Abstract
Salt tolerance and acclimation ability of two seashore lichens, Lecanora
pinguis and Niebla homalea, were examined to determine to what extent they are
actually marine adapted. Photosynthetic rate was used as an indicator of the
lichens' response to different conditions. Tolerance was tested by soaking
samples in distilled and various salt water concentrations for various lengths
of time, and acclimation ability was determined by giving preliminary treat¬
ments of distilled or salt water and then observing their effects on responses
to later treatments. L. pinguis was found to be more tolerant of salt water
than N. homalea, but N. homalea showed ability to acclimate to either marine
or terrestrial conditions, while L. pinguis could not adapt to terrestrial
conditions. Thus, L. pinguis is a true marine lichen, while N. homalea is more
accurately described as a salt-tolerant terrestrial lichen. Typical photo¬
sythetic rates for this study were 150 ul.g'ihr-1 for L. pinguis and 75 ul.g ihr
for N. homalea, as measured on a Gilson model IGRP 14 differential respirometer.
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Don Stallard
Introduction
Maritime or supralittoral lichens form communities close to
the sea, but salt tolerance of specific lichens has not been ex¬
amined. Perry and Sheard (1969) postulate that some maritime
lichens need regular washing with salt water. Other workers
(Fletcher (1973) and Hawksworth (1980)), for example, have arranged
lichens in various zones depending on their tolerance (halophilic)
or intolerance (halophobic) to salt. Distribution of species rela-
tive to exposure (Ballatine (1961)) or frequency of submersion
or spray (Weddell (1875)) has also been examined. Competion be¬
tween other organisms and lichens may also be important in deter-
mining their occurrence on seashore rocks.
A true marine lichen is defined here as one that can tolerate
life on the seashore and prefers to live there, being unable to
acclimate completely to another environment. Fischer-Piette and
Davy de Virville (1931) demonstrated that Caloplaca marina Wedd.,
occurring on seashore rocks, was not found on nearby estuary shores
with similar available substrates. The primary difference between
the two sites was the amount of exposure to salt spray of the ocean.
This investigation is designed to determine the effects of
various salt conditions on two other common supralittoral lichens.
Niebla homalea (Ach.) Rund. and Bowl. and Lecanora pinguis Tuck.
Samples were treated in distilled water, approximating rain which
is periodic (November through April) in this region, and were
soaked in various concentrations of seawater, approximating that
salt exposure to salt spray that is rapidly evaporated and there-
fore leaves very concentrated salt. Photosynthetic rate was the
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physiological response observed in all cases.
Materials and Methods
The experimental procedure was in two parts (refer to Fig. 1).
one designéd to test the salt tolerance of the two species and
the other to acclimate the species to different conditions. Six
to nine samples of each lichen for the tolerance test and four
samples for the acclimation ewperiment were collected in the morn-
ings when high natural water content of the thallus allowed easy
removal from the rocks. Squirts of distilled water were used to
raise the thallus water content of L. pinguis when field control
samples were collected in the afternoons and evenings. Care was
taken to choose samples with as little rock and sand imbedded as
possible.
As shown in the flow chart (fig. 1), the procedures for the
tolerance and acclimation studies were very similar. At step B
on the flow chart, the samples were soaked in different concentra¬
tions of artificial seawater, for 16 hours in the acclimation tests
and for periods of 24, 72, 144, and 264 hours in the tolerance tests.
Normal seawater concentration was set at 33 parts per thousand, and
all other concentations were based on this figure, which is an
average for Monterey Bay. The salt solutions were changed after the
samples were removed at 72 and 144 hours in the tolerance tests. In
both experiments, dry control and field samples were submerged for
one minute in distilled water and then air dried on a paper towel
for one hour before being measured for oxygen production in a
Gilson model IGRP 14 differential respirometer. All respirometer
chambers contained a bicarbonate buffer mixture of .035 M KHCO-
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and .065 M NaHCO, designed to keep C0 concentration constant
without affecting photosynthesis rates (Pratt, 1943).
The samples were run at 15°0 for alternating hour-long periods
of dark, light (290 uE.mfs-1), dark, and gas volume changes were
read at the end of each period. Following the three hour period,
the samples were oven dried at 100°0 and then weighed to give
absolute gas evolution rates.
The major difference in procedures came at step A of Fig. 1
in the acclimation experiment. Here samples of each species re¬
ceived twice daily treatments of either distilled or 2.5 X sea¬
water concentration, or were left untouched. These samples were
kept in a box outdoors so they would experience normal light,
temperature, and humidity cycles. Each spray treatment involved
three squirts from a spray bottle delivering 0.5 ml per squirt.
The tolerance test samples were kept indooors during the soaking
