Table Of ContentAmerican Fern Journal 93(2):56-69 (2003)
The pH, Temperature, Light Intensity, Light
of
Effects
on Spore Germination
and Moisture Levels
Quality,
Missouri
Southeast
Cheilanthes of
feei
in
Maria and Lucinda Swatzeli
Sarah Nondorf, Melissa A. Dooley, Palmieri,
L.
J.
MO
Cape Girardeau, 63701
Department of Biology, Southeast Missouri State University,
Abstract.—Cheilanthes feei is a xerophytic fern that is broadly distributed throughout the United
narrow
States west of the Mississippi. Although it has a broad distribution, it occupies a very
approx-
niche. In southeast Missouri, C. feei inhabits crevices of limestone bluffs, in full sun,
m
imately 0.5-1.0 from the top of the bluffs. The physiological basis for the fern's restriction to this
xeric environment unclear. In this study, C. feei spores were subjected to a broad range of
is
and moisture
temperatures, pH, and light intensities, to varied light qualities, to different levels.
However,
spores can germinate under a wide variety of conditions.
Results indicate that C. feei
data suggest that spore germination optima and optimal conditions for protonemal growth overlap
narrowly. The disparity in optimum conditions may be a partial basis for the broad distribution
and narrow niche of
C. feei.
common
Moore widely North
Cheilanthes a fern that distributed in
feei is is
from southwestern Canada, south
America. range extends primarily to
Its
north central Mexico, and east to the Mississippi and Ohio River valleys of
common,
Midwestern United Although un-
States (Mickel, 1979). C. feei is it is
somewhat
usual in several ways. First, C. feei is a xerophytic fern. This is of
an oxymoron, since fern gametes are typically free-swimming and most ferns are
many
moist environments. However, in and cheilan-
restricted to C. feei
thoid ferns, the need for water for reproduction circumvented by apogamy.
is
C
narrow
Secondly, although widely distributed, occupies a very
feei is it
grows limestone
niche. In southeast Missouri, C. feei typically in crevices of
m
south sun and approximately 0.5-1.0 below
bluffs, typically facing in full
bluff tops.
many
The However,
basis for this habitat restriction unclear. there are
is
more
cannot compete with
feasible explanations. possible that the fern
It is
more
vigorous species in mesophytic habitats, but can survive in xeric en-
vironments. Cheilanthes like other Cheilanthes species, characterized
feei, is
by some adaptations can reduce water such numerous trichomes
that loss, as
and volume
a small surface area to ratio (Hevly, 1963; Gratani et 1998).
a/.,
would which
This be analogous to the saguaro cactus, Cereus giganteus, is
restricted to areas of intense sunlight, since the thick hydrodermis causes to
it
be light-limited (Darling, 1989). Another possible explanation for habitat re-
may
striction in Cheilanthes feei is that the narrow niche that C. feei occupies
provide the optimal growth conditions for the fern, so that the incidence of
A
C. feei in other habitats very low. final possibility habitat specificity.
is is
may
Cheilanthes feei be restricted to habitat based on unique characteristics
its
of both the fern and environment.
its
NONDORF
ET SPORE GERMINATION CHE1LANTHES
IN
AL.: 57
This study addresses the physiological basis for the restriction of Cheil-
anthes feei to limestone bluff crevices. Optimal conditions for spore germi-
nation are often a reflection of optimal growth conditions for the entire
life
cycles of the ferns. Since the fern gametophyte the most vulnerable stage in
is
gametophyte and
the fern cycle, physiology the necessary condition ranges
life
for growth and development are limiting for ferns. Hence, optimal conditions
for gametophyte survival and development are typically congruous with
optimal spore germination conditions (Raghavan, 1980; notwithstanding
necessary changes in light qualities which spur developmental changes). In
environmental
addition, conditions that negatively affect spore germination
An
typically reflect the physiological limitations of a fern species. obvious
example the need for water. Most spores require the presence of water and
is
undergo imbibition prior to germination. The gametophyte stage and the
fer-
tilization process also require at least a film of water.
Previous studies demonstrate that cheilanthoid ferns, with respect to spore
may
germination requirements, be physiologically similar mesophytic
to ferns.
