[ 25 ]

Trans. Br, mycol. Soc. 53 (1),25-38 (1969)

Printed in Great Britain

THE LIFE-CYCLE OF STEMONITIS HERBATICA

By PATTADA U. INDlRA*
University Botany Laboratory, Madras, India

(With Plate 2 and 5 Text-figures)

The events from spore germination up to the formation of the mature

plasmodium in Stemonitis herbatica Peck are described in detail. Spore germina-
tion is by the pore method; the haploid phase is of the completely flagellate type,
undergoing· several successive divisions presumably by amitosis; the zygote is
formed by isogametic union and develops into the plasmodium. Light seems
to inhibit gametic union. A new method of food intake is described for the
swarmer,

Detailed studies of the life-cycle of individual species of myxomycetes

are relatively few in the literature, and they rarely include members of the
Stemonitales. Knowledge concerning this group, or the genus Stemonitis, is
gathered from studies on different aspects of the life-cycle in different
species, such as spore germination and early phases (Gilbert, 1928a-c;
Ross, 1957a; Benedict, 1962, 1963, 1964), the plasmodium (Alexopoulos,
1959; McManus, 1961) and sporangial morphogenesis (Bisby, 1914; Gray,
1937; Ross, 1957 b, 1960; Goodwin, 1961). This paucity of comprehensive
knowledge of the group has been due mainly to failure to grow the species
in vitro through their entire life-cycle, until Alexopoulos (1959) first
cultured Stemonitis flavogenita and opened up a promising field.
In the present study, a single species, Stemonitis herbatica Peck, which was
cultured in vitro from spore to spore (Indira & Kalyanasundaram, 196 3),
was studied in some detail. The stages leading to plasmodium formation
are described here. Details of sporangial morphogenesis will be published
later.
MATERIALS AND METHODS

Spores developed in culture, less than a month old, were used in the
studies. The original culture was grown from spores of a specimen collected
by the author (Herb. MUBL/PUI I). Sporangia were immersed in a few
drops of a 0'001 %aqueous solution of Tween 80 (polyoxythylene sorbitan
monoleate). After 5- 10 min, they were crushed out with dissecting needles,
the skeletal structures removed and the spore suspension diluted with the
desired amount of culture medium. One sporangium per ml of the medium
was commonly used.
Three media were used in these studies: sterilized tap water, distilled
water and 1-2 % carrot decoction. Portions or drops of the spore suspen-
sion were placed in (a) watch glasses, (b) cavity slides, (c) plain glass
* Present address: Pharmacology Laboratory, Indian Institute of Science, Bangalore-
12, India.
26 Transactions British Mycological Society
slides and (d) hanging drops over cavity slides, all incubated in moist
chambers.
Most of the microscopical observations were made of living cultures in
drops on slides; for observation under higher magnifications or oil
immersion, the drop was covered with a coverslip supported at the edges
by fragments of no. I coverslip. Tests with vital stains like neutral red,
methylene blue and Janus green showed that these did not greatly
enhance observational facility and hence they were not generally used.
Fixed and stained preparations were used mainly to study division and
conjugation of the swarmers. Two methods were used. The first was a
modification of Cotner's method: a drop of the culture fluid at the desired
stage was placed on a clean slide and fixed by inverting over a vial of 2 %
osmic acid for about 30 s. A very small drop of 0'02 % aqueous crystal
violet was added to this and stirred well. The drop was allowed to settle
for a few minutes, a coverslip was placed over it, and scaled with fingernail
varnish. Examination of this preparation had to be completed within 2
days as it was not permanent but the method was easy and convenient.
In the second method, the slides were dried overnight under cover after
osmic acid fixation, and then stained with Heidenhain's iron-alum
haematoxylin and mounted in balsam (Johansen, 1940).
For placing single spores, or a limited number of spores, for germination,
a very dilute spore suspension was observed through the microscope;
individual spores were picked out with a micropipette and transferred to a
drop of the culture medium on a slide.

