Nuclear Migration in the Basidiomycete Schizophyllum commune

Author(s): Philip J. Snider and John R. Raper

Source: American Journal of Botany, Vol. 45, No. 7 (Jul., 1958), pp. 538-546
Published by: Botanical Society of America
Stable URL: http://www.jstor.org/stable/2439577 .
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 NUCLEARMIGRATIONIN THE BASIDIOMYCETE SCHIZOPHYLLUM COMMUNE1
PhilipJ. Sniderand JohnR. Raper
MIGRATION OF NUCLEI fordistancesofa fewmicra by the productof the numbersof distinctA and B
is commonly associatedwiththetransfer of gametic factors.
nuclei in sexuallyreproducingorganisms,but the Extensivenuclear migrationis knownto occur
extentof nuclearmigrationin the higherfungiis in doubly heterozygousmatings (AXBx X AYBY)
quite exceptional. In various Ascomycetesand and homozygousA matings(AXBOX AOfY), but it
Basidiomycetesimmigrantnuclei of one geniotype is not knownin homozygousB matings(AXBxX
oftenundergoextensiveintercellular migration with AYBx) or doubly homozygousmatings (AXBX X
astonishingspeed while progressivelycolonizing AXBx)of S. commune. These four classifications
an establishedmyceliumof anothergenotype.The include all possible matingsbetweenhaploid my-
existenceof nuclearmigrationin these two large celia. Both types of mycelial interactionsthat
groupsof fungiwas establishedbeyondall reason. involveextensivemigrationhave been used in the
able doubt by Reginald Buller and his students presentstudy. Nuclear migrationin homozygous
(1931, 1940). The presentpaper concernsthe A matingsleads to the establishment of sterile,
effectsof incompatibility and temperatureupon common-Aheterokaryons (Papazian, 1950; Raper
the rate of nuclear migrationin Schizophyllyumand San Antonio,1954), whiledoublyheterozygous
commune; informationabout such effectsmay be matingsyieldfertileheterokaryons. the wellknown
valuable to the investigation of the mechanismof dikaryoticmycelia. The patternof sexualityin
nuclear migration. S. communeand relatedfungiis reviewedin detail
Previous investigations of nuclearmigrationin elsewhere(Raper, 1953; Burnett,1956).
fungihave led to the generalconclusionthat the MATERIALS AND METHODS. -- Experiments for
migratorvrate for a givenspecies is severaltimes measuring the rate of nuclear migrationinvolve
the maximallinear growthrate of the mycelium. threeessentialcomponents: a mobile nucleus or
Buller (1931) estimatedthe migratoryrate in nuclei, a course for migration,and a means for
Coprinus lagopus to be at least 0.9 mm./hr.at detectingmigrantnucleiat specificpointsin space
22C., and Fulton(1951) obtaineda value of about and time. The migratorycourse used here is an
0.4 mm./hr.in Cyathutsstercoreus.The rate in extensive mycelium,the resident,grown on a com-
Gelasinospora tetrasperma,an Ascomycete,was plete medium in agar. Migrant nuclei, or migrants,
foundto be 4.0 mm./hr.by Dowding and Buller are supplied by a small inoculumplaced at the
(1940) and is claimedto have reached10.5 mm./ centerof the resident. Each residentstrainhas a
hr. in one instance (Dowding and Bakerspigel, specificbiochemicaldeficiency, while the migrant
strainis always wild type. Afterhyphal fusion
1954). The linear growthratesof the hyphaltips
between the two strains, migrants can move
of thesespecies rangefrom10-50 per centof the
throughoutthe hyphae of the resident,in which
migratoryrates. the migrantspass fromcell to cell throughsmall
Matingbehavior,nuclearmigration,and certain holes in the hyphalsepta betweencells. The pro-
othereventsleading to fruitingare governedby gressof migrationis determined by removingsmall
inheritedincompatibility factorsin S. commune. pieces of myceliumfrommeasuredpoints in the
The matingtypeof each haploid,or homokaryotic,residentand transferring theseto a minimalmedi-
myceliumis determined by one A and one B incom- um. The pieces of myceliumthat grow contain
patibilityfactor,and the specificpair of factors mijrants; those that contain onlv residentnuclei
in a givenhomokaryonmay be any of the many fail to grow. I'he details of this techniqueare
distinctA and B factorsexistingin the natural givenibelow.
populationof Schizophyllum.The A and B factors The type of matingsystem,the migrantstrain,
assort randomlyduring meiosis; a cross hetero- and theresidentstrainare all specificallvidentified
zygousfor bothkinds of factors,such as AXBxX by a singlenotationinvolvingthe matingtypesof
AYBY,yields four classes of F1 progenyin equal the two strainsin each experimental combination.
frequency,AxBx, BY AxBY, AYBX. The many Doubly heterozygous combinations,such as A1B1
distinctA and B factorsand theprocessof random - A2B2a-, producedikaryotizing systems,and
assortmentmake possible a large numberof dif- common-Aheterokaryotizing systemsare produced
ferentmatingtypes,the total being limitedsolely by combinationshomozygousfor A factorsonly,
such as A1B1 >-AB2 a-. The arrow always
1 Received for Publication January 17, 1958. pointsfromthe symbolof the migrantstrainto-
This work was supportedby Research grantsNSF-G3334 ward that of the
from the National Science Foundation and C-2221 from residentstrain,e.g., A1B1nuclei
the National Cancer Instituteof the National Institutesof in the last exainplemigratewithinthe mycelium
Health, U. S. Public Health Service. of the residentstrain A1B2a-. The four homo-
538
July,1958] SNIDER AND RAPER NUCLEAR MIGRATION IN SCHIZOPHYLLUM 539

