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There is a growing body of literature using transcriptomic data to study how tropical cnidarians and their photosynthetic endosymbionts respond to environmental stressors and participate in metabolic exchange. Despite these efforts, our understanding of how essential genes function to facilitate symbiosis establishment and maintenance remains limited. The inclusion of taxonomically and ecologically diverse endosymbionts will enhance our understanding of these interactions. Here we characterize the transcriptomes of two very different symbionts found within the temperate sea anemone Anthopleura elegantissima: the chlorophyte Elliptochloris marina and the dinoflagellate Symbiodinium muscatinei. We use a multi-level approach to assess the diversity of genes found across S. muscatinei and E. marina transcriptomes, and compare their overall protein domains with other dinoflagellates and chlorophytes. Our analysis identified several genes that are potentially involved in mitigating stress response (e.g., heat shock proteins pathways for mediating reactive oxygen species) and metabolic exchange (e.g., ion transporters). Finally, we show that S. muscatinei and other Symbiodinium strains are equipped with a high salt peridinin-chl-protein (HSPCP) gene previously identified only in free-living dinoflagellates. The addition of these transcriptomes to the cnidarian-symbiont molecular toolkit will aid in understanding how these vitally important symbiotic relationships are established and maintained across a variety of environmental conditions.

Associating with algal symbionts is considered largely beneficial for cnidarians such as corals and sea anemones, yet there are potential costs of hosting symbionts, such as the production of reactive oxygen species. We compared the photophysiology and H 2 O 2 production rates of Symbiodinium muscatinei and Elliptochloris marina , the dinoflagellate and chlorophyte symbionts, respectively, of the temperate sea anemone Anthopleura elegantissima. Analyses of photosystem II ( PSII ) function in the two symbionts, including maximum quantum yield and relative electron transport rates, were consistent with prior studies indicating that E. marina has lower photosynthetic performance than S. muscatinei at high temperature and irradiance. The efficiency of PSII in both symbionts was positively affected by the addition of exogenous catalase, suggesting that both symbionts experience H 2 O 2 -mediated declines in PSII efficiency. We found that S. muscatinei produced more H 2 O 2 than E. marina across all treatments, with the highest production under high light and temperature. Results were similar in experiments involving both isolated symbionts and symbionts residing within intact tentacles, indicating that the potential stress of symbiont isolation was not a significant factor biasing our results. Despite the lower typical densities of S. muscatinei relative to E. marina , extrapolations of cell-specific H 2 O 2 production rates to the intact symbiosis suggest that S. muscatinei imposes a greater H 2 O 2 burden on A. elegantissima. Even with this burden, however, the considerably higher productivity and fitness of S. muscatinei- bearing anemones documented in prior studies suggests that the net benefit of hosting S. muscatinei exceeds that of E. marina.

iv ACKNOWLEDGEMENTS vi TABLE OF CONTENTS vii LIST OF FIGURES viii LIST OF TABLES x INTRODUCTION 1 METHODS 4 Field sampling 4 Preparation of host and symbiont fractions 4 Lipid extraction 5 Lipid analysis 7 Statistical analyses 7 RESULTS 9 Fatty acid profiles of Symbiodinium muscatinei and Elliptochloris marina 9 Fatty acid profiles of zooxanthellate, zoochlorellate, and aposymbiotic hosts 15 Fatty acid profiles of hosts and their algal symbiont 20 DISCUSSION 24 Fatty acid profiles of Symbiodinium muscatinei and Elliptochloris marina 24 Fatty acid profiles of zooxanthellate, zoochlorellate, and aposymbiotic hosts 26 Fatty acid profiles of hosts and their algal symbiont 29 Conclusions and future research directions 30 REFERENCES 32