SPORE GERMINATION

The spore of Stemonitis herbatica is purple-brown, spherical and minutely

spinulose (Text-fig. 1 A). Electron micrographs indicate that the spines are
scattered, and appear to have a layered structure. It is about 8 Jim diam,
with centrally situated nucleus. A few globular bodies are occasionally seen
in the cytoplasm, but generally it appears homogeneous (Text-fig. I B).
The time required for the first protoplast to emerge, from th e time of
placing the spores in water, depends mainly on the age of spores, and to
some extent on external factors. The time required for the various stages
of germination is also highly variable. The following account is based on a
spore which required 2 h for the complete emergence of the protoplast,
from the time of placing in water (PI. 2, figs. 1-10; Text-fig. I).
Within about an hour of being placed in water, the protoplasm becomes
heterogeneous, and several rounded, refractive bodies may be seen (Text-
fig. I C). One or two vacuoles may also appear. In neutral red, several
deeply stained bodies are seen, usually aggregated towards a side (Text-fig.
1 D). Presently, a small area of the spore wall becomes thin and very pale,
indicating softening (PI. 2, fig. I; Text-fig. 1 D). This area does not seem
to be predetermined, as the wall in ungerminated spores appears uniformly
thick and dark-coloured. The spore may remain in this state for a period of
up to 30 min, towards the latter half of which time active movement of
the protoplasmic granules begins, particularly near the pale area, where
the granules appear to be in a state of agitation.
 Stemonitis. Pattada U. Indira 27
The protoplast begins to swell, resulting in bulging of the softened area
(PI. 2, figs. 2, 3; Text-fig. I E). Continued pushing causes the wall to
rupture rather irregularly in the centre of this area. The protoplast begins
to s.queeze out this ?pening, the edges of which are jagged.
As It squeezes out It retracts Itself from the wall on the opposite side.
During this. process the spore may rotate slightly (PI. 2, figs. 4-6).
In about 15-20 min , the entire amoeboid protoplast has emerged (PI. 2,
figs. 7,8; T ext-fig. I F-H). Soon after emergence it shows strong move-
ments which ma y cause th e spore case, to which it is still attached, to

D E

10 Jlm

T ext-fig. I. Spore germination in Stemonitis herbatica. A, B, surface and sectional views;

C, after about 10 min in water; D, after about 25 min, showing softening of wall on
ri ght side; E, protoplast pushing against the wall on softened side; Y, emergence of
protoplast through ruptured wall ; G, surface view showing ruptured wall ; H,
quiescent protoplast after em ergence; I, j, protoplast becoming amoeboid and
releasing itself from the spore case.

rotate, but soon it becomes quiescent and rounds off to a diameter roughly
equal to that of the spore, about 8 p,m (PI. 2, fig. 9; Text-fig. I H).
It may remain in this state for about 15 min, after which it releases itself
and moves away from the spore case by slow amoeboid movements
(PI. 2, fig. 10; Text-fig. I I, J). The exact time of appearance of the
flagella is not known, but the y are seen within 15-30 min after emergence.
The amoeboid swarm cell (T ext-fig. 2A-C;) continues to move over the
surface of the slide or watch glass for 15-30 min, towards the end of which
it becomes linearly oriented and moves with a glidi.ng motion. Presently it
leaves the glass surface with a jerk, becomes comma-shaped and revolves
at great speed, finally swimming away with the characteristic wriggling-
rotating movement of the swarmer.
28 Transactions British Mycological Society

THE SW ARMI NG PHASE

The comma-shaped, actively swimming stage will, for the sake of

convenience, be referred to as a swarmer (Text-fig. 2D), to distinguish it
from th e flagellated, elongate-amoeboid stage, which will be referred to as
the swarmcell or elongated swarmcell (T ext-fig. 2F-H). A third stage, in

