Phylogenetic Study of Tad3 Evolution

Transcription

1 Phylogenetic Study of Tad3 Evolution Yizhu Lin Introduction trna-specific adenosine-34 deaminases (ADAT) are an essential enzymes found in Bacteria and Eukarya that catalyze the conversion of adenine (A) to inosine (I) at trna s wobble position 34. I 34 is able to wobble with adenine, cytosine, and uridine, enlarging codon recognition capacity during protein synthesis. In Eukarya, a heterodimeric form of ADAT is formed by Tad2p and Tad3p. The two subunits are sequence-related, both of which contain cytidine deaminase (CDA) motifs. [1] Interestingly, the two are separately encoded, yet function and sequence related. Previous studies presented many cases in which the genes encoding such kind of heterodimer are coevolving, exerting positive or directional selection to each other, like integratin α and βchain genes in vertebrate. [2] Therefore, here in this project, we expect the functional intimidation of Tad3p and Tad2p indicates a co-evolutionary relationship of their encoding genes. Notably, TAD3 gene is one of the 10 genes that have multiple introns in S. cerevisiae [3]. It contains a non-canonical branchpoint sequence 5 -AACTAAC-3 instead of 5 - TACTAAC-3, indicating an alternative-splicing-related regulation of TAD transcription. Inspired by the unique 2 intron feature, the evolution of TAD3 introns will be studied in this project. The TAD3 intron phylogenetic tree is expected to correlating with genome duplication and may point the origin of intron. Method 1. Sequence acquirement Saccharomyces cerevisiae Tad3p and Tad2p s amino acid sequence were obtained from Saccharomyces Genome Database (SGD) [4]. By Blastp Saccharomyces cerevisiae s Tad3p and Tad2p s amino acid sequence with fungi taxa in NCBI, we were able to obtain Tad3p and Tad2p s amino acid sequences of other fungi species. To enlarge taxa coverage, we obtained some sequences by searching NCBI with keyword TAD3 or TAD2 under protein catalog. Since we aim to compare Tad3p s phylogenetic tree with Tad2p s, only fungi species that have available sequences of both Tad3p and Tad2p were included in our study. However, we didn t find Tad3p or Tad2p s sequences of Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces paradoxus and Saccharomyces bayanus by the two methods above. But TAD3 and TAD2 DNA sequences of these four species can be found by blastn with Saccharomyces cerevisiae s DNA sequences in SGD, and their sequences show high similarity with Saccharomyces cerevisiae s DNA sequences. So we aligned DNA sequences of these 5 species, cut off TAD3 s intron sequences according to Saccharomyces cerevisiae s verified intron region, then translate TAD3 and TAD2 gene into protein sequences (using ApE). These DNA sequence are also used in TAD3 s intron analysis. In total, we selected 36 protein sequences of both Tad3p and Tad2p for further analysis, including 33 fungi sequences and 3 animal species as outgroup. Five Saccharomyces species were used in intron sequences analysis. (See Appendence for sequences used in this project) 2. Multiple protein sequences alignment Multiple protein sequences alignment of Tad3p and Tad2p were conducted using Jalview. We aligned the sequences using each of the four major algorithms (Mafft, Muscle, Tcoffee and

2 Probcons) with default parameters. After alignment, columns with more than 50% gaps were trimmed for further study. From alignment we can see that Tad3p has one highly conserved region ( S. cerevisiae Y240~L272) and several other shorter or less conserved region. The C-terminal of Tad3p is much less conserved. All the four algorithms successfully aligned the highly conserved region consistently, except Mafft introduced 2 big gaps in Cryptococcus neoformans. Tcoffee seems to introduce many small gaps at the C-terminal. Mafft, Muscle and Probcons also show highly consistency at other conserved sites such as S. cerevisiae W140, P141, C88. Tad2p seems to be more conserve than Tad3p. The N-terminal of Tad2p is relatively more conserved ( S. cerevisiae M7~P167), and the four algorithms aligned this conserved region in a similar way. The C-terminal of Tad2p is much less conserved, and several species lost these C- terminal sequences completely. The alignments of Tad2p s C-terminal region from four algorithms are very different from each other. However, following tree building results show that trimming of the C-terminal unconserved region will not change the topology of phylogenetic tree. Such, we consider alignment of Tad2p from these four algorithms are equally reliable. 3. Model selection and tree building Based on multiple alignment results, all the 4 alignments of Tad3p and Mafft alignment of Tad2p (with and without C-terminal unconserved region) were used for model selection and tree building. Model selections were conducted by TOPALi to obtain parameters for Maximum Likelihood tree building. Models that had minimal AIC or BIC were used for PhyML tree building in Seaview. We also constructed Neighbor Join tree using Seaview. Results of TOPALi model selections show that in all the 6 alignments, WAG+I+G model is always the one has minimal AIC and BIC. The parameters for PhyML tree building are summarized in the following table. Protein Alignment pinv alpha Mafft(with C-terminal) Tad2p Mafft(without C-terminal) Mafft Muscle Tad3p Tcoffee Probcons Species tree Species tree is from NCBI Taxomony common tree. 5. TAD3 intron analysis TAD3 gene sequences of five Saccharomyces species were aligned using Clustal in Jalview. The five sequences are highly similar to each other. Based on verified intron sequence of S. cerevisiae,we were able to find introns of other 4 sequences. Since the 5 splice sites, 3 splice sites and branchpoint sequences are all well aligned, we think the introns obtained by this method is reliable. Each of the two introns was cut off for tree building in Seaview.

