Molecular Detection of Northern Leatherside Chub (Lepidomeda Copei) DNA in Environmental Samples

Western North American Naturalist

Volume 78 Number 1 Article 8

4-4-2018

Molecular detection of northern leatherside chub (Lepidomeda copei) DNA in environmental samples

Joseph C. Dysthe U.S. Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT, [email protected]

Kellie J. Carim U.S. Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT, [email protected]

Thomas W. Franklin U.S. Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT, [email protected]

Dave Kikkert Stantec, Inc., Salt Lake City, UT, [email protected]

Michael K. Young U.S. Forest Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, MT, [email protected]

SeeFollow next this page and for additional additional works authors at: https:/ /scholarsarchive.byu.edu/wnan

Recommended Citation Dysthe, Joseph C.; Carim, Kellie J.; Franklin, Thomas W.; Kikkert, Dave; Young, Michael K.; McKelvey, Kevin S.; and Schwartz, Michael K. (2018) "Molecular detection of northern leatherside chub (Lepidomeda copei) DNA in environmental samples," Western North American Naturalist: Vol. 78 : No. 1 , Article 8. Available at: https://scholarsarchive.byu.edu/wnan/vol78/iss1/8

This Note is brought to you for free and open access by the Western North American Naturalist Publications at BYU ScholarsArchive. It has been accepted for inclusion in Western North American Naturalist by an authorized editor of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Molecular detection of northern leatherside chub (Lepidomeda copei) DNA in environmental samples

Authors Joseph C. Dysthe, Kellie J. Carim, Thomas W. Franklin, Dave Kikkert, Michael K. Young, Kevin S. McKelvey, and Michael K. Schwartz

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. 94 WESTERN NORTH AMERICAN NATURALIST (2018), V OL . 78 N O. 1, P AGES 92 –99 f o

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) We were not able to obtain tissue from M

n several important species, specifically the (

o rare haplotype of northern leatherside chub i t a

r and 3 congeneric taxa of spinedace (White 0 0 5 5 t 0 0 2 2 n 3 9 1 1

e River spinedace L. albivallis; Virgin spine -

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a spinedace, L. m. pratensis ). To test the ability

F of the assay to detect these specimens, we

developed and screened 2 synthetic plasmid

DNA fragments encompassing the assay’s

amplicon in the cytb region (Carim et al.

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C 2016a). The plasmids were synthesized by

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KJ175010.1) and of the White River spine-

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into ampicillin vectors containing PvuI cut

sites (pIDTSMART-AMP; Integrated DNA

Technologies). The White River spinedace se -

Q Q

quence was used to represent the Big Spring

(accession: AF452091.1) and Virgin spine- B

daces (accession: AF452092.1) as all 3 species

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C C 3 end of the reverse primer, and 5 bases from A ) ፱ G T T ፱ T G 3 C C T – the 3 ፱ end of the probe. The sequence for G T T ፱ T T 5 C C ( C each plasmid construct was verified on both C G G e G T c A A . G G n strands via Sanger sequencing by the sup - T T - - e A T T u T T N M M plier, Integrated DNA Technologies, using q C G A A e D S C A F F e M13 primers (Integrated DNA Technolo -

gies). We linearized the plasmids with PvuI

(Invitrogen catalog # 25420-118) according e

s to the manufacturer’s protocol, purified the

products using the GeneJET PCR Purifica - t

tion Kit (ThermoFisher Scientific), quanti -

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r high end-point fluorescence relative to the n t t t h a a a o P t e e e a . p l l l highest (900 nM for each primer) concentra -

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t B h h h h y t t t t A a r r r r value (the earliest cycle at which the amplifi - s T o o o o

A N N N N cation curve crossed the threshold). Using the 96 WESTERN NORTH AMERICAN NATURALIST (2018), V OL . 78 N O. 1, P AGES 92 –99

