Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

Abstract

18 19 1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few 20 large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 212) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. 22The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. 23Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which 24 identification was feasible in the routine national monitoring. 253) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded 26 a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological 27 method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of 28 specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R 2 were low. 29With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding 30 datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as 31 expensive as morphology-based identification. 32 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment 33 results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable 34 method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological 35 identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock 36 the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems 37 with current laboratory protocols and reference databases. 38 39 40

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

“…After samples are collected using a standardised protocol ( Figure 1A, Buss et al, 2015), invertebrate specimens are usually separated from any debris such as substrate and non-target organic matter as part of the morphological identification process ( Figure 1B). While this increases the chance that some taxa and specimens will be overlooked (Haase, Pauls, Schindehütte, & Sundermann, 2010), in most metabarcoding studies of freshwater macroinvertebrates, specimens were separated from debris (Carew et al, 2013;Elbrecht, Vamos, et al, 2017;Emilson et al, 2018;Gibson et al, 2015). This is often done as part of preceding morphological identifications, or out of the concern that homogenising an entire sample might introduce polymerase chain reaction (PCR) inhibitors and complicate standardisation.…”

“…Over the past few years, several studies have demonstrated the feasibility of metabarcoding-based monitoring of freshwater macroinvertebrates (Andújar et al, 2018;Carew, Pettigrove, Metzeling, & Hoffmann, 2013;Gibson et al, 2015;Hajibabaei, Shokralla, Zhou, Singer, & Baird, 2011). Despite some methodological limitations (presence/absence data, primer bias) (Piñol, Mir, Gomez-Polo, & Agustí, 2014;Elbrecht & Leese, 2015), assessment results are at least comparable if not superior to conventional morphology-based stream monitoring approaches (Elbrecht, Vamos, Meissner, Aroviita, & Leese, 2017;Emilson et al, 2018;Gibson et al, 2015). Some macroinvertebrate reference databases are already fairly comprehensive especially for common taxa (Carew et al, 2017;Curry, Gibson, Shokralla, Hajibabaei, & Baird, 2018).…”

“…Routine stream monitoring requires the collection and identification of thousands of kick samples (Buss et al, 2015); however, current metabarcoding studies are typically limited to a few dozen samples (Andújar et al, 2018;Elbrecht, Vamos, et al, 2017;Emilson et al, 2018). If DNA metabarcoding is to be used in routine largescale monitoring projects, a substantial scale up of laboratory protocols is needed in a way that ensures a high level of reliability and quality of data.…”

“…We propose a streamlined metabarcoding approach that runs up to 288 individual samples on a single Illumina sequencing run ( Figure 1), using the BF2 + BR2 fusion primer system , which has been shown to work well with macroinvertebrate monitoring samples (Elbrecht, Vamos, et al, 2017) but is currently limited to multiplexing of up to 72 samples per sequencing run . Our new extended primer set allows for flexible multiplexing of samples in up to three 96-well plates thereby simplifying sample handling and reducing the risk of crosscontamination, while allowing for replication, as well as negative and positive controls.…”

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

“…DNA metabarcoding allows high-throughput and reliable identification of macroinvertebrate species biodiversity in multiple samples, simultaneously (e.g. Carew, Kellar, Pettigrove, & Hoffmann, 2018;Elbrecht, Vamos, Meissner, Aroviita, & Leese, 2017;Porter & Hajibabaei, 2018). Unlike morphological identification, DNA metabarcoding has the benefit of being less susceptible to the suite of potential errors identified above and provides easy access to species level information (Carew et al, 2017;Curry, Gibson, Shokralla, Hajibabaei, & Baird, 2018).…”