BarbaraL. Wilson, Department
of Botanyand PlantPathology,
OregonStateUniversty Corvalis Oregon
DavidL. Doede,USDAForestServce Mt AdarnsRangerStaton.TroutLake.Washington
afo
Laboratory2375Fruitridge
Road
ValerieD. Hipkinsi,USDAForestServlce,NatonalForestGenetc E ectrophoresis
Camno Ca lorn a 95709
fsozymeVariationin Sisyflnchium sarmenfosum(lridaceae)
Abstract
Sis.\tinchnm sarn(ntosutl is a rarc. duodccaploidplant endenric to the Mt. Adams and Mt- Hood areasof Washington and
Oregon. Isozlme v.rialion in six [hshingron populalions was assessed1ojnfer geneticvariation in ihis species.Five ofthe sir
srmpled populationsuere nonomorphic ibr thc \ixlccn cnzymes suneyed. although at one putative locus one ofthese popula
tions $as nonomorphic lbr an cnz),mc band pattem noi seen in any other pofulation. The sixrh population had 1o\l' levels of
vari|lion in two cnr,"-nics. Th is loll lcvel of \,'ariation hasoften beenobservedin vefy f e speciesand high polyploids.Appdrently.
thc spccicshas rcccntly gone through a botrleneck,perhapsdufing its receni origin as a speeics.
lntroduction
ldeally. managementpracticesdesignedto presen'e rure plant biodiversity are bascdin parl on
an understandingof the population geneticsof
the rare species(Ftrlkand Holsinger1991).For
practicalreasons,suchpracticesareusuallybased
on the gereralizationthat narrowly endemicspeciesusuallyexhibitlow geneticvariation(Hamrick
et al. 1991).However,individualendemicspeciesvary widely in their geneticdivcrsity (Kanon
1991.Gitzendanner
and Soltis2000)and in the
degreeto which thatvariationis panitionedamong
populations(Hamrick et al. 199i ). This studyuses
isozyme electrophoresisto characterizethe genetic variationin the narrowly endemic,high
polyploid plant. SiJl,"irclilrlfi sqtnentosum
S u k s d o r f e x C r e e n e . a n d t o i l d d r c s ss o m e
questions.
nranagcnlcnt-related
Sisyrincltiunr
sernefltosunLPale Blue-eyed
Grass.is endemicto a smallareaofsouth central
Washingtonand northernOregol, in the vicinity
of Mt. Adams and Mt. Hood (Henderson1976,
JohnGamonpers.com..).Two thirdsof theknown
populationsoccur in tlte Gifford PinchotNational
ForestofWashington(AndreaRavcnpcrs.com.).
Its habitat includes mesic to $et meadowsand
tbrestopeningsat clevationsof 480 to 1220m
(Gamon and Raven, pers. com). Sistrinchium
Aurhor ro !lhom corre\pondence\hould be addfessed
3,16 NorthwestScience.Vol.7,1.No.4,2000
oloL!Nrhe\orh{enS.ienrificA$.c
i.n
A Ingh^rc\eneil
sartnentosumis characterizedby glaucouslbliageandpaleblue,rathersmallflowers with tepals
that arc not emarginate(Henderson1976).It reproducesvegetatively.spreadingby rhizomes
at leaston a scaleof many centimeters(Raven
pers.com.).It alsosetsseed.and is highiy selt'compatible(Henderson1976).
Sislrinchium surmerlosrm is a duodecaploid
(l2X) thoughtto have originatedwithin the S.
iduhoenseE. P Bicknell complex (Henderson
1916). Silrrint:hium idaloense is widely distributed from British Columbia south to northern
California, eastto the Rocky Mountains.All Pa
cific Nothwest Si.rlrlnc,4iuz speciesare tetraploid to duodecaploid(Henderson1976).Sirlrlr
chium idahoenseis usuallyoctoploid(8X), but
duodecaploidpopulationsare known from cen
tral Oregon(Henderson1976).No duodecaploid
spcciesor populationsof Si.rlri,?criilm areknown
to be sympatricwith S. sarmentosum,but we are
not awareofchromosomecountsfor S. idarosr.re
liom the rangeof S. sannentosutn.Artificial hybridizationexperimentsindicate
thatmanyPacific
Northu,estspeciesare interfertile, and artificial
crossesbetweenS.r^.Jnnerlosilnandduodecaploid
individuals of S. idahoense produce seed
(Henderson1976).Pollinationamong Sis-\rinchiunt
speciesresults in seedset only when the pollen
chronrosomenumber equals or exceedsthat of
the ovule (Henderson1976).
Management-relatedissuesled to this study.
First, censusingof Sislrinchiutn s.trmentosunis
complicatedby the rhjzomatousgrowth folm of
theseplants.The inconspicuousshoots(ranrets)
can be counted,but many of them may belong to
the same genetic individual (genet). Therefbre,
censusinggenetsis impossible, and the gcnetic
effect of a reduction in the populationof nmets
is not obvious.Sccond,the obsenation that cattle
cat and tran.rplethe flowering and fiuiting stens
of S. sarmentosum,significantly reducing seed
set(Ravenpers.com.), led to qucstionsaboutthe
role of seedproductionin S. ilrn etlr:rt? populations.Sccdprcductionaffectsbothdemographics
and genetic diversity. Two demographiceffects
of seedsare obvious: they are the only method
tbr producing new genetic individuals (genets),
and seeds,rather than rhizomes,are the units of
long-distancedispersal.The geneticefltct of seed
production in this vegetatively spreading,selfcompatible,potentiallylonglived, perennialplant
dependson its geneticdiversity,its breedingsys
tem. andthe rate of seedlingestablishment.None
is knov,tnlor S.samentosum,but
of these1'actors
isozymeanalysiscan oticn shedlight on the first
two. An additional concern is the potential 1br
(BamettandKohn 1991)
outbreeding
depression
or blurring of speciesboundariesdue to hybridization between S. sannentosumand sympaffic
S. itlahoense.Therefore,we evaluatedthehypoth-
esisthat hybridization hasoccurredin the one S.
sarmentosunpoptlation observedto be molpho
logically diverse.
