Fusion of the Subunits Α and Β of Succinyl-Coa Synthetase As A

Home , Dikarya, Pezizomycotina, Saccharomycotina, Taphrinomycotina

Research Article

Fusion of the subunits a and b of succinyl-CoA synthetase as a phylogenetic marker for Pezizomycotina fungi

Amanda M. Koire and Andre R.O. Cavalcanti Biology Department, Pomona College, Claremont, CA, USA.

Abstract Gene fusions, yielding the formation of multidomain proteins, are evolutionary events that can be utilized as phylogenetic markers. Here we describe a fusion gene comprising the a and b subunits of succinyl-coA synthetase, an enzyme of the TCA cycle, in Pezizomycotina fungi. This fusion is present in all Pezizomycotina with complete genome sequences and absent from all other organisms. Phylogenetic analysis of the a and b subunits of succinyl-CoA synthetase suggests that both subunits were duplicated and retained in Pezizomycotina while one copy was lost from other fungi. One of the duplicated copies was then fused in Pezizomycotina. Our results suggest that the fusion of the a and b subunits of succinyl-CoA synthetase can be used as a molecular marker for membership in the Pezizomycotina subphylum. If a species has the fusion it can be reliably classified as Pezizomycotina, while the absence of the fusion is suggestive that the species is not a member of this subphylum. Key words: fused genes, fungi phylogeny, evolutionary marker. Received: March 19, 2011; Accepted: June 23, 2011.

