Adherence, growth of R. affine carpospores.
Page 2
Joel D. Reimnitz
ABSTRACT
The percentage of carpospores of Rhodoglossum affine (Harvey)Kylin
adhered to glass plates was shown to attain a maximum level at 20 minutes
following artificial release and drop to a significantly lower level
at 4 hours.
Early morphological development of tetrasporophyte embryos is described,
One-week growth of embryos in response to gradients of temperature and
salinity have been investigated. In all treatments embryos grew signifi¬
cantly faster if settled first at 14°/330/oo then moved to environmental
growth baths than if settled directly in those baths.
The results suggest that it may be useful to study environmental
effects on early embryonic development and growth of R. affine carpospores
when considering the geographical distribution of the adult tetrasporophyte,
Key words: adherence, carpospore, embryo, Rhodoglossum affine, coalescence,

tetrasporophyte.
Adherence, growth of R. affine carpospore
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Joel D. Reimnitz
INTRODUCTION
An easily overlooked fact is that the algae seen growing along
coastal boundaries have not always existed in their macroscopic adult
forms, but begin their life cycles as spores. When considering the
distribution of an alga it is common to observe the effects of environmental
stresses on adult plants. What is seldom considered is the possibility
that macroalgal distribution may be just as much a function of the early
activity of the plant (that is, while it is a spore or sporeling) as it
is a function of the activity of the adult plant.
Red algal spores are purportedly non-motile. They enter the water
column often by forcible ejection from the cystocarp and are carried along
by water currents, which have been shown to be far more of an influence
on their distribution than any sinking due to gravity effects (Coon, et. al.,
1971). Where spores settle, how fast they settle, and how well the grow
where they settle could possibly have broad influences on the distribution
of a given species in the field. McLachlan (1974) suggests that distri¬
bution of Fucus in nature is largely controlled by factors acting on the
embryo and that responses of the embryo may be different than those of
the mature plant.
Adherence rate studies indicate a strong relationship between the
amount of time spores are allowed to settle onto glass plates and the
percentage of spores attached. Adherence percentages may improve with
time in one species and decrease with time in others (Suto, 1950; Charters,
et. al., 1972).
Temperature and salinity effects on early embryonic development have
been documented for species of Fucus (Munda, 1977; McLachlan, 1974; Bird
and McLachlan 1974), Hypnea (Mshigeni, 1976), and in Chondrus crispus
(Prince & Kingsbury, 1973). None of these studies have noted the effects
Adherence, growth of R. affine carpospores.
Page
Joel D. Reimnitz
of local environmental conditions during settlement on early growth.
I here report on adherence rate studies undertaken in the lab with
R. affine carpospores. Also reported are the effects of temperature and
salinity on the one-week growth of R. affine tetrasporophytes settled in
"normal" environments (Monterey Bay water conditions during May) versus
"stressful" environments (various temperature and salinity conditions).
** -- -
Adherence, growth of R. affine carpospores.
Page 5
Joel D. Reimnitz
MATERIALS AND METHODS
Female R. affine were collected at low tide at Pt. Pinos, California,
rinsed briefly in filtered seawater and air-dried for one hour. All
seawater used was first passed through a 54 filter. After drying, fronds
were placed cystocarp down in plastic dishes filled 1 cm. deep with
seawater at 22.5 C. Pink patches of carpospores began appearing on the
dish bottom within 2 hours. Spores were withdrawn within ½ hour after
the cystocarp began to shed using a pasteur pipette, deposited in a 10 cc.
beaker, and diluted to a workable (7-10 spores per field when a drop of
spore solution was examined at 100X) density with 14°C seawater.
For adherence tests a paper grid with 10 prominently-marked 1.3 x 1.3
mm. squares was epoxied between two glass slides. A 1 mm.high lip was
built on the top slide around the area of the grid using silicone glue
to prevent water run-off in the grid area. Slides were placed in 10 x 150
mm. petri dishes and covered with 25 cc. seawater. Tests were performed
at 14 C at a light intensity of 100W/cm'. One drop of a freshly-prepared
