Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies

Authors: DOORENWEERD, CAMIEL - University Of Hawaii; SAN JOSE, MICHAEL - University Of Hawaii; LEBLANC, LUC - University Of Idaho; BARR, NORMAN - Animal And Plant Health Inspection Service (APHIS); Geib, Scott; CHUNG, ARTHUR - Consultant; DUPUIS, JULIAN - University Of Kentucky; EKAYANTI, ARNI - Niogret Ecology Consulting; FIEGALAN, ELAIDA - Luzon State University; HEMACHANDRA, KENNANTUDAWAGE - University Of Peradeniya; AFTAB HOSSAIN, MOHAMMAD - Bangladesh Atomic Energy Commission; HUANG, CHIA-LUNG - Minjiang University; HSU, YU-FENG - National Taiwan University; Morris, Kimberly; MUSTAPENG, ANDI MARYANI - Consultant; NIOGRET, JEROME - Niogret Ecology Consulting; HONG PHAM, THAI - Vietnam Academy Of Science And Technology (VAST); THI NGUYEN, NHIEN - Vietnam Academy Of Science And Technology (VAST); SIRISENA, UDA - Rajarata University Of Sri Lanka; TODD, TERRENCE - Animal And Plant Health Inspection Service (APHIS); RUBINOFF, DANIEL - University Of Hawaii

Submitted to: Molecular Ecology Resources
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2024
Publication Date: 7/2/2024
Citation: Doorenweerd, C., San Jose, M., Leblanc, L., Barr, N., Geib, S.M., Chung, A.Y., Dupuis, J.R., Ekayanti, A., Fiegalan, E., Hemachandra, K.S., Aftab Hossain, M., Huang, C., Hsu, Y., Morris, K.E., Mustapeng, A.A., Niogret, J., Hong Pham, T., Thi Nguyen, N., Sirisena, U.G., Todd, T., Rubinoff, D. 2024. Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies. Molecular Ecology Resources. 24(6). Article e13987. https://doi.org/10.1111/1755-0998.13987.
DOI: https://doi.org/10.1111/1755-0998.13987

Interpretive Summary: DNA barcoding is the process of sequencing the same region of the mitochondrial genome (Cytochrome C Oxidase I) and using variation of the sequence of that region to distinguish species from each other (typically at 2% variability). Applying state of the art barcode sequencing to over 5,000 individuals of the pest group Dacini (True fruit flies), we evaluate the utility of DNA barcoding to define species across this important group. We find in some cases, we are able to use DNA barcoding to refine our assignments of species, such as Dacus transversus and D. perpusillus, or alternatively combine (synonymize) species such as D. maculipterus and D. satanas. Alternatively, there are some species for which COI is not suitable, or explore which fragments are best suited to be used for species identifications. We recommend specific fragments of COI to be used for Dacini flies to generate the most robust data sets for species identifications, but they are not without some limitations and considerations that should be maintained during analysis.

Technical Abstract: The utility of a universal DNA ‘barcode’ fragment (658 base pairs of the Cytochrome C Oxidase I [COI]) has been established as a useful tool for species identification – and not for understanding the evolutionary history of a group. Since the first barcoding paper in 2003, large amounts of COI sequence data have been produced that hold promise for rapid species identification, e.g., for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which eighty are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5,601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we find a ‘soft’ barcode gap around 2% parwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We additionally compared the information content of four different fragments of COI that are regularly used for Dacini fruit fly identification, and found that all fragments >450 base pairs long provide similar resolution. After extensive efforts of minimizing operational error, 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is likely mostly due to introgression and/or incomplete lineage sorting of the mitochondrial DNA. We conclude with recommendations for future generation and use of COI libraries. Based on both morphology and COI data, we revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.