758
AMERICAN JOURNAL OF BOTANY
[Vol. 40
illuminatedcholoroplasts.III. Simultaneousphototorycharacteristics
of the blue-green
alga Anabaena.
and consumption
of oxygenstudiedwith
production
Plant Physiol.28: 63-69.
oxygenisotopes. Arch.Biochem.Biophys.38: 365-370. WEIGL,J. W., P. M. WARRINGTON,
AND M. CALVIN.1951.
VAN NORMAN, R. W., ANDA. H. BROWN. 1952. The relaThe relationof photosynthesis
to respiration.Jour.
tive rates of photosynthetic
assimilationof isotopic
Amer.Chem.Soc. 73: 5058-5063.
forms
of carbondioxide. PlantPhysiol.27: 691-709.
WH[ITTINGHAM,
C. P. 1952. The chemicalmechanism
of
WEBSTER, G. C., AND A. W. FRENKEL. 1953. Somerespiraphotosynthesis.
Bot. Rev. 18: 245-290.
THREE NEW UNICELLULAR CHLOROPHYCEAE FROM SOIL'
FrancisR. Trainorand HaroldC. Bold
THE IMPORTANCE of careful
andprolonged
study Hormotilopsis gelatinosa sp. nov.
of certainunicellularalgae in culture,beforetaxoCellulaesphericae,
ad 18, diam,maturae,
Zoosporae
long.,0.6-2.5A
nomic dispositionof themis made, has been em- 8.5-15.OA
lat.,formam
mutantes
dummovegetativae
abundans
amylum
phasizedby one of us (Bold, 1950) and reiterated vent. Cellulaevetustiores
habentes.Habitat.:In soloex
byStarr(1952). The organismsto be describedin atqueoleumluteo-flavum
(Zea m4rys),
in loco dictoStockbridge,
this paper were isolatedand studiedas a segment agrofrumentario
Massachusetts,
Aug.1951.
of a long rangeinvestigation
of soil algae in progHormotilopsis
gelatinosa(fig.1-19, 59), a memress at VanderbiltUniversity. They have been
ber of the familyPalmellaceaeof the orderTetracultivatedin a varietyof inorganicmedia such as
modifiedBristol'ssolutionin liquidformand solidi- sporales,was isolatedfroma soil samplecollected
Mass. It has
fied with agar (Bold, 1949), 'Volvox solution" froma cornfieldnear Stockbridge,
been
(Moewus,1940) and in soil-waterculturesaccord- and cultivatedin modifiedBristol'ssolution,with
withoutagar, "Volvoxsolution"and soil-water
ing to the methodof Pringsheim(1946, 1950).
tubes.
Aftermanyunsuccessful
attempts,
bacteriaBacteria-free
culturesof the organismsare mainfree
cultures
(No. FRT-1) of the organismwere
tainedat VanderbiltUniversity
and at Indiana Uni- finally
obtained;thesegrowwell on modifiedBrisversityand have been sentto theCultureCollection
tol's
agar
and on "Volvax solution"solidifiedwith
at the Botany School, Cambridge,England. In
1.5 per cent agar.
addition,herbariumspecimensof the organisms,
The organismwas at firstprovisionallyconsidprepared from uni-algal cultures,have been deered
to be a Chlorococcum-like
alga, inasmuchas
positedat the ChicagoMuseumof NaturalHistory.
culThe algae were studiedin the livingconditionin it has this appearancein recentlytransferred
tures. Sometimelater,the same cultureswereexfreshmountsand hangingdroppreparations
and in
permanentsmears stained, after fixationin 3:1 amined,and it becameevidentat oncethattheprovisional diagnosiswas incorrect,for the alga had
absolutealcoholand aceticacid mixture,
in Haidensecreted
hain's iron-alumhaematoxylin
and by the Feulgen cells, considerablegelatinewhichconnectedthe
formingamorphousmasses. Furthermore,
technique.
abundant
vegetativecell divisionswere observed,
Hormotilopsis gelatinosa gen. et sp. nov.
an attributewhichremovesthe organismat once
Hormotilopsis gen. nov.
Cellulae in massis gelatinosisaggregatae,e tubis eis fromthe Chlorococcales.
Young vegetativecells of HormotilopsisgelatiHormotilaeconsimilibus
typicecompositis.
Tubi per secretionemdefinitam
gelatinaee cellulisvegetativis
enascentes, nosa measurefrom8-9, in diameter,have a thin
saepe dichotome
ramosiatque stratifacti.Cellulaeiuvenes wall,a stigma,singlepyrenoidand a hollow,spherisine gelatina.
cal chloroplastid
witha peripheralopeningthrough
Cellulae chromatophorum
poculiformem,
pyrenoideum,whichtwocontractile
vacuolesand a singlenucleus
nucleumunicum,vacuolas pulsantesduas habentes,per can be seen (fig.14k16). Oldercells
eitherlack the
membranam
delicatamcircumdatae.
