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EXPERIMENTAL INVESTIGATION OF TIDAL AND FRESHWATER INFLUENCE ON SYMBIODINIUMMUSCATINEI ABUNDANCE IN ITS HOST, ELEGANTISSIMA

A Thesis submitted to the faculty of San Francisco State University Z o lQ In partial fulfillment of the requirements for H A t X the Degree

Master of Science

In

Marine Science

by

Daniel John Hossfeld

San Francisco, California

May 2019 Copyright by Daniel John Hossfeld 2019 CERTIFICATION OF APPROVAL

I certify that I have read Experimental investigation of tidal and freshwater influence on

Symbiodinium muscatinei abundance in its host, Anthopleura elegantissima by Daniel John

Hossfeld, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in

Marine Science at San Francisco State University.

Sarah Cohen, Ph.D.

Professor

Lorraine Ling, Ph.D. Postdoc Researcher at Stanford University EXPERIMENTAL INVESTIGATION OF TIDAL AND FRESHWATER INFLUENCE ON SYMBIODINIUMMUSCAT1NEI ABUNDANCE IN ITS HOST, ANTHOPLEURA ELEGANTISSIMA

Daniel John Hossfeld San Francisco, California 2019

Controlled experiments testing effects of temperature, salinity, and aerial exposure were paired with field observations to investigate symbiont expulsion in the abundant intertidal anemone, Anthopleura elegantissima. In the study region, A. elegantissima hosts a single symbiont, the Symbiodinium muscatinei. The San Francisco outflow creates a tidally influenced low-salinity plume that impacts adjacent coastal sites.

Salinity, temperature, and aerial stress induce a bleaching response similar to corals where symbionts are expelled, causing further energetic stress. Using field observations of environmental conditions and symbiont abundance at sites on a gradient of exposure to estuarine outflow, along with fully crossed multifactorial lab experiments, we tested for changes in symbiont abundance in response to various combinations of three stressors.

Lab experiments were designed to mimic short term outflow events with low salinity, high temperature, and aerial exposure treatments. The aerial exposure treatment was a statistically significant factor in suppressing symbiont repopulation (ANOVA, p=0.017).

Symbiont density decreased with increasing tidal height (ANOVA, p=0.036), suggesting that aerial exposure may affect symbiont density more than sea surface temperature and salinity. Unanticipated documentation of survival in 9 months of burial and subsequent repopulation of symbionts is reported in comparison to results from past observations. The study of this symbiosis is useful in examining predicted changes in ocean conditions in tidepool communities.

I certify that the Abstract is a correct representation of the content of this thesis.

Chair, Thesis Committee Date PREFACE AND/OR ACKNOWLEDGEMENTS

Thank you to Dr. Sarah Cohen and fellow Cohen lab students for assistance in fieldwork, wet lab, and logistical planning. Dr. Lorraine Ling assisted with flow cytometry and statistical analyses. Dr. Ed Carpenter, Dr. Frances Wilkerson, and Dr. Colin Leasnre shared expertise in developing methods. Dr John Pringle and lab offered generous use and training on the flow cytometer Dr Phil Cleves assisted with statistical analysis design, Mattie Mrlik assisted with initial laboratory work through the Vassar Internship

Grant Fund. Gabe Edralin provided critical field assistance. Thanks to Steve Hossfeld and

Jackson’s Hardware for controlled facility design.

This project is partially funded by awards to DH from the Dr. Earl H. Myers and Ethel M.

Myers Oceanographic and Marine Biology Trust, The SFSU Rosenblatt Award, The

SFSU Romberg Tiburon Center Award, and the SFSU Institutional Research Award.

Collections and surveys were carried out under California Department of Fish and

Wildlife (Permit #1042178169) and National Park Service (Permit GOGA-2017-SCI-

0025) Permits. Thanks to Darren Fong (NPS) for facilitating access to NPS sites.

