The Auk 116(3):781-791, 1999
HARLEQUIN
DUCK RECOVERY FROM THE EXXON VALDEZ OIL
SPILL: A POPULATION
GENETICS
PERSPECTIVE
RICHARD LANCTOT,TMBUDDY GOATCHER,2'5KIM SCRIBNER• L6SANDY TALBOT? BARBARAPIERSON,• DANIEL ESLER,• AND DENNY ZWIEFELHOFER 3 •AlaskaBiological Science Center,U.S.Geological Survey,1011 EastTudorRoad,Anchorage, Alaska99503,USA; 2Katmai NationalParkandPreserve, KodiakCoastalUnit Office,202 CenterAvenue #201, Kodiak, Alaska 99615, USA; and
3Kodiak NationalWildlifeRefuge, 1390BuskinRiverRoad,Kodiak,Alaska99615,USA
ABSTR^CT.--Concerns aboutHarlequinDuck (Histrionicus histrionicus) populationrecovery followingthe ExxonValdez oil spill led biologiststo askwhetherbirdslocatedin different molting and wintering areasbelongto geneticallydistinctand, thus, demographicallyindependentpopulations.Owing to the lack of directobservations of movementsamongmarine areas,three classesof geneticmarkersthat differed in modeof inheritancewereused
to evaluatethe degreeof geneticdifferentiation amongwinteringareaswithin PrinceWilliam Sound(PWS)and the AlaskaPeninsulaand KodiakArchipelago(APKA).We couldnot rejectthenull hypothesisthat the winteringaggregations within eachregionare composed of a singlegeneticallypanmicticpopulation.Differencesin genotypefrequenciesamong winteringlocations withinPWSandAPKAwerelow andnonsignificant for all threeclasses of markers.Furthermore,we sawno evidencefor deviationsin Hardy-Weinbergequilibrium or gameticdisequilibriumbetweenlociwithin a winter collectionsiteaswouldbe expected if theselocaleswere composedof individuals from reproductivelyisolated(and genetically distinct)breedinglocales.Finally,no evidencefor significantstructuringwasnotedbetween PWS and APKA. Lackof spatialgeneticstructuringcouldbe due to the cumulativeeffects of low levelsof geneflow overlong time periods,low levelsof geneflow by immaturebirds
movingbetweenmarinehabitats,or to episodicdispersalcausedby habitatalteration(e.g. volcaniceruptions).HarlequinDucksarelikelyto recolonizeor enhancepopulationsin areas recoveringfrom environmentaldamage via emigration of birds from non-affectedareas.
Demographic studiessuggest,however,that levelsof movements arelow, and that populationrecoveryby emigrationis a long-termprocess.Received 11 May 1998,accepted 14January 1999.
ON 24 MARCH 1989, the T/V Exxon Valdezran
aground on Bligh Reef in Prince William Sound,Alaska, spilling approximately41 mil-
vironment
of Prince
William
Sound
received
about40%of the oil thatwasspilled(Galtet al. 1991). The effectsof this oil spill on resident
fish and wildlife havebeen dramatic (Piatt et al. Piatt et al. 1990). Subsequentwind and ocean 1990,ECI 1991)and the subjectof extensiveincurrents spread the oil southwestthrough vestigations. Harlequin Ducks (Histrionicushistrionicus) PrinceWilliam Sound(PWS),along the Kenai lion liters of crude oil (Piatt and Lensink 1989,
and Alaskapeninsulas,and alongthe Kodiak are year-roundinhabitantsof nearshoreenviArchipelago. Muchof the oil wasdeposited in ronmentswithin the oil spill zone (Isleib and nearshoreintertidal and subtidalhabitats(Neff Kessel1973,Agler et al. 1994).Two hundred et al. 1995), which are important to a large and twelve Harlequin Ducks were recovered number of vertebrates,includingmolting and from beachesafter the oil spill (J. Piatt pers. winteringwaterfowl.Indeed,thenearshore en- comm.),but the actualnumberthat died probablywasmuchhigherbecause therecoveryrate of waterbirdswas low due to birds being scav-
4E-mail:
[email protected]
enged,sinking,or notbeingfoundalongshorelines (Ford et al. 1987,ECI 1991).Adjusted estimatesthatcorrectfor theserecoveryproblems USA. 6Present address: Department of Fisheries and range from 1,298 to 2,650 Harlequin Ducks Wildlife, Michigan State University,East Lansing, killed from oiling (ECI 1991, Piatt and Ford Michigan48824,USA. 1996, J. Piatt pers. comm.). Post-spill studies sPresentaddress:USFWS,EcologicalServices,825 Kaliste Saloom Road, Lafayette, Louisiana 70508,
781
782
LANCTOT ETAL. Alaska Peninsula /
[Auk,Vol.116
Prince William Sound
KodiakArchipelago
Afognak
Green Island
50
0
50Km
20
0
20 Km
FIG.1. HarlequinDucksampling locations in theAlaskaPeninsula / KodiakArchipelago andPrinceWilliam Sound.
