Experimental Studies on Megaspore Viability, Parthenogenesis, and Sporophyte Formation in

Marsilea, Pilularia, and Regnellidium

Author(s): Paul G. Mahlberg and Margaret Baldwin
Source: Botanical Gazette, Vol. 136, No. 3 (Sep., 1975), pp. 269-273
Published by: The University of Chicago Press
Stable URL: http://www.jstor.org/stable/2473611
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BOT.GAZ.136(3):269-273. 1975.
(D 197Sby The Universityof Chicago.All rights reserved.

EXPERIMENTAL STUDIES ON MEGASPORE VIABILITY, PARTHENOGENESIS, AND

SPOROPHYTE FORMATION IN MARSILEA, PILULARIA, AND REGNELLIDIUM

ANDMARGARET
PAULG. MAHLBERG BA1>DWIN
Departmentof Plant Sciences,IndianaUniversity,Bloomington,Indiana47401

ABSTRACT
Megasporesof Marsilea, Pilularia, and Regnellidium possess an averageviability of 50%,-75%0 when
grownin the presenceor absenceof sporocarpcontents. Approximately96% of the viable megasporesin
Regnellidium formedsporophytesuhen grownin the presenceof microspores.In Marsilea and Pilularia
in contrast sporophytesformedon 58% and 38CXo respectively of the viable megaspores.Factorsinfluenc-
ing fertilizationremainunknown but it was determinedfor Marsilea that archegoniumreceptivity to
fertilizationprogressivelydecreasedwhen archegoniawere not fertilized within approximately12-24 h
and no fertilizationoccurredafter 24 h. The appearanceof sporophyteson nearlyall isolatedmegagameto-
phytes of Regnellidilem indicated the occurrenceof parthenogenesisrather than apogamy. In contrast
this phenomenonM as absentfor isolatedmegasporesof Pilularia and for nearlyall megasporesof Marsilea.
Parthenogenesisappearsto performa majorrole in the reproductionof Regnellidium but not in Marsilea
and Pillularia.