karyoticstrainsused as residents,A2B2a- (667), TABLE 1. Culture media for Schizophyllum commune

A2B2u- (665), A1B1a- (232), A2B2n- (661), Grams/liter Standard Migration
and thethreeused as migrantstrains,A4B4 (672), distilled water complete complete Minimal
A2B4 (671), A1B4 (20), 'wereall isolated from
laboratorycrosses. The single gene mutantsa- Bacto-peptone 2.0 5.0
(arginineless),n- (niacinless), and u- (uracil- Asparagine 1.0
less) are non-leakydeficiencies(Raper and Miles, Glucose 20.0 2.0 20.0
Thiamin.HCl (jug.) a 120 120
in press). KH,PO4 0.46 0.46 0.46
The residents are grownupon'a completemedium K2HPO4 1.0 1.0 1.0
in 15-cm. Petriplates in eitherof two ways. The MgSO4 0.5 0.5 0.5
choice of methodis importantbecause migration Agar 20.0 20.0 20.Ob
differsconsiderablyin the resultingpreparations. a As supplied in peptone
A radiateresidentis a myceliumgrownby placing b
Nobel's agar (Difco)
inoculumat the centerof a plate and incubating
the culturefor 10-14 days, so that the mycelium
coverspracticallythe whole plate by the timethe complete. Although immigration occurs freely at
migrantinoculumis added. A reticulateresident the peripheryof radiate residentson standard com.
is formedby multipleinoculation,growth,and plete, it either fails to occur or is unpredictable in
anastomosisof hyphalfragments in the following the older portions of radiate residents. Correlated
way. A,myceliumone weekold on agar is macer- with this difference,the hydrogen-ion concentra-
ated thoroughly in a WaringBlendor,and 105-106 tion of standard complete at to is near the initial
viable fragments are pipettedinto60 ml. of molten pH of 6.8 at the peripheryof radiate residentsbut
agar mediumat 45?-50?C.; afterdispersingthe below pH 3.0 at the center. The concentrationof
fragments thoroughly, the mixtureis poured into H-ions irn migration complete medium (table 1)
a sterileplate and allowed to solidifv. Innumer- remains between pH 7.2 and 6.0 for at least 2
able hyphalfusionsbetweenthe viable fragments weeks of growth,and immigrationon this medium
bring about the synthesisof a single mycelium seems to occur as well at the center as at the pe-
throughoutthe plate within72 hr. of incubation riphery.
at 220C. The growth test for detecting the presence of
The migrantstrainis implantedas a specially migrant nuclei is done on a minimal medium of
preparedinoculum,themigrantdisc. The condition asparagine, glucose, and thiamin (table 1). Test
of the migrantdisc is criticalto achievingsuitable plugs 2 mm. in diameter are cut from the resident
entryof migrantsat the centerof the resident. and transferred to minimal medium. Since the
Discs 5 mm. in diameterare cut fromthe fringe resident strain always possesses a nutritional de-
of activelygrowingmycelia on agar and trans- ficiency, the minimal medium cannot support
ferredin an uprightposition to fresh medium. growth of the resident mycelium unless wild type
The exposed surfacesof the discs become fuzzy nuclei, supplied by the migrant strain, are present
with new growthafter 12-24 hr. on the fresh (fig. 1). Test plugs containing only one migrant
medium; they are then removed,inverted,,and nucleus are assumed to give positive growth tests.
pressed firmlyupon the center of the resident In rare instances a positive growth test may be
mycelium.The transferof the migrantdisc to the obtained from one or more test plugs as a result
residentis used as timezero (to) for the experi- of accidental contamination from an external
ment.Placing the migrantdisc at the center of source. Whenever this infrequent possibility was
theresidentwas expectedto resultin two desirable suspected, the presence of migrant nuclei was veri-
features: the patternof migrationwithinthe resi- fied independentlyby mating tests specific for the
dent should have the shape and area determined A and B factors of the migrant strain.
by an expandingcircle,and the migrantsshould The rate of nuclear migration is defined in this
encountersimilar conditionsat equal distances paper as the rate of change in length of the mean
along all radii. The latteris especiallyimportant radius of the area occupied by the migrant popu-
in radiate residents,in whichthe hyphaevary in lation. Since few, if any, of the many possible
age fromthe centeroutward. routes for migration in a resident mycelium are
The mediumused forculturingthe residentsand actually along straight lines, the value for migra-
migrantdiscs is a peptone-glucose medium. The tory rate as defined here must generally be less-
mediumis similarto thestandardcompletemedium and never more-than the rate of migration per
routinely used forS. commune(table 1; Raper and unit length of hypha. The quantityto be measured
San-Antonio,1954), but thereare important quan- is thus the effectivemigratoryrate of whole popu-
titativedifferences betweenthe two. The medium lations of nuclei and is not necessarily equal to the
used here (table 1) containsmore peptone,more rate of individual nuclei per unit length of hypha.
thiamin,and less glucose than does the standard The effectivemigratoryrate is a measurable quan-
540 AMERICAN JOURNAL OF BOTANY [Vol. 45

