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Introduction

The mission of the United States Environmental Protection Agency (EPA) is to protect public health andsafeguard and improve the natural environmentCthe air, water, and land upon which life depends. Achievement ofthis mission requires the application of sound science to the assessment of environmental problems and to theevaluation of possible solutions. The National Center for Environmental Research (NCER) at EPA is committed toproviding the best products in high-priority areas of scientific research through significant support for long-termresearch.

The Office of Research and Development’s (ORD) Ecological Research Strategy identifies monitoring researchfocused on biological indicator development at the molecular, community, and landscape levels of biologicalorganization as a high-priority research program. These indicators will be used for the monitoring of ecosystemcondition as well as for exposure evaluation. The development of new characterization methods and the improvementof multiscale monitoring designs also are high-priority research components. This research represents the extramuralcomponent of ORD’s Environmental Monitoring and Assessment Program (EMAP).

In 1997, NCERQA issued a Request for Applications (RFA) on Ecosystem Indicators. The purpose of thissolicitation was to support research leading to the development of techniques and indicators that characterize andquantify the integrity and sustainability of ecosystems at local, regional, national, and/or global scales. In 1998 and1999, the RFAs focused on molecular and landscape indicators, while maintaining their previous emphasis on integrityand sustainability. In 2000, the program’s focus switched to estuarine indicators, with the expectation of fundinggroups of researchers on the East Coast, West Coast, Gulf of Mexico, and Great Lakes. Additionally, in FY 2000, newresearch on the development of ecosystem classification systems and associated reference conditions will be funded.

Annual progress reviews such as this allow investigators to interact with one another and discuss progress andfindings with EPA and other interested parties. If you have any questions regarding the program, please contact theprogram manager, Barbara Levinson, by telephone at (202) 564-6911, or by e-mail at [email protected].

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Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Section 1. Molecular Genetics Research

Genetic Diversity in California Native Fish Exposed to Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Susan Anderson, Andrew Whitehead, Bernie May, Mark Bagley, Kathryn Kuivila, Barry Wilson, David Hinton

Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Katharine G. Field

Intraspecies Genetic Diversity Measures of Environmental Impacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Dan E. Krane, G. Allen Burton, Keith Grasman

Demographic and Genetic Factors Affecting Population Viability of Lupinus perennis, an Indicator Species of Oak Savanna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Helen J. Michaels, R.J. Mitchell

Are Genetic Diversity and Genetic Differentiation Bioindicators of Contaminant Impact on Natural Populations? Fundulus heteroclitus as a Model Estuarine Species . . . . . . . . . . . . . . . . . . . . . . . . . 11Michael C. Newman, Margaret Mulvey, Michael A. Unger, Wolfgang K. Vogelbein

Multilevel Indicators of Ecosystem Integrity in Alpine Lakes of the Sierra Nevada . . . . . . . . . . . . . . . . . . . . . . . . . . . 12James T. Oris, Sheldon I. Guttman, A. John Bailer, John E. Reuter, Glenn C. Miller

Ecosystem Monitoring via Genetic Diversity Surveys of Dandelions Using VNTR Multilocus DNA Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Steven Rogstad, Brian Keane

Section 2. Multiscale and Landscape Indicators

Foliar Chemistry as an Indicator of Forest Ecosystem Status, Primary Production, and Stream Water Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19John Aber, Richard Hallett, Mary Martin, Marie-Louise Smith, Scott Ollinger, Scott Bailey

Environmental Factors That Influence Amphibian Community Structure and Health as Indicators of Ecosystem Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Val Beasley, Lucinda Johnson, Carl Richards, Patrick Schoff, Rebecca Cole, Camilla Lieske, Anna Schotthoefer, Cathy Johnson, Joseph Murphy, Marvin Piwoni

Modeling Ozone Flux to Forests Across an Ozone Concentration Gradient in the Sierra Nevada Mountains, CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Allen Goldstein, Jeanne Panek

Effects of Forest Fragmentation on Community Structure and Metapopulation Dynamics of Amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Lucinda Johnson, Catherine Johnson, Randall Boone, John Gross

Land Use and Geomorphic Indicators of Biotic Integrity in Piedmont Streams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25David S. Leigh, B.J. Freeman, M.C. Freeman, E.A. Kramer, M.J. Paul, C.M. Pringle, A.D. Rosemond, R. Cifaldi, A. Roy, D. Walters

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Table of Contents (continued)

Developing Effective Ecological Indicators for Watershed Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Duncan T. Patten, Wayne Minshall, Rick Lawrence, Andrew Marcus

Development and Evaluation of Multiscale Mechanistic Indicators of Regional Landscapes . . . . . . . . . . . . . . . . . . 27Carl Richards, Lucinda B. Johnson, George E. Host

Development and Testing of a Multi-Resource Landscape-Scale Ecological Indicator: Forest Fragmentation, Structure, and Distribution Relative to Topography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Steven W. Seagle, Philip A. Townsend

Ecological Indicators for Large River-Floodplain Landscapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Monica G. Turner, E.H. Stanley, M.D. Dixon, R.E. Freeman, S.E. Gergel, H. Kang, J.R. Miller, J. West

Characterization of the Ecological Integrity of Commercially Grazed Rangelands Using Remote Sensing-Based Ecological Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Neil E. West, Robert A. Washington-Allen, R. Douglas Ramsey

Section 3. Aquatic Indicators

Using Bioindicators To Develop a Calibrated Index of Regional Ecological Integrity for Forested Headwater Ecosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Robert P. Brooks, Robert S. Mulvihill, Terry Master, Timothy J. O’Connell

Stream Plethodontid Assemblage Response (SPAR) Index: Development, Application, and Verification in the MAHA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Robert P. Brooks, Gian L. Rocco

Soil Enzyme Stability as an Ecological Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Richard P. Dick

Integrative Indicators of Ecosystem Condition and Stress Across Multiple Trophic Levels in the San Francisco Estuary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Richard C. Dugdale

Developing an Indicator for Nutrient Supply in Tropical and Temperate Estuaries, Bays, and Coastal Waters Using the Tissue Nitrogen and Phosphorus Content of Macroalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Peggy Fong

Microbial Indicators of Biological Integrity and Nutrient Stress for Aquatic Systems . . . . . . . . . . . . . . . . . . . . . . . . . 44James P. Grover, Thomas H. Chrzanowski

Foraminifera as Ecosystem Indicators: Phase 1. A Marine Benthic Perturbation Index; Phase 2. Bioassay Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Pamela Hallock, Heidi Crevison, Thomas Dix, Helen Talge, Dana Williams

Biogeochemical Indicators of Watershed Integrity and Wetland Eutrophication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48K. Ramesh Reddy, W.F. DeBusk, A. Ogram, W. Graham, M.M. Fisher, E.F. Lowe, L.W. Keenan

Development and Evaluation of Ecosystem Indicators for Urbanizing Midwestern Watersheds . . . . . . . . . . . . . . . . 49Anne Spacie, Jonathan M. Harbor, Midhat Hondzo, Bernard A. Engel

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Table of Contents (continued)

An Integrative Aquatic System Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Richard S. Stemberger, Eric K. Miller

Effects of Interacting Stressors in Agricultural Ecosystems: Mesocosm and Field Evaluation of Multilevel Indicators of Wetland Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Stephen T. Threlkeld

Index of Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Section 1.

Molecular Genetics Research

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Genetic Diversity in California Native Fish Exposed to PesticidesSusan Anderson 1, Andrew Whitehead 1, Bernie May 2, Mark Bagley 2, Kathryn Kuivila 3, Barry Wilson 2, and David Hinton 21University of California at Davis, Bodega Marine Laboratory, Bodega Bay, CA; 2University of California atDavis, Davis, CA; and 3U.S. Geological Survey, Sacramento, CA

The overall goal of this project is to determine theeffect pesticide exposure may have on genetic variationin a California native fish. Objective 1 is to examinepopulations of Sacramento sucker (Catostomus occi-dentalis) exposed to landscape-scale pesticide releases todetermine whether changes in genetic diversity are as-sociated with indicators of pesticide exposure and aredistinguishable from natural genetic variation. Molecu-lar techniques to be utilized include Amplified FragmentLength Polymorphisms (AFLP) and microsatellites.Objective 2 is to compare the AFLP technique with theRandomly Amplified Polymorphic DNA (RAPD) tech-nique to determine which produces the most informativeand reproducible DNA fingerprints. Objective 3 is toevaluate potential linkages between any observedchanges in genetic variation and fitness parameters inindividuals and populations.

Pesticide exposure patterns are being characterizedas one step in separating natural genetic variation frompesticide-related variation. An existing pesticide-usedatabase is being used in combination with GeographicInformation System (GIS) applications, as well as pest-icide analysis in rivers and creeks, to quantify historicalpatterns of exposure, and thereby, to select samplingsites.

Fish are being sampled from sites inferred to beimpacted by agriculture and from upstream clean ref-erence populations (see Figure 1). Fin clips are archivedfrom individual fish for genomic analysis. AFLP andmicrosatellite analyses will be performed on archived finclips, and the resulting genomic patterns will be com-pared among exposed and reference populations.

Biomarkers indicating genetic damage (DNA strandbreaks) and enzyme inhibition (acetylcholinesteraseactivity) in fish exposed to pesticides are beingexamined at selected reference and exposed sites as anadditional method of discriminating pesticide-exposedpopulations from reference populations. An assessmentof the infor- mativeness and reliability of RAPD andAFLP finger- printing methodologies has beencompleted for population genetic analyses using fishwith a well-established pedigree. Numbers ofsegregating bands were compared and tested fordifferences in reproducibility.

Whether exposed and reference populations differin average sensitivity to pesticide exposure in laboratoryexperiments will be tested using fitness parameters and

biomarker responses as endpoints. Correlations betweengenotype and tolerance also will be tested.

A definitive study design was devised using dataon pesticide exposure and information regarding fishavail- ability. Seven hundred fin-clip samples ofSacramento sucker from multiple watersheds werearchived. At this time, field sampling for AFLP andmicrosatellite analyses are approximately 75 percentcomplete. The DNA strand break assay has been furtherdeveloped this year. The acetylcholinesterase enzymeinhibition assay also has been optimized with anexamination of variation in response among tissues andcharacterization of different isoforms.

In comparing the relative merits of RAPD and AFLPtechniques, it was found that the number of segre- gatingbands is higher using RAPD than AFLP, but that thereproducibility of RAPD bands is far lower. Only 1percent of the AFLP bands were judged to be irrepro-ducible. In contrast, 16.1 percent of the RAPD bandswere irreproducible. At present, there is no reason tobelieve that any criterion that can be developed willincrease the reliability of RAPD methodology to a levelthat is comparable to AFLP analysis.

GIS analyses coupled with chemical analyses haveresulted in the selection of sampling locations that to-gether represent a strong field experimental design. Thisdesign allows for field replication, extensive character-ization of background genetic variation, and examina-tion of genetic differences over a very large geographicscale.

This study is unique because genetic variation as-sociated with contaminant exposure has not been eval-uated in fish populations on a large geographic scale.The RAPD technique is significant because it is used inongoing U.S. EPA Environmental Monitoring and As-sessment Program (EMAP) studies; however, the newlyavailable AFLP technique permits examination of moreof the genome per unit effort and has been demonstrat- edto be more informative and reproducible.

Tasks for the year 2000 are directed entirely atprogress on Objective 1. The first task is completion ofapproximately one-half of the AFLP and microsatelliteanalyses. The second task is evaluation of DNA strandbreaks and acetylcholinesterase enzyme inhibition inlab- oratory exposures to diazinon as well as in fieldcaging studies.

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Figure 1. Locations of sampling sites in the Central Valley of California.

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Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water Katharine G. FieldDepartment of Microbiology, Oregon State University, Corvallis, OR

Fecal pollution in water is a threat to ecosystemintegrity that also poses health risks to humans. Oftenthe problem is not mitigated because the source of thepollution cannot be determined. For example, runofffrom nonpoint sources such as farm manure and failingseptic systems may be implicated. The standard indica-tors for fecal pollution, fecal coliforms, do not distin-uish between human and animal sources.

An indicator system based on molecular markersfrom the anaerobic bacterial group Bacteroides-Prevo-tella was developed as part of this research project. Theindicators are not grown, but instead, molecular markersamplified from filtrate from water samples are measured.Using this method, human fecal pollution is dis-tinguished from cow fecal pollution in estuarine andriver waters (see Figure 1).

The objectives are to: (1) develop additionalmarkers from other important polluting species; (2) iden-tify the indicator strains or species; and (3) make thesystem quantitative, allowing for estimation of the pro-portions of different sources of fecal pollution.

Fecal genetic markers are amplified out of feces orwater samples by polymerase chain reaction, using fluo-rescently tagged primers specific for 16S rDNA genesfrom Bacteroides-Prevotella. The amplification pro-ducts are digested with restriction enzymes and rapidlyscreened on a DNA automated sequencer in GeneScanJmode, which estimates the proportions of each fragmentbased on relative fluorescence. Fluorescence data areconverted into an electropherogram diagnostic for spe-cific bacterial constituents. Work on this research pro-ject using this approach resulted in the identification oftwo Bacteroides-Prevotella diagnostic markers for hu-man feces and three markers for cow feces.

