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Development of novel molecular methods for accurate and economical identification of species has become critical both for pure biological research and for a wide range of applied areas. The most widely used current molecular diagnostic tool, the mitochondrial cytochrome c oxidase subunit 1 gene (COI), the so-called DNA barcode, has been highly criticised and is known to be ineffective at distinguishing species in many groups. Alternative markers are needed to circumvent these issues and provide diagnosticians with a greater range of tools for making accurate identifications. To address this, we describe here a novel analytical workflow for diagnostic marker development that utilises near-genomic-scale data to search for potential informative loci. The workflow takes advantage of orthologous gene databases, in combination with tests of phylogenetic resolution, and benchmarking of nucleotide variation against COI, to determine putative loci that might outperform COI. We use transcriptomes of 14 tephritid fruit flies and especially the taxonomically complex genus Bactrocera, as a case study. Of 1646 orthologues searched, our workflow retained a total of five loci following our conservative filtering strategy. One locus, POP4, had strong potential as a novel diagnostic marker for Bactrocera fruit flies. POP4 discriminates most species in the training set of taxa, but like COI fails to separate the sibling species Bactrocera tryoni and Bactrocera neohumeralis. Further validation of this potential new marker against a broader taxonomic sample is ongoing. We advocate that this simple and efficient workflow is, with minor modification, customisable for diagnostic development in almost any taxonomic group.

Bactrocera dorsalis, the Oriental fruit fly, is one of the world's most destructive agricultural insect pests and a major impediment to international fresh commodity trade. The genetic structuring of the species across its entire geographic range has never been undertaken, because under a former taxonomy B. dorsalis was divided into four distinct taxonomic entities, each with their own, largely non-overlapping, distributions. Based on the extensive sampling of six a priori groups from 63 locations, genetic and geometric morphometric datasets were generated to detect macrogeographic population structure, and to determine prior and current invasion pathways of this species. Weak population structure and high genetic diversity were detected among Asian populations. Invasive populations in Africa and Hawaii are inferred to be the result of separate, single invasions from South Asia, while South Asia is also the likely source of other Asian populations. The current northward invasion of B. dorsalis into Central China is the result of multiple, repeated dispersal events, most likely related to fruit trade. Results are discussed in the context of global quarantine, trade, and management of this pest. The recent expansion of the fly into temperate China, with very few associated genetic changes, clearly demonstrates the threat posed by this pest to ecologically similar areas in Europe and North America.

Bactrocera dorsalis, the Oriental fruit fly, is one of the world's most destructive agricultural insect pests and a major impediment to international fresh commodity trade. The genetic structuring of the species across its entire geographic range has never been undertaken, because under a former taxonomy B. dorsalis was divided into four distinct taxonomic entities, each with their own, largely non‐overlapping, distributions. Based on the extensive sampling of six a priori groups from 63 locations, genetic and geometric morphometric datasets were generated to detect macrogeographic population structure, and to determine prior and current invasion pathways of this species. Weak population structure and high genetic diversity were detected among Asian populations. Invasive populations in Africa and Hawaii are inferred to be the result of separate, single invasions from South Asia, while South Asia is also the likely source of other Asian populations. The current northward invasion of B. dorsalis into Central China is the result of multiple, repeated dispersal events, most likely related to fruit trade. Results are discussed in the context of global quarantine, trade, and management of this pest. The recent expansion of the fly into temperate China, with very few associated genetic changes, clearly demonstrates the threat posed by this pest to ecologically similar areas in Europe and North America.

1. Major global horticultural and food security tephritid fruit fly pests, Bactrocera papayae (papaya fruit fly) and B. invadens (invasive fruit fly), were synonymised with B. dorsalis (Oriental fruit fly) by Schutze et al. (2015a) based on extensive integrative taxonomic evidence from multiple sources. This synonymy was peer reviewed by eight independent experts. 2. Drew & Romig (2016) withdrew B. papayae and B. invadens from synonymy based on opinion drawn primarily from disparate geographical distribution, morphological, and host use information. This reversal was not subjected to peer review. 3. We consider the withdrawal from synonymy as invalid due to significant errors and misrepresentations of the literature provided in the arguments of Drew & Romig (2016) that we propose would not have withstood peer scrutiny. 4. This case reflects a broader issue of individual taxonomic authorities using opinion to challenge extensive evidence generated via scientific hypothesis-testing methods by discipline specialists. 5. We recommend that taxonomic acts not subjected to peer review, especially of pest species, be actively discouraged by the broader scientific and regulatory community.

Understanding the molecular mechanisms of organismal response to human-derived ecosystem change is recognised as a critical tool in monitoring and managing impacts, especially in freshwater systems. Fundamental to this approach is to determine the genes involved in responding to ecosystem change and detect modifications to their expression and activity in natural populations. Potential targets for this approach include well-known detoxification genes that are upregulated in response to stress. Here, we tested whether expression of such genes varied in association with differences in ecosystem health and could be detected in the field. We sampled populations of the freshwater midge, Cricotopus draysoni, from two geographically proximate sites in southeast Queensland, Australia, which differed in their ecosystem health, at multiple time points. We assessed transcriptome-level differential gene expression and predicted greatest differential expression between sites, associated with organismal responses to local physico-chemical factors. In contrast, we observed a clear and dramatic difference in gene expression – including of known detoxification genes – between time points, specifically between periods at the start and end of the austral summer rainfall when in-stream water levels are most different. These data suggest that these waterways experience greatest pollution load when water levels are high following rainfall events.