TABLE 3. Species used for in vitro testing of the northern leatherside chub eDNA assay. For samples of the northern leatherside chub, origin refers to the Idaho waterbody where the samples were collected. For samples of all other species, origin is listed as U.S. state, Canadian province, or synthetic plasmid. Family name Species name Common name Sample size Origin Cyprinidae Lepidomeda copei Northern leatherside chub 4 Goose Creek, ID 2 Squaw Creek, ID 11 Tin Cup Creek, ID Cyprinidae Agosia chrysogaster Longfin dace 1 NM Cyprinidae Cyprinella lutrensis Redside shiner 1 UT Cyprinidae Cyprinus carpio Common carp 1 MT Cyprinidae Gila atraria Utah chub 1 ID Cyprinidae Hybognathus argyritis Western silvery minnow 1 MT Cyprinidae Lepidomeda albivallis White River spinedace 1 Synthetic plasmid Cyprinidae Lepidomeda aliciae Southern leatherside chub 2 UT Cyprinidae Macrhybopsis gelida Sturgeon chub 1 MT Cyprinidae Macrhybopsis meeki Sicklefin chub 1 MT Cyprinidae Meda fulgida Spikedace 2 AZ, NM Cyprinidae Pimephales promelas Fathead minnow 1 NM Cyprinidae Platygobio gracilis Flathead chub 1 MT Cyprinidae Ptychocheilus lucius Colorado pikeminnow 1 WY Cyprinidae Rhinichthys cataractae Longnose dace 2 MT, UT Cyprinidae Rhinichthys cobitis Loach minnow 1 NM Cyprinidae Rhinichthys osculus Speckled dace 4 AZ, NM, UT Catostomidae Catostomus catostomus Longnose sucker 1 MT Ictaluridae Ictalurus punctatus Channel catfish 1 MT Salmonidae Oncorhynchus clarkii bouvieri Yellowstone cutthroat trout 1 ID Salmonidae Oncorhynchus clarkii utah Bonneville cutthroat trout 1 ID Salmonidae Oncorhynchus mykiss Rainbow trout 1 ID Salmonidae Salmo trutta Brown trout 1 CO Salmonidae Salvelinus fontinalis Brook trout 1 Quebec optimal concentrations of 300 nM of forward known distances downstream of caged north - primer and 900 nM of reverse primer and the ern leatherside chubs. Northern leatherside same PCR recipe and thermal profile above, chubs were placed into each stream in a cage we tested assay sensitivity by analyzing sepa - (similar to Jane et al. 2015), and eDNA sam - rate 7-level standard curves created from tar - ples were collected before the cages were get qPCR product and from the linearized placed into the streams. Then, 2 days later synthetic plasmid DNA of the rare haplotype. eDNA samples were taken at 50, 100, 200, The qPCR product and linear plasmid DNA 300, 400, 500, and 1000 m downstream in were purified using PureLink™ PCR Micro each creek. This sampling design was repli - Kit (Invitrogen), quantified on a Qubit 2.0 cated in 2 other reaches of each creek on dif - fluorometer, and serially diluted in sterile ferent days using different specimens. Because TE to 31,250, 6250, 1250, 250, 50, 10, and 2 the purpose, in this context, was to affirm the copies per 4 mL. Both standard curves were ability of the assay to detect northern leather - analyzed across 6 replicates at each level on side when eDNA was present, we selected the same 96-well plate. In addition, because samples collected at 100, 200, 300, and 400 m synthetic plasmid DNA of the White River from one reach of each creek. Here, 7 northern spinedace was detected with the assay, we leatherside chubs (84 to 118 mm in length and created and analyzed a 7-level standard curve weighing 5.5 to 15 g in Jensen Creek; 95 to using the methods described above. 112 mm in length and weighing 8 to 12 g in Finally, we validated the assay in vivo by Trout Creek) were placed into each stream screening eDNA samples collected from 4 in a cage before eDNA samples were col - streams in the western United States with lected. Previous studies using similar caged known patterns of occupancy by northern fish experiments detected eDNA perfectly at leatherside chub (Table 4). In 2 of the streams distances up to 250 m downstream from the where northern leatherside chubs were ini - cages (Jane et al. 2015) including during a flood tially absent (Jensen and Trout Creeks), eDNA event, indicating that 250 m was a conserva - samples were collected for a separate study at tive detection distance. However, the ability DYSTHE ET AL . ♦ NORTHERN LEATHERSIDE CHUB EDNA A SSAY 97

TABLE 4. Collection information for eDNA samples used for in vivo validation of the northern leatherside chub eDNA assay. Waterbody (state) Latitude Longitude Expected a Dectected a