Introduction quence analysis (Alexopoulos et al., 1996; Spatafora et al., Amongst eukaryotes, Fungi is the kingdom with most 2006). sequenced genomes. The sequenced genomes cover fungal In recent years, comparative genomics rather than species broadly and include agriculturally, medically, and gross morphology has been used to analyze specific sub industrially significant species with very diverse genomes. groups of fungi. Notably, analysis of the phylum The fungal kingdom is divided in Chytridiomycota, Neo- Saccharomycotina has revealed major events of evolution callimastigomycota, Glomeromycota, Ascomycota, Basi- such as recent whole genome duplication and subsequent diomycota and Blastocladiomycota, and Microsporidia gene loss or subspecialization (Arvas et al., 2007; Dietrich (Hibbett et al., 2007). Ascomycota, which diverged from et al., 2004). In Pezizomycotina, on the other hand, only a Basidiomycota in the region of 741-1195 million years ago subset of protein families related to plant biomass degrada- (Scannel et al., 2007), is the largest phylum of the Fungi tion and secondary metabolism revealed signs of recent ex- kingdom and is again divided into Saccharomycotina, pansion, likely because of a repeat-induced point mutation Taphrinomycotina, and Pezizomycotina (Lumbsch and mechanism for the active removal of duplicated sequences Huhndorf, 2007). Pezizomycotina is the largest subphylum that has been identified in varying levels in Pezizomycotina of Ascomycota and contains more than 33,000 described species (Arvas et al., 2007). Gene duplication significantly species (Spatafora et al., 2006). influences evolution, as duplicated genes may be retained if Pezizomycotina, often referred to as filamentous they provide an advantage, either because of an increased fungi, grow primarily in a filamentous form and degrade gene dosage effect or because of increased functional di- biomass to free sugars that are used as source of energy and versification, or may alternatively be lost (Wagner, 1998; carbon (Arvas et al., 2007). Most species exhibit a domi- Li and Graur, 2000; Cornell et al., 2007). Multiple copies of nant hyphal growth form, with almost all of the sexually re- the genes coding for the a and b subunits of succinyl-CoA producing forms possessing ascomata (Spatafora et al., synthetase have been shown to exist in several species of 2006). Filamentous ascomycetes have been shown to be a Aspergillus and in Neosartorya fisheri, placing succinyl- monophyletic group distinguished from Taphrinomycotina CoA synthetase among the subset of proteins showing gene and Saccharomycotina on the basis of ribosomal DNA se- expansion in Pezizomycotina (Flipphi et al., 2009). Succinyl-CoA synthetase, also known as succinate- Send correspondence to Andre R.O. Cavalcanti. Biology Depart- CoA ligase and succinate thiokinase, is an acid-thiol ligase ment, Pomona College, 175 W 6th Street, Claremont, 91711 CA, that forms carbon-sulfur bonds. Succinyl-CoA synthetase USA. E-mail: [email protected]. is an enzyme of the citrate cycle, a major metabolic path- 670 Koire and Cavalcanti way in which acetyl-CoA derived from the catabolism of ies of the protein, the fusions were artificially split into a’ sugars and lipids is oxidized to carbon dioxide and energy and b’ subunits. To increase the diversity of fungal species in the form of GTP, NADH, and FADH2 (Hanson, 2008). analyzed, we also included the sequences of the a and b This enzyme catalyzes the reversible transformation of succinyl-CoA synthetase subunits of two Neocallimasti- succinyl-CoA, orthophosphate, and GDP to succinate, gomycota fungi. As an outgroup we used the a and b sub- GTP, and the four-carbon metabolite coenzyme A in a units of succinyl-CoA synthetase from three mammals, three-step substrate-level phosphorylation reaction (Kane- Homo sapiens, Mus musculus and Canis familiaris. hisa and Goto, 2000; Hamblin et al., 2008). Succinyl-CoA synthetase is composed of two subunits, a and b, which Phylogenetics analyses generally form dimers in eukaryotes and tetramers in pro- An alignment of the a subunits of succinyl-CoA karyotes. Nucleotide specificity of succinyl-CoA synthe- synthetase for 53 organisms, including 25 Pezizomycotina, tase is thought to be determined by the N-terminal domain 14 Saccharomycotina, 2 Taphrinomycotina, 7 Basidiomy- of the beta subunit (Hamblin et al., 2008), and the alpha cota, 2 Neocallimastigomycota as well as 3 metazoans to subunit may contain a targeting sequence for the enzyme provide an outgroup, was created in ClustalX v.2.0.11 (Lar- (Birney and Klein, 1995). While prokaryotes are capable of kin et al., 2007) using default settings. A total of 80 se- using both types of nucleotides, eukaryotic succinyl-CoA quences were aligned, including the region corresponding synthetase enzymes usually occur as