Corresponding author: Adam R. Wall (
Academic editor: I.S. Wehrtmann
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
Wall AR, Campo D, Wetzer R (2014) Genetic utility of natural history museum specimens: endangered fairy shrimp (Branchiopoda, Anostraca). In: Wehrtmann IS, Bauer RT (Eds) Proceedings of the Summer Meeting of the Crustacean Society and the Latin American Association of Carcinology, July Costa Rica, July 2013. ZooKeys 457: 1–14. doi:
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 (
At least 15 plant species are recognized as threatened or endangered in California vernal pool habitats, but only a few invertebrates are similarly recognized (
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
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.
We first inventoried, digitized, and georeferenced our entire anostracan collection — approximately 5,000 lots. We selected 50 specimens from across the taxonomic range that had been contributed by different collectors and consulting companies using a range of different field preservatives prior to deposition at the LACM (at the LACM, all specimens are transferred from the field preservative into fresh museum-grade not denatured ethanol). We then attempted to amplify a ~550 bp mt16SrDNA fragment (see Table
Extractions and amplifications attempted for this publication. Taxa arranged in alphabethical order. Locality, specimen collection date, collector, and preservative are as transcribed from specimen labels. Specimen condition and body part used in extraction are indicated if this information was recorded. Double-stranded DNA concentration in ng/µL. Qubit value indicated as low,
Taxon | Date of collection | Description of preservative on label | Locality | Collector | Part of specimen used | Extraction number | Outcome | dsDNA ng/µL 0<0.05 | |
---|---|---|---|---|---|---|---|---|---|
1 |
|
06-Jul-90 | 70% ethanol | California, Mono County, Mono Lake, south Tufa Reserve | H. Kuck | 1 broken specimen | 2013 | contaminated; blasts as |
0 |
2 |
|
01-Jan-10 | fixed and preserved in 95% ethanol | California, Mono County, Mono Lake | M. Hauser | 1 whole squished specimen | 2008 | *beautiful sequence | 39.1 |
3 |
|
23-Apr-92 | 70% ethanol | California, Lassen County, Hog Flat Reservoir | King, Gluesenkamp, Tritt | 1 broken specimen | 2003 | failed | 0 |
4 |
|
23-Mar-92 | 70% ethanol | California, Shasta County, Fall River | King, Gluesenkamp, Kloock | 2 broken pieces | 2017 | failed | 0.2 |
5 |
|
unknown | acetone | California, San Bernadino County, Mojave Desert | J. Martin, J. Plum | 2 phyllopods only | 1990 | failed | 0.17 |
6 |
|
unknown | not indicated | Washington, Grant County | unknown | 1 small whole specimen | 2006 | failed | 0.13 |
7 |
|
unknown | not indicated | Washington, Grant County | unknown | dissected off egg sack with eggs | 2007 | failed | 0 |
8 |
|
27-Dec-12 | fixed and preserved in 95% ethanol | California, San Diego County, Marine Corps Base Camp Pendleton | L. Woolley | not recorded | 2036 | failed | no data |
9 |
|
27-Dec-12 | fixed and preserved in 95% ethanol | California, San Diego County, Marine Corps Base Camp Pendleton | A. Fisher | not recorded | 2037 | failed | no data |
10 |
|
29-Dec-12 | fixed and preserved in 95% ethanol | California, San Diego County, Marine Corps Base Camp Pendleton | A. Fisher | not recorded | 2038 | failed | no data |
11 |
|
28-Dec-11 | fixed and preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook, et al. | egg sac only | 1992 | *beautiful sequence | 6.62 |
12 |
|
02-Apr-12 | fixed and preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook | 1 gravid female | 2026 | *beautiful sequence | 13 |
13 |
|
02-Apr-12 | preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook | 1 gravid female | 2027 | *beautiful sequence | 11.3 |
14 |
|
02-Apr-12 | preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook | 1 gravid female | 2034 | failed | 0 |
15 |
|
02-Apr-12 | preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook | 1 squished male | 2028 | *beautiful sequence | 29 |
16 |
|
23-Mar-10 | preserved in 70% ethanol | California, San Luis Obispo County, California Valley | Chris Powers | posterior half of single broken specimen | 2005 | failed | 39.6 |
17 |
|
27-Feb-01 | fixed and preserved in 95% ethanol | California, San Luis Obispo County, Paso Robles | M. Dallas | 1 specimen, not gravid, not obviously male | 2032 | contaminated; blasts as cladoceran | 18.7 |
