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



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.
Source: Northwest Science, 92(2):149-157.
Published By: Northwest Scientific Association
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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
Bass(Micropterus dolomieu)
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
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.
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.
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-
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
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 (
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.
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
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.
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).
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
whitesh Prosopium williamsoni 2HQ557336, 337 5 35
Muskellunge Esox maquinongy 4 EU524600-602, 659 7 5 7
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.
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
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
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
macrolepidotum 2JN027298; KF930145 6 6 4
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
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.
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.
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
Location Latitude Longitude Date
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
... These and other types of data can be collected by autonomous vehicles, filling holes between (but not replacing) ship-based surveys. These methods provide data in key areas, including population abundance [453], the presence of invasive species [454][455][456], a history of thermal stress [457], habitat issues such as barriers to passage [458], and community composition [459]. While these techniques have limitations [460], we predict that new genetic approaches will change our entire perspective on freshwater and marine communities and the spatial and temporal overlap among species. ...
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As we confront novel environmental challenges, a full understanding of the physical and biological processes that govern species responses to climate change will help maintain biodiversity and support conservation measures that are more robust to irreducible uncertainty. However, climate impacts are so complex, and the literature on salmon and trout is so vast that researchers and decision makers scramble to make sense of it all. Therefore, we conducted a systematic literature review of climate impacts on salmon and anadromous trout as a resource for stakeholders, managers, and researchers. We reviewed studies published from 2010 to 2021 that address climate impacts on these fish and organized them in a database of 1169 physical and 1853 biological papers. Papers are labeled with keywords across eight categories related to subject matter and study methods. We compared the literature by biological process and life stage and used these comparisons to assess strengths and weaknesses. We then summarized expected phenotypic and genetic responses and management actions by life stage. Overall, we found the largest research gaps related to species interactions, behavioral responses, and effects that carry over across life stages. With this collection of the literature, we can better apply scarce conservation resources, fill knowledge gaps, and make informed decisions that do not ignore uncertainty.
... Smallmouth bass (Micropterus dolomieu) are a non-native species known to reduce abundance, alter habitat use, and extirpate small prey fishes (MacRae and Jackson, 2001). Smallmouth bass are known to feed with increased efficiency in warmer water on a variety of taxa including insects, crayfish, and small fish (Schultz et al., 2017;Franklin et al., 2018). Smallmouth bass are a hypothesized novel predator of Umpqua chub (Oregonichthys kalawatseti), a small warm water minnow endemic to the Umpqua basin (Simon and Markle, 1999) that became a state and federal sensitive species (U.S. Fish and Wildlife Service [USFWS], 2020; Oregon Department of Fish and Wildlife [ODFW], 2021). ...
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A major challenge in ecology is disentangling interactions of non-native, potentially invasive species on native species. Conditional two-species occupancy models examine the effects of dominant species (e.g., non-native) on subordinate species (e.g., native) while considering the possibility that occupancy of one species may affect occupancy and/ or detection of the other. Although conditional two-species models are useful for evaluating the influence of one species on presence of another, it is possible that species interactions are density dependent. Therefore, we developed a novel two-species occupancy model that incorporates multiple abundance states (i.e., absent, present, abundant) of the native species. We showcase the utility of this model with a case study that incorporates random effects and covariates on both occupancy and detection to help disentangle species interactions given varying occupancy and detection in different abundance states. We use snorkel survey data from the Umpqua basin, Oregon, where it is hypothesized that smallmouth bass Micropterus dolomieu, a non-native piscivore, exclude Umpqua chub Oregonichthys kalawatseti, a small endemic minnow. From our two-species multi-state (2SMS) model, we concluded that average occupancy was low for both fishes, and that when non-native bass were present, overall native chub occupancy in the present (0.18 ± 0.05 SD) and abundant (0.19 ± 0.03) states was higher than when non-natives were absent (0.14 ± 0.02/ 0.08 ± 0.02), indicating the non-native was not excluding the native species. By incorporating a species interaction factor, we found a positive association (6.75 ± 5.54 SD) between native chub and non-native bass. The covariates strongly related to occupancy were elevation, algae, and land cover type (urban and shrub). Detection probability for both species (0.21–0.82) was most strongly related to the covariates day of year, water temperature, gravel substrate, and stream order/ magnitude. Incorporation of detection probability and covariates enabled interpretation of interactions between the two species that may have been missed without their inclusion in the modeling process. Our new 2SMS occupancy model can be used by scientists and managers with a broad range of survey and covariate data to disentangle species interactions problems to help them inform management decisions.
