Heat-evolved strains of the coral symbiont Cladocopium proliferum have diverged in exon usage

Experimental evolution at 31{degrees}C produced strains of the coral symbiont Cladocopium proliferum (Symbiodiniaceae) that show increased thermal tolerance in culture, and some of them also raise the bleaching tolerance of coral. Here we investigated how exon usage and alternative splicing have diverged in the heat-evolved strains (SS3, SS5, SS8) from their wild-type cultures (WT10) by reanalysing published transcriptomes of the strains in symbiosis with Acropora kenti larvae at 27{degrees}C using limma, DEXSeq and rMATS. Exon usage separated the strains from an unselected wild-type strain, with 1,308 genes with differential exon usage for SS8, 4,931 for SS5 and 10,464 for SS3, and 59% to 72% of these genes showed no differential expression. SS8, the only strain that also raises the bleaching tolerance of coral larvae, was the closest to the wild type, while a shared set of 757 genes changed in all three heat-evolved strains together. Junction level splicing events (rMATS) did not follow this order (721, 687 and 847 events against WT10), and skipped exons were the most common type, followed by alternative 5' splice sites. Between SS8 and the non-protective strains, alternative 5' splice sites were the most common type (91 of 291 events against SS5 and 68 of 207 against SS3), and retained introns were nearly as common as skipped exons. No GO term was enriched in the shared set or the SS8 lists, but both held genes with a possible link to heat tolerance, among them a mitochondrial glutathione peroxidase in the shared set and a glutathione S-transferase in which SS8 differed from the non-protective strains. Future studies of heat-evolved symbionts should therefore include exon level and splicing analyses to resolve the fine scale differences between strains that gene level analyses miss.