Jenson Creek (ID) 43.184053 −111.165938 Nb N 43.176676 −111.153279 Nb N 43.200062 −111.190858 Y Y 43.199314 −111.190092 Y Y 43.198735 −111.189428 Y Y 43.198109 −111.188915 Y Y Trout Creek (ID) 43.158117 −111.071187 Nb N 43.142916 −111.080279 Y Y 43.143605 −111.079783 Y Y 43.144306 −111.079307 Y Y 43.144902 −111.078632 Y Y Rattlesnake Creek (MT) 46.945720 −113.945220 N N Tincup Creek (ID) 42.980710 −111.281300 Y Y aN (no) and Y (yes) refer to occupancy of northern leatherside chub based on traditional surveys and eDNA-based detection. bSamples were taken prior to placement of northern leatherside chub into the stream. to detect eDNA at greater downstream dis - curve for the rare haplotype resulted in an tances is unknown. eDNA samples were also efficiency of 102.6% (r2 = 0.995, y-intercept collected from 2 other streams; one where = 38.8, slope = −3.3) and a limit of detection northern leatherside chub has been observed at 2 copies per reaction. The DNA of the during historical surveys (Tincup Creek; Table common and rare haplotypes was detected in 4), and one where northern leatherside chub all 6 replicates at concentrations averaging 2 has never been observed (Rattlesnake Creek; copies per reaction. Technically, an assay can - Table 4). The eDNA samples were collected not achieve an efficiency greater than 100%, by filtering 5 L of water using methods out - as that would indicate a more than doubling of lined in Carim et al. (2016c). DNA was ex - the target amplicon during each cycle. How - tracted from the filters with the DNeasy Tis - ever, the reported efficiency value is calcu - sue and Blood Kit (Qiagen, Inc) following a lated based on the slope of a linear regression modified protocol (Carim et al. 2016b). The which, due to associated error, can result in an extracts were then analyzed using the opti - estimated efficiency of >100%. Assays should mized PCR conditions described above with a have an efficiency as close to 100% as possi - TaqMan Exogenous Internal Positive Control ble; however, an efficiency between 90% and (1.5 mL of 10X IPC assay and 0.15 mL of 50X 110% is generally acceptable (Thermofisher IPC DNA per reaction; Life Technologies) used Scientific 2016), and the calculated efficiency in place of some of the water to screen for PCR should be reported even if that value exceeds inhibition. All eDNA analyses were performed 100%. The standard curve for the plasmid across 3 replicates for each eDNA extract and DNA representing the 3 spinedace taxa was included a PCR no-template control substi - also efficient (98.9%, r2 = 0.984, y-intercept = tuting distilled water for DNA template. 40.2, slope = −3.3), and DNA was detected in The assay detected DNA in vitro from all 4 of 6 replicates at concentrations averaging northern leatherside chub samples, including 2 copies per reaction. Northern leatherside the plasmid DNA of the rare haplotype. The chub DNA was not detected in any environ - assay also detected DNA of the plasmid rep - mental samples taken where the species was resenting the Big Spring, Virgin, and White expected to be absent. Furthermore, the assay River spinedaces. The assay did not detect detected northern leatherside DNA in all DNA from any of the other nontarget species samples where the species was expected or or in the no-template controls. The standard known to be present, including at 100, 200, curve for the common haplotype resulted in an 300, and 400 m downstream from caged fish efficiency of 100.8% (r2 = 0.996, y-intercept in Jensen and Trout Creeks (Table 4). = 38.8, slope = −3.3) and a limit of detection The ability of the qPCR assay to consis - (defined as the lowest concentration with tently and reliably detect low concentrations >95% amplification success; Bustin et al. of northern leatherside chub DNA, as indi - 2009) at 2 copies per reaction. The standard cated by the in vitro standard curve analysis, 98 WESTERN NORTH AMERICAN NATURALIST (2018), V OL . 78 N O. 1, P AGES 92 –99 provides support for its application as a sensi - ACKNOWLEDGMENTS tive survey tool. Furthermore, the assay did not detect the DNA of potentially sympatric We thank Lee Mabey (Caribou-Targhee nontarget fish species, or of southern leather - National Forest), Trout Unlimited, and the side chub, the taxon from which it was split Bureau of Land Management for collecting during previous genetic analyses (Johnson et tissue samples. al. 2004). On the other hand, it did detect a DNA fragment representing 3 congeneric LITERATURE CITED spine dace species: Big Spring, Virgin, and BELK , M.C., AND J.B. J OHNSON . 