(a) PHO84 ASlncRNA expression levels. From left to right: Cladogram of N. castellii and the genus Saccharomyces budding yeasts, and expression levels of PHO84 lncRNA for each species as determined by RT-qPCR. Expression level is relative to N. castellii, whose value was set to 1. The data is presented in logarithmic scale. (b) Expression levels of GAL10 ASlncRNA for each species as determined by RT-qPCR. As in (a), the expression level is relative to N. castellii, which was set to 1. For (a) and (b), mean and standard error of the mean were determined using RNA isolated from 2 different cultures, 3 technical replicates per culture. The data is presented in logarithmic scale. (c) Principal Component Analysis (PCA) of ASlncRNA transcriptomes in budding yeast. (d) Neighbor-joining tree based on pairwise distance matrices (Jensen-Shannon distance metric) for the genus Saccharomyces budding yeasts and N. castellii. Bootstrap value showing N. castellii as an outgroup (out of 100). Highlighted in blue are all yeast species of the genus Saccharomyces.

(a) Global ASlncRNA levels among budding yeast species. From left to right: Cladogram of N. castellii and the genus Saccharomyces budding yeast. N. cas, S. uva, S. kud, S. mik and S. cer denote N. castellii, S. uvarum, S. kudriazevii, S. mikatae, and S. cerevisiae, respectively. Boxplots of distributions of normalized read counts (log2 scale) mapping antisense 5031 orthologous open reading frames for budding yeasts. For all box plots, the midline represents the median value, the borders of the box represent the values at the 25^th (first quartile) and 75^th percentiles (3^rd quartile), and the whiskers represent the following:upper whisker = min(max(x), Q_3 + 1.5 * IQR), lower whisker = max(min(x), Q_1 − 1.5 * IQR), where IQR = 3^rd quartile value – 1^st quartile value . The notches surrounding the median value represent the 95% confidence interval estimation for the medians. Data for all RNA-sequencing experiments was collected from RNA extracted from two different isogenic cultures. (b) Global sense RNA levels among budding yeast species. As in (a), except reads mapping in the sense orientation. (c) Heatmap representation of pair-wise Wilcoxon-rank-sum tests for ASlncRNA transcriptomes. (d) Heatmap representation of pair-wise Wilcoxon-rank-sum tests for mRNA transcriptomes. (e) Antisense read density at convergent genes in Saccharomyces species as compared to N. castellii. Ribbon Plots of antisense read density in log2-scale at genes arranged in convergent orientation for (top to bottom) S. uvarum (n = 3172 genes), S. kudriavzevii (n= 3208 genes), S. mikatae (n= 3438 genes), S. cerevisiae (n = 3656 genes). N. castellii (n = 3064 genes) is represented in all the plots by the blue ribbon. The lines represent the antisense RNA-seq signal, while the outer borders of the ribbon represent 1 standard-error of the mean away from the mean. (f) Antisense read density at tandem genes in Saccharomyces species as compared to N. castellii. Ribbon Plots of antisense read density in log2-scale at genes arranged in convergent orientation for (top to bottom) S. cerevisiae (n= 3046 genes), S. mikatae (n= 3366 genes), S. kudriavzevii (n= 3141 genes), S.uvarum (n=3146 genes), N. castellii (n= 2846 genes).

(a) Kernal density estimates of the length distributions of ASlncRNAs in the indicated species of budding yeast. The number of identified ASlncRNAs is shown in parentheses (see Methods). (b) Boxplot representation of the distribution of the amount of overlap in base-pairs between ASlncRNA-mRNA pairs in S. cerevisiae (n = 2543), S. mikatae (n = 810), S. kudriavzevii (n = 525), S. uvarum (n = 431), N.castellii (n = 177). P-value (p ≪ 2.2e⁻¹⁶) was determined using a two-sided Wilcoxon rank-sum test. See legend for description of boxplot features.

(a) The effects of the exosome on global ASlncRNA levels in S. cerevisiae and N. castellii. Boxplots of distribution of normalized read counts at CUT-ASlncRNAs (ASlncRNAs that increase levels in rrp6 mutant) in control and rrp6 strains of S. cerevisiae (n = 2420), p ≪ 2.2e⁻¹⁶ determined using a two-sided Wilcoxon rank-sum test (S. cerevisiae WT vs rrp6). and N. castellii (n = 2481), p ≪ 2.2e⁻¹⁶ determined using a two-sided Wilcoxon rank-sum test (N. castellii dcr1 vs dcr1 rrp6). (b) The effects of RNAi on global ASlncRNA levels at tandem genes in S. cerevisiae and N. castellii. Boxplots of the distribution of normalized read counts of ASlncRNAs at tandem oriented genes for wild type and RNAi+ S. cerevisiae (Top, n = 3656 genes) or wild type and dcr1 N. castellii (Bottom, n = 3064 genes). (c) The effects of RNAi on global ASlncRNA levels at convergent genes in S. cerevisiae and N. castellii. As in (b), except at convergent oriented genes, S. cerevisiae (n = 3046 genes), N. castellii (n = 2846 genes). See legend for description of boxplot features. (d) Growth defects of N. castellii rrp6 mutant are partially recued by dcr1 mutation. A spot test of 5-fold serial dilutions of N. castellii dcr1, wild type, rrp6, dcr1 rrp6 strains on YEPD at an elevated temperature (N. castellii grows optimally at 25°C.)