ValerieD.Hipkinsl,(ISDAForestServce PacI c Southwest
Fesearch
Staton.NatlonaForestGenetc E ectrophoresis
Laboratorv2480CarsonRoad P acerv e, Ca fornia95667
BarbaraL. Wilson, nsttutefor App ed Ecology,227 SW 6th Street,Corva s Oregon97333
RichyJ. Harrod,USDAForestServce Okanoganand Wenatchee
NationaForests2l5MeodyLane Wtrnatchee
Washngton98801
ano
C a r o l A u b r yU, S D AF o r e sSt e f v l c eO y r . p c N a t o n a F o r e s tl B 3 5 B a c k L a k e B v d . S W
O y m p a W a s h i n g t o9n8 5 1 2
lsozymeVariationin ShowyStickseed,a WashingtonEndemicPlant,
and Relatives
Abstract
Iltltk(1id \'.nustd, a te plant endemicto Chelan Counl). Washingbn. consistsoftlvo entitiesdrat djffcr most nohbly bv flo\ief
color and gcogrlphic localion. $t evaluaredrhe hvpothesisrhat thcsc cnlilics arc separatespeciesu\ing a phenot,vpicanalysisol
i s o T y m c \ l r i a t i o n l o c o m p a r e b o t h l b r m s o f H . r . / 1 r r l n \ \ i t h s v p a t r i ca n da l l o p r t r i cp o p u l a t i o nos f t h e c l o s e l yr e l a t c dH . . / i f i i l r . l
\ar. .r.i./.r, and $ilh fhc tnlopatric taxa fl. d/flrJd vr. attntnii and H. dtJfu-vr!ar. l/il.rd. Enz)me band phenottpesrerealcd lhal
ali populalions $ere urirble. $rth 60% to 80'/i polymorphic cnTynrcsystcns. \{on variation was lvithin population.$ilh onlrl2t; amons population \ariation detected.The level of variarion was mo(lerarelo high in ll. rcrrsrzr populationsconlpilrcd rL)
o$cr,ry.ll k?/i.r popul:rtions.Enz) me band patterndata sLrpportlhc lcpararion0117.r. rrt / fion H. din(d. Howc\,cr.datado not
pro!idc c\idcncc 1br lhe taronomic separalionof rhe t$'o color forrns of H. rl,rasta at t\e specieslevel. Regardlesrof lhc laxo
nomic rccognition oJ H. renurr.r color lbrms, they should be con\er|ed as rare c\lrcnes of l/dci.lnl morpholog) and habilat
lntroduction
Hqckelia \)efiusta(showy stickseed),of the family Boraginaceac.
is a 2-4 dm. insect-pollinated,
perennialplant endemicto steeprocky slopcs
coveredwith granite scrcc in Chelan County.
Washington.The specieswasfirst descdbedfrom
a population in Tumwater CaDyonal 488 m clevation(Pipcr 19211).
The TumwaterCanyonpopu
lation haswhite flowers with corolla limbs averaging7.4 nrnrlong,andis theor v extantfl yer&,rtr
populationthathaswhitc flowers.ThreeadditionaLl
Chr:lanCounty populationsof 2 3 dm plantswith
smallerblueflowers(colollalimbsavcraging4.2
mm loDg) have been included in the H. \rwtstu
speciesconcept(Catr 197,1.
GentqrandCan 1976).
Thesebluc-llowcredpopulationsare located16
km southsouthwest
ofthe whitc-floweredpopulation at ca. 2000 nr elevation.The morphological and habitat differencesbetwccn white-flowered and blue-flowctcd H. wnu.\ta support the
hypothesisthatthe$ hite-andblue floweredpopulationsrepresenttwo taxa (Gamon 1988).The
tAuthor to $ho concspondenceshould be addre\sed.