A dated molecular phylogeny is proposed for the Tanypodinae, a diverse subfamily of Chironomidae (Diptera). We used molecular data from fragments of one ribosomal gene (28S), one nuclear protein-coding gene (CAD), and one mitochondrial protein-coding gene (COI), analysed using mixed model Bayesian and maximum likelihood inference methods. All proposed tribes were sampled, namely, Anatopyniini, Clinotanypodini, Coelopyniini, Fittkauimyiini, Macropelopiini, Natarsiini, Pentaneurini, Procladiini and Tanypodini. A multilocus dataset of 1938 characters was compiled from 123 individuals including outgroups. Monophyly was supported for all tribes although some relationships were not robust. Relationships between tribes and some genus groups are highly congruent with a morphology-based estimate. Relationships within tribe Pentaneurini mostly find weak support, yet previously hypothesised groupings and monophyly or lack thereof in well-sampled genera are revealed. The tempo of diversification of the family was deduced by divergence time analysis (BEAST). Origination of a subfamily stem group in the late Jurassic to early Cretaceous was inferred, with all tribes and many genera of Pentaneurini originating and diversifying in the Cretaceous. Some nodes are biogeographically informative. Gene sections supported the backbone, but more extensive sampling is needed to estimate shallower phylogenies and to better understand the tempo and diversification of Tanypodinae.

The Thai-Malay peninsula represents a well-known transition zone between distinct floral and faunal assemblages of the Sundaland and mainland Asian regions and regional biogeographical patterns are known to be complex. The region represents the mid-point of the native range of the tephritid fruit fly Zeugodacus cucurbitae (Coquillett), which has existed in the region for a substantial proportion of its evolutionary history. Here, we integrate data from multiple markers (morphological: wing shape, male aedeagus length; molecular: cox1, microsatellites) to investigate the relative impacts of historical biogeographical features on Z. cucurbitae across Thailand and the Thai-Malay peninsula. Overall, morphological datasets revealed subtle population structure across the Thai-Malay peninsula that approximates a latitudinal cline rather than abrupt disjunctions associated with biogeographical barriers. Molecular markers provided finer resolution of biogeographical patterns: microsatellites suggested significant structure associated with the Khorat Plateau, both molecular markers indicated populations are structured across the Kangar-Pattani Line, but there was no effect of the Isthmus of Kra. Together, this study suggests that mountainous biogeographical barriers (Khorat Plateau), island isolation, and transitions in climate and vegetation (Kangar-Pattani Line) have significantly influenced population structure in this species. These data contribute vital evidence toward understanding non-traditional biological transition zones along the Thai-Malay peninsula.

Invasion into new areas by already widespread pest organisms often occurs through non-obvious routes, with the origins of such invasions difficult to determine. Understanding population structure using multiple datatypes can help untangle past dispersal events and reveal putative contemporary invasion pathways. The tephritid fruit fly, Zeugodacus cucurbitae (Coquillett), is a serious pest of cucurbits and other commercial crops and is considered native to the Indo-Oriental region, but is invasive in both Africa and the Pacific. Here, we combine molecular (microsatellites and COI) and morphological (male genetalia length and wing shape geometric morphometrics) data within an integrative taxonomic framework to test hypotheses concerning levels of Z. cucurbitae population variation observed in Southeast Asia (native range, 10 sites, ~200 individuals) versus the West Pacific (invasive range, 4 sites, ~80 individuals), and whether single or multiple introductions of Z. cucurbitae have occurred into the West Pacific. We also use this case to explicitly test if using an integrative approach provides more information about hypothesized invasion pathways than either genetic or morphological approaches would do alone. All datasets support Z. cucurbitae as being more variable in Southeast Asia than the West Pacific, and within these regions populations appear to be structured geographically. In particular, mainland and Sundaic Southeast Asian locations formed separate clusters, and New Guinea and Solomon Islands were not closely related to Guam and Hawaii. Evidence supports a separate single origin for New Guinea from the Melanesian arc, the Solomon Islands from Malaysia/Singapore, and Guam from mainland Asia, but multiple introductions into Hawaii from mainland Asia. Taken together, we argue that there is great value in integrating evidence from multiple sources as it can provide finer resolution of population relationships than any single data sou

Ecosystem monitoring, particularly of freshwater ecosystems, is critical for long-term management of impacted areas. However, current approaches do not account for sublethal effects on populations and detection of impact can only be made after the fact. One method to resolve this is to use an ecotoxicogenomic approach to assess changes in expression of stress response genes in target species. Cricotopus Van Der Wulp is a diverse genus of non-biting midges that in Australia occupies a wide range of lotic freshwater ecosystems, with marked variation among species in their pollution tolerance. The species C. albitarsis, C. draysoni and C. parbicinctus are considered to be particularly tolerant and often co-occur, thus presenting an ideal system for exploring transcriptome-level differences to understand and identify potential candidate molecular proxies for stream impact. This first requires a critical mass of transcriptome data from natural populations. This project addresses this by sampling the three target species from two locations in southeast Queensland in which they occur in sympatry. Larval transcriptomes were sequenced using either 50bp single end or 100bp paired end chemistries and assembly following read quality control produced 46 380, 17 167, and 27 125 transcripts for C. draysoni, C. albitarsis, and C. parbicinctus, respectively. Comparative analyses suggest broad similarities among species, reflecting their common heritage; however some key differences are noted. Cricotopus albitarsis and C. draysoni appeared to lack expression of chemorepellent genes, generally involved in predator avoidance. Cricotopus parbicinctus possessed an apparently unique cluster of proteins associated with antioxidant activity – generally involved in mediating oxidative stress. Additionally, C. draysoni possessed a unique cluster associated with storage of nutrients in the fat body, a body part whe

