Lateral Transfer of Mating System in Stemphylium

Lateral transfer of mating system in Stemphylium

Patrik Inderbitzin†‡§, Jennifer Harkness†, B. Gillian Turgeon¶, and Mary L. Berbee†

†Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4; and ¶Department of Plant Pathology, 334 Plant Science Building, Cornell University, Ithaca, NY 14853

Edited by Barbara A. Schaal, Washington University, St. Louis, MO, and approved June 16, 2005 (received for review March 8, 2005) The fungal genus Stemphylium (Ascomycota) contains selfing species physical linkage of the MAT alleles could be accounted for by a that evolved from outcrossing ancestors. To find out how selfing crossover event, for other arrangements the mechanism remained originated, we analyzed the Stemphylium MAT loci that regulate unknown (7). Phylogenies from housekeeping loci were congruent sexual reproduction in ascomycetes and compared MAT structures with one another, and they supported the MAT gene arrangements and phylogeny with a multigene Stemphylium species phylogeny. in showing that each self-fertile Cochliobolus species evolved inde- We found that some Stemphylium species’ MAT loci contained a pendently from a self-sterile ancestor. single gene, either MAT1-1 or MAT1-2, whereas others contained a To investigate the origin of self-fertility in Stemphylium,we unique fusion of the MAT1-1 and MAT1-2 regions. In all fused MAT compared the position of self-fertile species in an organismal regions, MAT1-1 was inverted and joined to a forward-oriented phylogeny and in a phylogeny of the mating-type genes. We MAT1-2 region. As in the closely related Cochliobolus, Stemphylium analyzed the mating-type loci to assess homology of the gene species with fused MAT regions were able to self. Structural and arrangements in selfing and outcrossing species. We had expected phylogenetic analyses of the MAT loci showed that the selfing- that, as in Cochliobolus, self-fertility would have originated in conferring fused MAT regions were monophyletic with strong sup- different species by independent mating-type gene fusions. Instead, port. However, in an organismal phylogeny of Stemphylium species we found evidence for a single fusion that was transferred across based on 106 isolates and four loci unrelated to mating, selfing arose lineages. in two clades, each time with strong support. Isolates with identical fused MAT regions were present in both clades. We showed that a Materials and Methods one-time origin of the fused MAT loci, followed by a horizontal Fungal Strains Used. The 106 isolates of Stemphylium used were from transfer across lineages, was compatible with the data. Another the collection of P.I. or from other culture collections. Isolates were group of selfers in Stemphylium only had forward-oriented MAT1-1 chosen to represent the genetic diversity of known Stemphylium at their MAT loci, constituting an additional and third origin of selfing strains, with an increased sampling around the type species S. her- in Stemphylium. barum. See Table 1, which is published as supporting information on the PNAS web site, for details. filamentous ascomycetes ͉ nonvertical inheritance ͉ evolution ͉ Pleospora ͉ horizontal transfer Assessment of Self-Fertility. Self-fertility of the fungal strains was evaluated in the laboratory. Single ascospores or conidia were mong bacteria, lateral gene flow has redistributed charac- germinated on water agar, transferred to V8 agar plates upon Aters of evolutionary importance (1). In eukaryotes including germination (8), stored at room temperature or at 4°C, and fungi, evolution through lateral gene transfer is often proposed, periodically examined for the formation of sexual spores (ascos- but alternative explanations for character distributions have pores) (9). been difficult to rule out (2). The mating type locus in the ascomycetous fungal genus Stemphylium has provided an op- Molecular Work. Mycelium was scraped off the surface of a Petri portunity to detect an ancient lateral transfer that changed a plate, and DNA was extracted by using a standard phenol- fungal breeding system. We used phylogenetic analyses based on chloroform extraction method (10). PCR reactions were performed five loci in conjunction with gene rearrangement data to show by using a variety of conditions (see Supporting Methods in Sup- that the distribution of mating strategies in Stemphylium is best porting Text, which is published as supporting information on the explained by a lateral transfer of the genes controlling mating. PNAS web site). In haploid filamentous ascomycetes, self-sterility and self-fertility Loci sequenced for species phylogenies were the ribosomal depend on the configuration of genes at MAT, the mating type internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehy- locus (3). Each self-sterile individual has only one of the two drogenase (GPD), elongation factor-1 ␣ (EF-1 ␣) and the noncoding alternative single-copy genes, MAT1-1 or MAT1-2,atitsMAT region between the vacuolar membrane ATPase catalytic subunit A locus. Like large portions of the human X and Y chromosomes, the gene (vmaA), and vpsA, a gene involved in vacuolar biogenesis (11). DNA and amino acid sequences of MAT1-1 and MAT1-2 are no Primers were from refs. 12–14 or designed for this study based on more similar than expected by chance. Outside of the Ϸ1,100- to DNA sequences available from GenBank (see Table 2, which is 4,200-bp MAT region, sequence similarity between homologous published as supporting information on the PNAS web site). chromosomes reappears and haploid individuals of opposite mating Primers for the intergenic spacer between vmaA and vpsA were types have syntenous flanking genes (4, 5). In contrast to self-sterile species, haploid self-fertile species in Stemphylium (this study), as in the closely related Cochliobolus (6), This paper was submitted directly (Track II) to the PNAS office. contain both MAT1-1 and MAT1-2 genes in the same genome. Abbreviation: ITS, internal transcribed spacers. Demonstrating that the presence of a MAT1-1͞MAT1-2 fusion was Data deposition: The DNA sequences reported in this paper have been deposited in the enough to confer self-fertility, transformation of a mating-type GenBank database (accession nos. AY316968–AY316973, AY316975–AY316978, AY316980, AY316982–AY316989, AY316991–AY317074, AY324671–AY324776, AY329168–AY329270, deletion strain of the self-sterile species Cochliobolus heterostrophus AY329271–AY329323, AY335164–AY335180, AY339853–AY339864, and AY340940– with the fused MAT genes produced self-fertile individuals (7). AY340945). Alignments have been submitted to TreeBASE (study accession no. S1313; Matrix Suggesting that self-fertility originated convergently, each of the accession nos. M2301–M2305). four selfing species of Cochliobolus had unique splice sites adjacent ‡Present address: Department of Plant Pathology, 334 Plant Science Building, Cornell to its MAT1-1 and͞or MAT1-2 genes, and the direction of tran- University, Ithaca, NY 14853. scription of the two genes along with the length of the intergenic §To whom correspondence should be addressed. E-mail: [email protected]. intervening sequences differed across species. In some cases, the © 2005 by The National Academy of Sciences of the USA