3 Results 1. Comparison of trees derived from Maximum Likelihood Method and Neighbor Joining Method. Typically, PhyML tree and NJ tree generated from same alignment have same topology. However, NJ tree fails to distinguish different branch length, while PhyML tree does better in branch length determination. For example, in the NJ tree generated from Tad3p s Mafft alignment, the Cryptococcus neoformans var. neoformans branch is very close to animal group, while PhyML is able to separate Cryptococcus neoformans var. neoformans from the animal group. Given this result, our further analysis will based on only PhyML tree but not NJ tree. 2. Comparison of Tad2p tree with and without C-terminal unconserved sequences Compare the Tad2p Mafft - PhyML tree with C-terminal unconserved sequences and without C-terminal unconserved sequences, we can see that both the topology and branch length of the two trees are almost the same. Since the alignments generated by four different algorithms are very similar in N-terminal conserved region, we assume that different alignment will not affect tree building results of Tad2p. We will use Tad2p s PhyML tree based on Mafft alignment for further analysis. 3. Comparison of Tad3p trees based on different alignment methods. From results we can see that topology of these four trees is similar but still has some disagreement. Probcons and Muscle put Saccaromycetales group and leotiomyceta group together, while Schizosaccharomyces derived from Ascomycota earlier. This is consistent with species tree obtained from NCBI Taxonomy database. However, Mafft and Tcoffee put leotiomyceta group and Schizosaccharomyces group together. All of the four alignments generated a tree with a branch that has a low bootstrap when trying to distinguish these three groups. Within Saccharomyces group, only Mafft put S.mikatae and S. paradoxus together, while others put S. paradoxus closer to S. cerevisiae. Compare Probcons with Muscle, Muscle shows bigger bootstrap values on more branches than Probcons. Based on analysis above, we conclude that Muscle alignment is a more suitable alignment for Tad3p. 4. Comparison of Tad3p tree, Tad2p tree and species tree. The species tree from NCBI is not a binary tree and it cannot distinguish the speciation order of most species in Saccharomycetaceae group except Saccharomyces. Tad3p tree and Tad2p tree differ a lot from each other in these species. Besides, in Tad2p tree, Yarrowia lipolytica is far away from other Saccharomycetales species. This disagrees with both species tree and Tad3p tree. Both Tad3p tree and Tad2p tree put Candida group within Debaryomycetaceae. Interestingly, both Candida and Debaryomycetaceae belong to CTG clade, which translate CTG as serine instead of leucine [5, 6]. The topology of our trees is consistent with this trna-related trait. However, we cannot see obvious evidence for Tad3p and Tad2p s co-evolution. 5. TAD3 intron analysis. Since we only have 5 species for alignment and intron sequences is relatively short, the trees generated from intron are not so robust. Trees generated by PhyML and NJ method are almost the same. Trees based on both first intron and second intron show that S. mikatae has longer

4 distance to others. And S. mikatae have two substitutes in first intron s BPS (CACTAAC instead of AACTAAC) and 3 SS (TAG instead of CAG). Other TAD3 gene, such as Candida glabrata, Candida albicans etc. that closely related to Saccharomyces don t have introns, indicating these two introns originated with the speciation of Saccharomyces. Discussion Through this study, we generated gene trees of both Tad3p and Tad2p. Generally, Tad3p tree and Tad2p tree are consistent with species tree, and are consistent with CTG clade. However, we cannot infer Tad3p and Tad2p co-evolution from our study, since there are no significant unique topology similarity between Tad3p tree and Tad2p tree. As for TAD3 intron analysis, only five species and relatively short sequence length seems to be not enough for tree analysis. But we can still see that introns are much less conserved than exons, and these TAD3 introns may originate independently in Saccharomyces.

