITS region lengths. The ITS sequence of C. luteolus CBS943 was 432 bp (EMBL accession number AJ493576), C. curvatus CBS570 was 382 bp (EMBL accession AJ493577) and C. laurentii WM601 was 431 bp (EMBL accession AJ493578).

The sequences were aligned and searched for variety or molecular type-specific sequence signatures. All C.n. var. grubii (serotype A) strains revealed identical ITS sequences. The same was found for all C.n. var. neoformans (serotype D) strains. In addition the C.n. var. neoformans strains had identical sequences to the serotype AD hybrid strains investigated when the obtained PCR products were sequenced direct. In contrast to the low variation found in the serotype A, D and AD strains, revealing only two ITS sequencing types, the 68 strains studied from C.n. var. gattii exhibited five different sequence types. The following ITS types were found within the C. neoformans complex: ITS type 1=C.n. var. grubii (serotype A) and the serotype A allele of the AD hybrid; ITS type 2=C.n. var. neoformans (serotype D), the serotype D allele of the AD hybrid and the serotype AD hybrid (if sequenced directly from the obtained PCR product); ITS type 3, ITS type 4, ITS type 5, ITS type 6 and ITS type 7 all correspond to C.n. var. gattii (serotypes B and C). Fig. 1 shows a sequence alignment including one representative sequence for each observed ITS type. The sequences representing C.n. var. grubii and C.n. var. neoformans were distinguishable from C.n. var. gattii by one nucleotide at position 11 of the ITS1 region (T in C.n. var. grubii and C.n. var. neoformans or C in C.n. var. gattii). C.n. var. grubii showed sequence variation at nucleotide positions 19 and 108 of the ITS1 region from those of C.n. var. neoformans (C or T in position 19 and T or A in position 108 in C.n. var. grubii or C.n. var. neoformans respectively). C.n. var. grubii showed a characteristic base A at nucleotide position 221 in the 5.8S rDNA gene where there was a G in the other two varieties. Based upon the sequence difference at positions 11, 19, 108 and 221 of the ITS1 and 5.8S rRNA gene, the three varieties were found to be delineated by the combination of these four bases:CCTG for C.n. var. gattii, TCTA for C.n. var. grubii and TTAG for C.n. var. neoformans (Figs. 1 and 2).

1

Alignment of ITS sequences of all three varieties of C. neoformans, representing a sequence of each ITS type. Discriminatory positions 10, 11, 15, 19 and 108 in the ITS1 region, 221 in the 5.8S rRNA gene, and 298 and 346 in the ITS2 region are indicated.

1

Alignment of ITS sequences of all three varieties of C. neoformans, representing a sequence of each ITS type. Discriminatory positions 10, 11, 15, 19 and 108 in the ITS1 region, 221 in the 5.8S rRNA gene, and 298 and 346 in the ITS2 region are indicated.

2

ITS sequence signatures which make it possible to separate between C.n. var. grubii, C.n. var. neoformans and C.n. var. gattii, as well as between the molecular types of C.n. var. gattii, based upon eight combinations of variable nucleotides at positions 10, 11, 15, 19 and 108 in the ITS1 region, 221 in the 5.8S rDNA gene, and 298 and 346 in the ITS2. VNIIIA corresponds to the ITS type 1 allele sequence and VNIIIB corresponds to the ITS type 2 allele sequence obtained from the AD hybrid strains after cloning the amplified ITS1-5.8S-ITS2 region.

2

ITS sequence signatures which make it possible to separate between C.n. var. grubii, C.n. var. neoformans and C.n. var. gattii, as well as between the molecular types of C.n. var. gattii, based upon eight combinations of variable nucleotides at positions 10, 11, 15, 19 and 108 in the ITS1 region, 221 in the 5.8S rDNA gene, and 298 and 346 in the ITS2. VNIIIA corresponds to the ITS type 1 allele sequence and VNIIIB corresponds to the ITS type 2 allele sequence obtained from the AD hybrid strains after cloning the amplified ITS1-5.8S-ITS2 region.

Cloning of serotype AD ITS regions

The ITS1-5.8S-ITS2 regions amplified from one serotype AD strain (IFM46138), representing the URA5 hybrid between VNI and VNIV [15], were cloned and then randomly selected clones were individually re-sequenced, to determine whether the hybrid strains are haploid or diploid at the ITS locus. All cloned sequences were found to harbor either the ITS type 1 or the ITS type 2 allele sequence, corresponding to the serotype A or D ITS sequence, respectively. This confirmed that the serotype AD hybrid is diploid at the ITS1-5.8S-ITS2 locus.

