crytococcus heveanensis 的交配型位点和有性繁殖：真菌中的性决定染色体位点及性别进化

The Mating Type Locus(MAT)and Sexual Reproduction of Cryptococcus heveanensis:Insights into the Evolution of Sex and Sex-Determining Chromosomal Regions in Fungi

Banu Metin,Keisha Findley,Joseph Heitman*

Department of Molecular Genetics and Microbiology,Duke University Medical Center,Durham,North Carolina,United States of America Abstract

Mating in basidiomycetous fungi is often controlled by two unlinked,multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors.In contrast to this tetrapolar organization,Cryptococcus neoformans/ Cryptococcus gattii have a bipolar mating system,and a single biallelic locus governs sexual reproduction.The C.neoformans MAT locus is unusually large(.100kb),contains.20genes,and enhances virulence.Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus,converting an ancestral tetrapolar system to a bipolar one.Here we tested this model by studying Cryptococcus heveanensis,a sister species to the pathogenic Cryptococcus species complex.

An extant sexual cycle was discovered;co-incubating fertile isolates results in the teleomorph(Kwoniella heveanensis)with dikaryotic hyphae,clamp connections,septate basidia,and basidiospores.To characterize the C.heveanensis MAT locus,a fosmid library was screened with C.neoformans/C.gattii MAT genes.Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters:one corresponding to the homeodomain locus and the other to the pheromone/receptor locus.Strikingly,two divergent homeodomain genes(SXI1,SXI2)are present,similar to the bE/bW Ustilago maydis paradigm,suggesting one or the other homeodomain gene was recently lost in C.neoformans/C.gattii.

Sequencing MAT genes from other C.heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus,similar to known tetrapolar species.Taken together,these studies reveal an extant C.

heveanensis sexual cycle,define the structure of its MAT locus consistent with tetrapolar mating,and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of

a pathogenic clade.These studies provide insight into convergent processes that independently punctuated evolution of

sex-determining loci and sex chromosomes in fungi,plants,and animals.

Citation:Metin B,Findley K,Heitman J(2010)The Mating Type Locus(MAT)and Sexual Reproduction of Cryptococcus heveanensis:Insights into the Evolution of Sex and Sex-Determining Chromosomal Regions in Fungi.PLoS Genet6(5):e1000961.doi:10.1371/journal.pgen.1000961

Editor:Hiten D.Madhani,University of California San Francisco,United States of America

Received December17,2009;Accepted April20,2010;Published May20,2010

Copyright:?2010Metin et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:This study was supported by NIH/NIAID R01grants AI39115and AI50113to JH.The funder had no role in study design,data collection and analysis, decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:heitm001@https://www.wendangku.net/doc/7216918378.html,

Introduction

Although many organisms,in particular microorganisms,can reproduce both asexually and sexually,the vast majority appears to undergo sexual reproduction during their life cycles.The hypotheses advanced as to why sex is so ubiquitous are myriad,but center on several recurrent themes[1,2].First,the admixture of genetic material from two genetically distinct individuals that occurs during sexual reproduction may give rise to novel gene combinations that result in offspring better suited to novel or changing environments.Second,sexual reproduction may serve to remove deleterious mutations,such as transposons,that have arisen within the genome.Finally,sex might serve both roles, facilitating combinations of successful alleles and simultaneously purging the genome of deleterious ones.

Until recently it has been difficult to test these models for the possible benefits of sex in experimentally tractable model organisms.However,recent studies with the model yeast S. cerevisiae have provided direct experimental evidence for a benefit of sexual reproduction.Isogenic diploid strains were engineered that are either capable of sporulation with meiotic recombination(sexual)or were able to sporulate but not undergo meiotic recombination(asexual,spo11,13mutant)[3]. Under a variety of different stressful environments,the sexual strain had a competitive advantage compared to the asexual strain.A series of related studies in both S.cerevisiae and the model alga Chlamydomonas provide additional support for a benefit conferred by sex in response to novel or challenging environments[4–6].In response to constant environments, asexual reproduction is also likely to be of relative benefit by relieving strains from the metabolic and genetic costs associated with sex[7].Thus,a balance between sexual and asexual reproduction may be struck in response to different environ-mental conditions.

While the vast majority of sexually reproducing organisms occur as just two sexes or mating types,transitions in sexuality from two to multiple mating types,and vice versa,have occurred in the fungal kingdom.Sexual reproduction is common in fungi,and mating type occurs in two general patterns:bipolar and tetrapolar mating type(reviewed in[8–10].In the bipolar systems,a single genetic locus known as the mating type locus(MAT)occurs in two alternate forms,known as idiomorphs(a or a,a or A,+or2,P or M),and these govern the identity of the cell[11–13].Co-incubation of isolates of opposite mating type under suitable conditions leads to sexual reproduction.Species with bipolar mating systems are found in the ascomycete,basidiomycete,and zygomycete phyla,providing evidence that this is an ancestral organization.In the basidiomycete phylum,many species instead have a more complex sex determining system,known as tetrapolar,in which two unlinked sex determining loci are present [8,10,14,15].One locus encodes homeodomain transcription factors and the other locus encodes pheromones and pheromone receptors,and both loci must differ for sexual reproduction to occur.In many species,these loci are multiallelic,resulting in literally thousands of different mating types,which promotes outcrossing[16].Transitions from tetrapolar to bipolar mating type determination have occurred multiple independent times, and in several examples result from fusions of the two unlinked loci to form one contiguous region(reviewed in[17,18]),potentially illustrating common evolutionary pressures limiting mating-type/ sexes to just two.

Although many fungi are currently classified as asexual, genomics has revealed that many such fungi retain the mating type locus and machinery necessary for both mating and meiosis [19–24].In some cases,population genetics studies also reveal an equal distribution of opposite mating types in nature,and evidence for recombination[25–28].In some notable recent examples,such as Aspergillus parasiticus and Aspergillus fumigatus,an extant sexual cycle has been discovered[29,30].Thus,there may be few truly asexual fungi,and many fungi,and perhaps most,for which cryptic sexual cycles remain to be discovered [31,32].

Sexual reproduction in the basidiomycetous Cryptococcus patho-genic species complex involves a well-defined bipolar mating type system with two opposite mating types,a and a[33–38].Cell-cell fusion leads to the production of dikaryotic hyphae with fused clamp connections,and ultimately the hyphal tips differentiate to form basidia in which nuclear fusion and meiosis occur.Budding from the basidial surface then produces long chains of basidio-spores,the suspected infectious propagules[39,40]for reviews see [41–43].

Molecular analysis of the Cryptococcus mating type locus reveals it to be unusual,spanning.120kb and encompassing.20genes [44–49].With the exception of the homeodomain genes,SXI1a and SXI2a,which govern cell type identity,the a and a mating type alleles are otherwise composed of divergent alleles of a common gene set[50,51].A genomic approach comparing the extant,diverged mating type alleles in C.neoformans var.grubii (serotype A),C.neoformans var.neoformans(serotype D),and C.gattii revealed that the MAT locus is composed of four gene strata of distinct evolutionary ages,including an ancient set of ancestral genes,two gene strata of more intermediate evolutionary origin, and a set of apparently recently acquired genes that are species-but not mating type-specific[47].The finding that the Cryptococcus bipolar MAT locus contains all of the genes normally found in the two unlinked tetrapolar loci suggests that a fusion of the two loci might have been involved in the origins of this unusual gene cluster linked to virulence and differentiation.The hypothesized evolutionary model posits that an ancestral tetrapolar mating system expanded by a series of acquisitions of genes of related function to form two large gene clusters that subsequently underwent fusion via a chromosomal translocation to form an unstable tripolar intermediate stage that subsequently underwent recombination or gene conversion to form the bipolar state.A series of more recent gene conversions and inversions then occurred,giving rise to the extant bipolar mating type alleles and resetting the molecular clock for some genes within distinct strata and even evicting genes from MAT in other examples[47].The recent discovery that the MAT locus is flanked by and contains recombination hotspots provides evidence that the expansion and fusion of these loci may have been driven by activation of recombination[52].In a recent study,the homeodomain genes were relocated to an unlinked genomic location,providing direct experimental support that Cryptococcus can be engineered to complete a tetrapolar sexual cycle and that the tripolar intermediate may have conferred an evolutionary disadvantage and therefore have been transitory[53].A central goal of the studies presented here was to further rigorously examine the proposed MAT evolutionary model by comparative studies with related but divergent fungal species.

In a recent study,a multi-locus gene phylogeny was developed with species closely related to the pathogenic Cryptococcus species complex,which includes C.neoformans var.neoformans,C.neoformans var.grubii,and C.gattii[54].In this study,the yeasts analyzed defined two clades.One contains the pathogenic species complex as well as Cryptococcus amylolentus,Tsuchiyaea wingfieldii,and Filobasidiella depauperata,and was termed the sensu stricto clade. The other clade encompasses yeasts more distantly related to the pathogenic species complex,and was termed the sensu lato group.

