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Aquatic Toxicology 61 (2002) 195 – 209 www.elsevier.com/locate/aquatox Genetic structure of Fundulus heteroclitus from PAH-contaminated and neighboring sites in the Elizabeth and York Rivers Margaret Mulvey, Michael C. Newman *, Wolfgang Vogelbein, Michael A. Unger Department of Enironmental Science, Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, VA 23062, USA Received 5 February 2002; received in revised form 25 March 2002; accepted 8 April 2002 Abstract Population genetic characteristics of mummichog, Fundulus heteroclitus, from the heavily industrialized Elizabeth River and nearby York River (Virginia USA) were assessed relative to sediment PAH concentrations. Allozyme genotype frequencies for all loci were consistent with random mating expectations at each locality and age class. Fish from all sites had comparable levels of enzyme polymorphism and heterozygosity regardless of the associated sediment PAH concentrations. Allozyme frequencies for 12 of 15 loci were homogeneous for mummichog from all localities except that allozyme frequencies were significantly different for the Idh -2 locus of (adult and juvenile) mummichog at the heavily-contaminated Atlantic Wood site relative to all other sites. Additionally, allele frequency differences were noted for Ldh -C and Gpi -1 among juvenile mummichog. Values for F st were 0.0254 and 0.0141 in the juvenile and adult samples, respectively, indicating greater among-locality genetic differentiation for juvenile mummichog than for adults. Juvenile mummichog are more likely to remain in their natal area while adult samples reflect movement of fish during two or more winter seasons. Correlation analysis suggested that genetic differentiation was not correlated with geographic distance at the spatial scale studied here; however, there was a significant correlation between genetic distance and differences among sites in organic carbon-normalized PAH concentrations. Mummichog collected at the heavily PAH-contaminated AW locality were genetically distinct from those at neighboring sites. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Fundulus heteroclitus ; Fish; Population Genetics; PAH; Allozymes 1. Introduction The Elizabeth River Estuary in Virginia, USA has a long history of physical and chemical per- turbation. Dredging, industrialization, and urban- ization led to significant fragmentation of habitat. * Corresponding author. Tel.: +1-804-684-7105; fax: +1- 804-684-7097 E-mail address: [email protected] (M.C. Newman). 0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0166-445X(02)00055-3
Transcript
Page 1: Genetic structure of Fundulus heteroclitus from PAH-contaminated ...

Aquatic Toxicology 61 (2002) 195–209 www.elsevier.com/locate/aquatox

Genetic structure of Fundulus heteroclitus fromPAH-contaminated and neighboring sites in the Elizabeth

and York Rivers

Margaret Mulvey, Michael C. Newman *, Wolfgang Vogelbein,Michael A. Unger

Department of En�ironmental Science, Virginia Institute of Marine Science, College of William & Mary, Gloucester Point,VA 23062, USA

Received 5 February 2002; received in revised form 25 March 2002; accepted 8 April 2002

Abstract

Population genetic characteristics of mummichog, Fundulus heteroclitus, from the heavily industrialized ElizabethRiver and nearby York River (Virginia USA) were assessed relative to sediment PAH concentrations. Allozymegenotype frequencies for all loci were consistent with random mating expectations at each locality and age class. Fishfrom all sites had comparable levels of enzyme polymorphism and heterozygosity regardless of the associatedsediment PAH concentrations. Allozyme frequencies for 12 of 15 loci were homogeneous for mummichog from alllocalities except that allozyme frequencies were significantly different for the Idh-2 locus of (adult and juvenile)mummichog at the heavily-contaminated Atlantic Wood site relative to all other sites. Additionally, allele frequencydifferences were noted for Ldh-C and Gpi-1 among juvenile mummichog. Values for Fst were 0.0254 and 0.0141 inthe juvenile and adult samples, respectively, indicating greater among-locality genetic differentiation for juvenilemummichog than for adults. Juvenile mummichog are more likely to remain in their natal area while adult samplesreflect movement of fish during two or more winter seasons. Correlation analysis suggested that genetic differentiationwas not correlated with geographic distance at the spatial scale studied here; however, there was a significantcorrelation between genetic distance and differences among sites in organic carbon-normalized PAH concentrations.Mummichog collected at the heavily PAH-contaminated AW locality were genetically distinct from those atneighboring sites. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Fundulus heteroclitus ; Fish; Population Genetics; PAH; Allozymes

1. Introduction

The Elizabeth River Estuary in Virginia, USAhas a long history of physical and chemical per-turbation. Dredging, industrialization, and urban-ization led to significant fragmentation of habitat.

* Corresponding author. Tel.: +1-804-684-7105; fax: +1-804-684-7097

E-mail address: [email protected] (M.C. Newman).

0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0166 -445X(02 )00055 -3

Page 2: Genetic structure of Fundulus heteroclitus from PAH-contaminated ...

M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209196

High and spatially heterogeneous concentrationsof polycyclic aromatic hydrocarbons (PAH), trib-utyltin (TBT), metals, and other toxicants havebeen reported from water, sediments, and organ-isms (Bieri et al., 1986; USEPA, 1999). For exam-ple, PAH concentrations of sediments from asuperfund site adjacent to the former AtlanticWood Industries (AW) wood treatment plant areas much as 2700 times higher than those of othersites along the Elizabeth River.

Mummichogs (Fundulus heteroclitus) are abun-dant in shallow marshes throughout the ElizabethRiver. Discrete patches of suitable habitat areseparated by unsuitable areas of shipyards andconcrete debris along highly-modified reaches ofthe Elizabeth River such as the Southern Branch.Some contaminants reported in Elizabeth Riversediments are directly toxic to mummichog athigh concentrations (Williams, 1994) and chroni-cally stressful at lower concentrations (Vogelbeinet al., 1990; Huggett et al., 1992). Yet, mummi-chogs are consistently present despite the veryhigh concentrations of PAHs in AW sediments.

Mummichogs are reported to exhibit strong sitefidelity (Lotrich, 1975; Meredith and Lotrich,1979). Summer home ranges in Delaware saltmarshes were approximately 36 m along a creekside and few fish were observed to cross to theopposite bank (Lotrich, 1975). Although long-dis-tance movement occurs occasionally (Lotrich re-ported recapture of fish 375 m from release), localmummichog can behave as semi-isolated subpop-ulations. Mummichog inhabiting heavily contami-nated areas likely spend all or most of their livesexposed to potential stressors.

Isolated subpopulations, especially if small, canexperience genetic drift and inbreeding. Popula-tion genetic theory predicts that, on average, suchpopulations would exhibit a loss of genetic varia-tion and an increase in homozygosity relative tolarger non-fragmented populations. This predic-tion would be especially important in pollutedenvironments if contaminant exposure increasedmortality and reduced population size. Relativeisolation of organisms in discrete habitat patchesmight also enhance acquisition of tolerance tolocal contaminant conditions. Thus, genetic varia-tion might be further reduced for organisms in

heavily contaminated habitat patches and differ-entiation of fish among habitat patches would beenhanced.

