Table Of ContentAmerican Fern Journal 97(3):149-154 (2007)
The Gametophyte Huperzia selago Culture
of in
Dean
Whittier
P.
TN
37235-1634
Department of Biological Sciences, Vanderbilt University, Nashville,
Helena Storchova
Experimental Botany CAS, Rozvojova 263, 16500 Prague, Czech Republic
Institute of
—
They
and
Abstract. Cultured gametophytes of Huperzia selago are dorsiventral strap-shaped. are
basically the same as cultured gametophytes of other terrestrial Huperzia species and also
essentially the same as gametophytes of H. selago collected from loose soil. Besides shape, the
cultured gametophytes have anatomical features found in gametophytes of H. selago from loose
Bruchmann
These gametophytes are Type gametophytes as characterized by in 1898.
soil. III
Although both gametangia occur on individual cultured gametophytes, the meristematic groove
The number Huperzia
produces them different times and they are not intermixed. of terrestrial
at
number
species with described gametophytes is still small (six), but that has tripled as a result of
the recent studies on these gametophytes in culture. Because all the terrestrial Huperzia species to
have Type gametophytes, would not be unexpected for the undescribed gametophytes of
date
III it
Type gametophytes.
other species in this group to also be
III
Bruchmann
In early studies on gametophytes of the Lycopodiaceae, (1898)
Lycopodium The shapes and type of nutrition of
recognized types
five for s.L.
ame
Type
shaped Type disk-shaped (mycorrhizal); elongated
(mycorrhizal);
III
II
and
Type IV pincushion-shaped (photosynthetic);
uniaxial (mycorrhizal);
V These recognized
Type branched cylindrical (mycorrhizal). types are still
gametophytes
and they demonstrate the range of variation in the of this family.
=
Lycopodium Huperzia selago Mart. & Schrank) gameto-
selago
L. (L.)
[
Type gametophytes (Bruchmann,
phytes from were the basis for 1898).
soil III
amet
Prior culturing
to
was Lycopodium lucidulum Michx. [=Huperzia lucidula (Michx.)
that of
To understanding
Trevisan) by Spessard (1922). provide a better of terrestrial
Huperzia
gametophytes
Huperzia gametophytes, of four tropical, terrestrial
(Whitt
Hup
Bruchmann
Because was one of the shapes that (1898)
strap-shaped. this
gametophytes Huperzia selago from these tropical
described of soil,
for
Huperzia gametophytes were considered Type gametophytes.
terrestrial III
conclusion would be provided cultured
Additional support
for this if
To examine
gametophytes of H. selago had a dorsiventral shape. this
were grown
gametonhvtes H. selago in culture.
nnssihilitv. of
Methods
Materials and
September
Spores Huperzia selago were obtained from plants collected in
of
and May in the Jeseniky Mts. of the Czech Republic. Vouchers are on deposit at
AMERICAN EERN VOLUME NUMBER
150 JOURNAL:
97
3 (2007)
Tabu: Huparzia known
Terrestrial species with gametophvles.
I.
Species Source
Reference
H. salogo Bruchmann,
1898
soil
lucidula
II. soil Spessard, 1022
H. lucidula &
culture Whittier Webster, 1986
H. crassa culture Whittier, 2000
H. cumingii
culture Whittier, 2000
hypogaea
H.
culture 2006
Whittier,
H. saururus
culture Whittier. 2006
selago
//. culture present study
TENN.
The spores were sown within one month To
of their collection. redtice
the incidence of contamination, the spores were wetted and water
stored in
for
hours and 20%
24 surface sterilized with Clorox (1.1% sodium hypochlorite)
by method The
the of Whittier (1964). spores were then suspended
in sterile
water, and sown on ml medium X mm)
13 of nutrient culture tubes 125
in (20
with screw caps were The
that then tightened. cultures were maintained
in
_1
darkness under hour umol-m~ 2
or a 12 photoperiod (50 from cool white
-s
)
lamps ±
fluorescent 22
at 1°C.
NH
The medium mg mg MgS0 mg
nutrient contained 100
C1, 50 -7H 0, 25
4 4
2
mg HP0
K
CaClv, and 50 per Minor elements and FeEDTA completed
liter.