Coloniaper divisionem peripheralopeningof the
chloroplastid
or it may
cellularumvegetativam
magnitudine
aucta.
be thatit is obscuredby starchand oil. Theyhave
Reproductio
per fragmentationem
coloniarum
atque per
no stigmaand contractilevacuoles. Adult vegetabipartitionem
cellularum
vegetativarum
endogenam
iteratam
as demonstrated
in (plerumque) 4 zoosporasquadriflagelatas.Cellulae tivecells are uninucleate,
by Haiquae zoosporasformant
structura
nonpraecipueimmutatae, denhain'siron-alumhaematoxylin
and the Feulgen
non papillatae. Zoosporaeduas vacuolaspulsantes,stigma nucleartechnique.
anterius,nucleumunicum,pyrenoideum
unicum,chroma- Whenthecellshavereacheda size of 12-14y,the
tophorum
parietalemhabentes,sine membranis.
cell wall can be morereadilyobservedand an exReproductio
sexualisnon observata.
ternalgelatinouslayer can be detectedevenlydistributedaroundthe cell (fig. 15). When stained
1 Receivedforpublication
July2, 1953.
The authorsare indebted
to theNaturalScienceResearch with a dilute solutionof methyleneblue or imFund of VanderbiltUniversity
for a grantto covercost mersedin India ink,theextentof thislayercan be
of excesspagination.
demonstrated
clearly. Gradually,as thecell grows
December,19531
TRAINOR AND BOLD-NEW
CHLOROPHYCEAE
759
older and larger,thereis a changein the manner 9, 10). As the flagelladisappear,thecells become
of depositionof thismaterial,in thatit is secreted sphericaland formdelicatewalls (fig.14-19).
more abundantlyat one pole of the cell, so thata
DISCUSSION. - Hormotilopsisgelatinosa in the
tubeis formed(fig.1-4, 17, 59). Aftercytokinesis vegetativestateexhibitsa strongsuperficialresemhas occurred,the daughtercells also secretegela- blance to Hormotilamutcigena
Borzi as well as to
tine, and a mass of cells connectedby tubes is Urocofccustropicus West, W. and G. S. West
formedin thisway (fig.1-3). If thetwodaughter (1907), thelatternow includedin Hormotila.It is
cells deposit their gelatine at contiguouspoles, unlikethe plant describedby the Wests because
branchingoccurs. Because the groupstendto ad- their alga possesses firmer"stalks," thickercell
hereto each otherin cultures,no reliableestimate walls, and the colonies do not containmorethan
could be made of the maximumnumberof cells eightcells.
In appraisingBorzi's (1883) descriptionand
forminga group,althoughas many as fifteento
figuresof H. mucigena,one mustkeepin mindthe
thirtywereseen frequently.
Both starchand oil are presentin the cells of fact that his observationswere based on material
Hormotilopsisgelatinosa. A few grainsof starch collecteddirectlyfromnature,wherethe alga was
can usuallybe detectedin youngvegetativecells, growingon moistrocks. The probability
thatmore
but oldercells frequently
becomefilledwithstarch than one alga was presentis thus enhanced,and
grains(fig.3-6). Whena cultureon agar is about this indicatesstronglythat the cells depictedby
a monthold, the color of the plants gradually Borzi in fig. 3-5 in P1. VIII probablyare of an
changesfromgreento orangebecauseofthedeposi- alga otherthanHormotila.
The similarityin generalconfiguration
tion of a pigmentedoil in the cells. This same
of the
but it beginsmuch colonyin Hormotilaand the presentorganismhas
changeoccursin liquid cultures,
later,probablybecause growthis slowerthan on been noted above. However,the organismsdiffer
in a numberof important
solid media.
attributes.Not theleast
Zoosporeformationcould be obtainedregularly of theseis the factthatthezoosporesof Hormotla
and with ease by inoculatingmaturevegetative are biflagellatewhile those of Hormotilospsisare
cells into,freshmedium. However,old, oil-filled quadriflagellate.
Furthermore,
in thelattertheyare
cells oftenrequireseveraldays to formzoospores, regularlyproducedin foursfromunmodified
vegeaftertransfer
to freshmedium. Such old cells can tativecells, while Borzi figurespapillatezoosporbe made to formzoosporesmoreeasilyby flooding angia liberatingas manyas 64 zoospores.
One mightbe inclinedto inferthatBorzi erred
the agar plate on which they are growingwith
apparently
occurs in depictingthe zoosporeof Hormotilaas biflagelliquid medium. Zoosporogenesis
late,wereit not thathis drawingsillustrateexcepperiodicallyin soil-watercultures.
is thatof re- tional keennessof perceptionwithregardto other
The methodof zoosporeformation
peated bipartition(fig. 5-7). The singlenucleus morphological
details. Borziwas unableto discern
of thevegetative
cell does not undergodivisionun- the finerdetailsof cell structure
in the vegetative
til just priorto cell division. Four zoosporesare cells of Hormotilaotherthanthepyrenoid.Whethregularlyformedfromeach cell. Zoospore-forminger thecellsof Borzi'sHorrmotila
weretetrasporalean
cells are usually 16-18k in diameterand may be in structurelike those of Hormotilopsismust remainuncertain.
eitherisolatedcells or membersof a group.
