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Under pressure : biomechanics of buoyancy in Bull Kelp (Nereocystis leutkeana) Liggan, Lauran 2016

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UNDER PRESSURE: BIOMECHANICS OF BUOYANCY IN BULL KELP (NEREOCYSTIS LUETKEANA)  by Lauran Liggan  BSc, Humboldt State University, Arcata, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2016  © Lauran Liggan, 2016   	 ii	Abstract 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 	 iii	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.   	              	 iv	Preface  Chapter 1 and 2 are based on work conducted in the Martone Laboratory (Botany Department, University of British Columbia) and at the Bamfield Marine Sciences Centre on Vancouver Island. I was responsible for collecting all data reported in the lab and the field. Research was funded by an NSERC Discovery grant to Dr. Patrick Martone.   My research supervisors, Dr. Patrick Martone and Dr. Margo Lillie (Zoology Department), aided in data analyses and chapter revisions during the writing of the thesis. Dr. Christopher Harley (Zoology Department) also contributed to the final revisions of the thesis.              	 v	Table of contents Abstract……………………………………………………………………………….… ii Preface…………………………………………………………………………………... iv Table of contents.…………………...…………………………………………………... v List of tables …...……………………………………………………………………... viii List of figures …...……………………………………………………………………… ix Acknowledgements…………………………………………………………….....…... xiii Introduction …………………………………………………………………………….. 1 Chapter 1:  Effects of hydrostatic pressure on pneumatocyst growth and development……………………………………………………………………………... 5 1.1 Introduction ...………………………………………………………………... 5 1.1.1 Ecology and life history ...………………………...……...………... 5 1.1.2 Understanding responses to hydrostatic pressure ...………...……... 6 1.1.3 Pneumatocyst material stress and the risk of breaking ...…...……... 9 1.1.4 Study objectives ...…………………………….……………...…... 10 1.2 Methods ...…………………………………………………………………... 11   1.2.1 Sample collection ...…………………………..…………………... 11   1.2.2 Measuring internal pneumatocyst pressure..……………….……... 12   1.2.3 Calculating wall stress ...………...………………………...……... 15   1.2.4 Compressive modulus ...………………………………………….. 16   1.2.5 Quantifying changes in pneumatocyst geometry ...……...……...... 19   1.2.6 Calculating pneumatocyst environmental safety factor...……...…. 20  	 vi	1.3 Results ...…………………………………………………………..………... 21   1.3.1 Changes in pneumatocyst volume and internal pressure ...……..... 21   1.3.2 Calculated wall stress ...…………………………………………... 26 1.3.3 Pneumatocyst wall material properties ...…………..……...……... 27 1.3.4 Pneumatocyst geometry ...………………………………………... 27 1.3.5 Pneumatocyst safety factor ...…………………………...………... 31 1.4 Discussion ...…………………………………………………………...…… 32 1.4.1 Changes in pneumatocyst internal pressure and volume ......…….. 32 1.4.2 Pneumatocyst wall stress and the risk of buckling ...…………...... 34 1.4.3 Reducing wall stress ...………………………………….………... 35 1.4.4 Pneumatocysts at risk of buckling ...……………………………... 36 1.4.5 At what depth will a small pneumatocyst break? ...………….……37 1.4.6 Conclusion ...……………………………………...………...……. 38 Chapter	2:	Developmental	changes	in	gas	composition	and	buoyancy	of	Nereocystis	pneumatocysts	...……………………………………………………………………….	40	2.1	Introduction	...…………………………………..………………………………….…………...	40 2.1.1 Growth and development ...…………………...…...……………... 40 2.1.2 Pneumatocyst gas composition and production ...………………... 41 2.1.3 Pneumatocyst buoyancy ...………………………………………... 43 2.1.4 Study objectives ...……………………………………...….……... 43 2.2 Methods ...……………………………………………………………....……44   2.2.1 Specimen collection ...………………………………..……....…... 44   2.2.2 Internal gas composition ...…………………………………...…... 45 	 vii	  2.2.3 Pressure and CO2 fluctuations ...……………………...…………... 47   2.2.4 Buoyant force ...…………………………………………………... 50   2.2.5 Buoyant safety factor ...…………………………………….…...... 52 2.3 Results ...………………………………………………………………….… 53   2.3.1 Pneumatocyst gas composition ...………………………….……... 53   2.3.2 Pressure and CO2 fluctuations ...………………..…………….…... 57 2.3.3 Pneumatocyst buoyancy ...……………………………...…….…... 59 2.4 Discussion ………………...…………………………….……...…………... 61 2.4.1 Pneumatocyst gas composition ...…………………………….…... 61 2.4.2 Pressure and CO2 fluctuations ...…………………………..….…... 64 2.4.3 Pneumatocyst buoyancy ...…………………………………….….. 65 2.4.4 Conclusion ...………………………………………………….….. 67 Conclusion ...…………………………………………………………………………... 69 Future directions ...………………………………………………….…………….…... 72 References ...…………………………………………………………………………… 76  				  	 viii	List of tables Table 1: Table showing calculated ESF values for 4 pneumatocysts collected between 2.7 m and 6 m………….…………….….…………….……………………….......……. 31 Table 2: Estimated critical breaking depth and breaking stress for 2 small pneumatocysts (with critical geometry) recorded to have the greatest wall stresses and the greatest ri: t found between 7 and 8 m of seawater……………….…….…………………….……… 31 Table 3: Table showing average gas concentrations of air and a pneumatocyst. Standard deviation of gas concentrations in a pneumatocyst is expressed in. Pneumatocyst gas concentrations were compared to atmospheric gas concentrations by using information available online from the National Oceanic and Atmospheric Administration (NOAA.gov, USA) ……………………………………………….………………….… 56  Table 4: Table showing buoyant factor (BSF) calculated from 5 large pneumatocysts. Average BSF is 2.18±1.3.…………….….………………………………...…………… 60             	 ix	List of figures Figure	1:	Diagram	of	Nereocystis	luetkeana	sporophyte	(Denny	et	al.	1997).	Scale	bar	is	1	m	(Denny	et	al.	1997).….…………...…………….…………….……………... 3 Figure 2: Diagram of a pneumatocyst under pressure. Arrows indicate direction of force where pressure is applied. The force differential  (ΔF) is expressed in the equation above where Po= external hydrostatic pressure, ro = the outer radius, Pi = internal pneumatocyst pressure, and ri= the internal radius..………………………...………………….……….. 7 Figure 3: Model of how a balloon would act to changes in hydrostatic pressure using eqn. 1.1. Expected changes in internal balloon pressure and depth decreases (A). Expected changes in balloon volume as depth decreases (B)…………...……….………. 8 Figure 4:  A) Location of Victoria (circled), British Columbia Canada on the southern end of Vancouver Island B) Map of Ogden Point Breakwater. White line represents area where whole thalli were collected between 0 and 9 meters of seawater (provided by Google Maps)…………………………………………………………..……………….. 11 Figure 5: Diagram of water manometer. Pneumatocysts were punctured using a lubricated syringe needle. Gauge pressure was calculated by the equation above where ρ = density of water 1000 kgm-3, g= 9.81 ms-2, h = height of water moved in both arms post puncture, and PATM = atmospheric pressure………………...….…….….……………… 14  Figure 6: Instron design for tension-compression tests. Top cross-beam is 9 cm width x 0.8 cm length and the base block is 8 cm width x 8 cm length. Pneumatocyst samples cut into doughnuts or cubes are glued to both the cross beam and the block. Black arrows indicate direction of tension and compression during data collection.…………………. 17 	 x	Figure 7: Stress-Strain curves for one sample showing 4 cycles of compression and tension. The black bracket indicates where compressive modulus was measured (slope of the line) at 1% (0.01mm/mm) compression during the 2nd cyclic cycle.…………...…... 18 Figure 8: Pneumatocyst volume at various depths (A). Pneumatocyst internal pressure at various depths (B). Red dotted line indicates atmospheric pressure.……...…………… 22 Figure 9: Average internal pressure for small (8-20 cm3) pneumatocysts, found shallow (<2 m) and at depth (>8 m). Error bars indicate standard deviation of internal pressure (Shallow ± 16.86 kPa; Deep ± 16.82 kPa)) for each pneumatocyst depth group (t = 1.03; df=13.6; P>0.1).………………………………………………....…………….………... 23  Figure 10: Internal pneumatocyst pressure and total pneumatocyst gas as a function of pneumatocyst volume.…………….…………….……...…………….………………… 24 Figure 11: Blue data points: internal pneumatocyst pressure plotted with pneumatocyst depth (0-9m). Green data points: external hydrostatic pressure plotted with depth (0-9m).…………….………………………………………………………...………….….. 25 Figure 12: Pneumatocyst wall stress at depths between (0-9m).……………...…….…. 26 Figure 13: Young’s modulus for different pneumatocyst volumes.………...……….… 27 Figure 14: Estimated wall stress at depths from 0-9m for a given ri:t ratio (1:1,3:1,and 6:1; A). Dashed line indicates estimates of wall stress for a given ri:t ratio at 6 m. ri:t ratio at depths between 0-9m (P>0.05; B) .................………….....………….……………… 28 Figure 15:  Pneumatocyst wall stresses for small pneumatocysts with different ri:t ratios. All pneumatocysts were collected between 7 and 8.7 m …...………………...….…….. 29 Figure 16: Inner pneumatocyst radius at different depths (A). Grey data points are samples that began growing below 9 m and thus growth data is missing from the 	 xi	substratum to 9 m. These data were not included in analyses of pneumatocyst development. Pneumatocyst wall stress with an inner pneumatocyst radius (B). Black dotted line indicates overall trend in wall stress as inner radius increases. ri:t ratio with inner radius from (C). Black circle indicates pneumatocysts with the greatest wall stresses shown in part B. Inner pneumatocyst radius with pneumatocyst tissue volume (D)………. .…………….…………………………………………………………………….……… 30 Figure 17: Map of southern Vancouver Island, British Columbia Canada. The yellow circle indicates location of Bamfield (A). Map of field sites where Nereocystis was collected (B) (provided by Google Maps).…………….……………………………..… 45 Figure 18: Diagram of Q trak sensor. CO2 and CO concentrations in the pneumatocyst were calculated using the equation above. Where C1= the concentration read on the monitor (ppm), V1= volume of the calibration wand (ml), C2= unknown pneumatocyst concentration (ppm), and V2= 0.5 ml sample………………………………………..…. 46 Figure 19: Diagram of water manometer. Pneumatocysts were punctured using a lubricated syringe needle. Gauge pressure was calculated by the equation above where  ρ = density of water 1000 kgm-3, g= 9.81 ms-1, h = height of water moved in both arms post puncture, and PATM = atmospheric pressure. …………..…….………..…………… 48 Figure 20: Diagram of contraption designed to measure net buoyant force of the intact thallus. Whole kelp is placed in basket and applies an upward force that is detected by a force transducer.……………………………………………………………………..….. 51 Figure 21:  Concentrations of CO, CO2, O2, and estimated N2 for different pneumatocyst volumes (P>0.05). Dotted lines indicate average gas concentration…………………… 54 Figure 22:  Log total gas for different pneumatocyst volumes (10-1000ml).…...……... 55 	 xii	Figure 23: Average concentrations of gases in air compared to average concentrations of gases in a pneumatocyst.  Pneumatocyst gas concentrations were compared to atmospheric gas concentrations by using information available online from the National Oceanic and Atmospheric Administration (NOAA.gov, USA).……….………….……. 56    Figure 24:  Log inner surface area to volume ratio (SA:V) for various pneumatocyst volumes (A). The total change or fluctuation of CO2 for experimental pneumatocysts of various pneumatocyst volumes (B). SA:V is estimated with the regression model in part A. Grey dotted line indicates the average fluctuation of CO2.………………….………. 57  Figure 25: Changes of light (A), internal pneumatocyst pressure (B), and CO2 concentration (C) during a 24 hour time period starting at 21:30. All data points represent average values. Error bars indicate standard deviation.…….…………..………………. 