Exposure of mummichog to high PAH concen-trations has been associated with an increasedfrequency of liver lesions and cancers (Vogelbeinet al., 1990). Williams (1994) reported that mum-michog from the AW location tolerated levels ofPAHs that resulted in 100% mortality of fish froma reference locality on the nearby York River.Additionally, mummichog embryos from the AWsite have a lower incidence of cardiac abnormali-ties than mummichog from other Elizabeth Riverand York River localities if exposed to AW sedi-ments (Ownby et al., 2002). Taken together, theseobservations indicate that AW mummichog pos-sess genetically-based enhanced tolerance toPAHs relative to fish from reference localities.

Several hypotheses may be forwarded to ac-count for the persistence of mummichog in heav-ily-contaminated habitat. First, PAH exposuremight not adversely affect mummichog. Thisseems unlikely. Second, the high levels of PAHsare harmful to fish and mortality of AW mummi-chog is high; however, fish are replenishedthrough immigration from elsewhere in the estu-ary. The AW site acts as a sink in a metapopula-tion. A third possibility is that AW fish might bea locally stable subpopulation that is tolerant ofhigh sediment PAH concentrations and immigra-tion from neighboring subpopulations might beuncommon.

To discriminate among these hypotheses, weinitially studied the microgeographic populationgenetic structure in mummichog along the highly-modified Southern Branch of the Elizabeth River.Mummichog from nine sites ranging from heavilycontaminated to less contaminated were subjectedto protein electrophoresis to assess the impact ofstochastic processes, such as gene flow and drift,as well as the possibility of toxicant-associatedselection. Allozymes are highly polymorphic andinformative for mummichog in this region(Cashon et al., 1981). Allozymes should act asmarkers of population genetic structure and pro-cesses underlying it. None of the isozymes studiedis explicitly related to contaminant tolerance al-though frequencies of allozyme genotypes (Gpi-A,

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 197

Ldh-B, Idh) have been associated with environ-mental variables and performance differencesamong allozyme genotypes have been described.

Two years after the survey of the SouthernBranch of the Elizabeth River, the scale of thisstudy was expanded to include a second branch ofthe Elizabeth River and sites in the adjacent YorkRiver estuary. The intent of this expansion was todetermine whether the findings could be extendedto include more geographically distant popula-tions and populations with more extreme contam-inant differences. Also, defining the geneticqualities of York River mummichog relative toElizabeth River mummichog populations was im-portant because past and ongoing studies com-pare with AW mummichog qualities to those ofYork River watershed reference populations.

Specifically, the following questions were ad-dressed using the allozyme data. First, does thelevel of contamination influence the genetic struc-ture of mummichog populations within a land-scape mosaic of sites varying in sediment PAHconcentrations? Genetic distance among mummi-chog would be determined only by geographicaldistance if differentiation among mummichogwere unrelated to contamination. If high contami-

nant concentrations lead to high mortality in pol-luted habitat and mummichog were migratinginto these areas from neighboring areas, we mayexpect high estimates of gene flow and little ge-netic differentiation among mummichog from dif-ferent localities. Alternately, if mummichogexhibit strong site fidelity and minimal migrationamong patches, migration estimates would be lowand mummichog from localities might be geneti-cally differentiated. Second, does genetic variabil-ity decrease with increasing levels of PAHcontamination? Third, is there evidence suggestingthat potential or existing reference sites near theYork River are genetically distinct from ElizabethRiver sites? An affirmative answer to this lastquestion would suggest that reference sites forstudying AW mummichogs should be selectedfrom within the Elizabeth River, not from theYork River watershed.

2. Methods

2.1. Fish collection

Mummichogs were collected initially in October

Fig. 1. Collection sites for mummichog on the Elizabeth and York Rivers. Panel A, York River and Panel B, Elizabeth River.

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209198

Fig. 2. PAH concentrations (ng/g dry weight) measured in duplicate sediment samples at each site. York River sites are identifiedwith a (Y) below their site abbreviation.

1998 from the Southern Branch of the ElizabethRiver (Fig. 1). Sites were chosen along a 12 kmreach of the river. Each site was a discrete habitatpatch separated from the others by industrialareas unsuitable for mummichog. Sample locali-ties differed markedly in sediment PAH contami-nation (Fig. 2). Two sites (Atlantic Wood andRefueling Station, RS) were chosen specificallybecause of their proximity to historical sources ofPAH. Several others were selected from nearbylocations having notionally lower concentrationsof PAHs. Approximately 30 juveniles (mean=1.5g wet weight, standard deviation (S.D.)=0.6 g,n=286) and 60 adults (mean=7.9 g wet weight,S.D.=2.5 g, n=537) were collected with baitedminnow traps for analysis. Equal numbers ofmale and female fish were sampled.

Additional mummichog populations were sam-pled in October 2000 from the Eastern Branch ofthe Elizabeth River and from the adjacent andrelatively clean York River (Fig. 1). One EasternBranch site (Colonnas Shipyard, CS) was selectedbecause of the high sediment PAH concentrationsreported earlier for that site. The York Riverwatershed populations represented reference pop-

ulations used in past studies of PAH exposureconsequences to AW mummichog, e.g. Vogelbeinet al. (1990), Williams (1994), Armknecht et al.(1998). Sixty adult fish (mean=6.6 g wet weight,S.D.=3.3 g, n=480), but no juveniles, were col-lected from these eight additional sites. The pri-mary intent of this expanded collection was toincrease the number and range of populationsrelative to geographical distance and PAH con-centrations. A secondary intent was to generatebackground genetic information with which toassess the appropriateness of using York Riverreference populations in studies of ElizabethRiver mummichog populations.

2.2. Sediment collection

Sediments were collected from all 17 stations inthe Elizabeth and York Rivers during the periodsof fish sampling. At each station, three surfacegrabs were taken from F. heteroclitus habitat witha stainless steel Ponar grab and were then homog-enized in a stainless steel bucket to produce acomposite sample. Duplicate composite samples

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 199

were collected at each location and analyzed forcontaminants. Sediment samples for organic com-pound analyses were placed in cleaned glass jarswith teflon-lined lids, placed on ice in the field,and stored frozen until analyzed.

2.3. Analytical methods

Sediment samples were analyzed for organiccontaminants using the protocol described byGreaves et al. (1991). Briefly, sediments werefreeze-dried, spiked with surrogate standards, andextracted with dichloromethane by acceleratedsolvent extraction. The resulting extracts werefractionated by sequential gel permeation and sil-ica gel chromatographies, and analyzed for aro-matic or heterocyclic compounds by capillary gaschromatography with flame ionization detectionand gas chromatography-mass spectrometry inthe full scan electron ionization mode. Blanks,duplicates, and standard reference materials wereanalyzed in tandem with the samples. Polycyclicaromatic hydrocarbons were summed as per theappendix of Horness et al. (1998).

