Comparative Genomics of Biotechnologically Important Yeasts Supplementary Appendix

Home , Debaryomyces hansenii, Eremothecium gossypii, Hanseniaspora, Lipomyces, Pachysolen tannophilus, Pichia anomala

Comparative genomics of biotechnologically important yeasts Supplementary Appendix

Contents Note 1 – Summary of literature on ascomycete yeasts used in this study ...... 3 CUG-Ser yeasts ...... 3 Other Saccharomycotina ...... 5 Taphrinomycotina ...... 10 Note 2 – Genomes overview ...... 11 Yeast culturing, identification, DNA and total RNA extraction ...... 12 Genome sequencing and assembly ...... 12 Transcriptome sequencing and assembly ...... 13 Table S1. Genome statistics ...... 14 Table S2. Annotation statistics ...... 15 Figure S1. Genome size, repeat content, and protein conservation measures for the yeasts...... 16 Figure S2. Intron occurrence in 303 predicted orthologs in the yeasts...... 17 Figure S3. Distribution of TY-LTR elements in the yeasts...... 18 Figure S4. Organization of rDNA genes in yeast species...... 19 Note 3 – Organism phylogeny ...... 20 Figure S5. Phylogenetic tree inferred with ExaML ...... 21 Figure S6. Phylogenetic tree inferred with FastME ...... 22 Note 4 – Alternative genetic codes: CUG coding for Ser and Ala...... 23 Figure S7. Translation of CUG codons to Ser or Ala ...... 24 Table S3. Results of Bagheera predictions of codon usage for selected yeasts...... 25 Dataset S1. Genetic code of P. tannophilus ...... 25 Note 5 – Correlation of genomically encoded enzymes to metabolic traits ...... 26 Assigning genes to enzyme functions ...... 27 Figure S8. Fermentative, metabolic, osmotolerance, and temperature-dependent growth of yeasts...... 28 Figure S9. Loss of Complex I genes and evolution of fermentative lifestyle ...... 29 Figure S10. Correlation between disaccharide metabolism and genome content ...... 30

Note 6 – Gene clusters ...... 31 Dataset S2. Phylogenetic distribution of gene clusters ...... 32 Dataset S3. Pairwise associations of ECs ...... 32 Note 7 – MAT locus organization and mating-type switching ...... 33 Pan-ascomycete synteny at the MAT locus...... 33 Homothallism and heterothallism...... 33 Origin of mating type switching in Saccharomycotina...... 34 Loss of H3K9me2/3 heterochromatin...... 34 Figure S11. Mating-type locus organization and synteny in Ascomycota...... 37 Figure S12. MAT locus isomeric structures in Pachysolen tannophilus and Ascoidea rubescens...... 38 References ...... 40

Note 1 – Summary of literature on ascomycete yeasts used in this study

The organisms used in this study were selected to characterize the genomes of yeasts important or potentially important from a biotechnological perspective, and to fill in gaps in our understanding of phylogenetic relationships. Many of the yeasts were selected because of their abilities to ferment or metabolize xylose, cellobiose, arabinose, galactose, maltose and other substrates such as methanol or to produce useful fuels and advanced chemicals. Even though many yeasts and fungi will grow on various carbon sources aerobically, fewer will convert these substrates into ethanol or other chemicals with meaningful yields at significant rates. Some species were selected for their capacities to make lipids and others were selected for their acid- thermo- cryo- or osmotolerance. Even though we understand some of the mechanisms determining these traits, we do not know how widespread they are, nor do we know the constellation of metabolic activities supporting these properties. By sequencing the genomes of several species having the traits of interest, and by comparing those genomes to other species without those traits, we expected to discover new mechanisms. We also sought to better understand diverse species that occupy habitats such as insect guts, soil or exudates of plants. Finally, we wanted to broaden knowledge about yeast species that could be adapted to new uses. Increasingly, biotechnology is seeking new yeasts as “platform organisms” for the production of natural or engineered metabolites. Synthetic biology and metabolic engineering are increasing the capacities of microbes to make specialized, rapid xylose fermentation could provide novel routes for the bioconversion of agricultural and wood harvest residues into renewable fuels and chemicals. This literature summary provides a summary of the major physiological or genetic properties, and growth habits as documented in the existing literature. The text is organized to follow our overall taxonomic structure as determined by whole genome comparisons. Selected members of Pezizomycotina and Basidiomycota have been included in our study for phylogenetic comparisons, but they are not covered in this literature summary.

CUG-Ser yeasts

Scheffersomyces stipitis Scheffersomyces stipitis(1) is a predominantly haploid, heterothallic yeast related to Candida shehatae, Spathaspora passalidarum and several other pentose metabolizing ascomycetous yeast species(2, 3). Strains of Sch. stipitis are among the best xylose-fermenting yeasts in type culture collections(4-7). Fed batch cultures of Sch. stipitis produce up to 47 g/L of ethanol from xylose at 30°C under low aeration conditions(5). Sch. stipitis belongs to a group of yeasts that uses code 12 in which CUG codes for serine rather than leucine(8). This makes correct translation of many heterologous genes problematic. Sch. stipitis CBS 6054 is related to several yeasts found as endosymbionts of beetles that inhabit and degrade white-rotted wood(9-11). Unlike Saccharomyces cerevisiae, which regulates fermentation by sensing the presence of fermentable sugars such as glucose, Sch. stipitis induces fermentative activity in response to oxygen limitation(12). A description of this genome was published in 2007(13). The comparative

3 analysis presented in the current publication helps to identify the genomic features underlying its physiological traits.

Spathaspora passalidarum Spathaspora passalidarum(10) like P. stipitis and Candida tenuis (see below), can ferment and assimilate xylose, cellobiose, glucose and maltose. Efficient fermentation of xylose, a five- carbon sugar that is a major component of plant cell walls, is a major step towards effective biofuel production from plant materials. These three yeasts are also unusual in that they are found in symbiotic association with wood-boring beetles, and comparative study may give new insight into the genetic underpinnings of symbiotic relationships(9, 14). A number of other species of Spathaspora have been described with similar properties(15-17). However, not all related species ferment xylose well(18). S. passalidarum is unusual in that it has a strong capacity for xylose fermentation in the absence of or with very low aeration(19). Moreover, it will co-ferment glucose, xylose and cellobiose, which makes this yeast particularly useful for ethanol production from mixed cellulosic and hemicellulosic sugars(20). An analysis of the S. passalidarum genome was previously published(21).

Candida tanzawaensis Candida tanzawaensis is likewise representative of the large and mostly unstudied group of yeasts that are often associated with insects, particularly fungus-feeding beetles(22). The phylogenetic classification of C. tanzawaensis was previously studied by rRNA gene sequencing(23), and a number of related yeasts have been identified(24). C. tanzawaensis has mainly been studied from a taxonomic perspective, and relatively little is known about its physiology or biochemistry. One of the best sources of microbial enzymes that can degrade polysaccharides and other components of mushrooms are the microbes growing on basidiomata, which are the fruiting bodies of basidiomycetes. The basidiomata of shelf fungi and mushrooms are comprised of highly complex polysaccharides that are difficult to degrade enzymatically, so the present study examined the genome of this yeast to see if it contains enzymes capable of degrading unusual polysaccharides as well as other traits.

Hyphopichia burtonii Hyphopichia burtonii is best known for its ability to grow on starch(25). It is halotolerant(26), and has been found in cured meat(27, 28). The species is widespread in nature and appears to be strongly competitive(29). Hyphopichia burtonii along with Wickerhamomyces anomalus and Saccharomycopsis fibuligera, are spoilage yeasts that cause chalk mold defects on partially baked breads and traditional cookies(30). H. burtonii and it appears to have fairly strong amylolytic activity.

Debaryomyces hansenii Debaryomyces hansenii is a cryotolerant, osmotolerant and halotolerant marine yeast that can tolerate salinity levels up to 24%. Its halotolerance is largely attributable to its highly inducible activity of glycerol-3-phosphate dehydrogenase(31), and its consequential ability to produce large amounts of glycerol(32). D. hansenii is the most common species of yeast found in all types of cheeses and is common in dairies and in brine due to its ability to grow in the presence of salt at low temperature and to metabolize lactic and citric acids. It is widely studied mainly for its role in cheese production and for its physiological properties. D. hansenii is able to overproduce riboflavin(33) and is normally considered non-pathogenic. The genome of D. hansenii has been previously published(34).

Candida tenuis Candida tenuis is another xylose fermenting yeast belonging to the CUG-Ser clade that is found in association with wood or bark beetle larvae(35, 36) – in this case as an endosymbiont of the gut in longhorn beetles(14, 37) (Cerambycidae) where they are found in specialized structures termed mycetomes(37). C. tenuis has been long recognized and well-studied for its ability to ferment xylose(38). It is perhaps best studied for its xylose (aldose) reductase(39), which has been engineered to increase activity with NADH as a cofactor(40-42).

Metschnikowia bicuspidata Metschnikowia bicuspidata is an aquatic yeast(43) that has been reported to infect freshwater prawns(44, 45) and brine shrimp and to cause mortality when infected shrimp are fed to salmon(46). A number of yeasts such as Wickerhamomyces anomalus(47), Kluyveromyces siamensis(48), Pichia membranifaciens(43), and Williopsis saturnus(49) all produce killer toxins effective against M. biscuspidata.

Babjeviella inositovora Kurtzman and Suzuki recently reclassified Pichia inositovora (Golubev & Blagodatskaya) and Yamadazyma inositovora (Billon-Grand)(50) into a new genus, Babjeviella inositovora(51).One unusual feature of this yeast species is that it harbors linear DNA plasmids with a novel killer toxin activity(52, 53). Sequencing showed that the smaller of these elements has four ORFs that account for 95% of the sequence: a putative immunity gene, a B-type DNA polymerase, and proteins similar to the alpha- and gamma-subunits of Kluyveromyces lactis killer toxin, zymocin(54). In common with other linear extrachromosomal DNA elements, they have terminal inverted repeat sequences(55). Chitin-binding and hydrophobic domains enable a separate anticodon nuclease element to enter the target cell and cleave specific tRNAs 3' of the wobble nucleotide(56). Recently, Kast et al. have shown that ORF4 of B. inositovora causes specific fragmentation of the 25S and 18S rRNA in the target yeast(57).