For example, most germinate and develop pH,
ferns best at a slightly acidic
at 25°C, in moist conditions, under red light phytochrome response), and
(a
_1
2
moderate light intensity (100 |imolm~ (Raghavan, 1980). With respect to
-s
)
pH, temperature, light quality and light intensity, these are also the optimal
conditions for spore germination in several cheilanthoid species (Hevly, 1963;
Raghavan, 1973). Still, these ferns have very different distributions than C.
may
and
feei, optimal spore germination conditions for C. feei be different as
we
examined
well. Therefore, the effects of pH, temperature, light intensity,
and on
light quality, moisture levels germination rates of C. feei spores. In
we
measured and
addition, the potential water content porosity of rock sub-
strate in C. feei habitat.
Materials and Methods
—
Plant Collection. Cheilanthes sporophylls were collected in the and
feei fall
MO.
winter of 2000 from Reis Biological Station, Steeleville, To harvest spores,
sporophylls were crushed using a mortar and Cheilanthes spores
pestle. feei
average 67.0 in diameter (Knobloch, 1969) and spores were separated from
yvxn
mesh C
the plant material using a 75.0 jim brass sieve and stored 4° in the
at
dark.
—
Culture Conditions. Although study addresses optimal spore conditions
this
for Cheilanthes there previously no information available on optimal
feei, is
medium
culture conditions, growth contents or osmolality, with the exception
of anecdotal information (Siegler, 2002). Therefore, a standard growth
medium, Knudson's-C phosphate
(C-Fern, 2001), that contains iron,
salts,
buffer, sugar as an osmoticum, and agar for solidification, was selected. The
effects on spore germination of any of these contents, such as sucrose, on C.feei
are unknown, and have varying effects on germination in other species
we
make
(Raghavan, 1989; Sheffield et 2001). Therefore, strove only to
al.,
them consistent through of the treatments. All treatments then, were cul-
all
VOLUME NUMBER
AMERICAN FERN
JOURNAL:
58 93 2 (2003)
pH
Table Standard conditions within experiments were 25°C, continuous white
5.5, at
1.
100 umol-m identifiably saturated agar-based Knudson's-C. the exceptions were the light
-s~
,
quality and light intensity experiments, in which 25°C or 100 umolirT^s" 1 was difficult to
some
maintain in light intensities or qualities.
Li{^ht intensity Moisture level
pH 2 1
Temperature (umol-m
Variable s- Light quality of substrate
)
pH NA
25°C 100 White Saturated
NA
Temperature 5.5 100 White Saturated
NA
Light intensity 5.5 33°C White Saturated
NA
Light quality 5.5 33°C 75 Saturated
NA
Moisture 25°C 100 White
level 5.5
of substrate
Dark min
plus 5.5 25°C 100 (45 pre -stimulus) White/dark Saturated
True min
dark 5.5 25°C 0.04 (45 pre -stimulus) White/dark Saturated
tured on the same growth medium lot. In addition, in the absence of infor-
mation on on we
the affects of surface sterilization germination, also used a
standard procedure and
(Guiragossian-Kiss
Kiss, 1998).
With
the exception of the moisture level experiment, spores were surface
7%
sterilized in a (v/v) commercial bleach solution with 0.1% Triton X-
(v/v)
ddH
100 for 20 min. Spores were then rinsed in sterile 0, sown on a modified
2
mM mM
medium
Knudson's-C (NH S0 Ca(N0
(C-Fern, 2001; 3.7 4.2 )2-4
4 2 4 3
mM ) ,
mM
H KH P0 uM
FeS0 uM H B0
0, 1.8 27.0 -7H 0, 17.5 sucrose, 10
2 2 4 4
, 2 3 3
,
uM MnS0 H uM ZNS0 Mo0
uM uM
Na
10 0, 3 -7H 0, 0.1 -2H 0, 0.01
4 2 4
2 2 4 2
CuS0
mM)
-5H 0, 0.01 |iM CoCl -6H 0) with 1.2% (w/v) sucrose 9-cm
(35 in
4
2 2 2
Petri dishes (Guiragossian-Kiss and Kiss, 1998). All plates were prepared and
poured from the same Knudson's-C
preparation to ensure a consistent con-
centration of sucrose. Dark control plates were wrapped in aluminum and
foil
same
incubated in the conditions as other plates within the same experiment.
