Uneven Distribution of Mating Types among Genotypes of Candida glabrata Isolates from Clinical Samples

In order to shed light on its basic biology, we initiated apopulation genetic analysis of Candida glabrata, an emergingpathogenic yeast with no sexual stage yet recognized. A worldwidecollection of clinical strains was subjected to analysis usingvariable number of tandem repeats (VNTR) at nine loci. The clusteringof strains obtained with this method was congruent with thatobtained using sequence polymorphism of the NMT1 gene, a locuspreviously proposed for lineage assignment. Linkage disequilibriumsupported the hypothesis of a mainly clonal reproduction. Noheterozygous diploid genotype was found. Minimum-spanning treeanalysis of VNTR data revealed clonal expansions and associatedgenotypic diversification. Mating type analysis revealed that80% of the strains examined are MATa and 20% MAT ${alpha}$ and that thetwo alleles are not evenly distributed. The MATa genotype dominatedwithin large clonal groups that contained only one or a fewMAT ${alpha}$ types. In contrast, two groups were dominated by MAT ${alpha}$ strains.Our data are consistent with rare independent mating type switchingevents occurring preferentially from type a to ${alpha}$ , although thealternative possibility of selection favoring type a isolatescannot be excluded.

INTRODUCTION

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INTRODUCTION

Candida glabrata has emerged as significant among the etiologicagents of invasive candidiasis and is of particular concernbecause of its natural resistance to azole derivatives (17,39). This and/or the severity of underlying conditions of patientsinfected with C. glabrata may explain the higher mortality associatedwith this species compared to other Candida species (27). Todate, knowledge about the basic biology and the traits thatmake C. glabrata a human pathogen remains limited in comparisonwith the amount of data accumulated for Candida albicans, thepredominant pathogenic yeast. The population structure and modeof reproduction of a pathogen are highly relevant to questionssuch as those about the evolution of virulence and drug resistanceor the relationship between sexual reproduction and virulence.Recent works on the population structure of C. glabrata concludedthat reproduction is predominantly clonal, although the possibilityof rare recombination events has been suggested (9, 12). Incontrast to Saccharomyces cerevisiae, C. glabrata is an obligatehaploid which has apparently lost the ability to reproduce sexually,like many other fungal pathogens or symbionts (31).

As a part of a large comparative yeast genomics project, thecomplete genome of C. glabrata type strain ATCC 2001 (CBS138)was sequenced, assembled, and annotated (13). In contrast tothe lack of an observed sexual stage, this and other work haveshown that C. glabrata possesses three genetic cassettes, homologsof the HML, HMR, and MAT loci involved in mating type determination(MATa or MAT ${alpha}$ ) in S. cerevisiae (36, 40). In S. cerevisiae, homothallism(self-fertility) is the result of a sophisticated mating typeswitching mechanism in which the MAT locus is converted froma to ${alpha}$ and vice versa at each generation (25). This relies ona DNA double-strand break created by the HO endonuclease, followedby homologous recombination with one of the silent cassetteslocated in subtelomeric regions harboring either the a or ${alpha}$ allele,namely, HMR and HML, respectively (37). Interestingly, in additionto an HO endonuclease gene homolog, C. glabrata also possesseshomologs of the majority of genes involved in the mating andmeiosis processes, as is the case for other fungi with no observedsexual cycle (31).

The presence of these genes in the genome does not, however,provide information on the existence and frequency of sexualreproduction in nature. Although the existence of both MATaand MAT ${alpha}$ types in natural populations of C. glabrata was described(5, 29), the distribution of mating types among genotypes andthe possibility of mating type switching within populationshas not been studied in depth.

Multilocus-based genotyping methods such as multilocus enzymeelectrophoresis, multilocus sequence typing (MLST), and multilocusvariable number of tandem repeats (VNTR) analysis are valuabletools for population structure analysis and phylogenetic reconstruction.Multilocus VNTR analysis allows improved discrimination (2),which can be useful for fine typing and short-term evolutionaryquestions, although phylogenetic relationships must be inferredwith caution because of the homoplasy inherent to microsatellitemarkers (10). Length polymorphism analysis of such regions hasbeen successfully applied for typing other fungi, such as C.albicans, Aspergillus fumigatus, Coccidioides immitis, and Saccharomycescerevisiae (4). Like all eukaryotic genomes analyzed so far,the genome of C. glabrata reveals a substantial number of microsatellitesequences.