Methods
This study used plants from the six populations
ol S.sar-merLtosum
in the Gifford PinchotNational
Forest that could be relocated and were large
enoughfor sampling(Table | ). Leaveswere collected in 1997 and tansported oD ice to the National ForestGeneticElectrophoresisLaboratory
(NFGEL). Basedonlbliagecolor,the Little white
Salmon River sampleswere believed to include
S. idahoense,S. sarmentosum,and their hybrids.
A tentativeidentification was recordedfor each
samplefuom that population.
Preparat
on
Sarnple
Sampleswere preparedusing standardNFGEL
procedures(Anonymous 1995). A leaf sample
approximately8-11cm long wasgroundin a cold
mortar with 400 microliters of a Tris buffer pH
7.5 (Gottlieb1981).The resultingslurrywastransferred to wells in a microtiter plate and storedat
70 'C. In preparationfor electrophoresis,slurry
was thawedand absorbedonto 3 mrn wide wicks
preparedftom whatman 3MM chromatography
paper.
TABLE l. Sirr r.ri",, .1aD,r"rr ,r? populationsused ir this s!ud). All populationsi'ere locatedin SkamaniaCounty, ['ash
"Outside
"inside
and
relers 1()lhe
inglon. in rhe \,1t.Adam\ RangerDislricl ofthc Cifford Pinchol NationalForest.
location relative to the grazing erclosure at Cavc Crcek. Except as noted, the most imponanl grazing aninals wcre
cattlc. FS Rd. = Forest Service Road.
Latitudc/
Longitudc
Collection
Date
Grazing
Siatus
Cave Creek
inside
n o n h o f F S R D 8 6 3 1 .c a . 0 . 5 m i l e s o u t ho f
t h c j c ! . o f R d s .8 6 1 I & 8 6 2 0
45.9333'N
2 Ju],""1998
121.6,137"w
light for
three ]ears
Cave Creek
norlh of FS RD 861l. ca. 0.5 mile south ()1
t h e j c r .o f R d s . 8 6 l l & 8 6 2 0
45.9333'N
t 2 r .6.137"W
3 0 J u n e1 9 9 8
Intense
Cxyuse
ca. ,100fcc! north of Catu,ie l\'leadowand
w c s t o f M e a d o wC r e e k
,16.1076'N
121.7892'W
21Ju]y 1998
l i e h t( b i g
ganrel
Liitle whire
Salmon Ri!el
snrall meadow south ofFS Rd. 18. 0.25
m i l e s o u r ho l j c t . o f R d s . l 8 a n d 1 8 . 0 6 8
.15.8117"N
121.6645"W
1 9J u l y 1 9 9 8
light
the northwc\l sccrionof PetersonPfairie
:15.9769'N
9 July 1998
121.66,15'w
n o n eu n i i l
fa11
nordl end oflhe prairic. ca. 0.6 mile south
o l j u n c t i o no f F S R d r . 8 6 0 & 8 6 2 0 . 1 3 0
'15.9188'N
1 0J u l y 1 9 9 8
111.7061"W
ligh!to
moderate
Prairie
South Pfairie
Sarnple
Size
Sirr rirrt/iiim Iroz) me Variation 347
Electrophoresis
Methods of electrophoresisfbllowed the general
methodologyofConkle et al. (1982)exceptthat
most enzyme stains are somewhat modified
(Anonymous 1995).A lithium borate elecrode
buller (pH 8.3) was used with a Tris citrate gel
butfer (pH 8.3) (Conkleet at. 1982)to resolve
(ADH), fluothe enzymesalcoholdehydrogenase
rescentesterase(FEST), leucineaminopeptidase
(LAP), malic enzyme (ME), phosphoglucomu(PGI).
isomerase
tase(PGM). andphosphoglucose
A sodium borate electrodebuffer (pH 8.0) was
usedwith a Tris citrategel bul1er(pH 8.8) (Conlle
et al. 1982)to resolveglutamate-oxaloacetate
transaminase(GOT). glucose-6-phosphate
dehydrogenase(G6PDH), triosephosphateisomerase(TPI),
pyrophosphorylasc
and uridinediphosphoglucose
(UGPP). A morpholine citrate electrodeand gel
buffer (pH 6.1) (Conkleet al. 1982)was usedto
resolve diaphorase(DIA), isocitratedehydroge'
(MDH), and
nase(IDH), mxlatedehydrogenase
phosphogluconatedehydrogenase(6PGD). A11
enzymeswere resolvedon I l6lostarchgels.Stain
rccipcsfor enzymesfollow Anonymous(1995)
excaptthatGOTwas stainedusingtherccipefrom
Wendeland Weeden(1989).Two peopleindependentlyscoredeachgel. When they disagreed,
a third personresolvedthe conflict. For quality
control. 10clrofthe individuals were mn and scored
twice.
