Abstract
The algal-cnidarian symbiosis is a useful system for investigating patterns of selection
and adaptation different from standard single-organism models. Current research, mostly focused
on tropical ecosystems, indicates that the host/symbiont relationship can be flexible: hosts can
house different symbionts under different conditions. This flexibility may allow for adaptation of
the entire symbiotic system (holosymbiont) rather than the individual players. However, less is
known about the behavior of temperate algal-cnidarian systems. In this study, 1 examine the fine
scale dinoflagellate symbiont biogeography in the temperate sea anemone Anthopleura
elegantissimam, as first observed by LaJeunesse and Trench (2000). Restriction Fragment
Length Polymorphism (RFLP) analysis of small subunit (SSU) ribosomal DNA indicates a
complex transition zone from Cape Mendecino (N40° 24’, W124° 23’) to Salt Point (N38° 33’,
W123° 20’), with anemones to the north harboring Symbiodinium muscatinei and those to the
south co-harboring S. muscatinei and S. californium. S. californium is present in the north and
south of the transition zone, but absent from the center. A new RFLP signature was found in
anemones from the center of the transition zone, suggesting the presence of a third dinoflagellate
species endemic to that area.
Introduction
The algal-cnidarian symbiosis offers an important example of the potential for non¬
genotypic adaptation in the natural world: current understanding suggests that multiple algal
symbiont species may exist in flexible, adaptive relationships with their cnidarian hosts
(Buddemeier and Fautin 1993; Baker 2003). These symbionts, dinoflagellates of the genus
Symbiodinium, represent an ancient and deeply diverged group (Rowan and Knowlton 1992;
Rowan 1998) in which closely related types can differ in their response to such important
environmental factors as temperature and irradiance (Rowan and Knowlton 1995; Savage et al.
2002; Baker 2003). While host-symbiont associations that exhibit high degrees of specificity
appear to be range-limited by the physiological limitations of their symbionts (lglesias-Prieto et
al. 2004), other, more flexible relationships may allow for greater and faster adaptation to
different environmental conditions (Rowan et al. 1997; Baker 2001).
The widely distributed temperate sea anemone Anthopleura elegantissima may use
symbiont-flexibility to exploit a range of ecological niches. In addition to dinoflagellate
zooxanthellae, A. elegantissima can host chlorophyte zoochlorellae (Muscatine 1971; Lewis and
Muller-Parker 2004). Zoochlorellae and zooxanthellae differ physiologically with respect to
temperature and irradiance (Verde and McCloskey 2001, 2002) and are associated with
anemones in different environments: zooxanthellate anemones are found preferentially in the
exposed upper intertidal, while zoochlorellate anemones tend to be in lower, shaded habitats
(McCloskey et al, 1996). In transplant experiments, aposymbiotic anemones grew symbiont
populations adapted to the local microhabitat (i.e. zooxanthellae in bright light, zoochlorellae in
partial light), suggesting that environmental factors can select for symbiont population type in A.
elegantissima (Secord and Muller-Parker, 2005).
A. elegantissima also hosts at least two species of Symbiodinium zooxanthellae
(LaJeunesse and Trench 2000). S. muscatinei is found throughout A. elegantissima’s range from
southern California to northern Washington, while S. californium has been reported in
association with S. muscatinei only south of Carmel, California. These data suggest that
environmental factors may also account for variation of zooxanthellae type in A. elegantissima,
and the authors suggest temperature as the limiting factor. However, the large distance between
sample sites in that study limits the utility of the results. Knowledge of the biogeography of A.
elegantissima’s symbionts on a finer spatial scale could be useful in assessing the actual factors
involved in creating the variation described above, as well as tracking and predicting the effects
of changing environmental conditions on the Pacific coast.
In this study, I utilized the methods of LaJeunesse and Trench (2000) to develop a finer-
scale biogeography of A. elegantissima’s dinoflagellate symbionts. I sampled anemones at 30-80
kilometer intervals between Bodega Bay, CA and the Oregon border, amplified a portion of the
18s ribosomal gene using Symbiodinium specific primers, and characterized genotype using
RFLP analysis.