Other Saccharomycotina This clade includes the methylotrophs, Ogataea polymorpha, Candida arabinofermentans, and Komagataella pastoris. As mentioned in the main text, methylotrophy is distinguished by the presence of methanol oxidase (EC 1.1.3.13), but some genes for other enzymatic activities are also present in relative abundance. These include formaldehyde transketolase (EC 2.2.1.3), which is completely absent in the CUG yeasts, along with higher levels of ubiquinone-linked NADH dehydrogenase (EC 1.6.5.3), and transaldolase (EC 2.2.1.2).

Komagataella phaffii The yeast strains commonly called Pichia pastoris, which are frequently used for heterologous protein production(58-61), are now recognized to come from two different species(62) in the genus Komagataella. As with Ogataea polymorpha, these yeasts can produce large amounts of heterologous proteins. These are often expressed using the alcohol oxidase (AOX1) promoter following induction on methanol, although other promoter systems have also been developed(63). Most of the widely-used biotechnology strains of "Pichia pastoris", including CBS 7435, its derivatives GS115 and X-33, and the host strains for the popular Invitrogen expression system, are the species Komagataella phaffii(62). Others, including stain DSM 70382 which is also used in biotechnology, are the species Komagataella pastoris(62). Complete genome sequences of K. phaffii CBS 7435(64) and GS115(65), and a draft genome sequence of K. pastoris DSMZ 70382(66) have previously been reported. The genomes of K.

5 phaffii and K. pastoris are significantly different in sequence, approximately 10% divergent at the nucleotide level. Our phylogenomic analyses were done using the GS115 (K. phaffii) genome sequence(65).

Ogataea polymorpha Ogataea polymorpha(67), which is still commonly known as Hansenula polymorpha, is one of two methylotrophic yeasts frequently used for heterologous protein expression – the other being Komagataella species ("Pichia pastoris")(58, 59). Most of the interest in these two yeasts stems from their abilities to produce large amounts of proteins from genes driven by alcohol oxidase (AOX1) promoters following induction on methanol. One advantage of O. polymorpha over K. phaffii is its higher level of thermotolerance(68). From a physiological perspective, most of the research on O. polymorpha has centered on its thermotolerance(69) alcohol oxidases(70, 71) and development of technologies for improved genetic manipulation(72, 73), although some studies have used O. polymorpha as a platform for metabolic pathway engineering(74). The group of strains previously called "Hansenula polymorpha" is now recognized to consist of two different species(67, 75): strains NCYC 495 and CBS 4732 are Ogataea polymorpha, while strain DL-1 is Ogataea parapolymorpha. We sequenced the genome of O. polymorpha strain NCYC 495 in this study, and the genome of O. parapolymorpha strain DL-1 was previously sequenced by Ravin et al. (76). The O. polymorpha and O. parapolymorpha genomes are approximately 10% divergent in genome sequence.

Candida arabinofermentans Candida arabinofermentans(77) was isolated from insect frass and related strains have been found in association with bark beetles(78). Like O. polymorpha and K. phaffii, it is methylotrophic(79). However, its ability to ferment L-arabinose to ethanol(80) makes it almost unique among the various classified yeast species(81). Prior to its genome sequencing in order to identify genes related to arabinose utilization(82), it had not been well studied.

Pichia membranifaciens Pichia membranifaciens produces organic acids and is a common food and wine spoilage yeast. It is the type species of the genus Pichia, which was previously a polyphyletic group based largely on ascospore morphology. P. membranifaciens is often found as a film on the surface of table wine, olives and cheeses(83-85), where it carries out oxidative metabolism. It will tolerate up to 11% ethanol by volume and has occasionally been reported as a contaminant in sugar cane juice, sulfite fermentation plants and natural tequila fermentations(86). It produces acetaldehyde, ethyl acetate, and iso-amyl acetate(87). Strains of P. membranifaciens have been reported with a strain dependent tolerance of 2 to 3 M NaCl(88), which probably accounts for its occurrence in olive brines(84, 89, 90). P. membranifaciens produces killer toxins active against Brettanomyces bruxellensis and grey mould disease of grapevines(91, 92). Despite its prevalence and apparent osmotolerance, P. membranifaciens has not been well studied from genetic or molecular biological perspectives prior to its genome sequencing.

Dekkera bruxellensis Dekkera bruxellensis (anomorph Brettanomyces)(93) is a hemiascomycetous yeast with a genome size of about 13 Mb. D. bruxellensis is best known for its wine spoilage activity(94, 95), in which it produces volatile ethylphenols(96-98). D. bruxellensis is particularly difficult to deal with because like S. cerevisiae, it is a facultative anaerobe(99-101) and it has a high tolerance for ethanol(101). D. bruxellensis is found in sourdough starters where it contributes to the acidity of the bread(102). D. bruxellensis will grow at low pH. It uses a wide range of carbon sources

6 under aerobic conditions and at least some strains will produce ethanol from xylose(103) and cellobiose(104) under oxygen limitation or anaerobiosis. These features have led some researchers to suggest D. bruxellensis as a promising yeast for the fermentation of lignocellulosic hydrolysates(103, 105).

Pachysolen tannophilus Pachysolen tannophilus is a rare and unusual yeast, having been isolated by Boidin and Adzet in 1957(79), and reportedly once again in 2010(106). It is best known as the first yeast shown to ferment xylose directly to ethanol(107, 108). James et al. developed a genetic system for P. tannophilus very early(109). Aside from xylose and galactose, P. tannophilus has the unusual ability to ferment glycerol to ethanol(110, 111). Early in its study, the presence(112) and the induction(113, 114) of xylose reductase (XOR, XYL1) and xylitol dehydrogenase (XID, XYL2) were shown to be instrumental in xylose assimilation. Multiple forms of XOR were demonstrated(115), and cultivation of P. tannophilus on nitrate was shown to enable aerobic growth on xylose while blocking fermentation(116). By selecting for rapid growth on xylitol and nitrate while preventing ethanol oxidation mutants with significantly higher xylose fermentation properties can be obtained(38). Soon, however, Scheffersomyces shehatae (previously Candida shehatae), a yeast known to be associated with wood boring insects(117) eclipsed the capacities of the best P. tannophilus isolates(118), and later, however, a broad screening of unconventional yeasts for xylose metabolism showed that S. stipitis, which is closely related to S. shehatae, had even greater xylose fermenting capacity(119). New publications on P. tannophilus have fallen in recent years, but it remains of interest mainly because of its unusual abilities to ferment xylose and glycerol, while resisting compounds in tanning liquors and various waste streams. A draft genome of P. tannophilus was previously reported(120) in addition to the complete, annotated genome sequence reported here.

Kluyveromyces lactis Kluyveromyces lactis (formerly Saccharomyces lactis) is a heterothallic species with a predominantly haplontic cycle (anamorph: Candida sphaerica). Many strains were originally isolated from milk-derived products and its name is derived from its ability to assimilate lactose and convert it to lactic acid. The natural habitat of K. lactis, however, is diverse. K. lactis has become attractive to science and biotechnology owing to its distinct metabolic and physiological properties. One of the most defining features of K. lactis is its ability to ferment galactose with high efficiency(121), and its consequent use in producing ethanol from cheese whey(122, 123). K. lactis has been used extensively as a probiotic (124). Aside from its use in dairy applications, K. lactis is well-recognized for carrying linear DNA plasmids encoding for killer factors(125, 126). Like the closely-related S. cerevisiae, K. lactis carries circular plasmids that enabled the early development of an autonomous replication-based genetic system(127-129). Dujon, Sherman and Fischer, et al. sequenced and published its genome in 2004(130), and that sequence was used in the present comparative analysis.

Eremothecium (Ashbya) gossypii The filamentous fungus Eremothecium gossypii, previously known as Ashbya gossypii, was first described in 1929 as a cotton pathogen transmitted by sucking insects(131). In addition to cotton, it infects other agricultural crops such as citrus fruits. It is likely better known as an appealing model to study filamentous growth(132) because it is haploid with a small genome, has efficient gene targeting, propagates plasmids and grows on defined media(133). E. gossypii is used in industry for the production of riboflavin (134-136). The E. gossypii genome has been

7 used to annotate the genome of S. cerevisiae, to which it is closely related. The genomic sequence used in the present study was previously published (133, 137).

Saccharomyces cerevisiae Saccharomyces cerevisiae is so well known that proverbially, it needs no introduction. It holds the distinction of being the first fully sequenced eukaryotic genome(138). This fundamental work, which is employed in the present analysis, has been cited more than 1500 times. Probably one of the most defining features of S. cerevisiae is its capacity for anaerobic growth with only minimal nutritional supplements (139-141) and its simultaneous production of ethanol. S. cerevisiae lacks the native capacity for fermentation of cellobiose, xylose or arabinose, but all three of these features have been engineered (142-144). S. cerevisiae’s capacity for anaerobic growth on xylose has been demonstrated, but redox balancing and ATP production appear to be limiting when compared to anaerobic growth on glucose (145-150). S. cerevisiae has a very active glycolytic pathway for ethanol production, a high capacity for glucose uptake by facilitative transport and the capacity for anaerobic growth, but it lacks mitochondrial complex 1 (NADH:ubiquinone oxidoreductase), which is responsible for the oxidation of NADH coupled to proton translocation, and consequently for ATP synthesis. S. cerevisiae’s extensive genetic system(151, 152), the creation of a nearly complete collection of deletion mutants(153, 154), extensive analysis of protein-protein interactions(155-157) and expression studies(158, 159) have made S. cerevisiae the best understood eukaryotic model organism(160, 161).