2006. Biological status of White River spinedaces. This is not surpris - leatherside chub: a framework for conservation of ing given how closely related these species western freshwater fishes. American Fisheries Soci - are to the northern leatherside chub (Johnson ety Symposium 53:67–76. et al. 2004), resulting in few mismatches be - BLAKNEY , J.R., J.L. L OXTERMAN , AND E.R. K EELEY . 2014. Range-wide comparisons of northern leatherside tween the assay and the spinedace sequences chub populations reveal historical and contemporary screened (Table 1). However, we do not rec - patterns of genetic variation. Conservation Genetics ommend this eDNA assay for the detection of 15:757–770. spinedace because the nucleotide mismatches BUSTIN , S.A., V. B ENES , J.A. G ARSON , J. H ELLEMAN , J. may lead to reduced detection rates in field HUGGETT , M. K UBISTA , R. M UELLER , T. N OLAN , M.W. PFAFFL , G.L. S HIPLEY , ET AL . 2009. The MIQE guide - applications, especially if PCR inhibitors are lines: minimum information for publication of quanti - present (Jane et al. 2015). Importantly, there tative real-time PCR experiments. Clinical Chemistry are no instances of range overlap between 55:611–622. these spinedace species and northern leather - CARIM , K.J., K.R. C HRISTIANSON , K.S. M CKELVEY , W.M. PATE , D.B. S ILVER , B.M. J OHNSON , B.T. G ALLOWAY , side chub (Jezorek and Connolly 2013), so M.K. Y OUNG , AND M.K S CHWARTZ . 2016a. Environ - false positive results (de tection of northern mental DNA marker development with sparse bio - leatherside DNA where the species is absent) logical information: a case study on opossum shrimp would be unlikely across the northern leather - (Mysis diluviana ). PLOS ONE 11:e0161664. side range. CARIM , K.J., J.C. D YSTHE , M.K. Y OUNG , K.S. M CKELVEY , AND M.K. S CHWARTZ . 2016b. An environmental As long as robust eDNA sampling protocols DNA assay for detecting Arctic grayling in the upper (such as Carim et al. 2016c, McKelvey et al. Missouri River basin, North America. Conservation 2016) are paired with field surveys that Genetics Resources 8:197–199. address the ecological characteristics influenc - CARIM , K.J., K.S. M CKELVEY , M.K. Y OUNG , T.M. W ILCOX , AND M.K. S CHWARTZ . 2016c. Protocol for collecting ing the distribution of this species (Dauwalter eDNA samples from streams. General Technical et al. 2014, Schultz et al. 2016), the assay will Report RMRS-GTR-355, U.S. Department of Agri - be effective at detecting target DNA in low culture, Forest Service, Rocky Mountain Research concentrations, as demonstrated by eDNA Station, Fort Collins, CO. 18 pp. http://www.fs.fed .us/rm/pubs/rmrs_gtr355.pdf assays designed for other taxa (Wilcox et al. DAUWALTER , D.C., S.J. W ENGER , AND P. G ARDNER . 2014. 2013, 2015). Results from such surveys could The role of complexity in habitat use and selection be instrumental in helping biologists and man - by stream fishes in a Snake River basin tributary. agers target conservation efforts and evaluate Transactions of the American Fisheries Society 143:1177–1187. the success of northern leatherside reintro - DOWLING , T.E., C.A. T IBBETS , W.L. M INCKLEY , AND G.R. duction efforts and other management activi - SMITH . 2002. Evolutionary relationships of the plagop - ties (UDWR 2009). The presence of a rare terins (Teleostei: Cyprinidae) from cyto chrome b haplotype from Muddy Creek provides a cau - sequences. Copeia 2002:665–678. tionary note. These rare haplotypes do occur JANE , S.F., T.M. W ILCOX , K.S. M CKELVEY , M.K. Y OUNG , M.K. S CHWARTZ , W.H. L OWE , B.H. L ETCHER , AND in many species (see Wilcox et al. 2015) and A.R. W HITELEY . 2015. Distance, flow and PCR inhi - will lead to false negative results if the haplo - bition: eDNA dynamics in two headwater streams. types are unknown and fixed within a local Molecular Ecology Resources 15:216–227. JEZOREK , I.G., AND P.J. C ONNOLLY . 2013. Distribution and population. Therefore, when entering a new movement of Big Spring spinedace ( Lepidomeda area, we suggest either testing the assay by mollispinis pratensis ) in Condor Canyon, Meadow first collecting samples in areas where the tar - Valley Wash, Nevada. Western North American get species is known to be present or by Naturalist 73:323–336. sequencing the primer/probe region from JOHNSON , J.B., T.E. D OWLING , AND M.C. B ELK . 2004. Neglected taxonomy of rare desert fishes: Congru - locally derived tissue samples prior to relying ent evidence for two species of leatherside chub. on assay results for management. Systematic Biology 53:841–855. DYSTHE ET AL . ♦ NORTHERN LEATHERSIDE CHUB EDNA A SSAY 99

