Table Of ContentGametophyte and
Development, Sex Expression
Antheridiogen System incompleta
in Pteris
Cav. (Pteridaceae)
Carmen Prada*, Vanessa Moreno, and Maria Gabriel y Galan
Jose
o de Biologia Vegetal Facultad de Biologia, Universidad Complutense,
I,
Ciudad Universitaria, 28040 Madrid, Spain
Abstract.—The gametophytic phase of several species of Pteris has been well studied, but for
others, due perhaps to their more restricted distribution known. Agar and cultures of
soil
little
is
different spore samples of P. incompleta were established in order to analyze developmental
features of its gametophytes. Gametophyte development followed the Ceratopteris pattern, but
resulted in a slightly different morphology from that of other more common species of the genus.
Sex expression was variable among gametophyte populations, and was affected by culture
An
medium.
antheridiogen system was present and promoted both male precocity and dark
germination. Antheridiogen response was variable among gametophyte populations. Positive
antheridiogen response in interspecific gametophyte pairings suggests a common antheridiogen
system and
in Pteris vittata P. incompleta.
Woros.—
Key Gametophyte, morphology,
sexual expression, antheridiogen, Pteris incompleta
and
Pteris is a large diverse genus of about 250 terrestrial and epilithic species
(Tryon and Tryon, 1982) of predominantly and
tropical subtropical distribution.
Despite being one of the largest genera of homosporous known
ferns,
little is
about the sexual generation and
of Pteris species (Atkinson, 1973; Laird
Mendoza
Sheffield, 1986; Chiou, 1992; 1996-97]
et al, except for P. vittata L.
which many
has been studied from morphological and
physiological aspects
(Ito, 1962; Kato, 1963; Kato, 1969; Gemmrich, 1986 Tu and Ma,
a-b; 2003).
Only
a few species of Pteris grow in temperate one them,
areas; of P.
incompleta
Cav., is native to the Macaronesian and Mediterranean regions
where
represents
it a relic of a Tertiary flora that virtually disappeared with the
first glaciations (Pichi Sermolli, 1979; Salvo, 1990). a protected species in
It is
Spain where
populations
its are, at present, very scarce and vulnerable, in
great part because of the fragility of the ecosystems where the species grows.
Our was
goal to study gametophyte development and
sex expression of
P.
incompleta under
Some
laboratory
conditions. of our preliminary
results
suggested
the possibility of an antheridiogen system
operating
in this species.
An
antheridiogen system
similar to that of Pteridium aquilinum Kuhn,
(L.)
which
induces
the formation of antheridia young and
in prothalli substitutes
for the light requirement in spore germination, known be produced by
is to P.
(Gemmrich,
vittata 1986 a-b; Raghavan,
1989). This antheridiogen active in
is
GAMETOPHYTE
OF
PTEH1S
.:
I
intheridiogen assays in light conditions, de
of female gametophytes spores. Cultures n
/
n VA:
the dark. P. vittatu (Valencia): C.\: P.
GO: incompleta Gomera).
P. (La
and Gemmrich,
other species oiPteris in different fern genera (Schneller, 1979;
1986b; Schneller et al, 1990; Nester-Hudson et ah, 1997; Chiou and Farrar,
1997). Therefore additional assays were undertaken hypothesis.
to test this
Materials and Methods
Spores used study were taken from sporophytes
for this collected in the
locations listed in Table Voucher specimens are deposited in the
1.
-
Universidad Complutense de Madrid herbarium (MACB).
Biologia
Spore samples for cultures were taken from single sporophytes kept dry at
room temperature since the plants were collected. Gametophytes were grown
under fluorescent light on a 12-h light, 12-h dark cycle at 20 ± 2°C, in plastic
cm
Petri dishes 6 in diameter.
—
and
Spore germination gametophyte development. Multispore cultures on
mineral agar (medium described in Dyer, 1979) were established by sacking
pinnae on piece of weigh paper and placing the obtained spores in the
fertile a
Sowing sample was
Petri dishes. of each replicated twice. Percentage
germination was recorded a random sample of 50 spores from each of
for
two plates, every three days until there was no further increase.
To development from
study the different represented stages of spore
random
germination until sexual maturity (which lasted ca. 9 weeks), samples
were taken weekly. Gametophytes were stained with chloral hydrate acetocar-
mine (Edwards and Miller, 1972), mounted in water and observed under a light
were used young
microscope. These agar cultures also as a source of presexual
on
gametophytes in transplant studies of sex expression soil cultures.
