Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis.

Abstract

Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.Unlike most plants and animals, the vast majority of eukaryotic microorganisms can reproduce asexually, and this together with their small size can make it very difficult to determine the relative importance of sexual reproduction in nature by direct observation alone. Rather, molecular markers must be used to test for the clonal population structure expected if sex is absent and the recombinant genotypes expected if sex is present (1-3). It is important in such studies that marker identity reflect common descent and that identities due to convergences, parallelisms, and reversals be minimized; in this respect, the most informative markers are DNA sequences (4,5). However, most studies of human pathogens have used allozymes or random amplified polymorphic DNAs (RAPDs) (1-3, 6). Neither approach is completely satisfactory, as allozyme patterns can be misleading because of natural selection (7), and RAPD patterns can be difficult both to repeat and to interpret in terms of Mendelian loci (8)(9)(10) (coccidioidomycosis) in California, with case reports 10 times more frequent than normal (13,14). Like some 20% of fungi (15), no sexual stage has ever been reported in C. immitis (16). Unfortunately, the inability to cross strains has hindered basic and applied research on this species.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. MATERIALS AND METHODSFor our study we have analyzed 30 clinical isolates from 25 patients at a single hospital in Tucson, Arizona. Three patients contributed multiple samples, collected up to 3 weeks, 16 months, and 8 years apart, respectively (Table 1). ¶ All isolates were collected in [1979][1980][1981][1982][1983][1984][1985][1986][1987][1988][1989][1990], before the epidemic.Our strategy for finding molecular markers begins with low-stringency PCR amplification from genomic DNA using arbitrary primers (-20-mers) in various pairwise combinations (11). Genomic DNA was isolated following heat treatment to k...

Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis

“…Only 6 out of 10,000 such randomizations yielded the observed length of 25 (P ϭ 0.0006; informative sites only; range of sums of tree lengths from randomized data is 25-32; test performed using PAUP‫0.4ء‬d52), showing significant incompatibility among the five loci. This incompatibility indicates sexual reproduction in C. immitis, in agreement with data from a single locale (15). Note that the very low divergence among isolates and complete lack of multiple alleles in any of the sites suggest that homoplasy due to multiple hits is highly unlikely in our data set.…”

“…Yet such knowledge is essential for locating the sources of new outbreaks, defining the relatedness of isolates recovered from different patients, and developing effective vaccines against the fungus. Recently, a molecular genetic analysis of nucleotide polymorphisms among isolates from a single hospital in Tucson, Arizona, provided strong evidence for sexual reproduction (15). To test whether the species is panmictic in its entire range or whether there is geographic differentiation among different populations, we have used a sample of 17 isolates, from California, Texas, Arizona, Mexico, and Argentina, covering the entire known species range (see Table 1).…”

Disease surveillance in recombining pathogens: Multilocus genotypes identify sources of human Coccidioides infections

“…Within both species, F st is significant between most pairs of populations (Table 1), and isolates tend to cluster in phylogenetic analyses according to where they were found, showing that pronounced phylogeographical structure exists (4). Previous analyses have shown that levels of linkage disequilibria are low within these populations (barring South American isolates), indicating that recombination has caused local panmixia (17,18). Within South America, the likelihood that this population results from a postpleistocene radiation from North America, and has therefore undergone a population bottleneck, means that levels of linkage disequilibria are high relative those observed in North America (4).…”

Inbreeding levels of twoUstilago maydispopulations

“…F is values were estimated using only strongly polymorphic markers, defined as markers with allele frequencies of 30-70% (Burt et al 1996). Markers that showed significant linkage disequilibrium were not used in calculations of F is .…”