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Transactions of the American Fisheries Society 118:317-330. 1989
Genetic Differentiation among Lake Trout StrainsStocked into Lake Ontario
CHARLES C. KRUEGER AND J. ELLEN MARSDENDepartment of Natural Resources. Fernow Hall, College of Agriculture and Life Sciences
Cornell University, Ithaca. New York 14853. USA
HAROLD L. KJNCAIDNational Fishery Research and Development Laboratory, U.S. Fish and Wildlife Service
Rural Delivery 4. Box 63, Wellsboro, Pennsylvania 16901, USA
BERNIE MAYCornell Laboratory for Ecological and Evolutionary Genetics
Department of Natural Resources, Cornell University
Abstract.—The lake trout Salvelinus namaycush is the focus of an international effort by fisherymanagement agencies to restore this once-native species to Lake Ontario. Evaluation of repro-ductive success and comparisons among stocked lake trout strains require genetic markers. Weused allozyme variation to make genetic comparisons among strains of lake trout stocked intoLake Ontario. Forty-two proteins presumed to be encoded by 102 loci were resolved electropho-retically; 18 loci were polymorphic. Among 16 samples from five principal origins (Finger Lakes,Great Lakes basin, northern New York-Adirondack, Manitoba, and mixed origins-Lake Ontario),the average observed heterozygosity was 0.029, and the proportion of polymorphic loci was 0.125.Significant differences occurred among the 16 samples at all 18 possible locus comparisons. Theaverage /%, value was 0.14. Hierarchical analysis indicated that the variation among samples withinan origin was greatest within the Great Lakes basin, which included the greatest number of samplesand represented the largest geographic area. Most variation observed among samples, however,occurred among origins. The 1983 and 1984 hatchery year classes produced from the 1978 Senecabrood stock did not differ significantly. The 1981 Seneca brood stock more closely resembled thesample of wild lake trout from Seneca Lake than progeny from the 1978 brood stock. The closeraffinity of the 1981 brood stock to wild Seneca lake trout may be due to the larger number of wildadults from Seneca Lake used to found these fish than was used to establish the 1978 brood stock.Siscowet ("fat") and "lean** lake trout from Lake Superior were significantly different from eachother; however, the level of variation between them was not greater than that among samples fromother origins, and thus did not support recognition of siscowet lake trout as a distinct subspecies.The Jenny Lake strain possessed a genetic affinity to the siscowet sample from Lake Superior.Historical reports about the origin of this strain suggest that siscowet lake trout from northernLake Michigan may have been used to found this strain. Therefore, the Jenny Lake strain mayserve as a gene source for the establishment of the siscowet lake trout in Lake Ontario. Thesubstantial differentiation among lake trout strains reported here supports the feasibility of usingallozyme markers to identify the parental sources of naturally produced young.
The lake trout Salvelinus namaycush was for- Pritchard 1931). By the late 1950s, wild, naturallymerly an abundant native species in Lake Ontario, spawned lake trout were presumed to be extinct;Commercial fishing for the species began as early catches in the 1960s were supported by hatcheryas the 1830s after the collapse of the fishery for plantings. The eventual demise of lake trout inAtlantic salmon Salmo salar (Pritchard 1931). Few Lake Ontario has been attributed to overharvest-records are available, however, that document ing, predation by the sea lamprey Petromyzon ma-commercial catches during the 1800s. During the rinus, cultural eutrophication, and habitat de-1900s, records of annual commercial catches of struction and degradation (Christie 1972, 1973).lake trout indicated a peak harvest of 515,000 kg Current management of lake trout focuses onin 1925 followed by a steady decline to less than several strategies intended to achieve the goal of500 kg in 1963 (Baldwin et al. 1979). The decline reestablishment of naturally reproducing, self-sus-of inshore stocks of lake trout probably began in tain ing populations (Schneider et al. 1983). Man-the mid-1800s due to overharvest by seining dur- agement actions are conducted cooperatively bying the autumn spawning season (Koelz 1926; provincial, state, and federal fishery agencies and
317
318 KRUEGER ET AL.
include environmental protection, angling regu-lation, sea lamprey control, and the stocking ofseveral strains of hatchery-reared yearling laketrout. The choice of strains for stocking has fol-lowed a strategy designed to maximize the geneticvariability among lake trout planted into LakeOntario (Schneider et al. 1983). As a result, laketrout from eight different sources (or strains) havebeen stocked into Lake Ontario since 1973. Fivestrains, Clearwater, Killala Lake, Lake Manitou,Seneca Lake, and Superior, composed 91% bynumbers of the lake trout stocked from 1973 to1986. In addition, gametes are collected annuallyfrom mature lake trout originally stocked as year-lings into Lake Ontario; eggs are fertilized andincubated in a hatchery, and resultant fish arereared and stocked back into the lake as yearlings.These fish are known as the Lake Ontario strainand represent a mixture of pure strain and inter-strain matings.
The management agencies conduct studies toevaluate strains that survive to maturity, spawn,and produce young (Schneider et al. 1983). Thestrains found to be most successful are then stockedin greater numbers because it is presumed thatthey possess better fitness for this environment.This presumption is based on historical, genetic,and life history evidence that indicates the exis-tence of genetically different strains. Historicalevidence from the Great Lakes region suggests thatimportant differences in spawning time, food pref-erence, and depth distribution have developed inlake trout strains (Brown et al. 1981; Goodier1981). Genetic differences among hatchery andwild populations have been reported on the basisof allozyme and mitochondria! DNA data (Dehr-ing et al. 1981; Dehring 1985; Grewe and Hebert1988; Ihssen et al. 1988). Life history and perfor-mance characteristics after stocking have also sug-gested that genetic differences exist among strainsin terms of survival (Plosila 1977), hatching andemergence times (Horns 1985), gas retention inthe swim bladder (Ihssen and Tait 1974), seasonalbathythermal distribution (Elrod and Schneider1987), and dispersal after stocking (Elrod 1987).
Evaluation of the survival of different stockedlake trout strains to maturity is relatively straight-forward through the use of well-established pro-cedures of marking hatchery fish (e.g., with codedwire tags) before stocking for later recovery withfishing gear such as trawls or gill nets. In contrast,procedures to determine which strains successful-ly spawn and produce viable young after stockingare not established. Young must be captured and
in some way compared to their potential parentalstrains. We describe the first of four steps in thedevelopment of a procedure to determine the strainorigins of lake trout young captured from LakeOntario: that of genetically characterizing theirpotential parental sources. The other steps wereto confirm the genetic basis of new allozyme poly-morphisms identified in the first step (Marsden etal. 1987), to capture young from a Lake Ontariospawning reef (Marsden et al. 1988), and to com-pare the allelic frequencies of the young to thoseof the potential parents through a mixed-stockanalysis procedure.