spore solution was dropped at each of the four corners of the grid area.
At scheduled times, slides were withdrawn from the dishes and the number
of spores within the limits of each of the 10 marked squares was tabulated.
Next the slide was rinsed by dipping it gently into a basin of seawater
at 14 C, holding it vertically, and moving it 8 times across the distance
of 15 cm. in approximately 5 seconds. Charters, et. al. (1972) noted
little change in the percent of spores adhering to plates under a wide
range of force. Following this rinse the same 10 squares were again counted
for spores and the ratio of spores present after the rinse to before the
rinse was multiplied by 100 to get the percent adhering.
Adherence, growth of R. affine carpospores.
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Joel D. Reimnitz
Temperature and salinity experiments were performed by first
settling spores on 2 (salinity) or 4 (temperature) coverslips at each of
two different conditions: (1) Base-settled spores were settled for
hour in trays at 14°0, 330/00 salinity, and light intensity of 65/cm?,
then transferred to growth trays; (2) Bath-settled spores were settled
directly in the growth trays. The growth trays were 10 x 150 mm. plastic
petri dishes filled with 25 cc. of seawater adjusted to the various
experimental temperatures and salinities and enriched with Provasoli's
medium. After 1 week in the growth trays the coverslips were removed and
the diameter of the discoid holdfast base measured with an ocular micro¬
meter at 450X. At least 20 embryos were measured for each salinity con¬
dition except for 230/00 (14 counts), and at least 250 measurements at
each temperature condition.
The salinity experiment was performed in a 14°C constant-temperature
room with a 16:8 light:dark photoperiod at 65W/cm. All saline environ-
ments were created by mixing appropriate amounts of Instant Ocean (tm)
with distilled water.
The temperature experiment was performed by placing 16 x 30 cm.
plastic trays filled 5 mm. deep with distilled water in constant tempera¬
ture baths. The growth trays were floated on this distilled water. With
this arrangement the temperature inside the growth trays precisely matched
the temperature of the distilled water, which was monitored at 24 hour
intervals. The photoperiod was 16:8 light:dark at 40W/cm2.
Adherence, growth of R. affine carpospores.
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Joel D. Reimnitz
RESULTS
Early morphological development of R. affine carpospores grown at
14°0/330/00 proceeds as illustrated in figure 1(a-i). The zygote divides
asymmetrically, the first 5 or 6 cell divisions occurring without
enlargement of the embryo. Subsequent cell divisions produce an embryo
which on glass appears discoid from overhead view. After 4-6 days uniseriate
hairs appear and project away from the holdfast. These hairs are lost
or resorbed after the embryo begins growth ofga thallus from the base.
Rhizoids typically project upwards and laterally from the growing thallus
(see figures 2(a) and 3). Carpospores settling within 100-200 um of one
another will overlap in territory as their holdfasts expand. In such
cases first the outermost cells of each embryo mingle and eventually the
two embryos coalesce into one. The resulting embryos are indistinguishable
from embryos decending from a single carpospore except that they are
larger in size (see figures 1(k,1) and 2(b)).
Figure 4 shows adherence of carpospores as a function of time. The
percentage of spores adhering increases rapidly until 20 minutes after
spores are artificially released when the greatest adherence percentage
was observed. Adherence percentages dropped to a low at 4 hours; the
mean low value at four hours is significantly below the combined mean
of the 20 through 60 minute values (Student's t-test, p£.01). During
early experimental trials if the glass slides were tipped, water would
run off of the slide and by capillary action all of the water covering a
newly-settled spore would be withdrawn. Spores so exposed would die
immediately as evidenced by dispersal of their organelles outside of
cell boundaries.
Results of the salinity experiments are shown in figures 5, 6, and 7.
......
Adherence, growth of R. affine carpospores.
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Joel D. Reimnitz
Spores in both settlement conditions died within 1 week at 130/00 salinity.
Fastest growth of the embryos occurred at 330/00. Carpospores withstood