—
Sex expression. Cultures on soil (a 3:1 mixture of commercial compost and
CA VA
sand) were used study sexual expression of samples GC, and
to (see
Table In order to observe the sequence of formation of gametangia on each
1).
individual gametophyte, 50 young gametophytes from the multispore
agar
15-30 and
cultures were transplanted at the cell stage separately arranged in a
AMERICAN FERN VOLUME NUMBER
JOURNAL:
98
1
Percentages of spore germination each sample, time
for
r
ion and, in brackets, weeks which those wen
at
^
p I
time passed from
e collecting the fronds to spore sowing.
%
TIME FOR GERMINATION OF GERMINATION
two
regular pattern in Petri dishes as follows: six row
dish, the row with two gametophytes,
first the following four rows with
five
gametophytes and row
per row, the with The was
last three. final density
ca.
cm" 2
one
prothallus Examinations were made two week
at intervals for
.
18 weeks. For these observations, gametophytes were mounted
individually
in
examined compound
water, with a microscope and replaced back
into culture.
VA
Multispore on GC
cultures mineral agar were used samples and
also
for
in order to test if sex expression affected by culture medium. Random
is
samples
of ca. 50 gametophytes from both cultures were taken two-week
at
during
intervals the 18 weeks, stained described above and
as the percentages
of male, female and
sterile, bisexual gametophytes
calculated.
Antheridiogen assays.—
-To test antheridiogen activity in P. incompleta, two
different aspects were studied: induction of male precocity and induction
of
spore germination in darkness. Several were made
using incompleta
tests
P.
and
was
P. vittata. Pteris vittata used as reference organism because
its
antheridiogen system has been described and
reactions antheridiogen
its to are
known
well (Gemmrich,
1986a-b; Raghavan, was used
1989). Pteris vittata in
two
ways:
source
as a of antheridiogen
a) to test antheridiogen response in
and
spores gametophytes
of incompleta; and
P. bj as a bioassay to measure
response
antheridiogen
to
activity of female incompleta
P. gametophytes.
we
As
had
access to several samples
of P. incompleta, multiple combina-
tions of female gametophytes/spores were arranged shown
as in Table All
is
1.
the cultures were on
established mineral
agar, as described above spore
for
germination, placing one female gametophyte
in the center of the dish
Petri
and
sowing
the corresponding spores around
Controls for the antheridiogen
it.
response of both species were made
in similar cultures without female
gametophytes
(Table and
cultures
5
1, 1,
8).
To
detect male random
precocity, samples
of 50 gametophytes each
ca. of
culture were taken
at intervals of two weeks during 18 weeks,
stained
as
described above
for spore germination and
percentages
of male, female
sterile,
and
bisexual gametophytes were
calculated (Table
cultures 4 7 9
1, 2, 3, 6,
and
10)
.
To
carry out the test of spore germination
in the dark, the combinations
of
samples
of cultures and
4, 9 10 (Table were used. Six were made
1) replicates
each
for culture, three with only
spores as control, and three with a female
gametophyte and
the corresponding
spores around were wrapped
Plates
in
it.
PRADA GAMETOPHYTE
ET AL: OF PTERIS INCOMPLETA
17
two aluminium
folds of paper, kept in a box and placed in the same growth
room
with the other cultures. Each pair of plates (with and without female
gametophyte) was unwrapped
and
the content examined 4 and
after
2,
6 weeks.
Results
—
Spore and
germination gametophyte
morphology. Spore
germination
in
incompleta common
Pteris is of the Vittaria type, the most in homosporous
ferns (Nayar and Kaur, Table shows
1971). 2 rates of spore germination
obtained each
for sample, as well as the time passed from sowing
the
to
beginning which was when
of germination, evident the rhizoid emerged.
first
After spore germination, the rhizoid and 1-4
first a short filament of
cells
were The
formed.
filament divides apical or subapical
its cell starting to
produce narrow and
a plate later a spatulate one, following the Ceratopteris
type of development described by Nayar and Kaur The
(1971). plate lacks a
meristem
but most
at this first stage, for of the prothalli the meristem
is
organized weeks growth
after 1 to 3 of following germination.
The
meristem an
is in apical or slightly lateral position. Activity of
meristematic cells gives rise after a few days to a cordate but somewhat
asymmetrical prothallus with two wings almost same By
of the size
(Fig. la-f).
many
the time that of the gametophytes reached the cordate shape other
gametophytes remained
of the cultures with without
irregular, or a defined
meristem. Mature gametophytes were naked
(without hairs), either cordate
(Fig. lg-h) or irregular
(Fig.
li).