isoforms specific for to the a subunit in the fusion genes in Pezizomycotina. A either GTP or ATP (Hamblin et al., 2008). Multiple copies b b of the succinyl-coA synthetase genes tallies with the find- separate alignment of and ‘ subunits of the same organ- ing that duplications are most common in Pezizomycotina isms was created in the same manner. In total, 83 sequences b when the proteins produce secondary metabolites (Arvas et were used in the subunit alignment, including the region al., 2007), and with the common belief that natural selec- corresponding to the b subunit in the fusion genes in Pezi- tion on metabolic phenotypes mainly targets enzymes that zomycotina. control the rate of turnover of molecules through a meta- Each set of aligned sequences corresponding to the a bolic pathway, such as succinyl-CoA synthetase (Flipphi et and b subunits was used as input to infer phylogenetic trees al., 2009). using maximum likelihood and Bayesian analysis. For the In Pezizomycotina, one set of the duplicated a and b maximum likelihood analysis we first used the program gene copies for succinyl-CoA synthetase has become ProtTest (Abascal et al., 2005) to select the amino acid sub- fused. Gene fusion, yielding formation of multidomain pro- stitution model that best fits the protein alignments. In both teins, is a major driving force in protein evolution and is a datasets, the best model of amino acid substitution was the rare evolutionary event in comparison to the frequency of LG model (Le and Gascuel, 2008), with invariable sites and point mutations. Consequently, gene fusion events were re- across site rate variation. We used PhyML 3.0 (Guindon cently utilized as an evolutionary marker to pinpoint the and Gascuel. 2003), as implemented in SeaView (Galtier et root of the eukaryotic tree (Nara et al., 2000; Stechmann al., 1996) to create maximum likelihood trees using the LG and Cavalier-Smith, 2002, 2003). Here we examine how model of amino acid substitution (Le and Gascuel, 2008) the presence of the succinyl-CoA synthetase fusion gene with optimized number of invariable sites and optimized can be used as a simple phylogenetic marker for Pezizo- across site rate variation. Bootstrap support was calculated mycotina, and attempt to trace the origin and nature of this using the aLRT model (Anisimova and Gascuel, 2006). fusion in Fungi. The Bayesian analysis was performed using MrBayes (Ronquist and Huelsenbeck, 2003) with four Monte Carlo Materials and Methods Markov Chains and variable rates of substitution. Each chain was run for 1,000,000 generations and trees were GenBank sequence data sampled every 1,000 generations. The first 100 trees were excluded as burn-in and we used the remaining 900 trees to We used the fungi genome BLAST tool from NCBI calculate bootstrap values. The trees were visualized and to search all complete fungal genomes with predicted pro- prepared for publication using Dendroscope (Huson et al., teins in GenBank. We started with the sequence of a Pezi- 2007) and FigTree. zomycotina succinyl-CoA synthetase a and b fusion protein, and used blastp to identify all other succinyl-CoA Results synthetase proteins in different fungal genomes. a b With the exception of Microsporidia species, we were The fusion of the and subunits of succinyl-CoA can serve as a phylogenetic marker for able to identify at least one a and one b subunit in each fun- Pezizomycotina fungi gal genome available in the Genomic BLAST page (Table S1). The searches returned both fused and non-fused genes. We used the sequence of the fused a and a subunits For aligning the fused proteins with non-fused a and a cop- of succinyl-CoA synthetase from Pyrenophora tritici- Succinyl-CoA synthetase in fungi 671 repentis, a Pezizomycotina, to search all the complete fun- the remainder of subunit a and the full length of subunit b. gal genomes with protein predictions available in the We aligned both genes with the fusion genes and deter- genomic blast webpage form NCBI. mined the boundaries between the a and b subunits assum- As of August 1st 2009, the genomic blast webpage al- ing the proteins are fused and used these sequences in the lowed the search of the protein sequences of 51 fungal analyses. Like in A. niger, the putative fusion proteins genomes: 41 Ascomycota, 7 Basidiomycota and 3 Micros- groups with the fusion proteins in the other Pezizomycotina poridia. The 41 Ascomycota are divided in 25 Pezizo- in the phylogenetic trees (Figures 1 and 2, Table S1). mycotina, 14 Saccharomycotina, 2 Taphrinomycotina. All Finally, while the fusion is not annotated in Giberella genomes analyzed had both the a and b subunits of zeae, in this organism a subunit b gene follows a subunit a succinyl-CoA synthetase, with the exception of the three gene. Both subunits are in the same strand but are 3 kb apart