the highest salinity condition (730/00) although nearly all were still
undivided after one week. Observed embryos at 230/oo and 330/oo all had
disc-shaped holdfasts. At 430/00 rhizoids were noted in approximately
20 percent of observed embryos while at 530/0o at least 50 percent of
observed embryos had developed at least one rhizoid growing from the
holdfast base (see figure 1(j)). Contrasting the bath-settled population
of spores with the base-settled spores for each salinity shows them to
be growing at significantly different rates for each condition but
330/0o and 730/00 (Student's t-test, p2.01). Except at 330/oo the base¬
settled spores grew faster than bath-settled spores.
Results of temperature experiments are shown in figures 8 and 9 and
table 1. Greatest growth was observed at 20.5°C, with a percentage of
spores alive at 3 and 26 C and no spore surviving 28° exposure. All
temperature-treated embryos demonstrated disc-shaped holdfasts. For the
14° through 24 range base-settled spores were tested against bath-settled
spores and found to be growing at a significantly faster rate at each
temperature (Student's t-test, p.01).
In glass tubes filled with spores suspended in seawater and
placed in a light-sealed box, settlement by the spores after 4 days was
observed to occur only on the half of the tubes closest to a point light
source placed in the box.
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Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
DISCUSSION
Early morphological development of R. affine carpospores is similar
to development of the related red alsa Chondrus occellatus Holm examined
by Inoh (1947). A question not yet investigated is what is the
orientation of the asymmetrically-dividing embryo with respect to its
attachment orientation on the substrate.
Coalescence of algal embryos has been described in Chondrus crispus
(Tveter and Mathieson, 1976; Ring, 1970; Taylor and Chen, 1973), and
Gracilaria verrucosa (Jones, 1956). Coalesced sporelings have been shown
to produce fronds that grow more rapidly than non-coalesced sporelings
(Tveter and Mathieson, 1976). Because carpospores may yield either male
or female tetrasporophytes, such coalescence could have interesting
morphological and possibly physiological consequences in the resulting
plant. Further investigations into the morphology, physiology, and
reproduction of coalesced embryos are being planned.
How quickly a spore adheres to a substrate could influence where
or when they settle. That a percentage are attached after 5 minutes
and the highest percentage attached is observed at 20 minutes indicates
that settlement in R. affine carpospores can occur relatively rapidly.
They thus appear capable of adhering given the right local conditions
regardless of tidal conditions.
Adherence percentages for R. affine carpospores are highest between
O and 60 minutes from release then decrease with time up to 4 hours.
Christie, et. al. (1970) have shown that green algal zoospores have a
chemical adhesive contained in vesicles on the outer walls of the spore.
Adhesion is proposed to be a process of release of this adhesive and
hardening of the adhesive over time by chemical changes. Variation may
.. ...
Adherence, growth of R. affine carpospores.
Page 10
Joel D. Reimnitz
occur from plant-to-plant and possibly from season-to-season in the
quality of this adhesive. They also reported, as the three runs shown
on figure 4 demonstrate, that there is considerable variation between
spore batches in the ability to adhere to glass.
What I suggest is that adhesive is released when the spore first
comes in contact with a surface and serves to anchor it initially.
Possibly then it does not harden effectively over time and proves inade¬
quate for keeping the spore attached when the spore is later exposed
to the forces of water flowing past it. Boney (1966) indicates that
water motion is necessary for optimum algal growth. As all experiments
here were performed under still-water conditions on an artificial
laboratory surface, the results may be a result of some missing
environmental "stimulus" promoting spore adhesion over time.
The observation that glass-attached spores die when the water surround-
ing them is withdrawn seems in a practical sense maladaptive on the part
of the spore, given that it could often be uncovered and left exposed
in the field at low tide. The observations refered
to were noted
in the early periods after spore settlement and it is unknown whether the
spore develops resistance to this type of exposure over time. If so this