Text-fig. 2. Swarming phase of Stemonitis herbatica. A-C, am oeboid swarmcell soon after
germi nation; D, E, free-swimming swarmer ; F-H, elongated swarmcell, showing one
flagellum at F and G, and three flagella at H; I,], am oeboid swarmeell following
elongated swarmeell stage , showing pseudoflagellum (p) ; K-N, ingestion ofbaeterium
by the free-swimming swarme r; 0, P, amoeboid ingestion ofbaeterium by amoeboid
swarmeell (flagella no t shown ); Q-T, repeated ingestion by swarmcell at definite spot
near flagellar end .

which the swarmcell is not elongated but completely amoeboid, though

still ret aining its flagella, will be referred to as the amoeboid swarmcell
(Text-fig. 21-J). The term myxamoeba is generally used for a swarmcell
which has withdrawn its flagella and become amoeboid, but such a stage
was hardly ever seen in cultures of this species.
 Stemonitis. Pattada U. Indira 29
The medium in which spores are germinated is literally swarming with
swarmers by the end of 24 h. These are seen swimming through the medium,
and appear in large numbers at the surface. When a drop is placed on a
slide the swarmers tend to move towards the edge of the drop.
Under optimum conditions of temperature, depth of fluid, spore con-
centration, etc., the swarmers may remain active for longer than 48 h, but
generally in water cultures most of the swarmers turn into swarmcells by
the end of this period. By the end of 72 h, mostly spherical cysts and a few
amoeboid swarmcells are seen, and even the latter eventually encyst.
The activity of the swarmers or swarmcells is maintained for several days,
and further development takes place, only in a nutrient medium such as
1-2 % carrot decoction agar, and not in water.

Structure, from living cultures

The swarmer is typically inverted comma-shaped, being broad at the
posterior end and narrow at the anterior end, about 15 p,m in length and
5-6 p,m in thickness. At the narrow anterior end are situated two flagella,
one of which appears longer than the body of the swarmer and is regularly
seen (PI. 2, fig. II). The other flagellum, only slightly shorter than
the first, is not always readily seen. At the posterior end is often seen a
characteristic slender, curved tail-like projection. One or two vacuoles
may be seen at the posterior end (Text-fig. 2 D). Further details cannot be
seen in living swarmers because of the active movement but can be clearly
seen in the swarmcell.
The swarmcell is generally elongate pyriform, but the shape is main-
tained only at the anterior end. The posterior end is constantly changing,
with numerous slender pseudopods being constantly formed (Text-fig.
2 F-H). These are often filiform, resembling flagella but distinguished from
them by the absence of any curvature or lashing movements, and may be
called pseudoflagella (Cohen, 1959). They are seen more often in amoeboid
swarmcells (Text-fig. 2 I). The two flagella are clearly seen at the anterior
end, and although not actively used in locomotion they are constantly
lashing. Sometimes a third flagellum is also seen close to these (Text-fig.
2H).
The protoplasm of the swarmcell is differentiated into a clear, trans-
parent ectoplasm, which is firm at the anterior end but plastic at the
posterior, and finely granular endoplasm. The nucleus is situated at the
narrow anterior end, almost filling up the diameter at this end so that only
a thin layer of cytoplasm surrounds it, and, following the contour of the
body, it is usually elongate-ovoid, though occasionally spherical. The
flagella are connected to the nucleus bya broadlyconical,hyaline structure.
A nucleolus is present in the centre of the nucleus. The endoplasm contains
several large granules. A highly refractive, crystalline body is occasionally
seen. One to four vacuoles are usually seen at the posterior end, and some
of these may be food vacuoles, enclosing bacteria, yeast cells, etc.
The amoeboid swarmcell is of indefinite shape, constantly changing
(PI. 2, figs. 12, 13). The nucleus is usually seen at some point near the
surface, with the flagella attached and constantly lashing.
 Transactions British Mycological Society