C.heveanensis lies in the sensu lato clade,together with Bullera dendrophila,Cryptococcus bestiolae,Cryptococcus dejecticola,and a recently described sexual species,Kwoniella mangroviensis[55].The fact that C.heveanensis is located in a sister clade to the pathogenic Cryptococcus species complex makes it an excellent candidate to provide insights into the evolution of the MAT locus and sexual reproduction in pathogenic Cryptoccocus species.

Author Summary

Comparative genomics provides an approach to under-stand evolution of sex-determining regions in animals and fungi.One example is the unusual mating-type(MAT)locus of pathogenic Cryptococcus species(.100kb,.20genes). The fungal MAT locus is a model for gene cluster evolution and shares features with sex chromosomes of plants/ animals.In previous studies with C.neoformans/C.gattii,a model for this unusual locus was proposed in which two ancestral unlinked loci were fused into one contiguous locus.C.heveanensis represents an evolutionary window to test this model,since it is related to but distinct from pathogenic Cryptococcus species.The organization of MAT in two probably unlinked loci in C.heveanensis provides evidence supporting the model and additional novel insights into how serial gene acquisitions created evolu-tionary strata.We also discovered an extant sexual cycle for C.heveanensis and correlated fertility with MAT alleles. These findings provide direct experimental validation of the central tenants of sex-chromosome evolution originally proposed by Ohno involving sex determinants arising on autosomes,formation of gene clusters with coherent functions in sex,and mechanisms by which specialized recombinationally suppressed genomic regions expand to capture an entire chromosome as a sex chromosome.

The closest yeast with a defined sexual cycle to C.heveanensis is K. mangroviensis,which is also located in the sensu lato clade containing yeasts more distantly related to the pathogenic Cryptococcus species [54].The basidial structure of K.mangroviensis differs from that in C. neoformans and C.gattii and contains septa(phragmobasidia) whereas the pathogenic Cryptococcus species have nonseptate basidia(holobasidia)[33,55].When pairs of compatible K. mangroviensis strains are co-cultured on CMA medium,dikaryotic hyphae with clamp connections and basidia are produced[55].Of 19K.mangroviensis strains isolated from the Bahamas,7were fertile (37%)and12were sterile(63%).Among the seven fertile isolates, six were assigned to the a mating type and one to the A mating type.From these results,the authors proposed that the mating system appeared to be bipolar and biallelic.The nature of the K. mangroviensis mating system will be further clarified by analysis of additional fertile strains,progeny from genetic crosses,and the MAT locus.

In this study,we analyzed the mating behavior and MAT locus structure of C.heveanensis.First,we discovered an extant sexual cycle for C.heveanensis involving dikaryotic hyphae with clamp connections and basidia with cruciate septa and termed this teleomorph Kwoniella heveanensis.Second,we determined the structure of the MAT locus involving two probably unlinked gene clusters corresponding to the homeodomain locus and the pheromone/receptor locus.The presence of two divergently transcribed homeodomain genes in the homeodomain locus suggests this MAT locus arrangement is ancestral to the pathogenic Cryptococcus species,and one or the other homeodomain gene has been lost recently from MAT in C.neoformans and C.gattii.In addition,the presence of numerous other genes around the C. heveanensis MAT locus that correspond to genes located on the same chromosome but far away from MAT in C.neoformans suggests intrachromosomal rearrangements occurred during the evolution of the C.neoformans MAT locus and flanking regions.Character-izing MAT alleles from other C.heveanensis isolates suggests that the homeodomain locus is multiallelic and the pheromone/receptor locus is at least biallelic.Additionally,the MAT-specific region is likely to be restricted to the SXI1and SXI2genes in the homeodomain locus and spans at least the STE3,STE12, MFA1/2,CNB00600,and CNG04540genes in the pheromone/receptor locus,consistent with a tetrapolar mating system.This analysis reveals an evolutionary intermediate with two apparently unlinked MAT loci into which genes have been acquired,lost,and translocated,providing insights into the events that punctuated the evolution of a contiguous sex determining gene cluster in C. neoformans and C.gattii.These studies serve as an evolutionary window both for the evolution of mating type loci in fungi,and also into similar events that have driven the evolution of sex chromosomes in fungi,plants,and animals including insects,fish, and mammals[18,56–58].

Results

Discovery of the sexual cycle of C.heveanensis

To study mating behavior and the MAT locus of C.heveanensis, the isolates(Table1)were first analyzed to validate their species assignment.When the D1/D2and ITS regions were sequenced and compared to the type strain CBS569,all9strains were found to have identical D1/D2sequences(Table1,Figure S1).Seven isolates also had identical ITS sequences whereas two isolates (BCC8396,BCC11754)had one nucleotide difference in the ITS region(Table1,Figure S1).These findings are in accord with the assignment of these isolates as C.heveanensis.Two other isolates (CBS9459,CBS6097)appear to represent related,but distinct species based on a higher level of divergence in the D1/D2and ITS sequences(see Figure S1).All strains appear to be haploid based on fluorescence-activated cell sorting(FACS)analysis by comparison to C.neoformans haploid and diploid reference isolates (Table1,Figure S2).

To test if this species has an extant sexual cycle,isolates were co-cultured and examined morphologically for production of mating specific structures.Strains were inoculated on V8(pH5or7)or MS media alone and in every pair-wise combination and incubated at room temperature in the dark or light.Five strains were self-filamentous when incubated alone while the other strains were not(Table1).The filaments produced during monoculture were highly branched,irregular in shape,lacked clamp connec-tions,and monokaryotic(nuclei stained with Sytox green)(data not shown).In contrast,dikaryotic hyphae with clamp connections and basidia fruiting bodies indicative of sexual reproduction were

Table1.Description of C.heveanensis isolates used in this study.

Strain number Location of

isolation Substrate

Sequence difference

compared to CBS569

Ploidy based on

FACS analysis

Self-filamentation on

V8medium(pH5)in

monoculture Fertility Mating type

D1/D2ITS

CBS569T Indonesia Sheet rubber00Haploid+fertile A1B1

BCC8398Thailand Insect frass00Haploid+fertile A2B2

BCC8396Thailand Insect frass01Haploid+fertile A1B3

BCC11754Thailand Insect frass01Haploid+fertile A1B3

BCC8384Thailand Insect frass00Haploid+sterile A1B2*1

BCC8305Thailand Insect frass00Haploid-sterile A1B4*

BCC8313Thailand Insect frass00Haploid-sterile A1B4*

BCC11757Thailand Insect frass00Haploid-sterile A1B5*

BCC15000Thailand Flower00Haploid-sterile A1B6*

T:type strain.

*:inferred mating type from molecular analysis.

1:the B locus of this strain was found to be very similar to BCC8398(92%for Sxi1and94%for Sxi2,with just one amino acid difference in the N-terminal dimerization region of Sxi1and four amino acid differences in this region of Sxi2),but it could represent a different allele(B7).

doi:10.1371/journal.pgen.1000961.t001

produced by three mating combinations(BCC83986CBS569, BCC83986BCC8396,and BCC83986BCC11754).Optimal mating conditions were on V8medium(pH5)in the dark.By10 days incubation,compatible mating pairs had begun to produce dikaryotic filaments with clamp connections(Figure1A and1B), and basidia embedded in the agar were first apparent at2to3 weeks of incubation(Figure1C),and later developed into clusters in a matrix reaching the agar surface and visible by eye (Figure1D–1G).The basidia are globose with a diameter of 7.5–10.5m m and cruciate septa,similar to the related species K. mangroviensis and Tremella mesenterica(Figure1H–1K,[55]). Basidiospores(subglobose,diameter2.3–3.3m m)were associated with the basidial clusters(Figure1J and1K)and were clearly distinguishable from the larger elliptical budding yeast cells(3.5–4.9m m65.0–7.2m m)(Figure1L).Fecundity was associated with self-filamentation(4/5self-filamentous strains were fertile;4non-filamentous isolates were sterile(Table1)).These morphological observations define an extant sexual cycle for this species.The dikaryotic sexual state that formed when compatible pairs of C. heveanensis were co-cultured was named Kwoniella heveanensis because the basidial morphology of this species is quite similar to that of K. mangroviensis(for Standard and Latin descriptions,see Materials and methods).Both species are phylogenetically related in a sister species clade to the pathogenic Cryptococcus species complex[54]. Characterization of the C.heveanensis MAT locus

To elucidate the structure of the C.heveanensis MAT locus, probes to MAT-specific genes of C.neoformans and C.gattii were generated by degenerate PCR.These probes(LPD1,RPO41, CAP1,ZNF1,STE20,IKS1,FAO1,and NOG2)were used to screen a fosmid library for the type strain C.heveanensis CBS569. Sequencing of positive fosmids and assembly of the sequences resulted in two large gene clusters:a region containing the homeodomain locus and flanking regions spanning,80kb and a region containing the pheromone/receptor locus and flanking regions spanning,180kb(Figure2).The homeodomain locus of C.heveanensis contains two divergent homeodomain genes(SXI1 and SXI2)encoding an HD1and an HD2class product,similar to other basidiomycete MAT loci[59–61].Because the MAT locus alleles in C.neoformans and C.gattii contain only SXI1a or SXI2a but not both,this suggests that one or the other homeodomain gene has been lost recently in these pathogenic species.

