Molecular and Phenotypic Description of Coccidioides Posadasii Sp. Nov., Previously Recognized As the Non-California Population of Coccidioides Immitis

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Mycologia, 94(1), 2002, pp. 73–84. ᭧ 2002 by The Mycological Society of America, Lawrence, KS 66044-8897

Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis

M. C. Fisher1 dance (GCPSR) to detect genetically isolated groups The Institute of Zoology, Regent’s Park, London NW1 by comparing the gene trees from a number of dif- 4RY, UK ferent loci (Avise and Ball 1990). Different genes will G. L. Koenig have different genealogies within a species due to re- T. J. White combination, but between species the genealogies Roche Molecular Systems, 1145 Atlantic Avenue, will be concordant due to the effects of genetic iso- Alameda, California 94501, USA lation and drift causing lineage sorting and coales- J. W. Taylor cence. Detecting the common branches between Department of Plant and Microbial Biology, University these gene trees is the key to GCPSR. of California at Berkeley, Berkeley, California 94720- GCPSR is finding increasing usage within both 3102, USA meiosporic and mitosporic fungal taxa (Taylor et al 2000), for instance the Gibberella fujikuroi complex (O’Donnell et al 1998), Ajellomyces capsulatus (Kwon- Abstract: Coccidioides posadasii sp. nov., formerly Chung) McGinnis and Katz (Kasuga et al 1999), As- known as non-California (non-CA) Coccidioides immi- pergillus flavus Link (Geiser et al 1998) and Coccidi- tis, is described. Phylogenetic analyses using single oides immitis Rixford and Gilchrist 1896 (Koufopanou nucleotide polymorphisms, genes, and microsatellites et al 1997, 1998). Here, we use GCPSR to demarcate show that C. posadasii represents a divergent, genet- barriers to gene flow between individuals of the path- ically recombining monophyletic clade. Coccidioides ogenic fungus Coccidioides immitis. Our analysis and posadasii can be distinguished from C. immitis by nu- those of others clearly show the existence of two ge- merous DNA polymorphisms, and we show how ei- netically isolated and deeply divergent clades within ther of two microsatellite loci may be used as diag- C. immitis and we use this as the basis for describing nostic markers for this species. Growth experiments a new species, Coccidioides posadasii. Knowledge of show that C. posadasii has significantly slower growth rates on high-salt media when compared with C. im- genetically defined species enables workers to look mitis, suggesting that other phenotypic characters closely for previously undetectable morphological may exist. and phenotypic differences. We use this approach to Key Words: allele, Coccidioidomycosis, microsat- show that C. immitis has a tendency to grow faster ellite, Onygenales, phylogeny, systematics than C. posadasii on high-salt media. This demonstrates that other, perhaps clinically important, characters may exist. Coccidioides immitis is a dimorphic pathogenic fun- INTRODUCTION gus found in the southwestern United States, Mexico, Species can be defined as groups of organisms that Central and South America (Pappagianis 1988). In share a common evolutionary history and, as a con- the saphrobic phase C. immitis is characteristically sequence, share exclusive characters. This is known found inhabiting the arid, sandy soils of the Lower as the evolutionary species concept (ESC; Simpson Sonoran Life Zone. Inhalation of arthroconidia caus- 1951, Simpson 1961, Wiley 1978) and is the most in- es a chronic pulmonary infection in humans and oth- clusive species concept to date (Mayden 1997). Mo- er vertebrates. In ca 0.5% of cases, secondary coccid- lecular genetics (Reynolds and Taylor 1991) and cla- ioidomycosis occurs, a serious disseminated infection distic analyses provide a method of diagnosing spe- that is often fatal (Rippon 1988). Immunity gener- cies under the ESC by describing shared exclusive ated from resolving the infection is specific and usu- characters (apomorphies) using an operational ally lifelong. method known as phylogenetic species recognition Coccidioidomycosis was originally described by (PSR). A subset of PSR uses genealogical concor- Alejandro Posadas (and later confirmed by Robert Wernicke) from a soldier, Domingo Ezcurra, who ac- Accepted for publication June 4, 2001. 1 Corresponding author, phone: 020-7449-6617, Fax: 020-7586-2870, quired his infection in the Argentine pampas (Posa- Email: [email protected] das 1892, Wernicke 1892). Posadas and Wernicke rec-

73 74 MYCOLOGIA ognized the presence of an organism, likened to a This observation suggests that the species described protozoon of the order Coccidia. Formal description here are evolutionary species and could be recog- of C. immitis was performed by Rixford and Gilchrist nized as biological species, as well as phylogenetic from a case observed in California (Rixford and species, if a teleomorph were to be found. Gilchrist 1896). However, the parasite was then still Recently, the sampling of the C. immitis biogeo- thought to be a protozoan. The correct taxonomic graphic distribution was extended to include previ- status of C. immitis as an ascomycete fungus was dem- ously unsampled populations from Southern Califor- onstrated by Ophu¨ls and Moffit (1900) by culture on nia, Central and Southern Mexico, Venezuela, and artificial media of the fungal mycelia using arthro- Brazil, and analyzed using a suite of microsatellite spores isolated from laboratory infections of guinea markers (Fisher et al 1999, Fisher et al 2000b). Phy- pigs. The etiological relationship between C. immitis logenetic analyses showed that, despite the increased and coccidioidomycosis was also demonstrated by breadth and depth of sampling, the C. immitis phy- showing that arthroconidia cause infection in several logeny still contained two major clades (Fisher et al types of laboratory animal. The lack of any known 2000c). Here, we use our dataset of microsatellite al- meiosporic state in vitro or in vivo hampered further leles to show that these clades correspond to the pre- classification