INTRODUCTION
The giant chiton, Cryptochiton stelleri (Middendorff, 1846) is
known from northern Japan to southern California for its thick brick-
red girdle covered by tufts of crimson calcareous bristles (Smith, 1975).
Close examination of this mollusk often reveals that Cryptochiton's
dorsal surface also maintains a minature forest of red algae. Pleonosporium
squarrosum Kylin (Ceramiaceae), the 2-3 cm tall red alga is a leading
member of the chiton's epizoic community. The distribution of P. squarrosum
is limited to pilings of docks at Friday Harbor, Washington (Kylin, 1925)
and the backs of a few subtidal invertebrates in Monterey, California
(Abbott and Hollenberg, 1976). P. squarrosum appears very selective in
its habitat, yet common on the Cryptochiton despite an earlier report by
MacGintie and MacGintie (1968) which indicated that Cryptochiton backs did
not carry a distinctive flora or fauna. The purpose of this study was to
investigate the obvious chiton-alga association-its incidence, distribution.
causality and natural history.
MATERIALS AND METHODS
Work on this interrelationship was carried out between April and
June 1980. The Cryptochiton stelleri populations at several locations were
sampled: in the kelp bed off Mussel Point, Monterey Bay at a depth of
25-30 ft; in the Point Alones kelp bed east of Mussel Point at a 25 ft depth:
from the tidepools at Pescadero Point at a -0.9 tide level; and from the
intertidal zone on a -1.h tide at Stillwater Cove, Carmel Bay. These
sites were chosen because they represent varying conditions of exposure to
waves and of water depth.
Cryptochiton stelleri population density was estimated by swimming
or walking quadrats 5 me and the number of Cryptochitons with Pleonosporium
squarrosum was noted. Random specimens were collected, tagged, weighed
and measured. Percent cover by P. squarrosum was analyzed by generating
50 random polar coordinate points marked on a transparent sheet of plastic
that was pinned to the center of each chitonts back. The presence or
absence of P. squarrosum was recorded under these 50 points. Other epizoic
life was also noted. While in the field collecting chitons, a search was
conducted for P. squarrosum not on Cryptochiton.
In the laboratory, samples of Pleonosporium squarrosum were picked
off the backs of Cryptochitons. Counts were made of female, male, poly-
sporangia-bearing and sterile plants. Some vegetative axes were cut for
culture and experimentation. Others were chosen to drop spores for spore
germination tests. All the cultures were grown in disposable plastic petri
dishes with filtered (.22/m) sea water, a nutrient sea water mixture and
germanium dioxide to retard diatom growth. Substrate, temperature and
light varied with the experiment.
SULT
Weight, Size and Distribution
The 39 collected chitons ranged in length from 15-28 cm with wet
weights between 260 g and 1330 g. These individuals were similar in size
and age to others previously studied (MacGintie and MacGintie, 1968).
Most obvious was that some Cryptochiton stelleri were algae-covered and
some were bare-backed (fig. 1). Pleonosporium squarrosum was absent on the
backs of the Cryptochiton st Pescadero Point and at Stillwater Cove.
However, the populations of Cryptochiton from Mussel Point and Point Alones
kelp beds had P. squarrosum in varying amounts.
Percent Cover of Pleonosporium squarrosum
The two populations from Mussel Point and Point Alones kelp beds not
only showed a difference in percent of Pleonosporium-covered Cryptochitons,
but also a difference in percent cover by P. squarrosum on the individual
chitons (fig. 2). The eleven sampled Mussel Point Cryptochitons had
percent cover ranging from 26% to 0% with a mean of 138; whereas, the 13
Point Alones chitons had Pleonosporium cover ranging from 918 to 0% with
a mean of 60%.
Of the chitons that did show Pleonosporium squarrosum algal cover,
the presence of the alga seemed most common within a circle of radius
6.5 cm centered at the center point of the chiton back. Sixteen of the 18
collected Cryptochiton with P. squarrosum demonstrated this centralized
concentration of algae. The alga also was prevalent on the dorsal humps
made by the underlying plates.
Other Epizoic Life
During the search for Pleonosporium squarrosum on Cryptochiton
stelleri, much other epizoic algae was discovered. Included was: Lyngbya
sp., Bryopsis sp., Cladophorasp., Enteromorpha linza (Linnaeus) J. Agardh,
Ulothrix sp., Giffordia sandriana (Zanardini) Hamel, Sphacelaria sp.,
Goniotrichum sp., Heterosiphonia japonica Yendo, Platythamnion sp.,
Polyneura latissima (Harvey) Kylin, Polysiphonia pacifica Hollenberg,
Pterosiphonia dendroidea (Montagne) Falkenberg, Rhodoptilum plumosum (Harvey
& Bailey) Kylin, and colonial diatoms. Epizoic organisms included gastropods
(Tegula brunnea and Ocenebra spp.), nematodes, tiny hermit crabs and
occasionally, amphipods and copepods.
Host Specificity
