LIFE CYCLE DYNAMICS OF THE HARMFUL BLOOM FORMING MACROALGAE ULVA SPP. IN NARRAGANSETT BAY, RI

Macroalgal blooms occur worldwide and have the potential to cause severe ecological and economic damage. Narragansett Bay, RI is a eutrophic system that experiences summer macroalgal blooms composed mostly of Ulva compressa and Ulva rigida. All Ulva species have isomorphic, biphasic life cycles, and the relative contribution of the haploid and diploid life history stages to bloom formation is poorly understood. In this study, we used flow cytometry to assess ploidy levels of U. compressa and U. rigida populations from five sites in Narragansett Bay, RI, USA. Both haploid gametophytes and diploid sporophytes were present for both species. Sites ranged from a relative overabundance of gametophytes to a relative overabundance of sporophytes, compared to the null model prediction of √2 gametophytes to 1 sporophyte. We also assessed growth rates and measured cell sizes to investigate potential differences between life history phases. We found no significant differences in growth rate between ploidy levels for either species. Sporophyte cells were significantly larger than gametophyte cells in U. compressa. Our results indicate the presence of both phases of each of the two dominant bloom forming species throughout the bloom season, and represent one of the first studies of in situ Ulva life cycle dynamics.

v DEDICATION I would like to dedicate this thesis to John Hall. He is one of the most caring and passionate teachers I have had the pleasure of knowing, and I am forever inspired by his unparalleled dedication to ecology and environmental conservation.
vi PREFACE This thesis is being submitted in manuscript format. It consists of one chapter which will be submitted for peer-reviewed publication in the journal Marine Ecology Progress Series. vii

FIGURE PAGE
Chapter 1 Figure 1. Figure 1. Isomorphic biphasic life cycle of Ulva. Ulva cycles between two morphologically similar multicellular adult phases, a haploid gametophyte and a diploid sporophyte. Diploid sporophytes produce haploid zoospores that develop into gametophytes. Haploid gametophytes produce haploid gametes. When a "+" and "-" gamete fuse they form a zygote, which develops into a diploid sporophyte.……………13  x

INTRODUCTION
Macroalgal blooms typically consist of large accumulations of ephemeral macroalgal biomass. These blooms occur worldwide, often in shallow areas with relatively low water mixing that are affected by coastal eutrophication, and they have the potential to cause severe ecological and economic damage (Rosenberg 1985, Thomsen & McGlathery 2006, Deacutis 2008. The largest documented bloom on record occurred four weeks before the 2008 Beijing Olympics, with a bloom of an estimated 20 million tons of Ulva prolifera in the Yellow Sea near Qingdao, China (Gao et al. 2010, Ye et al. 2011, Liu et al. 2013. The ecological effects of macroalgal blooms are often far reaching and indirect; algal blooms negatively affect seagrass, sessile invertebrates and perennial algae (Hauxwell et al. 1998, Hauxwell et al. 2003, Worm & Lotze 2006. Large blooms can create hypoxic environments that contribute to mass fish and invertebrate die-offs (Management 2003, Deacutis et al. 2006) and hydrogen sulfide from decaying algal mats can cause symptoms such as difficulty breathing and nausea in humans (Fulton et al. 2003). Blooms have increased worldwide over the years in frequency and intensity (Morgan et al. 2003, Smith et al. 2005, Lyons et al. 2009).
The green macroalgal genus Ulva forms large and dense sheets, a phenomenon known as green tides, and can proliferate by asexual (fragmentation) and sexual reproduction. Green tides include many genera of green algae, such as Chaetomorpha, and affect at least 37 countries worldwide (Morand & Merceron 2004, Merceron et al. 2007). Ulva is one of the most common macroalgal bloom-forming genera present in green tides and is the focus of this study.
Like many algae with complex life cycles, Ulva has a biphasic life cycle consisting of an alternation between two free-living forms, a haploid phase (1N, gametophyte) and a diploid phase (2N, sporophyte; Figure 1). These phases of Ulva are isomorphic, meaning that the gametophyte and sporophyte are morphologically similar and cannot be visually distinguished in the field. If the phases are ecologically equivalent, we expect a ratio √2 gametophytes to 1 sporophyte at equilibrium (Thornber & Gaines 2004). This predication is due to the reproductive success of the sporophyte phase, resulting in a relative overabundance of adult gametophytes. For isomorphic algal species, however, a wide range of distributions of ploidy ratio in have been documented in the field (for review see Thornber 2006).
There are two published studies on the in situ life cycle dynamics of Ulva; Hiraoka and Yoshida (2010) found a non-seasonal alternating dominance of the two phases for U. pertusa, and Alström-Rapaport and colleagues (2010) found a seasonal shift in the phases of U. intestinalis, although sporophytes were always more abundant.
This lack of a broader understanding of Ulva life cycle dynamics may be due to the difficulty of discerning between isomorphic phases; however, ploidy can be rapidly determined using flow cytometry (Ulrich & Ulrich 1991, Le Gall et al. 1993, Doležel et al. 2007a. Flow cytometry quantitatively analyzes the DNA content of nuclei in a suspended solution and can allow for a more rapid and less expensive analysis than molecular techniques.
Although isomorphic sporophytes and gametophytes appear identical, they can occupy different ecological niches (Destombe et al. 1993, Dyck & De Wreede 1995. For example, one phase may be responsible for forming blooms, while the other may occur during non-bloom forming months. In addition, the two phases may vary in growth rates, temperature optima, or susceptibility to herbivores . Similarly, phases could vary in their response to environmental variables such as temperature and nutrients (Hannach & Santelices 1985, Destombe et al. 1993. If there are ecological differences between Ulva gametophytes and sporophytes, the distribution of life history phases will be partially dependent upon the physical and biological factors of the system.