Hanseniaspora valbyensis Hanseniaspora valbyensis is an unusual yeast that is often found in traditional balsamic vinegar and cider fermentations. Balsamic vinegar is made by a natural fermentation of cooked juice, which is rich in pectins, has a high sugar content and a low pH. As such, H. valbyensis will propagate in a stressful environment(162). H. valbyensis is known for its significant pectinolytic activity(163), which could account for its prevalence in balsamic and natural cider fermentations(162, 164-166). It also possesses endo-glucanase activity(166). It does not produce high levels of ethanol but does form significant amounts of ethyl and phenethyl acetate(166). Its osmotolerance, acid tolerance and capacities for polysaccharide depolymerization, make it of potential biotechnological interest.

Cyberlindnera jadinii The asexual state of Cyberlindnera jadinii is the correct name for the well-known taxon Candida utilis, which has been used since the early 1900s as a fodder yeast for livestock and as a dietary supplement for humans. The species grows well on pentoses and tolerates lignin by- products, which has made it attractive for fermentation of spent sulfite liquor from paper processing(167, 168) or wood hydrolysates(169). It shows significant lipase activity and has been used to add value to the waste oil processing industry(170, 171). C. utilis has also been reported to synthesize (R)-phenylacetylcarbinol, a pharmaceutical precursor for L-ephedrine production(172-176). C. jadinii has been isolated from a variety of substrates, but the natural habitat of this species is uncertain. Some strains are from clinical sources, and the species appears to be a low-grade opportunistic pathogen(177). From a physiological perspective, C. jadinii is Crabtree negative, which means that cell yields can be high under aerobic conditions(178-180).

Wickerhamomyces anomalus Wickerhamomyces anomalus, previously known as Pichia anomala and Hansenula anomala, is frequently associated with spoilage or processing of food and grain products. Its capacity for

8 growth on a wide range of carbon sources at low pH under high osmotic pressure and with little or no oxygen enables it to propagate in a wide range of environments(181). It is a non- Saccharomyces wine yeast that contributes to wine aroma through the production of volatile compounds. In recent years it has been used as a biocontrol agent against other fungi due to its ability to produce mycocin killer toxins(182-184). It has been studied for its cyanide-resistant alternative oxidase activity(185, 186), and it possesses an active beta-glucosidase that plays a role in wine fermentations(187-190). It also produces phytase(191) and an exo-beta- (1,3)- glucanase(191). Schneider et al. published a draft genome of this yeast in 2012(192), but the genomic sequence, annotation and analysis reported here were performed at JGI.

Ascoidea rubescens Lindeau collected Ascoidea rubescens from sap flowing from felled beeches in the royal forest at Wolbeck. Brefeld studied the isolate and gave a comprehensive description in 1891. Ascoidea species live in close association with bark beetles, in slime fluxes of trees or behind bark. All species appear primarily to be disseminated by insect vectors(193-195). The A. rubescens genome was sequenced principally for phylogenetic comparisons.

Yarrowia lipolytica Yarrowia lipolytica is well recognized for its ability to produce lipids from glycerol or other carbon sources(196, 197). Its lipogenic activity along with roles in other processes has made it very important from a biotechnological perspective(85, 198-200). Genetic transformation, expression and secretion systems are well developed for Y. lipolytica(201-207). These tools have been used to engineer Y. lipolytica for the production of a wide range of products such as citric(208), succinic(209) and arachidonic(210) acids(211), erythritol(212) along with higher value eicosapentaenoic (EPA) and docosahexaenoic (DHA) essential fatty acids (213-217). Y. lipolytica mutants have been used extensively in the study of peroxisome biogenesis and function(218, 219). The copy of the genome of Yarrowia lipolytica strain CLIB122 used in the present study was obtained from Génolevures [Release Version: 2012/02/09](130).

Nadsonia fulvescens Nadsonia fulvescens var. elongata has no significant biotechnological applications. It is a relatively rare soil yeast(220, 221) that is characterized by a unique life cycle(222). N. fulvescens is one of the few yeasts that divide by bipolar budding, but it is not closely related to other species with similar budding characteristics(223, 224). Following conjugation between the parent and the first budding cell, the zygote moves into a second bud formed at the opposite end of the parent cell. A septum then arises between the parental cell and the second bud and the new bud becomes an ascus. This species forms ubiquinone with a side chain of six isoprene units, but neighboring species form ubiquinone with nine isoprene units. Other yeast species form ubiquinone with nine isoprene units, thus providing an unusual contrast. We sequenced its genome mainly to clarify the phylogeny of basal ascomycete yeast species.

Tortispora caseinolytica Tortispora caseinolytica (previously Candida caseinolytica) (225, 226) is notable for its apparent taxonomic diversity when its gene sequences from the large-subunit rRNA, the mitochondrial small-subunit rRNA, and cytochrome oxidase II are compared to those of other yeasts(79). It occurs in the rotting tissues of opuntia cactus in the Sonoran desert. It does not ferment any sugars and it assimilates only a limited number of carbon compounds, including 2- and 5- ketogluconic acids. It exhibits strong extracellular proteolytic activity on casein at pH 6.5, but gelatin is not hydrolyzed or is only weakly hydrolyzed by a few strains(225). When cultivated on

9 a medium containing casein, it secretes an active, broad-range protease. Like N. fulvescens and A. rubescens, the genome of T. caseinolytica was sequenced mainly to clarify phylogeny and to discover new genes from a taxonomically unusual organism.

Lipomyces starkeyi Lipomyces starkeyi has been studied extensively, and its capacity for lipid production has been recognized since the 1950’s(227-229). In recent years it has been studied mainly for its capacity to produce and accumulate large amounts of lipid from a wide range of carbon sources(230) including starch(231, 232), xylose(230, 233-237) and even sewage sludge(231, 238). With optimal ratios of carbon to nitrogen, L. starkeyi will accumulate > 60% lipid by weight(233), making it a prime candidate for biodiesel production from renewable biomass(230, 231, 236- 238). A transformation system for L. starkeyi has recently been described(239). Its genomic sequencing was included in this study for its biotechnological and biofuel potential as well as to clarify its taxonomic status.

Taphrinomycotina

Pneumocystis jirovecii Pneumocystis jirovecii is a fungus that causes severe pneumonia in immune-compromised patients. This genome lacks virulence factors and most amino acid biosynthesis enzymes and presents reduced GC content and size. Together with epidemiological observations and inability to grow in culture, these features suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs. This genome sequence will boost research on this deadly pathogen, a major cause of mortality in patients with impaired immune systems. The genome of P. jirovecii was published previously(240) and is included here principally for phylogenetic comparisons(235).

Schizosaccharomyces pombe Schizosaccharomyces pombe or "fission yeast" is a model organism in molecular and cell biology. The sequence of the S. pombe genome was published in 2002, by a consortium led by the Sanger Institute, becoming the sixth model eukaryotic organism whose genome has been fully sequenced(241, 242). Approximately 120 natural strains of S. pombe have been isolated. The genetic system of S. pombe is highly developed, which has allowed the collection of almost 5,000 heterozygotic diploid deletion mutants that have enabled annotation of function through comparisons with budding yeast(243).

Taphrina deformans Taphrina deformans is a fungus responsible for peach leaf curl, an important plant disease. It is phylogenetically assigned to the Taphrinomycotina subphylum, which includes the fission yeasts and the mammalian pathogens of the genus Pneumocystis. Its role in peach leaf curl has been recognized since about 1926(244), and it has been studied principally as a plant pathogen. Its production of indol-3-acetic acid and cytokinin are responsible for the deformation of plant leaves following infection(245-247). The genome of T. deformans was published in 2013 and is used here for phylogenetic comparisons(248).

Saitoella complicata Saitoella complicata was included in this study for comparative analysis of poorly sampled yeast lineages. This rare fungus occupies a unique position as one of a small group

(Taphrinomycotina) of the earliest diverging Ascomycota(249-251). Its distinct position and saprobic life style – capable of living in an environment rich in organic matter that lacks oxygen – while belonging to a phylogenetic group containing plant and animal pathogens, makes this species important for sequencing. A draft genome of S. complicata has been previously published(252), but the complete, annotated sequence performed by JGI has been used in the present analysis. Note 2 – Genomes overview The genomes of Saccharomycotina yeasts are, in general, compact (13.2 Mb on average) (Table S1, Fig. S1) with low repeat content (3% repetitive on average; Table S1). The smallest genome in Saccharomycotina is O. polymorpha at 9.0 Mb, and the largest is L. starkeyi at 21.2 Mb, followed by Y. lipolytica at 20.5 Mb. The same pattern holds true for the Taphrinomycotina yeast genomes (Table S1). The two ascomycete yeast subphyla differ in these respects from the filamentous fungi of Pezizomycotina, Basidiomycota, and early diverging fungi, which generally have larger, more repetitive genomes. Predicted proteomes of the ascomycete yeasts are, similar to their genomes, compact at 3.5k to 8.2k proteins (Table S2, Fig. S1), which again contrasts the filamentous fungi, which tend to have more predicted proteins. Using MCL clustering(253), we inferred protein family relationships among some 342 fungi, including the ascomycete yeasts, and found a core proteome 2.5k to 4.8k that is conserved throughout the fungi (Fig. S1). A substantial fraction of yeast proteomes consists of proteins specific to Ascomycota (absent from Basidiomycota and early diverging fungi), and generally a small fraction consists of organism-specific proteins (Fig. S1). We noticed that the predicted coding genes of the yeasts tend to be intron-poor (Table S2). In order to remove potential bias caused by differing genome completeness and gene calling methods, we used a set of 303 single copy orthologs from the 30 yeast genomes to quantify introns in yeast genomes (Fig. S2). Our data showed that these genes in Saccharomycotina yeasts have few introns, consistent with previous work(254) showing intron-rich ancestors to the fungi, and early-diverging fungal lineages and intron losses in later derived species. Lipomyces starkeyi is an exception with an average of three introns per gene. In contrast to Saccharomycotina, the yeasts of Taphrinomycotina tend to have introns, most notably Pneumocystis jirovecii, which has an average of six introns per gene in these 303 orthologs (Fig. S2). We used the methods described by Carr et. al.(255) to identify Ty family long terminal repeat (LTR) retrotransposons in the yeast genomes. As previously reported, S. cerevisiae has a large number of complete Ty elements and signatures of partial insertions as seen by LTR only elements. While Ty elements are relatively rare in other yeasts, their distribution shows lineage specific expansion suggesting horizontal transfer as previously proposed. S. cerevisieae primarily shows Ty1 and Ty2 elements are rare in other yeasts while Ty3 and Ty5 are rare in S. cerevisieae, but found in several other lineages. Since the ribosomal DNAs are among the most abundantly transcribed genes in the genome, their organization and stoichiometry is expected to be under strong evolutionary constraint. However, the rDNA loci have been reorganized several times during yeast evolution. In all species except Pneumocystis(256), tens to hundreds of copies of the 18S, 5.8S and 26S rDNA genes are arranged as a tandem array (or multiple arrays), but in some such as S. cerevisiae the 5S genes are also included within the repeating unit of this array, whereas in others such as Y. lipolytica the 5S genes instead occur at dozens of dispersed loci around the genome(257).

