Beyond barcoding: A mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Abstract

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117-200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.

The complete mitochondria genome ofCalliphora vomitoria(Diptera: Calliphoridae)

“…All values for maximum intraspecific variations of the Calliphoridae species were no more than 2%. The interspecific variations between species were larger than 5% except for L. cuprina and L. sericata which were no more than 2%, the results was consistent with the previous study (Nelson et al 2012), which indicated that phylogenetic analysis of whole mt genome sequences resulted in much stronger support for discrimination between C. vomitoria and other five species, but week for discrimination between the L. cuprina and L. sericata.…”

“…The complete genome was amplified in eight fragments (Nelson et al 2012), and the double-stranded sequence was verified by prime walking within each long polymerase chain reaction (PCR) product. The entire fragments were amplified by TaKaRa MightyAmp Taq (Takara, Dalian, China) and performed on an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany) in 20 lL reaction volume.…”

“…The sequence data from GenBank which are L. porphyrina, C. vomitoria, C. rufifacies, H. ligurriens, four L. cuprina, two L. sericata of Calliphoridae and S. africa, S. similis from Sarcophagidae family. Except C. vomitoria, the other Calliphoridae genomes originally published in Nelson's paper (Nelson et al 2012), S. similis published in mitochondrial DNA (Yan et al 2016b) and S. africa published in mitochondrial DNA (Fu et al 2016).…”

Utility of COI, CAD and morphological data for resolving relationships within the genus Sarcophaga (sensu lato) (Diptera: Sarcophagidae): A preliminary study

“…The monophyly of the family and its three subfamilies, Miltogramminae, Paramacronychiinae, and Sarcophaginae, has been well supported (Giroux et al, 2010;Kutty et al, 2010;Pape, 1992Pape, , 1996. Little is known about the placement of the Sarcophagidae within the Oestroidea; it has been proposed that the sister family may be either the Tachinidae or the Calliphoridae, or even the still unnamed McAlpine's fly (Kutty et al, 2010;McAlpine, 1989;Nelson et al, 2012;Pape, 1992;Rognes, 1997).…”

The complete mitochondria genome of Aldrichina grahami (Diptera: Calliphoridae)

“…The entire genome was amplified in eight fragments (Nelson et al 2012a(Nelson et al , 2012b. All of the fragments were amplified by TaKaRa MightyAmp Taq (Takara, Dalian, China) and performed on an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany).…”