In this work, we have characterized by VNTR analysis the diversityand relationships at a microevolutionary level of a collectionof 198 C. glabrata strains isolated mostly from clinical samples.Phylogenetic relationships at a deeper evolutionary level wereassessed by NMT1 gene sequencing, while determination of themating type was performed to investigate the evolutionary historyof mat switching.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains. One hundred ninety clinical isolates collected from distinctindividuals and eight environmental or nonhuman isolates weretested (see Table S1 in the supplemental material). Strainshad been isolated from Africa, Oceania, North and South America,and Europe from 1932 to 2004. One hundred thirty-seven clinicalstrains were collected during a European study of candidemiainitiated by the European Confederation of Medical Mycology(coordinator, R. Grillot). In all cases, identification wasconfirmed by biochemistry using the Api 32 C identificationsystem (BioMérieux, Lyon, France). All strains were storedat –80°C before testing.

Microsatellite analysis. (i) Selection of loci. Version 20 of the assembly of the C. glabrata genome (type strainATCC 2001) was used for the search of monotonous, perfect microsatellitesusing the Tandem Repeat Finder software from G. Benson (3) asimplemented on Institut Pasteur's web services (http://www.pasteur.fr/).Nine regions were selected, and primer pairs were designed andsynthesized; the forward primers were labeled with a fluorophore(Applied Biosystems, Courtaboeuf, France) (Table 1).

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TABLE 1. Characteristics of the nine microsatellite-containing loci and respective oligonucleotide primers for amplification used for the analysis of length polymorphism

(ii) Length polymorphism analysis. After a 2-day incubation of yeasts on Sabouraud agar plates(BioMerieux, Marcy l'Etoile, France), DNA was obtained fromcolonies using a one-step DNA extraction method with Chelex100 (Sigma-Aldrich, St. Quentin-Fallavier, France) resin (24),adjusted to a 50-ng/µl concentration. Amplification wasperformed in a volume of 15 µl with 1x buffer, a 5 µMconcentration of each oligonucleotide primer, 0.2 mM deoxynucleosidetriphosphates (Pharmacia, Uppsala, Sweden), 0.6 U of Taq GoldDNA polymerase (Eurogentec, Liege, Belgium), and 1.2 µlof DNA. Thermal cycling parameters were as follows: 7 min at94°C; 3 cycles of 30 s at 94°C, 30 s at 69°C, and30 s at 72°C; 3 cycles of 30 s at 94°C, 30 s at 65°C,and 30 s at 72°C; 3 cycles of 30 s at 94°C, 30 s at60°C, and 30 s at 72°C; 28 cycles of 30 s at 94°C,30 s at 55°C, and 30 s at 72°C; and a final extensionof 10 min at 72°C.

PCR products for a given strain were then pooled together witha molecular marker (400HD-Rox; ABI) and sized in an automatedsequencer (ABI377; ABI, Courtaboeuf, France). Sizes of the ampliconswere automatically assigned using GenScan Analysis software(vs3.1; ABI). Multilocus patterns were then determined usingthe Genotyper vs2.5 software (ABI).

Population genetic analysis. (i) Population structure based on VNTR and NMT1 sequence data. In a preliminary study of sequencing one example of severalalleles observed for each locus (data not shown), we did notobserve length changes associated with insertion/deletion withinthe flanking regions of the microsatellite loci. Consequently,fragment sizes were converted into deduced repeat sizes at eachlocus. Each unique allelic combination composed of the deducednumber of repeats at the nine VNTR loci was considered a newrepeat type (RT). Phylogenetic relationships were estimatedbased on RT using the minimum-spanning tree (MStree) methodas implemented in BioNumerics v5.10 (Applied-Maths, Sint-Martens-Latem,Belgium). The MStree algorithm links RTs so that the sum ofthe distances (number of distinct alleles between two RTs) isminimized (33); variants with the same number of mismatchesto several RTs were preferentially linked to RTs with the highesttotal number of single-locus variants (SLVs), or double-locusvariants in case of a match (16). Importantly, RT frequencywas not taken as a criterion for determining links in casesof matches; hence, the founder genotypes of clonal groups werenot identified by taking into account their frequency. In contrastto the unweighted-pair group method using average linkages (UPGMA)or phylogenetic methods, which place all extant genotypes (consideredleaves) at the tips of the branches, in an MStree graph boththe nodes and leaves correspond to observed genotypes (15).Therefore, in the graphical representation, strains with thesame allelic profile fall in the same circle, whose size isproportional to the number of strains with that particular RT.