bh77

Fig. 1. Growthfromtestplugson minimalmediumindicatesthe presenceof wild type migrantnuclei (A4B4).

The argininelessresidentstrain (A2B2a-) is unable to growon minimalmedium. Seventeen of the 24 plugscon-
tainmigrants.-Fig.2. The radialsample,a patternof testplugs used fordeterminingthe extentof migration.The
migrantdisc (center)is 5 mm.in diam. The testplugs,alreadyremoved, are 2 mm.in diam.and exactly.5mm.apart.
Migratory distanceis measuredfromthe edge of the migrantdisc to the proximaledge of the furthestpositivetest
plug on each row. The preparation is a reticulateresident.

tityunderthe followingessentialconditions:(1) are quite unlikecommon-Aheterokaryons. In cul-

the movingboundaryof the migrantpopulation
mustbe locatedat severalsuccessivetimes,(2) the
boundarymustmovecontinuously duringthistime,
and (3) the boundarymustbe narrowlydefined
at each time. If a sampleof testplugs consistsof
all the plugs takenfromone residentat one time,
a seriesof suchsampleswillsupplythedata needed
forcalculatingmigratory rate,provided,of course,
the requirements just outlinedare met.
The populationdynamicsof the migrantnuclei
place certainlimitationsupon the kind of sample
of testplugsthatcan be used to locatethebounda-
ry of the migrantpopulationaccurately.Two types
of sample have been used in this study. The
radial sample consistsof 13 equally spaced test
plugson each of 4 radii orientedat rightanglesto
one another(fig.2). The grid sampleconsistsof
about 150 plugs taken fromthe intersections of
a 1 cm. grid coveringthe wholeresident(fig. 4).
A singleresidentmay be sampled8 timesby slight
rotationof successiveradial patternsor 5 timesby
sliaht translationof the coordinatesof successive
grid samples; in both cases migratorytractsare
leftketweenall holes. The radial sample permits
the analysisof a large numberof residentsat fre-
quentintervals, but it requiresmigrantpopulations
of relativelyhigh densityin comparisonto the
grid sample.
RESULTS.-Dikaryoticmycelia of S. commune
tures of dikaryoticmycelia growthis abundant,
fruitbodies generallydevelop,and the cells are
functionally diploid in that geneticcomplementa-
tion is fullyeffective;in common-Aheterokaryons
growthis less and is morphologically aberrantin
comparisonto dikaryoticmycelia,fruitbodies are
unknown, and geneticcomplementation is impaired.
Clamp connectionsare characteristic of dikaryotic
mycelia,and a stableratioof 1:1 forthetwotypes
of nucleiis also characteristic.Clamp connections
are rare in common-Aheterokaryons, and the nu-
clear ratio varies widelyfroma stable 1:1 ratio.
The characteristics just mentionedfor common-A
heterokaryons all result from the disruptionof
orderlyprocessesby theincompatibility mechanism
in this peculiartypeof heterokaryon (Raper and
San Antonio,1954). The rate of nuclearmigra-
tion in common-Aheterokaryons was examinedin
the firstsectionof results,in orderto determine if
migratoryrate was among the activitiesaffected
by the incompatibility mechanism. No evidence
was obtainedto supportthis possibility.
The second sectionconcernsthe effectof tem-
peratureon the rate of nuclear migration. The
rate of a complexprocess under a given set of
conditionsis usuallylimitedby the rate of a single
step in the process. The rate limitingstep may
he eithera physicalor chemicalprocess,and the
temperature quotientfor the rate of the over-all
July,1958] SNIDER AND RAPER-NUCLEAR MIGRATION IN SCHIZOPHYLLUM 541

NUCLEAR MIGRATION AND INCOMPATIBILITY

A4 B4 - t-4 A' B' a aA

AB4 A' 81
-ooOo 99 o ) QQo Qq9<) ?9 Q999; 999 _
60-
50-
E40-
48
48
Z 30-
0
z

cn u-60
L 0
50w
40-
1JJ72 Z

10

RADIAL ROWS OF TEST PLUGS

RESIDENT PLATES

Fig. 3. Nuclear migrationin a dikaryotizingand a common-A heterokaryotizing system,each containing the same
resident strain (radiate residents). The four radii of test plugs comprisingeach radial sample have been oriented
vertically to facilitate comparison. Each black spot representsone test plug that contained migrants; the open circles
are negative plugs. The 12-hr.and 24-hr. samples were entirelynegative and have been omitted.