Methods and materials
Collecting
Tissue samples from 83 A. elegantissima were collected in May of 2005 between Pacific
Grove and the Oregon border. A small piece of tissue was taken from each anemone, ranging
from a few tentacles to a radial section, and preserved in 70% ethanol.
Specific locations included Pelican Point (PP; N41° 57’, W124° 12’); Wilson Creek
Beach (WC; N41° 36’, W124° 07’); Trinidad Head, north and south sides (TN and TS; N41° 02’,
W124° 08’); Humboldt Bay north jetty, interior and exterior (HI and HE; N40° 46’, W124° 14’);
Mattole Road on Cape Medocino (MR; N40° 24’, W124° 23’); Shelter Cove(SC; N40° 01’,
W124° 04’); Van Damme State Park, near Albion (AL; N39° 16’, W123° 47’); Point Arena Cove
(PA; N38° 54’, W123° 43’); Salt Point (SP; N38° 33’, W123° 20’); Bodega Bay (BB; N38° 19’,
W123° 04’); and Pacific Grove (PG; N36° 37’, W121° 37’). Preliminary analysis of Bodega Bay
specimens indicated the presence of S. californium, so no samples were taken between there and
Pacific Grove.
Between four and ten anemones were sampled at each site (Except PG, n-2). Samples at
each site were taken from different tide heights and light exposure levels, and where possible,
from different sides of potential biogeographic break points (e.g. Trinidad Head).
DNA Extraction, Amplification, and RFLP Analysis
I extracted DNA from the samples using a NucleoSpin6 spin-column kit from BD
Biosciences. Between 1 and 3 tentacles from each anemone were extracted according to
manufacturer instructions, except at half reagent volumes and spinning at 6000 rpm in a
microcentrifuge for every step except elution. Columns were eluted with 50 ul warmed elution
buffer as supplied in the kit and microcentrifuged at 3000 rpm for one minute.
PCR amplification of an approximately 1800bp piece of thel 8s Small Sub-Unit
ribosomal DNA (SSUFDNA) was performed using the following Symbiodinium-specific primers
developed by Rowan and Powers (1991): sssz (GCA GTT ATA RTT TAT TTG ATG GTY
RCT GCT AC) and ss3z (AGC ACT GCG TCA GTC CGA ATA ATT CAC CGG). 1:10
dilutions of the extracted genomic DNA were amplified in 25 ul reactions on a DYADTM
thermocycler (MJ Research) under the following conditions: an initial denaturing at 92°C for 3
minutes; 35 cycles of 92°C denaturing for 30 seconds, 50°C annealing for 1 minute, and 72
extension for 1 minute; and a final 5 minute extension at 72°C. Amplifications were visualized
on 1.5% agarose gels stained in ethydium bromide.
Successful amplifications were digested with aTagl restriction enzyme in New England
Biolabs Buffer 3 with BSA. 5 ul of the restriction solution, containing 1 unit of enzyme, were
added to 20 ul of PCR product and incubated at 65°C for four hours. The product was run out on
3% agarose gels at 100V for two hours, then stained in ethydium bromide solution and
photographed.
I also amplified a sequence of approximately 650bp spanning part of 18s, internally
transcribed spacer (ITS) 1, 5.8s, ITS2, and part of 28s using the primers s-DINO (CGC TCC
TAC CGA TTG AGT GA) and DIR-rev (ATA TGC TTA AAT TCA GCG GGT). 25ul
reactions were performed on a DYADTM thermocycler (MJ Research) under the following
conditions: an initial denaturing at 92° for 3 minutes; 35 cycles of 92° for 30 seconds, 55° for 30
seconds, and 72° for 2 minutes; and a final extension at 72° for 5 minutes.
Cloning, DNA sequencing, and analysis
SSU amplifications from sample BB4 and ITS amplifications from samples BB4, PAS,
and PP6 were cloned in competent E. coli cells using the pGEM-TG vector kit from Promega.
