Supporting Information for: “Monitoring Endangered Freshwater Biodiversity by Environmental DNA” Fig. S1. Accumulated probabilities of detecting the targeted species in the field studies when taking 1, 2 or 3 samples. The number of ponds positive for the respective species when considering all 3 samples is set to 100%. P. fuscus (blue, n=14), T. cristatus (red, n=10), L. pectoralis (black, n=9), M. fossilis in stagnant water (green, n=11), M. fossilis in running water (purple, n=8), L. apus (yellow, n=10) and L. lutra (brown, n=4). Fig. S2. fit on the DNA concentration in individual containers of the mesocosm experiment through time. PF1.1 denoting density of one individual of P. fuscus in replicate 1, TC4.2 denoting 4 individuals of T. cristatus in replicate 2 etc. Fig. S3. Modeled parameter estimates for A: DNA excretion and B: DNA degradation by the two species T. cristatus (TC) and P. fuscus (PF) in mesocosm experiment at densities of 1, 2 and 4 specimens pr. 80 l.
Fig. S4. QQ-plot for mesocosm experiments demonstrating fulfillment of the assumption of normality after log10 transformation. TC: T. cristatus; PF: P. fuscus. Field experiment Species Locality Conventional DNA Success rate qPCR replicates Coordinates Pelobates fuscus GRIB29 + + 1/3 4 N55.98225, E12.24362 GRIB30 + + 3/3 4,4,4 N55.98246, E12.24107 PFDE1 + + 2/3 3,3 N49.81814, E8.378260 + + 3/3 3,3,3 N49.82278, E8.374980 + + 3/3 4,3,3 N55.98929, E12.20933 + + 3/3 3,3,3 N55.99674, E12.15944
MAL1 - - 0/3 - N55.83233, E12.52472 Triturus cristatus JD1 + + 2/3 3,3 N55.78913, E12.57943 JD2 + + 2/3 4,4 N55.78860, E12.57722 JD3 + + 3/3 3,4,4 N55.78705, E12.56818 JD4 + + 3/3 4,4,4 N55.78702, E12.56808 JD5 + + 2/3 3,5 N55.79373, E12.56872 JD6 + + 2/3 3,3 N55.79307, E12.58687 JD7 + + 3/3 4,4,4 E12.57713 JD8 + + 3/3 4,4,4 E12.56059 JD9 + - 0/3 - N55.79574, E12.55170 JD10 + + 3/3 4,4,4 N55.79847, E12.54997 JD11 + + 2/3 4,3 N55.79799, E12.58399
controls BOT1 - - 0/3 - N55.68651, E12.57432 BRO1 - - 0/3 - N55.70701, E12.50857 MAL1 - - 0/3 - N55.83233, E12.52472 Misgurnus fossilis (ponds) KAR1 + + 3/3 3,3,3 N57.69979, E26.45889 MFP1 + + 3/3 3,3,3 N53.22960, E21.87083 MFP2 + + 3/3 3,3,3 E21.87074 MFP3 + + 3/3 3,3,3 N53.22970, E21.87130 MFP4 + + 2/3 3,3 N53.24053, E21.96304 MFP5 + + 2/3 3,3 N53.24008, E21.96298 MFP6 + + 3/3 3,3,3 N53.24576, E22.00651 MFP7 + + 3/3 3,3,3 N53.24590, E22.01087 MFP8 + + 2/3 3,3 N53.24710, E22.00922
