Notes Eight Novel Microsatellite Loci Developed from Vernal Pool Fairy Shrimp Kristy Deiner, Joshua M. Hull, Bernie May Genomic Variation Lab, Department of Science, One Shields Avenue, Davis, 95616 Present address of J.M. Hull: Sacramento Fish and Wildlife Office, U.S. Fish and Wildlife Service, 2800 Cottage Way, W-2605, Sacramento, California 95825

Abstract We developed primer sets for eight di-, tri-, and tetranucleotide microsatellite loci for the threatened vernal pool fairy shrimp lynchi from CA, CAA, ATG, CAGA, and TAGA enriched genomic libraries. We tested primers in 74 individuals of B. lynchi from California, as well as 19 individuals from six other Branchinecta species: B. coloradensis, B. constricta, B. dissimilis, B. lindahli, B. mesovalensis, and B. oriena. The eight loci showed sufficient variability for investigations of B. lynchi evolution, population structure, and genetic diversity and may provide a new tool for assisting in delineation of management areas.

Keywords: Branchinecta lynchi; evolution; fairy shrimp; microsatellite; population; vernal pool Received: November 21, 2012; Accepted: March 28, 2013; Published Online Early: March 2013; Published: June 2013 Citation: Deiner K, Hull JM, May B. 2013. Eight novel microsatellite loci developed from vernal pool fairy shrimp. Journal of Fish and Wildlife Management 4(1):134–138; e1944-687X. doi: 10.3996/112012-JFWM-096 Copyright: All material appearing in the Journal of Fish and Wildlife Management is in the public domain and may be reproduced or copied without permission unless specifically noted with the copyright symbol ß. Citation of the source, as given above, is requested.

The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service. * Corresponding author: [email protected]

Introduction we present results from tests of cross-amplification in six other Branchinecta species. The vernal pool fairy shrimp Branchinecta lynchi is adapted to the ephemeral aquatic conditions of vernal Methods and Results pool habitats of southern Oregon and central and southern California (Figure 1). Although widely distribut- Genetic Identification Services (Chatsworth, CA) creat- ed, B. lynchi is uncommon throughout its range (Eng et ed eight libraries enriched with microsatellite motifs (CA, al. 1990), and it is listed as threatened pursuant to the CAA, ATG, CAGA, and TAGA) by using DNA from B. lynchi U.S. Endangered Species Act (ESA 1973, as amended) and that came from Riverside County, California. A cocktail has an IUCN ranking of vulnerable/A2c (IUCN 2011). of seven blunt-end cutting enzymes (RsaI, HaeIII, BsrB1, Primary threats to B. lynchi populations include habitat PvuII, StuI, ScaI, EcoRV) partially restricted genomic DNA. degradation, habitat loss and fragmentation, altered We adapted fragments in the size range of 300 to 750 bp hydrology, and invasive plant and animal species (USFWS and subjected them to magnetic bead capture (CPG, Inc., 1994, 2005). Lincoln Park, NJ) using biotinylated capture molecules. Management of B. lynchi and other closely related We prepared libraries in parallel with Biotin-CA, Biotin- species of conservation concern may benefit from an CAA, Biotin-ATG, Biotin-CAGA, and Biotin-TAGA as understanding of the evolutionary and population capture molecules. We amplified captured molecules genetic relationships among vernal pool complexes at and used HindIII to remove the adapters. We then ligated a range-wide scale, as well as from a better understand- the resulting fragments into the HindIII site of pUC19 and ing of the landscape features that structure populations electroporated recombinant molecules into Escherichia on a more local level. Here we describe the development, coli DH5a. We selected random recombinant clones for characterization, and genetic diversity of eight novel sequencing and expressed enrichment levels as the microsatellite loci for B. lynchi. We tested loci for diversity fraction of sequences that contained a microsatellite. We from samples from throughout the species’ range, and obtained sequences on an ABI 377 DNA Sequencer/

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Figure 1. Branchinecta lynchi specimens from the collection at the California Academy of Sciences (CAS) in San Francisco, California (CA). Top is a male (CAS161073) collected from Placer County, California, and bottom is a female (CAS180431) collected from Solano County, California. Photo credit: Kristy Deiner.