5 References 1. Novoa, E.M., et al., A role for trna modifications in genome structure and codon usage. Cell, (1): p Hughes, A.L., Coevolution of the vertebrate integrin alpha- and beta-chain genes. Mol Biol Evol, (2): p Hossain, M.A., C.M. Rodriguez, and T.L. Johnson, Key features of the two-intron Saccharomyces cerevisiae gene SUS1 contribute to its alternative splicing. Nucleic Acids Res, (19): p Cherry, J.M., et al., Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res, (Database issue): p. D Mallet, S., et al., Insights into the life cycle of yeasts from the CTG clade revealed by the analysis of the Millerozyma (Pichia) farinosa species complex. PLoS One, (5): p. e Wang, H., et al., A fungal phylogeny based on 82 complete genomes using the composition vector method. BMC Evol Biol, : p Appendence 1. List of species studied Ashbya_gossypii Aspergillus_clavatus Aspergillus_flavus Candida_albicans Candida_glabrata Candida_dubliniensis Candida_orthopsilosis Candida_parapsilosis Cryptococcus_neoformans_var._neoformans Debaryomyces_hansenii Homo_sapiens (outgroup) Kazachstania_africana Kluyveromyces_lactis Lachancea_thermotolerans Leptosphaeria_maculans Millerozyma_farinosa Nasonia_vitripennis (outgroup) Naumovozyma_dairenensis Pan_troglodytes (outgroup) Penicillium_marneffei Pyrenophora_tritici-repentis Saccharomyces_cerevisiae Saccharomyces_paradoxus Saccharomyces_mikatae Saccharomyces_kudriavzevii Saccharomyces_bayanus

6 Scheffersomyces_stipitis Schizosaccharomyces_pombe Schizosaccharomyces_japonicus Talaromyces_stipitatus Tetrapisispora_blattae Tetrapisispora_phaffii Torulaspora_delbrueckii Vanderwaltozyma_polyspora Yarrowia_lipolytica Zygosaccharomyces_rouxii 2. Sequences for alignment Tad3p sequences: see Tad3p_modified.fa Tad2p sequences: see Tad2p_modified.fa TAD3 gene sequences: see Tad3gene.fa 3. Multiple Sequences alignment See fasta files for trimmed and untrimmed alignments. 4. Species Tree from NCBI taxonomy

7 Seaview TAD3fortreeML.nwk Wed Dec 12 16:27: TAD3 Mafft PhyML tree TAD3_Cryptococcusneoformansvar.neoformans TAD3_Pantroglodytes TAD3_Homosapiens TAD3_Nasoniavitripennis TAD3_Schizosaccharomycesjaponicus TAD3_Schizosaccharomycespombe 0.27 TAD3_Saccharomyceskudriavzevii TAD3_Saccharomycesbayanus 0.52 TAD3_Saccharomycesmikatae TAD3_Saccharomycesparadoxus 0.82 TAD3_Saccharomycescerevisiae TAD3_Naumovozymadairenensis TAD3_Vanderwaltozymapolyspora TAD3_Yarrowialipolytica TAD3_Pyrenophoratritici-repentis TAD3_Leptosphaeriaaculans TAD3_Candidaglabrata TAD3_Tetrapisisporablattae TAD3_Kazachstaniaafricana TAD3_Ashbyagossypii TAD3_Kluyveromyceslactis TAD3_Zygosaccharomycesrouxii TAD3_Torulasporadelbrueckii TAD3_Lachanceathermotolerans TAD3_Tetrapisisporaphaffii TAD3_Candidaparapsilosis TAD3_Candidaorthopsilosis TAD3_Candidadubliniensis TAD3_Candidaalbicans TAD3_Aspergillusclavatus TAD3_Scheffersomycesstipitis TAD3_Millerozymafarinosa TAD3_Debaryomyceshansenii 0.5 TAD3_Aspergillusflavus TAD3_Talaromycesstipitatus TAD3_Penicilliummarneffei

8 1 TAD3 Mafft PhyML tree TAD3_Cryptococcusneoformansvar.neoformans TAD3_Candidaglabrata TAD3_Tetrapisisporablattae TAD3_Saccharomycesmikatae TAD3_Saccharomycescerevisiae TAD3_Naumovozymadairenensis TAD3_Saccharomycesbayanus TAD3_Saccharomycesparadoxus TAD3_Saccharomyceskudriavzevii TAD3_Nasoniavitripennis TAD3_Homosapiens TAD3_Pantroglodytes TAD3_Pyrenophoratritici-repentis TAD3_Leptosphaeriaaculans TAD3_Talaromycesstipitatus TAD3_Penicilliummarneffei TAD3_Aspergillusclavatus TAD3_Aspergillusflavus TAD3_Schizosaccharomycespombe TAD3_Schizosaccharomycesjaponicus TAD3_Kazachstaniaafricana TAD3_Ashbyagossypii TAD3_Kluyveromyceslactis TAD3_Lachanceathermotolerans TAD3_Zygosaccharomycesrouxii TAD3_Torulasporadelbrueckii TAD3_Vanderwaltozymapolyspora TAD3_Tetrapisisporaphaffii TAD3_Candidaparapsilosis TAD3_Yarrowialipolytica TAD3_Debaryomyceshansenii TAD3_Millerozymafarinosa TAD3_Scheffersomycesstipitis TAD3_Candidaalbicans TAD3_Candidadubliniensis TAD3_Candidaorthopsilosis