until work by Sigler and Carmichael vious classifications of CA (Group II) and non-CA (1976) recognized the similarity between the asexual (Group I) C. immitis. Species rank is proposed for spores (arthroconidia) of C. immitis and those (aleu- the two clades. rioconidia) found in the mitosporic genus Malbran- chea Sacc., placing C. immitis in the order Onygena- ceae. This relationship was confirmed by molecular MATERIALS AND METHODS phylogenetic methods (Bowman et al 1992, Pan et al One hundred and sixty-seven isolates identified by clinical 1994, Bowman et al 1996), and Uncinocarpus reesii laboratories as Coccidioides immitis were used in this study Sigler and Orr was shown to be the sister group to (APPENDIX). These isolates represent the entire known C. immitis. geographical distribution of the pathogen and are cryogen- Research on the intraspecific relationships of C. ically preserved in the Roche Molecular Systems Culture immitis was first attempted by Zimmerman et al Collection (RMSCC) for future reference (Roche Molecu- (1994), who compared RFLPs of total genomic DNA lar Systems, 1145 Atlantic Avenue, Alameda, California 94501, USA). Liquid cultures of each isolate were grown in and showed that 15 clinical isolates formed two a BL3 containment facility and total genomic DNA extract- groups, referred to as Group I and Group II. Group ed from lyophilized mycelia according to the protocol de- I contained the isolate ‘Silveira’ that is extensively scribed by Burt et al (Burt et al 1995). Polymerase chain used in laboratory studies. Subsequent work by Burt reaction (PCR) amplification of nine microsatellite-contain- et al (1997) using RFLPs of 10 DNA loci demonstrat- ing loci (GAC, 621, GA37, GA1, ACJ, KO3, KO7, KO1, ed the occurrence of highly significant differences in KO9) was performed for each isolate using the fluores- allele frequencies between clinical isolates from Cal- cently labelled primers and conditions described previously ifornia, Arizona and Texas, the Californian popula- (Fisher et al 1999). The multilocus genotype of each isolate tion being the most divergent. This result was cor- was determined by electrophoresing the PCR products roborated by Koufopanou et al (1997, 1998) who through a 6% denaturing polyacrylamide gel using an au- used genealogies of five nuclear genes to show that tomated sequencer (Applied Biosystems), the alleles pre- sent at each locus being determined by reference against a C. immitis consists of two non-interbreeding taxa, CA TAMRA-labelled internal size standard. A standardized (centered in California) and non-CA (represented by method for typing the alleles at each locus is available as a. clinical isolates from Arizona, Texas, Mexico, and Ar- pdf file at http://plantbio.berkeley.edu/ϳtaylor/mf.html. gentina). The ‘Silveira’ isolate was included in non- Phylogenetic analyses were performed using the micro-

CA C. immitis, showing that Zimmerman’s Group I satellite genetic distances DAS (Stephens et al 1992, Bowcock 2 and Koufopanou’s non-CA were synonymous. Nucle- et al 1994) and (␦␮) (Goldstein et al 1995). Here, DAS ϭ otide sequence divergence between CA and non-CA 1 Ϫ (the total number of shared alleles at all loci / n) where showed that they had been reproductively isolated n is the number of loci compared. Pairwise distances cal- from one another for the past 11 million years, a culated using the mean character distance option in PAUP* result that was subsequently corroborated using a 4.0b1 (Swofford 1998) are identical to DAS and were used separate set of loci and C. immitis isolates (Fisher et here. The neighbor-joining algorithm in PAUP clustered these user-defined distances using the minimum evolution al 2000b). That independent loci were randomly as- option, and support for each clade was estimated by 1000 sorting with respect to one another within CA and neighbor-joining bootstrap replications of the dataset. Ge- non-CA showed that genetic recombination had oc- netic distance between populations was assessed using the curred between individuals within the two groups, microsatellite distance (␦␮)2. This measure more closely re- despite no teleomorph ever having been described flects the genetic distance that has accrued within loci by for C. immitis (Burt et al 1996, Fisher et al 2000a). accounting for the size of alleles, as well as their frequen- FISHERETAL:DESCRIPTION OF COCCIDIOIDES POSADASII sp. nov. 75 cies. (␦␮)2 equals the square of the difference in mean al- (black) are shown in FIG. 1. These show that two loci, lele size (x) between two populations A and B such that GAC2 and 621, are diagnostic for CA and non-CA. 2 2 2 (␦␮) ϭ (xA Ϫ xB) . Confidence intervals for (␦␮) were The other microsatellite-containing loci all show calculated by bootstrapping over loci using the program MI- overlapping distributions in the sizes of alleles be- CROSAT (Minch et al 1995). Alignments were deposited in tween CA and non-CA, however, in all cases the allele Tree Base, and are available under study accession number frequencies are dramatically different illustrating the S692. We looked for differences in the phenotype of CA and deep genetic divergence between these two clades. non-CA by (i) comparing the growth rates of colonies on The results of taking into account allele size as well media with salt concentrations increasing to near-inhibitory as frequency are shown in FIG. 3. The tree inferred levels and by (ii) comparing spore-size. Pilot experiments using (␦␮)2 illustrates a tenfold increase in genetic were performed where 8 isolates were chosen (four of the distance between CA and non-CA genotypes when CA and four of the non-CA genotype) and grown on YEG compared to the distance seen between populations agar (1% yeast extract, 1% glucose, 1.5% agar, Difco, BD within each clade, and has strong (99%) bootstrap Microbiology Systems, Sparks, Maryland 21152) containing support. the following concentrations of NaCl; 0.034 M (2%), 0.068 Non-CA C. immitis has a much wider biogeograph- M (4%), 0.102 M (6%). Each isolate was initially grown on ical distribution when compared to