Structure, fromfixed andstained preparations

In fixed and stained preparations of swarmers and swarmcells, a single
flagellum is seen in most cases, being in line with the body and held away
from it, the second one being obscured because it is irregularly and
variously oriented. The length of the flagellum varies, 12-17-24 pm,
usually exceeding the length of the body which is 10-15 flm, the diameter
being 5-8 flm. Granules of varying size are seen in the protoplasm but the
region immediately surrounding the nucleus is relatively free of them.
The nucleus when spherical is 4-5 flm diam and, when elongated, 6-7 flm
in length. A definite nuclear membrane is evident. The nucleolus is
central, about I pm diam. The rhizoplast and structures associated with
the flagella are seen as a triangular cap over the nucleus. Two basal
granules can sometimes be distinguished.
In 48-h-old cultures, many of the swarmcells are seen to have increased
considerably in size, and this is particularly evident in fixed and stained
preparations. These swarmcells are 16 flm or more in length and 8-10 flm
diam, The nuclei are larger, being a little over 5 flm diam, and more
spherical than elongated. The nucleolus has enlarged to over twice the
original diameter, being a little over 2 flm, and stains very deeply.

Locomotion
Movement of the swarmer is very fast, and fascinating to watch. It
moves with the flagellar end forward, rotating around its own axis either
clockwise or anticlockwise and traversing a fairly wide circle in this
process. The actual course of movement, however, appears to be in straight
lines and not spirals. The posterior tail-like projection sways to and fro
during these movements. Often the swarmer is seen to pause in its course
and change direction.
The swarmcell moves with a characteristic gliding, worm-like move-
ment in which unidirectional protoplasmic flow is involved. The swarmcell
attaches itself to the substratum by the posterior end and elongates
considerably, becoming correspondingly narrower. The anterior end is
then held on the substratum, and the posterior end released and drawn
forward by protoplasmic flow. The flagellar end is always held forward.
The flagella are lashing continuously in all directions and are apparently
sensory in function.
Truly amoeboid movement is displayed by the amoeboid swarmcell and
sometimes by the elongated swarm cell also. During such movements, the
entire ectoplasm, with the nucleus and flagella, seems to be very plastic,
and able to glide over the endoplasm, in any direction. The entire organelle,
including nucleus and flagella, is seen to change its position in the body
with remarkable alacrity, enabling the swarmcell to change direction.
The direction seems to be determined by the flagellar end, which glides
over and projects in that direction, followed by the rest of the protoplasm.
Thus in this movement also, the flagellar end is held forward.
 Stemonitis. Pattada U. Indira 31
Nutrition
A very peculiar mode of feeding, hitherto not reported in myxomycetes,
has been observed in the swarmer. In the course of its swimming move-
ments the swarmer occasionally pauses near a bacterial colony. There is
next a vigorous lashing movement of the flagella, which seem to help not
only in the detection of the bacteria but also in sweeping them down close
to the body. When a bacterium has reached the curve of the comma just
below the flagellar end, the swarmer bends over, trapping the bacterium
in the bend, and revolves round and round at a rapid rate. After a while it
stops rotating and relaxes, and then the bacterium is seen within the body,
enclosed in a food vacuole. Thus food seems to be ingested not by means of
pseudopodia but by movements of the swarmer as a whole, and at a
definite region of the body (Text-fig. 2 K-N).
In the swarmcell, food particles are taken in by pseudopodial ingestion.
The pseudopodia which surround the food particle are very slender and
the process of ingestion very rapid (Text-fig. 2 0, P). Occasionally,
pseudopodia are seen to form and close over as if ingesting food, repeatedly
in quick succession at a definite region slightly below the flagellar end
(Text-fig. 2Q-T). No solid particle was seen to be taken in during this
process. The amoeboid swarmcell has often been seen trying to ingest spore
cases or cysts, and then giving up and moving away, as these structures are
too large to permit ingestion.
Excretion
The number of vacuoles in the swarmcells is variable, and constantly
changing. Usually, the comma-shaped swarmer contains only one or two
vacuoles whereas the swarmcell may enclose as many as four or five.
Regular pulsating movements of the vacuoles have not been observed but
the dimensions of individual vacuoles are seen to change, and coalescence
of small vacuoles has been noticed. Occasionally, a vacuole enlarges
considerably, moves towards the surface and then suddenly diminishes and
disappears, perhaps emptying its contents to the exterior. In some cases an
entire vacuole may be thrown out of the body.

