Matthew Perl
ABSTRACT
Spore packets from Smithora naiadum (Anderson)
Hollenberg were shown to settle preferentially on its
natural host Phyllospadix torreyi (S. Wats.) over several
other natural and artificial substrates.
Flourescein dye was introduced to both intact and
rhizome-severed basal portions of Phyllospadix blades but
showed little translocation beyond point of entry. This
dispelled the possibility of distant translocation of
nutrients through Phyllospadix and into its epiphytes.
An attempt to qualitatively determine if Smithora
receives biochemical communicants from its seagrass host
yielded no such products. A possible procedure for approaching
the question was developed though.
A possible circadian variation in photosynthetic rate
in the red alga Smithora was demonstrated.
INTRODUCTION
The red alga Smithora naiadum has been only described
growing on two closely-related seagrasses, Phyllospadix
torreyi and Zostera marina L. (Hollenberg, 1959). Harlin
(1973a) reported culturing immature Smithora on a synthetic
seagrass, "Olefern," but reproductive maturity was reached
on natural hosts alone (Harlin, 1973b).
Harlin (1973b) further demonstrated the movement of
labelled carbon from seagrass host to Smithora epiphyte
in culture. Possibly the red alga receives some nutrients
or a biochemical developmental cue from its host. This
"messenger" might well be derived from the host's photo-
synthetic process. Sugars have been found to be passed
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from autotroph to parasite (Smith, et al, 1969). Other
plants have been demonstrated to leach photosynthetic
products, some of which are subsequently reabsorbed (Tukey,
1970; Tukey & Mechlenburg, 1964). Brown algae actually
absorb sugars from their environment which are later
processed by the plant (Drew, 1969). Penhale and Smith
(1977) found that Zostera released photosynthates into
surrounding water and suggested that these products might
be used for nutrition by the adjacent epiphytic community.
Electronmicroscopy has shown there are no cytoplasmic
connections between the laminae of the host seagrass and
the rhizoids of the alga (McBride & Cole, 1969). Seagrass
release, followed by algal reabsorbtion, presents itself
as a candidate for the Smithora-Phyllospadix association.
My investigations were aimed at helping to explain the
alga's host specificity through nutritional requirements.
In addition other experiments were also directed
towards clarifying the symbiosis. Spore settling studies
followed substrate preferences of the loosed marginal sori
(spore packets). Daily tests were conducted to see if
the algal sori preferentially land on their natural hosts
or if random chance alone dictates where the spore bundles
settle. Dye movement in the seagrass was examined as a
possible indicator of nutrient flow between host and epi-
phyte. Final experiments measured photosynthetic activity
of the red alga over 24 hour periods to further describe
Smithora itself. Circadian cycles, demonstrated in innu-
merable plant and animal species, have received sparse
attention in the algae until recently. Daily variation
in photosynthetic capacity for a red alga Iridaea flaccida
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reaches a maximum near noon, when available light also
peaks (Harris, 1978). A daily cycle was shown for Ulva
lactuca, and seasonal variations above and beyond the
daily one were also discovered (Mishkind, et al, 1979).
SPORE SETTLING
am
MATERIALS AND METHODS
A special frame was built from 1 by 2 redwood with
central horizontal strips to sandwich the substrates under
examination (Figure 1). Phyllospadix, another sea plant
Gracilaria, and a land plant of the cat-tail family Typha
sp. were the organic substrates chosen. Rubber and glass
were also tested. The substrates were prepared as thin
strips to resemble the Smithora's natural hosts. Yellow
flags attached to vertical posts facilitated location
of the apparatus during high tides.
The frame was freshly prepared with the substrates
each morning for four consecutive days. Phyllospadix was
cleaned of all epiphytes before being inserted; other
substrates were examined to remove any blotches which
might later be confused with algal spores. Four strips
of each substrate served as replicates for each trial.
The frame was tied to stakes in the lower intertidal such
that it would remain submerged for all but the lowest
tides. The tie stakes were planted adjacent to densely
Smithora-covered Phyllospadix to insure availability of
algal sori.
The apparatus remained in the field for 24 hour inter-
vals. At the time of collection it was removed from the
stakes and brought into the lab where spore packets could
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be counted. Each substrate strip was examined with a
hand lens, and a record was made of the number of packets
which had settled on it. The strips were then cleaned
or replaced before the frame was returned to the field.