Spores were incubated d under
for 7 various experimental conditions, with the
exception of the moisture experiment, during which were
spores
treated
for
When
10 days. feasible, conditions for each (Table were maintained
test 1) at
_1
pH
nmolm"2
5.5, 25°C, 100 s of white light, and saturated (agar medium).
However, the parameters of some experiments required different conditions.
For example, with
respect to light quality, spores were incubated 33°C and
at at
_1
umolm"2 maximum
75 achieve and
s to consistent each
light intensity in
light quality.
Temperature, Q
Light Intensity, and Light To examine
the
Knud
pH
was To
not used.
test the effects of temperature on spore germination,
spores were incubated and
at 4°C, 25°C, 33°C. Light intensity was established
by GE PAR
using Halogen XTRA
either Ultra 38 (90 watts) or Sylvania Halogen
PAR
38 (120 watts) and by varying the distances between and
the source
light
the was
spores. Light intensity verified with a Li-Cor Quantum/Radiometer/
Photometer, model
LI-185. Light intensities were:
(dark), 10, 50, 75, 100, 125,
NONDORF
ET SPORE GERMINATION CHEILANTHES
AL.: IN 59
_
-2
-l
150 (imolm Treatments were contained within chambers avoid
-s~ to
.
incidental light and cooled with an electric fan system. Dark treatments were
prepared by wrapping Petri dishes in foil after they were inoculated and sealed
with Parafilm. Light qualities tested were blue, red, white, green, far red, and
These were
dark. light qualities established using plexiglass (Cadillac
filters
Wavelengths were measured
Plastics, Southfield, MI). for light using
filters
Data Logger Pro software and were: white (420-710 nm), blue (420-570 nm),
green (500-595 nm), red (600-695 nm), red (650-705 nm).
far
—
Dark Control (Dark Plus True Dark). During the surface and
vs. sterilization
sowing process, spores are typically exposed white approximately 45
to light for
min
and this characterizes spore preparation for experiments. Even dark
all
To
treatments receive this white light prestimulus (Dark Plus). test the affects of
this white pre-stimulus, the germination rate in the standard dark control
was compared
preparation (Dark Plus) with the germination of spores
rate that
_1
sown 2
were and
surface sterilized in 0.04 (.imolm~ s of white light (True Dark).
—
To
Moisture Levels. avoid introduction of additional moisture, spores were
not surface sterilized and were sown on sterilized paper (Whatman #1, 9
filter
cm) that were wetted with Knudson's-C (no sucrose avoid contamination
to
would no
that hinder germination, agar). Moisture levels were: 10, 20, 30,
0,
2
40, and 50 |ilcm~ Germination rates were counted at 7 d, but protonema were
.
allowed to develop to the tenth day for observation and measurement.
—
Data Analysis and Sampling. To ensure that spores, which germinate quickly,
were counted within only a few hours of each other, but to also obtain large
sampling, experiments were conducted independently. Testing
variables
all at
one time would sacrifice the integrity of the counts. Spores were sown on 4—10
plates per treatment, depending on the parameters of the experiment. For
example, only 4 plates were used in light quality experiments to ensure that
all
were and same
plates placed within the center of the received the
filters light
intensity. Spores were scored as germinated or non-germinated on a haphazard
basis up to 300 spores per plate, depending the number of plates allowed by
the parameters of the experiment (n = 400-1200 spores per treatment). Spore
when
germination was counted the exine had ruptured and protonemal
cell
was
extrusion Because germination/non-germination binomial
visible. a
is
were method
score, the data transformed using the arc sine transformation to
An =
ensure normality. analysis of variance was then performed
(a 0.05) to
determine and
significance of differences in results. For clarity continuity, the
transformed means and standard deviations were used in figures.
—
Porosity, Specific Retention, and Actual Retention. Rock samples were
obtained with special permission from Reis Biological Station (RB), Steele-
MO, Mammoth
ville, from bluffs along Big River at Road (MR), approximately
MO
MO
mi Highway and
1.5 south of H, from private land in Cedar Hill (CH),
Highway Highway
approximately 1.5 east of 30 along BB. Because collection
was destructive, rock sample were purposely limited 10 approximately
sets to
cm 3 Two
2.5 pieces. sample sets were collected from each One was
site. set
VOLUME NUMBER
AMERICAN FERN JOURNAL:
60 93 2 (2003)
200
180
160
c
140
o
120
c
100
80
CD
60
5?