E - m a i l :v h i p k i n s ( a l f s . i i d . u s
170
NofihwestScience.Vol.77. No. 2.2003
taxonomiccontroversyis of particularcuncnt
interestbecause1L r,r:nri.itais consideredendan
gcredin Washington(GamoD1988,Washington
NaturalHedtageProgram1991)andhasbeenproposcdfbr endangered
speciesstatus(U.S. Fish
and Wildlife Service 20001.The blue-flowered
taxon is being dcscribedin anotherpaper,but has
no publishednameat this time.Therefbre.in this
paperwe consistentlyreterto theseplantsaswhiteand blue flowered forms of H. venusta.
The taxonomicstatusof1L r,etastais compli
catedby morphologicalvariationin thewidespread
tax<JnH. d|]hsq \ix. arirlzr(sagebrushd iffu sestickseed).Hat:keliu di/fusa var. aritla is a I 6 dm
specics.
andusuallyhassmall.whiteflowersthat
rnay be lightly washedwith blue. However,thrcc
H. diflusa var. urida populationsgrowing 5 -:[0
km lrom the white-floweredH. rez&rtd populati,,n rrre'imilar to uhite-flouererlH. ( u\tLtfr
flou,er and fruit characteristics.Can (1974) and
Gentry and Can (1976) suggestedthat the observedmorphological similarities resultedfrom
i ntrogrc.'ionliom w hite-llou elcdH. fi tttt rIa ink)
neatbyH. dilfusa var.arida. Ha<:keLiu
di.ffusot ar.
arida Jlso intergrades with crcanr-colored 11.
dtjfusuvar..otto,?ll(Columbiasticksced)in the
Yakima area of Washington.In rum. H. clilfusa
\Jr. cottorii intergradeswith blue-flowered l/.
di/J sa vtr. dilfusa (wcstefn diffuse stickseed)in
thc ColumbiaGorye (Gcntryand Can 1976).A
rccentanalysisof lldc*elia morphologysuppons
reccrgnizing
H. diffusavar.drila, andthe*hiteand blue-floweredcotrponents of H. venustoas
separatebut closely related taxa (Hamod et al.
1999).
Starch-gcleleotrophoresiswas usedto deter
mlne whethcr genetic data suppon recognition
of white- and blue-flowered color tbrms of 11.
ler&.rlcilsdistinctspecies.Enzymeelectrophorcsis
has been successfullyemployedin systematic
studiesof plants.largelvbecauseof its ability to
quantifv genetic similarities(Crawford 1989,
Cra*,fbrd 1990. Briggs and Walters 1997). To
cstabli.h
: t u n t i l r d sf o r e rc l u t t i n gl i r r o n o m
icsiElnificanceofelecftophoreticvariationin 11.rer&.rti.
we sampledsevenpopulationsof 11.dilfusa y.Jr.
arlrla, and one populationeachof1L diJJusovar.
cottonii and H. difiusa vu. dffisa. We scored
enzyne vadation in terms of phenotypicband
similarity. and usedthesedatato assessthe magnitudeof geneticvariationwithin populationsand
thedegreeof similadly amongpopulations.Variation ard similarity \\,ithin H. dtl:fusdservedas a
templateforjudging A r.rrurtd taxonomicrank.
U n d e r l p h e n e t i e\ p e c i e \( o n c c p l -s p e c i e sa r e
distinctand distinguishable
bascdon similarity
f W i n s t o nl o c ) q rU
. \ i n Fa b i o l o g i ( r l\ p e \ ' i e \ c o n
cept. a speciesis deflnedas a feproductivelyisolated system of breeding populations (Winston
l999). Therctbre.besidesbeing characterizedbv
Iower genctic similarities(asin the pheneticspccicsconcept),congenericspeciesusuallyhavefixed
orconsistentdifferencesin isozymcallelesat some
loci (biological speciesconcept)(Avise 199,1).If
the H. venustucolor forms are simply divergent
populationsof H. tlilfustt yar. aricla, we expcct
one or both foms will be more similar to the l?.
difusa v'ar.arklapopulationsincludedin thc study
(all collectedwithin 80 km of theH. yeru"r/./populations) than they are to H. diJfusuvar. cottortii
and,H. dffisa tar. difi sa.
