Phenotypic Plasticity and Genetic Variation of Vaccinium Macrocarpon, the American Cranberry. I. Reaction Norms of Clones from C

Home , Vaccinium macrocarpon

Phenotypic Plasticity and Genetic Variation of Vaccinium macrocarpon, the American Cranberry. I. Reaction Norms of Clones from Central and Marginal Populations in a Common Garden Author(s): C. Neal Stewart Jr. and Erik T. Nilsen Source: International Journal of Plant Sciences, Vol. 156, No. 5 (Sep., 1995), pp. 687-697 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/2475048 . Accessed: 15/04/2011 16:44

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http://www.jstor.org Int.J. PlantSci. 156(5):687-697.1995. ? 1995 byThe Universityof Chicago. All rightsreserved. 1058-5893/95/5605-0012$02.00

PHENOTYPICPLASTICITY AND GENETIC VARIATION OF VACCINIUMMACROCARPON, THEAMERICAN CRANBERRY. 1. REACTION NORMS OF CLONESFROM CENTRAL AND MARGINALPOPULATIONS IN A COMMONGARDEN

C. NEAL STEWART JR. AND ERIK T. NILSEN Departmentof Biology, Virginia Polytechnic Institute and StateUniversity, Blacksburg, Virginia 24061-0406

Vaccinium macrocarponAiton (Ericaceae) cranberry,a dwarfshrub and a typicaltaxon in temperate peatbogs, has its central distribution incool temperate regions in eastern North America. Isolated southern marginalpopulations are distributedalong the Appalachian corridor and on theNorth Carolina coastal plain.A commongarden (Blacksburg, Virginia) was utilizedto determinewhether marginal cranberry clonesexhibit greater phenotypic plasticity than central clones. Three central clones from Massachusetts (MA), Wisconsin(WI), and New York (NY) and threemarginal clones from North Carolina (NC), Tennessee(TN), and WestVirginia (WV) weretested. A suiteof phenotypictraits was measuredin responseto edaphicvariation in thecommon garden. An analysisof reaction norms took the form of an analysisof covarianceto testfor significant differences among clones and to estimateregression slopes (plasticity)when compared with environmental (nutrient) variation. There was no regionalvariation in phenotypicplasticity, but there was significantclonal differentiation for77% ofnonintercorrelated traits. However,in mostcases the differences were seemingly random, with little biological importance. Hence littledifferentiation in relation to populationorigin was observedamong clones. Matrix comparisons wereperformed using a Manteltest to checkfor pairwise correlations among the followingmatrices: geographicdistances, trait means, plasticity, and molecularvariation assessed by randomamplified polymorphicDNA (RAPD) profiling.No correspondencewas foundamong matrices. The recentpost- glacialdistribution of cranberry may account for the absence of phenotypic and geneticheterogeneity.

Introduction and Bazzaz 1983; Silander1984, 1985a; Mac- Donald and Chinnappa1989; Thompsonet al. Geneticdifferentiation among ecologically di- 1991). An alternativeviewpoint is thatpheno- versepopulations has been widelydocumented typicplasticity and geneticvariation may be pos- (Turesson 1922a, 1922b; Clausen et al. 1940; itivelyassociated. If strongdirectional or stabi- Quinn 1978; Silander1985b). Phenotypicplas- lizingselection were pervasivein populations, ticity,the morphological and/or physiological re- thenphenotypic plasticity could shieldgenetic sponsesof a genotypeto spatialor temporalen- vironmentalheterogeneity, has been hypothe- variationfrom the effectsof selection,thereby maintaininggenetic variation (Gillespie and Tur- sizedto be an importantaspect of genetic differentiation elli 1989; Goldsteinand Holsinger1992). withinpopulations (Bradshaw 1965; DeKroon and Schieving(1990) have provided Sultan that 1987). Gause (1947) proposed phe- a frameworkfor characterizing clonal plantlife notypicplasticity could be an alternativemode histories.Most vegetatively spreading facultative of adaptationcompared to "genoadaptation"or intrapopulationalgenetic differentiation. This in- clonalshrubs are classified as conservativegrowth verserelationship between phenotypic plasticity plants.These are homologousto thecategory of "stress variationhas been on em- tolerators"(Grime 1979; Chapin 1980). and genetic rejected This categoryincludes arctic, boreal, and tem- piricalgrounds (Moran et al. 1981; Hume and peratepeatland bog dwarfshrubs, such as Vac- Cavers 1982; Scheinerand Goodnight1984; Woodand Degabriele1985; Schlichting and Lev- cinium macrocarpon,which tolerate a suite of stressessuch lownutrient avail- in environmental as 1986;Taylor and Aarssen1988; Counts1993) and low has ability,physiological drought, temper- and on thefindings that phenotypic plasticity atures.Conservative growers respond plastically a geneticbasis (Bradshaw 1965; Schlichting 1986; to increasednutrient availability, potentially re- Scheinerand Lyman 1991; Scheiner 1994). How- sultingin rapidsite filling by one or a fewgenets, ofa ever,some research supports the hypothesis althoughChapin (1980, 1987) arguesthat phe- trade-offbetween phenotypic plasticity and ge- notypicplasticity would not be an importantmode neticvariation (Cook and Johnson1968; Ped- of adaptationfor these plants. Guerilla growth ersen1968; Jain 1978; Wu and Jain 1978; Zangerl (LovettDoust 1981) wouldallow a singleclone to spatiallyexclude possible competitors from a 'Authorfor correspondence and reprints.Present address: site,thereby rendering a possible selectivead- Departmentof Biology,University of North Carolina, vantageto plasticgenets. This scenarioindicates Greensboro,North Carolina 27412-5001. thatin long-lived perennial plants, plasticity could Manuscriptreceived January 1995; revisedmanuscript re- indeedbe an importantmode of adaptationin ceivedAnril 1995. locationswith heterogeneous microsites. Theo-

