Dietary DNA metabarcoding enables researchers to identify and characterize trophic interactions with a high degree of taxonomic precision. It is also sensitive to sources of bias and contamination in the field and lab. One of the earliest and most common strategies for dealing with such sensitivities has been to filter resulting sequence data to remove low-abundance sequences before conducting ecological analyses based on the presence or absence of food taxa. Although this step is now often perceived to be both necessary and sufficient for cleaning up datasets, evidence to support this perception is lacking and more attention needs to be paid to the related risk of introducing other undesirable errors. Using computer simulations, we demonstrate that common strategies to remove low-abundance sequences can erroneously eliminate true dietary sequences in ways that impact downstream dietary inferences. Using real data from well-studied wildlife populations in Yellowstone National Park, we further show how these strategies can markedly alter the composition of individual dietary profiles in ways that scale-up to obscure ecological interpretations about dietary generalism, specialism, and niche partitioning. Although the practice of removing low-abundance sequences may continue to be a useful strategy to address a subset of research questions that focus on a subset of relatively abundant food resources, its continued widespread use risks generating misleading perceptions about the structure of trophic networks. Researchers working with dietary DNA metabarcoding data—or similar data such as environmental DNA, microbiomes, or pathobiomes—should be aware of potential drawbacks and consider alternative bioinformatic, experimental, and statistical solutions. We used fecal DNA metabarcoding to characterize the diets of bison and bighorn sheep in winter and summer. Our analyses are based on 35 samples (median per species per season = 10) analyzed using the P6 loop of the chloroplast trnL(UAA) intron together with publicly available plant reference data (Illumina sequence read data are available at NCBI (BioProject: PRJNA780500)). Obicut was used to trim reads with a minimum quality threshold of 30, and primers were removed from forward and reverse reads using cutadapt. All further sequence identifications were performed using obitools; forward and reverse sequences were aligned using the illuminapairedend command using a minimum alignment score of 40, and only joined sequences retained. We used the obiuniq command to group identical sequences and tally them within samples, enabling us to quantify the relative read abundance (RRA) of each sequence. Sequences that occurred ≤2 times overall or that were ≤8 bp were discarded. Sequences were considered to be likely PCR artifacts if they were highly similar to another sequence (1 bp difference) and had a much lower abundance (0.05%) in the majority of samples in which they occurred; we discarded these sequences using the obiclean command. Overall, we characterized 357 plant sequences and a subset of 355 sequences were retained in the dataset after rarefying samples to equal sequencing depth. We then applied relative read abundance thresholds from 0% to 5% to the fecal samples. We compared differences in the inferred dietary richness within and between species based on individual samples, based on average richness across samples, and based on the total richness of each population after accounting for differences in sample size. The readme file contains an explanation of each of the variables in the dataset. Information on the methodology can be found in the associated manuscript referenced above.
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Comparative analysis of zooplankton diversities and compositions estimated from complement DNA and genomic DNA amplicons, metatranscriptomics, and morphological identifications
Abstract Community-based diversity analyses, such as metabarcoding, are increasingly popular in the field of metazoan zooplankton community ecology. However, some of the methodological uncertainties remain, such as the potential inflation of diversity estimates resulting from contamination by pseudogene sequences. Furthermore, primer affinity to specific taxonomic groups might skew community composition and structure during PCR. In this study, we estimated OTU (operational taxonomic unit) richness, Shannon’s H’, and the phylum-level community composition of samples from a coastal zooplankton community using four approaches: complement DNA (cDNA) and genomic DNA (gDNA) mitochondrial COI (Cytochrome oxidase subunit I) gene amplicon, metatranscriptome sequencing, and morphological identification. Results of mismatch distribution demonstrated that 90% is good threshold percentage to differentiate intra- and inter-species. Moderate level of correlations appeared upon comparing the species/OTU richness estimated from the different methods. Results strongly indicated that diversity inflation occurred in the samples amplified from gDNA because of mitochondrial pseudogene contamination (overall, gDNA produced two times more richness compared with cDNA amplicons). The unique community compositions observed in the PCR-based methods indicated that taxonomic amplification bias had occurred during the PCR. Therefore, it is recommended that PCR-free approaches be used whenever resolving community structure represents an essential aspect of the analysis.
