vernal pool
vernal pool

eDNA Resources to Monitor and Manage Vernal Pool Species in California

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.

The UC Reserve System’s Jepson Prairie Natural Reserve is one of the GVL’s field sites for this season. Vernal pools can be home to a huge variety of plant an animal species. This American Avocet is a native wading bird who nests on the banks of temporary pools like this one and eats the crustaceans and insects that live in it. Photo courtesy Amanda Coen.
America Avocet
Hawk awaits ground squirrel
A hawk waits patiently at the entrance to the burrow of a California Ground Squirrel. The open space and rolling landscape of vernal pool complexes provide an ideal place for ground squirrels to burrow.
While vernal pools can be very large in area, they tend also to be very shallow, rarely exceeding 4ft in depth.
Vernal Pool

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).

The Vernal Pool Tadpole Shrimp (L. packardi), with the California Tiger Salamander (CTS) and Conservancy Fairy Shrimp (CFS) in the background. Photo courtesy Amanda Coen 2017.
Vernal Pool Tadpole Shrimp
Branchinecta lynchi caught in a dip-net during a post-eDNA survey.
California Tiger Salamander larvae are regular vernal pool residents. Photo courtesy USFWS.
Vernal Pool Tadpole Shrimp, California Tiger Salamander and Conservancy Fairy Shrimp
Branchinecta lynchi under a microscope in the GVL laboratory.
Branchinecta lynchi

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.


GVL Undergraduate Student Henry Hwang prepares the eDNA Sampling rig





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.


Future Work

In addition to the species-specific and community eDNA analysis, this project has the overall goal of understanding the diversity and divergence of these species. Consequently, future work will be aimed at identifying and characterizing that diversity across the range of these species.


US Fish and Wildlife Service

US Bureau of Reclamation

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)

For more information contact Shannon Kieran: