Comparative analysis with other species
Using the annotated genomes, we constructed orthologous gene clusters ofB. l. tsinlingensis, with other salmonid fishes and other outgroup fishes. There were 31,296 gene families identified in these 11 species, and B. l. tsinlingensis contained 19,145 gene families. Because salmonid fishes have almost twice the number of genes compared with other species, the number of gene families is also twice that of other species (Figure 4A ). In these 31,296 gene families, 11,343 gene families were salmonid-specific. Salmonids shared 16,840 (53.81%) gene families with Esox lucius , 14,783 (47.24%) withDanio rerio , and 13,491 (43.11%) with Lepisosteus oculatus (Figure 4B ). A total of 1,216 1:1 single-copy orthologous genes were identified and used to construct phylogenetic trees, including a 4DTv tree, CDS tree, and protein tree. All of these trees recovered the same phylogenetic topology with high confidence (Figure S11-S13 ). B. l. tsinlingensis and 8 other salmonid fishes formed a Salmonidae cluster, and B. l. tsinlingensis was the most ancient salmonid lineage. The divergence time between E. lucius and salmonid fishes was 132.9 Mya, and the divergence time between B. l. tsinlingensis and other salmonid fishes was 41.4 Mya (Figure 4C ). Because of the Ss4R event in salmonids, we assessed the expanded gene families in salmonid lines andB. l. tsinlingensis , respectively. GO and KEGG enrichment analysis of salmonid expanded gene families indicated that most genes were related to protein binding, kinase activity, transferase activity, Toll signaling pathway, receptor activity, Glycolysis / Gluconeogenesis, citrate cycle, Pentose phosphate pathway, and Fatty acid biosynthesis, which indicates that metabolic processes in salmonids may differ from those in other fishes after the Ss4R event; the larger body size of salmonids might reflect differences in metabolic processes (Table S15-S16 ). The GO and KEGG enrichment analysis of B. l. tsinlingensis expanded gene families revealed that most genes were related to the regulation of apoptotic process, cell death, insulin receptor binding, lysozyme activity, biological regulation, ascorbate and aldarate metabolism, lipopolysaccharide biosynthesis, and mismatch repair, which indicates that the immune system of B. l. tsinlingensis might differ from that of other fishes (Table S17-S18 ). These unique expanded gene families can help us better understand the biology B. l. tsinlingensis and aid its conservation.