Incidcntalto the taxonomicgoal ofthis study.
we addressedthe concernthat white-flowercd11.
renusta may have extremely limited variation
comparcdto the blue-floweredform. as well as
to H. diffusapopulations.Geneticanalysisof
isozyne \ariation revealthat endemicplants often have limited variation (Hamrick and Godt
1990).However,measuresof isozyme variation
ln populationsof rrLreplants havebeen shown to
be conelatedwith thosemeasuresin populations
oftheir widespread
congencricrelatives,andsometimes the endemic plants are ntore variable
(Gitzendanner
andSoltis2000).
Methods
Populationssampledincludedtheoneextantpopulirtion of white-flowercd H. tenusta, one of the
two extantpopulationsofblue-flowered 1L lrnrst4
the samesevenpopulatronsol H. difiu.ta y{. oida
examined in a moryhological study (Harod et
ai. 1999),and one populationeach of 1L d4ffisa
\ar. cottonii andH. dffisa var.diJJisa(Table l).
The H. dilJlsa va;;cottonii andH. dffisu'tar. dilJusa
populationswere collected190and 320 km fuom
TABLE L Hdr telnr populalidrs sampledin this stu(ly.Hack?lia t e,tunu (blue'flower) \amplcs uere micropropagatcdplanttets.
a n da l l o d e r s a m p l e sc o n s i s r e o
d f r i l d p l . r Dct o l l e c r i o n s .
# indi!iduals
,Y. 1cn,rr.r (white-flo\|er)
H. r .n,Jr.r (blue 1lo\\er)
H. dillutu \at. utkta
IL diltusatdt. ut u
H. dihtsd lat. driad
H. dl.ffutu \.r. andl
H. dtlh!sd \r. driAl
H. ltfuv \ar dtidtl
H tlillu\a |ar aridd
H. difriM \ar. totto ii
H. ttiflusa \r. di|itsa
I]FV
B\'I
DC
DE
MC
PE
sc
COT
DIF
Tum{alcr ClanyLrn.
9.6 km \les! ofLcayenwofh. \lA
Crystal Lake. 19.0lm southwestof Leavcnwonh,WA
Burch Mountain.,l.8 lJn nofthwest of Wenatchee.\A
D o u g l J .C f . e k . 2 6 . nl m r o h o t Q u r . l ' ' ) .U {
Derby Can].on. I 1.3 km southeanof Leavenworh. WA
Moses Coulee 2,1.0km north of Quincy. wA
PondcrosaEnarcs, 17.7 km north olLealenwofth. WA
SwakaneCanyon. 19.3km notheast ofWcnarchee.WA
Tun\r ater Canyon. 1.6km west ofLearen$orrh. WA
Radesnake Hills. near Ntoxee.WA
Oncota Corge near Multnonah Falls, OR
22
t2
25
25
25
25
25
:5
25
29
30
llac'ielia IsozymeVariation
17|
the white flowered H. renusta population.Due
to the rarity of blue-flowercd 11.rerirsla, small
samplesof plant tissue wcrc taken from the liv
(Edsonet al. 1996).
ing plantsandmicropropagated
Only 12 plantswere sampled,due to site inaccessibilityand a desireto minimizenegativeimpactsto the smxll populations.For the otherpopr
lations.threeto file leavesperplantwerecollected
in the field and transponedto the laboratoly on
icc. Voucherspecimensof the H. l,.,rir.i/cpopulationswere depositcdat the Universityof Washington herbarium. Vouchers tbr the H. dilfusu
populationswere depositedat the herbariumof
the LeavenworthRangerDistrict of thc Okanogan
and WenatcheeNational Forests.
Samp e Preparat on
Sampleswcrc preparedusing NFGEL standard
operatingprocedwes(USDA ForestServicc199-5).