DataAnalysls
Sisyrinchium sdrntentosumis duodecaploid
(Henderson1976).Therefbre,in the most simple
case,a single band may representthe activity of
enzymescoded by six loci on 12 chromosomes.
We treatedeachenzymeas if it were encodedby
one putative locus. except that we treated PGI
and TPI as encodedby two putative loci (actually two putative setsof homologousloci) each
(Table2). Theretbre,we analyzedthesedataasif
they consistedof sixteenisozymes(16 putative
loci) (Table 2), reterredto in the text and tables
as sixteenenzymes.Gels were scoredfor band
ing pattem differencesandband presence/absence.
In this type of analysis,an enzymebanding pattem is considereda singlephenotypictrait at which
variation can be assessed.
Samplesfrom the Little White Salmon River
were analyzed as a single population of S.
sarmentosum.
Phenotypicdiversitymeasureswerecalculated
lrom both bandpresence/absence
and multi-band
patterns (Table 3). For presence/absence
data,
TABLE 2. Enzyne band paitern fiequencies fot Sist\inchian sarmentor(,l populations. "Band Patterns are schematicdia
gfams of bandsobservedfor eachpattern; I = presenceof a band;0 = absenceof a band seenin other pattemsof !ha!
Enzymc
Paltcm
ADH
DIA
FEST
COT 1
G6PDH
IDH
LAP
MDH
ME
6PGD
PGI-I
PGI-2
PGM
TPI,1
B
TPI 2
B
348
Cavc Crcck
insidc
Cayr.rsc
Mcado\rs
1.0000
L0000
1.0000
1.0000
1.0000
1.0000
1.0000
i.0000
1.0000
0000
0000
0000
0000
1.0000
1.0000
1.0000
1.0000
r.0000
1.0000
1.0000
1.0000
0.0000
r.0000
1.0000
0.0000
1.0000
0.0000
1.0000
1.0000
1.0000
1.0000
1.0000
r.0000
1.0000
1.0000
r.0000
B
UCPP
Ca\,c Crcck
outsidc
1.0000
1.0000
0.0000
1.0000
1.0000
0.0000
1.00{10
0.0000
1.0000
Wilson.Doede.andHipkins
r.0000
r.0000
1.0000
1.0000
1.0000
0.0000
1.0000
0.0000
1.0000
1.0000
0.0000
1.0000
Pelenon
Prairie
1.0000
L0000
1.0000
1.0000
r.0000
1.0000
1.0000
1.0000
1.0000
1.0000
r.0000
1.0000
0.0000
t.ri00t)
1.0000
0.0000
1.0000
0.0000
1.0000
Lit0ewhite
Salmon River
1.0000
1.0000
1.0000
1.0000
r.0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
L0000
0.0000
1.0000
1.0000
0.0000
1.0000
0.0000
1.0000
South
Prairie
Band
Paitern
0000
0000
0000
0000
0000
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000
0.9091
0.0909
1.0000
r.0000
0.0000
0 . 9 1l 8
0.0882
1.0000
lt
I
|l0
lll
11
UI
lll0
llll
ll
TABLts 3. Genetic variation in Sisrrin(:hiun nmc,?/orvrr populalions.revealcdbv isozynes treatcdassixteenpu|ati\'c loci N
= nean samplesize/enzlne. q.P= percentpc'lvmorphicputative locii seemethodslbr dcfinition. PI = pc'lymorphic
"inside" felers 1olhc location
inder. S.W.=Shannon Wcaverdiversity indcx. s.d.=standarddc!iaiion. Outside and
reladvc !o the srazins cxclosureat Cave Crcck.
N
Population
S.W.
P
bands
18.75
TOTAL
Cave Crcek inside
Crve Crcek outside
CayuseMeadows
L. White Salmon Rivel
35
t5
26
27
27
2',7
South Prairle
l,{
29
)
0.00
0.00
0.00
0.00
0.00
12.50
21
(0.82)
0.5
(0.81)
2.08
(5.l)
U
I
0
Nlean-/population
33
(1.6)
phenotypicdiversitywasmeasuredbya polymorphic index (PI), basedon the tiequencyofoccurrenceofeach band.For multi-bandpatterns,phenotypicdivenity measurcsinclude:(1) thenumber
of bandsfbund in eachplot, (2) percentof stains
that yield more than one band pattem, (3) the
averagenumberofband pattemsper stainin each
plot, and (4) Shannon-WeaverDiversity Index
values (Shannonand Weaver 1949).The Shannon-WeaverDiversity Index usesthe frequency
of each band pattern in eachplot. The larger the
lndex,the moredivene the plot.
Shannon-Weaver
The distribution of thc total variation within and
among plots was determinedby partitjoning the
Diversity Index.Band paC
total Shannon-Weaver
tem frequenciesand the ShannonWeaverDiver
sity Index were calculatedusing Popgene(Yeh
et al. 1997).
Focusng
soelectrlc
Enzyme banding patternsin the Little White
Salmon River populations were analyzed with
isoelectriclbcusing(Acquaah1992,Westermeier
1997).Methodsof isoelectricfocusingfollow
HyPure procedures(Anonymous 1994-1998).