The genomic region containing the homeodomain(HD)locus of C.heveanensis also contains the FAO1,FCY1,and UAP1genes, which form the left boundary of the pathogenic Cryptococcus species MAT locus(Figure3).Also included are the RPL22,SPO14,and CAP1genes in addition to the homeodomain genes,which are part of the MAT locus in C.neoformans var.neoformans,C.neoformans var. grubii,and C.gattii[46,47,49].These three genes are also linked to the homeodomain genes of Tremella mesenterica(Figure3).

The remaining genes found in the MAT locus of pathogenic Cryptococcus species are present in the region containing the pheromone/receptor(PR)locus of C.heveanensis(Figure3).These include the pheromone receptor gene,STE3,the pheromone gene MFA1,the pheromone sensing MAPK cascade components STE11,STE20and STE12,and other genes whose functions are either unknown,essential,or may not be related to mating (including BSP1,ETF1,ZNF1,PRT1,NCP1,MYO2,IKS1,RPL39, and BSP3).This region also contains the LPD1,GEF1,CID1, RPO41,and BSP2genes,which were hypothesized to be present at the boundaries of the ancestral sex determining regions in the proposed evolutionary model(RPO41and BSP2in the ancestral homeodomain locus and LPD1,GEF1and CID1in the ancestral pheromone receptor locus)and to have been entrapped in MAT when the HD and PR loci were brought together via a chromosomal translocation[47].The presence of these genes in the PR locus region of both C.heveanensis and T.mesenterica implies that these genes were likely to have all been linked to the ancestral PR locus prior to fusion to a contiguous bipolar sex-determinant. The HD and PR gene clusters of C.heveanensis have numerous other linked genes that are not present in the MAT locus of C. neoformans or C.gattii.Most of these genes(such as CND01630, CND01640,CND05260,CND05390,CNN00870,CNE02670, CNI00160,CND01540,CNG02430,CNE02190,CND01240, CND01560,and CND01570)are also present in the T.mesenterica PR locus region suggesting these genes have recently been lost from the MAT locus and relocated to other genomic locations in C. neoformans and C.gattii(Figure4).Some of these genes do not have apparent homologs in C.neoformans or C.gattii,suggesting these genes may have been lost during the evolution of the MAT locus.It is also interesting that while a majority of these genes are on the4th chromosome of C.neoformans strains JEC21/B3501A,where MAT is also located,they correspond to the opposite telomeric region, suggesting intrachromosomal rearrangements may have occurred during MAT locus evolution(Figure4,[62]).

Homeodomain locus of C.heveanensis is multiallelic,and the MAT–specific region is likely to be restricted to the SXI1and SXI2genes

Southern blot analysis of the8additional C.heveanensis isolates and the type strain with SXI1and SXI2gene probes showed a highly divergent profile,suggesting the homeodomain locus structure of C.heveanensis might be multiallelic(Figure5A). Strains BCC8305and BCC8313showed the same hybridization profile and sequence analysis also showed that the SXI1,SXI2, LPD1,RPL22,ZNF1,and STE11genes in these strains are100% identical for the regions sequenced(data not shown).Strains BCC 8396and BCC11754also appeared identical.Therefore,in these cases,one isolate from each strain pair was selected for further studies.Sequencing of an,12kb region,spanning SXI1and SXI2,from seven strains provided evidence that the locus is multiallelic with6different alleles of SXI1and SXI2(Figure S3). Percent sequence identity plots comparing the homeodomain region of CBS569with the corresponding region from other isolates showed that there is a highly dissimilar core correspond-ing to the59ends of the SXI1and SXI2genes and spanning a fairly restricted region of,2kb(indicated by a blue box in Figure5B)followed by a more similar region(,80%),while the 39ends of the genes are almost identical.A representative dot plot comparing the homeodomain region of CBS569and BCC 11757also shows the divergence at the59ends of both genes (Figure5C).Percent identity plots comparing the homeodomain region from each strain with the others are shown in Figure S4. The homeodomain genes of BCC8384and BCC8398were found to be very similar(Figure S3,Figure S4).When pairwise comparisons were conducted,percent identity of predicted amino acid sequences between BCC8384and BCC8398was found to be92%for Sxi1and94%for Sxi2,while percent identity varied between71–77%for Sxi1,75–82%for Sxi2for the other pairwise combinations.Considering the absence of mating between these two isolates,although they are both self-filamentous,these strains may have the same allelic versions of SXI1and SXI2,preventing mating.

Homeodomain proteins have distinct domains for different functions[63].The homeodomain is the DNA binding region consisting of3alpha helices.Fungal homeodomain proteins involved in mating are either HD1or HD2type according to the length of this homeodomain region[14].In HD2type homeodo-

main proteins,the distance between the first and second helix is three amino acids shorter than HD1homeodomain proteins.The C.heveanensis Sxi1and Sxi2proteins are HD1and HD2-type homeodomain proteins,respectively,like their C.neoformans counterparts (Figure 6A,[50,51]).

Studies involving S.cerevisiae a1and a 2,Coprinopsis cinerea HD1and HD2,and U.maydis bE and bW homeodomain proteins showed that following cell-cell fusion the two homeodomain proteins from compatible partners dimerize via their N-terminal domains [59,64,65].These studies suggest that the interaction is through coiled-coils formed by alpha helices aligned such that the hydrophobic residues are adjacent.Although,we couldn’t detect extensive coiled-coil formation in the C.heveanensis homeodomain proteins using the COILS software,there are 3–4alpha helical regions in the N-terminal domains of both Sxi1and Sxi2,which might form coiled-coil interactions (Figure S3).

In C.cinerea ,two bipartite nuclear localization signals (NLSs)were found in HD1and this region targeted a reporter protein to the nucleus [66].In contrast,no NLS was apparent in the C.cinerea HD2protein and HD2-reporter fusion proteins were not nuclear localized.Similarly in U.maydis ,a bipartite NLS in bE (HD1)was predicted [67].We found one bipartite and one 7-residue pattern (pat7)NLSs in Sxi1(HD1),while only one pat7type NLS in Sxi2(HD2)using the PSORT II software (Figure 6B).The RPL22and CAP1genes are part of the MAT locus in C.neoformans and C.gattii [47,49].The RPL22gene shares 88–90%identity among mating types while the 59and 39regions of the CAP1gene show different profiles.For C.neoformans var.neoformans and C.neoformans var.grubii ,the identity is 90–92%for the 59region of CAP1and 84–85%for the 39region.For C.gattii ,the identity is 88%for the 59region of CAP1and 93%for the 39region.Sequences from RPL22and CAP1were analyzed from C.heveanensis to determine if the HD locus encompasses these genes.When 530bp of RPL22gene sequence from all 7unique strains was analyzed,97to 99%sequence identity was observed.Similarly,comparison of 212bp of CAP1gene sequence from strains CBS 569,BCC 8313,and BCC 15000resulted in 99.5–100%identity.Based on this analysis revealing the RPL22and CAP1genes are more similar between C.heveanensis isolates than the a and a alleles of C.neoformans ,it is likely that the HD locus of C.hevenensis only includes the homeodomain genes,SXI1and SXI2,although other linked genes should also be examined to further support this conclusion.

These results show that the HD locus of C.heveanensis is multiallelic with at least 6different alleles determined among 7strains.Thus,additional extant HD locus alleles likely remain to be discovered.In addition,the MAT -specific region is likely to be limited most prominently to the 59end of the SXI1and SXI2genes coding for the N-terminal regions of the proteins known to be involved in the specificity of heterodimer formation.

