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Evon Hekkala NERL Postdoctoral Fellow EPA Region 5 Chicago, IL 28 April 2005. Molecular Markers for Ecological Indicators. Genetic Methodologies to improve existing Ecological Indicators for Aquatic Ecosystems.

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evon hekkala nerl postdoctoral fellow epa region 5 chicago il 28 april 2005
Evon Hekkala

NERL Postdoctoral Fellow

EPA Region 5

Chicago, IL

28 April 2005

Molecular Markers for Ecological Indicators

genetic methodologies to improve existing ecological indicators for aquatic ecosystems
Genetic Methodologies to improve existing Ecological Indicators for Aquatic Ecosystems
  • development of accurate and precise methods for biological identification of aquatic species and subspecies
  • delineation of ecological assessment units through analysis of genetic structure across multiple species
  • assessment of changes in genetic diversity as an indicator of present and historical environmental condition
  • assessment of genetic diversity at diagnostic loci and across the genome as an indicator of vulnerability to future environmental perturbations
  • integrated assessments to link landscape-level stressors to population-level outcomes
slide4

Ecosystem

Species

Genes

Biodiversity

  • Genetic diversity is a fundamental component of biodiversity
  • Stressors affect genetic diversity in predictable ways (ecological indicator)
  • Genetic diversity limits potential responses to future stressors (sustainability indicator)
  • Understanding of genetic diversity patterns enhances the value and interpretation of other ecological assessment data
improved methods for species identification and enumeration
IMPROVED METHODS FOR SPECIES IDENTIFICATION AND ENUMERATION

RELEVANCE:

  • Understanding of ecological condition depends on accurate description of species assemblages
  • DNA provides the most accurate and precise information on species identity
  • EPA needs efficient and transferable “DNA ID” or “DNA barcoding” methods
delineation of ecological assessment units
DELINEATION OF ECOLOGICAL ASSESSMENT UNITS

RELEVANCE:

  • Measurement and evaluation of ecological condition must be performed at the correct environmental scale
  • Many assessments incorporate the watershed or ecoregion as the fundamental assessment unit
  • For biological resources, the fundamental unit that responds to and adapts to the environment is the biological population
change in genetic diversity as an indicator of ecological condition
CHANGE IN GENETIC DIVERSITY AS AN INDICATOR OF ECOLOGICAL CONDITION

RELEVANCE:

  • Environmental stressors that alter the genetics of populations have lasting effects
  • Genetic change is brought about by environmental alterations that affect the breeding population size, mutation rate, population connectivity, or selective forces
  • Genetic change is an indicator of population and species-level effects, scales at which we have few good indicators
slide9

How?Collect SamplesExtract DNAAmplify Sections of DNAAFLP/RAPDMicrosatellitesSequencingAnalyze Data

sample collection and extraction
Sample Collection and Extraction
  • DNA is everywhere!
    • Traditional vouchering
    • Non-invasive, non-destructive
      • Fin Clips, scales, swabs, feces, hair, shed antlers, egg shells, scrimshaw……
      • Museum Collections ( wet/dry)
slide11

AFLP

AmplifiedFragment

LengthPolymorphism

slide12

Much more repeatable

More polymorphisms

Dominant (presence/absence), anonymous markers

Amenable to automation

AFLP

slide13

TATATATATATA

TATATATATATATATA

TATATATATA

TATATATATATATATATA

Microsatellites

  • Highly polymorphic (high mutation rates)
  • Well-characterized, codominant single-locus markers
  • Highly amenable to automation
slide14

Microsatellites

Locus 1 Locus 2

slide15

DNA sequences

  • Intensive analysis of one locus (COI, Cytochrome B)
  • Most explicit genetic ID available
  • More costly, but allows different types of analyses
slide16

Characterization and Identification

of species diversity

Arthropoda

Sp. A

Chordata

Sp.B

Cytochrome Oxidase I mitochondrial gene sequences from GenBank provides a large

framework for assignment

of experimental data to

gross taxonomic groups

Sp.C

Mollusca

(redrawn from Hebert et al. 2003)

slide17

PCR primers for amplification of targeted species

  • Requires identification of primer binding sites that are:
    • identical among individuals within a target group
    • absent or ineffective among members of excluded group

Requires identification of gene regions that are:

consistent within the target group

variable among members of different target groups

current projects nerl ord
Current Projects- NERL/ORD
  • Regional profile of fish genetic diversity in Eastern Cornbelt Plains Ecoregion (Region 5 REMAP)
  • Genetic diversity of stream fish in a coal mining-impacted region.
  • Regional profile of fish genetic diversity in Mid-Atlantic Integrated Assessment (EMAP) area
  • Temporal trends in genetic diversity in relation to experimental whole-lake acidification (collaboration with DFO-Canada)
  • Temporal and spatial patterns of fish genetic diversity in a highly modified urban stream
  • Integrated ecological assessments using genetic, landscape, and population modeling methods (cross-NERL/ORD collaboration)
  • Development of rapid Genetic ID methods to enhance detection and enumeration of benthic invertebrates
genetics of central stonerollers in the eastern cornbelt plains ecoregion
Genetics of Central Stonerollers in The Eastern Cornbelt Plains Ecoregion