Genotyper, using ABI Prism Taq dye terminator cycle Screening reactions underwent electrophoresis at 120 V sequencing methodology (Applied Biosystems, Grand for 30 min on a 3% agarose gel in a 16 sodium borate Island, NY). From the eight libraries, we sequenced a total buffer, after which we visually inspected the results. Loci of 768 clones. The clone screening produced quality were accepted as working if a PCR produced banding sequence reads but resulted in a low success rate of 12% patterns expected based on di-, tri-, and tetranucleotide (89 sequences) that had inserts with a repeat region and repeats. Only these working loci underwent further enough flanking sequence from which to design primers analysis. Among the 89 primers that we tested, 19 for amplification of the loci. amplified a PCR product, and we considered 8 to be We used default settings in Primer3 (Rozen and working loci (Tables 1 and 2). Skaletsky 2000) to design 89 primer sets with melting To assess the utility of these eight loci, we tested 74 B. temperatures between 59 and 61uC. We used the lynchi samples from many pools in three additional QIAGEN DNeasy kit (Qiagen, Inc. Valencia, CA) following regions in California (Carrizo N = 24; San Joaquin Valley the manufacturer’s recommended protocol to extract N = 13; and southeastern Sacramento Valley, N = 37) DNA from Branchinecta tissue samples. Preliminary and estimated allele range and number of alleles at each screening reactions consisted of testing a series of locus (Data S1, Supplemental Material ). Additionally, we polymerase chain reaction (PCR) cycling protocols tested these loci in a total of 19 individuals from six other starting with protocol 1 and moving to the next if it Branchinecta species that are also present in North did not amplify a product (up to protocol 5, Table 1). We America: B. coloradensis (N = 3), B. constricta (N = 3), tested loci in a set of seven individuals from across the B. dissimilis (N = 3), B. lindahli (N = 3), B. mesovalensis species range in California: Butte County, N = 1; Shasta (N = 1), and B. oriena (N = 6). All PCRs were carried County, N = 1; Fresno County, N = 1; Tuolumne County, out in 10-mL volumes with final concentrations of 16 N = 1; Tehama County, N = 2; and Riverside County, supplied buffer (Faststart TAQ, Roche, Inc., Basel, N = 1, which was the same sample that we used to Switzerland) 16 bovine serum albumin, 0.2 mM create libraries from which we discovered the loci. dNTPs, 1.5 mM MgCl2 for all loci except Blynchi-C11

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Table 1. Polymerase chain reaction protocols for eight microsatellite loci optimized in Branchinecta lynchi. Numbers in cells indicate (in order) temperature in degrees Celsius/time in seconds/number of cycles at that temperature and time. Protocol 1 included a ramp protocol starting at a low temperature that increased by 0.1uC at each cycle to a final temperature. Protocols 2 through 5 included a touch-down (TD) in which the cycle started at a high temperature and decreased to a final temperature.

Cycling regime 1 Cycline regime 2 PCR Total protocola cycles Denature Annealing Extension Denature Annealing Extension

1 35 94/40/35 Ramp 56.5–60/40/35 72/30/35

2 30 94/30/15 TD 65–50/60/15 72/90/15 94/30/15 53/60/15 72/45/15

3 35 94/40/15 TD 65–60.5/40/15 72/30/15 94/40/20 60/40/20 72/30/20

4 40 94/30/15 TD 65–50/60/15 72/90/15 94/30/25 50/60/25 72/45/25

5 30 94/40/5 TD 70–65/40/5 72/30/5 94/40/15 TD 65–60/40/15 72/30/15 a All PCRs had an initial denature of 94uC for 4 min and a final extension of 72uC for 7 min and 60uC for 20 min.