In viewof theseconsiderations,
Motilityof the zoosporesbegins whentheyare
one is facedwith
stillinsidethemothercell. The zoosporesultimate- a dilemmain disposingof the organismdescribed
ly rupturethe wall and emergeenclosedin a deli- above as Hormotilopsis.Phycologists
have beenincate vesicle (fig.7). Aftermovingin thisrestricted consistentin theirappraisal of the importanceof
theybecomefree-swimming.morphological
area fora fewminutes,
criteriaas theydelimitgenera. For
The zoosporesvaryconsiderablyin size and shape example,Carteriaand Chlamydomonas,
of the Vol(fig. 8-12). Extremelylong and slendertypesas vocales are segregatedinto separate genera priwell as triangularzoosporeswithfour flagellaat marilyon thebasis thatthe formeris quadriflageltwoloci wereobserved;theseprobablyrepresented late and the latterbiflagellate. rn both Ulothrix
theproductsof incomplete
cleavage. The zoospores and Stigeoclonium
of theUlotrichales,
on theother
varyin size from8:5-15.0i in lengthand from0.6- hand,both quadriflagellate
zoosporesand biflagel2.5,uin width. Each zoosporehas fourflagellaof late gametesare producedby the organismin one
equal length,two contractile
vacuoles,a stigmain life cycle. Under the circumstances,
the authors
the anteriorp.artof the cell, a single nucleus,a have followedthe precedentavailablefromconsidpyrenoidand a parietal chloroplastid,the latter erationof the genericlimitsof Carteria,and Chlavariableas to extentand positionin the cell. The mydomonasand have erectedthe genusHormotilwall, as indicated opsisfortheorganismdescribedin thissection.
zoosporesseemto lack a definite
by plasmolyticagents,and can changetheirshape
somewhatduringtheir period of motility.They CHLOROCOCCUMoleofaciens sp. nov.
swimfor a fewhours and thencome to restwith
Cellulae maturae sphericae, ad 46,u magnitudine. Memtheir flagellaextendedin cruciformfashion (fig. brana in cellulis iuvenibustenuis,in cellulis maturise cul-
760
[Vol. 40
AMERICAN JOURNAL OF BOTANY
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December,1953]
TRAINOR AND BOLD
NEW CHLOROPHYCEAE
761
organismsdescribedin thispaper,forit is lighter
in color than that producedby them. Agar culturesof 1 month'sage and liquid culturesof 1?/2-2
month's age have a characteristicyellow color
whichreflects
the abundanceof oil produced.This
oil and the starchgrainsusuallyobscuretheopening in thechloroplastid,
if one is present.
The thicknessof thecell wall increaseswithage
from0.5-6.5,u(fig. 20-26, 33-37). Normally,in
activelygrowingcultures,it does not exceed 2.51A
in thickness.However,maximumwall thickness
characterizes
mostcells in old cultures(fig.21).
Whencells froman agar culture1-3 wk. old are
transferred
into freshmodifiedBristol'sagar or
liquid, zoosporesare formedwithin24-36 hr. by
repeated bipartitionof the vegetativecells and
aplanospores(fig.22-25, 61). Olderagar cultures
willundergozoosporogenesis
if the agar is flooded,
Chlorococcum
oleofaciens(fig.20-41, 60, 61), a but this process requiresmore than 36 hr. Zoomemberof thefamilyChlorococcaceae,
orderChlo- sporesusuallywill continueto be liberatedfor3 or
rococcales,was isolatedfroma soil samplefroma 4 days in such cultures. Inasmuchas almostall
plowedfieldnear Duanesburg,New York. Chioro- sizes of vegetative
cellsmayundergozoosporogenecoccum oleofaciensgrows readilyin a varietyof sis, thenumberof zoosporespercell is highlyvarimedia such as modifiedBristol'ssolution,withand able. As fewas twohave been observedwithinthe
withoutagar, "Volvox solution'" and soil-waterme- smallestcells, whilelargercells may producemore
dium. Uni-algal and bacteria-freecultures (No. than one hundred. The zoospores range in size
FRT-2) are maintainedon modifiedBristol'sagar. from8.0-10.6,tin lengthand 2.7-5.3,uin width.
