Rhinolophus affinis Raw sequence reads

Gene expression changes contribute greatly to phenotypic variations in nature. Studying patterns of regulators of gene expression is important to fully understand the molecular mechanism underlying phenotypic variations. To explore relative roles of different regulators of gene expression (differential gene expression, alternative splicing (AS) and non-coding RNAs (e.g. lncRNAs)) in phenotypic variation, we used two recently diverged subspecies of the intermediate horseshoe bat (Rhinolophus affinis hainanus and R. a. himalayanus) with great phenotypic variations as the system. The latter subspecies emits much higher ultrasonic frequencies than the former. With the combination of Illumina short-read and Nanopore long-read RNA-seq data, we identified much more differentially expressed genes (DEGs) at the transcript level than at the gene level. Compared to R. a. hainanus, R. a. himalayanus exhibited much more upregulated DEGs and multiple of them may play important roles in the maintenance and damage repair of auditory hair cells. We also identified over 400 differentially expressed lncRNAs and their target DEGs upregulated in R. a. himalayanus were also mainly involved in a protective mechanism for auditory hair cells, suggesting an important role of lncRNAs in acoustic divergence between the two subspecies. Using three different methods, we also performed AS analyses and identified several candidate alternatively spliced genes (ASGs) that expressed different isoforms which may be associated with phenotypic variations of the two subspecies. We observed significantly less overlap than expected between DEGs and ASGs, supporting complementary roles of differential gene expression and AS in generating phenotypic variations.