suggest thatnegative effects fromthespillcon- nessalsocanbeinferredusinggeneticmarkers tinue, based on differences in winter survival
publ. data), declinesin numbersof molting birdswithin the spill zone(D. Rosenberg unpubl.data),and detectable levelsof hydrocarbonsin HarlequinDucksand theirpreyfrom
(Slatkin1985,1995;SlatkinandBarton1989).In this study,we usedthreeclasses of molecular geneticmarkersthat differ in their modeof inheritance (biparental, maternal, and sexlinked)to determinewhetherpopulationstructuring occurredamongwinter aggregations of
1989 to 1993 (Patten1995).
Harlequin Ducks in coastalmarine habitatsof
To understandthe processof post-spillrecoveryof HarlequinDucks,andto identifyimpedimentsto recovery,it is criticalto determine whetheraggregations of individualswithinlo-
PWS and the Alaska Peninsula/Kodiak Archi-
betweenoiled and unoiledareas(D. Eslerun-
cal areas of the marine environment are dis-
pelago (APKA). Informationon population structurewasthenusedto assess thepotential constraintsto recoveryof Harlequin Ducks fromtheExxonValdez oil spill.
crete and demographicallyindependent,i.e. whetherthe populationis structuredwithin METHODS theoil spillregion.Forexample, if birdslocated in oiled and unoiled areasbelongto demoWecompared geneticcharacteristics of fouraggregraphicallydistinctpopulationsegments (i.e. gationsof moltingbirds(referredto aspopulations) capturedwithin each of two regions(PWS and APKA;Fig. 1). Because band resightingsand radiotelemetrydataindicatethat HarlequinDucksalmost alwayswinter at or neartheir moltingsites(Robertson1997,D. Eslerunpubl.data),we considered these moltingaggregations to representdiscretewintering populations. Samplingsiteswithin the PWSregion includedBay of Isles,GreenIsland,FoulBay,and MontagueIsland,whereassamplingsiteswithin the the marine environment. In the absence of diAPKA regionincludedUganikand Uyak baysand rect observationaldata, populationdiscrete- AfognakIslandof the KodiakArchipelago,andKat-
managementunits; Moritz 1994), then certain
segments of thepopulationmayhavebeen(and are continuingto be) influenceddisproportionately by oil. Alternatively, a lack of spatial structurewould imply that oil effectsare distributedthroughout a larger,panmictic population. Unfortunately,little is known about movements of HarlequinDucksamongareasof
July1999] mai on the Alaska Peninsula.
