Variation in Pollen Size, Fertilization Ability, and Postfertilization Siring Ability in
Erythronium grandiflorum
Author(s): Mitchell B. Cruzan
Reviewed work(s):
Source: Evolution, Vol. 44, No. 4 (Jul., 1990), pp. 843-856
Published by: Society for the Study of Evolution
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Evolution,44(4), 1990, pp. 843-856
VARIATION IN POLLEN SIZE, FERTILIZATION ABILITY, AND
POSTFERTILIZATION SIRING ABILITY IN
ER YTHRONIUM GRANDIFLOR UM
MITCHELL
B.
CRUZAN'
Department
ofEcologyand Evolution,
StateUniversity
ofNew Yorkat StonyBrook,
StonyBrook,New York11794 USA
TheRockyMountainBiologicalLab, CrestedButte,CO 81224 USA
Abstract.-The mean volume of pollen grainsand total pollen productionvaried both withinand
The second flowersof two-floweredplants tended to
among plants of Erythroniumgrandiflorum.
produce smallerand fewergrainsthan firstflowers,but therewas no overall relationshipbetween
mean pollen grainsize and productionper flower.I evaluated the effectsof pollen size differences
withinand among plants on two components of male reproductivesuccess: pollen tube growth
and postfertilization
siringability.
Pollen tubes grownin media were longerforsecond flowers,but were not correlatedwith the
mean size ofpollengrains,suggesting
that(1) internalresourcecontentofpollen (i.e., carbohydrates
plus lipids) was not associated with the hydratedsize of pollen, and that (2) pollen fromsecond
flowerscontainedmore resources.I analyzed the growthrate and the fertilizationabilityof pollen
growingin styles.Growthrate differedamong donors and recipients,but no effectsof pollen or
donor characters(i.e., pollen production,grain size, and flowerposition) were detected.In single
donor pollinations,pollen size was negativelycorrelatedwith fertilizationabilityacross donors,
and positivelycorrelatedwithpostfertilization
siringabilityof donors. A second experimentused
in pollen size and flowerposition had effectssimilar to
pairs of donors; within-plantdifferences
thesingledonorexperimenton fertilization
werenotsignificant.
ability,butamong-plantdifferences
The resultscorroborateearlierexperimentsthatsuggestthatthe growthof pollen tubes in the style
is probably controlledby the recipient,since donor charactershad minimal effectson pollen
fertilizationability.
in mean grainsize and
Postfertilization
siringabilitywas not affectedby within-plantdifferences
production.For among-donordifferences,
the numberof seeds set foreach donor was positively
correlatedwiththe mean grainvolume, and when a donor producinglargepollen fertilizedovules
in an ovary,therewas increasedseed abortionforseeds in the same ovarysiredby a second donor.
In addition, the total numberof seeds produced by a fruitwas decreased when both donors had
largepollen, apparentlydue to increasedpostfertilization
abortion.Postfertilization
processes apthat are expressedthroughcompetitionamong depear to be influencedby paternaldifferences
veloping seeds formaternalresources.
Received May 16, 1989. Accepted November 10, 1989.
indicatingthat floralcharacterscan be selected throughmale function.Delivery of
pollen to stigmasdoes not,however,ensure
male reproductivesuccess. Depending on
pollinator activity,the number of pollen
grainsdeposited on a stigmacan exceed the
number of ovules (Snow, 1986; Cruzan,
1989), and usually pollen fromseveral donors is present (i.e., pollen carryoverhas
been foundin severalspecies: Thomson and
Plowright,1980; Thomson and Thomson,
1989). It has been suggestedthat competition forovules among pollen fromdifferent
donors would select for high tube growth
rates and fertilizationabilities (Haldane,
1932; Willson and Burley, 1983; Stephenson
and Bertin,1983). On the otherhand,
1Present
address:Department
ofBotany,
University
ofToronto,25 WillcocksSt.,Toronto,Ontario,CAN- recipientstylesmayimpose incompatibility
ADA M5S 3B2.
barriersto certain pollen types, so pollen
Male charactersin some species can be
related to mating success throughan increase in the abilityof males to compete for
mates(intrasexualselection)(Darwin, 1871).
There has been considerable speculation
about intrasexuallyselected charactersin
plants (Janzen, 1977; Willson and Burley,
1983; Stephenson and Bertin, 1983), and
thereis some evidence thatplant characters
can affectpollen delivery(the firststage of
reproductionthroughmale function).Characteristicsof floraldisplays have been correlated with directmeasures of pollen donation(Willsonand Bertin,1979; Bell, 1985;
Stanton et al., 1986; Cruzan et al., 1988)
843
844
M. B. CRUZAN
charactersthat allow the circumventionof
these barrierswould be advantageous. In
eithercase, the average fertilizationability
of pollen donors mightdifferif therewere
variationin thecharactersaffecting
thepollen vigorof donors (the average fertilization
abilityof pollen when testedacross several
recipients).Pollen vigor is a statisticalattributeof plants; the actual charactersconferringan increased fertilizationabilityon
pollen would probably be strictlyphysiological, making them difficultto measure.
Certain more easily measured morphological charactersof donors and theirpollen,
however, representlikely candidates that
mightbe relatedto pollen vigor.In thiscontext,the average grainvolume (ifgrainsize
limitsresourcecontent)and the total number ofgrains(if limitedresourcesare divided among grains)produced by a plant may
be indicativeoftheavailabilityof resources
in pollen that are potentiallylimitingto
growth.Averagepollen size may also be indicative of a plant's overall metabolicvigor
if the volume that a grain achieves at maturityis dependenton itsgrowthrate.Either
greaterresourcecontentor increased metabolic vigor of pollen may increase its fertilization ability,affectingmale reproductive success.
In thispaper, I firstdescribevariationin
pollen grainsize and productionwithinand
among plants. I then use within-plantdifferencesin thesecharactersto examinetheir
potentialimpacton pollen tubegrowthrate,
fertilization
ability,competitiveability,and
postfertilization
siringabilityof pollen donors.