18 |
|
13-Jan-04 | fixed and preserved in 95% ethanol | California, Santa Barbara Co., Los Padres National Forest | T. Murphey | squished gravid female | 2030 | failed | 0.3 |
19 |
|
03-Feb-05 | fixed and preserved in 95% ethanol | California, San Luis Obispo County | D. Hacker | posterior half of gravid female | 2033 | failed | 51.4 |
20 |
|
17-Feb-05 | fixed and preserved in 95% ethanol | California, Santa Barbara Co., Los Padres National Forest, Branch Mountain Quad | T. Murphey | squished gravid female; all animals in this lot are pretty mangled | 2031 | failed | 17 |
21 |
|
unknown | 70% ethanol | Washington, Grant County | unknown | 1 specimen | 1991 | failed | 0.254 |
22 |
|
03-Apr-93 | 70% ethanol | California, San Bernadino County, Mojave Desert | C. Cash-Clark, T. Clark | 1 specimen | 2019 | failed | 0.16 |
23 |
|
22-Aug-02 | 95% ethanol | Mongolia, Dundgovi’ aimag, near Sangiyn Dalay (Erdenedalay) | R. Wetzer, S.L. Boyce, N.D. Pentcheff | 1 whole small specimen | 2004 | failed | 24.4 |
24 |
|
09-Mar-05 | preserved in 70% denatured ethanol, transferred to 70% ethanol | Mexico, Baja California, Tijuana, Jesus Maria Mesa | K.B. Clark | 1 gravid female | 2024 | failed | 0.7 |
25 |
|
13-Jan-11 | preserved in 70% denatured ethyl alcohol, transferred to 70% ethanol | California, San Diego County, Brown Field Municipal Airport | D. Wolff | posterior half of gravid female | 2029 | failed | 0 |
26 |
|
24-Nov-08 | preserved in 95% ethanol | California, San Diego County, Ramona Water District, Ramona Spray Fields | E. Ervin | eggsac + furca from female | 2023 | failed | 4.7 |
27 |
|
28-Dec-11 | preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook, et al. | anterior portion of female specimen | 1995 | failed | 25.7 |
28 |
|
17-Dec-07 | transferred to 95% ethanol Feb. 2011 | California, San Diego County, Otay Mesa, Dexstar Property | C. Powers | 1 male | 2025 | failed | 49.2 |
29 |
|
28-Dec-11 | preserved in 95% ethanol | California, San Diego County, San Diego, Carmel Mountain Preserve | J. Snapp-Cook, et al. | 1 specimen | 1993 | failed | 7.5 |
30 |
|
22-Aug-02 | 95% ethanol | Mongolia, Dundgovi’ aimag, near Sangiyn Dalay (Erdenedalay) | R. Wetzer, S.L. Boyce, N.D. Pentcheff | 1 small specimen | 2001 | failed | 18.8 |
31 |
|
22-Aug-02 | 95% ethanol | Mongolia, Dundgovi’ aimag, near Sangiyn Dalay (Erdenedalay) | R. Wetzer, S.L. Boyce, N.D. Pentcheff | 1 small specimen | 2002 | failed | 49 |
32 |
|
22-Aug-02 | 95% ethanol | Mongolia, Dundgovi’ aimag, near Sangiyn Dalay (Erdenedalay) | R. Wetzer, S.L. Boyce, N.D. Pentcheff | 1 whole squished animal | 2018 | *beautiful sequence | 57.9 |
33 |
|
07-May-40 | 70% ethanol | Canada, Nova Scotia, Edinberg [sic] | D. Belk | 1 male specimen | 2015 | contaminated; blasts as |
0 |
34 |
|
01-Apr-32 | 70% ethanol | Canada, Ontario, Saint Thomas | M.S. Ferguson | anterior end of broken specimen | 2014 | failed | 0 |
35 |
|
30-Apr-99 | 70% ethanol | Minnesota, Bloomington | A.B. Forbes | 1 female – doesn’t look well preserved | 2022 | failed | 0 |
36 |
|
15-May-12 | fixed and preserved in 95% ethanol | California, Lassen County, Poison Lake | M. Hauser, D. Striley | posterior half of the single mushy specimen | 2020 | *beautiful sequence | 27.3 |
37 |
|
19-Feb-92 | 70% ethanol | California, Tehama County, Tuscan Buttes | King, Mazzucco, Scuderi | 2 pieces broken specimen | 2000 | contaminated; blasts as |
0.14 |
38 |
|
24-Mar-92 | fixative unknown – transferred to 70% ethanol | California, Tehama County, Dale’s Plains, Dale’s Lake | King, Gluesenkamp, Kloock | 1 whole specimen | 1987 | failed | 0.225 |
39 |
|
26-Mar-04 | 70% ethanol | California, Riverside County, Murrieta, Mesa de Colorado, Santa Rosa Plateau | M. Angelos | 1 small female specimen | 1999 | contaminated; blasts as |
0.293 |
40 | not identified | 08-Jun-11 | fixed and preserved in 95% ethanol | Utah, Wallsburg, near Provo-Jordan River Pkwy | M. Hauser | 1 female specimen | 2021 | failed | 47.6 |
40 |
|
22-Aug-02 | 95% ethanol | Mongolia, Dundgovi’ aimag, northwest of Delgerhangay (Khashaat/Delger Khanay Uul) | R. Wetzer, S.L. Boyce, N.D. Pentcheff | posterior half of adult specimen | 2009 | *beautiful sequence | 10.3 |
42 |
|
13-Apr-36 | 70% ethanol | Tennessee, Reelfoot Lake | unknown | 1 specimen, this lot had previously dried and had been realcoholed | 2012 | failed | 0 |
43 |
|