Table 1. Extended. Cycling regime 3 Denature Annealing Extension Loci that worked for protocol Blynchi-B263, Blynchi-C11, Blynchi-D210 Blynchi-A273, Blynchi-C219 Blynchi-B107 Blynchi-B205 94/40/10 TD 60–55/40/10 72/30/10 Blynchi-H8 and Blynchi-H8, which had a concentration of 2.0 mM Using PCR protocols established for B. lynchi, cross- MgCl2, 0.05 U/mL Taq DNA polymerase (Faststart TAQ, amplification in six sister taxa was successful for two loci. Roche, Inc., Basel, Switzerland). Fluorescently labeled We were successful in amplifying the Blynchi-B205 locus forward and unlabeled reverse primers were at a con- in B. coloradensis (three of three individuals), B. oriena centration of 0.54 mM. We added 1 mL of template DNA, (five of six individuals), and B. dissimilis (one of three which ranged in concentration from 10 to 70 ng/mL. We individuals). Similarly, we amplified the Blynchi-C11 locus used a 3730 DNA Analyzer to separate PCR products and in B. lindahli (one of three individuals) and B. constricta visualized and scored the products with GENEMAPPER, (two of three individuals). (Applied Biosystems, Grand Island, New York) version 4.0 (Applied Biosystems, Grand Island, NY). Discussion The number of alleles per locus ranged from 3 to 14 with an average of 7.9 in pooled sample sites (Table 2). The eight microsatellite loci that we developed in this We tested Hardy–Weinberg equilibrium (HWE) and study showed diversity across B. lynchi sample sites and linkage disequilibrium (LD) within a single vernal pool may be useful for studies of population genetic structure from the southeastern Sacramento Valley (N = 26) with and range-wide genetic diversity to aid in identification ARLEQUIN version 3.5.1.3 (Excoffier and Lischer 2010). of areas of conservation importance. These loci may One locus, Blynchi-A273, was monomorphic for this also be helpful for testing hypotheses related to the sampling site and therefore we could not execute tests. molecular evolution and reproductive biology of this Expected heterozygosity for all seven polymorphic loci diverse group of . However, data ranged from 0.28 to 0.73, while their observed hetero- from these loci require careful interpretation in popula- zygosity ranged from 0.00 to 0.57. We found that five loci tion genetic studies because they deviate from HWE. Loci significantly deviated from HWE (all had heterozygosity that are out of HWE may indicate that their populations deficiencies, P , 0.05, Table 2) after Bonferroni correc- of origin violate assumptions of many population genetic tion for multiple tests (Rice 1989). One locus pair (C11 6 models. Therefore use of such loci has the potential to B205) was significantly out of linkage equilibrium (P , bias measures of population substructure. 0.05, Table 2) after Bonferroni correction for multiple Wild populations may deviate from HWE for several tests (Rice 1989). reasons, including population subdivision (spatial or To rule out potential genotyping errors we regeno- temporal), assortative mating, inbreeding, and selection. typed all southeastern Sacramento Valley (N = 26) Other studies have found Anostraca shrimp in natural samples three times from the same DNA extract using populations to deviate from HWE. For example, previous the multitube approach and calculated the mean error studies using other genetic loci indicated that Branchinecta rate per locus following that of Pompanon et al. (2005). coloradensis (Bohonak 1998), Branchinecta sandiegonensis The mean error rate per locus was zero for all loci except (Davies et al. 1997), and Branchipodopsis wolfi (Brendonck Blynchi-C11, which had an error rate of 2.6%. et al. 2000) all showed departures from HWE in natural

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Table 2. Characterization of eight microsatellite loci examined in 74 vernal pool fairy shrimp, Branchinecta lynchi sampled from Carrizo (24), San Joaquin Valley (13), and Sacramento Valley (37). Motif indicates the microsatellite repeat for each locus, number of gene copies indicates the number of alleles successfully amplified from the total sample (this number is not 148 in all cases because some loci in some individuals failed to amplify), and Na is the number of alleles observed for each locus among all samples. Observed heterozygosity (HO), expected heterozygosity (HE), Hardy–Weinberg equilibrium (HWE), and linkage disequilibrium (LD) were calculated from a subset of 26 individuals sampled from a single pool in southeastern Sacramento Valley. Of these, N indicates the number of individuals that were successfully amplified for each locus and subsequently used to estimate HO,HE, HWE, and LD.

