This is to certify that the
thesis entitled

Developmental

Studies

of

Two

Species

k o w s k i e l 1 a ( N o w a k . )S c h r o e t e r :N . r a m o s a
and

N.profusa

of

Butler

Karling.
presented by

John

Maurice

Roberts

has been accepted towards fu lfillm e n t
of the requirem ents fo r
Ph.D.

degree in

Botany

M a jo r professor

D ate

M -7 95

J u ly

1.QB

Nowa-

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DEVELOPMENTAL STUDIES OF TWO SPECIES OF NOWAK 0W5K IEL LA (NOWAK.)
SCHROETER: N. RAMOSA BUTLER AND N. PROFUSA KARLING

Thesis for degree of Ph. D.

Michigan State College

John Maurice Roberts

1946

DEVELOPMENTAL STUDIES OF TWO SPECIES OF NOWAK OWSKI ELLA (NOWAK •)
SCHROETER:

N. RAMOSA BUTLER AND N. PROFUSA KARLING

ByJohn Maurice Roberts

A THESIS
Submitted to the Sohool of Graduate Studies of Miohigan
State College of Agriculture and Applied Science
in partial fulfilment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology
1946

TABLE OF CONTENTS

Introduction.............. • • • • .............. • . 1
Materials and Methods#
............................ 3
Physiology# * • # • • • • # • • ................. # # 6
Solid Substrata and Liquid Media........... # 6
Temperature. • • • * • • • • • # • • • # • • 1 3
Oxygen# • • # • • • • • • • • # • • • • • •
15
Light# • • • • • # • • • • • « • • • • • • • 16
Miscellaneous# • • • # • • • • • • • • •• #16
Microchemical Tests.............• • • • • • 1 7
Development# ......... • • • • • # • • • • • • • • • 1 9
Structure of the Zoospore# • • • • • • • • # 1 9
Germination of the Spore.............
27
Establishment of the Thallus* • • • • • • •
30
Vegetative Portion.........
30
Flexuous Filaments# • • • # • • •
33
Formation of the Zoosporangium
• 36
Zoosporogenesis. • • # • • • • • • • • • • • 4 0
Zoospore Escape# • • ........... • • • • • 4 7
Fusion of Cells and the Resting Bodies
of N# ramosa. •• • • • • • • • • • • • •
49
Table of Measurements for Strains Studied* • • • • • • 52
Discussion.........
54
Summary* • # • • • • • • • • • • • • • • • • • • • • • 5 8
Acknowledgements* • # • • • • • • • • • • • • • • • •
59
Index to Authors# # • • • • • • • • # • • • • • • • •
60
Index to Genera and Species of Fungi Mentioned* • • • 61
Illustrations. # • # • • • • • • • • •
. • 62
Literature Cited* • • • • • • • • • • • • • • • # • • • 7 1

INTRODUCTION
The first species of the genus of polycentric chytrids,
now called Nowakowskiella, was described by Nowakowski (1876) as
Cladoohytrium elegans*

In 1893, Sohroeter established the opercu-

late genus NowakowslriLslla as distinct from the inoperoulate Cladochytrium#

Constantineanu (1901) questioned the validity of this genus,

but described a second species, N* endogena, which is now considered
to be a variation of the type species*

A valid second species,

_N. ramosa, characterized by a pseudoparenchyma preceding the
formation of resting spores was found by Butler (1907) on rotting
Triticum vulgare stems in India#

Domjan (1930) recalled

Constantineanu* s old specific name when he found Iff# ramosa in
Hungary on the rotting leaves of Typha# N. elegans and N* ramosa
were the only valid species which had been described in time to
be included in the preparation of Sparrow* s monograph on the
Aquatic Phycomycetes in 1943#
Since 1940, six new species of Nowakowskiella have been
described:

N* profusa Karling (1941), N* hemisphaerospora Shanor

(1942), N* delioa Whiffen (1943), N* granulate, N* elongate, and
N* maorospora Karling (1944 and 1945)•
The position of this genus in the phylogenetic scheme is
still undecided#

In calling his species Cladoohytrium, Nowakowski

placed it in a group which has been considered to contain possible
transition forms between the monocentric ohytrids and the filament­
ous Phycomycetes with true mycelium*

This entire group, the

oladochytriaceous fungi, has remained among the chytrids on the
basis of their zoospore structure, not possessing true mycelium,

p* 2

and other features which will he discussed later with reference to
the present report of findings with Nowakowskiella,
Karling (1932) proposed the descriptive term nrhizomyceliuI^l,,
for the thallus of all the polyoentrio members of the Chytridiales*
which were then included in the Cladochytriaceae,

Whiffen (1943)

questioned whether the swellings in the thallus of Nowakowskiella
were homologous with those in Cladochytrium,since the former were
non-septate.

She suggested that it might be better to separate

these two genera on the basis of the presence of septa in these
swellings, rather than on the presence of an operculum*
In his monograph on the aquatic phycomycetes, Sparrow
(1943) divided all the chytrids into two parallel series:

the

Operculatae and the Inoperculatae, thus further removing
Nowakowskie 11a from the family Cladochytriaceae*
It is true of all the lower phycomycetes that the final
decision as to the tenability of a phylogenetic scheme must await
further studies of the cytology, development, and reproduction of
all forms, especially those which appear to represent transitions*
At the present time, only five species of chytrids have been fully
investigated;

Polyphagus Euglenae (Wager, 1899, 1913: Dangeard,

1900-1901), Cladoohytrium replicatum (Karling, 1937), Endochytrium
operoulatum (Hillegas, 1940), Catenomyces persioinus (Hanson,
1945a) , and Rhizophydium ooronum (Hanson, 1945b) •
The purpose of this study is to fill in one more gap in
our knowledge of chytrid structure so that future workers may proceed
in greater clarity, rather than to criticise the existing theories
on the basis of investigating two species of a single genus.

p. 3

MATERIALS AND METHODS

The two species of Nowakowskiella examined in this study
were N, ramosa Butler and N* profusa Karling*

The original oolleotion

of N_. ramosa was found by Professor E. A. Bessey on grass leaves in
distilled water to which had been added a half teaspoonful of debris
from a rain water cistern*

During the course of the study additional

specimens were found on hemp achenes that had been submerged in a
garden pool*

The first and subsequent collections of N* profusa

were obtained on hemp achenes as bait in the same pool and from
the relatively still water along the edge of a river*
Stock cultures were found to persist longer and to develop
more luxuriantly if the water used contained some organic matter*
Sterilised river water fitted this purpose very well*

For sub­

stratum* both in maintaining the cultures and studying the forms
cytologic ally, cellophane sterilised in 80^ ethyl alcohol and
rinsed with sterile distilled water was used*

Sterilised hemp

achenes were added to the river water and cellophane medium to keep
the growth vigorous in stock cultures
Cultures were kept in petri dishes in ■which they could
easily be observed with the low power of the microscope.

For the

study of living specimens at greater magnifications (high dry and
oil), use was made of hanging drops suspended from the cover slips
resting on rubber washers*

The cover slips were sealed to the

washers with vaseline to prevent drying and the washers were
fastened to the slides with either vaseline or a water-proof cement*
Permanent preparations were stained at first with Harris’s

P. 4

or Delafield* s haematoxylin or with Mayer* s haemalum*

These stains

had been preceded by Belling* s modification of Navashin* s fluid.
Most of the cytologioal data included in this paper, however, are
taken from material which had been killed and fixed in Sass' modi­
fied Bouin* s fluid or Belling* s Navashin* fluid and stained by the
smear modification of the haematoxylin method developed by Tuan.
Also used and found helpful in some instances for fixing certain
images were Flemming's weak fluid, undiluted and one-half strength,
Camoy* s fluid, and the Zirkle-Ehrliki fluid.

Among the other

stains used was Flemming* s triple stain.
Since the material was so fragile, rather them wash the
specimens from the cellopheme chips on which they were being mounted
by passage through a dehydration series, that which was stained other
than by Flemming's method was mounted directly from the last water
rinse of the staining technique into glycerine jelly.

In this way,

all the slides studied were whole mounts, alleviating the difficulty
of matching cytological structures which is encountered in the use
of serial sections.

Extreme care was necessary to dehydrate

material stained by the Flemming1s method.

The dehydration solutions

in large enough volumes not to evaporate completely were run over the
specimens on a large glass plate.

Dilute balsam was used for

mounting.
A minimum of handling was imperative in staining the
specimens.

Whenever it was possible to do so without leaving a

tenacious residue to spoil the preparation, materials were kept
in Soyka or Syracuse dishes and treated and washed by adding the liquids
to the dish, rotating slowly to be sure of even exposure, and pouring
off the excess.

In pouring,oare was taken that the specimens did

p.

not pass the shoulder of the dish.

5

Rinsing was accomplished by moving

the specimens to a dish of distilled water and adding and decanting
several changes of distilled water to and from the dish.

Even after

a good rinsing in this manner, the material was dipped into another
dish of fresh distilled water whenever it was transferred from one
solution dish to another.
A drop of a 1% aqueous solution of Poirrier* 8 blue was
allowed to diffuse into the mount from the edge of the cover slip
in the study of living specimens.

This procedure was an aid in

the observation of internal and external structures, since it
seemed to outline the parts of the otherwise hyaline masses.
Zoospores were stained by the Feulgen reaction, Gram* s
stain, and the method described by Cotner (1930) in which the spores,
in a *005% aqueous solution of crystal violet, were dehydrated
over sulfuric acid.
All slides which are the bases of descriptions of cytological details were examined with a 9OX anastigmat oil immersion
objective coupled with 15X anastigmat oculars.

p. 6

PHYSIOLOGY*
Solid Substrata and Liquid Media
When the fungi (both species) were first isolated into
unifungal cultures, the medium employed was sterile distilled water
into which had been placed boiled grass leaves and hemp achenes.
The author repeatedly washed infected grass leaves in sterile
distilled water and transplanted these leaves into sterile dis­
tilled water containing sterile filter paper in order to attempt
to rid the cultures of contaminating bacteria and microscopic animals
that abounded in the debris from which the isolations were obtained.
The fungi grew well in this medium for about a week, then the growth
began to wane.

The fungi failed to become established after being

transferred on filter paper to another culture dish containing
sterile distilled water and filter paper.

Feeling that the diffi­

culty might be in the lack of nutrients or minerals, the author
transplanted other pieces of infected filter paper into sterile
water from a nearby river in which were pieces of sterile filter
paper and autoclaved cellophane.

The growth showed some degree of

regained vigor on the filter paper and no growth occurred on the
cellophane.

However, the opacity of the filter paper, as well as

the decolorized grass leaves, made it difficult to observe the
initial stages of growth*

Cellophane chips which were sterilized

in 80% ethyl alcohol and rinsed with sterile water were used as a
clear substratum.

The alcohol-sterilized cellophane showed more

luxuriant growth than the autoclaved and still remained clear enough
* Except where noted otherwise this portion on physiology is based
on the behaviour of N. ramosa.

P* 7

for the observation of minute details of structure and cytology.
It was a fortunate accident that the author used cellophane that
had been shirt and cigar wrappers for these initial experiments
with growth; since in later trials, growth was not obtained on any
cellophane which had come off packages of cigarettes of several
brands.

The stock cultures of the fungi were kept thereafter in

sterile river water containing cellophane chips and two or three
hemp achenes per petri dish.

Throughout the continuation of the

cultures, the best growth for fii. ramosa was on the hemp achenes,
while N. profusa grew better on the cellophane chips.
of

Zoosporangia

profusa usually developed on the hemp achenes from two days

to a week later than they did on the cellophane in the same
culture.
The author removed the pericarps from several hemp
achenes and placed them into dishes separate from those into
which the contained oily seeds were placed, in order to detect
if the pericarp was sufficient substratum for the maintenance of
N. ramosa. Some of these dishes contained sterile distilled water,
others sterile river water.

There was no growth on the true seeds

in either the distilled or river waters; whereas, growth was
sparse on the pericarps in distilled water and abundant on the
pericarps in the river water.
Since grass leaves are among the standard media for the
culture of chytrids, the growth of N. ramosa on cellophane,
quackgrass leaf, and corn leaf was studied comparatively in
different solutions.