11390–11395 ͉ PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501918102 Downloaded by guest on September 28, 2021 based on Cochliobolus heterostrophus sequences that G. Saenz and Generation of Mating-Type DNA Phylogenies. For MAT1-1 analyses, B. G. Turgeon obtained from the former Torrey Mesa Research the inverted parts of the fused MAT region from 11 selfing species Institute. were combined in an alignment with homologous regions from 11 Primers used for amplification of the MAT regions were de- species with separate MAT regions. Similarly, the MAT1-2 parts signed based on DNA sequences (15, 16), and later, on sequences from species with fused MAT regions were used for analyses from this study (see Tables 3–8, which are published as supporting together with homologous regions from five species with separate information on the PNAS web site). MAT regions. Settings for phylogenetic analyses were as for the To determine DNA sequences of the entire mating-type genes four loci species data set except that likelihood bootstrap support and flanking regions, the chromosome walking kit Vectorette was percentages were based on 500 replicates. used (Sigma-Genosys, The Woodlands, TX). Additional primers were designed from previously sequenced conserved DNA binding Generation of Mating-Type Protein Phylogenies. The MAT gene regions of the MAT genes and used together with the kit primers DNA sequences were translated into amino acids. To the MAT1-1 for PCR amplifications of MAT flanks (see Supporting Methods and protein alignment, five outgroup taxa from GenBank were added Tables 9–11, which are published as supporting information on the (Alternaria alternata, AB009451; Cochliobolus cymbopogonis, PNAS web site). AF129744; C. heterostrophus, AF029913; C. ellisii, AF129746; C. sa- PCR products were completely sequenced in both directions by tivus AF275373). The MAT1-2 alignment contained the same using a Big Dye Terminator Cycle Sequencing Kit v2.0 or 3.0 outgroup taxa as MAT1-1 (A. alternata, AB009452; C. cymbopogo- (Applied Biosystems, Foster City, CA) according to the manufac- nis, AF129745; C. heterostrophus, AF027687; C. ellisii, AF129747; C. turer’s instructions with a variety of sequencing primers (see sativus, AF275374). The alignments were generated by CLUSTALX Supporting Methods and Tables 9–11). 1.8 (17). For parsimony analyses, the PHYLIP 3.572 package was used (21). Most parsimonious trees were inferred by using PROTPARS. Generation of Species Phylogenies. DNA sequences were assembled Default options were used, except that input order was randomized by using AUTOASSEMBLER 1.4.0 (Applied Biosystems PerkinElmer) and 30 random sequence additions were used (jumble set to 30). and DNA sequence alignments were generated by CLUSTALX 1.8 Strict consensus trees were calculated and drawn by using PAUP* (17) by using default settings and manually optimized in SE-Al 1.D1 (19). For bootstrap analyses, 500 random data sets were created by (18). For some of the isolates used in this study, DNA sequences using SEQBOOT with default settings and analyzed in PROTPARS with were retrieved from GenBank (accession nos. AF442783, one random taxon addition per data set (jumble set to 1). For AF442788, AF442789, AF443882, AF443887, and AF443888). All likelihood trees, the quartet puzzling algorithm in TREE-PUZZLE 5.0 other sequences were generated in this study. Phylogenies were (22) was used with default settings, except that heterogeneous rates inferred with four algorithms by using PAUP* 4.0B10 for parsimony, were approximated with four gamma rate categories. For distance likelihood, and neighbor-joining (19), and MRBAYES 3.0B4 for Bayes- trees, the distance matrix obtained with TREE-PUZZLE 5.0 was ian analyses (20). imported into the FITCH component of PHYLIP 3.572 (21) to construct The most parsimonious trees were inferred by using 30 heuristic and optimize the tree topology by using a Fitch–Margoliash method searches with random addition of taxa but default settings other- with default settings, except that the global optimization option was wise, coding gaps as missing characters. Bootstrap support was from in effect and the input order was randomized with the jumble option 500 replicates by using random taxon addition but, otherwise, set to 30. The resulting tree was manipulated in MACCLADE 4.03 (23), default settings. and printed in PAUP* (19). For likelihood analyses, base frequencies, transition-transversion ratio, proportion of invariable sites, and gamma shape parameter were estimated from a most parsimonious tree. The gamma distribution was approximated by four rate categories. Most likely trees were estimated by using 30 heuristic searches with random taxon addition and, otherwise, default settings. Bootstrap support values were based on 185 replicates. Neighbor-joining analyses were done by using likelihood modeled distances with parameters estimated on a most parsimonious tree. Branch support was evaluated with 500 bootstrap replicates. For Bayesian analyses, a general time reversible model of evolution was used. Rate heterogeneity across sites was modeled with a gamma distribution. Four chains starting with a random tree were run for 1 million generations, retaining each 100th tree. The first 1,000 trees of the 10,000 collected trees were discarded, and the posterior probabilities were based on the remaining 9,000 trees. The 50% majority rule consensus tree was calculated in PAUP*. Combinability of data sets was evaluated by using the partition Fig. 1. Alternative arrangements of the opposite mating-type genes MAT1-1 homogeneity test as implemented in PAUP* 4.0B10 (19). and MAT1-2 at the mating-type locus in Stemphylium. Obligate outcrossers, in their dominant haploid phase, have the ancestral condition, either a forward- Mating Type Screening. All 106 Stemphylium strains were screened oriented MAT1-1 (1) or a forward-oriented MAT1-2 (2). Fused genes of self- EVOLUTION for the presence of MAT genes and MAT gene arrangement. At fertile species carry a reversed MAT1-1 (3) with its flank fused to a forward first, specific PCR primers were designed based on ref. 15. For MAT1-2. White boxes are genes, and half arrowheads indicate direction of MAT1-1, primer pair Jen1f͞Jen1r was designed on the conserved transcription. ORF1 is a flanking gene of unknown function (7). The fused DNA binding motif alpha box (see Supporting Methods). The mating-type region may have evolved through 1b, an inversion of a MAT1-1 MAT1-2 primer pair was Jen2f͞Jen2r, situated on the conserved region that had a short ‘‘ATGG’’ sequence producing 1c with a CCAT motif. The juxtaposition of the CCAT from MAT1-1 may have permitted crossover with a DNA binding high mobility group box motif. Later, new primers forward-oriented MAT1-2 region (2), leading to the fused MAT regions (3) were designed to fit all Stemphylium strains. For MAT1-1, these found in Stemphylium. The CCAT motif is always present at fusion sites where primers were Alpha181f, used together with Jen1r, and HMG85f, MAT1-1 is joined to MAT1-2 (Fig. 2). A CCAT motif also appears in the 5Ј flank used with Jen2r for MAT1-2 screening (see Supporting Methods). of unfused MAT1-2 regions, homologous to the putative crossover site (Fig. 2).

