Patrick Martone

Adhesion is a critical first step in the successful recruitment of seaweeds. Kelp (Laminariales) sporophytes secrete adhesives from specialized rhizoid cells to anchor themselves to the substrate and withstand immense forces applied by waves. The composition of macroalgal adhesive has primarily been described in the Fucales as a “modified extension of the cell wall”, but the extent to which kelp adhesives are compositionally similar to fucoids is unknown. Moreover, the cellular organization of the attachment interface of kelps is poorly understood. To address this, rhizoid cells and adhesive from developing Alaria marginata sporophytes were examined with brightfield, fluorescence, and scanning electron microscopy to describe the organization of the attachment interface and biochemical composition of adhesives and walls in developing holdfasts. The primary holdfast is composed of elongated, thick-walled cortical cells and a meristoderm layer. New rhizoids appear to differentiate from meristoderm cells by modification of the meristoderm outer wall and elongation by tip growth. Rhizoid cells contain small spherical plastids distributed evenly along the length of the cell. Physodes are found along their length and phenolics localize in rhizoid walls. The highest resolution chemical analysis of kelp adhesives to date was provided by confocal microscopy, demonstrating adhesives are composed of fucose containing sulfated polysaccharides (FCSPs), alginate, and phlorotannins and dominated by BAM3 and BAM4 epitopes, not alginates as expected. Adhesives are biochemical extensions of the cell wall with distinct but variable stoichiometries, suggesting adhesives are more complex and dynamic than expected. Chemical analysis demonstrated that adhesives are likely underestimated in their complexity and identified weak points in our current understanding of kelp adhesives, for example calling into question the roles of phenolics and proteins. Finally, these results provide a baseline understanding of adhesive composition in kelps and establish biochemical methods to allow further examination of the developmental, ecological, and evolutionary dynamics of adhesion.

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In land plants and algae, cellulose, a glucose polymer, is important for strengthening tissues and preventing breakage in the face of physical forces. Cellulose synthase enzymes (CESA) are responsible for producing cellulose. While our understanding of land plant CESAs has advanced for several species, it is unclear whether the systems surrounding cellulose synthesis are the same in all plant lineages. For example, no red algal CESAs have been functionally demonstrated. The objective of this thesis is to discover and characterize putative CESA encoding genes from the calcifying red alga Calliarthron tuberculosum and compare their function to those from the land plant eudicot Arabidopsis thaliana.Using a bioinformatics approach, I identified three candidate CESAs from Calliarthron tuberculosum’s transcriptome dataset (CtCESA1, CtCESA2, and CtCESA3). I explore the evolution of CESAs in gene tree analysis and find that while CtCESA1 was closely related to other red algal CESA sequences, CtCESA2 and 3 were more closely related to bacterial cellulose synthases (Ch 2). Using yeast and insect cell expression systems, I heterologously express, purify, and test the CtCESA1 protein in glucose tracer assays to look for polymer formation. CtCESA1 showed evidence of glucan synthase activity that was comparatively lower than plant (PttCESA8) and bacterial (BCSA/BCSB) cellulose synthases (Ch 3).Finally, I test for functional compatibilities between the land plant (A. thaliana) and red algal (C. tuberculosum) CESAs. A. thaliana encodes multiple non-redundant CESAs that function in primary cell wall and secondary cell wall regions as well as several other accessory proteins critical to cellulose synthesis. However, only some accessory proteins were recovered from C tuberculosum’s transcriptome in bioinformatics analyses (Ch 2). To ultimately test for functional differences, I introduced the CtCESA1 gene into A. thaliana cesa mutants deficient in cellulose production (Ch 4). The red algal CtCESA1 partially rescued the A. thaliana primary cell wall cesa6 mutant but not cesa3 or the secondary cell wall cesa7 mutant. This thesis collectively presents the first functional evidence of a red algal CESA and demonstrates a combination of both deeply conserved and largely distinct aspects of cellulose production between the red algal and land plant lineages.

