Mosquito Tree of Life: Jurassic–Cenozoic Culicidae Phylogenomics (194.8 to 0.0 Ma), Single Origin of Hematophagy, Salivary D7 & Apyrase Recruitment, and Secondary Loss in Toxorhynchites

Dual-pipeline empirical phylogenomics (MAFFT v7.526, IQ-TREE v2.4.0 UFBoot2 B = 1,000, LSD2 & PAML v4.10.10 codeml) across 100 complete Culicidae & Culicomorpha mitogenomes (T001–T100) and 48 RefSeq nuclear orthologs (1,648,476 bp raw; 16,032 bp supermatrix) To reconstruct the Mosquito Tree of Life (Diptera: Culicidae) and test whether vertebrate blood-feeding evolved once along the family stem or independently in Anophelinae and Culicinae, we executed an empirical bioinformatic synthesis across 120 primary NCBI RefSeq and GenBank sequence records totaling 1,648,476 bp (100 complete Culicidae and Culicomorpha mitochondrial genomes = 1,595,551 bp across circular phylogram tip nodes T001 – T100 , plus 20 core RefSeq nuclear transcripts = 52,925 bp drawn from 48 verified RefSeq orthologs across 6 nuclear gene families) using MAFFT v7.526 , IQ-TREE v2.4.0 ( B = 1,000 UFBoot2 replicates), IQ-TREE LSD2 relaxed molecular clock dating, and PAML v4.10.10 ( codeml ) . Third-codon purine/pyrimidine recoding ( RY3 ) reduces 100-species mitochondrial CDS Relative Composition Frequency Variability by 76.3% compared to raw third-codon positions (0.0737 → 0.0175), supporting a single origin of Culicidae hematophagy (crown Culicidae 145.00 Ma ; 95% CI: 145.00–160.23 Ma; 100% UFBoot2), nesting obligate non-hematophagous elephant mosquitoes ( Toxorhynchites , T060 , T062 , T063 ) inside crown Culicinae (stem 109.45 Ma , crown 53.36 Ma ; 100% UFBoot2), demonstrating 2.11×–4.48× relaxed purifying selection on Toxorhynchites rutilus salivary D7L1 ( ω = 0.2138 vs. 0.1011 in biting Culicinae) and secreted salivary apyrase ( ω = 0.1025 vs. 0.0229–0.0491) alongside 1.57× tighter purifying selection on yolk-precursor Vitellogenin-A1 ( ω = 0.0929 vs. 0.1457), and uncovering a published GenBank sample-swap error in PQ585799.1 ( "Toxorhynchites rutilus" ), which is 100.00% identical across 14,500 bp to Culex tarsalis ( PQ585801.1 ) from the same 2025 sequencing batch. Dual-pipeline empirical phylogenomic synthesis of the Mosquito Tree of Life across 120 primary NCBI RefSeq/GenBank records (1,648,476 bp raw; 100 complete mitogenomes T001–T100 + 20 core RefSeq nuclear CDS across 48 PAML orthologs) using MAFFT v7.526, IQ-TREE v2.4.0 (B = 1,000 UFBoot2), LSD2 relaxed clock dating, and PAML v4.10.10 codeml resolves a single origin of Culicidae hematophagy (crown 145.0 Ma; 95% CI: 145.0–160.2 Ma; 100% UFBoot2), demonstrates that obligate non-biting Toxorhynchites nests inside Culicinae (stem 109.45 Ma, crown 53.36 Ma; 100% UFBoot2) with 2.11x–4.48x relaxed purifying selection on salivary D7L1 (omega = 0.2138 vs 0.1011) and apyrase (omega = 0.1025 vs 0.0229–0.0491) alongside 1.57x tighter purifying selection on Vitellogenin-A1 (omega = 0.0929 vs 0.1457), and uncovers a published GenBank mitogenome sample swap (PQ585799.1 = 100% identical across 14,500 bp to Culex tarsalis PQ585801.1). Canonical Preprint URL: https://crvscience.com/labs/preprints/culicidae-phylogenomics-hematophagy-preprint Master (Concept) Zenodo DOI: 10.5281/zenodo.24039858 Open-Source Software Repository (GNU GPL-3.0): https://github.com/bpwhite/crvscience-labs