ABSTRACT
Endosymbiotic algae, commonly of the genus Symbiodinium, are important in the life
cycles of many marine invertebrates. Being aware of the diversity of algal symbionts in species
of symbiont-containing invertebrates is a first step towards understanding host-symbiont
interactions and what adaptive advantages these interactions provide. The symbionts of a
common Californian intertidal sea anemone, Anthopleura sola, were surveyed across a
latitudinal gradient of approximately 800 km. A. sola samples were collected at six sites from
Monterey Bay to San Diego, California. Analysis of the chloroplast 23s ribosomal RNA gene
showed three distinct types of Symbiodinium (types I, II, III). Data show previously unknown
diversity in Anthopleura symbionts in a tri-partite latitudinal replacement of Symbiodinium types.
Type I was the only Symbiodinium haplotype present in Monterey, the northernmost site. Going
south, it was still the dominant type in Big Sur, but type III was also present in low levels. Only
type III was present in the Santa Barbara Channel. The final three, low latitude sites, White's
Point, Dana Point, and Cabrillo Point, were populated with types II and III. Type II was more
common at White's Point and Dana Point, while type III was more common at the southernmost
site, Cabrillo Point. Results suggest that type I is a cold-adapted Symbiodinium, type II is cool¬
adapted, and type III is warm-adapted.
INTRODUCTION
Zooxanthellae, golden-brown endosymbiotic algae, commonly of the genus
Symbiodinium, are important symbionts in the life cycles of many protists, corals, sea anemones,
and bivalves (Droop 1963; McLaughlin and Zahl 1966; Trench 1987). The symbiotic
interactions between invertebrates and algal endosymbionts are an important source of primary
productivity in the world ocean. Studies of Symbiodinium have shown much diversity within
members of the genus (Rowan 1998). Being aware of the diversity of algal symbionts in species
of symbiont-containing invertebrates is a first step towards understanding host-symbiont
interactions and what adaptive advantages these interactions provide.
Along the North American Pacific coast, anemone species of the genus Anthopleura are
commonly found to be in symbiotic relationships with members of the genus Symbiodinium.
There are more than 30 named species within the genus Anthopleura (Duchassaing and
Michelotti, 1860), both clonal and solitary, zooxanthellate and azooxanthellate (Geller and
Walton 2001). Three sister species, Anthopleura elegantissima, A. sola and A. xanthogrammica,
are zooxanthellate and found cohabitating along the central California coast. A. sola was
previously known in the literature as the solitary form of A. elegantissima until it was proven to
be a separate non-interbreeding species (Pearse and Francis 2000).
Studies of the Symbiodinium communities in Anthopleura have focused on A.
elegantissima, both the clonal and solitary forms. Rowan and Powers (1991) identified only one
species of Symbiodinium, belonging to clade B, in samples collected from Pacific Grove,
California. Rowan (1998) noted that their analyses of the partial small subunit ribosomal DNA
(SSUrRNA) sequences underestimate diversity. LaJeunesse and Trench (2000) did a survey of
Symbiodinium in A. elegantissima at six sites from Washington to Southern California, sampling
27 solitary and 37 clonal specimens, citing the presence of two species of Symbiodinium, S.
muscatinei belonging to clade B and S. californium of unknown clade. S. californium was later
shown to be a contaminant free-living dinoflagellate (Jon Sanders, personal communication).
Sanders (2005) described the presence of three types of Symbiodinium in a survey of A.
elegantissima from southern Oregon to Monterey Bay, California.
Given the recent designation of Anthopleura sola as a separate species, work has yet to be
done to determine many baseline data among this species and its symbiont(s). This study
identified and compared populations of Symbiodinium within A. sola across about 800 kilometers
of A. sola's central range from Monterey Bay, California to San Diego California. Analysis of
the chloroplast 23s ribosomal RNA gene was used to delineate differences in populations of
Symbiodinium.
The main questions I hoped to answer were: Which Symbiodinium are symbiotic with A.
sola? Are the same Symbiodinium found in A. sola and A. elegantissima?
Based on the prior studies of A. elegantissima, which looked at both the clonal and
solitary forms (i.e. A. sola) and which found only a single Symbiodinium species present at any
one site, the naïve null hypothesis is that the Symbiodinium populations found in A. sola are the
same as those found in A. elegantissima.