... Unfortunately, while efforts are underway to classify the thermal and flow associations of many species, these are often developed at relatively small scales and therefore have limited applicability to large scale regional analyses (Myers et al., 2017;Krabbenhoft et al., 2020). Additionally, species may even vary in which thermal class they are associated with from region to region (i.e., smallmouth bass (Micropterus dolomieu) are considered a warmwater species in some areas (Sharma et al., 2009), and coolwater species in others (Franklin et al., 2018)), and many species (particularly non-game species) have not been evaluated for thermal or flow associations at all (For example the FiCli database contains only 41 manuscripts related to Cyprindidae, compared to 168 for Salmonidae; Krabbenhoft et al., 2020). The lack of a uniform classification of species associations with thermal and flow characteristics limits our ability to evaluate patterns across guilds at a broad scale. ...
Climate change is expected to alter stream fish habitat potentially leading to changes in the composition and distribution of fish communities. In the Northeastern and Midwestern United States we identified the distribution and characteristics of those fish communities most and least at risk of experiencing changes in climate which deviate from the climate they are associated with. We classified stream fish communities based on a suite of climate and environmental variables with multivariate regression trees under both recent and future conditions based on eight climate models. Our findings showed that some areas, such as the majority of the Illinois, Wisconsin, and Iowa), have high levels of risk of change in stream class, while much of Kentucky, West Virginia, Virginia, Pennsylvania, Eastern Ohio, Southern Michigan, and the Atlantic Coast are at relatively low risk. Stream class shifts ranged from over 75% of segments lost (associated with cooler temperatures) to gains of over 40% (associated with warmer temperatures). Common warmwater species such as green sunfish (Lepomis cyanellus), bluegill (Lepomis macrochirus) and largemouth bass (Micropterus salmoides) are expected to have the largest net gains in associated stream classes, while species associated with cooler streams such as Southern redbelly dace (Chrosomus erythrogaster), slimy sculpin (Cottus cognatus), and Eastern blacknose dace (Rhinichthys atratulus) were expected to experience the largest proportional losses. By pairing our climate risk predictions with other stressors such anthropogenic land use, habitat fragmentation, and water quality impairment, we identified opportunities for preservation (low risk due to all threats), restoration (low risk due to climate, high risk due to other stressors), and adaptation (high climate risk with low risk from other stressors). Understanding which communities are at risk due to climate change will aid in developing adaptation strategies to help sustain them in the future.
... As a check on the probability of type II error (i.e., false negative) where age-0 individuals may be rare, we collected environmental DNA (eDNA) samples in conjunction with all 2019 surveys using the field sampling and laboratory methods described by Franklin et al. (2018) and Rubenson and Olden (2019). Field and laboratory methods are described in the Supplementary Methods available in the online version of this article. ...
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Knowledge of potential spread by introduced species is critical to effective management and conservation. The Smallmouth Bass Micropterus dolomieu is an example of a fish that has been introduced globally, often spreads after introduction, and has substantial predatory impacts on fish assemblages. Nonnative Smallmouth Bass in the free‐flowing Yellowstone River, Montana, have expanded from warmer, downstream sections of river into colder, upstream sections containing socio‐economically valuable trout fisheries. We sought insight into mechanisms controlling upstream spread by evaluating whether progressively colder upstream climates physiologically constrained successful recruitment by limiting age‐0 growth and preventing overwinter survival (i.e., population establishment). We documented the phenology, growth, and overwinter survival of age‐0 Smallmouth Bass across a temperature gradient leading to their upstream extent in the Yellowstone River. The upstream extent of population establishment did not appear limited by water temperature alone. Age‐0 body size at the onset of winter did not differ significantly between colder, upstream reaches and warmer, downstream reaches. Instead, the earlier hatch timing exhibited by some age‐0 individuals in upstream sections allowed them to experience longer growing seasons than many individuals in downstream sections. This counter‐intuitive hatching phenology mediated much of the expected decreases in growth in colder, upstream climates. Furthermore, evidence of successful overwinter survival and simulations of age‐0 starvation mortality indicated that age‐0 individuals at the upstream extent of their distribution successfully recruited to the age‐1 year‐class during four consecutive years. However, age‐0 individuals were rare or absent throughout the uppermost upstream distribution of adults, suggesting that something other than temperature limits or discourages reproduction farther upstream. Taken together, our results suggest that Smallmouth Bass have not yet reached the thermal limit of their upstream distribution in the Yellowstone River and that future spread may challenge fisheries managers tasked with management of coldwater trout fisheries in this river.
