Recovery of DNA barcodes from blackfly museum specimens (Diptera: Simuliidae) using primer sets that target a variety of sequence lengths

Abstract

In this study, we evaluated the efficacy of various primers for the purpose of DNA barcoding old, pinned museum specimens of blackflies (Diptera: Simuliidae). We analysed 271 pinned specimens representing two genera and at least 36 species. Due to the age of our material, we targeted overlapping DNA fragments ranging in size from 94 to 407 bp. We were able to recover valid sequences from 215 specimens, of which 18% had 500- to 658-bp barcodes, 36% had 201- to 499-bp barcodes and 46% had 65- to 200-bp barcodes. Our study demonstrates the importance of choosing suitable primers when dealing with older specimens and shows that even very short sequences can be diagnostically informative provided that an appropriate gene region is used. Our study also highlights the lack of knowledge surrounding blackfly taxonomy, and we briefly discuss the need for further phylogenetic studies in this socioeconomically important family of insects.

DNA barcodes from century‐old type specimens using next‐generation sequencing

“…; Hernández‐Triana et al . ). PCR amplification and cycle sequencing employed standard CCDB protocols (Ivanova et al .…”

Tracking wetland community evolution using Diptera taxonomic, functional and phylogenetic structure

“…Sequences of the 658‐bp fragment of the cytochrome c oxidase subunit I (COI‐5P) gene of Eremoneura, Cyclorrhapha and Schizophora taxa for this phylogeny were obtained from the Barcode of Life Database (BOLD) (Ratnasingham & Hebert, ) or from our own sequenced specimens. Three hundred and fifty‐six specimens of 139 species not available in BOLD were sent for DNA barcoding at the Canadian Centre for DNA Barcoding, Guelph, Ontario, using C_LepFolF/C_LepFolR primer sets (Hernández‐Triana et al ., ). Sequences of Dolichopus brevipennis (Dolichopodidae), Sphaerophoria philanthus (Syrphidae), Chelipoda truncata (Empididae), Hypocera ehrmanni (Phoridae), and Lonchoptera furcata (Lonchopteridae) were used to root the tree (outgroup).…”

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

“…We also included three identified numt sequences from B. tryoni (JQ420918–JQ420920, Blacket et al ., ). We then aligned the following primers against the dacine sequences to assess primer matching: the universal primers LCO1490 and HCO2198 (Folmer et al ., ), the fruit fly‐specific forward primer FFCOI (Blacket et al ., ), the internal primers MF1 (Hajibabaei et al ., ; identical to MLepF1 of Hernandez‐Triana et al ., ), MH_MF1 (Hajibabaei et al ., ), MLepR2 (Hernandez‐Triana et al ., ), and H343 and L280 (van Houdt et al ., ). Based on this alignment, and the level of variation at primer binding sites, we modified LCO1490 and HCO2198 to be more dacine‐specific, and designed two dacine‐specific internal primers based on MH_MF1/MLepR2/H343 and MF1/MLepF1/L280 by either directly substituting bases or introducing degeneracy at some sites (Figs.…”

“…A solution to this problem is to utilize a series of primers that amplify shorter "minibarcode" portions of the desired barcode region and then assemble these minibarcodes to construct the full contig. This has proved an especially successful approach for obtaining COI barcode data from museum insect collections (e.g., Hernandez-Triana et al, 2014;Mitchell, 2015), including dacines (van Houdt et al, 2010), and a large suite of internal primers is now available.…”