Multi-marker molecular taxonomy and cladistic inference of curculionid stem borers as emergent pests of cucurbit horticultural production systems

Abstract:

Curculionid beetles rank among the least molecularly characterised stem-boring pests attacking cucurbit crops in the Neotropics, a gap that leaves growers and quarantine authorities without reliable diagnostic tools. This research applied a multi-marker molecular taxonomy framework — combining mitochondrial COI, nuclear ITS2, and 28S rDNA — to characterise curculionid stem borers collected from cucurbit production fields in coastal and highland valleys of Peru between April 2019 and March 2021. A total of 1,126 adult and larval specimens were recovered from four cucurbit hosts: butternut squash (Cucurbita moschata), pumpkin (C. maxima), melon (Cucumis melo), and cucumber (C. sativus) across eight districts in Lima and Ica regions. Morphological sorting separated specimens into four species groups, but overlapping pronotal sculpture and elytral striation patterns left 22.8% of adults unresolved. Multi-marker sequencing confirmed four species: Anthonomus grandis, Cosmopolites sordidus, Rhynchophorus ferrugineus, and Sitophilus abbreviatus. Concatenated phylogenetic analysis (maximum-likelihood and Bayesian inference) recovered two well-supported clades: a Curculioninae clade (A. grandis + C. sordidus; bootstrap 98%, posterior probability 0.99) and a Dryophthorinae-Entiminae clade (R. ferrugineus + S. abbreviatus; 96%/0.98). Interspecific K2P distances ranged from 6.2% to 13.2%, with a clear barcoding gap against intraspecific values (0.4-1.9%). A. grandis was the dominant species (34.6%), followed by C. sordidus (26.3%), S. abbreviatus (22.1%), and R. ferrugineus (17.0%). Damage incidence was highest on butternut squash (41.7% of sampled plants showing borer entry holes). These results establish a molecular reference for curculionid diagnostics in Peruvian cucurbit systems and highlight A. grandis as the priority management target.