For eachindividual,l0 mm2 ol lcaf tissuewas
ground il liquid nitrogen and proteins were exffactedin a Tris butfcr pH 7.5 (Goftliebl98l).
The resultingslulry was t[ansfered to microtiter
platewellsandstoredat -70"C.For electrophorcsis.
the slurry was thawed and absorbedonto 3 mm
*idc wicks preparedfromWhatman 3MM chromatographypaper.
E ectrophoresis
Methods of electrophoresisfollow the general
nethodologyof Conkleet al. (1982)exceptthat
most enzynre stains iue somewhatmodified as
Service( 1995).A lithium
outlincdin USDA F-orest
borateelectrodebuft-er(pH 8.3) *as usedwith a
Tris citrategel buffer.pH 8.3 (Conkleet al. 1982).
to resolve malic enzyme (ME). phosphogluco
phosphoelucose
i.omerase
Inulaser PCM r. .rrrt1
(PCI). A sodiumborateelectrodebuffer (pH 8.0)
was used with a Tris citrate gel butfcr, pH 8.8
(Conkleet al. 19U2).to resolveglycerate-2-dehydfogenasc(GLYDH). glutamate-oxaloacetate
transanrinasc(GOT). glucose 6-phosphatedehydrogcnase(G6PDH), tdosephosphateisomerase
(TPl). and uddine diphosphoglucoscpyrophosphorylase(UGPP).A morpholinecitrateelectrode
andgcl buffer,pH 8.0,wasusedtoresolveisocitrate
dehydrogenase(IDH), malate dchydrogenase
(MDH). phosphogluconate dehydrogenase
(6PGD),and shikimic acid dehydrogenase
(SKD)
(USDA ForcslService1995).Two zones,designatedF (laster)and S (slo\\,er),were resolvedlor
l'72
Hipkins et al.
eachofthe enzymestainsCOT, PGI. andUGPP.
fbr a total of 15 enzyme systems.AII enzymes
w e r er e ' o l r e d , ' nl l l s t u r c hg c l . . E n , , 1 r l e. m i n
recipesfollow USDA ForestService(1995)except that GOT was stainedusing the recipe from
Wendeland Weeden(19139).
Two peopleindcpendentlyscoredcachgel. When they disagreed.
a third personresolvedthe conflict. For quality
control,l0% ofthe individualswcrcrun ard scored
twice.
Data Analys s
We pcrformeda phenotypicanalysisof isozyme
vadation. We use the term enzyme systemhere
as a unit in ;r phenotypicanalysisof isozymes.
The enzyme systemis a region of activity on an
isozyme(starch)gel,which in a diploidplantwould
corespondto a singlelocus.In polyploidsit correspondsto a set of loci that are homologousor
homeologous(dependingon the method of inhcritance).All enzyme systemswere scoredfbr
A band
bandpattemsandbandpresence/absence.
plttem consistsofone to severalbands.Fol agiven
enzyme system,all bandsobservedat a specillc
migration distancearc treatedas identjcal. even
ifthey occur in difTerentpattems.Thesedataare
suitedfor aphenotypicinsteadofgenotypicanalydata as
sis. Scoring starchgel electrophoresis
enzyme band-pattemphenotypeshas been succ e s . f u l l ye m p l o y e dt o d e s r ' r i bheu u r r r i a t i . n i .
paftitionedwilhin andamongpopulations(Kahler
et al. 1980,Poverene
andCurvetto1989.Vickcry
1990,Chunget al. l99l,Dolan 1994.Rogers199,1,
Sammanct al. 2000. Wilson et al. 2000).