The inocula for the cultures were all

vigorously growing cultures on cellophane chips in sterile river
water.

Table I records the results.

p. 8

Table I. Growth of Nowakowskiella ramosa on various substrata
in different solutions
Substrata

River water

Cellophane
Corn leaf
Quack grass leaf
Filter paper
Hemp achene

Physiological
saline

Distilled
water

None
None
None
None
—

None
Slight
Good
Slight
Good

Good
Good
Good
Good
Luxuriant

Grains of barley, com, oats, rye, and wheat were germinated
in sterile river water; and the fungus on cellophane was added to each
culture in an attempt to find if N. ramosa is parasitic on cereals.
In only one of the oats cultures was there any suggestion that the
fungus was growing on the living plant.

In this one there were

several zoosporangia produced from filaments which appeared to be
growing from the primary root.

However, when the entire seedling

finally died, there was no evidence of filaments or sporangia on
or in the dead plant.

The fungus was not found to be growing on

the grain, stem, leaf, or root in any other cultures; although
there was good growth on pieces of sterile cellophane which had
been placed into the oulture dishes as checks on the viability
of the inocula.
It was decided to test the ability of JN. ramosa to grow
on the killed grains of the same species in different solutions
and to determine the portions of these grains attacked, since this
fungus did not attack living grains and seedlings of cereals.
Some grains had failed to germinate in preparation
for the parasitism experiment above.
dishes containing these grains.
rye and wheat.

The fungus was seeded into

Growth was observed only on

Whole grains of all five of those previously used

and rice were autoclaved in the dry state and then were tested for

p. 9

their ability to support the growth of the fungus in sterile river
water as an extension of this study.

It was interesting to note

that there was no growth on autoclaved rye and wheat grains.

The

growth was very good on barley and oats, good on com, and fair on
rice.

The degrees of growth used here are relative, based on the

subjective observation of the vegetative extension of the fila­
ments and the development of zoosporangia.
Extracts of barley, corn, oats, rice, rye, and wheat grains
and hemp achenes were made, to follow up the experiment testing
the relative usability of the pericarps and endosperm by this fungus*
The materials were sterilized in the autoclave at 10 pounds pressure
for 20 minutes and were added, at the rate of 1 ml. extract to 20 ml.
of the liquid medium in the petri plate.

The final percent endosperm

was 2,5/6, since the original extract was made by grinding 1 gram of
endosperm into 1 ml. distilled water.

The liquid and liquefiable-

solid media used were Knop*s solution, distilled water, sterile
river water, and 1% agar.

These media were seeded with cello­

phane chips bearing well-established vegetative beginnings of growth.
The growth obtained in this experiment was fairly constant
for all types of extract, with exception of the c o m and the oats
extracts.

There were few rhizoids formed in any of the liquid

cultures,but the erect sterile filaments became greatly extended
from the cellophane ohips that carried the inoculum.

The forma­

tion of zoosporangia was very sparse except in sterile river water,
in which there were many sporangia borne on short lateral branches*
In cases in

which the sporangia were numerous, the filaments

appeared to be appressed to the bottoms of the culture dishes and
not waving erect as was found in the stock cultures.

This appressed

p. 10

habit was so pronounced In cultures of N_. profusa that when the
substratum to which the rhizoids were attached was removed from
a plate, the werect" filaments and sporangia remained fast to the
bottom of the plate, as though they were cemented there.

There

was hardly any growth at all in the cultures containing c o m
extract.

There was little formation of zoosporangia except in

the river water in the oats cultures.

The sporangia were produced

on short compact filaments in which the isthmuses between the
swellings were much shorter than is the normal length.

The growth

seemed fairly abundant in Knop* s solution end in distilled water
to which oats extract had been added, but the filaments were very
much shortened and looked knobby because the swellings were formed
so close together.

Vegetative growth was more extensive than ever

observed in a completely liquid medium in all the cultures on the
plain agar to which the endosperm extracts had been added.
was a difference in sporangium production, however.

There

There were

numerous sporangia formed close to the cellophane inoculum in the
barley, rice, and hemp cultures*

The greatest extension of sterile

filaments among these three agars was 9mm. in the hemp agar.

No

sporangia at all formed in the oats, rye, and wheat agars; but in
the rye and wheat cultures the filaments extended so far and were
so thick that they gave the impression of being contaminants at
first observation.

The growth proved to be made up of slender

filaments of Nowakowskiella with swellings formed only at the bases
close to the cellophane inoculum.

The c o m cultures resembled

the last three in most respects, except that there were present a
few sporangia close to the cellophane chips.

p. XI

The next step in this study of substrata was to take the
pericarps of the above cereals and of hemp and attempt to grow the
fungus on them in the same solutions and on agar.

The data

obtained in this phase of the study are given in Table II.
Table II.

Growth of Nowakowskiella ramosa three weeks after
inoculation on,six different pericarps in three liquids
and on the surface of 1.0% plain agar

Pericarp

Agar

Barley Pair
Corn
Fair
Oats
Slight
Rice
Slight
Rye
Abund.
Wheat
Abund.
Hemp
Abund.

Vegetative Growth
Knop* s Distilled River
Water
Sol.
Water

Sporangium Production
Knop* s Distilled River
Agar
Sol.
Water
Water

Good
None
None
Abund.
None
Good
Good

Slight
Fair
Slight
Slight
Abund.
Abund.
Abund.

None
None
Good
None
Slight
None
Good

Good
Fair
Fair
Good
Fair
Slight
Good

Good
None
None
Abund.
None
Good
Good

None
None
None
None
Slight
None
Good

Good
Fair
Fair
Good
Fair
Slight
Good

The above data demonstrate that the pericarp of the hemp
achene is definitely superior to that of the cereal grains for the
growth of 1&. ramosa. This observation has practical value for the
mycologist who wishes to escape from excessive bacterial contamination
in a culture of this fungus.

It is also of interest to know that

in the hemp pericarp cultures the filaments of the fungus were
found to extend as far as 2 cm. from the pericarp into the agar.
A portion of the agar in which were some of the filament tips was
transferred to a dish containing cellophane and hemp achenes in
sterile river water.

By this means a pure culture of the fungus

was obtained.
The growth had become initiated by the end of the first

p. 12

week after inoculation in all the cultures used in the study above,
except those of the pericarps in Knop's solution*

Although growth

— * ramosa will occur in distilled water and in Knop's solution,
on the average the most uniform growth on each of the solid media
was found in river water*

This indicated to the author that al­

though much of the food matter is obtained from the carbohydrate
parts of the solid, something more than the inorganic salts is
obtained from the surrounding liquid.
The rhizoids have been found to penetrate the cellophane chips
and to lie free in channels of liquid digested in this solid, as an
indioation of the utilization of cellulose-like breakdown products
and the ability of N. ramosa and N* profusa to digest materials of
a higher polysaccharide nature.

This channeling of the cellophane

has been observed in the absence of microscopic animals or bacteria,
and the channels follow the same outline as the rhizoids.

Apparently,

there is an exoenzyme produced by the rhizoids which digests the
*polysaccharide** to a soluble substance or substances to which the
rhizoid wall is permeable.

The composition of the breakdown pro­

ducts is beyond the scope of this report.
The action of N. ramosa on oellulose was tested with ster­
ile cotton and a technical grade of cellulose acetate in agar, dis­
tilled water, Knop* s solution, and river water.

It was found that

zoosporangia were borne as far as 2.5 cm. away from the initial
inoculum in the agar containing cotton, whereas cellulose acetate
was not attacked at all.

Plates with nothing but the wrapping

cellophane inoculum showed good growth at the end of three weeks
when the experiment was ended.

Some vegetative growth and few

sporangia developed on the cotton in Knop* s solution, but none on

p. 13

the cellulose acetate.

Also in Knop*s solution, and in oats extract,

the thalli on the cellophane were made up of short knobby filaments
and bore zoosporangia in clusters on short erect filaments*

The

growth was sparse on cellophane in distilled water, while cotton
and the technical cellulose acetate were not attacked at all*
There was no growth on the cellulose acetate in river water, but
the filaments and zoosporangia were abundant on cotton and cello­
phane.

The presence of digested portions of the cotton fibers

and the cellophane chips resembled the effect in the channeling by
the rhizoids already described above*

Whether or not the rhizoids

actually entered the cotton fibers was difficult to ascertain*

In

most cases, the association appeared to be one of the rhizoids lying
along side of the fiber or the shadowy outline left by the digested
fiber*

Temperature
All of the above experiments with Ef. ramosa and _N. profusa
were done at room temperature varying from 20° to 25°C.

When the

first specimens of N* ramosa were obtained from their cistern source,
it was not possible to keep a culture alive for more than ten days
at a temperature above 20°C.

At this time the cultures were kept

in a water-cooled incubator that maintained a temperature between
16° and 18°C*

The incubator was made by keeping a constant flow of

tap water running through the jacket of an old slide dryer*

This

arrangement gave better results than a refrigerator set between these
temperatures, probably because the humidity was kept higher so that
drying-out was less rapid*

Repeated attempts during the first year

that this species was in culture were made to get cultures to develop

p. 14

normally at room temperature.

Satisfactory growth began to be

obtained in cultures kept on the sill of an open window in the
middle of September of that year.

As continuous sub-culturing

of these was carried on, there finally was found to have developed
a strain that on some occasions grew well for three to five days
in a room where the temperature varied between 25° and 28°C.
The author added fresh river water to an old culture that
had been completely dried up for six months, near the end of the
observations on N. ramosa.

This culture was left standing at

room temperature on a table top.

After three days, new filaments

appeared extending into the liquid surrounding one of the hemp
achene^ and cellophane chips were added to this culture and were
attacked by spores from the sporangia formed after a week.

The

strain arising from this culture continued to grow normally in the
stock medium described above through several sub-cultures for over
a year, at room temperature.

In fact, the growth at room temperature

seemed to be more abundant than that in the cooled incubator.
It would be well to mention here that this is the only
evidence of a rejuvenation of old cultures of IT. ramosa observed by
the author during the four years he maintained this species in
culture.

Never by accident or design has he observed the germination

of resting spores.

The resumption of growth in this case may have

been due to the germination of resting spores under the pericarp
of the hemp achene, but proof of this assumption was not found.
Karling (1944a) has observed the resting spores of a Brazilian
strain of N. ramosa to germinate, either directly as zoosporangia
or indirectly as prosporangia.
N. profusa grew readily from the first isolation at room

p. 15

temperature in summer or winter.

Growth in the cooled incubator

was definitely inferior to that on the table top.

The author* s

strain of this species even withstood the high temperatures
developed in the trunk of a black automobile standing in the sun
all day for two weeks, and grew readily in sub-cultures again
after this exposure.
The author sometimes used the quick change of temperature
method for forcing the release of the zoospores from the sporangium.
Some of the cultures were allowed to freeze solid and to remain fro­
zen for as long as three days.

When these cultures were thawed and

examined, the vast majority of the spores were formed with normal
structure.

Less than one percent of them showed the uneven cleavage

of the sporangial cytoplasm to form multiflagellate spores as
reported for Blastocladia by Cotner (1930)•

These abnormal spores

will be discussed further under '"Development” •

Oxygen
Indications were found that N. ramosa is dependent on the
presence of a ready supply of oxygen for normal growth.

The author

has never been able to get this fungus to continue growth in the
confines of a test tube.

All the cultures mentioned in this paper

were in petri or soyka dishes.

Even here, the growth was always

better in the more shallow, wider petri dish.

The fungus was found

to be growing most luxuriantly at the surface of the medium, in
almost all the cultures where growth and reproduction appeared
normal.
level.

Sporangia were hardly ever produced much below the water

p. 16

U* profusa differed from N. ramosa in response to oxygen
Also.

Although the former species was never grown in a test tube,

it grew well in 500 ml. flasks containing 200 ml. of river water.
In addition, the sporangia of N* profusa are produced in abundance
appressed to the bottom of a petri dish, and hardly ever were
found floating in the immediate surface of the culture liquid.