9 Seaview TAD3fortreeNJ.nwk Wed Dec 12 16:42: TAD3_Nasoniavitripennis TAD3_Pantroglodytes TAD3_Homosapiens TAD3_Cryptococcusneoformansvar.neoformans TAD3_Schizosaccharomycesjaponicus TAD3_Schizosaccharomycespombe TAD3_Talaromycesstipitatus TAD3_Penicilliummarneffei TAD3_Aspergillusclavatus TAD3_Aspergillusflavus TAD3_Pyrenophoratritici-repentis TAD3_Leptosphaeriaaculans TAD3_Tetrapisisporaphaffii TAD3_Naumovozymadairenensis TAD3_Ashbyagossypii TAD3_Tetrapisisporablattae TAD3_Vanderwaltozymapolyspora TAD3_Torulasporadelbrueckii TAD3_Zygosaccharomycesrouxii TAD3_Lachanceathermotolerans TAD3_Kazachstaniaafricana TAD3_Kluyveromyceslactis TAD3_Candidaglabrata TAD3_Saccharomycesparadoxus TAD3_Saccharomycescerevisiae TAD3_Saccharomycesmikatae TAD3_Saccharomyceskudriavzevii TAD3_Saccharomycesbayanus TAD3_Millerozymafarinosa TAD3_Debaryomyceshansenii TAD3_Candidaparapsilosis TAD3_Candidaorthopsilosis TAD3_Candidadubliniensis TAD3_Candidaalbicans TAD3_Scheffersomycesstipitis TAD3_Yarrowialipolytica TAD Mafft NJ tree

10 0.2 TAD3 Mafft NJ tree TAD3_Candidaglabrata TAD3_Kluyveromyceslactis TAD3_Kazachstaniaafricana TAD3_Lachanceathermotolerans TAD3_Zygosaccharomycesrouxii TAD3_Torulasporadelbrueckii TAD3_Tetrapisisporaphaffii TAD3_Ashbyagossypii TAD3_Vanderwaltozymapolyspora TAD3_Saccharomycescerevisiae TAD3_Saccharomycesparadoxus TAD3_Tetrapisisporablattae TAD3_Naumovozymadairenensis TAD3_Saccharomycesmikatae TAD3_Homosapiens TAD3_Saccharomyceskudriavzevii TAD3_Saccharomycesbayanus TAD3_Pantroglodytes TAD3_Nasoniavitripennis TAD3_Millerozymafarinosa TAD3_Debaryomyceshansenii TAD3_Talaromycesstipitatus TAD3_Penicilliummarneffei TAD3_Aspergillusclavatus TAD3_Aspergillusflavus TAD3_Pyrenophoratritici-repentis TAD3_Leptosphaeriaaculans TAD3_Schizosaccharomycesjaponicus TAD3_Schizosaccharomycespombe TAD3_Cryptococcusneoformansvar.neoformans TAD3_Candidaparapsilosis TAD3_Candidaorthopsilosis TAD3_Candidadubliniensis TAD3_Candidaalbicans TAD3_Scheffersomycesstipitis TAD3_Yarrowialipolytica

11 Seaview TAD3_Muscle_trimmed.nwk Wed Dec 12 16:56: TAD3 Muscle PhyML tree TAD3_Leptosphaeria_aculans/1-315 TAD3_Pyrenophora_tritici-repentis/ TAD3_Debaryomyces_hansenii/ TAD3_Naumovozyma_dairenensis/1-321 TAD3_Vanderwaltozyma_polyspora/1-318 TAD3_Schizosaccharomyces_pombe/4-306 TAD3_Schizosaccharomyces_japonicus/7-300 TAD3_Cryptococcus_neoformans_var._neoformans/ TAD3_Homo_sapiens/ TAD3_Pan_troglodytes/1-305 TAD3_Aspergillus_flavus/1-185 TAD3_Aspergillus_clavatus/2-321 TAD3_Yarrowia_lipolytica/1-314 TAD3_Scheffersomyces_stipitis/3-309 TAD3_Candida_albicans/1-314 TAD3_Candida_dubliniensis/1-314 TAD3_Candida_orthopsilosis/ TAD3_Candida_parapsilosis/1-304 TAD3_Millerozyma_farinosa/ TAD3_Saccharomyces_bayanus/1-322 TAD3_Saccharomyces_kudriavzevii/1-321 TAD3_Saccharomyces_mikatae/ TAD3_Saccharomyces_cerevisiae/ TAD3_Saccharomyces_paradoxus/1-321 TAD3_Candida_glabrata/1-317 TAD3_Tetrapisispora_phaffii/1-322 TAD3_Ashbya_gossypii/1-317 TAD3_Kluyveromyces/1-316 TAD3_Lachancea_thermotolerans/1-318 TAD3_Torulaspora/1-318 TAD3_Zygosaccharomyces/1-324 TAD3_Kazachstania_africana/1-321 TAD3_Tetrapisispora_blattae/1-325 TAD3_Nasonia_vitripennis/ TAD3_Penicillium_marneffei/3-324 TAD3_Talaromyces_stipitatus/