Thank you to Audriana Ossenberg for support, advice, field work assistance, rent subsidies, and reminders to walk away once in a while. Th;* thesis wouldn’t exist in the same capacity without her. The same can be said for Steve and Beth Hossfeld, thank you for many free dinners and advice. TABLE OF CONTENTS

List of Tables...... viii

List of Figures ...... ix

Introduction...... 1

Materials and Methods...... 6

Results...... 11

Discussion...... ’...... 13

References ,28 LIST OF TABLES

Table Page

1. Experimental design for wet lab treatments...... 19 2. ANOVA results for Experiment 1 ...... 20 3. ANOVA results for Experiment 2...... 20

viii LIST OF FIGURES

Figures Page

1. Map of field sites,...... 21 2. Field sampling protocol...... 22 3. Treatment setup for Experiment 1...... 23 4. Treatment setup for Experiment 2...... 23 5. Salinity and Temperature at the Fort Point Buoy...... 24 6. Symbiont density compared across tidal heights...... 24 7. Symbiont density at Point Bonita...... 25 8. Symbiont repopulation after burial at Mile Rock...... 25 9. Experiment 1 results...... 26 10. Experiment 2 results...... 26 11. Experiment 2 results by treatment...... 27 1

Introduction

Marine organisms living in the rocky are known to be hardy when coping with multiple stressors, but a changing climate is altering life even for the most robust species. The rocky intertidal zone is an accessible marine with a history of studies comparing population dynamics to changing ocean conditions (Sanford et al 1999,

Sanford et al 2008, Sagarin et al 2009, Harley 2011, Jeuterbock 2013). Recent studies show climate change impacts on communities of intertidal marine invertebrates, especially considering cascading effects from altered temperature regimes in both water and air (Barry et al 1995, Helmuth et al 2002. Walther et al 2002, Helmuth et al 2006,

Hawkins et al 2008, Miekowska 2008. Wetliey et al 2011). Zonation within the intertidal is described by regions across vertical height ranges that are stratified due to temporal variation of aerial exposure at low tide. Organisms at higher vertical zones experience more exposure which can alter body temperature, water temperature, desiccation time or salinity in isolated pools. Altered coastal freshwater inpul as a result of climate change can also affect organisms - for example, rocky intertidal predators such as sea stars are forced to alter behavior during high freshwater input to avoid exposure (Garza & Robles

2010). Freshwater input can occur from nearby coastal runoff due to snowmelt, precipitation, groundwater and sewage export. In tidepools, precipitation can also directly affect the salinity due to rainfall while the pool is exposed (Witman & Grange 1998). 2

Study system

The Anthopleura elegantissima (; ) harbors

Symbiodinium similar to many -building corals. Information on the symbiotic relationships between cnidarians and Symbiodinium species is crucial to understanding the implications of the ongoing widespread coral bleaching events, leading to death of many reefs worldwide. This study provides a temperate intertidal example of altered host-symbiont interactions under varied and multifactorial stresses related to anticipated coastal climate change. It further provides foundational knowledge for the potential of niche construction in the symbiosis between S. muscatinei and A. elegantissima (e.g. following Moczek et al 2015).

Anthopleura elegantissima is a commonly observed, abundant rocky intertidal organism found on the eastern boundary of the Pacific from Alaska to Baja California. This species can reproduce asexually (clonal fission) and sexually (broadcast spawning). Anthopleura elegantissima maintains a symbiotic relationship with up to two species of photosynthetic that live and reproduce within the tentacles, providing 13 to 45% of the energy in the anemone’s diet and altering its behavior (Fitt 1982, Pearse 19741, Pearse 19742,

Bingham et al 2014). In rocky intertidal near the San Francisco Bay in

California, A. elegantissima hosts one thermally tolerant symbiont, Symbiodinium muscatinei (Sanders & Palumbi 2011, Muller-Parker et al 2007, LaJeunesse & Trench

2000). Stressful conditions produce a bleaching response similar to that of corals 3

(McCloskey et al 1996, Saunders & Muller-Parker 1997) where the anemone expels the symbionts. Past laboratory studies indicate a relationship between environmental conditions including exposure, temperature, light and the density of symbionts present in

A. elegantissima (Bingham et al 2011, Saunders & Muller-Parker 1997, Verde &

McCloskey 2001). Expulsion of the symbiotic algae can cause further stress due to the loss of an energy source. The single stressor of low-salinity has been explicitly tested in field and laboratory studies on A. elegantissima in southern California (Martin et al

1996), showing significant (greater than 20% loss) bleaching effects when anemones were exposed to varying amounts of freshwater for five days and longer. In this study, we tested effects of freshwater alongside additional stressors that would occur in nature.

