DOI: 10.5061/dryad.m0cfxpp29
title: Sample site coordinates, environmental data, number of copies of target DNA/ul for each sample and limit of detection plot

description: Human activities in coastal areas are accelerating ecosystem changes at an
 unprecedented pace, resulting in habitat loss, hydrological modifications,
 and predatory species declines. Understanding how these changes
 potentially cascade across marine and freshwater ecosystems requires
 knowing how mobile euryhaline species link these seemingly-disparate
 systems. As upper trophic level predators, bull sharks (Carcharhinus
 leucas) play a crucial role in marine and freshwater ecosystem health.
 Telemetry studies in Mobile Bay, Alabama suggest that bull sharks
 extensively use the northern portions of the bay, an estuarine-freshwater
 interface known as the Mobile-Tensaw Delta. To assess whether bull sharks
 use freshwater habitats in this region, environmental DNA surveys were
 conducted during the dry summer and wet winter seasons in 2018. In each
 season, 5 x 1 L water samples were collected at each of 21 sites:
 five sites in Mobile Bay, six sites in the Mobile-Tensaw Delta, and ten
 sites throughout the Mobile-Tombigbee and Tensaw-Alabama Rivers. Water
 samples were vacuum-filtered, DNA extractions were performed on the
 particulate, and DNA extracts were analyzed with Droplet Digital™
 Polymerase Chain Reaction using species-specific primers and an internal
 probe to amplify a 237-base pair fragment of the mitochondrial NADH
 dehydrogenase subunit 2 gene in bull sharks. One water sample collected
 during the summer in the Alabama River met the criteria for a positive
 detection, thereby confirming the presence of bull shark DNA. While
 preliminary, this finding suggests that bull sharks use less urbanized,
 riverine habitats up to 120 km upriver during Alabama’s dry summer season.
description: 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.
description: All the information needed to use this dataset are provided in
 the Methods above.