ITS signature sequences for subtyping C.n. var. gattii

The ITS region including the 5.8S rRNA gene was sequenced for 68 strains of C.n. var. gattii and compared with those from C.n. var. grubii, C.n. var. neoformans and the AD hybrid. The ITS types were defined based upon the sequences at four variable positions, positions 10 and 15 in the ITS1 region, and 298 and 346 in the ITS2 region. Of the 68 strains, 14 were classified as AAGG=ITS type 5, 29 as AAAC=ITS type 4, 16 as GGGC=ITS type 3, and seven as AGGC=ITS type 7 (Fig. 1). Two strains (IFM50896 and B-5742) could not be assigned to any of these groups; the four-base sequence combination for these strains was AAGC. It was observed that the AAGC ITS type has greatest similarity to, and possible origins within, the AAAC and AGGC types. Therefore the AAGC sequence type was designated a new ITS type 6 (Fig. 1). ITS sequencing divided all 68 studied C.n. var. gattii strains into five ITS types.

ITS sequence signatures to subtype all varieties and molecular types of C. neoformans

Based on the combination of eight different bases at the positions of 10, 11, 15, 19, 108 (ITS1 region), 221 (5.8S region), 298, and 346 (ITS2 region), we found that the 96 strains of C. neoformans could be classified into seven ITS types, which could be correlated to molecular types previously determined by PCR fingerprinting [12,14,16] or RAPD analysis [17]: ITS type 1 (ATACTAGC=C.n. var. grubii; molecular type VNI and VNII and the serotype A allele of the AD hybrid (VNIIIA), ITS type 2 (ATATAGGC=the serotype D allele of the AD hybrid (VNIIIB), AD hybrid after direct sequencing of the PCR product and C.n. var. neoformans; VNIV), ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC) both=C.n. var. gattii; VGI=RAPD type III), ITS type 4 (ACACTGAC=C.n. var. gattii; VGII=RAPD type II), ITS type 5 (ACACTGGG=C.n. var. gattii; VGIII=RAPD type I), and the new ITS type 6 (ACACTGGC=C.n. var. gattii; VGIV) (Fig. 2).

Phylogenetic analysis of the ITS1-5.8S-ITS2 region

In order to determine the phylogenetic relatedness between the different molecular types and varieties of C. neoformans, representative sequences of each ITS type and previously identified molecular types were aligned and a phylogenetic analysis was carried out using the program PAUP* 4.0b10, generating six maximum-parsimony trees, each with a tree length of 173 steps, a consistency index (CI) of 0.9711, a retention index (RI) of 0.9609, and a homoplasy index (HI) of 0.0289. The consensus tree generated from this analysis clustered the obtained ITS sequences into two major branches with the first one comprising the ITS type 1 (C.n. var. grubii=molecular types VNI and VNII and the serotype A allele of the AD hybrid, VNIIIA) and ITS type 2 (serotype D allele of the AD hybrid (VNIIIB) and C.n. var. neoformans, VNIV) and the second cluster comprising all five C.n. var. gattii ITS types: ITS types 3 and 7 (both VGI=RAPD type III), ITS type 4 (VGII=RAPD type II), ITS type 5 (VGIII=RAPD type I) and the newly discovered ITS type 6 (VGIV) (see Fig. 3). The first cluster was subdivided into two branches, corresponding to ITS type 1 (C.n. var. grubii and the serotype A allele of the AD hybrid) or ITS type 2 (C.n. var. neoformans and the serotype D allele of the AD hybrid).

3

Phylogenetic analysis of the seven ITS sequences observed among C.n. var. grubii, C.n. var. neoformans and C.n. var. gattii. The heuristic search (PAUP* 4.0b10) resulted in six most parsimonious trees of which one is presented. Numbers above the branches represent the branch length and numbers below the branches indicate the proportions above 50 of the 100 bootstrap replications in which a given branch appears. Branch lengths are proportional to the character state changes as indicated on the marker bar. The tree consists of 173 steps and reveals a CI of 0.9711, RI of 0.9609, and HI of 0.0289. VNIIIA represents the ITS type 1 allele sequence and VNIIIB the ITS type 2 allele sequence obtained from the AD hybrid (molecular type VNIII) strains after cloning the amplified ITS1-5.8S-ITS2 region.