CA C. immitis, YEG plates and 3 mm diameter plugs removed from the being recovered from across the southwestern Unit- colony margin. These were then placed on the test media, ed States, southern California, northern, central and incubated in the dark at 30 C, and colonies were measured southern Mexico and South America. No isolates of across their diameters after 15 d of growth. Subsequently, an expanded experiment was performed where 20 clinical non-CA C. immitis have been recovered from the San isolates (10 each of the CA and non-CA genotypes) were Joaquin Valley, California, except the isolate ‘Silvei- grown in replicates of 4 on YEG agar containing either ra’, commonly used in laboratory studies. Non-CA 0.034 M (2%) or 0.136 M (8%) NaCl, colony growth being and CA C. immitis appear to be sympatric in southern measured after 4, 8, 10, and 15 d of incubation at 30 C. California, and northern and southern Mexico; how- The length of arthroconidia were measured for a selec- ever low numbers of samples from central Mexico do tion of strains of the CA and non-CA genotypes. Cultures not rule out the occurrence of CA C. immitis in this were grown on malt extract agar (4% malt extract, 1.5% region. agar, Difco, BD Microbiology Systems, Sparks, Maryland We tested four different culture media in order to 21152), stained with lactophenol cotton blue (Hardy Diag- examine the colony growth rates of CA and non-CA nostics, Santa Maria, California 93455) and sealed with nail isolates. In a pilot experiment, a trend was observed varnish. Lengths were then determined for a minimum of where non-CA isolates (filled circles) appeared to 20 arthroconidia from each culture. grow more slowly than CA isolates (open circles) as NaCl concentration in the medium was increased RESULTS (FIG. 4). We investigated this effect further by increasing the scale of the experiment; 10 isolates from CA All microsatellite loci were highly polymorphic, with and non-CA were grown in quadruple replicates on between 8 and 26 alleles found at each locus (FIG. media containing either 0.034 M (low salt) or 0.136 1). Pairwise groupings using DAS show that all isolates M (high-salt) NaCl. FIG. 5 shows the population occur within one of two major clades, illustrated in mean and 95% confidence intervals for the growth FIG. 2A, B. Other than the support found for genet- rates of CA (open bars) and non-CA (filled bars) on ically identical isolates (shown as terminal branches the two media. While CA grew initially faster on low joined by vertical lines), these are the only clades that salt medium, by day 10 there was no significant dif- are supported by bootstrap analysis. Mapping previ- ference between the two groups. On high-salt (inhib- ously genotyped isolates from Koufopanou et al itory) medium, growth of CA and non-CA was re- (1997) and Fisher et al (2000b) onto this tree shows stricted relative to the low-salt medium. However, on that the ‘upper’ clade (comprising 106 samples) con- this medium significant inter-group variation was tains isolates that are exclusively of the non-CA ge- seen; growth of CA was significantly faster than non- notype (FIG. 2A), and that the ‘lower’ clade (com- CA for the duration of the experiment. The ranges prising 61 isolates) contains isolates that are exclu- of each group overlap, showing that this character- sively of the CA genotype. This demonstrates that the istic is not diagnostic between CA and non-CA (FIG. original division of C. immitis into CA and non-CA 5). Therefore, growth on high-salt plates appears to (Koufopanou et al 1997) using GCPSR holds across reflect phenotypic differences that have accrued be- the entire distribution of fungi represented in this tween CA and non-CA, but these are not discrete and study, and is the phylogenetic basis for our proposing may not be used to distinguish between the two spe- species status for CA and non-CA. The distribution of cies. alleles at each locus for CA (white) and non-CA We measured the size of arthroconidia for four 76 MYCOLOGIA

FIG. 1. Allele frequency distributions showing the sizes of PCR products at nine microsatellite loci for C. immitis (white bars; n ϭ 61) and C. posadasii (black bars n ϭ 106). cultures of CA (RMSCC 2006, 2007, 2009, 2010) conidia was not a diagnostic character for CA and and three of non-CA (1037, 1038, 1039). Means of non-CA. the two groups were as follows: CA; 5.96 ␮m (SD ϭ 0.93) and non-CA; 5.88 ␮m (SD ϭ 0.85). Use of TAXONOMY a two tailed t-test showed that the means of these distributions were not signiﬁcantly different from Based on these and other published data, a new spe- each other (t ϭ 1.977, d.o.f. ϭ 138, P ϭ 0.602). cies is proposed for non-CA C. immitis. We have cho- From this we concluded that the length of arthro- sen to recognize and name this evolutionary species FISHERETAL:DESCRIPTION OF COCCIDIOIDES POSADASII sp. nov. 77

FIG. 2. Cladogram inferred using DAS and neighbor-joining showing A. the C. immitis (CA) clade and B. the C. posadasii (non-CA) clade. Bootstrap values Ͼ50% are shown as numbers above branches, terminal branches joined by vertical lines denote isolates with identical genotypes. The tree is mid-point rooted. * 2239 was isolated in Texas, but the patient became infected in California (Burt et al 1997). ** 2375 was isolated from a Chinese patient with a history of travel in the USA. † non-clinical isolates from armadillo burrows, Brazil. ‡ ‘blind’ duplicated isolates used as genotype controls. § 1932, 1933, and 2030, respectively are isolates of ‘Silveira’ from R. Cox. SILV is an isolate of ‘Silveira’ from D. Pappagianis and shows length mutations at 2 loci compared with 1932, 1933, and 2030. RMSCC 2233A/B were two separate genotypes recovered from a single clinical sample. Z’94, K’97, B’97 and F’00 are references to the original papers that describe genotypes of the isolate (see Appendix). 78 MYCOLOGIA

FIG. 4. Summary of mean colony size Ϯ 95% conﬁdence intervals after 15 d growth on YEG media containing increasing concentrations of NaCl. White circles ϭ C. immitis (CA; n ϭ 4) and black circles ϭ C. posadasii (non-CA; n ϭ 4).