GenBank No. Allele size accession gene range HWE P Name Primer sequences no. Motif copies Na (bp) N HO HE value LD Blynchi-A273a F: GCCTCAGGAAAACTGTTATTCAA KC170306 CA 144 3 132–136 — — — — — R: ATCACAGAACGGTTGGGAAGT Blynchi-B107 F: CGAGAGTTATCGTGCTTACAG KC170307 ACC 138 12 155–227 24 0.250.55 ,0.01* n.s.b R: AATCGGTGAAGTTGTTTCC Blynchi-B205 F: TTTATTGATAACCCGTCTAGGTAAAGA KC170308 ACC 132 5 117–131 24 0.540.49 0.68 w/C11c R: CGGGATGAAGGAAAATTGTAAA Blynchi-B263 F: CCTGCGGCATGATAGATAGATA KC170309 ACC 112 11 130–169 24 0.750.66 0.59 n.s. R: CCTCCAGATAGGGTTAGCACAC Blynchi-C11 F: TAGGATGACCCAGACCAGTA KC170310 ATG 110 7 249–267 23 0.350.62 0.01* w/B205c R: TTGCCACTTGTTAGAACCAC Blynchi-C219 F: TTCAGAAAGAGGCTGGAGGA KC170311 ATG 138 14 142–172 24 0.000.54 ,0.01* n.s. R: GGGAGGAGTGTCGTGATTTTT Blynchi-D210 F: ATGTTAGCGGTCAACCAAGG KC170312 CAGA 116 6 173–233 25 0.320.57 ,0.01* n.s. R: TTAGTTTGGTCGGGTTCCTG Blynchi-H8 F: ACTGCAGTATTTGCTACTGTGATT KC170313 TAGA 114 5 138–192 25 0.080.27 ,0.01* n.s. R: GCGCTACGAGTTGGATCAAT a HWE and LD were not tested for Blynchi-A273 because it was monomorphic for this subsample from a single pool. b n.s., not significant. c Significant LD between this pair of loci after Bonferroni correction with a P-value of ,0.01. * Significant at the 0.05 alpha level after Bonferroni correction for multiple tests. populations. Explanations for the deviations from HWE that that these loci may not be abundant in the genome or occur so broadly across this group of crustaceans are that other genomic searching methods such as deep related to their reproductive biology. Because many of sequencing would be useful in future discovery of loci in these species have evolved in ephemeral pools that may this species (Santana et al. 2010). experience multiple years of drought, they produce a resting egg bank capable of persisting for multiple years Supplemental Material similar to seed banks of many plant species (Brendonck and Please note: The Journal of Fish and Wildlife Management De Meester 2003). During years with favorable conditions, is not responsible for the content or functionality of any adults from multiple generations of eggs may emerge from supplemental material. Queries should be directed to the dormancy, resulting in temporal gene flow (i.e., admixture corresponding author for the article. of populations through time) and deviations from HWE. Because our HWE estimates are derived from 26 adult Data S1. Microsatellite genotypes for 74 individual Branchinecta lynchi from three regions (Carrizo = 24, San individuals from one pool in a single season, we cannot Joaquin Valley = 13, Sacramento Valley = 37) for the distinguish among the various possible causes of hetero- eight microsatellite loci developed in this study collected. zygosity deficiency, including those just described. There- Found at DOI: http://dx.doi.org/10.3996/112012- fore, a logical next step is to use these loci to evaluate both JFWM-096.S1 (16 KB XLSX). samples from multiple locations as well as time series samples from individual pools to begin to develop an Reference S1. [USFWS] US Fish and Wildlife Service. understanding of the relative influences of spatial and 2005. Recovery Plan for Vernal Pool Ecosystems of temporal isolation on population genetic structure. California and Southern Oregon. Subsection (pages Although these eight microsatellite loci are not in II-191 to II-203) of Recovery Plan for Vernal Pool HWE, they provide a tool that may be used to advance Ecosystems of California and Southern Oregon. Portland, conservation efforts in this endemic and threatened Oregon relating to Branchinecta lynchi. Portland, Oregon. North American species of fairy shrimp through the Found at DOI: http://dx.doi.org/10.3996/112012-JFWM- study the genetic diversity, evolution, and spatial and 096.S2; also available at http://www.fws.gov/sacramento/ temporal population structure. The low success rate of es/Recovery-Planning/Vernal-Pool/es_recovery_vernal-pool- finding microsatellite loci for B. lynchi, however, suggests recovery.htm (734 KB PDF).

