Leese, F.; Sander, M.; Buchner, D.; Elbrecht, V.; Haase, P. & Zizka, V.M.A. (2021): <b>Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification</b>. <i>Environmental DNA</i> <b>3</b>(1), 261-276<br>DOI: <a href="http://dx.doi.org/10.1002/edn3.177" target="_blank">http://dx.doi.org/10.1002/edn3.177</a>.
Resource Description
Title:
Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification
FOR816dw ID:
21
Publication Date:
2021-01-01
License and Usage Rights:
Resource Owner(s):
Individual:
Florian Leese
Contact:
email:
florian.leese.ude <at> uni-due.de
University Duisburg-Essen
Fakultät für Biologie
Aquatische Ökosystemforschung
Universitätsstr. 5
45141 Essen
Individual:
Mandy Sander
Contact:
email:
webmaster <at> lcrs.de
Individual:
Dominik Buchner
Contact:
email:
webmaster <at> lcrs.de
Individual:
Vasco Elbrecht
Contact:
email:
webmaster <at> lcrs.de
Individual:
Peter Haase
Contact:
email:
webmaster <at> lcrs.de
Individual:
Vera M. A. Zizka
Contact:
email:
webmaster <at> lcrs.de
Abstract:
Abstract 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.
Keywords:
| bioassessment | bioindication | biomonitoring | COI | eDNA | insects | LTER | metabarcoding | primer bias |
Literature type specific fields:
ARTICLE
Journal:
Environmental DNA
Volume:
3
Issue:
1
Page Range:
261-276
Metadata Provider:
Individual:
Maik Dobbermann
Contact:
email:
dobberma <at> staff.uni-marburg.de
Laboratory for Climatology and Remote Sensing
Faculty of Geography
Deutschhausstr. 12
Philipps University of Marburg
Room 02-A46
35032 Marburg
Germany