The primary purpose of our study was to com-pare genetically lake trout strains that are pro-posed to be, or have been, stocked into Lake On-tario. In addition to the primary purpose, weexamined the relatedness of some hatchery broodstocks to their wild progenitor populations. Thestudy was divided into two parts: first we con-ducted an extensive electrophoretic survey toidentify protein products of gene loci potentiallyuseful for strain characterization, and then wecompared allelic variation within and among laketrout strains. Specific comparisons were madeamong hatchery strains, between year classes ofprogeny from a single hatchery brood stock, be-tween hatchery brood stocks and their wild pro-genitor populations, and between "lean" and sis-cowet ("fat") lake trout from Lake Superior. Ourstudy documents the baseline data used to identifythe parental hatchery origins of naturally pro-duced lake trout young captured from a Lake On-tario reef.
Methods
Collections.— Sixteen collections of 40-94 fisheach from five principal origins (Finger Lakes,Great Lakes basin, Manitoba, northern New York-Adirondack, and mixed origins-Lake Ontario;Table 1) were analyzed electrophoretically. Four-teen of the collections were of yearling lake troutpropagated in hatcheries, one of the Finger Lakessamples was of brood-stock adults, and the Man-itoba sample was of a natural, unstocked popu-lation from Clearwater Lake.
The mixed origins-Lake Ontario samples (MX-ONT series) contained yearling lake trout propa-gated from gametes that were collected from feraladult lake trout originally stocked as age-1 fish inLake Ontario. These samples (MX-ONT83, MX-ONT84, MX-ONT85) represented a combinationof pure-strain crosses and intercrosses of seven
GENETIC DIFFERENTIATION AMONG LAKE TROUT STRAINS 319
TABLE 1.—Sample names, abbreviations, and origins of lake trout analyzed electrophorelically. The percentageof Lake Ontario stocking is given for only a single year class when the sample abbreviation designates a specificyear class. Samples unavailable for analysis (and their percentage of total Lake Ontario stocking) were Seneca 85(5.0%), Hills Lake (5.3%), Green Lake (0.7%), Mishibishu (0.1%), and Ontario 82 (0.1%). N.A. - not applicable.
LakeOntariostocking
Location and strain Year Parental 1973-1986or sample name Abbreviation class Sample type Hatchery gamete source number (%)
Seneca LakeSeneca 81Seneca 83Seneca 84Seneca Wild
Finger Lakes-New York
FL-SEN8IFL-SEN83FL-SEN84FL-SEN-W
1981198319841983
Brood-stock adultsBrood-stock progenyBrood-stock progenyWild progeny
Great Lakes Basin
Wild population1978 brood stock1978 brood stockWild population
Lake SuperiorGull Island ShoalSiscowetsSuperior
OtherJenny Lake
Killala LakeLake Manitou
GL-GISGL-SISGL-SUP
GL-JL
GL-KLGL-LM
1984Mixed1983
1984
19831983
Wild progenyWild adultsBrood-stock progeny
Hatchery progeny
Brood-stock progenyWild progeny
Wild populationN.A.1970 brood stock
Wild Lewis Lake malesx brood-stock females
1976 brood stockWild population
Mixed origins-Lake Ontario
1801,400
770>900
960N.A.
70
237> 1,600>9,000
Lake Ontario
Proposed0.44.52.3
NoneProposed
35.5
0.612.69.4
Ontario 83Ontario 84Ontario 85
Clearwater Lake
MX-ONT83MX-ONT84MX-ONT85
MN-CWL
198319841985
1983
Wild progenyWild progenyWild progeny
ManitobaWild progeny
Feral populationFeral populationFeral population
Wild population
564676
>400
700
0.30.80.7
21.7Northern New York-Adirondack
Lake GeorgeRaquette Lake
A-LGA-RL
19831983
Wild progenyWild progeny
Wild populationWild population
84>500
ProposedProposed
strains—Superior, Lake Manitou, Seneca Wild,Clearwater, Hills Lake, Green Lake, and Mishi-bishu.
Two brood stocks and their progenitor wildpopulation were represented among the FingerLakes samples (FD-SEN series; Table 1). The Sen-eca 81 sample (FL-SEN81) contained brood-stockadults of the 1981 year class, established with ga-metes from 34 females and 146 males collectedfrom Seneca Lake, New York (see Royce 1951 fordiscussion of this strain). The FL-SEN83 and FL-SEN84 collections were of two year classes (1983and 1984) of age-1 progeny propagated from the1978 Seneca brood stock, which was establishedfrom gametes from 7 female and 20 male wild fishcollected in Seneca Lake. The Seneca Wild sample(FL-SEN-W) consisted of hatchery yearlingspropagated from gametes collected directly frommore than 900 lake trout in Seneca Lake.
Both lean and siscowet lake trout were repre-sented in the Great Lakes collections from Lake
Superior (Table 1). The Gull Island Shoal sample(GL-GIS) originated from gametes collected fromwild lean lake trout captured from a spawning reefin the Apostle Islands region of Lake Superior(Swanson and Swedberg 1980). The siscowet laketrout sample (GL-SIS; see Eschmeyer and Phillips1965) contained adults captured from this sameregion of Lake Superior. Lean and siscowet formsof lake trout were identified by morphometric dif-ferences (Khan and Qadri 1971). Siscowet laketrout have been proposed for stocking into deep-water habitats of Lake Ontario. The Lake Superiorstrain fish were progeny of the 1970 hatchery broodstock. This brood stock was established in 1956with gametes collected from wild lean lake troutcaptured from Lake Superior out of the ApostleIslands (females) and from Copper Harbor, Mich-igan (males), regions (GL-SUP; J. Driver, Mich-igan Department of Natural Resources, personalcommunication). The GL-GIS and GL-SUPsamples represent lean forms that coexist in the
320 KRUEGER ET AL.
TABLE 2.—Tissue sources, locus designations, and eleclrophoresis buffers for lake trout proteins. Enzyme numbersare as recommended by IUBNC (1984). Isoloci are identified by a comma between their numeric designations.Reference to multiple loci in general is accomplished with parentheses around numeric designations. Tissues usedwere heart (H), white muscle (M), liver (L), kidney (K), and eye (E). Buffer systems used were as follows: A (Ayalaet al. 1973, as modified by May et al. 1979); C (Clayton and Tretiak 1972 as modified by May et al. 1979); C2(Clayton and Tretiak 1972, buffer adjusted to pH 7.0 with NaOH); H (Cardy et al. 1980); M (Markert and Faulhaber1965); R (Ridgway et al. 1970); S-4 and S-9 (Selander et al. 1971, tray buffer diluted 1:19 for gel buffer). Locussystems were designated as either monomorphic (M) or polymorphic (P).