1
RESUDI
Figure 2 shows the results of four runs. The
Phyllospadix had a far greater number of spores land and
attach than other substrates tested. The figures vary
from one day to the next but are consistent for each day.
All figures are lower estimates because spores which had
landed on other spores could not be resolved. During the
study it was observed that spore packets will wash off
most substrates on which they settled except Phyllospadix.
SION
This experiment showed a marked spore preference for
attachment to Smithora's natural host (Phyllospadix) over
several other substrates, whether natural or synthetic.
This phenomenon may be a derivative of the obligate rela-
tionship between the seagrass and the alga.
Smithora may
be bound by some sticky, mucus-like substance on the
Phyllospadix. Extracts of the seagrass, coated on glass
rods, could be used to test this possibility.
The frame received exposure to a full range of tides
during the experiment. Some mornings the apparatus lay
fully exposed. At other times it was submerged beneath
three feet of water. These widely variable circumstances
serve to substantiate results obtained rather than to com-
plicate them. The data represents full exposure to tide
fluctuations. These are the conditions under which sori
will encounter their host seagrass as well as other substrates.
Matthew Perl
While settling data from one day to the next may not be
compared quantitatively, the relative magnitudes of settling
within each trial, a qualitative demonstration of host
binding preference, stands up consistently. Thus I may
say that in the ocean the spores tend to attach to Phyllo-
spadix rather than to an old bottle or stray Gracilaria
strand.
That Smithora has a much greater binding rate to its
host makes for a survival rate higher than chance landings
alone. If there is a real physiological requirement,
provided by seagrasses, for Smithora to mature, then a
distinct preference for spores to settle on that host is
a tremendous reproductive advantage. Particularly in the
marine environment, where continual water motion is a rule,
a true binding capacity of symbiote for its host is beneficial.
NUTRIENT TRANSLOCATION
The possibility that nutrients are absorbed through
the roots of Phyllospadix and move up through the rhizome
structure into the blades, as a prerequisite for transfer
directly to the epiphytes, was examined.
TR
TERIALS AND M
THODS
A patch of the seagrass, lightly colonized, was uprooted
and rinsed in seawater to remove debris and detritus from
the intact root-rhizome system. Large blades of Smithora
which might interfere with dye position determination were
cut from the seagrass's laminae. The plants were divided
into two similar clumps for parallel studies. One half
had roots and rhizomes severed near the proximal portion
of their sheathes; the other clump remained uncut. Through-
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out the procedure the plants were submerged in ambient
seawater.
The basal ends of both seagrass portions were inserted
into a concentrated flourescein-seawater solution (Figure 3).
The blades were put into plain seawater. Tissue was draped
over the air-exposed sections to prevent drying out and
to prevent the dye from carrying from one vessel to the
other across the Phyllospadix's surface. The plants were
kept in the dye for four hours. Previous dry runs had
shown longer exposures (up to 24 hours) to have negligible
effect on the results of the experiment. The plant was
exposed to light of intensity 13 pEin/m/s for the duration
of the experiment. The baths varied in temperature from
17°C to 21°c.
After the set interval the plants were removed from
the apparatus and immediately washed in fresh water to
remove dye from their exterior. They were then laid out
on moist paper in a dark room. The plants were sectioned
into 5 cm. lengths to prevent further dye movement and to
simplify subsequent measurements. An ultraviolet lamp
(UVSL 25 Mineralight) was used to determine the longitudinal
distance the dye had penetrated each Phyllospadix blade.
Also, the total length of each blade was measured. Finally
a notation was made as to whether a particular blade was
intact (designated normal) or had been torn at its distal
end (broken).
SULTS
Table I summarizes the results of the simulated nutrient
movement. Little translocation of dye beyond the surface
of the flourescein solution was observed for normal blades.
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The liquid in the dyeing vessel was 12 cm. deep; these blades
had a mean dyed distance of 13 cm., both for sections with
and without root structures.
Broken strands of Phyllospadix (torn at the distal
leaf tip) showed much greater dye transfer. In the sample
clump which retained its root complex the dye moved an
average of 23 cm. beyond the surface of the coloring solution.
Most notable were the strands severed at both ends. These
laminae moved the dye an average of 12 cm. or 98% of the
average total length of all blades.