because the original description of C. immitis was from a patient in San Francisco who most likely con- tracted his infection within California (Rixford and Gilchrist 1896). Based on the geographical distributions of CA and non-CA, this infection was most likely to have been caused by CA C. immitis, which should therefore retain the name. Coccidioides posadasii Fisher, Koenig, White et Tay- lor, sp. nov. ϭ Coccidioides immitis, non-California population sensu Koufopanou et al, Proc. Natl. Acad. Sci. USA 94 5478– 5482 (1997). FIG. 3. Unrooted phylogram inferred using (␦␮)2 and Morphologia idem ac Coccidioides immitis, distinguibilis neighbor-joining showing the relationships of phylogeo- characteribus sequentibus nucleotiditis ﬁxationibus mutuis graphic populations within C. immitis and C. posadasii. inter Coccidioidem immitem et Coccidioidem posadasii: Bootstrap values Ͼ50% are shown. positiones synthase chitinis 192 (A), 288 (T); positiones dioxygenase 872 (C), 1005 (C), 1020 (G), 1179 (C), 1272 (T); positiones orotidine decarboxylase 473 (A), 506 (C), 606 (C), 647 (A); positiones serine 477 (G), 517 (C), 632 (C), 744 (G), 887 (C); positio chitinase 910 (T) (Koufo- panou et al 1997, Koufopanou et al 1998); positio z 134 (T) (Burt et al 1997). Loci sequentes microsatellitum distribu- FISHERETAL:DESCRIPTION OF COCCIDIOIDES POSADASII sp. nov. 79

FIG. 5. Summary of mean colony size Ϯ 95% confidence intervals (rectangles) and total ranges (lines) on A. low (0.034 M) and B. high (0.136 M) salt-containing YEG media. White bars ϭ C. immitis (CA; n ϭ 10) and black bars ϭ C. posadasii (non-CA; n ϭ 10). Day PI signifies day post inoculation. tionibus propriis allelones: GAC2, PCR amplitudo operis crosatellite loci have exclusive allele distributions: primeribus GAC2.1 et GAC2.2 ϭ 206 bp; 621, PCR ampli- GAC2, PCR product size using primers GAC2.1 and tudo operis primeribus 621U et 621L ϭ 397–401 bp. GAC2.2 ϭ 206bp; 621, PCR product size using prim- Coccidioides posadasii is morphologically indistin- ers 621U and 621L ϭ 397–401bp. Distribution: south- guishable from Coccidioides immitis. C. posadasii is di- western United States, Central and South America. agnosed by the following nucleotide characters (giv- HOLOTY PE: Pappagianis isolate ‘Silveira’ en as the gene, the nucleotide position in the gene, (RMSCC Silveira, appendix) isolated in the San Joa- and, parenthetically, the nucleotide fixed in C. posadasii) showing reciprocal fixation between C. im- quin Valley, California 1951. Widely used laboratory mitis and C. posadasii: Chitin synthase positions 192 isolate, maintained by the American Type Culture (A), 288 (T); Dioxygenase positions 872 (C), 1005 Collection (ATCC) #28868. This isolate is currently (C), 1020 (G), 1179 (C), 1272 (T); Orotidine decar- on regulatory hold at the ATCC due to C. immitis boxylase positions 473 (A), 506 (C), 606 (C), 647 being a controlled pathogen. A killed sample of (A); Serine proteinase positions 477 (G), 517 (C), RMSCC Silveira has been lodged in the Jepson Her- 632 (C), 744 (G), 887 (C); Chitinase position 910 (T) barium, University of California at Berkeley, Berkeley, (Koufopanou et al 1997, Koufopanou et al 1998). z California 94720, USA. Frozen samples of RMSCC position 134 (T) (Burt et al 1997). The following mi- Silveira are stored at Roche Molecular Systems, Ala- 80 MYCOLOGIA meda, California and the Centers for Disease Control and expense. However, this technique must be used and Prevention, Atlanta, Georgia. with caution as concerns exist when using microsat- Etymology. posadasii; after Alejandro Posadas who ellites for phylogenetic analyses due to their mode described the first case of coccidioidomycosis, which and rate of mutation. Constraints exist on the sizes was from Argentina (Posadas 1892) of microsatellites, causing a limit on the genetic distance that can accrue between genetically isolated taxa (Garza et al 1995, Lehmann et al 1996). Further, DISCUSSION the high mutation rates seen at microsatellite loci This study describes the division of Coccidioides im- cause the re-appearance of alleles that were previ- mitis into two species, C. immitis and C. posadasii, ously lost from populations, resulting in mutational using GCPSR. We have chosen to give species status convergence (homoplasy) of alleles that are identical to C. posadasii for the following reasons: (i) Genetic by size but not by descent. Such homoplasy can be isolation appears to be absolute as no incongruencies directly observed from the allele distributions illus- have been detected between C. immitis and C. posa- trated in FIG. 1, where C. immitis and C. posadasii dasii in the genealogies of the 12 nuclear loci studied share identical alleles at all loci except GAC2 and (five described by Koufopanou et al 1997 and seven 621. A cursory analysis of the data might lead to the described by Fisher et al 2000b), thus fulfilling the conclusion that genetic isolation between C. immitis criteria of GCPSR, (ii) the two species