687 688 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Central populations selectionand driftwould decrease the amount of geneticvariation. A facultativelysexual popula- tionmay turn exclusively clonal if fecundity de- creasesto zerobecause of a lackof available ger- minationsites (Eriksson 1992). If a population was exclusivelyclonal, one couldenvisage a sin- gleadaptively plastic clone (or severalclosely re- lated plasticclones) excludingnonplastic intra- specificcompetitors in such a setting.It would not be surprisingthat, if plasticityof growthis an importanttrait, one wouldobserve few genets NC in old isolatedclonal populations. For this study we used V macrocarpon,the commerciallyimportant cranberry that occurs naturallyin peatlandsin thenortheastern United o h rn arg al populations States,the Great Lakes region,and southeastern Canada, withdistributionally marginal popula- tionspocketed in the centraland southernAp- palachian mountainsand the North Carolina coastal plain (fig.1). These two typesof populations(central and marginal)correspond gen- Fig.1 The distributionof Vacciniummacrocarpon showing erallywith glaciation (central) and unglaciation thegeographic location of accessions. (marginal)of the last Pleistocene maximum. There are geneticand ecologicaldifferences as well. Amongcentrally distributed populations there are retically,phenotypic plasticity may be advanta- highergene flow and largersuitable habitats than geous in spatiallyor temporallyheterogeneous in marginalpopulations (Ogle 1984). Marginal habitats,whereas nonplastic or canalized phe- populationsare smaller,have lowersexual re- notypesmay be moreadvantageous in stableor production,and are thoughtto be relictsof the homogeneoushabitats (Sultan 1987). Opportu- Pleistoceneice age(Wieder et al. 1981;Ogle 1984; nityfor site filling by fewclones would be con- Stewart1993b). Moreover,marginal cranberry tingentupon longperiods between colonization populationshave been shownto have lowerge- events(i.e., low immigration),competitive ex- neticvariation than central populations (Stewart clusion,and smallsite area. and Excoffier,in press).Specifically, there were Althoughsubstantial research has recentlyin- fewerdiscernible clones and less interclonalmo- volvedthe nature of phenotypic plasticity and its lecularvariation in marginalpopulations com- rolein plant fitness, very little work has been done paredto central populations. Southern clones were usingclonal plants (Silander 198 5b; deKroon and largerand presumably older, indicating decreased Schieving1990). As a subsetof plasticity studies, recruitmentwithin marginal sites. Also, southern investigationsof geographicpatterning of clonal marginalsites are relativelymore heterogeneous plantplasticity have beenundertaken (Cook and withregard to phosphorusand nitrogenavail- Johnson1968; Hume and Cavers1982; Scheiner ability(C. N. StewartJr. and E. T. Nilsen,un- and Goodnight1984; Silander 1984, 1985a; Tay- publisheddata). lorand Aarssen 1988; Michaels and Bazzaz 1989; The objectiveof thisstudy was to determine Thompson et al. 1991; Vasseur and Aarssen whetherclones frommarginal populations are 1992).However, in manyof these cases, the clon- moreplastic than clones from central populations al speciesstudied have been almostexclusively inresponse to nutrient variability. Nutrient avail- foragersor consolidatorssensu deKroon and abilityhas beenshown to be an importantfactor Schieving(1990), not conservativegrowers. Ex- in dwarfshrub ecology (e.g., Chapin and Shaver amplesof conservative growth plants include most 1985; Stewartand Nilsen 1993). Furthermore, woodyclonal species and all dwarfshrubs typical thereare regionaldifferences in nutrientavail- of arctic,boreal, and temperatepeatlands. abilitythat are positivelyassociated with tem- Life historystrategies of long-lived,clonally peraturein peatlands(Billings et al. 1982). As a spreadingplants indicate that phenotypic plas- resultof low clonal variation because of historical ticitycould be adaptivelysignificant in island- and ecologicalfactors in southernmarginal sites, like populationssubject to littlegene flow.For did theexisting predominant clones survive be- example,for any given facultatively sexual pop- cause theyhad plasticphenotypes? Do presum- ulation,when emigration ceases, the only source ably old clones frommarginal populations re- of additionalgenetic variation would be by mu- spond differentlyto nutrientfluxes than young tationand recombination.On the otherhand, clonesfrom central populations? STEWART & NILSEN-PLASTICITY AND GENETIC VARIATION. I. 689