periods, but experienced normal light cycles through a large win-
dow.
Results
Lecanora pinguis showed 234.4 ul-gihr-1 for its highest
photosynthetic oxygen production rate (Fig. 2) in 2 X seawater
concentration. Intermediate concentrations of salt water in
general elicited higher rates than any other concentrations or
distilled water. By 144 hours, distilled water had greatly de-
pressed photosynthetic rates, while the salt water treatments
failed to reduce rates after the 72 hour mark. 10 X seawater
concentration (Fig. 2) depressed rates to a constant level by
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the 24 hour point.
On the other hand, in distilled water, Niebla homalea (Fig.
3) showed 90.2 ul.g:hr-l as its highest photosynthetic oxygen
evolution rate. Rates in distilled water were consistently equal
to or higher than those of samples treated with any concentration
of seawater. Concentrations of 5 X seawater or greater were photo-
synthetically limiting by the end of 24 hours soaking, while treat-
ment for 144 hours or more in any solution (distilled or salt
water) depressed rates to levels that remained constant through
264 hours.
Niebla homalea displayed ability to acclimate (Fig. 4) to
different water conditions, showing highest rates of photosynthe-
sis after experiencing a certain condition when previously exposed
to that condition. Lecanora pinguis, on the other hand, showed no
ability to acclimate to any specific condition.
Despite large standard deviations, rates of photosynthesis
(Fig. 4) from samples subjected to different treatments in the ac-
climation experiment substantiated the results of the tolerance
tests. Niebla homalea showed highest rates following a distilled
water treatment, while Lecanora pinguis produced the most oxygen
after a salt water treatment. Soak treatments in general elevated
photosynthetic rates over those of dry samples, except for L. pin-
guis soaked in distilled water. N. homalea samples from the con-
trol and field groups photosynthesized at much higher rates rela-
tive to soaked samples than did L. pinguis samples.
Discussion
The above results suggest that Lecanor
pinguis fulfills the
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two criteria of a marine lichen, namely tolerance to salt and
inability to acclimate to less marine conditions, while Niebla
homalea only meets one, that of salt tolerance. Thus, L. pinguis
appears well adapted for life in the supralittoral zone, while
N. homalea fits well in the seashore terrestrial region, having
characteristics of both halophilic and halophobic lichens (Flet-
cher (1973)).
Remarkable in itself is the fact that the algal component of
both lichens managed to survive even the harshest conditions and
produce a photosynthetic rate at least one-quarter of the average
field sample rate. Heat-killed and salt-soaked samples showed
plasmolyzed algae and zero photosynthetic rates, while the experi-
mental algae were normal in appearance. The lichen thallus as a
whole often appeared duller in color following prolonged soaking,
Further salt tolerance studies on terrestrial lichens would clear
up the question of whether the survival of Niebla homalea for 264
hours in 10 X seawater concentration is evidence of some marine
adaptation, or another example of the extreme hardiness of lichens
in general.
Both species showed higher photosynthesis rates when the
thallus percent water content was higher owing to long soaking
period. This result was surprising, for Lange (1980) found that
Ramalina maciformis (Del.) Bory had photosynthetic rates approxi-
mately equal at thallus percent water contents of 60 and 85. This
difference could possibly be due to the difference in soaking dura-
tions. 60% water content was reached by the field and control
samples, which were soaked for only one minute before the hour dry-
ing period, while the 85% water content was the result of a full
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day of soaking followed by a one hour dry-out.
The experiments were designed to eliminate as much variabil-
ity as possible. In the tolerance tests, the control group was
used to test the effects of room temperature and light levels as
compared to field conditions. The unsprayed control of the accli¬
mation tests was used to determine the effects of removal from
rocks and seashore conditions. In both cases, controls and field
samples showed no significant differences in photosynthetic rates.
The one hour drying periods on paper towels following soak
treatments were to allow thallus hydration to decrease somewhat
from saturation and bring percent water content to comparable
levels in all samples of a species. Also, it was assumed that any
rehydration respiration in field and control samples was stabilized
by the end of this period, making the average of the respiration
rates for the two dark periods in the respirometer approximately
correct for the light period, when photosynthesis was measured.
Studies done with Peltigera polydactyla (Neck.) Hoffm. found re-
wetting respiration after a one hour drying period to be approxi-
mately linear when plotted against time (Smith and Molesworth
(1972)).
All oxygen production figures have been corrected for temper-
ature and air pressure to give absolute values. Also, a correc-
tion factor to counterbalance added weight from salt deposition
was employed. Salt deposition increased linearly with solution
concentration for both species (Fig. 5). The concentrations of