ISCUSSION
While many marine plants have been described that absorb
nutrients through their root systems (McRoy & Goering, 1974),
this mode of action is apparently not significant for
Phyllospadix. Experiments on closely-related Zostera showed
good uptake of, but minor subsequent movement of either
"CO or 3P through the root-rhizome system (Penhale & Thayer.
1980). The absence of flourescein movement beyond the surface
of the dye suggests that Phyllospadix used an alternate method
of nutrient absorption as well. One might expect some residual
level of root uptake since seagrasses are submerged vascular
plants. However, it seems that as the sea permits the entire
plant to be bathed in a nutrient broth, absorption via the
entire leaf surface is a more efficient and effective way
for the seagrass to "feed." Nutrient translocation after
absorption by the root system would be minimized, as observed.
The results of the dye movement studies cannot be considered
absolutely conclusive. The flourescein molecule is larger
than many inorganic nutrients like nitrogen and phosphorous.
These smaller molecules may be more easily absorbed by the
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roots. Experiments indicate otherwise however, showing
negligible 'C uptake by roots or rhizomes when compared
"C absorbed through the leaves (Barbour & Radosevich,
1979).
Lateral sections of dyed Phyllospadix showed the dye
tended to travel by way of three air canals of the lamina,
probably by diffusion. These canals extend the length of
the leaf and may play a role in gas transport in the plant.
These tubes are sealed at the tip. When blades were severed
at both ends they acted as siphons. Water was observed to
move from a high reservoir to a lowerone via the blades.
This activity explains the near complete transfer of dye
through the broken, root-severed laminae. Most likely
though this activity doesn't reflect on any life-preserving
function in the real world.
LEACHATE ANALYSIS
An experiment was run to isolate and identify carbo-
hydrates released by Phyllospadix into surrounding water.
Later experiments would be aimed at demonstrating uptake of
these sugars by the epiphytic community, if these nutrients
could be located.
THODS
MATERIALS AND !
A portion of Phyllospadix was uprooted and transferred
with seawater into the lab. The plant was rinsed in fresh
water to remove microorganisms; all visible epiphytes were
removed. The plant was inserted into the apparatus shown in
Figure 4. The collection container into which the seagrass
ran was called "active;" the other, "control." Each contained
900 ml. of 0.22 um filtered "Instant Ocean.'
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The leaching experiment was conducted for five days.
Water in the two collection vessels was continuously agitated
by magnetic stirrers. The plant was exposed to 125 pEin/m/s
on a light-dark cycle which approximated the normal daylight.
Air-exposed seagrass parts were wrapped in plastic to reduce
desiccation. The baths were kept at about 11 C.
At the end of the experimental period the two collection
solutions were filtered to remove all debris; centrifuged
samples from each vessel showed no particulate impurities,
so 600 ml. of fluid was poured off of each one. In a control
experiment both active and control were spotted on paper
chromatograms to check for the presence of carbohydrates.
The solvent system used was n-butanol/ethanol/water (40:11:19);
ammoniacal silver nitrate was sprayed on finished chromatograms
to reveal any sugars (Benson, et al, 1950; Heftmann, 1967).
For the final run the control aliquot was refrigerated while
the active sample was lyophilized. The dried salts from
the active chamber were then stored in a desiccator while the
control was freeze-dried. Drying of both samples took five
days.
The next step in the analysis involved separating minute
amounts of photosynthates presumably released by the seagrass
from the large mass of sea salts to allow concentration of
the organic fraction. Carbohydrates and inorganic salts
share similar solubilities in most solvents, complicating
separation. A trimethysilyl (TMS) ether formation promised
to be the easiest to prepare and the most stable (Hammarstrand,
1968; Szymanski, 1968; Holligan & Drew, 1971). Derivative
preparation involved replacing the sugars' hydroxy groups
10
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hydrogens with TMS groups. The appropriate reagent Sigma Sil-A
(Sigma Chemical Co.) is a mixture of trimethylchlorosilane,
hexamethyldisilazane, and pyridine (1:3:9).
To each dry salt sample (active and control) 5 ml. of
TMS reagent was added under nitrogen, following the directions
supplied with the reagent. The major obstacle at this point
was that the instructions recommended O.1 ml. reagent be
used for each 1 mg. of sample. Because the reactant sugars
were in a mixture with sea salts, it was impossible to know
beforehand the concentration of sugars in each sample.
Thus an anticipated excess of reagent was used.