2.4. Protein electrophoresis

Eyes and caudal muscle tissue were taken forisozyme analysis and stored at −70 °C. Tissueswere prepared for electrophoresis by grinding inapproximately 250 �l of deionized water and cen-trifuging at 14 000 rpm for 45 s. Supernatant fluidwas absorbed onto filter paper wicks and insertedinto 12% (w/v) horizontal starch gels. Fourteenenzyme loci and one general protein were assayed.Tris-citrate pH 8.0 gels and buffer (Selander et al.,1971) were used to resolve lactate dehydrogenase(Ldh-A, -B, and -C), glucosephosphate isomerase(Gpi-1, -2), isocitrate dehydrogeanse (Idh-1, -2)using eye tissue, and malate dehydrogenase (Mdh-1, -2), NADP-dependent malate dehydrogeanse(Me-1), mannose phosphate isomerase (Mpi ),and a non-specific protein (Gp) using muscle tis-sue. The lithium hydroxide gel-buffer of Selanderet al. (1971) was used for phosphoglucomutase(Pgm) and aspartate aminotransferase (Aat). TheTris citrate pH 6.9 buffer (diluted 1:10 for buffertrays and 1:20 for gels) was used to resolve 6-

phosphogluconate dehydrogenase (Pgd). Afterovernight (16 h) electrophoresis, gels were slicedand stained following standard methods (Hillis etal., 1996; Selander et al., 1971).

2.5. Data analysis

Isozyme data were analyzed using BIOSYS-2(Swofford and Selander, 1981) to test for fit of theobserved data to random mating expectations,determine homogeneity of allele frequencies usingcontingency �2 statistics, and evaluate divergenceamong populations using genetic distance (Nei,1972) and F-statistics (Wright, 1978). The �2 testswere done only for cases where expected numbersof observations were greater than five and Bonfer-roni adjustments were made to maintain an exper-imentwise � of 0.05 if several statistical tests weredone. Fish migration among Southern Branchsites was estimated from Fst values for juvenileand adult samples. Because of small sample sizesfor individuals with many heterozygous loci, indi-vidual heterozygosity was grouped into the fol-lowing categories for analysis: 0, 1, 2, 3, and �3.A three-way Mantel test (Smouse et al., 1986) wasdone with ARLEQUIN software (Schneider et al.2000) using genetic distance, geographic distance,and contaminant distance between pairs of sites.Geographic distance was estimated ‘over water’using nautical chart software (Capn 45). Contam-inant concentration differences were square rootsof the absolute differences in total sediment PAHconcentration between sites. Subsequent correla-tion analysis of genetic distance and contaminantconcentration difference were done using thePROC CORR procedure of the SAS statisticalpackage (SAS Institute Inc., 1989) and PAH con-centrations normalized to organic carbon insediments.

3. Results

3.1. PAH in Elizabeth Ri�er sediments

Analysis of the non-polar aromatic fractionshowed a predominance of unsubstituted and sub-stituted PAHs. Total PAH concentrations ranged

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209200

from a low of 99 ng/g dry weight at the PP site toa high of 264 114 ng/g dry weight at the AW site.The PAH concentrations were arranged in Fig. 2from the lowest to the highest to illustrate the widegradient in PAH contaminations represented by thesites. Also shown in this figure, duplicate samplesfrom each site showed low variability relative tothat across sites.

3.2. Population genetics

Allozyme frequencies for the 15 loci for juvenileand adult mummichog from the Elizabeth RiverSouthern Branch sampling are provided in Table 1.The �2 tests for all 17 sites indicate that genotypicdistributions were consistent with random matingexpectations for fish from any site and, in the caseof the sites on the Southern Branch of the ElizabethRiver, any age. There were no significant differ-ences in allozyme frequencies between juvenile andadult mummichog at any Elizabeth River SouthernBranch sites. Significant heterogeneity in allozymefrequencies for the Southern Branch of the Eliza-beth River sites were observed for three of the 15loci examined. The �2 tests for homogeneity ofallozyme frequencies among juvenile mummichogfrom the Elizabeth River Southern Branch indi-cated significant differences at the Ldh-B, Gpi-1,and Idh-2 loci (all P�0.01). For Southern Branchadults, there were significant differences in thefrequencies of Idh-2 alleles among the sites (P�0.00003) but there was no simple relationshipbetween allozyme frequency and sediment PAHcontamination of sites. The frequency of the Idh-2allozymes was consistently different for AW mum-michog (Fig. 3). Both juvenile and adult fish fromAW had a significantly lower frequency of thecommon Idh-2 allozyme than fish from other sitesalong the Elizabeth River. Consistent with theseresults for the Southern Branch adult mummichog,�2 tests for homogeneity of allozyme frequenciesindicated significantly lower frequency of the com-mon Idh-2 allele at AW relative to all 16 sites(P�0.001).

There were no significant differences amonglocalities in any measures of overall genetic vari-ability (Table 2). The mean number of alleles perlocus was comparable for juveniles and adults for

the Southern Branch of the Elizabeth River sites.Mean heterozygosity was comparable among adultfish for all 17 sites. Genetic variability was notdifferent between highly contaminated (e.g. AW,CS and RS) and less contaminated sites (Table 2).

The three-way Mantel tests indicated a positive(0.407), but statistically nonsignificant (�=0.05),correlation between the difference in sediment PAHcontamination and genetic distance between popu-lations of the nine Southern Branch sites. Very lowand statistically nonsignificant correlation coeffi-cients indicated no relationship existed betweengeographic distance and genetic distance for fishalong the Southern Branch, nor for fish sampled atthe wider geographical scale. Because geographicaldistance was not correlated with genetic distancefor these sites, more focused correlation analyseswere done for genetic distance versus differences inPAH concentrations with PAH concentrations be-ing normalized to sediment organic carbon content(Fig. 4). These analyses were done with and withoutthe AW site to assess if a correlation was presenteven in the absence of this extreme site. There wasa highly significant correlation (Kendall � correla-tion coefficient=0.302, P�0.001) in analyses ofall 17 sites. After omitting the AW site, the corre-lation coefficient was lower (0.177) for the remain-ing 16 sites but still statistically significant(P=0.009).

Wright’s Fst value (Wright, 1978), a measure ofgenetic differentiation, was 0.025 for juvenile mum-michog and 0.014 for adult fish from the ElizabethRiver Southern Branch samples. These values weresignificantly different from zero (Workman andNiswander, 1970), indicating genetic structureamong the Southern Branch sampling sites. Thelarger Fst value for the juvenile samples suggestedthat migration among sites is more likely for fishof the older age classes. Wright’s Fis values (Fis=0.017 and 0.011 for juveniles and adults, respec-tively) indicated greater relatedness for thejuveniles within sites relative to that observed forthe adults.