Multi-stressor effects of the low-salinity plume stemming from rainy season runoff events out of the San Francisco Bay include salinity, seawater temperature and aerial temperature stress on the A. elegantissima / S. muscatinei symbiosis. The bleaching response to this combination of stressors is untested in lab or field studies, although photosynthetic capability is known to be reduced in extreme aerial exposure events

(Shick 1984). As a pervasive member of eastern Pacific intertidal habitats, A. elegantissima is an excellent candidate for understanding effects of common and predicted estuarine outflow and rainfall events including low salinity stress on rocky intertidal communities. 4

Environmental change in the intertidal zone

Continuing global-scale changes in rainfall patterns and increasing intensity of storm events are expected as anthropogenically-amplified climate change occurs (Milly et al

2008, Karoly 2014, Polade et al 2017). While uncertainties exist in projections of future rain and snowfall decreases in California (Cayan et al 2008), storm events are expected to increase in intensity (Cloem et al 2011, Dettinger 2011, Swain et al 2018). Average aerial temperatures are expected to increase in California as well as annual extreme heat days (Cayan et al 2008). Sea surface temperature is increasing as well (Rayner et al

2003).

The San Francisco Bay is the meeting point of the Pacific Ocean with rainfall, snowmelt, and groundwater seepage collected from a watershed covering 40% of California’s land.

This watershed outflow creates a low-salinity plume outside of the Golden Gate, exacerbated by a receding tide and freshwater runoff events. Predictions of extreme rainfall suggest more low salinity events at the Golden Gate in the future (Cloem et al

2011). The effects of this plume on local organisms are not well studied, although freshwater is a well-known stressor on saltwater organisms (Garza & Robles 2010,

Engbretson 1994). Previous studies indicate the ecological importance of the outflow in local marine habitats for populations of Leptasterias sea stars (Melroy et al 2017, Braun et al 2016). The freshwater plume stays on the ocean s surface, and therefore affects near-surface habitats including the rocky intertidal zone (Ruhl et al 2001). This plume is driven by snowmelt runoff and precipitation events from March to May (Largier 1996). 5

Due to the bathymetry in the Bay, the northern side of the Golden Gate tends to have lower salinity than the southern side (Martin tr al 2007).

Shifting environmental conditions alter the dynamic rocky intertidal habitats, and while significant long-term species-level monitoring exists (Multi-Agency Rocky Intertidal

Network, Partnership of Interdisciplinary Studies of Coastal Oceans) acute changes in community structure are potentially overlooked due to less frequent monitoring time scales (Murray et al 2006). A controlled experimental approach linked to field comparisons can quantify tolerance thresholds o f A. elegantissima to low-salinity, high- temperature water parcels and provide greater understanding of the stress accompanying large runoff events. The results give context to long term monitoring efforts. Due to its wide geographic distribution and common appcarance in upper intertidal rocky habitats,

A. elegantissima is a good indicator for marine habitats influenced by nearby freshwater input. Intertidal anemones are also subject to the fluctuation of sediment from wave action (Fitt 1982), and A. elegantissima have previously been observed surviving burial events as long as three months (Taylor & Littler 1982).

Warming aerial temperatures have the potential to drastically affect intertidal communities during low tide. Simulated exposure scenarios in the laboratory experiments were included to determine the relative effect of this known stressor to temperature and salinity stress. The experimental design tested the parameter range of environmental cues that lead to symbiont expulsion from A. elegantissima at sites near the San Francisco Bay 6

outflow. We evaluated the hypotheses that relatively high temperature and low salinity compared to local ocean conditions force the escalated expulsion of symbionts from the tentacles of A. elegantissima, and that aerial exposure forces further expulsion.

Experimental design reflected the importance of initial investigations into timescale of responses in both field and laboratory settings.