Starting with fecal DNAs from other species, thesame approach will be used to test for the cross-occur-rence of markers among different species, and to devel-

op markers from other species. 16S rDNA clone li-braries will be prepared from Bacteroides-Prevotellaamplification products and the marker strains in theclone libraries will be located and phylogenetically iden-tified. The proportional contribution from cattle, hu-man, and other sources will be measured by means ofreal-time quantitative PCR with a Perkin-Elmer Taq-Man.

The project is in its first year and significant pro-gress has been made towards the first two objectives.Fecal samples from 10 additional species have been test-ed for the presence of the human and cow markers. Ofthese, other ruminant species had some of the cow mark-ers. None had human markers. Markers for additionalspecies currently are being developed. 16S rDNA clonelibraries were prepared from PCR products from Bact-eroides-Prevotella-specific primers, and cow and humanmarker sequences were identified by row-and-columnPCR followed by GeneScanJ analysis. When the cloneswere sequenced, it was found that each marker repre-sented a gene cluster rather than a single sequence. Thehuman marker clones were related to Bacteroides unifor-mis and B. vulgatus. The cow markers comprised twonovel gene clusters within Bacteroides but were unre-lated to any previously characterized microorganisms.

Source identification of fecal pollution is a long-standing problem. Results from this research promise toprovide a solution that will be rapid and economical.The next steps will be to continue developing Bacter-oides-Prevotella markers from important polluting spe-cies, such as swine and waterfowl, and to survey the fre-quency of occurrence of the markers in individuals andpopulations, to make the assay quantitative. In addition,PCR primers specific to each individual marker are beingdeveloped to make the assay easier and more af- fordablefor water-quality laboratories that do not have access toa DNA automated sequencer.

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Figure 1. T-RFLP analysis of amplified Bacteroides-Prevotella 16S rRNA genes distinguishes human from cow fecal pollution. Solidlines represent combined human fecal DNAs; dotted lines represent combined cow fecal DNAs. The arrows indicate cow-or human-specific genetic markers.

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Intraspecies Genetic Diversity Measures of Environmental ImpactsDan E. Krane, G. Allen Burton, and Keith GrasmanDepartment of Biological Sciences, Wright State University, Dayton, OH

This project’s primary focus is an assessment of thesuitability of RAPD-PCR (randomly amplified polymor-phic DNA-polymerase chain reaction) genetic diversitymeasures as an alternative or adjunct for toxicity andbioaccumulation tests of ecological risk in a wide varietyof resource types.

Initial efforts will assess the amenability of a set oforganisms from a range of trophic levels to RAPD-PCRanalysis and will identify organisms suitable for use at avariety of sites. Subsequent work will use these organ-isms to determine the impact of a broad range of envi-ronmental stressors upon overall genetic diversity levelswithin exposed populations.

The initial stages of this project have focused onrefining the RAPD-PCR DNA profiling technique anddetermining which native organisms at a variety of localaquatic and terrestrial sites would be most suitable foranalyses. Profiles from six different aquatic species(crayfish, snails, fathead minnows [Pimephales pro-melas], mosquitofish [Gambusia affinis], damsel flies,and Hyalella azteca) and five terrestrial species (pillbugs, earth worms, spiders, garlic mustard, and violets)have been generated and evaluated. All organisms stud-ied to date have proved amenable to highly reproducibleRAPD-PCR typing.

The important roles played by the species consi-dered to date, in addition to their nonmigratory natures,normally high population densities, broad distribution,and sexual reproduction make them very well-suited forgenetic diversity studies. The extremely high geneticsimilarity seen in garlic mustard (probably due to itsrecent introduction and invasive nature) both within andbetween collection sites makes it unsuitable for sub-

sequent work. All other organisms display statisticallysignificant differences in genetic diversity between pop-ulations collected at contaminated and reference sites(see Figure 1 for an example in crayfish). Populationscollected at reference sites generally have had thehighest levels of diversity, and these measures arecorrelated with the diversity measures of other speciescollected at the same sites as well as with other measuresof ecosystem health, including IBI (the Index of BioticIntegrity) and ICI (the Invertebrate Community Index).Initial surveys of aquatic systems and their closelyassociated terrestrial sites have been made at Dick’sCreek in Middletown, Ohio, and the Little Scioto Riverin Marion, Ohio. Analyses of the DNA profiles from thepopulations of organisms will be completed and used todetermine which will be most suitable for use at othersites.

In Year 2 of the proposed study, genetic diversitylevels of the most informative sentinel species will beconsidered in surveys of a third aquatic system that hasbeen contaminated with a different set of pollutants.Specifically, organisms will be collected at one referenceand three contaminated sites along the Clark Fork Riverin southwest Montana.

This river transects a variety of agricultural areasalong its 120-mile reach. Mining, milling, and smeltingactivities have occurred extensively throughout itsheadwater and tributary streams, resulting in substantialheavy metal contamination of the watershed. The systemis particularly interesting, due to its harboring of apollution sensitive and high-quality benthic macro-invertebrate community despite extreme metal toxicitylevels.

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Genetic Similarity

Figure 1. Index of Biotic Integrity (IBI) values are inversely correlated with RAPD-PCR based measures of genetic similarity. Averagepairwise similarity of each organism relative to all others collected at its site are plotted against the independently obtainedIBI value (Ohio EPA, personal communication) for that site (N=144, r=-0.770, p<<0.001) and a solid line shows the bestfit linear regression. Crayfish collected from the impacted and reference Ottawa River sites are displayed as triangles (ª),those collected from sites along the Little Scioto River are displayed as circles (è) and those collected from Elk Creek andits reference stream, Dick’s Creek, are shown as squares (~). Large Xs correspond to the mean pairwise genetic similarityof crayfish at each site (N=8, r=-0.804, p<0.01) and a dashed line corresponds to the best fit linear regression for those points.

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Demographic and Genetic Factors Affecting Population Viability of Lupinus perennis, an Indicator Species of Oak SavannaHelen J. Michaels 1 and R.J. Mitchell 21Department of Biological Sciences, Bowling Green State University, Bowling Green, OH; 2Department of Biology,University of Akron, Akron, OH

Plants in small and sparse populations often havelow reproductive success, indicating that fragmentationof populations by human activities may prevent pop-ulations from being self-sustaining. This research is in-tended to elucidate whether and how population declinedevelops for a model organism, Perennial Lupine (Lup-inus perennis: Fabaceae). This plant species, an im-portant indicator species for the imperiled Oak OpeningsSavanna community of the Great Lakes ecosystem, notonly suffers from habitat loss and fragmentation, but alsois the only host plant for three endangered butterflyspecies. Thus, understanding how demographic andgenetic factors contribute to decline of this species willimprove understanding of this community and recoveryefforts for imperiled butterfly species.

The causes of declines in plant reproductive suc-cess in small and sparse populations include decrease inpollinator services, loss of genetic diversity, inbreedingdepression, and combinations of these factors. This re-search seeks to determine the importance of each factor,utilizing a blend of observational and experimental tech-niques and merging precise data from novel DNA-basedindicators of genetic diversity with classical ecologicaldata on reproductive ecology.

The observational studies focus on how existingvariation in population size and density affect the factorsabove. Experimental studies involve a reciprocal trans-plant between large and small populations to confirmthat population size is the cause of the observed patterns.These data will not only improve the understanding offundamental biological principles in small populations,but also will be informative for designing Oak Savannamanagement strategies.

Pollinator visitation increased significantly indense areas within populations, but did not differsignificantly among populations varying in size.Significant inbreeding depression of seed set andoffspring fitness was ex- hibited in a pilot experiment. Apreviously unnoticed seed predator (Megalotomusquinquespinosus; Hemipt- era; Alydidae) that mimics antbehavior and appearance

was detected. Feeding damage from this bug disruptsLupine seed dormancy, potentially affecting plant de-mography and population health. Several DNA micro-satellite markers with which to assess inbreeding rateshave been developed.

The pollinator visitation data indicates that eco-logical factors may influence population reproductivesuccess on a fine scale, such that within-population den-sity variation may be as or more important than pop-ulation size itself (see Figure 1). Should these trends besubstantiated in subsequent studies, management activ-ities may need to consider plant density as well as popu-lation size to improve the status of L. perennis, the spe-cies that depend upon it, and the overall health of theOak Savanna ecosystem.

The pilot study of inbreeding depression hashelped refine the methods and experimental design to beem- ployed in the larger study during Year 2. The resultsalso suggest that inbreeding depression will be detect-able. The occurrence of an ant-mimicking seed predatorappears to be unknown to most scientists and managersinterested in Lupine and the Karner Blue. Work to dis-seminate this information is underway through conver-sations and a manuscript in preparation.

For Year 2 of the project (2000B2001), the tasks tobe completed include: assessing genetic variation (iso-lating DNAs, running gels), analyzing the mating system(planting seeds, isolating DNAs, running gels), plantingphytometers in the field, continuing pollinator observa-tions, inbreeding depression pollinations, planting in-breeding depression seeds (fall), and analyzing and writ-ing up research results. During Year 3 (2001B2002), itis planned to assess phytometer success, complete themating system gels, assess inbreeding depression seed-lings, and further analyze and write up the researchresults.

More information can be found at the followingWeb Sites: http://www.uakron.edu/biology/mitchell/lupine.html and at http://www.bgsu.edu/departments/biology/people/faculty/michaels/research.html.

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Vis

its

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Lupine Abundance Class

Figure 1. Inflorescence visitation rates by bumble bees to observation plots in 1999 (mean ± se). Lupine density significantlyaffected visitation rate (F1, 80 = 9.6, P < 0.0027).

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AW (6)RS (5)CT (4)PC (6)SC (2)JC (2)CM (2)NM (3)PP (1)

PC (1)

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AW (6)RS (5)CT (4)PC (6)SC (2)JC (2)CM (2)NM (3)PP (1)

PC (1)

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Are Genetic Diversity and Genetic Differentiation Bioindicators of Contaminant Impact on Natural Populations? Fundulus heteroclitus as a Model Estuarine SpeciesMichael C. Newman, Margaret Mulvey, Michael A. Unger, and Wolfgang K. VogelbeinVirginia Institute of Marine Science, The College of William & Mary, Gloucester Point, VA

Molecular genetic traits of the mummichog arebeing evaluated as bioindicators of population-level ef-fects of pollution. Mummichogs were sampled from ninelocalities along the Elizabeth River in Virginia.Sediment PAH concentrations span four orders of mag-nitude among these sites. Localities vary also in sed-iment concentrations of chlorinated hydrocarbons, tri-butyl tin, and metals. The prevalence of proliferativeliver lesions was high in fish from one locality, theAtlantic Wood (AW) site, with a PAH concentration of371,212 ng/g of sediment.

Molecular genetic data currently are being used toaddress several predictions regarding population re-sponse to contaminants: (1) populations residing in con-taminated habitats are genetically distinct from populat-ions in neighboring clean sites; (2) populations at pol-luted sites exhibit lower genetic diversity than populat-ions taken from clean sites; and (3) the genetic structureof mummichog in the Elizabeth River reflects the mosaicof highly contaminated and clean habitat.

Genetic data consist of allozyme genotypes andDNA sequences for a 450 base pair segment of the

d-loop of the mitochondrial genome. Preliminary anal-yses of allozyme data do not indicate correlation withlevel of contamination. Analyses suggest that overallgenetic variability did not differ with PAH concen-tration. Highly polluted localities (AW, RS) were notgenetically depauperate relative to neighboring relativelynonpolluted localities (SC, JC). Indeed, mummichogfrom the heavily PAH-contaminated AW site show highdiversity in d-loop haplotypes.

Mummichogs from the AW site share the commonhaplotype found at other locations along the ElizabethRiver as well as several highly divergent haplotypes (seeFigure 1). Genetic analyses suggest that mummichogmay move among localities more than previously report-ed.

Hypotheses regarding contaminant tolerance andindividual and populations performance relative to gen-etic variability and contaminants will be examined in thenext phase of the project. Additional survey work ofmummichog inhabiting contaminated and clean habitatsmay be undertaken to evaluate the generality of theseresults.

Figure 1. Relationships among d-loop haplotypes and distribution among Elizabeth River sites.

12

Multilevel Indicators of Ecosystem Integrity in Alpine Lakes of the Sierra NevadaJames T. Oris 1, Sheldon I. Guttman 1, A. John Bailer 1, John E. Reuter 2, and Glenn C. Miller 31Miami University, Oxford, OH; 2University of California, Davis, CA; 3University of Nevada-Reno, Reno, NV

The overall objective of the project is to developprotocols for environmental assessments of alpine lakesin the Sierra Nevada with a range of human impacts.These assessments will be conducted over the range oflevels of biological organization (molecular to eco-system), utilizing currently available assessment tech-niques and with the addition of two new ecological in-dicators.