Inderbitzin et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11391 Downloaded by guest on September 28, 2021 Fig. 2. DNA sequence alignments (within boxes) showing the splice sites in the fused region containing mating-type genes MAT1-1 and MAT1-2 in self-fertile species of Stemphylium. The fused regions are aligned with their unfused homologs from other species. ORF1 is a ﬂanking gene of unknown function (7). The top part of the diagram is an ORF1 proximal splice junction; bottom part is an ORF1 distal junction. The sequences are arranged by phylogenetic groups asin Fig. 3. Dots indicate nucleotide identity to P2. In the upper sequence box, the column of spaces indicates the junction between the forward ﬂank and the inverted MAT1-1 region, where sequence homology between fused and unfused regions is lost. The shaded nucleotide positions indicate the ATGG that would, through inversion, produce a CCAT motif. In the lower sequence box, the shaded CCAT is the postulated crossover site with MAT1-1 to the left and MAT1-2 to the right.

Testing of Alternative Phylogenetic Hypotheses. Alternative phylo- regions (from 34–280 bp inside ORF1, a conserved ORF of genetic hypotheses were evaluated with the Shimodaira-Hasegawa unknown function, to 322–2,373 bp downstream of MAT1-1) and test as implemented in PAUP* 4.0B10 (19) by using default settings, 2,571–7,248 bp for the six MAT1-2 regions [from 21–321 bp inside except that the test was one-sided and full optimization was chosen. ORF1 to 397–4,804 bp downstream of MAT1-2, including 291 bp For each round of testing, the most likely tree was evaluated against of ␤ glucosidase gene (BGL1) in strain P232]. MAT1-1 and MAT1-2 the most parsimonious trees obtained given a topological con- were respectively 1,193 and 1,093 bp in length. straint. Topological constraints generated in MACCLADE 4.03 (23) were monophyly of the taxa with fused MAT regions in the species MAT Genes from Self-Fertile Isolates with Fused MAT Regions. PCR screens showed that 76 self-fertile isolates contained an inverted phylogeny and nonmonophyly of the taxa with fused MAT regions MAT1-1 gene less than Ϸ1.5kbfromaMAT1-2 gene (Fig. 1). A for the MAT1-1 and MAT1-2 DNA phylogenies. Constrained most representative selection of 17 fused MAT regions were sequenced. parsimonious trees were inferred as described for the species Sequencing coverage was 3,851–6,102 bp [from 255 bp inside phylogenies. GTPase activating protein gene (GAP1) in strain P1, for remaining isolates beginning 44–167 bp within ORF1, to 418-1300 bp down- Results stream of MAT1-2]. MAT genes from fused regions were equal in MAT Genes from Self-Sterile Isolates. In 23 self-sterile isolates, one length to the ones from separate MAT regions. of the two opposite mating type genes, either MAT1-1 or MAT1-2, was detected (Fig. 1). Presence of a single gene at the MAT locus MAT Genes from Self-Fertile Isolates with Only MAT1-1 Detected. The was confirmed by sequencing of the MAT locus in 17 representa- remaining seven isolates included in this study were also self- tives. Sequencing coverage was 2203–4269 bp for the 11 MAT1-1 fertile and all were confined to group SP (Fig. 3; group SP is

11392 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501918102 Inderbitzin et al. Downloaded by guest on September 28, 2021 Fig. 3. The self-fertile species with fused mating-type regions are monophyletic in a mating-type region phylogeny but polyphyletic in a phylogeny based on four other loci. The most likely phylogenetic tree from the Stemphylium mating-type MAT1-1 region (Left) is thus incongruent with the most likely, four-locus species tree (Right). Trees are rooted based on mating-type amino acid phylogenies by using outgroups (see Figs. 8 and 9). Lengths of horizontal branches are proportional to substitutions per site. Support values by the branches are from likelihood͞parsimony͞Bayesian͞neighbor-joining. Branches in bold were supported by 100% in all analyses. Phylogentic groups from ref. 24 and this study are indicated by the narrow vertical lines. Self-fertile species with fused mating-type regions are indicated by bold vertical lines. For unnamed taxa, only strain numbers are given.