Young vegetativecells are ovoidal in shape be- They are ellipsoidal,some withone side flattened
cause of theirrecentoriginfromwalledzoospores (fig. 29, 30), have a delicatewall, two flagellaof
of thatform,but,as thecellsmature,theyassumea equal length,a singlenucleus,a peripheralchlorosphericalshape. Maturecells (fig.20, 21) oftenat- plastid,two contractile
vacuoles,a singlepyrenoid
tain a maximumdiameterof 46,u. Each vegetative and an anteriorstigma (fig. 28-32). When the
cell possesses a hollow, spherical chloroplastid, zoosporesare formedfromold, oil-filled
cells,they
whichmay or maynot have an openingin theside have oil dropletsin additionto theabove-mentioned
opposite the place where the single pyrenoidis structures.The presenceof the zoosporewall was
embedded(fig.20, 37). The cells are uninucleate confirmed
by plasmolysiswithsalt solution.
at all times(fig.39, 40). Nucleiweredemonstrated Soon afterthe vegetativecell has completedits
by stainingwith Haidenhain's iron-alumhaema- bipartitions
intozoospores(fig.22-26, 61), thelattoxylin and by the Feulgen nuclear technique. ter begin to move. The outerwall layer ruptures
Starchis producedabundantly.
and the contentsooze out enclosedin a delicate
As in Hormotilopsisgela2inosaand Characium vesicle (fig.27). They thenbeginto swimwithin
polymorph
Um,2 abundantoil is producedby old this limitedarea and eventuallyemergefromthe
cells (fig.21). The oil in C. oleofaciensapparently vesicleand swimaway.
containsa different
pigmentfromthatin the other
The average lengthof time the zoospores are
2 Characiumpolymorphum
to determine
inasmuchas zoospore
is a new species to be de- motileis difficult
formation
scribedin thefollowing
section.
oftencontinuesfor3 or 4 daysin a given
turis veteribusad 6.5A crassitudine. Cellulae uninucleatae,
unum pyrenoideumhabentes. Chromatophorussphericus,
parietalis, cavus, cum vel sine apertura in uno latere.
Oleum flavumin cellulis maturise culturisveteribusabunde
productum,amylumnecnon adest.
Reproductio asexualis per formationem zoosporarum
aplanosporarumque,per biparitioneminteratumprotoplasti
cellularum vegetativarum productarum. Aplanosporae
aetate variantes zoosporas formarenecnon potentes. Duae
ad plures quam centum zoosporae per cellulam productae,
secundum magnitudinem.Zoosporae ellipsoidaeae, in una
superficie saepe subplanatae, cum membranis, 8.0-10.6,u
long., 2.7-5.3,ulat., duas vacuolas pulsantes habentes. Zoospora duobus flagellis longitudine aequis, nucleo unico,
chloroplastoparietali, pyrenoideounico, stigmate anteriori
praedita. Aplanosporae in culturis vetustioribusabunde
productae,zoosporas ad media nova translataeefficientes.
Reproductiosexualis non observata.
Habitat.: In agro arato, in loco dicto, Duanesburg, New
York, Sept. 1951.
Fig. 1-19. Hormotilopsis
gelatinosa.-Fig.1. Habit sketchof a smallportionof theplantas seenunderlow magnification.X381.-Fig. 2. Enlargedview showingdichotomous
of the gelatinoustubes.X871.-Fig. 3. Vegetative
branching
cells showingcup-likechloroplastid,
nucleusand contractile
singlepyrenoid,
vacuoles;noteeccentric
of geladisposition
tine.X2121.-Fig. 4. Surfaceviewof an oldervegetative
cell showingnumerous
starchgrainsand formation
of gelatinous
tubes.X2121.-Fig. 5-6. Successivestagesin zoosporeformation
in vegetative
cells. X2121.-Fig. 7. Liberationof zoosporesin vesicle.X212I.-Fig. 8. Liberatedzoospore.X2121.-Fig. 9. Posterior
polarviewof zoospore.X2121.-Fig. 10.
Telescopedviewof zoosporefromanterior
pole,showingrelationbetweenflagella,contractile
vacuoles,stigmaand pyrenoid. X2121.-Fig. 11-13.Variationsin zoosporeform.X212l.-Fig. 14. Youngvegetative
cell; contractile
vacuolesvisible, cell wall notyet demonstrable.
cell in surfaceview and medianopticalsecX2121.-Fig. 15-16.Young vegetative
tion,respectively.
X2121.-Fig. 17. Early stage in eccentricformation
of gelatine.X2121.-Fig. 18-19. Cells stained
withHaidenhain'siron-alum
in plastidand uninucleate
haematoxylin
showingpyrenoids
condition.
X2121.
Fig. 1-58werepreparedwitha Spencermicroscope
and Leitzcameralucida. The approximate
magnification
is included
in the legendof each figure.All figures
weredrawnfromlivingmaterial,exceptwhereotherwise
stated. The photoweremadewitha LeitzMicamcamera;theirmagnifications
micrographs
are givenin thelegends.
