Mitochondrial genomes of Psylloidea (Hemiptera): candidate broad-range primers for long-polymerase chain reaction, genewise variability, and calibration-sensitive time-calibrated phylogeny

Psyllids (Hemiptera: Psylloidea) are economically important sap-feeders, yet comparable mitochondrial genome (mitogenome) resources remain unevenly developed, partly because amplification success is often lineage-dependent. We designed 6 candidate broad-range long-PCR primer pairs from alignments of 60 published psyllid mitogenomes and evaluated them using a long-PCR/Sanger workflow and independent Illumina resequencing across 7 species representing 4 sampled families. The primer sets recovered near-complete mitogenomes across the sampled taxa, and cross-platform comparisons were fully concordant outside the control region, supporting recovery of protein-coding, ribosomal RNA, and transfer RNA (tRNA) regions. The control-region amplicon of Diaphorina citri showed multiple bands and was therefore interpreted separately. Comparative analyses of 67 mitogenomes identified genewise differences in nucleotide diversity, conserved primer-binding windows, and lineage-restricted mitochondrial tRNA structural variants involving trnCys, trnGlu, trnGly, and trnHis. Nucleotide- and amino-acid-based phylogenies recovered broadly similar family-level structure but differed at unstable deep nodes involving Diaphorina and Diclidophlebia paucipunctata. Saturation diagnostics indicated that the mitochondrial signal was not fully saturated overall, although third codon positions showed the strongest saturation tendency. Relaxed-clock analyses using 2 calibration strategies bracketed the crown age of Psylloidea at ∼49 to 129 Ma, indicating that the resulting dates should be viewed as calibration-sensitive hypotheses rather than definitive age estimates. This workflow provides a practical framework for generating comparable Psylloidea mitogenomes while highlighting the need for additional empirical validation in unsampled families and for nuclear data to resolve unstable deep relationships.