IntroductionInvertebrate communities are central to many environmental monitoring programs. In freshwater ecosystems, aquatic macroinvertebrates are collected, identified and then used to infer ecosystem condition. Yet the key step of species identification is often not taken, as it requires a high level of taxonomic expertise, which is lacking in most organizations, or species cannot be identified as they are morphologically cryptic or represent little known groups. Identifying species using DNA sequences can overcome many of these issues; with the power of next generation sequencing (NGS), using DNA sequences for routine monitoring becomes feasible.ResultsIn this study, we test if NGS can be used to identify species from field-collected samples in an important bioindicator group, the Chironomidae. We show that Cytochrome oxidase I (COI) and Cytochrome B (CytB) sequences provide accurate DNA barcodes for chironomid species. We then develop a NGS analysis pipeline to identifying species using megablast searches of high quality sequences generated using 454 pyrosequencing against comprehensive reference libraries of Sanger-sequenced voucher specimens. We find that 454 generated COI sequences successfully identified up to 96% of species in samples, but this increased up to 99% when combined with CytB sequences. Accurate identification depends on having at least five sequences for a species; below this level species not expected in samples were detected. Incorrect incorporation of some multiplex identifiers (MID’s) used to tag samples was a likely cause, and most errors could be detected when using MID tags on forward and reverse primers. We also found a strong quantitative relationship between the number of 454 sequences and individuals showing that it may be possible to estimate the abundance of species from 454 pyrosequencing data.ConclusionsNext generation sequencing using two genes was successful for identifying chironomid species. However, when detecting species from 454 pyrosequencing data sets it was critical to include known individuals for quality control and to establish thresholds for detecting species. The NGS approach developed here can lead to routine species-level diagnostic monitoring of aquatic ecosystems.

Geographical patterns for quantitative traits in Drosophila and other insects are commonly used to investigate climatic selection. They are usually determined from comparisons of populations over extensive areas and based on one collection per population. Here we consider patterns in the Australian endemic species Drosophila serrata established over a shorter transect with repeated sampling. Summer (prewinter) and spring (post-winter) collections were made from 10 to 14 localities, incorporating the southern border of D. serrata and extending approximately 1000 km northwards along the eastern coast of Australia. Linear or curvilinear associations with latitude were evident for development time, viability and cold resistance but patterns differed between collections. Some geographical (population) and genetic associations between traits were found and these also tended to differ between collections. Results confirm the importance of cold stress resistance over winter to the southern border of this species. Microsatellite markers were developed for D. serrata. These indicated a low level of genetic differentiation between populations, high levels of gene flow and no evidence that the most southerly populations were isolated. The results suggest that selection generated geographical patterns in cold resistance, development time and viability, and that substantial gene flow may prevent adaptation at the border to conditions beyond the current distribution of D. serrata.

1. We investigated the distribution of chironomid taxa in urban wetlands in the greater Melbourne area, Australia, to test if their distribution was influenced by sediment pollution and other environmental variables. 2. For identification of the Chironomidae, DNA markers generated via polymerase chain reaction-restriction fragment length polymorphism of cytochrome c oxidase sub unit I (COI) were validated against morphology and reference specimens for more than 5000 chironomids representing over 80 species. DNA-based identification generally concurred with morphological separation, but also indicated the existence of cryptic diversity in some genera. 3. Non-metric multidimensional scaling (NMS) and canonical correspondence analysis (CCA) showed chironomid assemblages were structured among wetlands and could be linked to several habitat characteristics. However, Chironomidae assemblages were only weakly linked to sediment pollution. 4. Logistic regressions identified potential bioindicators of sediment pollution. Riethia stictoptera, Tanytarsus inextentus, Coelopynia and Chironomus 'februarius' were negatively associated and Chironomus duplex was positively associated with sediment pollution. Thresholds for the pollution sensitivities of specific species were mostly similar to those established with previous microcosm tests. 5. Several other environmental factors influenced the distribution of specific chironomid taxa. Salinity, substratum type and submerged and riparian vegetation were particularly important. 6. We conclude that specific chironomid taxa rather than assemblages have potential as bioindicators of sediment pollution provided their ecological preferences are considered and their pollution sensitivities are characterized using multiple methods. The integration of DNA-based techniques should facilitate accurate and rapid identification of bioindicators species.

BackgroundHigh throughput DNA sequencing of bulk invertebrate samples or metabarcoding is becoming increasingly used to provide profiles of biological communities for environmental monitoring. As metabarcoding becomes more widely applied, new reference DNA barcodes linked to individual specimens identified by taxonomists are needed. This can be achieved through using DNA extraction methods that are not only suitable for metabarcoding but also for building reference DNA barcode libraries.MethodsIn this study, we test the suitability of a rapid non-destructive DNA extraction method for metabarcoding of freshwater invertebrate samples.ResultsThis method resulted in detection of taxa from many taxonomic groups, comparable to results obtained with two other tissue-based extraction methods. Most taxa could also be successfully used for subsequent individual-based DNA barcoding and taxonomic identification. The method was successfully applied to field-collected invertebrate samples stored for taxonomic studies in 70% ethanol at room temperature, a commonly used storage method for freshwater samples.DiscussionWith further refinement and testing, non-destructive extraction has the potential to rapidly characterise species biodiversity in invertebrate samples, while preserving specimens for taxonomic investigation.

The wheat curl mite (WCM), Aceria tosichella Keifer, is an eriophyoid pest of cereals, and the vector responsible for transmitting wheat streak mosaic virus. Several authors have suggested cryptic species of this mite identified through morphological variation, but this has never been conclusively demonstrated. Here, we use the mitochondrial 16S rRNA gene and two nuclear markers (internal transcribed spacer 1 and adenine nucleotide translocase) to show that WCM from Australia consists of at least two separate lineages that may represent putative species. In our study, both WCM variants were widespread and the only eriophyoids found on wheat varieties. The WCM variants were also found on alternate host plants, including some plants not known to host WCM. These results have implications for the control of this pest within Australian cereal crops.