3

Phylogenetic analysis of the seven ITS sequences observed among C.n. var. grubii, C.n. var. neoformans and C.n. var. gattii. The heuristic search (PAUP* 4.0b10) resulted in six most parsimonious trees of which one is presented. Numbers above the branches represent the branch length and numbers below the branches indicate the proportions above 50 of the 100 bootstrap replications in which a given branch appears. Branch lengths are proportional to the character state changes as indicated on the marker bar. The tree consists of 173 steps and reveals a CI of 0.9711, RI of 0.9609, and HI of 0.0289. VNIIIA represents the ITS type 1 allele sequence and VNIIIB the ITS type 2 allele sequence obtained from the AD hybrid (molecular type VNIII) strains after cloning the amplified ITS1-5.8S-ITS2 region.

PCR fingerprinting and RAPD analyses

PCR fingerprinting with the minisatellite-specific primer M13 and the microsatellite-specific primer (GACA)4 previously grouped over 1000 clinical and environmental isolates into eight major molecular types [12,14,16]. RAPD analysis has previously grouped isolates of C.n. var. gattii into three RAPD types. The PCR fingerprinting molecular type VGI (corresponding to the RAPD type III) was further subdivided by ITS sequence analysis into two ITS types: ITS type 3 and ITS type 7, which differed by one nucleotide at position 10 of the ITS1 region. Examples of PCR fingerprints obtained from ITS type 7 strains are shown in comparison to the eight molecular PCR fingerprinting standards in Fig. 4. No specific differences were found in the PCR fingerprinting profiles or RAPD patterns within the ITS type 7 isolates, when compared to those of ITS type 3.

4

PCR fingerprint profiles obtained from ITS type 7 isolates using the microsatellite-specific primer M13 (5′-GAG GGT GGC GGT TCT-3′) as a single primer in the PCR. Lanes 1–5=ITS type 7 isolates, lane 6=1-kb Gibco BRL molecular marker, lanes 7–14=the molecular type standard strains.

4

PCR fingerprint profiles obtained from ITS type 7 isolates using the microsatellite-specific primer M13 (5′-GAG GGT GGC GGT TCT-3′) as a single primer in the PCR. Lanes 1–5=ITS type 7 isolates, lane 6=1-kb Gibco BRL molecular marker, lanes 7–14=the molecular type standard strains.

The C.n. var. gattii strains IFM50896 and B-5742, previously assigned to molecular type VGIV by PCR fingerprinting, were observed to have a unique ITS1-5.8S-ITS2 sequence, and were assigned a new grouping, ITS type 6. This subgroup of C. neoformans strains had not been found in the RAPD analysis conducted previously in Japan [17]. RAPD analysis was carried out on the ITS type 6=VGIV strains in this study, and compared with the previously established RAPD patterns. The RAPD patterns of the ITS type 6 strains were different from representative strains of the other ITS types: IFM5879 (ITS type 5=VGIII=RAPD type I), IFM48218 (ITS type 4=VGII=RAPD type II), and IFM46640 (ITS type 7=VGI=RAPD type III). RAPD bands of approximately 380, 890 and 980 bp were observed to be characteristic for the ITS type 6 strains, and were not present in the RAPD profiles of strains belonging to any other ITS types (Fig. 5). A new RAPD patterns, RAPD type IV, was established, which corresponds to the PCR fingerprinting molecular type VGIV.

5

RAPD patterns of the new genotype RAPD type IV of C.n. var. gattii of strain IFM50896 (ITS type 6) compared to representative strains of other ITS types: IFM5879 (ITS type 5=RAPD type I), IFM48218 (ITS type 4=RAPD type II), and IFM46640 (ITS type 7=RAPD type III).

5

RAPD patterns of the new genotype RAPD type IV of C.n. var. gattii of strain IFM50896 (ITS type 6) compared to representative strains of other ITS types: IFM5879 (ITS type 5=RAPD type I), IFM48218 (ITS type 4=RAPD type II), and IFM46640 (ITS type 7=RAPD type III).