Materials and Methods

Field setting

Field surveys o f A. elegantissima colonies were used to test the prevalence of bleaching in relation to estuarine output and ocean conditions. Four sites were selected along the coastal region near the San Francisco Bay. The four sites north to south include Slide

Ranch (14.5km from outflow), Point Bonita (5.6km from outflow). Mile Rock (4.7km from outflow), and Rockaway (25km from outflow) (Fig 1). One near-outflow site, Mile Rock, was selected to determine the influence of burial and low light on tidepools and was excluded from other analyses. Sea surface temperatures were measured using buoy data from Fort Point (located within the San Francisco Bay) and on-site temperature logger readings (Onset HOBO Pendant Part # UA-002-64). 7

Colony sampling across sites

Anthopleura elegantissima forms colonies of genotypically identical individual polyps via asexual reproduction. To assess differences in colonies at sites with varying distance from the San Francisco Bay outflow three to five tidepools containing a single colony were sampled at varied tidal heights at each site (Fig 2). In each colony, at least five anemones were sampled, using dissection scissors to snip and collect three tentacles per anemone. Each of the 14 total colonies across four sites were sampled roughly monthly.

T dal height was measured following tidepool selection using a stadia rod along with a sight level. Corrected tidal heighl from NOAA (tidesandcurrents.noaa.gov) was used to calculate vertical tidal height. All pools containing anemones were above Mean Lower

Low Water (MLLW).

Colony selection criteria included individual counts greater than 10 anemones/colony, light exposure, and accessibility. Light availability was chosen as at least 50% of daylight exposure without shading (e.g., from a nearby face or nearby boulders) except for

“low light’’ colonies. Mile Rock samples were excluded from some statistical tests due to the lack of comparable light and tidal height settings.

Sampling scheme within colonies

Colonies of A. elegantissima were photographed, counted, and surveyed for signs of bleaching. Closed anemones in tidepools were shaded for ease in collection by manually adding algal cover, which enabled them to open. Colonies were divided into inner and 8

outer portions, which were then gridded, and anemones randomly selected into each category to avoid effects of intercolonial differences (Ayre and Grosberg 1995).

Controlled Setting

The wet lab experiment tested effects of altered aerial exposure, salinity and temperature on bleaching in A. elegantissima. Temperature was measured using Onset HOBO

Pendants (Part # UA-002-64). Salinity was measured using a calibrated YSI2030, which measures in units of parts per thousand (ppt). All salinity measurements and treatments are reported in ppt for consistency. Two experiments were done, each with different methods. In Experiment 1, anemones were in tanks with the constant water conditions described in the treatment group they were assigned. In Experiment 2, anemones were exposed to treatments for three hours a day and kept in control conditions otherwise.

Experimental Setup

Before treatments began, all collected anemones were divided into separate tanks and acclimated for one week in the wet lab under common ocean conditions found offshore of

San Francisco Bay, ~14°C and 32ppt. Twenty anemones were collected on each collection trip at +0.18m above MLLW and were subsequently randomly assigned to treatments shown in Table 1. Treatments and controls for Experiment 1 are shown

Figure 3. Treatments and controls for Experiment 2 are shown in Figure 4. Anemones were collected from the two sites north of the outflow: Slide Ranch (far from outflow) and Point Bonita (near/in outflow). Two seawater tables were set to constant temperatures (14°C in Table 1, 17°C in Table 2). Each individual was placed in isolated 9

circulation n a tank with strong aeration. Each treatment lasted five days to mimic a short-term low salinity pulse associated with a storm event. Anemones were held on a 24- hour light cycle (14 light, 10 dark) at a low clear daylight intensity (10^mol/m2/s).

Tentacles were collected on days one, three and five. Exposure treatments consisted of removing the anemone from water for a 3-hour period.

Symbiont Quantification

Measurement of symbiont densities required homogenization of the tissue and symbionts in artificial seawater with a Fisher Powergen 125 centrifuge, and dilution of 25j.il aliquots of homogenate into 250f.il samples in artificial seawater with 0.01% SDS. Samples were then loaded into a Guava easyCyte HT 2-laser flow cytometer and analyzed using accepted settings for counting Symbiodinium (Krediet et al 2011).