40
pH8.5
pH5.5 pH6.5
pH4.5
20
PH
pH
on
Fig. 1. Effects of spore germination, transformed data. Cheilanthes feei germinated best in
pH There was no difference between pH and Dark germination shown) were
4.5. 6.5 8.5. rates (not
than 5%.
less
from and
collected C. feei habitat one
but elsewhere on the bluff where
fi
=
n
determined as 100[l-(P /P where P (bulk density) defined as the
b d )], b is
original sample oven dried weight divided by the saturated pre-oven dried
(g)
3
volume (cm and P
(particle density) defined as the original sample oven
d is
)
dried weight divided by the mineral matter volume (cm 3
Specific retention
(g)
).
was
of the substrate determined as the amount that the substrate can retain
against gravity divided by the volume Actual
total (Fetter, 1988). retention
values were subsequently determined cm 2
for planes within the substrate. This
was amount 3
calculated as the of water (cm retained against gravity divided
)
further
comparison with
laboratory conditions.
Results
Temperature, Light and
Intensity, Light Qi Germination
=
400-1200)
pH
germinated pH
at the highest rate in acidic (pH 4.5 and Limestone
5.5).
M
since Knudson's-C does not buffer well pH. Dark
at this controls (Dark Plus;
800-1000)
spores
fi
germinated optimally 25°C Note
at (Fig. 2). that germination also occurs at 33°C
and
Dark
at 4°C. controls (Dark Plus) are not shown. Dark germination
rates at
25°C and 33°C were less than 5%. At 4°C in the dark no
(storage conditions),
=
germination occurs. Spores (n 600-1200) germinated under wide
a range of
light intensities (Fig. 3). All germination rates are low but comparable to
difficult
The umolm^s
intensities). optimal germination occurred 100 1 and
rate
at
were
there between
significant differences
the highest germination
(100
rate
NONDORF ET SPORE GERMINATION CHE1LANTHES
AL.: IN 61
O
c 50 -
£
40-
30 25°C
I
20
33°C
10
4°C
Temperature
The
Effects of temperature on spore germination, transformed data. optimal temperature
Fig. 2.
for spore germination is 25°C. Germination in 33°C and 4°C is significantly less. However, spores
germinated in temperatures. In the dark controls (not shown), no germination occurred at 4°C
all
5%
(storage conditions). In the dark, C. feei germinated below at 25°C and 33°C.
2
|imol-m'" 1 other light intensities, and the dark control (Dark Plus) rate.
-s"
),
Cheilanthes feei spores germinated under all light qualities, even in the Dark
Germination varied
Plus controls rates in different light qualities
(Fig. 4).
was no
but not For example, there significant difference
greatly, significantly.
A
was
between white, red and green. notable difference in the far-red
far
were
treatment in germinated spores. All germinated spores in this treatment
when
at the 2-cell protonemal stage scored. This was not observed in any other
Dark germinated 7.7%. Prepa-
quality treatment. controls (Dark Plus)
light at
Without
mm
40
35
30
c
o
25
03
C
20
g
•
15
e>
10
150
75 100
125
5
-2—
pmohm
-1
Light Intensity in # s
Effects of light intensity on spore germination, transformed data. Germination rates for
Fig.
3.
umolm
were predictably low in 33°C. The optimum rate was under 100 ^•s- 1 and there were
significant differences between light treatments and the dark controls.
~
AMERICAN FERN JOURNAL: VOLUME NUMBER
62 93
2 (2003)
120
100
c 80
o
c
60
E
40
20
Light Quality
Fig. 4. Effects of light quality on C. feei spore germination, transformed data. With the exception
of far red, results in each light quality were highly No
variable. significant difference exists
between
germination any
rates in light quality. However, spores germinated in far red light were
found at the 2-cell protonemal stage. This did not occur in any other light quality treatment. Dark
germination was 7.7%.
at
=
exceeded any
previous light treatments. Dark Plus: n True Dark
590;
n
4300.
