Research Article |
Corresponding author: Adam Wall ( adamrwall@gmail.com ) Academic editor: Ingo S. Wehrtmann
© 2014 Adam Wall, Daniel Campo, Regina Wetzer.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
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, Costa Rica, June 2013. ZooKeys 457: 1-14. https://doi.org/10.3897/zookeys.457.6822
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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 (
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 Ambystoma tigrinum (Amphibia: Caudata: Ambystomatidae) (
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, i.e., 0<0.05 ng/µL. Asterisk (*) indicates sequence was generated and is listed in Table
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 | Artemia monica | 06-Jul-90 | 70% ethanol | California, Mono County, Mono Lake, south Tufa Reserve | H. Kuck | 1 broken specimen | 2013 | contaminated; blasts as Homo | 0 |
2 | Artemia monica | 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 | Branchinecta coloradensis | 23-Apr-92 | 70% ethanol | California, Lassen County, Hog Flat Reservoir | King, Gluesenkamp, Tritt | 1 broken specimen | 2003 | failed | 0 |
4 | Branchinecta dissimilis | 23-Mar-92 | 70% ethanol | California, Shasta County, Fall River | King, Gluesenkamp, Kloock | 2 broken pieces | 2017 | failed | 0.2 |
5 | Branchinecta gigas | unknown | acetone | California, San Bernadino County, Mojave Desert | J. Martin, J. Plum | 2 phyllopods only | 1990 | failed | 0.17 |
6 | Branchinecta gigas | unknown | not indicated | Washington, Grant County | unknown | 1 small whole specimen | 2006 | failed | 0.13 |
7 | Branchinecta gigas | unknown | not indicated | Washington, Grant County | unknown | dissected off egg sack with eggs | 2007 | failed | 0 |
8 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta lindahli | 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 | Branchinecta longiantenna | 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 | Branchinecta lynchi | 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 | Branchinecta lynchi | 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 | Branchinecta lynchi | 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 | Branchinecta lynchi | 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 | Branchinecta mackini | unknown | 70% ethanol | Washington, Grant County | unknown | 1 specimen | 1991 | failed | 0.254 |
22 | Branchinecta mackini | 03-Apr-93 | 70% ethanol | California, San Bernadino County, Mojave Desert | C. Cash-Clark, T. Clark | 1 specimen | 2019 | failed | 0.16 |
23 | Branchinecta orientalis | 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 | Branchinecta sandiegonensis | 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 | Branchinecta sandiegonensis | 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 | Branchinecta sandiegonensis | 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 | Branchinecta sandiegonensis | 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 | Branchinecta sandiegonensis | 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 | Branchinecta | 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 | Branchipodopsis affinis | 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 | Branchipodopsis affinis | 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 | Chirocephalus | 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 | Eubranchipus holmanii | 07-May-40 | 70% ethanol | Canada, Nova Scotia, Edinberg [sic] | D. Belk | 1 male specimen | 2015 | contaminated; blasts as Homo | 0 |
34 | Eubranchipus | 01-Apr-32 | 70% ethanol | Canada, Ontario, Saint Thomas | M.S. Ferguson | anterior end of broken specimen | 2014 | failed | 0 |
35 | Eubranchipus | 30-Apr-99 | 70% ethanol | Minnesota, Bloomington | A.B. Forbes | 1 female – doesn’t look well preserved | 2022 | failed | 0 |
36 | Eubranchipus | 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 | Linderiella occidentalis | 19-Feb-92 | 70% ethanol | California, Tehama County, Tuscan Buttes | King, Mazzucco, Scuderi | 2 pieces broken specimen | 2000 | contaminated; blasts as Homo | 0.14 |
38 | Linderiella occidentalis | 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 | Linderiella santarosae | 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 Homo | 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 | Phallocryptus | 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 | Pristicephalus comptus | 13-Apr-36 | 70% ethanol | Tennessee, Reelfoot Lake | unknown | 1 specimen, this lot had previously dried and had been realcoholed | 2012 | failed | 0 |
43 | Streptocephalus sealli | 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 | Streptocephalus sealli | 15-Aug-55 | 95% ethanol | California, Mariposa County, Yosemite, May Lake Trail | unknown | posterior end of animal | 1989 | failed | 0.18 |
45 | Streptocephalus texanus | 27-Aug-56 | 70% ethanol | New Mexico, Cain Ranch | S.F. Wood | dissected egg sack | 2010 | failed | 0 |
46 | Streptocephalus woottoni | 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 | Streptocephalus woottoni | 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 | Streptocephalus woottoni | 29-Jan-03 | preserved in 70% ethanol | California, Riverside County, Temecula | unknown | 3-4 phyllopods removed from single specimen | 1997 | failed | 6.8 |
49 | Tanymastix stagnalis | 12-Aug-34 | 70% ethanol | Denmark, Raabjerg Mile | E.W. Kaiser | 3 broken pieces used | 1996 | failed | 0.224 |
50 | Thamnocephalus platyurus | 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 Anostraca sequences: taxonomy, Genbank number, and locality information. All specimens and DNA are deposited in the collections of the Natural History Museum of Los Angeles County. Required permits are on file at USFWS and/or LACM.