762
[Vol. 40
AMERICAN JOURNAL OF BOTANY
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22
X2
201-2
2
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243
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31
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27~~~~~~~~~~~~~~~~~~~~~~~~~~~~7
32
40
39
33
AA
~~
37~
~
~
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34
1953]
December,
TRAINOR AND BOLD-NEW
CHLOROPHYCEAE
763
maiores1OO,u,
culture. When the zoosporebecomesquiescent,it diametrointerdum
plerumquesphericae,raretainsits ellipsoidalshape (fig. 35-38). Young rius ovatae,semperstipitatae,stipitead 9,t longitudine.
vegetativecells alwayshave cup-shapedchloroplas- Cacumenstipitisdisciformeaut e bracchiisduobus ad
tidsand graduallybecomesphericalas theyenlarge quattuorcompositum.Membranacellulae ad 12A.crassitudinein cellulisveteribus;saepe stratifacta,
straitisimti(fig.33-40).
mis saepe irregularibus.Chromatophorus
parietalisproA majorityof the cells in old culturesproduce tuberatione
centraliunicumpyrenoideum
continente
praeaplanospores(fig. 41, 60) ratherthan zoospores. ditus,in cellulis vetustioribus
densus,amylo abundante
These are similarin appearanceto zoospores,when atque oleo luteo-flavo
celatus. Cellulae praeterzoosporas
firstformed,
buttheyare retainedwithinthemother multinucleatae.
cell, whose wall expands as the aplanosporesenReproductioper formationem
zoosporarum
per cellulas
variantesfactarum.Zoosporaeduo flagella
large (fig. 41). When a culturewith numerous magnitudine
unicumpyrenoaplanosporesis transferred
into freshmedia, the aequa, duas vacuolaspulsantesanteriores,
parietalemhabentes,8-19,a
aplanosporesdivide to formzoospores,regardless ideum,unumchromatophorum
of theirsize. Hence,aplanospores,stillsurrounded long.,2.0-5.4,ulat., sine membraniset stigmatibus.Zooadmodumantequamquiescuntstipitema polo anby the parentcell wall,may have zoosporeswithin sporae
terioreformantes;
formatio
stipitismotionetorquentecirthem.
cumaxemverticalem
comitata.
DISCUSSION.-This alga has been placed in the
Reproductio
sexualisnon observata.
genusChlorolcoccum
Meneghini(nomengenericum Habitat.: In solo agri, in loco dicto,LowndesCounty,
conservandunpropositum,
Silva and Starr,1953), Georgia.
the genericlimitsof whichhave been emendedby
Starr (1952) to includeonlythosesphericalchloroCharaciwm
polymorphum
(fig.42-58, 62-63), a
coccaceousalgae whichpossessvegetative
cellswith member of the family Characiaceae, order Chloroa hollow,sphericalchromatophore
and one or more coccales, was isolated from soil from a field in
pyrenoidsand whichreproduceby meansof walled LowndesCounty,Georgia.3The soil was extremely
zoosporeswithtwoequal flagella.
rich in bacteriaand fungi,and it was withsome
No attemptwas made to identifythis organism difficulty
thatbacteria-free
cultureswerefinallyobwith one of the older species of Chlorococcum tained. The latter(No. FRT-3) are nowmaintained
whichStarr (1952) considersto be nominadubia. on modifiedBristol'sand "Volvox" agars. Charaof cium polymorphum
Instead,thewritershave comparedtheattributes
can also be cultivatedin the
C. oleofacienswiththose of acceptablespecies of liquid of these media, withoutagar, and in soilas listed by Starr (1952, table 1). watertubes,but it growsmostabundantly
Chlorococcum.
on modiFromthiscomparisonit is obviousthatthepresent fiedBristol'sagar. This mayreflect
eithera special
organismdiffers
fromanyrecognizedspecies. None growthrequirementor the stimulatory
effectof
of thesespeciesproducesyellowoil in the quantity abundantoxygen.
characteristic
of C. oleofaciens.The latterdiffers The vegetativecells of Characiumpolymorphum
fromC. multinucleastum
Starrin thatit is uninucle- are variablein size and form. Althoughold vegeate throughout
its vegetativecycle. It is unlikeC. tativecells may measuremorethan1OO,u,
the averinfusionum(Schrank) Menegh.sec. Bold (1931), age cell size is 2545p,. Normallythe cells are
whose protoplastbecomesmultinucleate
just prior spherical, but when zoospores become quiescent,
to sporogenesisby progressivecleavage. Although they do not always round up completely,and, as a
themarkedthickening
ofthewall in oldercellssug- result,ovoidal cells develop (fig. 42, 43, 45). These
gestsC. minutuim
Starr,thelargersize,theproduc- probably form zoospores precociously,for none
tion of oil and the absenceof sexualityin C. oleo- morethan25-30,uin lengthwas observed.
faciens distinguishit fromthe former.C. oleoWiththeexceptionof thelargestcells,all vegetafacienslacks the spinyhypnosporesof C. hypno- tive cells possess a stalk which is apparentlyan
sporumStarrand exceedsin size C. echinozygotumextensionof the cell wall (fig.42-25, 55, 56, 62).