Figure 1.Sexual reproduction of C.heveanensis .(A,B)Hyphae with clamp connections.Scale bars represent 50m m.Representative clamp connections (CC)are shown in the enlarged images located in the lower left panels (arrows,scale bars =10m m).(A)Differential interference contrast (DIC)image,(B)fluorescence image showing cell walls,septa and nuclei stained with Calcofluor white and Sytox green.(C–E)DIC images of basidial clusters.Scale bars represent 50m m.(C)A young basidial cluster embedded in the agar,(D,E)mature basidial clusters on the surface of the agar.(F,G)Scanning electron microscopy images of basidial clusters,scale bar in (F)is 50m m,and in (G)is 10m m.(H)A developing basidial cluster with two mature and numerous young basidia,scale bar =10m m.(I and J)Globose basidia with cruciate septa.Scale bars represent 10m m.(K)A basidium and basidiospores,scale bar =10m m.(L)Vegetative yeast cells,scale bar =10m m.

doi:10.1371/journal.pgen.1000961.g001

Figure 2.C.heveanensis MAT locus structure.MAT gene probes (represented by blue bars in the figure)were used to screen the fosmid library of C.heveanensis CBS 569.Sequencing the positive fosmids,represented by black lines,and subsequent assembly of the sequences generated two clusters corresponding to the homeodomain locus (,80kb)and the pheromone/receptor locus (,180kb).The genes that are present in the MAT locus of the pathogenic Cryptococcus species are shown in black,while others are shown in yellow.The red line indicates an ,20kb region sequenced by primer walking.

doi:10.1371/journal.pgen.1000961.g002

Pheromone/receptor locus of C.heveanensis is at least biallelic

When the pheromone and the pheromone receptor gene sequences were amplified by PCR with CBS 569specific primers,PCR fragments of the expected sizes were obtained for all strains except BCC 8398.Sequencing of these PCR products showed that the pheromone and receptor gene sequences were identical for all of the strains that yielded PCR products (data not shown).For the atypical strain BCC 8398,degenerate primers were used to amplify the STE3,STE12,CNB00600,and CNG04540genes and the gaps between these genes were closed by primer walking.This sequence analysis resulted in an ,9kb sequence spanning the 59end of STE3,STE12,a pheromone gene,which was named MFA2,and the CNG04540and CNB00600genes.All of these genes were divergent between BCC 8398and CBS 569except CNB00600.Sequence identity values are 54%for STE3,53%for STE12,75%for MFA1/2,and 63%for CNG04540.In addition,the gene order also differed as indicated in the comparison and the dot plot (Figure 7A and 7B).Interestingly,the divergent region between BCC 8398and CBS 569starts at the 39end of the CNB00600gene.The majority of this gene corresponding to the 59portion (1285bases)is 98%identical,while for a small portion at

the 39end (139bases for CBS 569and 124bases for BCC 8398),the sequence identity is 53%suggesting this gene may have represented the border of MAT or have been subject to more recent gene conversion.

The pheromone genes of C.heveanensis ,MFA1(from CBS 569)and MFA2(from BCC 8398),encode 39amino acid-long pheromone precursors (Figure 7C).Both were identified by the characteristic C-terminal motif of lipopeptide pheromones:CAAX,where A is an aliphatic amino acid [68].While Mfa1ends with the motif CVIA like pheromone precursors from C.neoformans ,C.grubii ,C.gattii and T.mesenterica ,Mfa2has CIIA at its C-terminus.Tremerogen a-13,a T.mesenterica pheromone,was isolated and its structure determined [69].According to this study,after proteolytic cleavage,the mature pheromone starts with a glutamic acid (E)residue indicated with an arrow in Figure 7C.Since this residue is conserved in all Cryptococcus pheromones,the proteolytic cleavage might occur at the same site in other pheromones as well.

No or minimal sequence differences were observed for the LPD1(100%sequence identity in 530bases),STE11(99%in 470bases),ZNF1(100%in 410bases)or IKS1(99%in 1740bases)genes.Also,Southern blot analysis using STE11and MYO2

gene

Figure 3.Synteny between MAT locus regions of C.heveanensis ,T.mesenterica ,and a alleles of C.neoformans var.neoformans ,C.neoformans var.grubii ,and C.gattii .C.heveanensis and T.mesenterica genes that are present in the MAT locus of C.neoformans and C.gattii are shown in black,while others are shown in yellow.Syntenic blocks containing more than one gene are shown as pink bars.The T.mesenterica and C.heveanensis homeodomain locus contains two homeodomain genes homologous with SXI1and SXI2;whereas,C.neoformans and C.gattii contain a single homeodomain gene,namely SXI1for a ,and SXI2for the a allele.Upper scale bar refers to T.mesenterica and C.heveanensis ,while lower scale bar refers to C.neoformans var.neoformans ,C.neoformans var.grubii and C.gattii .JGI annotation IDs for T.mesenterica genes are listed in Table S1.The relative order and the orientations of the C.heveanensis and T.mesenterica contigs are unknown.doi:10.1371/journal.pgen.1000961.g003

fragments as probes showed the same hybridization profile among all strains tested (Figure 7D).This shows that the divergent region is likely to be more restricted than that of C.neoformans and C.gattii and contains at least the receptor gene STE3,the pheromone gene MFA1/2,STE12,and CNG04540.

These results suggest that the PR locus of C.heveanensis is at least biallelic similar to U.maydis and Tremella species,and it contains not only STE3and MFA1/2,but also other genes including at least STE12,CNB00600,and CNG04540.Sequence analysis of the genomic regions flanking this region from the isolate BCC 8398will be necessary to establish whether any additional regions of the PR MAT locus remain to be identified.

Mating type genes exhibit three different phylogenetic patterns

Phylogenetic analyses were conducted on selected mating type genes (CID1,GEF1,LPD1,BSP1,SPO14,ETF1,STE20,STE11,STE3and STE12)from C.heveanensis ,T.mesenterica ,C.neoformans var.neoformans ,C.neoformans var.grubii and C.gattii (Figure S5).These genes were classified into three groups based on their evolutionary history and two representative examples from each group are demonstrated in Figure 8.These analyses show that,STE3and STE12exhibit a mating type specific pattern,where C.heveanensis CBS 569and T.mesenterica show a closer relationship to the a mating type group,while C.heveanensis BCC 8398is closer to the a mating type group.BSP1,SPO14,ETF1,STE20,and STE11are mating type-specific in C.neoformans and C.gattii ,however,none of these genes from either C.heveanensis or T.mesenterica followed a mating type-specific pattern.The third gene group consists of CID1,GEF1and LPD1that do not show a mating type-specific pattern,but demonstrate a species-specific pattern for all species.

Discussion

We report here two advances with respect to understanding the evolution of sexual reproduction and mating type determination in fungi.First,we discovered and characterized the sexual cycle for C.heveanensis .Second,we cloned and sequenced the mating type locus for this organism.Unlike related species that commonly infect animals,C.heveanensis is not a pathogen and is instead associated with insects and flowers.This species represents an evolutionary window that provides novel insights into understand-ing transitions that occur between tetrapolar and bipolar mating type systems,and in model and pathogenic species.In particular,this analysis reveals how a tetrapolar ancestral species with two unlinked sex determining regions of the genome gave rise to bipolar extant species in which the sex determinants are linked,providing direct experimental validation of the central tenants of sex chromosome evolution originally proposed by Ohno [56].These models involve the emergence of sex determinants on autosomes,which are then captured into a sex determining region or chromosome by sequential gene acquisition into strata of different evolutionary ages involving genes of coherent function.The basidiomycete mating type locus represents an exceptional experimental paradigm to test,and validate experimentally,key features of this model.That similar events have transpired repeatedly,and independently,in plants and animals and other fungi,illustrates common underlying principles that are involved in the emergence,evolution,and plasticity of genomic sex determining regions as diverse as fungal mating type loci and sex chromosomes.

Recent multilocus studies support the assignment of C.heveanensis to a clade of species related to but distinct from

the

Figure 4.C.heveanensis MAT locus region shares synteny with the 4th chromosome of C.neoformans JEC21.C.heveanensis MAT locus includes numerous genes that are not present in the MAT locus of C.neoformans or C.gattii .Although many of these genes are on the 4th chromosome of C.neoformans strain JEC21where MAT is located,they correspond to the opposite telomeric region,suggesting intrachromosomal rearragements during the evolution of the MAT locus.Pink lines/bars depict the synteny between genes located within the MAT locus of C.neoformans and the corresponding genes in C.heveanensis .Blue lines/bars show the syntenic relationship of the genes that are linked to the MAT locus of C.heveanensis ,but not part of the MAT locus of C.neoformans .The C.heveanensis genes whose C.neoformans homologs are located on different chromosomes are indicated with purple arrows with chromosome numbers.The genes that do not have apparent C.neoformans homologs are indicated by NCH,which stands for ‘‘no C.neoformans homolog’’.doi:10.1371/journal.pgen.1000961.g004

pathogenic species C.neoformans and C.gattii [54].Importantly,this analysis revealed that C.heveanensis occurs in a sensu lato group of species,including K.mangroviensis ,which was recently discovered and found to have an extant sexual cycle that was defined as bipolar [55].A critical aspect in finding the sexual cycle for C.heveanensis was the recent discovery of several new isolates for the species,which were made readily available by their deposition in a public strain collection [70].Of three isolates previously reported to be C.heveanensis ,our D1/D2and ITS sequence analysis provides evidence that only the type strain CBS 569is in fact C.heveanensis ,whereas two other isolates (CBS 9459and CBS 6097)appear to represent isolates of closely related cryptic species (Figure S1).Neither of these isolates was fertile with the C.heveanensis type strain.All eight of the recently reported isolates are,based on D1/D2and ITS sequence analysis,bona fide C.heveanensis isolates.Pairwise incubation of all nine available C.heveanensis isolates under a broad set of conditions and media resulted in mating for three of the 36possible mating combinations.Thus,at least four of the nine C.heveanensis isolates are fertile.Mating resulted in the production of dikaryotic hyphae with fused clamp connections,basidia with cruciate septa,and basidiospores.Notably,in solo