The resulting colonies were then picked into strip tubes and screened for presence of the desired
sequence. Screens were run on a DYADTM thermocycler (MJ Research) using the vector primers
T7 and M13r under the following conditions: initial denaturing at 94° for 3 minutes, 35 cycles of
94° for 30 seconds, 55° for 30 seconds, and 72° for 1 minute 30 seconds, followed by a 5 minute
final extension. Some successful screens were reamplified using the internal SSU primers and
reaction conditions as described above.
Screens were visualized on 1.5% agarose gels stained in ethydium bromide, and those
yielding the desired band size were cleaned for sequencing using shrimp alkaline phosphatase
and exonuclease.
Cycle sequencing was performed on a DYADTM thermocycler (MJ Research) in 1Oul
reactions containing 6.5ul water, 1.75ul 5X sequencing buffer, lul cleaned PCR template,
0.25ul primer, and 0.5ul BigDye6 from Applied Biosystems, Inc. Reaction conditions were as
follows: initial denaturing at 96° C for 3 minutes followed by 40 cycles of 96° C for 10 seconds,
50° C for 10 seconds, and 60° C for 4 minutes. Sequencing was attempted using all primers
described above. DNA was precipitated from the product with 40ul of 75% isopropanol and
pelleted in a centrifuge at 12,500g for 1 hour. Pellets were dried, resuspended in formamide, and
sequenced on an Applied Biosystems 3100 Genetic Analyzer.
Sequences were cleaned and aligned in SequencherTM 4.2 (Applied Biosystems, Inc.).
Alignments were exported to PAUP* 4.0b10 (available from Sinauer Associates, Inc) and
analyzed for parsimony.
Results
Digestion with aTagl yielded three characteristic RFLP profiles distributed
geographically (Fig 1). The first, found north of Cape Mendecino, has distinct bands at
approximately 900bp, 500bp, and 200bp (Fig 2a), equivalent to the restriction sites for S.
muscatinei as described by LaJeunesse and Trench (2000).
The second, found south of Cape Mendecino, had the same three bands as the northern
populations with an additional band at approximately 800bp (Fig 2b). This additional band
coincides with the band found by LaJeunesse and Trench (2000) in southern anemone
populations, and may represent both large fragments (867bp and 794bp) predicted by the
sequence for S. californium in GenBank (AF225965). The 129bp fragment predicted by this
sequence is visible in digests of all samples from Pacific Grove and Bodega Bay and one from
Salt Point (Fig 3a), but not in those north of Bodega Bay (Fig 3b). This may be due to poor
amplification of S. californium from anemones with mixed populations (LaJeunesse and Trench,
2000). Alternatively, it may be due to the loss of a restriction site in some S. californium from
northern anemones. Digests displaying the 800bp band from Pacific Grove to Cape Mendocino
yielded an additional band at around 1000bp not described by LaJeunesse and Trench (2000), a
band especially strong in those digests that did not show the 129bp fragment. This 1000bp band
may represent bases 1-1000 of the amplified S. californium fragment, undigested either due to
loss of the 867bp restriction site in these populations or an incomplete digestion.
Notably, the band pattern representative of S. californium is absent in digests from
Albion and Point Arena, both of which are south of Cape Mendocino. All samples from these
two sites, as well as several from Mattole Road and two from the south side of Trinidad Head,
displayed a patter similar to that from northern samples but with two additional bands: one near
880bp, the other near 480bp (Fig 2c). This new restriction pattern is undescribed, and may
represent a new Symbiodinium type endemic to the central California coast.
In the two collection locations where multiple restriction patterns were present, samples
didn’t appear to be correlated to elevation above mean tide level or basic microhabitat type. At
the Mattole Road location, the only one to show all three restriction patterns, there appeared to
be at least one anemone (MR8) containing each Symbiodinium type. The full data set is given in
Appendix I.
To test the possibility that the third, novel restriction pattern represented a new
Symbiodinium type, I cloned and sequenced SSU and ITS sequences from sample PAS, which
displays the new restriction pattern; and PP6, which doesn’t. Unfortunately, the resulting SSU
sequence fragments, ranging from 125 to 350bp, were too short to include any of the restriction
sites. ITS clones, which yielded sequences between 500 and 650bp, mostly clustered with an ITS
sequence from S. muscatinei obtained from GenBank (AF333510). While one cloned sequence
from PAS grouped outside this cluster, a 100-replicate bootstrap search does not show
significance (Fig 4).