After two hours the reaction mixture was placed in 10 ml.
of pesticide-grade hexane and vigorously shaken. The hexane
would dissolve the sugars-turned-ethers; inorganic salts are
insoluble in hexane. The solution was decanted to isolate
organic fractions from salts. The liquid was placed on dry
ice and taken to Stanford's main campus. The next morning
10 ul aliquots of the hexane-soluble components of both active
and control solutions were to be gas chromatographed.
LTS
Paper chromatograms from the dry run developed poorly.
Only random splotches appeared on them.
The gas chromatogram of the active sample leachate
derivative showed no peaks beyond those of standard background
noise. The control was not processed because of the poor
showing of the primary sample and because of the prohibitive
cost of G.C. analysis.
DISCUSSION
Harlin's data suggest that there is some type of biochemical
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communication between host and epiphyte of the Smithora-
Phyllospadix complex (Harlin, 1973b). The preceding experiment
supposed the most likely candidates for such a transfer
would be carbohydrates. The primary suspects (fructose.
sucrose, and an unidentified trisaccharide) were described
by Drew data as the major free sugars of Phyllospadix leaves
(Phillips & McRoy, 1980). I was unable to demonstrate that
these or any other sugars are leached by the seagrass. This
result most certainly doesn't serve to discount the hypothesis
of a chemical messenger, nor does it discount sugars'
involvement. If present, sugars may have been in minute
concentrations, undetectable by the procedure which was
followed. The TMS reagent could have been absorbed by the
much greater mass of salts present in the mixture. Reagent
concentration may have been diluted beyond reactive and
detectable levels.
Time constraints prevented repetition of this experiment.
Should the general procedure be repeated, a more effective
method should be sought for separating the salts from the
suspected leachates. Radioactive carbon labelling also
presents itself as a useful tool for identifying the leachates.
Labelled compounds could be followed closely during many
phases of the separation and analysis. Aside from sugars.
other organic products are possible candidates for passing
from seagrass to Smithora. These may serve as nutrients for
epiphytes or in the capacity of hormone-like signals for
induction of algal maturation. The identification of such
transferred leachates should be actively pursued; this effort
would contribute much towards elucidating the precise nature
of the Smithora-Phyllospadix association.
Matthew Perl
12
CYCLIC VARIATION IN SMITHORA PHOTOSYNTI
STIC POTENTIAL
The photosynthetic capacity of the red alga was monitored
over two 24 hour periods. The results were correlated with
natural light availability and tides. Possible relations
between the data and the symbiosis under consideration were
proposed.
MA
ERIALS AND METHODS
The first half of this experiment consisted of the per-
iodic collection of heavily colonized Phyllospadix at four
hour intervals for a 24 hour period. Sections of Smithora
were removed, and the alga's photosynthetic capacity at that
particular time was measured. The second half of the exper-
iment was conducted two days later and differed in that a
large amount of epiphytized seagrass was collected at one
time (6:30 A.M.). Again the photosynthetic activity of the
alga was monitored at four hour intervals for one day. Smithora
not being tested at a particular time was kept intact on its
host in seawater at 12°0 with a light-dark regime to match
the natural diurnal one.
Photosynthetic rates of the epiphyte were measured on
a Gilson Differential Respirometer. Slices 1.2 cm in area
were made of Smithora blades. Six such sections were placed
in each reaction vessel with 6 ml. of filtered seawater.
0.75 ml. of 0.035 M KHCO,/0.065 M NaHCO, buffer solution was
placed in the flasks' sidearms to maintain a constant pressure
of CO gas. Five replicates were run for each time period.
along with a dark control (flask prepared as above but covered
with black plastic) and a plain seawater control.
Respirometer vessels were equilibrated for 30 minutes
prior to each run. The respirometer bath was held at ambient
Matthew Perl
13
ocean temperature of 12°0; shaking speed was 3. Lights for
photosynthesis were partially shielded by mesh screens to
exaggerate periodic fluctuations in the potentials; intensity
was 60 pEin/m/s.
Net change in oxygen within each reaction vessel was
measured at fifteen minute intervals for approximately two
and a half hours. The runs were stopped after two successive
readings showed significantly reduced gas production as
compared to earlier measurements. Mean oxygen production
as ul/15 min. was calculated for each replicate. A single
mean value was obtained for each time interval (6:00 A.M..
10:00 A.M., 2:00 P.M., etc.). These numbers are plotted in
Figure 5 along with daily light-dark cycle and tidal cycle
for comparison.