Starr,whose walls do not thickenwith age. For The lengthof the stalkvaries between5.S-9.0,uin
thesereasonsand others,whichare apparentwhen lengthand O.5-l.O,uin diameter. It usuallyreaches
moredetailedcomparisonsare made,the organism its maximumsize earlyin thelifeof thevegetative
describedabovemeritsrecognition
as a newspecies. cell (fig. 56). At its distal end thereis eithera
CHARACIUMpolymorphum sp. nov.
3 Thewriters
areindebted
to Professor
ElsieQuarterman
CelIulaevegetativae
magnitudine
formaque
variantes,forthisand othersoil samples.
Fig. 20-41. Chlorococcum
oleofacienssp. nov.-Fig. 20. Youngvegetative
cell in surfaceview; starchgrainsand single
pyrenoid
visible.X2121.-Fig. 21. Old, oil-filled
cell withthickened
vegetative
wall. X871.-Fig. 22-25. Outlinedrawings illustrating
zoosporeformation
by bipartition.
X212l.-Fig. 26. Vegetativecell transformed
into zoosporangium.
X2121.-Fig. 27. Liberationof zoosporesin vesicle.X2121.-Fig. 28-32. Variationsin zoospores.X2121.-Fig. 33-34.
Opticalsectionand surfaceviews,respectively,
of polarviews,of youngvegetative
cells. X212I.-Fig. 35-38. Stagesin
development
of vegetativecells,opticalsectionand surfaceview. X2121.-Fig. 39-40. Young and maturevegetative
cells stainedwithHaidenhain'siron-alum
haematoxylin,
showinguninucleatecondition.X212l.-Fig. 41. Developing
and distortion
of parentcell wall. X871.
aplanospores
764
[Vol. 40
AMERICAN JOURNAL OF BOTANY
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December,
19531
TRAINOR AND BOLD-NEW
CHLOROPHYCEAE
765
in theformof a disc (fig.55, 63) or two to zoospores. The latterare variable in number,
thickening
to fourprojections(fig.45) by meansof whichthe dependingon the size of the cell undergoingzoostalkis attachedto the side of theculturevesselor sporogenesis.Large, maturecells like those illusis tratedin fig.46 formmorethan100 zoospores.
to detritusin the culture. The stalkfrequently
not formedor is incompletely
formed,when the
The zoosporesthemselves
varyin size from2.0cells are cultivatedin agar media. Cells without 5.4,uin widthand from
in length.They
0.8-18.Ott
stalksare oftenquiteirregularin appearance.
are also variablein shape (fig.51-54). Theyhave
The cell wall is extremely
variable in thickness. a single pyrenoidand nucleus,two equally long
In activelygrowingcells it does notexceed5 or 6
flagella,two contractilevacuoles, and generallya
in thickness,
butin verylarge,old cellsit sometimes few,ratherlarge starchgrainsof parentalorigin,
becomesswollen,reachinga thicknessof 12gt. In buttheylack a cell wall and stigma(fig.51-54).
this state it is plainly stratified,
irregularon the
The zoospores have been observedto emerge
innersurfaceand somewhatseparatedfromtheprofrom
theparentcell in two different
ways. Sometoplast. Cells in whichthe major portionof the
times
it
be
can
seen
that
the
cell has been
parent
wereobservedto have
wall was of averagethickness
a portionthickenedconsiderably. This thickening subdividedwiththe potentialzoosporeswithinnot
sometimeswas at the proximalend of the stalk,so yet motile (fig. 49). When the wall ruptures,the
zoosporesare violently
ejectedand emergeseriatim;
thatthecells wereeccentricin appearance.
become
motile
they
and soon swim
immediately
The vegetativecells have a single,largepyrenoid
this
away.
In
of
the cell
type
zoospore
liberation,
(fig.42-47). The chromatophore
of youngcells is
empties
1-3
in
min.
At
other
times,
the
zoospores
parietalbut becomesmassivein oldercells. Starch,
whichundercertainconditionscompletelyfillsthe can be seen movingslowlywithinthe parentcell
maturecell,is formedaboutthepyrenoid(fig.46). beforetheirliberation. The wall thenrupturesand
Within3 or 4 wk.aftertransfer
intofreshmedium, thezoosporesemergeslowly,surroundedby a vesibe- cle, in whichtheyremainfor a few minutes(fig.
thevegetative
cells of Characiumpolymorphum
comeorangein color. This phenomenon
takesplace 50). They then graduallybecome freeand swim
in liquid culturesand on agar and is caused by the away.