The use of population genetics analysis as a re-sponse indicator and the use of molecular biomarkers ofexposure to contaminants as a diagnostic indicator willbe tested for incorporation into monitoring and assess-ment programs for surface waters. These indicators willprovide information concerning the status of populationdiversity and stability as well as the exposure to non-persistent, nonbioaccumulative contaminants. This in-formation is missing from current monitoring andassessment protocols.

Over the 3-year project period, standard environ-mental assessments of a select group of alpine lakes witha defined range of human impacts will be conducted.There will be a total of 16 assessment sites (see Figure 1).Those selected for assessment include four minimal- lyimpacted areas (Castle Lake, Eagle Lake, Marlette Lake,and Upper Angora Lake), eight areas with a range ofmoderate impacts (Fallen Leaf Lake, Gold Lake, LakeTahoe at Sand Harbor, Prosser Reservoir, SpauldingReservoir, Stampede Reservoir, Topaz Lake, and TwinLakes), and four highly impacted areas (Boca Reservoir,

Donner Lake, Lake Tahoe at Tahoe City, and Lake Tahoeat South Lake Tahoe).

In addition to the standard assessment, populationgenetic assessments will be conducted in fish and in-vertebrates at these same sites. Allozyme electropho-retic analysis and Randomly Amplified PolymorphicDNA (RAPD) analyses will be conducted on two organ-isms common to the lakes of the region (fish: Lahontanredside; invertebrate: Signal crayfish). Contaminantexposure assessments also will be conducted in fishusing molecular biomarkers of exposure in the gills offish. Five markers indicative of exposure to a widevariety of chemical contaminants (persistent andnonpersistent) and that can account for interactionsamong complex mixtures of contaminants will bemeasured over time in the assessment areas using cagedrainbow trout.

These additional techniques then will be applied tocurrent assessment protocols. Because a group of lakeswith defined levels of human impacts will be examined,the discriminatory ability of the assessment techniquesmay be analyzed using the current protocols compared tothe protocols with the two new indicators added.

It is hypothesized that because current protocols donot account for genetic diversity or nonpersistent con-taminants, the addition of these new indicators willgreatly enhance the monitoring and assessment programsfor surface waters.

13

Figure 1. Map of assessment site locations within the Sierra Nevada.

14

Ecosystem Monitoring via Genetic Diversity Surveys of Dandelions Using VNTR Multilocus DNA ProbesSteven Rogstad and Brian KeaneDepartment of Biological Sciences, University of Cincinnati, Cincinnati, OH

New methodologies in molecular genetics mayprovide novel types of ecological indicators for mon-itoring the integrity of natural ecosystems and the sus-tainability of ecosystems that are affected by anthropo-genic influences. One type of ideal ecological indicatorwould be an organism that grows in a wide variety ofhabitats that could easily be used to monitor for thepresence of mutagens or anthropogenic factors that alterpopulation genetics.

Dandelions (Taraxacum officinale: Weber; Aster-aceae) are a model ecological indicator organismbecause: (1) dandelions have an extremely wide ecolog-ical amplitude, growing almost worldwide from sea-levelto alpine biomes, and from the tropics to north-temperatehabitats; (2) dandelions grow rapidly, and populationscan be easily manipulated and monitored; (3) it has beendocumented that a variety of pollutants can be seques-tered in dandelion tissues; (4) dandelion seeds are pro-duced asexually, rendering the detection of mutationseasy; and (5) preliminary data have been generated usingvariable-number-tandem-repeat (VNTR) DNA probes thatsuggest that mutation rates and population geneticdiversity parameters can be analyzed successfully withdandelions.

The specific hypothesis to be tested is that dande-lions can be used as a sensitive ecological indicatorspecies via comparative analyses of VNTR genetic mark-ers used to examine anthropogenic changes in geneticdiversity of both mutation rates and population genetics,at pollution-impacted versus nonimpacted sites.

Dandelions will be sampled from 12 sites: 6 rel-atively pristine sites versus 6 sites with chronic, highlevels of contaminants. To determine whether rates ofmutation differ between these two types of sites, leaftissue and 8B10 seeds will be collected from each of 10plants per site. Dandelion seeds are produced via aga-mospermy, a nonsexual process in which all seed-

lings from a maternal plant should be geneticallyidentical to that maternal plant.

Preliminary results demonstrate that although theprevious statement is true, a low level of mutation (about0.005% of bands transmitted) is detectable. By survey-ing the parent-to-offspring transmission of tens of thou-sands of VNTR markers, whether mutation rates differbetween pristine versus contaminated sites is being test-ed (see Figure 1).

These markers also will be used to examine wheth-er populations differ in genetic diversity. Initial resultsdemonstrate that very local populations harbor a largeamount of genetic diversity, although certain clones maybe widely distributed. Soil and dandelion tissue havebeen analyzed from more than 20 sites for metals, and 12of these sites covering a range of contamination arebeing selected to compare dandelion mutation rates andpopulation genetics.

It is expected that a determination will be made asto whether VNTR markers in dandelions can be used assensitive indicators of anthropogenic changes in pop-ulation genetic diversity due to either altered mutationrates or stressor-induced selection. If mutation rates orgenetic diversity in dandelions are correlated withpollution history, dandelions provide an easily utilizedbiomonitor to survey the ecological integrity andsustainability of a wide range of habitats acrossmultiple spatial scales throughout the world.

Besides completing the above projects, furtherexperiments growing dandelions in controlled mediaspiked with mixtures of common pollutants will be con-ducted to investigate whether mutation rates increasewith increasing pollutant concentrations. Further, stud-ies have been initiated to determine whether dandelionsfrom the most heavily polluted sites have higher fitnesswhen competing with dandelions from more pristine siteswhen both are grown in polluted and nonpolluted soils.

15

Figure 1. Maternal plant-to-offspring transmission of VNTR genetic markers in dandelion detected with the TTCCA (= coresequence) PCR-STR probe. Markers for two different maternal parents are shown (lanes A and B), with 10 clonal offspringof each maternal parent also shown (5 offspring on each side of each parent). More than 30 VNTR markers are shownacross both parents, and parents clearly differ in the markers transmitted to their clonal offspring. One new mutant markeris detectable in the eleventh lane from the left (marked with a <). If an autoradiograph is interpreted to have a transmissionof approximately 30 markers to 20 offspring (= 600 markers transmitted), with one novel marker, the detectable mutationrate is 1/601 = 0.00166 for that particular gel-probe autoradiograph. Three micrograms of genomic DNA per lane weredigested with a five-fold excess of TaqI restriction endonuclease.

Section 2.

Multiscale and Landscape Indicators

19

Foliar Chemistry as an Indicator of Forest Ecosystem Status, Primary Production, and Stream Water ChemistryJohn Aber, Richard Hallett, Mary Martin, Marie-Louise Smith, Scott Ollinger, and Scott Bailey Complex Systems Research Center/EOS, University of New Hampshire, Durham, NH

Monitoring the biogeochemical status of forest andstream ecosystems is a key component of assessingenvironmental quality in the Northeastern United States.Any monitoring system that requires spatiallycontinuous capabilities will need to use some form ofremote sensing. Because forest canopies are the onlyportion of the system accessible to optical reflectanceremote sensing instruments, they offer the most likelytarget surface for monitoring forest health in this spatialmode.

It was hypothesized that forest productivity, soilmineralogy, and foliar chemistry at the whole-stand (notindividual tree) level are all tightly linked to the bio-geochemical status of a forest ecosystem. It was furtherhypothesized that the concentration of cations in forestcanopies will be measurable by high spectral resolutionremote sensing, as has been demonstrated for nitrogenand lignin, and that watershed-level stream chemistry,reflecting soil mineralogy, also will be predictable fromwatershed-level values of canopy chemistry derived byremote sensing.

The study area is the White Mountain NationalForest (WMNF), a 300,000 ha area in northern NewHampshire. At the intensive plot scale, the long-termsampling program at the Bartlett Experimental Forest andthe Hubbard Brook Experimental Forest, New Hampshire,will be used and augmented to examine and attempt topredict interannual variations in foliar chemistry as wellas woody and foliar production.

At the regional scale, canopy chemistry, soil min-eralogy, and forest productivity will be measured at aseries of existing experimental and monitoring researchsites. Work on the spatially continuous monitoring scaleacross the White Mountain region will include the devel-opment of algorithms for predicting canopy cation con-centrations using data from NASA’s Airborne Visible-Infrared Imaging Spectrometer (AVIRIS). At thewatershed scale, 50 streams will be sampled in the

WMNF covering a range in estimated mineralogicalrichness. A nested approach will be used to determinethe optimum scale at which stream water chemistry maybe predicted. In addition, high spatial resolution (3B4 m)spectral data will be collected for sampled watersheds vialow altitude AVIRIS data acquisition.

Preliminary findings include: (1) strong relation-ships between whole-stand level foliar canopy N, NPP,and forest floor C:N ratios exist; (2) foliar Ca can bemapped across the WMNF (300,000 ha) using remotesensing technology (see Figure 1); and (3) a GIS modelhas been developed for the WMNF that predicts elementcontent of glacial tillCthe predictions for till Ca havebeen compared to plot level foliar Ca data, and a signifi-cant relationship exists.

Remote sensing technology can be used to estimatefoliar chemistry (N, Ca) at a landscape scale. With thisinformation, forest productivity estimates can be made.Soil mineralogical characteristics also can be modeled atthe landscape scale. Relationships between stream waterchemistry, mineralogy, foliar chemistry, and forest pro-ductivity can be determined with this information. Thisprogram then would establish the scientific basis fordeveloping a satellite- or aircraft-based remote sensingprogram for monitoring forest health and stream waterquality.

The next steps are to: (1) continue the collection and analysis of stream water from 50 streams across theWMNF; (2) process and analyze new AVIRIS imageryfrom a low altitude platform for selected water-shedsCAVIRIS low altitude data (3B4 m resolution) forthe summer of 2000 has been requested through NASA’sAirborne Science Program; (3) collect data from newplots across the WMNF to validate foliar and glacial tillelement concentration maps; and (4) use soil and foliarelement coverages to develop relationships with mea-sured stream water chemistry and forest productivity.

20

Figure 1. AVIRIS predicted plot level canopy Ca concentration plotted against measured canopy Ca concentrations.

21

Environmental Factors That Influence Amphibian Community Structure and Health as Indicators of Ecosystem IntegrityVal Beasley 1, Lucinda Johnson 2, Carl Richards 3, Patrick Schoff 2, Rebecca Cole 4, Camilla Lieske 1, Anna Schotthoefer 1, Cathy Johnson 2, Joseph Murphy 1, and Marvin Piwoni 51College of Veterinary Medicine, University of Illinois, Urbana, IL; 2Natural Resources Research Institute,University of Minnesota, Duluth, MN; 3Minnesota Sea Grant, Duluth, MN; 4USGS National Wildlife HealthCenter, Madison, WI; 5Illinois Waste Management and Research Center, Champaign, IL

The overall goal of this research is to assess therelative influence of landscape patterns, biotic interact-ions, water quality, and contaminants on amphibianhealth and community structure. The following will beevaluated: (1) the relative influence of wetland- andwatershed-scale factors (i.e., landscape and ecologicaldata) on amphibian community structure and health; and(2) whether amphibian community structure and healthare indicative of ecological integrity.

During 1998, anuran community structure, wetlandhabitat, and landscape characteristics were quantified at64 sites distributed throughout northern Illinois, south-ern-to-central Wisconsin, and southern-to-central Min-nesota (see Figure 1). Site-specific surveys also wereconducted to determine the incidence and types ofanuran malformations and deformations.

Landscape data were summarized within a 2 km and10 km radius/buffer surrounding each wetland.Macroinvertebrates were identified to the family level,and dominant vegetation was characterized for each wet-land. Anuran data were summarized for 1998 and 1999,individual species-habitat relationships were examined,and site occupancy models were developed for severalfrog species based on local and regional characteristics.Logistic regression models were developed to predict theoccurrence of the wood frog, spring peeper, and gray treefrog. Models incorporating data at several spatial scaleswere better predictors of frog presence/absence thanthose incorporating variables from only one spatial scale.During 1999, 36 wetlands in central Minnesota wereanalyzed for anuran and macroinvertebrate communitystructure, wetland vegetation, and physical/chem- icalattributes, including habitat structure, water, andsediment quality. Parasitological studies were conductedon tadpoles, metamorphs, adult frogs, and gastropods.

In separate studies, full necropsies were performed on485 frogs. A comprehensive set of tissues was collect-ed, fixed in buffered formalin, and processed for histo-pathologic examination.

Malformations or deformations were identified in13 of 559 frogs from 8 wetlands in 1998, and in 7 of 544frogs from 6 sites in 1999. Among the malformationswere webbed skin between the femur and tibio-fibula,skin lacking spots, and hemimely (partial absence) of thehind limb. Parasites identified included: Echinostomaspp., Rhabdias ranae, Haematolechus sp., Megalodiscussp., and Ribeiroia sp. Echinostoma spp. were found inthe kidneys of frogs from all sites at prevalences of40B100 percent (mean 82%) and intensities of 8B321echinostomes/frog (mean 90).