1. Major global horticultural and food security tephritid fruit fly pests,Bactrocera papayae(papaya fruit fly) andB. invadens(invasive fruit fly), weresynonymised withB. dorsalis(Oriental fruit fly) by Schutzeet al.(2015a) based onextensive integrative taxonomic evidence from multiple sources. This synonymy waspeer reviewed by eight independent experts.2. Drew & Romig (2016) withdrewB.papayaeandB.invadensfrom synonymy based onopinion drawn primarily from disparate geographical distribution, morphological, andhost use information. This reversal was not subjected to peer review.3. We consider the withdrawal from synonymy as invalid due to significant errors andmisrepresentations of the literature provided in the arguments of Drew & Romig (2016)that we propose would not have withstood peer scrutiny.4. This case reflects a broader issue of individual taxonomic authorities using opinionto challenge extensive evidence generated via scientific hypothesis-testing methods bydiscipline specialists.5. We recommend that taxonomic acts not subjected to peer review, especially of pestspecies, be actively discouraged by the broader scientific and regulatory community.

1.Major global horticultural and food security tephritid fruit fly pests, Bactrocera papayae (papaya fruit fly) and B. invadens (invasive fruit fly), were synonymised with B. dorsalis (Oriental fruit fly) by Schutze et al. (2015a) based on extensive integrative taxonomic evidence from multiple sources. This synonymy was peer reviewed by eight independent experts. 2. Drew & Romig (2016) withdrew B. papayae and B. invadens from synonymy based on opinion drawn primarily from disparate geographical distribution, morphological, and host use information. This reversal was not subjected to peer review. 3. We consider the withdrawal from synonymy as invalid due to significant errors and misrepresentations of the literature provided in the arguments of Drew & Romig (2016) that we propose would not have withstood peer scrutiny. 4. This case reflects a broader issue of individual taxonomic authorities using opinion to challenge extensive evidence generated via scientific hypothesis‐testing methods by discipline specialists. 5. We recommend that taxonomic acts not subjected to peer review, especially of pest species, be actively discouraged by the broader scientific and regulatory community.

The influence of climatic changes occurring since the late Miocene on Australia’s eastern mesic ecosystems has received significant attention over the past 20 years. In particular, the impact of the dramatic shift from widespread rainforest habitat to a much drier landscape in which closed forest refugia were dissected by open woodland/savannah ecosystems has long been a focal point in Australian ecology and biogeography. Several specific regions along the eastern coast have been identified previously as potentially representing major biogeographical disjunctions for closed forest taxa. Initially, evidence stemmed from recognition of common zones where avian species/subspecies distributions and/or floral communities were consistently separated, but the body of work has since grown significantly with the rise of molecular phylogeographic tools and there is now a significant literature base that discusses the drivers, processes and effects of these hypothesised major biogeographical junctions (termed barriers). Here, we review the literature concerning eight major barriers argued to have influenced closed forest taxa; namely, the Laura Basin, Black Mountain Corridor, Burdekin Gap, Saint Lawrence Gap, Brisbane Valley Barrier, Hunter Valley Barrier, Southern Transition Zone and East Gippsland Barrier. We synthesise reported phylogeographical patterns and the inferred timing of influence with current climatic, vegetation and geological characteristics for each barrier to provide insights into regional evolution and seek to elicit common trends. All eight putative biogeographical barriers are characterised currently by lowland zones of drier, warmer, more open woodland and savannah habitat, with adjacent closed forest habitats isolated to upland cool, wet refugia. Molecular divergence estimates suggest two pulses of divergence, one in the early Miocene (~20–15 Mya) and a later one from the Pliocene–Pleistocene (~6–0.04 Mya). We conclude with a prospectus for future resear

Bactrocera correcta is one of the most destructive pests of horticultural crops in tropical and subtropical regions. Despite the economic risk, the population genetics of this pest have remained relatively unexplored. This study explores population genetic structure and contemporary gene flow in B. correcta in Chinese Yunnan Province and attempts to place observed patterns within the broader geographical context of the species’ total range. Based on combined data from mtDNA cox1 sequences and 12 microsatellite loci obtained from 793 individuals located in 7 countries, overall genetic structuring was low. The expansion history of this species, including likely human-mediated dispersal, may have played a role in shaping the observed weak structure. The study suggested a close relationship between Yunnan Province and adjacent countries, with evidence for Western and/or Southern Yunnan as the invasive origin of B. correcta within Yunnan Province. The information gleaned from this analysis of gene flow and population structure has broad implications for quarantine, trade and management of this pest, especially in China where it is expanding northward. Future studies should concentrate effort on sampling South Asian populations, which would enable better inferences of the ancestral location of B. correcta and its invasion history into and throughout Asia.