Tissuesamplesvariedfrom 0.12 to 0.51 gram,
preparedwith proportional amounts of loading
buff;r. Polyacrylamidegels were stainedfor the
(ACP), diaphorase
enzymesacid phosphatase
(DlA), esterase(EST), lnalatedehydrogenase
(MDH), peroxidase(PER), and phosphohexose
isomerase(PHI). Both buffeISFS5480(pH 3- 10)
and FS5080(pH ,1-5)were usedto resolveACP,
7. S.W among
populations
L 1875
(0.401)
1.0000
r.0000
1.0000
r.0000
1.0000
L1250
0.3.180
0.0387
0.000t)
0.0000
0.0000
0.0000
0.0000
0.3261
0.0000
0.0000
0.0000
0.0000
0.0001)
0.o311
1.02
(0.051)
0.05,1,1
(0.133)
(0.0rs)
0.1070
0.0064
EST, MDH, and PER. Buffer VGl l l0 (pH 3-7)
was usedfor DIA and PHI. Buffer VG1080 (pH
3 l0) was usedfor DIA, EST, MDH, PER, and
PGM. Furtherinformationon buffer composition
is availableinAnonymous(199,+1998).
Results
Two adjustmentswere made to the data set before final analysis. One individual from South
Prairie was removedfrom the analysisbecauseit
had unique pattemsat 15 of the 16 enzymes
sampled;it was prcsumedto be of a graminoid
speciesmtherthanS..t4lt enloium.Samplesfrom
the Little White Salmon River population were
all treated ts S. sarmentosumbecausetheir enzyme band pattemswere all identical.
Although most isozymepatternswere simple
and invariant, someenzymesexhibited multiple
bandsin all individuals(Table2). Becauseoflack
could
ofvariation in this species,bandsegregation
not be used to idertify specific alleles and loci.
Therefore,we treatedthe bandpatternasthe unit
ofanalysis. Howeveq eachgel stainedfor PGI or
TPI exhibited two regionsof activity, which we
consideredto representisozymesfrom different
(Gottlieb 1982).Theresubcellularcompadments
fore, we divided the PGI and TPI bandsinto two
units. We refer to theseunits as putative loci.
Five of the six S. sannentosumpopulations
werc monotestedusingstarch-gelelectrophoresis
morphic for all 16 enzymesexamined(Table 2).
Oneofthese(CaluseMeadow)wasmonomorphic
S i s y r i nh,i u n l . o / y r n c\ a r i a t i o n 1 4 q
TABLE'1. Genetic variation in Sirlr.ir./iirr,r populations.re|ealed bv isozynes. and in a rare l.lr. N = mean sample sizelenzyme. Pops= number ol populationsin the study.Enzymes= thc numberofdifferent stainsused.Loci* = the number
of different rcgions on fie sel lreatedas if they were loci. %P En7.= pcrcenl of el1zylnestainsthat revealcdpolymor
phisms. '/. P = perccn!polymorphic pulalive loci or enzymestreatedaslocji seemethodsfor definirion. Pattems/locus
= patternspef putatlve locus. S.W. = Shannon\\'eaver divefsity inder. Data for.t. m.rnrdrrfl and S. septentrioruLe
reanalyzedas band fattems by lhc aulhors. with all high polyploid populationsfcated as ^t_/rorldrxlr.
q.P
Pops Enzymes
36
200
6
5
6
Loci*
Enz.
21
6l
l.l
l3
1,1
1l
I
219
'/,P
locus
2l
1.9583
L1875
1.2500
0.2661
0.0387
0.1,117
P a v c k1 9 8 9
this study
P a v e k1 9 8 9
1.000
0.0000
S i m o n i c h&
Morgan 1994
21
10
tbr a TPII bandpattemfound in no other sampled
population(Table2). One population(SouthPmirie) was polymoryhic at two enzymes.PGI2 and
TPi2. Band pattems obtained were simple and
consistedofa singleband in 43% ofthe pattems.
An additional 37clcof the pattcrns consistedof
two bandseach,l0c/. weremadcup ofthree bands,
and l0c/cwere made up of four bands(Table 2).
t heCa1u.eMerJow\ populationhadoneunique
band, and the South Prtirie population had two.
Sis,""rinchium
sarnentoswn had very low levels
ofenzyme variation(Table3). The levelsofvariation observedin this specieswas lower than
thutohser\ed previou.lyin Slsldrc/rirrz 'pecies
(Table4).
S.W
ln theLittleWhite Salmonpopulation,isoelectric focusing revealcdapproximately186 bands,
countingbandson eachenzyme/buff'ercombination separately.
Eleven(6.57r)ofthesebandswere
polymorphic(Table5). No banddifferentiatedthe
putativepa!:entalsamplesor the putativehybrids.
Discussion
A geneticinterpretrtion
of5. .rcnrerlorrn isoz)me
band patterndatawould be preferredover a phenotypic analysis,becausea genetic analysis
proridesinformationaboutpopulariongenetic.
and resultscan be compareddirectly with compilationsof plant isozymegenetics(Hamrick and
TABLE 5. Polymorphic bandsrevealcdb) isocleclric ibc using in the Litd e Wh ite Salmonpopularionof Sl.rirlncrn,n. Sarnplcd
irdividuals are numbered. I = band presence:0 = band absence.* = samc band revealedon bulTersVGl080 and
FS5080. SeeMethods for enzvmc abbrcviations.