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Geniculate coralline algae are notoriously challenging to identify in the field due to confusing morphological variation. Consequently, former species delimitations based exclusively on morphology are often unsupported by sequence-based phylogenies. The purpose of my research was to determine whether Corallina chilensis Decaisne, basonym of C. officinalis var. chilensis, was a distinct species or should be considered a variety of C. officinalis; and consequently whether C. chilensis was distributed in two hemispheres. In order to answer these questions, I sequenced psbA, CO1, and rbcL genes from 76 voucher specimens representing Corallina collections from ~2000 to 2019. I applied names by comparing these sequences with published sequences and type specimen sequences, including an rbcL sequence from the specimen collected by Darwin (#2151 from Valparaiso, Chile), the holotype specimen for C. chilensis designated by Harvey. I used phylogeny with additional support from morphometric, Automatic Barcode Gap Discovery, and distance matrix analyses for species delimitation. DNA from the Chilean C. chilensis holotype matched an unnamed coralline species commonly found in the Northeast Pacific, and C. chilensis specimens formed a separate clade from C. officinalis specimens in my phylogenetic analyses. Corallina chilensis is a distinct species, not a variety of C. officinalis, and it is present in both hemispheres. Going forward, the name C. officinalis var. chilensis should be discontinued, and the older name C. chilensis should be used in its place.

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Kelp forests are one of the most productive ecosystems in the world, supporting a diverse assemblage of species. Throughout their history of study, kelp forests have undergone shifts between kelp forests and urchin barrens. Urchin barrens – rocky reefs which have no remaining kelp habitat due sea urchin grazing – have important consequences for species that rely on this habitat for survival. In Howe Sound, British Columbia Neoagarum fimbriatum is the dominant habitat-forming kelp and is the essential settlement habitat for commercially important juvenile spot prawns, Pandalus platyceros. Since 2013, the abundance of Neoagarum has declined following an increase in green urchins, Strongylocentrotus droebachiensis, due to loss of top-predator sea stars. This shift in the rocky reef community has led to concerns of continued decline in Neoagarum. This study looks at two aspects of Neoagarum persistence in the face of intense grazer pressure. First, seasonal growth patterns with respect to light and temperature variation are examined. Second, the impact of high densities of green urchins on Neoagarum loss are quantified. Seasonal growth patterns showed that Neoagarum grows throughout the year with maximal growth rates during summer, reaching up to 7% d-¹. Light was determined to be the main driver of these patterns. This was apparent particularly during spring plankton blooms when periods of minimal light at the depth of the kelp beds correlated with the lowest growth rates, while highest growth correlated with the longest days and highest light intensity. These growth rates suggest that in the absence of grazer pressure, Neoagarum has the capacity to develop dense kelp beds in a matter of months. Urchin grazing experiments showed that the relationship between urchin density and the rate of kelp biomass loss scaled linearly, but only at density below 36 urchins m-². Density-dependent restriction of green urchins was apparent at densities greater than 36 urchins m-². Despite lower per capita grazing rates at high densities it appears that so long as densities of green urchins densities remain high, their ability to consume greater than just 7% kelp biomass per day will lead to further declines in Neoagarum kelp beds.

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Maintaining buoyancy with pneumatocysts is essential for subtidal seaweeds with long flexible thalli, such as Nereocystis luetkeana (Nereocystis), to achieve an upright stature and compete for light. However, as Nereocystis grows, pneumatocysts are exposed to significant changes in hydrostatic pressure. Exposure to changing hydrostatic pressure could cause complications since the pneumatocyst is filled with gases that may expand or contract, potentially causing pneumatocysts to break, flood, and no longer be buoyant. This study explored how Nereocystis pneumatocysts resist biomechanical stress and keep the developing sporophyte upright in the water. Throughout development, pneumatocysts had an internal pressure consistently less than atmospheric pressure (3 – 100 kPa), indicating pneumatocysts always experience compressional loads. The structural integrity and design of the pneumatocyst to resist buckling was assessed by measuring compressional modulus (material stiffness), calculating material stress, analyzing critical geometry, and estimating critical buckling pressure. Small pneumatocysts found at depth (inner radius = 0.8 - 0.9 cm; wall thickness = 0.2 cm) were demonstrated to have reached a critical size in development and are at greatest risk of buckling. Pneumatocysts do not adjust material properties or geometry to reduce wall stress, but they are naturally resistant to hydrostatic loads. Critically small pneumatocysts are estimated to buckle at 35 m depth, which was observed to be sporophytes’ lower limit in the field. Data suggest that hydrostatic pressure, not just light limitation, might explain the maximum depth to which Nereocystis is capable of growing. Pneumatocyst gas composition did not change throughout development, and contrary to previous studies, internal gas concentrations were different from the atmosphere with O₂, N₂, CO, and CO₂ concentrations of 59%, 40%, 1.6%, and 0.6% respectively. Furthermore, pneumatocyst surface area to volume ratio did not correlate with the exchange of gases produced from photosynthesis and respiration. As sporophytes grow, total buoyant force is steadily outpaced by the weight of growing thalli, and the risk of the pneumatocyst sinking increases. Adult sporophytes are estimated to sink when pneumatocysts volume reaches 1.3 L, close to the maximum observed size in the field.