early lack of exposure-resistance could require timing of settlement
such that early exposure is not likely to occur.
As figure 7 shows, R. affine carpospores grow fastest in the salinity
range of 230/0o to 430/00. The effects of salinity on growth are much
more apparent after 11 days than after 7 days, with growth at 230/oo and
530/00 being repressed much more than the 330/oo and 430/oo conditions,
The temperature optimum for growth of R. affine carpospores occurs
somewhere in between 17 and 24°C (figure 8). There are seasonal varia-
Adherence, growth of R. affine carpospores.
Page 11
Joel D. Reimnitz
tions in the water temperature in the ocean off of the California coast.
Having a high temperature optimum for growth would imply faster growth
rates during those seasons when the water temperature approaches the
optimum. If size of the plant has any bearing on its ability to resist
grazing effects and other environmental stresses this could possibly
lead to seasonal variations in the survival capacities of the early
plant. Quantities of spores released has been noted in some species to
follow lunar and seasonal patterns (Christie and Shaw, 1969; Guzmán-del-
Proo, et. al., 1972). It would be interesting to compare such release
patterns with water temperature and temperature optimums for growth of
algal embryos.
In both the salinity and the temperature experiments there was
significantly greater survival of the carpospores and faster embryonic
growth if the carpospores were settled at base conditions than if settled
at bath conditions. In the temperature experiments this occurred, even
when the two settlement temperatures were the same (14°C), so this result
is not explained by temperature shock on the base-settled spores.
In the temperature experiments the light conditions were similar for the
two settlement conditions but not identical. McLachlan (1974) has shown
that widely contrasting light conditions can have significantly different
effects on the development of algal embryos. Because light conditions
were uniform for all conditions in the salinity tests yet the differential
growth was still observed, it is unlikely that light conditions could
be the sole cause of the faster growth of the base-settled spores. Given
that water motion effects are necessary for optimum development and
growth of algal plants, the possibility exists that sometime during the
transfer of the coverslips from one bath to the next sufficient jostling
. ... ...
Adherence, growth of R. affine carpospores.
Page 12
Joel D. Reimnitz
occurred to stimulate the embryos transferred to faster growth than
those never moved.
As the histograms of figure 9 show, the bath-settled spore popula¬
tions do not contain nearly as many large-size-class embryos at each
temperature as do the base-settled spore populations. If the size differ¬
ential between bath- and base-settled embryos were to be explained by the
washing away of less viable spores during the transfer process, fast-
growing embryos should still appear among the bath-settle embryos.
After one week of growth the combined average of bath- and base¬
settled embryos at 14°C/330/00 for the salinity test was 129/4m. and for
the temperature test 84um. There are several possible explanations for
this difference. The spores did come from difference spore batches, and
handling during the early collection processes was not identical. In
addition the salinity experiment was performed in a culture medium of
Instant Ocean plus Provasoli's medium; the temperature experiment was
performed in seawater plus Provasoli's. Mshigeni (1976) found large
differences in the growth of carpospore germlings as a function of the
culture media.
Red algal spores are reportedly non-motile. Observations with the
directional light study seem to indicate that spores selectively settle
or at least preferentially survive closest to a source of light. At
the very least, the conclusion to be drawn from this is that the place
of adherence and growth of the spores, given the environmental conditions,
was not strictly random. At the most one could hypothesize a response
on the part of the spores with respect to light. Tests of this hypothesis
are being planned.
If indeed the spores are somehow responsive to light this information
Page 13
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
supplies some further explanations as to what may be happening in the
adherence rate tests. The adherence rate tests were performed in condi¬