MATERIALS AND METHODS
Specimen collection
Anthopleura sola samples were collected at six sites along the California coast (Fig. 1.) at
China Point, Pacific Grove (N36° 37'; W121° 54); San Geronimo Road, Cayucos (N35
W120° 56); Coal Oil Point, Santa Barbara (N34° 25'; W119° 52'), White’s Point, San Pedro
(N33° 43'; W118° 182), Dana Point, Dana Point (N33° 28; W117° 43'), and Cabrillo Point, San
Diego (N32° 40'; WI17° 15’). At each site, twelve A. sola individuals were sampled from as
many habitats as possible by taking tentacle clippings, using scissors that were washed in
seawater between individuals to remove any tissue from the previous individual. Each sample
was placed in a 2.5ml tube. The tubes were then filled with 95% alcohol and placed in the
refrigerator once back at the laboratory
Genomic DNA extraction, PCR amplification, and DNA Sequencing
Genomic DNA was extracted from two to three tentacle clips (about 25 mg) using a
NucleoSpin OTissue kit and protocol for proteinase digestion and spin-column separation
(Macherey-Nagel Inc., Easton, PA).
The chloroplast 23s ribosomal RNA gene was amplified from 1 ul of genomic DNA
using primers of Zhang et al. (2000) and the following reaction volumes: 16.875 ul H2O, 2.5 ul
dNTPs, 2.5 ul MgClz buffer, 0.125 ul EconoTaq polymerase (Applied Biosystems), and 1 ul
each forward and reverse primers. PCR amplifications were done on a Peltier thermal cycler
with an initial denaturing step of 1 min at 95° followed by 35 cycles of 45 s at 95°, 45 s at 50°.
60 s at 72°, followed by a single cycle of 7 min at 72°. The PCR product was cleaned up by
adding 0.5 ul of shrimp alkaline phosphatase (SAP, Amersham Pharmacia), 0.5 ul of
exonuclease I (Exo I, Amersham Pharmacia), and 1 ul of dilution buffer and then incubated for
15 min at 37° followed by 15 min at 80°. Cycle sequencing of cleaned PCR product was
achieved with forward and reverse CP23s ribosomal RNA gene primers (Rowan and Powers
1991) and sequenced using an Applied Biosystems 3100 Genetic Analyzer.
Analysis
Sequences were grouped according to sample site and aligned using Sequencher 4.5
(Gene Codes Corporation). After sequences were visually cleaned and mixed sequences
removed, unique haplotypes were noted. Sample sequences were then compared across sites and
with A. elegantissima Symbiodinium sequences from Sanders (personal communication) using
PAUP*4.0 to generate maximum parsimony trees. Generic clade B Symbiodinium was used as
an outgroup and a heuristic bootstrap analysis of 20 replicates was run on the maximum
parsimony tree.
RESULTS
Five samples failed to amplify and several samples did not sequence cleanly or showed
mixed sequences, which were not analyzed. The number of clean sequences obtained from the
original 12 samples taken at each of the six sites are as follows (note that sites have been given
letter designations starting with A at the northernmost site to F at the southernmost): site A, 7;
site B, 11; site C, 10; site D,9; site E, 12; site F, 6 (Table 1).
Preliminary sequence data show much haplotype diversity both among and within sites
(Fig. 2). As shown on the phylogram, Symbiodinium populations can be grouped into two main
types, roughly equating to northern and southern populations (Fig. 2). Cladogram bootstrap
values indicate strong support (78) for the North-South population node (Fig. 3). The cladogram
also depicts moderate bootstrap support (65) for a second node within the northern type (Fig. 3).
These three distinct types of Symbiodinium are labeled types I, II, III and graphed according to
site (Fig. 4A). Data show tri-partite latitudinal replacement of Symbiodinium types (Fig. 4B).
Type lwas the only Symbiodinium haplotype present in the northernmost site (A). Going south.
it was still the dominant type in Big Sur (B), but type III was also present in low levels. Only
type III was present in the Santa Barbara Channel (C). The final three, low latitude sites,
White's Point (D), Dana Point (E), and Cabrillo Point (F), were populated with types II and III.
Type II was more common at White's Point (D) and Dana Point (E), while type III was more
common at the southernmost site, Cabrillo Point (F).
DISCUSSION
Much haplotype diversity exists within and among sites
A. sola Symbiodinium showed a surprisingly high degree of diversity considering there
were 7 unique haplotypes out of the 55 good sequences. A similar study by LaJeunesse and
Trench (2000) of Symbiodinium in Anthopleura that compared 64 individuals (37 A.
elegantissima and 27 A. sola) found only one type- and they sampled a latitudinal range more
than twice as large as the one in this study. Jon Sanders (personal communication), in a similar
study of A. elegantissima from Oregon to Southern California with about 100 samples, recorded
only 4 types of unique haplotypes, with only two types supported in a bootstrap analysis.