Introduction Material and methods

Heterosporousferns in which both megasporesand
microspores are easilSrseparated prior to gameto-
ph) te development can be emplow-edto advantage in
experimental studies of both gametophyte and
sporophvte formation. SHAW(1897) and NATHAN-
SOHN(1900) in studies on Marsilea(!rummondii, M.
macra,and M. vestitareportedthe formationof sporo-
phytes among isolated megaspores.Later STRASBUR
GER(1907) reportedapogamy in M. (Srummondii and
from developmental studies maintained that the
apogamous sporophytes were derived from diploid
megaspores that developed among haploid mega-
spores within a sporocarp.More recentl) BHARDWA-
JA and ABDULLAH (1972) reported parthenogenesis
in five of eight examined species of Marsilea.
Whereasfew studies on apogamy or parthenogene-
sis have been pursued on Marsileaceae, several
developmental investigations have been performed
on differentiation of spores, gametophxrtes, and
embrxos in Marsilea(MARSCHALL 1923; SCHULTZ
1936; FELLER19D3; BOTERBERG 1956; DEMALSY-
FELLERl957; RICEand LAETSCH 1967), Pilularia
(SCHULTZ 1936), and Regnelli(Sium (CHRYSLER and
JOHNSON1939; HIGINBOT9-IAM 1941). ATKINSON
(1943) examined the process of fertilization for
megasporesin Marsilea,and more recentlSrMACHLIS
and RAWITSCHER-KUNKEL (1967) described the or-
ganization of the enclosing tissues of Marsilea
megaspores.
In this study a comparative analysis is made of
megasporesfrom species of Marsilea,Pilularia,and
Regnellielium to evaluate megaspore viabilitw=,the
occurrenceof parthenogenesis or apogamy, and the
frequencsYof sporophyte formation among popula-
tions of megasporesgrown in the presence or absence
of microspores.
269
Sporocarpsof Marsilea testita H. and (;., Pilulatia
americana A. Br., and Regnellidium (liphyllum
Lindm. were surface sterilized for 20 min in 3<JO
potassium hypochlorite and rinsed in three changes
of sterile distilled water. Initially all experiments
were performed aseptically,, but subsequent data
showed this procedure unnecessary. A surface-
sterilized sporocarp was cut in half and placed in a
sterile plastic petri dish (100 X 15 mm) contain-
ing 20 ml of distilled water (pH 7). Each dish
contained onln one sporocarp or group of isolated
megaspores derived from a sporocarp. In one set of
experiments WHITE'S(1943) mineral salt solution
was substituted for water.
Megaspores were isolated, usually 40-SO, or 25 in
some experiments, under a stereomicroscopewith a
micropipet. They were freed from microspores by
transferring them sequentially through several
changes of distilled water prior to a final transfer to
the experimental condition in plastic petri dishes.
The original dishes containing the remaining mega-
spores and microspores served as controls. Each
experiment consisted of three plates for each condi-
tion being examined.
Cultures were grown in a culture room at 2z +
1 C under a 12 h light/12 h dark cycle and illumi-
nated at t50-650 lx with Sylvania Gro-luxfluorescent
lamps.
Data were recorded at the end of a 6-day culture
period for Marsilea and Pilularia. This interval was
selected during a series of initial studies on these
genera in which the number of developed mega-
gametophytes and sporophytes was recorded over a
14-day period; from this test it was determined that
all megagametophytes and sporophytes that de-
veloped in a culture were detectable after 3 da!js; a
4321 ......
...... 486
530
542
560 77.8
65.8
87.2
74.3
270
period twice this length of time was chosen as a
recordingperiod. RegnelliFliumrespondedmore slow-
ly and was evaluated at the end of 10 days.
Data accumulatedfrom these experimentsincluded
the numberof megasporesthat formedgametophytes,
here termed "viable megaspores," and the number
of sporophytes formed on megagametophytes under
differenttest conditions. Data were comparedamong
experiments on each genus and among genera to
interpret their significance and to identify trends in
megagametoph)te development and sporopht,-tefor-
mation.
Results
Regnelli(lium(liphyllum.-Regnelli(liumsporocarps,
which are spherical in form and approximately 6.1
mm in diameter, contained an average of 216
megaspores. These megaspores, round in shape and
averaging 660 ,um in diameter, possessed a similar
viability whether grown in the presence (76.2%6)or
absence (70.6So) of microspores (table 1). Mega-
gametophytes were evident on megaspores after the