58 Figure 26: Log thallus weight (downward force, N) as a function of pneumatocyst volume (A). Log net buoyancy (upward force measured in Fig 20, N) as a function of pneumatocyst volume (B). Log total buoyant force (upward force, N) as a function of pneumatocyst volume. Dotted line indicates average total buoyant force (C; P>0.05).... 59  Figure 27: Log buoyant safety factor (BSF) as a function of Log pneumatocyst volume.  The last 5 data points are represented in table 4.………………...…….……………..… 60        	 xiii	Acknowledgements  This study would have not been successful without the help and support from a handful of people. I would first like to thank my supervisor Dr. Patrick Martone, for giving me the much needed guidance, patience, and support to begin and accomplish my first attempt conducting research and developing my own ideas. His enthusiasm for science, seaweeds, and the marine world inspired me to be passionate about my work. I would also like to thank Dr. Margo Lillie who advised and guided me through out this project. Margo imparted her knowledge and experience conducting research in the field of biomechanics, which helped me frame and design my thesis.    The Bamfield Marine Sciences Centre provided much needed lab space for the majority of this study. All equipment used for the transport of pneumatocyst gas samples were graciously donated from Dr. Andreas Christen in the Department of Geography and Atmospheric Science Program. Equipment for measuring CO2 was donated by Dr. Santokh Singh and the Plant Physiology Lab.   My fieldwork was tedious and required a cohort of people to collect useful data in a timely manner. With out the SCUBA diving and snorkeling support of Kyra Janot, Sam Starko, Rachel Munger, Laura Borden, and Meagan Abele, this study would have been impossible. I am very grateful that the UBC Aqua Society provided me with endless training, SCUBA tanks, and other resources, which made these sub tidal collections possible. I would like to specially thank Sarah Calbick for being not only my field and lab assistant but also my confidant who helped me design better methods for collecting data and limiting bias. My lab mates are amazing and I am very thankful to have had the opportunity to befriend and learn from each of them. They spent precious time to read my manuscripts and listen to countless practice talks through out this study.   I would like to thank my parents, Susan and Stuart Liggan for financially supporting me throughout my educational career. They have been nothing but positive about my passion for marine science and I cannot thank them enough for exposing me to the marine world at a young age. With out them, my love for the sea would not exist.  And lastly, I would like to thank my partner and best friend Bartek Radziej. He kept me sane with just a smile and up beat attitude during my long absences conducting fieldwork, my late nights in the lab, and as I forced him to help me in the field. Without his unconditional support and kindness, I would have never been able to complete this degree.    	 1	Introduction	Photosynthetic	organisms,	such	as	trees	and	kelp,	have	evolved	morphological	characteristics	that	enable	them	to	compete	for	light.	Though	these	characteristics	are	diverse	and	cater	to	the	particular	ecosystem	where	each	species	thrives,	they	all	serve	the	same	purpose:	maintain	an	upright	posture	while	exposed	to	physical	stresses.	The	ability	to	remain	upright	allows	these	organisms	to	increase	light	interception	and,	ultimately,	increase	photosynthesis	to	fuel	growth	and	reproduction.		In	terrestrial	forests	trees	build	unique	three-dimensional	ecosystems	that	house	a	diverse	community	of	organisms	(Brandt	et	al.	2014).	Trees	gain	height	and	build	complex	environments	by	having	rigid,	reinforced	trunks	that	allow	them	to	grow	upright	against	gravity	(Woodward 2004).	Trees	augment	their	biomechanical	properties	by	creating	wood	and	adding	girth	through	secondary	growth	(Fournier	et	al.	2006;	Jung	et	al.	2016).	The	rigid	and	strong	structure	of	trees	allows	them	to	tolerate	environmental	stress,	resist	toppling	in	storms,	and	support	forest	ecosystems	(Sellier and Fourcaud 2009;	Jung	et	al.	2016).			 Similar	to	terrestrial	forests,	kelps	build	diverse	three-dimensional	forests	in	near-shore	marine	ecosystems	(Arzee	et	al	1995).	Kelps	also	grow	tall	(up	to	45	m;	Spalding	and	Foster	2003),	but	unlike	trees,	kelps	generally	do	not	develop	thick	trunks	for	structural	reinforcement.	Instead,	some	kelps	produce	slender,	flexible	thalli	that	are	highly	extensible	and	not	very	stiff,	yet	they	still	manage	to	remain	upright	(Koehl	and	Wainwright	1977).		Kelps,	such	as	the	Giant	Kelp	(Macrocystis	pyrifera)	and	the	Bull	Kelp	(Nereocystis	luetkeana),	buoy	their	lax	thalli	toward	the		 2	surface	by	developing	gas-filled	floats	(Stewart	et	al.	2007).	Like	trees,	kelps	are	exposed	to	environmental	stresses	such	as	hydrodynamic	forces,	and	the	ability	of	kelp	stipes	to	resist	breakage	has	been	well-studied	(Denny	et	al.	2007).	However,	the	ability	of	certain	kelps	to	produce	and	maintain	gas-filled	floats	underwater	has	been	poorly	studied	(but	see	Dromgoole	1981)	and	deserves	further	examination.		Study	system		Nereocystis	luetkeana	(K.	Mertens)	Postels	et	Ruprecht	(Nereocystis)	is	a	large	ecosystem-forming	kelp	in	the	Order	Laminariales.	Nereocystis	is	typically	found	from	depths	up	to	35	m	and	is	geographically	distributed	in	wave/current-exposed	areas	along	the	west	coast	of	North	America	(Abbott	and	Hollenberg	1976;	Spalding	and	Foster	2003).	Nereocystis	has	an	annual	life	history,	appearing	first	as	a	young	sporophyte	in	late	winter,	growing	to	adulthood	by	early	summer,	and	reproducing	and	becoming	senescent	in	autumn	(Koehl	and	Wainwright	1977).	Gametophytes,	which	are	microscopic	and	filamentous,	likely	persist	all	year	hidden	in	the	rocky	substratum	(Springer	et	al.	2007).	Nereocystis	is	capable	of	growing	about	6	cm	a	day	and	can	have	a	flexible	thallus	that	is	heavy	and	reaches	massive	sizes	(Setchel	1947).		Like all kelps, the sporophyte morphology includes a holdfast, stipe, and blade. However, unlike other kelps that lie prostrate or grow upright by stiffening their stipes, Nereocystis has a bulbous gas-filled float, called a pneumatocyst, which is buoyant and keeps the flexible thallus upright in the water (Fig 1). As Nereocystis grows and becomes heavier, the pneumatocyst provides structural support for the thallus, exposing blades to 	 3	maximum light (Stewart et al. 2007). Without pneumatocysts, thalli could not remain upright, and would be left growing near the substrate, near hungry herbivores and in low light, ultimately decreasing fitness (Chenelot and Konar 2007). Therefore, Nereocystis must maintain a resilient pneumatocyst that can tolerate biological and physical stresses, such as the pressure gradient between internal gases and external hydrostatic pressure.         										Figure	1:	Diagram	of	Nereocystis	luetkeana	sporophyte	(Denny	et	al.	1997).	Scale	bar	is	1	m	(Denny	et	al.	1997).  In Chapter 1, I investigate the structural design of Nereocystis pneumatocysts to resist buckling under hydrostatic loads. As sporophytes grow from 35 m depth, the developing pneumatocyst is exposed to changing hydrostatic pressure. This chapter explores how pneumatocysts change internal pressure and volume as they experience 	 4	different hydrostatic loads. Factors that would aid in reducing wall stress such as internal pressure, material properties, and geometry are measured to determine how pneumatocysts contend with applied loads and at what developmental stage they are at greatest risk of breaking. Furthermore, this study determines the maximum depth where pneumatocysts can develop. In Chapter 2, I characterize the gas composition of pneumatocysts and determine if it changes as they grow and develop.  I investigate whether physiological gases are added and subtracted to the pneumatocyst as a function of surface area to volume ratio, and predict which gases drive changes in internal pressure. Since the pneumatocysts are used to produce an upward buoyant force, this study determines how the buoyant force of the pneumatocyst changes to support the weight added to the fast growing sporophyte.  In summary, this study gives a comprehensive overview of how the developing pneumatocyst of Nereocystis experiences and tolerates the dynamic physical environment.  	       	 5	Chapter 1 Effects of hydrostatic pressure on pneumatocyst growth and development  1.1 Introduction 1.1.1 Ecology and life history Bull Kelp or Nereocystis luetkeana (Nereocystis) builds unique ecosystems in near-shore environments off the Pacific Northwest coast of North America. Nereocystis creates three-dimensional habitats by having a single gas-filled float called a pneumatocyst, which keeps the flexible thallus upright and vertical in the water column. Nereocystis as a primary producer has a large biomass that increases available nutrients which fuel not only marine, but also terrestrial ecosystems (Duggins and Simenstad 1989). Without this charismatic alga, the diversity of organisms in the subtidal world might otherwise be limited.   Nereocystis is an annual kelp, where fertilization, growth, and reproduction occur in one year. Young sporophytes are generally found no deeper than 30 to 35 m (Spalding and Foster 2003) and begin to grow rapidly toward the surface about 6cm a day (Scagel 1947). Like all kelp species, a young sporophyte begins its life as an upright with a simple holdfast, stipe and blade. Pneumatocysts begin to form within the transition zone between the stipe and the blade, about 2 weeks after germination (Nicholson 1970). During development, medullary cells along the transition zone begin to tear, releasing gas internally and creating the pneumatocyst (Dromgoole 1981). Once pneumatocysts have formed, sporophytes grow upwards and reach the surface in as little as 4 months (Duncan 1973). As thalli become larger, they increase biomass and become heavier, with blades 	 6	that can weigh up to 20 kg in air (Denny et al. 1997). The pneumatocyst increases volume as the thallus grows larger, extending into the stipe, increasing buoyancy to keep the heavy sporophyte upright. This upright posture helps the large flexible thallus increase light interception for the blades as the sporophyte grows from depth.    1.1.2 Understanding responses to hydrostatic pressure Hydrostatic pressure exerted on a pneumatocyst is a compressive load that is applied along the outer surface of the structure. Since Nereocystis begins its life at depth and must grow up toward the surface, the pneumatocyst is exposed to changing hydrostatic pressure. Generally, pneumatocysts first develop when hydrostatic pressure is greatest and decreases over its lifetime. Being exposed to hydrostatic pressure could cause complications since the pneumatocyst is filled with gas, and if the pneumatocyst breaks, the thallus would flood and would no longer be buoyant. Pneumatocyst gas is incased in a thick wall (1-10 mm) where pressure created by these gases is exerted on the inner surface of the wall (Fig. 2). Hydrostatic pressure acts on the outer surface of the pneumatocyst wall creating a pressure gradient (Fig. 2). Since the pneumatocyst is a thick-walled structure, differences between the force applied on the outer wall by external pressure and the force applied on the inner wall by internal pressure (force differential) influences material stress. Stress generally is an internal distribution of forces within a material that balances and reacts to external loads applied. For a spherical object, like a young pneumatocyst, the inwards force due to hydrostatic pressure is acting over the outer surface area of the pneumatocyst wall and the internal pressure is acting over the inner surface area, creating material stress (Fig. 2). This force differential in the wall 	 7	conceivably puts the pneumatocyst at risk of breaking.            