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Three lamprey eDNA field studies: what we learned by applying eDNA methodology
Carl OstbergWestern Fisheries Research Center, USGS
Overview
Three lamprey field studies1. Tracking colonization in the Elwha River following
dam removal2. Distribution and seasonal differences in eDNA
across Puget Sound watersheds3. Spatial distribution and occupancy in Chehalis
River tributaries
eDNA method
Filtration
Quantitative PCR (typically 3 replicates)
Extract DNA
3 replicate water samples/site (+ Negative controls)
1L 1L 1L
0 1 2 3 4 5 6 7 8 9Number positive qPCRs Quantify DNA copy #
Results
? ? ? ? ? ? ? ? ? Target species detected?
Study 1: Using eDNA to track recolonization and dispersal of fishes
following dam removal on the Elwha River, Olympic National Park, Washington
eDNA is a good method for monitoring recolonization in backcountry
Removed April 2012
Removed August 2014
Objective
Use spatial and temporal sampling of eDNA as a means for monitoring:• Spatial extent of recolonization by native species • Dispersal of non‐native Brook Trout
Chinook
Steelhead
Coho Pink Chum
Sockeye Coastal Cutthroat
Pacific Lamprey
Jeremy Monroe
Bull Trout
USFWS
Lampetra spp
Mike Hayes
Native anadromous species
Brook TroutNon‐native
1
2
34
5
10
6
78 9
1112
1314 15
16
17
1819
202122
23
2425
Spatial distribution of Elwha eDNA water collections (25 sites)
Temporal distribution of water collections
2014 Jul Aug Sep Oct Nov Dec
2015 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
2016 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
2017 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
2014 2015 2016
Jul
Sep
Nov
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov Jan
Feb
Mar
Apr
May
Jun
1 X X X X X X
23456 X X X X X X X X
7 X X X
8 X X X X X X X X X X
9 X X X X X X
10 X
11 X X
12 X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X
15 X X X X X X X X
16‐25 X X X X X X X X X X X X X X X X
1
2
34
5
10
6
78 9
1112
1314 15
16
17
1819
202122
23
24250 1 2 3 4 5 6 7 8 9
Number positive qPCRs (out of 9)
x Not sampled
Pacific Lamprey eDNA detection
2014 2015 2016
Jul
Sep
Nov
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov Jan
Feb
Mar
Apr
May
Jun
1 X X X X X X
23456 X X X X X X X X
7 X X X
8 X X X X X X X X X X
9 X X X X X X
10 X
11 X X
12 X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X
15 X X X X X X X X
16‐25 X X X X X X X X X X X X X X X X
1
2
34
5
10
6
78 9
1112
1314 15
16
17
1819
202122
23
24250 1 2 3 4 5 6 7 8 9
Number positive qPCRs (out of 9)
x Not sampled
eDNA detections increase over time
2014 2015 2016
Jul
Sep
Nov
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov Jan
Feb
Mar
Apr
May
Jun
1 X X X X X X
23456 X X X X X X X X
7 X X X
8 X X X X X X X X X X
9 X X X X X X
10 X
11 X X
12 X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X
15 X X X X X X X X
16‐25 X X X X X X X X X X X X X X X X
1
2
34
5
10
6
78 9
1112
1314 15
16
17
1819
202122
23
2425
• Detections progress upstream over time
0 1 2 3 4 5 6 7 8 9
Number positive qPCRs (out of 9)
x Not sampled
2014 2015 2016
Jul
Sep
Nov
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov Jan
Feb
Mar
Apr
May
Jun
1 X X X X X X
23456 X X X X X X X X
7 X X X
8 X X X X X X X X X X
9 X X X X X X
10 X
11 X X
12 X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X
15 X X X X X X X X
16‐25 X X X X X X X X X X X X X X X X
1
2
34
5
10
6
78 9
1112
1314 15
16
17
1819
202122
23
2425
• Single detections interpreted with caution
0 1 2 3 4 5 6 7 8 9
Number positive qPCRs (out of 9)
x Not sampled
2014 2015 2016
Jul
Sep
Nov
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov Jan
Feb
Mar
Apr
May
Jun
1 X X X X X X
23456 X X X X X X X X
7 X X X
8 X X X X X X X X X X
9 X X X X X X
10 X
11 X X
12 X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X
15 X X X X X X X X
16‐25 X X X X X X X X X X X X X X X X
1
2
34
5
10
6
78 9
1112
1314 15
16
17
1819
202122
23
2425
• Detections differ across months
0 1 2 3 4 5 6 7 8 9
Number positive qPCRs (out of 9)
x Not sampled
Study 2: Distribution and seasonal differences in Pacific Lamprey and Lampetra spp eDNA across 18 Puget Sound watersheds
Study questions:1. How well does eDNA reflect
known species distributions?
2. Are there seasonal differences in detection?
Sampling:Fall: October 9 – November 13, 2014Spring: May 6 – June 30, 2015,
Manuscript in review
Pacific Lamprey known occurrence• most data are from 2011
eDNA detected
Not present or eDNA not detected
• Application of eDNA replicated the known Pacific Lamprey distribution
How well does eDNA reflect Pacific Lamprey distribution?
1
10
100
1000
1
10
100
1,000
10,000
100,000
Flow
rate (m
3 /s)
Pacific Lam
prey eDN
Amean
copy no./L
Are there seasonal differences in eDNA detection?
