Effects of Spatial Distribution and Reproductive Biology on in situ Fertilization Rates of a Broadcast-Spawning Invertebrate

10 Pages·1997·4.2 MB·English

by  R Coma

Reference:Bin/ Bull 193:20-29.(August. 1997)
Effects ofSpatial Distribution and Reproductive
Biology on in situ Fertilization Rates of
a Broadcast-Spawning Invertebrate
RAFEL COMA1 AND HOWARD R. LASKER2
DepartmentofBiologicalSciences,StaleUniversityofNewYorkatBuffalo.
P.O. Box601300. Buffalo. New York'l4260-1300
Abstract. Insitufertilizationwasexaminedinthegor- Introduction
gonian Psendoplexaiira porosa during 1994 and 1995
spawningeventsintheSan BiasIslands. Panama,toas- Whetherornotfertilization ratesconstraintherepro-
sess spatial and temporal variation in fertilization suc- ductivesuccessoffree-spawningorganismshasbecome
cess and to determine whether in situ fertilization was a subject ofincreasing interest in the last 10 years (see
sperm limited. Fertilization rates did not differ signifi- Levitan. 1995,forreview).Despiteagrowingnumberof
cantlybetweenyears(60%vs.55%),butmonthlymeans studies on fertilization rates, the questions still remain
were significantly different, ranging from 22% to 66%'. whetherlowfertilizationratesarecommon,whetherlow
Fertilization rate variedamongdays, rangingfrom to fertilization rates are caused by sperm limitation,
85%;80%ofthisvariabilitywasexplainedbydailyvari- whether fertilization rates limit reproductive success,
ation in the number of colonies that spawned. A and whether morphologiesand behaviorsthat enhance
weightedaverageofinsitufertilizationratessuggeststhat fertilization actasconstraints on the life-history evolu-
67% or more ofspawned eggs are fertilized in nature. tion ofbroadcast-spawningtaxa. Weexamined the fer-
Spermlimitationdidnotoccuronthenightswhenmost tilization ratesoftheCaribbean gorgonian Pseiuloplex-
ofthecoloniessynchronouslyspawned andwhen most auraporosainanefforttoaddressthesequestions.
oftheeggswerereleased. Eggscollecteddownstream of Theconclusion that fertilizationsuccessisan impor-
thepopulation often had higher fertilization ratesthan tant factorcontrolling the overall reproductive success
eggs collected either adjacent to theirsource colony or ofbroadcast-spawningspeciescomesfromthreetypesof
eggscollectedinthemiddleofthepopulation,whichin- data: observationsoflowfertilizationratesduringnatu-
dicates that in dense populations, eggs may have ral spawning events; experimental manipulations indi-
multiple opportunities to be fertilized. Traits such as catingthatspermdensitycan limitfertilizationsuccess;
highlysynchronousspawning, high fecundity, largeegg andhydrodynamicmodelsofgametedilution. Lowfer-
size, large polyps, and large colonies directly and indi- tilization rateshavebeen reportedin taxarangingfrom
rectly enhance P. porosagamete production and fertil- coelenteratesto fishes(Petersen, 1991;Oliverand Bab-
ization. Theselife-historytraitsreducetheeffectsofga- cock, 1992; Babcockand Mundy, 1992; Babcockelal..
metedilutionduringspawningeventsandthusdecrease 1992; Petersen el al.. 1992; Brazeau and Lasker. 1992;
theimportanceofspermlimitationinthepopulationdy- and Levitan, 1995). Moreover, fertilization rates are
namicsofP.porosa. highly variable: even specieswith high averagefertiliza-
tionratesmayexhibitcasesofverylowfertilization(e.g.,
Sewelland Levitan, 1992),andconversely,specieswith
R1eCcuerirveendt7adAdurgesuss:tI1n9s9ti6t;utacdccepCtieednc2iJesundeel19M9a7r.-CSIC. PasseigJoan wlohwenferotbisleizravteidonorvaetresmsaonmyetdiamyess(ed.eg.m,onP.stkruantae;hLiagshkerarteesl
deBorbos/n.08039Barcelona.Spam. al.. 1996).
2Authortowhomcorrespondenceshouldheaddressed. Whentheyoccur,lowfertilizationrateshavegenerally
GORGONIAN FERTILIZATION
been attributed to sperm limitation. The only experi- fertilizationratesthathavebeenmeasuredinsiiu(Briar-
mentaldemonstration ofsperm limitationduringnatu- einii ashcstiniini <0.01%-6%. Brazeau and Lasker,
ral spawningevents isthat ofLaskercl al. (1996). who 1992; P/c.xaura kuna on average 26.4%, data from
foundthatincubatingnaturallyspawnedeggswithsup- Lasker cl al. 1996). Lasker el al. (1996) also provided
plementalspermenhancedthefertilizationratesofPle.\- data on fertilization success in Pseudoplexauraporosa.
awakitna. Fieldexperimentsthatreportdecreasingfer- noting that high fertilization rates (including some
tilizationrateswithdistancedownstreamfromspawning >80%)weremorecommoninP.porosathaninP.kuna.
malesagainsuggestthatspermdensitylimitsfertilization Thesespeciesappearedtodifferintheirfertilizationrates
success(Pennington, 1985;Yund, 1990;Levitan. 1991; andintheimportanceofspermlimitation. Inthispaper
Levitanrt/, 1991. 1992;Babcockela/., 1994;Brazeau weexpandourobservationsofP.porosaand showhow
and Lasker, 1992;Oliverand Babcock, 1992; Benzieet reproductive strategy affects fertilization success in this
a/.. 1994; Benzieand Dixon, 1994; Yund and McCart- species.
ney, 1994; Levitan, 1995; Levitan and Young, 1995;
Laskerclal.. 1996;Comaand Lasker. 1997). Although MaterialandMethods
the effectsofthedownstream dilution ofsperm can be
lessenedbysynchronousspawning,amongmanyspecies FieldworkwasconductedatKorbiskireef,apatchreef
onlyasmallproportionofthepopulationparticipatesin located near the Smithsonian Tropical Research Insti-
natural spawningevents(Randall cl ul.. 1964; Mosher. tute(STRI)fieldstationintheSanBiasIslands,Panama.