werecalculated
Phenotypicdiversitymeasures
from bothbandpresence/absence
andmulti-banded
patterns.For presence/absence
data.phenotypic
diversity was measuredby a polymorphic index
(PI). basedon thc frequencyoloccunenceofeach
bandamongindividualsin a population(Chung
et al. 1991).For multi-band patterns,phenotypic
include:(1) thenumberofband
diversitymeasures
pattcms found in each sampledpopulation, (2)
percentof enzyme systemsthat yield more than
onebandpattem.(3) the averagenumberofband
pattemsper enzymesystemin eachpopulation.
and (4) ShannonWeaverDiversity Index values
(Shannonand Weaver 1949). The frequency of
eachenzyme bandpattern was usedto calculate
Divcrsity lndex. Larger Shthe Shannon-Weaver
annon-Weaver
indicesindicatemorediversepopulations.The distributionofthe totd variationwithin
and amongpopulationswas determinedby partitioning the total ShannonWeaver Diversity lndex (Chunget al. l99l ). The phenotypicrelationships among populationswere determinedby
Hedricks identities(Hedrick l97l)
calcuJating
fbr rnulti-bandpatterndata.andcluster(LIPGMA)
analysis(Chung et al. l99l). Band pattem frequenciesandthe Shannon-Weaver
DiversityIndex
werc calculatedusing Popgenc(Yeh et al. 1997),
usrnglhe haploidcudtrrninant
markcrrctting..
Fesults
'fhe
Hackelia populationssampledwere all variable, with 60-807cof the enzymc systemspolymoryhic,38 50 bandspresent.and 2.00- 3.,10
multibandedpattemsper enzyme system(Table
l J . A l l h c f ( ' p u l a l i o nl e r e l .i ' o z ) m e r u r i l l i o ni n
white flowered1L ycrr.r/.?wasmoderateto high
populations.The blue
comparedto otherH.rc&e/irr
t'loweredl/. 1,e,1&srd
population was moderately
variable.At the species),eve),
H. dillhsa vlar.urida
wasmorc variablcthan H. yeruslc at threediversity indicators (# bands,# pattcrns per enzyme
system,andShannon-Weaver
diversityindex).The
isolatedpopulationsol H. difusa var.cottonii and
H. diffusa var. diJJusahad the lowest levels of
enzymevariability ilt all diversitymeasures.Over
thc entire study,most variationwas within popul r t i r r n r .u i t h o n 1 13 2 r , o I r h e \ a r i a l i o na m o n g
populations.Of thetotal variationmeasuredwithin
each species,20.37cof the variation was among
populationsin H. dilfusa var ttri)tt, and 14.21c
was sharcdbetweenthe blue- and white-flowered
populationsof 11.\,enusta(Txb]te2).
Most bandpattemswere sharedamongpopu
lations. but some were restrictedto single taxa.
Of the 100enzymepattemsobser,/ed,86 occurred
rt H. dilfustt var arida, and 78 (9I 7o)of the patterns that occurrcd tn H. dilJrsa var. arirla also
occuned in at leastone other taxon. The remaining 14 of the 100 total band pattemsoccurred
only in taxaotherthanH. diflusa ttar.arida.However,theindividualbandsmakingup eight olthese
pattems did occur in 1L diJJ sa var. erida. The
other six of th(] 14 patternsincludeda total of six
bandsnot obseryedn H. dilfusa tar. orida. One
of these six unusual pattems,occurring only in
white-flowered1L r,eaa.rla,lacked a TPI-F band
observedin all othersamples.
TABLE 2. Vdiation n Hd.'tclnr populations, revealedby starch-gelelecrophorcsis. N = mcan sample size/enzlme slsrem.
'Z P = percenrpolynrrphic enzyme s)stems.PL = pol)morphic indcx. S.\\'. = Shannon$'ea!er diversity index.
Taxon:
populatron
N
30.9
19.1
ll.7
15.,1
#
bands
5.1
50
11
.18.5
# unique
band\
%
P
# pattemvcnT_voc
s y s l e m( s . c . )
Pl.
S.\1'.