processcan differentiate betweenthesealternatives. pair of combinationshaving the saine resident

A temperature quotientforthe rate of nuclearmi- strain,A4B4-> AB'a- and A'B4--> A1B'a-
grationin reticulateresidentshas been determined. (fig. 3).
The completedata have been given whereverthis The progressof mnigrationin thisexprimentwas
could be done,but in mostinstancesit was neither not,and probablycould not have been,sufficiently
possiblenor necessaryto give morethanrepresent- chartedfrom radial samples to permita precise
ative results. determinationof migratoryrate,but the rate can
Migrationand incompatibility.-Several resident be estimatedwithincertainlimits. The lowestesti-
strainswereused forcomparingnuclearmigration mate of the rate is given by dividingmigratory
in dikaryotizing and common-Aheterokaryotizingdistance (see legend to fig. 3) by the total time
systerns.In orderto establisha sound basis for intervalfromto, e.g., the estimateis 60 mm./48
comparison,two differentmigrant strains were hr., or 1.3 mm./hr.,in plate 5 of A4B4 >
used witheach residentstrain; one migrantpro- A1B'a (fig.3). The highestestimateis derivedby
duced a dikaryotizing systemand the otherpro- assumingthatthe absence of positiveplugs in the
duced a common-Aheterokaryotizing systemin 12-hr. and 24-hr. samplesliterallymeansthatmi-
separatesetsof culturesof thesame residentstrain. grationbegan afterthefirst24 hr. This estimateis
A replicateseries of 6-10 radiate residentswas 60 mm./24hr., or 2.5 mm./br.(fig. 3, saine plate
used in each specificcombinationof strains,and as above). The low estimateis a limitin the sense
radial samplesof testplugs were takenfromeach thatit is not subjectto revisiondownwardas a re-
residentat 12, 24, 48, and 72 hr. afterto. The sultof samplingerror: the actual ratecannothave
experiment was done at 220C., the temperature at been less than 1.3 mm./hr.The high estimate.on
which Buller measuredthe rate of migrationin theotherhand,is nota limit:itsvalue is subjectto
Coprinus lagopus. The firstevidence of migration revisionupwardif migrationbegan later than 24
in the presentexperimentappeared in the 48-hr. hr. afterto or downwardif undetectedmigrants
samples,whenmigrantswere alreadyclose to the were in transitduringthe first24 hr. The actual
peripheryof the residents. Therewereonlya few rate,therefore, may be considerablymorethan2.5
positivetestplugs in the 48-hr.samples,and these mm./hr.but not less than 1.3 mm./hr.in this
were so widelyscatteredthat it was impossibleto example. The low estimatesof migratoryrate
draw sharp boundariesfor the area occupied by rangedfrom1-1.5 mm./hr., and thehighestimates
the nigrantpopulations. These findingsare illus- rangedfrom2-2.5 mm./hr.
trated by the complete data fromone representative The percentageof positivetest plugs was ex.
542 AMERICAN JOURNAL OF BOTANY [Vol. 45

PATTERN OF NUCLEAR MIGRATION higher,(3) the data do not indicatethatmigratory

rate is affectedby incompatibilityin matings
homozygousforA and heterozygous forB factors,
42 hrs. 54 hrs but (4) therateat whichthepopulationdensityof
migrantsincreases-and most likelyits finalden-
sity as well-is lower in common-Amatingsthan
in dikaryotizingmatings.
The directcause of thelow percentageof positive
00