Materialand methods cranberrycenters. When comparingclonal ori- gins,Blacksburg's climate most resembledthat SITES SAMPLED foundat theTN site(mean Julytemperature = The predominantclones fromthree disjunct 21.0?C,mean yearlyprecipitation = 116.4 cm), southernpopulations (NC, TN, WV), and from followedclosely by MA (meanJuly temperature threenorthern, centrally distributed populations = 22.20C, mean yearlyprecipitation = 120.7 cm) (NY, WI, MA) wereused in a commongarden and WV (meanJuly temperature = 20.0?C, mean experiment,which was performedin Blacksburg, yearlyprecipitation = 111.8 cm). The NC site Virginia.Sample sizes of populations were small (mean Julytemperature = 23.80C,mean yearly becauseof the extreme rarity of bogs in thesouth- precipitation= 111.8 cm) was warmer,and NY easternUnited States. In fact,Vaccinium macro- (mean Julytemperature = 17.70C,mean yearly carponis rarein NorthCarolina, Tennessee, Vir- precipitation= 96.5 cm)and WI (meanJuly tem- ginia,and WestVirginia. We used singleclones perature= 17.0?C,mean yearlyprecipitation = as physiologicalrepresentatives of populations. 78.7 cm) siteswere colder (Ruffner 1985). In marginalpopulations genetic variation is sig- nificantlylower than in central populations RAPD PROFILING (Stewartand Excoffier,in press).However, even Clonalidentities and geneticdistances were as- in centralpopulations, there is littlegenetic vari- sessedby randomamplified polymorphic DNA ation(Hugan et al. 1993),so usingsingle clones (RAPD) profiling(Williams et al. 1990). DNA to representpopulations is justifiedat thislevel was isolatedfrom leaves by either the Doyle and ofinquiry. The clonefrom Dare County,NC, was Doyle (1987) methodor usingthe Stewartand collectedfrom a 0.5-ha coastalpocosin. At the Via (1993) protocol,which were of equivalent site,small patches of cranberry grow among larg- qualityfor RAPDs. RAPD cyclingparameters er ericaceous(typical pocosin) shrubs.The TN have been describedby Stewartand Via (1993). clonewas froma smallnatural bog on a Johnson Reproducibleband states(bands presentin du- County,TN, farm.This 0.1-ha site had been plicate reactions)were scored as presence/ab- fencedfor 11 yrto excludecattle. The WV clone sencedata. Of the 40 primers(OPA and OPB kits was sampledfrom a smallbog on Droop Moun- fromOperon, Alameda, Calif.) screened, 25% re- tain,WV. TN and WV siteshave been studied vealedinterclonal polymorphisms. The following withrespect to vegetation(Stewart and Nilsen primerswere used to generate105 polymorphic 1993),phenotypic plasticity, and clonalvariation bands: OPA4 (5' AATCGGGCTG), OPA7 (5' (Stewartand Nilsen 1995). The NY clone origi- GGTCCCTGAC), OPA9 (5' GGGTAACGCC), natedfrom Featherston Haugh Lake in Schenec- OPA 1l (5' CAATCGCCGT), OPA13 (5' CAG- tedyCounty, NY. This clonewas collectedfrom CACCCAC),OPA18 (5' AGGTGACCGT),OPB4 thefloating mat on thenorth side of the lake. The (5' GGACTGGAGT), OPB 18 (5' CCACAGC- cranberryarea was small (< 1 ha) at this lake. AGT), whichresulted in a compositegenetic pro- These fourclones are predominantin theirre- filefor each sample. Reproducible characters were spectivepopulations, especially NC and TN, in scoredas presenceor absencedata. whichthe sitesare nearlymonoclonal. The TN and WV clonescorrespond to the predominant EXPERIMENTAL DESIGN clones(clone A in WV) describedin Stewartand Cloneswere propagated by homogeneouscut- Nilsen (1995). The WI and MA clonesare cul- tingsa yearbefore the studybegan to eliminate tivarsoriginally isolated fromnatural popula- home siteeffects and yieldmature plants. Cran- tionsand vegetatively propagated for commercial berryclones, each representing a population, were production.The WI clone ('Searles') was origi- randomlyassigned to 1600 cm2plastic pans con- nallygrown in WisconsinRapids in 1893 and tainingCanadian peat, so thateach pan had ra- was selectedfrom that vicinity(Dana 1983). metscontaining all sixclones. The sixclones were Likewise,a Cape Cod, MA, clone('Early Black') arrangedrandomly within pans. The experiment was firstcommercialized in 1857 in Harwich, was establishedSeptember 1991, fertilizer added MA (Dana 1983). Searles and EarlyBlack are March 1992,and above-groundtissue harvested unimprovedisolates selected from native bogs. September1992. Nutrienttreatments were also The Searlesand EarlyBlack accessionsused in randomlyassigned. The nutrienttreatments were theseexperiments came courtesyof Ocean Spray (1) nitrogenaddition: 1.1 g/m2supplied as slow Cranberries,Inc. releaseurea; (2) phosphorusaddition: 1.5 g/m2 suppliedas treblesuperphosphate; (3) nitrogen ENVIRONMENTAL CONDITIONS and phosphorusadded together at thesame rates The climaticconditions in Blacksburg,VA, the as above; (4) no nutrientadditions. The nutrient experimentalsite (mean July temperature= applicationrates were derived from Eck (1964, 21.30C,mean yearlyprecipitation = 108.7 cm) 1990) and Eaton (1971a, 1971b). The fertiliza- were similarto northeasternU.S. commercial tion rate representedamounts known to elicit 690 INTERNATIONAL JOURNAL OF PLANT SCIENCES growthresponses in a varietyof cultivars. Nitro- tive spreadonly (Eck 1990). Abovegroundbio- gen and phosphoruswere chosenbecause they masswas harvestedat theend ofthe experiment are thetwo most limiting nutrients to cranberry. (September1992). Leaves werecounted and bio- We establisheda large rangeof nitrogenand masspartitioned according to organand module phosphorusavailabilities in whichto measurere- type.We chose various traitsbecause of their actionnorms. The fertilizerwas appliedas rec- obviousimportance as indicatorsof growth habit ommendedfor commercialcranberry growers and rate (table 1). Midwinterleaf anthocyanin (Eck 1990). Means and standarddeviations for levels fromuprights were taken February 1993 soilnutrients were: pH, 5.37 + 0.69; ammonium, on incidentalleaves that remained after harvest. 