Enzyme or other proteinAspartate aminotransferase (AAT)
Adcnosinc dcaminase (ADA)Alcohol dchydrogenase (ADH)Adcnylate kinase (AK)Aldolase (ALD)Crcatinc kinase (CK)
Diphorasc (cytochrome-/>5 reductasc; DIA)Esterase (carboxylesterase; EST)Fructose-bisphosphaiasc (FBP)
a-L-fucosidase (aFUC)Fumarate hydratase (FUM)Galactosaminidase (GAM)Glyccraldchyde-3-phosphatc dchydrogenase (GAPDH)Guaninc deaminase (GDA)
Glycerate-2-dehydrogenase (G2DH)a-o-glucosidase (oGLU)Glycerol-3-phosphate dehydrogenase (G3P)
Glucose-6 -phosphate isomerase (GPI)
Gluiamic-pyruvic transaminase(alanine aminotransferase: GPT)
Glutathione reductase (GR)0-Glucuronidase (0GUS)Hexosaminidase (jV-acetyl-/3-glycosaminidase; HA)Isocitratc dehydrogenase (IDH)
L-laciate dehydrogenase (LDH)
a-Mannosidase (aMAN)Malate dehydrogenase (MDH)
'Malic* enzyme (ME)
Mannosc-6-phosphate isomerase (MPI)Mcthylumbelliferyl phosphatase (MUP)
Nucleoside phosphorylase(purinc-nucleoside phosphorylase; NP)
Peptidase with glycyl-leucine (PEP-GL)Pcptidasc with Icucyl-alaninc (PEP-LA)Peptidase with leucyl-leucyl-leucine (PEP-LLL)Pcptidasc with phcnyl-alanyl-prolinc (PEP-PAP)Phosphogluconate dehydrogenase (PGD)Phosphoglycerate kinase (PGK)
Enzymenumber
2.6.1.1
3.5.4.41.1.1.12.7.4.34.1.2.132.7.3.2
1.6.2.23.1.1.13.1.3.11
3.2.1.514.2.1.2No number1.2.1.123.5.4.3
1.1.1.293.2.1.201.1.1.8
5.3.1.9
2.6.1.2
1.6.4.23.2.1.313.2.1.301.1.1.42
1.1.1.27
3.2.1.241.1.1.37
1.1.1.40
5.3.1.8No number
2.4.2.1
3.4.11-133.4.11-133.4.11-133.4.11-131.1.1.442.7.2.3
Numberof loci
5*
2121«4
32*2»
1»21»1«2
122»
3
3
2«224
5
16
3
13
2
23*3a
212
LocusAat- 1,2Aat-3Aat -4Aat-5Ada-(1.2)AdhAk-(1.2)Ald-2Ck-(1.2)Ck-(3.4)Dia-(1.2,3)Est-B'(l,2)Fbp-3Fbp-4aFuc-1Fum-1.2Gam-2Gapdh-4Gda-1Gda-2G2dhaGlu-(1.2)G3p-lG3p-3Gpi-1Gpi-(2,3)Gpt-(1.2)Gpt-3Gr-(1.2)0Gus-(l,2)Ha-(1.2)Idh-(l,2)ldh-(3,4)Ldh-(l.2)Ldh-3Ldh-4Ldh-5a- ManMdh-1.2Mdh-3,4Mdh-(5,6)Me-1.2Me-3MpiMup-lMup-(2,3)Np-1Np-2Pep-gl-(l,2)Pep~Ia-( 1.2.4)Pep-m-d.3.4)Pep-pap- 1.2PgdPgk-1Pgk-2
TissueMELMELMEMEMMLELMLMKLLLMMMMLM, LHLEMEMELELEMEMMLMEMEEELK,EMMM
PreferredbufferRRRS-9CRC2ARMCRS-4S-4HC2RS-4CCCS-9AARRRR4S-9RS-4ARMS-4MMAA, S-4ACCRCS-4C2MCCMCRCC
Type oflocus
systemPMpbMMc
MMMc
MMMMMMMPMMc
MMMMc
PMPMMMMc
MMM«MMPMc
MMMc
PMPMMPMMc
MMMMPMPM
GENETIC DIFFERENTIATION AMONG LAKE TROUT STRAINS 321
TABLE 2.—Continued.
Enzyme or other protein
Phosphoglucomutase (PGM)
Pyrophosphatc phosphohydrolase(inorganic pyrophosphatase; PP)
Protein (PRO)Superoxide dismutase (SOD)
Sorbitol dehydrogenase (SDH)Triose-phosphate i so m erase (TPI)
Enzyme Numbernumber of loci Locus
5.4.2.2
3.6.1.1
No number1.15.1.1
1.1.1.145.3.1.1
5
3
4»2
13
Pgm-JPgm-2Pgm-3.4Pp-(1.2)Pp-3Pro-(1.2.3t4)Sod-1Sod-2SdhTpi-(J.2.3)
TissueE,LE, LELMMMMLM
PreferredbufferA,CA,CAS-9S-9RRRRC2.A
Type oflocus
systemMc
PPMMMMPMM
a Additional zones of activity sometimes observed but did not meet criteria for designation as separate locus products.b Null ailele.c Potentially polymorphic—either three or fewer variants were observed. In all individuals tested the variant was too poorly
resolved to be classified.
wild with siscowets (represented by GL-SIS) inLake Superior (Goodier 1981).
The other Great Lakes samples included JennyLake (GL-JL), Killala Lake (GL-KL), and LakeManitou (GL-LM; Table 1). The Jenny Lake fishwere intercrosses between 124 females of a broodstock held at the Jackson (Wyoming) National FishHatchery and 113 wild males collected from Lew-is Lake, Wyoming. The Lewis Lake strain is pre-sumed to have originated from stockings of fishpropagated from gametes collected from northernLake Michigan in the late 1800s (Krueger et al.1983). Killala Lake, which served as the originalsource of the Killala Lake strain, is in a Lake Su-perior drainage in Ontario (49°05'N, 86°32'W).Lake trout in Lake Manitou, which is located onManitoulin Island in Lake Huron, have served asa hatchery gamete source since before 1960 (Hen-derson 1982).