Discussion

Variation within the ITS regions including the 5.8S rRNA gene for the three varieties of C. neoformans and the AD hybrid was examined via sequencing to measure intraspecific variation. In the current study, five variable nucleotide (nt) positions were observed within ITS1, one within the 5.8S rRNA gene, and two within the ITS2 region. Previous comparisons of the cryptococcal ITS regions observed only three variable positions within the combined ITS1 and ITS2 regions between isolates representing serotypes A, D, B, and C [24]. Given that only four strains were used in that study, certainly the variable nucleotide positions observed with different molecular types, particularly within C.n. var. gattii, could not be discerned. In addition, the ITS1, 5.8S, and ITS2 regions were reported to be 124 nt, 158 nt and 187 nt in length (total region length=469 nt), respectively [24], whereas in the current study the ITS1 region was observed to be 121 nt, the 5.8S gene 169 nt, and the ITS2 region 173 nt in length (total region length=463 nt). Close examination of the sequences of Fan et al. [24] available from the NCBI database (http://www.ncbi.nlm.nih.gov) revealed that the ITS1, 5.8S, and ITS2 regions were 120 nt, 169 nt, and 173 nt in length (total=462 nt), respectively, despite the different lengths originally reported [24]. Alignment of their rDNA sequences (accession numbers L05428 and L14068) [24] to the ITS sequences in the herein reported study revealed a single deletion of a T nucleotide in the sequence obtained by Fan and co-workers, at position 76 of the alignment, corresponding to the ITS1. The sequences observed in the current study did not have any insertions or deletions causing length variability, and are in agreement with those sequences of the locus reported in previous studies [17]. The observed deletion within the sequence of Fan et al. [24], may be attributed to the relatively crude sequencing techniques available at that time.

In addition, the current analysis revealed that each variety of C. neoformans can be clearly differentiated based upon its ITS sequence. A single variable nucleotide was also found at position 19 of the ITS1 region, comprising a T in C.n. var. neoformans and the serotype D allele of the AD hybrid, and a C in the C.n. var. gattii, C.n. var. grubii and the serotype A allele of the AD hybrid. Comparison of the 5.8S gene sequences from 68 strains of C.n. var. gattii, seven C.n. var. neoformans strains and 10 serotype AD hybrid strains with those obtained from 11 C.n. var. grubii strains revealed a single nucleotide difference at position 221, with G for the first group and A for C.n. var. grubii. Hence, by determining the base composition at position 19 in the ITS1 region and position 221 of the 5.8S region, all three varieties could be differentiated with C.n. var. grubii CA, C.n. var. neoformans TG and C.n. var. gattii CG.

Nucleotide differences delineate C.n. var. gattii and C.n. var. grubii (including the serotype A allele of the AD hybrid) from C.n. var. neoformans (including the serotype D allele of the AD hybrid) at the ITS1 locus. From these results, we propose that the four nucleotide positions 11, 19, 108 and 221 should form the basis for the differentiation of the three varieties, having nucleotide combinations of TCTA=C.n. var. grubii, TTAG=C.n. var. neoformans, and CCTG=C.n. var. gattii. This single PCR step/sequencing method is simple and reliable due to the small amplified ITS band size of 463 bp.

It was observed that cloned ITS sequences from serotype AD revealed two ITS sequence types: ITS type 1=serotype A allele and ITS type 2=serotype D allele, indicating that the serotype AD is diploid at this locus. These results confirmed the results found in a previous study examining the ploidy of the serotype AD [22]. This study observed that strains of serotype AD comprised either two or three different copies of the URA5 gene, which corresponded to URA5 sequences from strains corresponding either to molecular types VNI, VNII and VNIV or VNII and VNIV, suggesting different recombination events between C.n. var. grubii and C.n. var. neoformans leading to diploid or triploid strains [16,22]. If cloned ITS sequences from serotype A, B or C strains were sequenced, only a single ITS type per sequenced strain was obtained, confirming the haploid nature of each of these strains. No heterogeneity in the sequences of this locus of the rRNA repeat unit was found within a single haploid serotype A, B or C strain (data not shown). For unknown reasons, direct sequencing of the ITS1-5.8S-ITS2 locus from the AD hybrid strains, without prior cloning, revealed always only the ITS type 2. The two alleles of this locus of the AD hybrid strains could only be obtained after cloning of the PCR product. A similar phenomenon was observed when sequencing other parts of the rRNA locus (data not shown) or the URA5 gene [22].

Interestingly, it was found that C.n. var. gattii strains belonging to molecular type VGI and RAPD type III could be further sub-typed based upon their ITS genotype. Strains in this group were observed to belong to either ITS type 3 or ITS type 7. Isolates from Australia and Papua New Guinea were observed to belong to ITS type 7 (Table 1), and most of the strains of ITS type 3 originated in Asian countries. Further investigations into the geographical associations of these ITS types would be of interest.