Variance component analysis of the genetic datafor all 17 sites indicated that 99.3% of the totalgenetic variation was associated with variationwithin a sample site, 0.5% was associated withvariation among sites within the river, and 0.2%was variation between the two rivers.

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 201

Tab

le1

Allo

zym

efr

eque

ncie

sof

mum

mic

hog

from

Yor

kan

dE

lizab

eth

Riv

ersi

tes

Gpi

-1G

pi-2

Aat

Riv

erG

pSi

te

34

57

23

41

23

45

92

34

12

0.58

0.99

0.08

0.01

0.23

0.75

0.01

0.01

0.99

Yor

kK

ings

Cre

ek(K

Y)

0.42

1.00

0.21

0.78

0.01

0.01

0.99

0.56

0.44

Car

ters

Cre

ek(C

C)

1.00

0.25

Que

ens

Cre

ek(Q

Y)

0.75

0.01

0.98

0.01

0.01

0.37

0.59

0.03

0.99

0.01

0.01

0.16

0.83

0.01

1.00

0.57

0.42

Cat

lett

Isla

nds

(CI)

0.50

Rad

ioT

ower

Nor

th0.

020.

980.

020.

230.

781.

000.

48E

aste

rnE

lizab

eth

(RN

)0.

51C

ampo

stel

laH

eigh

ts0.

980.

030.

180.

810.

010.

011.

000.

49(C

H)

0.01

1.00

0.23

0.78

0.52

1.00

0.48

Nor

thC

anal

(NC

)0.

021.

000.

010.

130.

861.

00C

olon

nas

Ship

yard

0.46

0.53

(CS)

1.00

0.28

0.72

0.59

Sout

hE

lizab

eth

1.00

0.42

Pow

erP

lant

(PP

)(0

.01)

(0.9

9)(0

.20)

(0.8

0)(0

.02)

(0.9

8)(0

.45)

(0.5

5)1.

000.

370.

63N

ewM

illC

reek

0.99

0.01

0.49

0.51

(NM

)(1

.00)

(0.3

0)(0

.70)

(0.6

3)(1

.00)

(0.3

7)P

arad

ise

Cre

ek(P

C)

1.00

0.30

0.70

1.00

0.33

0.67

(0.0

2)(0

.98)

(0.1

7)(0

.83)

(1.0

0)(0

.36)

(0.6

4)1.

000.

300.

680.

010.

010.

010.

420.

990.

010.

58Jo

nes

Cre

ek(J

C)

(1.0

0)(0

.20)

(0.8

0)(0

.97)

(0.0

3)(0

.42)

(0.5

8)0.

62C

hann

elM

arke

r2

1.00

0.30

0.70

1.00

0.38

(CM

)(1

.00)

(0.2

3)(0

.77)

(0.5

9)(1

.00)

(0.4

1)Sc

uffle

tow

nC

reek

0.48

0.01

1.00

0.27

0.73

1.00

0.51

(SC

)(0

.00)

(1.0

0)(0

.29)

(0.7

1)(0

.65)

(1.0

0)(0

.35)

0.63

Cro

wn

Tan

kF

arm

1.00

0.24

0.76

1.00

0.38

(CF

)(1

.00)

(0.3

8)(0

.62)

(1.0

0)(0

.48)

(0.5

2)1.

000.

190.

810.

63R

efue

ling

Stat

ion

1.00

0.37

(RS)

(1.0

0)(0

.18)

(0.8

2)(1

.00)

(0.5

0)(0

.50)

0.69

1.00

0.20

0.80

1.00

Atl

anti

cW

ood

(AW

)0.

31(1

.00)

(0.1

0)(0

.90)

(1.0

0)(0

.69)

(0.3

1)Id

h-2

Ldh

-AId

h-1

Ldh

-BL

dh-C

23

45

89

23

42

32

34

23

48

0.30

0.73

0.22

0.02

0.02

1.00

0.78

0.23

1.00

0.99

KY

Yor

kC

C0.

710.

280.

010.

980.

020.

770.

230.

010.

980.

011.

000.

010.

700.

270.

030.

010.

990.

800.

20Q

Y0.

980.

020.

010.

99C

I0.

700.

280.

021.

000.

800.

201.

001.

00R

N0.

780.

210.

011.

000.

860.

140.

990.

010.

010.

99E

ast

Eliz

abet

h0.

010.

830.

171.

000.

800.

201.

000.

990.

01C

H0.

850.

151.

00N

C0.

770.

231.

001.

000.

780.

231.

001.

000.

840.

161.

00C

SP

P0.

010.

790.

190.

010.

990.

010.

830.

171.

000.

020.

98So

uth

Eliz

abet

h(0

.01)

(0.7

6)(0

.23)

(0.0

1)(0

.97)

(0.0

2)(0

.83)

(0.1

8)(1

.00)

(1.0

0)0.

770.

231.

000.

780.

221.

00N

M1.

00

Page 8: Genetic structure of Fundulus heteroclitus from PAH-contaminated ...

M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209202

Tab

le1

(con

tinu

ed)

Ldh

-BL

dh-C

Idh-

1Id

h-2

Ldh

-A

48

23

45

89

23

42

32

34

23

(0.8

3)(0

.17)

(1.0

0)(0

.84)

(0.1

6)(1

.00)

(1.0

0)0.

850.

150.

980.

020.

84P

C0.

161.

000.

990.

01(0

.87)

(0.1

3)(1

.00)

(0.8

1)(0

.19)

(1.0

0)(1

.00)

0.02

0.67

0.31

JC1.

000.

850.

150.

010.

980.

010.

010.

99(0

.01)

(0.7

7)(0

.22)

(0.9

8)(0

.02)

(0.8

8)(0

.01)

(0.1

2)(0

.95)

(0.9

7)(0

.03)

(0.0

2)(0

.01)

CM

0.77

0.23

0.99

0.01

0.81

0.19

1.00

1.00

(0.7

3)(0

.27)

(0.9

8)(0

.02)

(0.7

3)(0

.27)

(0.9

7)(0

.03)

(1.0

0)0.

860.

140.

990.

010.

880.

12SC

1.00

1.00

(0.8

0)(0

.20)

(1.0

0)(0

.86)

(0.1

4)(0

.98)

(0.0

2)(1

.00)

0.78

0.22

1.00

0.85

0.15

1.00

CF

0.01

0.98

0.02

(0.7

8)(0

.22)

(1.0

0)(0

.65)

(0.3

5)(0

.02)

(1.0

0)(0

.98)

0.83

0.17

1.00

0.88

0.12

RS

1.00

1.00

(0.7

2)(0

.28)

(0.0

2)(0

.98)

(0.6

9)(0

.31)

(1.0

0)(1

.00)

0.01

0.58

0.42

1.00

0.83

0.17

0.01

0.97

0.02

AW

0.99

(0.5

8)(0

.42)

(1.0

0)(0

.87)

(0.1

3)(0

.02)

(0.9

7)(0

.03)

(0.9

8)M

e-1

Mpi

Mdh

-1M

dh-2

23

42

34

56

12

34

56

72

34

1

0.02

0.98

0.13

0.66

0.19

0.02

0.01

0.98

0.02

1.00

Yor

kK

YC

C1.