Lake trout from the Adirondack region of NewYork have been proposed for stocking into LakeOntario (Table 1). Lake trout in Raquette Lake,New York (A-RL), are believed to represent pre-dominantly original Adirondack fish. In contrast,lake trout in Lake George, New York (A-LG),may be derived from a combination of native LakeGeorge fish plus Finger Lakes and other Adiron-dack lake trout as judged from past stocking rec-ords (T. Pelchar, New York State Department ofEnvironmental Conservation, personal commu-nication).
Electrophoretic procedures and locus designa-tions.—Immediately after capture, lake trout werefrozen and shipped on dry ice or in liquid nitrogento the laboratory where they were stored at — 80°Cuntil analysis. Protein expressions in eye, heart,
liver, kidney, and muscle tissues were examinedby horizontal starch gel electrophoresis and his-tochemical staining (May et al. 1979). Fifty-threeproteins (list available from the authors) were ex-amined for resolution of separate locus products.Optima] electrophoretic conditions were deter-mined by screening possible protein-buffer-tissuecombinations. Interpretation of banding patternswas based on previous inheritance studies of en-zyme variation in lake trout (May et al. 1979,1980; Hollister et al. 1984; Marsden et al. 1987).Alleles for all enzymes were named on the basisof relative electrophoretic mobilities of their pro-tein products; the most common ailele was usuallyassigned a value of 100.
Statistical procedures.—Conformance to Hardy-Weinberg expectations within samples was as-sessed by the fixation index (Fts, estimate of de-viation from Hardy-Weinberg proportions due todifferentiation within subpopulations) and log-likelihood G-test (Levene 1949; Nei 1977; Sokaland Rohlf 1981). Six isoloci that share alleles-Aat-1.2, Fum-1.2, Mdh-3,4, Me-1,2, Pep-pap-1,2,and Pgm-3,4—v/ert not examined for confor-mance to these expectations because the allelicvariation observed could have occurred at bothloci that encode each enzyme (locus abbreviationsare given in Table 2). For purposes of analysis,variation at these loci was assigned as describedbelow. Variation was equally split between eachlocus at Aat-1.2. Fum-1.2, Mdh-3.4, and Pgm-3,4because three doses of the rarer alternate ailele wereobserved in some samples. A null ailele was sus-pected at Pgm-3,4. When scored in an individual,this ailele was classified for analyses as a dose ofthe most common ailele in the individual (e.g.,
322 KRUEGER ET AL.
700,100.94,0 was changed to 100,100,100,94) orthe most common allele in the sample (e.g.,100,94,91,0 was changed to 100.100,94,91). Allvariation was assigned to the second locus of Me-1,2 because no fish with more than two doses ofthe rare allele were observed. The distribution ofvariation at Pep-pap-1,2 suggested a partitioning ofalleles between the isoloci (e.g., nearly all individ-uals in the wild Manitoba-Clearwater Lake sam-ple, MN-CWL, were of the 100,100.179,179 phe-notype). Therefore, Pep,pap-2 was assigned twodoses of 179. and all other gene doses were as-signed to Pep-pap-1. The rare fish without twodoses of 179 were designated to be variable atPep-pap-2.
Comparisons of genetic differences among sam-ples were assessed with heterozygosities, the (7-test,F5t values (estimate of deviation from Hardy-Weinberg proportions due to differentiation amongsubpopulations), and genetic distance coefficients.Observed heterozygosities and their variance es-timates were calculated as described by Nei andRoychoudhury (1974) and Nei (1977). Allelecounts by locus were compared statistically bycontingency table analysis with the (/-statistic (So-kal and Rohlf 1981). A probability level of P <0.05 was used to reject the null hypothesis thatgenetic differences among samples were not sig-nificant. The significance level was modified toaccount for the increase in type-I error when mul-tiple tests of the same hypothesis were made (Coo-per 1968). Heterogeneity tests over all sampleswere partitioned by tests of subsets of samplesbecause (/-values are additive. This hierarchicalapproach to the analysis was also conducted withan Fsl analysis (Wright 1965; Nei 1977) to parti-tion genetic variation within and among popula-tions. Genetic distances (Nei 1972) were calculat-ed over polymorphic loci only and subjected tounweighted pair-group method cluster analysis(Sneath and Sokal 1973). Analyses of the data wereperformed by use of "Genes in Populations," amicrocomputer program designed by B. May andC. C. Krueger and written by W. Eng, CornellUniversity.
ResultsLoci Resolved and Allelic Variation
Forty-two proteins presumed to be encoded by102 loci were resolved electrophoretically (Table2). Eighteen polymorphic loci with frequencies ofalternative alleles of at least 0.05 were observedamong the 16 collections (all variation at the Me-
1,2 loci was assigned to Me-2\ Table 3). Severaladditional loci that were provisionally determinedto be polymorphic had too few variants to warrantfurther data analysis (Table 2). Of the 18 poly-morphic loci, 12 consisted of six pairs of isoloci.Null alleles were suspected to occur at Pgm-3,4.Average observed heterozygosity per locus was0.029 (range, 0.024-0.039) and the proportion ofpolymorphic loci was 0.125 (range, 0.098-0.157)across all samples. Heterozygosity was highest inthe Raquette Lake sample (A-RL) and lowest inthe 1981 Seneca brood-stock sample (FL-SEN81).No significant deviations from Hardy-Weinbergproportions were observed. Average F^ across allsamples was -0.016; 12 of 16 samples had neg-ative average values across loci; this indicates aslight tendency towards heterozygote excesses.
Differences among SamplesSignificant differences occurred among the 16
samples at all 18 possible locus comparisons (P <0.01; Table 4). The total (/-statistic summed overall loci also indicated significant differences amongsamples (P < 0.01). More than 71% of the total(7-value was attributable to differences amongorigins; the rest was contributed by differencesamong samples within origins (Table 4). Signifi-cant differences were evident among sampleswithin each of four origins: Finger Lakes, GreatLakes, mixed (Lake Ontario), and Adirondack (P< 0.01; Table 4). A test was not possible withinthe Manitoba origin because only one sample wasexamined. Variation among Great Lakes samplescontributed more to the total G-value (22%) thanthe differences observed within any other origin.The Great Lakes origin also was represented bymore samples and covered a larger geographic areathan other origins.