Modified Lowry Method

The Modified Lowry Method is regularly used to quantify anemone tentacle protein in comparison to Symbiodinium counts (Rastrick & Whiteley 2017, Saunders & Muller-

Parker 1997, McCloskey et al 1996). This method provided a symbiont count per gram of protein as a standard for symbiont presence and density. Tentacles were frozen and homogenized in artificial seawater, then animal protein was precipitated out and resuspended in ultrapure water, and the resulting protein concentration was quantified. 10

Feeding

Collected anemones were fed once per week by dropping limpets (Lottia spp. collected from field sites) directly into the oral disk. Shell length of limpets was measured using calipers and ranged from 4mm to 10mm. Success of feeding was evaluated the following day by looking for empty shells or live limpets. Anemones were not fed for the duration of experiments.

Statistical Analysis

Symbiont density was standardized to animal protein and calculated for all samples. A

Shapiro-W"k Test of Normality upheld the assumption of a normal distribution among the samples. One-way Analysis of Variance (ANOVA) tested the influence of tidal height on symbiont density. In all tests, the dependent variable was symbiont count per gram animal protein. Independent variables for wet lab experiments included water temperature, salinity, and aerial exposure. For field samples, independent variables included water temperature, salinity, aerial exposure during low tide, and site. A three- way ANOVA tested the influence of salinity, temperature and air exposure on wet lab data. This test is a fully crossed assessment of the relationship between

Symbionts/Temperature, Symbionts/Salinity, Symbionts/A::- Exposure, as well as the relationship of symbionts to multifactorial changes. For ANOVA, p<0.05 was considered sufficient to reject the null hypothesis. Tukey’s Honestly Significant Difference (HSD) tests determined site effects and inter-treatment differences. All statistical analysis was done in R with the dplyr package. 11

Results

Temperature and freshwater input

Daily average SST ranged from 10.7°C to 15.3°C at the Fort Point buoy. The buoy documented a decrease in average daily salinity during the survey period due to outflow from the San Francisco Bay and Delta (Fig 5). A 28ppt reading translates to the presence of roughly 10% freshwater.

Field setting

Symbiont density decreased with tidal height (ANOVA, F=4.5, p=0.036) at included sites

(see Methods section Colony sampling across sites) (Fig 6). The samples used ranged across three sites and across tidal heights ranging 0.3m to 2m above MLLW. Slide Ranch and Rockaway tidepools showed no change in symbiont abundance across sampling dates

(Tukey, p>0.1 for all pools).

The lidepools at Point Bonita are the most proximal to the bay outflow. The relationship of tidal height and symbiont density at Point Bonita changed over the three-month survey period (Fig 7). Symbiont density partitioned by tidal height in the first survey (ANOVA,

F=10.33, p=0.007) On the second survey, symbiont density no longer partitioned by tidal height (ANOVA, F^=0.9, p=0.35). On the third survey density remained unpavtitioned by tidal height (ANOVA, F=0.5, p=0.47). Between the second and third surveys, all colonies experienced more than a 50% decrease in mean symbiont density (Tukey HSD, p<0.05 for all colonies). This decrease is coincident with the documented decrease in salinity and 12

minor increase in temperature (Fig 5). Five inches of rain was recorded near Point Bonita from April 5th to April 7th, 2018. No other notable onsite precipitation was recorded during the survey period.

Coincident observation o f burial and repopulation at Mile Rock

Preliminary observations before conception of this study yielded the opportunity to observe how burial affects this system. A sampled pool at Mile Rock filled with a single colony of A. elegantissima was buried for over 9 months due to natural sand fluctuation.

Once unburied naturally, the anemones were found notably smaller, desiccated, yet alive.

Subsequent sampling of the same poo! in the following weeks showed a statistically significant 700% increase in mean symbiont density in the colony (ANOVA, F=5.7, p=0.044), due to three anemones with comparable densities to the “Not Buried” anemones in other colonies. Figure 8 shows the repopulation of symbionts in the recently unburied anemones compared to two colonies in pools that were not buried.