Resolving species relationships and confirming diagnostic morphological characters for insect clades that are highly plastic, and/or include morphologically cryptic species, is crucial for both academic and applied reasons. Within the true fly (Diptera) family Chironomidae, a most ubiquitous freshwater insect group, the genera CricotopusWulp, 1874 and ParatrichocladiusSantos-Abreu, 1918 have long been taxonomically confusing. Indeed, until recently the Australian fauna had been examined in just two unpublished theses: most species were known by informal manuscript names only, with no concept of relationships. Understanding species limits, and the associated ecology and evolution, is essential to address taxonomic sufficiency in biomonitoring surveys. Immature stages are collected routinely, but tolerance is generalized at the genus level, despite marked variation among species. Here, we explored this issue using a multilocus molecular phylogenetic approach, including the standard mitochondrial barcode region, and tested explicitly for phylogenetic signal in ecological tolerance of species. Additionally, we addressed biogeographical patterns by conducting Bayesian divergence time estimation. We sampled all but one of the now recognized Australian Cricotopus species and tested monophyly using representatives from other austral and Asian locations. Cricotopus is revealed as paraphyletic by the inclusion of a nested monophyletic Paratrichocladius, with in-group diversification beginning in the Eocene. Previous morphological species concepts are largely corroborated, but some additional cryptic diversity is revealed. No significant relationship was observed between the phylogenetic position of a species and its ecology, implying either that tolerance to deleterious environmental impacts is a convergent trait among many Cricotopus species or that sensitive and restricted taxa have diversified into more narrow niches from a widely tolerant ancestor.

The informal taxon ‘genus Chile’ of Brundin, based solely on pupal exuviae of a podonomine Chironomidae, has remained inadequately known for half a century. New collections reveal life associations, and provide molecular data to hypothesise a precise phylogenetic placement in the austral Podonominae. A densely sampled molecular phylogeny based on two nuclear and one mitochondrial DNA markers shows ‘genus Chile’ to be the sister group to Podonomopsis Brundin, 1966. Within Podonomopsis a clade of South American species is sister to all Australian species. We discuss how to rank such a sister group taxon and treat ‘genus Chile’ as a new subgenus Araucanopsis, subg. nov. with the new species, Podonomopsis (Araucanopsis) avelasse, sp. nov. from Chile and Argentina as genotype of the monotypic subgenus. We describe P. (A.) avelasse in all stages and provide an expanded diagnosis and description of Podonomopsis to include Araucanopsis. A dated biogeographic hypothesis (chronogram) infers the most recent common ancestor (tmcra) of expanded Podonomopsis at 95 million years ago (Mya) (68–122 Mya 95% highest posterior density), ‘core’ Podonomopsis at 83 Mya (58–108) and Australian Podonomopsis at 65 Mya (44–87). All dates are before the South America–Australia geological separation through Antarctica, supporting previous conclusions that the taxon distribution is ‘Gondwanan’ in origin. Podonomopsis, even as expanded here, remains unknown from New Zealand or elsewhere on extant Zealandia.

The rapid uptake of transcriptomic approaches in freshwater ecology has seen a wealth of data produced concerning the ways in which organisms interact with their environment on a molecular level. Typically, such studies focus either at the community level and so don’t require species identifications, or on laboratory strains of known species identity or natural populations of large, easily identifiable taxa. For chironomids, impediments still exist for applying these technologies to natural populations because they are small-bodied and often require time-consuming secondary sorting of stream material and morphological voucher preparation to confirm species diagnosis. These procedures limit the ability to maintain RNA quantity and quality in such organisms because RNA degrades rapidly and gene expression can be altered rapidly in organisms; thereby limiting the inclusion of such taxa in transcriptomic studies. Here, we demonstrate that these limitations can be overcome and outline an optimised protocol for collecting, sorting and preserving chironomid larvae that enables retention of both morphological vouchers and RNA for subsequent transcriptomics purposes. By ensuring that sorting and voucher preparation are completed within <4 hours after collection and that samples are kept cold at all times, we successfully retained both RNA and morphological vouchers from all specimens. Although not prescriptive in specific methodology, we anticipate that this paper will assist in promoting transcriptomic investigations of the sublethal impact on chironomid gene expression of changes to aquatic environments.

Our attention has been drawn to lapsi and errors in a recent publication in this journal concerning Cricotopus Wulp (Diptera: Chironomidae) (Drayson et al., 2015).

In the century since the description of the orthoclad genus Paratrichocladius Santos-Abreu (Diptera: Chironomidae), separation in any life stage from the cosmopolitan, diverse Cricotopus Wulp has been problematic. Molecular analysis reveals the presence of two species in Australia that conform in morphology to Paratrichocladius and which form a well-supported clade including Paratrichocladius micans (Kieffer) from Africa and a distinct southern African larva. This clade clusters with taxa allied with Cricotopus albitibia (Walker), in turn nested within all other sampled Australian Cricotopus. Relevant nodes strongly support Cricotopus as nonmonophyletic without inclusion of Paratrichocladius. We synonymize Paratrichocladius with Cricotopus syn.n, treating Paratrichocladius as a subgenus. Cricotopus (Paratrichocladius) australiensis Cranston sp.n. is described for Trichocladius pluriserialis Freeman from Australia, which is not the same species under that name in New Zealand. Cricotopus (Paratrichocladius) bifenestrus Cranston sp.n. from Australia is described, also in all life stages. The many new combinations, listed in an Appendix, include three replacement names for new secondary homonyms, namely: Cricotopus (Paratrichocladius) sinobicinctus Cranston & Krosch nom.n. for Paratrichocladius bicinctus Fu, Sæther & Wang, Cricotopus draysoni Cranston & Krosch nom.n. for Cricotopus brevicornis Drayson, Krosch & Cranston, and Cricotopus (Paratrichocladius) sikhotealinus Makarchenko & Makarchenko nom.n. for Cricotopus orientalis Kieffer. We conclude with comments on wider issues in the taxonomy of Paratrichocladius, especially concerning New Zealand species.