81.9 ? 15.8,g/cm3; phosphorus,0.85 ? 0.34 Leaf anthocyaninlevels are hypothesizedto be ,ug/cm3;potassium, 5.42 ? 0.85,ug/cm3.Soil nu- physiologicalindicators of stressand maybe as- trientcontent fell within the ranges of commercial sociatedwith latitudinal patterns correlated with cranberrybogs (Fisher 1951; Eck 1990).The pans growth(Rabino and Mancinelli1986). werein two raisedbeds (blocks).Thus, the ex- perimentaldesign was 6 clonesx 4 nutrienttreat- STATISTICAL ANALYSES mentsx 2 blocksx 4 replicates= n = 192plants, An ANCOVA and multiplecomparisons (Tu- withclones nested within pans. We employeda key'sHSD) wereused to assess clonal differen- totalof 32 rametsfrom each genet(eight per nu- tiation,plasticity, and differencesin reaction trienttreatment). Although this was a balanced norms.Clonal identitywas the main effectand completefactorial design, because of plant over- soilnutrients was thecovariate in themodel. The winteringmortality (probably transplant shock) clonemain effect tested for significant differences thedata setwas notbalanced. amongclones and means of traits, i.e., tested when The originalexperimental design assigned the clonalreaction norms had differentheights. Phe- fertilizationregimes as classificationvariables in notypicplasticities were determinedby simple a profileanalysis of variance.However, unex- linearregression by clone, i.e., tested the null hy- pectedcomplications demanded another statis- pothesisthat slope = 0. To stabilizevariance, and ticalapproach. The panshad holesin thebottom as a normalizationprocedure, log log (base 10) forwater drainage to avoid solublesalts buildup. transformationswere performed.Regression As a result,an unexpectedalteration of pH and analysis(by clone), against one nutrientat a time, macronutrients,presumably caused by capillarity was performedto determinesignificant traits and fromthe mineralsoil underlyingthe pans, oc- nutritionalfactors and to estimateplasticities curred.These nutrients swamped the effect of the (slopes).The slopes(reaction norms) were com- experimentalnutrient additions. This resultwas pared by using an ANCOVA homogeneityof confirmedby an analysis of variance,which slopesmodel; i.e., slopeswere different ifthe nu- showeda nonsignificantrelationship between ap- trient*cloneinteraction term was significant(SAS plied fertilizertreatments and themeasured soil Institute1990). Reactionnorm analysis is ap- nutrientlevels (data notshown). Therefore, data propriatesince we used clonal replicates. We chose wereprimarily analyzed by analysis of covariance linearmodels to modelplasticity since nonlinear (ANCOVA), usingthe measurements of soil nu- models(e.g., quadratic) did not improvethe fit trientconcentrations of root medium in eachpan of the data significantly.Total plasticityamong at theend ofthe experiment as thecovariate and cloneswas assessedby using a one-wayANOVA cloneidentity as theindependent variable. in whichestimates of the linear regression slopes Soil fromthree places from each pan was bulked of all nonintercorrelatedtraits in responseto and pH and soil macronutrient(P, K, Ca, Mg) edaphicvariables were used as responsevariables concentrationswere measured at the beginning byclone. Character correlations were determined and end ofthe experiment by theVirginia Poly- by usinga Spearmanrank correlation procedure technicInstitute and StateUniversity soil labo- (Zar 1984).Intercorrelated characters (r > 0.8; P ratory,using a doubleacid extraction.Ions were < 0.001) wereexcluded from global analyses (see measuredwith an inductivelycoupled plasma nextparagraph). Type III sums of squares are spectrophotometer(Donahue and Gettier1988). reportedin all analyses. Ammoniumpools were analyzedby the indo- RAPD data were analyzedby scoringband phenolblue method(Keeney and Nelson 1982) presenceor absencefor each clone.Genetic dis- on a secondset of soil samplescollected on the tanceswere estimated using the euclidean squared same date. distance(Excoffier etal. 1992)adapted for RAPDs (Huffet al. 1993). Excoffieret al. (1992) have GROWTH shownthat for profile data the euclidean distance The functionalplant modules in V. macrocar- indicatesthe number of mismatch bands between pon are uprights(phalanx traits), which are im- clones(i.e., the numberof mutationsnecessary portantfor sexual reproduction,and runners to explainprofile differences between two clones). (guerillatraits), which are importantfor vegeta- So in thiscase, a euclideandistance of 20 indi- STEWART & NILSEN-PLASTICITY AND GENETIC VARIATION. I. 691