incubations all four of the fertile strains were self-filamentous and produced monokaryotic hyphae,whereas only one of the sterile isolates was self-filamentous.Thus,the formation of monokaryotic hyphae by mating partners appears to be associated with and may precede successful mating,and these may facilitate location of mating partners or fusion of isolates.Based on the similarities of the sexual cycle between C.heveanensis and K.mangroviensis ,we have assigned the teleomorphic designation Kwoniella heveanensis .In addition to providing insights into the evolution of mating and MAT ,the discovery of the sexual cycle for C.heveanensis will also facilitate further laboratory investigations and strain construction.How might the self-filamentous phenotype that may be necessary for sexual reproduction be controlled?Five of the nine C.heveanensis isolates are self-filamentous,and four of these are fertile in genetic crosses.The filaments produced may represent something similar to a conjugation tube,however as these occur during mono-culture,they are not induced by pheromones produced by a mating partner of compatible mating type.Possibilities include that elements of the pheromone response pathway have been constitutively activated by mutations,or in response to nutritional cues or limitation alone.Alternatively,

it

Figure 5.Homeodomain region of C.heveanensis is multiallelic and likely to be limited to the SXI1and SXI2genes.(A)Southern blot analysis showing hybridization patterns of SXI1and SXI2gene probes to EcoRV digested genomic DNA from C.heveanensis https://www.wendangku.net/doc/7216918378.html,ne 1,CBS 569,2,BCC 8305,3,BCC 8313,4,BCC 8384,5,BCC 8396,6,BCC 8398,7,BCC 11754,8,BCC 11757,9,BCC 15000.(B)Percent sequence identity plots comparing an ,12kb region containing the SXI1and SXI2genes from CBS 569with the corresponding region from other isolates.The blue box shows the highly dissimilar region corresponding to the N-terminal regions of Sxi1and Sxi2.The black bars under the genes depict the exons.(C)A representative dot plot comparing the homeodomain region of CBS 569(x-axis)and BCC 11757(y-axis).doi:10.1371/journal.pgen.1000961.g005

may be that these isolates are self-responsive to their own pheromone as a partial agonist of their Ste3receptor homolog.In such a model,co-culture leads to more robust pheromone signaling necessary to enable cell-cell fusion and generation of a heterodimeric homeodomain complex that enables completion of sexual reproduction.Future studies to address these and other models will be necessary to test the activity and specificity of the mating pheromones and the Ste3homologs involved in sensing them,and to isolate mutations that abrogate the self-filamentous phenotype testing if,for example,the pheromone and pheromone receptor genes are involved and whether this is necessary for mating.

Analysis of the mating type locus for C.heveanensis revealed two apparently unlinked genomic regions,one corresponding to the homeodomain gene locus (B )and the second to the pheromone/pheromone receptor gene locus (A )(Figure 9).Notably,the B locus (HD)includes a pair of divergently oriented genes encoding homeodomain proteins related to Sxi1/Sxi2from C.neoformans and

bE/bW from U.maydis .The A locus (PR)includes genes encoding a Ste3pheromone receptor homolog and a mating pheromone.Both loci are embedded in larger gene clusters,including some previously identified in the C.neoformans MAT locus and novel ones.To establish which genes in the ,80and ,180kb assemblies are mating type specific,representative regions were isolated by PCR and sequenced from fertile and sterile isolates.This analysis revealed striking diversity in the intergenic region and 59coding regions of the SXI1/SXI2genes,flanked by a less divergent region (,80%identity)and followed by nearly identical 39coding regions (Figure 5).The divergent sequence region spans ,2kb.Importantly,this locus appears to be multi-allelic,based on sequence divergence,and at least 6alleles are present in the 7isolates examined,suggesting additional alleles may remain to be discovered.This MAT organization is quite similar to the U.maydis b MAT locus [59,71,72]corresponding to the diverse N-terminal coiled-coil dimerization domain that dictates self-vs.non-self recognition.The RPL22and CAP1genes linked to SXI1/SXI2

in

Figure 6.Sxi1and Sxi2from C.heveanensis align with HD1and HD2homeodomain proteins,respectively.(A)Alignment of the homeodomain regions of S.cerevisiae (Sc),C.cinerea (Cc),C.neoformans var.neoformans (Cnn),C.neoformans var.grubii (Cng),C.gattii (Cg),C.heveanensis (Ch)homeodomain proteins.These proteins are classified as either HD1or HD2according to the length of the homeodomain region.There are three extra amino acids between helix 1and helix 2of HD1proteins.C.heveanensis Sxi1and Sxi2proteins are of type HD1and HD2,respectively.(B)Schematic representation of the Sxi1and Sxi2domain structures.DD,HD,and NLS denote dimerization domain,homeodomain,and nuclear localization signal,respectively.The types of nuclear localization signal are indicated in parentheses:Bipartite and pat7,pat7denotes the 7-residue pattern.

doi:10.1371/journal.pgen.1000961.g006

C.heveanensis had very similar sequences from the strains analyzed (97to 100%identity),suggesting these genes are outside the mating-type specific region.Similar to other basidiomycetes,including U.maydis ,https://www.wendangku.net/doc/7216918378.html,mune ,and C.cinerea ,we suggest dimerization occurs between HD1and an HD2class homeodo-main proteins encoded by different mating type locus alleles [65,73,74].

The A locus (PR)of C.heveanensis encodes a Ste3pheromone receptor homolog,a mating pheromone,and other genes involved in pheromone sensing (Ste11,Ste12,and Ste20)are present or linked.Thus far,our sequence analysis for this large gene cluster spans ,180kb for the CBS 569type strain and representative sequence from other isolates.This analysis allows us to conclude that the gene sequences,and the gene organization,differ substantially between two different alleles present in the popula-tion (Figure 7).The conclusion that at least two alleles are present is also supported by consideration of which strain combinations are fertile and which are infertile (Table 1).The finding that the C.heveanensis A MAT locus might be biallelic is similar to the tetrapolar species U.maydis ,in which the a mating type locus is also biallelic [75].The C.heveanensis A locus is distinguished from that of

U.maydis in that additional genes are present within the locus,and the linked or flanking genes are also quite distinct.While further analysis of additional genomic regions from other isolates will be required to define the full extent of these MAT locus alleles,it is apparent that highly conserved sequences are embedded within more divergent ones (see Figure 7B).This may illustrate that gene conversion occurs within the locus,as is apparent in the MAT locus of C.neoformans ,or that this MAT locus allele represents a transitional form into which related genes have been recruited but have not yet diverged into mating type specific allelic forms.Recent studies of the ectomycorrhizal fungus Laccaria bicolor ,a tetrapolar species,have illustrated differences in the evolutionary trajectory for the two mating type loci [76].Whereas the homeodomain locus appears to be in a region of the genome where syntenic gene organization is conserved,the pheromone and receptor gene locus is more dynamic and subject to different selective pressures,leading to dramatic changes in gene order and organization and insertion of many transposable elements.Similar properties may hold for the two mating type locus alleles of C.heveanensis and may be responsible for some of the unusual features of MAT in this and related

species.

Figure 7.Analysis of the pheromone/receptor region of C.heveanensis isolates.(A)Comparison plot of the PR locus of CBS 569and BCC 8398.The red and blue bands represent the forward and reverse matches,respectively.The genes are mostly in the same orientation except MFA1/2.The intensity of the color is proportional to the percent identity of the match,where red bands show regions of high sequence identity,while pink bands indicate lower sequence identity.(B)Dot plot analysis of the PR locus of CBS 569and BCC 8398.(C)Sequence alignment of pheromone sequences from CBS 569,BCC 8398,C.neoformans var.neoformans MF a ,C.neoformans var.neoformans MF a ,C.neoformans var.grubii MF a ,C.neoformans var.grubii MF a ,C.gattii MF a ,C.gatii MF a and T.mesenterica pheromone tremerogen a-13.The arrow indicates the N-terminal amino acid of tremerogen a-13after proteolytic cleavage.(D)Southern blot analysis showing hybridization patterns of STE11and MYO2gene probes to EcoRV digested genomic DNA from C.heveanensis https://www.wendangku.net/doc/7216918378.html,ne 1,CBS 569,2,BCC 8305,3,BCC 8313,4,BCC 8384,5,BCC 8396,6,BCC 8398,7,BCC 11754,8,BCC 11757,9,BCC 15000.

doi:10.1371/journal.pgen.1000961.g007

The molecular organization of the C.heveanensis MAT locus allows us to propose that this species has a tetrapolar mating type determination system.This conclusion is based on the finding of two apparently unlinked loci that correspond to the A and B mating type locus alleles of other tetrapolar fungal species [16,77,78].Second,a high level of genetic sequence divergence is found for genes within each of the two loci,consistent with a role in mating type determination.Third,the B locus is multiallelic,as is the case of other tetrapolar fungi but not in bipolar species.Fourth,the specific alleles found for the isolates are well correlated with those that are in fact fertile in that all three successful combinations differ at both MAT loci,whereas for at least some of the infertile strain combinations,they share a closely related or identical allele at one or both MAT loci (see Table 1).Fifth,the finding that in the population that the A1locus allele occurs in isolates with at least six different highly diverged B locus alleles is consistent with two unlinked loci in linkage equilibrium defining mating type.Given the dramatic sequence divergence between the different B alleles,models in which these arose by divergence while linked to the A1locus seem less plausible.Put another way,if the A and B loci were to be linked,we would expect a higher proportion of the population would consist of A1linked to,for example,B1,than is observed.

