Sample site coordinates, environmental data, number of copies of target DNA/ul for each sample and limit of detection plot

Water samples were collected at a series of estuarine, deltaic, and freshwater sites in Alabama once in the wet winter season (February 19–20, 2018) and once in the dry summer season (August 21–22, 2018). In total, five estuarine sites were sampled from Dauphin Island to the northern extent of Mobile Bay, six sites were sampled within the MTD, and ten freshwater sites were sampled in two river systems: the Mobile-Tombigbee and the Tensaw-Alabama. In each river system, sites spanned ~190 km north of the mouth to just south of the Coffeeville Lock and Dam and Claiborne Lock and Dam in the Tombigbee and Alabama Rivers, respectively (Fig. 1A). All sampling sites were spaced 15–25 km apart (Fig. 1A). At each site, 5  1 L water samples and environmental data, including temperature (C°), salinity (psu), dissolved oxygen (mg/L), and depth (m), were collected approximately 0.5 m below the surface of the water. While bull sharks are known to use the entire water column, previous studies have identified surface water sampling as the most suitable method for eDNA collection in headwaters (Katano et al. 2017). In Mobile Bay, water samples were collected in proximity to structures (e.g., oil rig, lighthouse), and at all riverine sites, samples were collected across the width of the river. All eDNA field and laboratory protocols and controls followed Schweiss et al. (2020), and new gloves were used at each sampling site. Water samples were collected in the field using sterile, 1 L high-density polyethylene Nalgene® bottles and stored on ice in clean marine coolers, or frozen, until water filtration (see Schweiss et al., 2020). In addition to the filtration, DNA extraction, and PCR negative controls described in Schweiss et al. (2020), negative collection controls were also included. The negative collection controls consisted of autoclaved deionized water, which was taken onto the boat and placed in a clean marine cooler on ice with field samples to test for field contamination (e.g., Jerde et al., 2011). All negative control samples (collection, filtration, DNA extraction, and PCR) were processed and analyzed in replicates of five, according to the protocols of Schweiss et al. (2020), and were defined as negative if they did not meet any of the criteria for positive detections. Water samples were vacuum-filtered in a laboratory using 47-mm-diameter, 0.8-μm nylon filters (Cole Parmer®, Illinois, USA) and preserved in 95% ethanol at room temperature (see Schweiss et al., 2020). Total eDNA was extracted from ¼ of each filter following the Goldberg et al. (2016) QIAGEN® DNeasy® Blood & Tissue Kit protocol incorporating the QIAshredder™ spin columns. A species-specific bull shark Droplet Digital™ PCR (ddPCR™) assay was used to target a 237-base pair fragment of the mitochondrial NADH dehydrogenase subunit 2 (mtDNA ND2) gene using the reaction mixtures and ddPCR™ cycling conditions described in Schweiss et al. (2020). Five replicates (5% of the total eDNA extract) were run for each sample on the Bio-Rad® QX200™ AutoDG™ Droplet Digital™ PCR System (Droplet Generator instrument no. 773BR1456, Droplet Reader instrument no. 771BR2544) platform. Positive detections were defined as samples with at least one ddPCR™ replicate that met all three analysis criteria: 1) droplets were above the manual threshold of 3,000 amplitude, 2) droplets were within the known positive droplet range for the target species (e.g., 4,500–6,000 amplitude), and 3) the concentration (copies/μL) was greater than or equal to the refined Limit of Detection (LoD) of 0.09 copies/μL for the assay, using the Rare Event Detection (RED) analysis in Bio-Rad® QuantaSoft™ software.