Western North American Naturalist 78(1), © 2018, pp. 92–99 Molecular detection of northern leatherside chub (Lepidomeda copei ) DNA in environmental samples JOSEPH C. D YSTHE 1,* , K ELLIE J. C ARIM 1, T HOMAS W. F RANKLIN 1, D AVE KIKKERT 2, MICHAEL K. Y OUNG 1, K EVIN S. M CKELVEY 1, AND MICHAEL K. S CHWARTZ 1 1U.S. Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT 59801 2Stantec, Inc., Salt Lake City, UT 84107 ABSTRACT .— The northern leatherside chub ( Lepidomeda copei ) is a cyprinid fish native to the Snake River, Green River, and Bonneville basins of the western United States. Population declines prompted the development of a multi - state conservation agreement and strategy, which emphasized the need to reliably delineate its current distribution and monitor its status. To facilitate species monitoring, we developed a quantitative PCR assay to detect northern leatherside chub DNA in environmental samples. The assay consistently detected northern leatherside chub DNA in concentra - tions as low as 2 copies per reaction and did not amplify DNA of potentially sympatric fish species. The assay amplified a synthetic DNA template representing 3 congeneric species: White River spinedace ( L. albivallis ), Virgin spinedace, (L. mollispinis mollispinis ), and Big Spring spinedace, ( L. m. pratensis ); however, none of these are sympatric with northern leatherside chub. Field tests of the assay accurately reproduced expected patterns of species occupancy. RESUMEN .— La especie Lepidomeda copei es un pez ciprínido nativo de las cuencas del Río Snake, Río Green y del Lago Bonneville del oeste de los Estados Unidos. La disminución de su población impulsó el desarrollo de un acuerdo de conservación multi-estado, que enfatiza la necesidad de delinear con precisión su distribución actual y de monitorear su estado. Para facilitar el monitoreo de las especies, aplicamos la técnica cuantitativa de Reacción en Cadena de la Polimerasa (PCR, por sus siglas en inglés) que permitió detectar ADN de L. copei en muestras ambientales. La técnica detectó de manera consistente ADN de L. copei en concentraciones menores a 2 copias por reacción, sin amplificar ADN de otras especies de peces potencialmente simpátricas. La técnica amplificó un templado de ADN sintético que representa tres especies congenéres: L. albivallis, L. mollispinis mollispinis, y L. m. pratensis. Sin embargo, ninguna de estas especies es simpátrica con L. copei . Los muestreos de campo reprodujeron con precisión los patrones previstos en cuanto a la ocupación de las especies. Fishes of the genus Lepidomeda (family Bonneville and Snake River basins. They also Cyprinidae) are patchily distributed through - concluded that northern leatherside chub is out warm- and cold-desert streams of the not the sister taxon to southern leatherside Bonneville, Colorado River, and Snake River chub, but is more closely related to Virgin basins (UDWR 2009, Blakney et al. 2014). spinedace ( L. mollispinis ) and White River This group has been the subject of some taxo - spinedace ( L. albivallis ) (Johnson et al. 2004). nomic revision. Until 2004, the leatherside Due to this taxonomic revision and the chub ( Snyderichthys copei or Gila copei; understanding of the northern leatherside’s Johnson et al. 2004) was considered a broadly taxonomic uniqueness, there has been greater distributed taxon of the intermountain western interest in the evaluation of its current distri - United States. Considering genetic, morpho - bution. While some populations that were logical, and ecological evidence, however, John - regarded as introduced (UDWR 2009) were son et al. (2004) split this taxon into 2 species actually likely to be indigenous (Blakney et al. and placed them in the genus Lepidomeda: 2014), suggesting a wider range than previ - the southern leatherside chub ( L. aliciae ) in ously thought, there is a broad consensus that the southern Bonneville basin and the north - the northern leatherside chub has declined ern leatherside chub ( L. copei ) in the northern across its range and has been extirpated from *Corresponding author: [email protected] JCD orcid.org/ 0000-0002-6790-7841 KJC orcid.org/ 0000-0002-9622-9146 MKY orcid.org/ 0000-0002-0191-6112 92 DYSTHE ET AL . ♦ NORTHERN LEATHERSIDE CHUB EDNA A SSAY 93 several basins (Belk and Johnson 2006, UDWR tool, we further examined the specificity of 2009). To mitigate further range contractions, each component of the assay in silico to deter - this taxon was petitioned for listing under the mine potential sources of non target detection. U.S. Endangered Species Act (USFWS 2011) We then compared the candidate assay and was designated as a species of conserva - with all northern leatherside chub cytb tion