In pathological studies on 485 frogs, intersex wasidentified in 6 percent of all frogs (from 11 sites). At leastone lesion was identified in 55 percent of frogs.Condition scores from slightly poor to emaciated werenoted in 8 percent of frogs from 12 sites. Parasitism wasnoted in the body cavity, GI tract, kidney, liver, lung,muscle, and skin. The most common abnormality forbody cavity, kidney, liver, and lung was inflam- mation(52B84%); for GI tract it was no digesta (50%); formusculoskeletal system it was trauma (52%); and forspleen it was splenomegaly (73%). Diagnoses and sexdeterminations will be confirmed histologically.

Examinations of metamorphs, identification ofparasites, histopathology studies, and chemical analysesfrom the 1999 field season will be completed in thecoming months. Additional ecological, water quality,contaminant, and anuran community structure and healthdata will be collected in 2000 before the assessment ofthe potential use of amphibian community structure andhealth as indicators of wetland conditions.

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Figure 1. Amphibian community study areas.

23

Modeling Ozone Flux to Forests Across an Ozone Concentration Gradient in the Sierra Nevada Mountains, CAAllen Goldstein and Jeanne PanekDepartment of Environmental Science, Policy, and Management, University of California, Berkeley, CA

Tropospheric ozone is a pollutant that is responsi-ble for forest damage worldwide. Extensive ozone mon-itoring networks currently measure ozone concentrat-ions throughout the United States and Europe; however,the physiologically relevant measure of ozone for foresthealth is not concentration, but rather ozone flux, theamount of ozone that actually enters the foliage.

A model is being developed to estimate ozone fluxfrom ozone concentration utilizing routinely measuredozone and meteorology. It is hoped that this model willbe adapted for monitoring networks. The utility of *13Cas a proxy for stomatal conductance in the estimation ofozone deposition also is being explored.

In 1997 and 1998, an initial study was conductedat Blodgett Forest to investigate the influence of typicalMediterranean summer drought on the uptake of ozoneand general physiological response of ponderosa pine.Fluxes of ozone, CO2, water, and energy were measuredby eddy covariance from May through October. Usingthis eddy flux data, typically reported ozone exposuremetrics were compared against direct measurements ofozone deposition to document that ozone metrics arepoor predictors of ozone uptake in California pine eco-systems.

In May 1999, the field campaign was expand-edCsites were established at three additional locationsalong an ozone injury gradient in the Sierra NevadaMountains, California. Site locations and characteristicsare presented in Table 1. These sites take advantage ofexisting ozone monitoring (National Park Service andCalifornia Air Resources Board Environmental Protect-ion Agency) and an ongoing forest damage assessmentproject. Year-round continuous measurements of eco-system-scale ozone, carbon dioxide, water, and energyfluxes started in May at Blodgett Forest. Measurements

of leaf-level physiology were made monthly at each sitefrom May through September and included diurnal netphotosynthesis, stomatal conductance and transpiration,predawn and afternoon water potential, photosynthesisresponse curves to light and carbon, and dark res-piration. Foliage was collected for starch *13C analysisevery month. At the end of the season, foliage sampleswere collected for cellulose *13C measurement

Another study at Blodgett Forest to gain furtherinsights into the limitations imposed by typical summerdrought on the uptake of carbon and ozone in the pon-derosa pine ecosystem was completed. Two sites were setupCone control and one wateredCin a ponderosa pineplantation. Carbon uptake in 1-year-old control fo- liagewas reduced compared to the watered treatment duringthe 3 measurement days following treatment by 39percent, 35 percent, and 30 percent, respectively, per unitleaf area. Stomatal conductance was lower at the controlsite, leading to a reduction in estimated ozone deposition(ozone concentration times stomatal conductance) of 36percent, 46 percent, and 41 percent of the watered site,respectively. This experiment demonstrated that sitemoisture is one of the most important factors controllingozone uptake in California forests. Expected changes inclimate will profoundly affect the ozone uptake byCalifornia forest ecosystems.

The field campaign will continue during the grow-ing seasons of 2000 and 2001. Models that estimate sto-matal conductance and ozone flux into foliage will beevaluated and compared for their appropriateness in theSierra Nevada ponderosa pine ecosystem. The selectedmodel will be developed further to apply to this project’sspecific needs and compare modeled ozone depositionwith direct measurements of canopy-scale ozone depo-sition at Blodgett Forest.

Table 1. Site locations and characteristics.

Site Location SlopeElevation

(m)OII1 Ozone2

(ppb)

Sequoia/King’s Canyon National Park N36E33'55" W118E46'36" 30% 1920 41.3 63a

Yosemite National Park N37E42'43" W119E42'19" 10% 1220 14.7 41a

Blodgett Forest Research Station Control Site N38E53'43" W120E37'58" 2% 1315 N/A 49b, 57c

White Cloud N39E19'00" W120E50'45" 10% 1326 27.3 62a

1Ozone Injury Index (OII) is derived from a combination of the primary effects of ozone on pine (Arbaugh et al.,1998). 224 hour mean ozone concentration (in ppb) from June 1BOctober 31 were: (a) from Arbaugh et al.,1998, (b) from our measurementsin 1998, and (c) from our measurements in 1999.

24

Effects of Forest Fragmentation on Community Structure and Metapopulation Dynamics of AmphibiansLucinda Johnson 1, Catherine Johnson 1, Randall Boone 2, and John Gross 21Natural Resources Research Institute, University of Minnesota, Duluth, MN; 2Natural Resource EcologyLaboratory, Colorado State University, Fort Collins, CO

The goals of this research project are to quantify theeffects of forest fragmentation on amphibian com-munity structure and metapopulation dynamics in vernalpools, and to develop appropriate ecosystem indicatorsfor vernal pool ecosystems at multiple spatial scales.

The objectives of this study are to: (1) quantify themanner and extent to which forest fragmentation influ-ences amphibian community structure of vernal poolecosystems; (2) assess the extent to which landscape- andlocal-scale features reflect fundamental structural prop-erties of vernal pool habitats and their biotic com-munities and, conversely, the extent to which indices ofbiotic integrity (e.g., amphibian community structure)reflect local and landscape properties; (3) develop pre-dictive models that integrate landscape-scale factors withpond-scale attributes to quantify key compositional andstructural attributes of the amphibian community, andderive ecosystem indicators at multiple spatial scales;and (4) develop predictive models to quantify the extentto which forest fragmentation influences the metapop-ulation dynamics of woodland amphibians and predictthe consequences of landscape change on these metapop-ulations.

Recent evidence of declines and an increased rateof malformations in amphibian populations have prompt-ed much interest and research into the potential anthro-pogenic stressors associated with these occurrences.Results of some of this research indicate that fragment-ation may have a significant effect on the metapop-ulation dynamics of amphibian communities, whichcould result in declines and regional extinctions of pop-ulations. Forest fragmentation results in changes to for-est landscapes and habitats that may have both direct and

indirect effects on local and regional amphibian com-munities (e.g., direct habitat loss, disruption of dispersalcorridors, altered habitat structure, and microclimatechanges).

It was hypothesized that forest fragmentation canbe directly related to changes in landscape structure andlocal habitats (e.g., vernal pools) that have a measure-able effect on the integrity of amphibian communities.Landscape, local habitat, and biotic community variableswill be quantified to examine the effects of forest frag-mentation on pond-breeding woodland amphibians atthree spatial scales: the landscape scale, the local or"pond-scape" scale (including ponds and surroundingterrestrial habitat), and the aquatic pond habitat. Rela-tionships among these hierarchically nested scales willbe quantified using an integrated series of empiricalmodels. This approach of identifying relationships acrossscales and integrating them into a modeling frameworkwill allow the development of multiscale ecologicalindicators of the effect of forest fragmentation on vernalpool ecosystems and on regional amphibiancommunities.

It also was hypothesized that indices of communitydiversity and structure and amphibian species traits canbe used to assess effects of forest fragmentation on ver-nal pool systems and regional amphibian communities.Metapopulation models will be developed to assess theeffects of fragmentation on local and regional popula-tions, and to elucidate the mechanisms by which forestfragmentation may influence the composition and persis-tence of vernal pool communities. This is a new project,and to date there are no results to report.

25

Land Use and Geomorphic Indicators of Biotic Integrity in Piedmont StreamsDavid S. Leigh, B.J. Freeman, M.C. Freeman, E.A. Kramer, M.J. Paul, C.M. Pringle, A.D. Rosemond, R.Cifaldi, A. Roy, and D. WaltersGeography Department, University of Georgia, Athens, GA

The overall goal and objective of this project is todefine the predictive capabilities of scale-variable at-tributes of land cover (GIS-based) and geomorphology asrisk assessment indicators of biotic integrity of streamecosystems on the southern Piedmont.

The watershed under investigation is the upperEtowah River Basin north of Atlanta, GA. Given vari-ous aspects of historical landscape change, this researchis investigating the following three ancillary questions:(1) Do physical stressors and the corresponding eco-logical response vary as a function of land cover in thewatershed? (2) Is this relationship consistent withinwatersheds of vastly different sizes? (3) Do antecedentland cover conditions (>50 years ago) influence thephysical stressor and ecological response relationship?

The approach to answer these questions involvestwo main projects over a 3-year period. The first pro- jectinvolves a comprehensive field survey of the geo-morphic condition, habitat, water quality, and biolog-ical integrity in 30 streams draining watersheds of threedistinct size classes of about 15, 50, and 100 km2. Thesewatersheds have variable land cover of 50B100 percentforest, based on the 1993 multiresolution landcharacteristic (MRLC) databases. The stream reaches aresurveyed for a length of at least 15 stream widths.Regression analysis is used to develop predictive modelsof biotic conditions from geomorphic indicators, habitatassessment, water quality, and land cover characteristics.The second project involves a detailed analysis of geo-morphic conditions, stream habitat, and biotic integrityof five streams (out of the original 30) that appear as

unexplained residuals in the predictive models, whichwill be compared to five other streams that fit the models.This will involve an expansion of 10 surveyed reaches toa length of at least 1 km, and further assessment of thereach-scale versus watershed-scale controls on bioticintegrity.

During January 2000, raw data from all aspects ofthe field survey were finalized and are being analyzedalong with the land cover data. The preliminary findingssuggest that reach-scale geomorphic and habitat assess-ments are somewhat better indicators of biotic integritythan the 1993 watershed-scale land cover characteristics.For example, the average particle size of the stream bed(in phi units) is highly correlated with the relativeabundance of pool species of fish, exhibiting a correla-tion coefficient of 0.82 (see Figure 1). In contrast, thebest correlation between pool species and land cover isfound with percent forest, exhibiting a correlation co-efficient of -0.48, and all other land cover classes are notsignificantly correlated.

The significance of the preliminary findings may bethat objective reach-scale assessments of physical char-acteristics are better indicators of biotic conditions thanwatershed land cover. More analysis of linkages be-tween the watershed-scale land cover and reach-scalephysical conditions are needed to explore causalrelation- ships and develop multiple regression modelsthat may provide the best indicators of biotic conditions,however. The next steps involve continued analysis ofthe raw data from the 30 sites and identification of sitesfor the second phase of the analysis.

Figure 1. Relationship between mean Phi particle size and pool species (relative abundance) in streams of the upper EtowahRiver Watershed (10–150 km2 basins).

26

Developing Effective EcologicalIndicators for Watershed AnalysisDuncan T. Patten 1, Wayne Minshall 2, Rick Lawrence 3, and Andrew Marcus 3

1Yellowstone Ecosystem Studies, Bozeman, MT; 2Idaho State University, Pocatello, ID; 3Montana State University,Bozeman, MT

This project is designed to develop improvedindicators and innovative techniques for assisting andmonitoring ecological integrity at the watershed level inthe Western United States. The specific objectives of thisstudy are to develop practical, scientifically validindicators that: (1) span multiple resource categories; (2)are relatively scale independent; (3) address differentlevels of biological organization; (4) can be rapidly andcost-effectively monitored by remote sensing; and (5) aresensitive to a broad range of anthropogenic and naturalenvironmental stressors.

The study is based, in part, on the hypothesis thatstreams and riparian areas often reflect the ecologicalintegrity of the associated watersheds. Due to a "funneleffect," these areas are the accumulation zones of en-vironmental disturbances occurring in the watershed (seeFigure 1). For example, logging, overgrazing, and for-est fires influence sediment erosion rates that directlyinfluence downstream stream and riparian conditions.Monitoring of key indicators in these accumulationzones may provide an efficient, cost-effective way toevaluate and monitor the ecological integrity andsustainability of the surrounding watershed.

The Upper Yellowstone River and its tributarieswill be used because the watersheds of these streams in-corporate a broad range of environmental conditionsfrom relatively pristine in certain watersheds withinYellowstone National Park, to highly disturbed by forestfires and land uses such as mining, logging, and agri-culture in other watersheds. Identification, assessment,and validation of effective indicators will involve the in-tegration of results from research at various scales, in-

cluding: (1) analysis of hyperspectral and traditionalmultispectral imagery from both aerial and satellite plat-forms; (2) field surveys of stream morphology and ripar-ian habitat associated with remote sensing to assess in-dicators; and (3) intensive site-specific stream samplingof macroinvetebrate communities to validate the effec-tiveness of these indicators. Use and evaluation of re-mote sensing technologies is the primary research meth-odology. All indicators chosen must be able to be mon-itored by remote sensing.

