A Non-Invasive Sampling Method for Detecting Non-Native Smallmouth Bass ( Micropterus dolomieu )

Abstract

The smallmouth bass (Micropterus dolomieu) is a cool-water fish species native to central North America. Widespread introductions and secondary spread outside of its historical range have led to new recreational fisheries and associated economic benefits in western United States, but have also resulted in a number of ecological impacts to recipient ecosystems, including threats to Pacific salmon. Management of introduced smallmouth bass populations, now and into the future, relies on accurate detection and monitoring of this species. To address this need, we developed an environmental DNA assay that can detect smallmouth bass DNA extracted from filtered water samples in concentrations as low as 2 mtDNA copies per reaction. Field testing demonstrated that eDNA sampling produced results largely consistent with snorkel surveys, a traditional visual assessment, and gained a few additional positive detections. While this assay is robust against non-target detection, including the only other Micropterus in Pacific Northwest streams, largemouth bass (M. salmoides), the high genetic similarity within the sunfish family Centrarchidae made it unable to distinguish smallmouth bass from spotted bass (M. punctulatus) and some Guadalupe bass (M. treculii). The high sensitivity of this method and assay will be particularly useful for identifying the location of non-native smallmouth bass in the Pacific Northwest, quantifying its rate of spread, and aiding management actions.

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A Non-Invasive Sampling Method for Detecting Non-Native
Smallmouth Bass (Micropterus dolomieu)
Author(s): Thomas W. Franklin, Joseph C. Dysthe, Erika S. Rubenson, Kellie J.
Carim, Julian D. Olden, Kevin S. McKelvey, Michael K. Young and Michael K.
Schwartz
Source: Northwest Science, 92(2):149-157.
Published By: Northwest Scientific Association
https://doi.org/10.3955/046.092.0207
URL: http://www.bioone.org/doi/full/10.3955/046.092.0207
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149
NorthwestScienceNotes
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Northwest Science, Vol. 92, No. 2, 2018
ThomasW.Franklin and JosephC.Dysthe1, United States Department of Agriculture, Forest Service, National Genomics
Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula, Montana 59801
ErikaS.Rubenson, School of Aquatic and Fishery Sciences, University of Washington, 1122 NE Boat Street, Seattle,
Washington 98195
KellieJ.Carim,United States Department of Agriculture, Forest Service, National Genomics Center for Wildlife and Fish
Conservation, Rocky Mountain Research Station, Missoula, Montana 59801
JulianD.Olden, School of Aquatic and Fishery Sciences, University of Washington, 1122 NE Boat Street, Seattle,
Washington 98195
KevinS.McKelvey, MichaelK.Young, and MichaelK.Schwartz, United States Department of Agriculture, Forest
Service, National Genomics Center for Wildlife and Fish Conservation, Rocky Mountain Research Station, Missoula,
Montana 59801
ANon-InvasiveSamplingMethodforDetectingNon-NativeSmallmouth
Bass(Micropterus dolomieu)
Abstract
The smallmouth bass (Micropterus dolomieu) is a cool-water sh species native to central North America. Widespread
introductions and secondary spread outside of its historical range have led to new recreational sheries and associated eco-
nomic benets in western United States, but have also resulted in a number of ecological impacts to recipient ecosystems,
including threats to Pacic salmon. Management of introduced smallmouth bass populations, now and into the future,
relies on accurate detection and monitoring of this species. To address this need, we developed an environmental DNA
assay that can detect smallmouth bass DNA extracted from ltered water samples in concentrations as low as 2 mtDNA
copies per reaction. Field testing demonstrated that eDNA sampling produced results largely consistent with snorkel sur-
veys, a traditional visual assessment, and gained a few additional positive detections. While this assay is robust against
non-target detection, including the only other Micropterus in Pacic Northwest streams, largemouth bass (M. salmoides),
the high genetic similarity within the sunsh family Centrarchidae made it unable to distinguish smallmouth bass from
spotted bass (M. punctulatus) and some Guadalupe bass (M. treculii). The high sensitivity of this method and assay will
be particularly useful for identifying the location of non-native smallmouth bass in the Pacic Northwest, quantifying its
rate of spread, and aiding management actions.