% S.\\'. among
populations
I
.l
E6.7
80.0
66.1
'73.1
3 . 8 0( 0 . 5r )
3.10(0.58)
2.13(0.,r1)
0.380
0.164
0.337
0.351
0.111
0.697
0.577
0.617
t1.2
80.0
7l.l
'73.3
0.3'7'7
0.268
0.30E
0.307
0.231
0.371
0.3,19
o.Il',t
0.29:1
0.8,13
0.626
0.652
0.585
0.537
0.752
0.752
0..187
0.621
l0.l
66.1
60.0
80.0
80.0
60.0
70.5
5.73(0.38)
3.07(0.,11)
3.1l (0.,17)
3.00(0.60)
2.87(0.48)
3.13(0.55)
r.20(1..18)
2.6'1(0.5,r)
3.06
H. ditilsa
B\{
DC
DE
N'IC
PE
sc
lt,l.5
11.9
2r.6
l6.l
22.1
l5.l
r9.l
18.0
19..1
65
,tl
.1.1
.1IJ
:13
50
t2
3IJ
11.1
;
2t.1
39
I
60.0
2.13(0.38)
0.231
0.,160
19.9
38
0
66.7
2.00(0.24)
0.217
0.,110
205.3
13
93.3
6.67(0.38)
0.,1l,l
I
0
l
0
H. diffusa
H. diffusa
vr\r. dillusa
Tolal
31.8
llactella IsozymeVariation
Wc obscrved73 bandsin the 100enzymepatterns,aod65 (897.) oftheseoccurredin H. dfirsc
var.rrrida. One PGI S bandwasunique to 11.z/lfirsa
vilt. cottonii. Onc PGM band was unique to the
H. dilfusa var.cottonii/H. drl.fusavtr. dtflusa ptir
and occurredin all sampledindividuals in those
taxa. The white flowered 11.l,er!.!Ll population
had fbur uniquebandsplus the uniqueabsence
of one band, however,all pattemsunique to this
populationoccunedat < I 17cfiequency.In contrast.only two of the sevensampledpopulations
of H. diJJusavar.arft./ahad uniquebands(one in
BM andthreein onepatternin DE). AIJthebands
obscrvcdin bluc-flowercd1L velirsz alsooccuned
rn H. dilfusovN. arlda, but not necessarilyin the
(in PGI-S,TPI,
samecombinations:
threepatterns
andUGPP F) occuned in blue flowercd H. r?rrst r
and not in 11.diffusa var.arida.
Eight enzymebandpattemswere shlred only
by H. venustaand thosc samplcd1L diJfusavar.
zrrirlopopulations(PE, SC, and TW) which had
beenh; puthe.iueJtu sfiI'u .ign. of inlrrrgre:sion
(Can 1974).Threeof thesepattems(two in PGI
S and one in UCPP-S) occurredin moderatefiequency (0.2 0.5) in white-flowercd H. tenLtstL
and wcrc rarc or uncomnlon (ftequency 0.05
0.ll) in the H. diJlrsavar.arida populationsin
which the1,occurred.
Hedrick'ssimilalitiesamongpopulationsof
H. diffusu var.arida averaged0.786 (range0.667
to 0.934).Heddck'ssimilaritiesbetween1L dffisa
yar. arida and the other varieties of H. diJlhsa
a\eragedless.SimilaritiesloH. dill sovar.cottonii
averagcd0.694andthoscto H. diffnsa rrr. diflusa
ar,eraged0.706; none of the pairwise similarities
exceeded0.733.Hedrick'ssimilaritiesbetween
H. tliJlusavar.ttritla populationsand H. renLtsto
were lower ,yet,averaging0.660 (range 0.561
0. 746) for whiteJlowered 1L r,enrislaand 0.63,1
(range0.520-0.76:l) tbr blue-1lou'ered
1L r,err.rla.