1982; Pennington, 1985; Michin. 1987; Levitan. 1988; Pseudoplexaura porosa is a gonochoric broadcast
Pearseclal., 1988;McEuen. 1988;BabcockandMundy, spawnerwitha 1:1 sexratio;itspawnsin highlypredict-
1992;Babcocketa/., 1992;Gladstone. 1992;Beiringand ableeventsthatoccurshortlyaftersunsetafterthesum-
Lasker.unpubl.data). mer full moons (Lasker cl al.. 1996; Ross and Lasker.
Hydrodynamic models and measurements ofturbu- unpubl. data). P. porosa is the second most abundant
lentdiffusionandthedilutionofspermalsopredictthat gorgonianon Korbiskireef(Laskerelal.. 1988)and.be-
fertilization rates should be low under many circum- causeofthesizeofitscolonies(upto250cm in height),
stances(Denny, 1988;DennyandShibata, 1989;Lasker one ofthe dominant members ofthe benthos. The P.
and Stewart. 1993; Levitan and Young, 1995; Lasker porosapopulationatKorbiskiismostdenseina 150-m:
and Kapela. 1997). However, the predictions ofthose areaontheeasternsideofthereefalongachannel that
models are dependent on variables such as the rate of separates Korbiski from an adjacent reef. The colonies
gamete release and net flow, and several authors have atKorbiskiwerelabeledandmapped.Allcoloniestaller
reported cases in which high fertilization ratesare pre- than50cmweremeasured,andafragmentwascollected
dicted(Dennyetul., 1992;Benzieelal.. 1994;andLevi- forsexidentification.
tanandYoung. 1995). Field collections were made during all spawning
Spermlimitationisnottheonlycauseoflowfertiliza- events in 1994 (June to September) and during June.
tion rates. Forexample. Levitan (1996a) reportsdiffer- July,and Augustin 1995. Eggswerecollected from the
ences in quality among sea urchin eggs, and similar watercolumnshortlyaftertheywerereleasedfromcolo-
effects have been observed among gorgonians (Lasker, nies. Scubadiverspositioneddownstream ofeitherspe-
unpubl.data).Inaddition.MeadandDenny(1995)have cificcoloniesorthewholepopulationcollected theeggs
identified hydrodynamic conditions that interfere with (700j/m in diameter) in 60-ml plasticsyringes. Thesy-
sperm-egginteractionsregardlessoftheconcentrationof ringescontainingthecollectedeggswerebroughttothe
sperm.Thusthecommonnessandcausesoffertilization STRI fieldstationandafter0.5-1.5 hoursalleggswere
limitationareunclearandmaybespeciesspecific. counted, placed in 120-ml polypropylene specimen
Caribbean gorgonians are among the few taxa in cups, and incubated with seawater that had been col-
which fertilization ratescan bepredictablyand directly lected priortothestart ofspawning(sperm-freeseawa-
measuredduringnaturalspawningevents. Manygorgo- ter). Containers were suspended from the field station
niansexhibitsynchronousspawning(Kinzie, 1970;Bra- docktomaintaintemperatureandprovidesomestirring.
zeauandLasker, 1989, 1990).andthelargeeggsreleased After 12 h the developingembryos were counted with
by octocorals can be collected from the water column theaidofastereomicroscope.Thatvaluewasusedasthe
(Lasker et a/.. 1996) or from the surface ofthe colony estimatorofthenumberofeggsthathadbeen fertilized.
(Brazeau and Lasker, 1992). Although there are some Results are expressed as percent fertilization and were
overlaps between species in the timing of spawning, arctantransformedpriortostatisticalanalysis. Laskeret
manytaxaspawn ondifferentdaysoratdifferenttimes al. (1996) discussed the biases inherent to these proce-
ofday,allowingthecollectionofmonospecificgroupsof duresandconcluded thattheproceduresdid not intro-
gametes.Thegorgoniansstudiedtodatehavethelowest ducealargesystematiceffect,buttheydid notethatob-
22 R. COMA AND H.R. LASKER
served fertilization ratesmayslightly underestimatethe incubated with sperm-free water and another 50 with
truefertilizationrate. waterfromanaquariumcontainingamalecolony.Asin
In 1994weassessedoverallfertilizationratesaswellas thefieldsamples,eggswereassayedthefollowingmorn-
spatial variation in fertilization. Overall fertilization ingtodeterminetheproportion fertilized. Asapositive
rates were determined from eggs collected 5m down- control.50virgineggswereincubatedinwaterfromthe
stream ofall ofthe P. porosacolonieson each night of maleaquariumcolony.Threereplicatecontrolswereset
spawning. Theexactpositionofthedivercollectingthe upat the beginningandagain attheend oftheenrich-
eggs was based on the prevailing current. Lasker ct til. mentprocess(about25 min later). Thecolonyexplants
(1996) have previously shown that in the absence of usedtoobtainvirgineggsandspermwerecollectedfrom
downstream male colonies, fertilization rates do not Korbiski reef2-3 daysbeforespawning. Explantswere
differovertherangeof1 to5mfromaspawningfemale. maintainedin 18-literaquaria,andthewaterwasvigor-
Farther downstream eggs became too dilute to be col- ouslyexchangedevery2hbetween0600and 1800hand
lectedinasignificantnumber.Todeterminewhereeggs thenonceintheevening(2200-2400h).Watersamples
arefertilized,eggswerealsocollectedeitherataposition fromthe maletankwerecollectedatthetimeoftheex-
0.5-1 mdownstream fromafemalecolony(June 1994) periment,andspermdensitywasestimatedfollowingthe
orfromthecenter(labeledCinFig. 1)ofthepopulation protocolofSwainetal.(1997).
(July 1994). Positional effectswerealsostudied in Sep-
tember 1994. when eggs were collected both upstream Results
anddownstreamofamalecolony.
