Analysis of the complete mitochondrial DNA from Anopheles funestus: An improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution

Virtually no information regarding timing of deep lineage divergences within mosquito family (Culicidae) exists, which poses an important problem in the postgenomic era. To address this issue, the complete 15,354 bp mitochondrial genome of Anopheles funestus was assembled from both mtDNA and cDNA sequences generated from transcripts of the mtDNA-encoded protein and rRNA genes. Analysis of the transcript information allowed an improved genome annotation, revealing that the translation initiation codon for the cox1 gene is TCG, rather than atypical, longer codons proposed in several other insects. The 5'ends of nad1 and nad5 transcripts begin with TTG and GTG triplets, respectively, which apparently serve as the translation initiators for those genes. We used all the A. funestus mtDNA gene sequences and three other publicly available mosquito mtDNA genomes for the estimation of divergence time points within Culicidae. The maximum likelihood date estimates for the splits between Anopheles and Aedes (approximately 145-200 Mya), between Anopheles subgenera Cellia and Anopheles (approximately 90-106 Mya), and between lineages within subgenus Anopheles (approximately 70-85 Mya) inferred from protein-coding genes are roughly twice as high as the dates based on RNA gene sequences. Although existing evidence does not unequivocally favor one of the alternatives, fossil-based predictions of the age of the family Culicidae are in better agreement with dates inferred from protein-coding genes.