Division
That division of swarmcells does occur within 24 h was established by
the following experiment. A very dilute spore suspension was prepared,
and small numbers of spores transferred to drops of nutrient solution on
glass slides. The number of spores in each drop was counted, and the
slides incubated in moist chambers. After 24 h, the slides were removed
and the swarmcells counted. The results (Table I) indicate that more than
one division, and perhaps as many as three, may take place within 24 h, at
least in some of the swarmers.
In living cultures, division could be observed in about 4-5 h after
placing the spores for germination. The culture at this stage consists
largely of swarmcells which are assuming fantastic shapes. The process is as
follows: the body of the swarmcell elongates, and constantly changes
 Transactions British Mycological Society
Table I. Counts ofswarmers following spore germination in
Stemonitis herbatica Peck
Original no. No. of swarmcells
Slide no. of spor es at end of 24 h
1 5 23
2 5 12
3 16 77
4 10 14
5 27 23
6 24 47
7 21 52
Total 108 248

A B

F H

(:/:\) L-.J
10 ,u m

Text-fig. 3. Division of swarmcell in Stemonitis herbatica. A-H, living cultures: A, appear-

ance of constriction in the middle of the cell; B-D, daughter protoplasts pulling apart;
E-G, daughter protoplasts drawing together again (process repeated several times);
H, eventual separation of daughter protoplasts. I-N, fixed and stained preparations:
I, swarm-cell with enlarged nucleus; J, nucleus with nucleolus divided into two;
K, nucleus constricting in the middle; L, nucleus after division, the daughter nuclei
still interconnected, flagella have already appeared at the opposite pole ; M, daughter
nuclei separated and the protoplast constricting in the middle; N, daughter protoplasts
separated.

shape. A constriction appears in the middle, causing the bulk of the proto-
plasm to shift to the two ends. Finally, two protoplasmic masses are seen
connected by a thin strand. These masses are pulled apart and brought
together in succession several times. Eventually the connecting strand
breaks in the middle and the daughter cells move apart (Text-fig. gA-H).
 Stemonitis. Pattada U. Indira 33
In fixed and stained preparations, no mitotic figures could be seen but
there was some indication of amitotic division. In some cells, the nucleus
was elongated, and two nucleoli were often seen. In some cells the nucleus
was slightly constricted midway between the two nucleoli while in others
the constriction was deeper (Text-fig. 3 J-L). A few cells showed two
nuclei in close proximity, while in others the two nuclei were at opposite
poles, the body of the cell being constricted in the middle. Two flagella
were sometimes observed, at opposite ends of the swarm cell.

Encystment
Some or all swarmcells in a culture usually encyst after varying periods
of activity depending upon the conditions. Prior to encystment, the
flagella are withdrawn, and the swarmcell revolves, eventually becoming
spherical and then quiescent. While encystment can to some extent be
prevented by the regular addition of fresh nutrient medium, revival of the
cysts has not been observed.