plugs in both typesof matingsduringthe first48

hr. afterto in radiate residentswas soughtin a
separateexperiment.The low recoverymay have
resultedeitherfroma low populationdensityor
froman asymmetrical patternof migration. Grid
66 hrs. T2 hris sampleswere used insteadof radial samples,and
0 0 the intervalbetweensampleswas shortenedto 12
hr.,so thatthe wholepatternof migrationin each
residentcould be recordedat severaltimes. Two
importantresultswere obtained. A few positive
plugs were found well isolated from the main
groupof positiveplugsin mostof thegridsamples,
and the patternof migrationin the radiate resi-
dentswas veryirregular. It followsthat,even for
dikaryotizing systems(fig. 4), both asymmetrical
FEig. 4. Illustration of the irregular pattern of nuclear patternsof migrationand low densitiesof the
migration and the scattered distriboution of migrants char-
acteristic of radiate residents. Black spots indicate the
migrantpopulationcontributeto the low percent-
presence of migrant nticlei; the negative test plugs are
age of positiveplugs recoveredfromradiate resi-
omitted. A 30-hlr. sample is omitted also, as it was com- dents. Migratingnuclei in radiate residentsmust
pletely negative. Each grid sample (ca. 150 test plugs) pass throughmany cells withoutcolonizingthem
is from the same radiate resident of A4B4 > A 2B2a - immediately;this impliesratherstronglythat the
incubeated at 22?C. mere existenceof two compatiblenuclei in one
cell does not mean the two nuclei necessarilycon-
stitutea dikaryonin the strictsense of being a
tremelylow in the 48-hr.samplesboth fromcom- pair of conjugatenuclei. In spite of the shorter
mon-24heterokaryotizing and from dikaryotizinlgintervalbetweensamples,it is once again impos-
systems,even in plates wheresome migrantshad sible to do morethan estimatethe migratory rate.
almostreachedthe edge of the plate. Beyond483 The low estimatein fig. 4, for example, is 1.55
hr. the percentagerose swiftlyto 100 per cent in mm./hr.(65 mm./42hr.), and the high estimate,
dikaryotizingsystemsbut lagged far behind in based upon the entirelynegative 30-hr. sample
common-Asystems,the percentagein the latter (omittedin fig.4), is 5.4 mm./hr.(65 mm./12
rarelyreachinghigh values by the end of the ex- hr.). The reliabilityof these estimatesis con-
periment. Analyses of nuclear ratios in hetero- sidered in the discussion and is the subject of
karyons of S. commune (Raperand San Antonio, currentexperiments.
1954; Sniderand Raper,unpublished)have shown Migrationand temprerature.-The rate of a mul-
that the ratio in establishedcommon-Ahetero- tiple step, biochemicalprocess has a temperature
karyonsrangeswidelyfromthe 1:1 ratio charac- quotient,or Qio, of about 1.0 if a physicalprocess
teristicof establisheddikaryons. Althoughit is is therate-limiting stepor a quotientof significant-
probable that the low frequencyof positivetest ly more than 1.0 if a chemicalreactionis rate
plugsactuallymeansthatthedensityof themigrant limiting. The single dikaryotizingsystemA4B4
populationwas quite low, it is also possiblethata A2B2 a- was used in an experiment to estab-
highlyasymmetricalpatternof migrationdevel- lish the Qlo of migratory rateforthe interval220-
oped by a dense;population of migrantscould have 320C., and the resultsindicate that a chemical
caused the low yield of positiveplugs. These pos- processis ratelimiting, providedthatthe principle
sibilities are examined in another experimzentof limitingfactorsdescribedabove can actuallybe
below. appliedto theprocessof nuclearmigration.Reticu-
The main conclusionsfromthe firstexperiment late residentswereused in this experiment instead
are-that ( 1) the minimalestimateof migratory of radiate residents,as the populationdynamics
ratein radiateresidenits of S. commune is similar of migrantsin reticulateresidentsare muchmore
to the 0@nm.0hr. estimatereporte0l by Buller for favorablefor measuringthe rate of migrationby
C. lagopus,(2) theacLual ratemaybe considerably use of radial samples. A replicateseries of 10
July,1958] SNIDER AND RAPER-NUCLEAR MIGRATION IN SCHIZOPHYLLUM 513

MIGRATION AT 220C.
t 15 872 hr. 96 hr. 120 hr. 144 hr. 168 hr. 192 hr.
I0-

20 40 60 0 20 40 60 0 20 40 60 0 20 40 60

?1
M 0 20 40 60 0 20 40 60 0

WD
cc
LU.
48 hr. 54 hr. I X
1- MIGRATION
60 hr. XX
AT 320C.
66 hr. 72 hr. 78
hr.a

l0

0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
DISTANCE OF MIGRATION (mm.)
Fig. 5. Variation in migratorydistance used to estimate the symmetryof the area occupied by migrantpopulations
in reticulate residents. The smaller the standard deviation of each mean migratorydistance, the closer the areas of
the migrant populations approximate a circle. The data are from one trial of the temperatureexperiment.