The conclusion that C.heveanensis has a tetrapolar mating type determination system can be further tested by two approaches.

First,additional physical evidence that the two MAT loci are unlinked in the genome,based on electrophoretic separation and demonstration by chromoblot that the two lie on different chromosomes,whole genome sequence analysis,or independent segregation following meiosis,would all provide evidence the two are unlinked.Thus far it has not been possible to resolve chromosomes of this species by pulsed field gel electrophoresis,even employing conditions that work effectively for the closely related species C.neoformans ,C.gattii ,and C.amylolentus .Whole genome analysis may prove to be a more facile approach.The evidence that the two MAT loci are unlinked is that we have been unable,thus far,to find fosmids that physically span both loci or provide evidence the two are linked.It is formally possible that the PR locus could be linked to the right most end of the HD locus,which would place the two ,20kb apart.We view this as unlikely as we found no evidence for fosmids in the library that would span such a theoretical region.It is formally possible that the two loci are on the same chromosome but far enough apart to be unlinked genetically,or that the two loci are partially linked.Future studies will be necessary to examine this in further detail.

Second,additional genetic evidence involving the isolation and analysis of meiotic progeny could further establish this species as tetrapolar.The sine qua non of a tetrapolar mating system is the production of four types of meiotic progeny (A1B1crossed with A2B2yielding four mating types:A1B1,A2B2,A1B2,and A2B1

).

Figure 8.Mating type genes exhibit three phylogenetic patterns.(A)Species-specific profile exhibited by CID1and GEF1,(B)mating type-specific pattern exhibited by STE20and SPO14from C.gattii and C.neoformans ,but not from C.heveanensis or T.mesenterica ,(C)mating type-specific profile demonstrated by STE3and STE12.doi:10.1371/journal.pgen.1000961.g008

Thus far we have not been successful in isolating basidiospores by micromanipulation,and this is a challenge as these are often embedded in a matrix together with basidia.Moreover,in our experience attempts to purify progeny from fungal mating mixtures often leads to a high rate of contamination from the parental isolates,confounding analysis.Future studies could approach this using genetically marked strains to enable selection of meiotic progeny to determine their mating type.A further implication of our findings and conclusions is that the mating type determination system for the closely related species K.mangroviensis ,currently classified as a bipolar species with a and A mating types,may also turn out to be tetrapolar with further detailed analysis.Our findings provide insight and direct experimental support for key features of an evolutionary model that was proposed earlier for the origins of the unusual bipolar mating type locus of the pathogenic Cryptococcus species complex based on a comparative genomic analysis of three closely related species (Figure 10)[47].The proposed model hypothesized that the ancestral state was a tetrapolar organism with two unlinked loci,one encoding homeodomain genes and the other pheromones and the pheromone receptor that evolved in a stepwise fashion to the large bipolar mating type alleles in C.neoformans and C.gattii .In this model,two unlinked MAT loci first expanded by acquiring additional genes related to sexual reproduction,resulting in a series of gene strata of different evolutionary ages.Second,the two MAT loci fused via a translocation event in one of the two mating types,resulting in an intermediate ‘‘tripolar’’state.Third,gene conversion between the linked and unlinked sex determinants

resulted in fusion of the two loci in the other mating type and collapse to a bipolar state.Finally,more recent gene conversions and rearrangements occurred giving rise to the extant MAT alleles observed today.

Our analysis of the C.heveanensis mating type locus alleles reveals two large gene clusters,one corresponding to the left half of the Cryptococcus mating type locus encoding homeodomain genes and the other to the right half encoding pheromones,pheromone receptors,and signaling components.This organization serves as a molecular window on the organization of the MAT locus in the last shared common ancestor to the two lineages and represents key features of the hypothesized intermediate stages in the MAT evolutionary model (Figure 10).First,we see that the two ancestral MAT loci are probably unlinked and in some cases appear to have acquired additional related flanking genes.Second,the two large gene clusters have been formed,but have not yet undergone fusion to the tripolar or bipolar state.Given that two related large unlinked gene clusters are also present in the related species C.amylolentus (Findley and Heitman,unpublished results),the most parsimonious model is that the organization of MAT in C.heveanensis represents the ancestral state prior to fusion,rather than the result of a recent translocation that separated a contiguous MAT allele.Third,the strata of most recently acquired genes (RPO41,BSP2,GEF1,CID1,and LPD1)are all present in the right gene cluster linked to the pheromone and pheromone receptor genes,and thus were already present prior to linkage of the two regions.This differs in detail from the originally proposed model in which RPO41and BSP2had been depicted as linked to

the

Figure 9.C.heveanensis is likely to have a tetrapolar mating type system with a multiallelic mating type locus and at least a biallelic pheromone/receptor locus.C.heveanensis has a multiallelic HD locus (B locus)including the genes SXI1and SXI2with at least 6alleles among 7unique strains examined.The PR locus (A locus)of C.heveanensis is at least biallelic and 6of the strains have the A1allele,while one of the strains,BCC 8398,has the A2allele.The PR locus contains at least the STE3,STE12,MFA1/2,CNB00600,and CNG04540genes.doi:10.1371/journal.pgen.1000961.g009

homeodomain gene cluster,and GEF1,CID1,and LPD1as linked to the pheromone/receptor locus [47].It also raises the likelihood that these genes are linked because they are functionally related in some way to MAT function,rather than being simply entrapped when the two unlinked MAT loci were fused by translocation.We note that the organization of the two gene clusters in C.heveanensis represents a juxtapositioning of MAT -specific genes (SXI1,SXI2)with genes that are part of the MAT locus in C.neoformans /C.gattii but not yet MAT -specific in C.heveanensis .Similarly,for the pheromone/receptor gene cluster,sequences that are not mating type-specific are embedded between mating type-specific sequenc-es.These gene clusters therefore represent transitional intermedi-ates representing MAT loci to which genes of related function have been recruited,but not yet captured into the mating type-specific regions of the genome or begun to diverge into mating type-specific alleles.

The model presented in Figure 10represents our model for the events that led to the formation of a large bipolar mating type

locus from a tetrapolar ancestral system.The proposed expansion of the ancestral HD and PR loci would likely have involved rearrangements facilitated by repetitive sequences that accumulate in recombinationally suppressed regions of the genome.If these genes have a function in sexual reproduction,as many do,this could have sustained the organization of the locus until the two loci fused.Some features of the gene strata apparent in the extant C.neoformans and C.gattii MAT alleles reflect episodic gene conversion events that occurred after the two ancestral MAT loci fused [44,47].Thus,multiple mechanisms operated involving acquisition of linked genes of related function into two expanding gene clusters,as is apparent here for the STE12gene linked to the PR locus,and gene conversion events between alleles both prior to and following linkage of the HD and PR loci that influenced the evolutionary trajectory and plasticity of this dynamic region of the genome.

A key finding of this comparative analysis is that two divergently oriented homeodomain genes are present in the MAT locus of

C.