000.

100.

750.

130.

020.

010.

950.

020.

021.

001.

000.

100.

720.

160.

030.

01Q

Y0.

980.

020.

010.

990.

030.

960.

010.

090.

740.

170.

010.

95C

I0.

030.

021.

00R

N1.

000.

080.

660.

220.

040.

880.

070.

051.

00E

ast

Eliz

abet

h1.

000.

130.

700.

150.

020.

020.

880.

040.

05C

H0.

010.

990.

011.

000.

080.

730.

150.

030.

940.

050.

011.

00N

C1.

000.

090.

640.

230.

030.

010.

92C

S0.

060.

021.

000.

990.

010.

180.

680.

110.

030.

94So

uth

Eliz

abet

h0.

030.

030.

990.

01P

P(1

.00)

(0.1

7)(0

.56)

(0.2

4)(0

.03)

(0.0

3)(0

.96)

(0.0

1)(1

.00)

0.04

0.95

0.14

0.68

0.14

0.04

0.01

0.01

0.01

0.93

NM

0.05

0.01

1.00

(1.0

0)(0

.08)

(0.6

7)(0

.23)

(0.0

2)(0

.97)

(0.0

3)(1

.00)

PC

0.02

0.98

0.15

0.66

0.16

0.03

0.01

0.93

0.05

0.01

0.99

0.01

(1.0

0)(0

.16)

(0.6

5)(0

.14)

(0.0

5)(0

.95)

(0.9

8)(0

.05)

(0.0

2)JC

1.00

0.17

0.58

0.18

0.05

0.01

0.02

0.95

0.03

0.01

1.00

(1.0

0)(0

.12)

(0.7

3)(0

.13)

(0.0

2)(0

.93)

(0.0

2)(0

.05)

(1.0

0)1.

000.

190.

570.

170.

070.

90C

M0.

031.

00(1

.00)

(0.0

9)(0

.63)

(0.1

9)(0

.09)

(0.9

2)0.

07(0

.08)

0.01

(1.0

0)1.

00SC

0.13

0.68

0.11

0.08

0.02

0.92

0.05

0.02

0.99

0.01

(1.0

0)(0

.14)

(0.5

4)(0

.29)

(0.0

3)(0

.97)

(0.0

1)(0

.02)

(1.0

0)C

F0.

010.

990.

120.

650.

160.

070.

920.

060.

021.

00(1

.00)

(0.1

4)(0

.59)

(0.2

2)(0

.05)

(0.0

2)(0

.90)

(0.0

5)(0

.03)

(1.0

0)0.

010.

990.

110.

630.

170.

090.

96R

S0.

030.

011.

00(0

.02)

(0.9

8)(1

.03)

(0.7

4)(0

.10)

(0.0

3)(0

.98)

(0.0

2)(1

.00)

1.00

0.12

0.64

0.17

0.07

0.92

0.07

0.01

AW

1.00

(1.0

0)(0

.25)

(0.4

9)(0

.19)

(0.0

7)(0

.94)

(0.0

2)(0

.04)

(1.0

0)P

gdP

gm

12

34

56

78

23

45

0.01

0.83

0.07

0.10

0.01

KY

Yor

k0.

99C

C0.

830.

080.

100.

010.

870.

13

Page 9: Genetic structure of Fundulus heteroclitus from PAH-contaminated ...

M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 203

Tab

le1

(con

tinu

ed)

Pgd

Pgm

12

34

56

78

54

32

QY

0.02

0.84

0.04

0.10

0.98

0.03

0.02

0.78

0.07

0.12

0.01

0.01

CI

0.03

0.97

RN

0.01

0.79

0.08

0.10

0.03

0.97

0.03

Eas

tE

lizab

eth

0.01

0.03

0.78

0.05

0.13

CH

0.01

0.91

0.08

0.01

0.83

0.04

0.11

0.01

0.03

0.98

NC

0.79

0.13

0.08

CS

0.99

0.01

0.75

0.10

0.14

Sout

hE

lizab

eth

0.07

0.92

PP

(0.8

2)(0

.11)

(0.0

7)0.

010.

01(0

.86)

(0.1

4)0.

010.

820.

090.

08N

M0.

090.

91(0

.03)

(0.8

1)(0

.03)

(0.1

3)(0

.80)

(0.2

0)0.

030.

780.

080.

11P

C0.

890.

11(0

.01)

(0.0

2)(0

.72)

(0.0

6)(0

.19)

(0.0

5)(0

.95)

0.01

0.80

0.04

0.15

JC0.

940.

06(0

.01)

(0.8

2)(0

.17)

(0.1

7)(0

.83)

CM

0.01

0.82

0.06

0.11

0.01

0.88

0.12

(0.7

2)(0

.03)

(0.2

5)(0

.02)

(0.1

2)(0

.86)

SC0.

010.

020.

770.

090.

110.

010.

910.

08(0

.01)

(0.0

2)(0

.83)

(0.0

8)(0

.06)

(0.0

1)(0

.82)

(0.1

7)0.

010.

780.

180.

030.

050.

95C

F(0

.76)

(0.1

0)(0

.12)

(0.0

2)(0

.95)

(0.0

5)0.

010.

800.

120.

060.

010.

130.

87R

S(0

.84)

(0.0

3)(0

.13)

(0.0

1)(0

.82)

(0.1

7)0.

800.

040.

140.

030.

07A

W0.

93(0

.01)

(0.8

1)(0

.06)

(0.1

2)(0

.92)

(0.0

8)

Dat

afo

rju

veni

les

are

plac

edin

pare

nthe

ses.

Page 10: Genetic structure of Fundulus heteroclitus from PAH-contaminated ...