12 TAD3 Muscle PhyML tree 1 TAD3_Scheffersomyces_stipitis/3-309 TAD3_Candida_albicans/1-314 TAD3_Candida_dubliniensis/1-314 TAD3_Candida_orthopsilosis/ TAD3_Cryptococcus_neoformans_var._neoformans/ TAD3_Schizosaccharomyces_pombe/4-306 TAD3_Schizosaccharomyces_japonicus/7-300 TAD3_Yarrowia_lipolytica/1-314 TAD3_Nasonia_vitripennis/6-297 TAD3_Pan_troglodytes/1-305 TAD3_Talaromyces_stipitatus/ TAD3_Homo_sapiens/ TAD3_Penicillium_marneffei/3-324 TAD3_Aspergillus_clavatus/2-321 TAD3_Aspergillus_flavus/1-185 TAD3_Pyrenophora_tritici-repentis/ TAD3_Leptosphaeria_aculans/1-315 TAD3_Candida_parapsilosis/1-304 TAD3_Debaryomyces_hansenii/ TAD3_Saccharomyces_bayanus/1-322 TAD3_Millerozyma_farinosa/ TAD3_Saccharomyces_kudriavzevii/1-321 TAD3_Saccharomyces_mikatae/1-317 TAD3_Saccharomyces_cerevisiae/1-319 TAD3_Saccharomyces_paradoxus/1-321 TAD3_Candida_glabrata/1-317 TAD3_Naumovozyma_dairenensis/1-321 TAD3_Tetrapisispora_blattae/1-325 TAD3_Kazachstania_africana/1-321 TAD3_Zygosaccharomyces/1-324 TAD3_Torulaspora/1-318 TAD3_Lachancea_thermotolerans/1-318 TAD3_Kluyveromyces/1-316 TAD3_Ashbya_gossypii/1-317 TAD3_Vanderwaltozyma_polyspora/1-318 TAD3_Tetrapisispora_phaffii/1-322

13 Seaview TAD3_Probcons_trimmedML.nwk Wed Dec 12 16:59: TAD3 Probcons PhyML tree TAD3_Leptosphaeria_aculans/1-315 TAD3_Ashbya_gossypii/1-323 TAD3_Kluyveromyces/1-322 TAD3_Torulaspora/1-321 TAD3_Zygosaccharomyces/1-329 TAD3_Kazachstania_africana/1-325 TAD3_Tetrapisispora_blattae/1-329 TAD3_Vanderwaltozyma_polyspora/1-326 TAD3_Tetrapisispora_phaffii/ TAD3_Saccharomyces_bayanus/1-324 TAD3_Saccharomyces_kudriavzevii/ TAD3_Saccharomyces_cerevisiae/1-321 TAD3_Saccharomyces_paradoxus/ TAD3_Saccharomyces_mikatae/1-322 TAD3_Naumovozyma_dairenensis/1-328 TAD3_Candida_glabrata/1-322 TAD3_Debaryomyces_hansenii/1-323 TAD3_Millerozyma_farinosa/1-321 TAD3_Candida_orthopsilosis/1-302 TAD3_Candida_parapsilosis/1-301 TAD3_Candida_albicans/1-312 TAD3_Candida_dubliniensis/1-312 TAD3_Scheffersomyces_stipitis/1-313 TAD3_Yarrowia_lipolytica/1-319 TAD3_Schizosaccharomyces_pombe/1-309 TAD3_Pyrenophora_tritici-repentis/1-327 TAD3_Lachancea_thermotolerans/1-322 TAD3_Schizosaccharomyces_japonicus/1-301 TAD3_Cryptococcus_neoformans_var._neoformans/1-319 TAD3_Homo_sapiens/1-306 TAD3_Pan_troglodytes/1-296 TAD3_Aspergillus_clavatus/1-321 TAD3_Nasonia_vitripennis/ TAD3_Aspergillus_flavus/1-185 TAD3_Talaromyces_stipitatus/1-322 TAD3_Penicillium_marneffei/1-322