Discussion
This study shows a clear shift in dinoflagellate symbiont types inhabiting A.
elegantissima on the California coast, and characterizes the primary transition zone between the
two major symbiont types as an approximately 240 kilometer section between latitudes N40° 24'
(Cape Mendecino) and N38° 23’ (Salt Point). This transition zone is not a gradual cline, showing
instead at least one alternation between northern- and southern-type symbiont communities. A
new RFLP profile endemic to the transition zone suggests but does not confirm the presence of
an undescribed Symbiodinium species inhabiting anemones in this transition zone.
While the large-scale results—specifically the loss of S. californium in anemones
between central California and Oregon—are consistent with previous observation (LaJeunesse
and Trench, 2000), the finer scale of sampling reveals a greater degree of complexity than was
previously understood.
It has been proposed that colder water temperatures are the primary selective force
limiting S. californium’s range to the south (LaJeunesse and Trench, 2000). Although local water
temperature has been well established as the most important factor in determining Anthopleura
symbiont type at the phylum level (Saunders and Muller-Parker, 1997; Secord and Augustine,
2000), its effects on symbiont population at the species level have not been directly studied.
Most studies of temperature effects on symbiont population composition in cnidarians focus on
tropical subtidal species, and most directly demonstrated physiological species boundaries are for
maximum rather than minimum thermal tolerances (Baker, 2003). Since A. elegantissima is an
intertidal species exposed to a much wider range of environmental extremes, those studies may
have limited applicability.
The biogeographic complexity of the transition zone raises questions about the
hypothesis that cold water temperatures are the major factor controlling the distribution of
Symbiodinium type in A. elegantissima. How can temperature variation account for the
ubiquitous representation of S. californium in samples from Salt Point and Shelter Cove and its
complete absence from samples at two points in between? The relative lack of sites showing
multiple symbiont community profiles suggests that either competition between symbiont types
is strong enough to act uniformly on small differences, or that the local environmental
differences controlling symbiont community structure are large enough to generate a sharp cline.
If water temperature is the controlling variable, measurements at those locations should track the
symbiont distribution. Such an observation would be the first strong evidence in favor of that
hypothesis.
Because A. elegantissima is a mid- to upper-intertidal organism, however, its most
significant thermal stresses are likely to occur when exposed to air. Studies of another Pacific
10
coast intertidal species, the California mussel, have shown that interactions between tide timing,
wave splash, and terrestrial conditions can generate selectively significant "hot spots" at northern
latitudes (Helmuth et al., 2002). Such a phenomenon could account for the transition zone
observed in this study: Cape Mendecino could represent an area of reduced cold stress during the
most selectively important times, thus allowing for the presence of S. californium farther north
than a simple temperature gradient would predict.
This study also suggests, but does not confirm, the presence of a third Symbiodinium type
present in California A. elegantissima. The additional RFLP signal in some samples is difficult to
explain by poor amplification because of the clarity and uniformity of the bands, and because of
the geographic consistency of the signal. However, the smaller size of both new bands implies
the presence of a third new band, potentially in the 300-400bp range, which is not visible in any
of the digests. Sequence analysis, if consistent with the new RFLP signal, should provide ample
evidence either way. More extensive molecular investigation will be required, but if
substantiated as an additional member of the A. elegantissima symbiont group, this third type
would be unique in its limited range.
Conclusion
In this study, I used RFLP analysis of 18S Small Subunit Ribosomal DNA to assess the
previously undescribed fine scale biogeography of Symbiodinium species in symbiotic
relationships with Anthopleura elegantissima on the California coast. Results were consistent
with a single species (Symbiodinium muscatinei) inhabiting all anemones, with a second species
(S. californium) found only in southern hosts. The transition appeared to take place between
Cape Mendocino and Salt Point, a distance of approximately 240 kilometers. Symbiont
populations within the transition zone did not follow a simple cline, instead displaying near-
ubiquitous S. californium presence at the north and south ends of the zone and complete absence
in the center. RFLP digests from anemones in the transition zone which lacked S. californium
displayed a new restriction pattern, suggesting the presence of a previously unknown
Symbiodinium type endemic to the transition zone.