are deeply di- and C. posadasii was not absolute, due to the occur- vergent, estimations of the time of genetic isolation rence of alleles that are shared between the two taxa. ranging from 11 (Koufopanou et al 1998)— 12.8 We have addressed these concerns in a previous study (Fisher et al 2000b) million years ago, (iii) C. immitis by comparing the phylogeny inferred using the mi- and C. posadasii have been shown, separately, to ge- crosatellites against the phylogeny inferred using netically recombine in nature (Burt et al 1996, Fisher genes, and there showed that (i) they were identical, et al 2000a) and (iv) we have sampled the entire and that (ii) microsatellite alleles shared between the worldwide distribution of this pathogen, therefore we two taxa were a result of mutational convergence and feel that the possibility is small of discovering further not interbreeding (Fisher et al 2000b). This demon- cryptic species of Coccidioides that would change the strated that the Coccidioides microsatellite loci were taxonomic relationships described here. correctly diagnosing taxonomic units that had been Coccidioides immitis appears to have the smaller bio- identified using the GCPSR, and were therefore suit- geographic distribution of the two species, centered able for extending the GCPSR to include the isolates on the San Joaquin Valley, California. In this region, used in this study. However, it should be recognized C. immitis appears to be the only species, however that, without performing this initial study, using mi- two clinical isolates of C. posadasii, Silveira and K-727, crosatellite phylogenies alone to describe a species were recovered from this area. Silveira was isolated may not be valid. from a patient in Bakersfield in 1951 and K-727 from Recognition of the two species has enabled us to a patient in 1992; however, it is not known whether show that C. posadasii grows more slowly on media either patient had a history of travel to other parts of the southwest where these infections may have containing high salt concentrations. The range in been acquired. Such movements of patients suffering growth rates overlaps between the two species, show- from coccidioidomycosis have been previously de- ing that, although the difference in growth rates is tected using molecular markers (Burt et al 1997). significant, this phenotype is not diagnostic. The Coccidioides immitis and C. posadasii appear to be presence of differences in the amino acid composi- sympatric in southern California and Mexico. Here, tion of proteins between the two species (Koufopanou significant intraspecific differences in allele frequen- et al 1997, Peng et al 1999) suggests that other phe- cies between populations show that these are bona notypic differences may exist. This difference may ex- fida populations, and are not solely due to the im- tend to variation in the antigenicity of key proteins migration of patients with infections that were gained or pathogenicity factors. Recognizing the existence of from other areas (Fisher et al 2000c). C. posadasii is instrumental in allowing researchers In this paper, we have used phylogenetic analyses and physicians to investigate these possibilities. of microsatellite loci to identify isolates as C. immitis or C. posadasii. This approach was used because ac- ACKNOWLEDGMENTS quiring multiple gene genealogies for all the isolates described in this paper would be very difficult due We thank Jim Trappe for providing the Latin diagnosis, and to the amount of sequencing involved. Instead, the Jamie Platt for advice. The National Institutes of Health and microsatellites provided a means of acquiring multi- the California Healthcare Foundation contributed to fund- locus genotypes for many isolates with far less effort ing this study. FISHERETAL:DESCRIPTION OF COCCIDIOIDES POSADASII sp. nov. 81

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Proc Natl Acad Sci USA 98:4558–4562. and some other hypomycetes with arthroconidia. My- Garza JC, Slatkin M, Freimer NB. 1995. Microsatellite allele cotaxon 4:349–488. frequencies in humans and chimpanzees, with impli- Simpson GG. 1951. The species concept. Evolution 5:285– cations for constraints on allele size. Mol Biol Evol 12: 298. 594–603. ———. 1961. Principles of animal taxonomy. New York: Co- Geiser DM, Pitt JI, Taylor JW. 1998. Cryptic speciation and lumbia University Press. 