METHODS
GeneralBiology
Erythronium
grandiflorum
Pursh (Lili-
aceae) is a perennial herb arising from a
buried corm. Plants appear in early spring
shortlyaftersnowmelt,and flowering
plants
produce fromone to several flowers.Each
floweris relativelylarge (2.5-4.0 cm), with
six anthersoccurringin two whorlsof three
antherseach. On multiflowered
plantsflowers open sequentially,with approximately
24 hr between the anthesis of each flower
on an inflorescence.Anthersin the outer
whorldehisce shortlyafteranthesis,and the
anthersof the inner whorl usually dehisce
within24 hr of the outeranthers.The three
lobes of the stigma correspond to three
channelswithinthelumen ofthefluid-filled
style.The channels are open to each other,
but pollen tubes from pollen germinating
on each lobe tendto growalong one channel
(Cruzan, 1990), allowingme to compare the
growthof pollen fromdifferent
sources in
the same style.When pollen fromthe same
source is applied to the two separate lobes
the two pollen populations performsimilarly(unpubl. data). Afterpollination,pollen tubes require between 24 and 72 hr to
reach the ovary, but no additional tube
growth takes place after 72 hr (Cruzan,
1989). When styleswere collected 5 days
afterpollination,therewere oftenover 100
tubes presentin the stigmasof flowers,but
20-80% of theseneverreach the ovary(stylar attrition;Cruzan, 1989). Both stylarand
ovarian attrition(failureof pollen tubes enteringthe ovary to produce seeds) contribute to variation in seed productionin natwithstylarattrition
urallypollinatedflowers,
havinga largerinfluencethanovarian (Cruzan, 1989). In all of the experimentswhere
tube growthwas measured in the style,I
used measurementsonly on tube populationsthathad at least 10 tubes. I conducted
my experiments during the summers of
1985, 1986, and 1987 near the Rocky
Mountain Biological Lab, Gothic, Colorado.
Pollen Size Variation
I collected anthers from two-flowered
plantsto determinewithin-and among-plant
variation in pollen production and grain
volume. I collectedundehiscedanthersfrom
10 plantswithin30 m of each otherin each
of fourpopulations(Table 1). From each of
the two whorls of each flowerI removed
two anthers,placed theminto separate 1.5ml microcentrifuge
tubes, allowed them to
dehisce, and stored them in 70% EtOH. I
diluted each sample with 100 ml of 0.1%
saline, and used a 16 channel Coulter
Counter (model TA II; see Harder et al.,
1985 forfurtherdetails) to take three2-ml
subsamples fromthis solution. I estimated
theaveragegrainvolume and thetotalnumber of grains presentforeach antherfrom
a size frequencydistributionacross five
845
VARIATION IN POLLEN SIZE
TABLE 1. Meanpollengrain
diameter
andpollenproduction
forone-flowered
Erythronium
grandiflorum
plants
intheEastRiverValleynearGothic,Colorado.Dateofcollection,
infourpopulations
growing
elevation,
number
ofplantsused in theanalysis,meanpollengraindiameterin micrometers,
meantotalpollenproduction
per
inparentheses
plant,andthemeanlength
oftheoutertepalofflowers
aregiven.Numbers
arestandard
deviations.
Withina column,meansfollowedby the same letterare not significantly
different
fromeach other(Tukey
multiplerangetest).
N
Date
Elevation (m)
403 Trail Head
6/21
2,950
8
Bellview
6/21
3,060
10
Baldy
6/24
3,130
9
SchofieldPass
7/2
3,260
9
Site
CoulterCountersize channelsthatspanned
a size range of 3,000-97,000 ,im3. I calculated a total volume for each channel by
multiplyingthe average frequencyfor a
channel by the predeterminedmidpoint
volume of thatchannel. Beforemakingcalculations,I took the naturallog of the midpointvolumesto normalizethem,and backtransformed
the values obtained to give the
average volume in cubic micrometers.I
analyzed the data in a nested analysis of
variance using the Varcomp procedure of
SAS (SAS Inst., Inc., 1985).
in Vitro
PollenTubeGrowth
Previous measurementsindicated that a
considerablepollen size difference
could exist between flowersof an individual plant
(Cruzan, unpubl. data). I used thisattribute
of E. grandiflorum
to determinewhat effect
pollen size mighthave on germinationand
pollen tube growthin vitro.I collected undehiscedanthersfromeach flowerof 35 twofloweredplants from a single population,
and pooled thesix anthersfromeach flower.
I storedanthersin air-tightvials to prevent
them fromdehiscing,and opened all vials
at the same time to forcethe anthersfrom
the two flowersof a plant to dehisce simultaneously.Afterthe anthershad begun
to dehisce,I placed a small amountofpollen
fromeach flowerin an aqueous germination
mediumcontaining20% sucrose,15% Knox
gelatin,and 5% nutrientsolution (containingboric acid, 100 ppm; calcium, 300 ppm;
potassium, 100 ppm). I firstmeltedthegermination medium and spread a small
amount into a thin layer on a microscope
Pollen diameter
39.06 a
(16.35)
40.38 b
Pollen production
75,762 a
(9,414)
97,338 b
Tepal length
29.5 a
(1.1)
31.6 b
(15.83)
39.72 c
(16.65)
(19,230)
91,962c
(15,450)
(1.1)
28.8 a
(1.1)
38.83 a
(17.85)
105,006 d
(18,807)
31.6 b
(1.1)
slide. I placed the pollen fromboth flowers
of an individual plant at the same time on
different
areas of the same slide, placed the
slide into a petridish containingmoistened
tissue paper, and kept the slide in the dark
at room temperaturefor48 hr.I stainedthe
slides with Alexander's stain (Alexander,
1969), and mounted them by meltingthe
gelatinmixturebeforeadding a cover slip.
I analyzedtheslidesforgraindensity,germination, and tube length. I determined
grain densityand percentagepollen germination by censusing slides under a compound microscopeat 100 x. I made a transectacrossthecenterofeach slideand scored
all of the grains encounteredforgermination. I scoredabnormallyshorttubes as ungerminated,because theywere much shorter than the median pollen tube length. I
calculatedgraindensityforeach slide based
on the area sampled as the total numberof
grains encounteredand percentagegermination for each floweras the fractionof
grains encounteredthat had germinated.I
measuredtube lengthsundera stereoscopic
microscopeat 12 x. Using an opticaltracing
device in conjunctionwith a DigitBit digitizer,I recorded the coordinates of points
alongthelengthofa tubeincludingitsorigin
and tip, and then added the distances between adjacent pairs of points to give the
total pollen tube length.The large number
of tubes made tubes toward the center of
the pollen aggregationdifficultto trace, so
I measured tubes extendingoutward from
the main aggregation.I measured the 10
longest traceable pollen tubes present for
each flower.