Figure 10.A model depicting the evolution of the bipolar MAT locus in the pathogenic Cryptococcus species from ancestral unlinked homeodomain and pheromone/receptor loci.First,mating-related genes together with other genes were acquired into two unlinked loci.Second,the mating type specific region including only the homeodomain genes in the HD locus and the pheromone and the pheromone receptor gene in the PR locus expanded serially by rearrangements or insertions leading to suppressed recombination.During expansion,some of the genes were relocated to different parts of the genome and others were lost.Third,the two loci were combined to a single locus in one mating type by a translocation event forming a transient tripolar system.Fourth,recombination between the mating types occurred to form the bipolar system.Ongoing inversions and rearrangements fashioned the extant C.neoformans /C.gattii MAT locus.Genes shown with black or white arrows are those found in the C.neoformans /C.gattii MAT locus,those in white exhibit a species-specific phylogeny,and those in yellow are not linked to the extant MAT locus in the pathogenic Cryptococcus species complex.doi:10.1371/journal.pgen.1000961.g010

heveanensis,whereas only one or the other homeodomain gene is present in the a or a MAT alleles of C.neoformans and C.gattii.This provides evidence that,like other tetrapolar basidiomycete fungi such as U.maydis,Schizophyllum commune,and C.cinerea,the ancestral Cryptococcal MAT locus contained two paired,divergent homeodomain genes and one or the other was lost recently in the C.neoformans/C.gattii lineage.Examples of homeodomain gene loss are also seen in MAT of pathogenic Candida species[79–81]. For example,the alpha2homeodomain gene has been lost in two haploid emerging yeast pathogenic species,Candida lusitaniae and Candida guillieromondii,both of which have retained complete sexual cycles,and in addition C.guilliermondii has also lost the a1 homeodomain gene.As discussed elsewhere[17],the transition from paired to solo homeodomain genes further serves to restrict outbreeding potential.In species with paired homeodomain genes, there are two opportunities to form functional heterodimeric pairs following cell fusion,whereas species with only a single MAT encoded homeodomain protein have only one chance to form a productive heterodimer required for completion of sexual reproduction.A second key finding is that the C.heveanensis A locus(PR)is at least biallelic;when additional isolates become available it will be possible to test if other extant alleles are present (triallelic or multiallelic).Previous studies of U.maydis,which is tetrapolar with a biallelic pheromone/pheromone receptor locus [75],and the closely related species Sporisorium reilianum[77],which is tetrapolar and triallelic at the pheromone/pheromone receptor locus,provide evidence that transitions have occurred in other fungi from multiallelic/multiallelic to multiallelic/biallelic mating type determination systems.These transitions involving the loss of allelic diversity at one of the two unlinked mating type alleles result in a relative decrease in outcrossing frequency but do not alter the restriction to inbreeding engendered by the tetrapolar state[17]. Thus,a loss of allelic diversity at the A locus encoding pheromone and pheromone receptors may have similarly occurred in the lineages leading to C.heveanensis.The outcome for the species then in both cases is a restriction in outbreeding potential,which may serve to further promote inbreeding and clonality in the population,and this transition in sexuality may have contributed to the emergence of this clade of organisms as successful pathogens of animals,including humans.

Materials and Methods

Strains and media

Strains used in this study are listed in Table1.C.heveanensis type strain CBS569,CBS9459,and CBS6097were obtained from the Centraalbureau voor Schimmelcultures(CBS),Netherlands. Other strains of C.heveanensis were from the BIOTEC Culture Collection(BCC),Thailand[70].Strains were grown in YPD broth or on YPD solid medium.Mating assays were performed on V8medium(pH5and pH7)[82]and Murashige and Skoog(MS) medium minus sucrose(Sigma-Aldrich,[83])by pairwise inocu-lation of strains and incubation at room temperature in the dark on dry upright media without parafilm.

Standard and Latin descriptions of the sexual stage Standard description:Kwoniella heveanensis Metin,Findley,& Heitman sp.nov.

Etymol.:The epithet is chosen to be identical with that of Cryptococcus heveanensis(Groenewege)Baptist&Kurtzman comb. nov[84].

Heterothallic fungus.Hyphae dikaryotic,clamp connections fused.Basidia clustered,submerged initially,finally on aerial hyphae,globose,7.5–10.5m m,cruciately septate,sterigmata not observed.Basidiospores subglobose,2.3–3.3m m,germinating by conidia.

Latin description:Kwoniella heveanensis Metin,Findley,&Heit-man sp.nov.

Species heterothallica.Hyphae dikaryoticae,fibulae fusae.Basidia aggregata,primum submersa,deinde superficialia,globosa,7.5–10.5m m diam,septis cruciatis divisa,sterigmatibus carentia,Basidios-porae subglobosae,2.3–3.3m m diam,cito conidia proferentia. Fluorescence-activated cell-sorting(FACS)analysis

The protocol for the preparation of cells for FACS was based on a previous protocol[85].Briefly,,50m l of cells scraped from overnight grown cultures of YPD plates were washed with PBS and fixed in1ml of70%ethanol overnight.Then the tubes were centrifuged and the supernatants were discarded.After washing the cells with1ml of NS buffer(10mM Tris-HCl pH7.2,0.25M sucrose,1mM EDTA,1mM MgCl2,0.1mM CaCl2,0.1mM ZnCl2),they were resuspended in180m l NS buffer.Then,20m l RNase A(10mg/ml,Qiagen)and5m l of propidium iodide (0.5mg/ml,CALBIOCHEM)were added.After incubating at room temperature in the dark with mixing for at least2hours, 17.5m l of the cells were mixed with700m l of50mM Tris-Cl pH7.5and the tubes were sonicated for45seconds.Flow cytometry was performed on10,000cells and analyzed on the FL1 channel with a Becton-Dickinson FACScan.

Microscopy

For microscopic examination of mating,either the mating mix was inoculated into V8medium(pH5)medium plates,and later a block of agar containing the hyphae and basidia was transferred to microscope slides,or the mating mix was directly inoculated onto slides containing a thin layer of mating medium.

For staining,a modified version of a previously published protocol was used[86].Calcofluor white(0.05%solution,BD)and Sytox green(5mM,Invitrogen)were used to stain the cell wall and nuclei,respectively.The slide containing the agar piece with hyphae or basidia was first washed with PBS,then,5–10m l of Calcofluor white was added and mixed gently with a cover slip. After incubating for15min,the excess dye was washed away with PBS.Then fixing was performed by incubating the slide in fixing solution(3.7%formaldehyde,1%triton-X100in PBS)for15min and the slide was washed with PBS.After that,5to10m l of1:10 diluted Sytox green was spotted on the slide,mixed with a cover slip,and incubated for15min.Then the slide was washed with PBS,mounted with a cover slip,and examined under the microscope.Microscopy was performed with an Axioskop2plus upright microscope(Zeiss).Images were captured using an AxioCam MRm camera and AxioVision application software (Zeiss).Scanning electron microscopy was performed with an XL30environmental SEM(FEI).

Fosmid library preparation

For library production,the CopyControl Fosmid Library Production Kit(Epicentre)was used according to the manufac-turer’s instructions.In each library,a total of,16,000clones were picked into96well plates.Purified genomic DNA(,40kb fragments)was first sheared,end-repaired,and separated using contour-clamped homogenous electric field(CHEF)on a CHEF DR-II apparatus(Bio-Rad).The following conditions were used: 1-to6-sec switch time,6V/cm,14u C for14to15hrs.The size-fractionated DNA was recovered by gel extraction and precipi-tated.The insert DNA was then ligated into the CopyControl pCC1FOS cloning-ready vector and incubated overnight at room

temperature.The ligated DNA was packaged and plated on E.coli phage-resistant cells overnight at37u C.Fosmid clones were then picked into96well plates and eventually transferred to384well plates,which were stored at280u C.

DNA extraction,degenerate PCR,and library screening For DNA isolation,strains were inoculated in50ml YPD medium,grown for two days at room temperature with shaking and centrifuged.After freezing at280u C,the pellet was freeze-dried overnight and DNA was isolated as previously described [87].

Degenerate primers were designed for LPD1,RPO41,CAP1, ZNF1,STE20,IKS1,FAO1and NOG2based on known gene sequences from C.neoformans var.neoformans,C.neoformans var.grubii, and C.gattii.These primers(Table S2)were used in PCR reactions with C.heveanensis genomic DNA using Ex Taq polymerase (Takara)and the products obtained were gel extracted with QIAquick Gel Extraction Kit(Qiagen).The PCR fragments were cloned into pCR2.1-TOPO using the TOPO TA Cloning Kit (Invitrogen).From the resulting colonies,plasmid isolation was performed with QIAprep Spin Miniprep Kit(Qiagen)and the plasmids were sequenced with M13F and M13R vector specific primers.After obtaining MAT gene sequences,specific primers were designed using a web-based program,Primer3(http://frodo. https://www.wendangku.net/doc/7216918378.html,/).After PCR with specific primers,the resulting gene fragments were gel-extracted as previously described and used as probes to screen the library.

For library screening,colony lifts were performed on Hybond membranes(Amersham)using standard protocols[88].The PCR products were labeled with Prime-It II Random Primer Labeling Kit(Stratagene)with dCTP.Hybridization was performed using ULTRAhyb hybridization buffer(Ambion)according to the manufacturer’s instructions.

Sequencing and assembly

Positive fosmids were isolated from200ml of overnight grown cultures using the Qiafilter plasmid midi kit(Qiagen).The fosmid DNA obtained was sheared using a hydroshear apparatus (Genomic Solutions)to1.6–3kb fragments.The gel extracted sheared DNA was then ligated to adapters.To prepare the vector, pUC18plasmid DNA was cut with EcoRI and HindIII removing the polylinker site,and ligated to single stranded oligos that contain one end complementary to the restriction enzyme site,and the other non-complementary to itself to prevent self-ligation.The sheared and adapter-ligated DNA was then ligated to the pUC18-derived vector and the reaction was used to transform DH5a competent cells(Invitrogen).White colonies were inoculated onto 96well plates and plasmid isolation was performed with DirectPrep96MiniPrep kit(Qiagen).For each positive fosmid, four to five96well plates were used.