M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209204

Table 2Genetic variability in adult mummichog from the York and Elizabeth Rivers

River Mean sample sizeLocality Mean number of Percentage of loci Meanpolymorphic heterozygosityper locus alleles per locus

Power Plant 32.9 (0.1) 2.1 (0.2)Elizabeth River–Southern 73.3 0.185 (0.052)Branch Juveniles (PP)

31.9 (0.1) 1.8 (0.3) 53.3New Mill Creek 0.163 (0.051)(NM)

32.0 (0.0) 2.0 (0.3) 66.7Paradise Creek 0.162 (0.048)(PC)

30.0 (0.0) 2.3 (0.2)Jones Creek 80.0 0.176 (0.043)(JC)

32.0 (0.0) 2.0 (0.3) 60.0Channel Marker 0.189 (0.054)2 (CM)

32.8 (0.2) 2.1 (0.3)Scuffletown 60.0 0.199 (0.059)Creek (SC)

29.0 (0.0) 2.0 (0.3) 60.0Crown Tank 0.189 (0.054)Farm (CF)

30.9 (0.1) 2.0 (0.2)Refueling 73.3 0.161 (0.043)Station (RS)

33.7 (0.2) 2.0 (0.3) 66.7 0.192 (0.058)Atlantic Wood(AW)

59.5 (0.5) 2.3 (0.3)Power Plant 80.0Elizabeth River–Southern 0.185 (0.052)(PP)Branch Adults

59.9 (0.1) 2.2 (0.3) 66.7New Mill Creek 0.189 (0.049)(NM)

59.7 (0.2) 2.2 (0.3)Paradise Creek 80.0 0.161 (0.042)(PC)Jones Creek 59.9 (0.1) 2.5 (0.3) 73.3 0.189 (0.059)(JC)

60.0 (0.0) 2.1 (0.3)Channel Marker 60.0 0.197 (0.055)2 (CM)Scuffletown 59.4 (0.6) 2.4 (0.3) 66.7 0.177 (0.051)Creek (SC)Crown Tank 59.9 (0.1) 2.1 (0.3) 66.7 0.170 (0.048)Farm (CF)

56.7 (0.8) 2.0 (0.3)Refueling 60.0 0.159 (0.045)Station (RS)Atlantic Wood 57.0 (0.0) 2.1 (0.3) 66.7 0.191 (0.052)(AW)

59.7 (0.2) 2.5 (0.3)Kings Creek 80.0York River Adults 0.165 (0.050)(KY)

59.9 (0.1) 2.4 (0.3) 73.3Carters Creek 0.171 (0.045)(CC)

59.8 (0.2) 2.5 (0.3)Queens Creek 86.7 0.180 (0.054)(QY)

59.7 (0.2) 2.4 (0.4) 66.7Catlett Islands 0.171 (0.048)(CI)Radio Tower 59.9 (0.1) 2.3 (0.3)Elizabeth River–Eastern 73.3 0.178 (0.052)North (RN)Branch Adults

59.8 (0.1) 2.5 (0.4) 73.3Campostella 0.175 (0.045)Heights (CH)North Canal 59.7 (0.3) 2.0 (0.3) 53.3 0.157 (0.048)(NC)

60.0 (0.0) 2.0 (0.3) 53.3Colonnas 0.144 (0.045)Shipyard (CS)

Standard errors for estimates are provided in parentheses.

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 205

Fig. 3. Idh-2 allele frequencies for loci with significant heterogeneity among adult mummichog at sites along the Elizabeth and YorkRivers. Consistent with Fig. 2, sites are arranged in order of increasing sediment PAH contamination. The common allele (3) andallele 4 are separated from the remaining rare alleles (‘other’=2, 5, 8 and 9 alleles in Table 1).

4. Discussion

Insufficient data are currently available to de-termine the possible impact of environmental con-taminants on the genetic qualities of naturalpopulations. Mummichog occur in discrete habi-tat patches and are thought to exhibit strong sitefidelity. These characteristics make them usefulfor testing the hypothesis that markedly differentlevels of environmental contamination have ge-netic consequences in exposed populations overrelatively short distances.

The microgeographic genetic structure of mum-michog on a spatial scale encompassing the Eliza-beth and York Rivers presents a mixed picturerelative to predictions that fish residing in con-taminated localities would be genetically distinctfrom fish in neighboring, uncontaminated habitat.Mummichog from the most heavily contaminatedlocality, AW, were distinct from fish at otherlocalities. Fish from the moderately contaminatedRS site were less distinct from other fish. Astatistically significant correlation was observedbetween genetic distance and sites differences incarbon-normalized contaminant concentrations(Fig. 4).

The minor genetic differentiation between theYork and Elizabeth Rivers suggested that refer-ence sites from York River are justifiable in stud-ies of AW mummichog. No evidence wasproduced that suggested the need to select anElizabeth River reference site in such studies.

Mummichogs from AW or other highly con-taminated sites were not genetically depauperate.All measures of genetic diversity (percent of locipolymorphic, number of alleles per locus, andheterozygosity) were comparable at AW andother localities in the Elizabeth and York Rivers.

Genetic distinction was heavily influenced bydifferences in the frequencies of allozymes in theAW mummichog compared with fish from else-where in the Elizabeth and York River landscape.Allozyme frequencies for the Idh-2 locus for themummichog at the AW locality were consistentlyand significantly distinct from other localities. Es-

Fig. 4. Plot of mummichog genetic distance, geographic dis-tance, and square root of absolute difference in organic car-bon-normalized PAH concentrations.

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209206

timates for migration rates of mummichog in thisstudy and previously published work (Brown andChapman, 1991; Smith et al., 1998) indicate sig-nificant amounts of gene flow at this geographicscale. High rates of migration act to homogenizeallozyme frequencies among fish from throughoutthe South Branch of the Elizabeth River. Thedistinct allele frequencies at the AW locality sug-gest that, although the potential for migration ishigh, migrants do not persist at the heavily con-taminated location or do not participate success-fully in reproduction. Although the mechanismsunderlying genetic differentiation of mummichogat heavily contaminated localities remains un-known, population genetic processes have beenimpacted by contamination at AW.

Measures of performance in organisms experi-encing environmental stress have been associatedwith metabolic efficiency, especially if resourcesare limited (Koehn and Bayne, 1989; Parsons,1997). Lactate dehydrogenase-B has been linkedto rates of cellular metabolism (Powers et al.,1991). Swimming endurance (DiMichele and Pow-ers, 1982b) and hatch times (DiMichele and Pow-ers, 1982a,) in mummichog differ among Ldh-Bgenotypes. Isozymes of glucosephosphate iso-merase have been related to organism perfor-mance during abiotic stress. Several studies havesuggested that the relationship occurs because thelocus regulates metabolite flux through glycolysis(Watt, 1985; Riddoch, 1993). In the present study,there were no significant correlations between al-lozyme frequencies at specific loci and PAH con-centration. However, genetic distance wascorrelated with site differences in organic carbon-normalized PAH concentration. The overall ge-netic divergence of the AW mummichog fromother mummichog collected in the Elizabeth Riverwas greater than divergences detected amongother sites. Mummichogs from RS, the secondmost contaminated site, were also genetically di-vergent from other Elizabeth River localities butless divergent than mummichog at AW (Fig. 4).

