A molecular phylogeny of Pythium insidiosum Andrew M. SCHURKO 1 , Leonel MENDOZA 2 , C. Andre´ LE ´ VESQUE 3 , Nicole L. DE ´ SAULNIERS 3 , Arthur W. A. M. DE COCK 4 and Glen R. KLASSEN 1 1 Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. 2 Medical Technology Program, Department of Microbiology, Michigan State University, East Lansing, Michigan, 48824-1031, USA. 3 Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario KIA 0C6, Canada. 4 Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands. E-mail : umschur3@cc.umanitoba.ca Received 24 September 2002 ; accepted 6 March 2003. Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among 23 isolates of Pythium insidiosum, the etiological agent of pythiosis in mammals. The isolates were divided into three distinct clades that exhibited significant geographic isolation. Clade I consisted of isolates from North, Central, and South America, while clade II contained isolates from Asia and Australia. Also present in clade II was an isolate from a patient in the USA, but the origin of the infection may have been in the Middle East. Clade III was comprised of isolates from Thailand and the USA. All 23 P. insidiosum isolates were more closely related to each other than to any other Pythium species in this study. Additionally, all Pythium isolates formed a clade separate from both outgroup species, Phytophthora megasperma and Lagenidium giganteum. The ITS sequence results tend to support the existence of geographic variants or cryptic speciation within P. insidiosum. The sequence information obtained also provides an abundance of data for applications in the diagnosis of pythiosis and identification of P. insidiosum from clinical samples. INTRODUCTION Pythium insidiosum is the etiological agent of pythiosis, a granulomatous disease that occurs in tropical and subtropical regions, with some cases having also been reported in temperate regions. Pythiosis is character- ized by cutaneous and subcutaneous infections (Miller, Olcott & Archer 1985, Bentinck-Smith et al. 1989, Dykstra et al. 1999), bone lesions (Mendoza, Alfaro & Villalobos 1988, Alfaro & Mendoza 1990), esophagitis (Patton et al. 1996), gastrointestinal disease (Allison & Gillis 1990, Buergelt 2000), and pulmonary infections (Goad 1984) in mammals including equines, canines, felines, cattle, and a captive spectacled bear (Tre- marctos ornatus). In humans, symptoms such as arter- itis (Sathapatayavongs et al. 1989, Thitithanyanont et al. 1998), keratitis (Virgile et al. 1993, Badenoch et al. 2001), and cutaneous or subcutaneous infections (Shenep et al. 1998) have been reported. P. insidiosum has also been isolated from a mosquito larva (CBS 777.84). Pythium comprises over 120 described species, the majority of which are saprophytic or pathogenic to plants (van der Plaats-Niterink 1981, Dick 1990). P. insidiosum (de Cock et al. 1987) is the only species in the genus capable of infecting mammals. Included in this set of isolates are two of P. destruens (Shipton 1987) that have been shown to be conspecific with P. insidiosum based on immunodiffusion (ID) tests (Mendoza & Marin 1989) and restriction fragment length polymorphism (RFLP) analysis of the ribo- somal DNA (rDNA) intergenic spacer (IGS) (Schurko et al. 2003) ; the name P. insidiosum has priority (de Cock et al. 1987). Although fluorescent antibody and ID tests con- cluded that isolates of P. insidiosum from around the world were antigenically identical (Mendoza, Kaufman & Standard 1987), other studies revealed the existence of physiological and molecular variation. For instance, McMeekin & Mendoza (2000) showed varying effects of streptomycin on the growth of P. insidiosum iso- lates in vitro. Streptomycin inhibited or had no effect Mycol. Res. 107 (5): 537–544 (May 2003). f The British Mycological Society 537 DOI: 10.1017/S0953756203007718 Printed in the United Kingdom.