Genus/species | Genbank No. | Locality |
---|---|---|
Artemiidae: Artemia monica | KF790567 | USA, California, Mono County, Mono Lake, ~38.011°N ~119.012°W, hypersaline lake, 95% ethanol. 1 Jan 2010. Coll. M. Hauser. RW12.244.2008 |
Branchinectidae: Branchinecta lindahli | KF790568 | USA, California, San Diego County, San Diego, Carmel Mountain Preserve, 32.929°N, 117.22°W, vernal pool 4 in. deep, 8 ft. wide, 28 ft. long, water slightly murky, 63 µm net, 95% ethanol. 28 Dec 2011. JS pool #21, MBPC 11637. Coll. J. Snapp-Cook, C. Lieberman, A. Wall, P. Sun, R. Wetzer. RW13.042.1992 |
Branchinectidae: Branchinecta lindahli | KF790569 | USA, California, San Diego County, San Diego, Carmel Mountain Preserve, 32.933°N, 117.215°W, vernal pool in dirt road, 95% ethanol. 2 Apr 2012. City ID # 22, js_fs_37, MBPC13258. Coll. J. Snapp-Cook. RW13.047.2026 |
Branchinectidae: Branchinecta lindahli | KF790570 | USA, California, San Diego County, San Diego, Carmel Mountain Preserve, 32.932°N, 117.215°W, vernal pool in dirt road, 95% ethanol. 2 Apr 2012. City ID # 20, js_fs_38, MBPC13259. Coll. J. Snapp-Cook. RW13.048.2027 |
Branchinectidae: Branchinecta lindahli | KF790571 | USA, California, San Diego County, San Diego, Carmel Mountain Preserve, 32.928°N, 117.22°W, vernal pool in dirt road, 95% ethanol. 2 Apr 2012. City ID # 26, js_fs_35, MPBC13256. Coll. J. Snapp-Cook. RW13.046.2028 |
Chirocephalidae: Chirocephalus sp. | KF790572 | Mongolia, Dundgovi’ aimag, near Sangiyn Dalay (Erdenedalay), 46.135°N, 105.106°E, 2 acre pond, 0-1 ppt, 23.2°C, 63 µm mesh net, 95% ethanol. 22 Aug 2002. GPS#016, Mongolia Expedition 2002, MBPC 431. Coll. R. Wetzer, S.L. Boyce, N.D. Pentcheff. RW13.034.2018 |
Chirocephalidae: Eubranchipus sp. | KF790573 | USA, California, Lassen County, Poison Lake, 40.659°N, 121.197°W, temporary lake, hand, 95% ethanol. 15 May 2012. Coll. M. Hauser and D. Striley. RW12.242.2020 |
Streptocephalidae: Streptocephalus woottoni | KF790574 | USA, California, San Diego County, Carlsbad, Poinsettia Lane Commuter Station Vernal Pools, large pool at southern end of complex, 33.108°N, 117.318°W, vernal pool 15 m x 30 m, 12-24 inches deep, murky water, 1 Apr 2005. MBPC 10061. Coll. J. Snapp-Cook. RW13.007.2016 |
Thamnocephalidae: Phallocryptus sp. | KF790575 | Mongolia, Dundgovi’ aimag, northwest of Delgerhangay (Khashaat/Delger Khanay Uul), 45.424°N, 104.481°E, large lake reduced to tiny watering hole, 11 ppt, 28°C, 63 µm mesh net, 95% ethanol. 22 Aug 2002. GPS#020, Mongolia Expedition 2002, MBPC 435. Coll. R. Wetzer, S. L. Boyce, N. D. Pentcheff. RW13.036.2009 |
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 — not denatured ethanol or other alcohols. If not denatured ethanol is unavailable, we recommend fixation and preservation in 100 proof (or higher) vodka, rum, Everclear™, or similar drinking alcohol, rather than any sort of denatured alcohol. This method, although the next best choice, has been successfully used during expeditionary work by one of us (RW) since the mid-1980s. Although 100 proof spirits are only 50% ethanol by volume, the quality of the alcohol matters more than the concentration — if you cannot drink it, it’s not good for specimens. Second, specimens should always be in a volume ratio of at least 3:1 alcohol:specimens to avoid degradation from dilution of preservative by body fluids. Third, once specimens are returned from the field, ethanol should be replaced with fresh 95% not denatured ethanol to compensate for dilution of the preservative by water extracted from specimen tissue.
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 (e.g. whether endangered or threatened, or not). For example, simply accessioning both the listed and non-listed species will make it possible to definitively address questions about hybridization between B. sandiegonensis and B. lindahli. Furthermore, depositing all specimens collected for a survey, not just a single voucher specimen for each species, will increase sample sizes to enable population level molecular studies.
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.