14 1 TAD3 Probcons PhyML tree TAD3_Ashbya_gossypii/1-323 TAD3_Kluyveromyces/1-322 TAD3_Zygosaccharomyces/1-329 TAD3_Torulaspora/1-321 TAD3_Lachancea_thermotolerans/1-322 TAD3_Cryptococcus_neoformans_var._neoformans/1-319 TAD3_Schizosaccharomyces_pombe/1-309 TAD3_Schizosaccharomyces_japonicus/1-301 TAD3_Nasonia_vitripennis/1-283 TAD3_Pan_troglodytes/1-296 TAD3_Homo_sapiens/1-306 TAD3_Leptosphaeria_aculans/1-315 TAD3_Pyrenophora_tritici-repentis/1-327 TAD3_Talaromyces_stipitatus/1-322 TAD3_Penicillium_marneffei/1-322 TAD3_Aspergillus_clavatus/1-321 TAD3_Kazachstania_africana/1-325 TAD3_Tetrapisispora_blattae/1-329 TAD3_Vanderwaltozyma_polyspora/1-326 TAD3_Tetrapisispora_phaffii/1-323 TAD3_Saccharomyces_bayanus/1-324 TAD3_Saccharomyces_kudriavzevii/1-322 TAD3_Saccharomyces_paradoxus/1-322 TAD3_Saccharomyces_cerevisiae/1-321 TAD3_Naumovozyma_dairenensis/1-328 TAD3_Yarrowia_lipolytica/1-319 TAD3_Scheffersomyces_stipitis/1-313 TAD3_Candida_dubliniensis/1-312 TAD3_Candida_albicans/1-312 TAD3_Candida_parapsilosis/1-301 TAD3_Candida_orthopsilosis/1-302 TAD3_Debaryomyces_hansenii/1-323 TAD3_Candida_glabrata/1-322 TAD3_Saccharomyces_mikatae/1-322 TAD3_Millerozyma_farinosa/1-321 TAD3_Aspergillus_flavus/1-185

15 Seaview TAD3_Tcoffee_trimmedML.nwk Wed Dec 12 16:58: TAD3 Tcoffee PhyML tree TAD3_Schizosaccharomyces_pombe/1-305 TAD3_Schizosaccharomyces_japonicus/1-297 TAD3_Yarrowia_lipolytica/1-319 TAD3_Scheffersomyces_stipitis/1-307 TAD3_Candida_albicans/1-306 TAD3_Candida_dubliniensis/1-306 TAD3_Candida_parapsilosis/1-294 TAD3_Debaryomyces_hansenii/1-321 TAD3_Millerozyma_farinosa/ TAD3_Saccharomyces_bayanus/1-321 TAD3_Saccharomyces_kudriavzevii/1-320 TAD3_Saccharomyces_mikatae/ TAD3_Saccharomyces_cerevisiae/ TAD3_Saccharomyces_paradoxus/1-319 TAD3_Candida_glabrata/1-321 TAD3_Naumovozyma_dairenensis/1-324 TAD3_Kazachstania_africana/1-322 TAD3_Tetrapisispora_blattae/1-327 TAD3_Ashbya_gossypii/1-316 TAD3_Kluyveromyces/1-318 TAD3_Lachancea_thermotolerans/1-319 TAD3_Torulaspora/1-318 TAD3_Zygosaccharomyces/1-325 TAD3_Vanderwaltozyma_polyspora/1-322 TAD3_Tetrapisispora_phaffii/1-319 TAD3_Cryptococcus_neoformans_var._neoformans/1-318 TAD3_Homo_sapiens/1-302 TAD3_Pan_troglodytes/1-292 TAD3_Leptosphaeria_aculans/1-313 TAD3_Pyrenophora_tritici-repentis/1-326 TAD3_Aspergillus_clavatus/1-320 TAD3_Candida_orthopsilosis/1-296 TAD3_Nasonia_vitripennis/ TAD3_Aspergillus_flavus/1-204 TAD3_Penicillium_marneffei/1-322 TAD3_Talaromyces_stipitatus/1-326

16 1 TAD3 Tcoffee PhyML tree TAD3_Candida_orthopsilosis/1-296 TAD3_Candida_parapsilosis/1-294 TAD3_Scheffersomyces_stipitis/1-307 TAD3_Candida_albicans/1-306 TAD3_Candida_dubliniensis/1-306 TAD3_Debaryomyces_hansenii/1-321 TAD3_Cryptococcus_neoformans_var._neoformans/1-318 TAD3_Yarrowia_lipolytica/1-319 TAD3_Nasonia_vitripennis/1-295 TAD3_Pan_troglodytes/1-292 TAD3_Homo_sapiens/1-302 TAD3_Talaromyces_stipitatus/1-326 TAD3_Penicillium_marneffei/1-322 TAD3_Aspergillus_flavus/1-204 TAD3_Aspergillus_clavatus/1-320 TAD3_Pyrenophora_tritici-repentis/1-326 TAD3_Leptosphaeria_aculans/1-313 TAD3_Millerozyma_farinosa/1-318 TAD3_Saccharomyces_bayanus/1-321 TAD3_Schizosaccharomyces_japonicus/1-297 TAD3_Schizosaccharomyces_pombe/1-305 TAD3_Saccharomyces_kudriavzevii/1-320 TAD3_Saccharomyces_mikatae/1-319 TAD3_Saccharomyces_cerevisiae/1-318 TAD3_Candida_glabrata/1-321 TAD3_Saccharomyces_paradoxus/1-319 TAD3_Naumovozyma_dairenensis/1-324 TAD3_Tetrapisispora_blattae/1-327 TAD3_Kazachstania_africana/1-322 TAD3_Tetrapisispora_phaffii/1-319 TAD3_Vanderwaltozyma_polyspora/1-322 TAD3_Zygosaccharomyces/1-325 TAD3_Torulaspora/1-318 TAD3_Lachancea_thermotolerans/1-319 TAD3_Kluyveromyces/1-318 TAD3_Ashbya_gossypii/1-316

