incorporating dna barcodes into assessments of the biological integrity of aquatic ecosystems
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Incorporating DNA barcodes into assessments of the biological integrity of aquatic ecosystems. Mark Bagley, United States Environmental Protection Agency Charles Spooner, US EPA Ronald Klauda Maryland Dept of Natural Resources David Schindel, CBOL Lee Weigt - PowerPoint PPT PresentationTRANSCRIPT
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Incorporating DNA barcodes into assessments of the
biological integrity of aquatic ecosystems
Mark Bagley, United States Environmental Protection Agency
Charles Spooner, US EPA
Ronald Klauda Maryland Dept of Natural Resources
David Schindel, CBOL
Lee Weigt
Robert Hanner, University of Guelph
2
Structure of Talk
1. Bioassessments, biological integrity and taxonomy
2. Application of DNA barcodes to stream bioassessments
3. From species to populations: Expanding from barcodes in bioassessments
3
Chemical
Integrity
Biological
Integrity
Physical
Integrity
Section 101 of the United States
Clean Water Act requires federal
and state governments to
"restore and maintain the
chemical, physical and
biological integrity of the
Nation's waters."
“Biological Integrity” and Environmental regulations
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Biological Integrity The condition of an aquatic community inhabiting
unimpaired waterbodies of a specified habitat as measured by an evaluation of multiple attributes of the aquatic biota. Three critical components of biological integrity are that the biota is
(1) the product of the evolutionary process for that locality, or site
(2) inclusive of a broad range of biological and ecological characteristics such as taxonomic richness and composition, trophic structure
(3) found in the study biogeographic region.» (USEPA 1996)
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Bioassessment• An evaluation of the biological condition of a
waterbody using biological surveys and other direct measurements of the resident living organisms Fish, macroinvertebrates, periphyton communities Important component of the majority of water quality
monitoring programs indicators of cumulative impacts on biological integrity from
aquatic stresses • nonpoint source pollution and other stressors.
Quantitative assessments of what the community looks like compared to “what it is supposed to look like”
• Indicator species, multimetric indices, predictive models
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Bioassessments
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Bioassessments
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Source: EPA822-F-02-006, 2002
Biological Assessment and the US Clean Water Act
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Bioassessments depends critically on good
taxonomic identifications
• Accuracy• Precision• Comparability of
findings• Credibility
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US EPA National Stream Survey
Design
• National assessment of the condition of wadeable streams
• 10 different taxonomic ID laboratories• 749 stream macroinvertebrate samples• Organisms identified to genus• 10% random re-identification by
independent taxonomist• Data quality objective – 85% repeatability
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• 77% of random re-identifications were to the same genus (1st try)
• 87% of random re-identifications were to the same genus after reassessment of data and correction of systematic errors
– Good enough?
US EPA National Stream Survey
Data Quality
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So many bugs, so few experts, such a pain to identify
Taxonomy of North American Aquatic Flies (Diptera)
Family Known species
Probable species
Known to genus as larvae
Known to species as larvae
Taxonomic experts
Ceratopogonidae 600 >800 <20% <5% 1-2
Thaumaleidae 26 >30 100% ~20% 1-2
Psychodidae 70 >200 ~30% <5% 0
Tipuloidea >1500 ~2000 ~80% <3% 3-4
“Empidoids” ~1400 ~2000 <20% <3% ?
Chironomidae** ~1000 >2000 ? ? ?
Sources:G. Courtney, L. Ferrington
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And progress in identifying aquatic larvae to species is slow
1966 - 40% of species known to science as larvae
2006 - 38% of 1418 described species with larvae known to science
Source: John Morse
Taxonomy of North American caddisflies (Trichoptera)
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DNA Barcodes
• A short DNA sequence from a standardized portion of the genome that is used to identify species
• DNA barcodes provide an objective standard for species identification
• Yada yada yada
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Possible Advantages of DNA Barcodes for Bioassessment
• Increased taxonomic resolution • Provide a common QA standard• Increase pool of expertise for
taxonomic identification• Pathway towards more fully automated
analyses• Objectivity, speed, cost (?)
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Advanced Monitoring Initiative
Project Goals• Develop a DNA barcode library for important
aquatic indicator species (EPT)• Ephemeroptera (Mayflies)• Plecoptera (Stoneflies)• Trichoptera (Caddisflies)
• Compare DNA barcodes to traditional bioassessments for EPT taxa
• Cost, Speed, Objectivity, Accuracy, Precision• How important is increased taxonomic precision?
• Determine how to efficiently incorporate DNA barcodes into a state bioassessment program
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Barcode development
• Develop barcode with COI sequences from taxonomically resolved adult specimens (Smithsonian collection)
• Add COI sequences for larval samples collected as part of Maryland’s state bioassessment
• Link larval and adult forms by DNA and incorporate ancillary data to complete barcode record
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Repeatability and barcode development
Morphology
MarylandDNR
EPA Lab
Guelph
Smithsonian
EPA
DNA
ReferenceBarcodeDatabase
Taxon Experts(Smithsonian)
RepeatabilityPrecisionCost
RepeatabilityAccuracyPrecisionCost
(taxonomic agreement)
(disagreementor MOTU)
SpeciesDescription
(adult specimens)
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Tech Transfer is Important!