Acknowledgements
Many thanks to George Somero and Steve Palumbi, my mentors and advisors throughout
this project; to the entire Palumbi lab for their kindness and patience in hosting me, especially
Tom Oliver and Adam McCoy; to Jim Watanabe for his assistance in editing. Special thanks to
Chris Harley of Bodega Bay Marine Lab for collecting the Bodega Bay samples and generously
hosting me on my own collecting trip.
Literature Cited
Baker, A. C. 2001. Reef corals bleach to survive change. Nature 411: 765-766.
Baker, A. C. 2003 Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and
biogeography of Symbiodinium. Annual Review of Ecology, Evolution, and Systematics
34: 661-68.
Buddemeier, R. W. and D. G. Fautin. 1993 Coral bleaching as an adaptive mechanism.
Bioscience 43: 320-326.
Helmuth, B.S., C. D. G. Harley, P. Halpin, M. O’Donnell, G. E. Hofmann, and C. Blanchette.
2002. Climate change and latitudinal patterns of intertidal thermal stress. Science 298:
1015-1017.
Iglesias-Prieto, R., V. H. Beltran, T. C. LaJeunesse, H. Reyes-Bonilla, and P. E. Thome. 2004.
Different algal symbionts explain the vertical distribution of dominant reef corals in the
eastem Pacific. Proceedings of the Royal Society of London 271: 1757-1763.
LaJeuness, T. C. and R. K. Trench. 2000. Biogeography of two species of Symbiodinium
(Freudenthal) inhabiting the intertidal sea anemones Anthopleura elegantissima (Brandt).
Biological Bulletin 199: 126-134.
Lewis, L. A. and G. Muller-Parker. 2004. Phylogenetic Placement of "Zoochlorellae
(Chlorophyta), Algal Symbiont of the Temperate Sea Anemone Anthopleura
elegantissima. Biological Bulletin 207: 87-92.
McCloskey, L. R., T. G. Cove, and E. A. Verde. 1996. Symbiont expulsion from the anemone
Anthopleura elegantissima (Brandt) (Cnidaria; Anthozoa). Journal of Experimental
Marine Biology and Ecology 195: 173-186.
Muscatine, L. 1971. Experiments with green algae coexistent with zooxanthellae in sea
anemones. Pacific Science 25: 13-21.
Rowan, R. 1998. Diversity and ecology of zooxanthellae on coral reefs. Journal of Phycology 34:
407-417
Rowan, R. and D. A. Powers. 1992. Ribosomal RNA sequences and the diversity of symbiotic
dinoflagellates (zooxanthellae). Proceedings of the National Academy of Sciences of the
United States of America 89: 3639-3643
Rowan, R. and N. Knowlton. 1995. Intraspecific diversity and ecological zonation in coral-algal
symbiosis. Proceedings of the National Academy of Sciences of the United States of
America 92: 2850-2853.
13
Rowan, R., N. Knowlton, A. Baker and J. Jara. 1997. Landscape ecology of algal symbionts
creates variation in episodes of coral bleaching. Nature 388: 265-269.
Rowan, R. and D. A. Powers. 1991. Molecular genetic identification of symbiotic dinoflagellates
(zooxanthellae). Marine Ecology Progress Series 71: 65-73.
Saunders, B. K. and G. Muller-Parker. 1997. The effects of temperature and light on two algal
populations in the temperate sea anemone Anthopleura elegantissima (Brandt, 1835).
Journal of Experimental Marine Biology and Ecology 211:213-224.
Savage, A. M., M. S. Goodson, S. Visram, H. Trapido-Rosenthal, J. Wiedenmann and A. E.