Figure 2: Diagram of a pneumatocyst under pressure. Arrows indicate direction of force where pressure is applied. The force differential  (ΔF) is expressed in the equation above where Po= external hydrostatic pressure, ro = the outer radius, Pi = internal pneumatocyst pressure, and ri= the internal radius.   Kelps that produce pneumatocysts for support prevent these structures from breaking under pressure by adjusting their structural and mechanical properties (Neushul and Haxo 1963). This suggests that investment, whether initial or continuous, is needed to ensure that the structural integrity of the pneumatocyst is not compromised when exposed to varying hydrostatic pressures. However, there is little knowledge of how these kelp grow and maintain the pneumatocysts’ buoyant function despite varying hydrostatic pressure gradients.  Pneumatocyst development poses questions as to how hydrostatic pressure influences changes in volume and internal pressure. To understand how the forces on a pneumatocyst change as it grows from depth towards the surface, I begin by considering a very simple model: a thin-walled balloon, which is filled with air at a depth of 9 m, sealed, and then brought towards the surface. How changing hydrostatic pressure affects Internal	Pressure Hydrostatic pressure ΔF = Poro2 – Piri2 	 8	the volume and internal pressure of this balloon can be calculated using Boyle’s Law  (eqn. 1):  P1V1=P2V2                                                                                                       (1) where P1 is the initial pressure and  V1 is the initial volume at 9 m, and P2 and V2 are the pressure and volume at any other depth. The temperature and number of moles of gas remain constant. In this example, I set P1 at 192 kPa, which is hydrostatic pressure at 9 m, and V1 at 1 cm3. I assume that the wall of the balloon is infinitely compliant so that the balloon passively responds to equilibrate internal pressure with the external hydrostatic pressure (Fig. 3A). As the balloon moves up through the water column from 9 m depth, hydrostatic pressure would decrease. Applying eqn. 1 shows how the balloon’s volume would increase by a factor of 2 at the surface (Fig. 3B). If volume increases to the balloon’s maximum capacity, then the balloon is at risk of tearing.                        Figure 3: Model of how a balloon would act to changes in hydrostatic pressure using eqn. 1.  Expected changes in internal balloon pressure and depth decreases (A). Expected changes in balloon volume as depth decreases (B). 100 120 140 160 180−8−6−4−20External pressure (kPa)Depth (m)1.0 1.2 1.4 1.6 1.8−8−6−4−20Pneumatocyst volume (cm3)Depth (m)A B 	 9	1.1.3 Pneumatocyst material stress and the risk of breaking  As sporophytes grow from depth towards the surface, pneumatocysts experience different pressure gradients, ultimately causing stress on the pneumatocyst wall material. Unlike the balloon, the gas content is not constant in the pneumatocyst (see Ch.2) and the material properties of the wall are capable of withstanding material stresses, since they are found at depth. Since pneumatocysts found at depth are not smashed by hydrostatic pressure, the material is able to resist deformation and hinder changes in volume, preventing internal pressure from equilibrating with external pressure.  Theoretical analyses of optimally designed spherical capsules have yielded formulas to estimate critical breaking pressure (Huston and Josephs 2008). These formulas are generally estimates and disregard geometrical discontinuities and material imperfections (Huston and Josephs 2008).  Regardless, these theoretical equations can be used to estimate the upper limits of critical breaking pressure or load. In this case, maximum hydrostatic pressure could be estimated for a spherical object such as a young pneumatocyst. Critical pressure is defined as the maximum force to break an object, and is a function of material stiffness (modulus), material deformity when loaded (Poisson’s ratio: change in material volume when loaded), and the objects geometry/shape. For the case of a sphere, critical breaking pressure can be generalized using the following equation (Pan and Cui 2010; Pan et al 2010; Kármán and Tsien 1941):                 !!" =  0.37!/ !! !                                                                                     (2) Where Pcr is the critical breaking pressure, E is the modulus, 0.37 is a constant assuming a standard Poisson’s ratio of 0.3 (changes in material volume when loaded) and deformation at the buckling point, t is the wall thickness, and r is the radius.  	 10	A pneumatocyst’s geometry, material properties, and deformation due to the force differential (Fig. 2) directly influences the critical breaking pressure and thus the accumulation of material stress in the wall. To minimize wall stress, Nereocystis thalli could control the rate of hydrostatic pressure changes by adjusting the rate of stipe growth. However, stipe elongation is essential and allows the blades of Nereocystis to have maximum light availability. Therefore, pneumatocysts could adjust gas production to control internal pressure, maintaining a low force differential, or reduce wall stress by altering material properties such as modulus or geometry as they move through the water column.  1.1.4 Study objectives In this study, I examine changes in volume and internal pressure as pneumatocysts develop from depth toward the surface. I investigate the extent to which pneumatocysts act like a passive balloon, changing volume and internal pressure as a function of the physical environment. I determine the pneumatocyst’s pressure gradient and evaluate the forces it experiences. I also estimate at what point in the life of a sporophyte is pneumatocyst material stress greatest and, at this stage, I evaluate if pneumatocyst geometry, material properties, or the force differential help reduce over all wall stress, minimizing the risk of breaking. Finally, I determine if pneumatocysts are capable of tolerating high hydrostatic loads, which would determine if pneumatocysts are ever at risk of breaking at any given stage of sporophyte development.    	 11	1.2 Methods 1.2.1 Sample collection            Nereocystis thalli of varying volumes from 3 to 1200 cm3 were collected at Ogden Point Breakwater (Coordinates: 48.413542, -123.387235) in Victoria, British Columbia, Canada (n=72) (Fig. 4). Whole plants were haphazardly sampled with SCUBA from depths up to 9 m (Fig. 4).   Figure	4:		A)	Location	of	Victoria	(circled),	British	Columbia	Canada	on	the	southern	end	of	Vancouver	Island	B)	Map	of	Ogden	Point	Breakwater.	White	line	represents	area	where	whole	thalli	were	collected	between	0	and	9	meters	of	seawater	(provided	by	Google	Maps).	  All samples were detached from the substratum at the holdfast so that the pneumatocyst was fully intact. Collection depth of the holdfast, day, tidal height (at 0 m chart datum), and time were recorded for each sample. This was used to calculate the depth of a pneumatocyst based on the 0 m tide line (eqn. 3).                                                                          Collected depth (m) – Tidal height (m) = Depth of Holdfast (m)                                 (3) Depth of Holdfast (m) + Length of Stipe (m) = Depth of pneumatocyst at a 0 m tide  B	A		 12	External hydrostatic pressure (Po) was estimated from pneumatocyst depth using eqn. 4: Po=(ρgh)+Patm                                                                                                      (4) where Po = calculated external hydrostatic pressure, ρ is the density of seawater  (1025 kgm-3), g is acceleration due to gravity (9.81 ms-2), h is pneumatocyst depth, and Patm is atmospheric pressure. Pneumatocyst volume was recorded by carving a hole in the pneumatocyst, filling the inside with water and pouring it into a graduated cylinder. An average of 3 measurements was used to determine volume. Pneumatocyst length and width was measured using Image J 64 version 1.49 (National Institutes of Health) by taking photos of each sample with a meter stick. All statistical analyses were conducted using R version 0.99.467 (R Core Development Team).   1.2.2 Measuring internal pneumatocyst pressure            Water manometers (diameter: 4-8 mm; length: 20-60 cm) (Fig. 5) were used to measure internal pressure of the pneumatocysts directly after collection (n = 64). Water manometers were attached to pneumatocysts using plastic tubing, a syringe, and a 21 gauge (0.7 mm) needle that was used to puncture pneumatocysts and measure the gas pressure inside (Fig. 5). All needles were lubricated with Vaseline to avoid air leaking during the manometer reading. Initial pressure readings were recorded no more than 30 seconds after the pneumatocysts were punctured. Internal pneumatocyst pressure was then calculated by using the dimensions of the manometer and the Ideal Gas Law with eqn. 5:  P!"#!$V!"!#$ = !RT  (Ideal Gas Law) 	 13	= !!" + !!" RT = P!"V!"RT + P!"V!"RT RT =  P!"V!" + P!"V!"                                                          P!" = !!"#!$!!"!#$ ! !!"!!"!!"                                            (5) Where V!"!#$ =  V!" +V!" where PPn is the unknown pneumatocyst pressure, Pgauge is the pressure reading from the manometer, Vmf is the total volume of the manometer from the water line to the needle after the pneumatocyst was punctured, Pmi is the pressure of the ambient air in the manometer, Vmi     is volume of the manometer arm before the pneumatocyst is punctured and VPn  is the volume of the pneumatocyst. The ability to accurately measure internal pressure depended on differences in volume between the manometer arms and pneumatocysts: When pneumatocyst volume was significantly less than the manometer arm, the water level in the manometer arm barely moved, introducing error. The resolution of all manometer readings was 1 mm. Therefore, manometer error was estimated by adding and subtracting 1 mm from each measurement of water height (h), and then propagating this measurement error through the equation shown in Fig. 5 to estimate variation in Pgauge and pneumatocyst pressure (PPn) in eqn. 5. Larger differences between manometer arm and pneumatocyst volume yielded greater propagated error. Error bars therefore represent the maximum and minimum pressure for each recorded pneumatocyst pressure.  Measuring internal pressure at the surface could generate error if the pneumatocysts expand and change volume as they are brought from depth. Pneumatocyst 	 14	diameter was measured using calipers for each sample at depth and at the surface. There was a minimal change in diameter by 0.5 to 1 mm, which was mostly due to error using calipers while diving and handling other equipment underwater. Therefore, I assumed that pneumatocyst volume did not change enough to make pressure measurements at the surface inaccurate.              Figure 5: Diagram of water manometer. Pneumatocysts were punctured using a lubricated syringe needle. Gauge pressure was calculated by the equation above where ρ = density of water 1025 kgm-3,  g= 9.81 ms-2, h = height of water moved in both arms post puncture, and PATM = atmospheric pressure.    The confounding effects of pneumatocyst depth and volume could both affect internal pressure. Thus, by keeping pneumatocyst volume constant, I determined if internal pressure varied with depth. Pressure differences between small pneumatocysts found at depth and small pneumatocysts found near the surface (n=28) were tested using a t-test. All calculated pneumatocyst pressures were also plotted against pneumatocyst h 	 15	depth and analyzed using linear regression analyses.  