Codon Usage Bias and Phylogenetic Analysis of the Mitochondrial Genomes in Two Enicurus Species

Background: Codon usage bias (CUB), which is shaped by mutation pressure, natural selection, and genetic drift, provides valuable insights into phylogenetic relationships and molecular evolution. This study investigated the patterns and determinants of mitochondrial genome codon usage in two Enicurus species (Enicurus scouleri and Enicurus schistaceus) and provided a foundation for understanding codon optimisation mechanisms and genetic relationships within this avian genus. Methods: Complete mitochondrial genome sequences were retrieved from GenBank, and ten protein-coding sequences were selected for CUB analysis. Evolutionary relationships across the studied species were investigated using phylogenetic trees and relative synonymous codon usage (RSCU) clustering diagrams. Results: GC1, GC2, and GC3 contents were below 50% in both species, with the third-position nucleotides exhibiting A3s > C3s > T3s > G3s composition. The average effective number of codons (ENC) value was >35, indicating a weak bias for codon usage. CUB reflects the combined effects of natural selection and mutational pressure, with the former exerting a stronger influence. Four shared optimal codons were identified with a strong bias towards A/C-ending triplets. Subsequent phylogenetic analysis validated the close kinship of the two Enicurus species, although RSCU-based clustering yielded results that diverged from the phylogenetic relationships. Conclusions: Comprehensive mechanistic analysis revealed natural selection as the dominant force shaping mitochondrial CUB in Enicurus species. The findings offered valuable insights for future research on the reproductive biology, environmental adaptation, and conservation of Enicurus birds while providing new perspectives on the molecular evolution and systematic development of Muscicapidae.