The Utility of DNA Barcoding for Specimen Identification in Tephritidae

COI sequences (DNA Barcodes) were obtained for 128 specimens (out of 202 specimens attempted, see Appendix I), representing 41 species in 22 genera. Of these 128 successful specimens, 44 yielded full DNA Barcodes (658bp), 59 yielded near-complete DNA Barcodes (561bp or more), and 25 specimens yielded sequences of less than 350bp.

When sequences were compared using a neighbor-joining phenogram (Appendix II), most, but not all, specimens formed distinct clusters which accurately reflected morphological species. Two clusters incorrectly “identified” morphologically distinct specimens. Euaresta festiva (TEPH012, TEPH098) clustered with a single Euaresta aequalis specimen (TEPH010) and a single Euphranta canadensis specimen (TEPH014), despite remaining disjunct from the main Euaresta aequalis and Euphranta canadensis clusters. All sequences in this Euaresta festiva cluster were longer than 601bp, so we postulate a possible contamination of the TEPH010 and TEPH014 sequences.

A single Rhagoletis cingulata specimen (TEPH 123) was clustered with Campiglossa sabroskyi (TEPH0117), while the remaining R. cingulata specimens successfully clustered together away from the Campiglossa group. This anomaly is possibly an artifact of insufficient data, as the TEPH123 sequence is only 288bp, or perhaps a contaminant.

The sequences identified above as potential cases of contamination were annotated in the BOLD database to reflect these concerns.

In multiple instances, DNA barcodes of closely related species provided insufficient information for distinguishing between these species (i.e. Tephritis candidipennis and T. pura; Eutreta frontalis and E. novaeboracensis; Urophora affinis and U. cardui). Further taxon sampling and inclusion of more specimens may be required for differentiating these species using DNA Barcodes or use of another marker may be necessary.

The objective of the sequencing component of this project was to contribute to the DNA Barcoding database by providing CO1 sequences based on morphologically identified specimens. This was done to facilitate the potential use of a sequence database as an identification aid, and not to test species concepts and taxon limits. With this in mind, our limited dataset does show that most genera and species of Ontario Tephritidae possess distinct DNA Barcode clusters, and that most haplogroups reflect morphologically defined taxa. The database of fruit fly DNA Barcodes is currently being expanded through an international collaboration of researchers under the Tephritid Barcode Initiative.