Internal pneumatocyst pressure was plotted against pneumatocyst volume and tested with a linear regression. Total molar concentration of pneumatocyst gases  were plotted against pneumatocyst volume. Internal pneumatocyst pressure and external hydrostatic pressure were plotted against pneumatocyst depth to determine if the pneumatocyst’s pressure differential (ΔP) changed with depth.   1.2.3 Calculating wall stress  Older pneumatocysts have two different geometries: a cylindrical hollow stipe and a spherical end. As mentioned previously, stresses are influenced by geometry and thus the cylinder portion of the pneumatocyst would experience a different magnitude of material stresses than the spherical end. A preliminary study was conducted to determine which portion of the pneumatocyst broke first. This was done by dropping 5 weighted thalli down to 50 m of seawater until they buckled, and revealed that the spherical end of pneumatocysts were at greatest risk of buckling. Thus, this study only focused on the average pneumatocyst wall stress accumulated in the spherical region of the pneumatocyst (eqn. 6). Eqn. 6 takes into consideration that the pneumatocyst is a thick-walled structure and therefore the outer portion of pneumatocyst wall experiences different loads than the inner portion of the pneumatocyst wall.  Average wall stress in a pneumatocyst at a specific depth was calculated using eqn. 6:  σ!"# = !!!!!-!!!!!!!!-!!!                                                                                  (6) Where σavg is the average circumferential stress in the pneumatocyst wall, Po is the 	 16	external hydrostatic pressure applied to the outer surface of the pneumatocyst wall, Pi is the internal pneumatocyst pressure applied to the inner surface of the pneumatocyst wall, ri is the internal pneumatocyst radius, and ro is the outer pneumatocyst radius. Positive values indicated compressive stresses. All radii and wall thicknesses were measured using calipers to the nearest 1 mm. A linear regression analysis was conducted to determine if material stress changed with pneumatocyst depth.   1.2.4 Compressive modulus   Material properties of pneumatocyst walls were tested at the University of British Columbia in Vancouver, British Columbia Canada. Samples (n=33) were analyzed using an Instron 5500R (Model # 1122, Instron Inc. MA, USA). Pneumatocysts were cut into circular doughnuts or cubes to measure the elastic modulus of the material. Modulus is a material property and independent of volume or shape. The Instron was equipped with an 8 X 8cm block at the base and a 9 X 0.8 cm cross beam atop (Fig. 6). Samples were glued onto the top cross beam and the base block using super glue (Fig. 6). The integrity of the adhesion during a test was verified with a high-speed camera (Casio exilim. EX-FH25).  Each sample was sprayed and hydrated with salt water before each test. Stress was measured by dividing force applied to the sample by its cross-sectional area. The movement (up or down) of the Instron crossbeam was used to measure strain. The modulus was calculated by dividing material stress with material strain (mm/mm) (i.e. slope of the Stress-Strain curve).  Modulus was measured by putting each sample in compression and tension for 4 cycles. Based on preliminary tests (see below) compressional modulus was measured and recorded via the 2nd cycle at 1% compression 	 17	(Fig. 6; Fig. 7). A linear regression analysis was conducted to determine if modulus changed with pneumatocyst volume.  Figure 6: Instron design for tension-compression tests. Top crossbeam is 9 cm width x 0.8 cm length and the base block is 8 cm width x 8 cm length. Pneumatocyst samples cut into doughnuts or cubes are glued to both the cross beam and the block. Black arrows indicate direction of tension and compression during data collection.  Three preliminary tests were conducted to establish a suitable test protocol. Initially, all samples were subjected to tension and compression through 4 cycles (n=12). The modulus was higher for the first cycle (Mullin’s Effect), but was constant for the 2nd, 3rd, and 4th cycle. Therefore, the 2nd cycle was used to measure modulus of pneumatocyst material.  Tension Compression KELP	9 cm Fixed Block 	 18	 Doughnut-shaped samples were exposed to 1, 3, 5, and 8% extension and compression (n=12). Compressive slopes were measured for each extension/compression series to determine if any of the extensions damaged the samples. All moduli appeared to be similar for every extension/compression and therefore the 1% extension method was used to measure modulus for all samples (Fig. 7).            Figure 7: Stress-Strain curves for one sample showing 4 cycles of compression and tension. The black bracket indicates where compressive modulus was measured (slope of the line) at 1% (0.01 mm/mm) compression during the 2nd cyclic cycle.   Modulus measurements were tested for pneumatocyst samples cut into doughnuts and cubes with heights from 14 mm to 5 mm and widths from 14 mm to 5 mm (n=5). This determined if there was a size limitation to accurately measure samples glued in the Instron. Modulus measurement accuracy decreased once the sample was less than  8X8 mm.  Therefore, all samples were cut to have a height (if doughnut) and width (if cubed) length no less than 8 mm.  (MPa) (mm/mm) TENSION COMPRESSION 	 19	1.2.5 Quantifying changes in pneumatocyst geometry Measured pneumatocyst inner radius (ri) and wall thickness (t) was used to determine the inner radius to wall thickness ratio (ri:t). Changes in average material stress with increased depth (up to 9 m) were estimated for ri:t ratios from 1:1 to 6:1 using eqn. 6. The optimal ri:t ratio was determined by examining differences in average wall stress, arbitrarily at 5 m for the 1:1, 3:1, and 6:1 estimated ratio. These three ratios represented the total range of ri:t a pneumatocyst would have. Calculated ri:t ratios of all experimental pneumatocysts (n=32) were plotted against pneumatocyst depth and tested with linear regression analyses.  A linear regression was also performed on ri:t ratios and average wall stress of small pneumatocysts (n=12) found at depths between 7 and 8.7 m. These data helped to determine the specific ri:t of a small deep pneumatocyst with the greatest calculated wall stress. Pneumatocyst inner radius (n=72) was plotted against depth. Average pneumatocyst wall stress was plotted against pneumatocyst inner radius (cm).  The pneumatocyst ri:t ratio was plotted with pneumatocyst wall thickness (n=64). Pneumatocyst inner radius was lastly plotted with pneumatocyst tissue volume. Pneumatocyst tissue volume was calculated by estimating the total pneumatocyst volume (tissue and gas space) of a sphere from the pneumatocyst’s measured outer radius (ro) minus the pneumatocyst gas space spherical volume from the pneumatocyst’s measured inner radius (ri). These plots were made to demonstrate how pneumatocyst size, geometry, and tissue volume changed as they developed and grew larger.     	 20	1.2.6 Calculating pneumatocyst environmental safety factor  Four whole thalli were collected and average wall stress was estimated using eqn. 6. Each thallus was then placed into a weighted basket with a SCUBA analogue depth gauge (Scubapro MODEL # 28.019.900) and dropped down to 50 m. A video camera (Go-Pro Hero 3+ silver edition) recorded the sound and depth (in ft.) when each pneumatocyst buckled, detected by an audible “pop.” This depth was converted to hydrostatic pressure (eqn. 4) and then average breaking stress was calculated using eqn. 6.  The structural integrity of a pneumatocyst was assessed by calculating the pneumatocyst’s environmental safety factor (ESF). Generally, ESF is the ratio between the maximum load an object can withstand before breaking and the load it experiences day to day. Thus, by definition, ESF describes the difference between a structure’s design and its working stress. A structure can only support its maximum design load and no more. Any additional load will cause the structure to fail.  For example, an ESF value of 1 indicates that a structure is just capable of tolerating loads it experiences day to day. As the ESF becomes larger that structural integrity of an object also becomes greater. The ESF of pneumatocysts were estimated by using eqn. 7. ESF =  !!"#$%!!                                                                                                     (7) Where σbreak is breaking stress of pneumatocyst wall and σ0 is wall stress of pneumatocyst located at depths between 2 m and 6 m.   An average of the 4 previous ESF estimates were used to determine at what depth 2 collected small, young pneumatocysts (ri=0.8 cm and 0.9 cm, t=0.2 cm) would break. 	 21	The estimated average ESF (3.9) was used to determine the pneumatocyst’s breaking stress with eqn. 7. σbreak was then used in eqn. 6 to estimate the hydrostatic pressure where the pneumatocyst would break. These values were compared to theoretical projections using eqn. 2, assuming the modulus was 3.9 MPa. Modulus was determined by estimating modulus of small pneumatocysts with the regression line in Fig. 13.  1.3 Results 1.3.1 Changes in pneumatocyst volume and internal pressure Pneumatocyst volume increased from 3 cm3 to 1200 cm3 as they grew from 9 m depth towards the surface (Fig. 8A). Small pneumatocysts between 3 cm3 and 50 cm3 were found at every depth and large pneumatocysts between 200 cm3 and 1200 cm3 were only found near the surface, above 2 m (Fig. 8A).  Internal pneumatocyst pressure did not significantly change with depth (Fig. 8B; P>0.05).  All internal pressures were less than atmospheric pressure (101 kPa), ranging from 3 to 100 kPa (Fig. 8B). Error bars indicate standard error of the manometer reading for a given sample. Manometer reading error was generally greatest when pneumatocysts were small or collected from great depth (Fig. 8B).                	 22	  Figure 8: Pneumatocyst volume at various depths (A). Pneumatocyst internal pressure at various depths.  Red dotted line indicates atmospheric pressure (P>0.05; B).  The confounding effects of pneumatocyst depth and volume could both affect internal pressure.  However, there was no significant difference between the mean pressure values of small (8-20 cm3) pneumatocysts found both at depth (>8 m) or near the surface (<2 m) (Fig. 9; t-test: P>0.1). The mean internal pressure for small pneumatocysts at the surface was 75±17 kPa and the mean internal pressure for small pneumatocysts at depth was 61±17 kPa (Fig. 9).                 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 0 200 400 600 800 1000 1200 Depth (m) Pneumatocyst volume (cm3) -9 -8 -7 -6 -5 -4 -3 -2 -1 0 0 20 40 60 80 100 Depth (m) Internal pneumatocyst pressure (kPa) 	 23	                       Figure 9: Average internal pressure for small (8-20 cm3) pneumatocysts, found shallow (<2 m) and at depth (>8 m). Error bars indicate standard deviation of internal pressure (Shallow ± 16.86 kPa; Deep ± 16.82 kPa)) for each pneumatocyst depth group (t = 1.03; df=13.6; P>0.1).   As pneumatocyst volume increased from a minimum of 3cm3 to a maximum of 1200 cm3, pneumatocyst gas increased from 1.5X10-6 mol cm-3  to 4.25X10-5 mol cm-3 (Fig. 10), and pneumatocyst pressure increased from a minimum of 3 kPa, reaching an asymptote around 80 kPa (Fig. 10).  Pneumatocysts greater than 200 cm3 had an internal pressure of 84±7 kPa (Fig. 10). Small pneumatocysts between 3 and 50 cm3, had variable internal pressures from 3 kPa to 100 kPa (Fig. 10). 6 of the small pneumatocysts found between 4 and 9 meters (Figs. 8A-8B) had the lowest internal pressures ranging from 3 to 40 kPa (Fig. 10).     0 10 20 30 40 50 60 70 80 90 100  Shallow   Deep Internal pneumatocyst pressure (kPa) Pneumatocyst depth 	 24	                  Figure 10: Internal pneumatocyst pressure and total pneumatocyst gas as a function of pneumatocyst volume.  