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 30,983 bp + 7,390 bp = 38,373 bp full-length & 16,032 bp partitioned; Native Rust gotoh-msa 89,830 bp; IQ-TREE v2.4.0 UFBoot2 B = 1,000, LSD2 & PAML v4.10.10 codeml) across 100 complete Culicidae & Culicomorpha mitogenomes (T001–T100 = 1,595,551 bp) and 48 RefSeq nuclear orthologs (1,648,476 bp total) Did mosquito blood-feeding evolve once or multiple times, and why do elephant mosquitoes ( Toxorhynchites ) feed only on nectar? Full-length alignments of 120 NCBI records (1,648,476 bp; 38,373 bp MAFFT and 89,830 bp Native Gotoh MSA ) date a single hematophagy origin to 145.00 Ma (100% bootstrap), nest Toxorhynchites inside Culicinae (53.36 Ma) with relaxed salivary D7L1 / apyrase constraint, and uncover a GenBank swap ( PQ585799.1 = PQ585801.1 ). Dual-pipeline empirical phylogenomic synthesis of the Mosquito Tree of Life across 120 primary NCBI RefSeq/GenBank records (1,648,476 bp total; 1,642,055 bp primary FASTA on disk across 100 complete mitogenomes T001–T100 = 1,595,551 bp and 20 core RefSeq nuclear transcripts = 46,504 bp across 48 PAML orthologs = 52,925 bp), aligned across 100% of their full lengths in both MAFFT v7.526 (30,983 bp mitogenome in 61.27 s + 7,390 bp nuclear in 5.42 s = 38,373 bp full-length supermatrix in 66.69 s; 16,032 bp codon-partitioned supermatrix) and Native Rust gotoh-msa (89,830 bp in 0.96 s; 64.1× faster than MAFFT) across a 32-chain multi-model combinatorial sweep (IQ-TREE v2.4.0 B = 1,000 UFBoot2, LSD2 relaxed clock, and PAML v4.10.10 codeml), dates a single origin of Culicidae hematophagy to 145.00 Ma (95% CI: 145.00–160.23 Ma; 100% UFBoot2), nests non-biting Toxorhynchites inside Culicinae (stem 109.45 Ma, crown 53.36 Ma; 100% UFBoot2) with 2.11×–4.48× relaxed purifying selection on salivary D7L1 (ω = 0.2138 vs 0.1011) and apyrase (ω = 0.1025 vs 0.0229–0.0491) alongside 1.57× tighter purifying selection on Vitellogenin-A1 (ω = 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