Based on F5t values over all loci, the averageamount of genetic variation observed among sam-ples within origins was about 2.5% of the totalvariation (Table 4). The variation among originswas considerably higher and made up 11.1% ofthe total variation. Thus the average lake troutsample contained about 86.4% of the total vari-ation observed in this study.
Higher frequencies of alternative alleles at cer-tain loci were diagnostic for samples within anorigin or for a particular origin (Table 3). FingerLakes and Adirondack samples were distinguish-able from others due to a higher frequency of allele7/5 at Me-1,2. Variations at Aat-1,2, Pgm-3,4,and Sod-2 were higher in Manitoba and Adiron-dack collections than in those from other origins.
GENETIC DIFFERENTIATION AMONG LAKE TROUT STRAINS 323
Pgk-1 and Mup-1 variations were diagnostic forthe Manitoba sample (MN-CWL). The higher fre-quency of allele 144 at Mdh-3,4 set apart the Ad-irondack fish from other samples. The GL-KL(Killala Lake) and GL-LM (Lake Manitou) sam-ples were distinguishable from other samples ofGreat Lakes origins and from other origins on thebasis of variation at Fum-1.2. The levels of vari-ation at Pep-pap-1,2 were diagnostic among sam-ples from Lake Superior and also differentiatedthe Manitoba sample from other origins.
Cluster analysis of Nei's genetic distance coef-ficients distinguished three basic groupings by or-igin: the Adirondack samples (group 1), FingerLakes and mixed origin samples (group 2), andmost samples from the Lake Superior basin (group3; Table 5; Figure 1). The genetic affinity of theFinger Lakes and mixed origin samples was notsurprising because many of the adult lake troutcaptured from Lake Ontario and used as a gametesource for the Lake Ontario strain were wild Sen-eca strain fish stocked in past years (C. P. Schnei-der, New York State Department of Environmen-tal Conservation, personal communication). TheMN-CWL (Manitoba) and GL-LM (Lake Mani-tou) samples showed little genetic affinity to othersamples. The GL-JL (Jenny Lake) and GL-SIS(siscowet) grouped together but were not closelyrelated to any other samples. The average geneticdistance between collections was 0.029 (range,0.001-0.122; Table 5).
Differences between Hatchery Year ClassesNo significant differences were observed in al-
lelic variation between FL-SEN83 and FL-SEN84samples of yearlings derived from the 1978 Senecastrain brood stock (Table 4). The total (7-valuesummed over all loci also did not indicate differ-ences between the two year classes (P > 0.28; Ta-ble 4). The genetic distance between these twoSeneca samples was as low as any observed in thisstudy (genetic distance = 0.001; Table 5).
Brood Stocks versus Wild Progenitor PopulationsTwo Seneca strain (Finger Lakes) brood stocks
and the Superior strain brood stock (Great Lakes)were compared to their wild progenitor popula-tions. The 1978 Seneca brood stock was repre-sented by the pooled sample of offspring, FL-SEN83 and FL-SEN84, and compared to hatch-ery yearlings propagated from gametes collecteddirectly from Seneca Lake (FL-SEN-W). Allelicvariation at two loci, Mup-1 and Pgm-2, was sig-
nificantly different between the pooled sample andFL-SEN-W (P < 0.05; Table 4). The primary dif-ferences in the pooled sample compared to theFL-SEN-W sample were attributable to the ab-sence of allele -140 at Mup-1 and the increasedfrequencies of allele 115 at Me-2 and allele 150 atPgm-2 (Table 3). However, the actual amount ofallelic differences at these three loci between thesesamples was small (±0.03-0.07). The total G-val-ue summed over all loci was significantly different(P < 0.01; Table 4); average heterozygosity wasnot significantly different, and the proportion ofpolymorphic loci was similar between these sam-ples.
The 1981 Seneca brood stock (FL-SEN81) wascompared to the FL-SEN-W sample describedabove. No significant differences were observed atany loci nor indicated by the total (7-value summedover all loci (Table 4). Values for heterozygosityand percent of polymorphic loci were similar be-tween these samples (Table 3).
The 1970 Superior strain brood stock was rep-resented by the 1983 year class (GL-SUP) and wascompared to the 1984 sample of hatchery year-lings propagated from gametes collected near GullIsland Shoal, Lake Superior (LS-GIS). No signif-icant differences occurred among the polymorphicloci (Table 4). The total G-value summed over allloci, however, indicated significant differences be-tween the two samples (P < 0.01). The largestallelic differences occurred at Aat-1.2, Mup-1, andPep-pap-1.2 (Table 3). Heterozygosities were notsignificantly different and the percent of polymor-phic loci was identical between these samples (Ta-ble 3).
Lean versus Siscowet Lake TroutThe two Lake Superior samples of lean lake trout
(GL-SUP and GL-GIS) were pooled and com-pared to the siscowet lake trout sample collectedfrom Lake Superior (GL-SIS). Allelic variation atthree loci was significantly different between thesesamples (Pgm-3,4 and Pep-pap- 7; Table 4). Dif-ferences between these samples were especiallylarge at Pep-pap-1 (P < 0.001). The frequency ofthe 138 allele (0.23) was much higher in the sis-cowet lake trout sample than in the lean lake troutcollections (average 0.03; Table 3). The totalG-value summed over all loci also indicated sig-nificant differences between these samples (P <0.01; Table 4). Values for heterozygosity and per-cent of polymorphic loci were similar betweenthese samples (Table 3).