Hedrick's similaritiesbetween11.ferrJld andthe
H. diJlusavar.arida populationshypothesizedto
(Gentry and
show introgressionwith 11.r,cr?u-rl.r
Can 1976)were not statisticlly diff'erentfrom
the similadties between1L r,errsra and other 11.
diJfusu var. arlda populations. These three H.
diffitsa 'i'ar.arida populationsappearno more sllrxlxr LoH. renusta than do the remaining four 11.
dilJusavar.arida populations.Hedrick's similarity between the H. tlifusa \nr. .ottonii and H.
dilfitsuvat.dill supopulationssampledwas0.662.
The similarity betweenwhite-flor'",ered
H. r,erasra
174
Hipkinset al.
and the blue-floweredH. venustapopulation
sampledwas0.877(Figure1).
Discussion
Interpretation
of EnzymeBandPatterns
N , ' r 6 r 1 1 rJ. n i n i r i J l: l e f i n ; n r l l z i n g i r o z l m e
bandsis to infertheallelesthatproducethebands
and thus detemine genotypcsof thc individuals
studied.We were unableto infer allelesfrom the
Hackelia isozyme patternsfbr severll reasons,
includingcomplicatedbandpattems,
overlapping
Ioci, and difficult.v geminating seedsfor crossing studies(Hanod 1999). Both 11../?frr.i and
H. vetvst.l are tetraploid (2n = 48) (Can l9?,1).
We are unawareof publishedintbrmation about
theodgin offtatpolyploidy (allopolyploid
or autopolyploid) or the mode ofinhedtance ofalleles
(disomicor tctrasomic).and the evidencetrom
isozymephenotypes
wasinconsistcnt.
In enzyrne
systemsfor which genetic interpretationscould
be provided (Weedenand Wendel 1989). genotypes fell into two categories.The combination
oftwo differenthomozygous
states(one-banded
pattems)andat leastoneheterozygousstate(suggestiveof tetrasomicinheritanceand autopolyp
loidy) was observedin GOT-F (2 populations).
GOT-S(5 populations),
SKD (5 populationsot
H. diffusa),and UGPP-F(3 populations).
Fixed
or excessiveheterozygosity(suggesLiveof disomic inheritanceandallopolyploidy)wasobsened
fbr6PGD andTPI-Fin allpopulationsiurdfor SKD
and UGPP-Fin white-flowered1L renzsla.
Genotypicinteryretationswerenot rcadily infered from dimeric PGI S andTPI-F, which had
complicatedpattens often involving five to seven
bands,and monomericUGPP-F,which often had
threeor more bands.Additionally, presumedloci
overlappedin PGM (2 :l bands in a restricted
areaof the gel) and MDH (one band in 90% of
individuals).In theory, geneticsof Hacfteli.r
isozymcsmight havebeenclaritiedby examining progcnyarraysof wild plantsor by examining isozymesin seedlingsproducedby controlled
crosses.However. seedset is low in wild plants
andno seedhasbeenproducedin contolled crosses
(Hanod 1999).
Enzyme Variation
C o n s c ni n g h i o d i r , ' r . i t 1i n r , r l r e .p r e . e rirn g
within-tarondivcrsity.A11thcllcctelic populations
BM
MC
DC
TW
DE
SC
PE
DIF
coT
BFV
#
t.2.
1.0
0.8
0.6
0.4
0.2
0.0
A! erageHedrick s disrancebett\ een cluqrerr
Figurc L UPGMA dendroeramof H.r.*elt.Jpopulations basedon H cdrick's distances
amongpopulations,which werc calculaledliom bandfrequencydara.WFV
and BFV = $hite .rDdblue-floweredl/. renurrd, respecti\el,v.SeeTabtc I
fof acronvms.
studiedwerevariablc(Table2). Althoughendemic
plants often have limited isozyme variation
(Hamrick and Godt 1990), white-flo$,ered H.
yenusta rnd the nearby PE and SC populations
ol H. diJJusavr. dida had the highestmeasures
of variability.At the populationlevel. all mea
sureso1 variation \'"'erehigher in white-flowered
H. renustd than in the averagepopulation of H.
Jiliut.t nr. orida. The high lerel of r.rrirtion in
whitc-flowered H. i)er?r/rr.?