Buffer I
2 3 1 5 6 I
DIA
1080 0
0
0
0
0
0
EST
108[J l
l
l
l
l
l
8 9 10 ll
0
l
00
0
t
|
0
r
0
0
l
l
l
0
0
0
0 0 t )
l
l
I t )
0
l
l
0
1
1
1
00
l
0
0
0
0
0
0
0
1
1
0
0
0
1l
0 0
1
t
l
0
t
l
t
l
l
l
l
l
0
1
t
l
l
0
I
0
0
1 0
0
0 0
0 0
l-l
0
0
0
0
0
l
l
l
1
l l
l
l
l
0
0 0
0 0
0
0
0 0 t )
l 1
l l
l
t
l
0 0 t ) I
0
0
0 0
1
t
t
\,IDH
508t) 0
PER
50rJt) r
1
PER
5'180 0
0
PER
5'180 1
0
1
1 1
PHI
1080 0
0
0 0
0
0
1
0 0
0
PHI
Il )
r
r
l l
0
0
1
0 0
0
0
Wilson.Doede,andHipkins
0
0
1
350
3
U
1
0
I
0
l
L 2
0
0
0 0
0
0
l
12
00
1 0
0
1
0
l
ll
0U
0
508t) 0
l
0
J. rdDr.
l0
l
I08t-) 0
EST
EST
P u l r l \. < h \ b r i J ,
: b 7 b t
I
I
0
EST
0
:
1
1
0
1
r
1
1 t
t
l
0
0
0 0
0
0 I t ) 0
0
1
00
0
0 0
1
0 0 1 - )0
0
0 0
0
0 0
0
0 0
l
t
t
l
l
l
l
0
0
0
r
0
0
l
0
0
0
0
0 0
0
0
0
0
0
0
0 0
0
0
u
0
0
0
l
l
Godt 1990).Two factors madc genetic interpre
tation of S. sarnrcntosut enzyne band pattems
impossible. FiISt. almost no bands segregated
within or among populations,iind thercfbre the
numberof loci and thcir relationshipsto specific
alleles werc difficult to judge. Second,uncommon alleles may escapedetectior becauseS.
sonnentosuntis duodecaploid(l2X) (Henderson
1976).For cxample.the most obvious interpretation of a pattemconsistingof a singleband is
that the genotypeproducing it is AA AA AA AA
AA AA. Ho\\,ever thc true genotype might be
\ \ A A A A A A 0 0 0 0 i f g e n es i l e n c i n go c c u r s
(Crawford 1990).ln addition.the onevisible band
may resuit from the genotypeAA AA AA AA
A A A B i l ' d o \ i l g cr e g u l a t i oonl g e n ee r p r e s s i o n
occursandlimits the amountofenzyme produced
from all theselocijointly. The enzymesproduccd
by the B allelc would be so rare comparedto the
enzymesproducedby theAallele thatthey would
probabl),not showup on thegel (AB heterodimeru
cuuld hc producedbccru.eol interlocusinteractions. but thesewould still occur at a small frequency relative to AA homodinrers,xnd therefore likely be invisible). If the plant is
autopolyploid,an allele may be sharedamongall
the homologousloci, and the plant night have a
genotypesuchasAAAAAAAB BB (Soltisand
Ricseberg1986).If a two- or three-bandpattern
onemustwonderhowmany
showsup occasionally,
copies of the alternlrtiveallele must be present,
and/orhow many null (silenccd)allelesmustbe
present,beforc the enzymeproducedby that altemativeallelecanbe observed.Phenotypicanal),sisprovidesa way of usingisozymedata,suchas
that n S. sarmentoJirr?,which cannot be inter
pretedgenctically(e.g.Chunget al. l99l).
Enzymc variation ln S. sannentosum(,ts de$'ascxfemely
tectedin starchgel electrophoresis)
lirrited. Comparisonwith geneticanalysesofplant
isozymedata(Hamrick andGodt 1990)is inshuc
tivc, if certain trendsare kept in lnind. First, the
percentpolymoryhicenzymesis ottenhigherthan
the percentpolymorphicloci, becausea single
cnzymestainmay revealboth variableand monom o r p h i cl o c i .S e ( u n Jl.h c n u r n h c r ofli r l l e m .r e poded per enzyme (or per putative locus) is ot:
ten somewhatgreatcrthan the number of alleles
per locus. becauseevery combinationof t$ o alleles can produce a difl'erent pattem. Also. pattems are not indcpcndent.The addition of one
;illclc for a dimeric enzymewill producetwo ad-
ditional bands.Third. the Shannon-Weaver
diversity index may overestimatedifferences.because
patternsproduced by homozygotesare consideredcompletelydifferentfrom pattemsproduced
by heterozygotes.
Si,ry'rinchiunsemrentosuntappearsto have
much less enzyme vadation seenin the average
narowly endemictaxon (Hamrick and Godt I 990,
Gitzendanner
and Soltis2000).For example,S.
sarmentosutnhad l9clc polymorphic enzymes,
comparedwith thc averageof 26% polymorphic
loci for endemic species,and Ll9 patternsper
enzyme,comparedto 1.80 allclesper locus in
endemicspecies(Hamrick and Godt 1990).The
variation in S. sannentosumis also lowgr than
that reported fbr other Slsrrincii/rr speciesof
the Pacific Nofihwest, despitemuch slruller sample
sizesin thoseotherstudies(Table4), andthe vadation in those specieswas consideredlow (Pavek
1989).Like themonomorphichis 1ac&rtris
Nutt.