846
M. B. CRUZAN
I measured the mean grain volume for each channel as covariates. Untransformed
each floweras describedabove. I eliminated data were approximatelynormallydistribtwooftheplantswithunusuallylargepollen uted.
grainsfromthe analysissince theywere sigFertilizationAbility
nificantlylargerthan the mean size of the
other plants (based on single observation
I measured stylarand ovarian attrition
comparisonsto themean,P < 0.05 forboth). forpollen fromdifferent
donors aftercrossUsing the GLM procedureof SAS (SAS es among plantsin situ. I selected 35 threeInst., Inc., 1985), I compared graindensity floweredpollen donors and crossedthemto
and percentage germination for the two 2-7 three-flowered
recipientseach. To preflowerpositions in ANOVA models with vent extraneouspollination,I covered the
the identityof each plant enteredas a ran- pistilsof recipientflowerswithgrass straws
dom factor.I analyzedtubelengthwithplant whiletheywerestillin bud. I pollinatedone
identityas a random factor,flowerposition or two of the flowerson each recipientwith
as a fixedfactor,and mean grain volume, pollen fromone of the donors,and in most
pollen density,and percentagegermination cases used two donors on each recipient.I
as covariates. All data approximated nor- collected undehisced anthersfromthe first
mal distributions,so no transformations flowersof the donors several days before
were necessary.
making pollinations and stored them in
closed microcentrifuge
tubesat 2?C. On each
day I allowed anthersto dehisce by opening
Pollen Tube GrowthRate in Vivo
the vials to expose themto lower humidity
Using the same two-flowered
plants used and made pollinationsby evenlycoatingrein thein vitrotestsdescribedabove, I com- cipient stigmaswith pollen on the second
pared the growthof pollen from the two day afterrecipientflowershad opened. Afflowersof each donor in the same recipient tercompletingthe pollinations,I preserved
style.I collectedrecipientbuds, submerged the remainingpollen from each donor in
theirstems in tubes containingwater,and 70% EtOH. I collectedrecipientstyles5 days
allowed them to open before making pol- afterpollinatingthem and stored them in
linations.Using fivedifferent
recipientflow- 70% EtOH. Pollen tube growth typically
ers foreach donor, I pollinated one lobe of ceases after2-3 days, and harvestingstyles
each of the three-lobedstigmaswithpollen after5 days has no effecton fruitset (Crufrom one flowerof a donor plant, and a zan, 1989). I collectedfruitswhentheywere
second lobe ofeach stigmawithpollen from mature,and countedand weighedthe seeds
the second flowerof the same donor. To fromeach fruit.I determinedmean grain
provide a point of referenceI excised the volume for all donors using the Coulter
unpollinatedlobe. I fixedstylesin 70% EtOH Counter as describedabove. Afterstaining
24 hr afterpollinatingthem and measured stylesas describedpreviously,I countedthe
pollen tubes under a stereoscopic micro- tubes presentin the stigmaand at the base
scope at 50 x . Afterslittingstyleslongitu- of the style,and calculated stylarattrition
dinallyand flattening
them,I stained them as thefractionoftubespresentin the stigma
witha 1:1 mixtureof acidified0. 1% aniline not reachingtheovaryand ovarian attrition
blue and acetocarmine(40% acetic acid sat- as the fractionof the numberof tubes presurated with carmine) for 10 min (Cruzan, ent at the base of the stylenot producing
1989), censusedtubesin each stylarchannel seeds (Cruzan, 1989). Using GLM I deterat 1.5 mm intervalsalong the lengthof the mined the effectof mean grain volume on
style,and calculatedmean tubelengthsfrom stylarattrition,ovarian attrition,and seed
these distributionsby assuming that tubes production,in models which included the
ended halfwaybetweencensus points (Cru- recipientflowerposition as a fixed factor,
zan, 1986). Using GLM again, I enteredthe and the mean grain volume of the donor
pollen donor identityand theidentityofthe and thenumberof tubespresentin the stigrecipientas random factors,flowerposition ma as covariates.I also examined theeffects
as a fixedfactor,and mean grain volume of pollen tube growthparametersand the
and the numberof pollen tubes presentin mean grainvolume of donors on the prob-
VARIATION IN POLLEN SIZE
847
ofvariation
inpollensizeand
ability of fruitset using logisticregression TABLE 2. Components
in Erythronium
pollenproduction
grandiflorum.
Two
withthe CATMOD procedureof SAS.
anthers
werecollectedfromeachantherwhorlofeach
flowerof 10 two-flowered
plantsin fourpopulations.
Pollen CompetitiveAbility
Numbersrepresent
the percentage
of totalvariation
Using geneticmarkersto assignpaternity explainedby each component.
Variancecomponents
to seeds, I examined the effectof variation and significance
levelsare froma completely
nested
in pollen production and pollen size both modelusingtypeIV sumsofsquaresfromtheGLM
between and within pollen donors on the procedure
ofSAS.
competitive ability of pollen afterdouble
Volume
Production
d.f
pollinations.I used pollen fromboth flow- Component
3
12.73*** 16.93***
ers of pairs of donors to pollinatetwo lobes Population
32
48.06*** 18.72***
on each recipientstigma.In thisdesign,each Plant
Flower
36
22.27*** 50.62***
recipientfloweralwaysreceivedpollen from Whorl
72
3.80**
0.38 ns
two different
donors and fromtwo different Error
144
13.14
13.35
donor flowerpositions.I electrophoretically **P < 0.01.