reticulateresidentsof A2B2a- was prepared for smallerthe variationin the mean migratorydis-
each trial of the experiment,and migrantdiscs tancewill be each timea set of samplesis collected.
of A4B4wereimplantedas soon as the agar solidi- The standarddeviation(s.d.) of the mean migra-
fied. Half of the residentcultureswerethenincu- torydistancein each set of samplesshouldthusbe
batedat a constanttemperature of 22?C.; theother a fairestimateof the regularityin the shapes and
half,at 320. Four consecutivetrialsof the experi- areas of the migrantpopulations. The mean s.d.
ment gave a sufficient accumulationof consistent for all sets of sampleswas 6 mm. in each trial of
data to calculatea reliabletemperature quotient. the experiment.The data fromone trialare given
The boundariesof the migrantpopulationsmust in fig.5. The value of s.d. (fig. 5) closestto the
be circular,uniform, and sharplydefinedin data to meanwas 5.8 mm. (192-hr.samplesat 22?C.), and
be used for such a calculation. The sharpnessof the totalrange of the values for s.d. was from3.1
the boundaryis primarilya functionof the popu- mm. (72-hr.samplesat 220) to 9.6 mm. (66-hr.
lationdensityof the migrants. In 97.3 per centof samplesat 320). The experimental errorin pre-
the 960 radial rows of test plugs collectedin the cisely locating the boundaryof a dense migrant
four trials of the experiment, the rows contained populationis determinedlargely by the interval
onlypositiveplugsup to a pointand onlynegative betweentestplugs,whichis 5 mm. in radial sam-
plugs beyondthat point. The remaining2.7 per ples. In order for the data to be acceptable,the
centof the radial rowseach had a singlenegative s.d. should certainlynot be more than twice the
plug withinan otherwiseunbrokenline of positive intervalbetweentestplugs,on one hand, but it is
plugs. There were no rows withan isolatedposi- improbablethat it could be much less than one
tive plug more than one space removedfromthe interval,or 5 mm.,on theother.The areas occupied
restof thepositiveplugs.These resultsare so close by the migrantpopulationswere thus equal and
to the expectations based upon a dense population circular,withinexperimentalerror,at each time
that a literal interpretationis probablyjustified: that sampleswere collected.
theboundariesweresharplydefined. The meanmigratory distancesfromall fourtrials
Afterthe requirement of a sharp boundaryhas wereaveragedforthe 220 treatments.These over-
been satisfied,it is possibleto use migratorydis- all meanvalues giveessentiallya straight line when
tanceas a basis forestimating the regularityof the plottedagainsttime,and the line, upon extrapola-
shapes and areas of the migrant populations tion to zero migratory distance,indicatesthatmi-
throughout the experiment.Migratorydistanceis grationmust have startednot later than 63 hr.
takenas the distance,along each radial row of test afterto. Over-allmean distancesat 320 were de-
plugs, fromthe migrantdisc to the boundaryof terminedthe same way,exceptthatthe data of the
themigrantpopulation. The closerthe boundaries firsttwo trials were excluded; in these two trials
of all the migrantpopulationsare to congruent the migratoryrate at 320 was not measurable from
circles throughoutthe period of migration,the the data obtained. Contrary to requirements (1)
544 AMERICAN JOURNAL, OF BOTANY [Vol. 45

