Vasco Elbrecht

Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

Abstract Environmental bulk samples often contain many different taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high‐throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here, we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate bulk samples, collected from a low‐mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5 × 5, 5 × 10, and up to 10 × 20 mm). Tissue powder from each size category was extracted individually and pooled based on tissue weight to simulate samples that were not sorted by biomass (“Unsorted”). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass (“Sorted”). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately fivefold when sorting the samples into three size classes and pooling by specimen abundance. Even coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high‐throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass or size is a simple yet efficient method to reduce current sequencing costs.

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an important group for bioassessment as many taxa show specific responses to environmental conditions. However, generating accurate macroinvertebrate inventories based on larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples to the species level, has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for bioassessment critically relies on carefully evaluating primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 most globally relevant freshwater invertebrate groups for stream assessment. Using these sequence alignments, we developed four primer combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples, each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet all detected more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently used in metabarcoding studies may not be suitable for amplification of freshwater macroinvertebrates. Therefore, careful primer evaluation and more region / ecosystem specific primers are needed before DNA metabarcoding can be used for routine bioassessment of freshwater ecosystems.

The complete mitochondrial genome of the stonefly Dinocras cephalotes (Plecoptera, Perlidae)

Abstract The complete mitochondrial genome of the perlid stonefly Dinocras cephalotes (Curtis, 1827) was sequenced using a combined 454 and Sanger sequencing approach using the known sequence of Pteronarcys princeps Banks, 1907 (Pteronarcyidae), to identify homologous 454 reads. The genome is 15,666 bp in length and includes 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and a control region. Gene order resembles that of basal arthropods. The base composition of the genome is A (33.5%), T (29.0%), C (24.4%) and G (13.1%). This is the second published mitogenome for the order Plecoptera and will be useful in future phylogenetic analysis.

PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers

1.DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that can serve as a backbone for primer design is currently available. 2.We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in another available software for that purpose, ecoPCR. 3.We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. 4.With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU based reference alignments generated with PrimerMiner can be used for manual primer design, or processed with bioinformatic tools for primer development. This article is protected by copyright. All rights reserved.

Distinct sensitivity of fungal freshwater guilds to water quality

Multiple anthropogenic stressors have been shown to impact animal and plant communities in freshwater ecosystems, but the responses of aquatic fungi remain largely unknown. Stressor effects on fungal communities may, however, result in changes of decomposition of plant litter and, thus, impact nutrient cycling, a key process for ecosystem functioning. We tested the impact of increased chloride and sediment levels, as well as reduced water flow velocity, on eukaryotic freshwater communities, with an emphasis on fungi, in a mesocosm experiment. Each of the three stressors was applied individually and in all combinations in a full-factorial design. Litterbags with non-sterilised tree leaves and sterile ceramic tiles were added to the mesocosms, to analyse the responses of communities in decaying plant material and in biofilms. Fungi preferably occurring in biofilms were supposed to represent indigenous aquatic fungi, while litterbag communities should be predominantly composed of fungi known from terrestrial litter. Community composition was assessed by high-throughput sequencing of amplified barcoding regions. Similarity matrices of operational taxonomic unit (OTU) tables calculated by UCLUST and CD-HIT-OTU-Illumina were significantly correlated. Preferred occurrence in biofilm and litter communities, respectively, was used for the grouping of OTUs into three ecological guilds. Stressor sensitivity varied among the guilds. While non-fungal, in particular autotrophic, OTUs responded to several treatments, two of the fungal guilds, i.e. those exclusively colonising litter and those preferably occurring on the ceramic tiles, showed no response to any applied treatment. Only fungi preferably, but not exclusively, colonising litter significantly responded to chloride addition. Their distribution patterns again correlated significantly with those of non-fungal OTUs, indicating possible interdependencies between both groups. The results indicate that eukaryotic freshwater communities are composed of different guilds, with distinctive sensitivity and tolerance to anthropogenic stressors.

Multiple-stressor effects on stream macroinvertebrate communities: A mesocosm experiment manipulating salinity, fine sediment and flow velocity

Stream ecosystems are impacted by multiple stressors worldwide. Recent studies have shown that the effects of multiple stressors are often complex and difficult to predict based on the effects of single stressors. More research is needed to understand stressor impacts on stream communities and to design appropriate counteractions. We carried out an outdoor mesocosm experiment to assess single and interactive multiple-stressor effects on stream macroinvertebrates in a setup with controlled application of three globally important stressors, namely, reduced stream flow velocity, deposition of fine sediment and increased chloride concentration in a full-factorial design. Each mesocosm comprised three compartments (channel substratum, leaf litter bag and drift net) that were individually analyzed and also compared. We identified 102,501 specimens in total (mainly to family level), 36.5% of which were found in the substratum, 60.6% in litter bags and 2.9% in the drift. Added fine sediment and reduced flow velocity had strong negative single-stressor effects on the abundances of EPT taxa, i.e. Ephemeroptera (mayflies), Plecoptera (stoneflies) and Trichoptera (caddisflies), and a positive effect on chironomid abundances in the substratum. Increased salt concentration reduced abundances of Ephemeroptera. Chironomids migrated from litter bag to channel substratum when water velocity was reduced and Leptophlebiidae in the opposite direction when sediment was added. All three stressors caused higher drift propensities, especially added fine sediment. Both additive and complex multiple-stressor effects were common. A complex three-way interaction affected EPT richness in the substratum, demonstrating the need to evaluate higher-order interactions for more than two stressors. Our results add further evidence that multiple-stressor interactions, notably increased salinity with other stressors, affect a variety of invertebrate taxa across different habitats of stream communities. The results have direct implications for water management as they highlight the need to re-evaluate defined salinity thresholds in the context of multiple-stressor interactions.

The mitochondrial genome of the Arizona Snowfly Mesocapnia arizonensis (Plecoptera, Capniidae)

Abstract We assembled the mitochondrial genome of the capniid stonefly Mesocapnia arizonensis (Baumann & Gaufin, 1969) using Illumina HiSeq sequence data. The recovered mitogenome is 14,921 bp in length and includes 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. The control region could only be assembled partially. Gene order resembles that of basal arthropods. This is the first partial mitogenome sequence for the stonefly superfamily group Euholognatha and will be useful in future phylogenetic analyses.

Isolation, characterization and cross amplification of eleven novel microsatellite loci for the hydrozoan coral Millepora

Milleporidae are of high ecological and economic importance, as, together with the scleractinian corals, they belong to the main reef builders of tropical coral reefs. Coral reefs face severe threats mainly due to anthropogenic disturbance. Understanding their population structure and dynamics is crucial for any conservation effort. Here we report the first microsatellite loci for the Milleporidae. Eleven polymorphic markers were developed for the hydrozoan corals Millepora dichotoma from the Great Barrier Reef (Australia) and tested for amplification in M. dichotoma from the Red Sea (Egypt), as well as for Millepora platyphylla from the Pacific Ocean (Moorea, French Polynesia). All loci were variable with 4–15 alleles per locus. Nine loci were transferable between geographic regions and species. These are the first microsatellites for hydrozoan corals. They will provide valuable tools for characterizing the population structure and genetic diversity of the group thereby benefitting coral reef conservation.