Homoplasy along the branches of the MStree was calculated usingthe MultiLocus Analyzer program (S. Brisse, unpublished). Thehomoplasy was defined as 1 – (K – 1)/M, where Kis the number of alleles and M the number of changes along theMStree. Hence, if each allele was generated by a single evolutionarychange, the number of changes since the ancestral state wouldbe K – 1, and the homoplasy index would be equal to zero.In contrast, if alleles are often changing by convergent evolutionor reversion to an ancestral state, M would become much greaterthan K and the homoplasy index would increase toward 1. Thesame parameter was deduced along the UPGMA tree obtained basedon profile data using PAUP* v4 (38).

The sequence polymorphism of a fragment from the NMT1 gene (CAGL0A04059g)has been shown to be sufficient for the assignment of C. glabratastrains into clades as previously defined by Dodgson et al.(11). The sequences of this 607-bp fragment in a selection of148 strains were determined (see Table S1 in the supplementalmaterial). New allele sequences were submitted to the MLST.netwebsite (www.mlst.net). The distance matrices obtained withNMT1 typing and microsatellite typing were compared by use ofa Mantel test using the Cadm software, which tests the nullhypothesis of incongruence between matrices (28). One thousandpermutations were performed, with an equal weight assigned toeach matrix. A P value of <0.05 was considered significant.

The geographical structure of genetic variation was estimatedby hierarchical F statistics at the levels of the continent(F_{Continent/Total}), country (F_{Country/Continent}), and city (F_City/Country)by Yang's method (41) implemented in HierFstat v 0.04-4 (22)and tested with randomization of the appropriate subunits betweenunits of the higher level to be tested. For instance, to testF_City/Country, isolates were permuted across the different citieswithin countries, and F_{Country/Continent} was tested by permutingcities with all the isolates contained between the differentcountries within each continent. The statistic used was theG-based test as described by Goudet et al. (23), and the numberof permutations was set to 1,000. A simple description of HierFstatcan be found in reference 7. Note that F_City/Country could beassessed only for France and Brazil. Because a correlation betweenloci might bias population differentiation measures and testing,we repeated F_ST analyses using the multilocus genotype of eachindividual as a unique locus with as many alleles as definedby the multilocus genotypes.

(ii) Mode of reproduction. In order to test the reproductive mode, the proportion of significantlinkage disequilibrium among pairs of loci and the index ofassociation were estimated. Linkage disequilibrium tests wereimplemented by Fstat 2.9.3.2 [EC] . The statistic used is the log-likelihoodratio G. For each pair of loci, in each subsample, the G statisticis computed on each genotypic table of paired loci (28 pairs),and values are summed over all subsamples for each pair. Foreach pair of loci, in each subsample, genotypes are then randomlyassociated (free recombination), and the G statistic is computedand then summed over all subsamples for this randomized dataset. This randomization was repeated 10,000 times. The P valueobtained was the number of times a randomized G was as highas or higher than the one measured in the real data set (7,8). Because repetitive tests enhance type I error, we also correctedthese P values using the sequential Bonferroni procedure (26),where P values are ranked from the lowest to the highest andthe first one is multiplied by the total number of tests (n= 28), the second by n – 1, and so on until the correctedP values reaches 1. Multilocus linkage was measured with Agapowand Burt's r_D (1) in each subsample and its significance testedby 1,000 randomizations (random reassociation of alleles atthe different loci) with Multilocus v 1.3b (available at http://www.agapow.net/software/multilocus/and updated from reference 1). This provided a series of independenttests were that combined by the Fisher (20) procedure in whichthe n P values of a test series are combined as ${chi}$ ² = –2 Formula ln(P_i), a quantity that can be comparedto a chi-square distribution with 2n degrees of freedom.

In case of strong linkage disequilibrium, using each locus asan independent unit of information for population structureanalyses may lead to erroneous conclusions. We thus reanalyzedall our results by using the multilocus genotype as a singlelocus and each RT as a different allele at that locus.

Comparison of means was undertaken with a Wilcoxon rank sumtest and comparison of variances with a F test (34) with S-Plus2000 Professional release 3 (MathSoft Inc., 2000).