(Simonich and Morgan 1994). Sistrinchium
(self-compatibilsarmentosumhaschnracteristics
ity. restrictedrange,andmoist habitat)associated
with extremelylow isozymevariation (Cole and
Biesboer1992).
Why are enzymepattemsin S. sanne tosutn
populationsso nearly invariant?There are severalpossibilities.First.ahigh polyploid taxonthat
aroseonce would include a very limited sample
of the genetic variatjon presentin the parental
speciesbecauseit includesonly the variation
presentin the one (or two) individual(s)that were
parent(s)of the first high polyploid individual
(Crawfbrd1990).Second.low populationsizes
for severalgenerationswould reducevariation,
even if the populationwas initially variable.
Sis\'rinchiumsqrmentosumhas had small populationsanda very limited rangesinceit wascollected by Suksdorf (Gamon pers. com.). Third.
low variation could characterizea speciesthat
rcproducesentirely vegetatively.However, vegetatively reproducingtaxa are often highly variable (Ellstrand and Roose 1987), and S.
tarmentosum appearsto reproduce sexually. It
produccsabundantseed when grazing does not
prevent seedset (Ravenpers.com.). The seedis
probably produced sexually. because S.
\ a r m tn l o \ u ml u i l \ h ' p r o J u ( e: . e e Ji n e e n r i n. t r tificial crosses(Henderson 1976).A fourth hy
pothesisis that the obser\'edlow variationresults
tiom many generations
of selfing.Selfingproof homozygousindividuals,
ducesa prcponderance
Sisyrinchtun lsozyme Variation
351
but whether selfing populationsare genetically
variableornot dependson the variabilityoffound
ing individuals and whether the population has
gonethrougha proJonged
periodof low size.Some
largely selfing populations are highly variable
(Knapp and Rice 1996).We favor thc hypothesis
that duodecaploidS. ,r4rmel?loJrzaroseonce(or
rarely) liom an ancestorwith lower chromosome
numbet
Sometimesisozymeanalysiscan dctermineif
a speciesis an autopolyploid or allopolyploid,
becauseallopolyploids
usuallyexhibitlixed het
erozygosity at many ioci (Crawford 1990).
Sistrinchiun sarmelltosLtmhas consistentmultiple-band pattems for severalenzymes,which
may representllxed heterozygosityat homologous loci. However,that doesnol help determine
thc origin of polyploidy in this species.The an
ccstor of S. sqnnentosumwas probably polyploid itself. We are not awareofinformation about
the isozymc variation in S. idtthoense,but duplicated loci were inferred for tetraploid S.
septe trion.ile E. P.Bicknell (Pavek I989). DuodecaploidSls_r'rlricftium
sdrmentosun tnay be aI
autopolyploidderivedfrom anallopdyploid ances
lor.but isozymeanrlysisol Sl'r rinr'lrlirm
.r.rnaenl.r.rrn alone cannoteliminate othcr hypotheses.
The variationrevealedby isozymeelectrophoresis is not always rcpresentativeof vadation in
physiologyandotherftaits(BanettandKohn 1991,
Hamrick et al. l99l). but thereare severalreasons to think S. sannentosumhas little genetic
variation ofany kind. The speciesshowslessfloral variation than most other Pacific Northwest
speciesofSlslrlrcftiln (Henderson1976).Mor
phologicalvariationobservedduring recentfield
work is limited; singleplantswith whiteorrwelvetcpaledflowers were observcdin the CaveCreek
population(Ravenpcrs. com.), and foliage color
variedonly in theLittleWhite SalmonRiverpopulation.ln addition.all populationsoccupysimilar. open, wet to somewhatdry habitatswithin a
very limited range.The apparentlack of genetic
variation in S. .sarntentostun
doesnot imply that
the speciessuffersinbreedingdepresston.
because
invariant plants may lack harmful genetic variants(Allard et al. 1986,Husbandand Schemske
t99"t).
Variationin foliage color and tepal shapesuggestedthatS.ldaroense,S.sarmentosum,
|.ndtheit
hybrids might have been collected at the Lirrle
White Salmon River site. That hypothcsis was
352
Wilson,Doede,and Hipkins
not supportedby the results of this study. The
plantsall hadthe sameenzymebandsasrevealed
by starchgel electrophoresis(Table 2). Isoelectric focusingofenzymesknown to be highly variable and to exist in multiple forms revealedlimited variationamongindividualsin this population
but no consistentdifferencesamongthe samples
labeledas different species.If the sampleidentifications are correct, this uniformity of enzyme
variation among species suggests that S.
sonnentosunris the recently derived autopolyploid descendentofthe widespreadS. ickLhoense.
However,the observedvariation in lbliage color
may represent intraspecitlc vadation in S.
tctrmentoslrm.Other Sisyrinchiumspcciesin the
Pacific Northwestare known to exhibit variation
in foliagecolor (Henderson1976).This point could
be clarified by additional researchthat jncluded
sampleslrom single-species
standsofS. rdaloezse
and voucher specimenswith flowers pressedin
the field. to presen'ethe floral t'eaturesnecessary
to confirmspeciesidentitication(Henderson1976).
Although the Shannon-Wcaver
diye$ity index
indicates thilt populations ol SistrinchiLtnt
sdnnentosun are little differentiated,gene flow
amongpopulationsmay be 1ow(Table3). Al1 individuals in the CayuseMeadow populationhad
a bandat TPI I thatwasobservedin no othcr population (Table 2). This situation could be stable
only if gene flow betweenCayuseMeadow and
any other sampledpopulation were very low.