*** P < 0.001 .
screened 240 two-floweredplants from a
ns = P > 0.10.
population at the upper end of Washington
Gulch, about 5 km fromthe RMBL, forthe
MDH locus. From this group I chose 110 sired by each donor. I analyzed the effects
homozygousindividualsto act as pollen do- of both donors' characterson each donor's
nors and recipients.I used 18 pairs of do- stylarand ovarian attritionand the pronors, with one donor of each pair being portion of seeds each sired by alternately
homozygousfastand theotherbeinghomo- treatingeach donor as eitherthefocaldonor
zygous slow, to pollinate groups of five (measurements were treated as response
homozygousrecipients(eitherfastor slow) variables), or as the nonfocal donor (meaby doing single donor pollinations to each surementsweretreatedas independentvarioftwo stigmalobes of each flower.For each ables). I used differentmodels to analyze
ofgrain
recipientflowerI pollinatedone stigmalobe bothwithin-and among-planteffects
withpollen fromthe firstflowerof one do- size and production. For within-plantefnor, and pollinated the second lobe with fects,I held the identityof the focal donor
pollen fromthe second flowerof the other statisticallyconstantusing GLM and anaofgrainsize and production
donor. For the second flowerof the same lyzedtheeffects
recipientI reversedthe donor flowerposi- and flowerposition of both the focal donor
tions,and switchedthe flowerpositionsfor and thenonfocaldonor on thefocaldonor's
the pair of donors between the recipient pollen tube growth,ovarian attrition,and
flowerpositions for successive recipients. fertilizationsuccess. For the among-plant
Five days afterpollinatingthem,I collected analysis, I used path models that included
the recipientstylesand storedthemin 70% thenumberoftubespresentin the styleand
EtOH and determinedthe amount of stylar grain size and productionforboth donors
and ovarian attritionforeach donor's pol- with the focal donor's degree of stylaratlen in each recipient floweras described tritionand fertilizationsuccess as depenabove. I storedand analyzedthepollenfrom dent variables using the REG procedureof
each donorflowerusingtheCoulterCounter SAS. Prior to analysis I square root-arcsin
as described previously. I measured the transformedthe proportionof seeds sired
lengthof an undehisced antherfromeach and leftall othervariables untransformed.
donor floweras a measure of total pollen
RESULTS
production(r2 = 0.814, P < 0.0001 forthe
Pollen Size Variation
pollen production vs. anther length relaMean grainvolume and productionvartionship;J. Thomson, pers. comm.). After
countingand air dryingtheseeds fromfruits, ied withinand among plants. The size disI electrophoretically
determinedthe geno- tributionsof pollen fromplants of the four
typeofa sample of 15 seeds fromeach fruit, populationscensused wereunimodal for36
or all of the seeds forfruitshavingless than of the 40 plants testedand bimodal forthe
15 seeds,to estimatetheproportionofseeds others. The latter individuals had large
848
M. B. CRUZAN
in vitromeanlength
3. Meanpollendiameter
ofthelongest10 pollentubes(mm),thepercentage
(,um),
ofgrainsgerminating
and thegraindensityon theslide,and in vivo meanpollentubelengthand numberof
forpollenfromthefirst
ofErythronium
pollentubespresent
and secondflowers
grandiflorum.
Pollendiameters
are in micrometers
and pollentubelengthsare in millimeters.
Means followedby the same letterare not
fromeachother.Standarddeviationsarein parentheses.
significantly
different
TABLE
In vivo
Flower position
First
Second
Mean diameter
Length
In vitrodensity
Germination
Length
Number
56.12a
(21.38)
49.53 b
(23.75)
4.18 a
(1.20)
5.17 b
(1.27)
90.68 a
(43.01)
84.45 a
(33.82)
47.47%a
(16.97)
56.38%b
(14.46)
3.17 a
(0.86)
3.07 a
(0.82)
19.1a
(5.8)
18.4a
(8.0)
numbersof small, apparentlyaborted pollen grainsand wereremovedfromtheanalysis.The mean pollen diameterrangedfrom
36.2 to 41.8 ,um(mean = 39.5, standard
deviation = 17.1, N = 36). Pollen production varied among plants much more than
pollen volume (mean = 15,519.6 grains/anther;standarddeviation = 446.6, N = 288),
and therewas no relationshipbetweenmean
pollen productionand mean pollen volume
among plants(r = 0. 12, P > 0.40, N = 36).
Pollen size and productionvaried among
the-four
populationscensused (Table 1), althougha greaterproportionofthetotalvariation in these characterswas among plants
than among populations (Table 2). Pollen
size and production also varied within
plants.Pollen volume varied betweenflowers of plants to a much greaterextentthan
withinflowers(between or withinwhorls),
but not as much as among plants (Table 2).
Pollen productionvaried between the two
flowersof plants to a much greaterextent
than either within flowersor between inTABLE 4. Analysis of in vitro pollen tube lengthof
pollen from firstand second flowersof Erythronium
grandiflorum.The identityof the donor was entered
as a random factor,donor flowerposition as a fixed
factor,pollen tubes measurednestedwithinthe flower
position,and mean pollen grainvolume, graindensity
on the slide, and percentagegerminationentered as
covariates. Parametersare based on type III sums of
squares fromthe GLM procedureof SAS.
Source
Donor identity
Donor flowerposition
Pollen tube (flower)
Mean pollen grainvolume
Grain density
Percentagegermination
d.f
F value
26
1
18
1
1
1
4.84
14.18
0.82
0.17
0.64
1.41
Probability
P
0.0001
0.0002
0.6813
0.6786
0.4253
0.2357
dividual plants (Table 2). Overall, the second flowersof plants in these populations
producedless pollen (mean = 82,470 grains
per flower)than theirfirstflowers(mean =
103,620 grains per flower),and the pollen
fromsecond flowerstendedto be smallerin
diameter(mean = 39.2 ,im) than pollen of
theirfirstflowers(mean = 39.9 ,im).
PollenTube Growth
in Vitro
Second flowersof the plants used in this
experimentand thein vivo experimenthad
smaller grains than firstflowers(Table 3).
The difference
betweenthemean pollen size
of firstand second flowerswas not consistent for all plants, so effectsof pollen size
on pollen vigor can be determinedseparatelyfromeffectsdue to flowerposition.
The mean lengthof the longest 10 pollen
tubes on germinationslides was affectedby
both the identityof the donor, and the positionof the source flower(Tables 3 and 4).
The proportionof grains germinatingwas
positivelycorrelatedwith theirdensityon
TABLE 5. Analysis of in vivo pollen tube lengthof
pollen fromthefirstand second flowersofErythronium
Donor identityand recipientidentitywere
grandiflorum.
enteredas random factors,flowerposition as a fixed
factor,and the mean grainvolume and numberofpollen tubes presentwere enteredas covariates. Parameters are based on type III sums of squares fromthe
GLM procedureof SAS.
