1

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