To determine Pleonosporium squarrosum specificity for Cryptochiton
stelleri, other substrate possibilities were carefully scrutinized (fig. 3).
Examined were wharf pilings at Monterey Harbor and Stillwater Cove.
carapaces of the decorator crab Loxorhynchus crispatus (Stimpson, 1857),
colonies of hydroids, tubes of the worm Diopatra ornata (Moore, 1911)
found in the subtidal Cryptochiton habitat, clam and abalone shells, subtidal
rock faces, subtidal algae, and even the sandy sea bottom on which Crypto-
chiton crawl. Although Abbott and Hollenberg (1976) report P. squarrosum
on decorator crabs and hydroids, no new specimens were observed on these
substrates. Neither was P. squarrosum found on the other non-chiton
substances. There were certainly a variety of other small algae, but no
P. squarrosum.
Pleonosporium squarrosum Population Structure
To estimate population structure of Pleonosporium squarrosum, a
random sample of 167 plants were picked. Counts indicated 68% sterile
plants, 11% polysporangia-bearing plants (fig. 1), 11% male plants with
spermatangia (fig. 5), and 8% female plants with procarps (fig. 6).
Since the distribution of Pleonosporium squarrosum was found to
vary among the Cryptochiton populations (none on intertidal chitons and
different amounts on subtidal chitons), laboratory experiments were set up
to test hypotheses about the reasons for P. squarrosum specificity and
selectivity. Is it the substrate itself, the temperature, the light level.
the air exposure or another epizoic creature that determines P. squarrosum
incidence and growth?
Experimental
Spores from polysporangia were presented with various substrates in
the first experiment on germination preferences. Spores successfully
germinated on plastic, clamshell, worm tube, felt, glass and brick (fig. 7),
but no germinated spores were observed on sandpaper, living Loxorhynchus
carapaces, hydroid or red algae. Unfortunately, multiple attempts to
germinate spores on living Cryptochiton backs were unsuccessful. The
greatest amount of germination occurred when the polysporangial frond was
placed on the substrate and allowed to drop spores directly on the desired
substrate, rather than dropping the spores in a separate petri dish to be
pipetted up and then distributed.
A second experiment was conducted to check growth of Pleonosporium
squarrosum on non-chiton substrates. P. squarrosum, when cut, develops
rhizoids at the severed end (fig. 8). Transplants of these tiny cuttings
were attempted on a variety of possible substrates, including glass, plastic,
worm tube, hydroid fronds, red algae, brick and felt. The rhizoids were
observed entangled on the hydroid and worm tube, but the thalli turned
pink and died. Fronds were observed to only briefly stick to plastic and
glass, and no attachment was successful with red algae, brick or felt.
Plants transplanted onto bare-backed Cryptochitons did not die, but never
became firmly attached.
Temperature levels fluctuate from warm in tidepools to cold in
subtidal areas. Does temperature influence Pleonosporium squarrosum
distribution? Fronds were cut, put in sterile vials and held in temperature
baths to determine the effects of elevated temperature on P. squarrosum.
The alga had been maintained in the laboratory at 18.5°0, so 20°C was
chosen as a starting point with additional temperatures of 21°, 21°, and
25 C. Samples were placed in the baths at set times so that a continuum
from 1-6 h was achieved (fig. 9). The fronds turned bright pink (leakage
of photosynthetic pigments) or showed plasmolysis. There was no significant
change observed in the plants held at 20° and 21°0, nor in the control
held at 120. However after 2 h at 21°C 10% plant damage was noted, and
similarly at 25°0 plant damage rose after 2 h until 90% death was reported
after 6 h of exposure.
Light levels vary between tidepools, shallow subtidal and deep
subtidal areas. Is Pleonosporium squarrosum distribution and growth
affected by light? Growing tips were cut and placed in sterile vials in
the dark (O lux), under illumination equal to that at a depth of 25 ft
(86 lux), and under high illumination (1668 lux). The total length of the
fronds (thallus plus rhizoids) was measured and the total gain in length
was plotted against time (fig. 10). Most impressive was the difference
between increase in length of plants in low subtidal-like illumination
and those at a high intensity light level. The low illuminated fronds
grew over three times as much as the highly illuminated fronds. Much of
the growth of the plants in the dark was rhizoidal.
Intertidal Cryptochiton stelleri are often exposed to the air
during the tidal cycle. Is Pleonosporium squarrosum tolerant of exposure
to air? Fresh tips were cut and kept in 12°0 sea water. At set times the
tips were exposed to air by placing the fronds on dry plastic or damp
filter paper. A range of air exposure time was obtained-from 5 to 360 min
(fig. 11). The control which remained underwater showed no change. On
