Mitochondrial genomes of Psylloidea (Hemiptera): Highly variable genes, phylogeny and divergence time estimation

Abstract Psyllids (Hemiptera: Psylloidea) are recognized as economically important sap-feeders. To enable broad mitogenome recovery, universal long-PCR primers were designed from alignments of 60 published mitogenomes and were validated by benchmarking Sanger-based assemblies against Illumina resequencing across seven species ( Blastopsylla occidentalis , Cornegenapsylla sinica , Macrohomotoma gladiata , Cacopsylla citrisuga , Cacopsylla chinensis , Diaphorina citri , and Bactericera cockerelli ). Single amplicon bands were obtained for each primer set, excepting for multiple bands with the control region (CR) of Diaphorina citri ; each assembly using Sanger sequences was confirmed by Illumina sequencing, independently. Resulting mitogenomes (14,499–15,220 bp) showed high cross-platform concordance, with read depths 193–72,903×. Sliding-window nucleotide diversity across 67 mitogenomes (CR excluded) indicated nad2 , atp8 , nad5 , and nad6 as most variable, whereas cox1 , cox2 , and both rRNAs were most conserved; primer sites lay within low-diversity windows (mean Pi  ≤ 0.07). Four lineage-restricted tRNA secondary-structure variants were observed: trnCys , trnGlu , trnGly , trnHis . Phylogenies inferred by maximum likelihood (ML) and Bayesian inference (BI) were largely congruent and resolved family clades as strongly monophyletic; however, the placement of Diaphorina and Diclidophlebia paucipunctata was sensitive to data type, with amino-acid PCG analyses supporting Diaphorina within Psyllidae (Fig. S4) while nucleotide all-gene analyses yielded an alternative Bayesian placement. Relaxed-clock analyses dated crown Psylloidea to the Early Cretaceous (~ 130 Ma; 95% HPD 114–146), with major family divergences concentrated in the Cretaceous (~ 105–98 Ma). This validated workflow expands mitogenomic resources and provides practical marker guidance for barcoded surveillance, pathway inference, and vector risk assessment within integrated pest management.