Environmental DNA tools to inform conservation of desert fishes at Ash Meadows National Wildlife Refuge

Authors: Dana Shellhorn, Caren Goldberg, Jesse Brunner, Mary Sterling, David Navarro, Ambre Chaudoin, Michael Schwemm, Kevin Guadalupe, Michael Bower

Year: 2024

Abstract

Ash Meadows National Wildlife Refuge (AMNWR) is an internationally important biodiversity reserve in the Mojave Desert that encompasses over 23,000 acres and contains multiple warm springs systems. These springs support populations of three federally endangered endemic fish: the Ash Meadows Amargosa Pupfish, Cyprinodon nevadensis mionectes, the Warm Springs Pupfish, C. n. pectoralis, and the Ash Meadows Speckled Dace, Rhinichthys osculus nevadensis. Restoration of the aquatic systems at Ash Meadows National Wildlife Refuge has been ongoing since the land was acquired in 1984, but these fishes are still threatened by established invasive fish populations as well as new introductions of released aquarium fish. The implementation of restoration and monitoring efforts is challenging due to the large number of springs and size of the refuge. Additionally, the spring systems at the refuge have unique invertebrate assemblages that are sensitive to introductions from species both outside and within the refuge, so any monitoring equipment must adhere to strict decontamination protocols. To help address these challenges, we are developing a multi-pronged approach using environmental DNA (eDNA) methods for detection and monitoring of aquatic species at the refuge. In this project, we developed and validated quantitative PCR (qPCR) assays for the three endemic fishes as well as invasive Green Sunfish, Lepomis cyanellus. Additionally, we are developing a metabarcoding workflow tailored to detecting 205 potential invasive fish species at the refuge including common aquarium pets, as well as other fishes. We will be employing these eDNA analysis tools using a pilot study to develop a rigorous framework for long-term surveillance of non-native fish invasions and monitoring of native fish status in the unique habitats found at the refuge. As part of the development of methods for using eDNA to inform restoration programs at the refuge, we conducted a live cage experiment in April 2024 to investigate eDNA transport in Crystal Spring, which has invasive Green Sunfish that impact the system’s Ash Meadows Amargosa pupfish population. We first cleared the experimental reach of Green Sunfish using hoop netting and snorkel surveys. Then we placed caged fish at the spring head for 24 hours before eDNA sampling, followed by 24 hours of undisturbed conditions between caged fish treatments. We collected eDNA samples every 80 m from the spring head to 400 m downstream in three conditions in the following order: control with no sunfish, cage with one sunfish, and cage with three sunfish. Samples were collected on-site using Smith-Root self-preserving filter packs. We extracted DNA from the samples in the lab and analyzed them in triplicate using the Green Sunfish qPCR assay. We found that the eDNA of Green Sunfish was detectable at water temperatures greater than 30⁰C and could be reliably detected up to 160 m downstream. We will use this information to characterize Green Sunfish distribution in the system during repeated eDNA sampling and Green Sunfish eradication efforts in 2025.