For cnidarians that can undergo shifts in algal symbiont relative abundance, the underlying algal physiological changes that accompany these shifts are not well known. The sea anemone Anthopleura elegantissima associates with the dinoflagellate Symbiodinium muscatinei and the chlorophyte Elliptochloris marina, symbionts with very different tolerances to light and temperature. We compared the performance of these symbionts in anemones maintained in an 8-11.5 month outdoor common garden experiment with simulated intertidal conditions and three levels of shading (2, 43, and 85% ambient irradiance). Symbiont densities, mitotic indices, photophysiology and pigments were assessed at three time points during the summer, a period of high irradiance and solar heating during aerial exposure. Whereas S. muscatinei was either neutrally or positively affected by higher irradiance treatments, E. marina responded mostly negatively to high irradiance. E. marina in the 85% irradiance treatment exhibited significantly reduced Pmax and chlorophyll early in the summer, but it was not until nearly 3 months later that a shift in symbiont relative abundance toward S. muscatinei occurred, coincident with bleaching. Symbiont densities and proportions remained largely stable in all other treatments over time, and displacement of S. muscatinei by E. marina was not observed in the 2% irradiance treatment despite the potentially better performance of E. marina. While our results support the view that rapid changes in symbiont relative abundance are typically associated with symbiont physiological dysfunction and bleaching, they also show that significant temporal lags may occur between the onset of symbiont stress and shifts in symbiont relative abundances.

The temperate sea anemone Anthopleura elegantissima is facultatively symbiotic with unicellular algae. Symbiotic A. elegantissima can supplement heterotrophic feeding with excess photosynthate from their algal partners, while asymbiotic individuals must rely solely on heterotrophy. A. elegantissima individuals were collected from Swirl Rocks, Washington (48°25′6″ N, 122°50′58″ W) in July 2010, and prey capture and feeding characteristics were measured to determine whether asymbiotic individuals are more efficient predators. Feeding abilities were then measured again after a 3-week exposure to full sunlight or shaded conditions. Freshly collected asymbiotic anemones had larger nematocysts, but symbiotic individuals showed greater nematocyte sensitivity. Sunlight enhanced digestion and reduced cnida density in all anemones regardless of symbiotic state. Results suggest that the phototropic potential of A. elegantissima, as influenced by symbiotic condition, has little effect on heterotrophic capacity. The anemones appear to maximize heterotrophic energy input independent of the presence or identity of their algal symbionts.

The sea anemone Anthopleura elegantissima is a common member of intertidal communities along the west coast of North America, and can experience extended periods of increased temperature during summertime low tides. Internal body temperatures of emersed individuals of A. elegantissima were monitored in a laboratory wind tunnel and in the field, and factors influencing the anemones’ thermal experience were examined. Larger body size and aggregation with conspecifics slowed body temperature increases in controlled wind tunnel conditions. In the field, anemones in the interior of an aggregation stayed cooler than those on the edges, and microhabitat features related to light exposure and surface orientation overshadowed any direct effects of body size. In the warmest month only (July), aggregations of A. elegantissima were significantly larger at the upper limit of their distribution than they were at the mid and lower limits, suggesting aggregation in high intertidal zones may be a behavioral response to desiccation and temperature stress. As this sea anemone can host multiple species of symbiotic algae with different thermal tolerances, the ability to slow body heating may affect the type of algae hosted and thus the potential contribution of this abundant anemone to primary production in the intertidal zone.

We evaluated the seasonal stability of two algal symbiont populations in the temperate intertidal sea anemone Anthopleura elegantissima on San Juan Island, Washington, where the relatively thermally tolerant dinoflagellate Symbiodinium muscatinei coexists with the less thermally tolerant chlorophyte Elliptochloris marina. Random collection of anemones along repeatedly sampled transects over four seasons and three shore heights revealed S. muscatinei to be the dominant symbiont, with E. marina mostly limited to anemones in the lower intertidal zone. At the lowest shore height sampled (+0.2 m), the proportion of E. marina was between 40% and 50% of the total symbiont population throughout the year. Symbiont distribution patterns persisted despite considerable seasonal variation in aerial exposure, temperature, irradiance, nutrients, and phytoplankton concentration, as well as a high potential for symbiont shuffling, with mixed-symbiont assemblages occurring in 51% of all anemones sampled. Symbiont density in anemones also changed little despite three- to fourfold-higher division frequencies of both symbionts during July and November. Although the intertidal zonation of these symbionts was stable over an annual period, we predict that their spatial distributions will be responsive to longer-term environmental change, and anticipate that this anemone symbiosis will be a useful and highly tractable barometer for future climate change, with this study serving as a baseline. A diverse array of shallow-water cnidarians, including sea anemones, corals, jellyfish, and hydroids, engage in endosymbioses with phototrophic dinoflagellates (Symbiodinium spp.) that translocate photosynthetic products to their host. Many of these symbioses exhibit plasticity with respect to both the population density and genetic identity of the dinoflagellate partner, allowing them to exploit a wide range of conditions that vary spatially and temporally. In tropical systems, where cnidarian symbioses are particularly well studied, many hosts show depth-related differences in symbiont density and physiology, and also undergo predictable seasonal fluctuations in symbiont density and physiology that are correlated with environmental conditions (Fitt et al. 2000; Warner et al. 2002). In