spores were in culture for 36 h and were identifiable
by the development of a green cushion of cells with
numerous rhizoids at the papillate end of the spore.
The size of the cushion with its many rhizoids was
similar for most gametophytes. However, a few
megagametophytes developed small cushions pos-
sessing very few, sometimes only one, rhizoid. These
megagametophytes did not produce sporophytes.
Nonviable megaspores, although they appeared
similar to others, developed neither a cushion nor
rhizoids.No morphologicaldifferencesthat could ex-
plain the variation in spore viability were detected
among megaspores as they emerged initially from
the sporocarp.
Sporophytes became evident on the megagameto-
phytes after 5 days in culture and were identified bwr
the appearanceof the first leaf and, after 6 days, bSr
the appearance of the first root from the surface of
the cushion. The percentage of sporophyte formation
TABLE 1
MEGAGAMETOPHYTE AND
REGNELLIDIUM DIPHYLLUM
THE PRESENCE OR ABSENCE
MEGASPORES AND MICRO SPORES
EXPERI- Total (FO
MENT mega- gameto-
NO. spores phytes
Average 529 76.2
+ 8.3 + 9.4
72.5
59.9
86.5
74.3 120
130
120 70.0
66.3
80.0
65.6 65.8
57.1
80.0
65.6
BOTANICALGAZETTE [SEPTE MBER
closely paralleled megagametophyte development in
each experiment and averaged 73.3%0when grown
in the presence of microspores (table 1) . Each
sporophyte possessed a single leaf and root, approxi-
mately 5 and 3 mm long, respectively, when recorded
at the end of the 10-day culture period.
The small number of megagametophytes that
remained unfertilized appeared green and healthy.
Archegonia, although evident on the cushions
apparently were nonfunctional. These gametophytes
continued to remain unfertilized during an observa-
tion period of several weeks, after which time they
became brown and subsequently degenerated.
RegnelliFliummegaspores, when grown in the
absence of microspores,showed a viability similar to
the control. An average of 70.6%omegagametophytes
were developed (table 1). These gametophytes were
identical in appearance to the controls grown in the
presence of microgametophytes and showed no
physical damage from the transfer procedures em-
ployed in these studies.
Sporophytes formed on the megagametophytes in
the absence of microgametophytes.The average per-
centage of sporophyte formation was 67.1%^,which
was significantly similar to that (73.3%0)of the
controls (table 1). It appears, therefore, that
sporophytes formed parthenogenetically or apoga-
mously in Regnellidium.
The percentage of sporophytes formed on gameto-
phytes in these experiments was 95(6-96%owhether
the gametophytes were grown in the presence or
absence of microsporesand indicates that nearly all
viable megasporeswere able to develop sporophytes.
Pilularia americana. The small and spherical
sporocarps of Pilularia, which averaged 2.4 mm in
diameter, contained an average of 94 megaspores.
The percentage of megagametophytes formed on
these spores in the presence (68.5%6)of microspores
was similar to that found among megasporesisolated
(60.5%6)from microspores (table 2). Megagameto-
phyte cushions of chlorophylloustissue were evident
at the papillate end of the small oblong megaspores
(390 X 350 ,um)after a culture period of 48 h. As in
SPOROPHYTE F ORMATION OF Regnellidium,the cushion development was variable
MEGASPORES GROWN IN amongdifferentmegagametophytesin the population.
OF MICROSPORES Sporophytes formed on only slightly more than
one-half (58¢7o)of the total megaspore population
ISOLATED MEGASPORES grown in the presence of microspores, while for
megaspores isolated from microspores, no sporo-
NO Total NO NO
sporo- mega- gameto- sporo-
phytes were formed (table 2). Sporophyte formation
phytes spores phytes phytes resulted from egg fertilization in our strain of
Pilularia.
Marsilea testita.- Marsilea sporocarps contained
approximately 122 oblong megaspores (700 X 500
,um) which possessed an average viability of 59.5%0
73.3 122 70.6 67.1
in the presence and 52.3(Join the absence of micro-
+ 6.8 + 8.4 spores (table 3). These values were relatively high
compared with the viability observed from M.
432431 ......
....
.... 211
225
199
302
316 73.8
67.9
53.4
79.0
50.8¢
65.3 73.4
54.0
40.6
64.0
44.9
26.9 7575
75 52.0
61.3
56.0
73.0 () 0
75 49.3
48.0 0 36
024
12 ..........
..... 150
150 150 49.9+4.0
49.3_1.3
48.0+4.0
45.3_5.3 37.9+2.0
25.9+1.7 0