ESULT
Smithora showed a pronounced, regular variation in
photosynthetic capacity over 24 hour periods. The alga
produced oxygen at a maximum rate in early morning. The rate
drops during daylight hours and then climbs again during
the night. There was a 25% difference between maximum and
minimum production rates in the first trial (samples collected
from the field for each time run). An overall change of 10%
was observed for the second run (algal collection kept in
controlled conditions for the duration of the experiment).
Controls showed negligible changes in gas within their
vessels, never producing nor consuming more than 1 ul of gas
during any 15 minute period. Because of this insignificant
rate as compared to those measured for Smithora in light, the
controls were discontinued for the latter half of the second
run. They do not appear on Figure 5. No measurement was made
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between the hours of 5:00 P.M. and 12:30 A.M. on the first
run because extreme high tide and darkness prohibited collection
of samples.
TTTSSION
Drset
There should be no surprise to find a daily rhythm in the
photosynthetic capacity of the red alga. Insufficient data
are available to call this particular rhythm circadian,
but circadian rhythms have been demonstrated in many plants
(as mentioned in the introduction). When plotted in con-
junction with tidal and daylight cycles though, the variations
take on an interesting perspective. The highest rates of gas
production match times when the tides are lowest. As mean
tide level climbs, the photosynthetic capacity declines.
This phenomenon may be related to the position of Smithora
on its seagrass host. The alga is found on distal portions
of Phyllospadix. At high tide periods both host and epiphyte
tend to lay well below the water's surface. During low tide
Phyllospadix is often exposed to the air. However, epiphytic
Smithora, because of its position on the host, usually floats
just on the surface. Only during extreme low tides is
Smithora ever exposed to air. While floating at the surface
during low tides, the alga receives a maximum amount of
light. It would be sound biological strategy for the alga
to peak in its photosynthetic potential at these times. This
is just what I observed. Finally, as the available sunlight
declines during the day, the photosynthetic activity of
Smithora drops also, preserving production energy for better
conditions.
The conclusions drawn above should be read with care.
Photosynthetic measurements are tremendously variable. In
15
Matthew Perl
order to standardize measurements, equal area sections of the
alga were required. These could only be taken from the blades
of Smithora. Presumably these structures possess the major
photosynthetic apparatus in the mature algal specimens.
Additionally, the procedure required that the largest blades
of the alga be used. This may have biased my results to some
extent. Finally, the season of the year could have a critical
influence on the capacity for production of photosynthetic
metabolites. One might expect higher rates would be measured
in the Spring and Fall than in other seasons. The importance
of these considerations (portion of plant used, age of samples,
season) has been recently demonstrated by Littler and Arnold (1980)
and deserves to be kept in mind whenever data on photosynthetic
production is considered.
SUMMARY
I have attempted to demonstrate a few physiological
implications of the symbiosis between the epiphyte Smithora
The
naiadum and its natural host Phyllospadix torreyi.
information which I have presented includes:
1. The spore bundles of the algae show a settling
preference for the host over several other organic and inorganic
substrates. This would be important for host location and to
insure survival of the species.
2. A dye was absorbed through the roots and rhizomes of
the seagrass but showed negligible rates of translocation.
This information leads one to conclude that nutrients are more
likely taken up by all surfaces of the plant and used mainly
where they are absorbed.
3. Carbohydrates may be leached out by the seagrass and
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16
subsequently taken up by the surrounding epiphytic
community. However, I was unable to demonstrate either phenomenon.
4. The red alga Smithora shows a definite rhythm in its
photosynthetic capacity which may be important in explaining
its presence and position on the host Phyllospadix.
ACKNOWLEDGMENTS
My sincere thanks go to the entire faculty and staff
at H.M.S. for their friendship and support this quarter.
These fine people bring to Hopkins a warm, receptive
stimulating climate for research and learning. Special
thanks go to three people for guiding me along through
the quarter. Dr. Robin Burnett, Dr.-to-be Celia Smith, and
Dr. Isabella Abbott provided me with inspiration and insight.
And in the end I have to thank Kimberly too, for she kept
my spirit buoyed as it bobbed up and down with the tides.
Matthew Perl
17
LITERATURE CITED
Barbour, M.G. and Radosevich, S.R., 1979. 0 Uptake by the
marine angiosperm Phyllospadix scouleri. Amer. J. Bot.