The Australian species of the Orthocladiinae genus Cricotopus Wulp (Diptera: Chironomidae) are revised for larval, pupal, adult male and female life stages. Eleven species, ten of which are new, are recognised and keyed, namely Cricotopus acornis Drayson & Cranston sp. nov., Cricotopus albitarsis Hergstrom sp. nov., Cricotopus annuliventris (Skuse), Cricotopus brevicornis Drayson & Cranston sp. nov., Cricotopus conicornis Drayson & Cranston sp. nov., Cricotopus hillmani Drayson & Cranston, sp. nov., Cricotopus howensis Cranston sp. nov., Cricotopus parbicinctus Hergstrom sp. nov., Cricotopus tasmania Drayson & Cranston sp. nov., Cricotopus varicornis Drayson & Cranston sp. nov. and Cricotopus wangi Cranston & Krosch sp. nov. Using data from this study, we consider the wider utility of morphological and molecular diagnostic tools in untangling species diversity in the Chironomidae. Morphological support for distinguishing Cricotopus from Paratrichocladius Santo-Abreu in larval and pupal stages appears lacking for Australian taxa and brief notes are provided concerning this matter.

Bactrocera papayae Drew & Hancock, Bactrocera philippinensis Drew & Hancock, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta & White are four horticultural pest tephritid fruit fly species that are highly similar, morphologically and genetically, to the destructive pest, the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). This similarity has rendered the discovery of reliable diagnostic characters problematic, which, in view of the economic importance of these taxa and the international trade implications, has resulted in ongoing difficulties for many areas of plant protection and food security. Consequently, a major international collaborative and integrated multidisciplinary research effort was initiated in 2009 to build upon existing literature with the specific aim of resolving biological species limits among B. papayae, B. philippinensis, B. carambolae, B. invadens and B. dorsalis to overcome constraints to pest management and international trade. Bactrocera philippinensis has recently been synonymized with B. papayae as a result of this initiative and this review corroborates that finding; however, the other names remain in use. While consistent characters have been found to reliably distinguish B. carambolae from B. dorsalis, B. invadens and B. papayae, no such characters have been found to differentiate the latter three putative species. We conclude that B. carambolae is a valid species and that the remaining taxa, B. dorsalis, B. invadens and B. papayae, represent the same species. Thus, we consider B. dorsalis (Hendel) as the senior synonym of B. papayae Drew and Hancock syn.n. and B. invadens Drew, Tsuruta & White syn.n. A redescription of B. dorsalis is provided. Given the agricultural importance of B. dorsalis, this taxonomic decision will have significant global plant biosecurity implications, affecting pest management, quarantine, international trade, postharvest treatment and ba

The invasive fruit fly Bactrocera invadens Drew, Tsuruta & White, and the Oriental fruit fly Bactrocera dorsalis (Hendel) are highly destructive horticultural pests of global significance. Bactrocera invadens originates from the Indian subcontinent and has recently invaded all of sub-Saharan Africa, while B. dorsalis principally occurs from the Indian subcontinent towards southern China and South-east Asia. High morphological and genetic similarity has cast doubt over whether B. invadens is a distinct species from B. dorsalis. Addressing this issue within an integrative taxonomic framework, we sampled from across the geographic distribution of both taxa and: (i) analysed morphological variation, including those characters considered diagnostic (scutum colour, length of aedeagus, width of postsutural lateral vittae, wing size, and wing shape); (ii) sequenced four loci (ITS1, ITS2, cox1 and nad4) for phylogenetic inference, and; (iii) generated a cox1 haplotype network to examine population structure. Molecular analyses included the closely related species, Bactrocera kandiensis Drew & Hancock. Scutum colour varies from red-brown to fully black for individuals from Africa and the Indian subcontinent. All individuals east of the Indian subcontinent are black except for a few red-brown individuals from China. The postsutural lateral vittae width of B. invadens is narrower than B. dorsalis from eastern Asia, but the variation is clinal, with subcontinent B. dorsalis populations intermediate in size. Aedeagus length, wing shape and wing size cannot discriminate between the two taxa. Phylogenetic analyses failed to resolve B. invadens from B. dorsalis, but did resolve B. kandiensis. Bactrocera dorsalis and B. invadens shared cox1 haplotypes, yet the haplotype network pattern does not reflect current taxonomy or patterns in thoracic colour. Some individuals of B. dorsalis/B. invadens possessed haplotypes more closely related to B. kandiensis than to conspecifics, su

Knowledge on the evolution of the New Zealand insect fauna is reviewed and outstanding questions are highlighted. The New Zealand insect fauna is a composite of old and recent lineages and many spectacular examples of evolutionary processes are evident, including species radiations, hybridisation and unusual adaptations. We discuss the origins and evolution of four prominent communities within the insect fauna: terrestrial lowland insects, alpine insects, aquatic insects and insect communities from offshore islands. Within each of these communities, significant lineages are discussed, and in particular the crucial adaptations that enable these lineages to thrive and diversify. Glacial history has had a dramatic impact on the New Zealand insects, and the effects on different lineages are discussed. The New Zealand insects are unique, yet many are threatened with extinction, and efforts to preserve the fauna are reviewed. Despite the accumulating knowledge, major gaps still exist and these are outlined, as are opportunities to address key questions. The review concludes with a synthesis and a discussion of how systematics, new technologies and integrative approaches have the promise to improve dramatically our understanding of New Zealand insect evolution.