Mating type determination. Based on the sequence of the type strain ATCC 2001 and on previouswork (36), six primers were designed to test the presence ofa (a1 and a2) genes or ${alpha}$ ( ${alpha}$ 1, ${alpha}$ 2) genes specifically at the MATlocus: CgMATF, 5'-TGGTGAAGAACCCAGTTACG; CgMATR, 5'-TCGAAGGTGGTGATCTCTTG;CgMATalpha1R, 5'-ATGAAGTATACTGCCACAAAA; CgMARalpha2F, 5'-CTGAGAGAATGACGGAGAGTGTA;CgMATa1R, 5'-CCCAATACAAGATCTACGCA; and CgMATa2F, 5'-TGCGTAGATCTTGTATTGGG.These primers were used in pairs in four independent PCRs.

RESULTS

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RESULTS

VNTR-based genotypic diversity of C. glabrata. We selected nine microsatellite regions from the complete sequenceof the type strain ATCC 2001 (CBS138) (13) and analyzed theirlength polymorphism in 198 C. glabrata isolates. For the ninetested loci, all strains tested gave a unique PCR product, thesizes of which were used to deduce the repeat sizes, as indicatedin Table S1 in the supplemental material and available throughthe MLVA-NET website data system at www.pasteur.fr/mlva. Locus1 did not exhibit any polymorphism among the 198 tested strainsand was excluded from further analyses. For the other loci,three to eight different alleles were found per locus, witha total of 41 different alleles. The combination of allelesfrom the eight loci generated 90 different genotypes, or RTs,numbered RT1 to RT90. The four most frequent RTs were RT15 (n= 28 strains), RT13 (n = 19), RT52 (n = 13), and RT41 (n = 11).A total of 63 RTs were represented by a single strain. Overall,the genetic diversity (probability that two strains taken atrandom have different genotypes) was 0.96. When the eight lociwere incorporated successively in the analysis, the geneticdiversity reached a plateau, indicating that testing more lociwould not significantly enhance the discriminatory power (notshown).

Population structure of C. glabrata deduced from VNTR data. In order to visualize the genotypic diversity, an MStree analysisof the 90 distinct RTs was performed (Fig. 1). In this graphicalrepresentation, strains with the same allelic profile fall inthe same circle, the size of which is proportional to the numberof strains with that particular RT. We observe that most linkscorrespond to only one allelic mismatch and could thereforebe considered as corresponding to single evolutionary steps.Such pairs of RTs are defined as SLVs. However, the homoplasyindex calculated along the MStree was 0.73 (range, 0.67 to 0.81per locus), indicating that inferred phylogenetic links betweengenotypes that are not very closely related are probably unreliable.Similarly, the homoplasy index calculated along the UPGMA dendrogram(not shown) was 0.78.

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FIG. 1. MStree analysis of 198 C. glabrata strains based on allelic profiles at the eight VNTR loci. Each circle corresponds to an RT, the number of which is indicated inside the circle. The lines between circles indicate the similarity between profiles (bold, seven alleles in common; normal, six alleles; dotted, five alleles) Cross-links corresponding to a single allelic mismatch are indicated. Gray zones between some groups of circles delineate the six CCs (see text). In order to illustrate the concordance of VNTR data with NMT1 sequences, pie chart sections of circles were colored according to the NMT1 allele and number of isolates sequenced (white parts of pie charts correspond to the proportion of strains for which NMT1 was not sequenced).

The mode of reproduction was tested by the calculation of linkagedisequilibrium between loci, which is a major criterion fortesting clonality of haploid microorganisms. Within countries,among the 28 possible pairs of loci, 23 (82%) were in significantlinkage disequilibrium, with each locus being in significantlinkage with several other loci. At the sequential Bonferronilevel, 19 pairs (68%) remained significant and all loci wereinvolved several times in a significant linkage, except forvntr9, which was never found statistically associated with anylocus at this level; the latter result may be explained by theslightly increased homoplasy level of vntr9 (0.81) comparedto the other loci (range, 0.67 to 0.79). Differentiation wasmet between countries but not at smaller scales and not betweendifferent dates. Consequently, for linkage disequilibrium analyses,all strains from each country were considered as subsamples.Multilocus tests provided six subsamples, out of eight wherethe test was possible, with a significant linkage (r_D > 0,P < 0.005). Overall, the mean multilocus linkage (weightedfor sample size) was r_D = 0.155, with an associated combinedP value of <0.001 (obtained with Fisher's procedure). Overall,these results indicate a strong and global (genomic) associationbetween all loci in the different subsamples. Closer examinationof the MStree shows that the most frequent RTs (the largestcircles in Fig. 1) were often placed in a central position,with multiple SLVs that were generally much less frequent (smallercircles). This pattern is in agreement with the clonal expansion-diversificationmodel (15), according to which in clonal organisms, expandingsuccessful genotypes result in generation of a number of variants,each being more closely related to the founder genotype thanto other variants. Thus, each central frequent RT and its associatedSLVs may be considered as descending from a single ancestor.Given the high homoplasy index, the progressive diversificationof two independent expanding clones may result in similar oridentical genotypes by evolutionary convergence. Therefore,we chose to restrict the definition of clonal complexes (CCs)to the founder RTs with at least four isolates and their SLVs.With this definition, there were six CCs, which were named accordingto the number of the founder RT (CC6, CC15, CC41, CC52, CC64,and CC77), as shown in Fig. 1. Altogether, these CCs include143 strains out of 198 (72%). It must be noted that in CC15and CC52, one SLV itself included more than four strains (RT13and RT48, respectively), and therefore its own SLVs were alsoincluded in the complex. CC77 was a particular case, as it hadno SLV.

