4.4. Construction of a Phylogenetic Tree

To investigate structural differences in venom proteins, we aligned the deduced amino acid sequences obtained from the transcriptome data with full-length sequences using CLC Main Workbench 7 (CLC Bio, Waltham, MA, USA). Based on amino acid alignment of the orthologous venom genes, we constructed a phylogenetic tree using maximum likelihood method based on the JTT matrix-based model [63] with 1000 bootstrap replications. The percentages of trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown on each node. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. Phylogenetic analyses were conducted in MEGA X (Pennsylvania State University, PA, USA). Acid phosphatase ({"type":"entrez-nucleotide","attrs":{"text":"AF321918","term_id":"12958659"}}AF321918), defensin 1 ({"type":"entrez-protein","attrs":{"text":"AAA52303","term_id":"181529"}}AAA52303), defensin 2 ({"type":"entrez-protein","attrs":{"text":"AAC69554","term_id":"3818537"}}AAC69554), hyaluronidase ({"type":"entrez-protein","attrs":{"text":"AAC70915","term_id":"2935328"}}AAC70915), metalloproteinase ({"type":"entrez-protein","attrs":{"text":"AAA58658","term_id":"435970"}}AAA58658), phospholipase A2 ({"type":"entrez-protein","attrs":{"text":"AAF09020","term_id":"6453793"}}AAF09020), and serine protease ({"type":"entrez-protein","attrs":{"text":"CCA61110","term_id":"326684020"}}CCA61110) of Homo sapiens were used as outgroups. The following were also used as outgroups: arginine kinase ({"type":"entrez-protein","attrs":{"text":"JAV48307","term_id":"1178091772"}}JAV48307), phospholipase A1 ({"type":"entrez-protein","attrs":{"text":"JAV47955","term_id":"1178091068"}}JAV47955), and phospholipase B ({"type":"entrez-protein","attrs":{"text":"JAV47950","term_id":"1178091058"}}JAV47950) of Hadrurus spadix; mastoparan of an uncultured bacterium (AP032449) [64]; venom allergen 5 ({"type":"entrez-protein","attrs":{"text":"XP_022659921","term_id":"1253751876"}}XP_022659921) of Varroa destructor; carboxylesterase 6 ({"type":"entrez-nucleotide","attrs":{"text":"KK119399","term_id":"605031007"}}KK119399) of Stegodyphus mimosarum; icarapin ({"type":"entrez-nucleotide","attrs":{"text":"FX985504","term_id":"1446067172"}}FX985504) of Odontomachus monticola; dipeptidyl peptidase 4 ({"type":"entrez-protein","attrs":{"text":"XP_021001451","term_id":"1192757284"}}XP_021001451) and major royal jelly protein ({"type":"entrez-protein","attrs":{"text":"XP_021000670","term_id":"1192742764"}}XP_021000670) of Parasteatoda tepidariorum; and endocuticle structural glycoprotein ({"type":"entrez-protein","attrs":{"text":"PSN39565","term_id":"1370630402"}}PSN39565), neprilysin ({"type":"entrez-protein","attrs":{"text":"PSN37766","term_id":"1370628420"}}PSN37766), serine carboxypeptidase ({"type":"entrez-protein","attrs":{"text":"PSN48057","term_id":"1370639448"}}PSN48057), serine protease inhibitor ({"type":"entrez-protein","attrs":{"text":"PSN55974","term_id":"1370647654"}}PSN55974), tachykinin ({"type":"entrez-protein","attrs":{"text":"PSN47497","term_id":"1370638861"}}PSN47497), and vitellogenin ({"type":"entrez-protein","attrs":{"text":"CAA06379","term_id":"7009577"}}CAA06379) of Blattella germanica. Major venomic genes of other Hymenopterans, Dipterans, Hemipterans, Lepidopterans and Dictyopterans were included and used as outgroups. Any robust phylogenetic comparative analysis was not feasible due to the limited number of Aculeate wasps and bumblebee species tested in this study.