To check assembly correctness, we used the University of California, Santa Cruz (UCSC) genome browser’s Blat to align the assembled P. parnellii transcripts against the high-quality genome assembly of the little brown bat (myoLuc2). The longest transcripts of Casq1, Atp2a1, and Myl1 and their paralogs align with >90% identity to the orthologous gene according to the Ensembl and CESAR annotation (44, 45), showing that they are correctly assembled. In contrast, we found Myh2 to be misassembled as its transcript aligns to various parts of different myosin heavy chain genes. This misassembly is caused by the very high sequence similarity between the coding exons of the different myosin heavy chain genes, which is likely due to purifying selection.

Therefore, we used the following rationale to manually assemble the different myosin heavy chain (Myh) genes. The different fast-twitch fiber Myh genes were already present before the divergence of the amniote ancestor approximately 310 million years ago. Thus, intronic sequences that typically evolve neutrally will be diverged between Myh2 and other paralogs. In contrast, speciation between the little brown bat and P. parnellii happened much more recently around 53 million years ago (http://timetree.org/). Consequently, the introns between the Myh2 ortholog of both species evolved neutrally for a much shorter amount of time and thus still align (fig. S7A). Therefore, the amount of aligning intronic sequence distinguishes a genomic locus encoding an orthologous Myh gene from a locus that encodes a paralogous Myh gene.

To quantify the amount of aligning intronic sequence, we built alignment chains, as previously described (46), between the little brown bat genome (myoLuc2, reference) and the P. parnellii genome. Then, we selected the alignment chains that best align the intronic regions to obtain P. parnellii loci encoding parts of an orthologous Myh gene (fig. S7B). By extracting the aligning exons from these loci, we manually assembled the Myh1/2/4/6 genes. Blat of the translated transcript sequences against the little brown bat genome shows clear alignments to the orthologous genes (Ensembl and CESAR annotation), confirming the correct assembly of these four Myh genes. These assembled transcripts were used to quantify the Myh expression below.

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