Abstract
Amphalius spirataenius belongs to Arthropoda, Insecta, Siphonaptera, Ceratophylloidea, Ceratophyllinae, Amphalius. Only 2 species from the subfamily Ceratophyllinae have been sequenced for mitogenomes to date. The genus Amphalius mitogenome research was still blank. The A. spirataenius mitogenome was determined, annotated and analysed for the first time in this study. The 14 825 bp long genome has the typical metazoan of 37 genes with insect ancestral genome arrangement pattern. There was no significant difference in codon usage of 13 protein-coding genes: UUA, UCU, GUU, ACU and GCU were the most frequently used codons. It was found that the reason for codon preference mainly contributed to natural selection base on PR2, ENC-plot and neutrality curve analysis. Evolutionary rate, conserved sites, variable sites and nucleotide diversity analysis indicated that nad6 of A. spirataenius had the fastest evolutionary rate, while cox1 had the slowest evolutionary rate. Phylogenetic trees were reconstructed based on 13 protein-coding genes and 2 rRNA genes datasets using Bayesian inference and maximum likelihood method. The phylogenetic tree supported that both Siphonaptera and Mecoptera were monophyletic, and were sister groups to each other. This study filled gap of the genus Amphalius mitogenome sequences and was of great significance for understanding evolution of the order Siphonaptera.