represented by strains P56 and P107). However, based on PCR and Fig. 6, which is published as supporting information on the screening of all isolates in SP, only MAT1-1 was detected, in PNAS web site, for additional details). A representative taxon forward orientation as in outcrossers. A total of 15 isolates from sampling for the species tree in Figs. 3 and 4 was achieved by meiotic spores (ascospores) in group SP were screened for the choosing isolates from the phylogenetic species as follows. If the presence of MAT genes by using PCR primer sets Alpha181f͞ species had fused MAT regions, one MAT region was used for final Jen1ri and Jen2f͞Jen2r (Tables 3–6). These seven isolates were analyses. If the species had MAT1-1 and MAT1-2 in separate in Fig. 5, which is published as supporting information on the individuals, then one of each was chosen, if available. For ease of PNAS web site, and eight additional isolates of Stemphylium sp. interpretation and to facilitate analyses, the MAT1-2 strains P306, strain P56 were not listed in Table 1. Assuming that opposite P310, and P252 were excluded from species analyses and Figs. 3 and mating-type genes MAT1-1 and MAT1-2 segregate in equal 4. In preliminary phylogenetic analyses, these isolates had identical proportions as expected after meiosis, the odds that all 15 four locus genotypes to the respective cospecific MAT1-1 strains ascospores selected at random are MAT1-1 mating type would be P247, P239, and P240 (see Fig. 6). one in 32,768, a rare event (P Ͻ 0.0001). Thus, MAT1-2 might The representative most likely species tree (Ϫln likelihood ϭ be absent in this group, or we were unable to detect it. In the 8,197) containing 24 strains is illustrated in Fig. 3 as a phylogram three isolates sequenced, MAT1-2 was not present 2,450 bp and in Fig. 4 as a simplified diagram to highlight well supported upstream or 4,813 bp downstream of the MAT1-1 gene (isolates branches relevant to the conclusions of this study. Branches sup- Stemphylium sp. strains P56, P107, and P342; GenBank accession porting the polyphyly of isolates with fused MAT regions received nos. AY339851, AY339852, and AY339862, respectively). maximal support in all four methods of analysis (Figs. 3 and 4). In the MAT DNA analyses, the MAT1-1 data set consisted of Phylogenetic Analyses and Taxon Selection for Figs. 2–4. Two rounds 22 strains representing 22 species and the MAT1-2 data set of phylogenetic analyses attributed the 106 Stemphylium isolates to contained 16 strains from 16 species. Eleven strains from 11 EVOLUTION 22 phylogenetic species represented by 28 strains in Figs. 2–4. species were present in both data sets. The MAT1-1 data set of Preliminary parsimony analyses of the initial 106 taxa in the 1,688 characters was analyzed by using likelihood, parsimony, combined ITS, GPD, and EF-1 ␣ data set was used to select 53 Bayesian, and neighbor-joining methods. The most likely tree is isolates for sequencing of the fourth locus, vmaA-vpsA (see Fig. 5). shown in Figs. 3 and 4 (Ϫln likelihood ϭ 8,389). Parsimony, After a partition homogeneity test indicated that the 53 taxa, ITS, Bayesian, and neighbor-joining analyses were congruent for GPD, EF-1 ␣, and vmaA-vpsA data sets were not significantly branches relevant in this study, with 100% support from all different (P ϭ 0.179), they were pooled into a 2,371 character data analyses for the monophyly of MAT1-1 from fused regions (Figs. set and analyzed by using likelihood, parsimony, Bayesian, and 3 and 4). MAT1-2 also strongly supported the monophyly of the neighbor-joining methods (see Supporting Results in Supporting Text fused MAT regions. The MAT1-2 data set consisted of 1,632