the plastic, partial plant damage was evident after only 15 min and the
amount of damage climbed rapidly to 100%. The fronds on the damp filter
paper showed damage after 30 min and seemed more resistant to death by
drying.
A final test investigated the influence of herbivory on Pleonosporium
squarrosum. Herbivorous snails are commonly seen on intertidal and shallow
subtidal (10 ft) Cryptochiton stelleri. Could the grazing behavior of
these gastropods account for the paucity of P. squarrosum at these tidal
levels? Two chitons with known P. squarrosum percent cover were placed
in an aquarium with 12 Tegula brunnea (Philipp, 1848). The snails crawled
all over the chitons and seemed to be eating. After 19 days the chitons
seemed barer and the remaining P. squarrosum wasstraggly. Fecal matter
of the Tegula brunnea was also collected and observed to be brown and
riddled with red chiton bristles. In another test, fronds of the alga
were put in containers, each containing one T. brunnea. This experiment
was repeated three times and each time one quarter of the fronds would
disappear or look nibbled. All the T. brunnea were fed Macrocystis pyrifera
(Linnaeus) C. Agardh for 3 days previous to testing so that the results
of this herbivory testing would not be artifacts of snail starvation and
thus indiscriminate eating.
Since Pleonosporium squarrosum did not seem to grow efficiently as
a transplant and the spores are non-motile, germinating best directly
under the polysporangial fronds, how is P. squarrosum spread throughout a
Cryptochiton population? Chitons are sometimes found in twos or threes.
but do they move enough to act as a dispersal agent for P. squarrosum?
On May 20, two tagged chitons were released at a 10 ft depth in the Mussel
Point kelp bed and two at a 25 ft depth. Two days later these released
chitons were relocated and the distance from their starting point was
measured. At 10 ft, one chiton had moved 2.l m and the other, O.3 m.
At 25 ft, one had moved 0.6 m and its companion, 1.0 m. Cryptochiton
movement is great enough to be measurable, although in a previous study off
the Oregon coast, the Cryptochitons moved only 20 m from the point of
release in two years (Palmer and Frank, 1974).
DISCUSSION
The distribution of Pleonosporium squarrosum is still an enigma,
but plausible explanations can be derived. P. squarrosum's intertidal
appearance at Friday Harbor and strictly subtidal existence in Monterey
Bay may be due to temperature zonation. Much marine life from high
latitudes is found deeper as the equator is approached (Cullinery, 1976).
In experiments, P. squarrosum survived at such temperatures as 20°C and
21°0, but only long term experimentation would determine whether the
alga actually thrives and completes its life cycle at elevated temperatures.
P. squarrosum has never been reported in areas of warm water (Abbott and
Hollenberg, 1976), nor is its distribution as wide as that of its host.
A careful search for a northern Cryptochiton-Pleonosporium association
should be undertaken since Cryptochiton are abundant intertidally along
the Oregon coast (Palmer and Frank, 1974), at Friday Harbor and northward,
and may again support a P. squarrosum population.
On the Monterey Peninsula, why aren't all Cryptochiton stelleri
carriers of Pleonosporium squarrosum? Tidepool water may get too warm.
In addition, light intensity differs by magnitudes between 25 ft under
a kelp canopy and in the direct sunlight at low tide. Experimental results
did show a difference in growth rate of vegetative axes at different light
levels. In stress conditions of high light intensity the phycobilins, the
major photosynthetic pigments in red algae, are bleached out and photosyn¬
thetic rate and plant growth would diminsh(fStrain, 1951). Air exposure
and desiccation is probably a major factor limiting the Cryptochiton-
Pleonosporium association. The delicate alga showed no tolerance for
drying out in the laboratory and a chiton back, although damp, would be
unsatisfactory at low tide. Another possible reason for bare-backed
chitons intertidally and shallow subtidally (10 ft) is the existence of
herbivorous gastropods, specifically Tegula brunnea, that crawl over
chiton backs. In the laboratory they appear to be potential devastators
of epizoic P. squarrosum. The fecal matter filled with bristles alone
indicates that they do rasp Cryptochiton backs. Field studies would be
necessary to truly test the herbivory effects on C. stelleri with P.
squarrosum.
Since Pleonosporium squarrosum seems so selective for subtidal
living in Monterey, and since it does germinate on non-chiton substrates
in the laboratory, why is P. squarrosum no found subtidally without its
Cryptochiton? Perhaps the spores have little or no chance to drift
around and onto other substrates before germination (2-1 days after release).
Possibly P. squarrosum not on chitons is more easily grazed off. Maybe