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Organisms in the intertidal zone are regularly exposed to wave action, emersion, and competition. Competition for space may have been a factor leading to the evolution of epiphytes which have circumvented this problem by growing on other algae. Epiphytism is generally considered deleterious to hosts but, is this always true? This study explored costs and benefits of interactions between the epiphyte, Soranthera ulvoidea, and its host, Odonthalia floccosa, involving biomechanics, light acquisition, desiccation, and herbivory. Drag on epiphytized and unepiphytized hosts was measured in a recirculating water flume. Epiphytes increased drag on hosts by approximately 50% at each test velocity. Increased drag caused epiphytized hosts to be more likely to break from the substratum than hosts without epiphytes. Epiphytes experienced reduced drag when attached to hosts but sometimes broke before hosts. In fact, epiphytized hosts and epiphytes were equally likely to dislodge; this suggests that drag added by epiphytes may not be entirely harmful to hosts if epiphytes dislodge half the time, reducing overall drag on epiphytized hosts. The effects of epiphytism on light acquisition, desiccation, and herbivory were also investigated. Photosynthesis versus irradiance curves were constructed for hosts and epiphytes; saturation irradiances for both were approximately 50μmol m⁻²s⁻¹, and were not significantly different from irradiances under submerged algal canopies in the field. Thus, it was inferred that these epiphytes do not likely affect host light acquisition. Also, these epiphytes may not have arisen in response to light limitation as they reached photosynthetic saturation when exposed to light levels under other algae. When hosts with and without epiphytes were exposed to air, epiphytes doubled the time required for hosts to lose 50% of the water originally associated with their thalli. By delaying desiccation, epiphytes likely reduce physiological damage of emergent hosts. Lastly, invertebrate herbivores common to this study’s field site preferred grazing epiphytes over hosts. This feeding preference could benefit hosts by diverting herbivores away from host tissue and toward epiphytes. In sum, this study demonstrates that hosts and epiphytes often benefit by closely associating; these complex interactions could help explain the evolution and persistence of intertidal algal epiphytism.

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Intertidal macroalgae endure stresses associated with submerged and emerged conditions on a daily basis. Differences in physiology at high tide, low tide, and during recovery underlie spatial separation of species along the shore. Tidepools provide refugia from physical stresses associated with the low tide, and species with low stress tolerance may be restricted to these habitats. Species that survive emergence employ physiological and morphological strategies to survive exposure to pseudo-terrestrial conditions. I explored how tidepool and non-tidepool macroalgae respond to and recover from intertidal stressors, such as light, temperature, and desiccation. I investigated whether differences in physiology could explain differences in habitat distributions. To answer these questions, I explored the physiological responses of the coralline algae, Calliarthron tuberculosum (Postels and Ruprecht) E.Y. Dawson and Corallina vancouveriensis Yendo, to simulated tidal conditions. Calliarthron is restricted to tidepools, while Corallina can survive emersion during low tide. First, I documented physiological differences between the two species at high tide. Corallina performed similar to a high light adapted plant, while Calliarthron’s performance resembled that of a low light adapted plant. Surprisingly, their pigment composition did not differ, suggesting that both species are able to harvest light similarly but that other metabolic processes are at play. Second, I compared morphological and physiological strategies employed by Calliarthron and Corallina to resist stress during low tide. I found differences in the physiological responses of the two species to increased light and temperature, two chief stressors present in the tidepool microhabitat. Unlike Calliarthron, Corallina exhibited high tolerance to increasing water temperatures and was more effective at resisting desiccation via morphology. However, neither species photosynthesized in the air, regardless of hydration level. Finally, I quantified recovery upon the return of the tide. Both species recovered from warm tidepool temperatures. However, only Corallina recovered from the combination of temperature and desiccation stress associated with emergence.This study describes the variation in physiological performance of two intertidal macroalgal species during the tidal cycle, and documents several morphological and physiological strategies employed by species to survive stresses associated with low tide. Results help to explain the habitat differences between the two species.

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