A COMPARATIVE STUDY OF THE CLONAL AND SOLITARY FORMS
OF THE SEA ANEMONE ANTHOPLEURA ELEGANTISSIMA
The sea anemone Anthopleurd elegantissimg is referred
to as the aggregating anemone of the middle intertidal
region with an average column diameter of 1.5 inches by
Ricketts and Calvin (1968 ed.). This description overlooked
solitary occuring individuals of A. elegantissimg (Hand 1955),
which I have found to have a vertical distribution that
extends subtidally and a pedal disc diameter up to 15 cm.
Prelimingry field observations suggested to me that there
are two distinct forms of A. elegantissimg, an aggregating
form and a solitary form. Distinctive features of the two
forms include whether the anemone occurs in a dense clonal
aggregation created by asexual reproduction (Francis 1973-a)
or as solitary individuals, size, vertical distribution,
and habitat.
This study had the following objectives: 1) To examine
the differences between the clonal and solitary forms,
2) To determine if the differences are genotypic or pheno¬
typic, and 3) To determine if there are any controlling
influences on the occurence or expression of one form over
the other.
GENERAL MATERIAL AND METHODS
This study involved the protected rocky intertidal
zone (Ricketts and Calvin 1968 ed.) at Hopkins Marine Sta¬
tion and Pt. Pinos in Pacific Grove, Californig. All field
observations and laboratory animals were collected from these
areas.
The following definitions of clonal anemones (hereafter
clonal) and solitary anemones (hereafter solitary) were
used in this study. Clonal was defined as any anemone
failing to show aggression (Francis 1973-b) to a minimum
of one near neighbor or having the characteristic scar and
shortened tentacles indicative of recent fission. A solitary
was defined as any anemone which did not meet either criterig
of clonal.
The anemones used in the laboratory studies were collected
by carefully removing them from the rock substrate with a
spatuld or fingernails. Anemones were allowed to settle
in glass or plastic containers with a continuous flow of
sed water at approximately 13?C. Nonclonemates and solitary
anemones were kept separate to prevent aggression (Francis
1973-b). Anemones were not fed during the study,
Methods used in comparing the clonal and solitary were;
1) Tissue grafting to determine tissue compatibility,
2) Starch gel electrophoresis for isozymes, 3) Morphological
studies of nematocysts, 4) Field observations of distribution
and habitat, and 5) Measurements of surface and mass. Each
method and its results is treated in a separate section.
METHODS AND RESULTS
Tissue grafts
To determine if there is a difference in tissue compa¬
tibility between the clonal and solitary forms, two methods
of tissue grafting were employed using the following crosses:
1) Self to self, 2) Nonclonemate to nonclonemate, and
3) Clonal to solitary.
The first method involved culturing tissue sections,
The anemones were relaxed in an isotonic MgCly solution
and strips of body wall approximately 10mm X 4 mm X 1 mm
were excised from the pedal disc and column. Tentacle
sections were prepared by excising a whole tentacle.
clipping 1 mm of the tip to remove the aggression receptors
(Francis 1973-b), then slitting one side to produce a sheet
of tissue in a pie wedge shape. The two tissue pieces were
placed side by side with both ectodermal surfaces facing the
same way and the long cut edges in contact. This arrange¬
ment was held firmly between two frames supporting 1 mm nylon
mesh which allowed circulation of water over the tissue,
The frames were incubated in running sea water at about 13
for 7 days. In this manner, 1 series of pedal disc grafts,
1 series of column grafts, and 2 series of tentacle grafts
were prepared.
Grafts were evaluated after 7 days using the following
criterig, fusion was characterized by the tissue sections
remaining intact along 50% of the graft seam and rejection
was characterized by anything less than 50%. The results
showing 100% rejection for all crosses and tissue types
are presented in Table 1.
Problems encountered with the first method were tissue
necrosis and muscular action within the tissue which dis¬
rupted the edges in contact. Attempts to correct these
problems were made in the second method of tissue grafting,
In the second method, halves of anemones were used
instead of tissue sections and the halves were stitched
together to maintain contact between the cut edges. The
anemones were relaxed in an isotonic Mgcly solution, then
cut longitudinally in half. For each cross, halves of simi¬
liar size were paired. The column halves were sewed together
with square knots at 6 mm intervals using a curved needle and
cotton thread. The grafts were incubated in running sed
water at approximately 13'C. Two grafts were prepared for
each of the three possible crosses using this method,
The same definitions of fusion and rejection were used
as in the first grafting method. Table 2 shows that after
7 days, rejection was observed in all crosses. The events
leading to the rejection are similiar to asexual division
(Davis, 1972). The halves pinched into two autonomous