Clear associations were observed between the ITS types observed herein, and PCR fingerprinting and RAPD profiles observed in previous studies. In addition, by correlation of known PCR fingerprinting, RAPD and amplified fragment length polymorphism types [10], it has been possible to infer the relationship of ITS types to the genotypes determined in a study of the intergenic spacer (IGS) of C. neoformans[25]. ITS type 1 corresponds to the IGS genotypes 1A, 1B, and 1C, ITS type 2 corresponds to IGS genotypes 2A, 2B, and 2C. The ITS types 3 and 7 correspond to IGS genotype 4, ITS type 4 corresponds to IGS genotype 3, and ITS type 5 to IGS genotype 5. Representatives of ITS type 6 (PCR fingerprint molecular type VGIV, RAPD type IV) were not included in the published IGS study. However, the currently estimated phylogenetic relationships between isolates of each of the seven observed ITS types are in concordance with those determined using the IGS region [25].

C.n. var. gattii was first reported to be associated with Eucalyptus trees and the dispersal of this agent has been proposed to be related to eucalypt exportation from Australia throughout the world [7,12,26]. It has been found that clinical and environmental isolates of C.n. var. gattii from Australia and the USA could be classified into three major profiles (VGI, VGII and VGIII) using the RAPD method and PCR fingerprinting [7,13]. Among the three profiles, a predominance of type VGI was reported [7,13]. Our analysis of C.n. var. gattii showed that the clinical and environmental strains with the VGI molecular type from the USA [12,14,16] and RAPD type III profile [17] belong to ITS type 3, and other strains with the VGIII molecular type [12,14,16]=RAPD type I [17] belonged to ITS type 5. These data support previous suggestions of the possibility of a recent fungal transfer due to eucalypt exportation from Australia to the USA [7,26].

Of the 68 C.n. var. gattii strains, two (IFM50896 and B-5742) revealed a novel ITS genotype. These isolates, originating from Johannesburg, South Africa, and Punjab, India, respectively, both belong to the PCR fingerprinting molecular type VGIV [12,15]. VGIV has been isolated infrequently only among isolates from South Africa, India, and some countries within South America [16,27]. Our data support these PCR fingerprinting results, and we propose the designation of the new ITS type 6 for these strains. Since both strains were also found to belong to serotype C, the ITS genotypes among the other serotype C strains in the study were investigated. ITS typing results from the four other serotype C strains in the study (CBS6993, IFM5873, IFM5875, and IFM5883) revealed that they belonged to ITS type 5, corresponding to molecular type VGIII=RAPD type I. These data suggest that the serotype does not necessarily conform to ITS genotype delineations within C.n. var. gattii. Similar observations were made in molecular epidemiology studies using PCR fingerprinting [12,14,16,28], which suggests that serotyping is not a reliable typing method for C. neoformans.

From the point of view of DNA sequence similarity, ITS type 6 (ACACTGGC) (VGIV=RAPD type IV) has greater similarity to ITS type 5 (ACACTGGG) (VGIII=RAPD type I) and ITS type 4 (ACACTGAC) (VGII=RAPD type II) than to ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC) (both VGI=RAPD type III), based upon the rate of nucleotide substitutions. This level of heterogeneity was not observed among C.n. var. neoformans and C.n. var. grubii ITS sequences. Xu et al. [29], in a study of several cryptococcal genes, including the ITS1-5.8S-ITS2, estimated the divergence of C.n. var. gattii from C.n. var. grubii and C.n. var. neoformans to be 37 million years ago, whereas the divergence of C.n. var. grubii and C.n. var. neoformans was estimated at 18.5 million years ago. The divergence of serotypes B and C was estimated at 9.5 million years ago. Our observations of significantly greater diversity among C.n. var. gattii compared to that observed in C.n. var. grubii or C.n. var. neoformans support these estimates. C.n. var. grubii has been recently established [6] and the findings of this study support a phylogenetically fixed position of C.n. var. grubii, being distinct from the other two varieties of C. neoformans, but more closely related to C.n. var. neoformans than to C.n. var. gattii (see Fig. 3).

This study revealed further evidence for the delineation of three varieties within the C. neoformans species complex by providing a fast and informative technique to identify C. neoformans isolates in clinical laboratories to the variety level. It also indicates evidence for ongoing speciation within the C. neoformans species complex, by further subtyping of C.n. var. gattii isolates to the molecular type level and demonstrating the origin of the two alleles of the ITS1-5.8S-ITS2 locus of the AD hybrid strains.

Acknowledgements

This work was supported by the Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FNGRPF) program, through the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, of the Japanese Government in 2001 to Y.M. and the National Health and Medical Research Council of Australia, NH and MRC Grants 960792 and 990738 to W.M. S.K. and W.M. would like to thank Matthew Huynh for his help with the ITS cloning and sequencing.

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