2 4
medium
composition
the of The was
this (Whittier, 1998). carbon source
provided by
the addition of 2.5 of glucose per spore germination and
g liter for
gametophyte growth
early or 5 g of glucose per liter for the growth of older
gametophytes. The medium was pH
with 1.1% and was
solidified agar
5.9
at
before autoclaving.
A
of 6000 spores were examined determine
total to the percentage of spore
germination. The sample
for calculating the average sizes of the gametangia
and paraphyses was
30.
The
gametophytes were
fixed with Randolph's modified Navashin
fluid
(CKAF;
Johansen, 1940). After fixation, the gametophytes were embedded
in
and
paraffin sectioned by conventional techniques The
(Johansen, 1940).
were
sections stained with Heidenhain's hematoxylin, and
safranin O,
fast
green.
Results
About 0.1% May
of the spores from both and September
the
collections
germinated months
after 6 the dark and none germinated months
in 13
after in
illuminated cultures. Gametophytes began mature year sowing and
to a after
a total of 42 young and mature gametophytes developed from sown
the spores.
Mature gametophytes
are long, narrow, axial structures with distinct dorsal
and Some
ventral surfaces (Figs. 1, 2, 3). are straight (Figs. 1, 2, 3) and others
more
are sinuous. Lateral grooves along the sides of the gametophytes separate
and
the dorsal ventral regions (Figs. Most gametophytes have an
1, 2).
STORCHOVA: GAMETOPHYTE OF HUPERZIA SELAGO
WHITTIER & 151
view narrow gametophyte with
1-6. Cultured gametophytes of Huperzia selago. Lateral of
Figs. 1.
groove (small arrows) and meristematic region (large arrow). 2. Lateral view of apical region
lateral
view
with lateral groove (small arrows) and meristematic region (large arrow). 3. Dorsal of strap-
shaped gametophyte. Ventral view of apical region of wider gametophyte with meristematic
4.
groove (arrows). Non-median sagittal section of apical region with mature archegonium (small
5.
narrow gametophyte with
arrow) and meristematic region (large arrow). Cross section of
6.
mm
=
antheridia (arrows) at dorsal surface. Bars 1 for Figs. 1-4 and 100 |im for Figs. 5-6.
152 AMERICAN WAIN JOURNAL: VOLUME NUMBER
97
3 (2007)
hourglass shape
in cross section but a smaller ventral region can cause
narrower gametophytes
to lose this shape.
The
meristematic tissues are not terminal the gametophytc apex
at (Figs.
4,
The immature
dorsal tissues overarch the meristematic groove
shifting
5). to
it
the lower subterminal region of the apex. The derivatives the upper side
to
form
the dorsal tissues including the gametangia and paraphyses. Derivatives
to the lower side form the ventral region of the gametophyte with the rhizoids.
The
meristematic groove is continuous with the lateral groove on the sides of
gametophyte. However,
the no
there is essentially meristematic activity in the
grooves
lateral of these cultured gametophytes.
The
cells of the central region, which encompasses most of the internal
medium
and somewhat
tissue, are sized elongated longitudinally The
(Fig.
5).
above which
cells just the ventral surface, contain the mycorrhizal fungus
in
gametophytes from and
are small isodiametric. There no
soil, are vertically
elongated near
cells the ventral surface of the gametophytes.
The
paraphyses
are uniseriate filaments with an average length of 189.3
|im.
They composed
are of 2-3 with an average number
cells
cell of 2.9.
Each antheridium
contains an ellipsoidal mass of gametes with an
(Fig.
6)
opercular cell in the jacket layer at the gametophyte surface. The average size
of the gamete mass was 72.4 |im wide and 95.5 |im long.
The
archegonia are normal Huperzia The
for terrestrial species
(Fig.
5).
average length from egg base to neck tip 136. 8 jum and the neck protrudes
is
from the gametophyte an average The number
of 79.4 average
\im. of of
tiers
four neck cells in the neck is 4.9 and there are 4 neck canal cells in the neck
canal above
the egg.
and
Antheridia archegonia same
are in different regions of the gametophytes
ermixe
antheridia or archegonia There no evidence and
(Figs. antheridia
that
5, 6). is
archegonia are produced simultaneously from the meristematic groove.