Microsporidial genomes that completely lack both subunits and there seems to be a small intervening gene in the oppo- of the protein and were not analyzed further. The genome of site strand between the subunits. A large intron between the the Basidiomycota Postia placenta also seems to be lacking subunits could have confounded the gene prediction soft- both subunits of the gene; however, an additional search of ware, and it is not unknown for a gene to be nested inside an the genome page for this organism in the Joint Genome In- intron of another (Kumar, 2009). It is worth noting that the stitute (JGI), revealed that the genes are present, and we version of the genome available in GenBank is based on a used the sequences obtained from JGI in the analyses for preliminary whole-genome-shotgun that has been super- this organism. ceded by a new assembly of the genome. Unfortunately, the In addition to the sequences obtained from the new assembly doesn’t seem to be available at the time of genomic blast searches we included the sequences of 2 this writing. Interestingly, these two proteins group with Neocallimastigomycota fungi in the analysis to increase the the fusion proteins in the phylogenetic trees (Figures 1 and diversity of the fungal species. The Ascomycota, together 2, Table S1), indicating that they could be fused. The alter- with Basidiomycota, form the subkingdom Dikarya, and native explanation would be that the proteins were once are more closely related to each other than to Neocalli- fused and that the insertion of a gene in between the a and b mastigomycota or Microsporidia. Finally, we used the se- subunits broke the fusion into two working copies of the quences of the a and b subunits of succinyl-CoA synthetase subunits; while plausible, this second explanation seems to from three Metazoans as outgroup. be unlikely as the insertion would have to have occurred Out of the 25 Pezizomycotina fungi with complete right at the junction between the two subunits. This organ- genomes available in NCBI, we immediately detected the ism also has an extra copy of the subunit a that groups with fusion gene in 22 genomes. According to the annotation in the fusions, suggesting that the fusion gene was duplicated NCBI the fusion appears to be absent from Aspergillus in this organism and the subunit b was eliminated in one of niger, Coccioides immitis and Gibberella zeae. Interest- the copies of the fusion. ingly, each of these species has at least two a and two b These results indicate that, even though it might not subunits of succinyl-CoA synthetase. Thus, we further ana- have been annotated as such, the fusion of the a and b sub- lyzed these genomes to determine if the fusions are really units of succinyl-CoA synthetase is present in all 25 Pezi- absent or if they are just not annotated. zomycotina genomes available at GenBank, with the possi- In Aspergillus niger, there are two subunits a and two ble exception of that of G. zeae in which the fusion protein subunits b annotated in the genome; a tblastn search of the seem to at least have once been present. In addition, the fu- fusion protein against A. niger genome showed that a sub- sion gene was not detected in any other fungal genome and unit b gene immediately follows a subunit a gene in the a blast search of the entire NR database revealed that the fu- same strand, indicating that the genes could be fused in this sion is only present in Pezizomycotina fungi. organism. Furthermore, a blastp search of the gene predic- The apparent presence of the fusion gene in most tions in this organism’s genome webpage in JGI revealed Pezizomycotina and its absence in all other organisms sug- that a fusion gene is present (gene model number gest that the fused succinyl-CoA synthetase gene can serve jgi|Aspni1|176118). To maintain consistency, we used the as a phylogenetic marker for Pezizomycotina. If a species proteins in GenBank in the analyses. The proteins involved has the fusion it can be reliably classified as Pezizomy- in the putative fusion protein in A. niger group with the fu- cotina; if a species does not have the fusion it most likely sion proteins in the other Pezizomycotina in the phylogen- does not belong to this subphylum. etic trees, further confirming that these two proteins are a b indeed fused in this genome (Figures 1 and 2, Table S1). Evolution of the and subunits of succinyl-CoA synthetase in fungi A similar problem occurs in Coccidioides immitis.A tblastn search revealed that two consecutive genes in the We used the sequences of the succinyl-CoA synthe- genome hit the fusion gene. In this case, however, the first tase of 53 species to build phylogenetic trees for both the a gene only hits part of subunit a while the second gene hits and b subunits of the protein. The data set contained 48 672 Koire and Cavalcanti