In 1995 thesamplingprogram wasdesigned to mea- Spawning
sure overall fertilization ratesand test for the presence Spawning in P. porosa was highly synchronized in a
ofsperm limitation in the field. All measuresofin xiiu restricted2-4dayperiodthatstarted5-6daysafterthe
pfeorptuillaiztaitoino.n wWeerealcsaorrmieodniotuotreddowtnhsetrneuammbeorftohfefwehmoallee tfuelrledmoobosnerevaacthionmsonftrhomfrtohemsJuunmemetro Smeopnttehmbsero.f S1c9a8t7-
coloniesspawningbyexaminingeachfemalecolonyon through 1995 (36 observation nights) showed that all
thereefevery 15 minandrecordingthestartandendof spawningeventsoccurred5-10daysafterthefullmoon.
spawning for each colony. Male spawning was not Monthly spawning events were characterized by 1-2
readily observed; to verify that it had occurred, water nights of intense spawning and 1-2 nights of weak
samples were collected adjacent to arbitrarily selected spawning. Usually,thefirstandthelastnightshadweak
malecoloniesand sperm densitiesweredetermined by spawning.Mostcoloniesstartedspawningbetween 1815
meansofamodification(Swainela/.. 1997)oftheacri- and 1915handendedbetween 1915and 1945h. Differ-
dineorangedirectcounttechnique(Hobbieela/.. 1977). encesin thetimingofeggreleasebetweenspecificcolo-
An InterOceanS4currentmeterwasusedtoestimate nieswereconsistentbetween monthsandyears. Female
twhaessppleaecdedanwditdhiirnectthieonpoopfuwlaatteironf.low1..5Tmheacbuorvreentthemebtoetr- cfoorloanbioeustb1e0gamnins.pEagwgnirnegleabsyersetleeaadisliynginecgrgesaastedaolvoewrr1a5t-e
tom, atthe heightofmostcolonies. TheS4 metersam- 30 min and then rapidly decreased at the end ofthe
pledcontinuouslyat0.5-sintervalsstarting 15 min be- spawning. Sometimesfemalecolonieshadtwopeaksof
fore spawning and continuing until egg release had intensespawningduringasinglenight.
ended.
Sperm limitation was tested in enrichment experi- Spatialdistributionofcolonies
mentssimilartothosedescribedby Laskerelai (1996)
forPlexaiirakunu. Oneexperimentwasconducteddur- In the study area the density ofcolonies taller than
ingeachmonthofspawningin 1995(3nights).Eggswere 50cmwas0.1coloniespersquaremeter.Thepopulation
collectedonthereefinsyringesasdescribedabove.Two ofcolonieswithidentifiablegonadconsistedofsix male
divers collected side by side, downstream ofthe whole colonies(height = 125-230cm, mean = 194cm), and
population.Duringallthreeexperimentsthecurrentran six female colonies (height == 101-201cm. mean =
to the south, and the divers were located at position S 142cm). Samples collected at either ofthe two down-
(Fig. 1). Theeggs,which float,werecapturedabout3m streamcollectingpositions(NandS;Fig. 1)hadsimilar
abovethesubstratum inthe6-mwatercolumn. Collec- meandistancestothesixmalecolonies(N distance=
tionbeganatthestartofthespawningeventandcontin- 4-19.7 m.mean= 13m:S 6-23m,mean= 14.8m).
uedforabout60min,yielding 13to 15pairsofsamples Collectionsatthecentral position(C; Fig. 1)alwaysex-
(55-60 eggs per sample). The samples were then cluded halfofthemalepopulationasaresultoftheuni-
transported to the STRI field station where the paired directional flowregime. Thecentercollectionsitehada
sampleswerepooled. Fromeachgroupofeggs, 50were similarmeandistancetomalecolonies(centerposition
GORGONIAN FERTILIZATION 23
S4
3O20 A o
316
324
306 O
O 314*
294 313n** 321
315
94-126
295 94-135
303
1m
94-120
Figure I. MapofPseiidiiplexauranorosacolonies(tallerthan50cm)atKorhiski reef.San BiasIs..
Panama.Femalecolonies(tilledcircles),malecolonies(emptycircles),andS4currentmeter(triangle).
Collectionsweremadeatpositionsdownstreamoltheentirepopulation(NorS)andonepositioninthe
centerofthepopulation(C).
withcurrentfromthenorth 3coloniesatadistanceof fertilizationabove50%.Themodalfertilizationratewas
3.4-10m. mean = 5.6nr. centerposition with current 80%-90% in both years (Fig. 2). Fertilization ratesdid
from the south 3 colonies at a distance of 1.8-7 m, not differ significantly between years, but did vary sig-
mean = 5.1 m). nificantly between months and days (Tables I and II).
Thelowest monthly mean wasobserved in September.
Variabilityinfertilization Fertilization washigh on at leastone nightduringeach
majIonrsiittyu(f6er6t%i)liozafttiohnerdaonwgnesdtrbeetawmeseanm0pl%esanhdad98l%ev.elTshoef em1ao9c9nh5thnwiag(shTteasbbtlyiemtaIhtIe)e.dnAubnymnbwueearilghoftfeirfntegimltiazhlaeetifcoenortlioslnuiiczecasetisoosnbsdreuartrevienodgf
spawning(Table II; Fig. 3), yieldingan estimated fertil-
25r ization rateof67%. Sperm releasewasnotvisibleinthe
field, and measures ofsperm density were highly vari-
w 20
0) 1994
Q. D1995 TableI
,n 15 Nestedanalysis /variance<>/lcrlili:ationrule's/>/Pseudoplexaura
porosabetweenyear*,amongmonths,andamongdays(months
ncsleitinyears,anddaysnestedinmonllis)
0)
a. Source
10 20 30 40 50 60 70 80 90 100
Fertilization(%)
Figure 2. Frequency distribution offertilization ratesamongall
samplesofP.ieudoplexaurapornsaeggsfromallnights.
24 R COMA AND H.R. LASKER
Table11
Insitufertilizationrate<>/ PscudoplexauraporosaalKorhiski
Date
GORGONIAN FERTILIZATION 25
On two nights, the fertilization rate 1 m from a female 100r
colonywaslowerthanthedownstream fertilizationrate
(29June 1994:26%at 1 mvs.71%downstream;30June
1994: 3% at 1 m vs. 9% downstream), but on a third
night, fertilizationclosetothefemalecolonywashigher
dtohawnnstfreretaimli)z.atOionn 3d0owJnusntere1a9m94,(6a3t%hiradtdi1vmeravls.so4c6ol%- |.o 60
lected samples immediately downstream ofthe spawn- I
ing female. The difference between the two collections "g 40
made at the female colony. 38% and 3%, is indicative co.
ofthespatial variability in fertilization rates. Given the
tvhaartiattihoenrebewtewreeennodivseirgsniaftictahnetcodliofnfeyr,eintciessnobtetswurepernistihneg 20 - AD AJJuuulgnyues11t9917
dsaomwpnlsetsrecaomlleocftetdheaptoptuhleatcioolnon(yANaOndVAt,hoFse:yco=lle3c.t0e2d, 20 _4L0 _l60 80L 100
P = 0.099). However, it is also important to note that ControlFertilization(%)
relatively few samples were collected that night, which Figure5. Fertilizationralesamonginsitusamplesof/ViH/o/'/f.v-
wasthelastnightofspawning. aunifn<nnaeggsincubatedwith(enrichedlandwithout(control)addi-
On29and30July 1994,apositioneffectwasobserved tionalsperm.
