(A) Genomic locations of wtf genes in the reference genome and in the CBS5557 genome. The 25 wtf genes in the reference genome have been named according to their order in the genome (Bowen et al., 2003). Their locations are depicted as 20 blue vertical bars, including five thick bars denoting five tandem pairs. Our PacBio sequencing analysis revealed that in the CBS5557 genome, there are 32 wtf genes, whose locations are depicted as 23 vertical bars, including three thin red bars at locations where no wtf genes exist in the reference genome and four thick red bars at locations where compared to the reference genome one extra wtf gene is found. The wtf genes in the CBS5557 genome are named with the prefix cw and a number from the name of the syntenic gene in the reference genome. Two genes of a tandem pair corresponding to a singleton in the reference genome are distinguished using the suffixes a and b. Among the three genes of the triplet, two are named cw11 and cw12 based on their homology to wtf11 and wtf12, respectively, and the gene situated between cw11 and cw12 is named cw11x. Genes at new locations are named cw26, cw27, and cw28. Chromosome lengths are not drawn to scale. (B) Diagrams depicting the eight genomic locations with more than one wtf gene in at least one of the two genomes. Genes are shown as arrows. Introns are not shown. wtf17 is depicted not according to its annotation at PomBase, with its 5’ boundary revised based on sequence alignment. (C) Maximum likelihood phylogenetic tree of 57 wtf genes of the reference genome and the CBS5557 genome. DNA sequences including the conserved_up regions, predicted coding sequences, and associated introns were aligned using the L-INS-i iterative refinement algorithm of MAFFT (Katoh and Standley, 2014) (Figure 5—source data 1). Maximum likelihood analysis was performed using IQ-TREE (Nguyen et al., 2015). The tree was rooted by midpoint rooting (Hess and De Moraes Russo, 2007). Red dots on nodes indicate IQ-tree-calculated ultrafast bootstrap (UFBoot) support values < 95%. Colored rectangles highlight phylogenetic neighbors. Two genes are considered phylogenetic neighbors if they are separated by a single internal node with a support value >= 95%. Green rectangles indicate the 11 pairs of neighbors each composed of a reference wtf gene and a syntenic CBS5557 wtf gene. Yellow rectangles indicate the five pairs each composed of two wtf genes locating at different genomic positions. Magenta brackets denote the 12 genes that share a 150 bp sequence within the predicted intron 1 (see Figure 5—figure supplement 3). These 12 genes are divided into two subtypes, Intron-1-ATG genes and Exon-2-ATG genes, based on the locations of the first ATG codons downstream of the 150-bp-long sequence. The brown dashed box indicates genes with six exons. Scale bar, 0.1 nucleotide substitutions per nucleotide site.