247 p. recombination in the aflatoxin producing fungus As- Stephens JC, Gilbert DA, Yuhki N, O’Brien SJ. 1992. Esti- pergillus flavus. Proc Natl Acad Sci USA 95:388–393. mation of heterozygosity for single-probe multilocus Goldstein DB, Ruiz Linares A, Cavalli-Sforza LL, Feldman DNA fingerprints. Mol Biol Evol 9:729–743 MW. 1995. Genetic absolute dating based on microsat- Swofford DL (1998). PAUP*. Phylogenetic analysis using ellites and the origin of modern humans. Proc Natl parsimony (*and other methods). Version 4. Sunder- Acad Sci USA 92:6723–6727. land, Massachusetts: Sinauer Associates. Kasuga T, Taylor JW, White TJ. 1999. Phylogenetic relation- Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, ships of varieties and geographical groups of the hu- Hibbett DS, Fisher, MC. 2000. Phylogenetic species rec- man pathogenic fungus, Histoplasma capsulatum Dar- ognition and species concepts in fungi. Fungal Genet ling. J Clin Microbiol 37:653–663. Biol 31:21–32. Koufopanou V, Burt A, Taylor JW. 1997. Concordance of Wernicke R. 1892. Ueber einen Protozoenbefund bei My- gene genealogies reveals reproductive isolation in the cosis fungoides. Zentralbl Bakteriol 12:859–861. pathogenic fungus Coccidioides immitis. Proc Natl Acad Wiley EO. 1978. The evolutionary species concept reconsid- Sci USA. 94:5478–5482. ered. Syst Zool 27:17–26. ———, ———, ———. 1998. Concordance of gene gene- Zimmerman CR, Snedker CJ, Pappagianis, D. 1994. Char- alogies reveals reproductive isolation in the pathogenic acterization of Coccidioides immitis isolates by restric- fungus Coccidioides immitis (vol 94, pg 5478, 1997). tion fragment length polymorphisms. J Clin Microbiol Proc Natl Acad Sci USA 95:8414. 32:3040–3042. 82 MYCOLOGIA Locus 206206 ? 400206 22 218 400 262 260206 215 186206 186 399 261 237 399 241 218 186 294 215 294 261 241 235 262206 239 186 294 154 206 186 154 400 241206 235 400 241206 220 294 154 400206 220 294 400 262206 239 214 401 262206 239 215 186 154 399 260206 214 186 154 399 262 241 218 186 399 261 239 219 186 294 263 239 214 188 299 261 237 237 188 301 261 239206 235 186 294 152 252206 239 188 301 149 400 239206 235 294 147 400 241 237 233 294 154 400 235 216 297 150 262206 239 216 143 262 247 186 152 399 262 186 150 241 232 186 241 294 262 237 292 235 186 294 235 154 241 235 154 294 154 235206 154 400 215 ? 188 239206 299 399 237 220 151 260 186 239 299 237 147 GAC2 621.2 GA37 GA1 ACJ KO3 KO7 KO1 KO9 used in this study Coccidioides H18436993700 Z.3701 Quin (DNA)3737 R. Negroni3738 R. Negroni3740 R. Negroni2372 B. Wanke China 2375 B. Wanke2376 B. Wanke2377 R. Negroni Catamarca, Argentina2378 R. Negroni Jujuy, Argentina 2379 R. Negroni Catamarca, Argentina3795 R. Negroni3796 Piaui, R. Brazil Negroni3695 Piaui, R. Brazil Negroni (from3696 armadillo Piaui, G. burrow) Brazil San-Blas Argentina (from3704 armadillo G. burrow) San-Blas Argentina 2101 Naval Hospital Argentina2103 Naval Hospital ArgentinaKober UCSD Medical Argentina Center2214 T. Kirkland Argentina2215 T. Kirkland San Venezuela T. Diego, Kirkland2216 CA Barstow, CA Venezuela 2217 R. San Cox Diego, CA 2130 R. Cox2131 R. Cox2132 R. San Cox Diego,2133 CA R. San Cox Diego, San2233A CA R. Diego, Cox CA 2233B R. Cox Koufopanou et2234 al R. (’97) Cox Koufopanou M. et Rinaldi2235 al San (’97) Antonio, Koufopanou M. TX et Rinaldi2236 al San (’97) Antonio, Koufopanou TX et2237 al M. San (’97) Rinaldi Antonio, TX2238 M. San Rinaldi Antonio, TX2240 M. San Rinaldi Antonio, TX 2241 M. 206 San Rinaldi Antonio, TX 2062127 M. 206 San San Rinaldi Antonio, Antonio, TX TX 4002128 M. San San Rinaldi Antonio, Antonio, 400 TX 400 TX 2062129 M. 215 Rinaldi San2133 Antonio, R. 215 TX 215 Cox 400 San Burtcox4 Antonio, 262 206 R. et TX Cox al San 262 (’97) BurtSilveira1 Antonio, 261 206 R. 215 et TX Cox al 186 399 San (’97) BurtSilveira2 Antonio, 206 R. et TX 186 Cox al 186 400 R. San 262 (’97) BurtSilveira3 Cox Antonio, 206 241 R. et 215 TX Cox al 400 R. San (’97) 241Silveira4 Cox Antonio, 241 215 TX 186 294 400 R. San1036 262 Cox Antonio, 215 TX 294 294 D. 241 Burt1037 262 Pappagianis 235 et 215 186 al (’97), 239 Fisher1038 262 Koufopanou 235 San et 294 et Antonio, al 186 154 al TX (’00)1039 262 (’97) 241 J. San 154 Galgiani Antonio, Burt 186 154 TX et 2391040 241 al J. San (’97) Galgiani Antonio, Burt 186 294 TX et1041 241 al J. San 154 (’97) Galgiani Antonio, Burt 294 San TX San et1042 Joaquin 241 237 Joaquin al J. San Valley, Valley, (’97) CA Galgiani Antonio, Burt CA 294 San TX et1043 239 Joaquin al J. 206 Valley, (’97) Galgiani Burt CA 294 152 San et 239 Joaquin al J. Valley, (’97) Galgiani Burt CA 154 et 400 237 al J. (’97) Galgiani Burt 154 206 Tucson, et AZ al J. (’97) Galgiani 232 152 206 Tucson, AZ 400 206 Tucson, Burt AZ 262 et 400 al 206 Tucson, (’97), 216 Burt AZ Fisher et 400 Koufopanou et al et al 186 Tucson, (’97), 215 Burt al (’00) AZ Koufopanou et 400 (’97), et 262 al Zimmerman al Tucson, (’97), et 220 Burt (’97) AZ Fisher al et 241 et (’94) 259 al al Tucson, (’97) 232 206 (’00) AZ 186 263 206 294 Tucson, AZ 186 262 400 241 186 206 400 235 241 186 294 206 219 206 239 154 399 216 262 206 237 235 206 400 262 399 294 206 Burt 215 260 235 et 400 al 292 154 206 (’97), 399 Burt 186 247 247 Koufopanou 237 et 206 et al 262 186 147 al 206 (’97) ? Burt 219 (’97) 239 et 400 241 232 al 262 147 262 206 (’97), 399 Burt Fisher 253 186 et 400 et al 260 154 al (’97), Burt 292 219 219 (’00) Fisher 262 186 et 186 399 et 219 al 301 241 al (’97), 216 (’00) Fisher 186 et 235 237 al 186 241 Burt 216 (’00) ? ? 