animals and moved away from each other. Local necrosis
ground the stiches enabled the thread to cut through the
softened tissue freeing the anemone halves as they separgted.
Results for both grafting methods showed no differential
grafting response among the three crosses. The common
event observed was rejection.
Isozymes
Since electrophoresis for isozymes allows direct
visuglization of the gene products, this technique was used
as a measure of the genetic difference between clonal and
solitary A. elegontissima.
The method used was Brewer's (1970) vertical starch
gel electrophoresis for esterase.
Tissue extracts were prepared from mesenterial filaments
of five clonal and five solitary A. elegantissimg. As an
interspecies and intragenus comparison, two A. xanthogram¬
mica were also run. The mesenterial filaments were excised,
blotted of excess moisture and homogenized in a ground glass
hand homogenizer. The homogenate was centrifuged at
10,000 g for 10 minutes and the liquid supernatant used in
the electrophoresis.
Electrophoresis was carried out for 5 hours at 150
volts and 10 milliamps in a 3°C cold room following Brewer's
(1970) procedure for the esterase system. The gel was sliced
and stained for acid phosphatase activity (Shaw 1970) and
esterase activity (Brewer 1970).
Acid phosphatase activity was located in a faint band
common to both clonal and solitary A. elegantissimg and the
A. xanthogrammicg. These results indicated the similigrities
within the genus Anthopleura and did not resolve any genetic
differences within the species A, elegontissimg.
Results for the esterase activity are shown in Figure 1.
The A. xanthogrammica has one intermediate band which makes
its esterase activity pattern distinctly different from the
multi-banded pattern of the A. elegantissimg. The clonal
and solitary A. elegantissimg have a common fast band, but
show some variance in the two slower banding positions. These
two slow banding positions have the characteristics of one
locus with either a homozygous allele resulting in one
band or a heterozygous allele resulting in two bands,
the variation between the clonals and solitaries in the two
slower banding positions is genetic, the differences can
be attributed to any of the following: 1) The clonals and
solitaries have two different loci, 2) It is the same locus,
but different alleles, or 3) The esterase activity is an
inducible isozyme. Support for this last reason of induci¬
bility is seen in slot 14 where a clonal has a slow band
at a position similiar to a solitary's slow band. This
clonal animal has a pedal disc diameter of 8 cm which is
more common of solitaries than clonals.
Morphology
Nematocysts were used as the morphological charac¬
teristic to compare clonal and solitary, since it is a
taxonomic feature used in the classification of seg gne¬
mones (Hand 1955). Nematocysts types were identified and
percentages of the different types determined for the
mesenterial filaments and tentacles.
For the nematocyst study of the mesenterial filaments,
six clonals and five solitaries were used. A piece of mesen¬
terial filament was placed on a glass slide and squashed
firmly with the coverslip yielding a slide with identifiable
unfired and fired nematocysts resolvable with a light micro¬
scope. Nematocyst types within random fields were counted
until a total of 200 nematocyst were identified. The following
classifications by Hand (1955) were used to divide the
nematocysts into four groups: 1) Short basitrich 9.0-18.5 X
1.5-2.0 u, 2) Large basitrichs 25.0-39.0 X 3.5-6.0 u, 3) Thin
basitrichs 27.0-48,0 X 0.5-1.5 u, and 4) Microbasic p-masti¬
gophores 11.0-27.0 X 2.0-4.5 u and 18.5-47.5 X 3.5-4.5 u.
The results are compiled in Table 3. There is some
indication that clonals have a greater percentage of small
basitrichs and a lower percentage of large basitrichs than
solitaries, (p slightly greater than 0.05 using Student t test),
Nematocysts of the tentacles were studied in five clonal
and five solitary. The method involved snipping the tip of
the tentacle and treating this piece of tissue in the same
manner as the mesenterial filament preparation. Nematocysts
were counted until 100 were identified. The nematocysts
were classified as either spirocysts 9.0-29.0 X 1.5-3.0 u
or basitrichs 14.0-24.5 X 2.0-3.0 u (Hand 1955).
Results are summarized in Table 4 and show that the
clonals have more spirocysts relative to basitrichs than the
solitaries (p less than 0.05, Student t test).
To determine if the nematocyst frequencies are affected
by the size of the anemone and not necessarily its identity
as clonal or solitary, the nematocysts in the tentacles of
a clone containing varying sizes of animals were examined,
Nematocyst counts of tentacles were taken for two gnemones
at each 1.2 cm increment in pedal disc sized from 2.5 cm to
8.8 cm. Figure 2 shows that as the pedal disc size increases
the relative number of spirocyst decreases (r - 0.89, p is
less than O.01).
Field observations
Field studies were conducted to answer such questions
as the difference in vertical distribution and habitat that