MFP9 + + 1/3 3 N53.24729, E22.01024 MFP10 + + 3/3 3,3,3 N53.24295, E22.01768 Misgurnus fossilis (streams) SH2 ? + 3/3 3,3,3 N53.80837, E9.54235 SH3 ? + 1/3 3 N53.80003, E9.49517 SH4 ? + 1/3 3 N53.81951, E9.51973 SH5 ? - 0/3 - N53.82235, E9.49389 SH6 ? + 1/3 3 N53.84867, E9.49438 SH7 ? - 0/3 - N53.84240, E9.50423 SH8 ? + 1/3 3 N53.78150, E9.49425 SH10 ? - 0/3 - N53.76588, E9.51403 SH11 ? - 0/3 - N53.76135, E9.51103 SH12 ? - 0/3 - N53.76235, E9.46593
SKA7 + - 0/3 - N55.76740, E13.83848 SKA8 + + 1/3 3 N55.76749, E13.83607 SKA9 + + 1/3 5 N55.76749, E13.83607 SKA10 + + 1/3 4 N55.69911, E13.36750 SKA11 + + 2/3 4,4 N55.55074, E13.35331 + + 3/3 4,4,4 N55.53414, E13.26749 controls GRIB21 - - 0/3 - N55.98831, E12.18037 GRIB29 - - 0/3 - E12.24362 GRIB30 - - 0/3 - N55.98246, E12.24107 Lepidurus apus LEP1 + + 3/3 3,3,3 N53.24848, E21.96302 LEP2 + + 1/3 3 N53.24889, E21.96283 LEP3 + + 3/3 3,3,3 N53.24904, E21.96311
LEP4 + + 2/3 3,3 E21.91604 LEP5 + + 2/3 3,3 N53.23867, E21.99447 LEP6 + + 3/3 3,3,3 N53.22165, E22.05579 LEP7 + + 3/3 3,3,3 N53.23973, E21.99547 MFP8 + + 2/3 3,3 N53.24710, E22.00922 MFP9 + + 3/3 3,3,3 N53.24729, E22.01024 MFP10 + + 3/3 3,3,3 N53.24295, E22.01768 control DM1 - - 0/3 - N55.69999, E12.50484 HER1 - - 0/3 - E9.57437 HER6 - - 0/3 - N55.55949, E9.58360 lutra OD10 ? - 0/3 - N57.04169, E8.76599 OD12 ? + 2/3 3,3 N57.04839, E8.71714
OD13 ? - 0/3 - N57.04626, E8.71756 OD14 ? - 0/3 - E8.71738 OD15 ? - 0/3 - E8.71666 OD18 ? - 0/3 - N57.03394, E8.58624 OD2 ? - 0/3 - N57.10795, E8.91368 OD22 ? + 2/3 3,3 N57.03943, E8.75691 OD23 ? + 1/3 3 N57.04726, E8.72327 OD27 ? - 0/3 - N56.88909, E8.40157 OD28 ? + 1/3 3 N56.81362, E8.34860 OD6 ? - 0/3 - N57.07795, E8.81398 OD7 ? - 0/3 - N57.04404, E8.84741 OD8 ? - 0/3 - N57.04312, E8.84879 OD9 ? - 0/3 - E8.76587
Pelobates fuscus 2 1 3/3 4,3,4 2 2/21 4,4 4 3/3 4,4,4 9 1 3/3 4,4,4 2 2/21 4,4 4 3/3 4,4,4 23 1 3/3 4,4,4 2 2/21 4,4 4 3/3 4,4,4 44 1 3/3 4,4,4 2 2/21 4,4 4 1/12 8 64 1 3/3 4,4,4 2 2/21 4,4 4 1/12 10 After 66 1 1/3 3 2 2/21 4,3 4 1/12 4 73 1 0/3 - 2 0/21 - 4 0/12 - 79 1 0/3 - 2 0/21 - 4 0/12 -
Triturus cristatus CGTAAACTACGGCTGACTAGTACGAA 81 cyt-b CCGATGTGTATGTAGATGCAAACA CATCCACGCTAACGGAGCCTCGC Misgurnus fossilis MFCBL AGGTGGAGTCCTGGCCCTAT 70 cyt-b TTTTGAGGTGTGTAAGATGGGAACT MFCB.probe TTCTCTATCCTGGTCTTAATAG Leucorrhinia pectoralis LPCOIbL GCTTTCCCACGATTAAATAA 90 COI LPCOIbR TGCACCTCTTTCAACTATACTT ATAAGATTTTGACTTTTGCCTCC Lepidurus apus LACOIL TCGGATCCCCTTGTTTGTATG 85 COI LACOIR AGTGATGGCTCCTGCAAGGA TCTGTAGCAATCACAGCAT Lutra lutra LLCBL AAAGCCACCCTGACACGATT 80 cyt-b LLCBR AGCAGGTGGATTGTTGCTAGTG LLCB.probe TTCGCTTTCCACTTTAT Amphibians AmpCBLb AGTCCTGTTGGGTTGGTTGACCCNGTTT 119 cyt-b AmpCBR AATGCAACTCTCACCCGATTCTT Amphibians/Pelobates PFcytbL TCGCCCTTTCTCACAATTTC 80 cyt-b