betweencollectionsfromthecenterofthepopulationand
those downstream. Fertilization was higherat the down-
stream position than at the position in the center ofthe Sperm-limitationexperiments
spFfeitorgrpt.euiall4)ima.ztaoiAtfoinoanontm(rhataelwtreoes-cewboxalepyotenwrAyeiNmeaOennnVdetAgt(gh;2so6sFceoSle,clpoMeltcl=te1ec9d3t93e4id.)m1d0mco,eowdPmnipsa<atterr0lee.y0da0mut0pho1-ef; TevoeEnneirnnsigucrheemaecanhntmaeodxnepqteuhraitdmeuernsitunspgpwlteyhreeof1c9eo9gng5dsu,scpttahewedneionnnrgiacevhseminnetgnslt.e
cti<zhaaeu0ts.ime0oa0ntl0hed1i.so).wTwTnhasehsrteterhleewoaawlmassf(tetroonttniiaegll-ihztwfaaatoiyilfounAsrepNraauOtwpeVnsipAtrnr,ogebaiFamnbl/Say,en,pod=tcec1um16rb1%r7ee.rf2de2ar,btnieldP-- eaeessxnttpddener1uni7smmiAbetuenygtruossfotwef)eg.rsgepTsahciwononsntiedhnuewgcewtarfeteedemtroahlnceeodtslhaeuwyesmnrinogenh(to1ws9bhswJieiucrtnhvhee,ttdhh1eea9ggnJrrdueelaaytt//,--/
csfmoehalwlooewnceiocdelosoltnohsianaetymstpwhrlieetelchdeoamls3aoetdnduyagryuaessmeesbdtpeeefisronm.ratehrEeitxeheasexm.piSenerapittmieeomnnbteowrfasspoptlahyewpnosinnloygf dwas3ii9ent,drduePnsfonpet=roetrois0mcli.-icg1zeun7anirt4fri;oiiocnncFaihnrtgetah.dtedes5iss)efa.fwmteiheprnrlredeneeiecsceoans(vtipgiebahnreitgtrs8e.wt0deh%Cae./otntne(tssfTrtpia,eoelblr/dlm=eifenlrIc1Iit.u)im.3bli9ait.Tztahaidtteofiirnoo=esnn
carriedout in thelaboratoryat thebeginningoftheex-
100 perimentalwayshad fertilization rateshigherthan 80%
(81%-82%)anddidnotdifferfromcontrolsinitiatedat
80 t1h.2e5,enPd=o0f.2t9h6e).experiment (two-way ANOVA, Flfi =
Thelimiteddataonflowspeedsuggeststhatflowover
60 therangeofobservedspeeds had little impacton fertil-
ization. Forinstance, fertilization wasgreaterthan 80%-
= onboth 19Julyand 17August 1995despitethefourfold
01 40 differenceincurrentspeedonthetwonights(TableII).
20 O Center-45% Discussion
Downstream-83%
Fertilization success in P. porosa was often substan-
1900 1930Time 2000 ttihaallnytlhesossethraenpo1r0t0e%d-,foarndot/h/;ersibluroraadtcesaswte-rsepaowfntiennglotwaexra
(Petersen, 1991;OliverandBabcock, 1992;Babcockand
ilantdFiiiovgniud(ruceaeln4t.sear)mPparlnoedpsodcrootlwilnoesncttoerdfeaoPmnseo2uf9dtoJhpuelleeynxt1ai9ur9re5apaoptpoutnlihaxectciieonentgeg(rsdoofwfenrtsthitelripezeaodmp)ui.-n M19u9n2d;y,Pet1e9r9s2e;nBaetbcao/.c.k1e9l9u2)L.1A9s92;weSewperlelviaonudslLyevniottaend,
Meanfertilizationrateateachcollectionsiteisalsoshown. (Laskerela/., 1996),thissuggeststhatP.porosafertiliza-
26 R. COMA AND H.R. LASKER
tion ratesare sperm limited at times. There also was a Temporalvariahilityinfertilization
gatehnandterfmaelrotsictloirzeargtegissopnaorrnaedteersnec(leTeaabsbeledetwIueIn)e,dnbeurstpcoeournrmdidrtaeitloaenaasslesionisnuwgh1gi9ec9sh5t tnhifeFieclratoniwtlifdzeiarfttfiieolrnieznawctaiesonbveretarwtyeeesininmiSmleoapnrtteihnmsbbeowrta.hsMymoeaanirtsnh.llyTyhdveuaersiitago--
esefneaprtceFtiihrirrlmesnitzis,aagprtwheaiteownnbndoyiretantttlgeehisreme,mivntiweiunennmetgwdb.aeertierhgahftoetfr6etcd7iol%litzohoenedfi.feeeTsgrgotrsielclriaeezlalacsetuiialnosangetdreegaoagtvseve.efrrTrathoghmeee tatkiiinnulodAninzcaaLactaan(isLntoknahepsarkkrs.ieotnrbueeantrabpipnlculdsybalnsSb.ictemeidiwa(alattBartaertr)ni,tzboouir1te9ttea9hodn3atdmtoaoDonngbidtasxhmeoulrennyvt.peeucd1bh9rlae9i.nl4neg)da.ePasslteeaix)(naRauofenrsrdas-
anummobnegrniogfhtes,ggasndbeiitnpgrorbealbealsyedpepaekrsocnoltohneysaalmseo nviagrhitess nifIincdainvtiedfufaelctssiozenafnerdtilloiczaaltipoonprualtaestiaomnodnegnseictyhihnaovdeersimgs-
that the greatest numberofcolonies spawn. Therefore, (Levitan, 1991: LevitanandYoung, 1995).presumably
ourweightedaveragesprobably underestimate fertiliza- through theireffectson sperm density in the watercol-
tion. umn.Sessileorganismscannotaggregate,butsynchroni-
Second,wedidnotdetectanyevidenceofspermlimi- zation ofspawning increasesthe likelihood offertiliza-
tation in sperm-enrichment experiments, which were tion.Theimportanceofspawningsynchronizationisev-
conductedonthenightsonwhichmostspawningoccurs. ident in /*. porosa because most of the variance in
That 100% fertilizationwasneverobservedinthoseex- fertilizationamongnightswasexplainedbythenumber
perimentsmaybeafunctionofbiasesintroducedbythe ofspawning colonies. Although our measure ofsperm
sampling and incubation techniques (Levitan, 1995; density didnotcorrelatewithfertilizationrates,thecor-
Laskereltil.. 1996),butthosebiaseswouldhaveaffected relationbetweenfertilizationandthenumberofspawn-
boththecontrolsandtreatments. The failuretoseeen- ingcoloniesislikelyareflectionofthedensityofgametes
richment in samplesthat yieldedonly 50%. fertilization in the water column. This is well illustrated in the 26
rates(Fig. 5)suggeststhateggsinthoseparticularincu- September 1994datawhichshowthatwhenfewcolonies
bationswerelesscapableofdeveloping,dueeithertonat- spawned, most eggswere not fertilized. Conversely, on
uralvariation ingametequality, ortotheexperimental nightswhenmostofthespawningoccurred,fertilization
technique. Ineithercasetheconclusionthatspermwere rateswere highest and the enrichment experiments re-
notlimitingisnotaffected. vealednoevidenceofspermlimitation.