exist between clonal and solitary. The distribution of the
clonal was from 2,5 m in exposed greas with strong wave
action to ,6 m above mean lower low water level, while the
solitary were found from 1.2 m to subtidal depths. In
studying the clonal and solitary habitats the zone of
coexistance became a focus point.
A salient relationship was noted in surge channels in
which both solitaries and clonals were found. These surge
channels ranged in width from 10 to 50 cm and were of yarying
widths, a typical surge channel is illustrated in Figure 3.
Solitary anemones were found more frequently at the mouth of
these channels than within the channel. The reverse rela¬
tionship was seen for clonals. Twelve surge channels were
observed and anemones scored according to whether clonal or
solitary, and whether at the mouth or within the channel,
This data is summarized in Table 5 and shows a preferential
diestribution of solitary at the mouth and clonal within the
channel.
Another feature of the surge channel habitat appegred
to be a size gradient as a function of distance from the
channel mouth. Five surge channels were observed. Measure¬
ments of pedal disc size of both solitaries and clonals were
made. Locations of the anemones measured were 15 cm apart
extending from the mouth to a distance of 45 cms. The pedal
disc size of the anemone was plotted against its distance
from the channel mouth in Figure 4. The data fit a linear
regression (r = 0.87, and p is less than 0.01). It is
apparent that as the distance from the mouth increased,
the pedal disc size decreased,
Variations in size within a clone were noted for several
clones occuppying surge channels or vertical faces. The
clonal members negrest the surge channel mouth or at the
lowest position on a vertical surface approached the size
of solitary anemones. Data for this gradient of size as
a function of distance from the mouth was collected from
4 clones along surge channels. Pedal disc megsurements of
the anemones were taken at locations of 10 cm increments
from the mouth to a distance of 30 cm into the channel.
Results gre graphed in Figure 5 and show a linear increase
in pedal disc size as the distance from the mouth decregses
(r = 0.84, and p is less than O.01).
The sizes of animals within clones on sloping surfaces
were measured as a function of vertical height for four
clones. The lowest clonals were considered O with the
rest of the clone's position relative to this marker.
Measurements of the anemones' pedal disc size were taken gt
locations 10 cm apart, from O to 30 cm in vertical height.
The results are graphed in Figure 6 and show a linegr
increase in pedaldisc size as the vertical height decregses
(r = 0.73, p is less than O.01).
Surface to mass relationships
The morphology of the clonal and solitary anemone was
studied further by computing the following surface to mass
measurements: 1) Tentacle grea per mass, 2) Oral disc gred
per mass, and 3) Exposed surface ared per mass. Five clondl
and five solitary animals were used, and the data was
obtained in the manner described below.
Tentacle greas and oral disc greas were taken on
anemones induced to expand in a dilute solution of the liquid
supernatant from homogenized squid. For the tentacle greas,
five tentacles were randomly selected from an anemone and
the base diameter and length measured. Using the formuld
for the surface area of a cone, the average surface gred
per tentacle was determined. This value was than multiplied
by the total number of tentacles to givethe total tentacle
surface area for the anemone. The oral disc surfac gred
11
required an oral disc diameter measurement which was con¬
verted to area by the formuld for the grea of a circle,
The exposed surface area was measured in submerged
animals forced to contract by prodding them with a probe until
no further contraction was noted. The surface gred of
clonals was approximated as a half sphere since their
columns are protected by clonemates. The half sphere gred
was computed by using half the contracted column digmeter
as the value for the radius. The solitary surface grea
was approximated as that of a half sphere plus a cylinder
equal to the exposed column. The method of weighing employed
was reduced weight obtained by weighing the anemone suspended
in sea water. The results are summarized in Table 6. The
clonals have more tentacle grea, oral disc gred, and
exposed grea per mass than the solitary,
DISCUSSION
The results showed differences between clonal and
solitary in their isozyme activity, nematocyst frequencies,
distributions, habitat, and in several measures of surface
area to mass ratios.
The variance in esterase activity and nematocyst per¬
centages are indicative of possible genetic differences
between clonal and soiitary, however there is a strong
possibility that both results are inducible by size. This
influence is suggested by the large clonal with a solitary
esterase banding pattern. Clearer support for size influence
12
was the decrease in tentacle spirocyst percentages with increasing
anemone size within a single clone. Since the enzymes and