Bactrocera papayae Drew & Hancock, Bactrocera philippinensis Drew & Hancock, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta & White are four horticultural pest tephritid fruit fly species that are highly similar, morphologically and genetically, to the destructive pest, the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). This similarity has rendered the discovery of reliable diagnostic characters problematic, which, in view of the economic importance of these taxa and the international trade implications, has resulted in ongoing difficulties for many areas of plant protection and food security. Consequently, a major international collaborative and integrated multidisciplinary research effort was initiated in 2009 to build upon existing literature with the specific aim of resolving biological species limits among B. papayae, B. philippinensis, B. carambolae, B. invadens and B. dorsalis to overcome constraints to pest management and international trade. Bactrocera philippinensis has recently been synonymized with B. papayae as a result of this initiative and this review corroborates that finding; however, the other names remain in use. While consistent characters have been found to reliably distinguish B. carambolae from B. dorsalis, B. invadens and B. papayae, no such characters have been found to differentiate the latter three putative species. We conclude that B. carambolae is a valid species and that the remaining taxa, B. dorsalis, B. invadens and B. papayae, represent the same species. Thus, we consider B. dorsalis (Hendel) as the senior synonym of B. papayae Drew and Hancock syn.n. and B. invadens Drew, Tsuruta & White syn.n. A redescription of B. dorsalis is provided. Given the agricultural importance of B. dorsalis, this taxonomic decision will have significant global plant biosecurity implications, affecting pest management, quarantine, international trade, postharvest treatment and basic research. Throug

Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predominantly occur in south-east Asia and the Pacific. Identifying molecular diagnostics has proven problematic for these four taxa, a situation that cofounds biosecurity and quarantine efforts and which may be the result of at least some of these taxa representing the same biological species. We therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide geographic range; sequencing six loci (cox1, nad4-3′, CAD, period, ITS1, ITS2) for approximately 20 individuals from each of 16 sample sites. Data were analysed within maximum likelihood and Bayesian phylogenetic frameworks for individual loci and concatenated data sets for which we applied multiple monophyly and species delimitation tests. Species monophyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, Rosenberg's reciprocal monophyly measure, P(AB), Rodrigo's (P(RD)) and the genealogical sorting index, gsi. We specifically tested whether there was phylogenetic support for the four 'ingroup' pest species using a data set of multiple individuals sampled from a number of populations. Based on our combined data set, Bactrocera carambolae emerges as a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are unresolved. These data add to the growing body of evidence that B. dorsalis s.s., B. papayae and B. philippinensis are the same biological species, which poses consequences for quarantine, trade and pest management.

Many insect clades, especially within the Diptera (true flies), have been considered classically ‘Gondwanan’, with an inference that distributions derive from vicariance of the southern continents. Assessing the role that vicariance has played in the evolution of austral taxa requires testing the location and tempo of diversification and speciation against the well-established predictions of fragmentation of the ancient super-continent. Several early (anecdotal) hypotheses that current austral distributions originate from the breakup of Gondwana derive from studies of taxa within the family Chironomidae (non-biting midges). With the advent of molecular phylogenetics and biogeographic analytical software, these studies have been revisited and expanded to test such conclusions better. Here we studied the midge genus Stictocladius Edwards, from the subfamily Orthocladiinae, which contains austral-distributed clades that match vicariance-based expectations. We resolve several issues of systematic relationships among morphological species and reveal cryptic diversity within many taxa. Time-calibrated phylogenetic relationships among taxa accorded partially with the predicted tempo from geology. For these apparently vagile insects, vicariance-dated patterns persist for South America and Australia. However, as often found, divergence time estimates for New Zealand at c. 50 mya post-date separation of Zealandia from Antarctica and the remainder of Gondwana, but predate the proposed Oligocene ‘drowning’ of these islands. We detail other such ‘anomalous’ dates and suggest a single common explanation rather than stochastic processes. This could involve synchronous establishment following recovery from ‘drowning’ and/or deleteriously warming associated with the mid-Eocene climatic optimum (hence ‘waving’, which refers to cycles of drowning events) plus new availability of topography providing of cool running waters, or all these factors in combination. Alternatively a vicarianc

Bactrocera dorsalis (Hendel) and B. papayae Drew & Hancock represent a closely related sibling species pair for which the biological species limits are unclear; i.e., it is uncertain if they are truely two biological species, or one biological species which has been incorrectly taxonomically split. The geographic ranges of the two taxa are thought to abut or overlap on or around the Isthmus of Kra, a recognised biogeographic barrier located on the narrowest portion of the Thai Peninsula. We collected fresh material of B. dorsalis sensu lato (i.e., B. dorsalis sensu stricto + B. papayae) in a north-south transect down the Thai Peninsula, from areas regarded as being exclusively B. dorsalis s.s., across the Kra Isthmus, and into regions regarded as exclusively B. papayae. We carried out microsatellite analyses and took measurements of male genitalia and wing shape. Both the latter morphological tests have been used previously to separate these two taxa. No significant population structuring was found in the microsatellite analysis and results were consistent with an interpretation of one, predominantly panmictic population. Both morphological datasets showed consistent, clinal variation along the transect, with no evidence for disjunction. No evidence in any tests supported historical vicariance driven by the Isthmus of Kra, and none of the three datasets supported the current taxonomy of two species. Rather, within and across the area of range overlap or abutment between the two species, only continuous morphological and genetic variation was recorded. Recognition that morphological traits previously used to separate these taxa are continuous, and that there is no genetic evidence for population segregation in the region of suspected species overlap, is consistent with a growing body of literature that reports no evidence of biological differentiation between these taxa.