... The effectiveness of an eDNA assay for detecting a species also relies on a taxonomy that corresponds to evolutionary history, because the sensitivity and specificity of a qP-CR-based eDNA assay are correlated with the levels of divergence between the target species and all potentially sympatric taxa. In some cases, eDNA assays were recognized as being effective for detecting occupancy across most but not all of a species' range because high intraspecific diversity prevented development of a more comprehensive assay (Dysthe et al., 2017) or because the assay was intended for use in a limited area with a small pool of nontarget species (e.g., Franklin et al., 2018). When a named taxon, however, includes unrecognized biodiversity in the form of cryptic species, patterns of occupancy based on qPCR-based eDNA sampling may be spurious. ...
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A lineage of the Mountain Sucker species complex (Pantosteus jordani) exists as a genetically and morphologically distinct taxon restricted to the Missouri River basin. This species is thought to be declining throughout its range and is assumed to be extirpated from the southern portion of its distribution. We developed a quantitative PCR-based environmental DNA assay for P. jordani to help define and monitor its current range. The assay is both specific to P. jordani and sensitive to low amounts of DNA, with a detection limit of 10 DNA copies per reaction. In vitro experiments involved testing DNA from twenty tissue samples, collected from the Missouri River basin in Montana and Wyoming. The assay efficiently detected DNA of all P. jordani samples and did not amplify DNA of any closely related nontarget species. Additionally, 29 environmental DNA samples were taken in 19 waterbodies within P. jordani range and its presence or absence was determined prior to sampling at six of 29 sites. All sites where P. jordani was known absent produced negative results, and all sites where it was known present were confirmed with environmental DNA detections. The new assay was able to detect P. jordani at ten sites which were not previously known to contain individuals, demonstrating that this tool has the potential to rapidly expand the current understanding of this taxon's distribution.
Technical Report
Smallmouth Bass (Micropterus dolomieu), a non-native fish species in the Maritime Provinces, was first observed in Miramichi Lake (NB, Canada) in 2008. Since its discovery, Fisheries and Oceans Canada (DFO) has been leading containment, control, and monitoring activities with the support of non-government organizations and the Province of New Brunswick in an attempt to control the Smallmouth Bass (SMB) population within the lake. In August 2019, SMB was reported in the Southwest Miramichi River (SWM River), about 8 km downstream from Lake Brook, the outflow of Miramichi Lake. This led to a rapid mobilisation of resources by various partners and levels of government, including DFO, in an attempt to evaluate the spread and distribution of SMB within the SWM River system. Environmental DNA sampling in conjunction with species-specific qPCR testing was one of the methods used in both 2019 and 2020, as a means of gaining insight into the distribution of SMB. A total of 47 sites were sampled in both years and SMB DNA was found at multiple sites, with results classified as detected and suspected at sites downstream of McKiel Pond, where a total of 108 SMB were caught in 2019 and 2020. Results classified as inconclusive were also obtained upstream of Lake Brook, in McKiel Lake, and McKiel Brook, as well as a few other sites in the SWM River located between the outflow of McKiel Brook to Blackville. These inconclusive results warrant further investigation to confirm the presence of SMB in different portions of the watershed.