Keywords: eDNA, qPCR, invasive species, non-invasive sampling, aquatic species monitoring
Introduction
Smallmouth bass (Micropterus dolomieu) are
members of the sunsh family (Centrarchidae)
and are native to large areas of the midwestern
US and south-central Canada (Scott and Cross-
man 1973, Page and Burr 2011). Its popularity as
a sport sh has led to widespread introductions,
and it is now found in 41 US states and over 20
other countries (Loppnow et al. 2013). While these
introductions have formed important recreational
1Author to whom correspondence should be addressed.
Email: jdysthe@fs.fed.us

150 Franklin et al.
shing industries across the country, they have
also been associated with rapid declines in prey
species (generally small sh and craysh), with
potential cascading effects throughout entire eco-
systems (Jackson 2002, Vander Zanden et al. 2004,
Loppnow et al. 2013). Within the northwestern US,
the consequences of smallmouth bass predation
on salmonids is of particular concern (Carey et al.
2011), since smallmouth bass have been shown to
consume up to 35% of a single salmon run under
certain conditions (Fritts and Pearsons 2004,
Sanderson et al. 2009). The predicted warming
of water temperatures are likely to benet small-
mouth bass, both creating more suitable habitat
and increasing its metabolic efciency throughout
Pacic Northwest streams (Petersen and Kitchell
2001; Lawrence et al. 2014, 2015). Consequently,
developing rapid, repeatable, and cost-effective
techniques for assessing the distribution of non-
native smallmouth bass is critical for targeting
conservation and management activities (Brewer
and Orth 2015).
Studies have repeatedly demonstrated that
environmental DNA (eDNA) is a reliable, efcient
and sensitive tool for identifying the presence and
delimiting the distribution of aquatic species in
low abundance (e.g., Dejean et al. 2012, Goldberg
et al. 2013, Wilcox et al. 2013, Sigsgaard et al.
2015, Carim et al. 2016a, McKelvey et al. 2016).
To assist management of non-native smallmouth
bass, we developed an eDNA assay that detects
low concentrations of smallmouth bass DNA
extracted from ltered water samples.
Methods
To develop and validate the smallmouth bass
eDNA assay, we considered four commonly
sequenced regions of the mitome: cytochrome b
(cytb), cytochrome oxidase I (COI), mitochondrial
control region (mtCR), and NADH dehydroge-
nase subunit 2 (ND2), and two nuclear regions:
S7-ribosomal protein (S7-r) and rhodopsin. Of
these COI provided the best combination of good
geographic coverage of smallmouth bass, sufcient
nucleotide differences to distinguish smallmouth
bass from most non-target species, and sufcient
samples associated both with sympatric bass and
other non-target species. We therefore compiled
publicly available DNA sequences of a fragment
of the cytochrome oxidase I (COI) mitochondrial
region of the smallmouth bass and 50 closely re-
lated or potentially sympatric taxa (Table 1). The
smallmouth bass COI sequences (n = 38) were
from sh originating in Alabama (n = 1), California
(n = 1), Illinois (n = 2), Kentucky (n = 2), New
Mexico (n = 2), New York (n = 2), Ohio (n = 1),
and Pennsylvania (n = 1) in the United States; in
Ontario (n = 10) and Quebec (n = 10) in Canada; in
two locations in Japan (n = 2); and four of unknown
origin. We screened these sequences in silico us-
ing the DECIPHER package (Wright et al. 2014)
in R v. 3.0.3 (R Core Team 2013) and obtained
candidate primers to amplify smallmouth bass
DNA. We aligned the candidate primers with the
sequence data in MEGA 6.0 (Tamura et al. 2013)
and modied primer lengths (forward: 5’-CAGC-
TATTTCCCAGTATCAGACACC-3’; reverse:
5’-TTGAGGTTTCGATCCGTAAGRA-3’) to
optimize annealing temperatures in Primer Express
3.0.1 (Life Technologies; forward: 59.1 °C, reverse:
57.5–59.5 °C). Primers were designed to maximize
nucleotide mismatches with non-target species and
to amplify a 130-nucleotide fragment of the small-
mouth bass COI region. Within this fragment, we
designed a FAM-labeled, minor-groove-binding,
non-uorescent quencher (MGB-NFQ) probe
(FAM-TTATCGCTCCCAGTCCT-MGBNFQ) that
likewise minimized identity with non-targets. We
assessed the annealing temperature of the probe
in Primer Express 3.0.1 (69 °C) and screened the
primer-probe set for secondary structures using
IDT OligoAnalyzer (https://www.idtdna.com/
calc/analyzer). To evaluate the specicity of the
smallmouth bass assay, we compared primer and
probe sequences with sequences in the NCBI
database using a nucleotide BLAST search.