Pneumatocyst internal pressure is less than external hydrostatic pressure at all depths (Fig. 11). Internal pneumatocyst pressure was generally 100 to 200 kPa less than external hydrostatic pressure below 4 meters (Fig. 11), and 1 to 50 kPa less above 3 m (Fig. 11).     	 25	                Figure 11: Blue data points: internal pneumatocyst pressure plotted with pneumatocyst depth (0-9m). Green data points: external hydrostatic pressure plotted with depth (0-9 m).     0 50 100 150 200−8−6−4−20 Pressure (kPa)Depth (m)−8−6−4−20Depth (m)	 26	1.3.2 Calculated wall stress  Pneumatocyst wall stress significantly increased with depth (Fig. 12; P<0.001, R2=0.69). Pneumatocyst wall stress was maximum 460 kPa at 9 m depth and minimum 102 kPa at the surface (Fig. 12).     Figure 12: Pneumatocyst wall stress at depths between (0-9 m).      100 150 200 250 300 350 400−8−6−4−20Wall stress (kPa)Depth (m)y	=	-0.03x+3.05	R2	=	0.69	P<0.001		 27	1.3.3 Pneumatocyst wall material properties The modulus of smaller pneumatocysts (3-50 cm3) varied from 0.9 to 10 MPa (Fig. 13). Modulus increased significantly (up to 12 MPa) as pneumatocyst volume increased from 8 cm3 to 1200 cm3 (Fig. 13; P<0.001, R2=0.28).            Figure 13: Young’s modulus for different pneumatocyst volumes.  1.3.4 Pneumatocyst geometry According to eqn. 6, wall stress at a given depth increases with increasing inner radius to wall thickness ratios (Fig. 14A). At 6 m of depth, the estimated wall stress increased by 30% when the inner radius to wall thickness ratio increased from 1:1 to 3:1, to 6:1(Fig. 14A). The pneumatocyst’s inner radius to wall thickness ratio varied between 1:1 and 6:1, and did not significantly change with depth (Fig. 14B; P>0.05).    y	=	0.01x	+	3.5	R²	=	0.28	P=0.002		 28	 Figure 14: Estimated wall stress at depths from 0-9 m for a given ri:t ratio (ri = inner radius, t= wall thickness; 1:1(blue), 3:1(green), and 6:1(red); A). Dashed line indicates estimates of wall stress for a given ri:t ratio at 6 m. ri:t ratio at depths between 0-9 m (P>0.05; B).   Linear regression analysis indicated that small pneumatocysts at depth (between  7 m and 8.7 m) significantly increased in wall stress (from 187 kPa to 371 kPa) as pneumatocyst geometry (ri:t) increased from 0.5 to 4.4 (Fig. 15; P<0.001, R2=0.67).        100 200 300 400 500−8−6−4−20Wall stress (kPa)Depth (m)0 1 2 3 4 5 6 7−8−6−4−20Geometry (ri : t)Depth (m)A B 	 29	                         Figure 15:  Pneumatocyst wall stresses for small pneumatocysts with different ri:t ratios. All pneumatocysts were collected between 7 and 8.7 meters.    Inner radius increased from 0 cm to 3.5 cm as pneumatocysts grew from 9 m to  0 m (Fig. 16A). Wall stress increases (from 150 to 400 kPa) when the internal radius was between 0 cm and 1 cm (Fig. 16B).  Wall stress decreased from 400 to 150 kPa when the internal radius increased from 1 cm to 2 cm (Fig. 16B). Wall stress remained invariant when the inner radius was greater than 2 cm (Fig. 16B). The ratio between inner radius and wall thickness increased from 0 to 6 as internal radius increased from 0 cm to 3.5 cm (Fig. 16C; P<0.001, R2=0.48).  Data collected with an inner radius below 1 cm and an ri:t value around 4 had the highest wall stress values (Figs. 16B-C).  Generally an inner radius of 0.8 cm to 0.9 cm and a wall thickness of 0.2 cm had the highest stress values (Fig. 16B; Fig. 16C circled portion).   As inner radius increased from 0 cm to 1 cm, the total pneumatocyst tissue volume did not greatly increase (average volume =  0 1 2 3 4 5200250300350Geometry (ri : t)Wall stress (kPa)y	=	27x+194	R2	=	0.67	P<0.001		 30	2.3 ± 1.7 cm3; Fig. 16D). Pneumatocyst tissue volume then gradually increased from  2.3 cm3 to 200 cm3 as the internal radius increased from 1 cm to 3.5 cm (Fig. 16D). Overall pneumatocyst tissue volume increased by a factor of 142 once the inner radius was greater than 1 cm (Fig. 16D).   Figure 16: Inner pneumatocyst radius at different depths (A). Grey data points are samples that began growing below 9 m and thus growth data is missing from the substratum to 9 m. These data were not included in analyses of pneumatocyst development. Pneumatocyst wall stress with an inner pneumatocyst radius (B). Black dotted line indicates overall trend in wall stress as inner radius increases. ri:t ratio with inner radius from (C). Black circle indicates pneumatocysts with the greatest wall stresses shown in part B. Inner pneumatocyst radius with pneumatocyst tissue volume (D).     0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5−8−6−4−20 Internal radius (cm)Depth (m)−8−6−4−20Depth (m)0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5100200300400Internal radius (cm)Wall stress (kPa)0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.502468Internal radius (cm) Geometry (ri:t)0 50 100 150 2000.01.02.03.0Tissue volume (cm3) Internal radius (cm)A B C D y	=	1.3x+1.4	R2	=	0.53	P<0.001		 31	1.3.5 Pneumatocyst safety factor Table 1 shows the calculated Environmental Safety Factor (ESF) of collected pneumatocysts using eqn. 7. Average ESF was 3.9±0.92 (Table 1). Table 2 shows the estimated critical breaking depth and breaking stress of 2 small pneumatocyst with critical geometry (ri=0.8-0.9 cm, h=0.2 cm) found between 7 m and 8 m.  These pneumatocyst were estimated to break at 21 m and 35 m with a breaking stress of 1263 and 1100 kPa respectively (Table 2).   Table 1: Table showing calculated ESF values for 4 pneumatocysts collected between 2.7 m and 6 m.          Table 2: Estimated critical breaking depth and breaking stress for 2 small pneumatocysts (with critical geometry) recorded to have the greatest wall stresses and the greatest ri:t found between 7 m and 8 m of seawater.  Collected Depth (m)  σ0 (kPa)  σbreak (kPa) Depth Break (m) ESF2.7 132.8 590.5 16.8 4.42.1 136.9 632.8 18.0 4.63.5 124.2 470.1 18.0 3.86 178.5 464.5 16.8 2.6Average: 3.9±0.92Pneumatocyst A BDepth (m) 7 7.9ri (cm) 0.9 0.8t (cm) 0.2 0.2Internal pressure (kPa) 86 91ESF 3.9 3.9σo (kPa) 324 282σbreak (kPa) 1263 1100Breaking depth (m) 37 35	 32	1.4. Discussion 1.4.1 Changes in pneumatocyst internal pressure and volume   Unlike a passive balloon, whose volume was expected to increase two-fold as it rose to the surface, pneumatocyst volume increases by a factor of 400 (Fig. 8A). Clearly, Nereocystis is expanding more than expected for a passive gas-filled structure, which suggests that the pneumatocyst is not passive but rather increases volume significantly through growth and development. In the absence of mitigating responses, pneumatocyst expansion due to decreasing internal pressure would thin, strain, and potentially weaken the wall. The pneumatocyst overcomes this obstacle, in part, by extending into the stipe and having a meristematic region in the tissue, which allows the wall to become thicker as volume increases (Nicholson 1970; Fig. 2B).  Manometer readings reveal that pneumatocysts consistently have an internal pressure less than atmospheric pressure (Fig. 8B). These results support previous studies showing that older pneumatocysts had internal pressures less than 101 kPa (Langdon 1917; Rigg and Swain 1941). Dromgoole (1981) argued that lower internal pneumatocyst pressures of Carpophyllum spp. and Macrocystis pyrifera were a result from shading at depth, slowing oxygen production from photosynthesis. But, if this were true, the proportion of O2 in the pneumatocyst would vary with depth. Results discussed in Ch.2 suggested that there was no significant change in O2 concentrations as pneumatocysts grew larger and therefore it is unlikely that smaller pneumatocysts have lower pressures due to reduced oxygen production (See Ch.2). Rigg and Swain (1941) suggested the low pressure found in adult pneumatocysts was caused by rapid development, where pneumatocyst volume increases at a rate faster than the gas being put into it. This study 	 33	suggests that pneumatocysts continue to have a pressure less than 101 kPa throughout development, supporting the ideas presented by Rigg and Swain (1941).   Since sporophytes start growth at various depths, there are two confounding variables that can affect internal pneumatocyst pressure: depth and volume.  To test the effect of depth and to control for the effect of size (volume), I compared small pneumatocysts growing at >8 m depth and <2 m near the surface. At a single size, depth did not have a significant effect on pressure, and thus it is likely internal pressure changes independently of changing depth (Fig. 9).  As sporophytes grow and pneumatocyst volume increases, internal pressure and the total amount of gas reach an asymptote (Fig. 10). Young pneumatocysts have variable internal pressures and total gas. Although the sample size for large pneumatocysts  (>200 cm3) is low, these data indicate that internal pressure becomes closer to 80 kPa as pneumatocysts grow in size. According to the Ideal Gas Law (shown in eqn. 5), gas molecules released into to the developing pneumatocyst would directly influence its internal pressure. Thus, as suggested by Rigg and Swain (1941), though gas is being added to the pneumatocyst as it becomes larger, the pressure is still below atmospheric pressure due to a rapid increase in volume. It is also possible that pressure is controlled and deliberately held at 80 kPa to ensure that the material stays in compression, as the force differential decreases during pneumatocyst development. If internal pressure is higher than outer ambient pressure, the pneumatocyst material would need to be capable of tolerating tension. Since the material is already built to tolerate compression, it would likely be less cumbersome to continue resisting compression rather than producing a material that tolerates 2 different types of loads.  	 34	In sum, these data suggest that pneumatocyst internal pressure is not influenced by depth as it grows towards the surface, but rather changes in volume directly affect the internal pressure. Therefore when a sporophyte begins its life at depth, internal pressure changes at a rate slower than its rapidly growing pneumatocyst. These results reveal that pneumatocysts are not passive but rather actively regulated, and biological processes such as photosynthesis, respiration, and gas composition attribute to lower pressures found in pneumatocysts (See Ch.2).  1.4.2 Pneumatocyst wall stress and the risk of buckling   Contrary to what we expected in the balloon model, the risk of breaking decreases as pneumatocysts move towards the surface. Because internal pneumatocyst pressure is less than external hydrostatic pressure, pneumatocysts are constantly exposed to a positive pressure gradient (ΔP; Fig. 11) and therefore always under compression. The difference in external hydrostatic pressure and internal pneumatocyst pressure becomes greater with depth (Fig. 11), resulting in younger, smaller pneumatocysts experiencing the greatest loads and greatest risk of buckling.   Furthermore, since the pressure gradient is positive, the pneumatocyst experiences compressive forces at every stage of development.  Thus under compressive forces, pneumatocysts can be at risk of buckling as the pressure gradient increases, making the average wall stress increase with depth (Fig. 12). The contribution of pneumatocyst geometry also does not prevent changes in material stress with depth (Fig. 14B), and thus pneumatocysts that are found at depth are at greatest risk of buckling. As mentioned previously, deeper pneumatocysts tend to be small and young, therefore sporophytes 	 35	reach a critical survival point early in their development, where success in reaching adulthood depends on either tolerating or reducing material stress.   