Remote sensing techniques, which will enable se-lection of indicators to be used for rapid, cost-effectiveecological monitoring on the regional and local scale,also will be used to help identify key ecological indicat-ors in both streams and riparian areas. These also will becorrelated with ecological indicators of disturbances inthe surrounding watersheds, resulting in a set of ef-fective ecological indicators and the development of in-novative techniques for efficient watershed-levelanalysis at various scales in the Western United States.

As this project is in its initial phase, the sequentialsteps to be taken over the duration of this project in-clude: (1) coarse watershed characterization for initialsite selection; (2) stream and riparian site selection;(3) ground evaluation and selection of indicators atstream and riparian sites; (4) fine watershed char-acterization using remotely sensed data to determinestressors on study sites; (5) collection of remotely senseddata for study sites; (6) validation of watershed conditionusing aquatic biota; and (7) analysis of riparian andstreams conditions as indicators of watershed condition,and remote sensing as measurement of indicators.

Figure 1. Conceptual model of interrelationships among watershed conditions, stream and riparian indicators, and watershed "health" indicators (aquatic insects), showing drivers andstressors. Remote sensing will be used for watershed characterization and evaluated as a technological tool for measuring ecosystem indicators in the upper YellowstoneRiver Watershed, the study area of this project which represents a western North American landscape.

27

Development and Evaluation of Multiscale Mechanistic Indicators of Regional LandscapesCarl Richards 1, Lucinda B. Johnson 2, and George E. Host 2 1Minnesota Sea Grant College Program, University of Minnesota, Duluth, MN; 2Natural Resources ResearchInstitute, University of Minnesota, Duluth, MN

This project aims to develop suites of ecologicalindicators that cross spatial scales, mechanistically re-flect ecosystem states and processes, are statistically ro-bust, and are applicable across regional landscapes.These indicators also will be based on readily accessibleinformation available in a real-time framework.

The specific objectives are to: (1) develop pre-dictive models that integrate landscape-scale factors withreach-scale physical and chemical stream attributes toquantify key compositional and structural attributes ofstream biota and derive ecosystem indicators at multiplespatial scales; (2) evaluate the appropriate scale of ter-restrial and aquatic data necessary to resolve regionaland local aquatic resource questions; (3) improve theability to distinguish and quantify natural variation inindicators from that derived from anthropogenic stress-ors; (4) assess the extent to which regional and local-scale indices reflect fundamental ecosystem processesand structural properties of stream habitats and biota; and(5) quantify confidence limits and evaluate the geo-graphic transferability of regional- and local-scale in-dicators.

A multitiered sampling and modeling strategy wasused to integrate data collected at regional, local, andsite scales. These data will be used to identify indicatorsat each scale that reflect critical ecosystem processes orstate variables related to the integrity and sustainabilityof those ecosystems. These data also will be used to de-velop and test indicators representing fundamental driv-ing variables and processes at multiple spatial scales, andintegrate them into a system for identifying positive ornegative trends in ecosystem health.

The basic configuration of the landscape variedsubstantially between the two primary study regions. The

grain of the landscape was finer in Michigan with respectto both surficial geology and land cover characteristics.Watersheds in Minnesota were largely ho- mogeneouswith respect to these categories, while Mich- iganwatersheds exhibit more variation.

Strong (albeit seasonal) relationships were foundbetween landscape-scale features and dissolved nutrientconcentrations; however, results of nutrient bioassaysindicated that nutrients rarely limited primary produc-tion, and landscape-scale variables describe only a mod-erate amount of the variability in net primary production(less than 50%). This trend also was observed in macro-invertebrate communities that were weakly related tolandscape-scale attributes but strongly related to local(reach-scale) attributes.

Different parameters of interest are driven by dif-ferent scales and factors within the landscape. Land-scape-scale information can be very useful in predictingdifferences in stream water nutrient concentrations. Incontrast, it is difficult to predict major patterns of mac-roinvertebrate assemblages or primary production with-out understanding the reach-scale characteristics thathave strongest influence on these variables; however, thereach-scale variables are themselves partially governedby landscape-scale variables. The project will continueby examining other stream assemblages, and employingcorrelative and mechanistic analyses to identify relation-ships among ecological indicators operating at local andregional scales. This approach will be directed by theconceptual model presented in Figure 1. A critical partof the next phase will be model evaluation, particularlywith respect to placing confidence limits on predictivemodels. Following this will be an integration of indivi-dual models into a larger predictive framework.

Figure 1. Conceptual model illustrating the organization of mechanistic factors operating at multiple spatial scales. Components summarize the development

of predictive models, ecosystem attributes, and fundamental ecosystem processes. Indicators are derived at all spatial scales.

28

Development and Testing of a Multi-Resource Landscape-Scale Ecological Indicator: Forest Fragmentation, Structure, and Distribution Relative to TopographySteven W. Seagle and Philip A. TownsendAppalachian Laboratory, University of Maryland Center for Environmental Science, Frostburg, MD

The goal of this project is to understand how waterquality and avian habitat quality vary across landscapesas a function of forest distribution and topography (seeFigure 1). More specifically, the project seeks to deter-mine whether water and avian habitat quality covary sothat they can be simultaneously extrapolated across theMid-Atlantic Highlands using existing databases of to-pography, forest cover, and remotely sensed forest struc-ture.

Efforts thus far have focused on: (1) taking an in-tensive spring/summer field season census of forestinterior bird diversity and reproduction; (2) collectingfield data on forest structure within intensive field sites;and (3) obtaining and analyzing radar and Landsatsatellite imagery. Field sites were chosen to represent theclimatic and topographic variation of the Mid-AtlanticHighlands. Twenty 10-ha study sites were est- ablishedto equally represent dry and wet topographic positionswithin the Appalachian Plateau and Ridge-and-Valleyprovinces. Intensive studies on these sites will form theempirical basis to test the hypotheses concerning avianhabitat quality, and then provide the statistical basis toextrapolate across the Mid-Atlantic High- lands.

During 1999, each study site was intensively sur-veyed for breeding bird reproductive success. An arrayof forest structural data also was collected for each site.Preliminary results from the avian survey indicate thatforest interior bird reproductive success is higher on theAppalachian Plateau than in the Ridge-and-Valley, andwithin each province reproductive success is greater atlower slope positions. These results support the hypo-thesis that reproductive success, and thus habitat quality,is related to topography, with wetter provinces and

topographic positions having higher success. Radar im-ages of the intensive field sites are being analyzed toidentify predictors of variation in forest structural char-acteristics. It is planned to collectively use degree offorest fragmentation, structural diversity, and topograph-ic position to map the quality of avian habitat across theMid-Atlantic Highlands.

Amount of forest cover within multiuse watershedsis negatively correlated with the export of nitrogen fromthe watershed. Watersheds within the Mid-Atlantic arebeing analyzed to test the hypothesis that topographicposition of forest improves the correlation of forest coverwith water quality. If this hypothesis is correct, and pre-liminary findings regarding the effect of topography onavian habitat quality withstand further statisticalscrutiny, then forest cover and forest spatial distributionwill to- gether form a powerful indicator of both waterand avian habitat quality that can be mapped over largeareas from existing databases.

Efforts currently are focused on three tasks. First,the relationship between forest structure and character-istics of remotely sensed radar images are being anal-yzed. This relationship will help refine predictions ofavian habitat quality. Second, processing of Landsatland cover data and integration of forest cover with topo-graphical data are aimed at developing simple measuresthat describe the relationship between forest spatial pat-tern and topography. These measures will be calculatedat the watershed level and correlated with water qualityparameters. Finally, a second field season to extend theunderstanding of spatial variation in avian reproductionand forest structure is being prepared.

29

Figure 1. Multiple characteristics of forests in the Mid-Atlantic are being examined as indicators of both surface water quality andforest 0interior bird reproductive success. Hypothesized relationships (dark arrows) are being tested through eitherreanalysis of existing databases or field studies. In contrast, the effect of fragmentation on forest-interior bird reproductionis already quantified (white arrow). As indicator components, both forest fragmentation and forest topographic distributionare readily mapped from existing databases. The importance of forest vertical structure for forest bird diversity (whitearrow) is well-known, but the relationship is difficult to extrapolate from existing data. Thus, remotely sensed radarimagery is being examined for extrapolating forest structure over large areas.

30

Ecological Indicators for Large River-Floodplain LandscapesMonica G. Turner, E.H. Stanley, M.D. Dixon, R.E. Freeman, S.E. Gergel, H. Kang, J.R. Miller, and J. West Department of Zoology, University of Wisconsin, Madison, WI

Identifying landscape indicators that are well-correlated with specific ecological functions remains acrucial research need. Ecological indicators (population,community, ecosystem, and landscape) are being devel-oped and tested along reaches of the Wisconsin River. Aconceptual framework of this research is presented inFigure 1. Two questions are being addressed: (1) Whichlandscape metrics are most useful for monitoring pop-ulation, community, and ecosystem processes in largeriver-floodplain landscapes? (2) What are the constraintson extrapolating relationships between landscape metricsand ecological processes in large river-floodplain land-scapes?

Spatially extensive field sampling is being com-bined with landscape indicators to predict ecologicalvariables over broad scales. Field sampling (n = 220plots) was conducted during MayBJuly of 1999 withinsix 10B15 km study reaches. Vegetation (trees, shrubs,saplings, tree seedlings, and herbaceous cover), soils, andlitter accumulation were sampled within each 10 x 20 mplot, and soil cores were obtained to estimate potentialdenitrification. A census of birds was taken twice at eachplot using 8-minute point counts.

Preliminary results suggest that microbiologicalindices can be explained by ecological processes thatoperate at different spatial scales. At the landscape scale,microbial activity appears to be influenced by landcover, whereas the presence of levees may be a moreimportant determinant at the scale of individualtransects. At finer scales, different enzyme activities werefound in soils under different types of trees. Microbialprocesses are important for the ecological integrity offloodplain ecosystems; these results suggest that changesin land use patterns, levee construction or removal, or

tree species can affect these processes. Initial analyses of4,870 bird observations (70 species) revealed similarityamong the bird communities associated with each studyreach, with an average of 70B80 percent of the speciesshared between reaches on a pairwise basis. However,numbers of individual birds were quite variable, with thethree southernmost reaches containing approximately 30percent more birds than the three northernmost reaches(e.g., the number of woodpecker species and individualsdecreased from south to north). S o m e s p e c i e sgenerally associated with northern Wisconsin habitatswere only detected at the southern-most reaches,suggesting a strong influence of land use or river flowmodification. From a total of 39 tree speciesencountered, some floodplain species (e.g., Acer-saccharinum, Fraxinus pennsylvanica) were more abun-dant in the less human-modified southern reaches,whereas some upland species (Populus tremuloides,Quercus velutina) were more abundant in the moremodified northern reaches.

During the coming year, the following activities areplanned: (1) analyzing the field data obtained in sixriver reaches during the 1999 field season (bird censuses,vegetation sampling, denitrification, and microbialactivity) in relation to landscape indicators; (2)interpreting historical and recent aerial photography forstudy reaches in the 1930s, 1960s, and 1990s; (3)conducting the second season of field sampling; and (4)beginning to address Question 2 by predicting andtesting ecological indicators in new study landscapes,determining whether there are thresholds in landscapepattern beyond which ecological processes changequalitatively, and assessing the sensitivity of ecologicalindicators to landscape changes.

31

Figure 1. Conceptual framework for research in the Wisconsin River floodplain landscape showing relationships among societal values,assessment endpoints, ecological processes, and indicators.

32

Characterization of the Ecological Integrity of Commercially Grazed Rangelands Using Remote Sensing-Based Ecological IndicatorsNeil E. West, Robert A. Washington-Allen, and R. Douglas RamseyDepartment of Rangeland Resources, Utah State University, Logan, UT

The purpose of this retrospective study was tocharacterize the ecological integrity of a semi-arid land-scape using satellite remote sensing and Geographic In-formation System (GIS) technologies.

The ecological basis of the research was the tran-sition threshold hypothesis. A transition threshold is de-fined as the boundary in space and time between two se-ral states (e.g., the change of a grassland to woodland).A transition is the process (e.g., climate or grazing in-tensity) that brings about a change in state. An ecolog-ical risk assessment framework was used to determine theassessment endpoints of rangeland degradation as achange in plant growth form composition, a decrease inplant productivity, a reduction in soil quality,accelerated soil erosion, and a change in landscapecomposition and pattern.

Measurement endpoints, which relate to these fiveassessment endpoints, were derived from 27 years(1972B1998) of wet and dry season Landsat satellite im-agery and 2 years of annual scenes (1985 and 1986).Measurement endpoints were the soil-adjustedvegetation index (SAVI), a surrogate for vegetationparameters and soil quality; the soil stability index (SSI),a surrogate for soil erosion; and thematic maps, wherelandscape metrics were used to measure changes inlandscape structure and configuration. The study site was54,000 ha of the sagebrush steppe portion of the 108-year-old mixed grazing operation (wildlife and beefcattle) Deseret Land & Livestock Ranch (DLL) innortheastern Utah.