Campostoma anomalum

Photo courtesy of Ohio Dept. Natural Resources

goals
Goals
  • Define meaningful population units for ecological assessments
  • Assess relationship between genetic diversity and ecological condition
slide22

Study Sites

  • 91 sample sites
  • Part of Regional EMAP
  • Mostly agricultural
  • First-third order streams

Genetic Analysis

  • RAPD fingerprints
  • mtDNA Sequences
  • Assess genetic
  • differences within/
  • among sites
slide23

Genetic

Relatedness

Among sites

slide24

Genetics of Creek Chubs in a Mining-Impacted Region

Semotilus atromaculatus

Photo courtesy of Ohio Dept. Natural Resources

slide25

Mitochondrial

DNA

Population

genetic structure

stepwise multiple regression nuclear dna diversity
Stepwise multiple regression – nuclear DNA diversity

98% of the differences in genetic diversity within populations explained by geographic and environmental factors!

regional profile of fish genetic diversity in mid atlantic integrated assessment emap area
Regional profile of fish genetic diversity in Mid-Atlantic Integrated Assessment (EMAP) area

White SuckerCatostomus commersoni

  • How is Diversity distributed?
  • How accurate is Morphological ID in the field?
  • How do we identify Hybrids?
  • How do IDs affect IBIs?
slide28

99

DNA Taxonomic Identification

Semotilus atromaculatus Group 1

(303 sequences plus Genbank reference sequence)

97

100

99

Semotilus atromaculatus Group 2 (34 sequences)

100

Semotilus corporalis (11 sequences)

Sample 9576

100

Rhinichthys atratulus

Notropis stilbius

Notropis girardi

97

Luxilus cornutus

50

Sample 3786

100

Luxilus chrysocephalus

85

0.10

0.08

0.06

0.04

0.02

0.00

Linear sequence divergence

slide29

How accurate is field identification

of stream fishes?

  • 96% of white suckers were morphologically identified correctly
  • All creek chub were morphologically identified correctly, but the taxon is composed of two morphologically similar but genetically distinct groups in the MAIA region
  • A minimum of 85% of fallfish were morphologically identified correctly
  • All central stonerollers were morphologically identified correctly, but the taxon is composed of four morphologically similar but genetically distinct groups in the MAIA region
  • Field morphological identification seemed to be reasonably accurate for these taxa but morphological identification under-represented the actual biological diversity uncovered
  • Morphological analysis supplemented with genetic identification is recommended for future ecological assessments ie. DNA QA
slide30

100

Group1 (47sequences)

78

100

100

Group 2 (16 sequences)

100

Group 3 (11 sequences)

85

100

Group 4 (8 sequences)

Outgroup

(S. atromaculatus)

0.10

0.08

0.06

0.04

0.02

0.00

Linear sequence divergence

  • Multi-species assessment of fish genetic diversity in the MAIA region
  • Microsatellite diversity of white sucker was strongly associated with agricultural impacts and human population density.
  • Creek chub diversity was associated with stream substrate condition and geochemistry.
  • Central stoneroller diversity was associated with agriculture, human population density, runoff, pH, stream substrate and geochemistry.
  • Different species and genetic groups within recognized species appeared to respond to different environmental dimensions.
who are the culprits
Who are the Culprits?

European green crab

Where do they come from?

Zebra mussels

slide33

Daphnia sp.

Bosmina sp.

??

European

green crab

Zebra

mussel

Polychaete

slide34

Identifying species found in ballast

Traditional:

Morphological taxonomy

  • technologically simple (ie. microscopy…)
  • classification dependent on adult traits
    • larval and egg forms poorly characterized
  • requires broad knowledge of major taxonomic groups
    • or requires assistance from a range of experts
  • identification typically limited to family or genus level
  • limited treatment of cryptic or difficult taxa
  • no standard for comparison across studies
  • data have limited applicability (ie. species inventories…)
slide35

DNA extraction and purification

Resting eggs or tissue

in ballast water or sediment

www.glerl.noa.gov/res/task._rpts/nsreid10-1.html

“from sludge to sequences”

Sequencing of

cloned amplicons

Allele-specific PCR

amplification

Bacterial cloning

of amplicons

slide36

Collaboration

  • Research supported by the Regional Methods program
    • ORD partnering with Regions 5, 9, 10 and GLNPO
  • Novel application of allele-specific PCR methods and DNA sequencing technology
  • Development and application of bioinformatic databases
  • Research objectives:
    • Exploratory characterization of species diversity in ballast
    • Targeted screening of ballast for invasive species
slide37

Regional Implementation

Marker Development Laboratory

Regional Laboratory

  • Develop and test microsatellites, other markers
  • Design Assessment
  • Field Sampling
  • DNA extraction
  • (PCR)

Ecological

interpretation

  • (PCR)
  • Marker screening
  • Genetic Diversity assessment

Genetic Analysis Laboratory