We then evaluated the eDNA assay in vitro by
screening DNA extracted from 38 smallmouth
bass tissues (from 10 locations) and 30 additional
non-target species (Table 2). Smallmouth bass
tissues from Oregon were collected under Or-
egon Scientic Take Permit 19450 for Fish and
Freshwater Invertebrates; tissues from Colorado
were collected from Cheesman Reservoir under
written permission from the Denver Water Board.

151
Northwest Science Notes: Smallmouth Bass Environmental DNA Assay
All other tissues and DNA used in this study were
from archived samples collected for other projects
under appropriate state or federal permits. We
extracted DNA from tissue using the DNeasy
Blood and Tissue Kit (Qiagen, Inc.) following
the manufacturer’s protocol.
We tested smallmouth bass eDNA assay using
a StepOne Plus Real-time PCR Instrument (Life
Technologies) in 15-µl reactions containing 7.5
µl of Environmental Master Mix 2.0 (Life Tech-
nologies), 900 nM of each the forward and reverse
primer, 250 nM of probe, and 4 µl of DNA template
(~ 0.4 ng), with the remaining volume composed
of PCR-grade water. Thermocycling conditions
were as follows: initial denaturation at 95 °C for
10 min followed by 45 cycles of denaturation at
95 °C for 15 s and annealing and extension at 60
°C for 1 min. We optimized primer concentrations
using methods outlined in Wilcox et al. (2015),
which resulted in a nal concentration of 600 nM
each for the forward and reverse primer, and 250
nM for the probe. We then tested the sensitivity
of the marker by analyzing it with a seven-level
standard curve dilution series (31 250, 6 250, 1
250, 250, 50, 10, and 2 copies per 4 μl) made
from purified smallmouth bass PCR product
diluted into sterile TE. Each dilution was run in
sextuples using the aforementioned marker con-
centrations and cycling conditions. For all qPCR
experiments, a reaction was considered positive
if the amplication curve crossed the threshold
during the exponential phase.
Finally, we tested the assay in vivo by analyzing
environmental samples collected from 14 sites also
surveyed for smallmouth bass via snorkeling (Table
3). Environmental DNA was collected from water
samples using the protocol described in Carim et
al. (2016b). Briey, we ltered 5 l of subsurface
water through a glass microber lter (pore size
1.5 µm) with a peristaltic pump and the lter was
folded into quarters and immediately placed in
a clean 1 l plastic bag with silica desiccant. We
then stored the samples in a cool, dark location
until shipping them to the lab for processing (see
Carim et al. 2016b for details). After eDNA water
samples were collected, snorkel surveys were
conducted by two snorkelers on opposite shore-
lines of each river segment. Snorkelers surveyed
a 200 m segment of river immediately upstream
of the eDNA sampling location proceeding in an
upstream direction and counting all bass observed
within the segment. Where bass were present in
numbers too large to accurately tally, the count
was recorded as “abundant”. The snorkel surveys
were conducted as part of a larger study examin-
ing smallmouth bass occupancy across the Pacic
Northwest.
Environmental DNA samples were extracted
with the DNeasy Blood and Tissue Kit (Qiagen,
Inc., Valencia, CA) following a modied protocol
(Carim et al. 2016c). Using optimized assay con-
centrations and the qPCR prole described above,
we analyzed these eDNA samples in triplicate
reactions and included a TaqMan Exogenous
Internal Positive Control (1.5 µl of 10X IPC as-
say and 0.30 µl of 50X IPC DNA per reaction;
Life Technologies) in place of some of the water
to screen for inhibition. All analyses included a
no-template control where distilled water was
substituted for DNA.
Results
The in silico analyses revealed that smallmouth
bass are divergent from the majority of Centrar-
chidae species that overlap in range and we do
not expect to see cross amplication with large-
mouth bass (M. salmoides), the sole co-occurring
Micropterus in Pacic Northwest streams (Table
1). However, smallmouth bass differed little from
spotted bass (M. punctulatus; median difference
= 2 nucleotides; range = 0–4 nucleotides) and
25 of 32 Guadalupe bass sequences (M. treculii;
median difference = 3 nucleotides; range = 2–5
nucleotides) in the 623-nucleotide COI fragment
considered for this assay. As a result, all spotted
bass and most (25/32) Guadalupe bass sequences
were identical to smallmouth bass in the primer-
probe region and cross amplication would be
expected (Table 1).