Collection of Ulva
We We also determined Ulva biomass data from monthly subtidal surveys of the same sites, following the protocol in Guidone (2012). Briefly, at each site, we collected all algae in each of 0.16 m 2 subtidal quadrats placed 1 m apart along a transect line. All plots were < 2 m deep at mean lower low water.
Prior to thallus destruction for flow cytometry, we took a microscopic photograph at 400X of each individual that was analyzed for ploidy content. Using ImageJ (www.nih.gov), we created an overlying grid on the microscopic photograph, and measured the perimeter and area of the first ten cells at the intersections of the points of the grid to assess cell size differences between phases.

Flow Cytometry
We used flow cytometry to determine the relative abundance of gametophytes and sporophytes in U. rigida and U. compressa. Based on the C-values (haploid genome sizes) of U. compressa (0.13 pg, Le Gall et al. 1993) and U. rigida (0.16 pg, Le Gall et al. 1993), we used the freshwater unicellular alga Chlamydomonas reinhardtii, as an external flow cytometry control (C-value of 0.12 pg, Merchant et al. 2007). We specifically selected the cell wall-deficient mutant CC-400 cw15 mt+ as our control (University of Minnesota Chlamydomonas Center, chlamycollection.org).
We used an enzyme solution developed specifically for efficient production of We weighed all Ulva samples to 0.50 g wet weight, rinsed with them raw seawater to remove debris and epiphytes, and then thoroughly scrubbed them manually in 20 µm filtered seawater to remove smaller particles. Ulva samples were chopped with a razor blade in a large (85 mm x 25 mm) plastic Petri dish for one minute, and then the tissue was transferred into a small (55 mm x 15 mm) Petri dish that contained 5 mL of enzyme solution (Reddy et al. 2006).
Protoplasts were released by placing samples on a shaker at 50 rpm in the dark for two hours at room temperature (~21⁰C), then filtered with a 30 µm nylon mesh into a 5 mL polypropylene tube and spun for five minutes at 120 x g at 4⁰C. A total of 2 mL of supernatant was then removed and replaced with 2 mL of sterile filtered seawater.
Centrifugation with subsequent replacement of fluid was repeated twice, and after the last round of centrifugation, all supernatant was removed and replaced with 1 mL of sterile filtered seawater. We observed successful protoplast isolation via microscopic examination at 400X. In preparation for the flow cytometer samples were spun for five minutes at 120 g at 4⁰C, the supernatant was removed, and samples were kept refrigerated or on ice.
To liberate the nuclei, we added 1 mL of modified LB01 nuclear buffer kept on ice to each sample, vortexed and tapped the tube occasionally for eight minutes, and then to the small genome size and the preponderance of PI to bind to remaining cell wall polysaccharides from the extraction of Ulva protoplasts, which makes obtaining CV values less than 3% challenging (Kagami et al. 2005, Doležel et al. 2007b.