Within individual sites there was much diversity, with 4 of the 6 sites containing mixed
populations of Symbiodinium. A study of A. elegantissima showed no mixing of Symbiodinium
types at any sites (Fig. 5, Jon Sanders, personal communication).
The reason for elevated diversity in Symbiodinium in A. sola compared to A.
elegantissima can only be hypothesized. One possibility relates to the habitats in which the two
species are found: A. sola inhabits the mid- to low intertidal and extends subtidally as well, while
A. elegantissima has a range higher up in the intertidal. The higher intertidal experiences more
severe environmental conditions, which might explain why there is less Symbiodinium diversity.
Another possibility is difference in habitat for symbionts within the host: A. sola has a maximum
diameter of about 25 cm, while at its largest A. elegantissima is only 10 cm in diameter, but
usually is closer to 6 cm. That means A. sola has more than five times as much tissue volume
which could host more Symbiodinium types in niches that may not exist in A. elegantissima. The
diversity of Symbiodinium in A. sola could also be a factor of elevated diversity in A. sola, a
strictly sexually reproducing species, compared to A. elegantissima, a clonal species. It stands to
reason that in populations of equal number, a population which contains no clones would have
more genetic diversity than a population that contains clones. This elevated diversity in host
might well mean elevated ability to form symbiotic relationships with more diverse symbionts,
and might also be a factor contributing to A. sola's larger habitat range.
Latitudinal replacement, with one anomalous site
Data show tri-partite latitudinal replacement of Symbiodinium types. Results suggest that
type I is a cold-adapted Symbiodinium, type II is cool-adapted, and type III is warm-adapted.
The trend of latitudinal replacement is marred by a homogenous population of type III
Symbiodinium, all of the same haplotype, at Coal Oil Point (site C). A plausible reason for this
anomaly is the ocean circulation patterns in the Santa Barbara Channel around Coal Oil Point
during June, when A. sola spawns (McFadden et al. 1997). A map of surface currents in the
Santa Barbara Channel from the UCSB Ocean Surface Currents Mapping Project on a typical
June day shows strong eddy formation, which isolates water in the Santa Barbara Channel from
the California Current (Fig. 6.). These currents might have caused the isolated population of
type III Symbiodinium at Coal Oil Point, since there would appear to be little interaction with
populations coming from the North or South.
Different Symbiodinium in A. sola and A. elegantissima
Jon Sanders (personal communication) identified 4 haplotypes of Symbiodinium in
Anthopleura elegantissima. Of those four, only the "southern" haplotype shares the same
sequence as any of A. sola’s Symbiodinium. Coincidentally, the matching haplotype happens to
be the most common haplotype among the Symbiodinium populations identified in this study,
and it occurs predominantly in Southern California, which is the middle of A. sola's range.
Since genetic evidence suggests A. elegantissima and A. sola are recently diverged sister species
(Geller and Walton 2001), I hypothesize that this matching symbiont haplotype is a carry-over
from when the two species were not yet diverged. I posit the two species diverged in Southern
California, A. elegantissima being the basal species, and A. sola has diversified in habitat and
symbiont, expanding its range from Southern California up to the North, where it is evolving
new symbiotic relationships. Possibly the Symbiodinium and A. sola are co-evolving.
CONCLUSIONS
Preliminary analyses of Symbiodinium in A. sola led to rejection of the null hypothesis
that the Symbiodinium populations found in A. sola are the same as those found in A.
elegantissima. More than two-fold magnitude of diversity in haplotypes was found in A. sola
symbionts compared to A. elegantissima symbionts. Symbiodinium were classified into three
main types, which showed tri-partite latitudinal replacement. The anomalous site to the tri¬
partite latitudinal replacement can be explained by isolating ocean currents. The presence of
only one common haplotype between A. sola and A. elegantissima suggest possible historical
populations and co-evolution between symbiont and host.
Symbiodinium in the two sister species A. sola and A. elegantissima seems a good system
in which to study host-symbiont interaction and diversity.