the tiny alga loses in the competition for space, as on rock faces. On
Cryptochiton backs another species of algae for instance, Rhodoptilum
plumosum (Harvey) Kylin, may dominate and crowd out the P. squarrosum.
Decorator crabs, Diopatra worms and hydroids may be selective in what they
allow to grow on their backs, tubes or fronds, and perhaps P. squarrosum
rarely is given a start. More experiments should be done to follow the
laboratory spores that germinated—how long do they live? do they flourish?
or do they stagnate like the transplanted growing tips?
An important question remains—why the great discrepancy in amount
of Pleonosporium squarrosum on Cryptochitons inttwo kelp beds separated
by only a sand channel? Mussel Point and Point Alones kelp beds are
different. The Point Alones kelp bed has less exposure to swells, has
calmer water, is generally deeper, has a more sandy bottom, has higher
salinity and a lower Cryptochiton density. Later in the season will
Point Alones chitons migrate to Mussel Point kelp bed and spread more
P. squarrosum? Observations demonstrated that chitons do travel and
further underwater tracking would be interesting.
The association between Cryptochiton stelleri and Pleonosporium
squarrosum is clearly evident. P. squarrosum prefers C. stelleri as a
source of space and refuge. However, new questions and research
possibilities increase with each Cryptochiton back found covered with
Pleonosporium squarrosum.
SUMMARY
1. Pleonosporium squarrosum varies in distribution and density with
geographical location of Cryptochiton stelleri population: P. squarrosum
is absent on intertidal Cryptochitons from Pescadero Point and Sti
water Cove and on shallow subtidal (10 ft) Cryptochitons; P. squarrosum
occurs on 928 of the Point Alones Cryptochitons with a mean of 60%
cover on individual chitons; P. squarrosum is present on the whole on
51% of Mussel Point Cryptochitons with a mean individual percent
cover of 13
On Cryptochiton dorsal surfaces, P. sauarrosum tends to be centrally
located.
3. P. squarrosum spores germinate on many non-chiton substrates, but
transplanting cut portions is not successful.
1. P. squarrosum dies at elevated temperatures of 21°C and above.
P. squarrosum seems best adapted for growth at underwater light levels
P. squarrosum can not tolerate exposure to air and its drying effects.
Tegula brunnea are found crawling on Cryptochiton backs, will eat
P. squarrosum, and may be a limiting factor in the alga's distribution.
6.
Cryptochiton stelleri is a probable dispersal agent for Pleonosporium
squarrosum.
Kerla Metermid
12
ACKNOVLEDGEMENTS
Many thanks to all my friends: Dr. Isabella Abbott—enthusiastic
mentor, Jim Watanabe—dive buddy nonpareil, Roger Phillips-patient
cameraman, Bill Magruder—spore settling expert, Chris Thornton—seawater
filterer, and a special thank you to my herd of willing chitons.
LITERATURE CITED
Abbott, Isabella A. and George J. Hollenber.
1976. Marine Algae of California. i-xii+ 827 pp.; illus.
Stanford, Calif. (Stanford Univ. Press)
Cullinery, John L.
1976. The Forests of the Sea. i-x+290 pp. San Francisco,
Calif. (Sierra Club Books/
ylin, Harald
1925. The Marine Red Algae in the Vicinity of the Biological
Washington. Lunds Universitets Arsskrift
Station at Friday Harbor.
N.F. Avd. 2 21:1-87; plt.
MacGintie, George Eber and Nettie MacGintie
1968. Notes on Cryptochiton stelleri (Middendorff, 1816). The
Veliger 11(1): 59-61; 1 plt.
(1 July 1968)
Palmer, John Beach and Peter Wolfgang Frank
1974. Estimates of Growth of Cryptochiton stelleri (Middendorff,
1846) in Oregon. The Veliger 16(3): 301-301
Smith, A.G.
1975. Smaller Molluscan groups... In: R.I. Smith and J. Carlson (eds.),
Light's Manual:
Intertidal Invertebrates of the Central
California Coast. i-xvii +710 pp.; plts. 1-156. Berkeley,
Calif. (Univ. of California Press)
Strain, Harold H.
1951. The Pigments of Algae. In: G.M. Smith (ed.), Manual of
Phycology vol. xxvii: 213-260. Waltham, Mass. (Chronica
Botanica Co.)
EXPLANATIONS of FIGURES
1. Table of Cryptochiton density and percent of chitons with
Pleonosporium squarrosum.
2. Graph of percent cover by P. squarrosum on Cryptochitons from
Mussel Point and Point Alones.
3. Table of substrates examined for P. squarrosum growth.
4. P. squarrosum with polysporangia, 10X.
5. Male P. squarrosum with spermatangia, 10X.
6. Female P. squarrosum with procarp structure, 20X.
7. Germinated P, squarrosum spore, LOX.
8. P. squarrosum frond showing rhizoids, IX.
9. Graph of P. squarrosum response to elevated temperature.
10. Graph of P. squarrosum growth in different light levels.
11. Graph of P. squarrosum response to air exposure.
0
g.
LOCATION
Mussel Point Kelp Bed
10 feet deep
25 feet deep
35 feet deep
Point Alones Kelp Bed
25 feet deep
Pescadero Point
Stillwater Cove
AVERAGE DENSIT!
(chitons/m2)
.10
10
.15
.06
.29
.21
PERCENT of CHITONS with
P. squarrosum
87.5  mean - 54.25
100
901
80
70
60
0
a 50
2 40
30
20
—
10

