(A) More than half of all methylated positions in S. pistillata are located in annotated introns. (B) Introns have proportionally more methylated positions (11.3%) than exons (8.6%) or intergenic regions (3.3%) even when accounting for the different amounts of CpG dinucleotides in the respective regions. (C) Distribution of methylated positions across a standardized gene model with flanking 4-kb regions. Solid lines depict transcription start site (left) and transcription termination site (right). Exons and introns in the plot have normalized lengths that correspond to their respective mean lengths in S. pistillata (from left to right: 373, 1258, 363, 1114, 302; 253, 967, 312, 1037, and 669 bp). arb. units, arbitrary units.

(A) The expression of methylated genes is significantly higher than that of unmethylated genes (P < 10⁻³⁰⁰, Student’s t test). (B) Expression values for methylated genes are exponentially proportional to methylation level; however, the relationship of expression to methylation density is nonlinear. Methylation density at low levels is exponentially proportional to expression values, but it plateaus at ~40%. (C) Expression levels of the first six exons were calculated as natural logarithms of fold changes (FC) relative to the expression of the first exon. The difference in expression levels is likely driven by the reduction of cryptic transcription initiation in methylated genes. The difference is greater in highly methylated genes (median methylation level >80%). Asterisks represent P values from t tests of methylated (orange) or highly methylated genes (red) against unmethylated genes (peach), and colored accordingly. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) There is a linear relationship between the inverse of the coefficient of variation and the log₁₀-transformed mean expression values from all samples. The coefficient of variation, defined as the SD of measured expression values divided by their mean, is consistently lower in methylated genes than unmethylated genes.

(A) Most of the general growth– and stress response–related Gene Ontology (GO) terms are significantly enriched at pH 7.2. GTPase, guanosine triphosphatase. (B) A composite pathway diagram consisting of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways “ko04010”: “MAPK signaling pathway” and “ko04110”: “cell cycle” was produced in Cytoscape. At pH 7.2, more genes respond with a significant increase (red) than with a decrease (blue) in methylation; unshaded genes are methylated but not differentially regulated. (C) The methylation of genes that negatively regulate MAPK and JNK (red lines) increases, whereas the methylation of genes that positively regulate the same kinases (blue lines) decreases in a reciprocal manner (error bars denote ±1 SE).

(A) Mean methylation levels were significantly increased at pH 7.2. (B) Cell sizes were significantly larger in nubbins grown in the pH 7.2 tank. (C) Larger cell sizes were associated with skeletal structure that contained larger calyxes. (D) The skeletal porosity was significantly higher at lower pH. (E) Representative longitudinal sections of S. pistillata skeletons under pHs of 7.2 and 8.0. Error bars represent 1 SE. Asterisks denote significance of t test P values. **P < 0.01; ***P < 0.001.