“…Based on literature reports for species diversity and known species ranges, we defined a list of 161 amphibian species that could be detected in our aquatic eDNA samples and known to occur either presently or historically at each sampling site (Almeida, Gasparini, & Peloso, 2011;Folly et al, 2016 2017). We amplified the 12s rRNA mitochondrial fragment using the primers MVZ59 (5′-ATAGCACGTAAAAYGCTDAGATG-3′) (Graybeal, 1997); tVal (5′-TGTAAGCGARAGGCTTTKGTTAAGCT-3′) (Wiens, Fetzner, Parkinson, & Reeder, 2005); 12SA-L (5′-AAA CTGGGATTAGATACCCCACTAT-3′) (Palumbi, Martin, McMillan, Stice, & Grabowski, 1991); 12SF-H (5′-CTTGGCTCGTAGTTC CCTGGCG-3′) (Goebel, Donnelly, & Atz, 1999); 12S-H978 (5′-CTTA CCRTGTTACGACTTRCCT-3′) and 12S L148 (5′-ATGCAAGYMT CMGCRYCCCNGTGA-3′) (Walker, Lyra, & Haddad, 2018), following previously published amplification conditions (Faivovich et al, 2004;Walker et al, 2018).…”