Quantitative PCR
For C. sivickisi , the RT-PCR analysis was based on a TaqMan hydrolysis probe (Cop_siv_16S_P - 5’-AACTATTCGCCTCAC-3’) assay (Table 1). Each qPCR reaction was run on a QuantStudio 5 Real-Time PCR machine (Thermo Fisher Scientific) and consisted of 5.0 μL 1x TaqPath, 0.9 μM Copula_16S_F (Forward), 0.9 μM Copula_16S_R (Reverse), 0.25 μM Copula_16S_P probe, 2.5 μL sample and 0.7 μL of MilliQ for a final reaction volume of 10 μL. Results were obtained using optimised thermocycling for 40 cycles for 5 min at 95° C, 30 s at 95° C, 15 s at 60° C.
For all other species (C. fleckeri, C. xaymacana and C. barnesi ), eDNA detection was via a SYBR Green Power Up assay, with each reaction consisting of 1.4 μL MilliQ, 10 μL 2x PowerUp SYBR Green, 0.9 μM Forward primer, 0.9 μM Reverse primer, 5 μL sample for a final reaction volume of 20 μL. This reaction volume was chosen due to the lower specificity when compared to the hydrolysis probe and ran at optimised thermocycling for 50 cycles of 2 min at 50 °C, 2 min at 95 °C , 15 s at 95 °C, 1 min at 65 °C, utilising a Melt Curve of 15 s at 95 °C, 1 min at 60 °C, 15 s at 95 °C. Each sample was run in triplicate. Water samples that produced a PCR product were sent for Sanger sequencing (AGRF, Brisbane, Australia) to confirm the species-specificity of the resulting PCR product. For all species, amplification of a single technical replicate deemed the sample replicate as a positive detection.