Reproductivestrategyandfertilizationsuccess
Spatialvariabilityinfertilisation
Fertilizationrateswerelowonsomenightsandsperm
Laskereltil. (1996)showedthateggscollectedwithin limitationprobablydidoccuronthosenights:neverthe-
1 mofacolonyofPseudoplexawasp. hadsimilarfertil- less,theoveralleffectofspermlimitationonfertilization
ization ratestoeggscollected downstream. Presumably successin Pseudoplexauraporosa may below. Thisre-
sperm are continuously diluted asthey aretransported sult isin markedcontrasttothelow insitufertilization
downstream, soeggsreleased intothewatercolumn in- rates reported for Briareitm asbestinuni (Brazeau and
teract with ever decreasing concentrations of sperm. Lasker, 1992) and Plexaura kuna (Laskerel a/.. 1996).
Therefore, eggs that are not rapidly fertilized will have Furthermore,thesperm-enrichmentexperimentsfailed
ever decreasing probabilities of being fertilized. Of to detect sperm limitation with P. porosa. whereas the
course,downstream additionsofspermcanaffectfertil- results ofidentical experiments at the same study site
ization.Fertilizationratesthatwemeasureddidnotcon- stronglysuggestedspermlimitationwithP.Auna(Lasker
sistently vary between eggs collected within a meter of eltil.. 1996).
thecolonyandthosecollecteddownstreamoftheentire Althoughfewoctocoralshavebeenexamined,wecan
population, but collections made in the center ofthe compare gorgonians for which there are data to assess
population regularly underestimated the fertilization theeffectsoflife-historytraitsonfertilizationsuccess.At
success of the population. Whether fertilization in- Korbiski and at many sites at which it is common, P.
creased downstream ofindividual female colonies was porosa dwells at relatively low density in habitats with
probablyafunction ofthe behaviorofthenearby male fast(>10cm/s)unidirectionalcurrents. Modelsoffertil-
colonies, and that effect appears to have varied among ization processes predict low fertilization rates under
nightsandcolonies.Distancehadapositiveeffectonfer- theseconditions(Levitanand Young, 1995). However,
tilization when downstream transport enabled eggs to fertilization ratesare higher in P. porosa than in other
travelpastadditionalmalecolonies. gorgonians,afeaturethatisprobablyexplainedbytraits
GORGONIAN FERTILIZATION 27
suchasgameteproduction,fertilizationkinetics,spawn- is occupied by gonad. so polyp size may limit gamete
ingpattern,eggsize,polypsize,andcolonygrowthrate. production.
Gametedensity and in particularsperm density- Largecolonysizealsocontributestototal gamete re-
is one ofthe principal factors determining fertilization lease, and P. porosacoloniesare large relative to other
success(eggdensity hasonly a small effect on fertiliza- reefgorgonians.P.porosaalsohasthefastestgrowthrate
tion:Lillie. 1915:Vogelt'M/.. 1982;Levitanetat. 1991). observedamongstudiedgorgonianspecies(upto20cm/
P. porosa. which is not sperm limited, has spermaries year,unpubl.data). Energeticconstraints(Harrisonand
that are an orderofmagnitudegreater in volume than Wallace. 1990)orpolypinternalspacemaylimitcolony
thoseofPlcxaurakiina, which issperm limited (Lasker reproductiveoutput. Regardlessoftheproximal factors
(i-0i.8ill.m. m1939/6p)o:lyapn;dRoP.sspoarnodsaLahskaesr,thuenplaurbgle.stdastpae)rmfaoruineds cionnhtarboiltleinngvigrroonwmtehnatnsdthraetpreondaubclteiotnh,emP.tpooprroosdauccoelolnairegse
amongthestudiedgorgonianspecies(Briarcuniashcsti- coloniesthroughfastgrowth;this,inturn, promotesthe
num,0.05-0.15 mm'/polyp,BrazeauandLasker. 1990; production of a large number of gametes at a single
Paranniricca clavatu, 0.4mirr/polyp. Coma et a/., point.