Table1

KEY TO TRAIT ABBREVIATIONS

Abbreviation Trait Phalanxtraits: L/UP ...... Averageleaf number per upright ULWT ...... Averageupright mass per leaf (mg) LW/UP ...... Averagemass of all leavesper upright (mg) SW/UP...... Averagemass of stemper upright (mg) TOTUPW ...... Mass ofupright per plant (mg) UPWa ...... Averagemass of upright(mg) UP/PTa ...... Numberof upright per plant Guerillatraits: L/R...... Averageleaf number per runner RLWa ...... Averagerunner leaf mass (mg) LW/R...... Averagemass of all leavesper runner (mg) SW/R...... Averagemass of stemper runner (mg) RWa ...... Averagemass of runners (mg) TOTRW ...... Weightof runnersper plant (mg) R/PTa...... Numberof runners per plant Othertraits and ratios: UW/RW ...... Ratioof uprightmass to runnermass U/Ra...... Ratioof uprightnumber to runnernumber PTWT ...... Mass ofaboveground individual plant (mg) ANTHO ...... Midwinterleaf anthocyanin concentrations (optical densitiesat 510 nm [Lloydet al. 1992]) STOM ...... Averagestomatal density per 0.04 mm2of upright leaf

a Traitsnot included in globalanalyses, e.g., tables 4, 5 and fig.3, becauseof trait intercorrelations.See table2. catesabout 8 1%RAPD profilesimilarity between ammoniumconcentrations and pH meanswere clones.Trait means and plasticity distances among higherthan those found in ombotrophicor mi- cloneswere determined using the Canberra met- nerotrophicbogs, and phosphorusand potassium ric(Rohlf 1988). Distancematrices (geographic, concentrationswere higher than those found in RAPD profile,trait means, plasticities) were stan- ombotrophicbogs but comparableto minero- dardizedto a meanof 0 and a standarddeviation trophicbog (fen) soil (Heinselman1970). In con- of 1 (SAS Institute1990). Pairwisematrix com- trastto themeans, the standarddeviations rep- parisonswere performed using the nonparamet- resentedfield-level variation. ric Mantel test (Mantel 1967; Rohlf 1988; NTSYS-PC). In thisprocedure one matrixis re- PLANT RESPONSES arrangedand one matrixis keptintact; then a CLONAL DIFFERENTIATION. Clonal differenti- Pearsoncorrelation statistic is calculated.This ation was more pronouncedthan phenotypic permutationalprocedure is reiteratedmany times plasticity.A Spearmanrank correlation proce- (in thiscase, 1000),yielding a null distribution. dure (Zar 1984) revealedthat, of the 19 traits Then the actual correlationcoefficient between measured,several strong intercorrelations existed thetwo matricesis comparedto thenull distri- amongcharacters (table 2). Seventy-sevenper- butionas a significanttest. The Manteltest was centof nonintercorrelated traits were significantly performedon the followingdistance matrices: geographic,genetic, trait means, and plasticity distancesamong clones. Trait means and plas- Table2 ticitiesdistance matrices were based upon traits INTERCORRELATIONSAMONG TRAITS withno strongintercorrelations (where r - 0.8). Relationshipsamong the variousmatrices were Trait r Trait depictedby unweighted paired-group method us- LW/UP ...... 0.93 UPW ing arithmeticaverages (UPGMA) phenograms SW/UP ...... 0.85 UPW (Rohlf1988). TOTUPW ...... 0.88 UP/PT LW/R ...... 0.80 RLW Results LW/R ...... 0.90 RW SW/R ...... 0.90 RW SOIL NUTRIENTS R/P ...... 0.91 TOTRW Nutrientsand pH weresignificantly higher than UW/RW ...... 0.91 U/R thoseof mostnorthern bogs and even marginal Note. Spearman correlations(Zar 1984), where r 2 0.80, bogs (Stewartand Nilsen 1993). Experimental are shown. In such cases P < 0.001. 692 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Table3