JOHNSON , J.B., AND S. J ORDAN . 2000. Phylogenetic diver - dam/LifeTech/ Documents/PDFs/PG1503-PJ9169 gence in leatherside chub ( Gila copei ) inferred from -CO019879-Re-brand-Real-Time-PCR-Understanding mitochondrial cytochrome b sequences. Molecular -Ct-Value-Americas-FHR.pdf Ecology 9:1029–1035. THOMSEN , P.F., J. K IELGAST , L.L. I VERSEN , C. W IUF , M. MCKELVEY , K.S., M.K. Y OUNG , E.L. K NOTEK , K.J. C ARIM , RASMUSSEN , M.T.P. G ILBERT , L. O RLANDO , AND E. T.M. W ILCOX , T.M. P ADGETT -S TEWART , AND M.K. WILLERSLEV . 2012. Monitoring endangered fresh - SCHWARTZ . 2016. Sampling large geographic areas water biodiversity using environmental DNA. Molec - for rare species using environmental DNA: a study ular Ecology 21:2565–2573. of bull trout Salvelinus confluentus occupancy in [USFWS] U NITED STATES FISH AND WILDLIFE SERVICE . western Montana. Journal of Fish Biology 88: 2011. 12-month finding on a petition to list northern 1215–1222. leatherside chub as endangered or threatened. Fed - SCHONHUTH , S., D.K. S HIOZAWA , T.E. D OWLING , AND eral Register 76:63444–63478. R.L. M AYDEN . 2012. Molecular systematics of [UDWR] U TAH DIVISION OF WILDLIFE RESOURCES . 2009. western North American cyprinids (Cypriniformes: Rangewide conservation agreement and strategy Cyprinidae). Zootaxa 3586:281–303. for northern leatherside ( Lepidomeda copei ). Utah SCHULTZ , L.D., P.A. C AVALLI , H. S EXAUER , AND D. Z AFFT . Department of Natural Resources, Division of Wild - 2016. Habitat and fish assemblage associations and life Resources – Native Aquatic Species. Salt Lake current status of northern leatherside chub Lep - City, UT. Publication no. 09–11. idomeda copei in western Wyoming. Western North WILCOX , T.M., K.J. C ARIM , K.S. M CKELVEY , M.K. Y OUNG , American Naturalist 76:427–440. AND M.K. S CHWARTZ . 2015. The dual challenges of SIGSGAARD , E.E., H. C ARL , P.R. M OLLER , AND P.F. T HOM - generality and specificity with developing environ - SEN . 2015. Monitoring the near-extinct European mental DNA markers for species and subspecies of weather loach in Denmark based on environmental Oncorhynchus. PLOS ONE 10(11):e0142008. https:// DNA from water samples. Biological Conservation doi.org/10.1371/journal.pone.0142008 183:46–52. WILCOX , T.M., K.S. M CKELVEY , M.K. Y OUNG , S.F. J ANE , SPEAR , S.F., J.D. G ROVES , L.A. W ILLIAMS , AND L.P. W AITS . W.H. L OWE , A.R. W HITELEY , AND M.K. S CHWARTZ . 2015. Using environmental DNA methods to improve 2013. Robust detection of rare species using envi - detectability in a hellbender ( Cryptobranchus alle - ronmental DNA: the importance of primer speci - ganiensis ) monitoring program. Biological Conserva - ficity. PLOS ONE 8(3):e59520. https://doi.org/10 tion 183:38–45. .1371/journal.pone.0059520. TABERLET , P., E. C OISSAC , M. H AJIBABAEI , AND L.H. WILCOX , T.M., K.S. M CKELVEY , M.K. Y OUNG , A.J. S EPUL - RIESEBERG . 2012. Environmental DNA. Molecular VEDA , B.B. S HEPARD , S.F. J ANE , A.R. W HITELEY , W.H. Ecology 21:1789–1793. LOWE , AND M.K. S CHWARTZ . 2016. Understanding TAMURA , K., D. P ETERSON , N. P ETERSON , A. F ILIPSKI , AND environmental DNA detection probabilities: a case S. K UMAR . 2013. MEGA6: molecular evolutionary study using a stream-dwelling char Salvelinus fonti - genetics analysis version 6.0. Molecular Biology and nalis. Biological Conservation 194:209–216. Evolution 30:2725–2729. THERMO FISHER SCIENTIFIC . 2016. Real-time PCR: under - Received 5 October 2017 standing C t. Real-time PCR Application Note. Revised 25 January 2018 Applied Biosystems, ThermoFisher Scientific, Life Accepted 29 January 2018 Technologies. http://www.thermofisher.com/content/ Published online 4 April 2018