Controlled setting

Experiment 1 (See Methods) results indicate that the effects of 17°C (High Temp) alone,

25ppt (Low Sal) alone, or the interaction of the two (High Temp/Low Sal) did not significantly affect symbiont density after five days when compared to the control treatment of 14°C and 32ppt (ANOVA, p>0.05 for all treatments) (Fig 9, Table 2).

Results from Experiment 2 (Methods) show a statistically significant effect of mimicked low tide exposure on symbiont density populations (ANOVA, F=6.3, p=0.017) (Figs 10 13

& 11, Table 3). No significant effect of altered temperature (17°C) and salinity (25ppt) was detected, consistent with Experiment 1. The interaction between any two or three of the independent variables tested did not affect symbiont density during the course of

Experiment 2 (ANOVA, p>0.05 for all interactions).

Discussion

Results fit expectations of systematic bleaching events brought on by estuarine outflow and low tide series. The combination of field observations and controlled experiments suggest that the A. elegantissima / S. muscatinei symbiosis is susceptible to ongoing environmental variability, and will be affected by climate change. Field observations suggest a systematic bleaching event when low-salinity water parcels persist at intertidal sites. Anemones survived burial events lasting nine months, up to three times longer than previously recorded, and showing resilience to a common disturbance event (Airoldi

2003). Results from controlled multifactorial experiments show the importance of aerial exposure over moderate changes to sea surface temperature and salinity, suggesting that increased aerial temperatures due to climate change (Perkins et al 2012, Cayan et al

2008) are likely to spur future bleaching events. The inclusion of tidal simulation in controlled experiments is recommended due to field and lab results - exposure is an important stressor and must be used in future lab experiments. 14

Temperature and freshwater input

The freshwater pulse documented at Fort Point for the wet spring season of 2018 was moderate compared to past years, but the timing presents an opportunity to compare plume presence to symbiont density at Point Bonita, the site most directly influenced by bay runoff. Symbiont density across the sampled tidepools decreased by 50% with persistent presence of the plume, and tidepools that had previously been partitioned by tidal height all decreased to a common '‘low" density. The low density found for these tidepools that had previously partitioned with tidal height suggests that the disturbance was enough to reset local zonal partitioning. The influence of tidal height on symbiont density is consistently shown in the results of the field observations, but the significant decrease in symbiont density due to freshwater input suggests the effect of these low salinity water parcels on the symbiosis. Freshwater input will continue to affect the symbiosis as climate change will increase the intensity of major freshwater runoff events.

Field setting

The influence of tidal height on symbiont density is consistently shown in the results of the field observations. The surveyed tidepools ranged more than five feet of vertical height in the intertidal zone, which is extremely broad compared to many intertidal studies and demonstrates the hardiness of the A elegantissima/S. muscatinei symbiosis.

The results suggest that exposure and tidal height affect symbiont density as hypothesized, as tidepools at the higher vertical sampling limit had lower symbiont densities (colony means as low as 760 cells/pg protein) compared to equivalent pools at 15

lower vertical sampling heights (colony mean as high as 14200 cells/|xg protein). While tidal heigh t was expected to be of import to symbiont densities, these results are the first to present quantitative evidence of a relationship between tidal height and symbiont density in A. elegantissima.

Coincident observation o f burial and repopulation at Mile Rock

Previous studies have documented burial survival in A. elegantissima up to three months

(Taylor & Littler 1982, Pineda & Escofet 1989). The incidental observation in this study shows the extreme hardiness and survival capabilities of this species, as colonies were buried for nine months under sand (at +2ft above MLLW) that was well-trodden and multiple feet deep at some locations. The anemones exhibited extreme desiccation and lethargic movement upon unburial, but subsequent surveys showed that unburied individuals began repopulation of their symbionts. Within three weeks, between sampling surveys, the unburied colony went from near-undetectable amounts of symbionts to amounts comparable to many other colonies with “low” density, around 5000 symbionts per microgram of protein (Fig 8). This repopulation is cither rapid horizontal integration or, as found in previous laboratory work with Aiptasia anemones, a repopulation by a residual Symbiodinium population that was maintained even in prolonged total darkness

(Steen 1986). 16

Controlled Setting

Experimental testing of temperature, salinity and aerial exposure at amounts comparable to conditions near the San Francisco Bay confirms the importance of tidal height and aerial exposure over temperature and salinity when considering realistic predicted parameter ranges. Aerial exposure treatments provoked statistically significant deterrence of symbiont repopulation, while slightly elevated temperatures and quick pulses of freshwater did not affect symbiont density La Experiment 2.