324 KRUEGER ET AL.
TABLE 3.—Allelic frequencies observed at lake trout isozyme loci described in Table 2. No statistically significantdeviations occurred from Hardy-Weinberg proportions (P > 0.05). Sample abbreviations are defined in Table 1.
tr:__ . -,— KI»... X/--1, Great Lakes Basin
FL-Locusor SEN81statistic Allclc(AT*40)
Aat-1.2
Fum-1.2
G3p-l
Gpi-1
Ldh-3
Mdh-3.4
Me- 1, 2
Mup-I
Pep-pap-1.2
Pgk-1
Pgtn-2
Pgm-3.4
Sod-2
Average F^Average hetero-
zygositySE
8510011510090
10035
100200100
78too144100115100140100179138100167100150100
9491
10085
Proportion of poly-morphic loci
0.320.680.000.940.060.930.071.000.001.000.001.000.000.830.170.980.020.110.860.031.000.001.000.000.670.330.001.000.00
-0.053
0.024(0.008)
0.098
FL~SEN83
(N = 94)
0.310.690.000.900.100.890.111.000.001.000.001.000.000.830.171.000.000.110.850.041.000.000.930.070.650.350.000.980.02
-0.034
0.029(0.010)
0.108
FL-SEN84
(N = 80)
0.290.710.000.890.110.910.091.000.001.000.001.000.000.890.111.000.000.070.860.071.000.000.940.060.670.330.00LOO0.000.183
0.026(0.009)
0.098
FL-SEN-W(N = 80)
0.340.660.000.880.120.930.071.000.001. 000.000.990.010.930.070.970.030.080.890.031.000.000.990.010.610.390.001.000.000.022
0.025(0.008)
0.118
Lake SuperiorGL-G1S(N = 80)
0.360.630.010.910.091.000.00LOO0.00LOO0.000.980.020.990.010.990.010.210.750.040.990.01LOO0.000.460.420.120.940.06
-0.026
0.028(0.010)
0.137
GL-SIS(AT = 76)
0.310.690.000.900.10LOO0.00LOO0.00LOO0.000.980.020.940.060.900.100.200.570.230.990.01LOO0.000.440.540.020.990.010.020
0.029(0.010)
0.137
GL-SUP</V = 64)
0.470.510.020.900.10LOO0.000.990.010.980.020.980.020.990.010.930.070.180.800.02LOO0.00LOO0.000.460.480.060.940.06
-0.043
0.030(0.010)
0.137
GL-JL(N = 80)
0.480.490.030.900.10LOO0.00LOO0.00LOO0.000.990.010.990.011.000.000.050.600.35LOO0.00LOO0.000.410.560.030.910.09
-0.096
0.026(0.010)
0.098
OtherGL-KL
(AT - 80)
0.470.530.000.740.26LOO0.000.990.01LOO0.000.990.010.960.04LOO0.000.180.800.02LOO0.00LOO0.000.410.520.070.990.01
-0.009
0.032(0.011)
0.127
GL-LM(AT - 80)
0.230.770.000.790.21LOO0.000.920.08LOO0.000.960.04LOO0.000.960.040.350.590.06LOO0.00LOO0.000.640.340.020.980.02
-0.051
0.027(0.009)
0.118* Estimate of deviation from Hardy-Weinberg proportions within subpopulations.
DiscussionLoci Resolved and Allelic Variation
We made a special effort to examine more lociin this study than in past lake trout studies becauseof the low levels of genetic variability within andamong samples reported in previous studies(Dehring et al. 1981) and the management im-portance of developing the capability to differen-tiate stocks genetically and to identify naturallyproduced young. An additional 52 loci were re-
solved over those reported by Dehring et al. (1981);of these, 13 were polymorphic, and three of thepolymorphic loci, Pgk, Mup-1. and Pep-pap-1,2,were especially diagnostic for stock identification.
The genetic variability observed in lake troutpopulations is difficult to compare among studiesdue to the differences in numbers of loci and typesof proteins examined and the different geographicorigins of samples analyzed. Populations of laketrout in Lake Superior have been reported to con-tain an average heterozygosity of 0.015, based on
GENETIC DIFFERENTIATION AMONG LAKE TROUT STRAINS 325
TABLE 3.—Extended.
Ontario-mixed originsLocus orstatistic Allclc
Aat-1,2
Fum-1,2
G3p-l
Gpi-1
Ldh-3
Mdh-3.4
Me- 1,2
Mup-1
Pep-pap-1.2
Pgk-l
Pgm-2
Pgm-3.4
Sod-2
Average f'ua
Average hetero-zygosity
SEProportion of poly-
morphic loci
85100115100
9010035
100200100
781001441001151001401001791381001671001501009491
10085
MX-ONT83(A' = 80)
0.330.670.000.840.160.970.031.000.00
0.990.010.990.010.900.100.960.040.090.880.031.000.000.990.010.540.450.010.980.02
-0.022
0.027(0.009)
0.157
MX-ONT84(N = 80)
0.330.670.000.860.140.970.030.990.01
1.000.000.990.010.960.040.960.040.130.830.040.980.021.000.000.480.490.030.980.020.078
0.027(0.009)
0.147
MX-ONT85(N = 80)
0.330.670.000.880.120.950.050.990.01
1.000.000.970.030.940.060.990.010.100.850.051.000.001.000.000.470.460.070.970.03
-0.024
0.027(0.009)
0.127
ManitobaMN-CWL(N = 78)
0.520.470.010.910.091.000.000.890.11
0.990.010.950.051.000.000.880.120.510.490.000.860.141.000.000.360.560.080.740.26
-0.101
0.034(0.010)
0.147
AdirondackA-LG
(#=60)0.570.430.000.990.011.000.001.000.00
1.000.000.870.130.470.530.950.050.100.880.021. 000.000.990.010.340.650.010.730.27
-0.028
0.032(0.010)
0.118
A-RL(N = 60)
0.730.270.000.990.011.000.001.000.00
0.930.070.670.330.620.380.940.060.030.870.101.000.001.000.000.350.620.030.530.47
-0.068
0.039(0.012)
0.127
50 loci (Dehring et al. 1981). Lake trout popula-tions from Manitoba to eastern Ontario have beenreported to contain an average heterozygosity of0.047, based on 28 loci (Ihssen et al. 1988). Ourstudy is geographically most similar to the Man-itoba-Ontario study except that 74 additional lociwere examined. Average observed heterozygositywas 0.029 in our study, or 38% less than that re-ported by Ihssen et al. (1988). The average het-erozygosity among just the Lake Superior originsamples in our study was 0.029 (GL-GIS,
SIS, GL-SUP; Table 1) or about double that es-timated for the same geographic region by Dehr-ingetal. (1981).
Differences among SamplesDifferences among samples within an origin were
greatest for the six Great Lakes basin samples (Ta-ble 4); however, this result was not surprising be-cause the number of Great Lakes samples waslarger than the number of samples from otherorigins and covered a broader geographic area.
326 KRUEGER ET AL.
TABLE 4.—Average Fst* and G-test statistics calculated over 18 polymorphic allozyme loci observed in samplesof lake trout. Asterisks denote P < 0.01** for the heterogeneity G-test; for values without asterisks, P > 0.05.Sample abbreviations are defined in Table 1.