The in vitro analyses produced positive de-
tections in all smallmouth bass samples, and
negative results for all non-target samples. The
standard curve analysis resulted in an amplica-
tion efciency of 98.8% (r2 = 0.997; y-intercept

152 Franklin et al.
TABLE 1. Species, number of sequences, and GenBank accession number for DNA sequences used for in silico marker devel-
opment. Also included is the minimum number of nucleotide mismatches between each sequence and the forward
primer (F), reverse primer (R), and probe (P).
Nucleotide
mismatches
Name nGenBank accession numbers F R P
Smallmouth bass Micropterus dolomieu 38
AB378749, 750; EU524131, 810-828;
HQ557267, 268; JN027219-227; KC819888;
KF558298; KJ843438-440
0 0 0
Spotted bass Micropterus punctulatus 9 HQ579041; JN027232-235; KJ843420-423 0 0 0
Guadalupe bass Micropterus treculii 25 HQ557528, 529; KJ843386, 387, 393;
KJ843396-415 0 0 0
7aKJ843388-392; KJ843394, 395 4 4 2
Alabama bass Micropterus henshalli 4 KJ843374-377 4 2 2
Florida large-
mouth bass Micropterus oridanus 12 HQ557526, 527; JN027228, 229; KC684999;
KC789544-547; KJ843378-380 425
Largemouth bass Micropterus salmoides 40
EU524132, 834-838; HQ557265, 266, 285, 286,
411; JN027236-241; KC819886; KF558299-301;
KF930132, 133; KJ843416-419;
KP112310-317; KR477066, 222; KT248859;
KT307155; KX459325
42 3
Redeye bass Micropterus coosae 10 HQ579042-044; JN027215-218; KJ843435-437 4 2 2
Shoal bass Micropterus cataractae 9 JN027211-214; KJ843381-385 4 4 2
Suwanee bass Micropterus notius 8 HQ557325; JN027230, 231; KJ843441-445 5 3 3
Choctaw bassbMicropterus cf.
punctulatus 52 KJ843424-434; KT806130-170 4 3 2
Brook lamprey Lampetra planeri 1 KM286716 9 7 5
Brook trout Salvelinus fontinalis 4 HQ960794; HQ961027; KM287121, 123 37 5
Brown trout Salmo trutta 4 KC501168; KM287114, 116, 119 5 6 6
Bull trout Salvelinus conuentus 4 EU522399, 401, 403; EU524365 4 5 5
Channel catsh Ictalurus punctatus 2EU524685; JN026912 7 5 4
Chinook salmon Oncorhynchus
tshawytscha 4 EU524234; FJ164931; HQ712706; KF558293 6 5 5
Common carp Cyprinus carpio 2KF929811; KM286637 5 4 7
Cutthroat trout Oncorhynchus clarkii 4 EU524198, 201; HQ557150; JN027854 7 5 5
Dollar sunsh Lepomis marginatus 4 JN027021-025 4 34
Dolly Varden Salvelinus malma 4 EU522411, 413, 415, 417 4 5 4
European river
lamprey Lampetra uviatilis 1 KM286704 9 7 5
Flathead chub Platygobio gracilis 2JN028256, 259 5 5 4
Flier Centrarchus macropterus 4 JN024957-960 4 35
Freshwater drum Aplodinotus grunniens 2EU522444; EU523922 36 6
Goldeye Hiodon alosoides 2EU524650; KF929971 4 35
Green sunsh Lepomis cyanellus 4 JN026981-984 4 4 3
Kern brook
lamprey Entosphenus hubbsi 1 HQ557301 9 7 5
Klamath lamprey Entosphenus similis 1 JN025330 7 7 5
Longear sunsh Lepomis megalotis 4 JN027035-037, 042 24 4
Mottled sculpin Cottus bairdii 4 HQ557189; JN025020, 023, 026 5 5 5
Mountain
whitesh Prosopium williamsoni 2HQ557336, 337 5 35
Muskellunge Esox maquinongy 4 EU524600-602, 659 7 5 7

153
Northwest Science Notes: Smallmouth Bass Environmental DNA Assay
= 38.776; slope = –3.352), and DNA was detected
in all six replicates at two copies per reaction,
the lowest concentration tested. In vivo tests of
the eDNA assay were consistent with the results
of the snorkeling surveys. Specically, small-
mouth bass DNA was detected at all sites where
smallmouth bass were observed by snorkelers. At
two of these sites (MF John Day and Clark Fork
Rivers), three bass were observed at each site;
at the other three sites (Grande Ronde, Lochsa,
and NF John Day Rivers), bass were abundant.