1995b; Plcxaura kiina. 0.07mm'/polyp, Lasker and P.porosaiscommoninareasofhigh(low.Flowaffects
Stewart, 1993',Plexauraflexuosa,0.05 mmVpolyp.Beir- processes such as feeding, metabolic rate, fertilization,
ingand Lasker,unpubl.data). fragmentation, and survival. Although flow may en-
Spawningsynchrony also playsan important role in hancetraitssuchasfeeding,andthustheenergybalance
determining gamete density and fertilization success ofacolony,highflowprobablyreducestherateoffertil-
(Thorson, 1946; Harrison etal.. 1984;Sewell and Levi- ization. The adverse effectsofflow rateon fertilization
gwetromhaiiiaaonontgcmrd,hhheee.iea,t1nrSmce9mpoh9eg2opanaa)rostwcm.ofthhned.iP4tums.necpoAngtpaniciowgotoosrnhvnnhhotieo,cssnrrieawtgnndschtsueorserprvecpareaiactaernwsuiawagenrlosnsirneinemtsandghotpcgehnahowawtcpnanhnohesfairosaninnl4ocaligldlemyomolisno2tosvgnoewheoftdsorsfhusptgssltporae.hdaevweaaewnbgFtrn4ieoeuainnrlrunnetgtitdgrhgoaaehpt1oytretanse-l-hs earhnLHYfeneaoouflvvdwuemeiceenabtt1vsgeba7ee,tenrdrAhea,u1eoenln9ggf9oguoas5fsnblsp.;lst.aoaetwLnwhr1.1ednev99vi9eF9ifnn5dot2ei,ga;rrgitnahcni,Pitollensmilats1zorate9wangar9tneeinis6yeocepasnemn;es,orawpCnecpeaotieocpsnntieameltooa.tsaarv.rhgeee(eeard1dPn9noe8td9ifnfo02egntr;%Lhihhtta,nieassLlgvkyepiteoeezvooaftripnat,tui,f1agoll91rnnao1,9etwJ99aaiu87ttonol)5hendn.ye;r
nigPhrteslwiimtihnafrayvoerxapbelreicmoenndtisti(ounnspufobrl.ferdtaitlai)zathiaovn.e shown thiihzsaettoitrowynoatrnnaidigthstthpsursvoabrraeibdeludycferoetudhruefcoielmdtph(oeTraetbfaflneeccteIsI)oo.ffPsf.lppoeowrrmoonslaifmeriltitifael---
that,inadditiontoproducingmanysperm,atleastsome
tion.
gorgonians produce gametes that are able to fertilize a The Korbiski population produced an estimated 2.1
tliarogne(p1e0r2cesnpteargme/molf)e.ggTshi(s>7s0u%gg)esattslothwatsptehremfecrotnicleiznattriao-n eXst1i0m6ateigognso(f83re9prbordaunccthievseXtis5s0uecpme/rbrbraanncchh][mXi7n0impoulm-
kineticsofP.porosacouldyieldhigherlevelsoffertiliza- yps/cm [SD = 14, /; = 10] x 4.3 eggs/polyp [Rossand
tionatlowerspermconcentrationthancan beachieved Lasker, unpubl. data]). The six mature colonies ofthe
by otherbroadcast-spawningtaxa (sea urchins Vogel studied population were distributed over an area of
eBtabacl.o.ck1,98129;92L)e.vitan et al.. 1991; corals Oliver and b1r5y0osm/i:r;rifw6e7r%eopfraollduecggesdwaenrneuaflelrtyi.liTzehdi,stehsetnim5a6t,e00o0felmar--
Levitan (1993, 1996a,b) has argued that large eggs val production issimilartothatforEunice/lasingu/aris
present a larger target for sperm and thus increase the (60,000 larvae/m2; Theodor. 1967). The production of
probabilityoffertilization.P.porosaeggsareamongthe eggsinP.porosa(84,000eggs/nr)atKorbiskiisanorder
largest (700-750^m, Ross and Lasker, unpubl. data) ofmagnitude smaller than that documented for Para-
o3b3s0er/vume.dViignni,go1r9g7o0n;iaMnnsric(Ceoarac/al/iiufnoimicrau:h7r0u0m:^m,30M0.- Pn.utrcilcaevaatcalahvaatsaa(73r0ep,r0o0d0uectgigvse/mb:i;oCloogmya(eCtoalm..a19et95bal)...
fruclicosa: 600^m, Grigg, 1977; Plexawa homomalla: 1995a)similartothatofB. asbestinnm. andB asbesti-
315-640Mm, Martin. 1982; Plexawa kiina: 500- numhasfertilizationratesthatareanorderofmagnitude
600^m, Brazeau and Lasker, 1989; Briareum asbesti- lower than those of P. porosa (Brazeau and Lasker.
num: 600-900^m, Brazeau and Lasker, 1990; Para- 1992).
muricea clavuta:400-500^m. Coma et al.. 1995a). P. ThelifehistoryofP.porosaisaffectedbyawidevari-
porosapolypsarethelargestamonggorgonianspeciesin etyoffactors. Forinstance, polypsizeaffectsfeedingas
whichreproductionhasbeenstudied.Thistraitisimpor- wellasgonad volume;eggsizemayaffectlarval longev-
tantbecausealargeproportionofthefullymaturepolyp ity;growthratesaffectmaturationandrelativeprobabil-
28 R. COMA AND H.R. LASKER
itiesofsurvival. Itissimplistictoarguethatthelifehis- Brazeau,D.A.,and II.R. I.asker. 1992. Reproductivesuccessina
tabloeornybea,olfabnePcc.eadupsoberyossseaelleecictstiiobonunitlootnianorctroheueansrdelifffeeerrt-tihililisiztzaoatrtiyioontnrraisattuseccaweinsldsl Comiama.za,\arhteiRi\.nol,neianisnbiudeicnncHte.hMsisaRc.riinLnaBvasiekorbletrreo.b1ar1d1a4c9t:ae9,s71t.5t7hs-epS1am6Caw3aln.rliinsbgcbaeloaecnthoecotocertraoolcg.oern.a/elitF.yB\/roy.fiufMemrtmiilm-
limited by morphological constraints (Levitan, 1995). Biol.Ecol (inpress).
Nonetheless, it isclearthat many P. porosa life-history Coma,R.,M.Ribes.M.Zabala.andJ.M.Gili.I995a. Reproduction
traitsaresuccessfulatenhancingfertilizationcompared andc\cleofgonadaldevelopmentintheMediterraneangorgonian
wwietrhetnhoattofferstpieliczieedsawintdhofuerttisluiczhatitroanitrsa.tAesltwheorueghvaarlilaebglge,s ComePafa,froraRtm.,uinrMit.cheea/Macebladalivata,elararnaMdnaerJa.nEMgc.oorlGgioPlniri.oagnI.9SP9ea5r.br.a1m1u7Sr:eix1cu7ea3al-1crl8ea3vp.arload.ucMtairv.e
spermlimitationwasnotregularlyobserved.Spermlim- Ecol.Pn>K Scr.117:185-192.
itationprobablyplaysalesserroleinthepopulationecol- Denny,M.\V.1988. BiologyandMechanicso/liicIIV/vc-SncyvEn-
ogyofP.porosathanofothergorgonians. vironment PrincetonUniversityPress.Princeton.NJ.
Denny,M.\\'.,andM.F.Shibata. 1989. Consequencesofthesurf-
zoneturbulenceforsettlementandexternalfertilization.Am..\al
Acknowledgments 134:859-889.
