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eDNA Resources to Monitor and Manage Vernal Pool Species in California

Using environmental DNA (eDNA) to develop efficient survey protocols that will help to identify and protect vernal pool habitats of high conservation value.

eDNA Resources to Monitor and Manage Vernal Pool Species in California

Previous Work

Background and Significance of Study

Vernal pools in California support diverse assemblages of species, including waterfowl, amphibians, endemic plants, insects, and at least 67 species of crustaceans (most of which are endemic and rare). Among the crustaceans are vernal pool branchiopods, known commonly as “fairy shrimp”. There are approximately 25 species of branchiopods in California, eight of which are endemic to California. Californian Vernal pool branchiopods are extremely vulnerable to the loss and alteration of vernal pool habitat, and are at risk of further declines due to the effects of climate change.

Efforts to conserve rare and endangered vernal pool branchiopods depend on prioritizing conservation of remaining vernal pool habitats, with the goal of establishing a network of functioning vernal pool ecosystems that supports rare and endangered species and maximizes protection of biodiversity. To achieve this goal, managers need information about 1) which vernal pool habitats support rare and endangered species; 2) which are associated with increased biodiversity; and 3) which biotic characteristics are associated with listed species and/or increased biodiversity. One way of gaining this information is to develop a method of habitat assessment that can rapidly determining the presence of rare and endangered species and associated biodiversity (such as surrogate species, or those that are common and easily identified that may indicate the presence of listed or rare species). This project will use advances in the analysis of environmental DNA (eDNA) to develop efficient survey protocols that will help to identify and protect vernal pool habitats of high conservation value.

What is environmental DNA?

All organisms leave genetic material behind in their environments throughout their lives. The genetic material can originate as hair, skin cells, blood, reproductive material, or tissue. Scientists have recently acquired genetic material of target organisms from soil, water, fecal matter, and the gut contents of predators (Koizumi et al. 2016; Fahner et al. 2016; Valentini et al. 2015; Brandl et al. 2015). We call this genetic material “environmental DNA” because it comes from the environment rather than from the organism. Environmental DNA can come from many sources and many types of environment. So far, water has been the most common environment for eDNA research.

Why vernal pools?

Vernal pools are a great system to test the utility of environmental DNA. In this project, we are filtering vernal pool water to detect six target vernal pool denizens: Branchinecta lynchi, the Vernal Pool Fairy Shrimp (Threatened); Branchinecta conservatio, the Conservancy Fairy Shrimp (Endangered), Branchinecta mesovallensis, the Midvalley Fairy Shrimp, Linderiella occidentalis, the California Fairy Shrimp, Lepidurus packardi, the Vernal Pool Tadpole Shrimp (Endangered), and Ambystoma californiense, the California Tiger Salamander (Endangered).

Various combinations of these six species co-occur in vernal pools throughout California’s Central Valley. This project is developing molecular assays (that is, species-specific genetic markers that can be tested to determine species presence) for all six species. To do so, we have sequenced specific genes (Cytochrome Oxidase I [COI], the 16s ribosomal RNA gene, and the 28S ribosomal RNA gene) from these species and other, non-target species that live with them. We have found specific unique sites in their genome for each species and designed a qPCR assay around those sites.

Materials and Methods

We have gone out to vernal pool complexes throughout California to test our assays using vernal pool pond water.

At each pool, we filter a small amount (250-500mL) of pond water over a sterile, single-use cellulose nitrate or glass fiber filter. We do this three times at each pool, and also take a negative field control – clean, sterile water that we carry out with us filtered in the same method as the sample filters. After filtration, we do a visual dip-net survey in order to compare results.


83 pools have been sampled during the 2015-2016 and 2016-2017 wet seasons. Most were sampled more than once during the season. In total, there 130 sampling events, and 390 sample filters (plus 130 field negative control filters!)

We have extracted the DNA from these sample and control filters and run the assay for each species to compare the results between visual and molecular surveys. Early results suggest that our assays have strong concordance with visual surveys, and may be sensitive enough to detect target organisms earlier and later in the year than visual surveys are able to do.

This is an exciting project that has the potential to add an entirely new management tool for these delicate and vulnerable organisms! Stay tuned to this page for project updates and publications.


For more information contact Shannon Kieran:

US Fish and Wildlife Service

US Bureau of Reclamation

Current Work 

Project Background & Significance

California vernal pools are ephemeral waterbodies that support a vast diversity of endemic flora and fauna through distinct seasonal phases. Many specially adapted organisms, such as the endangered vernal pool fairy shrimp and California tiger salamander, inhabit these ecosystems during the wet season in fall and winter. While over 200 species of plants thrive in vernal pools, rare and endemic plants like the vernal pool goldfields bloom when the pools begin to dry during the spring and summer. This project aims to assist federal land managers and biologists by using genetic techniques to establish and improve biological inventory and monitoring methods for vernal pools within national wildlife refuges. The Identification of many different types of organisms, from tiny invertebrates to various wildflowers, requires specialized training and devoted resources. By leveraging environmental DNA from vernal pools and using metabarcoding, this study will inventory species across taxa groups, specifically for species of management concern, over multiple years. Results will include characterizing biodiversity through time for natural and man-made pools, development of a localized reference sequence database, and rapid in-field identification of eDNA using CRISPR-based technology, or SHERLOCK, as a monitoring tool for refuge biologists.


Graduate Student: Anderson Tate 

This work is done in collaboration with the U.S. Fish and Wildlife Service and the National Wildlife Refuge System.

More Information:


Deiner, K, J.M. Hull, and B. May. 2013. Eight novel microsatellite loci developed from vernal pool fairy shrimp. J. Fish & Wild. Man. 4:134-138. (pdf)

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