Sex expression.—
Progression of sex expression on
soil cultures for the
samples studied of P. incompleta (GC and CA) and (VA) summarized
P. vittata
is
GC
Sample showed
in Figure a small proportion (12%) of female cordate
2.
weeks Two
sowing, remaining weeks
prothalli 8 after the being
sterile. later
male gametophytes were abundant (62%) and a moderate proportion (28%) of
became Male
sterile female. gametophytes were small, irregular, and without a
When
defined meristem. cultures were 18 weeks bisexual gametophytes
old,
were produced most them
(24%), of developing from the males, which
in a
meristem differentiated and archegonia formed near the meristem
area
(Fig. 2c).
Bisexual gametophytes were cordate but smaller than the females.
CA
Sample showed simultaneously male and
cordate female gametophytes
weeks sowing 42% and 46% 4%
8 after in a proportion of respectively; only
8%
were sterile by that time and the remaining were cordate female
gametophytes from which an irregular basal lobe was formed and produced
became
antheridia, then such gametophytes functionally bisexual. At
10 weeks few became male and some
the prothalli females formed
sterile
the
The
lobes with antheridia. proportions of each sexual type had not change
at
weeks
18
(Fig. 2e).
78% 12%
gametophytes were
In P. vittata of female, bisexual and only
the
Two
remaining 10% by week weeks
presexual
presexual gametophytes
8. later
and most became and 6%
of the females bisexual, only remained
female.
PRADA
ET GAMETOPHYTE
AL: OF PTERIS INCOMPLETA
1
II
'/A
I
ins
11
HI
Progression of sex expression in the following weeks involved the
transfor-
mation of females into bisexuals by production of antheridia near the apical
notch where archegonia were placed, so that by week 18 100%
of
gametophytes were
bisexual
(Fig. 2a).
was
Progression of sex expression also observed in the cultures on
agar of
VA
GC
samples and and GC
(Table cultures 5 respectively). In sample
1, 1,
only male and female gametophytes were detected over the same period of
observation; the gametangia produced were antheridia
first instead of
archegonia and their formation started two weeks later with respect
to soil
VOLUME NUMBER
AMERICAN FERN JOURNAL:
20 98 1 (2008)
A
cultures (Fig. 2d). similar temporal displacement was observed in cultures
on medium VA, where male, female and bisexual
agar of P. vittata
gametophytes were detected simultaneously two weeks later with respect to
soil cultures; male gametophytes were detected in a low percentage (Fig. 2b)
which were
not seen in soil cultures.
on
Antheridiogen activity.—Results of assays antheridiogen activity are
segregated response endogenous or exogenous antheridiogen sources,
in to
tested through induction of male precocity and germination in the dark.
we
Response endogenous antheridiogen (which consider equivalent
to to
same
intraspecific the gametophytic individuals being of the population
effect,
produced
or different) considered here as the influence of the antheridiogen
is
by female gametophytes on male precocity and/or germination in the dark of
own
spores of species. Response to exogenous antheridiogen activity
its
we
(which consider equivalent to interspecific effect) is defined as the
on male
influence of antheridiogen of female gametophytes of a species
precocity and/or germination in the dark of spores of another species.
—
Response endogenous showed
to antheridiogen. Pteris vittata a particular-
A
vigorous response endogenous antheridiogen high proportion
ly to activity.
week
of prothalli formed antheridia in very early stages of development: by
6,
50%
gametophytes were 40-50
nearly of the male, spatulate, ameristic, cells in
90%
and nearly by week proportion changed over the
size; this
8; little
following weeks; gametophytes grew slowly and never reached cordate shape.
Control cultures, those without added female gametophytes, reached a level of
50%
only of male gametophytes by week and proportion dropped
14, this
drastically afterwards (Fig. 3a) due to the formation of archegonia in male
gametophytes which became bisexual, grew more than the ones and
last
reached normal
their cordate shape.
Cultures of P. incompleta had variable responses to endogenous antheridio-
gen activity depending on sample combinations. Female gametophytes from
GO
sample seemed induce middle response own
to a in spores since there
its is
a significant increase of male gametophytes by weeks 8 10 but no
to (Fig. 3c),
GC
response in the case of sample Gametophytes from samples
(Fig. 3b). other
of P. incompleta, particularly from sample CA, promoted a response almost as
high
as in the case of P. vittata (Fig. 3b).