262 239 292 et 206 al 262 220 (’97), Burt 154 Fisher 292 et 237 294 et al 262 152 al 186 235 (’97) 399 (’00) 206 186 188 299 186 235 292 235 186 237 147 220 228 241 237 220 399 152 239 152 206 241 294 260 292 294 150 299 219 206 206 154 292 237 399 206 206 186 247 237 237 262 399 400 206 235 154 220 239 147 152 150 399 400 186 206 218 216 154 400 206 261 299 215 216 241 399 261 260 216 186 399 237 260 260 292 215 186 186 260 249 147 215 235 186 186 262 237 253 186 294 260 154 255 253 188 299 301 206 253 235 188 292 301 239 237 237 301 150 399 237 237 237 297 152 151 237 215 301 150 151 233 151 260 237 143 188 152 255 299 237 147 . Isolates of Species RMSCC Source Geographic origin References PPENDIX A C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii FISHERETAL:DESCRIPTION OF COCCIDIOIDES POSADASII sp. nov. 83 Locus 206 399 218 260 188 237206 299 399 247206 220 147 399 260 217 186206 263 239206 399206 186 299 399 216 400 237 237 213 260 215 299 147 262 186 260 239 188 253 210 152 ? 301 237 237 299 299 150 239 237 151 147 218224 419226 416218 231 419222 233 416 253 210 418 252 229 188 256 229 212 252 239 212 252 239 212 294 239 212 294 239 243 294 239 247 294 145 243 294 243 ? 164 243 147 160 GAC2 621.2 GA37 GA1 ACJ KO3 KO7 KO1 KO9 1044104510461047 J. Galgiani1048 J. Galgiani1049 J. Galgiani1050 J. Galgiani1051 J. Galgiani1052 J. Galgiani1053 J. Galgiani Tucson,1054 AZ J. Galgiani Tucson,1438 AZ J. Galgiani Tucson,1439 AZ J. Galgiani Tucson,1440 AZ J. Galgiani Tucson,1441 AZ J. Galgiani Tucson,1443 AZ J. Galgiani Tucson,1444 AZ J. Galgiani Tucson,1445 AZ J. Galgiani Tucson,1446 AZ J. Galgiani Tucson,1447 AZ J. Galgiani Tucson,2334 AZ J. Galgiani Tucson,3692 AZ J. Burt Galgiani et Tucson,3694 al AZ J. (’97) Burt Galgiani et Tucson,3480 al AZ J. (’97), Burt Galgiani Koufopanou et Tucson, et3487 al AZ Naval al (’97) Burt Hospital (’97) et Tucson,3488 al AZ Naval (’97) Hospital Tucson,3489 AZ I. Burt Gutierrez et Tucson,3561 al AZ I. (’97) Burt Gutierrez et Tucson,5239 al AZ I. (’97) Burt Gutierrez et 206 Tucson,5248 al AZ I. Yuma, (’97) Burt Gutierrez AZ et Tucson,5256 al AZ I. Arizona, (’97), 399 Burt Gutierrez AZ Fisher et et5275 al I. al (’97) Burt Gutierrez (’00) et3490 218 al I. (’97) Sonora, Burt Gutierrez North et Mexico3503 al I. (’97) Sonora, Burt Gutierrez 260 North et Mexico3506 al I. (’97) Sonora, Gutierrez North Mexico3507 I. 188 Sonora, Burt Gutierrez North et Mexico3509 al 206 I. (’97) Chihuahaua, Fisher Gutierrez North et 237 Mexico2343 al I. (’00) Chihuahaua, Gutierrez North 399 Mexico2344 206 I. 206 294 Chihuahaua, Burt Gutierrez North et Mexico2345 al 206 I. (’97) 216 Chihuahaua, Burt Gutierrez North et 399 238 399 Mexico2346 al R. (’97) Chihuahaua, Burt Diaz North et 399 Mexico2347 260 al 206 R. 153 (’97) 218 Coahuila, 218 Diaz North Mexico2348 206 R. 216 Coahuila, Diaz 188 North 399 Mexico5258 260 206 259 R. Coahuila, Diaz North 399 Mexico5268 260 253 R. 216 Coahuila, Diaz 188 North 188 399 Mexico5250 206 R. 249 Coahuila, Diaz 186 299 North Mexico5274 260 237 206 I. 239 220 Gutierrez 3993472 260 239 206 237 I. Gutierrez 186 299 299 3993473 261 Nuevo 206 I. Leon, 219 Gutierrez 186 295 154 Central 399 Mexico3477 Nuevo 239 247 I. 237 Leon, 219 Gutierrez 186 Central 399 Meixco3478 259 Nuevo 228 206 231 I. Leon, 218 206 Gutierrez 292 147 Central 152 Mexico3693 261 Nuevo 239 I. Leon, 218 Gutierrez 188 292 152 Central 399 Mexico3698 260 Nuevo 399 241 I. Leon, San Gutierrez 188 299 Central Luis Mexico Potosi,3703 260 Nuevo 237 206 241 I. Central Leon, 218 Durango, Koufopanou Gutierrez Mexico 188 152 Central Central 216 et Mexico Mexico3705 al 237 206 237 Naval (’97) Tamaulipas, Hospital 186 Central 152 399 Mexico3706 260 237 ? 206 Naval 263 Aguascalientes, Hospital 294 152 Central 397 Mexico 239 UCSD 216 Michoacan, Koufopanou Medical 188 299 South 399 et Center 188 Mexico al 237 235 UCSD (’97) 217 Michoacan, Medical 299 South Center Mexico 263 255 237 UCSD 219 Michoacan, 239 Medical San 152 150 South Center Diego, Mexico 260 237 CA San Michoacan, Diego, San 188 299 152 South 206 CA Diego, 292 Mexico 260 CA Barstow, CA San 188 152 206 Diego, 237 237 CA 399 237 186 206 206 255 399 299 152 206 206 152 233 243 399 399 299 206 250 237 399 399 299 262 206 248 206 232 247 399 152 206 219 239 ? 186 ? 152 261 399 260 ? 219 399 152 262 241 206 206 188 188 187 218 262 262 206 219 219 294 187 399 399 226 257 241 262 206 187 187 399 261 261 239 237 206 294 296 294 220 248 186 399 238 239 206 187 154 187 233 399 246 237 ? 235 258 262 241 294 294 216 399 257 258 262 206 147 147 218 152 186 186 294 237 235 262 206 ? 294 248 186 399 ? 262 237 241 239 150 152 186 206 399 237 261 241 206 219 186 301 292 154 ? 237 241 206 400 150 218 186 294 399 255 237 235 262 150 206 206 206 399 215 239 ? 235 262 218 294 143 154 186 400 399 399 247 262 294 154 186 234 235 262 241 206 220 219 216 239 262 187 241 206 154 186 294 399 ? 264 206 260 260 152 237 186 294 399 237 239 206 219 187 399 187 187 294 255 239 218 292 156 399 262 259 229 241 249 235 294 154 239 262 249 186 294 154 235 261 ? ? 