Harlequin DuckPopulation Genetics All of these sites were
locatedinsidethe areaaffectedby the oil spill. MoltingHarlequinDuckswerecapturedat eachof the eightsamplingsitesduringJulyto Septof 1995, 1996,and 1997by herdingthe flightlessbirds into
783
UMITH primer. Mitochondrial DNA was PCR-am-
plified,purified,and sequenced usingSequitherm's EXCELDNA sequencing kits (EpicentreTechnolo-
gies) and 1.5 mM of HADUMITH primer. Sequences were visualized using autoradiography,manually pens(seeClarksonand Goudie1994).The sexof each scored,and aligned. duckwasdeterminedby plumagecharacteristics and Geneticanalyseswere restrictedto birds at least the ageby bursalprobing(Matherand Esler1999). one year of age. Data from males and femaleswere
Bloodwasextractedfrom thejugularor tarsusveins used for the biparentallyinheritedloci, and data and was preservedin lysis buffer (Longmireet al. wererestrictedto femalesfor the maternallyinher1988). DNA was extractedusing PuregeneDNA extractionkits andstandardproteinase-Kphenol-chloroformmethods(Sambrooket al. 1991). Thirty-threemicrosatellite loci (Fieldsand Scribner 1997, Buchholtzet al. 1998, Cathy et al. 1998) were examinedfor variationby screeningtwo individualseachfrom four populationson the westcoast of North America.Of these,fourbiparentallyinherited loci (Sfib4,Hhib2, Hhib5, and Bcab10)andtwo sex-linkedloci (Stihl and Bcab4)were variable.The sex-linked(Z-specific)loci provide an estimateof male-mediatedgeneflow from the precedinggenerationif samplingis conductedusingfemales.Microsatelliteloci were assayedusingthe polymerase chainreactionand end-labeled(32P-•ATP) primers. Specificconditionsfor eachlocusare availablefrom the senior author. Products were visualized
on 6%
denaturingpolyacrylamidesequencing gel after autoradiography. An M13 sequencing reaction(Amersham Life SciencesSequenasekit) and individual standardsof knowngenotypeswere run adjacentto samplesto provide an absolute-sizemarkerfor determiningthe sizeof microsatellitealleles. Primersspecificfor mitochondrialDNA (mtDNA) were designedso that mtDNA sequences, and not nuclearDNA sequences originatingfrom transposed mtDNA (i.e. numts; Sorensonand Fleischer1996), would be amplified.We verifiedthis by sequencing DNA obtained from mitochondrial-rich
heart tissue
and mitochondrial-poorblood from the same individual as describedin Sorensonand Quinn (1998).
Using the mtDNA-specificprimers,we amplifieda fragment(ca.385basepairs)of the 5' endof thecontrol regionthat is comparableto the hypervariable region 1 of the mammalianmtDNA controlregion (Vigilant1990,Wakely1993).Theseprimersincluded
ited mtDNA
and sex-linked
microsatellite
loci.
Foreachpopulation,all biparentallyinheritedloci were testedfor linkage (all two-locuscomparisons)
and Hardy-Weinberg disequilibriumusingthe Fisher'sexacttest in the GeneticsData Analysis(GDA) program(Lewis and Zaykin 1998).P-valueswere ad-
justedfor the numberof statisticaltests(Sokaland Rohlf 1995).Mean numberof allelesper locuswere calculatedusing BIOSYS(Swofford and Selander 1981).We estimatedobserved(Ho)and expected(H,.)
heterozygosity underHardy-Weinberg assumptions for eachlocus(BIOSYS)and for eachpopulationand locus(GDA). Theseestimatesof Hoand H,.were used to generateinbreedingcoefficients(F - 1 - [Ho/He]) combinedacrossloci for each population(Wright 1951) and testedfor significanceas describedin Li and Horivitz (1953). Statisticalanalysesof spatial heterogeneity in genefrequencybetweenandwithin regionsfor eachlocuswere assessed using hierarchicalF-statistics(Weir1996)in the GDA programat three levels:(1) amongindividualswithin popula-
tions,(2) amongpopulations withinregions,and(3) betweenregions.We alsocalculatedRo,,an analogue of F• (MichalakisandExcoffier1996),usingthe analysis of molecularvariance(AMOVA) program (Excoffier et al. 1992). Significanceof Fstvalues was based on 95% confidenceintervalsdeterminedby
bootstrappingacrossloci. Confidenceintervalsthat included zero were considerednonsignificant.We used allele frequenciesto calculateCavalli-Sforza