nematocysts are an integral part of the feeding process,
these features may be influenced also by diet and feeding
behavior. There is not enough evidence to support genetic
differences as maintaining the two distinct forms,
The existance of the two forms if not genetic, suggests
a phenotypic expression controlled by external forces,
Hand (1955) and Francis (1973-a) attribute the solitary
form to a more protected environment since its size is
more vulnerable to wave shock. I also found solitgries
in protected environments, but found them more frequently
at the mouth of surge channels than clonals, a distribution
pattern which may reflect differences in feeding habits,
I postulate that in surge channels a solitary at the mouth
is feeding on incoming food laden water, the water is
depleted of some of it particles by the solitary and can
support only smaller more efficient clonals further up the
channel. The size gradient observed in clones along surge
channels and vertical faces also fits this model. The
anemones nearest the mouth or lowest in vertical height
are feeding on the most food laden water and thus can grow
larger per unit food gathering greg.
The genetic variation of esterase activity and nema¬
tocysts are also compatible with the model since they
dre involved in the feeding process and could be influenced
by food availability. Of particular interest is that
small clonals have more spirocysts than large solitaries.
Spirocysts when discharged create a mesh of entangling
fibers thought to be used in trapping prey (Mariscal 1974).
It would be advantageous for the small clonal to have more
spirocysts since they would be feeding on water relatively
poorer in large pieces of food than the large solitary,
The morphological surface areas to mass can be applied
to the food availability scheme. Tentacle grea per mass
is d measure of feeding efficiency, and the oral disc greg
per mass is a measure of farming efficiency in providing sun¬
light for its algal symbionts. Thus the clonal form is a
more efficient feeder and farmer than the solitary, which
is in keeping with clonal's position in greas of possibly
decreased food availability,
This study best supports the model that solitary and
clonal are phenotypic expressions controlled perhaps by
food availability. The solitary individuals may be less
efficient feeders and are found at surge channel mouths and
at a lower tidal level than the clonals where the availability
of large amounts of food may be greater. The small clongl
is d more efficient feeder and farmer, and is a phenotype
expressed when food availability is low.
I wish to thank the faculty, staff and students at
Hopkins Marine Station; particularly my advisor Nathan
Howe for his endless help and encouragement, and Phil Murphy
for his expertise with starch gel electrophoresis.
SUMMARY
Clonal and solitary forms of A. elegantissima were
comparatively studied and found to have differences in
isozyme activity, frequencies in nematocyst types, and
intertidal location. These differences suggested that food
availability was the determining factor in the distribution
and expression of the two forms.
14
LITERATURE CITED
Davis, D. 1962. One equals two: a seaanemone achieves its
plurality in singular fashion. Nat. History 71(2): 61-63.
Francis, L. 1973-a. Clone specific segregation in the sed
anemone Anthopleurg elegantissimg. Biol. Bull. 144: 64-72.
Francis, L. 1973-b. Intraspecific aggression and its effect
on the distribution of Anthopleurd elegantissimg and
some related sed anemones. Biol. Bull. 144: 73-92.
Hand, C. 1955. The sea anemones of central Californig,
Wasmann J. Biol. Part 2 13: 37-99.
Mariscal, R. N. 1974. Nematocysts, p. 129-178. In Muscatine
and Lenhoff (eds.), Coelenterate biology: reviews and
new perspectives. Academic Press, N.Y. 501 p.
Ricketts, E. F., and J. Calvin. 1968. Between Pacific Tides
4th ed., rev. by J. W. Hedgpeth, Stanford University
Press.
TABLE CAPTIONS
Table 1: Grafting results using the first method of tissue
sections.
Table 2:
Grafting results using the second method of
stitching gnemone halves together,
Table 3:
Numbers of each nematocyst type counted per 200
total nematocysts in the mesenterial filaments
of 6 clonals and 5 solitaries. Nematocyst types
designated by A, B, C, and D are: A) Small basitrichs,
B) Large basitrichs, C) Thin basitrichs, and
D) Microbasic p-mastigophores.
Numbers of spirocysts and basitrichs per 100
Table 4:
nematocysts counted in the tentacles of 5 clonals
and 5 solitaries.
Table 5:
Precentages of the number of clonals and solitgries
situated at the mouth and within the channel of
surge channels.
Surface area to mass ratios for 5 clonals and 5
Table 6:
solitaries.
e
COMBINATION
SELF TO SELF
NONCLONEMATE
TO
NONCLONEMATE
CLONAL TO SOLITARY
TABLE 1
TISSUE
TYPE
PEDAL DISC
COLUMN
TENTACLE
PEDAL DISC
COLUMN
TENTACLE
PEDAL DISC
COLUMN
TENTACLE
% REJECTION
100
100
100
100
100
100
100
100
100
SAMPLE
SIZE
1
1
1
COMBINATION
SELF TO SELF
NONCLONEMATE TO
NONCLONEMATE
CLONAL TO SOLITARY
TABLE 2
% REJECTION
100
100
100
SAMPLE
SIZE
2
S.D.
115
126
92
104
105
72
102.3
18.75
B
40
28
72
58
48
52.3
17.49
TABLE 3