The sea anemone Anthopleura elegantissima (Brandt) hosts two species of symbiotic dinoflagellates, known as zooxanthellae, which coexist within the host at southern latitudes only. One of these species, Symbiodinium muscatinei LaJeunesse et Trench, has a broad latitudinal distribution, occurring in intertidal anemones from Washington state to Southern California. To investigate whether high thermal tolerance contributes to the ability of S. muscatinei to inhabit anemones from northern and southern regions, the upper thermal tolerance limit for photosynthesis of symbionts in northern (48°24′ N) populations of A. elegantissima was determined by subjecting anemones to a gradual increase in temperature from 12°C to 30°C over a 10-week period. Light-saturated photosynthetic rates of isolated zooxanthellae were the same over the range of 12°C–24°C and declined significantly at 26°C, which is 14°C and 5°C above average summertime seawater temperatures in northern Puget Sound and Southern California, respectively. At 28°C, zooxanthellae isolated from the anemones, and those expelled by their hosts, exhibited extremely low rates of photosynthesis and highly reduced chl content. The photosynthetic rates and chl content of expelled zooxanthellae were lower than those of retained zooxanthellae. The high thermal tolerance of S. muscatinei isolated from northern populations of anemones supports the broad latitudinal distribution of this symbiont, allowing it to coexist with S. californium (#383, Banaszak et al. 1993) in southern populations of anemones.

The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbi- otic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastroder- mal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across spe- cies and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

SUMMARY The susceptibility of algal–cnidarian holobionts to environmental stress is dependent on attributes of both host and symbiont, but the role of the host is often unclear. We examined the influence of the host on symbiont light stress, comparing the photophysiology of the chlorophyte symbiont Elliptochloris marina in two species of sea anemones in the genus Anthopleura. After 3 months of acclimation in outdoor tanks, polyp photoprotective contraction behavior was similar between the two host species, but photochemical efficiency was 1.5 times higher in A. xanthogrammica than in A. elegantissima. Maximum relative electron transport rates, derived from rapid light curves, were 1.5 times higher in A. xanthogrammica than in A. elegantissima when symbionts were inside intact tissues, but were not significantly different between host species upon removal of outer (epidermis and mesoglea) tissue layers from symbiont-containing gastrodermal cells. Tissues of A. xanthogrammica were 1.8 times thicker than those of A. elegantissima, with outer tissue layers attenuating 1.6 times more light. We found no significant differences in light absorption properties per unit volume of tissue, confirming the direct effect of tissue thickness on light attenuation. The thicker tissues of A. xanthogrammica thus provide a favorable environment for E. marina – a relatively stress-susceptible symbiont – and may explain its higher prevalence and expanded range in A. xanthogrammica along the Pacific coast of North America. Our findings also support a photoprotective role for thicker host tissues in reef corals that has long been thought to influence variability in bleaching susceptibility among coral taxa.

Along the North American Pacific coast, the common intertidal sea anemone Anthopleura elegantissima engages in facultative, flexible symbioses with Symbiodinium muscatinei (a dinoflagellate) and Elliptochloris marina (a chlorophyte). Determining how symbiotic state affects host fitness is essential to understanding the ecological significance of engaging in such flexible relationships with diverse symbionts. Fitness consequences of hosting S. muscatinei , E. marina or negligible numbers of either symbiont (aposymbiosis) were investigated by measuring growth, cloning by fission and gonad development after 8.5–11 months of sustained exposure to high, moderate or low irradiance under seasonal environmental conditions. Both symbiotic state and irradiance affected host fitness, leading to divergent life-history strategies. Moderate and high irradiances led to a greater level of gonad development in individuals hosting E. marina , while high irradiance and high summer temperature promoted cloning in individuals hosting S. muscatinei and reduced fitness of aposymbiotic anemones. Associating with S. muscatinei may contribute to the success of A. elegantissima as a spatial competitor on the high shore: (i) by offsetting the costs of living under high temperature and irradiance conditions, and (ii) by promoting a high fission rate and clonal expansion. Our results suggest that basic life-history characteristics of a clonal cnidarian can be affected by the identity of the endosymbionts it hosts.