Growth Experiments
We assessed growth rates of gametophytes and sporophytes of U. rigida and U.
compressa in outdoor flow-through ambient temperature seawater tanks on the University of Rhode Island's Narragansett Bay campus. We collected healthy Ulva individuals from the shallow subtidal zone in Greenwich Bay in the summer of 2013. In total, we used 90 U. compressa individuals (62 sporophyte and 28 gametophyte) and 61 U. rigida individuals (38 sporophyte and 23 gametophyte) for this analysis. We conducted growth experiments in June, July, and August to assess differences in growth over the peak bloom-forming months.
In the lab, we determined the species identity of each specimen via microscopic examination. We placed one 1.0g Ulva individual in each 2.5 L bucket with mesh sides; after 14 days, all growth experiments concluded and the Ulva was re-weighed. For each month, we had a sample size of at least five (up to a maximum of 36) individuals of each phase of each species, except for U. rigida sporophytes in August, when we only had three individuals. All Ulva were spun 20 times in a salad spinner prior to each weighing to ensure consistent mass, and all individuals were analyzed using flow cytometry for ploidy content (see above).

Statistical Analyses
To assess ploidy ratios in field populations of U. compressa and U. rigida, we Based on the results for the logistic regression model described above, we then selected the three significant continuous variables (salinity, salinity two weeks prior to specimen collection, and total Ulva biomass) and analyzed each individually in separate models for representation in graphical models. Data analyses were conducted in R (Wickham 2009, Team 2013) and JMP (JMP ® , Version 10. SAS Institute Inc., Cary, NC, 1989-2013.
Growth data were analyzed with a two way fixed factor ANOVA to measure differences across ploidy levels and months. Cell sizes were compared between gametophytes and sporophytes for each species using t-tests with unequal variances in JMP.

Ploidy
We found both gametophytes and sporophytes of each species present at each of the sampling location sites. There were significant differences among the relative ploidy levels at each site (Figure 3), compared to the null model prediction of √2 gametophytes to 1 sporophyte (χ 2 likelihood test, Table 1). U. compressa in Oakland Beach Cove (OBC) and Sandy Point (SP) differed from this null prediction with a relative overabundance of sporophytes. U. rigida in Warwick City Park (WCP) and Sandy Point (SP) differed from the null prediction with a relative overabundance of sporophytes in WCP and dominance of gametophytes in SP.
Based on AIC values, the strongest predictive model for ploidy relative abundance included the variables species, site, salinity at time of sampling, and total Ulva biomass ( Table 2) and not temperature, month of sampling, date of sampling, or total algal biomass. While salinity measurements with a time lag of two weeks prior were significant, they were not included in the model with the strongest AIC.
When we analyzed the significant continuous variables individually for their correlation to ploidy ratios, we found that the relative abundance of sporophytes was positively correlated with higher Ulva biomass at the time of collection ( Figure 4A; χ 2 3 = 16.10, p<0.01). We found increasing proportions of Ulva sporophytes at higher salinities at the date of sampling for both species ( Figure 4B; χ 2 3 = 13.36, p<0.01). Interestingly, salinity measurements with a time lag of two weeks prior yielded significantly increasing proportions of Ulva gametophytes at higher salinities ( Figure 4C; χ 2 3 = 10.54, p=0.01) for both species.