It is well established that the traditional taxonomy and nomenclature of Chironomidae relies on adult males whose usually characteristic genitalia provide evidence of species distinction. In the early days some names were based on female adults of variable distinctiveness – but females are difficult to identify (Ekrem et al. 2010) and many of these names remain dubious. In Russia especially, a system based on larval morphology grew in parallel to the conventional adult-based system. The systems became reconciled with the studies that underlay the production of the Holarctic generic keys to Chironomidae, commencing notably with the larval volume (Wiederholm, 1983). Ever since Thienemann’s pioneering studies, it has been evident that the pupa, notably the cast skins (exuviae) provide a wealth of features that can aid in identification (e.g. Wiederholm, 1986). Furthermore, the pupae can be readily associated with name-bearing adults when a pharate (‘cloaked’) adult stage is visible within the pupa. Association of larvae with the name-bearing later stages has been much more difficult, time-consuming and fraught with risk of failure. Yet it is identification of the larval stage that is needed by most applied researchers due to the value of the immature stages of the family in aquatic monitoring for water quality, although the pupal stage also has advocates (reviewed by Sinclair & Gresens, 2008). Few use the adult stage for such purposes as their provenance and association with the water body can be verified only by emergence trapping, and sampling of adults lies outside regular aquatic monitoring protocols.

Population genetic studies of freshwater invertebrate taxa in New Zealand and South America are currently few despite the geologically and climatically dynamic histories of these regions. The focus of our study was a comparison of the influence on realized dispersal of 2 closely related nonbiting midges (Chironomidae) of population fragmentation on these separated austral land masses. We used a 734-base pair (bp) fragment of cytochrome c oxidase subunit I (COI) to investigate intraspecific genetic structure in Naonella forsythi Boothroyd in New Zealand and Ferringtonia patagonica Edwards in Patagonia. We proposed hypotheses about their potential dispersal and, hence, expected patterns of genetic structure in these 2 species based on published patterns for the closely related Australian taxon Echinocladius martini Cranston. Genetic structure revealed for both N. forsythi and F. patagonica was characterized by several highly divergent (2.0–10.5%) lineages of late Miocene–Pliocene age within each taxon that were not geographically localized. Many were distributed widely. This pattern differed greatly from population structure in E. martini, which was typified by much greater endemicity of divergent genetic lineages. Nevertheless, diversification of lineages in all 3 taxa appeared to be temporally congruent with the onset of late Miocene glaciations in the southern hemisphere that may have driven fragmentation of suitable habitat, promoting isolation of populations and divergence in allopatry. We argue that differences in realized dispersal post-isolation may be the result of differing availability of suitable habitat in interglacial periods.

Background Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. Results Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a ‘non-Euclidean’ scenario which used geographical distances within a biogeographical ‘Sundaland context’ (r2 = 0.772, P < 0.0001) as compared to a ‘Euclidean’ scenario for which direct geographic distances between sample sites was used (r2 = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. BEAST analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the ‘non-Euclidean’ scenario (r2 = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level. Conclusions Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into t

With well over 700 species, the Tribe Dacini is one of the most species-rich clades within the dipteran family Tephritidae, the true fruit flies. Nearly all Dacini belong to one of two very large genera, Dacus Fabricius and Bactrocera Macquart. The distribution of the genera overlap in or around the Indian subcontinent, but the greatest diversity of Dacus is in Africa and the greatest diversity of Bactrocera is in south-east Asia and the Pacific. The monophyly of these two genera has not been rigorously established, with previous phylogenies only including a small number of species and always heavily biased to one genus over the other. Moreover, the subgeneric taxonomy within both genera is complex and the monophyly of many subgenera has not been explicitly tested. Previous hypotheses about the biogeography of the Dacini based on morphological reviews and current distributions of taxa have invoked an out-of-India hypothesis; however this has not been tested in a phylogenetic framework. We attempted to resolve these issues with a dated, molecular phylogeny of 125 Dacini species generated using 16S, COI, COII and white eye genes. The phylogeny shows that Bactrocera is not monophyletic, but rather consists of two major clades: Bactrocera s.s. and the ‘Zeugodacus group of subgenera’ (a recognised, but informal taxonomic grouping of 15 Bactrocera subgenera). This ‘Zeugodacus’ clade is the sister group to Dacus, not Bactrocera and, based on current distributions, split from Dacus before that genus moved into Africa. We recommend that taxonomic consideration be given to raising Zeugodacus to genus level. Supportive of predictions following from the out-of-India hypothesis, the first common ancestor of the Dacini arose in the mid-Cretaceous approximately 80 mya. Major divergence events occurred during the Indian rafting period and diversification of Bactrocera apparently did not begin until after India docked with Eurasia (50–35 mya). In contrast, diversification in Dacus

