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When the vas t majorit y of DNA in the sampl e is no ntarget DNA, univers al p rimer s for COI will amplif y a maj ority of COI fra gment s fro m no ntarge t or ganisms even if prime rs d o no t match well.

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 coamplification 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 BF2BR2 on samples collected throughout 15 months from a German LongTerm Ecological Research site (RhineMainObservatory, Kinzig River) to identify most abundant nontarget taxa.

Based on these data, we designed a new reverse primer (EPTDr2n) with 3specificity 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, BF2BR2 and fwhF2fwhR2n (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.

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The advanta ge of COI is its taxon omic re solution and the availability of an extensive reference database.

However, when universal primers are used on environm ental D NA (eDNA ) isolated f rom water, bent hic inver tebrat e read and OTU numbers are typical ly watered down, that is, under represente d, compared to whole specimen b ulk sampl es due to greate r co-amplific ation of abun dant nont arget tax a (e.g., fungi, a lgae, and ba cteria).

Because benthic stream invertebrate taxa a re of prime impor tance for regu latory biom onitoring, more effect ive ways to capture their diversit y vi a eD NA is olated from water are impor tant.

In this study, we ai med to improve benthi c i nvertebr ate assessme nt from eDNA by mini mizing nonta rget am- plificat ion.

Therefore, we generated eDNA dat a u sing universal primers BF2BR2 on samp les coll ected throughout 15 months from a German Long-T erm Ecologic al Research site (Rhine-Main- Obser vator y, Kinzig River) to identify most abundant non- target t axa.

B ased on these dat a, we d esigned a new revers e prime r (EPTDr2n) w ith 3-specificit y toward be nthic inver tebrate t axa and validated its specificit y in silico together with u niversal forward pr imer f whF2 using ava ilable data from GenBank and BOLD.

W e then pe rform ed in sit u test s using 20 Kinzig River e DNA sam ples.

W hile few t axa, su ch as flatworms were not detected, we show that the optimized primer avoids the nontar - get amplifi cation bia s and thus s ignifica ntly improve s benthic i nvertebr ate detect ion from eDNA.

The refore, t he major ity of rea ds are on-ta rget ver tebr ate read s.

Als o owed to this f act, eDNA-ba sed assessm ents for fish and amphibians are in principle at the level of routine application.

The advant age of COI is its ta xonomic reso lution and the avai labilit y of extensi ve refere nce dat a (An djar et al., 2018; Cla rke et al., 2017; Ratnasin gham Heb ert, 2007; Weigand et a l., 2019).

However, in contras t to 12S or 16S, COI as a protein-co ding gene show s codon degenerac y that limits taxon-spec ific primer design (Clarke et al., 2014; Deagl e et a l., 2014; Sh arma Kobayashi, 2014) maki ng it almos t impossi ble so fa r to desig n target-spe cific primers for di- verse gro ups such as inver tebr ates but also f ish and amphibians.

Elbrecht and Leese (2017a) proposed an approach using degenerate primer s fo r sp ecific ta xon g roups or taxa of a s pecifi c geo graphi cal region, depe nding on the rese arch o r appl ied qu estio n.

It should be noted, however, that primer degeneracy is no main concern when metaba rcoding is used for bul k t issue sampl es, as this biodi versit y soup ( Yu et al., 2012) mostl y contai ns DNA of t he tar get orga nisms (Elbrecht et al., 2016, 2017).

Sam ples are dominate d by DNA fro m living bacteria, fu ngi, phy toplan kton, and other nont arget DNA.