Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present ...study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8-7 taxa) but differences in read numbers assigned to taxa of interest (33.8%-93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.
Insects are the most diverse group of animals on Earth, but their small size and high diversity have always made them challenging to study. Recent technological advances have the potential to ...revolutionise insect ecology and monitoring. We describe the state of the art of four technologies (computer vision, acoustic monitoring, radar, and molecular methods), and assess their advantages, current limitations, and future potential. We discuss how these technologies can adhere to modern standards of data curation and transparency, their implications for citizen science, and their potential for integration among different monitoring programmes and technologies. We argue that they provide unprecedented possibilities for insect ecology and monitoring, but it will be important to foster international standards via collaboration.
Due to increasing human transformation of virtually all habitats on Earth, setting global priorities for conservation is essential. The emerging disciplines of macroecology and macroevolution (MEE) ...can provide a global perspective and information for such prioritization but remain relatively separated from conservation prioritization, partly because MEE researchers are unaware of the requirements for effective conservation prioritization and existing approaches implementing these. Indeed, existing approaches for conservation prioritization at large scale are scattered across literature. Here, we systematically review this literature and provide a guideline for researchers in MEE as to which of approaches might be suitable for their needs. From >11 000 scientific publications, we identified 134 methods suitable for commonly used MEE data and geographic scale. Furthermore, we use a ‘trait matrix' to identify families of similar approaches and to design a guideline to select the most suitable method, given a user‐defined set of data and analysis scope. The guidelines are freely available via the conserveR R‐package (<https://github.com/azizka/conserveR>). Finally, we used our review to identify increasing scalability, continuous monitoring and the integration of molecular, remote sensing and animal movement data as key areas for future impacts of MEE in conservation prioritization. We anticipate that our review can serve as a guide to MEE researchers interested in linking their data to conservation as well as for conservation scientists interested in using MEE approaches.
Metabarcoding is increasingly used to assess species diversity by high‐throughput sequencing where millions of sequences can be generated in parallel and multiple samples can be analysed in one ...sequencing run. Generating amplified fragments with a unique sequence identifier ('tag') for each sample is crucial, as it allows assigning sequences to the original samples. The tagging through so‐called fusion primers is a fast and cheap alternative to commercially produced ligation‐based kits. However, little is known about potential bias and inconsistencies introduced by the long nucleotide tail attached to those primers, which could lead to deficient reports of community composition in metabarcoding studies. We therefore tested the consistency and taxa detection efficiency of fusion primers in (1) a one‐step and (2) two‐step PCR protocol as well as (3) a commercially manufactured Illumina kit using mock communities of known composition. The Illumina kit delivered the most consistent results and detected the highest number of taxa. However, success of the two‐step PCR approach was only marginally lower compared to the kit with the additional advantage of a much more competitive price per library. While most taxa were also detected with the one‐step PCR approach, the consistency between replicates including read abundance was substantially lower. Our results highlight that method choice depends on the precision needed for analysis as well as on economic considerations and recommend the Illumina kit to obtain most accurate results and the two‐step PCR approach as a much cheaper yet very robust alternative.
DNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. The advantage of COI is ...its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from water, benthic invertebrate read and OTU numbers are typically “watered down,” that is, under represented, compared to whole specimen “bulk samples” due to greater co‐amplification of abundant nontarget taxa (e.g., fungi, algae, and bacteria). Because benthic stream invertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve benthic invertebrate assessment from eDNA by minimizing nontarget amplification. Therefore, we generated eDNA data using universal primers BF2/BR2 on samples collected throughout 15 months from a German Long‐Term Ecological Research site (Rhine‐Main‐Observatory, Kinzig River) to identify most abundant nontarget taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3’‐specificity toward benthic invertebrate taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in situ tests using 20 Kinzig River eDNA samples. We found that the percentage of target reads was much higher for the new primer combination compared to two universal benthic invertebrate primer pairs, BF2/BR2 and fwhF2/fwhR2n (99.6% versus 25.89% and 39.04%, respectively). Likewise, the number of detected benthic invertebrate species was substantially higher (305 versus 113 and 185) and exceeded the number of 153 species identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa, such as flatworms, were not detected, we show that the optimized primer avoids the nontarget amplification bias and thus significantly improves benthic invertebrate detection from eDNA.
Capturing benthic invertebrate biodiversity from environmental DNA (eDNA) isolated from water is difficult because of abundant nontarget taxa PCR amplification. A new primer combination maximizes target‐specific eDNA amplification for freshwater macroinvertebrate species. It detects especially more species than reported for two decades of extensive morphotaxonomic research at the studied Long‐Term Ecological Research (LTER) site.
DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in ...use, although standardization is key to its application in large‐scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size‐sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol‐based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.
Small and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most ...of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient.
Three Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored.
The small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample.
We demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80-90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.
Background
Chironomids, or non-biting midges, often dominate stream invertebrate communities in terms of biomass, abundance, and species richness and play an important role in riverine food webs. ...Despite these clear facts, the insect family Chironomidae is often treated as a single family in ecological studies or bioassessments given the difficulty to determine specimens further. We investigated stressor responses of single chironomid operational taxonomic units (OTUs) to three globally important stressors (increased salinity, fine sediment and reduced water flow velocity) in a highly replicated mesocosm experiment using a full-factorial design (eight treatment combinations with eight replicates each).
Results
In total, 183 chironomid OTUs (97% similarity) were obtained by applying a quantitative DNA metabarcoding approach. Whereas on the typically applied family level, chironomids responded positively to added fine sediment and reduced water velocity in the streambed and negatively to reduced velocity in the leaf litter, an OTU-level analysis revealed a total of 15 different response patterns among the 35 most common OTUs only. The response patterns ranged from (a) insensitivity to any experimental manipulation over (b) highly specific sensitivities to only one stressor to (c) additive multiple-stressor effects and even (d) complex interactions.
Conclusion
Even though most OTUs (> 85%) could not be assigned to a formally described species due to a lack of accurate reference data bases at present, the results indicate increased explanatory power with higher taxonomic resolution. Thus, our results highlight the potential of DNA-based approaches when studying environmental impacts, especially for this ecologically important taxon and in the context of multiple stressors.
Due to its remote and isolated location, Antarctica is home to a unique diversity of species. The harsh conditions have shaped a primarily highly adapted endemic fauna. This includes the notothenioid ...family Channichthyidae. Their exceptional physiological adaptations have made this family of icefish the focus of many studies. However, studies on their ecology, especially on their parasite fauna, are comparatively rare. Parasites, directly linked to the food chain, can function as biological indicators and provide valuable information on host ecology (e.g., trophic interactions) even in remote habitats with limited accessibility, such as the Southern Ocean. In the present study, channichthyid fish (
:
= 25,
:
= 33,
:
= 3,
:
= 4,
:
= 15) were collected off South Shetland Island, Elephant Island, and the tip of the Antarctic Peninsula (CCAML statistical subarea 48.1). The parasite fauna consisted of 14 genera and 15 species, belonging to the six taxonomic groups including Digenea (four species), Nematoda (four), Cestoda (two), Acanthocephala (one), Hirudinea (three), and Copepoda (one). The stomach contents were less diverse with only Crustacea (Euphausiacea, Amphipoda) recovered from all examined fishes. Overall, 15 new parasite-host records could be established, and possibly a undescribed genotype or even species might exist among the nematodes.
Worldwide, multiple stressors affect stream ecosystems and frequently lead to complex and non-linear biological responses. These combined stressor effects on ecologically diverse and functionally ...important macroinvertebrate communities are often difficult to assess, in particular species-specific responses across many species and effects of different stressors and stressor levels in concert. A central limitation in many studies is the taxonomic resolution applied for specimen identification. DNA metabarcoding can resolve taxonomy and provide greater insights into multiple stressor effects. This was detailed by results of a recent multiple stressor mesocosm experiment, where only for the dipteran family Chironomidae 183 Operational Taxonomic Units (OTUs) could be distinguished. Numerous OTUs showed very different response patterns to multiple stressors. In this study, we applied DNA metabarcoding to assess multiple stressor effects on all non-chironomid invertebrates from the same experiment. In the experiment, we applied three stressors (increased salinity, deposited fine sediment, reduced flow velocity) in a full-factorial design. We compared stressor responses inferred through DNA metabarcoding of the mitochondrial COI gene to responses based on morphotaxonomic taxa lists. We identified 435 OTUs, of which 122 OTUs were assigned to EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa. The most common 35 OTUs alone showed 15 different response patterns to the experimental manipulation, ranging from insensitivity to any applied stressor to sensitivity to single and multiple stressors. These response patterns even comprised differences within one family. The species-specific taxonomic resolution and the inferred response patterns to stressors highlights the potential of DNA metabarcoding in the context of multiple stressor research, even for well-known taxa such as EPT species.
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•Multiple stressor responses of stream invertebrate species are often complex.•Replicated mesocosm experiments are ideal to disentangle multiple stressor effects.•DNA metabarcoding was used to obtain high taxonomic resolution.•435 macroinvertebrate OTUs with different stressor responses were assessed.•DNA metabarcoding holds great promise to improve multiple stressor inferences.
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