andEdwards(1967)chorddistances amongthepopulations and constructedbootstrappedpopulation
HADUM1L (L16744;Desjardinsand Morais 1990):5'
treesusingsubroutines within thePHYLIPprogram (version3.572c;Felsenstein 1993).Thisdistancemetric hasbeendescribedas producingrobusttree topologiesfor groups separatedevolutionarilyover timeperiodscomparable to thepopulations we stud-
TGC CCG AGA CCT ACG GCT C 3'; HADUM2L
ied (Takezakiand Nei 1996).We usedbirds sampled
(L12;DesjardinsandMorais1990):5' TCT AAA ATG ACT CAA CAG TGC C 3'; and HADUMITH (H737; Desjardinsand Morais 1990):5' TGA GTA ATG GTG TAG ATA TCG 3'. Nuclearspecificprimersincluded HADUN1L (L16744;Desjardinsand Morais 1990):5'
at Shemya,on the outerAleutianIslandsof Alaska, as an outgroupin this analysis.For the SfipAand
TAC CCG
Bcab4 sex-linkedloci, we calculatedallele frequenciesand a measureof geneticdiversity(D; Nei 1987: equation8.3). Estimatesof varianceamong individ-
AGA CCT ACA GCT T 3' and HADUNuals within populations(•c), amongpopulations UCH (H737; Desjardinsand Morais 1990):5' TGA within regions(•ct), and betweenregions(•s•) were GTT ATG GTG TAGATA CTA 3'. MtDNA sequences derivedusingthe AMOVAprogram. (GenBank accession numbers AF101372 to MitochondrialDNA sequence haplotypeswereasAF101381)were obtainedby amplifying either the signedbasedon at least a singlebase-pairsubstituHADUM1L or HADUM2L primerswith the HAD- tion or insertion/deletionacrossthe 163 base-pair
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TABLE 2. Measures of genetic diversityestimated forHarlequinDucksfromfourpopulations eachwithin two marinecoastalwinteringregionsin south-central Alaska. Prince William
Variable
Sound
Alaska Peninsula and
Bayof
Foul
Green Montague
Isles
Bay
Island
Island
KodiakArchipelago
KatmaiAfognakUganik
Uyak
l•iparental microsat•llit• loci
Observedheterozygosity Expectedheterozygosity a Mean no. of alleles
Inbreedingcoefficient b
0.491 0.465
0.479 0.465
0.483 0.471
0.494 0.492
3.00
3.75
3.75
3.75
-0.056
-0.030
-0.025
-0.004
Sex-linked
microsatellite
0.463 0.474
0.427 0.464
0.462 0.481
3.50
3.00
3.75
0.023 0.080 0.040
0.482 0.470 4.00
-0.026
loci
Genetic diversityc(no. alleles) Sfi•l Bca•4
0.426
0.582
0.392
0.555
(3)
(3)
(3)
(3)
0.851
0.857
0.865
(13)
(8)
(10) Maternal
Haplotype diversity (•r)a Nucleotidediversity (h)ct
No. of haplotypes
0.427 0.004
4
0.629 0.005 4
mitochondrial 0.432 0.003 3
0.333
0.357
0.410
(3)
(3)
(3)
0.441
0.852
0.821
0.816
0.856
0.861
(14)
(10)
(6)
(10)
(11)
0.635 0.006 6
0.745 0.007 5
0.661 0.007 5
0.677 0.006 5
(3)
DNA 0.726 0.007 5
• AssumingHardy-Weinberg equilibrium. bWright(1951)inbreedingcoefficient. Significance of F at a specificlocusandpopulationweretestedasdescribed by Li andHorvitz(1953). ½GeneticdiversityNei (1987).FortheBca•,4locus,alleles190and194werecombined; andalleles214,216,218,220,222,224,226,230,and 238 were combined.
dHaplotype andnucleotide diversity indices fornonselfing populations (equations 8.4and10.4,respectively, ofNei1987),ascalculated by theREAPprogram (McElroy et al. 1991)frompairwise haplo•ype divergences calculated bytheMEGAprogram (Kumaret al.1993).
RESULTS segment.Pairwisehaplotypedistances werecalculatedby the MEGAprogram(Kumaret al. 1993)and usedto estimatehaplotype(h)andnucleotide diverEach populationof Harlequin Ducks had sity (•r) indicesfor nonselfingpopulations(equations threeto eightallelesat eachbiparentally inher8.1 and 10.4, respectively, of Nei 1987)usingthe ited locus(Table1). The meannumberof alleles
REAPprogram(McElroyet al. 1991).Wetestedhet-
erogeneityof genotypedistributionsamongsamples per locus,and observedand expectedhetero-
usingMonteCarlo resamplingand the chi-square zygosities,were moderatelyhigh and concortestofRoffandBentzen(1989).Thisapproachis suit- dant acrossall populations(Table2). The biableforgenetic-data matrices in whichmanyormost parentalloci.didnot deviatesignificantlyfrom elementsare very small (