Inderbitzin et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11393 Downloaded by guest on September 28, 2021 Fig. 4. The Stemphylium MAT1-1 mating-type region phylogeny contradicts the four-locus species phylogeny. Fused mating-type region sequences were monophyletic in the consensus tree (Left), but the species with the fusions were polyphyletic in the four-locus species consensus phylogeny (Right). Branches with Ͻ70% support in bootstrap and Bayesian analyses were collapsed. Branches in bold had 100% support in all analyses. Distribution of fused and separate mating-type regions is given by long vertical arrows. Short arrows represent conﬂicting inferred evolutionary origins of fused mating-type regions between the MAT1-1 region phylogeny and four-locus species phylogeny. Phylogenetic groups from ref. 24 and this study are given by the narrow vertical lines. The MAT1-1 tree has fewer taxa than the species phylogeny because of the absence of isolates of the opposite mating type.

characters. It was analyzed as MAT1-1, and the most likely tree Stemphylium was monophyletic and that the fused MAT regions of is illustrated in Fig. 7, which is published as supporting infor- the self-fertile species were monophyletic within Stemphylium and mation on the PNAS web site (Ϫln likelihood ϭ 6,215). were derived from separate MAT regions (see Supporting Results in Supporting Text; see also Fig. 9, which is published as supporting Phylogenetic Analyses of MAT1-1 Proteins. The MAT1-1 data set information on the PNAS web site). contained 26 representatives reflecting the total diversity of the 31 Stemphylium strains for which MAT1-1 was sequenced and five Testing of Alternative Phylogenetic Hypotheses. The Shimodaira– outgroup species. The amino acid alignment of 429 characters was Hasegawa test showed that constrained monophyly of the taxa with analyzed by using parsimony, likelihood, and distance algorithms fused MAT regions in the species phylogeny yielded significantly yielding congruent results (see Supporting Results in Supporting worse trees. Of the six constrained most parsimonious trees of 902 Text). The Fitch–Margoliash distance tree in Fig. 8, which is steps each, all were significantly worse than the most likely tree (P Ͻ published as supporting information on the PNAS web site, shows 0.05). In MAT DNA analyses, constraining taxa with fused MAT that Stemphylium was monophyletic with 100% parsimony and 92% regions to be polyphyletic yielded 24 and 2 most parsimonious trees quartet puzzling support. A monophyletic clade (groups B, C, and of 1,176 and 784 steps each for MAT1-1 and MAT1-2, respectively, TP) of MAT1-1 proteins from fused MAT regions was the sister all worse than the corresponding most likely tree (P Ͻ 0.05) (data group, with 88% parsimony bootstrap support and 81% quartet not shown). puzzling support, to a clade of proteins from unfused MAT regions (groups SP plus E). A second, basal clade also consisted of proteins Discussion from unfused mating type regions (groups F, A, and D). This The genus Stemphylium is closely related to Cochliobolus, sug- phylogenetic configuration indicated that the fused MAT region gesting that the mating systems of the two genera might have was derived from ancestral separate MAT regions (Fig. 8). evolved in similar ways. Our results showed that, as in self-sterile isolates of Cochliobolus, each of the 23 self-sterile isolates of Phylogenetic Analyses of MAT1-2 Proteins. The MAT1-2 data set Stemphylium that was tested had only one mating-type gene, contained 19 representatives reflecting the total diversity of the 23 either MAT1-1 or MAT1-2 (Fig. 1). Unexpectedly and unlike Stemphylium isolates for which MAT1-2 was sequenced and five Cochliobolus,inStemphylium, only one type of fused MAT outgroups. The amino acid alignment of 365 characters was ana- region was found and it was present in 76 self-fertile isolates, all lyzed with the same three algorithms as MAT1-1. MAT1-2 protein lacking separate MAT regions (Fig. 1). In all selfers with fused analyses were congruent with MAT1-1 analyses in showing that MAT regions, an inverted MAT1-1 gene was inserted between a