Source
Donor identity
Donor flowerposition
Recipient identity
Donor x recipient
interaction
Mean pollen grainvolume
Number of tubes present
Probability
P
d.f
F value
19
1
3
4.04
0.10
7.33
0.0004
0.7554
0.0008
24
1
1
4.76
0.40
0.71
0.0001
0.5325
0.4070
VARIATION IN POLLEN SIZE
849
6. Analysesof thenumberof pollentubesin thestigmaand at thebase of thestyle,theamountof
and thenumberofseedssetin Erythronium
stylarand ovarianattrition,
grandiflorum.
Independent
variables
are thepositionoftherecipient
flower(fixedfactor),
meanpollengrainvolumeofthedonor,and numberof
pollentubesin thestigmaand at thebase ofthestyle(covariates).
NumbersareF valuescalculatedfromthe
typeIII sumsofsquaresfromtheGLM procedure
ofSAS (SAS Inst.,1985).
TABLE
Pollen tubes
Attrition
Source
Stigma
Base
Stylar
Ovarian
Recipientflower
position
Donorpollengrainvolume
Pollentubesin stigma
3.53*
0.38 ns
-
1.73ns
7.44**
80.05***
2.82t
15.26***
10.44**
3.73*
2.36 ns
3.02t
-
-
-
Pollen tubes at stylebase
tP < 0.10.
0.15 ns
Seeds sired
6.47**
9.17**
5.41*
14.21***
*p < 0.05.
**p < 0.0 1.
*** p < 0.00 1.
ns = P > 0.10.
theslide (r = 0.21, P < 0.05, N = 56). Pollen in air-tightvials does not affectpollen tube
tubesfromsecond flowersweresignificantly growth(unpubl. data). The numberoftubes
longerthanpollen tubes of pollen fromfirst presentin stigmaswas not affectedby either
flowers(Table 3). Pollen density,percentage the mean grainvolume of the donor or the
germination,and mean volume had no sig- position of the recipientflower(Table 6).
nificanteffects
on tubelengthin vitro(Table The numberof tubes at thebase of the style
and stylar attritionwere significantlyaf4).
fectedby the position of the recipientflower, the numberof tubes presentin the stigPollen Tube GrowthRate in Vivo
ma, and themean grainvolume ofthedonor
After24 hr,tubes usually extendedfrom
(Table 7). Plants thatproduced small pollen
one-halfto three-quarters
the lengthof the
tended to have less stylar attritionthan
style.Mean tube lengthsafter24 hr ranged
plants that produced large pollen grains (r
from1.69 to 5.32 mm. Tube lengthsin vivo
= 0.48, P = 0.0016).
werenot correlatedwithpollen tube lengths
Only 26 of the 57 flowerspollinatedproforthe same plantsobtained in growthmeduced
a fruit,apparently because many
dium (r = 0.084, P > 0.5). Pollen fromthe
ovules were leftunfertilized.The amount
two flowerpositions growingin the same
ofstylarattritionwas a fairlygood indicator
style produced similar numbers of tubes,
of the probabilityof fruitretention(P <
whichgrewat similarrates(Table 3). Mean
from a logistic regressionmodel).
0.008;
tubelengthsforthesepollinationsweremost
Pollinations resultingin a small degree of
strongly
affected
bytheidentityofthepollen
stylarattritionhad a greaterprobabilityof
donor, the identityof the pollen recipient,
a fruit(holding the number of
producing
and theirinteraction(Table 5). Mean grain
pollen
tubes
at thebase ofthe styleconstant
volume and the number of tubes present
this
variable into the model as
by
entering
had no effecton mean tube lengthin vivo
a covariate). For flowersthat set a fruit,
(Table 5).
postfertilizationovule abortion was most
stronglyaffectedby the recipientflowerpoFertilizationAbility
sition and the number of tubes presentin
The mean grain diameter of the donors the stigma(Table 6). Seed set was affected
(firstflowers)rangedfrom24.4 to 33.0 ,um by therecipientflowerposition,thenumber
(mean = 29.5, standarddeviation = 1.76). of tubes at the base of the style,and the
Approximately 25% of the pollinations average pollen grain volume of the donor
failed due to loss of pollen viability.This (Table 6). Overall, plants producing large
was probablydue to prolongedexposure to pollen grains sired more seeds than plants
dryair sinceErythronium
pollen is sensitive producingsmall grains(r = 0.49, P = 0.035
to desiccation(unpubl.data). Previouswork forthe correlationbetweenpollen size and
indicatesthatstorageofErythronium
pollen numberof seeds sired).
M. B. CRUZAN
850
variationwithdonoridentity
TABLE 7. Variancecomponents
fromanalysesof covarianceforwithin-donor
in Erythronium
pollinations
grandiheldconstantforseveralmeasuresofdonorperformance
aftertwo-donor
was treatedas a randomfactorand recipient
was nestedwithindonor
identity
florum.The donoridentity
identity.
Effects
listedincluderecipient
and sire'sflower
positions,and thenumberofpollentubespresentin
ofthevariance
thestigma,pollenproduction,
and meanpollengrainvolumeforbothdonors.The percentage
ofSAS is given
fromtypeIV expectedmeansquaresfromtheGLM procedure
explainedby each parameter
(SAS Inst.,1985).
Attrition
Source
Recipientidentity
flower
position
Sireidentity
Flowerposition
Tubesin stigma
Grainvolume
Pollenproduction
Nonsire
Tubesin stigma
Grainvolume
Pollenproduction
Pollen tubes at stylebase
Stylar
Ovarian
Seeds sired
0.0*
0.0
0.0
2.3*
92.4***
3.3**
0.0
1.2t
0.6
.1lt
25.1***
18.6**
24.9**
0.0
60.0
0.0
40.Ot
0.0
0.0
0.0
0.0
24.7*
5.7
40.0*
4.8
4.8
4.3
2.5
0.6
0.0
1.2t
7.7*
0.8
21.1**
0.0
0.0
0.0
6.5
3.2
3.7
tP < 0.10.
* P < 0.05.
**p < 0.01.
***p < 0.001.
PollenCompetitive
Ability
In flowerspollinated with two donors,
each donor's tube growthin the style(when
acting as the focal donor) was affectedby
the charactersof both donors present(the
focal donor and the nonfocaldonor; Table
7). For the focal donor, stylarattritionincreasedwhenthereweremoretubespresent
in thestigma,and whenthemean grainvolume was greater(Table 7). The position of
the donor floweralso significantly
affected
the degree of stylarattrition,with pollen
fromsecond flowers(mean = 52.76%) having slightlyless attritionthan pollen from
firstflowers(mean = 53.29%) (Table 7). The
focaldonor's totalpollen productionhad no
effecton tube growth,but stylarattrition
was greaterwhen the nonfocal donor had
greaterpollen production(Table 7). Higher
numbersof nonfocaldonor tubespresentin
the stigmaalso tended to increasethe focal
donor's stylarattrition.In a path model examining among-donor variation in focal
donor's tube growth, the only covariate
having a strongeffectwas the number of
focaldonor tubespresentin thestigma(Fig.