A GIS database of site biological, physical, and ad-ministrative characteristics, including historical and cur-rent ranch management records, was developed. The datawere analyzed at the waterpoint, paddock, and land-scape scales using graphical timeseries and multiple re-

gression analyses. Sagebrush steppe is a two-phasemosaic of shifting dominance between shrubland andgrassland due to herbivory, fire, and climate change. Itwas hypothesized that at the landscape-level, SAVIwould respond to climate change (i.e., La Niña/El Niño).It also was hypothesized that at the community level,coincident periods of drought and intense grazing wouldlead to a landscape dominated by shrubland andincreased erosion.

Two validation studies were conducted that confir-med that SAVI, an indicator of phenology, was consis-tent with field data and that interannual SAVI can dis-criminate between grazing effects. It was found that atthe landscape-scale, the ranch had become more frag-mented and was an unstable limit cycle responding to ElNiño (wetting) and La Niña (drying), respectively.

At the community-level, shrubland dominatedsince 1974, grassland was declining, and there is noindication of increased erosion. Three-dimensionalsurface analysis suggests that in terms of SAVI response,increased grazing and climate are factors in shrubincrease, but grazing appears to be the main factor ingrass cover decline (see Figure 1).

Ecosystem measures such as SAVI can missfundamental changes in composition that are importantto ranch management. Limited field datasets from federalagencies had assessed the ranch in good condition.However, these datasets were temporally limited (6 yearsversus the 27 years of imagery), inconsistent, or measuredinappropriate indicators. The following tasks remain tobe completed: validation of the SSI, accuracyassessment of dry season thematic maps, comparison ofmetrics within 20 paddocks, and completion of thetimeseries piosphere study.

33

Figure 1. The top left figure suggests a fold catastrophe and is the relationship between the independent factors: number of cows andthe Utah Region 5 Palmer Drought Severity Index (PDSI), and the dependant factor: landscape-level lagged wet seasonmean soil-adjusted vegetation index (SAVI). PDSI < 0 indicates drought conditions. The same independent factors arerelated to community measures of grass cover (top right) and shrub cover (bottom center).

Section 3.

Aquatic Indicators

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Using Bioindicators To Develop a Calibrated Index of Regional Ecological Integrity for Forested Headwater EcosystemsRobert P. Brooks 1, Robert S. Mulvihill 2, Terry Master 3, and Timothy J. O’Connell 11Penn State Cooperative Wetlands Center, University Park, PA; 2Powdermill Nature Reserve, Carnegie Museum ofNatural History, Rector, PA; 3East Stroudsburg University, East Stroudsburg, PA

The objective of this project is to develop a region-al index of ecological integrity for forested headwaters inthe Mid-Atlantic States, with particular emphasis on theLouisiana waterthrush (Seiurus motacilla), referred to as"LOWA." LOWA are forest-breeding warblers that alsoare riparian obligate. They are dependent both on largepatches of mature forest and high-quality instreamconditions. The index is intended to integrate andcalibrate existing indicators of integrity that addressstream health and upland forest condition individually,and at different scales.

Beginning in 1998, LOWA were monitored on 23forested headwater streams in three physiographic pro-vinces of Pennsylvania (see Figure 1). These streamsrepresent either: (1) high water quality in large patchesof interior forest; (2) high water quality in a relativelyfragmented forest setting; or (3) low water quality in largepatches of interior forest. Established indices of bioticintegrity (IBIs) will be applied to the same streams whereLOWA reproductive success already has been measured.The degree to which attributes of LOWA reproductivesuccess can link small scale IBIs, such asmacroinvertebrate IBIs, to landscape scale IBIs (i.e., theBird Community Index) is being investigated.

Early analysis confirms that LOWA populationvariables correspond with large-scale condition as de-fined by the Bird Community Index. Correlates to in-stream condition are more complex to generate, and the

results are pending. For example, the 1998 macroin-vertebrate characterization for the Central study area in-cluded 9,862 individuals in 180 genera, 83 families, 18orders, 9 classes, and 4 phyla.

In 1999, unusually high nest predation rates in allthree study areas occurred. Nest predation rates wereelevated on both reference and impacted streams. In theCentral study area, the evidence suggests that mink(Mustela vison) are the culprit predators. It is speculat-ed that mink have become locally (and perhaps region-ally) abundant following a succession of mild winters.

Preliminary results of this research already havedisplayed the importance of "offstream" wetland seepsand springs as vital to the foraging efficiency of LOWAnesting on acidified streams. On acidified streams withabundant offstream foraging opportunities, LOWA arereproducing at a rate comparable to that observed in themost "pristine" streams.

This work also highlights the need to conduct re-search at large scales and across physiographic bound-aries. For example, had all of the sites been located inone region in 1999, it would not be possible to demon-strate that the elevated nest predation observed was awidespread phenomenon. The 2000 field season is thethird and final season on this project. In the upcomingyear, the IBI will be developed, and its relationship to theland cover pattern and the habitat attributes that havebeen measured from the ground will be examined.

Figure 1. Mean habitat suitability index (HSI) scores for Louisiana Waterthrush on reference and degraded streams in three physiographicprovinces of Pennsylvania.

38

Stream Plethodontid Assemblage Response (SPAR) Index: Development, Application, and Verification in the MAHARobert P. Brooks and Gian L. RoccoPenn State Cooperative Wetlands Center, Environmental Resources Research Institute, Pennsylvania StateUniversity, University Park, PA

Amphibians generally are considered to be valu-able response indicators (Vitt et al. 1991, Dunson et al.1992). In small lotic systems that largely exclude pre-dacious fish, small, lungless salamanders of the FamilyPlethodontidae, can exist in surprisingly high numbers,functioning as both invertebrate predator and vertebrateprey.

By virtue of their diverse and complex life his-tories and abundant and stable populations, stream sala-manders offer the opportunity of serving as importantecological indicators for the assessment of headwaters,especially where traditional indicator species assem-blages (fish, macroinvertebrates) may be poorly devel-oped or absent.

In a study of 14 Central Appalachians headwatersin Pennsylvania, Rocco and Brooks (in preparation)document significant stream plethodontid responses toacidified and degraded stream conditions, attesting totheir potential as bioindicators. Various useful metrics,based on species composition, abundance, and lifestage,also are proposed. In consideration of the effectiveness,relative ease, and low cost of the sampling technique,stream plethodontid assemblage responses (SPAR), usedalone or in combination with other small stream assess-ment criteria, promises to be an effective assessment andmonitoring tool for headwaters.

At the moment, however, the ability to interpretSPAR in similarly impaired streams across a largergeographical area is hampered by the relatively smallarea sampled. A reference base ideally would be geo-graphically widespread, represent the entire range ofconditions, and consist of a representative and unbiasedsample of the population of headwaters in the region ofinterest.

To address this need, the primary objectives of thisresearch are to: (1) describe the range and variability ofSPAR across commonly encountered gradients of anthro-pogenic degradation (e.g., stream acidification, forest andriparian corridor fragmentation and degradation,pollution) in the Mid-Atlantic Highlands Area (MAHA);(2) develop and adjust SPAR for use in MAHA head-

waters; and (3) evaluate the reliability and resolution ofSPAR by application and verification.

The development, application, and verification ofSPAR within MAHA will entail a two-phase process.Phase I aims to document and study plethodontid assem-blage responses to as many different stream conditions aspossible, across a large geographical area. This data- setwill enable the development of the SPAR index for theMAHA. In Phase II, a random set of headwaters ofunknown condition will be sampled and its impairmentdetermined by the SPAR index. Once applied, an inde-pendent assessment of the stream by traditional methods(macroinvertebrates, water quality, surrounding land-scape) will allow the verification of the reliability andconsistency of the index, and if necessary, its cal-ibration. The use of volunteers during the second phasewill permit evaluation of the method for nonspecialistsand long-term amphibian monitoring efforts.

The response composition of free-ranging, nat-urally occurring stream salamander populations in de-graded and nondegraded watersheds will be studied byintensive sampling. Relevant abiotic and biotic var-iables at the plot, stream reach, and watershed will bemeasured and correlated to SPAR metrics.

The expected findings include: (1) description ofstream salamander assemblage response along disturb-ance and pollution gradients commonly encountered inthe MAHA Region; (2) identification of species mostresponsive to the environmental degradation investi-gated; (3) recommendations on the use of stream sala-mander assemblages in the assessment of headwaters; and(4) development and testing of stream salamander metricsthat could be implemented separately or in con- junctionwith other criteria when performing small streamassessments.

Ultimately, it is hoped that this work leads to theimprovement of existing headwater assessment protocolsfor the MAHA by the addition of a bioindicator that isabundant, widespread, ecologically important to severaltrophic levels, and interfaces between aquatic andterrestrial components of riparian areas.

39

Soil Enzyme Stability as an Ecosystem IndicatorRichard P. DickDepartment of Soil Science, Oregon State University, Corvallis, OR

Sustainability assessment of ecosystems to assistland managers and policymakers in promoting long-termsustainability is a national priority, but quantifying envi-ronmental sustainability remains an elusive goal. Oneapproach is to use the soil as an indicator of ecosystem"health."

Soil enzyme activity assays are advantageous aspotential indicators because they are: (1) operationallypractical; (2) sensitive integrative "biological finger-prints" of past soil management; and (3) apparently re-lated to soil aggregation, linking enzymes with soil tilth.

In the first year of the project, the initial screeningof three promising soil enzyme assays as indicators tookplace at three experimental sites in Oregon (located indivergent forest and agroecosystems) where detailedmanagement history is known and soils vary widely insoil "health" (i.e., biological activity and organic mattercontent) because of past soil management. This includedrefinement of sample handling and laboratory protocolsto increase sensitivity and reproducibility of the indica-tors. Samples were taken in early May, June, and Sep-tember of 1999 to assess the seasonal temporal var-iability of these indicators. It was found that the forest

soils generally have much higher activities than agri-cultural soils. This makes sense, because agriculturalsoils are regularly disturbed. Forest soils, even withrecent logging, would only have had surface disturbanceonce, and even then, there would not be tillage. There isa very consistent pattern in the agricultural soils whenwithin-site treatment effects are examined, in that thosereceiving greater C inputs (as cover cropping, manure,and green manure) have higher activities than the plotsreceiving lower C inputs.

Across agricultural soils, the biological measure-ments such as ergosterol, fungi biomass, and biomass Care very consistent with soil enzyme activities. Bacterialcounts, however, show very few significant differences asa function of soil management in either forest or agri-cultural soils. This suggests that soil enzyme activitiesare good indicators of the soil biological component andare reflecting the differences due to fungal biomass.

The second year of the project will involve expan-ding to a wider range of soil types and soil managementsystems. A detailed data analysis will be performed andthe potential for relative soil quality indicators that areindependent of soil type will be investigated.

40

Integrative Indicators of Ecosystem Condition and Stress Across Multiple Trophic Levels in the San Francisco EstuaryRichard C. DugdaleRomberg Tiburon Center for Environmental Studies, San Francisco State University, Tiburon, CA

The goal of this project is to evaluate a set of po-tential indicators of ecosystem condition for the SanFrancisco Estuary (SFE). The objectives of the researchand field effort are to: (1) devise indicators of ecosys-tem condition; (2) investigate their relationships tostressors, including salinity, turbidity, nutrients, and in-troduced species; and (3) assess the utility of these in-dicators.

This research is being conducted on the open-waterecosystem of the SFE, including the portion of thelandscape from freshwater to marine ecosystems. Thesepotential indicators represent key population andindividual-level processes in a variety of trophic levels.The benthic community structure also is being examinedfor possible indicators of stress. All indicators arerelatively simple to measure, significant to populationecology, expected to be sensitive to stress levels, andlikely to be transportable to other aquatic habitats.

These indicators are: (1) nutrient status and pro-ductivity performance of phytoplankton; (2) relativecon- tribution of diatoms to algal biomass andproductivity; (3) reproductive rate of common copepod(zooplankton) species; (4) condition indices of larvalherring; (5) condition indices of the dominant benthicbivalves; (6) several measures of benthic communitystructure; and(7) expression of stress proteins in the indicator animalspecies.

This project is very new, funded in October 1999,so only preliminary findings are available. The fieldwork has been initiated with monthly cruises to threesites in the SFE. Measurements of temperature, salinity,nutrients, and algal biomass as chlorophyll, particulatenitrogen, and particulate carbon were made. NO3 andNH4 uptake (fractionated by size class) with samples

incubated under natural turbidity and clarified waterconditions were obtained during each cruise.

