BackgroundAs apex and mesopredators, elasmobranchs play a crucial role in maintaining ecosystem function and balance in marine systems. Elasmobranch populations worldwide are in decline as a result of exploitation via direct and indirect fisheries mortalities and habitat degradation; however, a lack of information on distribution, abundance, and population biology for most species hinders their effective management. Environmental DNA analysis has emerged as a cost‐effective and non‐invasive technique to fill some of these data gaps, but often requires the development of species‐specific methodologies.AimsHere, we established eDNA methodology appropriate for targeted species detections of Bull Sharks, Carcharhinus leucas, in estuarine waters in the northern Gulf of Mexico.Materials and MethodsWe compared different QIAGEN®DNeasy® extraction kit protocols and developed a species‐specific Droplet Digital™ PCR (ddPCR) assay by designing primers and an internal probe to amplify a 237 base pair portion of the ND2 gene in the mitochondrial genome of C. leucas. To validate the developed methods, water samples were collected from known C. leucas habitat and from an ex situ closed environment containing a single C. leucas individual. The effectiveness of the assay in an open environment was then assessed by placing one C. leucas into a flow‐through mesocosm system and water samples were collected every 30 min for 3 hr.ResultsThe developed C. leucas ‐specific assay has the ability to detect target DNA concentrations in a reaction as low as 0.6 copies/μl. DdPCR reactions performed on water samples from known habitat and 30 min after a shark was added to the closed environment contained 1.62 copies/μl and 166.6 copies/μl of target C. leucas eDNA, respectively. Carcharhinus leucas eDNA was detected in the flow‐through system within 30 min, but concentrations remained low and variable throughout the duration of the experiment.

The Critically Endangered Smalltooth Sawfish, Pristis pectinata, was once widespread in the tropical and subtropical waters of the Atlantic Ocean, but following substantial declines over the past century, the core population is currently confined to southwest Florida in the U.S. and the Bahamas. Recent research and verified public encounter reports suggests that this core population may be stabilizing and, potentially, expanding into formerly occupied areas of their historic range in the Western Atlantic; however, the status of this species in noncore waters is not well understood. Environmental DNA (eDNA) methods provide a relatively cost effective and rapid assessment tool for monitoring species occurrence in aquatic habitats. Here, we have developed an eDNA tool: a species-specific Droplet Digital™ PCR (ddPCR™) assay targeting a 100-base pair portion of the mitochondrial NADH dehydrogenase subunit 2 gene in P. pectinata, with the ability to reliably detect as little as 0.25 pg of target DNA. The assay was validated by collecting and analyzing a water sample from known P. pectinata nursery habitat in Florida, which was found to contain an average of 11.54 copies of target DNA/µL (SE = 0.72) in the reaction. The assay was then further tested by placing a juvenile sawfish in an ex situ tank and analyzing water samples collected at time intervals. The implementation of this eDNA tool into field surveys will provide additional, reliable data to assess species recovery and aid in prioritizing localities beyond the core range in which to focus research and education initiatives.

AbstractThe Critically Endangered Smalltooth Sawfish, Pristis pectinata, was once widespread in the tropical and subtropical waters of the Atlantic Ocean, but following substantial declines over the past century, the core population is currently confined to southwest Florida in the U.S. and the Bahamas. Recent research and verified public encounter reports suggests that this core population may be stabilizing and, potentially, expanding into formerly occupied areas of their historic range in the Western Atlantic; however, the status of this species in non-core waters is not well understood. Environmental DNA (eDNA) methods provide a relatively cost effective and rapid assessment tool for monitoring species occurrence in aquatic habitats. Here, we have developed an eDNA tool: a species-specific Droplet Digital™ PCR (ddPCR™) assay targeting a 100-base pair portion of the mitochondrial NADH dehydrogenase subunit 2 gene in P. pectinata, with the ability to reliably detect as little as 0.25 pg of target DNA. The assay was validated by collecting and analyzing a water sample from known P. pectinata nursery habitat in Florida, which was found to contain an average of 11.54 copies of target DNA/µL (SE = 0.72) in the reaction. The assay was then further tested by placing a juvenile sawfish in an ex situ tank and analyzing water samples collected at time intervals. The implementation of this eDNA tool into field surveys will provide additional, reliable data to assess species recovery and aid in prioritizing localities beyond the core range in which to focus research and education initiatives.

1. Formerly common in tropical and subtropical waters of the Atlantic Ocean, the Critically Endangered smalltooth sawfish, Pristis pectinata, underwent severe declines over the past century, restricting population(s) to south and south-west Florida in the US, and Bahamian waters.2. Anecdotal evidence (e.g. encounter reports from the public) suggests that P. pectinata have recently been observed in historically occupied US waters; however, no directed surveys have been conducted to verify their extent of occupancy.3. Here, environmental DNA (eDNA) surveys were used to investigate the occurrence of P. pectinata in three formerly occupied estuaries in US waters.Water samples were collected in the summer from the Indian River Lagoon and

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