OurthankstoElizabethBeiring,MaryAliceCoffroth. Denny,M.\\'.,J.Dairiki,andS.Distefano. 1992. Biologicalconse-
Stephanie Holzward. Kino Kim. Kristin Kittle. Tyler qexuteenrcneaslfoefrttiolipzoagtiroanp.hByioolnBwuallve-1s8w3e:p2t20s-h2or3e2s.: I. enhancementof
Ross, and Tim Swain fortheirgood-natured assistance Gladstone, \V. 1992. Observations ofthe crown-of-thorns starfish
on fartoomanynightdives,andtoSaraWeatherupfor spawning.AUM J Mar l-'rcsliwaterRes.43:535-537.
thesperm-densitycounts.OurworkintheSan Biaswas Grigg, R.W. 1977. Population dynamicsoftwogorgonian corals.
madeeasierandmorepleasantbythestaffoftheSmith- Ecology58:278-290.
astgohenarin.aknsWegTortothopaiRnceakylnthaReledsKoeuaTnraacphiIan,IdntishateintsSutaaen,ndBaitanhsdeStRoaeutpriuobnslpmiecacinao-lf HHaarrrrRaiicnsscdoohnn.,,reZPcP..r.uDLLi.u.,t,bmRmea.snnCtkd.yo.CB'fa.ebdCsc..colEceWlrkasa,elcvltGiai.encrie,Da..nABm1ucs9lot9lr0.eal.rJsd..aKRmP.e.ppO.rliov1de3urc3,t-Ci2.o0nC7,.WidniaslplCeaorcrseaa,ll
Panamaforpermission towork intheSan Biasandare andB.I..Willis.1984. Massspawningintropicalreefcorals.Sci-
gratefulforthesupportoftheNationalScienceFounda- ence223: I186-1189.
tion grant OCE 9217014 (HRL). the National Geo- Hobble.J.K., R.J. Daley,andS.Jasper. 1977. UseofNuclepore
graphic Society (HRL), a Ministerio de Educacion y fviilrtoernsfMoirercoohuinotli.n3g3:ba1ct2e2r5i-a1b2y28f.luorescencemicroscopy..!/>/'/. En-
Ciencia ofSpain Postdoctoral Fellowship to RC. The kinzie.R.A. 1970. Theecologyofthegorgonians(Cnidaria.Octo-
manuscript was improved by the comments of E.A. corallia)ofDiscoveryBay,Jamaica.Ph.D.Thesis.YaleUniversity.
Beiring, M.A. Coffroth and K. Kim. D. Levitan. T. NewHaven,CT.
Ross,andananonymousreviewer. Lasker,H.R..andW.Kapela.1997. Heterogeneouswaterflowand
itseffectsonthemixingandtransportofgametes.InProe SillInt.
CoralReelSymposium. H.A. Lessios.ed. Smithsonian Tropical
LiteratureCited ResearchInstitute,Panama.(Inpress.)
Babcock, R.C, and C.N. Mund>. 1992. Reproductive biology, Lasker,H.R.,andK.M.Stewart.1993. Gametedilutionandfertil-
spawningandfieldfertilizationofAcanlhasterplanci.AII.\I../ Mar izationsuccessamongbroadcastspawningoctocorals.Pp.476-483
FreshwaterRes.43:525-534. inProc ~ihIni CoralReefSymposium,Vol. I.R.11.Richmond,
Babcock,R.C.,ON.Mundy.J.Keesing.andJ.Oliver.1992. Predict- ed.UniversityofGuam.Agana.Guam.
ableandunpredictablespawningevents: insitubehaviouraldata Lasker,H.R.,M.A.Coffroth,andL.M.Fitzgerald.1988. Foraging
fromfree-spawningcoralreefinvertebrates.Inverlehr.Rcprod.De\\ patterns ofCyp/ioniafiihfay.sum on octocorals: the roles ofhost
22:213-228. choiceandfeedingpreference.Biol.Bull 174:254-266.
Babcock,R.C.,C.N.Mundy.andD.Whitehead.1994. Spermdiffu- Lasker,H.R.,D.A.Brazeau,J.Calderon,M.A.Coffroth.R.Coma,
sionmodelsand;/;sunconfirmationoflong-distancefertilization and K.Kim. 1996. lit\iiiiratesoffertilizationamongbroadcast
inthefree-spawningasteroidAcanlhaster/'lanci Bin/ Bull 186: spawninggorgoniancorals.Biol Bull 190:45-55.
17-28. Levitan,D.R.1988. Asynchronousspawningandaggregativebehav-
Benzie,J.A.H..andP.Divon.1994. Theeffectsofspermconcentra- iorintheseaurchinDimlemaanlillanim(Phillippi).Pp. 181-186
tion. sperm:eggratio, and gameteage on fertilization success in inEchinodermBiology. Proc.6iliInt. Echinodermconference. R.
crown-of-thorns starfish (Acanlhaster /y/anci) in the laboratory. Burke,ed.A.A.Balkema.Rotterdam.
Biol.Bull 186:139-152. Levitan, D.R. 1991. Influenceofbodysizeandpopulationdensity
Benzie, J.A.H., K.P. Black, P.J. Moran, and P. Di\on. 1994. onfertilizationsuccessandreproductiveoutputinafree-spawning
Small-scaledispersionoftheeggsandspermofthecrown-of-thorns invertebrate.Biol.Hull 181:261-268.
starfish(Acanlhaster/ylanci) in ashallow coral reefhabitat. Biol. Le\itan,I).R.1993. Theimportanceofspermlimitationtotheevo-
Hull 186: 153-167. lutionofeggsizeinmarineinvertebrates.Am.Nat.141:517-536.
Brazeau,D.A.,andII.R.Lasker.1989. Thereproductivecycleand Levitan.D.R.1995. Theecologyoffertilizationinfree-spawningin-
Brazsepaauw,niD.ngA.i,naanCdarIIi.bRb.ealn.agsokcrrg.on1i9a9(n1..BioSlexuBualllre1p7r6o:duIc-t7i.onandex- vLe.rMtecbErdatweasr.dP,p.ed1.2C3-R1C56PrienssE,coBloocgayRoaftMoanr.iFnLe.InvertebrateLarvae.
ternalbroodingbytheCaribbeangorgonianBrian-urnusht-.tlinuin. Levitan,D.R. I996a. Effectsofgametetraitsonfertilizationinthe
MarBiol.104:465-474. seaandtheevolutionofsexualdimorphism.Nature382:153-155.
