Mitochondrial genomes of two subspecies of Citellophilus tesquorum: Insights into the molecular evolution and phylogenetic analysis within Siphonaptera

Background Citellophilus tesquorum is a major flea vector in natural plague foci across the Palaearctic region of China, representing a significant threat to veterinary and public health. Precise identification and sound taxonomic delimitation of its subspecies are critical for effective vector surveillance and evolutionary research. Methods We first performed morphological identification to guarantee specimen authenticity and accuracy, then sequenced and annotated the complete mitochondrial genomes of C. t. dzetysuensis and C. t. mongolicus using the Illumina NovaSeq platform. Comparative mitogenomic and phylogenetic analyses were conducted with 33 publicly available flea mitochondrial genomes to explore genomic evolution, codon usage patterns, and phylogenetic relationships. Results The two newly sequenced mitogenomes showed high structural conservation and a strong AT bias, consistent with typical features in Siphonaptera. Natural selection was identified as the dominant force driving codon usage bias. Phylogenetic reconstructions revealed that C. t. dzetysuensis clustered strongly with C. t. sungaris , while C. t. mongolicus formed a well-supported sister clade, indicating genetic divergence among these subspecies. Discussion The observed genetic differentiation is likely driven by long-term geographic isolation and host adaptation. This study establishes an integrative morphological and mitogenomic framework, enriches flea mitochondrial genomic resources, and provides key molecular evidence for the phylogeny, taxonomy, and surveillance of flea vectors.