Additionally, eDNA assays detected smallmouth
bass at four sites where they were not visually
observed (Table 3). There were no detections of
DNA in the no-template controls.
Discussion
The eDNA assay we describe here efciently and
reliably detects low concentrations of smallmouth
bass DNA present in ltered water samples, and
will not amplify DNA of any non-target species
likely to be present in the Pacic Northwest.
While we did not evaluate the correlation between
eDNA quantity and smallmouth bass abundance,
this assay could be employed to do so in future
studies. This correlation has been examined for
TABLE 1. Continued
Nucleotide
mismatches
Name nGenBank accession numbers F R P
Northern pike Esox lucius 5EU524589; HM563699; HQ961033; KM224846;
KM286646 5 7 2
Olympic mud
minnow Novumbra hubbsi 4 HQ557339; JN027849-851 4 5 6
Pacic lamprey Entosphenus tridentatus 2GU440367; KF918874 7 7 5
Pit-Klamath
brook lamprey Entosphenus lethophagus 1 HQ579097 7 7 5
Rainbow trout Oncorhynchus mykiss 4 FJ999086, 088, 090; KM373668 6 5 4
Redear sunsh Lepomis microlophus 4 JN027043-046 4 3 3
River carpsucker Carpiodes carpio 2JN024862, 865 6 5 4
Sauger Sander canadensis 4 EU524368-071 4 35
Shorthead
redhorse
Moxostoma
macrolepidotum 2JN027298; KF930145 6 6 4
Shovelnose
sturgeon
Scaphirhynchus
platorynchus 2JN028406, 07 7 6 6
Slimy sculpin Cottus cognatus 3JN025088, 097, 099 5 5 5
Sockeye salmon Oncorhynchus nerka 4 EU524223, 225; FJ999233; HQ712703 5 6 4
Stonecat Noturus avus 2JN027790, 97 6 3 3
Three-spined
stickleback Gasterosteus aculeatus 3EU254634; HQ712384; KR862768 5 6 4
Walleye Sander vitreus 4 EU524374-377 5 35
Western brook
lamprey Lampetra richardsoni 1 JN026960 8 8 5
Western silvery
minnow Hybognathus argyritis 2EU524071, 074 5 6 5
White sucker Catostomus commersonii 2HQ579108; KF929688 5 7 4
Yellow perch Perca avescens 3JX516993; JX517139, 165 5 5 6
aThese seven samples are listed in Tringali et al. (2015) Figure 3 as “Lineage B”
bSamples in Genbank listed as Micropterus cf. punctulatus belong to a proposed species, the Choctaw bass (Tringali et al. 2015).

154 Franklin et al.
TABLE 2. List of species used for in vitro screening of the primers and probe in this study. Origin refers to the waterbody for
smallmouth bass; for all other samples, origin is listed by state.