1975] MAHLBERG& BALDWIN- MARSILEA,PILULARIA,REGNELLIDIUM

271
TABLE 2 percentage of sporophytes were formed (2%6-5%6),
MEGAGAMETOPHYTE AND SPOROPHYTE FORMATION OF suggesting a very low level of parthenogenesis or
apogamy ln thls specles.
. . .

PILULARIA AMERICANA MEGASPORES GROWN IN THE

PRESENCE OR ABSENCE OF MICROSPORES The less than lOOSoviability recorded for sporo-
phyte formation among an apparently normal popu-
MEGASPORESAND MICROSPORES ISOLATEDMEGA
SPORES lation of megagametophytes grown in the presence
of numerous functioning microgametophytes sug-
EXPERI- Total °,X0 °,X0 T otal °J0 °,X0
MENT mega- gameto- sporo- mega- gameto- sporo-
gests that anomalies may exist among megagameto-
NO. spores phytes phytes spores phytes phytes phvtes. A time-course study on a spore population
was performed to determine whether archegonia
became receptive to spermatozoidsat different times
during the megaspore incubation period. To test this
point, we added microspores to isolated megaspores
Average208 68.5 58.0 75 60.5 0 at different intervals following their initial isolation
+ 9.7 +12.1 + 8.3 0 in culture. Freshly released microsporeswere added
to cultures of isolated megaspores or developing
megagametophyte cultures at intervals of 0, 12, 24,
and 36 h. The controls consisted of sporocarpsfrom
TABLE 3 which spores were not separated. The percentage of
MEGAGAMETOPHYTE AND SPOROPHYTE FORMATION OF megagametophyte formation at all time intervals in
MARSILEA VESTITA MEGASPORES GROWN IN THE these experiments was similar and averaged between
PRESENCE OR ABSENCE OF MICROSPORES 45';X0and 55S0 (table 4). The percentage of sporo-
phyte formation in cultures where microsporeswere
MEGASPORESAND 1WICROSPOXES ISOLATEDMEGASPORFS added at time O (37.9(?Jo) was similar to that of the
control (40.7570),whereas when fresh microspores
F,XPERI- Total °50 °J0 Total % Sj
MENT mega- gameto- sporo- mega- gameto- sporo-
were added to 12-h cultures, the percentage of
NO. spores phytes phytes spores phytes phytes sporophytes formed decreasedsignificantly to 25.9{7o
(table 4). After 24 h in culture, the megagameto-
1 ...... 282 58.5 't0. 6 75 60.0 0
2 ...... 268 63 . 7 41.9 75 52.0 0
phytes did not respond to additions of microspores
and no sporophrtes were formed, although spermato-
zoids were released from the microgametophytes.
Ave.rage 292 59.5 38.s:; 75 52.3 ()
+ 6.3 + 7.3 + 4.9 Discussion
Approxirnately50(g70-75(g70 of the megaspore com-
plement in sporocarps from the three species ex-
amined formed megagametophytes when grown in
pubescens and M. hirsuta, in which we recorded distilled water. Substitution of WHITE'S(1943)
approximately 20%oor less for tested sporocarps.As mineral salt solution did not alter this response.
in the other species, some variation in size of the Viability appeared to be unrelated to the presence of
megagametophytes and abundance of rhizQidswas naturally occurring substances, whether stimulatory
evident in the population. Substitution of WHITE'S or inhibitory, in the sporocarp or the microspores,
(1943) mineral salt solution for distilled water did since a similar percentage of megagametoph-tes
not change the average viability of megaspores developed on spores grown in the presence or absence
grown in the presence of microspores. of sporocarp contents.
Sporophyte formation occurred on an average of Variation in viabilitS, which ranged from 48%oto
38.5%oof the megaspores grown in the presence of
microspores(table 3). Dififerencesin fertility may be
associated with archegonium development or recep- TABLE; 4
tivity to spermatozoids, rather than megagameto- LONGEVITY OFARCHEGONIUM RECEPTIVITY TO
phyte development, because many gametophytes FERTH,IZATION IN MARSILEA VESTITA
possessing numerous rhizoids, as well as those
possessing only a few, failed to form sporophytes. Time NO. % %
These gametophytes, as in Regnellidiumand Pilularia, (h) megaspores gametophytes sporophytes
remained green but unfertilized for several weeks (Control.. . 520 55. 8 _ 4. 1 40. 7 _ 2. 0
prior to their degeneration.
No sporophytes were formed in most experiments
where megaspores were isolated from microspores
(table 3). However, in a few experiments a low
272 BOTANICALGAZETTE [SEPTEMBER

87%67was evident in sporocarps from all genera.
Regnelli(:liumand Pilularia produced the most con-
sistent responses as measured in percentage of
megagametophyte formation. The strain of Marsilea
used in these tests had the lowest viability among
the genera examined, and some sporocarpscontained
no viable megaspores. Since we recorded similar
viability percentages for megaspores grown in the
presence and absence of sporocarp contents, no
factor directly associated with these contents ap-
peared to control megaspore viability. Ecological
factors may affect megaspore viability, and several
of these features are being examined at the present
time.
Rapidity of megagametophyteformationappeared
to be controlled by the megaspore rather than other
substances originating from the sporocarp contents.
Daily recordingof the numberof megagametophytes
of Marsilea and Pilularia indicated that all gameto-
phytes formed within 24 h at 25 C and few if any
formed subsequently, whether grown in the presence
or absence of sporocarp contents. Regnelli(:lium an earl) review of these phenomena in ferns, also
megagametophyte development was delayed, and
gametophytes did not become evident for several
days.
Archegoniain M. vestits were receptive to sperma-
tozoids for a relatively short period of time, approx- origin in Regnelli(liumis supported by the close
imately 12-24 h. It must be noted for this experi-
ment (table 4) that fresh microspores, rather than
spermatozoids, were added to isolated megaspores presence and absence of microspores. Further, the
or megagametophytes. An additional 8-12 h elapsed
before spermatozoids were released from the de-
veloping microspores.Nevertheless, there is a marked
and progressivedecrease in spermatozoidreceptivity
by archegoniaduring the gametophytic postgermina-
tion period. Nonfertilization of archegonia after
24 h or more in culture may be a result of degenera-
tion of the egg cell or closureof the neck canal, which the egg cell or a sterile archegonialjacket cell before
would prevent entrxrOfspermatozoids. One or both
mechanisms may serve as selection factors to syn-
chronize megaspore germination and megagameto-
phyte development with the rapid maturation and
release of spermatozoidsfrom microgametophytes.
Parthenogenesis was evident for isolated mega-
cultures of megaspores, it is possible that all sporo-
phytes in Regnellidiumwere formed partheno-
genetically. Our studies on M. testitaindicated that
parthenogenesis served only a minor role in this
species, while in Pilulariathere was no evidence for
the occurrenceof this phenomenon.
BHARDWAJA and ABDULLAH (1972) observed par-
thenogenesis in five of eight species of Marsilea:M.
brownii,M. di2Jusa,M. minuta,M. rajasthanensis,
and M. vestita.However, few data are presented in
their study . NATHANSOHN ( 1900), who studied the
effect of temperature on Marsileamegaspores, re-
ported that at 35 C sporophytes formed on up to
12go of the megagametoph)tes of M. testita and
M. macraand on over 90%0of those of M. drum-
monflii,whereasparthenogenesiswas absent in these
species at 18 C.
STRASBURGER (1907) referred to the development
of unfertilized egg cells in Marsileaas apogamS,
whereas SHAW (1897), perhaps more correctlA,
interpreted it as parthenogenesis. STEIL(1939), in
emphasized that parthenogenesis should be dis-
tinguished from apogam) in the origin of the sporo-
phD,tefrom the egg and sterile tissues, respectivels.
The parthenogenetic interpretation of sporophxte
correlation between the number of viable megaga-
metophs tes and sporophstes that develop in the
formation of only a single sporophxte on each
megagametoph)te and the development of the sporo-
ph) te in the position of the archegoniumrather than
elsewhereon the megagametophxte also support this
interpretationfor sporoph)te origin in Regnelli(lium.
Histological studies, however, are necessars to
determine whether the sporophste is derived from
it can be unequivocalls known whether the sporo-
phs tes are formed parthenogeneticall- or apog-
amousl-.
Acknowledgments
Ihe authors thank Dr. BLOOM for spc)ro- VV. VV.