Light
Although bright and direct sunlight seem to inhibit the
growth of these two species, the growth is as good in diffused
light as in complete daxkness.

Miscellaneous
An attempt was made to cut down the growth of the bacterial
contaminants by the bacteriostatic action of crystal violet.

The

author found that the strain of N. ramosa in question tolerated and
grew satisfactorily in a 1:100,000 dilution of this dye, but not in
1:10,000.
Halos were frequently formed around the fungal growth in
some of the agar cultures of the same species, which indicated that
N. ramosa may be slightly bacteriostatic itself.

The bacteria became

established in some instances around the site of inoculation before
the fungus did; and as the fungus developed a halo appeared in the
bacterial growth.

Bacterial contaminants rarely overran areas

where sporangia were being produced, in agar -where the fungus was
established.

This phenomenon was not exhibited in liquid cultures.

Bacterial zoogleal masses around the solid substrata would completely

p. 17

envelope the thalli.

The fungus responded to this condition by

exit papillae which extended beyond the limits of the slime.

Microchemioal Tests
Thallus walls of both species react to the chitosan
test as described by Johannsen (1941) • The walls of sporangia
and apophyses are stained red-violet by iodlne-potassium iodide
solution in conjunction with 1.0% sulfuric acid, if the thalli
have been previously boiled in saturated potassium hydroxide.
Picric acid gives, as Nabel (1939) described, a yellow coloration
that cannot be washed out.

When 75/6 sulfuric acid is added to

material treated in this way, the entire thallus dissolves.

The

sporangium and apophysis walls of both these species also reacted
slightly to ohlor-iodide of zinc, indicating the presence of some
cellulose.

Although an inner layer appeared to be present in

the sporangial wall of N. ramosa (this was not apparent in
N. profusa), this inner portion did not stain.

Both the chitin

etnd the celloluse seemed to be present in the same layer in both
species.

There was no evidence of an evanescent inner wall of

chitin such as Nabel described for his species, Rhizidiomyoes
bivellatus. These species of Nowakowskiella do agree with Rh.
bivellatus in that the walls of the filaments and rhizoids do
respond to tests for chitin and not for cellulose.

Eillegas

(1940) found the walls of the zoosporangia of Endoohytriuin
operculatum to give a weak cellulose reaction, as is true with the
two species being considered here, but does not mention their
behavior with tests for chitin*

p. 18

Sparrow (1943) writes, "The operculum is probably formed
of wall material.•••", in reference to the operculate chytrids in
general.

It is not clear to the present author whether he means

that the operculum is of the same composition as the wall or is
made of a transformed wall-substance.

In the case of both N. ramosa

and N. profusa, treatment with ruthenium red stains none of the
fungus except the operculum.

The reddish violet color acquired by

this structure indicates that it is primarily of a poetic nature.
The "oil" globules and refractive matter reacted similarily,
whether in the zoospores, filaments, spindle-organs, or rhizoids*
With Sudan IV they were stained golden yellow, became light brown after
standing in oamie acid, and were dissolved by acetone.

These find­

ings do not add any more information as to the composition of the
"oil" in the chytrids than Karling and Hillegas found with Cladochytrium replicatum and Endochytrium operculatum, respectively;
that is, they seem to be of a fatty nature but may be more complex.
However, treatment with saturated picric acid or with dilute eosin
did not show any indications of proteinaceous matter being associated
with the fatty globules.

This failure to stain may have been due to

the immiscibility of the water-dissolved stains and the fatty matter.
With picric acid the nuclear caps and cytoplasmic strands stained a
bright greenish yellow.

p. 19

DEVELOPMENT

Structure of the Zoospore
Figs. 1, 2, 3, 14, and 15
The zoospores of N. ramosa and N. profusa are typically
posteriorly uniflagellate, uninucleate, and spherioal.
The single refractive globule in the spore of N. ramosa
is colorless and excentric.

The position may be anterior or lateral

to the centrally located nucleus, but never posterior.

In this

respect the zoospore resembles that of Solutoparies Pybhii (Whiffen,
1942).

Posterior to the nucleus is a large shadowy body.

Because

of its lunate shape, this structure resembles a nuclear cap; ex­
cept that in the stained spores the nuclear cap is anterior or
lateral to the nucleus.

Occasionally one to three refractive

globules are found in the spore, instead of the usual one.

A

similar deviation from a typical single globule has been described
for N. elongate by Karling (1944a) and for Sporophlycti s roatrata
by San Chiun Sen (1944).

A single refractive globule is also typi­

cal of N. profusa. The position differs from that given above for
IT. ramosa in being lateral to and usually posterior to the central
nucleus.

Smaller refractive granules may be present in the area

posterior to the nucleus in some spores of N. profusa, as described
by Karling (1945) for N. macrospora. A lunate granular body such
as Ajello (1942) described in Polychytrium may also be seen in the
living cell.

The position of this body has been observed as pos­

terior, lateral, or anterior to the nucleus.

Whether this struc­

ture is homologous to the similar structure in N. ramosa, the

p. 20

vesicular side-body in Blastocladiella laevisperma and B^ asperosperma (Couch and Whiffen, 1942), or is a nuclear cap without a
fixed position was not determined.

Butler (1907) described the oil

globule of the N. ramosa zoospore as a very mobile one, and Hillegas
(1940) reported the globule in Endoohytrium operculatum as fluid
and changing shape when the amoeboid spore squeezes through a tight
space.

Neither of these characteristics was observed by the author

in the globules of N. profusa zoospores.

When the spore is escap­

ing from the zoosporangium or is resorting to amoeboid motion, as
it may do repeatedly, the globule remains in its posterior position
and keeps its spherical shape.

Under such circumstances the spore

extends clear, hyaline pseudopodia anteriorly or laterally while
the granular and refractive matter of the spore remains faintly de­
limited as an internal spherical mass.

Variability as to the re­

fractive globule in the genus Nowakowskiella is common.

Among the

other species, N. elegans, N. hemisphaerospora, and N. delica
(Matthews, 1928; Shanor, 1942a; Whiffen, 1943) have been described
as having a single spherical globule; N. elongata (Karling, 1944a)
usually has a single globule but may have two.

The refractive mater­

ial in N. maorospora (Karling, 1943) is composed of a disc-shaped
globule and minute granules; and N. granulata (Karling, 1944a) is
characterized by the presence of numerous golden brown granules.
Posterior to the nucleus a small vacuole in which may be
seen a minute granule is also visible in the living spore.

From

this granule the single flagellum extends out of the vacuole, through
a thin layer of cytoplasm and the plasma membrane, to the surrounding
medium.

In some of the spores of N. ramosa which contain more than

p. 21

one refractive globule, a second granule, located in the same or a
different vacuole as the first, is present.
or may not extend from this granule.
have flagella of unequal length.

A second flagellum may

A few of these biflagellates

Some of the biflagellate forms

are the same size as the typical uniflagellate, although they are
usually somewhat larger (10,6-21,$^ as compared to 4.8-9.7^ ).

It

is possible that these '"giant" spores with the two flagella are
formed by an upset in the cleavage of the sporangial cytoplasm, but
there is no evidence of two nuclei in them.

This biflagellate-

uninucleate condition of Nowakqwskiella sp. spores has also been
reported by Ellison (1945).

This lack of the extra nucleus may be

explained in that miniature, non-flagellated spores, consisting of
little more than a nucleus with a thin film of cytoplasm,are some­
times found.

It is admitted, however, that the attachment of the

flagellum of one spore to the nucleus of another seems improbable.
Since fusion of two spores has never been observed, it is believed
that these biflagellate spores are not evidence of sexuality in
this species.

Tri- and quadxi-flagellate giant spores have also

been observed with only one nucleus, and the author prefers to
feel that the multiflagellate condition may be due to improper cleav­
age, as reported by Cotner (1930) for Blastocladia and by other
workers for other chytrids.

"Whatever the reason for the align­

ment of more than one flagellum with a single nucleus, it appears
to be evidence that at least the initials of these appendages are
laid down between the times of division of the sporangial plasma
into the spore initials by furrowing and the final separation of
the mature spores preceding their escape.

The formation of the

flagellum while the spore is still in the sporangium is indicated

p.

22

by their presence being evident immediately on those spores not es­
caping through the orifice in the first mass to escape.

Likewise,

is this indicated in that spores attempting to separate themselves
from the mass at the orifice may be observed held by a fully formed
flagellum caught in the exit papilla by another spore.

In 31. profusa,

the author observed this entanglement of flagella in the papillae
so frequently that he wonders if this might not be the mechanism
explaining the massing of spores on discharge.

Some of these caught

flagella appear to have loops or vesicles at the ends.
no longer evident when the spore has freed itself*

The loop is

Hillegas (1940)

described similar loops on the flagella of immature spores of
Endochytrium operoulatumi Berdan (1941b) found loops in various
positions on the flagella of Catenochytridium carolinianum zoospores;
and Ajello (1942) reported them on the flagella of occasional
zoospores of Polyohytrium.

That these looped flagella are not results

of developmental irregularities but are homologous to the knobbed
modification of the whip-lash flagella found by Ellison in some of
the 2fycetozoa is doubtful.

Karling (1945b) and Hanson (1945a) have

recently described loop formation in the absorption of the flagella
by spores becoming sessile.

The observations on these two species

of Howakowskiella could not support or detract from Hanson's (1945b)
report that the flagellum of Rhizophydium coronum is wound about
the spore and unwinds as the spore escapes from the mass at the tip
of the exit papilla.

The author does feel that the belief that the

flagellum is formed within the sporangium is true.

This theory has

been advanced by Karling (1937), Hillegas (1940), and Couch (1945).
Usually there is a single vacuole, other than that contain-

p. 23

ing the blepharoplast, in the spores of both IT. ramosa and IT. profusa.
In N* profusa this vacuole most often is about the same size as the
refractive globule or slightly smaller.

Its position is lateral or

anterior, frequently opposite the globule in relation to the nucleus.
When a spore is kept In distilled water or is exposed to a solution
of Poirrier* s blue at a slightly toxic concentration, this vacuole
begins to swell until the spore is much distended, as little more
than a tonoplast about a huge (in relation to usual spore size) vac­
uole.

Seldom is it the small vacuole containing the blepharoplast

which undergoes this great increase in volume*

In this distended

condition of the spore, the nucleus and nuclear cap are compressed
into the posterior end of the spore near the point of insertion of
the flagellum.

"When the vacuole is quite large the spore ceases to

swim or to move by amoeboid action.

The flagellum of a spore in this

condition waves weakly but with insufficient power for locomotion.
Whether this is due to the great size of the spore or is due to the
effect of pressure on the internal motor structures is not con­
jectured here.
The most conspicuous structure in stained preparations
of the zoospores of both IT. ramosa and _N. profusa is a darkly-stain­
ing crescent which resembles a nuclear cap, surrounding a third or
more of the centrally-located nucleus#

This body is usually tilted

so that it Is oriented with the thicker part of the orescent to the
side and not directly anterior to the center in the spores of these
two species.

A-nuclear cap in a similar position has been figured

for Endochytrium operculatum by Hillegas (1940).

The finding of

nuclear caps in the zoospores of the chytrids prompted Ajello (1942)

p. 24

to suggest that these extra—nuclear structures are not as important
phylogenetic ally as once thought.

The shape of this structure may

vary from a more or less thin crescent to a cup or sphere in
Nowakowskiella. The spherical, form appears to be a hollow ball which
is filled with the nucleus.

Lateral to the nucleus is a large clear

space, the origin of which is not certain.

This space may be left

by the dissolution of the refractive globule found in the living
spore.

If this space does represent the position of the globule,

it indicates a fixed position for that structure relative to the
nucleus and nuclear cap.

Hillegas considers a similar clear space

to be the nucleus in E. operoulatum. The deeply-staining portion
of the crescent may be less heavy, showing a second clear sphere
containing a nucleolus-like structure, all surrounded by the oap9
in IT. profusa, especially.
In most of the stained spores, the flagellum is found to
be attached to a granule located in a small vacuole posterior to
the center of the spore with a rhizoplast passing through the cyto­
plasm from this granule to the nucleus.