SYNGAMY AND PLASMODIUM FORMATION

Syngamy
The presence in 48-h-old cultures in nutrient medium of zygotes and
young plasmodia indicates the possible occurrence of syngamy within that
period. In living cultures, however, the process could not be observed, as
the fusing gametes appeared to be highly photophobic. Frequently,
swarmcells were seen attached in pairs by their posterior ends or some-
times laterally, but they separated while under observation, in spite of the
use of a blue filter.
Cultures were therefore incubated in the dark. After 40 h, drops of the
culture fluid were transferred at intervals to microscope slides, fixed and
stained. Fusing swarmcells, zygotes as well as young plasmodia, could be
distinguished in the preparations at different stages (PI. 2, figs. 16-23;
Text-fig. 4).
Gametes were seen to be attached in pairs in several cases and these
were identical with the enlarged swarmcell, with a large body and large
nucleus. One or both gametes were seen to be flagellated in most cases.
They were attached by their posterior ends, their sides, or sometimes end to
end (PI. 2,figs. 16, 17; Text-fig. 4A-C). In some cases plasmogamy had been
completed and the two nuclei were in close promixity, sometimes actually
fusing (PI. 2, figs. 17, 18; Text-fig. 4D-H). Traces of flagella were still
seen in some cases where the nuclei were fusing. In a few cases, karyogamy
had been completed, and a zygote which had withdrawn the flagella and
assumed a spherical shape could be found (PI. 2, fig. 19; Text-fig. 4 I).

The {Ygote andyoungplasmodium

In living cultures, the newly formed zygote can be distinguished from
unfused amoeboid swarmcells by its larger sizes, abence of flagella and
frequent presence of ingested cysts or swarmcells in food vacuoles. In
3 MyC.53
34 Transactions British Mycological Society
stained preparations, it appears spherical, up to 20 usn diam with a large,
centrally situated nucleus having a deeply staining nucleolus (PI. 2,
fig. 19). Sometimes, there is some indication of coalescence between two
zygotes or between zygote and swarmcells (PI. 2, fig. 23).

10 pm
L-.J

G H

Text-fig. 4. Syngamy in Stemonitis herbatica, from fixed and crystal-violet stained

preparations. A, swarrncells (gametes) attached laterally, showing large nucleus with
enlarged nucleolus; B, gametes attached posteriorly; C, posterior end of one gamete
attached laterally to the other; D, E, plasmogamy completed, nuclei approaching each
other; F, contact of nuclei, with nuclear membrane breaking down at the point of
contact; G, karyogamy, structure at right a food vacuole; H, zygote, still retaining
flagella; I, zygote after shedding flagella and becoming spherical ; J, young, binucleate
plasmodium.

The zygote grows and enlarges rapidly, and here it may be called the
young plasmodium. As it enlarges, it puts forward numerous long, finger-
shaped pseudopods all over, which swing about and constantly change
shape. Soon the young plasmodium begins to ingest cysts and unfused
swarmcells. When a swarmcell is near, a filiform pseudopod is put forward,
which attaches itself to the swarmcell and then retracts, drawing the
swarmcell close to the body, where more pseudopods close over the
swarmcell and engulf it, forming a food vacuole (Text-fig. SA-D).
Numerous vacuoles are seen in the protoplasm besides the food vacuoles.
Sometimes, undigested swarmcells are egested (Text-fig. SE-G).
Side by side with enlargement, the nucleus seems to divide, and young
plasmodia with 2, 4, 16 and 32 nuclei have been found in a single prepara-
tion (PI. 2, figs. 21, 22).
Sometimes the young plasmodium may remain irregularly lobed until it
 Stemonitis. Pattada U. Indira 35
attains considerable size (PI. 2, fig. 24), but usually, a linear orientation
is established at an early stage, and protoplasmic streaming begins (PI. 2,
figs. 25, 26). The linear plasmodium elongates in both directions and
branches; the branches coalesce and give rise to the characteristic reticulum.
As the plasmodium grows and spreads out, it may contact an adjacent
plasmodium, in which case its branches coalesce with those of the latter.
This process may continue until finally a single extensive plasmodium is
seen in the culture. Under unfavourable conditions such as lack of aeration
or nutrients, however, the plasmodium may encyst, by withdrawing its
veins into a few compactly aggregated strands which are irregularly lobed
(PI. 2, fig. 27).
At this early stage, the young plasmodium has already begun to form
swarmers in the manner described previously (Indira, 1964). The structure
and activities of the mature plasmodium will be described elsewhere.