154 186 261 228 233 154 186 255 237 241 186 299 154 228 294 163 154 228 241 294 235 294 154 229 154 229 154 154 . Continued Species RMSCC Source Geographic origin References PPENDIX A C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. posadasii C. immitis C. immitis C. immitis C. immitis C. immitis 84 MYCOLOGIA Locus 226 416228228 229 419222 252216 ? 227 419216 208 416 252216 231 229 419 239 212 419 256 252 ?226 228 252 ?226 228 210 210 419 256224 239 202 256 239 239 243 ? 231 210 294 426 239 210 294 147 256 239 ? 243 210 231 294 239 243 210 294 156 234 256 243 243 294 164 239 243 210 210 145 147 243 294 162 239 239 162 243 294 294 164 243 239 156 160 224 422 228 254 208 239 292 243 166 GAC2 621.2 GA37 GA1 ACJ KO3 KO7 KO1 KO9 2102210421052106 T. KirklandMart T. KirklandWolf T. KirklandJunior T. Kirkland2393 T. Kirkland2394 T. Kirkland T. Kirkland2395 San Diego,2396 CA A. San Catanzaro Diego,2397 CA A. San Catanzaro Diego,2239 CA A. San Catanzaro Diego,2002 CA A. San Catanzaro Diego,2006 CA A. San Catanzaro Diego, San2007 CA Diego, M. CA Rinaldi San2009 R. Diego, CA Talbot San2010 R. Diego, CA Talbot San2011 R. Diego, CA Talbot San2012 R. Diego, CA Talbot San2014 Fisher R. Diego, et CA Talbot al2015 (’00) R. Talbot San2017 Antonio, Fisher R. TX et Talbot al2267 (’00) R. San Talbot Joaquin Valley,2268 CA R. San Talbot Joaquin Valley,2269 CA R. San Talbot Joaquin Valley,2270 CA R. San Talbot Joaquin Valley,2271 CA R. San Talbot Joaquin Valley,2273 CA R. San Talbot Joaquin Fisher Valley, et2274 CA al R. San (’00) Talbot Joaquin Valley,2275 CA R. San Talbot Joaquin Valley, Burt2276 CA R. San et Talbot Joaquin al Valley, (’97), Burt2277 CA Fisher R. San et et Talbot Joaquin al al Valley, (’97)2278 CA (’00) R. San Talbot Joaquin Valley, Burt2279 CA 224 R. San et Talbot Joaquin al Valley, (’97), Burt2280 CA Fisher R. San et et Talbot Joaquin al al 419 Valley, (’97) Burt2281 CA (’00) 226 R. San et Talbot Joaquin al Valley, (’97) Burt3526 CA R. San et 229 Talbot Joaquin al 419 Valley, (’97), Burt3527 CA Fisher R. San et et Talbot Joaquin al al Valley, (’97) Burt3529 CA 252 (’00) R. San et 231 Talbot Joaquin al 224 Valley, (’97) Burt3530 CA R. San et Talbot Joaquin al 212 Valley, (’97) Burt3531 CA 256 R. San et 416 Talbot Joaquin al Valley, (’97), Burt3532 CA 239 Koufopanou R. San et et Talbot Joaquin al 220 210 218 al Valley, (’97) Burt 2283311 CA (’97) R. San et Talbot Joaquin al 294 Valley, (’97)3312 CA 239 R. 419 San 414 Talbot Joaquin 252 Valley, Burt3261 CA 243 R. San et Talbot Joaquin al 294 222 Valley, 231 (’97) Burt 2273377 CA R. 204 San et Talbot Joaquin al 220 147 Valley, (’97) Burt5252 CA 243 R. San et 220 420 Talbot 254 Joaquin al 252 239 Valley, (’97) Burt5273 CA R. San et 418 Talbot Joaquin al 145 Valley, (’97), 426 Burt 2283560 CA 210 Fisher D. 210 294 San et et Pappagianis Joaquin al 224 al Valley, (’97) 227 Burt3505 CA (’00) I. San et Gutierrez Joaquin 228 al 239 224 264 239 243 Valley, (’97) Burt3474 CA I. San et 416 Gutierrez 252 Joaquin al Valley, (’97) Burt3475 CA 294 I. 252 208 160 San et 418 Gutierrez Joaquin al 222 224 ? Valley, (’97) 231 Burt3476 CA 204 I. San et Gutierrez Joaquin al 243 220 239 206 Valley, (’97) 227 Burt3479 CA Monterey, I. San et 426 CA Gutierrez 252 Joaquin al 222 245 ? Valley, (’97),RS CA 164 ? Fisher I. 294 San 418 239 et Gutierrez 252 Joaquin al Valley, 229 CA (’00) 210 I. 424 156 Baja, Gutierrez 224 North 222 245 296 227 228 Mexico 208 I. Baja, Gutierrez 239 220 ? North 228 Mexico ? 156 418 243 422 Chihuahaua, 254 252 239 North T. Mexico 293 Kirkland 418 Coahuila, 254 222 243 North 150 240 Mexico 228 208 206 294 204 Michoacan, 220 243 South 228 Mexico 210 156 418 Michoacan, 252 239 222 239 255 243 239 South Mexico 154 424 Michoacan, 252 239 South 222 229 Mexico 212 294 294 156 294 227 416 Guerrero, 222 218 South 208 Mexico 210 294 424 252 239 218 243 243 239 235 ? Vaccine 418 414 strain-origin 254 239 218 243 unknown 240 206 294 160 160 294 414 254 224 228 228 210 294 160 149 414 239 222 252 243 243 208 204 416 252 254 239 239 228 294 166 210 162 420 254 239 227 210 294 202 154 254 243 239 228 210 294 254 239 243 239 202 145 294 252 239 243 206 294 160 294 239 243 206 294 149 239 247 224 243 294 149 239 243 224 294 168 162 426 243 222 294 156 416 245 220 211 162 416 243 218 229 160 416 252 216 241 147 418 252 224 208 212 418 255 222 229 207 424 252 239 222 209 212 418 252 239 226 210 187 294 414 254 239 222 231 187 294 419 252 239 243 226 231 226 203 294 222 416 252 239 245 229 206 294 156 224 419 252 239 245 420 420 206 241 294 164 216 239 243 256 426 210 231 294 166 228 218 232 245 226 416 ? 294 154 ? 209 229 239 243 256 256 414 156 256 424 228 243 239 294 294 154 252 212 212 230 210 ? 162 228 254 245 247 ? 210 240 239 252 239 210 239 252 154 154 239 294 243 294 ? 214 239 294 210 293 243 164 243 239 294 243 245 239 243 154 166 294 243 164 145 294 164 243 145 243 150 156 . Continued Species RMSCC Source Geographic origin References PPENDIX A C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis C. immitis