is consistentwith the
obsenation that endemic plants are sometimes
more \rriable thln their u iderpreldcongcncriL
relatives(Gitzendannerand Soltis 2000).Phenotypic analysisof enzyne bandpatlernsprovides
no reasonto believc that lack of overall genetic
variationlimits survival of white-flowered11.
venusta.The population may be specializedin
someway.however,perhapsrequiring high mag
(St.John 1929).
nesiumsubstrates
Blue-1loweredH. venu.\tqwas also variable,
butmostmeasures
ofenzymevariationwerelower
than the averagefor H. dffisavar. arida populations (Table 2). This may be an artifact of sampling becauseonly 12micropropagated
blue-llow
ered plantletswere availablefor this study.
T a x o n o m i cR a n k o f W h i t e -a n d B l u e llawered Hackelia venusta
In gcnerrl.population.of difl'erenrcongencri,..
speciesrLredistinguishedby low genetic identities(Nei 1978).Geneticidentitiesamongconge
neric speciesaverage0.68. while those between
populationsofthe sane speciesare usually > 0.9
(Crawford 1989). However, not ail pairs of related speciesmeet this expectation.Some pairs
of congenericspecicshave genetic identities >
0.99andaslow as0.25(Crawford1989).We use
Hedrick'ssimilarities(Hedrick 1971) asaphenetic
Hacielia lsozymeVariation
115
n]easureofpopulationsimilarityto assess
thetaxo
n o m i ( r c n L , , l H . r c n t r . , t a . o l o lrb r m . u s i n gr
pheneticspeciesconcept(Winston1999).
Hedrick's similarities clearly separate11.
renustd fto:n1H. diJJisa (Figure l). Similarities
averagehighestamongH. dillusa vdr.arida popts,
lations and lowest betweenpopulationsof 11.
rcnusta and H. dilfusa yar. arida. Therefore,H.
renrsl4 populationsclusterfartherfrom 11.dlfz.ia
var arlda than the taxonomically recognized11.
dilJ sa rar. cottonii aDdH. dilfusa vat. dilfusa
(Figure l). Theseenzyne datasuppoftthe morphdogical datathat recognizeH. renLlst(litndH.
dffisa as closely rclated taxa (Carr 197.1.Gentry and Carr 1976.Harrodet al. 1999),despite
the intennediateappearanceof a few H. diJfusa
var.arido populations(Carr 197,1,
GentryandCan
1976).Blue and white-flowered H. venustaare
nroresimilar (Hedrick's similarity = 0.877) than
most pairs of H. dilfustt yar. arida populations.
Also, the 1L yerir.rtacolor fbrms are lesssimilar
to H. diffusa var.arida than are the namedvariet
iesH. difusa var.cottonii utd H. tlilfusatat difusa
(Figure1).Enzymephenotypedataalonedo not
provide evidencc for a taxonomic separationof
the forms using a pheneticspeciesconcept(Winston 1999).
When comparing band presence/absence
bclween H. tenusto color forms. the white-flowered fbrm had tbur unique bandsand one unique
absenceofa band.Ho*ever, aJItheseuniquebands
occunedat low frequencies.ln fact,no fixed band
difttrence distinguishedH. diffusu yar. cottonnii
frort H. tliflusa vardifusa,or H. d(J satar. aridu
from H. ventrsttt,providing no evidencefor taxonomic separationof the color forms using a biological speciesconcept(Winston 1999).Although
the enzymephenotypic data do not supportspecieslevelrecognitionofthe l/. uenrrtacolor forms.
blue- and white flowered 1L |en,sla are readily
and consistentlydifferentiatedfrom each other
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Received2l November2002
Actepted.for publitation I 3 Februan 2003
Hacftell.rIsoz; me Vrriation
177