Transcript Development of an Environmental Metagenetics Approach for

Nathan Evans, Mark Renshaw, Cameron Turner,
Brett Olds, Yiyuan Li, Christopher Jerde,
Gary Lamberti, Michael Pfrender, and David Lodge
University of Notre Dame
Develop a novel and practical method for
monitoring aquatic species richness
using molecular genetic tools, that:
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has better detection limits than
traditional tools
does not require handling of organisms
produces results rapidly
provides input into statistical models
that can produce rigorous estimates of
species richness
1
Collect
water
sample
2
Filter
water
sample
3
4
Extract
all DNA
Amplify
target
DNA
12s
Cytb
16s
Amplicon
Lengths
Cytb
#1
~413 bp
12s
#1
#2
~241 bp
~385 bp
16s
#1
#2
#3
~202 bp
~310 bp
~330 bp
Suite of 6
Markers
1
Collect
water
sample
2
Filter
water
sample
3
4
Extract
all DNA
Amplify
target
DNA
5
Sequence
amplicons &
Screen for
species
Data Prep Step
Raw reads
from Miseq
Cytb
#1
12s
#1
12s
#2
16s
#1
16s
#2
16s
#3
Filtering of
reads
Data Prep Step
Analysis Step
Raw reads
from Miseq
Known
species
Cytb
#1
12s
#1
12s
#2
16s
#1
16s
#2
16s
#3
Filtering of
reads
Map to
reference
Unmapped
reads
Data Prep Step
Analysis Step
Raw reads
from Miseq
Cytb
#1
12s
#1
12s
#2
16s
#1
16s
#2
16s
#3
Filtering of
reads
Known
species
Unknown
species
Map to
reference
Create OTUs
Unmapped
reads
Compare to
Genbank
Report of
species
Testing Scenarios
• Mesocosm communities
• Well censused natural communities
• Stream ecosystem
• Pond ecosystem
• Natural communities with unknown
species assemblages
Objective: Determine if our metagenetic
approach can be used to accurately measure
species richness in mesocosms with differing
assemblage structures
Treatment Population Densities
Even Abundance
Skewed Abundance
50
Population Number
Population number
50
40
30
20
10
0
40
30
20
10
0
FHM
BLG
GAM
KOI
Even-High
BFT CKC
Species
CSR
BST
WSK
FHM
BLG
Even-Low
GAM
KOI
BFT CKC
Species
Skewed-High
CSR
BST
WSK
Skewed-Low
All nine species were detected!
Fathead
Minnow
(FHM)
Bluegill
Sunfish
(BLG)
Eastern
Mosquitofish
(GAM)
Common
Carp
(KOI)
White
Sucker
(WSK)
Creek
Chub
(CKC)
Central
Stoneroller
(CSR)
Blackstripe
Topminnow
(BST)
Bullfrog
Tadpole
(BFT)
Objective: Determine if our metagenetic
approach can accurately describe the fish
species richness of a highly censused natural
stream ecosystem
Number of years (out of 17) each species was sampled
during annual electrofishing collections
# of years
Species
Creek chub (Semotilus atromaculatus)
Mottled sculpin (Cottus bairdii)
Johnny darter (Etheostoma nigrum)
Blacknose dace (Rhinichthys atratulus)
White sucker (Catostomus commersonii)
Green sunfish (Lepomis cyanellus)
Rainbow trout (Oncorhynchus mykiss)
Brown trout (Salmo trutta)
Rock bass (Ambloplites rupestris)
Smallmouth bass (Micropterus dolomieu)
Bluegill sunfish (Lepomis macrochirus)
Largemouth bass (Micropterus salmoides)
Hybrid sunfish (L. macrochirus X L. cyanellus)
Pumpkinseed sunfish (Lepomis gibbosus)
Yellow perch (Perca flavescens)
Central mudminnow (Umbra limi)
Rainbow darter (Etheostoma caeruleum)
Warmouth (Lepomis gulosus)
Hybrid sunfish (L. gibbosus X L. cyanellus)
Golden shiner (Notemigonus crysoleucas)
17
17
17
17
17
17
15
13
11
11
10
6
5
4
3
2
2
1
1
1
Four 60-m reaches with block nets
• Triple-pass backpack electrofishing
Flow
Electrofishing
eDNA
Additional eDNA
Blacknose dace
Largemouth bass
Creek chub
Johnny darter
Rainbow darter
Mottled sculpin
Bluegill sunfish
Green sunfish
Rock bass
Smallmouth bass
Brown trout
Rainbow trout
White sucker
Yellow bullhead
Common carp
 All 12 species detected by
electrofishing were
detected by metagenetic
sequencing. 3 species
were detected only by
eDNA.
Species richness (S) is the number of unique
species found in a defined area. We are using
the bias-corrected Chao II estimator for
incidence data:
𝑛 − 1 𝑞1(𝑞1 + 1)
𝑆𝐶ℎ𝑎𝑜2 = 𝑆𝑜𝑏𝑠 +
+
𝑛
2(𝑞2 + 1)
Estimated species richness
Correction for small
sampling effort
Observed species richness
Estimated number
of unobserved
species from rare
occurrences
q1= Number of species found in only one sample
q2 = Number of species found in only two samples
Electrofishing
eDNA
eDNA (15)
Electrofishing (12)
Objective: Determine if our metagenetic
approach can accurately describe the fish and
amphibian species richness of a thoroughly
sampled natural pond within a military property
Common Name
Black crappie
Blackchin shiner
Blacknose shiner
Bluegill sunfish
Bluntnose minnow
Bowfin
Brown bullhead
Central mudminnow
Common carp
Golden shiner
Grass pickerel
Green sunfish
Iowa darter
Lake chubsucker
Largemouth bass
Northern Pike
Northern redbelly dace
Pugnose shiner
Pumpkinseed sunfish
Redear sunfish
Sand shiner
Tadpole madtom
Warmouth
White sucker
Yellow bullhead
Yellow perch
Scientific Name
Pomoxis nigromaculatus
Notropis heterodon
Notropis heterolepis
Lepomis macrochirus
Pimephales notatus
Amia calva
Ameiurus nebulosus
Umbra limi
Cyprinus carpio
Notemigonus crysoleucas
Esox americanus vermiculatus
Lepomis cyanellus
Etheostoma exile
Erimyzon sucetta
Micropterus salmoides
Esox lucius
Phoxinus eos
Notropis anogenus
Lepomis gibbosus
Lepomis microlophus
Notropis stramineus
Noturus gyrinus
Lepomis gulosus
Catostomus commersonii
Ameiurus natalis
Perca flavescens
Lawler Pond
eDNA Samples
Traditional
eDNA
Additional eDNA
Pumpkinseed sunfish
Green sunfish
Bluegill sunfish
Warmouth
Blackchin shiner
Central mudminnow
Yellow bullhead
Brook stickleback
Largemouth bass
Creek chub
Golden shiner
Grass pickerel
White Sucker
Common carp
Fathead minnow
•
We have identified additional locations at
Ft. Custer and Camp Pendleton to serve as our
first application of the metagenetic approach
in natural ecosystems with unknown species
richness. eDNA sampling is planned for 2015.
•
We hope to refine our approach to provide the
DoD with an accurate and cost-effective tool
for monitoring aquatic biodiversity at military
sites across the nation.
Field and lab assistance:
Crysta Gantz, Matt Dougherty,
Mary Hupka, and Karen Uy
Contact Information:
Nathan Evans
University of Notre Dame
[email protected]