tions of diffuse light. Investigators of green algal zoospore attachment
perform their tests in the dark because the spores respond to light
and make the results uninterpretable (Christie and Shaw, 1968). If
R. affine carpospores respond to light, an initial contact with a
horizontal surface would release the chemical adhesive in the area on
the wall of the spore touching the surface. No further vesicles would
come in contact if the spore were seeking settlement in the direction
of light and no more adhesive would be released. Over time the spore
would simply not have enough adhesive present to be able to remain
attached to the plate when subject to water motion effects.
Growth of embryos in high salinity water yields abberrations in
early growth morphology. Variations in growth forms depending upon the
environmental exposure of the embryo have been well documented (McLachlan,
1974; Chen and McLachlan, 1972).
In this study growth at many temperature and salinity conditions
vields data indicating that there are differences in the growth rate
of embryos depending upon the environmental conditions they are subject
to. Whether these differences lead to later variations in the size and
morphology of adult plants is a question well worth further investigation.
As the bath-base studies indicate, small changes in the history of the
embryo may stimulate plants to different growth rates. It might be well
to consider these observations when analyzing distribution of growth
forms of a species in the field and the geographical distribution of
a given species.
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
ACKNOWLEDGEMENTS
Thanks Robin
Thanks Bill.
Page 14
-.....
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Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
REFERENCES
Bird, C. J., and McLachlan, J. 1974. Cold-hardiness of zygotes and
embryos of Fucus (Phaeophyceae, Fucales). Phycologia 13(3): 215-225.
Boney, A. D. 1966. A Biology of Marine Algae. 216 pp. London,
Hutchinson Educational Ltd.
Charters, A. C., Neushul, M., and Coon, D. A. 1972. Effects of water
motion on algal spore attachment. In Proc. Seventh Int. Seaweed Symp
Tokyo: Univ. of Tokyo Press.
Chen, L. C., and McLachlan, J. 1974. The life history of Chondrus
crispus in culture. Can. J. Bot. 50: 1055-1060.
Christie, A. O., Evans, L. V., and Shaw, M. 1970. Studies on the ship
fouling alga Enteromorpha. Ann. Bot. 34: 453-82.
Christie, A., O., and Shaw., M. 1968. Settlement experiments with
zoospores of Enteromorpha intestinalis (L.)Link. Br. Phycol. Bull. 3: 529-34.
Coon, D. A., Neushul, M., and Charters, A. C. 1972. The settling
behavior of marine algal spores. In Proc. Seventh Int. Seaweed Symp.
Tokyo: Univ. of Tokyo Press.
Guzman-Del Próo, S. A., Guzman, S., and Pineda-Barrera, J. 1972.
Shedding rhythm and germination of spores in Gelidium robustum. In Proc.
Seventh Int. Seaweed Symp. Tokyo: Univ. of Tokyo Press.
Inoh, S. 1947. (Development of algae) (in Japanese) 256 pp. Hokuryukan,
Tokyo.
1956. Effect of spore coalescence on the early development
Jones, W. E.
of Gracilaria verrucosa (Hudson) Papenfuss. Nature (London) 178: 426-7.
McLachlan, J. 1974. Effects of temperature and light on growth and
development of Fucus eduntatus and F. distichus ssp. distichus. Can. J
Bot. 52: 943-951.
Munda, I. M. 1977. Combined effects of temperature and salinity on
growth rate of germlings of three Fucus species from Iceland, Helgoland
and the North Adriatic Sea. Helgolander wiss. Meeresunters 29: 302-310.
Mshigeni, K. E. 1976. Effects of the environment on developmental rates
of sporelings of two Hypnea species (Rhodophyta: Gigartinales). Marine
Biology 36, 99-103.
Prince, J. S. and Kingsbury, J. M. 1973. The ecology of Chondrus
crispus at Plymouth, Massachusetts. III. Effect of elevated temperature
on growth and survival. Bio. Bull. 145(3): 580-588.
Ring, P. D. 1970. Developmental and ecophysiological studies of
Chondrus crispus (L.) Stackhouse. M.Sc. Thesis, University of Maine,
Orono, 73 pp.
.--...
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Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
Suto, S. 1950. Studies on shedding, swimming, and fixing of spores of
seaweeds. Bull. Japan. Soc. Sci. Fish. 16: 1-9.
Taylor, A. R. A., and Chen, L. C-M. 1973. The biology of Chondrus
crispus Stackhouse: systematics, morphology and life history. Proc.
Nova Scotia Inst. Sci. 27 (suppl.): 1-21.
Tveter, E., and Mathieson, A. C. 1976. Sporeling coalescence in
Chondrus crispus (Rhodophyceae). J. Phycol. 12: 110-118.

Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 1
251m
25um
25
25 um
100 um
0m
25 m
25 Am
200
25 um
Page 17
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 2
Page 18
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 3
Page 19
. . . . -
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 4
X



8
DNIAZHGV INSSaad
8
Page 20
Page 21
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 5
140
120
SETTLED AT
LABATH SALINITY
£100
SETTLED
— AT 33%0
2 80
6 60
O
40
20





A
23%o 33%o 43%o 53% 63% 73%0
SALINITY OF BATH
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 6
60
50
40
30
23%00
20
10

33%00
20
10
a

43%00
U 20
10

zO
30
53%00
20
10

50 100 150 200
O
DIAMETER OF HOLDFAST (um)
Page 22
. . .
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 7
300
1 200
100
43%0
33%0
23%0
SALINITY OF BATH
V11 DAYS
7 DAYS
53%0
Page 23
. ..
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 8
150
D 100
50

17
20.5
14
TEMPERATURE (°c)
24
Page 24
SETTLED
AT 14°
SETTLED AT
BATH TEMP.
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
FIGURE 9
17
20.5
140
SETTLED
AT BATH
TEMP.
d

p
SETTLEL
AT 14°


lu
100 200 100 200
O 100
SIZE OF EMBRYO (um)
24°
100
Page 25
Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
TABLE 1
NUMBER
BATH/
OBSERVATIONS
BATH
EMBRYOS
BASE
TEMP
COUNTED
SETTLED
472
2.3% (11) alive.
Bath
30
33% (20) alive.
Base
10
6.6% (52) at 2 2 cells.
791
Bath
70
696
817 (565) at 2 2 cells.
Base
100
327
10% (34) at 2 2 cells.
Bath
94% (478) at 2 2 cells.
100
Base
508
260
33 total present.
Bath
33
357
357 total present.
Base
26
——------
Bath
280
—--------
280
Base
Page 26
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Adherence, growth of R. affine carpospores.
Joel D. Reimnitz
CAPTIONS OF FIGURES
Figure 1: (a-h), Early morphological stages in the growth of R. affine
carpospores. (i) A one-week-old embryo pictured on the edge
of a coverslip. Note the uniseriate hairs ascending from the
holdfast region. (j) An embryo grown at 530/00 for 1 week.
Compare to (i) which was grown for 1 week at 330/00. (k,1).
2 and 3 embryos showing coalescence after one week of growth.
(a). A 2 week old embryo, showing beginning signs of thallus
Figure 2:
growth from the holdfast. (b). Coalescence of two embryos which
have almost completely merged into one plant.
Scanning EM of a three-week old embryo. Note the numerous
Figure 3:
rhizoids projecting from the holdfast area.
Adherence of carpospores as a function of time. The three
Figure 4:
lines represent three separate tests run with three different
spore batches. Not that although there is considerable
variation between batches in the percent adhering at a given
time they all demonstrate a high adherence percentage in
the 20-60 minute range and a lower adherence percentage
3 hours later.
Size of embros after one week as a function of growth bath
Figure 5:
salinity.
Histograms of embryo sizes in selected growth bath salinities
Figure 6:
after 11 days.
Size of embryos as a function of growth bath salinity at 7 and
Figure 7:
11 days.
Figure 8: Embryo size as a function of temperature at / days.
Figure 9: Histograms of embryo size after one week at selected temperatures.
CATIONS OF TABLES
Table 1: Observations on the growth of carpospores of R. affine at
selected temperatures after 1 week.