Correspondence of VNTR data with NMT1 sequences. In order to establish the correspondence of the six CCs withphylogenetic groups previously defined by MLST (12) and to definethe phylogenetic relationships among the six CCs, an internalportion of the NMT1 gene in representative strains of each CCand other strains was sequenced (see Table S1 in the supplementalmaterial). Indeed, alleles of this gene were previously shownto be strongly associated with MLST groups (11). Twenty distinctNMT1 alleles were found. In addition to eight alleles alreadydescribed, new allele sequences were found, which were referencedas NMT1-35 to NMT1-39 (http://cglabrata.mlst.net). NMT1 alleleswere mapped on the MStree (Fig. 1). Remarkably, the NMT1 alleledistribution was highly concordant with the VNTR data. Indeed,CC6, CC15, CC41, CC52, CC64, and CC77 were each characterizedby a predominant NMT1 allele (NMT1-2, NMT1-8, NMT1-3, NMT1-6,NMT1-16, and NMT1-4, respectively). These alleles had previouslybeen shown to characterize groups II, I, III, ST22, ST25, andIV, respectively (12). One or a few isolates of four of theseCCs (CC6, CC15, CC52, and CC64) contained variant NMT1 alleles.Examination of these allele pairs (NMT1-5 and NMT1-32, NMT1-8and NMT1-39, NMT1-6 and NMT1-37, NMT1-6 and NMT1-35, and NMT1-2and NMT1-3) showed that variant alleles differed by only onesingle-nucleotide polymorphism from the dominant allele of theirrespective CC, with the exception of NMT1-2 and NMT1-3 in CC6,with three single-nucleotide polymorphisms between them (seeFig. 1). Alleles from the strains sequenced outside of CCs weregenerally distinct from those characterizing the CCs, with afew exceptions possibly attributable to homoplasy or to ourrestricted definition of CCs (Fig. 1). The correspondence betweenVNTR and NMT1 was further supported by the high level of significanceof the Mantel test comparing the two distance matrices (P <0.01).

Geographical and temporal distribution of isolates. The influence of geographical origin on the genotype distributionwas tested at three hierarchical levels, i.e., continent, country,and city. Hierarchical analyses were undertaken for all isolatessampled between 1993 and 2003 with at least two representativesper city. Subsamples with no date, older than 1993, or witha single isolate were removed. We observed a weak and nonsignificantdifferentiation between cities within countries (F_City/Country= 0.012, P = 0.315), which is confirmed by the multilocus genotypeanalysis (F_City/Country = 0.007, P = 0.486). Between countries,differentiation is medium but highly significant with the eightmicrosatellite loci or multilocus genotypes (F_{Country/Continent}= 0.07, P = 0.001 in both cases). With microsatellites, differentiationbetween continents is the strongest (F_{Continent/Total} = 0.11,P = 0.015), while multilocus genotypes provide here a contradictoryresult (F_{Continent/Total} = –0.03, P = 0.415). This geographicstructure is reflected by the uneven distribution of some RTsor CCs. For example, although the majority of strains were isolatedin Europe, CC6 and CC64 show a high proportion of isolates fromNorth America (see Table S1 in the supplemental material). OnlyRT15 was isolated from four different continents. In contrast,the group of related RTs, RT72 to RT75, originated solely fromNorth America.

Our strain collection was isolated from patients between theyears 1996 and 2003, in addition to six reference strains isolatedbetween 1932 and 1988. A high number of RTs and all six definedCCs were isolated across several years. One extreme case wasRT15, isolated over a span of 40 years (see Table S1 in thesupplemental material), which suggests that VNTR profiles canbe stable over this time scale.

Phylogenetic distribution of mating type. We determined the mating types of 162 strains by a four-PCRsystem specifically targeting the genes at the MAT locus (Fig.2). Nearly 80% (n = 132) of our strains corresponded to MATaand 20% (n = 30) to MAT ${alpha}$ (see Table S1 in the supplemental material).There was no difference in the distribution of mating type accordingto either continent or country. Nevertheless, the frequencyof multilocus genotypes defined by VNTR data appeared to beunevenly distributed between the two mating types. Mating typea was more often associated with repeated genotypes (mean =2.42) than was mating type ${alpha}$ (mean = 1.50), although the differencewas not statistically significant (Wilcoxon rank sum test, P= 0.602). However, the variance in genotype frequency of eachmating type was very different (14.17 for a and 1 for ${alpha}$ ; F test,P < 0.001). This is due to the fact that the a type was oftenassociated with highly frequent RTs, while ${alpha}$ was never associatedwith RTs encountered more than four times.

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FIG. 2. Mating type determination for C. glabrata strains. (A) Genomic organization of Mat cassettes in C. glabrata and primers designed to specifically amplify genes at the MAT locus. Terminology for the HML, HMR, and MAT loci was by homology with S. cerevisiae. Note that the HML and MAT loci are located on chromosome B, while the HMR locus is located on chromosome E. (B) Results of amplification targeting loci ${alpha}$ 1 (upper left), ${alpha}$ 2 (upper right), a1 (lower left), and a2 (lower right). Type ${alpha}$ strains (lanes 5, 7, and 8) are US02Bal12, EF1521Blo1, and EF0510Blo1. Type a strains (lanes 1 to 4, 6, 9, and 10) are EF1515Blo1 ES0085Blo1, US003NY110, US02Bal020, EF1213Blo1, EF1204Blo1, EI4101Blo1, EF1031Blo1, US02Bal003, and USTCC90030.

In order to establish the phylogenetic relationships of a numberof strains of both mating types, we used the NMT1 sequencesto deduce the long-term phylogenetic relationships and usedthe VNTR data to infer microevolution. Since there was a one-to-onecorrespondence between CCs and the branches in the NMT1 phylogeny,we placed the VNTR-based CCs in front of their correspondingbranch (Fig. 3). Clearly, many well-supported NMT1 branchescontained both mating types. In four of the CCs, strains werepredominantly MATa. Nevertheless, each of these contained atleast one isolate of mating type ${alpha}$ . The phylogenetic associationof these rare MAT ${alpha}$ isolates with MATa isolates, initially deducedbased on multilocus VNTR data, was thus fully confirmed by NMT1gene sequencing. Contrasting with the predominance of MATa inother CCs, CC64 and CC77 comprised 18 MAT ${alpha}$ strains out of 30total. Remarkably, CC64 was made up exclusively of MAT ${alpha}$ strains.CC64 and CC77 did not appear to be associated together in asingle branch based on NMT1 phylogeny, suggesting an independentorigin of the ${alpha}$ type in these two groups.

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FIG. 3. Phylogenetic distribution of mating types. The neighbor-joining tree is based on Jukes-Cantor corrected distances between NMT1 sequences. The mating types of NMT1-sequenced strains are indicated at the tips of the branches: green circles, MATa; red triangles, MAT ${alpha}$ . The mating types of isolates of the six CCs, each being positioned at the tip of its corresponding branch, are indicated as a color pattern (red, MAT ${alpha}$ ; green, MATa) on the MStree of each CC. Bootstrap values obtained after 1,000 replicates are given at the nodes.

DISCUSSION

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DISCUSSION

Population genetic studies of C. glabrata are of major importanceto understand the biology and evolution of this prominent emergingpathogen among the etiologic agents of candidiasis. We definedVNTR markers based on the entire genome sequence of strain ATCC2001 and have used these markers to analyze a collection of198 C. glabrata strains. VNTR typing is a powerful tool forfine-scale population genetic studies and has been used fora number of fungal pathogens, such as Coccidioides immitis,Penicillium marneffei, and C. albicans (18, 19, 21).