1).
Variationin ovarian processeswas mostly among donors and was not affectedby
within-donorvariationin pollen characters
(Table 7, Fig. 1). When among-plantdiffer-
ences were held constant,none of the covariatesmeasuredhad significant
effectson
donor performanceand most of the variationwas explainedbydifferences
amongdonors(Table 7). The pathanalysisforamongdonor variation in donor performanceindicates that average grain volume was
stronglycorrelatedwitha donor's abilityto
sire seeds (Fig. 1). There was also a strong
negativeeffectof the mean size of the nonfocaldonor's pollen on thenumberof seeds
produced that were fertilizedby the focal
donor. The nonfocal donor's total pollen
productionand numberoftubespresenthad
negativeeffectson the success of the focal
donor, but these were not significant.
SynopsisofResults
Consistenteffectsforthe donor and pollen charactersmeasuredwerefoundforpollen tubegrowthin mostexperiments(Table
8). Pollen from second flowersproduced
longertubesin vitro,did not differ
in pollen
tubegrowthratein vivo, and had less pollen
tube attritionthanpollen fromfirstflowers.
Pollen productiondid not have any effect
on any of the tube growthmeasurements,
exceptforitsinfluenceon thestylarattrition
of pollen fromotherdonors growingin the
same style.Mean pollen grain volume did
notaffect
eithertubelengthin vitroorgrowth
851
VARIATION IN POLLEN SIZE
PollenTubesinStyle
(-0. 293***)
PollenGrainVolume
(0.007)
o
o
a
0
0
LL
a
o
0
(0. 090)
o
PollenTubesinStyle
(0. 107)
PollenGrainVolume
o
z
PollenProduction
(0.030)
LE
o
a
PollenProductioni
_
O
_
(0.086)
Stylar
(0.045)
PollenGrainVolume
(0. 581*)
PollenPro
d uctio
n_
(0. 047)
o
0
U_
PollenTubesinStyle-(-0.264)
PollenGrainVolume /
751**)
o
z
PollenProduction
(-0.238)
Attritiono
OX
/
PollenTubesinStyle
-
Seeds
Sired
(-O.
FiG. 1. Pathmodelsfortheamong-donor
effects
of cients (path coefficients)
frommultipleregression
pollentubeparameters
and donorcharacters
on the modelsthatincludedparameters
fora donor(focal
amountofstylar
attrition
andthenumber
ofseedssired donor)and fortheotherdonorpresent(nonfocaldoafterpollinations
withpairsofdonorsin Erythroniumnor)aregiven.
grandiflorum.
Standardized
partialregression
coeffi-
rate,but smallerpollen had less attritionin
both experimentswhere attritionwas examined. For ovarian attrition,neitherdonor flowerposition nor pollen production
had any effects.
Plants producinglargerpollen, however,had greaterseed siringability
thanplantsproducingsmallerpollenin both
experimentswhere it was examined.
DIscusSION
Pollen Resource Content
Pollen size varies bothwithin(Bell, 1959;
Bragg,1969; Stantonet al., 1986) and among
species (reviewedby Muller, 1979). Debate
about the consequences of differencesin
pollen size between species began in the
1800s (Darwin, 1877), and continuestoday
(refs.in Cruden and Lyon, 1985). Many of
these accounts assume that differencesin
pollen size representfunctionalmatingbarriers between species and that pollen size
has coevolved withstylelength.This interpretation presumes that pollen contains
limitingresourcesforthe growthof pollen
tubes and is autonomous in its growthto
the ovary. Physiologicalevidence conflicts
with these hypotheses.As early as 1830 it
was recognized that pollen grains must
receive nutrition from the stylar tissue
(Amici, 1830; cited in Cruden and Lyon,
1985). More recently,carbon labelingstudies clearlydemonstratethat microgametophytesare usingexternalsubstratesforpollen tubeconstruction(O'Kelley, 1955; Johri
and Vasil, 1961; Campbell and Ascher,
1975; Vasil, 1974). Although pollen is
known to contain storage materials(Todd
and Bretherick,1942; Baker and Baker,
1983), it is thoughtthattheseare used large-
8. Summary
ofresultsfromexperiments
forpollensourceflower
position,
pollengrainproduction,
and
mean pollengrainvolumeeffects
on pollentubegrowthand postfertilization
siringabilityin Erythronium
grandiflorum.
Resultsfromthein vitroand in vivo experiments
and thefertilization
ability(singledonorin
situ)and thecompetitive
ability(doubledonorin situ)experiments
aregiven.
TABLE
Experiment
In vitrotubegrowth
In vivogrowth
rate
Singledonorin situ
Double donorin situ
Singledonorin situ
Double donorin situ
Flower position
Pollen production
A. PollenTube Growth
Second> first
Not tested
No effect
Not tested
Not tested
Not tested
Second> first
No effect1
B. Seed SiringAbility
Not tested
Not tested
No effect
No effect
Pollen size
No effect
No effect
Smaller> larger
Smaller> larger
Larger> smaller
Larger> smaller
No effectof a donor's pollen productionon the attritionof its pollen was detected,but pollen fromlargerantherstended to increasethe attrition
of pollen tubes fromotherdonors growingin the same style.
852
M. B. CRUZAN
ly forgerminationand earlygrowth(Vasil,
1974; Cruden and Lyon, 1985); the majorityof pollen tube growthis supportedby
the stylartissue (reviewed in Vasil, 1974).
There is still the possibility,however,that
thereis some limitingresourcecontainedin
pollen thatis criticalto a pollen tube's successfullyreachingthe ovary,and thatlarger
pollen grainscontainmore of thisvital substance.
Althoughit has been assumed thatpollen
grain volume is correlated with resource
content,no data directlyaddress this issue.
