We examined the potential utility of museum specimens as a source for genetic analysis of fairy shrimp. Because of loss of their vernal pool habitat, some fairy shrimp (including Branchinecta sandiegonensis and B. lynchi) are listed as threatened or endangered in Southern California by the United States Fish and Wildlife Service. Management of those species requires extensive population genetics studies and the resolution of important genetic complexity (e.g. possible hybridization between endangered and non-endangered species). Regulations mandating deposition of specimens of listed species have resulted in thousands of specimens accessioned into the Natural History Museum of Los Angeles County that have been preserved in a variety of solutions. We subsampled those specimens, as well as other Anostraca with known collection and preservation histories, to test their potential for genetic analysis by attempting DNA extraction and amplification for mt16SrDNA. Fixation and preservation in not denatured ethanol had a far greater sequencing success rate than other (and unknown) fixatives and preservatives. To maximize scientific value we recommend field preservation in 95% not denatured ethanol (or, if pure ethanol is unavailable, high-proof drinking spirits, e.g. Everclear™, or 151 proof white rum), followed by storage in 95% not denatured ethanol.

Museum specimens, Anostraca , Branchinectidae , Branchinecta sandiegonensis , B. lynchi , B. lindahli , endangered, threatened, vernal pool, California

The largest collection of endangered Southern Californian fairy shrimp in the United States of America is at the Natural History Museum of Los Angeles County (LACM). The LACM is working closely with the United States Fish and Wildlife Service (USFWS) to increase the scientific value of these specimens for both morphological and molecular studies. Fairy shrimp occur in ephemeral vernal pool habitats worldwide (Keeley and Zedler 1998). In densely human populated areas, their fragile habitats continue to be severely degraded and many have been destroyed by urbanization (Bauder and McMillan 1998, King 1998, Simovich et al. 2013).

At least 15 plant species are recognized as threatened or endangered in California vernal pool habitats, but only a few invertebrates are similarly recognized (USFWS 2005). Branchinecta conservatio, B. longiantenna, and B. sandiegonensis are listed as “Endangered”, and B. lynchi is listed as “Threatened” by the USFWS. In California, the USFWS issues permits for the collection of fairy shrimp and requires the deposition of endangered and threatened species in one of two repositories: the LACM or the California Academy of Sciences in San Francisco. Traditionally, Southern California specimens come to the LACM and northern California collections go to the California Academy. Since 1995 about 5,000 lots of B. lindahli, B. lynchi, and B. sandiegonensis have been accessioned into the LACM collections. This represents about 95% of our total anostracan holdings.

Simovich et al. (2013) suggest that human disturbance is increasing the generalist B. lindahli’s range, which in turn is eroding the native range of B. sandiegonensis. Due to increasing sympatric distribution of these species, these authors (and Fugate 1998 before them) claim that the endangered and non-endangered species (B. sandiegonensis and B. lindahli) are hybridizing, thereby threatening the genetic integrity and persistence of B. sandiegonensis. Using a PCR-based screen using mitochondrial DNA to determine maternal lineage, in conjunction with morphological examination, Simovich et al. (2013) claim putative hybrids share their maternal DNA with the more common species at a site. Unfortunately, their claims are not testable or reproducible as the specimens used in their study are unavailable. Aside from this study, only an unpublished master’s thesis exists that addresses genetic aspects of putatively hybrid populations of Southern California Branchinecta sandiegonensis (Andrews 2013). That study depended on prior researchers’ assessments of hybridization in individual pools. These claims of hybridization underscore the need for comprehensive molecular studies to characterize the actual genetic diversity and species boundaries of Southern California fairy shrimp before further management and remediation recommendations are made.

In contrast to the lack of work being conducted on endangered Southern Californian fairy shrimp, there has been a large amount of work studying the genetics and phylogeographics of the endangered Californian salamander Ambystoma tigrinum (Amphibia: Caudata: Ambystomatidae) (Ryan et al. 2009, Johnson et al. 2010, Johnson et al. 2011). These studies were made possible in large part by a very extensive collection of samples — tail clippings — of A. tigrinum that span the salamander’s geographic range through the last 25 years. Just as important as the breadth of the collection of tail clippings was that these samples were preserved with a method that made them accessible for molecular study decades later. The findings from these studies have already helped the management of A. tigrinum by identifying which populations have the greatest genetic diversity and allowing USFWS to target high value populations for increased protection (Johnson et al. 2011). The LACM is working closely with USFWS to assemble a collection of endangered Californian fairy shrimp necessary for similar genetic and phylogeographic studies. Both the LACM and USFWS fully expect that one day such studies will help better inform and shape the management of endangered fairy shrimp.

In this study we test whether preservation in pure not denatured ethanol makes anostracan museum specimens more readily accessible for molecular studies over anostracan museum specimens that had historically been fixed in denatured ethanol, isopropyl, or even acetone, then transferred into pure not denatured ethanol. Our study compares the success rates of amplifying a fragment of mt16SrDNA for specimens preserved in not denatured ethanol and for specimens in other preservatives. Because of their rarity and the difficulty in collecting fresh fairy shrimp specimens, being able to use specimens already in museum collections would be advantageous. To improve the utility of future collections, we suggest improvements in field and post-field preservation and handling based on our findings. If adopted, these improvements will greatly enhance the genetic usefulness of specimens and thereby allow more thorough assessments.

Of the 50 individual anostracan samples on which we attempted PCR amplification, 13 were known to have been fixed and preserved in pure 95% ethanol, and 37 samples had unknown preservation histories but were suspected of being fixed and stored in denatured ethanol sometimes for years, until they were incorporated into the LACM collection. Of the samples fixed and preserved in 95% ethanol, 62% (8 out of 13) yielded useable mt16SrDNA sequences. In contrast, of the samples with unknown fixative and preservative history, only 3% (1 out of 37) yielded useable mt16SrDNA. The nine sequences generated here are available on GenBank (see Table 2). Sequencing success between samples fixed and preserved in ethanol and other samples was significantly different (Fisher’s exact test, two-tailed, P < 0.0007).

The one-tailed Mann-Whitney U Test showed that there was a difference (at the α = 0.05 level) between Qubit measurements of double-stranded DNA concentration for successful sequences vs. failed sequences, when amplifications of contaminants were considered as failed amplifications. However, direct examination of the data (see Table 1) showed that DNA concentration was a very poor predictor of sequencing success (except for the case of 0 or near-0 readings, which invariably failed).

We thank Susie Tharratt, Jonathan Snapp-Cook, and Julie Vanderwier (United States Fish and Wildlife Service) for their encouragement, support, and liaison in increasing community engagement resulting in improved specimen preservation and documentation. The fruits of their labor benefit the amazing animals and fragile habitats for which these regulations were designed. Two anonymous reviewers, Joel Martin, and Dean Pentcheff contributed helpful comments from which this manuscript greatly benefited. University of Southern California students Mark Floro, Julia Garcia, Janie Chen, Christina Li, and Harleen Marwah curated, inventoried, and documented the LACM fairy shrimp collections as part of their undergraduate training at the LACM. Kathy Omura, Dean Pentcheff, and Phyllis Sun are thanked for the countless hours of student training they contributed in making natural history tangible to the many students passing through our lab.