TEMPERATURE AND HYPHAL GROWTH TEMPERATURE AND NUCLEAR MIGRATION

60- 60-
.72

50 2 5006

E~~~~~~~~~~~~~~~~~~~
E
~~~~~~~~~~~~~~~~~~
2 0C.
w~~~~~~~~~~~~~
30- 0

20C
20- 20-

10 10~~~~~~~~~~~~220C

20 40 60 80 100 20 40 60 80 100
TIME (hr.) TI ME (hr.)

Fig. 6 (lef-t). Effectof temperatureon the rate of hyphal tip growthfor the migrant strain A4B4. The thin lines
show the expected curves if the growthrate at 320C. had been 2, 3, 4. . .8 times faster than the rate at 220, and the
heavy lines represent the actual results. The Qlo value, or change in growthrate for this 10 degree change in tem-
perature is thus less than 2.0.-Fig. 7 (right). Effectof temperatureon the rate of nuclear migrationfor the migrant
A4B4. The data in this figureare plotted the same way as those in fig. 6, and the figure shows that the 10 degree
increase in temperaturemultiplied the rate of migrationby a factor of about 6.0. The plateau in the curve at 320 is
an interestingand unexpected result being investigatedcurrently.

and (2) specifiedin Materialsand Methods,the the Qlo value given by these calculated growth
boundaryof the migrantpopulationsin the ex- rates is 1.7. The growthrate of hyphaltips in
cluded data were not located at severalsuccessive S. communeis thus much slowerthan migratory
timesduringtheperiodof activemigration.Migra- rate,as is generallytruein fungi.
tion at 32? musthave begun no later than 41 hr. Not onlythe absoluterates,but also the change
afterto. The culturesthus completedthe steps in rates due to variationof temperature are quite
requisiteto the initiationof migration63 hr./41 different for nuclear migrationand hyphal tip
hr.,or 1.5, timesfasterat 320 thanat 220. growth. The effectof temperatureon migratory
The growthrate of hyphaltips for the migrant rate for the interval22?-320C. was determined
strain,A4B4, was determined at the same two tenm- fromthemeanmigratory distancesdiscussedabove.
peraturesand underthe same generalculturalcoIn- A graphicestimate,done similarlyto the one de-
ditionsused in the presentexperiment.The data scribedabove forhyphalgrowth,suggeststhatthe
forhyphalgrowthrate,as plottedon a graph (fig. rates of migrationwere about 0.5 mm./hr.at the
6) , show thatthe temperature quotientfor the in- low and 3.0 mm./hr.at the hightemperatures and
terval220-320C. is less than 2.0. In this graph thatthe Qio value of migratoryrate for this tem-
(fig. 6) the time-axisinterceptsfor zero distance peratureintervalmustbe near 6.0 (fig. 7). The
of hyphalgrowthhavebeen translatedto theorigin mean ratesof migration(plus or minusthe stand-
of the graph. This facilitatesestimatingthe tem- ard deviation),calculateddirectlyfromthe data of
peraturequotient,or Qio value, directlyfromthe mean distancesratherthan fromthe graph, are
graph. The growthcurve for 220 is drawn as a 0.42 ? 0.07 mm./hr.at 22?C. and 2.71 ? 0.65
heavy line, and the theoretically expectedgrowth mm./hr.at 320. The value of Qio based on these
curvefor 320, if the growthrate should be 2, 3, figuresis thus 6.4. Calculationsfromthe other
4. . .8 timesfasterthanat 22?, is representedby a parametersof migratorydistance are in close
series of thinlydrawn lines. The actual growtb agreementwith those determinedfromthe mean
curvefor 320 (a heavyline) fallssignificantly be- distances. If calculatedfromtheshortestdistances
low the theoreticalcurve that would indicate a in each trial,the Qlo value is 6.4; fromthelongest
doubling of the growthrate. The growthrates distances,5.5; fromthe mode,7.1; or the median,
calculated directlyfromn the data for the mean 5.6.
distances (n -20) of hyphalgrowthwere 0.13 The resultsof the temperature experimentare
mm./hr.at 220C. and 0.22 mm./hr.at 320, and given the followinginterpretation.The migrant
JU13T, 1958] SNIDERt AND RAPER NUCLEAR MIGRATION IN SCHIZOPHYL:LUM 545

and residentmust growva little and anastomosis untenableat present. The low estimatesof 1--1.5
must occur betweenthemn before migrationcan mm./hr.can accountforthe results,such as those
begin; once initiated,migrationcannot proceed in fig.3, by assumingthat (1) shortlyafterto one
morethana millimeter or so in reticulateresidents to severalmigrantsenteredeach residentand mi-
until growthand anastomosishas also occurred grated undetectedfor many hours, that (2) the
among many separate mycelia of the resident divisionrate of the migrantswas limitedvirtually
strain. Since the processesrequisiteto the initia- to zero untilabout 40 hr. afterto,by whichtime
tionof migrationare completed1.5 timesfasterat theyhad migratedabout 50 mm.,and that (3) the
32?C., a factorcomparableto thetemperature quo- divisionrate thenacceleratedrapidlyin this rela-
tientof hyphaltip growth,the beginnino,of mi- tivelyyoung zone of the residentmycelium,so
grationis probablygovernedlargelyby the rate thatthe probabilityof detectingthe firstmigrants
of hyphaltip growth. Once growthand anastomo- in thisratherdistantzone was high. The mostim-
sis are sufficient for migrationto begin at the portantassumptionfor the low estimatesis that
centerof the resident,however,the rate of migra- migrationmustoccur undetected. Statisticalesti-
tion is at once unlimitedby eithergrowthor an-as- mates,whichwill not be discussedin detail,sug-
tomosisvirtuallyeverywhere in the resident.Tem- gestthatundetectedmigrationis theoretically pos-
peraturehas a markedeffectupon the rate of mi- sible for a very small numberof inigrantsonly.
gration,and the high temperature quotientforthe There is a probabilityof about 0.70 thatno more
interval22Q-320C. is takento mean thata chemi- than 5-10 well scatteredmigrantsin each of 10
cal reactionlimitsthe rate of nuclear migration, residentswould totallyescape detectionin radial