The controlled setting experiments suggest that moderately (within 25% change from expected 32ppt) decreased salinity does not affect symbiont density within a five-day period, a quick response as compared to a past study documenting slower onsets of bleaching due to stress and environmental change (Dimond et al 2013). Past studies have tested major temperature and salinity alterations and have shown dramatic results for change in symbiont density (Martin 1996, Saunders & Muller-Parker 1997).

Significance

This study was designed to explore moderate temperature and salinity alteration, and to include for the first time an exposure treatment which more closely mimics field conditions compared to water bath treatments. It is clear, from the significant differences in exposed and not exposed treatments in such a short time period, that including exposure considerations in experiments is crucial to evaluate effects on this symbiosis. 17

Aerial temperature increase, sea surface temperature increase and changes in storm- induced salinity fluxes are predicted concerns for California coastlines as anthropogenic- forced climate change progresses. This study provides an example system that is directly affected by these changes. As climate changc progresses, systematic bleaching may become more prevalent and severe, causing further alterations to a sensitive food web.

Exploring these interactions near major discharge provides insight for estuarine and coastal freshwater outflow sites.

There are no studies outlining the effects of outflow plumes on A. elegantissima across its broad geographic distribution, although the species is found across the entire west of the United States and Canada up into Alaska and subject to freshwater input across the entire range. Results here suggest that the anemone-Symbiodinium symbiosis can be a reporter of freshwater plume presence in the intertidal zone. Anemone tentacle assays are a cheap method that provides indication of system health without lethal harvest and can be adapted for assessment across broad geographic regions. There is potential to provide the public with a quick, non-invasive method of investigating local intertidal community health and primary productivity. The study of this ecosystem health indicator could prove useful for baseline ecosystem monitoring, potentially through citizen science. 18

Future Directions

Future directions following this study include examining genetic linkages to stress resistance and symbiont relationships in A. elegantissima, as l'ound in certain coral species (Langdon et al 2018, Palumbi et al 2014, Desalvo et al 2008), especially with respect to differential exposure (Zardi et al 2011). Modeling S. muscatinei food web contribution in various climate scenarios could predict intertidal productivity changes

(Towanda & Thuesen 2012). Further questions related to heterogeneous salinity tolerance within A. elegantissima colonies can be answered by testing whether inner polyps are buffered from freshwater influence by outer polyps. Predictive models of freshwater presence in the intertidal can also suggest high-likelihood bleaching events and hotspots.

Due to the observational nature of the field portion of this study, further investigation into the effects of the seasonal large-scale ecological disturbance on local A. elegantissima / S. muscatinei symbiosis is warranted. 19

Tables

Experiment Duration Temperature Salinity Air N per Description treatment (°C) (PPO Exposure (Yes/No)

14 32 N

14 25 N 5 straight days of 1 5 Days 10 conditions described 17 32 N

17 25 N

14 32 N

14 32 Y

14 25 N

14 25 Y 3hrs daily o f treatment conditions, otherwise 2 5 Days 5 kept in control 17 32 N conditions (blue)

17 32 Y

17 25 N

17 25 Y

Table 1. Experimental design for wet lab treatments, "exposed ” meaning periodically exposed to air. In Experiments I & 2, the control conditions are shaded blue. 20

Stressor compared to control F value p value

High Temperature 3.2 0.08

Low Salinity 0.5 0.47

High Temp x Low Salinity 0.1 0.69 Table 2. ANOVA results from Experiment I

Stressor compared to control F value p value

High Temperature 0.71 0.404

Low Salinity 0.03 0.860

Exposure 6.3 0.017

High Temp x Low Salinity 0.24 0.623

High Temp x Exposure 0.1 0.75

Low Salinity x Exposure 0.007 0.93

High Temp x Low Salinity x Exposure 0.27 0.6 Table 3. ANOVA results from Experiment 2