Indirectsupport comes fromobservations
of the distancethattubesgroweitherin vitro or in vivo afterincompatible matings
(Glover and Barrett,1983; Anderson and
Barrett,1986); however, thereis also evidence of this type to the contrary(Barnes
and Cleveland, 1963). Pollen grownin media or in incompatible styles presumably
ceases growth when internal limiting resources are exhausted (Johri and Vasil,
mean grainvol1961). In E. grandiflorum,
ume was not related to pollen tube length
in vitro. When mean grain size was held
constant,pollen fromsecond flowerstended
to produce longertubes. Anthersfromthe
two flowerpositions dehisced at the same
in tubelengthshould
time,so thisdifference
not be due to a differencein pollen age.
Togetherthese resultssuggestthat the resource contentof pollen grains is not correlatedwiththe measurementof grainvolume used in this study. Pollen size in E.
grandiflorumcan vary substantially dependingon its state of hydration(pers. observation). The volumes obtained from
CoulterCountermeasurementsrepresentthe
fullyhydratedstate;thismeasureis not necessarilycorrelatedwith the minimal internal volume, whichoccursin thedehydrated
state.If pollen tube lengthattainedin vitro
is trulyindicativeofgrainresourcecontent,
pollen grain volume as measured by the
Coulter Counter does not correspond to
grainresourcecontent,and pollen fromsecond flowerscontainsgreateramounts of resources than pollen fromfirstflowers.
gestingthatplants may be able to influence
the vigor of the pollen that they produce.
Previous workindicatesthatpollen tube attritionin E. grandiflorum
may be mediated
by the recipientstyle (Cruzan, 1989), but
evidence forsome pollen-mediatedcontrol
of pollen tube growthvia interactionsbetween growingtubes has also been found
(Cruzan, 1990). Pollen vigorin this species
probablyrepresentsthe abilityof pollen to
circumventcompatibilitybarriersimposed
by the style, and contributesto pollensource-specificdifferencesin pollen tube
growth.Within-donordifferences
in pollen
tube growthrepresentevidence thatpollen
donors can influencetheirfertilizationsuccess. Mean pollen size and the position of
the donor flowerboth affectedthe amount
of stylarattrition,perhaps because of associated charactersthat influencedpollen
vigor.These effects
wererelativelyweak and
were not detectedin all of the experiments,
so the abilityof plants to influencepollen
vigor may be minimal. Among-donordifferencesin pollen vigor were greaterthan
within-donordifferences,
but thismay representgeneticratherthanenvironmentalinfluenceson pollen vigor.
The effectsof mean grainsize, flowerposition, and pollen productionfound probably representcharactersthatare correlated
withsome physiologicalcharactersthatdirectlyaffectthe fertilizationabilityof pollen. Because the primarycharactersaffectingpollen vigorwerenot measured,it is not
knownwhatthecost ofproducingmorevigorous pollen to the donor is, but we can
assess the potentialeffectsof the characters
measured.Previousworkindicatesthatsecond flowersofE. grandiflorum
tendto have
greaterrelativeinvestmentin male function
than firstflowersthrough higher pollen/
ovule ratios and a reduced probabilityof
settinga fr-uit
(Thomson, 1989; Cruzan, unpubl. data), so increasingthe amount of resources sequestered in pollen of second
flowersmightrepresentanothertrendin this
direction.Producing smaller pollen grains
also appeared to increase the fertilization
ability of pollen, but there may be an asPollenTubeGrowth
in theStyle
sociated cost in the expected longevityof
Within-plantdifferencesin pollen ap- smallerpollen. Lily pollen tends to be very
peared to have effectson its fertilization susceptibleto drying(Stanleyand Linskens,
ability after pollinations to flowers,sug- 1974), and under fieldconditions,Erythro-
VARIATION IN POLLEN SIZE
nium pollen loses viability within a few
hours after anther dehiscence (Thomson,
Cruzan, and Rigney, unpubl. data). Since
smallerpollen grainshave a highersurfaceto-volumeratio,theywould be expectedto
have reducedlongevity,but thishypothesis
remainsto be tested.Althoughit would appear that pollen donors can influencethe
vigorofthepollen theyproduce,thewithindonor differencesin fertilizationability of
pollen detected were relativelysmall, and
theadvantagesmightbe offsetby associated
costs to the donor.
853
droplets) to the endosperm tissue. An example of pollen tubes contributingcytoplasm to the endospermhas been observed
in Plumbago,wherethecontentsof thepollen tube are deposited in the eggsack (Russell, 1980). The observed differencesin
postfertilizationsiring ability that corresponded to differencesin mean grain size
may be due to among-plantdifferencesin
the amount of cytoplasmic material contained in theirpollen. This material might
not contributeto tube growth,and may be
reservedfordepositionintotheembryosack.
If so, within-plantvariation in mean grain
Seed SiringAbility
size would be expectedto affectovule matThere were relatively large differences uration; however, within-plantdifferences
among donors in theirsiringsuccess after in mean grain size may not be associated
both singleand double pollinations;plants with the amount of cytoplasmic material
that produced large pollen also sired more presentin pollen. On theotherhand, pollen
seeds. The differences
in siringsuccess ob- grainsare relativelysmall compared to the
served probably represent differencesin femalegametophyte(< 1% by volume; pers.
postfertilization
ovule abortionratherthan observation), so paternal contributions
selectivefertilization
ofovules. Selectivein- would be expected to be small. The possihibitionof pollen tubes in the ovary could bilityof an effectof cytoplasmicinclusions
in the number of seeds on ovule maturationcannot be eliminated
explain differences
sired; however, there was nearly a one to without furtherexperimentation,but it
one correspondencebetweenthenumberof seems unlikelythat paternal contributions
tubes at the base of the styleand the total to embryogrowthcould be responsiblefor
numberof ovules fertilized(seeds plus ap- the patternsof ovule abortion found.
parentlyfertilizedand aborted ovules; unThe average size of the pollen produced
publ. data). The differencebetween the by plants appears to be correlatedwithgenumber of tubes at the stylebase and the neticallybased differencesin the competinumber of ovules fertilizedwas unrelated tive abilityof the zygotessired by a plant.
to the mean size of a donor's pollen and to Plants producinglargergrainsnot onlyhad
the difference
in size betweenthe pollen of lower ratesof abortion forthe zygotesthey
the two donors present(unpubl. data). Ap- sired, but also appeared to induce greater
in the numberof seeds amountsofpostfertilization
parently,differences
abortionforthe
sired by different
donors were due to post- zygotes sired by the other donor present.