L.
ig. 2

individual chitons
from
MUSSEL POINT

.

.
..

L


.

-...



-
1
P
individual chitons
from
POINT ALONES
fig. 3
LOCATION
AMT. of P.
SAMPLE
ALGAE FOUND
squarrosum
SLE
Wharf pilings, Monterey Harbor
50
Platythamnion sp.,
Pterosiphonia dendroidea
Wharf pilings, Stillwater Cove
Ulva sp., Porphyra sp.,
Enteromorpha sp.
Loxorhynchus crispatus
Polysiphonia sp.,
ryopsis sp.
hydroid colonies
Pleonosporium vancouverianum
Diopatra ornata tubes
faniella sp.
10
clam and abalone shells
faniella sp.
subtidal Pagurus shells
subtidal rock faces
variety
subtidal algae
sandy sea bottom
3 quadrats Pterosiphonia sp.
5
fig.










S
Soam
fig. 5
304m
AA
fig 6
4

fig.


V





50m
fig. 8
fi

—
L
ppod
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0
J-0 1
0
---
fig. 10
41
5
9
08

20


LOW (86 lux)
DARK (O lux
HIGH (1668 lux
—


T
1 2 34 56 7 89
duration of exposure to light (days)
fg 11


1

OONODSO
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8
0
10
c
—