Text-fig. 5. Ingestion and egestion of swarmcells by young plasmodia of Stemonitis

herbatica. A, pseudopod from plasmodial veins extending towards swarmcell; B, pseu-
dopod making contact with swarmcell; C, pseudopod retracting with swarmcell
attached, and growing round the latter; D, pseudopod with engulfed swarmcell; E,
ingested swarmcell in a vein; F, swarmcell coming to the surface; G, swarmcell egested.

DISCUSSION

The method of spore germination in Stemonitis herbatica falls into one of

the two categories described by Gilbert (1928 b), i.e. by the dissolution of a
pore in the spore wall. While Gilbert suggests that the wall is softened and
weakened in the region of opening, he does not describe any visible
indication of such a weakening. Koevenig (1964) shows a slight thinning
of the wall at the point of germination in germinating spores of Physarum
gvrosumin electron micrographs, stating, however, that no thinning isvisible
through the light microscope. But in the present study, a change in colour
and consistency of the wall is clearly seen prior to the formation of an
opening. There seems to be an enzymic action bringing about a softening
and weakening, but not a complete dissolution, of a region of the wall,
the pore developing due to the pressure exerted by the protoplast within.
3-2
36 Transactions British Mycological Society
Gilbert (1928 b) and Smart (1937) have emphasized that spore germination
in myxomycetes is not a simple osmotic process but involves enzymic action.
In the present case, germination is independent of any external source of
energy, occurring as readily in distilled water as in nutrient medium.
Comma-shaped swarmcells, here referred to as swarmers, as well as the
posteriorly amoeboid swarmcells, are known to be of common occurrence
in the myxomycetes. The different types of movement described here have
been seen and reported in several species (Lister, 191 I; Gilbert, 1928a;
Smart, 1938). The constant lashing movements of the flagella, which are
especially marked when bacteria are present around the swarmcell,
sweeping them close to the body, suggest that the flagella, at least in this
species, are sensory in function, serving not only to detect the presence of
food particles but also mechanically assisting in the process of ingestion.
Such a function has not been described for the flagella in myxomycetes,
though Smart (1938), describing the creeping movement in swarmcells,
states that the swarmcell uses the flagellum as if it were an antenna.
It is generally believed that, during the free-swimming period, food is
absorbed in solution, and ingestion of solid particles occurs only during the
creeping stage (Gilbert, 1928c). This is in contrast to what is seen in
S. herbatica. In fact, the method of ingestion described here for the free-
swimming swarmer was previously unknown in myxomycetes. It is
reminiscent of certain flagellates, and may be of phylogenetic significance.
The number of flagella in the myxomycetes is a rather controversial
question. While the biflagellate condition is commonly reported, uni-
flagellate swarmers are also seen (Cohen, 1959; Olive, 1961; McManus,
196I). In the present study it was found that while the longer flagellum
was consistent, the shorter one might occasionally disappear and reappear.
A third flagellum may also sometimes appear. Apart from this, pseudo-
flagella also are of common occurrence, but they eventually disappear and
cannot be mistaken for true flagella.
The ultimate conversion of swarmcells into myxamoebae by a perma-
nent withdrawal of the flagella has been described in several species. But
such a phenomenon was not observed in S. herbatica, where the swarmers
retain their flagella until they form zygotes or encyst. There is some
indication that the flagella are retained during division, as well as fusion,
of swarmcells, being withdrawn only at the time of, or after, nuclear fusion.
In this regard the species fits into the' completely flagellate type' of Ross
(1957a), as stated by Ross himself. However, contrary to the statement of
Ross that plasmodial formation does not begin earlier than 3-8 days and
that the coalescence of zygotes does not occur, plasmodium formation has
been observed within 48 h of germination, and coalescence of zygotes has
been seen.
The division of swarmcells is generally considered to be mitotic. There
seem to be no reports of amitotic division of the swarmcell. While failure to
observe mitotic figures in the present study does not indicate the absence of
mitosis, it seems possible that amitosis may be of frequent occurrence in
this species.
The photophobic nature of fusing gametes has been indicated by
Benedict (1963) for S. fusca, in which species McManus (1961) also
 Stemonitis. Pattada U. Indira 37
mentions her inability to observe gametic union in living cultures.
However, the repeated observation of union in living cultures by other
workers in other species indicates that this photophobic phenomenon is
not universal but restricted to certain species, and may be common in the
genus Stemonitis.
Reports of gametic fusion in living cultures have often been subject
to criticism as a misinterpretation of the division of swarmcells (Luyet,
1950). In fixed and stained preparations of S. herbatica, it is impossible to
mistake a dividing swarmcell for a stage in syngamy. The cell, nucleus
and nucleolus are considerably smaller in a dividing swarmcell than in
fusing gametes, and the orientation in the daughter cells is always linear.
In living cultures, gametes which were seen attached in pairs, were
mostly posteriorly and sometimes laterally attached. In fixed and stained
preparations they were variously attached, posteriorly, laterally or end to
end. This again is at variance from the observations of Ross (1957a), who
states that the attachment in this species is always posterior.
The presence of nutrients seems to be necessary for the completion of
syngamy and plasmodium formation, though not for germination. A
similar statement was made by Wilson & Cadman (1928) with regard to
Reticularia lycoperdon.
Linear orientation at an early stage seems to be characteristic of the
stemonitoid plasmodium, as it has also been described by Alexopoulos
(1959) for S.Jlavogenita and by McManus (1961) and Benedict (1964) for
Si fusca.
This work formed part of a doctoral thesis, University of Madras, 1966
and is published as Memoir no. 49 from the Centre of Advanced Study
in Botany, Madras University. I am very grateful to Prof. T. S. Sadasivan,
Director, University Botany Laboratory, for his encouragement; to Dr R.
Kalyanasundaram, Reader in Botany, for guidance, and for critically going
through the manuscript; and to the University of Madras and the Indian
Council of Scientific and Industrial Research, for the award of scholarships.