17 Seaview TAD2fortreeNJ.nwk Wed Dec 12 16:12: TAD2_Nasoniavitripennis TAD2_Homosapiens TAD2_Pantroglodytes TAD2_Cryptococcusneoformansvar.neoformans TAD2_Yarrowialipolytica TAD2_Penicilliummarneffei TAD2_Talaromycesstipitatus TAD2_Aspergillusclavatus TAD2_Aspergillusflavus TAD2_Pyrenophoratritici-repentis TAD2_Leptosphaeriamaculans TAD2_Schizosaccharomycespombe TAD2_Schizosaccharomycesjaponicus TAD2_Ashbyagossypii TAD2_Kluyveromyceslactis TAD2_Lachanceathermotolerans TAD2_Saccharomycescerevisiae TAD2_Saccharomycesparadoxus TAD2_Saccharomyceskudriavzevii TAD2_Saccharomycesbayanus TAD2_Saccharomycesmikatae TAD2_Zygosaccharomycesrouxii TAD2_Kazachstaniaafricana TAD2_Naumovozymadairenensis TAD2_Candidaglabrata TAD2_Vanderwaltozymapolyspora TAD2_Tetrapisisporaphaffii TAD2_Torulasporadelbrueckii TAD2_Tetrapisisporablattae TAD2_Candidaalbicans TAD2_Candidadubliniensis TAD2_Candidaorthopsilosis TAD2_Candidaparapsilosis TAD2_Scheffersomycesstipitis TAD2_Debaryomyceshansenii TAD2_Millerozymafarinosa TAD2 Mafft NJ tree

18 0.2 TAD2 Mafft NJ tree TAD2_Naumovozymadairenensis TAD2_Kazachstaniaafricana TAD2_Zygosaccharomycesrouxii TAD2_Saccharomycesmikatae TAD2_Saccharomycesbayanus TAD2_Saccharomyceskudriavzevii TAD2_Saccharomycesparadoxus TAD2_Candidaglabrata TAD2_Saccharomycescerevisiae TAD2_Lachanceathermotolerans TAD2_Ashbyagossypii TAD2_Kluyveromyceslactis TAD2_Vanderwaltozymapolyspora TAD2_Pantroglodytes TAD2_Homosapiens TAD2_Tetrapisisporaphaffii TAD2_Nasoniavitripennis TAD2_Torulasporadelbrueckii TAD2_Cryptococcusneoformansvar.neoformans TAD2_Yarrowialipolytica TAD2_Candidaalbicans TAD2_Tetrapisisporablattae TAD2_Schizosaccharomycespombe TAD2_Schizosaccharomycesjaponicus TAD2_Penicilliummarneffei TAD2_Talaromycesstipitatus TAD2_Aspergillusclavatus TAD2_Pyrenophoratritici-repentis TAD2_Leptosphaeriamaculans TAD2_Candidadubliniensis TAD2_Candidaorthopsilosis TAD2_Candidaparapsilosis TAD2_Scheffersomycesstipitis TAD2_Debaryomyceshansenii TAD2_Millerozymafarinosa TAD2_Aspergillusflavus

19 Seaview TAD2fortreeML.nwk Wed Dec 12 16:09: TAD2 Mafft PhyML tree TAD2_Yarrowialipolytica TAD2_Aspergillusclavatus TAD2_Aspergillusflavus TAD2_Penicilliummarneffei TAD2_Talaromycesstipitatus TAD2_Pyrenophoratritici-repentis TAD2_Leptosphaeriamaculans TAD2_Schizosaccharomycespombe TAD2_Schizosaccharomycesjaponicus TAD2_Cryptococcusneoformansvar.neoformans TAD2_Homosapiens TAD2_Pantroglodytes 0.01 TAD2_Saccharomycesbayanus TAD2_Saccharomyceskudriavzevii TAD2_Saccharomycescerevisiae 0.84 TAD2_Saccharomycesparadoxus 0.92 TAD2_Saccharomycesmikatae TAD2_Ashbyagossypii TAD2_Candidaorthopsilosis TAD2_Candidaparapsilosis TAD2_Candidaalbicans TAD2_Candidadubliniensis TAD2_Scheffersomycesstipitis TAD2_Debaryomyceshansenii TAD2_Millerozymafarinosa TAD2_Nasoniavitripennis TAD2_Lachanceathermotolerans TAD2_Kluyveromyceslactis TAD2_Zygosaccharomycesrouxii TAD2_Naumovozymadairenensis TAD2_Kazachstaniaafricana TAD2_Vanderwaltozymapolyspora TAD2_Candidaglabrata TAD2_Tetrapisisporaphaffii TAD2_Tetrapisisporablattae TAD2_Torulasporadelbrueckii 0.5