Comparison nutrients and chlorophyll concen-trations from December 1999 with previous obser-vations in Central Bay suggest relatively low values,probably resulting from an exceptionally dry fall.Comparison of nutrient and chlorophyll concentrationsfrom December 1999 in Central Bay with the two newup-estuary stations, San Pablo and Suisun Bays, showsnutrients, especially Si(OH)4, increasing and chlorophylldecreasing in the freshwater direction. Copepod (zoo-plankton) reproductive rates (see Figure 1) weremeasured by collecting with gentle net tows, diluting thecatch in surface bay water, and incubating individualfemales for 24 hours in 125 mL polycarbonate bottlesfilled with bay water with eggs strained out.

The first herring larval collections have just beenmade at the three sampling stations. The contents ofeach tow were split, with half preserved in formalin andhalf in ethanol for measurements of body characteristicsincluding length, weight, head width, and total eye diam-eter.

The ethanol-preserved samples are stored for fu-ture studies outside the scope of this project. Sites havebeen selected for the benthos project at each of the threestudy areas and collections made for condition andglycogen analyses. Work on stress-induced proteins hasbeen impacted by the departure of the post-doc involved,who has taken a faculty position at another institution.Planning for the prosecution of this aspect of the projectis underway with the possibility of arrangements forcollections to be made at our institution and the analysesat the individual’s new post. Other options are beingexplored as well.

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Figure 1. Histograms of egg production rates from copepods (Acartia) at Stations 2 (top) and 3 (bottom) during the first cruise. Adifferent species, not shown here, was most abundant at Station 1. Vertical lines indicate medians. Differences in eggproduction rates such as these will be analyzed for relationships with salinity, temperature, chlorophyll, and otherenvironmental variables.

42

Developing an Indicator for Nutrient Supply in Tropical and Temperate Estuaries, Bays, and Coastal Waters Using the Tissue Nitrogen and Phosphorus Content of MacroalgaePeggy FongDepartment of Organismic Biology, Ecology, and Evolution, University of California, Los Angeles, CA

Coastal eutrophication that results from increases innutrient supply is a critical problem worldwide, causingmajor changes in marine populations and communities.The overall goal of this project is to develop an indicatorthat quantifies nutrient supply to tropical and temperatemarine ecosystems using the tissue nitrogen (N) andphosphorus (P) content of macroalgae. To com- plementthe present suite of indicators used to measureeutrophication, this indicator is targeted to be especiallyuseful in systems where nutrients are supplied in pulsesor those where nonpoint sources of nutrients such asgroundwater or fluxes from the benthos are important.

The five specific objectives are to: (1) continue toidentify and test potential species for use as indicators;(2) establish relationships between timing and magnitudeof nutrient supply and accumulation of N and P in algaltissue; (3) establish quantitative relationships betweenenvironmental conditions, N and P supply, and tissue Nand P; (4) develop a numerical simulation model basedon experimental results that may be used as a "standardcurve" for the indicator to hindcast nutrient supply in thefield; and (5) field test the indicator.

Macroalgae from two regions in the Caribbean(Southwest Puerto Rico and Caribbean Panamá) and twoin the Eastern Tropical Pacific (Gulfs of Panamá andChiriquí, Panamá) were collected to determine if thenitrogen (N) and phosphorus (P) content of their tissuesreflected differences in nutrient availability in these sys-

tems, making them good indicator species. Large dif-ferences were found in the tissue P content of Hypneamusciformis, an exotic red alga that has invaded mosttropical areas. H. musciformis from the Caribbean hadone-half the amount of P than in the ETP. There alsowere significant differences in N content, but these wereof a lower magnitude. Similarly, Dictyota spp. in theCarribean had only one-half the tissue P content found inthe ETP; however, tissue N of Dictyota spp. was notdifferent among these ocean areas, suggest ting it maynot be useful as an indicator of P (see Figure 1).

The differences in P content in algal tissue betweenthe Caribbean and ETP may reflect the greater per-centage of P-adsorbing carbonates in Caribbean sed-iments. Tissue P content also was significantly differentamong regions: Gulf of Panamá > Gulf of Chiriquí >Caribbean Panamá > Puerto Rico. This contrasts withtissue N where the pattern was Gulf of Panamá > Gulf ofChiriquí = Puerto Rico > Caribbean Panamá. Whilegreater N and P in the Gulf of Panamá may be attributedto seasonal upwelling, the larger N content in PuertoRico compared to Caribbean Panamá may be due toanthropogenic inputs.

The tissue N and P content of macroalgae mayprovide useful insight into nutrient availability in tropi-cal systems. Furthermore, cosmopolitan species such asH. musciformis may be especially good indicators ofnutrient regime.

43

Figure 1. Differe n c e s w e r efound between Hypnea musciformis and Dictyota spp. sampled from the Eastern Tropical Pacific (ETP) and the Caribbean interms of: a) tissue N, b) tissue P, and c) N to P ratio.

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Microbial Indicators of Biological Integrity and Nutrient Stress for Aquatic SystemsJames P. Grover and Thomas H. ChrzanowskiDepartment of Biology, University of Texas at Arlington, Arlington, TX

This project explores connections between bio-logical integrity and nutrient supply and limitation, fo-cusing on the microbial component of lakes and reser-voirs. Several hypotheses are being tested concerningpatterns and correlations among indicators of biologicalintegrity that are based on nutrients (i.e., carbon,nitrogen, phosphorus, and trace minerals) and microbialcommunities. The temporal and interregional variabilityof these indicators also are being assessed.

Two reservoirs in north Texas are being examinedyear-round, and two lakes in the Experimental LakesArea (Ontario, Canada) are being studied during the ice-free growing season (see Figure 1). One of the latter lakesis pristine, while the other has been experimentallyeutrophied with phosphorus additions. Much of thiswork involves measures of seston element compositionand measures of changes in microbial communities fol-lowing experimental manipulation of nutrients (bio-assays).

Seasonal patterns have been analyzed and compar-isons made among lakes for the first year of sampling. Itwas hypothesized that indicators based on the C:N:Pcomposition of seston would agree with growth re-sponses in bioassay experiments.

In Texas lakes, temporal correlations betweenindicators of P-limitation and seston C:P and N:P ratioswere all in the hypothesized directions. Correlationsbetween indicators of N-limitation often were opposite topredictions. Indicators of algal and bacterial nutrientlimitation were positively correlated in Texas lakes, ashypothesized. Bioassay experiments in Canadian lakeswere generally uninformative, largely due to high var-iability and a number of unexpected negative effectsfrom nutrient enrichment.

Physiological profiles of bacterial communitiesbased on the use of 95 carbon substrates support ahypothesis that bacterial community structure shifts inresponse to seasonal changes in nutrient limitation.Principal components analyses reveal relatively highresponse to amino and carboxylic acids in cool seasons,and to carbohydrates in warm seasons. The amplitude ofthis pattern was stronger in the experimentally eutro-phied Canadian lake than in the oligotrophic referencelake.

These results address the relative utility of bioassayexperiments and measures of seston C:N:P compositionas indicators of nutrient limitation. The two approachesperform more consistently when P-limitation is diag-nosed than when N-limitation is diagnosed. Indicatorsbased on seston C:N:P ratios appear to be more widelyapplicable than those based on bioassay experiments,because the latter performed poorly in Canadian lakes.On the other hand, where applicable (in Texas lakes),bioassay experiments yield insights to microbial pop-ulations that may be useful in modeling and lake man-agement. Physiological profiling of bacteria suggestsseasonal shifts in use of carbon substrates, which may belinked to seasonal changes known for other limneticorganisms. These seasonal patterns appear to be morepronounced in eutrophic than in oligotrophic lakes, andmay help characterize lakes of different trophic status.

As the additional data from Years 2 and 3 areanalyzed, interannual variations in the indicators andthe patterns of correlation summarized above will be as-sessed. To obtain greater insight from indicators basedon seston composition, size-fractionated sampling ofseston in Canada during 1999 was instituted, thoughdata are not yet completely analyzed.

45

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Figure 1. Microbial indicators of biological integrity and nutrient stress for aquatic systems.

46

Foraminifera as Ecosystem Indicators: Phase 1. A Marine Benthic Perturbation Index; Phase 2. Bioassay ProtocolsPamela Hallock, Heidi Crevison, Thomas Dix, Helen Talge, and Dana Williams

Department of Marine Science, University of South Florida, St. Petersburg, FL

The objectives of this project are to develop pro-cedures for the routine use of foraminifera as indicatorsof biological integrity in field and laboratory appli-cations. In Phase 1, sediment cores are being used to testan index for assessing perturbations of marine benthicecosystems based on changes in key taxa of fora-minifera. In Phase 2, protocols for assessing stress re-sponses in foraminifera induced by elevated temperatureand visible and ultraviolet radiation are beingdeveloped.

A prototype index to quantify change in benthicecosystems that can be applied to historical, sediment-core, and surface-sediment datasets has been developed.The model ranks relative abundances of key foramin-iferal taxa and morphogroups, and total abundances,generating an index value when foraminiferal assem-blages are compared temporally or spatially. Tests of theindex using sediment cores from Florida Keys reefs andTampa Bay are in progress.

Bleaching has been observed in populations ofAmphistegina in all subtropical oceans since 1991. Fieldsampling in the Florida Keys in 1997B1999 revealedcontinued stress, with a significant increase in the sea-sonal bleaching peak in 1998. Stress symptoms in Am-phistegina can be documented using visual, physio-logical, and cytological techniques. Visual rankings,which can be verified by cytological analyses using elec-tron microscopy and image analysis, remain the mostcost-effective method of documenting stress responses.Protocols for measuring ATP to determine viability andmetabolic activities were adapted and results comparedwith visual and cytological assessments. Highvariability and standardization limitations indicate thatATP analysis is not cost-effective.

Exposure to elevated intensities of photosynthet-ically active radiation (400B700 nm), alone or in combi-nation with added UVB (280B320 nm), induced symbi-

ont loss in A. gibbosa similar to that seen in field pop-ulations. Comparison of responses to two spectrally dif-ferent visible light sources revealed that, although ex-perimental intensities of blue and white light were thesame in photons (7.76 µE/m2s), the higher-energy bluelight (7.21 x 10-5 W/m2) both induced faster growth andmore symbiont loss than the lower-energy white light(5.69 x 10-5 W/m2) (see Figure 1).

Mass bleaching devastated corals nearly worldwidein 1998. Bleaching in A. gibbosa in the Florida Keysalso peaked in the summer of 1998. A key differencebetween bleaching in corals and foraminifers is that coralbleaching correlates most consistently with elevated seasurface temperatures, which can induce photoinhibitorystress, while bleaching in Amphistegina appears to bedirectly associated with photoinhibitory stress. Otherresearchers using molecular biomarkers have found evi-dence of stress in corals 3 months prior to visible bleach-ing, indicating that corals may be responding to the samestresses that are bleaching the foraminifera. Recog-nizing similarities and differences between these twophysiologically similar, though taxonomically differentsymbiotic systems, should facilitate understanding theglobal decline of coral reefs. Visual techniques remainthe most dependable and cost-effective methods of doc-umenting and analyzing bleaching symptoms and shellbreakage in Amphistegina spp.

Goals for the year 2000 are to standardize visualprotocols using digital photography and image analysis,while continuing to document responses to photic andtemperature stress. Contacts also have been establishedwith scientists applying molecular biomarker protocolsto a variety of reef organisms, with the goal of deter-mining whether these techniques can be applied to Am-phistegina. The results of the core analyses and stess ex-periments will be prepared for publication.

47

Figure 1. Influence of spectral quality on growth and symbiont loss. Mean diameter of 45 A. gibbosa individuals exposed to twointensities (7.76 and 2.81 µE/m2) of two spectrally different light sources (blue lamp-photons are concentrated in the higherenergy portion of the electromagnetic spectrum, white lamp-photons are more concentrated in the lower energy portion). Shaded boxes show the percentages of individuals showing visible symbiont loss after 4 weeks (grown in 10 cm petri dishesat 25o C in nutrient-enriched Erdschreiber medium).

48

Biogeochemical Indicators of Watershed Integrity and Wetland EutrophicationK. Ramesh Reddy 1, W.F. DeBusk 1, A. Ogram 1, W. Graham 1, M.M. Fisher 2, E.F. Lowe 2, and L.W. Keenan 21Soil and Water Science Department, Agricultural and Biological Engineering Department, University of Florida,Gainsville, FL; 2St. Johns River Water Management District, Palatka, FL

The central hypothesis of this research is that ratesof biogeochemical cycling of C, N, and P in wetlands canbe used to indicate the ecological integrity of wetlands,and that the concentrations of certain forms of theseelements can accurately predict the rates of ecolog- icallyimportant processes.

The objectives of this research are to: (1) identifythe key biogeochemical processes impacted by nutrientloading and measure the rates of these processes alongthe nutrient gradient; (2) develop relationships betweena "process" and its related, easily measurable "indica-tor"; (3) determine the spatial and temporal distributionof easily measurable indicators for a test wetland ecosys-tem; (4) determine the spatial variations in biogeochem-ical processes, and develop spatial maps for variousprocesses to determine the extent of impact and risk as-sessment; and (5) validate the predictability of empiricalrelationships by making independent measurements ofbiogeochemical processes in different wetland ecosys-tems. It is planned to test the hypotheses presentedabove in the Blue Cypress Marsh Conservation Area(BCMCA), located within Upper St. Johns River Basin,FL. Some areas of the BCMCA have been impacted overthe years as a result of nutrient loading from adja- centuplands, resulting in distinct nutrient and vegetationgradients. The BCMCA provides the benefit of estab-lished gradients of high-nutrient (impacted) to low-nutrient systems (unimpacted), to test the hypotheses ofthis proposal.