The zoosporesdo not remainmotilemore than
synthesisof abundantoil, in the dropletsof which
1/2hr. undertheconditions
prevailingin laboratory
an orangepigmentis dissolved(fig.62).
is multinucleate(fig. cultures.Whentheybeginto movein rotaryfashCharaciumpolymorphum
47, 48); youngcellsat firstpossessa singlenucleus, ion about theirlongitudinalaxes, it is a sign that
butthissoon divides. Continuednucleardivisionis theywill soon become attachedand sessile. They
accompaniedby increasein cell size. Nuclei were oftengo throughthisrevolvingmotionin one place
observedin smear preparationsstainedwith Hai- and thenswimawayto another,buttheyultimately
by theiranteriorend and rapidly
denhain's iron-alumhaematoxylinand with the attachthemselves
Feulgennucleartechnique.The clear,vacuole-like generatetheir stalks,which immediatelyachieve
areas observedin youngcells in the livingcondi- theirmaximumlength.
tion,whose contentswere not obscuredby starch
There is evidencethatthe blepharoplastplays a
grains,probablyindicatethe positionof the nuclei dynamicrole in the formationof the stalk,for it
appearsthatit is by meansofthisstructure
thatthe
(fig.44).
Zoosporeformation
could be obtainedreadilyin zoosporefirstadheresto thesubstratum.The stalk
12-18 hr. by transferring
cells fromagar cultures is alwaysformedat the anteriorend. A free-swim1-3 wk. old into eithermodifiedBristol'ssolution mingzoosporethus becomesa youngsessilevegeor "Volvox solution." Transfersfromolder plates tativecell within2 or 3 mmn.
(fig.55, 56). Further
requireat least 36 hr. and sometimeswill not re- stagesin development
are illustratedin fig.42-45.
spond by formingzoosporesin liquid culturesun- Once the stalkhas been formed,thecell assumesa
less -transferred
two or threetimes. Division into sphericalform.The fateoftheflagellais notknown
by progressive
zoospores,apparently
cleavage,takes with certainty.Inasmuchas they disappear soon
place bothin lightand in darkness.Cells as small afterthezoosporesdevelopstalks,and becausethey
as 16,tin diameterhave been observedgivingrise have not been observedto be abscissed,it seems
Fig. 42-58. Characium polymnorphum
sp. nov.-Fig. 42. Youngvegetative
cell,medianopticalsection.X2121.-Fig. 43.
Young vegetative
cell, surfaceview; starchgrainsvisible.X2121.-Fig. 44. Slightlyolder vegetativecell illustrating
globoseformand vacuolarareas whichpresumably
indicatelocationof nuclei.X2121.-Fig. 45. Relatively
maturevegetativecell whichhas retainedthejuvenileovoidalshape,medianopticalsection.X2121.-Fig. 46. Surfaceviewofmature
vegetative
cell filledwithstarch;stipenotvisible.X2121.-Fig. 47-48. Matureand youngvegetative
cells stainedwith
Haidenhain'siron-alum
haematoxylin
showingmultinucleate
condition.
X2121.-Fig. 49. Immature
zoosporangium
stained
withIKI solution.X2121.-Fig. 50. Dehiscenceof zoosporangium;
stainedwithIKI solution.X2121.-Fig. 51-52. ZoosporesstainedwithIKI solution.X2121.-Fig. 53-54. Livingzoospores.X2121.-Fig. 55. Youngvegetative
cell containing starchgrainsfromparentcell,stainedwithIKI solution.X2121.-Fig. 56. Youngvegetative
cell withextremely
long
stalk.X2121.-Fig. 57-58.Old vegetative
cells containing
starchand oil; notethickened
walls.X871.
766
[Vol. 40
AMERICAN JOURNAL OF BOTANY
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of dihto y c.fi.-2'an
eeaie
^'-.,4:.
X 5
Fi
6)Potnofalv
cell of Cldorococcum
oleof.cee . S6.0.-Fig:;62.
gcuur
ofCd--'';
Old domat thick
the cell has been crushedby pressureof thecoverglass; notabundantdroplets
walledcell of Characium
polyrnwrphum;
showingstipe.X277.
cell of C. polymorphumn
of oil. X277.-Fig. 63. Maturevegetative
into the
probablethattheyare rapidlywithdrawn
cell body.
DISCUSSION.-Asurveyof the literaturereveals
no previouslydescribedspeciesof Characiumwith
attributesidenticalwiththose or the organismdescribed above. Kanthamma's(1940) figuresof
fromIndian soil are strongly
Characiumterrestris
suggestiveof the organismisolatedby the writers.