Douglas. 2002. Molecular diversity of symbiotic algae at the latitudinal margins of their
distribution: dinoflagellates of the genus Symbiodinium in corals and sea anemones.
Marine Ecology Progress Series 244: 17-26.
Secord, D. and G. Muller-Parker. 2005. Symbiont distribution along a light gradient within an
intertidal cave. Limnology and Oceanography 50: 272-278.
Secord, D. and L. Augustine. 2000. Biogeography and microhabitat variation in temperate algal¬
invertebrate symbioses: Zooxanthellae and zoochlorellae in two Pacific intertidal sea
anemones, Anthopleura elegantissima and A. xanthogrammica. Invertebrate Biology 119:
139-146.
Verde, E. A. and L. R. McCloskey. 2001. A comparative analysis of the photobiology of
zooxanthellae and zoochlorellae symbiotic with the temperate clonal anemone
Anthopleura elegantissima (Brandt). I. Effect of temperature. Marine Biology 138: 477-
489.
Verde, E. A. and L. R. McCloskey. 2002. A comparative analysis of the photobiology of
zooxanthellae and zoochlorellae symbiotic with the temperate clonal anemone
Anthopleura elegantissima (Brandt). II. Effect of light intensity. Marine Biology 141:
225-239.
Figures Legend
Figure 1. Symbiont populations of anemones on the Northern California coast. Pie graphs
illustrate relative abundance of each RFLP profile at each collecting site; pie size represents
sample size.
Figure 2. Three RFLP digests displaying the three different signatures. A) Northern symbiont
community; three bands indicate the sole presence of S. muscatinei. B) Southern symbiont
community C) Transitional symbiont community. Note the presence of additional bands
(arrowed) approximately 20-40 base pairs smaller than the two larger bands in a). This pattern is
indicative of S. muscatinei, possibly mixed with an unknown Symbiodinium type.
Figure 3. RFLP digests for anemones containing S. californium at two locations. A) Samples
from Bodega Bay. Note clear presence of a 129bp band, indicated by an arrow. B) Samples from
Shelter Cove, the northernmost location in which all samples harbored S. Californium. Note
absence of 129bp band and presence of a stronger band at around 110Obp, indicated by an arrow.
Figure 4. 100-replicate bootstrap tree of cloned ITS sequences from the symbiont populations of
anemones from Pigeon Point (PP6) and Point Arena (PA5). PP6 displays a typical S. muscatinei
RFLP signature. PA5 displays the intermediate, unknown RFLP signature in additions to the S.
muscatinei. Clone itsPA5-B7 may represent an additional Symbiodinium species.
Figure 1
PP
W
TI
T5
H
HE
MR
Sc
AC
PA (
Sp
B8C
S. muscatinei
S. muscatinei + unknown
S. muscatinei + S. californium
PGC
16
gure 2
Figure 3
18
Figure 4
Bootstrap

73


— ItSPA5-A8-f D10 08.ab1
— itSPPG-C7-fG10 14.ab
— itsP A5-B8-f F1O 12.ab1
—itsPA5-H8-fG08 14.ab1
— ItSPA5-E7-1 B08 04.ab1

— itsPA5-G7-f EOS 10.ab1
itSPP6-A5-f 007.13 ab
— S.nuscatnei
—itSPA5-B7-f E10 10.ab
S.-califomium
Appendix: RFLP symbiont types by sample
Pelican Point 1
4 -
Wilson Creek 1
M
Trinidad North 1
M, I
M, I
Trinidad South 1
Humbolt Interior 1
Humbolt Exterior 1
4
Mattole Road 1
M, C
M, C
M, I
Legend
Unsuccessful amplification
Symbiodinium muscatinei present
Unknown RFLP signature present
Symbiodinium californium present
Shelter Cove 1
Albion 1
Point Arena 1
Salt Point 1
Bodega Bay 1
Pacific Grove 1
M, I
M, I
M, C
M, I
M, I
M, C
M, C
M, C
M, C
M, C
M, C
M, I
M, I
M, I
M, I
M, I
M, I
M, I
M, I
M, I
M, I
M, I
M, I
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
M, C
20