11394 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501918102 Inderbitzin et al. Downloaded by guest on September 28, 2021 MAT1-2 and its flanks (Fig. 1). Also in all fused MAT regions, Stemphylium is Ͻ10 kb and, thus, not unreasonably long to be the 5Ј and 3Ј splices were at homologous positions in the DNA transferred asexually. sequence alignment (Fig. 2). Further, in gene genealogies, all No other evolutionary event could have caused the discontinuous MAT1-1 genes from fused MAT regions were monophyletic with distribution of the fused MAT regions in the Stemphylium species 100% bootstrap support in all analyses (Fig. 3). Similarly, in phylogeny (Fig. 3). We found no evidence for sources of phyloge- genealogies of MAT1-2, the genes from fused MAT regions were netic error such as extreme substitution rate heterogeneity or monophyletic with 100% bootstrap support (see Fig. 7). Phylo- paralogy (26). Convergent evolution of the fusion is ruled out by the genetic testing supported monophyly of the fused MAT regions, monophyly of the individual MAT1-1 (Figs. 3 and 4) and MAT1-2, because constraining the fused MAT regions to be polyphyletic right of gray box components of the fusions and by the identical resulted in significantly worse trees. In both cases, direction of arrangement and splice sites of all of the fused MAT genes (Fig. 2). evolution from separate to fused MAT regions was suggested by The high similarity of the MAT1-1 (see sequences in Fig. 2 Upper, phylogenetic analyses. Using amino acid translations of the MAT right of gray box, and Lower, left of gray box) sequences of sequences and including outgroup sequences in the analyses, self-fertile species in clusters B, C, and TP indicates that the fusion separate MAT regions were basal to fused MAT regions for both originated recently relative to the divergence of B and C from TP MAT1-1 and MAT1-2, suggesting ancestry of the separate MAT in the species tree (Figs. 3 Right and 4 Right). Its recent age suggests regions (see Figs. 8 and 9). Evolution of fused MAT regions from that the fusion had not persisted as an ancient polymorphism during separate progenitors was supported by DNA sequence compar- the evolution of the shared ancestors of B, C, E, and TP. In addition to selfers with fused MAT regions, Stemphylium ison showing that selfers with fused MAT regions could have contained another, not closely related, clade of selfers. This clade originated from a self-sterile ancestor by the inversion of an was group SP, where only MAT1-1 was detected, present in forward ancestral MAT1-1 gene plus flanking regions, creating a cross- orientation as in self-sterile isolates. Group SP may represent a third over site allowing the fusion of the inverted MAT1-1 region to lineage that evolved selfing independently, with unknown genetic MAT1-2 (Fig. 1). However it arose, in Stemphylium, unlike changes responsible for its evolution. Self-fertile isolates with only Cochliobolus, the fused mating-type genes characterizing the one detectable MAT allele have also been reported in Neurospora self-fertile isolates had a single origin. The Stemphylium species africana (27). phylogeny, on the other hand, suggested otherwise. Our data thus show convincingly that selfing in Stemphylium In contrast to the single origin of the fused mating-type genes, the evolved three times from self-sterile ancestors. One time was by four-locus Stemphylium species phylogeny showed two independent fusion of MAT1-1 and MAT1-2, a second time by horizontal origins of the self-fertile isolates with fused MAT regions (Fig. 3). transfer of the fused MAT locus across lineages, and a third time The self-fertile species with the mating type gene fusion in the TP by unknown means in the presence of only MAT1-1. cluster (Fig. 4) form a strongly supported monophyletic group with the species with separate mating-type genes in cluster E rather than We thank the following institutions and individuals: the former Torrey with the self-fertile species with the fusion in cluster C. Constraining Mesa Research Institute͞Syngenta for access to the vmaA-vpsA genes in clusters C and TP to be monophyletic resulted in significantly worse the unpublished C. heterostrophus genome sequence; G. Saenz for trees, lending additional support to the polyphyly of the fused MAT assistance with annotation of the vmaA-vpsA genes; A. and R. Bandoni, regions. The contradiction between MAT gene trees and the S. Landvik, and G. Zhang (University of British Columbia, Vancouver), M. E. Barr (Sidney, BC, Canada), N. O’Neill (U.S. Department of species phylogenies can be resolved by invoking a lateral transfer of Agriculture Research Service, Beltsville, MD), E. Simmons (Crawfords- the MAT1-1 and MAT1-2 gene fusion across lineages in the ville, IN), and the Culture Collection of Fungi, Technical University of evolution of Stemphylium. Denmark, Department of Biotechnology, Lyngby, Denmark, for gener- The mechanism and direction of transfer of the mating-type ous contributions of fungi and͞or help with field work; Seara Lim for region between lineages is unknown. A rare sexual or asexual laboratory assistance; L. Sigler of the University of Alberta Microfungus hybridization event, followed by lineage sorting, might have led to Collection and Herbarium and T. Merkx of the Centraalbureau voor the gene distribution found in selfers. In our studies, we have used Schimmelcultures for incorporating our strains into their collections. This work was supported by Natural Sciences and Engineering Research 106 strains of Stemphylium and found no evidence for any other Council of Canada (NSERC) operating grants (to M.L.B.), National hybridization across lineages. Kondo et al. (25) showed that a Science Foundation Grant DEB-9806935 (to B.G.T.) (subcontract fragment of at least 11 kb was transferred between a bacterial M.L.B.), and a University of British Columbia Graduate Fellowship and endosymbiont and an insect host. The fused MAT region in an NSERC postgraduate fellowship (to P.I.).