spores in all sporocarpsof our strain of Regnelli(Sium. carps of Regnelli(liumand MARIA MOOAKfor
Since there was no significant difference for the sporocarpsof Marsilea.This stud) was supported in
number of sporophytes formed in isolated or mixed part by the Indiana Universit) Foundation.

LITERATURE CITED
ATKINSON, Is. R. 1943.A preliminary report of fertilization in in Regnellidillm.
Bull. Torrey Bot. (Club66:263-279.
Marsilea testita.Amer. J. Bot.30:401-404. t)FMAtsY-FELLER, M. J. 1957. Gametophytes et gametogenese
BHARDWAJA, T. N., and S. ABDULLAH. 1972. Some observa- dans le genre Marsilea.Celltlle 58:171-207.
tions on parthenogenetic sporelings of the B ater fern FELLER,M. J. 1953. Sporocarpe et sporogenese che%Marsilea
Marsilea.
Nova HedWiga 21:521-528. 1lirsleta
R. Br. Cellule 55: 307-377.
BOTERBERG, A. 1956. Genese et differenciation des parois HrGINsoTHAM,N. 1941. Development of the gametolhvtes
sporales chez MarsileadigusaLepr. Cellule 58:81-106. and embro of Regnellidillmlliphyllllm. Amer. J. Bot.
CHRYSLER, M. A., and D. S. JOHNSON. 1939.Spore production 28: 282-300.
 197 S] MAHLBERG& BALDWIN MARSILEA,PILULARIA,REGNELLIDIUM
MACHLIS,L., and E. RAwlTscHER-KuNKEL.1967. The
hydrated megaspore of Marsilea vestila. Amer. J. Bot.
54: 689-704.
MARSCHALL,C. C. 1925. Differentiation of sporangia in
Marsileaqlladrifolia.BOT. GAZ.79: 85-94.
NATHANSOHN, A. 1900. Uber Parthenogenesis bei Marsiliaund
ihre Abhangigkeit von der Temperatur. Deut. Bot. Ges.
18: 99-109.
RICE, H. V., and W. M. LAETSCH.l967. Observations on the
morphology and physiology of Marsilea sperm. Amer. J.
Bot. 54:856-866.
273
SCHULTZ, A. 1936.Beitragezur Kenntnisder Entwicklungder
Macrogametophytenvon Pilularia globulifera L. und
MarsileaquadrifoliaL. Planta 25:703-719.
SHAW,W. R. 1897. Parthenogenesisin Marsilea.BOT.GAZ.
24:114-117.
STEIL,W. N. 1939. Apogamy,aposporyand parthenogenesis
in the Pteridophytes.Bot. Rev. 5:433-453.
STRASBURGER, E. 1907.Apogamiebei Marsilia.Flora97:123-
191.
WHITE,P. R. 1943. A handbook of plant tissue culture.
Ronald,New York.