• eDNA concentration higher in spring (better detection)• 14 streams positive in spring, 5 streams positive in fall
• False negative results for 9 streams
eDNA fall eDNA spring stream flow fall stream flow spring
Pacific Lamprey
1
10
100
1000
1
10
100
1,000
10,000
100,000
Flow
rate (m
3 /s)
Pacific Lam
prey eDN
Amean
copy no./L
Are there seasonal differences in eDNA detection?
• Possible causes for lower detection in fall:• Flow rate• Life history
eDNA fall eDNA spring stream flow fall stream flow spring
Pacific Lamprey
Study 3: Assessing the spatial distribution and occupancy of larval lamprey in Chehalis River tributaries
Black River (2015)
Skookumchuck River(2015)
Newaukum River(2015)
Wynoochee River (2017)Wishkah River
(2017)
• Evaluate performance of eDNA for detecting Pacific Lamprey and Lampetra spp compared to electrofishing.
• Determine if eDNA quantity is distributed differently for Pacific Lamprey and Lampetra spp within tributaries.
• Apply eDNA to an occupancy modeling framework
Objectives
2015 sampling (Black, Skookumchuck, Newaukum)Target larval habitat (10 sites/tributary)
Patch size = 50 meters• e‐fishing (late Sep – early Oct, 2015)• eDNA sampling (Oct 20 – 21, 2015)• Caveats
Pacific Lamprey: electrofishing vs. eDNA
Pacific Lamprey detections Electrofishing and eDNA
eDNA only
Electrofishing only
No detect by either method
0
20,000
40,000
60,000
5 10 15 20 25 30 35 40
eDNA copy no./L
Distance from Chehalis River (rkm)
Lampetra spp, Skookumchuck River
0
1,000
2,000
3,000
4,000
5 10 15 20 25 30 35 40
eDNAcopy no./L
Distance from Chehalis River (rkm)
Pacific Lamprey, Skookumchuck River
Is eDNA quantity distributed differently for Pacific Lamprey and Lampetra spp within tributaries?
R2 = 0.715F = 20.05, P < 0.005
R2 = 0.061F = 0.522, ns
• Combined temporal and spatial sampling• Allowed colonization to be tracked upstream over time.• Detections increased over time
• Single detections are interpreted with caution • replicated results provide confidence
• Seasonal differences in eDNA detection• Higher in spring/summer; lower in fall/winter
• Stream flow• Life history events
• eDNA accurately identifies species distributions
• eDNA performs comparably to electrofishing for identifying occupancy
• eDNA concentrations may inform on relative abundance/biomass• Further studies required
Lessons learned – summary
Acknowledgements
USGS NPSMarshal Hoy Sam BrenkmanDorothy ChaseJeff Duda USFWSMike Hayes Jeff Jolley (now with USGS)
Carrie Cook‐TaborNOAA Judith BarkstedtGeorge Pess
LEKTMike McHenry
Pacific Lamprey assay primer and probe sites are conserved among:• Vancouver Lamprey (E. macrostomus)• Miller Lake Lamprey (E. minimus)• Klamath Lamprey (E. similis)• Pit‐Klamath Brook Lamprey (E. lethophagus)
• forward primer has one mismatch with Pit‐Klamath Brook Lamprey
• Lampetra spp assay primer and probe sites are conserved among:• L. ayresii, L. richardsoni, L. pacifica, and L. hubbsi, although the assay may
have limited or no detectability of Lampetra spp in certain geographic locations.
• North Fork Siuslaw River, OR, Hunter Creek, CA, McGarvey Creek, CA, Kelsey Creek, CA, and Mark West Creek, CA
• Boguski et al. (2012) suggested the Lampetra spp in Siuslaw River, Kelsey Creek, and Mark West Creek could represent cryptic species.
Lampetra spp eDNA assay
Figure adapted from Boguski et al. 2012, Journal of Fish Biology 81:1891‐1914
Big River (1/4)
NF Siuslaw (5/5)
Hunter Ck (5/5)McGarvey Ck (5/5)
Kelsey Ck (4/4)Mark West Ck (4/4)
Lampetra distribution
• Agreement between eDNA and fish traps for 17/18 locations
• Lampetra eDNA detected in one additional stream
Lampetra
Mike Hayes
Lampetra known occurrenceeDNA detectedNot present or eDNA not detected
1
10
100
1000
1
10
100
1,000
10,000
100,000
1,000,000
Flow
rate (m
3 /s)
LampetraeD
NAmean copy
no./L
Seasonal difference in eDNA abundance
Lampetra spp detections Electrofishing and eDNA detect
eDNA only detect
Electrofishing only detect
Not detected by either method
Lampetra spp: electrofishing vs. eDNA
Linear regression: eDNA concentration on distance• Ho: slope is not different from zero
Distribution of Pacific Lamprey and Lampetra spp eDNA What do eDNA concentrations tell us?
110100
1,00010,000100,000
15 20 25 30 35 40 45 50 55 60Mean eD
NAcopy no./L
Distance from Chehalis River (rkm)
South Fork Newaukum River (8 sites)
Pacific Lamprey Lampetra sppR2 = 0.436 R2 = 0.007F = 4.63, P = 0.075 (DF = 1, 6) F = 0.04, ns (DF = 1, 6)