We report genetic and morphological evidence for the presence of Redeye Bass Micropterus coosae, in the Verde River of Arizona, previously thought to be Smallmouth Bass Micropterus dolomieu. We performed meristic measurements on 15 individuals sampled from the Upper Verde River Wildlife Area, Yavapai County, Arizona. Meristic data for lateral line scales, scales above lateral line, and scales below lateral line were all consistent with Redeye Bass and not Smallmouth Bass. We analyzed mitochondrial and nuclear genetic data to determine if one of the black bass (Genus Micropterus) species historically introduced to the Verde River was Redeye Bass and that they persist in the system. We extracted DNA from fin clips of five individuals for phylogenetic analysis of the NADH dehydrogenase subunit 2 (ND2) mitochondrial gene and for analysis of nuclear DNA using a diagnostic Single Nucleotide Polymorphism (SNP) panel. Results of the ND2 genetic sequencing and phylogenetic analysis indicated that these fish likely originated from native Redeye Bass stock from the Coosa River system of Alabama, Georgia, and Tennessee. Similarly, nuclear SNP data from the five individuals collected from the Verde River aligned with Redeye Bass reference genotypes based on STRUCTURE analysis. These results support the hypothesis that at least one of the introductions of black bass in Arizona’s Verde River founded a previously unrecognized population of Redeye Bass. Further work is needed to determine the extent of the Redeye Bass presence in Arizona, whether Smallmouth Bass are also present in the Verde River system, and if hybridization of Redeye Bass and other black basses is occurring.
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Over the last century, anthropogenic activities have caused substantial declines in the abundance of chum salmon (Oncorhynchus keta) throughout their range in western North America. As a result, chum salmon were listed as threatened under the U.S. Endangered Species Act in 1999. Recovery strategies have been developed, but limited baseline data on distribution impedes implementation of these strategies. Traditional methods for identifying spawning distribution (e.g., spawning ground surveys) may be inadequate for rare species like chum salmon because of a low detection probability. In contrast, environmental DNA (eDNA) sampling is extremely sensitive to species presence and has the potential to supplement or replace existing methods for monitoring listed species. However, legal and administrative issues associated with accurately describing distribution of a listed species put a premium on understanding factors that influence detection of a species by eDNA sampling. In this study, we (a) developed and tested a quantitative PCR‐based eDNA assay for chum salmon, (b) collected eDNA samples to describe the spawning distribution of this species in Columbia River tributaries between Bonneville and The Dalles Dams, and (c) tested whether spawn surveyors could inadvertently transport chum salmon DNA between streams on their boots and waders. The newly developed assay was specific and sensitive to chum salmon DNA in both tissue and eDNA samples. Chum salmon DNA was detected in the positive control site and in four streams, which increased known contemporary spawning locations in the study area. In the contamination trial, surveyors successfully introduced chum salmon DNA into the study area but eDNA was only detected intermittently and locally over the next 11 days. The accuracy of eDNA sampling, combined with success in detecting a rare species at the population scale, makes this technique well‐suited for monitoring recolonization of chum salmon during the recovery process.
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Four freshwater mussel species native to western North America, Gonidea angulata, Margaritifera falcata, Anodonta nuttalliana, and Anodonta oregonensis, have experienced dramatic declines over the last century and are currently threatened in many portions of their ranges. Therefore, improved tools for detecting and monitoring these species are needed. We developed multiplexed, species-specific, quantitative PCR assays for the detection of these species from environmental DNA (eDNA). We empirically tested species specificity and sensitivity of assays in the lab, and we also validated multiplex assays with field-collected eDNA samples. All assays were species specific, sensitive, and effective for detection from eDNA samples collected from streams and rivers. These assays will aid in the detection, monitoring, management, and conservation of these vulnerable mussel species.
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Environmental DNA (eDNA) is DNA that has been released by an organism into its environment, such that the DNA can be found in air, water, or soil. In aquatic systems, eDNA has been shown to provide a sampling approach that is more sensitive for detecting target organisms faster, and less expensively than previous approaches. However, eDNA needs to be sampled in a manner that has been tested and found effective and, because single copies of target DNA are detected reliably, rigorous procedures must be designed to avoid sample contamination. Here we provide the details of a sampling protocol designed for detecting fish. This protocol, or very similar prototypes, has been used to collect data reported in multiple peer-reviewed journal articles and from more than 5,000 additional samples at the time of publication. This process has been shown to be exceedingly sensitive and no case of field contamination has been detected. Over time, we have refined the process to make it more convenient. Our policy at the National Genomics Center for Wildlife and Fish Conservation is to provide collaborators with kits that contain all of the materials necessary to properly collect and store eDNA samples. Although the instructions in this protocol assume that the collaborator will have this same equipment, we also describe how users can create their own kit, and where we think there is flexibility in the equipment used.