The VNTR data were used to build an MStree that enabled us todefine subpopulations, or CCs. The six CCs were not clearlydemarcated based on VNTR data, as several of them were connectedon the MStree by a succession of links corresponding to singleallelic mismatches. This was in sharp contrast to the NMT1-basedphylogeny, in which all CCs appeared to be very distinct, withrestricted amounts of sequence polymorphism within CCs, comparedto sequence divergence among CCs (Fig. 3). We attribute thesecontrasting results to homoplasy in the VNTR data, which wasvery high. In contrast, homoplasy was virtually absent withinCCs. Therefore, VNTR and nucleotide sequences in housekeepinggenes clearly appear as complementary genetic markers that aresuitable at distinct evolutionary time scales. Nevertheless,concordance between VNTR-based groupings and NMT1 sequencessupports the idea that the disclosed CCs represent distinctivegenetic entities. NMT1 sequences were previously shown to correspondstrongly to phylogenetic groups, but two new groups were definedby our analysis, as was also the case in a recent study (29),highlighting the importance of representative strain samplingin order to achieve an accurate description of C. glabrata straindiversity.

It was interesting to observe that the central genotypes ofCCs, as identified by MStree analysis, were also the most frequentones. It is important to note that the central genotypes wereidentified by the MStree algorithm without taking into accounttheir frequency in the population but based only on the numberof their SLVs. Therefore, founder genotypes (RT52, RT41, RT15,and RT13) appear to represent the natural population of "infective"strains better than laboratory reference strains, which mayhave diverged genetically, perhaps because of nonoptimal modesof preservation in laboratories. For example, one can noticethat the type strain (ATCC 2001), which was used for the completegenome sequence project, is not included in any major CC andthus may not be the best representative strain of the species.Further attention should be given to these aspects with respectto virulence experiments. In addition, since biological differencessuch as resistance to flucytosine or alleles of the ALS genes(32, 35) have been previously demonstrated for genetic subgroupsof C. albicans, differences in clinically relevant propertiesshould also be evaluated for C. glabrata CCs or RTs. Selectingrepresentatives of each of the main CCs for the generalizationof any observation to the entire species is thus recommended.

The possible existence of mating and its impact at the populationlevel were also investigated. Many pathogenic hemiascomycetes,such as C. albicans, Candida tropicalis, and Candida parapsilosis,are considered to reproduce asexually most of the time (31).In contrast to the case for MLST analysis, incompatibility betweenloci cannot be tested with VNTR data because of homoplasy inherentto the method (10). However, the strong linkage disequilibriumbetween alleles for all pairs of loci and the significant indexof association demonstrated in this study support the hypothesisof a mainly clonal mode of reproduction in C. glabrata. Strongassociation of NMT1 alleles with VNTR-based CCs also suggeststhat homologous recombination of NMT1 alleles does not causefrequent replacement of alleles among distinct genetic backgrounds.Of note, we never observed the simultaneous presence of twoVNTR alleles. This could be explained either by an absence ofmating, by an instability of diploids, or by the presence ofhomozygote diploids resulting from inbreeding. Nonetheless,these homozygote diploids could still be expected to be heterozygousat the MAT locus, a situation we never encountered in our matingtype analysis. The pattern observed for several CCs, with apredominant genotype giving rise to closely related genotypes,is concordant with the clonal expansion-diversification modelof microbial evolution (15), according to which successful genotypesgenerate clonal families by multiple independent diversificationsfrom an ancestral genotype. Overall, these results are in accordancewith two previous studies, using multilocus enzyme electrophoresisand MLST, that concluded that the mode of reproduction of C.glabrata was mainly clonal (9, 12). Note that a predominantlyclonal mode of reproduction is not incompatible with rare eventsof genetic recombination, which were reported (12). The factthat the central genotype is also found to be the most frequentin the CC implies that the clonal expansion is recent or stillgoing on. Hence, the data suggest that as some genotypes (RTs)increase in frequency, they diversify by giving rise to a numberof variants, which can be revealed by VNTR analysis.

Even though no evidence for mating has ever been found in thisspecies, opposite mating types have been detected. In our collectionof strains, we found 80% MATa cells. This predominance of MATastrains is in accordance with the results reported by Srikanthaet al. (36), who found 58% MATa strains among 39 tested strains.In contrast, Lin et al. found only 32% MATa strains in a panelcollected from 37 patients, but since all of these originatedfrom Taiwan, this result may reflect differences in prevalenceof mating types and/or clones according to geography (29).