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Acknowledgments Available: http://www.fws.gov/endangered/esa-library/ pdf/ESAall.pdf. The work described in this article was conducted under a USFWS 10(a)(1)(A) research permit (permit number TE- Excoffier L, Lischer HEL. 2010. Arlequin suite ver 3.5: a FWSSFWO). We would like to thank Dave Kelly who new series of programs to perform population provided significant technical support. We also thank the genetics analyses under Linux and Windows. Molec- Journal Subject Editor and anonymous reviewers who ular Ecology Resources 10:564–567. greatly improved the manuscript. Financial support was [IUCN] International Union for Conservation of Nature. provided by the U.S. Fish and Wildlife Service, UC Davis 2011. IUCN Red List of Threatened Species. Version Dissertation Year Fellowship, The Society: 2011.2. Available: http://www.iucnredlist.org (January Denton Belk Research Award, and the Achievement 2012). Rewards for College Scientists Fellowship. Pompanon F, Bonin A, Bellemain E, Taberlet P. 2005. Any use of trade, product, or firm names is for Genotyping errors: causes, consequences and solu- descriptive purposes only and does not imply endorse- tions. Nature Reviews Genetics 6:847–846. ment by the U.S. Government. Rice WR. 1989. Analyzing tables of statistical tests. Evolution 43:223–225. References Rozen S, Skaletsky H. 2000. Primer3 on the WWW for Bohonak AJ. 1998. Genetic population structure of the general users and for biologist programmers. Methods fairy shrimp Branchinecta coloradensis (Anostraca) in in Molecular Biology 132:365–386. the Rocky Mountains of Colorado. Canadian Journal of Santana QC, Coetzee MPA, Steenkamp ET, Mlonyeni OX, Zoology 76:2049–2057. Hammond GNA, Wingfield MJ, Wingfield BD. 2010. Brendonck L, De Meester L. 2003. Egg banks in Microsatellite discovery by deep sequencing of freshwater zooplankton: evolutionary and ecological enriched genomic libraries. Biotechniques 46:217– archives in the sediment. Hydrobiologia 491:65–84. 223. Brendonck L, De Meester L, Riddoch BJ. 2000. Regional [USFWS] U.S. Fish and Wildlife Service. 1994. Determi- structuring of genetic variation in short-lived rock nation of endangered status for the conservancy pool populations of Branchipodopsis wolfi (Crustacea: fairy shrimp, longhorn fairy shrimp, and vernal pool Anostraca). Oecologia 123:506–515. tadpole shrimp, and threatened status for the Davies CP, Simovich MA, Hathaway SA. 1997. Population vernal pool fairy shrimp. Federal Register 59:

genetic structure of a California endemic branchio- 48136–48153. pod, Branchinecta sandiegonensis. Hydrobiologia 359: [USFWS] U.S. Fish and Wildlife Service. 2005. Recovery plan 149–158. for vernal pool ecosystems of California and Southern Eng LL, Belk D, Eriksen CH. 1990. Californian Anostraca: Oregon. Portland, Oregon: USFWS (see Supplemental distribution, habitat, and status. Journal of Crustacean Material, Reference S1, http://dx.doi.org/10.3996/ Biology 10:247–277. 112012-JFWM-096.S2); also available: http://www.fws. [ESA] U.S. Endangered Species Act of 1973, as amended. gov/sacramento/es/Recovery-Planning/Vernal-Pool/es_ 1973. Pub. L. No. 93-205, 87 Stat. 884 (Dec. 28, 1973). recovery_vernal-pool-recovery.htm (January 2013)

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