Discussion
H
shaped. This shape one
of the several shapes described H. selago by
is for
//
as those of H. selago from loose soil and as the other Huperzia
terrestrial
gametophytes grown
and
in culture (Whittier Webster, 1986; Whittier, 2006).
The
gametophytes
from culture are without mycorrhizal However,
fungi.
they same
are structurally the as the dorsiventral gametophytes of H. selago
described from (Bruchmann, The
soil 1898, 1910). similarities are easily seen
by examining Bruchmann's
and
illustrations of cross longitudinal sections
in
his 1898 and 1910 The
(Fig. 38) (Figs. 27, 28) publications. cultured
gametophytes have
longitudinally elongated and
central small
cells
iso-
diametric and no
cells vertically elongated cells in the area that correlates with
the mycorrhizal zone in gametophytes from soil. The apical meristem on the
is
lower side of the apex with overarching The medium-sized
dorsal
tissue.
GAMETOPHYTE OF HUPERZIA SELAGO 153
STORCHOVA:
WHITTIER
&
and archegonia with medium-sized necks are initiated in the
antheridia
form
maturin
on
paraphyses present the dorsal
mature gametangia, short are
the
connect with the
grooves
surface. Along the lateral surfaces there are that
meristematic groove of the apex.
ametopl
Hup
found the four other terrestrial
from they are in
selago
soil,
Whittie
H.
grown
Huperzia gametophytes in culture
paraphyses of the other
the
for
The same gametangia.
the
true for
(Whittier, 2006). is
on
The formation of archegonia and antheridia at different times these
Huperzia
gametophytes
ametophytes was observed in other terrestrial of
(Whitt
amet
The gametangia do not intermix on these cultured
gametophyte.
the
Hup
H
(Mak
miyoshiana
self-fertilization.
amou
hmann considerable
described
(1898] a
ame
some more
curved forms with being or less
elongated variously axial
The above forms were found in
and range intermediates.
cylindrical a of
He showed young
that
dense and another form occurred in loose soil.
soil
ame
and gametangia on
had paraphyses
The gametophytes
dorsiventral
loose
soil.
and
rhizoids ventrally.
dorsal surface
their
grown
Huperzia culture
in
The gametophytes of the terrestrial species of
growing
found with gametophytes of H. selago in soil
lack the variability
ameto
cultured
the
(Bruchmann, 1898; 1910). Less variability in
j
more
seems
This
gametophyte growth are absent on an agar surface. to
in soil
more
gametophyte shape culture because the agar surface is
explain the in
The structure of these
than other conditions. internal
similar loose soil soil
to
by
mycorrhizal fungus or
Huperzia gametophytes not altered by the lack of a
is
mm
Huperzia
gametophytes of H. selago and those of the other
cultured
e
Because
and Webster, 1986; Whittier, 2006) are alike.
species (Whittier
Type gametophytes
am ;toDhvtes of H. selago are characterized as III
Huperzia species are
(Bruchmann, the gametophytes of these other
1898),
Type gametophytes.
III
same shape and
have
Huperzia gametophytes from culture the
Terrestrial
from This occurs
gametophytes of H. selago loose soil.
the
internal structure as
growing without mycorrhizal
gametophytes a
even though the cultured are
154 AMERICAN FERN JOURNAL: VOLUME NUMBER
97
3 (2007)
The
fungus. morphological Type
stability of gametophytes under
those
III
conditions
indicates developmental
that this pattern from
distinctive those
is
of the other four types of gametophytes recognized by Bruchmann The
(1898).
Type
structural stability of gametophytes under
III different conditions
is
important to support the long held view gametophyte
that characters of
clubmosses have taxonomic
value (Bruchmann, 1898; Bruce, 1976; Wagner &
Had
morphology
Beitel, 1992). the of these cultured gametophytes been
different from the previously described Type gametophytes from
the
III soil,
validity of using gametophyte
characters taxonomic purposes would
for be
questioned.
Acknowledgments
We
thank James Hickey and Benjamin
Ollgaard for encouraging he earlier study of tropical,
I
We
terrestrial Ilupcrziu gametophytes
that led to this study. gratefully acknowledge Hans-Willi
Honegger
help
for translating sections of Bruchmann's
publications.
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