Germination in the dark due to endogenous antheridiogen activity was
GO
sown
tested in P. incompleta spores with female gametophytes of the same
A
low
sample. percentage (3%) of spores germinated by week Those
6.
gametophytes consisted of short, curved uniseriate filaments of 2-3 cells, some
bearing sown
antheridia. In control cultures, those in the dark without female
was
gametophytes, no
there germination.
—
Response exogenous was
to antheridiogen. Pteris vittata greatly influenced
CA
by
the antheridiogen of incompleta, from both samples and GC:
P.
gametophytes formed antheridia more than 4 weeks before control cultures,
and
the proportion of male prothalli rapidly reached nearly 100%. In the CA/
VA
22%
combination, gametophytes two
of the bore antheridia only
after
weeks
in culture (Figs. 3d, 4a).
INCOMPLETA
PTERIS
'
The reverse situation was detected also: P. incompleta was influenced by the
As
antheridiogen of female gametophytes of P. vittata. Fig. 3e shows, 4 weeks
GO
100%
sowing gametophytes sample developed
after near of in antheridia
(Fig. 4c); this proportion did not change in the following 14 weeks. This strong
maleness precocious since the male gametophytes appeared
first in the
is
weeks and lower
control culture four later in a initial percentage
(Fig. 3e).
Fig. 4. Precocious male gametophyt
a.
incompleta CA, two weeks
sowing:
after
gametophyte
— of P.
j \
~
J
produced -
in the !
'-
incompleta GC; GO
Xl
*,. lyte of P. incompleta induced by female
gametophyte
of P. GO
vittatc filamentous gametophyte
incompleta
of P.
produced
in the dark six v with
female gametophyte VA.
a of
P. vittata
;
PRADA ET AL: GAMETOPHYTE OF PTERIS INCOMPLETA
23
VA
Spore cultures of P. vittata grown in the dark and in the presence of
female incompleta gametophytes showed low
P. (GC), a percentage of spore
germination (3%) 4 weeks sowing. Gametophytes point consisted
after at this
of filaments 2-3 cells long. Plates maintained in the dark for 6 weeks showed
7%
germination and filamentous gametophytes, some which were
of
and some
branched, bearing 1-2 antheridia Similar gametophytes
(Fig. 4b).
developed from spores of P. incompleta (GO) sown in the dark with a female
gametophyte sown
of P. vittata (Fig. 4d). In control cultures, those in the dark
without female gametophytes, there was no germination.
—
To
Statistical significance of antheridiogen essays. check statistical
we
significance of the results performed chi-square analyses for each of the
experiments (7 in total, Fig. Data from control cultures were used as
3).
expected frequencies, while observed frequencies were obtained from cultures
< = we
with female gametophytes. In cases, p 0.05 therefore accept
all
(d.f. 7),
that observed male precocity due to the presence of female gametophyte.
is
The was GC/GC shown
only exception experiment, which,
for in as in
is
Fig. 3b, development of antheridia was delayed in time with respect to
its
control culture.
Discussion
Gametophytes of incompleta follow the same developmental pattern
P.
and and
described by Atkinson (1973) for P. tremula R. Br P. multifida Poir.,
young
form nearly symmetrical, cordate prothalli at a stage. This in contrast
is
comans
to other species of the genus, such as P. berteroana C. Agardh, P.
and have asymmetrical shape
that a strongly
Forst., P. grandifolia L., P. vittata,
gametophyte development (Stokey and Atkinson, 1952;
at early stages of
Mendoza
1996-97).
et
al.,
common development homosporous
The most sequence of sexual in ferns
followed by archegonia (Atkinson and
involves the formation of antheridia
Stokey, 1964). This sequence of gametangia development has been reported to
be fixed for several taxa (Herrero et al., 1993; Prada et al., 1995). In the case of
was
P. incompleta progression of sex expression slightly different in the
On
medium.
samples studied, and varied depending on the culture soil
cultures, sample GC produced initially female gametophytes and later male
CA
most which became cordate and bisexual. Sample
ones, of ultimately
which
produced male and female gametophytes concurrently, should favor
and
intergametophytic crossing (Klekowsky Lloyd, 1968), as well as a small
which were
proportion of other bisexual gametophytes in antheridia located
on irregular lobes of some of the cordate females. Similar female gametophytes
Bommeria and
with antheridial lobes were reported in (Haufler Gastony,
1978), a genus in which the existence of an antheridiogen system has been
demonstrated.
showed gametangia with
Agar delayed differentiation respect
cultures to soil
A
GC
was
sample sequence
cultures, and in the case of the different. culture