Growth
We found no significant differences in growth rate between phases for either species, (Figure 5; U.  Figure 6). Figure 1. Isomorphic biphasic life cycle of Ulva. Ulva cycles between two morphologically similar multicellular adult phases, a haploid gametophyte and a diploid sporophyte. Diploid sporophytes produce haploid zoospores that develop into gametophytes. Haploid gametophytes produce haploid gametes. When a "+" and "-" gamete fuse they form a zygote, which develops into a diploid sporophyte.    Table 2. Table for the best-fit logistic regression with a binomial distribution and ploidy as the independent variable. Model follows the form logit( ) = β 0 + β 1 x 1 + β 2 x 2 +… (e.g.

Ploidy Distribution
Macroalgal blooms are a problem worldwide and cause severe ecological and economic damage (Liu et al. 2013). Prior to this study, only two publications Hiraoka and Yoshida (2010) and Alström-Rapaport et al (2010) had assessed abundance of Ulva gametophytes and sporophytes, and found an alternating abundance of gametophytes and sporophytes that was seasonal only in the latter study. Here, our data indicate that both phases are present for each species throughout the peak bloom forming season, and that relative phase abundance is correlated with both abiotic and biotic factors. We found a high variability among sites in ploidy ratio among sites, with some sites matching the null model prediction of relative abundance, while others exhibited an overabundance of gametophytes or sporophytes. These deviations could be due to ecological differences among phases and/or environmental differences among sites. Sandy Point, which differed from the null hypothesis for both species, is a more exposed site and experiences more water mixing than the other sites (Sankaranarayanan & Ward 2006). However, as U.
compressa had an overabundance of sporophytes and U. rigida had an overabundance of gametophytes at this site, the relative impacts of environmental factors are challenging to assess and may represent specific environmental factors unique to each species. Warwick City Park and Oakland Beach Cove, which differed from the null hypothesis in U. compressa and U. rigida respectively, are more sheltered sites and experience less water mixing (Sankaranarayanan & Ward 2006).
We found a significant correlation of physical and biological factors on the relative abundance of gametophytes and sporophytes in our study system (Table 2, Figure  4). In this study system, low salinities are typically a result of increased freshwater flow from rivers caused by storms. In Narragansett Bay, increased flow in rivers yields higher concentrations of dissolved inorganic nitrogen and phosphorus (Nixon et al. 1995).
Therefore, although nutrient data are not available for our sampling period, low salinities can be used as a proxy for increased nutrients. Lower salinities from the date of sample collection were correlated with higher relative levels of gametophytes, while lower salinities from two weeks prior to specimen collection were correlated with more sporophytes (Figure 4). This shift in ploidy ratios may be due to several factors, such as salinity tolerance, positive response to nutrient availability from one phase over the other, or shift to asexual reproduction (Bliding 1963). While it is unlikely that a reproductive event would result in the presence of new adults after only two weeks (Leletkin et al. 2004), lower salinities may trigger more rapid growth of one phase from a microscopic to a macroscopic size (Hannach & Santelices 1985). Due to the biphasic life cycle, increased nutrients may either impact mortality and/or fecundity rates of either phase (Adams & Hansche 1974, Lewis 1985, with differential effects on the relative balance of phases. In addition, vegetative fragmentation of mature blades, germination of unfused gametes, and/or asexual production of diploid spores by sporophytes may impact the ploidy ratio (Van Den Hoek et al. 1995).
We also found a positive correlation between the relative abundance of sporophytes for total Ulva biomass for both species. One potential explanation is that gametophytes could be more successful when there is less competition for resources, while sporophytes are better competitors than gametophytes under higher competitive pressure for resources. While other environmental factors, such as temperature (Deacutis et al. 2006), have an impact on bloom abundance (Rivers & Peckol 1995, Kim et al. 2011 or growth rates (Guidone 2012), we found no impact of temperature on the relative abundance of gametophytes and sporophytes.
Previous studies have found a seasonal dominance of one ploidy phase (Dyck & De Wreede 1995) or a long term (11-20 month) non-seasonal cyclic dominance (Hiraoka & Yoshida 2010), or no seasonal trend (Thornber & Gaines 2003). As our sampling was limited to the bloom forming season, a cycling trend in ploidy for U. compressa and/or U. rigida could exist. However, due to the scarcity of Ulva specimens during non bloom forming periods , this would be challenging to assess.