ComparisonFinger Lakes origin
Seneca brood-stock samples (A)
Pooled Seneca brood stocks versus FL-SEN-W (B)
Combined6 (A) and (B)
FL-SEN83 versus FL-SEN84C
Pooled FL-SEN83 and -SEN84 versus FL-SEN-WC
FL-SEN81 versus FL-SEN-W
Great Lakes originLake Superior samples (C)
GL-SUP versus Gb-GIS
Pooled GL-SUP and -GIS versus GL-SIS
Other Great Lakes samples (D)
Pooled Lake Superior versus pooled "other" samples (E)
Combincdb (C). (D), and (E)
Mixed origins (MX-ONT83, -ONT84. -ONT85) (F)
Adirondack origin (A-LG and A-RL) (G)
Among origins (including MN-CWL) (H)
Combined6 (A) through (E)
Average Fst* overall loci (range)
0.003(0.000-0.023)
0.003(0.000-0.018)
0.005(0.000-0.029)
0.002(0.000-0.011)
0.004(0.000-0.024)
0.005(0.000-0.026)
0.030(0.000-0.106)
0.007(0.(XXM).028)
0.027(0.000-0.107)
0.088(0.000-0.288)
0.004(0.000-0.019)
0.064(0.000-0.205)
0.004(0.000-0.013)
0.027(0.000-0.061)
0.111(0.016-0.318)
0.103(0.026-0.252)
TotalG
39.6
33.9**
73.5**
15.8
40.2**
14.7
228.3**
46.5**
181.8**
530.3**
92.3**
850.8**
71.4**
101.0**
2,700.1**
3,796.9**
Number of lociwith significant
G-valuedf
28
15
45
13
15
14
44
21
22
40
22
110
42
24
96
360
P<Q.Q5 I
0
0
1
0
2
0
2
0
3
11
4
11
0
5
18
18
0
0
1
0
0
0
2
0
1
8
3
10
0
3
18
18
• Estimate of deviation from Hardy- Weinberg proportions due to differentiation among samples.b Average for Fst\ sum for G.c Nonorthogonal comparisons not included in totals.
Genetic affinity was observed between two LakeSuperior samples (GL-GIS and GL-SUP) and asample representing lake trout from Killala Lakein the Lake Superior drainage (GL-KIL; Figure 1;Table 5). The Gl^LM sample, representing laketrout from an island in northern Lake Huron,showed little genetic similarity to any other sam-ple. The two other samples of Great Lakes origin,GL-SIS and GL-JL, were grouped together on thebasis of cluster analysis of genetic distances butwere comparatively unrelated to other samples(Figure 1).
Based on the above discussion, we infer that thesiscowet lake trout of Lake Superior (GL-SIS) may
be related to the Jenny Lake fish of Lake Michiganorigin (GL-JL). Their genetic affinity was duepartly to their similarity in allelic frequencies atPep-pap-1,2 (Table 3). The Jenny Lake strainprobably originated in the late 1800s from theBeaver Island region of Lake Michigan (Kruegeret al. 1983). In a historical review of lake troutstocks in Lake Michigan, Brown et al. (1981) citedseveral records of the occurrence of siscowet laketrout from these waters. For example, Smith andSnell (1891) stated that the "siscowet or deepwatervariety of the trout" occurred "throughout thenorthern portion of the lake... especially betweenthe Manitou and Beaver Islands. In some places
GENETIC DIFFERENTIATION AMONG LAKE TROUT STRAINS 327
TABLE 5.—Nei's (1972) genetic distances between lake trout samples. Values are based on data from 18 poly-morphic allozyme loci. Sample abbreviations are denned in Table 1.
_FL- FL- SEN- GL- Gb- GL- GL- GL- MX- MX- MX- MN-
Sample SEN83 SEN84 W CIS SIS SUP GL-JL KL LM ONT83 ONT84 ONT85 CWL A-LG A-RLFL-SEN81FL-SEN83FL-SEN84FL-SEN-WGL-GISGL-SISGL-SUPGL-JLGL-KLGL-LMMX-ONT83MX-ONT84MX-ONT85MN-CWLA-LG
0.001 0.001 0.002 0.011 0.0250.00 1 0.002 0.010 0.023
0.001 0.011 0.0240.009 0.024
0.011
0.0120.0120.0140.0090.0030.017
0.0400.0380.0370.0360.0240.0130.026
0.0190.0170.0190.0130.0080.0210.0050.028
0.0260.0240.0250.0270.0150.0160.0250.0460.026
0.0040.0040.0040.0010.0080.0220.0070.0340.0080.026
0.0060.0060.0060.0030.0040.0150.0040.0280.0060.0210.001
0.0060.0050.0050.0020.0040.0170.0050.0280.0080.0250.0010.001
0.0640.0630.0710.0640.0310.0320.0320.0520.0380.0330.0590.0470.053
0.0390.0400.0480.0410.0410.0540.0320.0540.0390.0840.0360.0370.0350.069
0.0730.0750.0820.0720.0680.0830.0540.0650.0650.1220.0670.0680.0640.0890.016
fully half the trout taken are of this kind." Clearlythe possibility exists that the gametes collected tooriginate the Jenny Lake strain may have includedthose from siscowet lake trout. Siscowet whencompared to lean lake trout have been reportedto live in deeper waters (Van Oosten 1944), tohave different taxonomic characters (Khan andQadri 1970), and to have a higher fat content(Eschmeyer and Phillips 1965). Comparison ofsiscowet lake trout characteristics to those ob-served in Jenny Lake lake trout after stocking intoLake Ontario would provide additional evidenceto assess their proposed relatedness. If Jenny Lake
0.06 0.05 0.04 0.03 0.02 0.01 0.00
GENETIC DISTANCEFIGURE 1.—Dendrogram generated by cluster analysis
of Nei's (1972) genetic distance coefficients calculatedbetween lake trout samples from 18 polymorphic locionly. See Table 1 for definition of abbreviations.
fish are related to siscowets, the continued stock-ing of the Jenny Lake strain may encourage thedevelopment of populations that occupy greaterdepths and thus a different niche than that occu-pied by other strains (Elrod and Schneider 1987).