Species nOrigin
Smallmouth bass Micropterus dolomieu 2Cheesman Reservoir, CO
4 Clark Fork River, MT
3Missouri River, MT
4 River Rock Pond, MT
3Seeley Lake, MT
3MF John Day River, OR
13 NF John Day River, OR
2James River, VA
2Rocksh River, VA
2Upper Tye River, VA
Apache trout Oncorhynchus apache 1 NM
Atlantic salmon Salmo salar 1 F*
Bonneville cutthroat trout Oncorhynchus clarkii utah 1 UT
Brook trout Salvelinus fontinalis 1VA
Brown trout Salmo trutta 1 OR
Bull trout Salvelinus conuentus 1 OR
Chinook salmon Oncorhynchus tshawytscha 1 ID
Chum salmon Oncorhynchus keta 1 OR
Coastal cutthroat trout Oncorhynchus clarkii clarkii 2OR
Coho salmon Oncorhynchus kisutch 1 OR
Cutthroat trout Oncorhynchus clarkii 1WA
Dolly Varden trout Salvelinus malma 1AK
Gila trout Oncorhynchus gilae 1 NM
Lake trout Salvelinus namaycush 1 MT
Lake whitesh Coregonus clupeaformis 1 MT
Lamprey Lampetra sp. 1 OR
Largemouth bass Micropterus salmoides 3MT
Mountain whitesh Prosopium williamsoni 1 MT
Muskellunge Esox maquinongy 1 MN
Northern pike Esox lucius 1AK
Pacic lamprey Entosphenus tridentatus 1WA
Pink salmon Oncorhynchus gorbuscha 1 OR
Pit-Klamath brook lamprey Entosphenus lethophagus 1 OR
Rainbow trout Oncorhynchus mykiss 3ID, MT, OR
Sauger Sander canadensis 1 WY
Sockeye salmon Oncorhynchus nerka 2MT, OR
Walleye Sander vitreus 1WA
Westslope cutthroat trout Oncorhynchus clarkii lewisi 1 MT
Yellow perch Perca avescens 1WA
Yellowstone cutthroat trout Oncorhynchus clarkii bouvieri 1 WY
*F refers to a sample of farmed origin; location data not available for this sample.

155
Northwest Science Notes: Smallmouth Bass Environmental DNA Assay
other species (Wilcox et al. 2016) and has shown
promise as an assessment of species abundance in
lakes (Lacoursiere-Roussel et al. 2016) and large
streams and rivers (Doi et al. 2017). It is important
to note that publicly available sequence data for
spotted bass shows this species is nearly identical
to smallmouth bass across the COI gene (Tringali
et al. 2015), and identical in the primer and probe
regions (Table 1). Therefore, the assay will lack
specicity where these species co-occur, but can
also be used to reliably detect spotted bass where
smallmouth bass can condently be assumed to be
absent. Publicly available data for Guadalupe bass
displayed a range of haplotypes at the COI gene,
some of which are nearly identical to smallmouth
bass, and others that are highly (8.99–9.31%)
divergent (Tringali et al. 2015; Table 1). Thus,
this assay will not provide reliable results for
detection of smallmouth bass where they co-occur
with Guadalupe bass, nor will it provide reliable
detections of Guadalupe bass. Although this lack
of specicity may limit application of the assay
where both species are natively sympatric, it will
be a reliable and effective tool for assessing the
presence of nonnative smallmouth bass in sensitive
regions such as the Pacic Northwest. This assay
will facilitate the determination of smallmouth
bass occurrence and spread, making it a powerful
aid to any management actions.
Acknowledgments
We thank Bill Pate of Colorado State University,
Robert Humston of Washington and Lee Uni-
versity, and Ryan Kreiner and Mike Ruggles of
Montana Fish, Wildlife and Parks for providing
tissue samples. Funding support to ESR was
provided by the National Science Foundation
Graduate Research Fellowship Program, and to
JDO by the University of Washington H. Mason
Keeler Endowed Professorship.
TABLE 3. Collection information and detection results (Y = detected; N = not detected) of the snorkel surveys and eDNA samples
used for in vivo validation of the smallmouth bass eDNA marker.
Smallmouth bass
presence
Location Latitude Longitude Date
Snorkel
survey
eDNA
sampling
Clark River, MT 47.32260 –114.89276 7/28/16 Y Y
Lochsa River, ID 46.16267 –115.59100 7/27/16 Y Y
Grande Ronde River, OR 45.38432 –117.92917 7/19/16 Y Y
NF John Day River, OR 44.99097 –119.10401 7/20/16 Y Y
MF John Day River, OR 44.82513 –119.01089 7/21/16 Y Y
John Day River, OR 44.41832 –119.22548 7/22/16 NY
NF Malheur River, OR 43.75676 –118.06197 7/23/16 NY
Deschutes River, OR 45.38898 –120.87538 8/9/16 NY
Methow River, WA 48.04847 –119.92178 7/31/16 NY
Yakima River, WA 46.50535 –120.45632 7/18/16 N N
Payette River, ID 44.07372 –116.11997 7/24/16 N N
Salmon River, ID 45.35952 –113.94734 7/25/16 N N
Selway River, ID 46.09872 –115.54492 7/26/16 N N
Kootenay River, MT 48.60600 –116.04088 7/29/16 N N

156 Franklin et al.
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Received 06 October 2017
Accepted 22 January 2018