CLONAL
A
D
C
25
100
20
42
90
15
22
62
16
72
35
16
64
32.7
78.6
12.2
19.26
7.33
15.81
SOLITARY
B
C
73
10
70
84
83
84
78.8
7.4
2.80
6.76
D
17
32
38
42
35.2
10.34
X
S.D.
NUMBER OF
SPIROCYST
57
66
56
68
60.4
6.11
TABLE 4
CLONAL
NUMBER OF
BASITRICH
43
34
44
32
45
39.6
NUMBER OF
SPIROCYST
52
57
49
50
50.6
1.14
SOLITARY
NUMBER OF
BASITRICH
48
49
50
49.4

S.D.
X
S.D.
CLONAL n=286
% CLONALS
% CLONALS
AT MOUTH
IN CHANNEL
98%
TABLE 5
SOLITARY n=26
% SOLITARY
% SOLITARY
AT MOUTH
IN CHANNEL
81%
.

2

5
.



ON



o
a
oN
0

o
1

S 99

+ 0
0
0
Figure 1:
Figure 2
Figure 3:
Figure 4:
Figure 5
Figure 6:
FIGURE CAPTIONS
Starch gel electrophoresis stained for esterase
activity. Tissue extracts are from the mesenterial
fildments of 5 clonal and 5 solitary A. elegantissma,
and 2 A. xanthogrammicg. Two clonal anemone tissue
preparations were run in duplicate and the
identical results combined as seen in slot 1 & 2.
and slot 5 & 6.
Graph showing decrease in the percentage of
tentocle spirocysts as function of increasing size,
represented by pedal disc diameter. Sample size
is 10 gnemones from the same clone.
Illustration of a typical surge channel,
Size of gnemone determined by pedal disc diameter
as a function of distance from the mouth of a
surge channel. Data obtained from solitgries
and clonals at 12 surge channels,
Variation in size of clonals as a function of
distance from the mouth of a surge channel.
Data was collected from 4 clones along surge
channels.
Variation in size of clonals as a function of
location on a vertical surface. The lowest
clonal member is 0, with vertical heights being
relative to this marker. Data was obtained from
4 clones along vertical surfaces.
—





—
e
LE



L

LE

2

L
D
C

e

SIskooulas
Q
e

1
+



—

L

L


L
LL


—

eo

NId
—
LE
CHANNEL

FIGURE 3




MOUTA
83


LE
FIG.
10 000
SOLITARY
CLONAL

OO
O
45
30
DISTANCE FROM MOUTH IN CM
60
—

L
FIG
4
2
DISTANCE
O
O
30
20
FROM MOUTH IN CM
L
FIG.
O
O
20
VERTICAL HEIGHT
IN CM

30