the soaking and spraying solutions were kept within 5% of listed
values throughout the experimental period.
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Variability was considerable in this study, and if it were
to be repeated, certain methods could be changed to get more con¬
stant response values. Using only part of the thallus rather than
the whole plant would reduce any error introduced by the differences
in size or number of apothecia or other structures. Age of the
plants may be hard to estimate, but if it were possible, that fac-
tor, along with orientation in the field of samples gathered for
study, could be standardized, probably also resulting in less
variation. Finally, use of respiration rates as an indication of
stress or acclimation might provide more standard responses.
Photosynthesis was used as the indicator in this study because
of its higher rates, which make measurement easier.
A certain amount of variability is inherent in lichen photo-
synthesis rates, and the variability of photosynthesis rates in
this study seemed to follow certain trends as a function of soak-
ing solution concentration and regardless of soaking period length
throughout the tolerance experiment. Greatest variability in
rates for Niebla homalea were seen in lowest and highest concen-
trations of seawater, with less variability at the intermediate
concentrations. Lecanora pinguis, on the other hand, showed great-
est variability for one day soaking in distilled water and seawater
concentration, with all remaining concentrations and soaking dura-
tions having approximately the same variability.
Special thanks is given here to Dr. D.P. Abbott for his ad-
vice on graphics and lessons in enthusiasm, Chuck Baxter for his
examples in common sense, Dr. J. Watanabe for-explaining the num¬
bers, Mason Hale of the Smithsonian Institute for giving me species
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names to replace my pet names, all other members of the Hopkins
staff, and most especially, Dr
. 1. A. Abbott, who showed me it is
possible to overcome the formal bounds of professor-student re¬
lationships and become friends.
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Literature Gited
Ballantine, W. J. (1961). A biologically-defined exposure scale for the
comparative description of rocky shores. Fld Stud. 1: 1-18
Ferry, B.W., Sheard, J.W. (1969). Zonation of supralittoral lichens on rocky
shores around the Dale Peninsula, Pembrokeshire. Fld Stud. 1: 1-18
Fischer-Piette, E., Davy de Virville, A. (1931). Extracted from p. 265,
Johnson, T.W., Sparrow, F.K., Fungi in Oceans and Estuaries, (1961) J. Cramer
Fletcher, A. (1973). The ecology of maritime (supralittoral) lichens on
rocky shores of Anglesey. Lichenologist 5: 401-422
Hawksworth, D.L. (1980). Lichens of the South Devon coastal schists. Fld
Stud. 5: 195-227
Lange, O.L. (1980). Moisture content and C09 exchange of lichens. Oecologia
45: 82-87
Pratt, R. (1943). Studies on Chlorella vulgaris. VIII, Influence on photo-
synthesis of prolonged exposure to sodium bicarbonate and potassium
bicarbonate. Am. J. Bot. 30: 626-629
Smith, D.C., Molesworth, S. (1972). Lichen physiology XIII, Effects of re¬
wetting dry lichens. New Phytol. 72: 525-533
Weddell, H.A. (1875). Excursion lichenologique dans l’ile d’yeu sur la Cote
de la Vendei. Mém. Soc. natn. Sci. nat, math, Cherbourg 19: 257-316
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Captions
Eigure 1: Procedural flow chart of methods used for acclimation
and tolerance tests. Concentrations are times normal seawater
(33 ppt).
Figure 2: Photosynthetic response of Lecanora pinguis as a per-
centage of the highest rate (234.4 ul-g:hr-1) in response to
various salt and distilled water conditions. Bars represent
% confidence intervals. N-6-9. Bars over c and fare cont. & fiel
Figure 3: Photosynthetic response of Niebla homalea to various
salt and distilled water conditions as a percentage of the high-
est measured rate (90.2 ul.grihr-4). Bars represent 95% confidence
intervals. N=6-9. Bars over c and f are control and field.
Figure 4: Photosynthetic response of Niebla homalea and Lecanora
pinguis to various distilled and salt water conditions following
a two week acclimation period. All figures are percents of the
highest rate for each sample's own species (64.8 and 402.1 ul.gihr
for N. homalea and L. pinguis, respectively). Bars represent 95%
confidence intervals. N=4.
Figure 5: Oven-dry weight as percentage of fresh weight plotted
against concentrations of soaking solutions. Soaking was for
24 hours. Bars represent standard deviations.
—
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.
—
3

19-

5
S

—  —
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4
)
—

0
a
—
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——
(0
—
O
—
—
(9
6
—


— — —
5

O
Distilled
water
1X
SW
2X
SW
))(
SW
10X
SW
Tday 3days 6days 11days
-100


A
100
so
LA
LEd V
100
p.s. rate
50 as%of
A
greatest
ae
H
A
A

-100
50



AA
Salinity tolerance of Lecanora pinquis as
indicated by photosynthetic rate

Fig. 2
Distilled
water
IX
SW
2X
SW
5X
SW
IOX
SW
3 days
I days
6 days
day

W

L
V

A
D


L

W
A
A
Salinity tolerance of Niebla homalea as
indicated by photosynthetic rate
Fig. 3
.
100
50
100
50
100
p.s. rate
50 as % of
greatest
rate
100
100
50
—


E
0

L



1
5

8
8
H
+E
L

E
00

Dry wt.

as % 050
fresh wt.
O


—
Niebla homalea

Lecanora pinguis
—
10X
5X
Seawater Concentration
Fig. 5