Kirchoff et al. (1999) reported a discontinuityin the frequency of esterase allozymes down-stream of a bleach kraft pulp mill in MiramichiEstuary, New Brunswick, Canada and arguedthat there was limited gene flow. Although there

are numerous impediments to movement of mum-michog along the Elizabeth River SouthernBranch, no isolation-by-distance was observedand migration of fish among localities is probablynot limited at this spatial scale. The model, Fst=1/(1+4Nem), where Ne is the effective populationsize and m is the migration rate, was used toestimate the number of migrates per generation(Wright, 1978). For the Elizabeth River SouthernBranch mummichog, the estimate of effective mi-gration (Nem) was 9.6 and 17.5 from the juvenileand adult data, respectively. Brown and Chapman(1991) estimated Nem to be 24 for mummichogalong an 8.4 km shoreline. Kirchoff et al. (1999)reported estimates of Nem ranging from 3 to 125for mummichog at five estuarine locations (be-tween locality distances of 4 and 100 km) alongthe east coast of New Brunswick, Canada. Per-haps due to the numerous physical impedimentsto movement, migration estimates for the Eliza-beth River mummichog were at the lower end ofthe other available estimates. Regardless, geneflow is sufficient to prevent differentiation due togenetic drift if Nem is greater than 1. The widedistribution of rare alleles also indicates gene flowbecause rare alleles are most likely to be dispersedby migration (Slatkin, 1985). Rare alleles wereobserved in mummichog throughout the ElizabethRiver localities, including at the AW site.

Several characteristics contribute to the percep-tion that mummichog is a low dispersal species.Adults exhibit strong site fidelity and summermovements have been reported to be 36 m in aDelaware Bay estuary (Lotrich, 1975). Mummi-chog eggs possess adhesive fibrils and so remainon vegetation (Able, 1984) or empty bivalve shells(DiMichele et al., 1986) onto which they weredeposited. Taylor and DiMichele (1982) reportthat larval mummichog remain in shallow inter-tidal pools for as many as 6–8 weeks beforejoining adults in daily movements on and off themarshes. Winter movement of mummichog hasbeen reported to be greater and might mitigateagainst isolation of populations (Able and Felley,1986).

Our findings regarding mummichog populationgenetic structure are consistent with several pub-

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 207

lished studies of other mummichog populations.Brown and Chapman (1991) used mtDNA toinvestigate microgeographic population geneticstructure in mummichog along 8.4 km of a Mary-land salt marsh. They reported no significant ge-netic differentiation among samples and sufficientgene flow to prevent population differentiationassociated with genetic drift. In a study of thenorthern form of mummichog in Connecticut,Leamon (1999) found a high degree of gene flowover a 41 km range. Additionally, he reportedthat, although population-level differentiation waslow between sites, the population structure andallele frequency at a subpopulation (as little as500 m) was a poor predictor for neighboringsubpopulations. Leamon reported that the relativefrequencies of alleles varied widely within thesmall spatial (i.e. 41 km) or temporal scales (i.e. 2years) examined. Both of these studies argue thatmummichog may exhibit greater movement thanpreviously reported over scales ranging from 8 to41 km. Relative to the use of York River refer-ence populations in studies of AW fish, there wasno rationale based on genetic divergence due togeographic distance for arguing that an ElizabethRiver reference site would be preferable.

Factors contributing to significant differences inallele frequencies between sites include differencesin female reproductive success and high rates oflarval or juvenile mortality, possibly exceeding99% (Meredith and Lotrich, 1979). In the Eliza-beth River Southern Branch survey of this study,mummichogs were collected over approximately12 km. Estimates of population genetic structurewere similar to those reported in earlier studies.Genetic subdivision, as measured by Fst, was twotimes greater in adult mummichog than in juve-niles. This observation is consistent with greatersite fidelity among juveniles and movement ofmummichog adults during the winter. Populationgenetic structure of mummichog appears to reflectdiversifying forces (variance in reproductive suc-cess and low probabilities of reaching reproduc-tive age) and homogenizing forces (adult seasonalmovement).

Introduction of contaminants into the environ-ment is often associated with reduction in popula-tion size or elimination of organisms due to

toxicant-induced mortality. Populations mightfurther be altered by differential elimination ofparticularly sensitive individuals. Organisms thatpersist in contaminated habitats can exhibit toler-ance of conditions that would be deleterious toindividuals from non-polluted areas. Contami-nant-induced disturbance in populations may re-sult in reduction in genetic diversity in impactedareas. Despite high levels of PAH contaminationat AW, mummichog had genetic diversity com-parable to that of fish at neighboring cleanerlocalities. However, genetic distance was signifi-cantly correlated with organic carbon-normalizedPAH concentrations. Genetic distance was greaterbetween mummichog of the AW locality andother sites than among other sites. Significantdifferences in allele frequencies for the Idh-2 locusand greater overall divergence at the AW localitysuggest that, although the potential for migrationis high, allelic distributions were not homoge-nized. The highly contaminated habitat at AWapparently disrupts predicted population geneticprocesses. Additional studies have been completed(Ownby et al., 2002) that show that differencesreflect genetically-based differential success of fishexposed to the sediments at the AW.

5. Conclusion

The level of contamination did appear to influ-ence the genetic structure of mummichog popula-tions within a landscape mosaic of sites varying insediment PAH concentrations The mummichog atAW were genetically distinct from the other sam-pled populations despite high rates of migrationamong sites. However, genetic diversity was notcorrelated with the level of PAH contamination.There was no evidence that isolation by distancecauses a problem relative to using reference popu-lations from the York River watershed in studiesof the AW population.

Acknowledgements

This research has been supported by a grantfrom the US Environmental Protection Agency’s

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M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209208

Science to Achieve Results (STAR) program. Theauthors acknowledge the valuable comments oftwo anonymous reviewers and Dr Mark E. Hahn.This paper is contribution number 2457 of theVirginia Institute of Marine Science, The Collegeof William & Mary.

References

Able, K.W., 1984. Variation in spawning site selection of themummichog, Fundulus heteroclitus. Copeia 1984, 522–525.

Able, K.W., Felley, J.D., 1986. Geographical variation inFundulus heteroclitus : tests for concordance between eggand adult morphologies. Am. Zool. 26, 145–157.

Armknecht, S.L., Kaattari, S.L., Van Veld, P.A., 1998. Anelevated glutathione S-transferase in cresote-resistantmummichog (Fundulus heteroclitus). Aquat. Toxicol. 41,1–16.

Bieri, R.H., Hein, C., Huggett, R.J., Shou, P., Slone, C.,Smith, C., Chih-Wu, S., 1986. Polycyclic aromatic hydro-carbons in surface sediments from the Elizabeth Riversubestuary. Int. J. Environ. Anal. Chem. 26, 97–113.

Brown, B.L., Chapman, R.W., 1991. Gene flow and mitochon-drial DNA variation in the killifish Fundulus heteroclitus.Evolution 45, 1147–1161.

Computerized American Practical Navigator, version 4.5,1992–1996, Nautical Technologies, Ltd.

Cashon, R.L., Van Beneden, R.J., Powers, D.A., 1981. Bio-chemical genetics of Fundulus heteroclitus (L.). IV. Spatialvariation in gene frequencies of Idh-A, Idh-B, 6-pgdh-A,and Est-S. Biochem. Genet. 19, 715–728.