Surprisingly, we find evidence for at least five evolutionary transitions between these two modes of organization of the 5S genes (Fig. S4) rather than the single transition from dispersed to tandem organization that was previously postulated(257). We infer that the 5S genes were dispersed in the ancestor of Ascomycota, became incorporated into the main rDNA array around the time of the Ascoidea divergence, but then secondarily became dispersed again in several taxa including Komagataella (Fig. S4).

Yeast culturing, identification, DNA and total RNA extraction Yeast strains were grown in 500 ml Erlenmeyer flasks containing potato dextrose broth (CM0962, Oxoid, Thermo Fisher Scientific), and depending on taxon, incubated at 25 °C between 2 and 7 days until log-phase was reached. Cultures were spin-down, cell pellets quickly dissolved in approximately 10 ml sterile water and snap frozen in pre-folded aluminum bags and stored at – 80 °C until processed. DNA and total RNA extraction was carried out using Qiagen genomic tip 500/g and RNAeasy maxi kit columns respectively (Genomic tip 500/G, 13362; RNeasy maxi kit, 75162) following manufactures instruction. RNA integrity was assessed via Agilents bioanalyzer 2100 (Agilent Technologies) following manufactures protocols. Species (strain) identity prior genome sequencing was confirmed by subsequent PCR amplification and Sanger sequencing (3730XL, Thermo Fisher Scientific) of the universal fungal DNA barcode internal transcribed spacer region (ITS) employing standard protocols(258).

Genome sequencing and assembly Seven genomes (Ascoidea rubescens, Babjeviella inositovora, Candida arabinofermentans, Candida tanzawaensis, Cyberlindnera jadinii, Hyphopichia burtonii, and Nadsonia fulvescens var. elongata) were sequenced using Illumina platform only, combining Illumina fragment 270bp insert size and 4Kbp long mate-pair (LMP) libraries. The fragment library was produced from genomic DNA, sheared using the Covaris E210 (Covaris) and size-selected using Agencourt Ampure Beads (Beckman Coulter). The DNA fragments were treated with end repair, A-tailing, and adapter ligation using the TruSeq DNA Sample Prep Kit (Illumina) and purified using Agencourt Ampure Beads (Beckman Coulter). For the LMP libraries, genomic DNA was sheared to its desired insert size using the Hydroshear®. The ends of the fragments were ligated with biotinylated adapters containing loxP. The adapter ligated DNA fragments were circularized via recombination by a Cre excision reaction. The circularized DNA was digested using 4 base cutter restriction enzymes followed by self-ligation and inverse PCR. Both types of libraries were sequenced on Illumina HiSeq machines. Illumina data were QC ﬁltered for artifact/process contamination and subsequently assembled with AllPathsLG (259). The genome of Cyberlindnera jadinii was further improved by closing gaps with PacificBiosciences reads using PBJelly (260).

The remaining nine genomes were sequencing using hybrid approach, which combined 454 (Roche) standard and paired-end (PE) pyrosequencing with optional Illumina fragments and/or Sanger fosmid sequences. For 454 libraries, genomic DNA was fragmented by nebulization. The ends of the fragments were treated with end repair and ligated with adapters using the Titanium or 454 Rapid Library Preparation Kits (454). Adapter ligated products were immobilized on beads and treated with nick repair. Single strands were eluted to generate the final library. For 454 PE libraries, genomic DNA was sheared to its desired insert size using the Hydroshear®. The ends of the fragments were ligated with biotinylated adapters containing

12 loxP. The adapter ligated DNA fragments were circularized via recombination by a Cre excision reaction. The circularized DNA was randomly sheared using the covaris E210, ligated with adapters, and amplifed via PCR. Limited amounts of Illumina fragment data were produced as described above for all nine hybrid assemblies except for Candida caseinolytica and Ogataea (Hansenula) polymorpha and assembled with Velvet (261). In addition, fosmid libraries were prepared for O. polymorpha, Pachysolen tannophilus, and Pichia membranifaciens. For these, genomic DNA was sheared using the Hydroshear® and size selected for 40kb using a pulse field gel. The long inserts were cloned into pCC1FOS vector. The draft assemblies were produced from Roche (454) standard and PE data, with optional Sanger fosmid reads and/or shredded consensus from Velvet assembled illumina, followed by gapResolution automated gap closure (262).

Transcriptome sequencing and assembly The transcriptomes of Ogataea polymorpha, Nadsonia fulvescens, and Wickerhamomyces anomalus were sequenced using 454 (Roche) technology. mRNA was purified from total RNA using oligo dT beads, chemically fragmented, and reverse transcribed using random hexamers and SSII. The ends of the fragments were treated with end repair and ligated with adaptors using the 454 Rapid Library Preparation Kit (454). The libraries were sequenced using 454 (Roche) and assembled using Newbler (http://www.454.com/). For the remaining 13 transcriptomes, Illumina protocol was used. mRNA was purified from total RNA using oligo dT beads, chemically fragmented, and reversibly transcribed using random hexamers and SSII. The ends of the fragments were reverse transcribed using random hexamers and SSII. Second strand was synthesized using dNTP/dUTP mix. The ends of the fragments were treated with end repair, A-tailing, and ligration of adapters. The Second strand was removed using AmpErase UNG followed by PCR to enrich for the final library. The Illumina libraries were sequenced using HiSeq and assembled using Rnnotator (263).

portalid Scaffolds Assembly length Scaffold N50 Scaffold L50 Repeat covered regions Scaffold gaps GC % Scaffolds >=2kbp Organism MycoCosm Ascoidea rubescens NRRL Y-17699 Ascru1 63 17,503,998 6 1,035,938 17,300 38 32.85 47 Babjeviella inositovora NRRL Y-12698 Babin1 49 15,221,050 6 933,105 1,490 161 48.13 38 Candida arabinofermentans NRRL YB-2248 Canar1 62 13,234,078 6 701,640 6,984 91 34.74 52 Candida caseinolytica NRRL Y-17796 Canca1 6 9,180,161 2 3,113,199 485 43 45.42 6 Candida tanzawaensis NRRL Y-17324 Canta1 16 13,136,204 4 1,512,535 1,137 36 45.34 13 Cyberlindnera jadinii NRRL Y-1542 Cybja1 76 13,018,415 6 700,888 1,620 316 44.50 56 Hanseniaspora valbyensis NRRL Y-1626 Hanva1_1 647 11,464,179 11 332,595 20,073 516 26.71 646 Hansenula polymorpha NCYC 495 leu1.1 Hanpo2 7 8,974,850 4 1,302,532 742 1 47.86 7 Hyphopichia burtonii NRRL Y-1933 Hypbu1 27 12,403,110 3 1,853,527 5,071 78 34.99 16 Lipomyces starkeyi NRRL Y-11557 Lipst1_1 117 21,267,709 7 1,341,299 2,503 322 46.92 117 Metschnikowia bicuspidata NRRL YB-4993 Metbi1 48 16,055,203 3 2,544,904 3,529 373 47.85 48 Nadsonia fulvescens var. elongata DSM 6958 Nadfu1 20 13,746,944 4 1,627,684 4,388 44 39.25 16 Pachysolen tannophilus NRRL Y-2460 Pacta1_2 198 12,597,116 3 1,751,653 20,466 385 29.93 28 Pichia membranifaciens NRRL Y-2026 Picme2 11 11,584,685 3 1,676,949 2,867 134 45.12 11 Saitoella complicata NRRL Y-17804 Saico1 35 14,136,186 11 436,079 1,788 39 52.60 35 Wickerhamomyces anomalus NRRL Y-366-8 Wican1 46 14,145,566 3 2,188,197 2,641 161 34.55 46

Table S1. Genome statistics The MycoCosm portal id can be used to access the genome portals of the respective organisms on MycoCosm (http://genome.jgi.doe.gov/fungi) using the format http://genome.jgi.doe.gov/. A group page for the organisms used in this study can be found at http://genome.jgi.doe.gov/biotech_yeasts/.