Moisture Levels.—Although
Cheilanthes
feei spores germinated in the dark on
dry paper, germination were 2
filter rates optimized 20-50 ulcnT
in the
light at
There was no
(Fig. significant difference between
6). these light treatments, but
was
there a significant decrease in germination in the dark controls between
2
20-50
ul-cm as moisture increased. was
In addition,
there a substantial
difference in protonematal presence and 2
maturity. For reference, 20 ul-cm
mildew
will support growth and damp
merely
is to the touch. Microscopically,
no -2
liquid stands between
the fibers of the filter paper. At 30 ul-cm a film of
,
water coats the Above 40 ulcm"2
fibers. the filter paper saturated and water
is
,
stands between -2
the Protonema
fibers. that germinated 20 ulcm were
in
approximately when
200
urn in length scored and
exhibited planar growth, but
-2
protonema and
in 40 50 ul-cm were
only 100 and
urn in length
still
filamentous
(Fig.
7).
Porosity, Retention, and Actual
Retention.—
Reis
Biological
Sta-
MO
Mammoth
Road (MR)
rural site (approximately 30 mi from the Louis
metropolitan
St. area) that over-
ramp
looks a boat and fishing The worn
area. area by Cedar
is foot Hill
traffic.
(CH)
is private rural land that approximately mi
16
is outside of the
NONDORF ET SPORE GERMINATION CHEILANTHES
IN
AL.: 63
100
X
90
80
70
O
True
60
c Dark
50
E
40
Dark
30
Plus
20
10
Dark Treatments
The
Fig. Effects of white light prestimulus on dark germination in C. feei, transformed data.
5.
45 min white prestimulus (Dark Plus) strongly affects the germination rates of dark
light
germinated spores. Cheilanthes feei germination in True Dark scores between 80-100%.
MO
metropolitan area and the closest of the sites to the city (St. Louis,
metropolitan
area).
Average and retention increased with distance from the
porosity specific
Porosity the amount of pore space in a rock sample compared to the
city.
is
means
volume, and expressed as a percentage (Fig. 8A). Porosity
total is
120
100
O
80
c
mm
60
E
40
20
10 20 30 40 50
Moisture Levels ul*cm -2
in
on spore germination, transformed data. In the light
moisture
Fig. 6. Effects of levels C. feei
(white), germination optimizes at 20-50 ul-cm" 2 There is no significant difference between
.
2
germination rates in these treatments. Dark germination (Dark Plus; gray) at 20-50 ul-cm~ begins
2
between 20-50 ul-cnT Note that
and difference
decrease gradually there a significant
to is
.
germination occurs on dry paper in the dark.
filter
VOLUME NUMBER
AMERICAN FERN JOURNAL:
64 93
2 (2003)
-2
Protonematal maturity
Fig. 7. at different moisture levels. (A). Protonemata grown 20 ul-cm
at
are approximately 200 urn in length and exhibit planar growth. Protonemata grown
(B). in
-2
um
50 ul-cm are approximately 100 and
in length are filamentous.
Mean
Fig. (A). porosities Cedar Mammoth
8. at Hill (black), Road and
(white), Reis Biological
Station There was no
(gray). significant difference between areas within the same stratum that
contained Cheilanthes feei and areas which was
in C. feei absent. (B). Specific retention increased
with and
porosity with distance from
the metropolitan
area. Conversely, variation in specific
retention decreases with
distance. Actual amount 2
(C). retained was
in ul-cm" calculated as the
specifically retained water
distributed equally throughout
the pore space and mathematically
expressed within one Means
plane.
rock samples
for all taken from each between 20-30
site
fell
ul-cm
.
NONDORF ET SPORE GERMINATION CHEILANTHES
IN 65
AL.:
35%
30%
25%
20%
.£
8
o
15%
0.
10%
5%
0%
Present Absent
C. feei C. feei
all
(A)
14%
12%
o
10%
£ 6%
4%
a.
0)
2%
0%
Absent
Present
C. feei C. feei
all
(B)
90
80
N
o
70
60
c 50
a
40
30
o
20
£
<
10
Present Absent
C. feei C. feel
all
(C)