hut the rate is surelynot limitedby hyphal tip samples. Two or threetimesas manywoul,dcer-
growthper se. tainlynot migrateundetected.
DISCUSSION.-The data in this paper, although The high estimatesof migratoryrate are also
not concernedprimarilywith demonstrating the difficult
to discount. A possibleobjectionto them,
existenceof nuclearmigration,do in fact support otherthan the one of samplingerroralreadydis-
thisbasic premise. The migratory rateforS. com- cussed, concernsthe multiplicationrate for mi-
mune at 22?C. in radiate residentsis at least 10 grants. Totallv negativesamples,in experiments
timesfasterthan hyphaltip growth,whichis only notpresentedhere,have been followed24 hr. later
0.13 mm./hr.The temperature quotientformigra- by entirelypositivesamples. This indicatesthat
toryrate between220 and 32?C. is about 3 times in less than 24 hr. the residentshad becomesatu-
largerthan that for growthof hvphaltips. The ratedin the sense thattherewas now at least one
factssupportingthe spatial distribution of migrant migrantin everypossibletestplug area throughout
nuclei cannotbe accountedfor solelyon the basis the resident. One mightdoubt,momentarily, that
of hyphalgrowthfromthe migrantdisc. theresidentscould possiblybecomesaturatedwith-
The behaviorof migrantpopulationsis remark- in so shorta time. The factsavailable suggestthe
ably different in the two typesof residentprepara- contrary.The nucleargenerationcycleat 22?C. is
tion. Migratory rateas measuredat 22? is at least knownto be about one hr. in thegrowingfringeof
3 timesfasterin radiatethanin reticulateresidents, dikaryoticmycelia,as determinedin C. lagopus
but quite possiblythe explanationfor this differ- (Buller,1931). S. commu7neand otherHymenomy-
ence is thatthe migrantscan followa more direct cetes (Miles, P. G., unpublished). A single mi-
route within a radiate resident. The radially grantwhosedescendents divideonce an hourwould
orientedhyphaein this typeof residentmay pro- yield 8192 migrantswithin13 hr. The area of a
vide a routeonly one-thirdas long as that in the residentis equal to thecombinedareas of 4894 test
randomlyorientedhyphaeof a reticulateresident. plugs, after excludingthe area covered by the
The migratoryrate per unit lengthof hypha is migrantdisc. A residentcould thus becomesatu-
probablythe same in both. ratedin considerablyless than 24 hr. by only one
Migrantpopulationdensityalso differsimpor- or two initialmigrantsthatdividedabout once an
tantlyin thetwo typesof resident. This may be a hour and migratedat a mean rate of 5.0 mm./hr.
consequenceof the factsthat (1) thereis a great High estimatesof 5.4 mm./hr.have been obtained
difference in the age of the two typesof prepara- (fig. 4). Neitherthe low nor the high estimates
tionat thetimethemigrantinoculumis added and accordinglyappear impossible.
(2) thereare long age and physiologicalgradients SUMMARY
in the radiate residentwhich do not exist in the Biochemicalmutantshave been used as thebasis
reticulateresident. of a methodformeasuringthe rate of nuclearmi-
The migratoryrate in reticulateresidentshas grationwithinprestablished myceliaof theBasidio-
probablybeen measuredwitha reasonabledegree myceteSchizophyllumcommune. The values ob-
of accuracy,but thereliabilityof the rateestimates tained are effectiverates of migrationbased-upon
obtained in radiate residentsremains doubtful. the time required for the advancing frontof a
Neitherlow nor highestimates, however,are really populationof nuclei to travelfrompoint to point
546 AMERICAN JOURNAL OF BOTANY [Vol. 45

in a myceliumin whichthe hyphaebetweenpoints patibilitydue to common-Afactorsaffectsmigra-

rarelylie exactlyin a straightline. The effective toryratesignificantly.The effective rate of migra-
rate is not necessarilythe same as the rate of tion is at least severaltimesfasterthan hyphaltip
migrationper unitlengthof hypha,but the closer growthunderthe conditionsstudied,and the tem-
the hyphaeapproximatestraightlines, the closer peraturequotient,or Qio, of migratory rateforthe
rate approaches,as a limit,the rate of interval22?-32?C. is 3 timeslargerthan that for
the effective
migrationper unitlengthof hypha. The effective hyphaltip growth. The high Qio value of 6.0 for
ratesrangedfromabout 0.5-3.0 mm./hr.,depend- migratorymovementindicates that the rate is
ing upon certain assumptionsand upon various limitedby a chemicalratherthan a physicalproc-
experimental conditions. Estimatesof the effective ess. The limitingchemicalprocess has not been
rate in some experimentsof uncertainreliability identified,but the resultsand conditionsof the
ranged up to 5.5 mm./hr. A comparisonof mi- experiments dictatethatit is not hyphalgrowth.
gration in dikaryotizingand common-Ahetero- HARVARD BIOLOGICAL LABORATORIES
karyotizingmatingsgives no evidencethat incom- C,AMBRIDGE, MASSACHIJSETTS

LITERATURE CITED

BULLER, A. H. R. 1931. Researches on fungi,IV. Long-
man's, Green, and Co. London.
BURNETT, J. H. 1956. The mating systems of fungi I:
New Phytol. 55: 50-90.
DOWDING, E. S. AND A. BAKERSPIGEL. 1954. The migrating
nucleus. Canadian Jour. Microbiol. 1: 68-78.
, AND A. H. R. BULLER. 1940. Nuclear migrationin
Gelasinospora. Mycologia 32: 471-488.
FULTON, I. W. 1951. Nuclear migration anld the inter-
action of haploid mycelia in Cyathus stercoreu.s. Ph.
D. Thesis. Univ. Indiana.
PAPAZIAN, H. P. 1950. Physiologyof the incompatibility
factors in Schizophyllum commune. Bot. Gaz. 112:
143-163.
. 1951. The incompatibilityfactors and a related
gene in Schizophyllumcommune.Genetics 36: 441-459.
RAPER, J. R. 1953. Tetrapolar sexuality. Quart. Rev.
Biol. 28: 233-259.
- AND J. P. SAN ANTONIO. 1954. 'Heterokaryotic
mutagenesis in hymenomycetes.I. Heterokaryosisin
Schizophyllumcommune. Amer. Jour. Bot. 41: 69-86.