MULTIPLE COMPARISONS OF TRAIT MEANS (untransformed)WITH CLONES

Clone Trait NC TN WV MA NY WI Phalanx traits: L/UP ...... 30.9 ab 28.1 bc 28.0 bc 32.1 a 25.7 c 29.0 abc ULWT (mg) ...... 1.72bc 1.73 bc 1.41 d 1.70c 1.89b 1.96a LW/UP (mg) ...... 52.8 a 48.9 a 39.6 b 54.7 a 48.6 a 55.3 a SW/UP (mg) ...... 27.4 a 23.1 c 21.1 c 23.6 ab 25.0 ab 27.3 a TOTUPW (mg; ns) ..... 1701 1926 1756 2216 1229 1897 UPW (mg) ...... 80.0 ab 72.0 b 61.0 c 78.4 ab 73.6 ab 82.9 a UP/PT (ns) ...... 21.1 27.8 28.8 28.0 16.9 21.4 Runner traits: L/R ...... 40.3 b 36.8 bc 37.8 c 47.1 a 40.6 b 35.8 c RLW (mg) ...... 2.39 c 2.63 bc 2.07 d 2.76 b 3.10 a 2.58 bc LW/R (mg) ...... 95.7 b 97.1 b 78.5 c 129 a 128 a 93.9 b SW/R(mg) ...... 106abO 81.5c 81.8c 121 a 129a 88.7bc RW (mg) ...... 202 b 178 bc 160 c 250 a 256 a 188 bc TOTRW (mg) ...... 2282 ab 1827 b 1781 b 2887 a 3151 a 1582 b R/PT (ns) ...... 11.0 10.2 10.8 11.6 11.4 7.79 Other traitsand ratios: UW/RW ...... 1.17 c 2.84 a 1.32 bc 1.19 c 0.776 c 2.78 ab U/R (ns) ...... 2.81 6.24 3.26 3.32 2.26 5.30 PTWT ...... 4005 a 3991 a 3915 a 5536 b 4369 a 3950 a ANTHO (ns; optical den- sities at 510 nm) ..... 0.713 0.758 0.744 0.596 0.743 0.586 STO (ns) ...... 28.9 27.8 29.4 29.0 27.3 28.9

Note. Differentletters in rows denote significanceat a = 0.05, determinedusing Tukey's HSD (Zar 1984). ns = model reveals no significantdifferences at P = 0.05. Table lists traitabbreviations and textfor clone provenances.

differentamong clones tested at a = 0.05 (table exhibitedthe only positive slopes in responseto 3). CloneMA was largest(greatest PTWT, L/UP, N and pH variation(fig. 2). L/R [seetable 1 forabbreviations]) and was gen- erallymost vigorous vegetatively, whereas WV RAPD PROFILING was the smallestclone (least ULWT, UPW, The UPGMA clusteranalysis showed that RLW). There did not seem to be any obvious coastalclones NC and MA formeda clusterand centraland marginal patterns regarding clonal dif- werejoined successivelywith the WV and TN ferentiation.However, the MA and NY clones clones(fig. 3B). The twonorthern inland clones, had similarreaction norms, as did TN and WV WI and NY, also clusteredtogether but were rel- (table 3; fig.2). For some traits,such as LW/R, ativelydissimilar (euclidean distance of 20). SW/R,and RW, marginalclones had generally lowerrunner trait means than central clones (ta- MATRIX COMPARISONS ble 3). Pairwisecomparisons of the six standardized matricesshowed no significantcorrelations, with PHENOTYPICPLASTICITY. At the finescale of theexception that the nitrogen-response plastic- soilenvironmental manipulation, very little phe- ity matrixnegatively correlated with RAPD, notypicplasticity was noted in the measured traits. means,and geographicmatrices, but withlow r The typeIII generallinear models showedno (r < -0.5) (table5). significantdifferences, with the following excep- tions: TN clone (SW/R, RW; all independent Discussion variablesand interactions significant at a = 0.05), WI clone(PTWT, TOTUPW, STOM, RLW,LW/ GENETIC HETEROGENEITY UP, SW/UP;all covariatesand interactionssig- The seeds of Vaccinium macrocarpon,a tem- nificantat a = 0.05). The ANCOVA homogeneity peratebog mat plant,are presumedto be bird of slopes model (nutrient*cloneinteraction) re- dispersed,potentially for long distances(Ogle vealed no significantdifferences at P = 0.05. 1984). FollowingPleistocene glaciation V. ma- Cloneshad significantlydifferent levels of total crocarponmigrated from now distributionally phenotypicplasticity (table 4). WI and NC had marginalsites to currentlycentral sites (Stewart thegreatest positive plasticities in responseto N 1993b).Genetic distances estimated from RAPD and pH. Afterlog transformation these two clones phenotypesshow that the clones with the largest STEWART & NILSEN-PLASTICITY AND GENETIC VARIATION. I. 693 distancesbetween them (WI, TN) are genetically about 75% similar.This hypothesizedintraspe- cific phylogenyis not well modeled by the UPGMA clusteranalysis (data not shown), which , 1 02 had a dissimilarrelative topology from that of a treeconstructed when a largersampling of clones E g -_-=- is used (Stewart1993b; C. N. StewartJr., un- Ct) publisheddata). 7 WI -..-- NY It is recognizedthat the species is autogamous MA 6 --- WV and very homozygous,as allozyme variation _ _ - TN withinthe species is depauperate(Hill and Van- NC der Kloet 1983; Huganet al. 1993). In contrast, 6 6.5 7 7.5 a 8.5 9 1 02 blueberries(section Cyanococcus), which are out- Log Ammonium (ug/c in3) crossers,contain many allozyme polymorphisms (Breuderleet al. 1991). The genetichomogeneity of V. section is macrocarpon Oxycoccus, proba- 3 bly,at leastin part,a resultof thebreeding system,the recentdistributional history, and the currentisland-like distribution. The latteris not 2 Re