15-Aug-55 | 70% ethanol | California, Tulare County, Yosemite, Tioga Pass | unknown | 1 specimen, these had been previously dried and realcoholed | 1998 | failed | 0.213 |
44 |
|
15-Aug-55 | 95% ethanol | California, Mariposa County, Yosemite, May Lake Trail | unknown | posterior end of animal | 1989 | failed | 0.18 |
45 |
|
27-Aug-56 | 70% ethanol | New Mexico, Cain Ranch | S.F. Wood | dissected egg sack | 2010 | failed | 0 |
46 |
|
30-Mar-06 | 70% ethanol | California, San Diego County, Camp Pendleton, Marine Corps Base | S. Baldwin | ~5 phyllopods dissected off single specimen (only 1 specimen in the lot) | 1994 | failed | 7.28 |
47 |
|
01-Apr-05 | not recorded | California, San Diego County, Carlsbad, Poinsettia Lane Commuter Station Vernal Pools | J. Snapp-Cook | posterior half of male (already broken) | 2016 | *good sequence | 16 |
48 |
|
29-Jan-03 | preserved in 70% ethanol | California, Riverside County, Temecula | unknown | 3-4 phyllopods removed from single specimen | 1997 | failed | 6.8 |
49 |
|
12-Aug-34 | 70% ethanol | Denmark, Raabjerg Mile | E.W. Kaiser | 3 broken pieces used | 1996 | failed | 0.224 |
50 |
|
01-Aug-56 | 70% ethanol | New Mexico, Gran Quivira | S.F. Wood | posterior portion | 2011 | failed | 0 |
The starting material for DNA extractions varied among samples, one thoracopod to an entire animal, depending on total animal body size. Tissue samples were placed on paper towel to dry. Precipitation Reagent (Epicentre MMP03750) was added to each sample and vortexed vigorously for 10 sec., then centrifuged at 4 °C for 10 min. at 14,000 rpm. The supernatant (~300 µl) was transferred to a 2 ml tube. Genomic DNA was extracted and purified with a Quick-gDNA™ MiniPrep Kit (Zymo Research) following the manufacturer’s instructions, and eluted in a final volume of 60 µl of distilled water (in two elutions of 30 µl). Double-stranded DNA concentration of extractions was quantified using a Qubit 1.0 Fluorometer (Life Technologies) (see Table
The mt16SrDNA fragment was amplified with universal 16Sar and 16Sbr primers (
Sequences were edited and contigs assembled in the software program Sequencher (
A Fisher’s exact test (two-tailed, α=0.05) was used to determine whether there was a statistically significant difference in sequencing success between the ethanol-preserved and other samples (
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
Nine new mt16SrDNA
Genus/species | Genbank No. | Locality |
---|---|---|
|
USA, California, Mono County, Mono Lake, |
|
|
USA, California, San Diego County, San Diego, Carmel Mountain Preserve, |
|
|
USA, California, San Diego County, San Diego, Carmel Mountain Preserve, |
|
|
USA, California, San Diego County, San Diego, Carmel Mountain Preserve, |
|
|
USA, California, San Diego County, San Diego, Carmel Mountain Preserve, |
|
|
Mongolia, Dundgovi’ aimag, near Sangiyn Dalay (Erdenedalay), |
|
|
USA, California, Lassen County, Poison Lake, |
|
|
USA, California, San Diego County, Carlsbad, Poinsettia Lane Commuter Station Vernal Pools, large pool at southern end of complex, |
|
|
Mongolia, Dundgovi’ aimag, northwest of Delgerhangay (Khashaat/Delger Khanay Uul), |
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
Specimens known to be collected and preserved in 95% ethanol were successfully extracted, amplified and sequenced at a much higher success rate than those with unknown preservation history (probably denatured alcohol). Although some specimens enumerated in Table
Our aim was to maximize the scientific value of specimens and their biological usefulness for future studies. First, the results of our study make a very compelling case that initial specimen fixation and preservation in the field should use 95% ethanol —
In addition to the changes we suggest for the fixation and preservation, we also suggest changes to the type and number of voucher specimens being deposited after an environmental impact report is completed. We recommend accessioning specimens of all species, whether listed or not (
These small improvements to collecting protocols will make it possible to derive high-quality data for future biodiversity and phylogeographic research. Since the sacrifice of endangered and non-endangered crustaceans is necessary to evaluate their presence and abundance in the wild, they can become a valuable historic resource if properly curated and deposited.
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.