1.4.3 Reducing wall stress Variation in overall geometry (ri:t)(Fig. 14B), less stiff materials (Fig. 13), and higher wall stress at depth (Fig. 12), suggests that young pneumatocysts could be at risk of buckling. Though the elastic modulus of seaweed tissues generally varies with species and habitat (Dromgoole 1981), previous studies have suggested that a large change in material modulus (stiffness) would be needed to resist breaking as a pneumatocyst increases volume (Charters et al. 1969; Delf 1932). Therefore, if Nereocystis pneumatocyst materials were adjusted to resist buckling, modulus would need to be greatest at depth to reduce strain or deformation from elevated wall stress. Contrary to this speculation, the pneumatocyst wall has a modulus that was lowest in young individuals and becomes stiffer as volume increases (Fig. 13). Since pneumatocyst material stress is greatest at depth, changes in the material’s modulus does not reduce wall stresses. Furthermore, if Nereocystis were adjusting pneumatocyst morphology to reduce stress, then ri:t would be smallest at depth where material wall stress and the risk of buckling is greatest. However, ri:t ratios of deep pneumatocysts vary from 1:1 to almost 6:1 and do not correlate with depth (Fig. 14B), which suggests that pneumatocysts do not adjust their geometry to reduce wall stress.     	 36	1.4.4 Pneumatocysts at risk of buckling   How then do pneumatocysts resist buckling? The present study demonstrates that ri:t increases during development (Fig. 16C). Dromgoole (1981) suggested that a decline in ri:t would potentially make older pneumatocysts more susceptible to buckling under hydrostatic pressure. However, data presented here argue against this idea for two reasons. First, pneumatocyst inner radius increases as thalli grow toward the surface and therefore larger pneumatocysts are not found at depth (Fig. 16A). This is because the stipe elongates, moving the developing pneumatocyst towards the surface. Second, pneumatocyst wall stress is generally decreases once the pneumatocyst reaches an inner radius greater than 1 cm (Fig. 16B). During stipe elongation, the pneumatocyst experiences decreasing hydrostatic pressure, contributing to the overall reduction of wall stress. This previously mentioned concept presented by Dromgoole (1981) could be applied to pneumatocysts that are ‘stationary’ and experience similar hydrostatic pressures throughout development. However, since the pneumatocysts of Nereocystis experience decreasing hydrostatic pressures as they develop, Dromgoole’s concept cannot be applied.  The critical buckling geometry during the life of a developing pneumatocyst is when the inner radius is around 0.8 cm - 0.9 cm and the wall thickness is around 0.2 cm (Figs. 16B-C).  This geometry is considered critical since the pneumatocyst has a ri:t around 4:1 and the greatest amount of material stress, about 300 kPa (Figs. 16B-C). These values reflect that the particular geometry of this pneumatocyst is not being utilized to reduce overall wall stress. Material stress tends to be greatest in small pneumatocysts with a ri:t ratio around 4:1(Fig. 15). This increase in material stress is due 	 37	to ri:t increasing beyond a geometry of 1:1 (Fig. 15; Fig. 16C). These changes in ri:t suggest inner radius increases and the pneumatocyst wall does not thicken (Figs. 16B-C). Therefore during this point in development, pneumatocyst tissue volume does not change as internal radius increases, and as a consequence, wall stress increases (Fig. 16B; Fig. 16D). A pneumatocyst generally reaches this critical size immediately after formation, when there is little to no change in the total pneumatocyst tissue volume. At this point, gas begins to fill the very small torn pneumatocyst space, separating the medullary tissue to form the pneumatocyst wall (Fig. 16D). These results indicate that pneumatocysts can experience critical breaking stresses early in development at depth. During pneumatocyst formation, individuals need be capable of tolerating these critical stresses since the force differential, material properties, and geometry do not aid in reducing stress. If the young pneumatocyst is not able to withstand these compressive forces, the structure will be compromised and it is unlikely that the sporophyte would reach adulthood.    1.4.5 At what depth will a small pneumatocyst break? Small pneumatocysts found at depth with the critical geometry is estimated to tolerate material stresses up to 35 m of seawater (Table 2). Theoretically, if a young pneumatocyst had no material imperfections or geometric discontinuities, the critical breaking pressure using eqn. 2 (and a modulus of 3.9 MPa; Fig. 13) would be between 31 m and 36 m. These estimates assume that the pneumatocyst material has a Poisson’s ratio of 0.3. Current estimates of buckling pressure calculated from eqn. 6 and eqn. 7 are reasonably close to values of critical buckling pressure from eqn. 2 (Table 2). Similarities between these theoretical projections and the actual stress calculations indicates eqn. 6 	 38	truly represents the structural design and compressive loads a young pneumatocyst would experience. Generally, pneumatocysts have an average safety factor of 3.9, suggesting that pneumatocysts are about 4 times stronger than they need to be (Table 1). We can estimate if a pneumatocyst at the critical moment in its life is at risk of buckling by calculating its breaking stress and depth. A young pneumatocyst with the hypothesized (mentioned previously) critical geometry of ri = 0.8-0.9 cm and t=0.2 cm, will experience 1100 to 1200 kPa of wall stress at the point of buckling (Table 2). These pneumatocysts are then estimated to buckle in about 35-37 m of seawater (Table 2). These predicted buckling depths are remarkably similar to the maximum observed depth of Nereocystis (Spaulding and Foster 2003). Spalding and Foster (2003) suggested the depth limitation of a growing sporophyte was due to light availability. The present study demonstrates that hydrostatic pressure, not just light attenuation, might also define the lower limit of Nereocystis in the field.   1.4.6 Conclusion  This study demonstrates how the pneumatocyst of Nereocystis is not passive like a balloon. As pneumatocysts move from depth towards the surface, volume increases 400 fold while internal pressure does not significantly change. However, the increase in pneumatocyst volume causes internal pressure to be less than ambient air pressure. Internal pressure is variable in younger pneumatocysts and begin to reach 80 kPa as they become adults. Therefore, internal pressure tends to be less than atmospheric pressure, possibly because the pneumatocyst develops at a rate faster than the gas being added. 	 39	 Pneumatocysts have an internal pressure less than the hydrostatic pressure they are exposed to. This results in a positive pressure gradient and therefore young pneumatocysts experience compressive forces throughout development. This positive pressure gradient increases with depth, which suggests that young, small pneumatocysts experience the greatest compressive loads, putting them at greatest risk of buckling.  Pneumatocyst material properties and geometry (ri:t) are not optimized to reduce wall stress and do not help reduce the risk of buckling. Furthermore, wall stress is greatest in young pneumatocysts that have an inner radius between 0.8-0.9 cm and wall thickness of 0.2 cm (ri:t ~ 4), revealing a critical size (directly after formation) where the pneumatocyst is at greatest risk of buckling. Pneumatocysts are approx. 4-times stronger than they need to be to resist buckling.  Theoretical and calculated estimates in this study indicate that small pneumatocysts will buckle at approximately between 31 m and 37 m depth. These data suggest that hydrostatic pressure, not just light limitation may contribute to the maximum depth of pneumatocysts.  In the future, critical buckling pressure should be measured on young pneumatocysts to verify predictions from this study.        			 40	Chapter	2 Developmental changes in gas composition and buoyancy of                           Nereocystis pneumatocysts  2.1 Introduction 2.1.1 Growth and development Nereocystis luetkeana (Nereocystis) provide marine habitats for an array of organisms in near shore environments (Nicholson 1970). This kelp creates dynamic subtidal forests (Arzee et al. 1985) that are upright and extend from 35 m deep towards the surface of the sea. Subtidal kelps use one of two strategies to remain upright in the water: Some species maintain an upright stature by having a tough, rigid stipe, and others, like Nereocystis have a flexible thallus with buoyant, gas-filled floats called a pneumatocysts. Buoyant pneumatocysts keep the flexible seaweeds vertical in the water.   At 10-35 m depth, young sporophytes start their life in an environment with little light (Spalding et al. 2003). During pneumatocyst development, medullary cells along the transition zone begin to tear, releasing gas and creating the pneumatocyst (Dromgoole 1981). Once the pneumatocyst has formed, the sporophyte begins to grow rapidly towards the surface (Duncan 1973). As thalli become larger, they also increase biomass and become heavier, having blades that can weigh up to 20 kg in air (Denny et al. 1997).  Sporophytes are able to grow towards the surface by adding gas to the developing pneumatocyst, which ultimately increases buoyancy (Kain 1987).   	 41	2.1.2 Pneumatocyst gas composition and production Gas composition and internal pressure of pneumatocysts were heavily studied in the early 20th century (Frye et al. 1915; Langdon 1917; Langdon and Gailey 1920; Zeller and Neikirk 1915.). These studies concluded that 20- 25% of gas sampled from pneumatocysts was oxygen (O2) and about 70% or more was nitrogen (N2) (Langdon 1917; Rigg and Swain 1941), with both gases present in concentrations similar to air. Up to 13% carbon monoxide (CO) (Langdon and Gailey 1920) and about 2-7% carbon dioxide (CO2) were also documented from pneumatocysts. 	 Pneumatocysts become thicker with age, making gas loss unlikely, and thus all gases produced throughout development are stored (Langdon 1917; Langdon and Gailey 1920). Pneumatocyst volume increases at a rate faster than gases are produced, resulting in an internal pressure that is less than hydrostatic pressure (negative pressure) (Frye et al. 1915; Rigg and Swain 1941; See Ch.1). Pneumatocysts are able to regulate the production of internal gas and therefore internal pressure (Brackenbury and Garbary 2006). As a result, the partial pressure of gases drives the overall internal pressure inside the pneumatocyst.   In macroalgae, nutrient uptake generally correlates with the surface area of the thallus per unit volume (surface area:volume ratio or SA:V) (Rosenberg & Ramus 1984; Taylor et al. 1998; Hein et al. 1995). Similarly, gas production may correlate with the SA:V ratio of cells that line the inner wall of the pneumatocyst. Throughout sporophyte development, pneumatocyst SA:V decreases as pneumatocysts become larger. In other words, the number of cells in one cubic centimeter decreases as pneumatocyst volume increases, since cells in the pneumatocyst begin to elongate (Duncan and Foreman 1980). 	 42	If the gases in the pneumatocyst are a product of surface area, then as pneumatocyst volume increases, the concentration (%) of biologically-produced gases will also decrease; this would result in a large pneumatocyst that is mostly filled with inert N2, with a gas composition similar to air (NOAA.com, USA).  Pneumatocyst gas composition has only been studied in adult sporophytes, and has never been adequately quantified throughout development. As mentioned in Ch.1, internal pneumatocyst pressure actually increases as pneumatocysts become larger. It is therefore possible that the total amount of gas increases as pneumatocysts become larger, but that the overall gas composition changes.  By conducting in-situ experiments, Rigg and Swain (1941) demonstrated that internal pressure of large pneumatocysts fluctuates, reaching a maximum around the late afternoon when pneumatocyst cells have been exposed to light for an extended period of time. Pressure reaches a minimum during the night when light is no longer available (Rigg and Swain 1941).  Though Rigg and Swain (1941) only measured the physiological gas composition of adult pneumatocysts (~1000 ml), these fluctuations could likely change in magnitude due to changes in SA:V as pneumatocysts develop. However, no one has ever measured changes in gas composition and pressure concurrently in pneumatocysts of various sizes. If the pressure depends upon activity of the pneumatocysts’ physiologically active cells, then the magnitude of pressure fluctuations should change as SA:V decreases with increased pneumatocyst volume.      	 43	2.1.3 Pneumatocyst buoyancy According to Archimedes principle, any object completely or partially immersed in a fluid is buoyed up by a force equal to the weight of the fluid displaced by the object. Since salt water is approximately 1000 times more dense than pneumatocyst gases, buoyant force can be generated by the pneumatocyst as it displaces seawater. As the pneumatocyst increases in volume, more water is displaced, creating an upward buoyant force that keeps the flexible thallus upright.   Nereocystis have a large thallus that is more dense than seawater, posing potential problems as the dense tissue becomes heavier with increased size. These heavy thalli can weigh up to 20 kg in air, creating a substantial negative force that acts against the pneumatocyst (Denny et al. 1997). The pneumatocyst can no longer support the flexible thallus if its positive buoyant force is less than or equal to the weight of the tissue in seawater. It is important that the increase in pneumatocyst volume paces or outpaces the increase in thallus mass as Nereocystis grows. If the pneumatocyst is unable to support the heavy thallus, the sporophyte is at risk of sinking. If pneumatocyst buoyancy is increased at a rate greater than the mass being added to the sporophyte, then it could potentially waste energy by producing excess gases. Ideally, the sporophyte should increase pneumatocyst buoyancy at a comparable rate to the increase in mass during growth.   2.1.4 Study objectives In this study, I measured total gas composition of Nereocystis pneumatocysts of varying sizes. This allowed me to determine if pneumatocyst gas composition changes 	 44	during pneumatocyst development and growth. Since pneumatocyst SA:V decreases as pneumatocysts become larger, I predicted that the total proportion of physiological gases also decreases as pneumatocysts develop, leaving a pneumatocyst that is mostly comprised of N2. Using pneumatocysts of different sizes, I repeated experiments conducted by Rigg and Swain (1941), and investigated the diurnal changes in internal pneumatocyst pressure and CO2 in-situ in order to determine how gas fluctuations are influenced by changes in SA:V during development.  It is well known that pneumatocysts are used to produce an upward buoyant force, keeping the thallus upright in the water. In this study, I determined whether or not the buoyant force of the pneumatocyst changes to compensate for the fast growing thallus. I investigated if buoyancy is maintained by calculating the buoyant safety factor (BSF) for different life stages of the sporophyte. I also investigated whether the pneumatocyst’s BSF is maintained or changes with increased thallus size.  2.2 Methods 2.2.1 Specimen collection  Nereocystis thalli (n = 31) of various sizes were collected in Bamfield, British Columbia Canada, at three locations: Scott’s Bay (48.834687, -125.147232), Aguilar Point (48.839456, -125.140896), and Helby Island (48.855442, -125.168718; Fig. 17).  Plants were collected no deeper than two meters below chart datum. All samples were detached from the substratum at the holdfast so that pneumatocysts were fully intact. All specimens were taken back to the Bamfield Marine Sciences Centre for further evaluation. After experimentation, pneumatocyst volume was measured by carving a hole 	 45	in the pneumatocyst, filling the inside with water and pouring it into a graduated cylinder. An average of 3 measurements was used to determine volume. Pneumatocyst length and width was measured using a measuring tape (mm). Pneumatocyst wall thickness was measured using calipers (mm). All statistical analyses were conducted using R version 0.99.467 (R Core Development Team).  A                                                                            B Figure 17: Map of southern Vancouver Island, British Columbia Canada. The yellow circle indicates location of Bamfield (A). Map of field sites where Nereocystis was collected (B) (provided by Google Maps).                                                                         2.2.2 Internal gas composition 12 ml of gas was extracted with a 26 gauge (0.45 mm) needle and syringe from each pneumatocyst and stored in an Exetainer (Labco Limited 12ml, Lampeter UK). All Exetainers were vacuumed sealed and contained a septum to allow gas to be stored without air contamination. Exetainers were punctured no more than 3 times to avoid air contamination (Glatzel and Well 2008). Each Exetainer was stored upside down in distilled water to further prevent air contamination (Sturm et al. 2015).  Samples were cooled to 5°C during transit and in the lab prior to gas analyses.  Aguilar Point Scott’s Bay Helby Island 	 46	CO2 and CO concentrations in gas samples extracted from pneumatocysts were analyzed at room temperature (in ppm) using a Q-trak indoor air quality monitor 7565 (TSI Inc. MN USA).  Q-trak sensors were calibrated to 50 ppm of CO and 447 ppm of CO2 (CO2 in air) using a plastic case (calibration wand) that covered the sensors on the Q-Trak wand (Fig. 18). The calibration wand was also used in gas analyses by attaching rubber tubing at the end of the wand injecting 0.5 ml pneumatocyst samples through the tubing. CO2 and CO was electronically read after the gas sensor ran for 45 seconds, recording a peak concentration.  N2 was used to flush out any excess air in the sensor that would alter measurements.           Figure 18: Diagram of Q trak sensor. CO2 and CO concentrations in the pneumatocyst were calculated using the equation above. Where C1 = the concentration read on the monitor (ppm), V1 = volume of the calibration wand (ml), C2= unknown pneumatocyst concentration (ppm), and V2= 0.5 ml sample.   Neofox probes (Ocean Optics Inc. FL USA) were used to measure O2 concentrations in gas samples extracted from pneumatocysts (in %) diluted in a 50 ml sealed flask. Before each measurement, the flask was flushed with 100% N2 to evacuate 50	ml	0.5	ml	sample	Monitor	Calibra3on	Wand	C1V1=C2V2		 47	any residual air.  The flask had two openings, one of which was sealed using tape after the N2 flush, while the other was sealed by placing the Neofox probe into the flask, using tape to place a final seal around both openings.  2 ml of gas was extracted from the Exetainers using a 26 gauge (0.45 mm) needle and was placed into the flask with the probe. Each sample was mixed with the residual N2 using a magnetic stir bar during the reading. Data was recorded by measuring the peak concentration of O2 as the gas sample mixed with residual N2. Total gas concentration (ppm) of each sample was calculated using the following equation:                C! = !!!!!!                                                                                                           (8) Where C1 = the measured gas concentration (ppm), V1 = volume of the flask (50 ml), C2= unknown pneumatocyst concentration (ppm), and V2=  2 ml gas sample (ml). The same equation was used to calculate total CO and CO2 concentration in the pneumatocyst where C1= concentration measured from the 0.5 ml sample.  Gas concentrations (ppm and %) were plotted against pneumatocyst volumes and fitted with linear regressions. Log-transformed total gas (mmol) were plotted against pneumatocysts volume, and significant correlations were tested using linear regressions.   2.2.3 Pressure and CO2 fluctuations   Nereocystis (n = 12) were collected from Scott’s Bay from July 8 - 11, 2015 and stored in outdoor seawater bins with continuous flowing seawater between 11 and 13°C. Nine pressure manometers (diameter: 4-8 mm; length: 20-60 cm; Fig. 19) were placed vertically at a 90 degree angle on each bin. Experimental bins were side-by-side and exposed to direct sun with limited potential shading over the course of the day.  	 48	             For each round of experiments, pneumatocyst sizes varied from 5 to 400 ml. All samples were prepared by tying twine to the stipe and gluing it to the lip of the tank. This allowed for constant water flow without samples moving in the tank. Rocks were attached to kelp holdfasts to maintain the vertical posture they exhibit in their natural environment. The vertical posture also limited the effect of shading, allowing the full photosynthetic area to be exposed. Pneumatocysts were punctured with a 21 gauge  (0.7 mm) needle and equalized to atmospheric pressure so that the changes in pressure and CO2 would only reflect gas production and utilization of pneumatocyst cells. Water manometers were attached to pneumatocysts using plastic tubing, syringes, and needles (Fig. 19).          Figure 19: Diagram of water manometer. Pneumatocysts were punctured using a lubricated syringe needle. Gauge pressure was calculated by the equation above where ρ = density of water 1025 kgm-3,  g= 9.81 ms-1, h = height of water moved in both arms post puncture, and PATM = atmospheric pressure.               h 	 49	 Pneumatocysts were towel-dried before puncturing and the inserted needle was sealed with super glue and Vaseline. Initial pressure readings were recorded an hour after the pneumatocysts were punctured. Internal pneumatocyst pressure was then calculated by using the dimensions of the manometer and the Ideal Gas Law to derive eqn. 9:  P!"#!$V!"!#$ = !RT  (Ideal Gas Law) = !!" + !!" RT = P!"V!"RT + P!"V!"RT RT =  P!"V!" + P!"V!"                                                          P!" = !!"#!$!!"!#$ ! !!"!!"!!"                                          (9) Where V!"!#$ =  V!" +V!" where PPn is the unknown pneumatocyst pressure, Pgauge is the pressure reading from the  manometer, Vmf is  the total volume of the manometer from the water line to the needle after the pneumatocyst was punctured, Pmi is the pressure of the ambient air in the manometer, Vmi is the volume of the manometer arm before the pneumatocyst is punctured, and PPn is the volume of the pneumatocyst.  Every 4 hours, starting at 21:30, pressure (Ptotal) was measured using the manometer and a 1mL gas sample was extracted using a 21 gauge (0.7 mm) needle and syringe. Each time the pneumatocyst was punctured for a gas sample, the punctured area was sealed using Vaseline. CO2 concentration was measured in the gas sample by injecting the gas into plastic aquarium tubing (diameter: 4mm; length 10 cm) attached to an A S157 CO2 Analyzer (Qubit Systems Research, ON Canada), calibrated up to  2000 ppm. Six paired pressure and gas samples were taken for each pneumatocysts over 	 50	24 hours. Light intensity and water temperature were recorded at each sampling time using a LI-250A light meter (LI-COR Inc. NB, USA) and aquarium thermometers.  SA:V was estimated for pneumatocysts of various sizes by using previously collected volumes and inner radii of the spherical top (see Ch.1). These estimates were then plotted with previously collected pneumatocyst volumes and log transformed. A linear regression analysis was then performed with the transformed data. The projected linear equation suggested by the regression model was then used to further estimate the SA:V of the pneumatocysts used in this experiment.  