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.
Acknowledgments:We would like to thank Marlen Mährlein, Nathalie Kaffenberger, Cristina Hartmann-Fatu and Arne Beermann for help with sample collection or processing. Furthermore, we thank Jan-Niklas Macher for helpful discussions, Beatrice Kulawig for compiling the taxonomic data from the Rhine-Main-Observatory, and Martina Weiss for valuable comments on the structure of the manuscript. This work has been funded by a grant of the Bode Foundation to FL. This work has been conducted as part of COST (European Cooperation in Science and Technology) Action DNAqua-Net (CA15219). AbstractDNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (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 stream water, macroinvertebrate read and OTU numbers are typically "watered down", i.e. diluted, compared to whole specimen 'bulk samples' due to greater co-amplification of abundant nontarget taxa such as algae and bacteria. Because stream macroinvertebrate 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 macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3'-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.
Free-living olive thrushes Turdus o/ivaceus were offered a choice of wild olive Olea africana fruit of four colours, representing four ripeness categories. The thrushes ate mostly black fruit (ripest), followed by maroon (ripe) and olivecoloured (partially ripe) fruit. When riper fruit was unavailable, the thrushes selected more maroon or olive-coloured fruit. Green fruit (unripe) were never eaten, even when these were the only ones available. When offered pieces of dyed pear Pyrus communis, the thrushes preferentially selected orange pieces, followed by red and black pieces. Green pieces were never eaten. The results indicated that olive thrushes selected fruit on the basis of fruit colour, and that colour preferences differed between the two types of fruit offered.
Environmental DNA (eDNA) extracted from water is routinely used in river biodiversity research, and via metabarcoding eDNA can provide comprehensive taxa lists with little effort and cost. However, eDNA-based species detection in streams and rivers may be influenced by sampling season, location, and other key factors such as water temperature and discharge. Research linking these factors and also informing on the potential of eDNA metabarcoding to detect shifts in ecological signatures, such as species phenology and functional feeding groups across seasons, is missing. To address this gap, we collected 102 water samples every two weeks for 15 months at a long-term ecological research (LTER) site and at three different positions in the river’s cross section, specifically the water surface, riverbed, and riverbank. We analyzed macroinvertebrate species and molecular Operational Taxonomic Unit (OTU) richness and temporal community turnover across seasons and sampling positions based on COI metabarcoding data. Using Generalized Additive Models, we found a significant influence of sampling season but not sampling position on community composition. Community turnover followed a cyclic pattern, reflecting the continuous change of the macroinvertebrate community throughout the year (‘seasonal clock’). Although water temperature had no influence on the inferred community composition, higher discharge reduced the number of Annelida and Ephemeroptera species detectable with eDNA. Most macroinvertebrate taxa showed the highest detection rates in spring, in particular merolimnic species with univoltine life cycles. Further, we detected an increase in proportion of shredders in winter and of parasites in summer. Our results show the usefulness of highly resolved eDNA metabarcoding time series data for ecological research and biodiversity monitoring in streams and rivers.
Environmental DNA (eDNA) extracted from water is routinely used in river biodiversity research, and via metabarcoding eDNA can provide comprehensive taxa lists with little effort and cost. However, eDNA-based species detection in streams and rivers may be influenced by sampling season, location, and other key factors such as water temperature and discharge. Research linking these factors and also informing on the potential of eDNA metabarcoding to detect shifts in ecological signatures, such as species phenology and functional feeding groups across seasons, is missing. To address this gap, we collected 102 water samples every two weeks for 15 months at a long-term ecological research (LTER) site and at three different positions in the river’s cross section, specifically the water surface, riverbed, and riverbank. We analyzed macroinvertebrate species and molecular Operational Taxonomic Unit (OTU) richness and temporal community turnover across seasons and sampling positions based on COI metabarcoding data. Using Generalized Additive Models, we found a significant influence of sampling season but not sampling position on community composition. Community turnover followed a cyclic pattern, reflecting the continuous change of the macroinvertebrate community throughout the year (‘seasonal clock’). Although water temperature had no influence on the inferred community composition, higher discharge reduced the number of Annelida and Ephemeroptera species detectable with eDNA. Most macroinvertebrate taxa showed the highest detection rates in spring, in particular merolimnic species with univoltine life cycles. Further, we detected an increase in proportion of shredders in winter and of parasites in summer. Our results show the usefulness of highly resolved eDNA metabarcoding time series data for ecological research and biodiversity monitoring in streams and rivers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.