Number of Number of Genes Median Intron Length Avg. of number Introns pfam with proteases transporters secretome sscp Ascoidea rubescens NRRL Y-17699 6,802 109 0.38 4,782 167 667 269 41 Babjeviella inositovora NRRL Y-12698 6,403 73 0.26 4,833 187 710 260 36 Candida arabinofermentans NRRL YB-2248 5,861 95 0.33 4,495 152 617 221 20 Candida caseinolytica NRRL Y-17796 4,657 73 0.20 3,875 160 548 172 21 Candida tanzawaensis NRRL Y-17324 5,895 82 0.24 4,577 163 618 233 22 Cyberlindnera jadinii NRRL Y-1542 6,038 67 0.34 4,835 179 739 296 38 Hanseniaspora valbyensis NRRL Y-1626 4,800 94 0.19 3,417 119 493 149 10 Hansenula polymorpha NCYC 495 leu1.1 5,177 46 0.20 4,382 163 664 187 7 Hyphopichia burtonii NRRL Y-1933 6,002 82 0.22 4,769 169 694 269 20 Lipomyces starkeyi NRRL Y-11557 8,192 56 1.84 5,777 212 922 292 53 Metschnikowia bicuspidata NRRL YB-4993 5,851 93 0.26 4,396 159 579 261 60 Nadsonia fulvescens var. elongata DSM 6958 5,657 90 0.57 4,696 174 600 169 13 Pachysolen tannophilus NRRL Y-2460 5,675 97 0.32 4,616 165 643 189 13 Pichia membranifaciens NRRL Y-2026 5,546 114 0.22 4,309 147 618 216 28 Saitoella complicata NRRL Y-17804 7,034 56 1.23 5,404 225 710 290 36 Wickerhamomyces anomalus NRRL Y-366-8 6,423 58 0.42 5,160 198 843 288 22

Table S2. Annotation statistics Pfam annotations of predicted genes was performed using HMMER (v3.0) against the pfam database (v27)(264) and AntiFam (v3.0)(265). Proteases and transporters were identified using BLAST against the MEROPS database (v9.2)(266) and Transporter Classification Database (v.July 15 2011)(267) respectively. Secretome was predicted using a combination of SignalP (v4.1), TargetP (v1.1), TMHMM (v2.0) and WoLF-PSORT (v0.2)(268) using methods similar to those described by Emanuelsson et. al(269). Small secreted cysteine rich proteins (sscp) were identified as secreted proteins that were less than 300 amino acids, lacked pfam annotations, and had 2% or more cysteine residues.

Figure S1. Genome size, repeat content, and protein conservation measures for the yeasts. Yeasts newly sequenced for this study are shown in bold.

5 P.jirovecii

O.polymorpha

3 starkeyi L.

H.valbyensis

C.tanzawaensis

Average introns/gene Average

S.passalidarum

P.membranifaciens

D.bruxellensis

C.jadinii

2 T.caseinolytica

C.arabinofermentans

S.complicata

S.pombe

S.cerevisiae

H.burtonii

T.deformans

1 N.fulvescens

A.rubescens

W. anomalus

Y. Y. lipolytica

P.tannophilus

B. B. inositovora

S. stipitis S.

M.bicuspidata

K.phaffii D.hansenii gossypii E.

C.tenuis K.lactis 0 0 10 20 30 40 50 60 70 80 90 100 Percent intronless genes

Figure S2. Intron occurrence in 303 predicted orthologs in the yeasts.

Figure S3. Distribution of TY-LTR elements in the yeasts.

Figure S4. Organization of rDNA genes in yeast species. 5S rDNA genes are highlighted in red. For each species, BLAST searches were used to infer the organization of the tandem rDNA array and to search for dispersed 5S rDNA genes. Square brackets and the subscript n indicate that the structure of the complete repeating unit of the rDNA array was determined for that species. In many cases the 18S-5.8S-26S unit structure was not present in genomic scaffolds but was inferred from EST cluster data. ND, not determined. In Pneumocystis there is only a single copy of the 18S-5.8S-26S cluster, and we identified two 5S genes at different locations.

Note 3 – Organism phylogeny The 38 genome sequences were phylogenetically investigated using the DSMZ phylogenomics pipeline as previously described (270-277) using BLAST (278), OrthoMCL (279), MUSCLE (280), RASCAL (281), GBLOCKS (282), and MARE (283). Clusters of orthologs were generated using OrthoMCL, inparalogs were removed, the remaining sequences were aligned with MUSCLE and filtered with RASCAL and GBLOCKS. Three distinct supermatrices were compiled, (i) a ‘full’ supermatrix based on all filtered alignments comprising at least four sequences; (ii) a ‘MARE’ matrix obtained by removing relatively uninformative and low-coverage genes from the ‘full’ matrix using MARE under default values except that deleting organisms was disallowed; (iii) a ‘core’ matrix comprising only those genes present in all organisms. The ‘full’ supermatrix used 7,297 genes (2,094,063 characters), the ‘MARE’ matrix used 1,559 genes (364,126 characters), and the ‘core’ matrix used 418 genes (166,611 characters). Maximum likelihood and maximum-parsimony trees were inferred from the concatenated alignments with RAxML (284) and PAUP* (285), respectively, as previously described (270- 277). The selected model for all three matrices was PROTCATLGF (the LG model of amino acid evolution (286) in conjunction with the CAT approximation for modeling rate heterogeneity (284) and empirical amino-acid frequencies. The resulting trees had a log likelihood of (i) - 48,541,040.42, (ii) -11,399,822.76 and (iii) -5,676,545.75, respectively. The best maximum- parsimony (MP) trees found had a length of (i) 9,198,654 steps, (ii) 2,156,869 and (iii) 1,093,186 (not counting uninformative characters). In order to assess potential artifacts caused by model oversimplification and standard bootstrapping, the full matrix was also analyzed with ExaML version 3.0.7 (287) and one model per gene (estimated during search for the best tree) in conjunction with the partition bootstrap (288); TNT (289) was used for inferring MP trees and MP partition bootstrap values. Moreover, the entire proteomes were subjected to a Genome BLAST Distance Phylogeny (GBDP) analysis (290) using the greedy-with-trimming approach in -8 conjunction with formula d5, an e-value filter of 10 , and pseudo-bootstrapping (291). We performed a partition bootstrap analysis of the full matrix with ExaML(287) and TNT(289). In contrast to the trees shown in Figure 1 in the main text, this computationally expensive approach used (for ML) one model per gene and to obtain branch support bootstrapped entire genes, not individual alignment positions(288). The results shown in Figure S5 strongly support the findings depicted in Figure 1. The entire proteomes were also subjected to a Genome BLAST Distance Phylogeny (GBDP) analysis(292, 293). This method is of interest because the complete genomic information enters tree reconstruction, with corrections for paralogs on a pairwise basis, instead of a global determination of orthology(294, 295), which considerably decreases overall computation time. The tree shown in Figure S6 confirms most major groupings and yields no significantly supported conflicts compared to the previous phylogenetic analyses. Support is overall slightly lower, however; for instance, Taphrinomycotina receive only little support, and the relative positioning of Lipomyces and Tortispora is unresolved.

Figure S5. Phylogenetic tree inferred with ExaML with one individually estimated model per gene and rooted with Batrachochytrium dendrobatidis. Numbers on branches are ML (left) and MP (right) partition bootstrap support values. The branches are scaled in terms if the expected number of substitutions per site.

Figure S6. Phylogenetic tree inferred with FastME from interproteomic distances calculated with GBDP and rooted with Batrachochytrium dendrobatidis. Numbers on branches are GBDP pseudo-bootstrap support values. Long terminal branches are due to the distinct scaling used by GBDP in combination with formula d5.

Note 4 – Alternative genetic codes: CUG coding for Ser and Ala The CUG-Ser clade of yeasts, use an altered genetic code in which CUG codons are translated as Ser, rather than the canonical Leu (296-301). This change is due to alterations in the tRNA that decodes CUG (tRNACAG), which is recognized by seryl-tRNA synthetase and, to a lesser degree, leucyl-tRNA synthetase, resulting in a mixture of roughly 97% Ser and 3% Leu residues(302, 303) at CUG-encoded sites in proteins. To investigate the origins of this genetic code change, we inspected predicted tRNACAGs for the presence of three sequence features indicative of Ser translation(304): a G33 residue 5’ to the anticodon, which may lower rates of leucylation(305), a Ser identity element in the variable loop, and a G discriminator base (Fig. S7A). The assembled genomes of selected yeasts were submitted to the Bagheera web server(306) (Table S3). For an unambiguously “standard table” yeast like Lipomyces starkeyi, Bagheera predicts a 7:146 ratio of alternate codon usage to standard usage sequence similarity suggesting that L. starkeyi uses the standard table. Similarly, for a CUG-Ser yeast like Candida tanzawaensis, the ratio is 32:15 suggesting alternative codon usage. However, for P. tannophilus, that ratio is 14:12, only slightly in favor of alternative codon usage. This, combined with our observation of tRNA differences from CUG-Ser clade yeasts suggests that translation of CUG codons in P. tannophilus may be neither standard (Leu) nor the same as in the CUG- Ser clade (Ser). Two plasmids were designed to investigate usage of the alternate genetic code in P. tannophilus, both containing hygromycin resistance genes under the control of native glyceraldehyde-3-phosphate dehydrogenase promoters (1000 bp) and terminators (300bp). One version utilized a standard bacterial aminoglycoside phosphotransferase (APH) gene, while the other contained the same APH gene but was codon-optimized for expression in CUG yeasts. In particular, the codon-optimized APH replaced 9 CUG codons with alternate leucine codons, to preserve the integrity of the amino acid sequence in CUG yeasts. When introduced into P. tannophilus, thousands of transformants were obtained using the codon-optimized version of the construct (Fig. S7B), while none were obtained when treated under identical conditions with the version containing CUG codons (Fig. S7C). Oligonucleotides were obtained from Integrated DNA Technologies (IDT, Coralville, IA). Plasmids were constructed with the Gibson method for in-vitro enzymatic DNA assembly (307), using PCR-amplified products with 30 base pair overlapping regions. Plasmids were sequenced at Functional Biosciences Inc. (Madison, WI) to confirm that their assembly was successful. DNA was prepared for integrative transformations by digesting plasmids with BamHI and SfoI, followed by DNA purification. Transformations were carried out using a method similar to the standard lithium acetate transformation protocol (308). A dilute culture was grown in 50 mL of YPD overnight, and cells were harvested after reaching an OD600 near 0.2. After addition of the transformation mixture, cells were treated with a 30 minute incubation step at 30°C, a 20 minute heat shock step at 40°C, a 2 hour recovery step in 3 mL of YPD, and plated onto YPD media containing 125 µg/mL of hygromycin. Transformants were visible after about three days of outgrowth at 30°C. Our current success in demonstrating the ability to transform P. tannophilus with hygromycin resistance only in the absence of CUG codons highlights the utility and importance of considering alternate codon systems when attempting to engineer non-conventional yeast species.