Such distinct rhizoplasts

and blepharoplasts have been described in chytrid zoospores before
(Berdan, 1941; Karling, 1937; Hillegas, 1940).

Ellison (1945)

uses the presence of these structures for designating that the
swimming organelle of the phycomyeete zoospore is a true flagellum.
In most cases, the fixed spore lies so that the flagellum enters the
cytoplasm adjacent to one side of the darkly-staining crescent; and
in such cases,the thin rhizoplast can be seen passing diagonally
through the cytoplasm, so that the point of attachment to the nucleus
is at one of the pointed horns of the oap.

Karling (1942) described

p. 25

the nucleus tapering to the point of attachment of the flagellum in
the zoospore of Septochytrium macrosporum, in a similar manner to
the sub-triangular nuclei in the zoospores of Blastocladiella
simplex and Blastooladia (Matthews, 1937; Cotner, 1930).

Either a

similar tapering structure or two granular cytoplasmic strands
(one or both functioning as a rhizoplast) extend from an apex at
the blepharoplast to the posterior side of the tilted nuclear cap
in some spores.

Only one of the strands joins the central structures

at the tip of a horn of the cap.

If this really is a conical ex­

tension of the nucleus, a second opening in the cap must be pre­
supposed; if it be strands, the structure is like that found in
Monoblepharella Taylorii (Springer, 1945) and occasionally in
Blastooladia (Cotner, 1930).
The cytoplasm constituting the remainder of the internal
structure of the spore is made up of a minutely granular reticulum.
Atypical forms were present in most of the stained pre­
parations of zoospores.

Rarely were spores with two nuclei found,

more frequently with two flagella, two blepharoplasts, and/or two
rhizoplasts.

Ellison (1945) also found the biflagellate spores to

be uninucleate.

Another common atypical spore form was the "midget"

spore which measures 3)*. if spherical or 2x4 ^ if oblong and which
contains nuclear structures similar to the typical spore but very
little cytoplasm, no refractive globule or food body, and shorter
or no flagella.

The non-flagellate forms are those referred to

above as miniature spores which might have lost their flagella in
uneven cleavage.
The well-defined cap covering the nucleus disappears

p. 26

when the spore becomes sessile (between 4 and 12 hours after re­
lease from the sporangium for JR. ramosa. 6 and 24 hours for N.
profusa), in contrast to the occurrence in Cladochytrium replicatum,
Bndochytrium operculatum, and Polychytrium stromaphilum in which the
cap may persist during the early stages of gemination (Karling,
1937; Hillegas, 1940; and Ajello, 1942),

In place of the cap,

there is a deposit of large grannies around the outside of the
nuclear membrane.

Larger granules also become apparent in the

cytoplasm, radiating in elongated masses from the nuclear to the
plasma membrane, lining the inside periphery of the cell, and sur­
rounding small vacuoles.

Examination of spores which appear to be

in transitional stages in the disappearance of the cap indicates
that this structure is the source of the majority of these large
granules.

The clear space which is adjacent to the nucleus and

nuclear cap in the swimming spore may or may not be present or
may be partially filled in with granules.

Here again the behav­

iour of this clear space in the stained spore parallels that of
the globule in the living.

The nucleolus now takes a position in

the center of the nucleus, which is centrally-located in the
sessile spore.
fine reticulum.

The rest of the nuclear substance is marked by a
The nucleus rarely divides at this time, although

some sessile spores may be found which contain two nuclei.
were two nucleoli in one nucleus in one spore of N. ramosa.

There

p. 2 7

Germination of the Spore
Figs, 4, 5, and 16
Between 6 and 10 hours for N. ramosa and 6 and 24 hours
for N. profusa after having become sessile, the spore puts forth a
single slender germ-tube which usually sends off one branch before
it has grown longer than the diameter of the spore.

As the papil-

lary swelling which is the forerunner of the germ-tube becomes evi­
dent, a mass of densely-staining granules aggregates at this point.
As the tube develops the aggregation remains in the tip and becomes
less pronounced during the extension of the tube.

After the first

offshoot, the tube continues to branch more or less dichotomously
until the mature vegetative absorptive portion of the thallus is
established.

Although the germ-tube may branch two or three times

before there are any swellings formed, the most common development
results in a primary swelling, which may be homologous to the pri­
mary spindle organ in Cladochyfcrium replicatum (Karling, 1937) ,
before the second dichotomy occurs.

This enlargement is usually

first noticeable twenty-four hours after the spore germinates in
ramosa and up to seventy-two hours in N, profusa. During this
time the nucleus remains in the spore-case, where it may or may not
divide.

As the swelling enlarges, cytoplasm from the spore flows

to it, and the spore-oase begins to shrivel,

Berdan (1941a)

described the spore of Cladoohytrium hyalinum as empty before the
formation of the primary spindle organ.

Ifi/hen the swelling is about

2—4p across its broadest point, the nucleus leaves the spore-case
and passes through the narrow tube to the swelling.

A deviation

from the normal is that in this, and subsequent swellings formed,

p. 28

the nucleus may arrive in the filament before the swelling is
initiated, as Hillegas has described for Bndochytrium operculatum.
In the event that the nucleus has divided in the spore, only one
of the daughter nuclei passes to the swelling at this time.

These

species differ from Cladochytrium replicatum. Endochytrium opercula­
tum, and Iff. hemisphaeroapora (Karling, 1937; Hillegas, 1940;and
Shanor, 1942a) in the occurrence of nuclear division in ihe sporecase.

The nucleus becomes elongated and narrow in order to pass

through the narrow filament from the spore-case to the enlargement,
in the same manner as Karling reported for Cladochytrium replicatum.
After the primary swelling has formed there is no further growth
originating from the spore even though the spore-case may persist
and enlarge to a diameter of 5 to 12^. • Throughout the life of
many thalli of N. profusa the spore-case persists and enlarges
as an anucleate, rarely nucleate, spherical swelling Joined to
the functional primary swelling by a short, fine or broad isthmus.
The isthmus may be so short and broad that the spore-case appears
as a lobe of the functional swelling.

The persistence of the

spore-case may be ontogenetic evidence of rhisideaceous ancestry.
Although a nucleus left in the spore-case may divide, the
daughter nuclei, except as noted above in Iff* profusa, pass to
the primary swelling or to others which hhve begun to form in the
first branch of the germ-tube.

On rare oocasions as many as three

nuclei have been seen in the primary swelling.

The spore and the

branching of the gera-tube may be so close together that, with
the high-dry objective of the microscope, there appear to be as
many as three gem-tubes developing from the same spore.

However,

closer examination with oil-immersion shows this to be a false

p. 29

impression.

The fact that only one germ-tube is formed seems to be

important in light of the use of Catenaria* s forming two being one
of seven reasons given by Couch (1945) for moving that genus from
the Chytridiales to the Blastocladiales♦

Nuclear division during

these stages has demonstrated some typical mitotic figures.
An oborbive method of attempted germination was discovered
among some spores of N* ramosa that had become caught in the vessels
of a grass leaf.

These cells had assumed an amoeboid habit, then

the nuclei had divided one or more times, and at least one cell had
apparently begun to bud off bits of cytoplasm containing nuclei.

p. 30

8stabl iehmenb of the Thallus
V egetative Portion
Figs. 6 and 17
The spore-case may empty and the shriveled wall become
obscured, or as described above may persist after the primary swell­
ing and its rhizoidal offshoots have become established.

No cell

walls are foraed as the nuclei in the swellings divide and separate
so that the resulting thallus is without any septations, differing
in this respect from Cladoohytrium, Catenomyces. and Catenaria.
There may be one to four nuclei in each of these swellings.
The thallus continues to grow, branching dichotomously
as it does so.

Some of these branches remain appressed to the sub­

stratum and form additional nucleated enlargements from which, or
from the isthmuses, fine or coarse rhizoids are produced.

For one

or two days in Iff. ramosa and up to a week in Iff. profusa most of the
growth of the thallus consists of the establishment of spherical,
fusiform, and irregular swellings, appressed to and imbedded in the
substratum.

In the mature thallus numerous much-branched rhizoids

arise from the swellings and from the isthmuses between.

The swell­

ings may occupy part or all of the cavity of the host cell; and the
rhizoids dissolve their way into and ramify in the cell walls when
the fungi are grown in grass leaves.

These swellings when mature

may possess walls about the thickness of the walls of the mature
zoosporangia; usually, however, little thicker than the walls of
the larger filaments.

The rhizoids are short and may be expanded

in spots, especially at the scene of much-branching, into bladder­
like formations with thin walls and large vacuoles.

The resulting

p. 31

mature rhizoids are typically relatively short, blunt, and knobby.
Another polycentrio operoulate chytrid exhibiting thick, short rhi­
zoids in cellophane is Catenomyces persicinus (Hanson, 1945a)• Some
of the bladders are so great in diameter in places of pronounced
branching that they can be distinguished from the centers of develop­
ment only by the lack of nuclei, and some by the thinner wall.

This

network of rhizoids and swellings resembles the thallus of Megaohytrium Westonii (Sparrow, 1933) more closely than it does any of
the other polycentric chyfcrids.

The notable difference is that

Nowakowskiella contains no cross-walls delimiting the swellings
from the rest of the thallus.
The most pronounced inclusions in the living protoplasm
in the vegetative system described are the refractive globules.
These globules resemble closely the globules of the zoospores,
except for their great variety of sizes (0.2 to 2.0

in diameter).

The presence of similar refractive globules in the intramatrioal
portion of the thalli has been reported before for Iff* profusa
(Karling, 1944a) , as well as for Cladochytrium replicatum (Karling,
1937, Catenomyces persicinus (Hanson, 1945a), Cladochytrium
hyalinum, Catenochytiidium carolinianum (Berdan, 1941 a, b).

Few

scattered minute globules are present in the rhizoids which have
digested their way into the substratum; but wherever a swelling
occurs, there is an accumulation of globules.

The mass of globules

may completely fill the swelling in mature thalli.

The hyaline

cytoplasm appears to fill the thin absorptive rhizoids.

There

are large vacuoles, surrounded and divided diagonally by thin
strands of cytoplasm in the swellings.

The vacuolate nature of the

p. 32

cytoplasm of the vegetative portions of Clad, replicatum, Endochytrium, and Catenaria has been described by Karling (1937), Hillegas
(1940), and Couch (1945), respectively.

The globules are grouped

as to appear ixrtra-vacuolar, held in the cytoplasmic strands.

Stric­

tures in the isthmuses between the swellings may be filled with
cytoplasm, often of a denser appearance than that in the swellings,
forming a type of pseudoseptum.

These pseudo septa occur more

frequently in the older thalli.

Similar bands of material were

found in the rhizoids of Septochytrium variabile by Berdan (1942).
Fixing and staining dissolves out the refractive matter
and accentuates the structure of the cytoplasm and the nuclei.

If

osmic acid is used as a fixative and is not bleached out, the place
occupied by the refractive matter contains a brownish amorphous
mass.

Otherwise the spaces occupied by the globules are clear,

except for loose clumps of densely-staining granules.

Similar

granules are found scattered throughout the cytoplasmic strands.
The number of granules present in Iff. ramosa and Iff. profusa seems
to be greater than that for Clad, replicatum (Karling, 1937).
The nuclei are surrounded by masses of these granules in many
instances.

In their affinity for the nucleus these granules

resemble those which go to make up the nuclear cap, as will be
discussed under zoosporogenesis.

p. 33

Flexuous Filaments
Figs. 8 and 18
Long, flexuous, usually extramatrical filaments which
branch by repeated dichotomies arise from the sides or ends of the
vegetative swellings or from the isthmuses between.

These fila­

ments may be isodiametric throughout their entire length, especially
N. profusa (Karling, 1941), or may vary considerably in diameter.
Elongate fusiform swellings, which are even in outline, may usually
be found regularly in the proximal one-third of each filament in
the strains of both species studied.

Rarely, were secondarily

developed, typical absorptive-vegetative centers arising from these
swellings found.