Figure 1 - Phylogenetic trees of the a subunit of succinyl-CoA synthetase in fungi. Pezizomycotina sequences are in blue, Saccharomycotina in green, Taphrinomycotina in red and Basidiomycota in gray. Neocallimastigomycotina and Metazoans (outgroup) are in black. Dashed lines represent fused genes. Bootstrap support values are given in relevant nodes. (a) Mr Bayes tree; (b) PhyML Maximum likelihood tree.

Dikarya, 2 Neocallimastigomycota and 3 metazoans. The All sequences were aligned in ClustalW using default Dikarya are divided in 41 Ascomycota (25 Pezizomy- parameters and trees were built using MrBayes and cotina, 14 Saccharomycotina, 2 Taphrinomycotina) and 7 PhyML. The resulting trees are shown in Figures 1 and 2. In Basidiomycota (Table S1). The sequences of the fusion genes in the 25 Pezizomycotina were split into their a and b all trees the fused genes form a monophyletic group (with constituents. strong bootstrap support) and the non-fused copies in Pezi- Succinyl-CoA synthetase in fungi 673 zomycotina form another monophyletic group (with strong subunit present in Neocallimastigomycota and Basidio- bootstrap support). mycota groups with the fused copy in Pezizomycotina, while the a subunit present in Saccharomycotina and The phylogenetic trees of the a subunits (Figure 1) suggest that this subunit duplicated before the divergence Taphrinomycotina groups with the unfused copies of the of Neocallimastigomycota from Dikarya. Saccharomyco- subunit in Pezizomycotina. G. zeae has an extra copy of tina and Taphrinomycotina lost the copy that would later subunit a that groups with the fused genes, indicating that it become fused in Pezizomycotina, while Neocallimasti- probably resulted from a recent duplication of the fused gomycota and Basidiomycota lost the other copy. The a gene followed by the loss of the b subunit. This loss should

