Doctor of Philosophy
School of Biological Sciences
Meiklejohn, Kelly Ann, Taxonomy and systematics of the Australian Sarcophaga s.l. (Diptera: Sarcophagidae), Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2012. http://ro.uow.edu.au/theses/3729
The flesh flies (Diptera: Sarcophagidae) are a globally distributed family of over 3,000 species classified into three subfamilies and 173 genera. Almost 25% of sarcophagids belong to the genus Sarcophaga (sensu lato), which are further classified into 132 subgenera. The validity of, and relationships between these Sarcophaga s.l. subgenera remain questionable. Interestingly, many Sarcophaga s.l. species are of potential forensic importance, as they are attracted to and possibly breed in carrion. Despite this, the use of Sarcophaga s.l. specimens in forensic casework has been limited, as morphological species-level identification at any life stage is very challenging. Considering this, this PhD research was focussed on developing methods for the identification of the Australian Sarcophaga s.l., but also on evaluating the utility of various markers for resolving relationships within Sarcophaga s.l.
Prior to developing a range of tools for the identification of the Australian Sarcophaga s.l., it was necessary to define and clarify the fauna. Based on the current world catalogue of sarcophagids and various online catalogues for sarcophagids of the Australasian/Oceanian regions, 80 species have been documented from Australia, from the subfamilies Miltogramminae and Sarcophaginae. In 2009-2010, broad taxon sampling of sarcophagids across Australia was undertaken and locality information was documented from curated sarcophagids in a range of collections. From the information obtained, distribution records for 31 Australian species were updated and six new species records for Australia documented. Three new species as new to science from the large genus Sarcophaga s.l. have also been identified, with two of these described in this thesis. Additionally four new synonymies for the Australian flesh flies have been reported. Overall, the current Australian sarcophagid fauna comprises 84 species: 17 miltogrammines and 67 sarcophagines (with 55 being Sarcophaga s.l. species).
Little taxonomic work has been undertaken on the Australian Sarcophagidae since the 1950-70s, and the literature for the Australian species is now outdated. An updated key is provided for the Australian sarcophagids, allowing their separation into subfamilies and genera, along with the identification of all species of Sarcophaga s.l. A computer-based interactive LUCID key was also produced for the identification of the Australian Sarcophaga s.l., for use by non taxonomists. In a LUCID key, the user is able to choose morphological features that they are familiar with for identifications, instead of stipulated features which are found in traditional taxonomic keys. Included in both the updated taxonomic key and the LUCID key is a comprehensive database of illustrations and photographs of male terminalia, as well as updated biological and distributional information for each species of Sarcophaga s.l.
The molecular-based approach of DNA barcoding, which utilises a 648-bp fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene, was comprehensively evaluated for species-level identification of Australian sarcophagids. In a pilot study, barcoding effectively discriminated between 16 adult Australian sarcophagids. The current study evaluated barcoding on a larger taxon set of 588 adult Australian sarcophagids. A total of 39 of the 84 known Australian species were represented by 580 specimens, which includes 92% of species with potential forensic importance. A further eight specimens could not be reliably identified, but were included nonetheless as six unidentifiable taxa. A neighbour-joining (NJ) phylogenetic tree was generated and nucleotide sequence divergences were calculated using the Kimura-two-Parameter (K2P) distance model. All species except Sarcophaga (Fergusonimyia) bancroftorum, known for its high morphological variability, were resolved as reciprocally monophyletic (99.2% of cases), with most having bootstrap support of 100. Excluding bancroftorum, the mean intraspecific and interspecific variation ranged from 0-1.12% and 2.81-11.23%, respectively, allowing for species discrimination. This study also investigated whether DNA could be extracted and COI barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic extracts were prepared from all larval instars and puparia. Amplifications of the barcoding region were successful from all extracts, but puparial amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcode of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from the late second and early third instars. DNA barcoding was therefore validated as a suitable method for the molecular identification of all life stages of Australian sarcophagids.
This PhD research was also focussed on evaluating the usefulness of three sources of data for resolving relationships between 39 species from 14 Sarcophaga s.l. subgenera: the mitochondrial COI barcode region, ~800bp of the nuclear gene CAD and 110 morphological characters. Bayesian analyses were performed on all data combinations, but the most well resolved phylogeny was obtained when all three data sets were combined (78% of nodes with posterior probability (PP) of >0.90). Strong support at the species-level was provided by COI and the morphological data (PP of 1.00), and CAD facilitated high resolution at basal levels (PP of 0.93-1.00); support between most Sarcophaga s.l. subgenera was poor. Notably, the only Sarcophaga s.l. subgenera that were resolved as monophyletic were Liopygia and Parasarcophaga. The monophyly and relationships between the remaining Sarcophaga s.l. subgenera sampled remain questionable. The markers that were evaluated in this study provide greater phylogenetic signal when combined, rather than when used singly or as pairs. It is suggested that future phylogenetic studies on the genus Sarcophaga s.l. continue to combine data sets, and at least use COI and CAD, along with morphological data, for their analyses.