ACKNOWLEDGEMENTS
1 would like to thank George Somero for leading the Bio175H course and serving as an
advisor and editor throughout the quarter. Thanks also to Steve Palumbi for serving as an
advisor to my project, and allowing me use of his laboratory. Vicki Pearse was instrumental in
many aspects of this project from start to finish, as she is the resident expert on A. sola. Special
thanks to Kristy Deiner, Emily Jacobs-Palmer, Melissa Pespeni, Brent Lockwood, Tom Oliver,
and Jon Sanders for teaching me everything I know about laboratory genetics - the Palumbi lab
is the most fun and friendly lab I have come to know. And thanks again to Jon; he had the
permit, and the bio-diesel vehicle and the experience to make the collecting road trip - and the
project - happen. I hope we get a chance to finish what we have started.
LITERATURE CITED
Droop, M. R. 1963. Algae and invertebrate in symbiosis. Pp. 171-199. in P.S. Nutman and B.
Mosse, eds., Symbiotic associations. Cambridge Univ. Press, Cambridge, U.K.
Geller, J. B., and E. D. Walton. 2001. Breaking up and getting together: evolution of symbiosis
and cloning by fission in sea anemones (genus Anthopleura). Evolution, 55:
1781-1794
Lajeunesse, T. C., and R. K. Trench. 2000. Biogeography of two species of Symbiodinium
(Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima
(Brandt). Biol. Bull. 199: 126-134
McFadden, C. S., R. K. Grosberg, B. B. Cameron, D. P. Karlton, and D. Secord. 1997. Genetic
relationships within and between clonal and solitary forms of the sea anemone
Anthopleura elegantissima revisited: evidence for the existence of two species. Mar. Biol.
128: 127-139.
McLaughlin, J. J. A., and P. A. Zahl. 1966. Endozoic algae. Pp. 257-297. in S. M. Henry, ed.
Symbiosis. Vol. 1. Associations of microorganisms, plants, and marine organisms.
Academic Press, New York.
Pearse, V. B., and L. Francis. 2000. Anthopleura sola, a new species, solitary sibling species to
the aggregating sea anemone, A. elegantissima (Cnidaria: Anthozoa: Actiniaria:
Actiniidae). Proc. Biol. Soc. Washington 113: 596-608.
Rowan, R. 1998. Review—Diversity and ecology of Zooxanthellae on coral reefs. J. Phyc.
34: 407—417.
Rowan, R., and D. A. Powers. 1991. A molecular genetic classification of zooxanthellae and the
evolution of animal-algal symbiosis. Science 251: 1348-1351.
Sanders, Jon. 2005. Biogeography of Symbiodinium in Anthopleura elegantissima on the
California coast. Stanford University: Final Papers Biology 175H.
Trench, R. D. 1987. Dinoflagellates in non-parasitic symbioses. Pp. 530-570. in F. J. R. Taylor,
ed. The biology of dinoflagellates. Botanical Monographs no. 21. Blackwell Scientific
Publications, Oxford, U.K.
Zhang, Z., B. R. Green, and T. Cavalier-Smith. 2000. Phylogeny of ultrarapidly evolving
dinoflagellate chloroplast genes: A possible common origin for sporozoan and
dinoflagellate plastids. J. Mol. Evol. 51, 26-41.
Table 1: Sites listed with their respective designation letter. The numbers of total clean
sequences included in data analysis from each site are shown. Sequence counts by type of
Symbiodinium as delineated by phylogram and cladogram are recorded.
Site
Total 4 of samples
Type II
Typel
Type III
China Point-
San Geronimo-B
10
Coal Oil Point-C
White's Point-
Dana Point-
Cabrillo Point-F
FIGURE LEGENDS
Fig. 1. Map showing A. sola collection sites.
Fig. 2. Phylogram showing haplotypes of all samples of Symbiodinium from A. sola. Included
are all known haplotypes of A. elegantissima for comparison (Jon Sanders, personal
communication).
Fig. 3. Cladogram showing bootstrap values. Sequences are grouped into three major types:
types I, II, and III.
Fig. 4. Symbiodinium types are graphed by site, showing percentages of each type at each site in
a percentage bar graph (A) as well as in pie graphs on a map corresponding to sample location
(B).
Fig. 5. Phylogram showing haplotypes of all samples of Symbiodinium from A. elegantissima
(Jon Sanders, personal communication).
Fig. 6. Map of surface currents in the Santa Barbara Channel showing typical patterns for the
month of June (UCSB Ocean Surface Currents Mapping Project).
0
Fig. 1.
California Specimen Collection Sites
China Point
San Geronimo Road
Coal Oil Point
White's Point
Dana Point*
Cabrillo Pointe
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