Distal to this area, the swellings are irregular

in shape and length:

being fusiform, spherical, even triangular

at points of branching, to mere undulations in the otherwise parallel
filament walls.

The narrower portions range in diameter from 1.5

to 2.5^ in the more distal segments, while the swellings in these
segments are only 2.0 to 4.0^- • The length of the narrower portions
is 4.0 to 40

, as opposed to 11 •5 to 30^ for the swellings.

It is

difficult to draw a clear-cut differentiation as to what constitutes
a swelling and what an isthmus in this area.

If one overlooks the

small differences in diameter, it is possible to consider segments
as long as 300p as lacking true swellings.

The swellings and

isthmuses in the proximal portion are more easily differentiated,
since their sizes are 2.5 to 5.0^ x 6.0 to Ilf/ and 1.5 to 2.5^ x
4.0 to 30f* , respectively.

It is not uncommon to find one of these

flexuous filaments almost twioe as wide in the distal portion as it
is in the proximal.

Lateral, branches may form at almost right angles

and remain only 1.0 to 1.5^. in diameter.

Numerous extramatrical

p. 34

flexuous filaments may arise from five to eighteen main "trunks”
originating at a single rhizoidal system in Iff. ramosa.
These filaments may resemble true tubular coenocytes inter­
nally.

The oytoplasm in the younger thalli contains many deeply-

staining granules and small refractive globules, and is netted with
small vacuoles.

The vacuoles in older thalli are longer, surrounded

and crossed by thin strands of minutely-granular cytoplasm.

As in

the vegetative system, pseudo-septa of thick cytoplasm may be found
in points of constriction.
The presence of nuclei in these filaments is variable.
They are usually absent in the narrow lateral branches, and are
usually present in the swellings.
found in a single enlargement.

Up to six nuclei have been

Mature thalli may bear irregularly

swollen filaments lacking swellings, in which rounded nuclei (2.0
in diameter) are scattered throughout their lengths.

As many as

14 nuclei have been observed evenly distributed in a filament of
Iff. ramosa 485

long from its origin to the cross-wall separating

the zoosporangium from the rest of the tube.

The regular distribu­

tion and the rounded shapes and lateral nucleoli of the nuclei
make it appear that these structures are fixed in their places,
rather than passing through the filaments.

Karling (1937) described

the moving nucleus in Clad, replicatum as elongate and denselystaining.

Hillegas (1940) reported that the nuclei of Endochyt-

rium operoulatum elongate only when passing through a constric­
tion in a rhizoid.

Since the author has observed nuclei scattered

in the majority of the filaments which were alive at the time of
killing a*nd fixing, it is his opinion that these structures are

p. 35

typically nucleate in both Iff. ramosa and N. profuaa.

p. 36

Formation of the Zoosporangium
Figs. 9, 10, 19, and 22
Each of the flexuous filaments described in the preceding
section may bear one or more zoosporangia.

These reproductive bodies

can be terminal, intercalary, or on short lateral branches.

Most of

the zoosporangia develop extramatrically, rarely intramatrically.
However, even the intramatrical sporangia are outgrowths of short
specialized filaments.

The author has never observed a typical vege­

tative swelling to produce zoospores.

In this way these species of

Nowakowskiella differ from Clad, replicatum (Karling, 1937), and
Megachytrium Westonii (Sparrow, 1933).

Fhysocladia (Sparrow, 1932)

seems to exhibit similar specialization, even though the vegetative
swellings are extramatrical.

The differentiation of the thalli into

vegetative and reproductive portions is pronounced in N. ramosa and
2* Profusa. There is a superficial resemblance to Rhizopus
with shortened "stolons" in the general habit of a single thallus,
except for the presence of the intercalary swellings and zoosporangia.
The manner of bearing zoosporangia on the young, activelygrowing thalli differs slightly for Iff. ramosa and Iff. profusa. The
most readily observed difference is that the sporangia of Iff. ramosa
are more nearly terminal than those of Iff. profusa. The continuation
of the filament distal to the sporangium in N. ramosa is usually a
fine thread which shrivels as the sporangium matures.

In the few

instances where this thread has persisted, it has been found to be
anastomosed with another larger flexuous filament.

The same struc­

ture may be wide and branch one or more times before narrowing to
finely blunt ends or being terminated by a zoosporangium in Iff. profusa.

p. 37

Most often a filament of Iff. ramosa will branch near its end, and
each of the equal-lengthed branches will be terminated by a spor­
angium.

Both species may produce sporangia on the tips of short

lateral branches.

The filament below the sporangium of If. ramosa

is swollen into an infundibiliform apophysis-like structure.

If

such a swelling occurs in Iff. profusa, it is more nearly spherical;
and a constriction is found at the cross-septum between the swelling
and the sporangium.

The present strain of N. profusa, as with

Karling*s (1941) original strain, is rarely "apophysate”; but it is
not uncommon to find a short tenuous filament leading to an inter­
calary swelling as in N. elongate (Karling, 1944a).
True internal proliferation of the zoo sporangium has not
been observed by the author in either of the two species.

An

unusual type of proliferation is common in N. profusa. As the cul­
ture becomes older, the swollen or non-swollen filament above and
below the original sporangium begins to enlarge.

Cross-walls limit

these swellings from the remainder of the filament, the walls
thicken, and these become secondary sporangia.

Chains of two to

eight sporangia have been observed on old thalli.

If the swellings

form close together, their adjacent walls are flattened where they
have met in enlarging.

Less often there is a short isthmus between

two sporangia in a chain.

In either case, each individual sporan­

gium lays down its own thickened wall; so that if a sporangium were
pulled out of a chain, the walls of those adjacent to it could
remain intact.

There is extreme variability in the shapes of the

sporangia in such chains.

These chains of sporangia would resemble

the multiseptate sporangia of Iff. elongata as described by Karling
(1944a) except for the progressive formation and the double walls
in the present species.

p. 38

Elongate exit tubes are more common in N. ramosa than in
— • profusa* They develop on those sporangia that are intramatrieal or
surrounded by a zoogleal slime, and do not extend beyond the outer
edge of the enveloping material*

The zoosporangia of N. ramosa may

bear one to three exit tubes or papillae, one or all of which may be
branched; but only one is functional.
The swellings which develop into zoosporangia are usually
intercalary at their inception in both species.

The tip may be in­

cluded in the rounding out of the sporangial rudiment until the mature
sporangium is terminal, if the enlargement begins very near the tip
of the filament.

The swelling is fusiform-elongate at first, resembling

very much those in the lower third of the flexuous filaments.

These

swellings begin to increase in circumference more rapidly than they
do in length early in their development until they have become
spherical in shape.

Variations from the typical spherical form are

pyriform, ovate, obtusely-branched, obtusely-triangular, and hour­
glass shaped.

When the rudiment is about one-half its mature size

a cross-wall forms, dividing the swelling into two unequal parts in
N. ramosa and the "apophysate" sporangia of N. profusa, or at a
point near which the swelling begins in those lacking "apophyses".
The time of delimiting the incipient sporangia differs from that
observed by Whiffen (1943) in N. delioa. The smaller portion con­
tinues to develop into an evenly shaped funnel form in N. ramosa so
there is no constriction at the cross-wall.

The portions above and

below the septum continue to enlarge, the incipient sporangium the
more rapidly, in such a manner that there is a constriction between
the two unequal-sized spherical swellings in the "apophysate" forms

p. 39

on the thalli of N. profusa. The septum protrudes as a convex aro
into the sporangial cavity when it first begins to form, but is soon
inverted as a protrusion back into the filament.
The wall begins to thicken when the incipient sporangium
has reached mature size; and the cross-septum attains a thickness
equal to that of the rest of the sporangium wall.

At one point;

lateral, subapical, sub-basal, or rarely apical; the sporangium
wall forms a disc-shaped area thicker, but more refractile than the
rest, which is beginning to take a light tan cast.

Around the cir­

cumference of this disc the wall is very thin, seeming to consist
of little more than the external layer of the immature thin-walled
swelling.

If such structures as exit tubes or papillae have formed,

this disc is at the tip of a tube or a papilla; otherwise it is
flush with the surface of the wall.

This disc becomes the pec-

tinaceous operculum which bulges as a slight convexity on the
mature sporangium wall.

p. 40

Zoosporogenesis
Figs. 10, 11, 19, 20, and 23

Appearance of Living Material: The behavior of the protoplasm in
the developing sporangia of N. ramosa and N* profusa is essentially
the same as that described for other chytrids (Berdan, 1941a and b
and 1942; Couch, 1945; Hanson, 1945a and b; Hillegas, 1940;
Karling, 1937, 1944a and b, 1945a, b, and o; Whiffen, 1942 and 1943).
Because of this similarity, the author will refer to the observa­
tions of other workers only where differences in structure and
behavior of the organisms seem significant.
The refractive content of the otherwise hyaline cytoplasm
begins to increase in the enlargement as the swelling which develops
into a zoosporangium grows.

At the time of cross-wall formation

the refractive matter appears to be more concentrated in the center
of the swelling.

At this same time several large vacuoles may be

observed in the incipient sporangium, and a few large globules are
present.

Differing from the monocentric chytrids, the filaments

supporting the sporangia of these two species are not devoid of
cytoplasm when the cross-walls are formed.

The large globules in

the growing sporangium increase in size and number shortly after the
formation of the cross-wall.

Later these globules again disperse,

and the evenly granular appearance returns to the cytoplasm.

If

exit tubes are formed, they also are filled with the same type of
cytoplasm.

The sporangium attains its mature size during this stage

of evenly granular appearance.

Also during most of the granular

stage the vacuoles are not as evident as before.

Soon, however,

p. 41

elongate, narrow vacuoles can be seen forming in the cytoplasm,
dividing it into irregularly-shaped masses.

This cleavage

progresses primarily from the outer edge toward the center, but
additional vacuoles may be found arising independently of the
primary furrows, cutting the cytoplasm from the inside out, radially
or tangenti&lly.

The minute oil droplets coalesce into a single,

or at the most two, globules in each of the masses so formed.
Except for the refractive globules, the cytoplasm acquires a homo­
geneous appearance and swells until the vacuolar divisions are
obscured.

The cytoplasm completely fills the sporangial swelling,

in this stage, except for a clear space which has formed below the
operculum.

This space appears to be filled with a viscid substance

of low refractive properties, because of its spherical shape, main­
tained at the expense of the rest of the contents.

In the event

a long exit tube has been formed, it may be filled with cyto­
plasm such as in the body of the sporangium, up to the clear
space which is just below the operculum at the tip of the tube.
Appearance of Stained Material: The development of the sporangium
has been followed in the stained material by the study of cells
of different sizes, using the size as an indication of relative
maturity.

This method seemed to be practical because the size

of the swelling and the number of nuclei ordinarily paralleled
each other.
When the small intercalary swelling first appears it is
filled with a granular cytoplasm and exhibits one nucleus, about 2
in diameter.

The deeply-staining granules are small, with larger

granules massing in streaks through the cytoplasm.

Often these

p. 42

streaks are associated at one end with the nucleus, similar to the
observation of Karling (1937) in the newly nucleated spindle-organ
Clad, replicatum. The author* s observations on these two
species do not add or detract from the theory that the strand repre­
sents the line of passage of the nucleus.

As the swelling en­

larges, these strands disappear and the granules become evenly dis­
tributed throughout the sporangium rudiment.

The tapering portion

of the enlargement which is to become the subsporangial swelling
is filled with cytoplasm and may contain one or more nuclei like
those found in the sporangium rudiment, in N_. ramosa and in
Iff, profusa if such a swelling is formed.

Several large nuclei are

found in the rudiment before it has enlarged very much.

The first

nucleus in the swelling has been observed to be dividing in a few
preparations; but there was no proof discovered that all the
nuclei in the rudiment came from continued divisions of this single
nucleus or that some of them might not have migrated into the
swelling from the proximal or distal filaments.