Figure 2 - Phylogenetic tree of the b subunit of succinyl-CoA synthetase in fungi. Pezyzomycotina sequences are in blue, Saccharomycotina in green, Taphrinomycotina in red and Basidiomycota in gray. Neocallimastigomycotina and Metazoans (outgroup) are in black. Dashed lines represent fused genes. Bootstrap support values are given in relevant nodes. (a) Mr Bayes tree; (b) PhyML Maximum likelihood tree. 674 Koire and Cavalcanti not have a large phenotypic effect, as another copy of the ORFs bearing more similarity to other fungi than to fusion gene would be present in the genome. Saccharomycotina (Arvas et al., 2007). The trees of the b subunit (Figure 2) indicate a different evolutionary story for this subunit. The b subunit dupli- Discussion cated in Dikarya after their divergence from Neocallimas- tigomycota, and after the divergence of basidiomycetes Our results show that the sub-phylum Pezizomy- from ascomycetes, but before the divergence of Pezizomy- cotina is characterized by the presence of a fusion of the a cotina from Saccharomycotina and Taphrinomycotina. The and b subunits of succinyl-CoA synthetase that can be used copy that would become fused in Pezizomycotina was lost as a phylogenetic marker to determine if an organism be- from Saccharomycotina and Taphrinomycotina. longs in Pezizomycotina. If a species has the fusion it can A second unfused copy of the b subunit is present in be reliably classified as Pezizomycotina, while the absence three Aspergillus species (A. clavatus, A. flavus and A. of the fusion is suggestive that the species is not a member fumigatus) and in Neosartorya fischeri (Figure 2), all mem- of this subphylum. bers of Trichocomaceae, and groups with the fusion pro- Phylogenetic analyses show that the two subunits of teins. This suggests that the b subunit duplicated a second succinyl-CoA synthetase duplicated at different times in time before becoming fused in Pezizomycotina and was fungal evolution and then became fused in an ancestor of lost from most Pezizomycotina with the exception of some Pezizomycotina. The gene for the a subunit of succinyl- Trichocomaceae. CoA synthetase duplicated in an ancestor of the Neocalli- Interestingly, one Basidiomycota, Ustilago maydis, mastigomycota and Dikarya. One copy of the gene was also has two copies of each subunit of succinyl-CoA syn- then lost in all fungi with the exception of Pezizomycotina. thetase. One of the a and one of the b subunits group with The gene for the subunit b of succinyl-CoA synthetase the fusion gene in Pezizomycotina, which would suggest most likely duplicated in an ancestor of the Ascomycota af- that this gene could have been the result of a lateral gene ter the divergence of this group from Basidiomycota. One transfer between a Pezizomycotina and U. maydis. This copy of the gene was then lost from Saccharomycotina and would not be the first gene shown to have been transferred Taphrinomycotina, and kept in Pezizomycotina. from Pezizomycotina to U. maydis (Shen et al., 2009). We In Pezizomycotina, one copy of the a subunit fused to checked the location of these genes in the genome of U. a copy of the b subunit, generating a fusion protein while maydis and found that they are consecutive but in opposite retaining the unfused copies as well. As this fusion gene in- strands, and therefore cannot be not fused in this species. A volves two subunits of the same enzyme it is possible that it closer look at the trees reveals that the sequence of the U. provides a selective advantage by leading to greater cata- maydis subunits that group with the fusions has a long lytic activity or more efficient co-regulation of the expres- branch, and its grouping with the fusions could be ex- sion of the two succinyl-CoA subunits. plained by long branch attraction. The presence of two copies of each of the subunits in the genome of U. maydis Even though the presence of this fused gene could might have left one copy of each subunit free to accumulate lead to a more efficient enzyme, either by greater catalytic mutations, explaining why they would be in such a long activity or by more efficient co-regulation, Pezizomy- branch. However, it is also possible that the fusion gene cotina genomes maintained two copies (one fused one was horizontally transferred to U. maydis and a genome re- unfused) of each subunit of succinyl-CoA synthetase. arrangement broke the gene in two parts and reversed one This might indicate that this enzyme is needed in large of the subunits in relation to the other, although this expla- quantities in Pezizomycotina or, more interestingly, it is nation is less likely considering that such rearrangement possible that one of the copies of succinyl-CoA synthetase would need to have happened in such a way as to generate in Pezizomycotina has acquired an as yet unreported new working versions of the genes. function, as suggested by Flipphi et al. (2009) for other With the exception of U. maydis, the Basidiomycota duplicated genes in the primary carbon metabolism of form a monophyletic group in the trees. The Neocalli- Aspergillus species. mastigomycota, Taphrinomycotina and Pezizomycotina also form monophyletic groups. The Saccharomycotina Acknowledgments only form a monophyletic group in the maximum likelihood tree of the b subunit (Figure 2b); in all other trees This material is based upon work supported by the Yarrowia lipolytica groups with the unfused copy of the National Science Foundation under Award No. 0920697. proteins in Pezizomycotina and not with Saccharomy- AMK was funded with a grant of the HHMI to Pomona cotina. This unexpected placement of Yarrowia lipolytica College’s Biology Department. The authors thank Daniel has been seen in other analyses as well (Arvas et al., 2007, Martinez (Pomona College) for help generating and inter- Cornell et al., 2007) and has been attributed to Y. lipolytica preting the phylogenetic trees. Succinyl-CoA synthetase in fungi 675