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The spread of Mysis diluviana, a small glacial relict crustacean, outside its native range has led to unintended shifts in the composition of native fish communities throughout western North America. As a result, biologists seek accurate methods of determining the presence of M. diluviana, especially at low densities or during the initial stages of an invasion. Environmental DNA (eDNA) provides one solution for detecting M. diluviana, but building eDNA markers that are both sensitive and species-specific is challenging when the distribution and taxonomy of closely related non-target taxa are poorly understood, published genetic data are sparse, and tissue samples are difficult to obtain. To address these issues, we developed a pair of independent eDNA markers to increase the likelihood of a positive detection of M. diluviana when present and reduce the probability of false positive detections from closely related non-target species. Because tissue samples of closely-related and possibly sympatric, non-target taxa could not be obtained, we used synthetic DNA sequences of closely related non-target species to test the specificity of eDNA markers. Both eDNA markers yielded positive detections from five waterbodies where M. diluviana was known to be present, and no detections in five others where this species was thought to be absent. Daytime samples from varying depths in one waterbody occupied by M. diluviana demonstrated that samples near the lake bottom produced 5 to more than 300 times as many eDNA copies as samples taken at other depths, but all samples tested positive regardless of depth.
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The upper Missouri River basin in the northwestern US contains disjunct Arctic grayling (Thymallus arcticus) populations of conservation concern. To assist efforts aimed at understanding Artic grayling distribution, we developed a quantitative PCR assay to detect the presence of Arctic grayling DNA in environmental samples. The assay amplified low concentrations of Arctic grayling DNA consistently, and did not amplify non-target species, including sympatric salmonid fishes. © 2016, Springer Science+Business Media Dordrecht (outside the USA).
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This study tested the efficacy of environmental DNA (eDNA) sampling to delineate the distribution of bull trout Salvelinus confluentus in headwater streams in western Montana, U.S.A. Surveys proved fast, reliable and sensitive: 124 samples were collected across five basins by a single crew in c. 8 days. Results were largely consistent with past electrofishing, but, in a basin where S. confluentus were known to be scarce, eDNA samples indicated that S. confluentus were more broadly distributed than previously thought.
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Environmental DNA sampling (eDNA) has emerged as a powerful tool for detecting aquatic animals. Previous research suggests that eDNA methods are substantially more sensitive than traditional sampling. However, the factors influencing eDNA detection and the resulting sampling costs are still not well understood. Here we use multiple experiments to derive independent estimates of eDNA production rates and downstream persistence from brook trout (Salvelinus fontinalis) in streams. We use these estimates to parameterize models comparing the false negative detection rates of eDNA sampling and traditional backpack electrofishing. We find that using the protocols in this study eDNA had reasonable detection probabilities at extremely low animal densities (e.g., probability of detection 0.18 at densities of one fish per stream kilometer) and very high detection probabilities at population-level densities (e.g., probability of detection >0.99 at densities of ≥3 fish per 100m). This is substantially more sensitive than traditional electrofishing for determining the presence of brook trout and may translate into important cost savings when animals are rare. Our findings are consistent with a growing body of literature showing that eDNA sampling is a powerful tool for the detection of aquatic species, particularly those that are rare and difficult to sample using traditional methods.
Environmental DNA ( eDNA ) analysis for detecting the presence of aquatic and terrestrial organisms is an established method, and the eDNA concentration of a species can reflect its abundance/biomass at a site. However, attempts to estimate the abundance/biomass of aquatic species using eDNA concentrations in large stream and river ecosystems have received little attention. We determined the eDNA concentration and abundance/biomass of a stream fish, Plecoglossus altivelis , by conducting a snorkelling survey in the Saba River, Japan. Furthermore, we evaluated the relationship between eDNA concentrations and the estimated abundance/biomass of P. altivelis , and determined its spatial distribution within the river. Across the three seasons from spring to autumn, we found significant correlations between the eDNA concentration of P. altivelis and its abundance/biomass at study sites within the river. We detected the eDNA at the sites where we found only feeding traces on stones (where P. altivelis was not directly observed), but not at sites without feeding traces. Additionally, we tested the optimal number of qPCR replicates needed for the eDNA evaluation of P. altivelis abundance and biomass; only a small number of replicates was required when the eDNA concentration was high. Our findings suggest that eDNA analysis is a useful tool to estimate fish abundance/biomass as well as their spatial distribution in rivers.