This lack of

proof is further confused since there are several nuclei in the
swelling before the septum cuts it off from the rest of the filament.
The deeply-staining granules aggregate loosely in the
center, leaving the remainder of the cytoplasm less densely granu­
lar as the swelling continues to enlarge into a spherical form
and more nuclei are found in it.

When the rounded form of the

swelling is established, the adjacent portion of the proximal fila­
ment has swollen to be infundibuliform in Iff. ramosa, and spherical
or lacking in II. profusa. The distal continuation of the filament
begins to shrivel back to the rounded swelling, in those sporangia
which appear terminal at maturity although intercalary in origin.

p. 43

The distal filament may be swollen or not, if it persists.

The

flexuous filaments on which the sporangia are borne are usually
almost devoid of nuclei and highly vacuolate by the time the
sporangium is mature.

This appears to be more true for N. ramosa

than for IJ. profusa. Possibly the presence of more intercalary
swellings in the former is a factor affecting the presence of
nuclei in the non-swollen filaments*

The vacuolate nature of the

cytoplasm is less prominent nearer the sporangium; and in both
species nuclei may be present in the swollen or non-swollen filament
immediately below the sporangium.

This is also true of the distal

continuation of the filament if the sporangium is truly inter­
calary, or if this portion has anastomosed with another filament
as described above.

However, in the formation of chains of

secondary sporangia in N* profusa.the filaments become emptied of
nuclei and cytoplasm.
The granular mass in the center of the sporangium dis­
integrates as a densely granular reticulum spreads throughout the
swelling.

The nuclei become aligned on strands of this reticulum

in such a way that observation of subsequent nuclear divisions is
made difficult., Some strands of reticulum extend down to where
the tapering of the swelling begins; and at this place, a thick
plate of granules extends across the filament.

As this plate

becomes more apparent, the outer wall of the sporangium begins to
thicken, and the papillar swelling begins to grow through any
material present that will inhibit the free escape of the zoospores.
As the plate which will form the cross-septum becomes more compact
and densely granular, it bulges up from the filament into the

p. 44

sporangium, as though the pressure exerted in the filament were
greater than that in the sporangium itself.

The granules forming

the retieulum again become dispersed, and the cytoplasm is again
uniformly granular*
and deeply-staining.

The granules present at this time are minute
The area immediately below the exit papilla is

devoid of stainable cytoplasm at this stage.
The peripheral wall of the sporangium becomes thick and
deeply-staining, and the arched operculum is evident as a lightlystained dome which is thick in the center and tapers off toward the
edges.

At the point where the operculum is attached to the rest of

the wall, the wall is not thickened but has remained a thin ring.
By the time the operculum looks fully-developed, the cross-septum
between the sporangium and the filamexik has the same structure as
the rest of the sporangium wall.
The cytoplasm begins to be furrowed at the periphery by
the elongated vacuoles mentioned above* after the walls are completely
formed.

This furrowing by vacuoles progresses centripetally and

laterally until each nucleus has a mass of cytoplasm cut out around
it.

The rest of the cytoplasm in each of these masses becomes clearer

as the deeply-staining granular material oollects around each of the
nuclei.

The granular mass around.the nucleus consolidates and takes

the form of a loosely-packed, over-sized nuclear cap.

After the cyto­

plasm swells so as to obscure the divisions and fills the sporangium
case, the nuclear caps become more compact and more evenly and deeplystaining.

The nucleolus must function independently of the nuclear

cap, because it is evident as a small, dark body in the center of the nuc­
leus before and after the granules begin to clump about the nucleus.

p. 45

The above observations as to the formation of the nuclear
cap tend to support Karling* s (1937) belief that this structure
arises from chromatic bodies or granules in the cytoplasm and is
external to the nucleus itself.

As with Karling* s and Hillegas*

(1940) material, the present species demonstrate easily-stained
nuclear caps after non-chondriosomal fixatives.

It is of interest

to note the similarity of behavior between the granules which form
the nuclear cap and the refractive globules in their aggregation
and dispersion in the incipient sporangium.

The presence of

similar deeply-staining granules in the vegetative portion of the
thallus which in the living state contains a great deal of the
"oil globule” material indicates that the dispersed cap material and
the "oil" may be closely associated throughout the life of the
thallus.

It may be that the granular material is carried dispersed

in a lipoidal medium; and extrudes the lipoid as the "oil globule"
of the zoospore in the process of aggregating as the compact
nuclear cap.

The proximity of the cap and the globule in the

swimming spore appears to support this belief.

Since both the

globule and the cap lose their distinctive identity before and
during germination of the spore, the suggestions by Karling and
Hillegas that the cap is food-material which is used up in the
process seem to apply to Iff* ramosa and N* profusa.
Although the present author has not attained the per­
fection of staining the dividing nuclei demonstrated by the investi­
gators at Columbia University (Karling, Hillegas, and Hanson), he
was able to observe definite mitotic figures in both species of
Nowakowskiella studied.

The nuclear behavior differs in these

p* 46

species from that in Clad, replicatum and Endoohytrium operculatum
in that the nucleus does not increase appreciably in size before
division and divides repeatedly during the enlargement of the
incipient sporangium before and after the formation of the crosswall.

Division of all the nuclei in a single sporangium is not

absolutely synchronous although it occurs simultaneously, since
nuclei in various stages of division may be observed in a single
swelling.

p. 47

Zoospore Escape
Figs* 11 and 21
Wo further visible change takes place in the sporangium
for an indefinite time after maturation of the zoosporangium and
zoosporogenesis*

The entire contents begin to revolve in an un­

divided mass within the zoosporangium wall when conditions are
naturally or artificially conducive to zoospore discharge.

The

operculum is forced clear off or is thrown back as on a hinge
while the cytoplasm is in motion, and the subopercular material is
expelled.

After the operculum dehisces, the cleavage lines in the

eporangium again become distinct and the irregularly-shaped
individual spores are evident.
The spores escape as individuals in each gpeoies.

The

first to pass through the orifice flow through rapidly, apparently
forced from behind by those adjacent to them.

In both IT. ramosa and

N. profusa a mass of zoospores may form at the outside of the exit
papilla*

However, the individuality of these spores is evident

both before and after their escape.

The present author would like

to suggest that the spores escape into a mass, rather than en masse.
In these two species of Nowakowskiella a single spore must be
elongated end distorted to pass through the exit orifice; since the
opening is usually but one-half the diameter of the typical spheri­
cal zoospore, and the surrounding sporangium wall is rigid compared
to the naked protoplast of the spore.

A vesicle or any other con­

fining material was not found enclosing the mass of spores at the
tip of the papilla in either species.

Viftienever the outline of such

a structure was suspected, continued observation showed the line
to be the entangled flagellum of one of the spores in the mass*

p. 48

The existence of the mass is short-lived, only long enough for the
spores to round up, free their flagella, and swim away.
The number of zoospores formed per sporangium varies in
both species*

The zoosporangia producing the fewest spores have

been observed in cultures of N* ramosa when very small sporangia on
short filaments were formed early in the life of the thallus and con­
tained only four or eight spores*

The average number of spores

for a normal zoosporangium in this species has been determined as
about thirty-six.

Ho such small sporangia were observed on the

thalli of N. profusa, and the average number of spores in a typical
sporangium is between sixty and eighty.
All the secondary sporangia in a chain do not mature and
discharge their spores at the same time, even though they may be
adjacent on the same filament.

p. 49

Fusion of Cells and the Resting Bodies of N. ramosa
Fig. 13
Resting bodies were formed, developing from a pseudoparenchyma as described by Butler, in many of the older cultures.
These cells have yellowish walls of considerable thickness, which
are smooth in most cases.

However, it is not uncommon to find

resting bodies with the outer wall corrugated and with the inner
wall exhibiting striations.
Cultures were ordinarily transferred every two or three
weeks.

The flexuous filaments begin to produce short lateral out­

growths instead of numerous zoo sporangia, in cultures older than a
few weeks or in strains under laboratory cultivation for two or
three months.

These outgrowths may be merely irregularly-shaped

enlargements which are formed by the filament swelling on one side
only, or they may be short side branches consisting of thin isthmuses
terminated by club-shaped tips.

Two of these swellings come into

contact and become superimposed upon each other.

The

author had

difficulty in determining whether these cells actually fuse or if
their cell walls merely adhere to each other.

HSfhichever occurs, they

are found in this appressed condition often enough to indicate that
they are fastened together in some manner.

Both cells retain their

rounded shape, being flattened only where they are in contact.
cytoplasm in these cells is minutely granular, and each of them
contains a single nucleus.

The resultant structures are pretty

The

p. 50

definitely single cells with two rounded hemispheres, produced by
the fusion of the two swellings in some cases.

Whatever the case

may be, nuclear fusion has not been observed.

Karling (1944a) has

described the formation of pseudoparenchyma from single swellings
and single lateral branches, as well as from the fused tips of
branches in strains of N. ramosa collected in Brazil.

This "pooling*

of the contents of two cells from different filaments may be homolo­
gous with the behavior of the two cell-potentials from which arise
the resting spores of H. hemisphaerospora (Shanor, 1942a).

It is

possible that a procedure such as has been reported for H.
hemisphaerospora occurs during the formation of the pseudoparenchyma
in the Brazilian strains in which the pseudoparenchyma is produced
without the "fusion" of cells, but from a single cell.

The first

change that occurs in the "fused" cells is the formation of large
vacuoles with the tonoplasts extending across the cells so as to
separate them into four to eight clear areas.

A cell wall is laid

down where each line of cytoplasm is present, in such a way that
each of the "fused" cells is divided into as many daughter cells as
there are vacuoles.

Hot all of these daughter cells contain nuclei.

Those that do are either continuous with the mother filament or give
rise to apophyses as described below.
The nucleate cells in the pseudoparenchyma produce short
off-shoots which have clavate tips.

These tips are filled with a

granular cytoplasm as they enlarge.

After they have attained a

more or less spherical shape, the nucleus from the pseudoparenchyma
cell migrates into the apical swelling, and a plate of cytoplasm forms
aoross the base of the rounded resting-cell-initial.

The nucleus

p. 51

takes a central position in the cell, surrounded by a looselypacked mass of granules*

Strands of granules radiate from the

center to the periphery of the cell.

The presence of similar

chromatic granules has been reported for Polypfaagus Euglenae,
Clad, replioatum, and Endoehytrium operculatum (Wager, 1913;
Karling, 1937; Hillegas, 1940).

The nucleus in this resting

cell was observed not to divide but to remain single and centrally
placed.

The uninucleate condition until mature size is attained

has also been observed in Clad, replioatum and Endochytrium
operculatum (Karling, 1937 and Hillegas, 1940).
The living cell shows a dispersion of oil droplets evenly
throughout the cell and then a re-coalescing of the droplets into
about eight large globules.

Stained material shows the deeply-

staining granules to follow the same procedure.
remains reticulate.