GORCiOM\\ FERTILIZATION 29
Ievilan.I).R.I996b. Predictingoptimalanduniqueeggsizesinfree Pennington,J. 1.1985. Theecologyoffertilizationofechinoideggs:
spawningmarineinvertebrates.,l/n \ul 148:174-188. theconsequencesol sperm dilution, adult aggregation, and s\n-
Levitan.D.R.,andC.M.Young.1995. ReproductncMH.-a.-ssmlarge chronousspawning.Kinl Hull 169:417-4?!).
populations:empiricalmeasuresandtheoreticalpredictionsofler- I'etcrsen,C'.\\. 1991. Variation in fertilization rateintropical reef
tittzationintheseabiscuitClypeaslerrosaceus .1 i.\i> Mar Hint fish.lliiliilic>c\hivtiiunin correlatesandimplications.Bin/ Bull
/-;,,-/ i90:22i-:4i. 181:232-237.
Levitan,I).R.,M.A.Sewell,andF.S.Chia. 1991. Kineticsolfertil- Petersen,C.\V.,R.R.Warner,S.Cohen,H.C.Hess,andA.T.Sewell.
izationintheseaurchinSlrongylouenlrolua/w/iivViwin interaction 1992. Variablepelagicfertilizationsuccess:implicationsformate
Leviotfagn,amDe.teR.d,ilMu.tioAn.,Saegvev.elal,ndancdonFt.acSt.tCihmiea..B1i9o9l2.HullHo18w1:di3s7t1ri-b3u7t,ixo.n Randcahloli,ceJ.anE.d,sRp.atEi.alScphatrtoecrdnesr.ofamnadti\Vn.g.A./.SIt/aHrI,kM,J7r3.:139461-4-.401N.oteson
Salnrdonagbyluoncdeanntcroeluisnftlruaenmcme-sanfeurstil/i-z.aVtoi/oon^rs7u3c:ce2s4sN-in25t4h.e sea urchin 4th2e1-b4io3l3o.gyoftheechinoidn/nt/cnmanlillarwn Curihh../ Sci 4:
Lilliien.tFh.eR.fer1t9i1l5i.zatioSntupdioewserofofferstipleirzamtisonu.spVeInI.siAonnaslyosli".sIorfhmvairuiatBiionn/s Sewenlalt,urMa.lAs.p,aawnndinDg.oRf.tLheevidtaenn.dr1o9c9l2u.rot1eersteialiczuatciuonmbsuecrceCsuscduuirnianiguia
Martd3MBeiue5lnl.9,lx-cioF3c2r.a88anl01:o92.8b22lA4.an-n2d5ICon1is.ciP.lloeCxriaecupmrr:oadMuhcaotrmivoLemi,naiplmrliolup.o(riEcsnipneo:rn..)s\euextnua.lelluvlMnfiaeircuMnCcda\in:dba9ed: SwaiPiminlii,ecnixriToai.isuucrD:o.a.pHkvuuKl.nlianKMiaImmnn,:Ia'as.InSsIiHad/>I5,vIo1/.f/:jRslI./phueilr.-Camos1kr6dea6er.ln.sRi1et9ye9f7fS.oyrmtUphsoeesgiooufrmg,folnuHio.arnhe.s.ccLeosnrsacl-e
McFuen,F.S. 1988. SpawningbehaviorsoftheNortheastPacificsea sios. ed. Smithsonian Tropical Research Institute. Panama. (In
cucumbers(Holothuroidea:Echmodermata).Mar Binl98:565-585. press.)
Measdh,eaKr.sSt.r,esasnodnMfe.rt\i\li.zaDteinonnya.nd19e9a5rl.vdTehveeleotpt'meectnstofolhtvhderopdurvpnlaemsieca 'Iheeoldocri,imJp.o1r9t6e7m.entCodnetlraipbluatniuolna.aII'iie1tu\ldielidceug1o8:rg2o4ne1s-3(0V1II.):ecologie
Michuirnc,hiIn).Sl1r9o87n.gyloSceean-lwraotlemrptietmrppewariaittu^r.eBainn/d.sBuplalw-n1i8n8g:h4ie<hav56lourin Thormsaorni.neG.bo1t9t46o.m iRnveeprrtoedbruacttei,onwiathndspleacrivaalldreefveerleonpcmeetnottohfeDpalnainskh-
Mosttthlhhvceurriri.siiesaa.Pns./t'a.lI1rl'.nmMl1al9Ilr8nui2lil.rnuCiinvncSi/rpiiuia-icrw*Mc.nininc(n,cn/c.<g/i<Tbu(a/eLmnhu\apdvwaiMiogBmu)ar.y.oBPJfi.pn.t/Mh.e49(5aLi:sa7pw-Iir43de6o9ncN-cheiIi,r4noc1ldle.\lh\uonlno\-- VignSt(iCoc,mnrii/MclP.illiaairn1ivk9iai7te0on.niinh4:tnRhi1ienc-ise5o(r2Luc3.nh).)edd(eslOulrpeseriuocnmldooln.rteoMpcnrioloidlduicDtPuonnrotodlf-ei'lni\okc.-ollrAiaimlvliioiAicirlcoismr\slo.
Balkema.Rotterdam. l.incci(ROMA)Ser.8(1)10: I-26.
Oliu-r,J.,andR.Babeock.1992. Aspectsofthefertilizationecology Vogel, H.,G.Czihak, P.Chang,and VV. V\olf. 1982. Fertilization
ofbroadcastspawningcorals:spermdilutioneffectsand/';v/t/mea- kineticsofseaurchineggs.Muili Kn>\ci 58: 184-216.
surementsoffertilization,liinl Bull 183:404-417. \und.P.1990. Anin\uumeasurementofspermdispersalinacolo-
Pearse,J.S.,D.J.McClary,M.A.Sewell,\V.C. Austin, A. PITIV- nialmarinehydroid../ /-.'A/I /.mil 253: 102-106.
Ru/afa,andM.B>rne. 1988. Simultaneousspawningolsixspe- Yimd,P.,andM.A.McCartney.1994. Malereproductivesuccessin
ciesofechinodermsinBarklevSound.BritishColumbia.ln\'cnc/>i sessile invertebrates: competition for fertilizations. AiW<n,'i 75:
Rcprocl Ihr 14:279-288. 2151-2167.