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Table S1: Organisms and accession numbers of the proteins used in this study.

Accession numbers Species Fusion αβ

Pezizomycotina

Ajellomyces capsulatus gb|EEH08294.1 gb|EEH06102.1 gb|EEH03354.1

Ajellomyces dermatitidis gb|EEQ69414.1 gb|EEQ77311.1 gb|EEQ76061.1

Aspergillus clavatus gb|EAW13338.1 gb|EAW07752.1 gb|EAW12523.1 gb|EAW09110.1

Aspergillus flavus gb|EED44815.1 gb|EED50054.1 gb|EED46808.1 gb|EED53391.1

Aspergillus fumigatus gb|EAL84600.1 gb|EAL90410.2 gb|EAL91981.1 gb|EAL84389.2

Aspergillus nidulans gb|EAA65786.1 gb|EAA64406.1 gb|EAA61646.|

Aspergillus niger ref|XP_001392414.1 ref|XP_001398396. ref|XP_001392415.1 ref|XP_001400660.1 1 1

Aspergillus terreus gb|EAU37706.1 gb|EAU38921.1 gb|EAU30552.1 gb|EAU31032.1

Botryotinia fuckeliana gb|EDN22743.1 gb|EDN24969.1 gb|EDN23758.1

Chaetomium globosum gb|EAQ92530.1 gb|EAQ86176.1 gb|EAQ86581.1

Coccidioides immitis gb|EAS31442.1 gb|EAS30565.1 gb|EAS29800.1 gb|EAS31443.11

Coccidioides posadasii gb|EER27972.1 gb|EER27280.1 gb|EER28332.1

Magnaporthe grisea gb|EDK06465.1 gb|EDK06157.1 gb|EDJ98088.1

Microsporum canis gb|EEQ28748.1 gb|EEQ34080.1 gb|EEQ29517.1

Neosartorya fischeri gb|EAW20453.1 gb|EAW22789.1 gb|EAW24125.1 gb|EAW25715.1

Neurospora crassa gb|EAA34834.1 gb|EAA32357.1 gb|EAA34077.1

Paracoccidioides brasiliensis gb|EEH17930.1 gb|EEH16608.1 gb|EEH19675.1

Penicillium marneffei gb|EEA20408.1 gb|EEA24495.1 gb|EEA24589.1

Phaeosphaeria nodorum gb|EAT83662.2 gb|EAT77317.1 gb|EAT84267.1

Podospora anserina ref|XP_001911226. ref|XP_001908936.1 ref|XP_001909573.1 1

Pyrenophora tritici-repentis gb|EDU41918.1 gb|EDU40695.1 gb|EDU51437.1

Sclerotinia sclerotiorum gb|EDO04371.1 gb|EDO05039.1 gb|EDN91995.1

Talaromyces stipitatus gb|EED24140.1 gb|EED18241.1 gb|EED18353.1

Uncinocarpus reesii gb|EEP81085.1 gb|EEP79486.1 gb|EEP77760.1

Saccharomycotina

Ashbya gossypii ref|NP_985681.1 ref|NP_984650.1

Candida albicans gb|EEQ42982.1 gb|EEQ43728.1

Candida dubliniensis ref|XP_002416832. ref|XP_002422190.1 1

Candida glabrata ref|XP_447560.1 ref|XP_446321.1

2 Candida tropicalis gb|EER31682.1 gb|EER35932.1

Clavispora lusitaniae gb|EEQ39475.1 gb|EEQ38347.1

Debaryomyces hansenii ref|XP_461997.1 ref|XP_462565.1

Kluyveromyces lactis ref|XP_454091.1 ref|XP_451242.1

Lodderomyces elongisporus gb|EDK43263.1 gb|EDK45048.1

Pichia guilliermondii gb|EDK38689.2 gb|EDK39982.2

Pichia pastoris ref|XP_002493061. ref|XP_002491782.1 1

Pichia stipitis ref|XP_001387359. ref|XP_001382488.1 1

Saccharomyces cerevisiae ref|NP_014785.1 ref|NP_011760.1

Vanderwaltozyma polyspora gb|EDO15166.1 gb|EDO19215.1

Yarrowia lipolytica ref|XP_504333.2 ref|XP_502415.2

Taphrinomycotina

Schizosaccharomyces japonicus gb|EEB06483.1 gb|EEB08651.1

Schizosaccharomyces pombe ref|NP_594230.1 ref|NP_588466.1

Basidiomycota

Coprinopsis cinerea gb|EAU93204.1 gb|EAU93446.1

Filobasidiella neoformans ref|XP_571914.1 ref|XP_567043.1

Laccaria bicolor gb|EDR15553.1 gb|EDR15438.1

Malassezia globosa gb|EDP43341.1 gb|EDP42407.1

3 Moniliophthora perniciosa gb|EEB89615.1 gb|EEB90675.1

Postia placenta2 jgi|Pospl1|128778 jgi|Pospl1|120437

Ustilago maydis gb|EAK82784.1 gb|EAK81186.1

gb|EAK82289.1 gb|EAK82785.1

Neocallimastigomycota

Neocallmastix frontalis gb|AAP13544.1 sp|P53587.2

Neocallimastix patriciarum gb|AAP83350.1 gb|AAP83351.1

Metazoa - Outgroup

Homo sapiens gb|AAH00504.1 dbj|BAA91939.1

Mus musculus dbj|BAB22331.1 dbj|BAE29687.1

Canis familiaris ref|XP_532985.2 ref|XP_542566.2

1 Even though these two genes are annotated as independent genes, they are consecutive and in the same strand in the genome and group with the fusion genes in the phylogenetic trees, indicating that they might be fused. See text for a discussion.

2 Although both genes seem to be absent form, this genome in NCBI, further search in the organism’s genome webpage at JGI (http://genomeportal.jgi-psf.org/Pospl1/Pospl1.home.html) revealed that the genes are present. The accession numbers given in the table correspond to the

JGI accession numbers.