Sequencing reactions were performed with BigDye Terminator v3.1Cycle Sequencing Kit(Applied Biosystems)and analyzed on PE370096-capillary sequencer(Applied Biosystems)in the Sequencing Facility at Duke University.The sequence reads were assembled using Phred,Phrap,and Consed program packages [89–91].The gaps were closed by designing primers from contig ends using Primer3,amplifying across gaps by PCR and sequencing both strands.Short gaps were closed using iProof polymerase(Biorad)and long gaps were closed using LA Taq polymerase(Takara).

Southern blot analysis

For Southern hybridization of C.heveanensis strains,5m g of genomic DNA from each strain was digested with EcoRV,electrophoresed on a0.8%agarose gel,and blotted onto Hybond membranes(Amersham)using standard protocols[88].The membrane was then hybridized to MAT gene probes generated by PCR as described above.

Annotations and bioinformatic analyses

Annotations of the genes were done manually by comparing the 3-frame translated C.heveanensis sequences with the most similar protein sequences identified by BLASTX.Genbank accession numbers are,GU129941for CBS569homeodomain locus, GU205379for CBS569pheromone/receptor locus,GU129940 for BCC8398pheromone/receptor locus,GU129942,GU129943, GU129944,GU129945,GU129946,and GU129947for homeo-domain loci of the strains BCC8313,BCC8384,BCC8398,BCC 11754,BCC11757,AND BCC15000,respectively.Genbank accession numbers for D1/D2and ITS regions are GU585738and GU585749for CBS9459,GU585739and GU585750for CBS 6097,GU585740and GU585751for BCC8305,GU585741and GU585752for BCC8313,GU585742and GU585753for BCC 8384,GU585743and GU585754for BCC8396,GU585744and GU585755for BCC8398,GU585745and GU585756for BCC 11754,GU585746and GU585757for BCC11757,and GU585747 and GU585758for BCC15000,respectively.The accession number of the ITS region of CBS569is GU585748.

Percent sequence identity plots and dot plots were generated using the MultiPipMaker[92].Pairwise sequence identity values were calculated using Geneious4.6.4.

The comparison plot between pheromone/receptor loci of strains CBS569and BCC8398was produced using Artemis Comparison Tool Release8[93].The input comparison file was generated using WebACT with the tBlastx algorithm. Homeodomain regions of the proteins Sxi1and Sxi2were predicted by comparison to the previously characterized home-domain proteins.Helical content of the N-terminal domains of Sxi1and Sxi2were predicted with Jpred3[94].Nuclear localization signals were predicted with PSORT II[95]. Phylogenetic analysis

Phylogenetic analysis was conducted on coding sequences using MEGA4[96].The phylogenetic relationship was inferred using the Maximum Parsimony method.

Supporting Information

Figure S1Eight BCC isolates are confirmed to be C.heveanensis, while the strains CBS9459and CBS6097are closely related but different species.Eight isolates obtained from BCC have identical D1/D2sequence as the C.heveanensis type strain CBS569,while there are two differences between CBS569and CBS9459,and three differences between CBS569and CBS6097.Similarly,six of the eight BCC strains have identical ITS sequence as the type strain CBS569,and there is only one difference between the type strain and the strains BCC8396and BCC11754.However, there are eleven differences between CBS569and CBS9459, and eight differences between CBS569and CBS6097in the ITS region.

Found at:doi:10.1371/journal.pgen.1000961.s001(0.82MB PDF)

Figure S2 C.heveanensis isolates are haploid based on FACS analysis.The type strain CBS569and eight BCC isolates were found to be haploid based on FACS analysis conducted with the haploid control C.neoformans JEC20and the diploid control C. neoformans XL442.

Found at:doi:10.1371/journal.pgen.1000961.s002(0.24MB PDF)

Figure S3Sequence alignment of homeodomain proteins.(A) Sxi1,and(B)Sxi2sequence alignments.Purple boxes indicate the alpha helical regions at the N-terminal region of the proteins.Blue box shows the homeodomain region and green boxes indicate the predicted nuclear localization signals.

Found at:doi:10.1371/journal.pgen.1000961.s003(2.26MB PDF)

Figure S4Percent sequence identity plots of the homeodomain region.An extended version of Figure5B including percent sequence identity plots comparing an,12kb homeodomain region from(A)BCC8313,(B)BCC8384,(C)BCC8398,(D) BCC11754,(E)BCC11757,(F)BCC15000with the corresponding region from other isolates.The aligned regions are shown in green and well aligned regions(at least70%sequence identity)are shown in pink.

Found at:doi:10.1371/journal.pgen.1000961.s004(0.12MB PDF)

Figure S5Phylogenetic analysis of selected mating type genes. An extended version of Figure8.(A)Species-specific profile exhibited by CID1,GEF1,and LPD1,(B)mating type-specific pattern exhibited by BSP1,SPO14,ETF1,STE20and STE11from C.gattii and C.neoformans,but not from C.heveanensis or T. mesenterica,(C)mating type-specific profile demonstrated by STE3 and STE12,and(D)SXI1and SXI2sequence alignments from C. heveanensis strains,C.gattii and C.neoformans.Found at:doi:10.1371/journal.pgen.1000961.s005(0.14MB PDF)

Table S1JGI annotation IDs for T.mesenterica genes presented in Figure3.

Found at:doi:10.1371/journal.pgen.1000961.s006(0.02MB PDF)

Table S2Primer sequences.

Found at:doi:10.1371/journal.pgen.1000961.s007(0.06MB XLS)

Acknowledgments

We thank Jim Anderson for discussions,June Kwon-Chung and Rytas Vilgalys for advice and encouragement,Walter Gams for helpful comments and Latin translation,James Fraser for assistance with library construction,Alvaro Fonseca for advice and discussions,Wenjun Li for phylogenetic analysis,Leslie Eibest for scanning electron microscopy,Anna Floyd for technical assistance,and Marianela Rodriguez for critical discussions.We also want to thank BIOTEC Culture Collection(BCC), Thailand,and the investigators there for assistance with obtaining isolates central to this study.

Author Contributions

Conceived and designed the experiments:BM KF JH.Performed the experiments:BM KF.Analyzed the data:BM KF JH.Wrote the paper: BM JH.

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3、及时采种 仙人掌类果实一般50～60d成熟，有些种类为浆果，成熟时易遭鸟类等小动物啄食，应用网罩保护果实。有些果实果皮易开裂，要注意及时采收。采收的浆果应剥掉果肉，将种子洗净，再滤干水分。所得种子要装入纸袋，放于干燥冷凉处，并注明种子名称及采收日期。 4、消毒浸种 播种用土应是经过消毒的河沙或混沙腐叶土，必须用锅炒或蒸消毒，不能用药剂消毒。播种前，将盆土用浸盆法润湿，再用1％福尔马林或2.5％硫酸铜液浸种15min，取出清洗后即可播种。小粒种子可混沙均匀撒播于土面，不必覆土。大粒种子可按5mm×5mm的株行距点播。 5、适期播种

种子繁殖仙人掌的最佳时期是昼夜温差较大的春季和秋季。温差大不仅可提高出苗率，还可使幼苗加快生长。应避免阴雨天播种，宜选择晴好天气。若在温室内，四季均可进行种子繁殖。 6、播后精管 播后应用玻璃板盖于花盆上，每日必须定时露出一条缝以透气。一般仙人掌种类在15d之内可发芽。发芽后，应仔细观察盆内干湿情况，保持盆土表面有一定潮湿。幼苗必须控制光照，可将打孔白纸盖在玻璃上。随着小苗的长大更换白纸，使小孔相应增大、增多，以利光照。仙人掌的苗期管理应在温度、湿度、水分、光照、通风上严格把关。春秋生长旺季，要保持较大昼夜温差，并适时适量地给小苗以光照。夏季要加强通风，降低温度。冬季应放在室内暖和处，并适当增加光照，但不可过量。浇水应以浸盆法为主，盆面湿润即可。 7、防治病虫

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草 食 动 物 繁 育 技 术 班级：xxxxxxxx 姓名：xxxxxxx 学号：xxxxxxxxxxx

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（三）甲方挑选的犬只，如在产后天内因意外或不可预见的原因夭折，则乙方应于第 一时间内通知甲方并保留犬尸给甲方确认，并视情况由甲方重新选择犬只或做等值的售出处理。如乙方所产幼犬全部夭折，则待甲方确认后参考第三条第二点执行。 本协议一式二份，双方各执一份，具有同等法律效力。 甲方签字：乙方签字： 日期:年月日日期:年月日

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