• End users are participants in the project Maryland DNR, EPA-Water
• Tech transfer documents, hands-on workshops, and protocols are key products
• Chose end-users that will be influential in “converting” others
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TrichopteraNet spinning caddisflies (Hydropsychidae)
677 bp COI
102 specimens
NJ tree
Diplectrona modesta
Parapsyche apicalis
Cheumatophyche sp.
Potamyia sp.
Hydropsyche betteni
Hydropsyche aerata
Hydropsyche simulans
Hydropsyche betteni
Hydropsyche bidens/incomoda
Hydrophyche venularis/ scalaris
Symphitophyche sp.
Hydrophychidae sp.
0.1
21
59
Diplectrona
modesta
H 240
H 245
H 259
H 255
H 270
H 263
H 264
H 231
H 261
H 249
H 244
H 391
H 260
H 275
H 241
H 248
H 269
H 250
H 368
H 230
H 251
H 232
H 252
H 268
H 239
H 247
H 243
H 253
H 256
H 390
H 387
H 432
H 246
H 433
H 420
H 274
H 273
H 402
H 406
H 419
99
100
100
92100
99
99
64
0.02
“Hydropsychidae”
Diplectrona modesta
Parapsyche apicalis
Cheumatophyche sp.
Potamyia sp.
Hydropsyche betteni
Hydrophyche aerata
Hydrophyche simulans
Hydrophychidae betteni
Hydropsychidae bidens/ incomoda
Hydrophyche venularis/ scalaris
Symphitophyche sp.
Hydrophychidae sp.
0.1
22
Hydropsyche aerata H 703
H 485
H 525
H 452
Hydropsyche simulans
Symphitopsyche bronta/morosa H 519
Hydropsyche betteni H 314
Hydropsyche bidens/incommoda H 517
Hydropsyche venularis H 518
H 474
H 484Hydropsyche scalaris
Symphitopsyche bronta/morosa H 707
Symphitopsyche bifida H 846
H 844
H 845Symphitopsyche bronta/morosa
99
99
67
99
99
53
62
58
0.05
Diplectrona modesta
Parapsyche apicalis
Cheumatophyche sp.
Potamyia sp.
Hydropsyche betteni
Hydrophyche aerata
Hydrophyche simulans
Hydrophychidae betteni
Hydropsychidae bidens/ incomoda
Hydrophyche venularis/ scalaris
Symphitophyche sp.
Hydrophychidae sp.
0.1
23
0
2
4
6
8
10
12
00.
020.
040.
060.
08 0.1
0.12
0.14
0.16
0.18 0.
20.
22
w/in Species w/in Genus w/in Family
Preliminary comparison of
sequence divergences
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Summary• Bioassessments are critical
components of water quality programs
• Their wider implementation is hindered by lack of taxonomic expertise, poor resolving power of morphology, and expense.
• DNA barcodes can revolutionize biaossessments, removing previous roadblocks and bringing new sophistication to the field
• One key to success will be partnership with morphological taxonomists to find efficient ways forward
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But why stop at barcodes?
• The hard part is getting buy-in from bioassessment programs to transition to DNA surveys
• Once DNA is analyzed and archived for barcodes, it is available for more complex analyses of population structure and dynamics
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Mill Creek Watershed, Cincinnati, OH, USA
Ohio EPA aquatic habitat assessment
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Environmental characterization of mainstem sites
0
2
4
6
8
Tanner MC1 MC2 MC3 MC5
fish abundance/100 PAH (mg/ l) Pesticide (ug/ l)
0
2
4
6
8
Tanner MC1 MC2 MC3 MC5
fish abundance/100 PAH (mg/ l) Pesticide (ug/ l)
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Genetic Analysis
• 4 mainstem, 3 tributary, and one ‘control’ site sampled for central stonerollers (Campostoma anomalum) in 2001-2002
• 4 mainstem sites also sampled in 1994-1995
• Sample sizes ranged from n=10 to n=103
• All samples genotyped at 10 microsatellite loci
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Population structure analysis
MC1 MC2 MC6 MC7 MC3 MC8 MC5 TC
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Estimation of effective population sizes and connectivity (MLNE)
Site MC1, t-1(Resident Pool)
Site MC1, t-2
ImmigrantPoolGenetic
drift
migration
~ m, Ne
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Estimates of local effective sizes and immigration rates
Site Ne (95% CI) m (95% CI)
MC1 85 (55-140)a 0.29 (0.21-0.46)
MC2 12 (8-21)b 0.99 (0.53-1.00)
MC3 39 (28-105)a 0.99 (0.24-1.00)
MC5 86 (47-112)a 0.99 (0.43-1.00)
Source-sink dynamics?
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Mill Creek Watershed, Cincinnati, OH
A genetic tool for watershed management?
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Acknowledgements
• Advanced Monitoring Initiative Co-investigators and Collaborators Chuck Spooner, Suzanne Jackson, Eric
Waits, Mike Blum, US EPA Lee Weigt, Amy Driscoll, Smithsonian
Institution David Schindel, CBOL Ron Kluida, Ellen Friedman, Maryland DNR Robert Hanner, University of Guelph