BecauseS.sannenlasznhadsuchlow enzyme
patternvlLnationandmostpopulationsweremonomolphic, we cannot useenzymesto answercertain managemcnt-relatedquestions.
Enzymepat
ternscannotdelimit genets(clones),andthereforc
censusinggenetsremains problematic.Enzyme
patternscannotreveal thc relativeimportanceof
sexual and vegetativereproduction in this species,andthereforedecisionsaboutprctectingseed
set cannot be made on the basis of enzymepatterns. The differentiation between the Cayuse
Meadow population and the othershints at limited geneflow. suggestingthat populationestablishmcnt through long-distance seed dispersal
events (which requires seed production) is impofiant in the patteming of genetic diversity in
this spccies.Perhapsdiffercnt molecular techniques,suchas isoelectrictbcusing,RAPDs,or
ISSRs.can gencrateenoughvariation to address
thesequcstionsmoreeffectivcly.
The lack of observedvariation suggeststhat
few restrictionsneed be placed on prograDs to
increasethe S. sannentosumpopLrlationthrough
transplants.Moving plants (seedsor rhizomes)
among populationsmay be undesirablebecause
ir mightleadto Io.\ of rarcgenelic\ arialiun\no$
restrictedto singlepopulations.However,aslong
asgeneflow is aslimited asit appears,establishmentof new populationsat sitesnot now occupied by 5. sarmentosun needbe restraincdonly
by the pragmatictest that populationscan be establishedonly where the plants can grow.
This study suggeststhat prescrving enzyme
(andgenetic)diversityin S.sarmentosumrcqrires
preservingmany populationsbecausesome.like
CayuseMeadow, contain unique alleles.Also, a
large population should be maintainedat South
Prairie,thc only populationknown to be variable.
However,this study also suggeststhat management practices directed to$ard demographics,
ratherthangenetics,canbc effectivein long-terrn
LiteratureCited
Acquaah.C. 1991.Pfactical Pro|ein Elcctrophofesisfor Ce
ncric Research.Dioscolidcs Press,Portland.Oregon.
Allard. R. \\'., S. K. Jain. and P L. $'ofkman. 1968.Thc gc
nelicsol inbrccding polulations. Ad!ances in Cenet'
i c s 1 1 : 5 5l 3 l .
Anonymous. 1994 1998. H1-Pur€Procedures.HyPure l)jvisior ol Wallac. Inc., Akron. Ohio.
Anonymous. 1995.National Foresl Gencdc Elcclrophoresrs
LaboratoryStandardOperaling Procedures.\FGEL.
I SD\Iur<n\fl \rc.,arnrr.,C,,li'onriJ.
BarrettS
, . C . H . . a n dJ . R . K o h n . 1 9 9 1 .G e n e t i ca n d e v o l u ofsmall populationslTein plants:
tionaryconsequences
implicationsfor consenalion. Pages3 30 /n Falk, D.
A.. and K. E. Holsinger (edilors) Cereiics and Con'
sc arion of R e PlaDls. o),ford Lnilersity Pre\s.
Oxford. England.
. ndG. S. Derda.
C h u n g ,M . G . . J . L H a l l x i c k . S . B . J o n e s a
1991.Isorlilc lariationwirhin and anong popula
lions of Hl)rrd (Liliaceae)in Korea.S)llcnratic Botanr
l6:661-68.1.
Cole. C. T.. and D. D. Biesbocr. 1991.Monomorphism ir
duced gene flow. and clelslogamy rn rare and con1
mon speciesof tc.rpcrle:ri( F'abaceae
).Anerican Jour
n a l o f B o l a n v7 9 i 5 6 7 5 1 5 .
Conkle. NL T.. P D. tlodgrkiss. L. B. Nunnally. and S. C.
Huncr 1981.Starch Gel Eleclrophorcsisof Conifer
Sccds:a Lrborator,'-Mrnual. Gcn. Tech. Rep. PS\\'
g. Befkeley.CAiPaciiic South\\'estForestandRangc
Experiment Slation. ForestSer\ ice. U.S. Dcpartment
ofAgricullure. l8pp.
Cra$ibrd. D. J. 1990.Plant Molecular Syslcmatic!: \'lacroJohnWiley & Sons.NewYofk.
molccularAppfoaches.
Nc$ York.
presen'ationof S saraeilosriz biodivenity. Ur ess
populationsare extremelysmall (<50), demographic changesare morc important than loss of
geneticvariability for long-telm population suret al. 199,+).
This
vival (Menges1991,Schernske
is especially true in a species such as S.
sonnentosumthat haslittle geneticvadability.
Acknowledgements
Elecffophoresisand gel scoringwere petformed
by SuellenCanoll, PatriciaGuge, and Randy
Meyerat theNationalForestGeneticElectrophorcin
si. Laboraroryr NFCEL r. Othcr cooperators
cludeGregoryL. Cox.Narcy Fredricks,andSheila
Maninson ofthe ForestService,Ed Guenant and
AndreaRavenof the Berry BotanicalGarden,and
John Gamon of the Natural HeritageProgramof
theWashingtonDepaftmentof NaturalResourcas.
We aregrateful to anonymousreviewers for critical
commentson the manuscript.