1. Ochman, H., Lawrence, J. G. & Groisman, E. A. (2000) Nature 405, 299–304. 14. Berbee, M. L., Pirseyedi, M. & Hubbard, S. (1999) Mycologia 91, 964–977. 2. Rosewich, U. L. & Kistler, H. C. (2000) Annu. Rev. Phytopathol. 38, 325–363. 15. de Jesus Yanez Morales, M. (2001) Ph.D. thesis (Cornell University, Ithaca, NY). 3. Coppin, E., Debuchy, R., Arnaise, S. & Picard, M. (1997) Microbiol. Mol. Biol. 16. Berbee, M. L., Payne, B. P., Zhang, G., Roberts, R. G. & Turgeon, B. G. (2003) Rev. 61, 411–428. Mycol. Res. 107, 169–182. 4. Turgeon, B. G., Bohlmann, H., Ciuffetti, L. M., Christiansen, S. K., Yang, G., 17. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. Schafer, W. & Yoder, O. C. (1993) Mol. Gen. Genet. 238, 270–284. (1997) Nucleic Acids Res. 24, 4876–4882. 5. Cozijnsen, A. J. & Howlett, B. J. (2003) Curr. Genet. 43, 351–357. 18. Rambaut, A. (1995) SE-Al V1.D1 (Department of Zoology, University of Oxford, 6. Turgeon, B. G. (1998) Annu. Rev. Phytopathol. 36, 115–137. Oxford). 7. Yun, S.-H., Berbee, M. L., Yoder, O. C. & Turgeon, B. G. (1999) Proc. Natl. 19. Swofford, D. L. (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Acad. Sci. USA 96, 5592–5597. Other Methods) (Sinauer Associates, Sunderland, MA), Version 4. 8. Hawksworth, D. L., Kirk, P. M., Sutton, B. C. & Pegler, D. N. (1995) Dictionary 20. Huelsenbeck, J. P. & Ronquist, F. (2001) Bioinformatics 17, 754–755.

of the Fungi (CAB International, Wallingford, Oxon, U.K.). 21. Felsenstein, J. (2001) PHYLIP 3.572 (University of Washington, Seattle). EVOLUTION 9. Simmons, E. G. (1969) Mycologia 61, 1–26. 22. Strimmer, K. & von Haeseler, A. (1996) Mol. Biol. Evol. 13, 964–969. 10. Lee, S. B. & Taylor, J. W. (1990) in PCR Protocols, eds. Innis, M. A., Gelfand, 23. Maddison, D. R. & Maddison, W. P. (2001) (Sinauer Associates, Sunderland, MA). D. H., Sninsky, J. J. & White, T. J. (Academic, San Diego), pp. 282–287. 24. Caˆmara, M. P. S., O’Neill, N. R. & van Berkum, P. (2002) Mycologia 94, 660–672. 11. Tarutani, Y., Ohsumi, K., Arioka, M., Nakajima, H. & Kitamoto, K. (2001) 25. Kondo, N., Nikoh, N., Ijichi, N., Shimada, M. & Fukatsu, T. (2002) Proc. Natl. Gene 268, 23–30. Acad. Sci. USA 99, 14280–14285. 12. Gardes, M. & Bruns, T. D. (1993) Mol. Ecol. 2, 113–118. 26. Avise, J. C. (1994) Molecular Markers, Natural History, & Evolution (Chapman 13. White, T. J., Bruns, T. D., Lee, S. B. & Taylor, J. W. (1990) in PCR Protocols, & Hall, New York). eds. Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, T. J. (Academic, San 27. Glass, N. L., Vollmer, S. J., Staben, C., Grotelueschen, J., Metzenberg, R. L. Diego), pp. 315–322. & Yanofsky, C. (1988) Science 241, 570–573.

Inderbitzin et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11395 Downloaded by guest on September 28, 2021