The cytoplasm

Although the peripheral wall is thickened

slightly by the time the cross-septum is completed, after the
cell has become more stabilized as to its internal structure, a
striated and thicker inner wall is laid down inside the original.
These resting-bodies have not been observed to germinate in this
present strain, but Karling (1944a) has observed them to produce
zoospores directly or to function as pro sporangia.

p. 52

TABLE OF MEASUREMENTS FOR STRAINS STUDIED
^expressed in microns)

Structure

N. ramosa

Reproductive Structures
Zoospore
Diameter of spherical spore
Dimensions of amoeboid spore
Length of flagellum
Diameter of nuclear cap
Diameter of oil globule
Zoosporangium
Diameter of spherical sporangium
Dimensions of sporangium other
than spherical
Length of papilla
Usual form
Long variety
Diameter of papilla
Diameter of operculum
Thickness of operculum
Diameter of sub-opercular space
Thickness of wall
Diameter of nucleus
Subsporangial Swelling
Diameter
Length
Thickness of wall
Diameter of subtending filament
Resting Cell
Diameter
Thickness of wall
Diameter of nucleus
Flexuous Filaments
Swelling
Diameter of cell
Near vegetative oenter
In distal portion
Length of cell
Near vegetative center
In distal portion
Thickness of wall
Diameter of nucleus

N. profuse

4.85-9.7
5*82-7.7x
9.7-15.5
24-35
2 *0—2.3
2.5-3.0

3.9-4.7
3.6-7.73
3.5-4.5
20-30
1.5-2.0
1.0-2.0

27.16-31*37

17.0-40.C

35.9-56*3x
37.0-77.6
2.0-15.0
50.0-80.0
4*5-10.0
2.91-8.7
0.9-1.9
1.8-5.0
0.6-1.3
1.5-2.0
3.9-13.6
6.8-15.5
0.9
2.0-5.0

ll-20x
40-50
4.0-6.0
up to 30
4.0—8.0
3.0-4.0
0.6-1.0
1.5-6.0
0.6-1.0
1.5-2.0
0 or 2.0-6.5
7.0-16.C
0.5-0.7
1.5-4.0

7.5-15.0
0.5—0*6
2.0-5.0

-

7.0-10.0
2.8-7.0

2.5-6.0
2.0—2.5

7.0-12.0
11.0-35.0
0.2 or less
1.5-2.0

6 .0-11.0
11.5-30.0
0.2 or less
1*5-2.0

p. 53

TABLE OF MEASUREMENTS FOR STRAINS STUDIED
(expressed in microns)
Structure
Isthmus
Length
Near vegetative center
In distal portion
Diameter
Near vegetative center
In distal portion
Thickness of wall
Diameter of nucleus
Vegetative Center
Swelling
Diameter of cell
Length of cell
Diameter of globules
Thickness of wall
Diameter of nucleus
Isthmus
Length
Diameter
Thickness of wall
Rhizoids
Length
Branched
Unbranched
Diameter
Branohed
Unbranched
Extent of Vegetative Portion
Extent of Reproductive Portion

N. ramosa

IT. profusa

2.0-25.0
5*0—60.0

4.0-30.0
4.0-40.0

1.7-4.0
1.5-5.0
0.9
1.5-2.5

1.5-2.5
2.0-4.0
0.5-0.7
1.5-2.0

4.0-20.0
4.0-30.0
0.2-3.0
0.2 or less
1.5-2.0

3.0-12.0
4.0-24.0
0 .2-2.0
0.2 or less
2.0

0.0-7.0
0.5-1.5
0.2 or less

0.0—6.0
0.5-2.0
0.2 or less

9.0-60.0
2.0—30.0

9.0—48 *0
2 .0-20.0

0.5-1.5
0.5-0.8

0.5-2.0
0.5-1.0

35x50-35x200

40x40-16x160

up to 2 cm.

up to 2.5 mm.

p. 54

DISCUSSION
The long extramatrical filaments which arise from the
vegetative portion of the thallus, often in great enough numbers to
obscure it, are referred to here, collectively, as the "reproductive
portion" because each of these filaments may potentially bear inter­
calary or terminal zoosporangia or, in N. ramosa, resting cells.
The "vegetative" and "reproductive" portions of the thallus are
distinct from one another in external and internal structure in
both the N. ramosa and N. profusa strains studied.

The vegetative

portion as a separate unit superficially resembles the "rhizomycelium”
of the other polycentric chytrids with the nuclei confined to the
replicated swellings and the presence of enucleate Isthmuses and rhizoids.

However, these two species differ from the others in that no

reproductive organs are produced within the swellings of the vegeta­
tive portion.

A possible exception to this is that Karling* s orig­

inal description of N. profusa reports the formation of resting bodies
formed directly from the intramatrical swellings.
of N. profusa is sterile in this respect.

The present strain

It might be that the rest­

ing bodies described by Karling, as well as those by Whiffen (1945)
for N. delica, were not transformed true vegetative swellings, since
reproductive bodies may be formed intramatrically on short filaments
which do not extend out of the cells of decaying grass leaves.
The sterile swellings confined to the vegetative portion of
N. ramosa and N. profusa appear to be similar to the intramatrical
prosporangial (the term is used here in the sense of Whiffen, 1944)
swellings such as are found in Nephrochytritun, especially those forms
which are lobed.

Karling (1938) suggested that this monocentric

p. 55
genus might be a stage in the evolution of the polycentrie mode of
development.

If this conjecture were acceptable the flexuous fila­

ments on which the reproductive organs are borne would be elongated
and branched homologs to the isthmuses between the intramatrical
prosporangia and the extramatrical zoosporangia• Such a hypothesis
is substantiated to some degree by the occasional formation of a
single sporangium close to the substratum on a short filament which
extends only from the intramatrical swelling in the adjacent cell
of the decaying leaf*

The swellings in the extramatrical filaments

might be the result of progressive sterilization accompanying the
process of elongation*
The term "rhizomycelium" does not seem to characterize
adequately the reproductive portions of these two strains*

The

filaments are specialized outgrowths of the vegetative portion and
are not intimately associated with rhizoids in structure or function*
Unlike the typical rhizoids of the chytrids, they do not become
completely devoid of cytoplasm, nuclei, and food materials during
the production of a single zoo sporangium; but retain enough to
produce secondary, tertiary, and chains of zoosporangia in basipetal succession (especially in N* profusa which is lacking
numerous swellings in these filaments)*

Unlike in the "rhizomy-

eelium" of Cladochytrium replioatum, the nuclei are not confined
in the swellings of these sporangium-bearing filaments.
The vegetative portion persists throughout the life of
the thallus as an absorbtive and storage unit, so that a complete
change from a trophooentric to a genocentric thallus does not
occur*

In this manner the thallus of Nowakowskie 11a resembles

the true mycelium of the higher fungi, and continues to digest the

p. 56

substratum and produce asexual spores until the available food
supply has been diminished or the concentration of metabolic waste
in the medium inhibits the growth*
Similar to the higher fungi, N* ramosa produces fewer
asexual spores, and initiates resting-cell formation when the
culture has aged*

So far as has been possible to determine, the

production of resting-cells in this species is not preceded by
nuclear fusion, Karling (1945c) refers to the fusion of filaments
before the formation of the pseudoparenchyma as "vegetative anasto­
mosing” * Shanor (1942a) presented some evidence that a fusion
nucleus may be present in the resting-cell of N. hemisphaerospora*
The same might be true of N* ramosa, for the cytoplasmogamy in
N* hemisphaerospora bears some resemblance to the formation of the
first cell of the pseudoparenchyma in N* ramosa* The formation of
"parthenogenetic" resting spores has been reported for the genus
Siphonaria, a genus which supposedly demonstrates sexuality among
the chytrids (Karling, 1945c) *
In general, the two species of Nowakowskiella studied do
not differ greatly in the behavior of the nuclei and cytoplasm
during the development of the thallus and the formation of reproduc­
tive organs from that described for other chytrids*

Such differences

as do occur may be primarily due to the specialization of the
portions of the thallus and the extensive extramatrical development*
The most pronounced characteristic by which N. ramosa and
N* profusa differ from the other polycentric chytrids is the distinct
differentiation of the thallus into vegetative and reproductive
portions*

p. 57

As to the relationships of Nowakowskiellaj little can be
ventured until other members of the operculate series of the
Chytridiales are investigated more fully*

This gems might suggest

an incompletely known phylogenetic series between the Chytridiales
and the Zygomycetes in general habit and in the possible fusion of
filaments preceding the formation of resting-oells*

This hypothesis

can be considered merely as an interesting speculation until much
more is known about the trophic mycelium as compared with the repro­
ductive mycelium of the latter and until possible transition forms
have been investigated*

p. 5$

SUMMARY
1*

The growth of Nowakowskiella ramosa and .N* profusa is abundant
on solid substrata composed primarily of cellulose if the
surrounding liquid contains organic decomposition products,
such as found in river water, pond water, etc*

2*

The natural optimum temperature for the development of N. ramosa
is between 16° and 18°C. and that of N* profusa, 24° and 28°C*
Both species may be adapted to cultivation at the other
temperatures *

3* The cell walls of both species show a predominance of chitin
which is mixed with cellulose in the same layer*

The

operoula are pectinaceous»
4* The behaviour of the protoplasm during development of the
thallus and formation of the reproductive organs does not
differ greatly from that in other chytrids*
5* The flagellum appears to be formed within the sporangium and
is connected with the nucleus of the zoospore by a rhizoplast
near one of the points of a tilted nuclear cap*
6* The center of growth in the developing thallus is early
transferred from the spore case to a swelling in the
germination tube*
7*

The mature thallus is divided into two distinct portions which
function concurrently, the vegetative portion and the repro­
ductive portion.

8* The complete thallus is not adequately characterized by the
term " rhizomyoelium” whch has been applied to the thalli of
the polyoentric chytrids.

p. 59

Ac knowledgement s
The author wishes to express his gratitude to Distinguished
Professor of Botany Ernst A# Bessey of Michigan State College for
his helpful guidance and suggestions in the fulfilment of the
investigations and his careful correction of the manuscript*
Thanks are also given to Professor Richard de Zeeuw for his advice
in methods of staining and mounting specimens.

p. 60

INDEX TO AUTHORS

Name

Page

.

Ajello.
.............
20, 23, 25, 27
Berdan . . .. . . . . . . . . . . . . . 23, 25, 28, 32, 33, 41
Butler.
..................................... 1, 3, 21
Constantineanu..........
1
Cotner.
. . . . 5 , 15, 22, 26
Couch.......... .... .................... 21, 23 , 30, 33 , 41
Dangeard. . . . . . .
. . . . . . . .
2
Domjan.......................... . . . . * ............... 1
Ellison
22, 23, 25, 26
Hanson. • • • • • • * • ................
2, 23, 32, 41, 46
Hillegas 2, 18, 21, 23, 24, 25, 27, 29, 33, 35, 41, 46, 51, 52
Johannsen.
..........................
17
Karling
1, 2, 3, 15, 20, 21, 23, 25, 27, 28, 29, 32, 33, 34
....... 35, 37, 38, 41, 43, 46, 50, 51, 52, 55, 57
Matthews*
21, 26
Nabel............................................... 17, 18
Nowakcwski........... .......... * ...................
1
San Chiun Chen. • • • • .............. . • • • • • • • • • 2 0
Schroeter. • • • •
.. . .. 1
Shanor
. 1, 21, 29, 51, 57
.........
1, 2, 18, 32, 37
Sparrow.
Springer. • • • * . • • • • .........
26
Wager ............... . . . . . . . . . . . . . . . .
2, 51
Whiffen. . . . . . . . . . . . . . . .1, 2, 20, ^1, 39, 41, 55

p. 61

INDEX TO GENERA AND SPECIES OF FUNGI MENTIONED
(other than 9. ramosa and N. profusa)

Name

Page

Blastocladia. • • • . • • • * . * ............... 22, 26
Blastocladiella asperosperma. . . . . . . . ........ .21
laevisporma.
21
simplex. . . .
. 26
Catenaria. . . .
.30, 31, 33
Catenochytrium carolinianum.
....... . . • .23, 32
Catenomyces.
31
persioinus.
2, 32
Cladochytrium. . • •
.........
•• ,1, 2, 31
elegans. . . . .
. . 1
hy&linum. • • • • • • •
28, 32
replioatum......... 2, 18, 27, 28, 29, 32
. . . 33, 36 ,43, 47, 51, 52, 56
Endochytrium.............
33
operculatum........ 2, 18, 21, 23, 24, 25
27, 29, 35, 47, 51, 52
Megaohytrium Westonii.
.........
.32, 37
Monoblepharella Taylorii*
..........
26
Nephroohytrium.
..55
Nowakowskiella delioa. . . . . . . . . . . . . . 1, 21, 55
elegans*
1, 21
elongata
.1, 20, 21, 38
...........
1
endogena.
granulata. • •
• • • • • 1 , 21
hemisphaerospora. • . . . 1, 21, 29, 51, 57
macrospora. . . • • • • . • • • . 1 , 20, 21
Physocladia. . . . . .......... • • • • • • • • • . . 3 7
Polychytrium.
........... . . . . . . . . . . 2 0 , 23
stromaphilum. • • • • • • • • • • • • .
27
Polyphagus Euglenae. . . . . . . . . . .
2, 51
Rhizidiomyoes bivellatus......... • • • • ............ 18
Rhizophydium ooronum.
.................. . . . . . 2 , 23
Rhizopus. . . . . . . . . . . . . . ................. 37
SeptoohytriuiQ maorosporum
26
variabile. . . .
......
33
Siphonaria...........
.57
Solutoparies Pythii.
........... .
. . . . . . . .20
Sporophlyctis rostrata.
20

p. 62

ILLUSTRATIONS
(all except fig. 7, 9, and 22 approximately 25 microns / inch)
Fig. 1-13. Nowakowskiclla ramosa
Fig. 1.
Fig.