The Lake George (A-LG) and Raquette Lake(A-RL) samples of Adirondack origin showed lit-tle genetic similarity to Seneca strain samples (FL-SEN series; Figure 1). The A-LG sample was alsostatistically different from the A-RL sample; how-ever, these two Adirondack samples were moreclosely related to each other than to other samples(Table 4; Figure 1). Lake George has resident na-tive lake trout and has also been stocked withhatchery-reared fish. More than 600,000 Senecastrain lake trout of various sizes were stocked intoLake George between 1959 and 1970. In addition,more than 550,000 lake trout of Adirondack or-igin, mostly Raquette Lake strain, were stockedinto Lake George between 1959 and 1979 (Pel-char, personal communication). As a result, weanticipated but did not observe genetic affinitybetween the Seneca series and Lake George sam-ples. Raquette Lake has always had a large nativepopulation of lake trout that has served as a sourceof gametes for hatchery propagation for more than40 years. Haskell et al. (1952) reported that 1,000yearlings of Seneca origin were stocked into thelake in 1942. No Seneca strain lake trout are knownto have been stocked in Raquette Lake in the last30 years (Pelchar, personal communication). Likethe Lake George fish, the Raquette Lake sampleshowed little genetic affinity to the SEN series ofsamples. The genetic similarity observed betweenLake George and Raquette Lake samples could
328 KRUEGER ET AL.
indicate a genetic contribution from stocked fishof Adirondack origin in Lake George. Plosila(1977) reported that the survival of Adirondacklake trout stocked into Adirondack waters wasabout 16 times greater than that of Seneca strainfish stocked into the same waters. Similarly, Has-kell et al. (1952) reported that, based on recoveriesfrom spawning grounds in Raquette Lake of anequal stocking of Raquette Lake and Seneca Lakelake trout made several years earlier, hatchery fishof Raquette Lake origin were more than 23 timesmore common than Seneca lake trout. The lackof genetic affinity between the Seneca and LakeGeorge lake trout and the weak similarity betweenRaquette Lake and Lake George fish are probablyattributable to these differences in survival afterstocking.
Differences between Hatchery Year ClassesThe level of allelic variation was not different
between the 1983 and 1984 year classes of theSeneca strain that were progeny of the 1978 broodstock. Year-to-year stability in allelic variation isan important assumption when these data are usedfor parental origin analysis of naturally producedyoung (for further discussion see Marsden 1988).
Brood Stocks versus Wild Progenitor PopulationsDifferences between hatchery brood stocks and
their wild progenitor populations were only minor(Table 4). The 1981 Seneca brood stock (FL-SEN81) appeared more similar to the wild SenecaLake sample (FL-SEN-W) than to the pooledprogeny sample (FL-SEN83 and FL-SEN84) fromthe 1978 Seneca hatchery population. The closeraffinity of the 1981 brood stock may be due to theuse of a larger number of wild Seneca Lake adultsto found this brood stock (34 females and 146males) than were used to establish the 1978 hatch-ery population (7 females and 20 males). Sub-stantial genetic changes may occur between broodstocks and their wild progenitor populations sim-ply due to genetic drift and founder effects inducedby the use of too few parents. In general, 30 to100 pairs of wild fish (1:1 sex ratio) should be usedto establish hatchery brood stocks (Ryman andStahl 1980; Krueger et al. 1981; Allendorf andRyman 1987). Every effort possible should be usedto follow these guidelines when new hatchery pop-ulations are established. Due to the long matu-ration time of lake trout (about 6 years) and thelong period during which a brood stock can beused after maturity as a gamete source (5-10 years),most brood stocks currently used to propagate lake
trout for stocking into Lake Ontario are only oneor two generations removed from the wild. As aresult, domestication of these hatchery popula-tions due to intentional or unintentional selectionwill not be as rapid as in other salmonid species.
Lean versus Siscowet Lake TroutSome authors have recognized the siscowet lake
trout as a separate species (Agassiz 1850) or as asubspecies (Jordan and Gilbert 1882; Khan andQadri 1970) of lake trout. The results of our studyindicated that, based on 102 loci, the level of dif-ferentiation within Lake Superior (Fsl = 0.030) isnearly the same as that observed between the Ad-irondack samples (Fst = 0.027) and less than thatobserved among the three other samples of GreatLakes origin (Fsl = 0.088; Table 4). These resultsand the lack of fixed allelic variation provide littleevidence to support the recognition of siscowetlake trout as a separate subspecies.
In conclusion, substantial genetic differencesamong strains of lake trout were observed in ourstudy. Clearly, the strategy chosen by the fisherymanagement agencies to stock genetically differentgroups of lake trout into Lake Ontario has beensuccessfully implemented. A key to evaluating therestoration program for this species is to deter-mine which of the strains reproduce successfully.Such information would be helpful to guide de-cisions about the best strains to stock to achievethe program's goals. The substantial level of dif-ferentiation documented by our study supportsthe feasibility of using allozyme markers to iden-tify the parental sources of naturally producedyoung among lake trout strains.
AcknowledgmentsWe thank D. Ostergaard and H. Zumstein of
the Allegheny National Fish Hatchery for theirlong-term cooperation and assistance in this proj-ect. We also thank W. Miller, T. Pelchar, and G.Seeley of the New York State Department of En-vironmental Conservation for the Adirondacksamples and detailed stocking records, B. Swan-son of the Wisconsin Department of Natural Re-sources for the samples of Lake Superior origin,and J. M. Byrne and K. Steele of the Ontario Min-istry of Natural Resources for helping us obtainthe Killala Lake and Lake Manitou samples.Technical assistance in the laboratory was provid-ed by C. Azar and K. Henley. Special thanks toS. P. Gloss, formerly of the New York Fish andWildlife Cooperative Research Unit, for coordi-
GENETIC DIFFERENTIATION AMONG LAKE TROUT STRAINS 329
nating this work between the unit and CornellUniversity and for his support throughout thisproject. P. Grewe (Cornell University) and T. Toddof the U.S. Fish and Wildlife Service (USFWS)provided several helpful comments on earlierdrafts. This work was supported by the USFWSthrough research work order 6 from the New YorkFish and Wildlife Cooperative Research Unit andthe National Fishery Research and DevelopmentLaboratory in Wellsboro, Pennsylvania. This workis also a result of research sponsored by the Na-tional Oceanic and Atmospheric AdministrationOffice of Sea Grant, grant NA85-AADSG021. Ad-ditional support was provided by the New YorkAgricultural Experiment Station, New York StateCollege of Agriculture and Life Sciences, CornellUniversity, hatch projects 1476402 and 1477402.
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Received July 28, 1988Accepted February 21, 1989