- sharedwith the Cyanococcussection of Vaccin------ium. Unlikethe more generalist section Cvano- E 1.s coccus,section Oxycoccus are specializedto bog W habitats.It is likelythat springtimemigratory ti T3 WI birdsquickly dispersed V. macrocarponseeds to o 9--NY ENVIRONMENTAL ----SSAND MARGINMA availablebare suitable sites as glacialice retreated --- WV 7 ~~~~~~~TN (Ridley1930; Ogle 1984).This rapid colonization 6 - - NC and geneflow would have servedto homogenize 5 . . . . a genomethat had alreadybeen througha dis- 6 6.5 7 7.5 r.5 9 1s02n tributionalbottleneck. Although V. macrocarpon Log Ammonium (ug/c m3) is now foundin somewhatisolated populations, Fig.2 Representativenorms of reactionsof threetraits: especiallyat itssouthern limits, this distribution stemmass perrunner (SW/R), runner mass perplant (RW/ is onlya recent(last 5000 years)occurrence (Ogle PT), and runnermass (RW) versussoil ammoniumconcen- 1984). For an autogamousspecies with this dis- tration.Table 1 containstrait nomenclature. Traits and am- tributionalhistory, it is not unexpectedthat ge- moniumconcentration were log transformed tominimize and neticdistances between now geographicallydis- stabilizevariances. tantclones are relativelylow. Otherwork shows thatthe degree of geneticvariation of cranberry is low comparedto otherautogamous insect-pol- ENvIRONMENTALSTRESS AND MARGINALITY linatedperennials (Ellstrand and Roose 1987; Peatlandshave relativelylow nutrientstatus Hamrickand Godt 1989; Hugan et al. 1993; and dissolvedoxygen as a resultof highwater Stewartand Excoffier,in press).Strong differen- tables(Small 1972b). Sclerophyllousleaves may tiationin relationto habitatwould not be ex- providea highnutrient use efficiency to cope with pectedin V. macrocarponbecause of breeding chronicallylow nutrientstatus (Monk 1966; Small patterns,life history, and timelimitations. 1972a; Chapin 1980). In many commercialcran-

Table4 CLONAL DIFFERENTIATION FOR PLASTICITY (means of slopes) IN RESPONSE TO SOIL NUTRIENT CONCENTRATIONSAND LOG-TRANSFORMEDTRAITS AND NUTRIENTS

Soil nutrientfactor Log log transformed Untransformed Clone pH P N pH P N WI ..... 0.54 a 0.12 a 0.45 a 178.1a -2.76 b 2.73 ab NY ..... -0.09 abc 0.21 a -0.31 c 54.0 ab 5.61 ab -2.76 bc MA ..... -0.46 c 0.08 a -0.23 c -227.8 c 4.15 ab -6.43 c WV ..... -0.25 bc 0.29 a -0.16 bc - 120.4bc 13.72a -5.04 bc TN ..... -0.25 bc -0.24 c -0.24 c -96.3 bc 0.46 ab 0.78 abc NC ..... 0.33 ab 0.05 a 0.28 ab 105.5ab 0.56 ab 8.26 a

Note. Differentletters in columnsdenote significant differences at the a = 0.05 level. Geographic 6.00 4.50 3.00 1.50 0.00

A NY poNC I U ~TN 'WU WI

24.00 20.00 16.00 12.00 8.00 = 1: _ ~~NC B wu TN + -~~~~~~NY wI

Trait means 3.20 2.40 1.60 0.80 0.00

I ~ ~ ~ ~ ~ _LWU I~~~~~~~~~~~~~~~~ [TN _ ~~~~~~~~~~WI C f --- -NC NY

Plastic: N 3.20 2.40 1.60 0.80 0.00

LNC TN

Plastic: pH 3.20 2.40 1. 60 0.80 0.00

rEM E~~~~~~~~~~~~~~~ _TN STEWART & NILSEN-PLASTICITY AND GENETIC VARIATION. I. 695

Table5

PAIRWISE MATRIX COMPARISONS OF RAPD PROFILES, GEOGRAPHIC DISTANCES, TRAIT MEANS, AND PLASTICITIES; THE MANTEL TEST WAS USED TO TEST FOR PAIRWISE SIMILARITY BETWEEN EACH MATRIX