Fig.

Fig.
Fig.

Fig.
Fig.
Fig.

Fig.
Fig.

Fig.
Fig.
Fig.

Living zoospores: a-1, typicalspores; m-q, spores with more than
one globule, flagellum, and/or blepharopl&st
2* Stained zoospores: a-k, typical spores; 1-3, spores with more
than one nucleus, flagellum, rhi&oplast, and/or blepharoplast;
q, r, s, possibilies of structures in the event that two blepharoplasts are formed
3. Sessile spores (stained): a-g,typicalspores; h, binucleate
spore; i, two clear spaces left by oil globules; j, nucleus in
metaphase; k, two nucleoli; 1, three non-flagellate miniature
spores
4. Budding of amoeboid spores (stained): three spores which were
caught in the vessels of grass leaves in which germination
took place by budding
5. Germination of spores (stained) : a, typieal behavior showing
swelling in the branched germ-tube before the nucleus passes
from the spore-ease into the filament; d, e, and g, nuolei
dividing in the spore-cases; j, elongate daughter nucleus
passing through filament from spore-oase to primary swelling
6. Vegetative portion# rh, rhizoids; st, storage swelling; fl,
base of flexuous filaments
7. Habit sketch of thallus (about 240 microns/ inch) : S, sporan­
gium; R, resting-cell
8. Flexuous filaments (stained) : a and d, segments showing spacing
of rounded nuclei without swellings; o and e, nuclei dividing
in non-swollen filaments; f and g, binucleate swellings; b,
anucleate swelling
9. Zoosporangium shapes (about 150 microns/inch)
10. Development of zoosporangium (stained) : a-c, initiation of
swelling and multiplication of nuclei; d, nuolei aligned on
granular strands of cytoplasm and the pre-septum plate form­
ing; e, sporangium with mature walls in which cleavage by
+ vacuolation has begun; f, cleavage complete and granules mass­
ing about nuclei; g, cytoplasm swollen to obscure vacuoles and
the granular masses about the nuolei more compact
11. Mature zoosporangia: a, densely-stained material showing typi­
cal nuclear caps; b, material destained to show suggestions of
roticulo-granular constitution of nuclear caps
12. Zoospore escape (living); shows flagellum with terminal ves­
icle caught in exit orifioe and fully formed flagella within
the sporangium
13. Formation of the resting-oell: a, appressed swollen branches;
b, ”fused” branch tips; c, appressed branches* tips divided
into pseudoparenchyma; d, suggestion of nuclear division in
resting-cell; e, pseudoparenchyma bearing resting-cells; f,
resting-cell in late stage of maturation; g, mature resting­
cell

Fi

B. Germination of sp ores

P • $4

Fijr.

6.

V e j e t u t »v f t p o r

H abit

s k e tc h

of

thallus

r

Fig. 8. Flexuous filaments

Rg. 10* Development of zoosporangium

p • 6G

Fig- J-l . Mature

Fig.

13. Formation o-f restingcell

p. 67
Fig. 14-23 Nowakowskiella
Fig.
Fig.
Fig.
Fig.

14.
15.
16.
17.

Fig. 18.
Fig. 19.

Fig. 20.

Fig. 21.
Fig. 22.
Fig. 23.

Living zoospores: e, g, and h, spores assuming amoeboid habit
Stained zoospores: g and i, show signs of net-work in nucleus
terminating spores (stained)
Vegetative portion: a^and' b, drawings of living material in
cellophane; c and d, stained material (sp, spore-case; rh,
rhizoid; st, storage swelling; fl, base of flexuous filaments)
Flexuous filaments: a, living material showing pseudosepta;
b and o stained material
Development of the zoosporangium:(living material): a, inter­
calary; b# sub-terminal with shriveling distal filament; o,
on lateral branch showing mature wall and cleavage lines; d»
lateral and sessile (ss, sub-sporangial swelling)
Saturation of the zoosporangium (living) :a, a few large globules
with small globules massed centrally; b» small globules evenly
distributed leaving clear sub-opercular space; c, recoalescing
into large globules; d# completed coalescence and showing
cleavage lines
Zoospore escape (living); showing zoospores escaping by amoeb­
oid motion, held by caught flagella, and flagella bearing term­
inal loops
Zoosporangium shapes (about 60 microns/inch) : a, chain of
sporangia in which a filamentous isthmus occurs; b, "prolif­
erating” chain; d,and f, unevenly-shaped elongate forms
Zoosporogenesis (stained): a* granules massed in center; b,
nuclei aligned on granular strands of cytoplasm; c, cell show­
ing simultaneous but not completely synchronized mitosis; d,
late stage in the formation of nuclear caps; e, mature zoo­
spores in the sporangium

P.

Fig". 14.

Fig.,

Living

zoospores

16. Germi na.t i

Fig,\5. Stained zoospores

spores

Xy.»»
'V-*<v.
^ ^v/
<*>*o£
'*
* ;*
* ;•••
-“
U<i/— S*'*A *'V>

Fig.

17. V e g e t a t i v e p o rt i o n

68

F i g . 18 . Flexuous f i laments

o

o '.

F ig .

19* Developm ent o f zoosporangium

Fig. 20. M aturation of ^-oosporangium

o

Figr.

22-

Z o o sp o r^h g i'utn

shapes

F ig * 23. Zoospor ogehesis

p. 71

Literature Cited

1*

Ajello, L*

1942.

Polychytrium; a new oladoohytriaceous genus*

Mycologia, 34:442-451
2* Berdan, H, B,

1941a*

chytrids I*

A developmental study of three saprophytic

Cladoohytrium hyalinum, sp# nov, Amer* J* Bot.,

28:422-438
1941b.

A developmental study of three saprophytic

chytrids II*
4*

1942*
II*

Catenochytrium carolinianum* Ibid*, 28:901-911

A developmental study of three saprophytic chytrids

Septoohytrium variabile*

5* Butler, E* J*

1907*

Chytridiaceae.
6*

An account of the genus Pythium and some

Mem* Dept* Agr* India, Bot. Ser*, 1:1-160

Constantineanu, M* J. C*

1901*

mycologique de la Roumanie*
7* Cotner, P* B.

Ibid*, 29:260-270

Contributions a la flore

Revue Gen. Bot*, 13$369-389

1930.Cytological Study of the zoospores of

Blastooladia. Bot. Gaz., 89:295-309
8.

Couch, J. N.

1945.

Observations on the genus Catenaria*

Mycologia, 37:163-193
9# ____ and tthiffen, A.

J. 1942* Observations on the genus

Blastocladiella. Amer* J* Bot*, 29:582-591
10*

Dangeard, P* A*

1900-1901. Reeherehes sur la structure due

Polyphagus Euglenae Nowak, et sa reproduction sexuelle*
Le Botanists, 7:213-257
11. Domjan, A.

1936.

*Vizigombas*-Adatok Szeged es Tihany

Videic'erol (”Wasserpilzw-daten aus der Umbogung von Szeged
und Tihany).
Sparrow, 1943)

Folia cryptogam., 2:8-59 (Noted in review in

p. 72

12. Ellison, B. R.

1945.

Flagellar studies of zoospores on some

members of the Mycetozoa, Plasmodiophorales, and Chytridiales.
Mycologia, 37:444-459
13.

Hanson, A. M.

1945a.

A morphological, developmental, and cyto-

logieal study of four saprophytic chytrids X. Catenomyces
persicinus
14

. ______

Hanson. Amer. J. Bot., 32:431-438

1945b. A morphological, developmental, and cytological

study of four saprophytic chytrids II. Rhizophydium ooronum
Hans on.
15.

Ibid•, 32:479-438

Hillegas, A. B.

1940.

The cytology of Endoohytrium operculatum

(de Wildeman) Karling in relation to its development and
organization.
16.

Bull. Torrey Bot. Club, 67:1-32

Johannsen, D. A.

1941.

Book Company, Inc.
17.

Karling, J. S,

1932.

Plant Microtechnique.

New York
Studies in the Chytridiales VIII. the

organization of the ehybrid thallus.
18.

______

McGraw-Hill

Amer. J. Bot. 19:41-74

1937* The cytology of the Chytridiales with special

reference to Cladoohytrium replioatum. Mem. Torrey Bot. Club,
19:3-92
1938.

19.

A new chybrid genus;

Nephrochytrium. Amer. J.

Bot. 25:211-215
20

.

______ 1941. Cylindrochytrium .johnstonii gen. nov. et sp.
nov. and Nowakowskiel la profusum sp. nov Bull Torrey Bot,
Club, 68:381-387

21

. ______

1942. A new ohytrid with giant zoospores, Septochytrium

macrosporum, sp. nov. Amer. J. Bot., 29:616-622
22.

___ 1944a. Brazilian chytrids I. Species of Nowakowski ella.
Bull. Torrey Bot. Club, 71:374-389

p. 73

23.

1944b.

Brazilian chytrids IV".

Species of Rozella.

Mycologia, 36:638-647
24.

1945a. Brazilian chytrids V.

Nowakowskiella macrospora,

n. sp., and other polycentric chytrids.
25.

_ _ _ _ _ 1945b.

Brazilian chytrids VI.

Amer. J. Bot.

32:29-35

Rhopalophlyctis and

Chytripayeea, two new chitinophilio operculate genera.
Ibid., 32:362-369
26.

1945c.

Brazilian Chytrids VII.

Observations relative

to sexuality in two new species of Siphonarla.
27.

Matthews, V. D.
Pythium.

28.

1928.

Ibid., 32:580-587

Nowakowskiella and a new species of

J. Elisha Mitchell Sci. Soc., 42:229-232

______ 1937.

29 . Nabel, K.

A new genus of the Blastooladiaoeae. Ibid., 53:191-195
ii
1939. Uber die membrau niadorer Pilze, besonders von

Rhizidiomyoes bivellatus, nov. spez. Archiv. f. Mikrobiol.,
10&515-541
30.

Howakowski, L.

1876.

Beitrag zur Kenntnis der Chytridiaceen.

In Cohn, Beitr. Biol. Pflanzen, 2:92
31.

San Chiun Sen.

1944.

ciliated spores.
32.

Sohroeter, J.

A form of Spo rophlyot is roatrata with

Amer. J. Bot. 31:229-233

1893.

In Engler end Prantl, Naturlichen

Pflanzenfam. , 1:82
33.

Shanor, L.

1942a.

A new fungus belonging to the Cladochytriaceae.

Amer. J. Bot., 29:174-179
34.

1942b.

A new Monoblepharella from Mexico, Mycologia,

36:241-247
35.

Sparrow, F. K. Jr.

1932.

of Cold Spring Harbor.

Observations on the aquatic fungi
Mycologia, 24;268-303

p. 74

36*

1933.

Observations on operculate chytridiaceous

fungi collected in the vicinity of Ithaca, N. Y., Amer.
J. Bot., 20 j63-77
37.

___ 1943.

The Aquatic Phycomycetes*

University of

Michigan Press, Ann Arbor
38.

Springer, M. E.

1945.

Two new species of Monoblepharella.

Mycologia, 37:205-216
39.

Wager, H.

1913.

Life-history and cytology of Polyphagus

Euglenae. Ann. Bot., 27:173-202
40.

Whiffen, A. J.

1942.

Two new chytrid genera.

Mycologia,

34:543-557
41.

1943.

New species of Nowakowskiella and Blastocladia.

J. Elisha Mitchell Sci. Soc., 59:37-44
42.

1944.

A discussion of taxonomic criteria

Chytridiales.

Farlowia, 1:583-597

in the