fertilizationprocesses rather than an in- Since littlemore than nuclei are transferred
abilityto fertilizeovules.
frommicrogametophytesto the ovules in
Pollen parentsmay be able to manipulate fertilization,variation among developing
the probabilityof an ovule's maturationby seeds siredby different
donors probablyrecontributingextra nuclear material to the flects genetic differencesamong donors
embryoor endosperm(Willson and Burley, ratherthan differencesin the paternal en1983), whichmay explain thedifferences
in vironment(Mazer, 1988). Althoughthedifpostfertilizationsiring ability seen in E. ferencesbetweendonors in theirsiringabilTransmissionof pollen plas- ity are probably not directly due to
grandiflorum.
tids to embryosis known by biparentalcy- differencesin their grain volume, the avtoplasmic inheritancein some groups (Bir- erage size of the pollen grainsproduced by
ky, 1976), but this has not been observed a plant appears to be a fairlygood indicator
in closelyrelatedmembersof the Liliaceae. of its postfertilizationsiringability. If the
It is possible, however, that pollen tubes size thatpollengrainsattainbeforematurity
contributeplastids and other cytoplasmic is dependenton theirmetabolic vigorthen
inclusions (e.g., starch granules or lipid among-plantdifferences
in mean grainvol-
854
M. B. CRUZAN
The genotypeof the paternal parent apume may reflectdifferencesin metabolic
vigor.
parently affectedpostfertilization ovule
Theoretical considerations of maternal abortion,but it may be the zygote's genoinvestmentand returnsin offspring
fitness type that determinesits ultimate success.
predictthat maternalparents should have Ovule growthand competitionwith other
complete control over the relative success ovules may be viewed as the firstround of
and allocation ofresourcesto theiroffspring selection on newly formedzygotes(Naka(Smith and Fretwell, 1974). Observations mura, 1986; Wiens et al., 1987). If success
of paternal influence on seed weight (in in the ovary is a resultof metabolic vigor
Aralia hispida, Thomson, pers. comm.; and growthrate,thenpollendonorsthathad
Campsisradicans,Bertin,1982;Raphanus increasedsiringabilitywould also siremore
sativus, Marshall and Ellstrand, 1986; R. vigorous offspring.This implies that preraphanistrum,
Mazer, 1987; Phlox drum- and postdispersalsuccess may be positively
mondii,Schlichtingand Devlin, 1989) con- correlated.If this were the case, selection
tradictthispredictionand suggestthatthere would be predictedto minimizedifferences
may not be strictmaternalcontrolover zy- in siringability. In an earlier experiment,
gote success. Physiologicalmodels of plant ovarian processesdid not contributesigniforgan development also view fruitsand icantlyto seedlingvigor(unpubl. data), but
ovules as competitorsfora limitedresource pollen size differencesamong donors were
pool rather than being completely con- not assessed in thatexperiment.It remains
in
trolled by the maternal tissue; the success to be determinedwhetherthe differences
ofan individualorganis relatedto itsability competitiveability observed in the ovary
to maintain concentrationgradientsof as- will also be expressed afterseed germinasimilatesand growthsubstancesbetweenit- tion.
selfand thematernalplanttissueinfluenced
CONCLUSION
by othernearbyorgans(Giffordand Evans,
Pollen donors sometimesdiffered
in their
1981). In E. grandiflorum,ovule maturation patternswere more consistentwiththe pollen tube growthabilityin Erythronium
but only a small portion of
paternalcontrolhypothesis.When therewas grandiflorum,
a largediscrepancybetweenthe mean sizes the differencesin pollen vigor was associof the pollen produced by the two donors ated with mean pollen grain size or total
in a pair, theretended to be an increase in pollen production.Earlier experimentsinthe number of apparently fertilizedand dicated that tube growthmay be mediated
aborted ovules (slope = -0.20, P = 0.0299 by the style,possiblybecause of a multifacfroma torial incompatibilitysystem. Since there
forthe partial regressioncoefficient
model holdingthe numberof fertilizations were within-and among-donordifferences
and the total number of ovules constant). in fertilizationability, it is apparent that
Presumably,these abortions representless donors did have some influenceon their
vigorouszygotesthatwere outcompetedby fertilizationsuccess, but producing more
more vigorousones. In addition,total seed vigorouspollen may cost thedonor in terms
productionfromfruitswas negativelycor- of resourcessequesteredin pollen and polrelatedwiththesum ofthemean grainsizes len longevity.Althoughtherecan be amongof the two donors (slope = -0.13, P = donor differencesin pollen vigor, within0.0602 forthe partialregressioncoefficient donor differencesin pollen vigor were not
froma model as above). Apparently,when great,suggestingthatplants may have limtwo pollen donors that produce vigorous ited controlof this character.
Donor performancein the ovary,on the
zygotessiredovules in the same fruit,competitionamong ovules resultedin increased other hand, was more stronglyinfluenced
ratesofabortionforovules fertilized
byboth by differencesamong donors. Donors difsiringability,
donors, and reduced the overall fecundity feredin theirpostfertilization
ofthematernalparent.Such a patternwould whichwas correlatedwiththe mean pollen
nothave been predictedifthematernalpar- size, so that plants producinglargergrains
enthad controlover postfertilization
ovule also sired more seeds. The differencesobservedamong donors in theirsiringsuccess
abortion.
VARIATION IN POLLEN SIZE
were probably geneticallybased, since the
opportunityfor paternal contributionsto
offspring
is limited. When more than one
donor sired seeds in the same ovary, the
amount of ovule abortiondepended on the
mean pollen grain size of each donor, and
the differencein size between the donors.
The resultsare consistentwiththe hypothesis that zygotescompete with each other
for maternal resources,and that resource
allocation to ovules is not strictly
controlled
by the maternalparent.
855
Thumb. Theor. Appl. Genet. 46:
iumlongiflorum
143-148.
R. W., AND D. L. LYON. 1985. Correlations
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CRUZAN, M. B. 1986. Pollen tube distributionsin
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1989. Pollen tube attritionin Erythronium
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1990. Pollen donorinteractionsduringpollen
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CRUZAN, M. B., P. R. NEAL, AND M. F. WILLSON.
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Floral display in Phylaincisa:Consequences for
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Plants of the Same Species. JohnMurray,London.
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thesis,carbon partitioning,and yield. Annu. Rev.
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