20 TAD2_Cryptococcusneoformansvar.neoformans 0.5 TAD2 Mafft PhyML tree TAD2_Zygosaccharomycesrouxii TAD2_Tetrapisisporablattae TAD2_Vanderwaltozymapolyspora TAD2_Tetrapisisporaphaffii TAD2_Candidaglabrata TAD2_Torulasporadelbrueckii TAD2_Naumovozymadairenensis TAD2_Kazachstaniaafricana TAD2_Saccharomycescerevisiae TAD2_Saccharomycesparadoxus TAD2_Saccharomycesmikatae TAD2_Saccharomyceskudriavzevii TAD2_Saccharomycesbayanus TAD2_Kluyveromyceslactis TAD2_Lachanceathermotolerans TAD2_Ashbyagossypii TAD2_Candidaorthopsilosis TAD2_Candidaparapsilosis TAD2_Candidaalbicans TAD2_Candidadubliniensis TAD2_Scheffersomycesstipitis TAD2_Debaryomyceshansenii TAD2_Millerozymafarinosa TAD2_Yarrowialipolytica TAD2_Leptosphaeriamaculans TAD2_Pyrenophoratritici-repentis TAD2_Talaromycesstipitatus TAD2_Penicilliummarneffei TAD2_Aspergillusclavatus TAD2_Aspergillusflavus TAD2_Schizosaccharomycesjaponicus TAD2_Schizosaccharomycespombe TAD2_Homosapiens TAD2_Pantroglodytes TAD2_Nasoniavitripennis

21 Seaview TAD2fortree_trimmoreML.nwk Wed Dec 12 16:23: TAD2 Mafft ML tree without C- terminal TAD2_Yarrowialipolytica TAD2_Cryptococcusneoformansvar.neoformans TAD2_Aspergillusflavus TAD2_Aspergillusclavatus 0.93 TAD2_Talaromycesstipitatus TAD2_Penicilliummarneffei TAD2_Leptosphaeriamaculans TAD2_Pyrenophoratritici-repentis TAD2_Schizosaccharomycesjaponicus TAD2_Schizosaccharomycespombe TAD2_Pantroglodytes TAD2_Homosapiens TAD2_Lachanceathermotolerans TAD2_Ashbyagossypii TAD2_Kluyveromyceslactis TAD2_Kazachstaniaafricana 0.79 TAD2_Saccharomycesparadoxus 0.69 TAD2_Saccharomycescerevisiae TAD2_Saccharomycesmikatae TAD2_Saccharomycesbayanus 0.49 TAD2_Saccharomyceskudriavzevii TAD2_Zygosaccharomycesrouxii TAD2_Candidaglabrata TAD2_Torulasporadelbrueckii TAD2_Candidaparapsilosis TAD2_Candidaorthopsilosis TAD2_Candidadubliniensis TAD2_Candidaalbicans TAD2_Scheffersomycesstipitis TAD2_Debaryomyceshansenii TAD2_Millerozymafarinosa TAD2_Nasoniavitripennis 0.2 TAD2_Naumovozymadairenensis TAD2_Tetrapisisporaphaffii TAD2_Vanderwaltozymapolyspora TAD2_Tetrapisisporablattae

22 TAD2_Cryptococcusneoformansvar.neoformans TAD2 Mafft PhyML tree without C- terminal TAD2_Tetrapisisporablattae TAD2_Tetrapisisporaphaffii TAD2_Vanderwaltozymapolyspora TAD2_Candidaglabrata TAD2_Torulasporadelbrueckii TAD2_Zygosaccharomycesrouxii TAD2_Saccharomyceskudriavzevii TAD2_Saccharomycesbayanus TAD2_Saccharomycesmikatae TAD2_Saccharomycesparadoxus TAD2_Saccharomycescerevisiae TAD2_Naumovozymadairenensis TAD2_Kazachstaniaafricana TAD2_Kluyveromyceslactis TAD2_Ashbyagossypii TAD2_Lachanceathermotolerans 0.2 TAD2_Candidaparapsilosis TAD2_Candidaorthopsilosis TAD2_Candidadubliniensis TAD2_Candidaalbicans TAD2_Scheffersomycesstipitis TAD2_Debaryomyceshansenii TAD2_Millerozymafarinosa TAD2_Yarrowialipolytica TAD2_Pyrenophoratritici-repentis TAD2_Leptosphaeriamaculans TAD2_Talaromycesstipitatus TAD2_Penicilliummarneffei TAD2_Aspergillusclavatus TAD2_Schizosaccharomycesjaponicus TAD2_Aspergillusflavus TAD2_Schizosaccharomycespombe TAD2_Pantroglodytes TAD2_Homosapiens TAD2_Nasoniavitripennis

23 Seaview firstintron.nwk Wed Dec 12 22:42: S.mikatae 0.02 S.cerevisiae S.paradoxus S.bayanus S.kudriavzevii

24 Seaview secondintron.nwk Wed Dec 12 22:43: S.mikatae 0.02 S.kudriavzevii S.bayanus S.paradoxus S.cerevisiae