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EXPLANATION OF PLATE 2
Stemonitis herbatica
Figs. 1-10. Stages in spore germination. Time-lag photographs of a single spore. x 400.
Figs. 11-13. Fixed and stained preparations of swarmers and swarmcells. x 400.
Fig. II. Young swarmer with two flagella.
Fig. 12. Young amoeboid swarmcell,
Fig. Ig. Enlarged amoeboid swarmcell. The dark body is a spore coat.
Fig. 14. Dividing swarmcell, flagella at either end. x goo.
Fig. 15. Dividing swarmcell with two nuclei. x 300.
Figs. 16-23. Syngamy and plasmodium formation, from fixed and stained preparations. x 400.
Fig. 16. Paired gametes.
Figs. 17, 18. Plasmogamy. Note closely placed nuclei at one end in fig. 18 and a food vacuole at
the other end.
Fig. 19. Spherical zygote. Note nucleus, with dark, spherical nucleolus, on one side, and a
food vacuole on the other.
Fig. 20. Amoeboid zygote.
Fig. 21. Zygote after first division of the nucleus.
Fig. 22. Zygote after two divisions of the nucleus.
Fig. 2g. Coalescence of two zygotes.
Fig. 24. Developing plasmodia from living culture. Note irregular lobing, and large number of
food vacuoles. x 125.
Figs. 25. 26. Linear elongation, from living cultures. x 125.
Fig. 27. Encysting young plasmodium, unstained. x 80.

(Accepted for publication 20 January 1969)

 Trans. Br. mycol. Soc. Vol. 53. Plat e 2

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(Facing p. 38 )