Figure S7. Translation of CUG codons to Ser or Ala (A) tRNACAG features associated with translation of CUG codons to Ser is limited to CUG-Ser clade of yeasts. Basal to the CUG-Ser clade, M. bicuspidata and B. inositovora lack either the Ser identity element or discriminator base, respectively, possibly indicating Ser gradual acquisition of tRNACAG sequence features. P. tannophilus, like other yeasts related to it, lacks the characteristic features of Ser tRNACAG . (B-C) P. tannophilus cells were transformed with a hygromycin resistance construct either codon-optimized to utilize alternate leucine codons (B), or containing CUG codons (C). Variable colony sizes can reflect differences in the locus, copy number, or stability of genomic insertions.

Alternative Standard Indiscriminative

C. tanzawaensis 32 15 45

D. hansenii 26 17 44

P. tannophilus 14 12 54

A. rubescens 11 4 24

L. starkeyi 7 143 41

Table S3. Results of Bagheera predictions of codon usage for selected yeasts. Bagheera predictions were based on the following web server parameters. Reference species for gene prediction=Saccharomyces cereviseae S288C, Alignment method=MAFFT, Alignment configuration=End gap free.

Dataset S1. Genetic code of P. tannophilus In a separate excel file.

Note 5 – Correlation of genomically encoded enzymes to metabolic traits Growth profiles for the yeasts (309) are summarized in context of the phylogenetic tree in Fig. S8. In yeasts where the gene complement conflicted with the reported growth data (e.g. genes present but (-) on a substrate), showed variable growth, or in which the strain sequenced was not the strain with reported growth data, we performed tests of the sequenced strain’s growth on the substrate in question. Cultures were incubated in yeast nitrogen base (duplicate tubes) for one month at 25°C (or 20°C in the case of Ascoidea rubescens). Controls were carbon-free YNB and YNB with glucose. Results by substrate were: D-xylose: Candida tanzawaensis NRRL Y-17324 was confirmed D-xylose (-) as originally reported (309), Metschnikowia bicuspidata NRRL YB-4993 (+), Ogataea polymorpha NCYC 495 (+), Pichia membranifaciens NRRL Y- 2026 (-), Wickerhamomyces anomalus NRRL Y-366-8 (+); galactose: Ogataea polymorpha NCYC 495 (-), Wickerhamomyces anomalus NRRL Y-366-8 (+), Ascoidea rubescens NRRL Y17699 (-), Nadsonia fulvescens var. elongata DSM 6958 (+); L-rhamnose: Ascoidea rubescens NRRL Y17699 (-) in contrast to a previous report (309); maltose: Ogataea polymorpha NCYC 495 (+), Dekkera bruxellensis CBS 2499 (+), Eremothecium gossypii ATCC 10895 (+); raffinose: Dekkera bruxellensis CBS 2499 (-); sucrose: Ogataea polymorpha NCYC 495 (+), Dekkera bruxellensis CBS 2499 (+); melibiose: Debaryomyces hansenii CBS 767 (+); lactose: Scheffersomyces stipitis CBS 6054 (+), Debaryomyces hansenii CBS 767 (+), Candida tenuis NRRL Y-1498 (+), Lipomyces starkeyi NRRL Y-11557 (+); cellobiose: Ogataea polymorpha NCYC 495 (-), Dekkera bruxellensis CBS 2499 (+), Pachysolen tannophilus NRRL Y-2460 (+), Eremothecium gossypii ATCC 10895 (+). Our broad phylogenetic study confirmed and elaborated prior findings that loss of respiration Complex I (RC1) preceded the gain of bacterial dihydroorotate dehydrogenase (Ura1) in S. cerevisiae and closely related genera (Fig. S9). These two features along with other evolutionary changes such as expansion of genes for facilitated uptake of sugars enabled anaerobic growth in these yeasts. Unexpectedly, two other fermentative yeasts (Nadsonia fulvescens and Schizosaccharomyces pombe) showed diminished RC1 components, but did not acquire Ura1 (Fig. S9). The utilization of several disaccharides correlated broadly with the presence of genes encoding well-characterized enzymes, but several exceptions are noteworthy (Fig. S10). The genomes of many yeasts from the CUG-Ser clade lack recognizable SUC2 homologs, but each of these yeasts can still utilize sucrose. In each of these cases, their genomes encode multiple predicted alpha- or beta-glucosidases. An attractive hypothesis is that these enzymes have a broader specificity that allows them to bind to and hydrolyze glucose disaccharides more generally, a property known for some S. cerevisiae alpha-glucosidases(310). A similar relationship may exist among alpha-glucosidases for Sch. stipitis(311). In support of this hypothesis, within the CUG- Ser clade, only H. burtonii and D. hansenii encode beta-fructosidase (s), and these yeasts can utilize raffinose (a trisaccharide where a galactose monomer is linked to glucose monomer of sucrose, a configuration likely to block access from alpha- or beta-glucosidases). Surprisingly, A. rubescens scored positive on many disaccharide growth assays, but its genome is predicted to encode few of the enzymes characterized in disaccharide catalysis (Fig. S10). If the positive growth scores are not artifacts, this yeast species may be a fruitful source of novel genes. Notably, the L. starkeyi genome contains multiple homologs encoding each enzyme family, which is consistent with its broad spectrum of disaccharide utilization.

Assigning genes to enzyme functions Predicted protein sequences were assigned enzyme function by three methods: PRIAM (312) profile searches, TBLASTN searches using query protein sequences from the characterized pathways in model organisms vs. genome assemblies, and BLASTP searches of the same query proteins against the full predicted protein catalog of each organism. In the case of discrepancies between PRIAM/TBLASTN/BLASTP results, we manually inspected the results to resolve ambiguities. For example, to more thoroughly search the yeast genomes for homologs to known GAL pathway genes, we used TBLASTN to search the six translated reading frames of the genomes for matches to the known Gal1, Gal7, and Gal10 proteins. Using an e-value threshold of 10-5, we found the same complement of enzymes found by PRIAM, with one exception: TBLASTN detected one GAL10 gene in K. phaffii, whereas PRIAM detected none. The K. phaffii annotation (NCBI accession PRJNA39439) lacked any predicted protein sequence that we could assign to a Gal10 function (UDP-glucose 4-epimerase; EC 5.1.3.2), whereas the TBLASTN search identified a high-confidence hit from 1,583,243 to 1,584,238 on chromosome 4, where, presumably due to the inherent inaccuracies of automated annotation, a gene had not been predicted. We thus were able to assign a single putative GAL10 gene to K. phaffii (Fig. 3). XYL genes were detected by mapping the characterized proteins (313) (Xyl1: Picst3|89614; Xyl2: Picst3|86924; Xyl2: Picst3|68734) to their PRIAM assignments (EC 1.1.1.175, EC 1.1.1.9, and EC 2.7.1.17, respectively), and in turn finding these PRIAM assignments in the proteins of the study set. However, this approach was apparently not sufficiently sensitive because it resulted in Xyl1 and/or Xyl2 going undetected in several yeasts capable of growing on D-xylose (309). We therefore inspected the TBLASTN and BLASTP results, selecting TBLASTN (e-value threshold 10-5) results for Fig. 3 because it produced the fewest disagreements between gene complement and published growth data. Methylotrophy genes were detected similarly using K. phaffi queries: AOX (ALOX1_PICP7/ALOX2_PICP7), CAT (CCA37834), DAS (CCA39320/ACN76560), FDH (CCA39210), FGH (CCA39282), FLD (CCA39112), and TKL (CCA37054) to EC 1.1.3.13, EC 1.11.1.6, EC 2.2.1.3, EC 1.2.1.2, EC 3.1.2.12, EC 1.1.1.284, EC 2.2.1.1, respectively; Fig 3 shows the PRIAM results. Rhamnose genes were detected with BLASTP of queries Rha1 (Picst3|50944), Lra2 (Picst3|63908), Lra3 (Picst3|50672), Lra4 (Picst3|64442); Fig. 3 shows the results using an e-value threshold of 10-30. Genes involved in disaccharide metabolism in Fig. S10 were identified using TBLASTN and an e-value threshold of 10-5 with the following protein queries: alpha-glucosidase: S. cerevisiae CBS 2354 MAL32 (GenBank: CCB84898.1); beta-fructosidase: S. cerevisiae S288c SUC2 (NCBI Reference Sequence: NP_012104.1); alpha-galactosidase: S. pombe 972h- MEL1 (NCBI Reference Sequence: NP_595012.1); beta-galactosidase: S. stipitis CBS 6054 LAC4 (NCBI Reference Sequence: XP_001382569.2); beta-glucosidase: S. stipitis CBS 6054 BGL5 (NCBI Reference Sequence: XP_001387350.1).

Figure S8. Fermentative, metabolic, osmotolerance, and temperature-dependent growth of yeasts. Throughout this text we refer to the fermentative and metabolic abilities of the various yeasts. These abilities are summarized here. Adapted from Kurtzman et al. (75).