The research will focus on key biogeochemicalprocesses and microbial communities regulating the fateof nutrients in the soil and overlying litter layer at var-ious spatial and temporal scales. This will be accom-plished by conducting a series of laboratory and field ex-periments.

Statistical and modeling tools will be used foranalysis and synthesis of the biogeochemical process andindicator data, including: (1) descriptive statistics re-lated to frequency distribution and central tendency ofindicator values to make comparisons between areas oflow and high anthropogenic impact; (2) multivariateanalyses to evaluate the relationship between biogeo-chemical indicators, biogeochemical processes, and eco-logical integrity; (3) geostatistical analysis to comparethe characteristic spatial patterns and structure of biogeo-chemical indicators; and (4) temporal analysis of in-dicators in low- and high-impact areas.

The first stage of this research is expected to resultin the confirmation of the utility of simple chemicalmeasures as reliable predictors of rates of biogeochem-ical processes. The study also will produce statisticalmodels describing these relationships.

Such models would provide valuable research andmanagement tools. Because these measures would berapid and inexpensive, investigations of biogeochemicalprocesses could be expanded providing a better under-standing of these fundamental mechanisms of eco-systems.

49

I n c r e a s e d e n e r g y I n c r e a s e d e n e r g y èèèè d e s t a b i l i z e dd e s t a b i l i z e d

c h a n n e l , d e g r a d e d h a b i t a t ( Q H E I )c h a n n e l , d e g r a d e d h a b i t a t ( Q H E I )

U r b a n i z a t i o n l e a d s t o U r b a n i z a t i o n l e a d s t o

ii n c r e a s e d i m p e r v i o u s n e s sn c r e a s e d i m p e r v i o u s n e s s

C h a n n e l i n c i s i o nC h a n n e l i n c i s i o n C h a n n e l w i d e n i n gC h a n n e l w i d e n i n g

I n c r e a s e d r u n o f f v o l u m e a n d f r e q u e n c yI n c r e a s e d r u n o f f v o l u m e a n d f r e q u e n c y

I n c r e a s e d c h a n n e l s c o u rI n c r e a s e d c h a n n e l s c o u r

Development and Evaluation of Ecosystem Indicators for Urbanizing Midwestern WatershedsAnne Spacie, Jonathan M. Harbor, Midhat Hondzo, and Bernard A. EngelPurdue University, West Lafayette, IN

Urbanization is thought to have negative impactson stream ecosystems, and yet the actual causal relation-ships between land use change and stream communityresponse have not been well studied. This project focus-es on the development of predictive indicators of urban-ization that are applicable to Midwestern watersheds andstream ecosystems.

The objectives of this project are to: (1) quantifythe impacts of urbanization on hydrologic regimes, waterquality, and habitat structure of stream ecosystems usingpaired experimental watersheds, and to develop linkedmodels that accurately predict these impacts; (2) use thelinked models as a virtual laboratory within which togenerate and test indicators of urbanization and hydrol-ogic change in terms of responses of fish and macro-invertebrate communities; and (3) use these models andindicators to assess the response of stream communitiesto alternative urbanization scenarios with extension tolarger watersheds in the region.

The research examines seven watersheds in centralIndiana that are in transition from rural to urban. Link-ages between increased urban runoff, altered channelmorphology, water quality effects, and reduced bioticintegrity are being evaluated for three sites on eachstream. Percent urbanization of the watershed, derivedfrom 1997 SPOT satellite imagery, is used in a runoffmodel (L-THIA) to predict flow regime at each site.Intensive water quality sampling at selected sites pro-vides a dataset for the development and testing of aphysically-based water quality model. Stream cross-section measurements are used to determine the criticaldischarge (Qcrit) required to mobilize substrate.

Statistics describing the long-term exceedance ofQcrit characterize the level of habitat disturbance at thesite. Physical habitat at each site has been measuredusing standard procedures, including a qualitativehabitat evaluation index (QHEI). Macroinvertebrate,periphy- ton, and fish collections at each site are used tocharacterize stream community structure.

A dynamic hydrology model has been developedthat can simulate cross-sectional averaged velocities,shear stress velocities, and water depth variability duringstorm peaks. Water temperature dynamics and nutrienttransport also have been modeled with satisfactory re-sults. Stream habitat measurements (QHEI) and bio-logical collections have shown significant differencesacross the range of urbanization.

A suite of functional biological metrics currentlyare being evaluated. This work will provide a soundbasis for the use of specific indicators as tools in re-gional planning of watershed development. The riskanalysis portion of the work will provide a probabilisticmeasure as to whether a potential urbanization scenariocan achieve stream water quality and biological targets.The hydrologic and water quality models developed thusfar will be extended for use with dissolved oxygen andnutrient transport, and will be linked to the L-THIA run-off model. Stream biota and habitat quality, which al-ready have been correlated to general land use (seeFigures 1B3), will be further correlated to channel mor-phometry and flow variability.

Once this is accomplished, a risk analysis can beconducted on the effects of various runoff patterns on thestream community metrics.

Figure 1. A conceptual model of stream channel changes associated with urban watersheds.

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Figure 2. Hydrographs in urban streams are expected to show increased mean flow as well as increased frequency of storm peak flow.

Figure 3. Significant degredation of fish habitat quality, as measured by QHEI, has been observed in streams undergoing urbanizationin the region near Indianapolis, IN.

51

An Integrative Aquatic Ecosystem IndicatorRichard S. Stemberger and Eric K. MillerDepartment of Biological Sciences and Environmental Studies Program, Dartmouth College, Hanover, NH

This project uses measures of the relative supply ofcarbon (C), nitrogen (N), and phosphorus (P) to inte-grate watershed, lake, and pelagic zooplankton assem-blages in a multitier ecological indicator for monitoringlake integrity. Complex species assemblages are aggre-gated into simple consumer guilds that reflect relativeelemental N and P intracellular requirements of species.Element supply ratios relate a variety of potential risks tolake ecosystem function such as loss of the cool-waterrefuge, bioaccumulation of toxins, algal turbidity, acidi-fication, and UV-B toxicity. These risks are associatedwith zooplankton assemblage structure along the N:Pratio gradient.

Objectives of the project include the following:1. Evaluate metrics based on the C, N, and P sup-

ply of the total dissolved plus seston fraction of lakewater as indicators of the character of lake zooplanktonassemblage.

2. Establish the strength of the linkage betweenthe relative C, N, and P supply from the watershed to theC, N, and P supply to the lake. The hypothesis that N:P,C:N, and C:P ratios in lake water and watershed C, N, andP supply to lakes are significantly influenced bylandscape factors related to human land use activity,forest ecosystem composition, and regional air pollutiongradients will be tested (see Figure 1 for conceptualmodel).

3. Conduct a landscape characterization analysisof features in the lake-watershed basins which, in con-junction with concurrent watershed-associated streamand groundwater measurements, will be used to developproxy measures for expected relative C, N, and P supplyconditions for lake water.

4. Establish zooplankton assemblage-derived var-iables that reflect risks to ecosystem function, structure,or human health as a function of the elemental sup- plygradient.

5. Conduct a sensitivity analysis of the zooplank-ton metrics using existing and extensive temporal andspatial datasets. These analyses will establish statisticalconfidence and power to detect change for measures oflake integrity.

In the first field season, a total of 36 visits to 26

lakes in the Adirondack Mountain and Saint LawrenceValley regions of New York and Vermont were con-ducted. Fifteen of 75 selected tributary riparian zonelocations were sampled for field characterizations thatincluded vegetation survey, leaf-litter sampling, and soilcharacterization and sampling.

The foundation data layers (topographic factors,land cover/land use, soils, hydrography, climate, andatmospheric deposition) of a regional GIS that will beused for the characterization of the study watershedshave been acquired, converted to grid data, reprojected,and resampled as necessary. The generation of riparianzone overlays is in progress.

The EMAP dataset was analyzed for sensitivity ofvarious zooplankton metrics to distinguish differencesamong lakes on both regionwide and subregional scales.Sensitivity of metrices increased in one or more sub-regions relative to regionwide estimates for all metricstested. Richness and population metrics of calanoidcopepods and large cladocerans had 80B90 percent oftheir variance associated with the lake component rel-ative to variance from temporal and interaction sources.Metric performance improved at spatial scales, reflectingunderlying historical biogeographic associations.

Patterns in the structure of zooplankton assem-lages of the EMAP lakes in relation to effects of pre-dators and nutrient treatments in experimental meso-cosms also were evaluated. Fish predation reduced thebody sizes of comprising taxa in the mesocosms, but Nand P addition determined whether the assemblages werecalanoid (high N:P ratio), or cladoceran and rotifer (lowN:P) dominant. Nutrient effects were immediate andsubstantial.

The factors tested in the experiments are identicalto those identified in principal component analysis(PCA) of natural lakesCtotal N, total P, N:P ratio, andyoung fish. The results from these independent butmutually supporting studies greatly strengthens theunderlying importance of nutrient ratios in controllingstructure of zooplankton assemblages and furtherstrengthens the foundations for nutrient-basedzooplankton indicators.

52

Figure 1. Plot of N and P space showing how the zooplankton-NP ratio indicator is interpreted within different classes of terrestrialwatershed vegetation cover and land use. Open circles indicate lakes having Chl a values > 12 µg/L . These systems areassociated with P values > than 1 µmole/L and reflect riparian, urban, or agricultural P pollution. Dashed line in upperright panel delimits the N, P space (50 µmoles L-1 of N and 1 mole/L P) that characterizes the majority of Northeasternlakes. Broken vertical lines in lower panel delimit lake trophic state based on total-P criteria: O=oligotrophic lakes (<10µg/L total P), M=mesotrophic lakes (>10 < 30 µg/L total P), and E=eutrophic lakes (> 30 µg/L total P). Note allzooplankton groups may occur within each trophic state category and that total biomass will increase with increasing P.

53

Effects of Interacting Stressors in Agricultural Ecosystems: Mesocosm and Field Evaluation of Multilevel Indicators of Wetland ResponsesStephen T. Threlkeld Department of Biology, University of Mississippi, University, MS

The primary objective is to evaluate indicators ofmolecular, cellular, population, community, and ecosys-tem responses to multiple, potentially interacting, naturaland anthropogenic stressors at different spatial and tem-poral scales in agricultural wetlands.

The indicators are chosen to represent a selection ofmechanism-based and system-level integrative char-acteristics that might be amenable to cost-effective rou-tine monitoring. The null hypothesis is that indicatorsthat effectively characterize ecosystem responses to sin-gle stressors are also scale- and interaction-independent(i.e., useful even when there are multiple, interactingstressors with diverse operational scales). In the pro-ject’s first year, attention was focused on the devel-opment of a Geographic Information Systems (GIS) data-base for the watershed of the Little Tallahatchie River innorthern Mississippi. Physical features, agricultural landuse practices, and potential pesticide loads in the basinhave been characterized.

These datasets were incorporated into the GIS data-base that will be used to randomly select approximately150 field sites from about 18,000 potential samplingareas (ponds, oxbow lakes, impoundments, and inter-mittent stream segments) that have been identified.Physical features have been mapped from 1:100,000 and1:24,000 from U.S. Geological Survey data. Features notvisible on 1:100,000 data (already in digital form) weredigitized from 1:24,000 maps (e.g., intermittent streams,small ponds).

Land use practices have been characterized fromdata obtained through county Farm Services Agency of-

fices that maintain separate records for each farm in theirjurisdiction. For each square mile of the study area, cropdata has been gathered for all major and minor cropsplanted in 1998.

Potential pesticide loads were calculated for thefollowing chemicals: chlorpyrifos, atrazine, methyl para-thion, and monosodium methane arsonateCwhich com-prise the majority of pesticides applied to major crops inthe region (soybeans, cotton, corn, broadcast grains)Cbymultiplying the chemical application rate (amount ofchemical per acre) by the number of acres in a squaremile planted in a crop for which a particular chemical isused.

Application rates were obtained from resourceinformation at the Lafayette County (Mississippi) Ex-tension Office. This information incorporates data fromcompanies that produce the chemicals as well as datapertinent to Mississippi that may affect application (e.g.,weather patterns, soil types). From these chemical dis-tributions, watershed areas have been identified that aremost likely to have wetlands with agrichemical load-ings typical of the single or multiple stressor treatmentsplanned for the mesocosm experiment (see Figure 1),which is to commence in May 2000.

Many of the chemical combinations to be used inthe mesocosm experiment (even the four chemical com-binations) are represented in the GIS by multiple sectionswith numerous ponds that can be used for field eval-uation. In the first year, the analytical and sampling pro-tocols to be used in the upcoming mesocosm experimenthave been further developed.

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