With well over 700 species, the Tribe Dacini is one of the most species-rich clades within the dipteran family Tephritidae, the true fruit flies. Nearly all Dacini belong to one of two very large genera, Dacus Fabricius and Bactrocera Macquart. The distribution of the genera overlap in or around the Indian subcontinent, but the greatest diversity of Dacus is in Africa and the greatest diversity of Bactrocera is in south-east Asia and the Pacific. The monophyly of these two genera has not been rigorously established, with previous phylogenies only including a small number of species and always heavily biased to one genus over the other. Moreover, the subgeneric taxonomy within both genera is complex and the monophyly of many subgenera has not been explicitly tested. Previous hypotheses about the biogeography of the Dacini based on morphological reviews and current distributions of taxa have invoked an out-of-India hypothesis; however this has not been tested in a phylogenetic framework. We attempted to resolve these issues with a dated, molecular phylogeny of 125 Dacini species generated using 16S, COI, COII and white eye genes. The phylogeny shows that Bactrocera is not monophyletic, but rather consists of two major clades: Bactrocera s.s. and the ‘Zeugodacus group of subgenera’ (a recognised, but informal taxonomic grouping of 15 Bactrocera subgenera). This ‘Zeugodacus’ clade is the sister group to Dacus, not Bactrocera and, based on current distributions, split from Dacus before that genus moved into Africa. We recommend that taxonomic consideration be given to raising Zeugodacus to genus level. Supportive of predictions following from the out-of-India hypothesis, the first common ancestor of the Dacini arose in the mid-Cretaceous approximately 80mya. Major divergence events occurred during the Indian rafting period and diversification of Bactrocera apparently did not begin until after India docked with Eurasia (50–35mya). In contrast, diversification in Dacus, at

Barbadocladius n. gen. is erected and described in larval, pupal and adult stages for two species: B. andinus sp. nov. and B. limay sp. nov., from Andean streams. The larva is distinctive by virtue of the very large ventromental 'beard' and the anterior parapods with a 'sleeve' of hooklets in addition to apical pectinate claws. The pupa has hooklets on some tergal and sternal intersegmental membranes. The adult, reported only in teneral specimens has hairy eyes, no antennal apical strong seta, no acrostichals, bare and unmarked wings, cylindrical 4th tarsomere subequal in length to the 5th, pulvilli about half the claw length, and hypopygium with anal point, lacking a virga. Molecular phylogenetic analysis eliminates relationships directly to the Eukiefferiella complex (which also have pupal hooklets), or to the Cricotopus group (adults also with hairy eyes), suggesting instead a sister group relationship to a suite of predominantly austral genera of Orthocladiinae.

The eastern Australian rainforests have experienced several cycles of range contraction and expansion since the late Miocene that are closely correlated with global glaciation events. Together with ongoing aridification of the continent, this has resulted in current distributions of native closed forest that are highly fragmented along the east coast. Several closed forest endemic taxa exhibit patterns of population genetic structure that are congruent with historical isolation of populations in discrete refugia and reflect evolutionary histories dramatically affected by vicariance. Currently, limited data are available regarding the impact of these past climatic fluctuations on freshwater invertebrate taxa. The non-biting midge species Echinocladius martini Cranston is distributed along the east coast and inhabits predominantly montane streams in closed forest habitat. Phylogeographic structure in E. martini was resolved here at a continental scale by incorporating data from a previous pilot study and expanding the sampling design to encompass populations in the Wet Tropics of north-eastern Queensland, south-east Queensland, New South Wales and Victoria. Patterns of phylogeographic structure revealed several deeply divergent mitochondrial lineages from central and south-eastern Australia that were previously unrecognised and were geographically endemic to closed forest refugia. Estimated divergence times were congruent with late Miocene onset of rainforest contractions across the east coast of Australia. This suggested that dispersal and gene flow among E. martini populations isolated in refugia has been highly restricted historically. Moreover, these data imply, in contrast to existing preconceptions about freshwater invertebrates, that this taxon may be acutely susceptible to habitat fragmentation.

The potential restriction to effective dispersal and gene flow caused by habitat fragmentation can apply to multiple levels of evolutionary scale; from the fragmentation of ancient supercontinents driving diversification and speciation on disjunct landmasses, to the isolation of proximate populations as a result of their inability to cross intervening unsuitable habitat. Investigating the role of habitat fragmentation in driving diversity within and among taxa can thus include inferences of phylogenetic relationships among taxa, assessments of intraspecific phylogeographic structure and analyses of gene flow among neighbouring populations. The proposed Gondwanan clade within the chironomid (non-biting midge) subfamily Orthocladiinae (Diptera: Chironomidae) represents a model system for investigating the role that population fragmentation and isolation has played at different evolutionary scales. A pilot study by Krosch et al (2009) indentified several highly divergent lineages restricted to ancient rainforest refugia and limited gene flow among proximate sites within a refuge for one member of this clade, Echinocladius martini Cranston. This study provided a framework for investigating the evolutionary history of this taxon and its relatives more thoroughly. Populations of E. martini were sampled in the Paluma bioregion of northeast Queensland to investigate patterns of fine-scale within- and among-stream dispersal and gene flow within a refuge more rigorously. Data was incorporated from Krosch et al (2009) and additional sites were sampled up- and downstream of the original sites. Analyses of genetic structure revealed strong natal site fidelity and high genetic structure among geographically proximate streams. Little evidence was found for regular headwater exchange among upstream sites, but there was distinct evidence for rare adult flight among sites on separate stream reaches. Overall, however, the distribution of shared haplotypes implied that both larval and