Figure S9. Loss of Complex I genes and evolution of fermentative lifestyle Homologs to the catalytic components of Respiration Complex I (RC1; blue; EC 1.6.5.3 NADH:ubiquinone oxidoreductase; EC 1.6.99.3 NADH dehydrogenase; EC 1.6.5.11 NADH dehydrogenase (quinone)) were identified using the PRIAM pipeline(314). Alternative oxidase (Picst3|67332) was detected using BLASTP(315) and an e-value of 1e-50. URA9 (red) was detected using the K. lactis URA9 sequence (Q6CTX8|PYRD2_KLULA from Swissprot(316)) as a query and an e-value of 1e-50. URA1 (green) was detected using the 3610|Sacce1 sequence as a query and e-value of 1e-100. Fermentation capabilities (orange) are from Kurtzman et al(75).

Figure S10. Correlation between disaccharide metabolism and genome content Utilization (orange) was scored as follows: +, positive growth; v, variable among strains tested; s, slow growth; w, weak growth; -, no growth. The number of homologs encoding each enzyme (green) were detected using TBLASTN (e-value cutoff = 1.0e-5, no low complexity filtering) with the following query genes: S. cerevisiae CBS3254 MAL32, alpha-glucosidase; Sch. stipitis CBS 6054 BGL5, beta-glucosidase; S. cerevisiae S255c SUC2, beta-fructosidase; S. pombe 972h MEL1, alpha-galactosidase; K. lactis NRRL Y-1140 LAC4, beta-galactosidase.

Note 6 – Gene clusters Genes for enzymes in the same metabolic pathway are frequently adjacent to one another in genomes where they are coordinately regulated(317). Examples include clustering of genes for cellulose(311), urea, allantoin, galactose(318), and maltose degradation(310, 319, 320). Based on these earlier observations, we hypothesized that genes having related metabolic activities would be likely to remain proximal to one another in the various genomes if the physiological function of their combined activities depended on the presence of both metabolic activities(321). To test this hypothesis, we scanned the 38 genomes for genes encoding proteins with known enzymatic activities (i.e. EC numbers). We then scanned the scaffolds of each species and recorded the positions of genes for enzymatic activities that shared a common metabolite. Clusters were designated as two or more genes sharing one or more common metabolites, and separated by no more than six ORFs from one another on a scaffold, along with intervening genes. The combination of ECs found within a given cluster was designated as a cluster profile (CP), and many of these were found to recur in multiple genomes. The genes showing two or more metabolic and genomic associations were then plotted against our phylogenetic tree. We recorded 200 CPs that occurred in one or more species. We also performed a taxonomically independent analysis by counting the number of instances in which the gene for each enzyme occurred in any other pairwise cluster. Genes for 255 enzymes were found in CPs consisting of two or more related genes (Dataset S2). The number of CPs identified depends on the number of intervening ORFs allowed between genes for metabolically associated activities. Allowing for more intervening ORFs expands the number of clusters but also incorporates more proximal genes that could code for proteins metabolically unrelated to those in the CP. Genes often associated with activities for substrate assimilation include permeases, transporters or regulatory proteins, most of which are difficult to annotate. Examining the genomic context of CPs in specific taxa thereby can facilitate identification and annotation of other activities related to that metabolic activity. The number of CPs scored also depends on the numbers of subunits, orthologs, or isoforms of each enzymatic activity present in the genome. If multiple isoforms or subunits of a single enzymatic activity are present in the cell, there are more opportunities for the encoding genes to be paired with other related genes. For example, three separate DNA-directed RNA polymerases occur in the cell, each of which has multiple subunits – thereby increasing the frequency with which components of this activity can be paired with genes for other related enzymes. Genes for a particular enzyme occur in CPs with several other related genes, and some CPs occur across a taxonomic range. In some cases, the CPs occurred among multiple genes and across taxonomic ranges. Pairwise CPs in a single organism are common and scattered throughout the species examined. Other CPs were observed in up to 14 species over wide taxonomic ranges. Some CPs consist of genes for three or four metabolically related enzymes. These sometimes occur in a single organism, but sets of CPs consisting of three or four activities were identified in multiple species as well. Metabolic traits that depend on the presence of a few specific enzymatic activities can show close proximal associations over wide taxonomic ranges. Galactose metabolism is one of the best examples. Galactokinase [EC 2.7.1.6] occurs in 16 pairwise associations with EC 2.7.7.12 (UDP-glucose--hexose-1-phosphate uridylyltransferase), and 4 times in pairwise associations with EC 5.1.3.2 (UDP-glucose 4-epimerase), and all three enzymes must be present in the genome, along with galactose permease, in order for a species to metabolize galactose. The capacity to metabolize galactose is widely distributed among yeasts and other fungi, but is particularly prevalent in the CUG-Ser yeasts (Dataset S3). Genes for the metabolism of N- acetylglucosamine [EC 2.7.1.1, EC 3.5.1.25, and EC 3.5.99.6] are likewise frequently clustered

31 and frequently found among the CUG-Ser yeasts. As had been previously noted, genes coding for β-glucosidases [EC 3.2.1.21] are frequently found in association with cellulases (endo- glucanases; EC 3.2.1.4) and are occasionally found in association with EC 3.2.1.58 (glucan 1,3- β-glucosidases)(311). Surprisingly, genes encoding α-glucosidases (maltase; EC 3.2.1.20) were found more frequently in association with β-glucosidases than with glucan 1,4-α-glucosidases (glucoamylases; EC 3.2.1.3), but this might have been attributable to the greater abundances of α- and β-glucosidases in the genomes of the organisms studied. Also surprisingly, genes encoding glycogen synthases [EC 2.4.1.11] and 1,3-β-glucan synthases [EC 2.4.1.34] were frequently found in association with one another, but this might be attributable to their common precursor (UDP glucose). The presence of particular CPs can be indicative of how metabolic activities differ among taxonomic groups. For example, genes involved with biotin synthesis fell into two clusters that exhibited different phylogenetic and taxonomic distributions. The first, consisting of dethiobiotin synthase (EC 6.3.3.3) and adenosylmethionine---8-amino-7-oxononanoate transaminase (EC 2.6.1.62) was prevalent in the Saccharomycotina. The second, consisting of dethiobiotin synthase and biotin synthase (EC 2.9.1.6) prevailed among the other phylogenetic clades. These three genes function in sequence (EC 2.6.1.2, 6.3.3.3 and 2.9.1.6) in the biotin synthetic pathway, and in the Saccharomycota, EC 2.6.1.62 is found in a tandem bidirectional association with EC 6.3.3.3. In the CUG-Ser yeasts, biotin synthase (EC 2.8.1.6) is often clustered with genes for 60S ribosomal protein L22 and ribonuclease III, but the significance of this (if any) is not known. Genes coding for nitrate reductases (EC 1.7.1.3) (NADPH) occur in 10 pairwise clusters with EC 1.7.1.4 nitrite reductases [NAD (P)H] across a wide taxonomic range, and in some instances, these genes are adjacent or proximal to nitrate permease. The times that nitrate reductase or nitrite reductase occur alone in the genome of each organism were not counted, but because nitrite is toxic, the presence of nitrate permease and reductase would likely require the presence of nitrite reductase in the genome as well. Some of the most conspicuous CPs were found with DNA-directed RNA polymerase [EC 2.7.4.8], which occurred in 23 pairwise associations in the 38 organisms (Dataset S3). Almost all of these pairings [CPs 18, 43, 68, 78, 107, 140, 142 and 193] occur in purine metabolism where GTP and ATP are used to make RNA. In S. cerevisiae, the three forms of RNA polymerase (RNA polymerase I, II and III) are made up of a total of 31 subunits, so there are more opportunities for subunits of DNA-directed RNA polymerase to be proximal to genes for related enzymes. By comparison, DNA-directed DNA polymerase [EC 2.7.7.7], which has 3 genes associated with this function in S. cerevisiae, was found paired to related enzymes in only three instances. In Y. lipolytica, it was found proximal to genes for three other enzymes acting on its substrates: pyruvate kinase [EC 2.7.1.40], nucleoside-diphosphate kinase [EC 2.7.4.6] and UMP/CMP kinase [EC2.7.4.14]. It was also associated with pyruvate kinase in Komagataella phaffii and Cyberlindnera jadinii (Dataset S3). RNA-directed RNA polymerase activity [EC 2.7.7.48], which has 12 subunits in S. cerevisiae, was proximally associated with a related enzymatic activity, nucleoside-diphosphate kinase [EC 2.7.4.6] only once, in the basidiomycete Ustilago maydis.

Dataset S2. Phylogenetic distribution of gene clusters Dataset S3. Pairwise associations of ECs In separate excel files