66(3): 301-306.
Benson, A.A., Bassham, J.A., Calvin, M., Goodale, T.C..
Haas, V.A. and Stepka, W., 1950. The path of carbon
in photosynthesis. J. Amer. Chem. Soc. 72(4): 1710-1719.
Drew, E.A., 1969. Uptake and metabolism of exogenously
supplied sugars by brown algae. New Phytol. 68: 35-13.
Hammarstrand, K., 1968. Gas chromatographic analysis of
carbohydrates. Varian Aerograph, Walnut Creek, 6 pp.
Harlin, M., 1973a. "Obligate" algal epiphyte: Smithora
naiadum grows on a synthetic substrate. J. Phycol.
9: 230-232.
-1973b. Transfer of products between epiphytic marine
algae and host plants. J. Phycol. 9: 243-248.
Harris, J., 1976. Endogenous circadian rhythmicity of
photosynthesis in the marine alga Iridaea flaccida
(Rhodophyta) of the central California coast. Unpublished
paper on file at Hopkins Marine Station, Pacific Grove. CA.
Heftmann, E., 1967. Chromatography. Van Nostrand Reinhold
Company, New York, 851 pp.
Hollenberg, G.J., 1959. Smithora, an interesting new algal
genus in the Erythropeltidaceae. Pacific Naturalist,
1(8): 3-11.
Holligan, P.A. and Drew, E.A., 1971. Routine analysis by
gas-liquid chromatography of soluble carbohydrates in
extracts of plant tissues. New Phytol. 70: 271-297.
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18
Littler, M. and Arnold, K., 1980. Sources of variability
in macroalgal productivity: sampling and interpretation.
problems. Aq. Bot. 8: 111-156.
McBride, D. and Cole, K., 1969. Ultrastructural characteristics
of the vegetative cell of Smithora naiadum (Rhodophyta).
Phycologia 8: 177-186.
McRoy, C. and Goering, J., 1974. Nutrient transfer between
seagrass Zostera marina and its epiphytes. Nature
218(5444): 173-174.
Mishkind, M., Mauzerall, D., Beale, S.I., 1979. Diurnal
variation in situ of photosynthetic capacity in Ulva is
caused by a dark reaction. Plant Physiol., 64: 896-899,
Penhale, P. and Smith, Jr., W., 1977. Excretion of dissolved
organic carbon by eelgrass (Zostera marina) and its epiphytes.
Limnol. and Ocean. 22(3): 4oo-407.
Penhale, P. and Thayer, G., 1980. Uptake and transfer of carbon
and phosphorous by eelgrass Zostera marina and its epiphytes.
J. Exp. Mar. Biol. Ecol. 12(2): 113-123.
Phillips, R.C. and McRoy, C.P., 1980. Handbook of seagrass
biology: an ecosystem perspective. Garland STPM Press,
New York, 353 pp.
Smith, D., Muscatine, L., and Lewis, D., 1969. Carbohydrate
movement from autotroph to heterotroph in parasitic and
mutualistic symbiosis. Biol. Rev. 14: 17-90.
Szymanski, H.A, 1968. Biomedical applications of gas chromatography,
Plenum Press, New York, 198 pp.
Tukey, Jr., H.B., 1970. The leaching of substances from plants.
Ann. Rev. Pl. Phys. 21: 305-324.
Tukey, Jr., H.B. and Mechlenburg, R.A., 1964. Leaching of
metabolites from foliage and subsequent reabsorbtion and
: 737
ichate in plants. Am. J. Bot.
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7
30 CM
V
38 CM
—a
SUBSTRATES
——
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TET

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19
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20
10
40
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5 20
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20
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16
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12
DAY 1

DAY 2

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DAY 4
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SUBSTRATE
20
FIG 2
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21


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FIG 3
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G
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TOP VIEW
FIG 4
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2
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Matthew Perl
Root Condition
Intact:
Severed:
Table I DYE MOVEMENT STUDY
Ti
Distance Dye Moved
S
X(cm) s
f
13 3.1 36
Normal
35 11.5 12
Broken
Normal
13 2.3 33
Broken
42 8.4 10
% Length w/ Dye
S
20% 6.3 36
66% 22.2 12
23% 3.8 33
98% 3.3 10
0
Matthew Perl
TABLES
lable 1. Dye Movement Study.