Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species

Abstract

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.

Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment

“…Thus, studies on marine benthic communities that prove the suitability of DNA-based approaches for environmental biomonitoring are lacking. Using samples of known taxonomic composition, we show that an alternative barcode that targets a shorter region of the COI gene outperforms the 658 bp region that is commonly used for metabarcoding metazoans (Carew et al, 2013;Ji et al, 2013;Dowle et al, 2015;Elbrecht and Leese, 2015;Zaiko et al, 2015). Our data corroborate previous studies unveiling the lack of universality in the COI primers, which is translated to biases during PCR step (Pochon et al, 2013;Deagle et al, 2014).…”

“…Several studies analyzing the same samples with morphological and molecular taxonomy have been performed so far to benchmark COI based metabarcoding in animals, all focusing exclusively on freshwater or terrestrial macroinvertebrates Carew et al, 2013;Gibson et al, 2014;Dowle et al, 2015;Elbrecht and Leese, 2015) or carried out under morphological identifications limited to high taxonomic levels (Gibson et al, 2015). Thus, studies on marine benthic communities that prove the suitability of DNA-based approaches for environmental biomonitoring are lacking.…”

“…The accuracy of metabarcoding-based taxonomic inferences relies on the retrieval of a wide range of taxonomic groups from a given environmental sample using the appropriate barcode, primers, and amplification conditions (Deagle et al, 2014;Kress et al, 2015), and on the completeness of the reference database (Zepeda Mendoza et al, 2015). Some attempts have been performed to compare morphological vs. metabarcodingbased taxonomic inferences; yet, results are inconclusive as some studies do not apply both approaches to the same sample and/or have focused on a particular taxonomic group Carew et al, 2013;Zhou et al, 2013;Gibson et al, 2014;Cowart et al, 2015;Zimmermann et al, 2015). A recent study (Gibson et al, 2015) has performed morphological and metabarcoding-based taxonomic identification on the same freshwater aquatic invertebrate samples, but limited their visual identifications to family level.…”

Implementing and Innovating Marine Monitoring Approaches for Assessing Marine Environmental Status

“…Metabarcoding based on PCR cannot yet provide reliable biodiversity indices since, especially for eukaryotes, it does not supply information on the abundance of every single species detected (Lindeque et al, 2013;Hirai et al, 2015). Most of the studies aimed at evaluating the relationships between species abundance and metabarcoding data obtained looser associations (Carew et al, 2013;Zhou et al, 2013;Hirai et al, 2015). Conversely, stronger relationships have been reported between biomass and read proportions (Elbrecht and Leese, 2015).…”

Using DNA metabarcoding for assessing chironomid diversity and community change in mosquito controlled temporary wetlands

“…During recent years, DNA metabarcoding of whole communities has become a new powerful tool for environmental monitoring of aquatic ecosystems (Hajibabaei et al 2011, Carew et al 2013, Elbrecht and Leese 2015, Gibson et al 2015. The DNA-based assays to monitor species biodiversity proved to be a rapid and efficient tool that allows the recovery of a substantial amount of taxa (Sweeney et al 2011, Taberlet et al 2012, Yu et al 2012, Carew et al 2013.…”