\ 21

Figures

Figure 1. Field sites North to South. Slide Ranch, Point Bonita, Mile Rock Beach, and Rockaway Beach. Sites superimposed on a satellite image o f the freshwater plume, seen as lighter color, exiting the SF Bay on 04/21/2002. White clouds cover the ocean at Rockaway Beach and further south. Image: NASA, accessed at https://eol.jsc. nasa.gov/searchphotos/photo.pl?mission =ISS004&roll-E&frame=10288 22

Figure 2. Example of sampling regime in three pools at a single site. Three pools per site are chosen based on colony presence and tidal height o f each pool. Five anemones within pools are randomly selected (circled in red) and sampled for tentacles (3-4 per anemone) to assess symbiont density change over time. A) Example o f vertical height change across survey pools at Point Bonita B) Pool at Point Bonita with a single colony C) Close up view o f an A. elegantissima colony. 23

14°C 17°C Salinity *32 Salinity ■ 26 f Salinity > 32 Salinity >26 BB BB BH ■ ■ BB BB BB ■ ■ BB BB BB ■ i BB BB BB ■■ BB IB BB ■■ Control

Figure 3. Experimental wet lab setup for Experiment 1 - the leftmost group is the control (14°C, 32ppt).

14°C 17°C SUInlty s 32 Sillfllt* X 2C S-allnltv = 32 BB |B BB t o B BB B B BB

BL(w <* B C o n tro l .EafcMjjnt.

Figure 4. Experimental wet lab setup fo r Experiment 2 - in the leftmost group, the unexposed column is the control (14°C, 32ppt, unexposed). 24

Date Figure 5. Salinity and temperature measurements at the Fort Point Buoy during the survey period. Black dots show individual sampling measurements, the blue line shows daily average. Anemone sampling dates at Point Bonita are shown as red lines.

20000-

c

B 15000- 2 CL ro • | 10000- Slide Ranch ra S Point Bonita o> * H Rockaway

= 5000-

0-

0.5 1.0 1.5 2.0 Tidal height (m)

Figure 6. Individual anemones sampled for symbiont abundance shown with increasing tidal height. Bars show the mean at each tidal height sampled. 25

c 20000- A A

£ A A o ▲ rT 15000- • , _ • . * CO A E • a ■ High Pool 10000 - # < * • Middle Pool cn ▲ -5 5000- ^ * A Low Pool j n !) 1 ° 0- 2018-04-01 2018-04-15 2018-05-01 2018-05-15 Date Figure 7. Sampling measurements across tidal heights at Point Bonita. Symbiont abundance begins partitioned (3/29/2018), converges in the second survey (4/17/2018), and decreases by the third survey (5/17/2018).

c £ A O 6000- a CL A A g £ 4000- A • EZ Recently Unburied < • A a N0t Buried CD 3 2000-

\ ° 0 - ' 03/27/2018 04/18/2018 Date

Figure 8. Symbiont repopulation in anemones sampled after unburial at Mile Rock. On 4/18/2018, two pools were sampled that had never been buried, resulting in higher N for the “Not Buried”group. 26

c High Temp / Low Sal i2 5000- a> o 1 3 Experiment Day

Figure 9. Experiment 1 results -N = 10 per point, error bars show ±CI.

20000 -

•§ 15000- Control 2 -•* Exposed Q. -**- High Tem p i 10000- High Temp / Exposed O) Low Salinity D Low Salinity / Exposed High Tem p / Low Sal o 5000- -* » High Temp / Low Sal / Exposed

3 Experiment Day

Figure 10. Experiment 2 results - N=5 per point. Error bars show ±CI. 27

Exposure

12000

8000 Control Exposed 4000- * 3 c

0 O Salinityi

12000- 8000- 32ppt 25ppt 4000-

Experiment Day

Figure 11. Grouped results from Experiment 2. N=20 per point, error bars show ± CI. The aerial exposure treatment presence shows significant differences from the control at the third time point. 28

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