Ellstrand. N. C., and M. L. Roose. 1987. Patternsof genoAmericanJoumdl
tlpic di\ersily in clonalplant species.
o f B o r a n y7 4 : 1 2 3 - l 3L
F a l k . D . A . . a D dK . E . H o l s i n g e r( e d i t o r s ) .1 9 9 1 .G e n e t i c s
and Conservarionof Rare Planls. O brd Univcrslty
Press.Oxfofd, England.
N{. A., and P S. Soltis. 2000. Patternsof geCiitTendanner.
netic varirdon in rarc and widesprerd plant conge
n e r s .A c r i c a nJ o u r n aol f B o t a n y 8 7 : 7 8 17 9 2 .
Coltlicb, l-. D. I 9 8 L Gene number in spccicsof ^J/s|dft'd.
that hr!e dil1ercnl chronosome numbers. Proceed
ings of the National Acaden) ol Scicncc,USA
78:3726-3729.
1982.Consefvarionand duplication ol isozymcs
i n p l a n t s .S c i e n c el l 6 : 3 7 3 - 3 8 0 .
Hamrick. J. L.. and Nt. J. $'. Godt. 1990.AlloTyme dilersity
i n p l a n ts p e c i e sP. a g e s , + 3
63 /r Brorn. A. H. D., M.
T. Clegg. A. L. Kahler. and B. S. $tir (editors)Planl
PopulationGenetics,Breediru.andCcnclic Rcsources.
SinauerAssocialcs.Inc.. Sunderland,Maryland.
H a n r r i c k .J . I - . . M . J . W G o d l , D . A . M u r a w l k i . a n d M . D .
Loleless. 1991. Corrclatjons befl\een speciestraits
f u. rc o n ' c r r\ l o n
- n J - l l o / ) n e o i \e \ r r i : ' m p l r . : , l r o n
bntog). PagesT5861, Falk. D.A., andK. E. Holsingel
(cdjtors) Geneticsand CoNervadon ol Rarc Plants.
Oxford Univenitr- Prcss.Oxford. England.
Hcndcrson. D. Nl. 1976.,{ bios}srcmadc study of Pacific
N o r ! h w c s r e r nB l u e - e y e d G r a s s e s l S i s \ r i t k : h i u l n ,
lridaceae).Brillonia 28: I :19-I 76.
Husband,B. C., and D. W Schcnske. 1997.The effect of
inbreeding in diploid and teraploid populations of
L? ilah ium ansustifolium (Onagraceae):implicati(nrs
lbr thc gcncdc basi,iof inbreedingdepression.Elo
l u t i o n5 l : 7 3 7 - 7 . 1 6 .
Sts-trirtchiumIsozynteVariation
353
Karron. J. D. 1991.Pattems of genetic vdrialjon and breeding syslemsin rareplant species.Pages87 981, Falk.
D. A., and K. E. Holsinger (editors) Genetics and
Conservationof Rdre Pldnr\. Oxtord University Press,
Orford, England.
Knapp. E. E.. and K. J. Rice. 1996. Genetic sllucturc and
genefl o\\ in tArrrs gldr.rr (bluc wildrye): implications lbrnali!e grasslandrestoration.RestontionEcol
og,v,l:l-10.
Menges,E. S. 1991.The applicalionofminimum liable popu
lation theory to plarls. Pagcs 45 6l /n Falk, D- A..
and K. E. Holsjngef (editors) Cenelics and Conservarion of Rarc Plants.Orford Uni\ersily Press.Ox
ford, England.
Pavek.D. S. 19E9.Variadon\l ithh dnddnong sir Stn rrkitr,,?
(Iridaceae)Populationsin Northern Idaho and South
eancm Bntish Columbia. Ph.D. Disse adon. Uni!ersity of Idaho, Moscow.Idaho.
Schenskc. D. w. ts. C. Husband. NL H. Ruckclshaus.C.
Goodwillic.l. M. Parker and J. G. Bishop.199,1.
ElaluatiDg approachc\1otheconservationofrare and
endangeredplants. Ecolog) 75:58,16Uo.
Receiv'ed
2I Septenher1999
Actepted 14 August 20()()
35;l
Wilson, Doede,and Hipkins
Shannon, C. E.. and w. \veaver. 1949. The Mathematical
Theorl of Communication. Univ. of lllinois Press.
Urbana.lllinois.
Sindich. N!. T.. and N{. D. Morgan. 1994.Allozymic uni
lbmiry in lrir /4.rrr'ir (dwarf lake iris) in Wiscons i n .C a n a d i a D
J o u m a lo l B o l a n y7 2 i l l 2 0 - 1 7 2 2 .
S o l t i s ,D . 8 . . a n d I - . H . R j e s e b e r g1. 9 8 6 .A u t o p o l y p l o i d )i n
Tolniea nettaiesii (Saxiliagaccac):genetic inrights
iiLrn enzymcs electrophoresis.AmericaD Joumal ol
uotan)73:310-318.
Werdel. J. F.. and \. F. weeden. 1989.Visuaiizatior and in
terpreradonof plant isoTymes.Pages5-45 ,r Soltis.
D . E . . a n dP S . S o l t i s( e d i l o r s )I s o T y m e isn P I a n tB i olog!,. DioscoridesPfess.Poftland. Oregc'n.
Westermeier R. 1997. Elcctrophoresisin Practice (2nd edrtion). VCH Vedagsgesellshali,Weinhcim. Fcderal
Republic oi Cermany.
Ych. F. C., R. C. Yang, and T. Boyle. 1997.PopgcncVcrsion
1.20.Uni!ersil! of Alberta. Edmonton,A1befia.