CO1 barcodes resolve an asymmetric biphyletic clade for Diabrotica undecimpunctata (Coleoptera: Chrysomelidae) subspecies and provide fixed nucleotide variants for differentiation from related lineages using real-time PCR

Authors: TEMBROCK, LUKE - Colorado State University; WILSON, CHRISTINA - Colorado State University; ZINK, FRIDA - Colorado State University; Konstantinov, Alexander - Alex; TISHECHKIN, ALEXEY - California Department Of Food And Agriculture; TIMM, ALICIA - Colorado State University

Submitted to: Annals of the Entomological Society of America
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2023
Publication Date: 4/20/2023
Citation: Tembrock, L., Wilson, C., Zink, F., Konstantinov, A.S., Tishechkin, A., Timm, A. 2023. CO1 barcodes resolve an asymmetric biphyletic clade for Diabrotica undecimpunctata (Coleoptera: Chrysomelidae) subspecies and provide fixed nucleotide variants for differentiation from related lineages using real-time PCR. Annals of the Entomological Society of America. 3:1-13. https://doi.org/10.3389/finsc.2023.1168586.
DOI: https://doi.org/10.3389/finsc.2023.1168586

Interpretive Summary: Leaf beetles, especially spotted cucumber beetle and its relatives, are among the most important insects for U.S. agriculture. Many are serious pests and feed on crops destroying valuable plants costing millions of dollars annually. Others are important biological control agents that can be used to control unwanted and invasive weeds. This work provides an insight into classification of spotted cucumber beetle and its subspecies based on cytochrome oxidase 1 sequence data from intercepted US ports-of-entry and museum specimens. The study showed that generally accepted subspecies within spotted cucumber beetle species are not much different genetically and their status needs to be reevaluated. The study will be useful to biological control workers, pest managers, evolutionary biologists, ecologists, and anyone interested in plant feeding beetles.

Technical Abstract: Diabrotica undecimpunctata is a multivoltine polyphagous beetle species that has long been documented as a significant agricultural pest throughout its native range in North America. This beetle can vector bacterial and viral plant pathogens that result in major losses to crops such as cucumber and soybean. Many countries outside the Americas treat D. undecimpunctata as a species of quarantine importance, while in the USA only the subspecies D. u. duodecimnotata is subject to quarantine, to prevent introduction from Mexico. Identification of D. undecimpunctata on the basis of morphology alone can be complicated given the use of conflicting characters in the description of some subspecific taxa. To better understand relationships among D. undecimpunctata subspecies and other related species, we sequenced mitochondrial cytochrome oxidase 1 (CO1) and nuclear internal transcribed spacer 2 (ITS2) DNA from individuals in different subspecific taxa and across different parts of the species range using museum samples and interceptions. When our data were combined with publicly available Diabrotica data, no pattern of divergence consistent with the currently recognized subspecific designations was found. In addition, we compared phylogenetic patterns in CO1 data from the congener D. virgifera to demonstrate the utility of mitochondrial data in resolving subspecies. From the CO1 data, a diagnostic real-time PCR assay was developed that could successfully identify all haplotypes within the large D. undecimpunctata clade for use in surveys and identification at ports of entry. These findings underscore the need to resolve molecular and morphological datasets into cogent, lineage-based groupings. Such efforts will provide an evolutionary context for the study of agriculturally important attributes of Diabrotica such as host preferences, xenobiotic metabolism, and natural and anthropogenic patterns of dispersal.