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- Award ID(s):
- 1840868
- NSF-PAR ID:
- 10280697
- Editor(s):
- Fields, David
- Date Published:
- Journal Name:
- ICES Journal of Marine Science
- ISSN:
- 1054-3139
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Although metazoan animals in the mesopelagic zone play critical roles in deep pelagic food webs and in the attenuation of carbon in midwaters, the diversity of these assemblages is not fully known. A metabarcoding survey of mesozooplankton diversity across the epipelagic, mesopelagic and upper bathypelagic zones (0–1500 m) in the North Pacific Subtropical Gyre revealed far higher estimates of species richness than expected given prior morphology‐based studies in the region (4,024
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Zooplankton diversity in the deep “midnight zone” (>1000 m), where sunlight does not reach, remains largely unknown. Uncovering such diversity has been challenging because of the major difficulties in sampling deep pelagic fauna and identifying many (unknown) species that belong to these complex swimmer assemblages. In this study, we evaluated zooplankton diversity using two taxonomic marker genes: mitochondrial cytochrome oxidase subunit 1 (COI) and nuclear 18S ribosomal RNA (18S). We collected samples from plankton net tows, ranging from the surface to a depth of 5000 m above the Atacama Trench in the Southeast Pacific. Our study aimed to assess the zooplankton diversity among layers from the upper 1000 m to the ultra-deep abyssopelagic zone to test the hypothesis of decreasing diversity with depth resulting from limited carbon sources. The results showed unique, highly vertically structured communities within the five depth strata sampled, with maximal species richness observed in the upper bathypelagic layer (1000–2000 m). The high species richness of zooplankton (>750 OTUS) at these depths was higher than that found in the upper 1000 m. The vertical diversity trend exhibited a pattern similar to the well-known vertical pattern described for the benthic system. However, a large part of this diversity was either unknown (>50%) or could not be assigned to any known species in current genetic diversity databases. DNA analysis showed that the Calanoid copepods, mostly represented by
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Moreno-Hagelsieb, Gabriel (Ed.)Advances in the analysis of amplicon sequence datasets have introduced a methodological shift in how research teams investigate microbial biodiversity, away from sequence identity-based clustering (producing Operational Taxonomic Units, OTUs) to denoising methods (producing amplicon sequence variants, ASVs). While denoising methods have several inherent properties that make them desirable compared to clustering-based methods, questions remain as to the influence that these pipelines have on the ecological patterns being assessed, especially when compared to other methodological choices made when processing data (e.g. rarefaction) and computing diversity indices. We compared the respective influences of two widely used methods, namely DADA2 (a denoising method) vs. Mothur (a clustering method) on 16S rRNA gene amplicon datasets (hypervariable region v4), and compared such effects to the rarefaction of the community table and OTU identity threshold (97% vs. 99%) on the ecological signals detected. We used a dataset comprising freshwater invertebrate (three Unionidae species) gut and environmental (sediment, seston) communities sampled in six rivers in the southeastern USA. We ranked the respective effects of each methodological choice on alpha and beta diversity, and taxonomic composition. The choice of the pipeline significantly influenced alpha and beta diversities and changed the ecological signal detected, especially on presence/absence indices such as the richness index and unweighted Unifrac. Interestingly, the discrepancy between OTU and ASV-based diversity metrics could be attenuated by the use of rarefaction. The identification of major classes and genera also revealed significant discrepancies across pipelines. Compared to the pipeline’s effect, OTU threshold and rarefaction had a minimal impact on all measurements.more » « less
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Abstract Aim Our aim was to uncover patterns of distribution of marine subtidal rocky reef communities across six taxonomic groups and decompose the relative roles of species loss and turnover in total community variation. Additionally, we propose an easily calculated index that can be used to highlight areas with unique species composition for conservation planning. We estimated the strengths of associations between environmental factors and species richness and rarity.
Location Ilha Grande Bay, Brazil, covering about 150,000 ha harbouring different marine habitats.
Methods We used the Marine Rapid Assessment Protocol at 42 sites to gather information on environmental variables and species in six subtidal marine groups. We determined “singular” sites as the regions harbouring higher numbers of rare species. Then, we estimated the roles of species loss and turnover on the observed total variation among sites. We used Generalized Linear Model to partition the relative importance of the selected environmental factors in driving variation in species richness and singularity.
Results The singularity index and richness showed that the bay could be divided into three subregions for subtidal communities. Richness and rarity were structured at different spatial scales and associated with environmental variables related to water productivity and nutrients but varied among taxonomic groups. Community variation over space was largely associated with turnover of species.
Main conclusions Higher singularity and richness on the western side of the bay and around the main island suggested that these regions should be conservation priorities, but high species turnover across the whole bay indicated that portions of the central channel should be included in conservation strategies. This draws attention to the importance of community variation rather than just species numbers in conservation and management planning. The high species turnover indicated that these rocky reefs have high beta diversity when compared to other studied biological systems.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/icesjms/fsab084