However,in comparingtheirattributes,
a number
of importantmorphologicaldifferencesare revealed, whichindicatethattheyare different
species. The mostimportant
oftheseare maximumcell
size attainedby the vegetativecells,nuclearcondition, morphologyof the zoosporesand origin of
the stalk. The vegetativecells of C. polymo Awn
in the writers'culturesfrequently
exceed 1OO,uin
December,1953]
TRAINOR AND BOLD-NEW
CHLOROPHYCEAE
767
on the otherhand,38.5u so markedlyin theproductionof theirstalks.
diameter;in C. terrestris,
Becauseof thesedeviationsfromC. terrestris,
the
is citedas themaximumcell size. Furthermore,
the
dormantcells (fig. 57, previouslydescribedspecies of Characiumwhich
productionof thick-walled
58, 62), whose contentsare obscuredby the pro- the writers'organismmost closely resembles,the
ductionof large amountsof pigmentedoil, is an latterhas been classifiedas a new species,C. polynot listedby KanthammaforC. terrestris. morphum,with the attributesas set forthin the
attribute
The factthatKanthammastatesthatthevegetative formaldiagnosis. In conclusion,on the basis of
of thevariabilityof cell formof
contain6 or 7 nuclei,and that theirobservations
cells of C. terrestris
in laboratorycultures,theauthors
theyproduce 32-64 zoospores,indicatesa funda- C. polymorphum
mentaldifference
fromconditionsin C. polymor- questionthe feasibility
of accuratelyidentifying
all
phum,forthiswould necessitatethe occurrenceof Characiumspeciesin mixedcollectionsfromnature.
rapid and repeatednuclear division duringzoowhichare absent in
sporogenesisin C. terrestris,
SUMMARY
C. polymorphum.The nuclearnumberin cells of
The morphology
of threepreviouslyundescribed
alwaysfarexceeds6 or 7 (fig.47)
C. polymorphum
unicellular
isolatedfromsamplesof
Chlorophyceae
exceptin t-heyoungestcells.
are said to lack cultivatedsoil is describedand illustrated.The orThe zoosporesof C. terrestris
pyrenoidsand contractilevacuoles,both of which ganisms are: (FRT-1) Hormotilopsisgelatinosa
occur in those of C. polymorphum.However,the gen.et sp. nov.,Palmellaceae,Tetrasporales;(FRT.
mostcarefulexaminationhas failedto revealstig- 2) Chlorococcumoleofacienssp. nov., Chlorococ.
while caceae, Chlorocoocales;and (FRT-3) Characium
mata in the zoosporesof C. polymorphum,
theseare presentin C. terrestris.The mostfunda- polymorphunsp. nov., Characiaceae, Chlorococpredifference
between cales. The lettersand numberin parentheses
mental,and almostunbelievable,
these two organismslies in the methodof stalk ceding each species representthe culturenumber
formation.Accordingto Kanthamma,the stalkof underwhichthe organismsare maintainedin the
arises fromthe posteriorpole of the collectionat VanderbiltUniversity.Transfersof
C. terrestris
zoospore,whichis oftencolorlessand protuberant thesecultureshave been depositedin the Culture
whilethezoosporesare yet motile. In C. polymor- Collectionat the BotanySchool, Cambridge,Engphum, on the other hand, repeatedobservations land, and at Indiana University,and herbarium
showthatthe stalkarisesfromthe anteriorpole of specimenshave been sentto the 'ChicagoMuseum
in thezoospores ofNaturalHistory.
thezoospore,as does thehold-fast
of so many filamentous
algae. It seemsparadoxi- VANDERBILTUNIVERSITY,
NASHVILLE, TENNESSEE
cal thattwospeciesof thesame genusshoulddiffer
LITERATURE CITED
and cell structure
of Chlo- PRINGSHEIM,
BoLD,H. C. 1931. Life history
E. G. 1946. Pure cultures of algae. Univerrococcum
Bull. TorreyBot. Club 57: 577infusionum.
sity Press. Cambridge.
. 1950. The soil-water
604.
culturetechniqueforgrowing algae. In The culturingof algae, a symposium
1949. The morphology
of Chlamydomonas
chlamyKettering
FoundationYellowSprings,Ohio.
dogamasp. nov. Bull. TorreyBot. Club 78: 101-108.
in apply. 1950. Some problemsin the cultivation
of algae. SILVA,P. C., AND R. C. STARR.1953. Difficulties
ing the International
Code of BotanicalNomenclature
In The culturingof algae, a symposium.Kettering
to certainunicellularalgae, withspecial reference
to
Foundation.YellowSprings,Ohio.
Chlorococcum.
Svensk.Bot.Tidskr.47: 235-247.
BoRzi,A. 1883. Studialgologici.Fasc. L Messina.
STARR, R. C. 1952. Studiesin the Chlorococcales:
Cldoro.
KANTHAMMA,
S. 1940. On the life-history
of Characium
coccumFries and otherspherical,zoospore-producing
terrestris,
sp. nov. Jour.IndianBot. Soc. 19: 171-174.
genera.Thesis. Vanderbilt
Univ. Nashville,
Tennessee.
MOEWUS,
F. 1940. Die Analysevon 42 erblichenEigen. WEST, W., AND G. S. WEST. 1907. Freshwater
algae from
schaftender Chlamydomonas
eugametos-Gruppe.
Zeit.
Burma,includinga few fromBengal and Madras.
schr.Indukt.Abstam.Vererb.78: 418-522.
Ann.Roy.Bot.Gard.Calcutta6: 175-260.