Leigh A. Nelson

Integrative taxonomy, or iterative taxonomy?

The recently introduced term ‘integrative taxonomy’ refers to taxonomy that integrates all available data sources to frame species limits. We survey current taxonomic methods available to delimit species that integrate a variety of data, including molecular and morphological characters. A literature review of empirical studies using the term ‘integrative taxonomy’ assessed the kinds of data being used to frame species limits, and methods of integration. Almost all studies are qualitative and comparative – we are a long way from a repeatable, quantitative method of truly ‘integrative taxonomy’. The usual methods for integrating data in phylogenetic and population genetic paradigms are not appropriate for integrative taxonomy, either because of the diverse range of data used or because of the special challenges that arise when working at the species/population boundary. We identify two challenges that, if met, will facilitate the development of a more complete toolkit and a more robust research programme in integrative taxonomy using species tree approaches. We propose the term ‘iterative taxonomy’ for current practice that treats species boundaries as hypotheses to be tested with new evidence. A search for biological or evolutionary explanations for discordant evidence can be used to distinguish between competing species boundary hypotheses. We identify two recent empirical examples that use the process of iterative taxonomy.

Using COI barcodes to identify forensically and medically important blowflies

Abstract The utility of cytochrome oxidase I (COI) DNA barcodes for the identification of nine species of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae), from Australia, was tested. A 658‐bp fragment of the COI gene was sequenced from 56 specimens, representing all nine Chrysomya species and three calliphorid outgroups. Nucleotide sequence divergences were calculated using the Kimura‐two‐parameter distance model and a neighbour‐joining (NJ) analysis was performed to provide a graphic display of the patterns of divergence among the species. All species were resolved as reciprocally monophyletic on the NJ tree. Mean intraspecific and interspecific sequence divergences were 0.097% (range 0–0.612%, standard error [SE] = 0.119%) and 6.499% (range 0.458–9.254%, SE = 1.864%), respectively. In one case, a specimen that was identified morphologically was recovered with its sister species on the NJ tree. The hybrid status of this specimen was established by sequence analysis of the second ribosomal internal transcribed spacer (ITS2). In another instance, this nuclear region was used to verify four cases of specimen misidentification that had been highlighted by the COI analysis. The COI barcode sequence was found to be suitable for the identification of Chrysomya species from the east coast of Australia.

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

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.

Identification of forensically important Chrysomya (Diptera: Calliphoridae) species using the second ribosomal internal transcribed spacer (ITS2)

The identification of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae) may be hampered by their close morphological similarities, especially as immatures. In contrast to most previous studies, the utility of a nuclear rather than mitochondrial genetic marker was investigated to solve this problem. The second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) was amplified and sequenced from all nine Chrysomya species known from Australia. Difficulties encountered with direct sequencing of ITS2 for Chrysomya flavifrons necessitated cloning prior to sequencing for this species, which revealed a low level (0-0.23%) of intraindividual variation. Five restriction enzymes (DraI, BsaXI, BciVI, AseI and HinfI) were identified that were able to differentiate most members of the genus by polymerase chain reaction (PCR) restriction fragment length polymorphism (PCR-RFLP). The PCR-RFLP analysis revealed characteristic restriction profiles for all species except the closely related species pairs Chrysomya latifrons+Chrysomya semimetallica and Chrysomya incisuralis+Chrysomya rufifacies. Ch. incisuralis and Ch. rufifacies were able to be separated using the size differences resulting from amplification of the entire ITS region. The lack of intraspecific ITS2 sequence variation among eight Ch. incisuralis specimens was verified by the identical restriction profiles generated from these specimens. A DNA-based approach, such as PCR-RFLP, has the capacity to be useful for the identification of forensic entomological evidence in cases where morphological characters are unreliable.

Thermal attributes of Chrysomya species

The correct identification of forensically important arthropods for post‐mortem interval estimation is crucial, as the rate of larval development can vary substantially between species. The identification of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae) may be hampered by their close morphological similarities, especially as immatures. The aim of this study was to establish whether genetically closely related blowfly species would share similar developmental profiles. This could permit the application of developmental data to a number of closely related species, including those for which thermodevelopmental studies are lacking. If Australian Chrysomya were found to share developmental profiles, identification of the blowfly specimen to a level beyond genus may not be necessary, or at least it may not be necessary to distinguish morphologically similar sister species. The three Chrysomya species studied were collected from the same geographical location (Cairns, Australia), reducing the effects of acclimation and population‐level genetic variation. The experimental conditions in this study were virtually identical, which enabled direct comparisons to be made among the species. Blowfly larval lengths were obtained for 24‐hourly intervals at constant temperatures of 25, 30, and 35 °C. The thermal preferences of newly‐hatched feeding larvae were determined by their positions on a temperature gradient apparatus. This study established that all three species investigated differed significantly in their developmental profiles, despite the genetic closeness of the sister species Chrysomya megacephala (Fabricius) and Chrysomya saffranea (Bigot). Because of this, genetic distance was not considered to be a useful factor for predicting thermodevelopment profiles of closely related species within a genus, and highlights the necessity for correct species identification.

The complete mitochondrial genome of the gall-forming fly, Fergusonina taylori Nelson and Yeates (Diptera: Fergusoninidae)

The monogeneric family Fergusoninidae consists of gall-forming flies that, together with Fergusobia (Tylenchida: Neotylenchidae) nematodes, form the only known mutualistic association between insects and nematodes. In this study, the entire 16,000 bp mitochondrial genome of Fergusonina taylori Nelson and Yeates was sequenced. The circular genome contains one encoding region including 27 genes and one non-coding A+T-rich region. The arrangement of the protein-coding, ribosomal RNA (rRNA) and transfer RNA (tRNA) genes was the same as that found in the ancestral insect. Nucleotide composition is highly A+T biased. All of the protein initiation codons are ATN, except for nad1 which begins with TTT. All 22 tRNA anticodons of F. taylori match those observed in Drosophila yakuba, and all form the typical cloverleaf structure except for tRNA-Ser(AGN) which lacks a dihydrouridine (DHU) arm. Secondary structural features of the rRNA genes of Fergusonina are similar to those proposed for other insects, with minor modifications. The mitochondrial genome of Fergusonina presented here may prove valuable for resolving the sister group to the Fergusoninidae, and expands the available mtDNA data sources for acalyptrates overall.

Species diversity of Fergusonina Malloch gall flies (Diptera: Fergusoninidae) forming leaf bud galls on snow gum (Eucalyptus pauciflora Sieb. ex Spreng. complex), with a description of a new species from Tasmania.

A new species of Fergusonina (Diptera: Fergusoninidae) fly is described from terminal leaf bud galls (TLBGs) from the Eucalyptus pauciflora Sieb. ex Spreng. (snow gum) species complex from Australia. Fergusonina tasmaniensis Nelson sp.n. is the first species from the genus Fergusonina to be described from Tasmania and the fourth from this host complex. Fergusonina tasmaniensis sp.n. can be distinguished from the other snow gum Fergusonina species by differences in adult size, markings on the mesonotum and the male genitalia, and from all other described Fergusonina by host specificity and differences in adult colouration, setation, genitalia and the morphology of the larval dorsal shield. In a molecular phylogeny of the snow gum-inhabiting Fergusonina species, F. tasmaniensis sp.n. was resolved as monophyletic, and sister (mean distance = 3.82%) to a clade comprising F. daviesae Nelson and Yeates and F. omlandi Nelson and Yeates (mean interspecific distance = 2.48%).