Detecting invertebrate species in archived collections using next-generation sequencing

Invertebrate biodiversity measured at mostly family level is widely used in biological monitoring programmes to assess anthropogenic impacts on ecosystems. However, next-generation sequencing (NGS) could allow development of new more sensitive biomonitoring tools by allowing rapid species identification. This could be accelerated if archived invertebrate collections and environmental information from past programmes are used to understand species distributions and their environmental responses. In this study, we take archived macroinvertebrate samples from two sites collected on multiple occasions and test whether NGS can successfully detect species. Samples had been stored in 70% ethanol at room temperature for up to 12 years. Three amplicons ranging from 197 to 274 bps within the DNA barcode region were amplified from samples and compared to DNA barcoding libraries to identify species. We were able to amplify partial DNA barcodes from most samples, and species were often detected with multiple amplicons. However, some singletons and taxa poorly covered by DNA barcoding were missed. This suggests additional DNA barcodes will be required to fill 'gaps' in current DNA barcode libraries for aquatic macroinvertebrates and/or that it may not be possible to detect all taxa in a sample. Furthermore, older samples often detected fewer taxa and were less reliable for amplification, suggesting NGS is best used on samples within 8 years of collection. Nevertheless, many common taxa with existing DNA barcodes were reliably identified with NGS and were often present at sites across multiple years, showing the potential of NGS for detecting common and abundant species in archived material