Growth and Cell Size
We did not find any significant differences in growth rates of adult gametophytes and sporophytes of either species, but this does not preclude the possibility of differences at the germling stage (Hannach & Santelices 1985). In addition, growth rates can vary based on nutrient levels (Peckol et al. 1994); as nutrient levels shift in Narragansett Bay over seasonal cycles (Oviatt et al. 2002, Nixon et al. 2008, differences in Ulva growth rates between phases may emerge. Based on our U. compressa cell size data, future studies of U. compressa life cycle dynamics may be much more rapid and less expensive. Individuals can be predicted as gametophytes or sporophytes based on their cell size, with a subset confirmed using ploidy analysis. This would increase the ability to have larger sample sizes and more rapid assessment. Similarly, U. rigida trended toward larger sporophytes over gametophytes (significant at the 90 percent confidence level).
Differences in U. compressa cell sizes between phases can impact the surface area to volume ratio, allowing for faster uptake of nutrients in smaller cells (Lewis 1985). This is especially relevant in single-celled spores, gametes, and small juveniles, and may impact Ulva individuals in their early growth stages. U. rigida zoospores are 9-15 µm x 5-10 µm while gametes are 7-11 µm x 4-6 µm (Clayton 1992). Since gametes are much smaller than zoospores, they may have a survival advantage in their enhanced ability for nutrient uptake. There may also be other ecological differences between either phases across their lifespan, such as susceptibility to herbivores, light tolerance, salinity tolerances, and temperature optima (Destombe et al. 1993, Thornber 2006, Thornber et al. 2006, Guillemin et al. 2013, that may explain differences in ploidy ratios. Gametophytes do not have heterozygosity, so they do not have the genetic capacity to deal with environmental changes as efficiently as sporophytes. However, both beneficial and deleterious mutations will immediately be expressed in gametophytes, so deleterious mutations will be eliminated faster than in sporophytes, and if a beneficial mutation should arise in a gametophyte population, it will reach fixation faster than in sporophyte populations (Otto & Gerstein 2008).

Flow Cytometry Method
We designed our flow cytometry ploidy analysis methods from similar analyses in higher plants (Doležel et al. 1989, Doležel & Bartos 2005, Doležel et al. 2007a, which has been successful for other macroalgal studies (e.g. Le Gall et al. 1993). We first attempted chopping Ulva tissues with a razor blade in the presence of a nuclear isolation buffer to obtain isolated nuclei (essentially removing our protoplast isolation step). This method, which is successful in higher plants for flow cytometric analysis (Galbraith et al. 1983), was unsuccessful for Ulva. The amount of nuclei obtained was small and contaminated with other materials, likely organelle genomes and bacteria (Nakanishi et al. 1996). In addition, Ulva has high concentrations of anionic polysaccharides in its cell walls (Abdel-Fattah & Edrees 1972) which can interfere with obtaining a sufficient number of nuclei by binding to the positively charged nuleus, inhibiting the propidium iodide from attaching. Given these constraints, protoplast isolation was necessary to obtain sufficient numbers of nuclei for flow cytometry analyses (Kagami et al. 2005), which is successful yet time consuming and expensive (Reddy et al. 2006), thus limiting our abilities to obtain larger sample sizes.
Given the abundance of Ulva blooms worldwide (Morand & Merceron 2004, Merceron et al. 2007, it is crucial that we understand the basic ecology of Ulva, including the life cycle, to make more informed management decisions for bloom mitigation. This research presents an innovative step into further understanding Ulva life cycle dynamics and their relationships to associated abiotic and biotic factors, through the use of flow cytometry techniques.