DiMichele, L., Powers, D.A., 1982a. Ldh-B genotype specifichatching times of Fundulus heteroclitus embryos. Nature296, 563–564.

DiMichele, L., Powers, D.A., 1982b. Physiological basis forswimming endurance differences between LDH-B geno-types of Fundulus heteroclitus. Science 216, 1014–1016.

DiMichele, L., Powers, D.A., DiMichele, J.A., 1986. Develop-mental and physiological consequences of genetic variationat enzyme synthesizing loci in Fundulus heteroclitus. Am.Zool. 26, 201–208.

Greaves, J., Smith, C.L., Hale, R.C., 1991. Analytical protocolfor hazardous organic chemicals in environmental samples.Division of Chemistry and Toxicology, Virginia Instituteof Marine Science, School of Marine Science, College ofWilliam and Mary. Gloucester Point, VA. REF SH0001V48, no.131, p. 68.

Hillis, D.M., Moritz, C., Mable, B.K., 1996. Molecular Sys-tematics, second ed. Sinauer Associates, Inc, Sunderland,MA, USA.

Horness, B.H., Lomax, D.P., Johnson, L.L., Myers, M.S.,Pierce, S.M., Collier, T.K., 1998. Sediment qualitythresholds: estimates from hockey stick regression of liverlesion prevalence in English sole (Pleuronectes �etulus).

Environ. Toxicol. Chem. 17, 872–882.Huggett, R.J., Van Veld, P.A., Smith, C.L., Hargis, W.J.,

Vogelbein, W.K., Weeks, B.A., 1992. The effects of con-taminated sediments in the Elizabeth River. In: Burton, A.Jr (Ed.), Sediment Toxicity Assessment. Lewis Publishers,Boca Raton, pp. 403–429.

Kirchoff, S., Sevigny, J.-M., Couillard, C.M., 1999. Geneticand meristic variations in the mummichog Fundulus hetero-clitus, living in polluted and reference estuaries. Mar. Envi-ron. Res. 47, 261–283.

Koehn, R.K., Bayne, B.L., 1989. Towards a physiological andgenetic understanding of the energetics of the stress re-sponse. Biol. J. Linn. Soc. 37, 157–171.

Leamon, J.H., 1999. Gene flow and migration in populationsof Fundulus heteroclitus macrolepidotus located in south-eastern Connecticut. Ph.D. Thesis, University of Connecti-cut, p. 115.

Lotrich, V.A., 1975. Summer home range and movements ofFundulus heteroclitus (Pisces: Cyprinodontidae) in a tidalcreek. Ecology 56, 191–198.

Meredith, W.H., Lotrich, V.A., 1979. Production dynamics ofa tidal creek population of Fundulus heteroclitus (Lin-naeus). Estuar. Coast. Mar. Sci. 8, 99–118.

Nei, M., 1972. Genetic distance between populations. Am.Nat. 106, 238–292.

Ownby, D.R., Newman, M.C., Mulvey, M., Vogelbein, W.,Unger, M.A., Arzayus, 2002. L.F. Fish (Fundulus heterocli-tus) populations with different exposure histories differ intolerance of cresote-contaminated sediments. Environ.Toxicol. Chem., in press.

Parsons, P., 1997. Stress-resistance genotypes, metabolic effi-ciency and interpreting evolutionary change. In: Bijlsma,R., Loeschcke, V. (Eds.), Environmental Stress, Adapta-tion and Evolution. Birkhauser Verlag, Basel, Switzerland,pp. 292–305.

Powers, D.A., Lauerman, T., Crawford, D., DiMichele, L.,1991. Genetic mechanisms for adapting to a changingenvironment. Annu. Rev. Genet. 25, 629–659.

Riddoch, B.J., 1993. The adaptive significance of elec-trophoretic mobility in phosphoglucose isomerase (PGI).Biol. J. Linn. Soc. 50, 1–17.

SAS Institute Inc, 1989. SAS/STAT® User’s Guide Version 6,fourth ed. SAS Institute Inc, Cary, NC.

Schneider, S., Roessli, D., Excoffier, L., 2000. ARLEQUIN ver.2000: A software for population genetics data analysis.Genetics and Biometry Laboratory, University of Geneva,Switzerland.

Selander, R.K., Smith, M.H., Yang, S.Y., Johnson, W.E.,Gentry, J.R., 1971. Biochemical polymorphism and sys-tematics in the genus Peromyscus. I. Variation in theold-field mouse (Peromyscus polionotus). Stud. Genet. VI,University of Texas Pub., 7103, pp. 49–90.

Slatkin, M., 1985. Gene flow in natural populations. Annu.Rev. Ecol. Syst. 16, 393–430.

Smith, M.W., Chapman, R.W., Powers, D.A., 1998. Mito-chondrial DNA analysis of Atlantic coast, ChesapeakeBay, and Delaware Bay populations of the teleost Fundulusheteroclitus indicates temporally unstable distributions overgeologic time. Mol. Mar. Biol. Biotech. 7, 79–87.

Page 15: Genetic structure of Fundulus heteroclitus from PAH-contaminated ...

M. Mul�ey et al. / Aquatic Toxicology 61 (2002) 195–209 209

Smouse, P.E., Long, J.C., Sokal, R.R., 1986. Multiple re-gression and correlation extensions of the Mantel test ofmatrix correspondence. Syst. Zool. 35, 627–632.

Swofford, D.L., Selander, R.B., 1981. BIOSYS-1: a FOR-

TRAN program for the comprehensive analysis of elec-trophoretic data in population genetics and systematics.J. Hered. 72, 281–283.

Taylor, M.H., DiMichele, L., 1982. Spawning site selectionin Fundulus heteroclitus—utilization of empty musselshells. Am. Zool. 14, 928.

USEPA. 1999. Atlantic Wood Industries Superfund Site—March 1995 Fact Sheet. Accessed 10/20/99 1999. Webpage available from http://www.epa.gov/reg3hwmd/super/atl-wood/fs395.htm.

Vogelbein, W.K., Fournei, J.W., Van Veld, P.A., Huggett,R.J., 1990. Hepatic neoplasms in the mummichog Fun-dulus heteroclitus from a creosote-contaminated site. Can-cer Res. 50, 5978–5986.

Watt, W., 1985. Bioenergetics and evolutionary genetics: op-portunities for a new synthesis. Am. Nat. 125, 118–143.

Williams, C.A.H., 1994. Toxicity resistance in mummichog(Fundulus heteroclitus) from a chemically contaminatedenvironment. M.S. Thesis, College of William & Mary.

Workman, P.L., Niswander, J.D., 1970. Population studieson southwestern Indian tribes. II. Local genetic differen-tiation in the Papago. Am. J. Human Genet. 22, 24–29.

Wright, S., 1978. Evolution and Genetics of Populations.University of Chicago Press, Chicago.


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