concern throughout its range (UDWR sequences ( n = 47) available on GenBank 2009). Conservation efforts have emphasized (AF270885–AF270893, Johnson and Jordan the need to assess the distribution of northern 2000; AF452086–AF452087, Dowling et al. leatherside chub (Blakney et al. 2014, Schultz 2002; AY825431–AY825445, Johnson et al. et al. 2016), but its patchy occurrence and low 2004; JX443059, Schonhuth et al. 2012; and relative abundance (UDWR 2009, Dauwalter KJ175008–KJ175027, Blakney et al. 2014). et al. 2014) have sometimes made this task These sequences were obtained from fish challenging. Thus, developing a rapid and collected in 24 streams throughout the Bear reliable method for assessing presence and River, Green River, and Snake River water - distribution would be useful for evaluating sheds in Idaho, Nevada, Utah, and Wyoming. species status and prioritizing conservation We found that one of the 47 sequences (acces - efforts for this species. sion: KJ175010; Blakney et al. 2014), which Environmental DNA (eDNA) sampling has was not evaluated in the initial in silico step, proven to be an efficient and reliable method contained a single nucleotide polymorphism for delineating distributions of rare species (SNP) in which guanine replaced adenine 10 (McKelvey et al. 2016) and detecting sensitive bases from the 3 ፱ end of the probe. This fish species (Thomsen et al. 2012, Sigsgaard et al. originated in Muddy Creek within the Bear 2015, Spear et al. 2015) or species difficult to River basin in Wyoming, and was the only sample using traditional approaches (Taberlet fish with this SNP in 225 sequences exam - et al. 2012). Furthermore, analysis of eDNA via ined by Blakney et al. (2014), which included quantitative PCR (qPCR) is more sensitive and other northern leatherside chubs collected effective in detecting low DNA concentrations from Muddy Creek (Ernest Keeley, Idaho than traditional PCR methods (Wilcox et al. State University, personal communication). To 2013, 2016). Accordingly, we developed a ensure detection of this rare haplotype, we qPCR assay for northern leatherside chub for developed an additional probe incorporating eDNA-based detection throughout its range. this SNP; the assay is a mixture of both To develop an eDNA assay for northern probes (Table 2). leatherside chub, we examined 54 GenBank We tested the specificity of the assay in sequences of the cytochrome b (cytb) mito - vitro using a StepOne Plus Real-time PCR chondrial region of northern leatherside Instrument (Life Technologies) in 15- mL reac - chub and 14 sympatric or closely related non - tions containing 7.5 mL of Environmental target species (Table 1). We screened these Master Mix 2.0 (Life Technologies), 900 nM sequences in MEGA 6 (Tamura et al. 2013) each of forward and reverse primer, 125 nM of and identified candidate primer sites that each probe, 4 mL DNA template ( ~0.4 ng), would amplify an 80-nucleotide fragment and the remaining volume with PCR-grade unique to northern leatherside chub (Table 2). water. Thermocycler conditions were 95 °C for Within this fragment, we designed a FAM- 10 min followed by 45 cycles of denaturation labeled, minor-groove-binding, nonfluorescent at 95 °C for 15 s and annealing and extension at quencher (MGB-NFQ) probe (Table 2). We 60 °C for 1 min. We screened DNA extracted maximized within-primer and within-probe from 17 northern leatherside chub tissues nucleotide mismatches with nontarget species from 3 locations and from 22 additional to avoid instances of primer competition and species (Table 3). DNA used in this study was cross-amplification of the probe (Wilcox et al. obtained from archival samples, or from fin 2013). We adjusted primer and probe lengths to clips collected from fish that were immedi - optimize annealing temperatures in Primer ately released at the point of capture. Fin Express 3.0.1 (Life Technologies), and screened clips were stored in ≥95% ethanol until them for secondary structures using IDT DNA was extracted using the DNeasy Tissue OligoAnalyzer (https://www.idtdna.com/calc/ and Blood Kit (Qiagen, Inc) according to the analyzer). Using the NCBI nucleotide BLAST manufacturer’s instructions. 9 4 W E S T E R N TABLE 1. Species, number of sequences, and GenBank accession number for DNA sequences used for in silico assay development. Also included is the minimum number of N mismatches between the sequences and the forward primer (F), reverse primer (R), and probe (P). O R T H Nucleotide mismatches A ____________________ M Family name Species name Common name Sequences F R P GenBank accession number E R I C Cyprinidae Lepidomeda copei Northern leatherside chub 8 0 0 0 KJ175009.1; KJ175012.1; KJ175017.1; KJ175019.1; A N KJ175020.1; KJ175022.1;