Note 7 – MAT locus organization and mating-type switching Cell type in yeasts is specified by the genotype at the mating-type (MAT) locus. This locus commonly contains two genes (a1 and a2) in MATa haploid cells, and two different genes (α1 and α2) in MATα haploids(322, 323). The a2 and α1 genes code, respectively, for a-specific and α-specific transcription activators that are the master determinants of the two haploid cell types. The a1 and α2 proteins, which are simultaneously present only in diploid cells, are homeodomain proteins that form a heterodimer that represses transcription of haploid-specific functions such as mating. There are however many exceptions to this typical picture, with several losses of individual MAT genes having occurred in different species. For example, the a2 transcription activator was lost in an ancestor of S. cerevisiae. Pan-ascomycete synteny at the MAT locus. Our data reveal, for the first time, synteny conservation of genes near the MAT locus across the ascomycete phylogenetic tree, extending the known relationship between Pezizomycotina and Saccharomycotina(324) to Taphrinomycotina. The MAT locus is linked to each of NVJ2, APC5, APN2, SLA2 and SUI1 in at least one species in all three subphyla (Fig. 4 – main text). This conservation of context shows that cell type has been controlled during more than a billion years(325) of ascomycete evolution by a single orthologous locus (MAT), despite gross changes of the gene content at this locus such as the loss of all HMG domain genes in S. cerevisiae. The stability of MAT contrasts with the frequent turnover of sex-determination loci in animals and plants(326-328). Homothallism and heterothallism. The reproductive modes of yeast species can be characterized as heterothallic or homothallic, and the structures of their MAT loci correlate with these modes (Fig. 4 – main text). In heterothallic species each haploid strain is either MATa or MATα, and mating is possible only between haploids of opposite MAT genotypes. Fifteen genomes in our dataset appear to be from heterothallic species (Fig. 4C – main text) because their genome assemblies contain either MATa genes or MATα genes, but not both. Only one of these, C. jadinii is diploid (having MATa and MATα genes at allelic locations). Fourteen other species appear to be homothallic (Fig. 4D – main text), based on the presence of both MATa and MATα genes at non-allelic locations in their genome assemblies. The inferred homothallic or heterothallic structure of the genomes generally agrees with previous reports of the reproductive mode of each species(75) with four exceptions (D. bruxellensis, H. valbyensis, C. jadinii and N. fulvescens; Fig. 4B – main text). In homothallic species, any strain can mate with any other strain. At the cellular level, homothallism often occurs by mating type switching – a DNA rearrangement process by which the genes at the MAT locus of a cell are replaced by genes of the opposite type, converting a MATa haploid cell to a MATα or vice versa. Such homothallism is called secondary homothallism because mating still occurs only between MATa and MATα cells, as in heterothallism. Homothallic species in the family Saccharomycetaceae, such as S. cerevisiae and K. lactis, are secondary homothallics that switch mating types by a well characterized copy-and-paste mechanism wherein DNA at the MAT locus is replaced by DNA copied from one of two non- expressed ‘cassette’ loci called HMLα and HMRa(323, 329). Secondary homothallism was also recently described in two methylotrophic yeasts, O. polymorpha and K. phaffii(330, 331) (Fig. 4D – main text). These methylotrophs switch mating types by a flip/flop mechanism: a chromosomal region that has MATa genes at one end, and MATα genes at the other end, undergoes inversion to swap the MAT genes between an expression context and a repression context. Transcription of one set of MAT genes is repressed by a centromere (in O. polymorpha)

33 or a telomere (in K. phaffii) located just outside the invertible region. Inversion of the region moves the MATa and MATα genes relative to the repressing element, activating the previously silent genes and silencing the previously active genes. Inversion occurs by recombination between two identical ~2 kb sequences that form an inverted repeat (IR) at the ends of the region. Primary homothallism in yeasts is defined as the ability of any cell to mate with any other cell of the same species; in other words, these species do not have distinct ‘a’ and ‘α’ cell types. The molecular details of how cells of these species sense their ploidy, or avoid responding to their own pheromones, is unknown. Four yeasts in our dataset are possible primary homothallics: Scheffersomyces stipitis and Debaryomyces hansenii (CUG-Ser clade)(332), Lipomyces starkeyi (basal Saccharomycotina), and Pneumocystis murina (Taphrinomycotina)(333) (Fig. 4D – main text). Among these, only Sch. stipitis has been demonstrated experimentally to be haploid and homothallic, with Mendelian segregation of markers in meiosis. Origin of mating type switching in Saccharomycotina. Our new sequence data suggested that Pachysolen tannophilus and Ascoidea rubescens are secondary homothallics that can switch mating types by a flip/flop inversion mechanism similar to that of O. polymorpha and K. phaffii (Fig. 4D – main text). In both of these species, a chromosomal region containing MATa and MATα genes is flanked by an inverted repeat (IR) formed by two copies of a 2 kb sequence (blue triangles in Fig. 4D – main text). We confirmed that in P. tannophilus, the orientation of the 8-kb MAT region located between the two IRs can be inverted by growing cells in nitrogen limitation media (Fig. S12A), similar to the mechanism of mating type switching in the other methylotrophic yeasts O. polymorpha and K. phaffii. In A. rubescens, one copy of the IR is located at a telomere, similar to the organization in K. phaffii. We examined three strains of A. rubescens by PCR of genomic DNA, and confirmed that both possible orientations of the MAT region exist in this species (Fig. S12B). Genomic DNA isolations made from different colonies of the same A. rubescens strain appear to vary in the relative abundance of the two orientations, suggestive of frequent mating type switching in A. rubescens (Fig. S12). Because A. rubescens is an outgroup to the methylotroph and Saccharomycetaceae clades, the evolutionary origin of mating-type switching in Saccharomycotina can be traced to the point of divergence between this species and other Saccharomycotina (Fig. 4A – main text). The ancestral mechanism of switching in Saccharomycotina can be inferred to have been a flip/flop inversion system with two MAT-like loci (one expressed and one silent), which later evolved into a copy-and-paste system with three loci (one expressed and two silent) in the Saccharomycetaceae family(334). Surprisingly, we found that another yeast in the ‘methylotrophs’ clade, P. membranifaciens, has three MAT-like loci, which may indicate a parallel origin of a MAT/HML/HMR system in this species. Loss of H3K9me2/3 heterochromatin. In Schizosaccharomyces pombe, transcription of the silent MAT loci is repressed by di- or tri- methylation of lysine 9 of histone H3 (H3K9me2/3), forming a type of heterochromatin that is almost universal in eukaryotes but has been lost in S. cerevisiae and most other Saccharomycotina(334). We found that Lipomyces starkeyi is the only sequenced Saccharomycotina species with orthologs of genes involved in H3K9me2/3 heterochromatin processes (335). It has orthologs of S. pombe Swi6 (LSTA_112810), a chromodomain/ chromoshadow protein that binds to H3K9me2/3 nucleosomes and is central to heterochromatin-mediated gene silencing; of the H3K9 demethylase Epe1 (LSTA_69885); and a SET domain protein (LSTA_103455) that is a candidate ortholog of the H3K9

34 methyltransferase Clr4 although it lacks a chromodomain. L. starkeyi also has a family of retroelements coding for a protein fusion between a Tf1-like integrase and a Swi6-like chromodomain. Swi6-like chromodomains specifically bind H3K9me2/3, so the presence of these retroelements confirms that L. starkeyi has an H3K9 methyltransferase. Our results therefore suggest that mating-type switching, which cannot operate without a mechanism to silence the non-expressed MAT genes, evolved in the Saccharomycotina lineage relatively soon after the ancestral H3K9me2/3 form of heterochromatin was lost (Fig. 4A – main text). However, the mechanism of silencing by SIR proteins, as occurs in S. cerevisiae, did not appear until much later(334) (Fig. 4A – main text). Whether the first switching systems in Saccharomycotina silenced their extra MAT genes by proximity to centromeres or telomeres, or by some other mechanism, will be an interesting question for future studies. Independently, an analogous switching system using H3K9me2/3 silencing evolved in Schizosaccharomyces.

Figure S11. Mating-type locus organization and synteny in Ascomycota. (A) Phylogenetic tree showing inferred points of origin of mating-type switching, loss of H3K9me- mediated heterochromatin, and gain of Sir1-mediated silencing and HO endonuclease. (B) Summary of literature on homo- or heterothallism of each species (309). Discrepancies with genome data are highlighted in bold. (C,D) Schematic gene organization around MAT in selected species. Species were categorized as either heterothallic (C) or homothallic (D) depending on whether MATa and MATalpha genes are present in the same haploid genome assembly. Red outlines indicate species known or predicted to be capable of mating-type switching (secondary homothallism). Gene names in bold (NVJ2, APC5, SLA2, SUI1 and APN2) show conserved synteny among Taphrinomycotina, Pezizomycotina and Saccharomycotina near the MAT locus. To maximize data, for Wickerhamomyces and Pneumocystis we used different congeneric species for the MAT locus and phylogenomic analyses.

Figure S12. MAT locus isomeric structures in Pachysolen tannophilus and Ascoidea rubescens. (A) Mating-type switching in P. tannophilus by inversion of MAT genes. The schematic on the left shows the organization of the two copies of a 2-kb inverted repeat sequence (IR-L and IR- R), which includes the 3’ end of the gene SLA2, relative to the MATa and MATα genes, in the

38 type strain CBS4044. The orientation of the invertible region is assessed by PCR amplification of genomic DNA using primers A-D. Growth of CBS4044 in YPD media yields amplification products only from primer pairs AB and CD, indicating haploid cells with the chromosomal orientation as drawn. After growth of CBS4044 in nitrogen limitation media (NaKG: 0.5% sodium acetate, 1% potassium chloride, 1% glucose), PCR products are also seen from the AC and BD primer pairs, indicating recombination between the two copies of the IR in some cells in the culture. Strain ACY4044 (a) is isogenic to CBS4044 but has the opposite orientation of the invertible region. It was isolated by growing CBS4044 in NaKG for 24 h, followed by dilution and plating for single colonies on YPD agar and screening the MAT region orientation by colony PCR. The chromosome structure diagram is a composite of data from our assembly and from Liu et al.(336), which are both from the type strain of P. tannophilus. (B) Organization of the MAT region in A. rubescens. A 2-kb inverted repeat includes the 5’ end of the MATa1 gene. A telomere (TEL) is located immediately to the right of one copy of the IR (IR-R). The MATa and MATα genes are separated by approximately 45 kb of noncoding DNA. The 12 gel panels show PCR amplification, with 4 primer pairs as indicated, of genomic DNA isolated from three strains of A. rubescens including the one whose genome was sequenced (NRRL Y-17699). For each strain, we isolated genomic DNA separately from four colonies cut from YM agar (A. rubescens is hyphal and did not grow well in liquid culture). Most of the DNA preparations show a predominance of either the AB and CD products (corresponding to the orientation as drawn) or the AC and BD products (corresponding to recombination between the two IRs), but with a significant presence of the alternative orientation. These cultures could be either a mix of two haploid cell types (with a majority unswitched and a minority switched), or a mixture of diploid and unswitched haploid cells. The chromosome structure diagram represents a merge between scaffolds 22 and 25 of our genome assembly of the type strain of A. rubescens. This junction, and the presence of the telomere beside IR-R, were confirmed by PCR. The consensus telomeric repeat sequence of A. rubescens is 5’- (TGCTGGATGA)n.

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