Geo- graphic Trait Slope, Slope, Factor RAPD distance mean X= pH X= P Geographic distances ..... 0.18 Trait means ...... 0.34 0.36 Slopes, X= pH ...... 0.41 0.26 0.29 Slopes, X= P ...... 0.23 0.15 0.21 0.32 Slopes, X = N ...... 0.22 0.07 0.18 0.37 0.09

Note. P values of Mantel test (1000 iterations)are listed. For slopes, the variable on the X axis is listed. See textfor details about testsand factors.Note that all P values are not significantat a = 0.05. berrygrowing regions, such as inWisconsin, there foundlarge amounts of ecotypicdifferentiation is a yearlongpossibility of frost(Curtis 1959). as well. Since V. macrocarponis a dominantmember of In V macrocarpon,there generally seems to be the vegetationwhere these conditions are prev- no geographicpattern regarding phenotypic dif- alent,it is presumablyadapted to theseabiotic ferentiation.Although many clonal traitmeans factors.One couldargue that at itssouthern limit, are significantlydifferent, there is no patternin whereit existsas a relict,V macrocarponis ex- the differences,and the differencesin manyin- posedto stressesfor which it is notwell adapted, stancesare biologicallyslight (table 3). Thereare such as heat and interspecificcompetition with also no obvious patternswhen comparing geo- morevigorous plants. Therefore, the few clones graphic,genetic, trait mean, or plasticitydistance thatinhabit these marginal relict sites could con- matrices(table 5; fig.3). In otherinstances, where ceivablypossess traits not present in centralsites, thesetypes of comparisons have beenmade, the e.g., phenotypicplasticity, which have allowed matricesor treesfor genetic distances and geo- themto survivethis suite of "foreign"stresses. graphicand traitmeans also did not correlate Severalstudies with clonal plants have found (Schlichtingand Levin 1988; Vasseurand Aars- thatclones from ecologically marginal (stressful) sen 1992). These authorsconcluded that phe- siteshave greater phenotypic plasticity. Grant and notypicplasticity was independentof traitsand Antonovics(1978) found that Anthoxanthum was a traititself (Schlichting and Levin 1988),or odoratumplants from adjacent marginal sites re- thatplasticity was notnecessarily adaptive (Vas- spondedmore quickly to environmentalchange seurand Aarssen1992). The nonsignificanceof comparedwith a centralpopulation. However, matrixcorrelations could be a productof the lim- ecotypicdifferences were greater than plasticity. itedsample size. Perhapsif a largersample were Lotz and Blom (1986) foundthat in Plantago gathered,geographic patterns would emerge.In major,plants from stressful environments showed V. macrocarponthe most parsimoniousexpla-- greaterplasticity. Few studieshave examined bog nationis thatthe geneticand phenotypicsimi- plantplasticity, and nonehave compared genetic laritiesamong clones are a resultof the recent variationand phenotypicplasticity. Vaccinium clonalancestry and relativelylow geneticdiffer- vitis-idaeawas foundto be theleast plastic of bog encesamong clones. The lowgenetic divergences dwarfshrubs in a high arctic nutrientstudy are likely,in turn,to be a resultof a glacialbot- (Chapinand Shaver1985). In a commongarden tleneckand rapidcolonization coupled with au- study,Riebesell (1981) foundthat more stressed togamousbreeding habit. alpinepopulations of Ledum groenlandicum had higherplasticity in photosynthesisrates than cen- Acknowledgments tral bog populations.Ohlson (1989) compared We wishto thankC. E. Guyderfor collection low nutrient(stressed) and highernutrient pop- assistance,B. J. Turnerfor RAPD inspiration, ulationsof the mire plant Saxifraga hirculus and and OceanSpray Cranberries, Inc., and the North foundhigher plasticity in marginalclones. She CarolinaFish and WildlifeService at Alligator

Fig.3 UPGMA phenogramsshowing clonal relationships using various distance matrices: geographic (A), RAPD (B), trait means(C), and traitplasticity in responseto N (D) and pH (E). Euclideandistances (Excoffier et al. 1992) wereused for RAPDs, and theCanberra metric was used forphenotypic data (Rohlf1988). 696 INTERNATIONAL JOURNAL OF PLANT SCIENCES

River forcranberry accessions. Thanks to Carl by grantsfrom Sigma Xi, the ResearchSociety, Schlichting,James Hancock, Gregory Cheplick, and OceanSpray Cranberries, Inc. Full tables (e.g, and threeanonymous reviewers for helpful com- correlationand ANOVA) maybe foundin Stew- mentson earlierdrafts. This workwas supported art(1993a).

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