In the present study, the distribution of mating types amonggenotypes revealed two kinds of asymmetry. First, in most CCsthere were few MAT ${alpha}$ cells, whereas more than half of the totalMAT ${alpha}$ cells were clustered in two particular CCs. Hence, the distributionof mating types is highly nonrandom. This observation seemsincompatible with switching at frequencies in the range of VNTRmarker evolutionary rate, unless selection is acting againstMAT ${alpha}$ strains. Second, MAT ${alpha}$ was associated with rare genotypes(involving fewer than five isolates), in contrast to MATa, whichdominated frequent genotypes (up to 22 isolates per RT). Thisresult may be explained by a selective advantage of MATa isolatesin a clinical context or simply by the increased exposure ofpatients to MATa isolates. Regarding the phylogenetic distributionof mating types, our data support the hypothesis that matingtype switching occurred several times independently, at leastonce in each of CC6, CC15, CC41, CC52, and CC77. We observedthe existence of few MAT ${alpha}$ strains in the MATa-dominated CC6,CC15, CC41, and CC52. At first sight, this observation couldsuggest a preferential directionality of mating type switchingfrom MATa to MAT ${alpha}$ , rather than the reverse. Under this hypothesis,the maintenance of MATa isolates in the population could bedue to a selective advantage. However, it is not possible toexclude the possibility that switching occurred from ${alpha}$ to a inthese groups, followed by a selective advantage of MATa isolates.Nevertheless, the fact that the MAT ${alpha}$ -dominated CC64 did not showa single MATa variant, even if it had time to diversify intoseveral distinct RTs, could reflect the fact that switchingfrom ${alpha}$ to a is rare, at least in the CC64 genomic background.Indeed, we believe previous reports support our data: in thelaboratory, switching has been reported in PCR experiments butnever in live cells despite intensive searching (5, 6; our unpublishedresults). In human patients, switching is mentioned in two studies,where isogenic strains of both mating types were isolated froma single patient. In one study, it was speculated that switchingfrom a to ${alpha}$ occurred (5), and in the second, an MAT ${alpha}$ isolate wascollected later than two MATa isolates (29; C. Y. Lin, personalcommunication).

In S. cerevisiae, where switching occurs every other generation,it is driven by a DNA cut by the Ho endonuclease at the MATlocus. While the C. glabrata HO homolog does not contain anyof the point mutations known to inactivate HO in S. cerevisiae(6, 14), and while this gene is transcribed in growing cells(30), it is not known whether the enzyme is active, in whichcase its regulation could still be very different from thatin S. cerevisiae. Even in the absence of an active Ho, switchingcould occur by gene conversion between the MAT locus and theHML and HMR loci. Most C. glabrata cells harbor type ${alpha}$ informationat HML and type a information at HMR, like S. cerevisiae. Thegenome sequence has revealed that HML and MAT are located onthe same chromosome, while HMR is located on a different chromosome(14). This may explain the more frequent occurrence of switchingfrom MATa to MAT ${alpha}$ , while the reverse switch would involve twodifferent chromosomes and may be more difficult to achieve,thus providing a molecular hypothesis for the asymmetry in matingtype distribution reported here. Alternately, our sample, whichis dominated by clinical strains, may be biased in favor ofstrains of type MATa, while type MAT ${alpha}$ strains may predominatein other environments (that remains to be described). It isconceivable that ${alpha}$ strains have a selective disadvantage in humansor are less pathogenic and hence less frequently retrieved fromclinical samples. It is noteworthy that switching is not necessarilyindicative of sexual reproduction. For mating to occur, cellsof opposite mating types must meet, and this may be rare giventhe single-clone dominance found in infected patients. Matingtype switching in a single cell would theoretically providea possibility for mating between sister cells. This warrantsfurther studies of the possible link between mating and successfulevolution of C. glabrata strains as human pathogens, includingsystematic MAT typing during the course of infection in patientsat different body sites.

ACKNOWLEDGMENTS

This work was supported in part by ACI no. MIC0314 and GDR 2354from the CNRS. The platform Genotyping of Pathogens and PublicHealth receives financial support from Institut Pasteur andthe Institut de Veille Sanitaire. T. De Meeûs is supportedby the CNRS and IRD.

We thank colleagues from the Unité de GénétiqueMoléculaire des Levures and from Génolevures forstimulating discussions. We are indebted to Bill Merz (Baltimore,MD), D. Perlin (Newark, NJ), and G. Sanson (Sao Paulo, Brazil),who provided some strains tested in this work.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Hôpital St Antoine, 184 rue du Faubourg St Antoine, F75012 Paris, France. Phone: 33 1 49282186. Fax: 33 1 49283030. E-mail: christophe.hennequin{at}laposte.net

Published ahead of print on 16 January 2009.

Supplemental material for this article may be found at http://ec.asm.org/.

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