Recorded amplitudes of the minimum and maximum concentrations of CO2 was plotted with estimated SA:V to determine if SA:V influences diurnal pressure and gas fluctuations.  An average and standard deviation across all 12 samples was also calculated for both internal pressure and CO2 during each sampling time. Light intensity (μMol ms-1) was plotted with time (24 hour clock starting from 21:30). Both internal pneumatocyst pressure and CO2 concentration were plotted with time (24 hour clock starting from 21:30).  2.2.4 Buoyant force Nereocystis thalli (n = 26) were collected from Scott’s Bay and transported to the Bamfield Marine Sciences Centre. Stipe and blade densities were calculated by dropping tissue fragments (n= 26) of known mass into a graduated cylinder to measure volume as water displacement, and then by dividing mass by volume. Thallus mass was measured by hand-drying all blades and stipe and weighing them on a 100 kg scale. Thallus volume was calculated by dividing tissue mass by density. Thallus weight in water was then calculated using the following equation: 	 51	Wth = Vg(ρkelp – ρwater)                                                                                       (10) Where Wth is weight of the thallus in water (N), V is the volume of plant (m3), g is the gravity (9.81ms-1), ρkelp is the density of kelp (kgm-3), and ρwater is the density of seawater (1025 kgm-3).    Net buoyancy was measured as the upward force exerted by the whole kelp.  Force was measured with a single beam 5 kg force transducer (model #FORT5000, World Precision Instruments Inc. FL, USA) mounted to a basket (Fig. 20).               Figure 20: Diagram of contraption designed to measure net buoyant force of the intact thallus. Whole kelp is placed in basket and applies an upward force that is detected by a force transducer.   Measurements were recorded using Lab View/Signal Express. Total buoyant force of was then calculated using the following equation:  Force TransducerDirection of Force AppliedKelp Submerged in Water	 52	            Bforce =  Bnet +  Wth                                                                                              (11) where Bnet is the net buoyancy (N), Bforce is the total buoyant force (N), and  Wth is the weight of thallus (N). Log-transformed weight of thallus in seawater, log-transformed net buoyancy, and log-transformed total buoyant force were plotted against pneumatocyst volumes, and significant correlations were tested using linear regressions.   2.2.5 Buoyant safety factor   Calculating an environmental safety factor (in this context, a buoyant safety factor, BSF) is a method to understand if the pneumatocyst is at risk of sinking at any point during sporophyte development. Generally, environmental safety factor is a ratio of the maximum force a structure can withstand to the force it experiences naturally (Johnson and Koehl 1994; Stewart 2006). A pneumatocyst that has a total buoyant force slightly greater than the weight of the thallus, would have a buoyant safety factor (BSF) slightly greater or equal to one. Any more tissue weight added (i.e. blades), results in the pneumatocysts’ total buoyant force equaling the thallus weight, making the sporophyte sink or become neutrally buoyant.  The same 26 Nereocystis samples used to measure buoyant force were also used to calculate Buoyant safety factor (BSF).  Buoyant safety factor (BSF) was calculated as the ratio between buoyant force and weight of thallus, such that               BSF =  !!"#$%!!"                                                                     (12)   Where Bforce is the total buoyant force (N) which equals the maximum thallus weight (N) when the pneumatocyst is neutrally buoyant, and Wth is the actual weight of the thallus in seawater (N).  	 53	 5 additional thalli with pneumatocyst volumes between 800 and 1000ml were collected around docks at the Bamfield Marine Sciences Centre.  Thallus mass and weight in seawater was measured as mentioned above for all samples. Thallus volume was calculated by dividing tissue mass by density. Maximum weight that pneumatocysts could support before sinking (Wmax) was calculated by attaching 500 g or 200 g analytical weights on the blades with sewing twine, until the pneumatocyst obtained neutral buoyancy.  BSF was calculated using the following equation:   ESF = !!"#!!                                                                                                        (13) Where, W0 is the actual weight of the thallus in seawater (N), and WMax is the added weight (N) attached to the blades and the thallus weight to obtain neutral buoyancy. Log-transformed BSF for both the original 26 samples and the additional large 5 thalli were plotted against log-transformed pneumatocyst volume, and significant correlations were tested using a linear regression.  2.3 Results 2.3.1 Pneumatocyst gas composition           Relative gas composition did not change significantly with pneumatocyst volumes from 5 to 1000 ml  (P>0.05; Figs. 21A-D).  Average % concentration for CO, CO2, O2, and estimated N2 were 1.2 ± 0.78%, 0.6 ± 0.15%, 60 ± 17.2%, and 40 ± 13.7%, respectively. Average concentration of CO, CO2, O2, and estimated N2 were 12000, 6000, 590000, and 390000 ppm, respectively (Figs. 21A-D).      	 54	    Figure 21: Concentrations of CO, CO2, O2, and estimated N2 for different pneumatocyst volumes (P>0.05). Dotted lines indicate average gas concentration.               However, as pneumatocyst volume increased from 10 ml to 1000 ml, the amount of CO increased significantly from 0.002 mmol to 0.55 mmol  (P<0.001, F=61.93, R2 = 0.68; Fig. 22A); CO2 increased significantly from 0.003 mmol to 0.19 mmol (P <0.001, F=434.7, R2 = 0.94; Fig. 22B); O2 increased significantly from 0.29 mmol to 17.2 mmol  (P<0.001, F-stat=75.8, R2 = 0.96; Fig. 22C), and N2 was estimated to increased significantly from 0.11 mmol to 16.2 mmol (P<0.001, F-stat=277.5, R2 = 0.91;  Fig. 22D).    A B C D 	 55	  Figure 22:  Log total gas for different pneumatocyst volumes (10-1000ml).              The concentration of CO in Nereocystis pneumatocysts was approximately 48,000 times greater than in air (Fig. 23; Table 3). The concentration of CO2 in pneumatocysts was 15 times greater than in air (Fig. 23; Table 3).  The concentration of O2 was 2.8 times greater than in air (Fig. 23; Table 3).  The concentration of N2 was half as much as that in air (Fig. 23; Table 3).       		y	=	0.94x	-	1.5	R²	=	0.96	P<0.001		 y	=	1.1x	-	2.1	R²	=	0.92	P<0.001		y	=	0.91x	-	3.2	R²	=	0.68	P<0.001		 y	=	0.93x	-	3.5	R²	=	0.94	P<0.001		A B C D 	 56	 Figure 23: Average concentrations of gases in air compared to average concentrations of gases in a pneumatocyst.  Pneumatocyst gas concentrations were compared to atmospheric gas concentrations by using information available online from the National Oceanic and Atmospheric Administration (NOAA.gov, USA).           Table 3: Table showing average gas concentrations of air and a pneumatocyst. Standard deviation of gas concentrations in a pneumatocyst is expressed in. Pneumatocyst gas concentrations were compared to atmospheric gas concentrations by using information available online from the National Oceanic and Atmospheric Administration (NOAA.gov, USA).        CO  1.2%   CO2  0.6%  O2  60%  N2  40%  Pneumatocyst CO  0.000025% CO2  0.04% O2  21% N2  79% Air 	 57	2.3.2 Pressure and CO2 fluctuations          The SA:V decreases from 2.5 to 0.9 as pneumatocyst volume increases from 19 ml to 380 ml (R2 = 0.83; Fig. 24A) By using the line fitted in Fig. 24A, pneumatocysts in the pressure experiment were estimated to have a SA:V between 1.2 and 2.4 as pneumatocysts increased in volume from 19 ml to 300 ml (Fig. 24B). The amplitude, or difference between the highest concentration of CO2 and the lowest concentration of CO2 (per sample) did not significantly change with increased pneumatocyst volume (P>0.05; Fig. 24B). Throughout the experiment, CO2 diurnally fluctuated 78.1 ± 13 ppm (Fig. 24B).    Figure 24:  Log inner surface area to volume ratio (SA:V) for various pneumatocyst volumes (A). The total change or fluctuation of CO2 for experimental pneumatocysts of various pneumatocyst volumes (B; P>0.05). SA:V is estimated with the regression model in part A. Grey dotted line indicates the average fluctuation of CO2.              Light intensity increased throughout the day from 0 to 500 μmol m-2 s-1 (Fig. 25A).  Internal pneumatocyst pressure increased with increasing light exposure from 100.7 to 101.2 kPa (between 5:30 and 13:30) and then decreased from 101.2 to 100.8 kPa when light intensity decreased from 500 μmol m-2 s-1 to 0 μmol m-2 s-1 (between 13:30 and y	=	-0.3x	+	0.78	R²	=	0.83	P<0.001		A B 	 58	21:30; Figs. 25A-B). Internal pneumatocyst pressure was lowest (100.4 kPa) at 1:30 when exposed to darkness for 4 hours (21:30 to 1:30; Figs. 25A-B). As internal pressure increased from 100.7 to 101.2 kPa, concentrations of CO2 decreased from 75 ppm to 5 ppm (Figs. 25B-C). Concentrations of CO2  were greatest at 5:30 (75 ppm) when the pneumatocyst was exposed to darkness from 21:30 to 5:30 (Fig. 25C).                   Figure 25: Changes of light (A), internal pneumatocyst pressure (B), and CO2 concentration (C) during a 24 hour time period starting at 21:30. All data points represent average values. Error bars indicate standard deviation.  100.0 100.4 100.8 101.2 101.6 -5 0 5 10 15 20 Pneumatocyst pressure(kPa) 0 20 40 60 80 100 120 -5 0 5 10 15 20 CO2 (ppm) Time (24hr clock) 0 100 200 300 400 500 -5 0 5 10 15 20 Light intensity (µmol m-2 s-1) 20 20 20 DAY NIGHT A B C 	 59	2.3.3 Pneumatocyst buoyancy             Thallus tissue density was 1090 ± 0.1 kgm-3. Thallus weight increased from  0.003 N to 1.65 N (0.01 to 3.01 kg) as thalli grew and pneumatocyst volume increased from 5 ml to 207 ml (P<0.001, F=16.63, R2 = 0.32; Fig. 26A). Net buoyancy decreased from 2.21 N to 0.27 N with increasing thallus size (P<0.001, F=20.8, R2 = 0.38; Fig. 26B).  Therefore, total buoyant force did not change significantly with increasing thallus size (P>0.05; Fig. 26C), averaging 1.36 ± 0.5 N at all size classes.       Figure 26: Log thallus weight (downward force, N) as a function of pneumatocyst volume (A). Log net buoyancy (upward force measured in Fig II.4, N) as a function of pneumatocyst volume (B). Log total buoyant force (upward force, N) as a function of pneumatocyst volume. Dotted line indicates average total buoyant force (C; P>0.05).  0 50 100 150 200−2.0−1.5−1.0−0.50.0Pneumatocyst volume (ml)Log(Thallus weight N)0 50 100 150 200−0.6−0.4−0.20.00.20.4Pneumatocyst volume (ml)Log(Net buoyancy N)0 50 100 150 200−0.10.00.10.20.3Pneumatocyst volume (ml)Log(Total buoyant force N)y	=	0.007x	-	1.7	R²	=	0.32	P<0.001			y	=	-0.003x	+	0.2	R²	=	0.38	P<0.001		A BC 	 60	          Buoyant safety factor (BSF) was estimated to decrease from 432 to 1.6 with an increasing thallus size from 5 ml to 207 ml (Fig. 27). Average BSF of large individuals was 2.18±1 (Table 4). Weighted down thalli had measured BSF values (grey data points) consistent with calculated BSF values (P<0.001, F=22.56, R2 = 0.41; Fig. 27).  According to the linear regression, growing thalli are estimated to sink when pneumatocyst volume exceeds 1.3 L (Fig. 27).                    Figure 27: Log buoyant safety factor (BSF) as a function of Log pneumatocyst volume.  The last 5 data points are represented in table II.2.        Table 4: Table showing buoyant safety factor (BSF) calculated from 5 large pneumatocysts. Average BSF is 2.18±1.3. 0.0 0.5 1.0 1.5 2.0 2.5 3.00.00.51.01.52.02.5Log(Pneumatocyst volume ml)Log(BSF)0.00.51.01.52.02.5Log(BSF)Grey	data	points	are	from	Table	II.2	y	=	-1.05x	+	3.1	R²	=	0.41	P<0.001			 61	2.4 Discussion 2.4.1 Pneumatocyst gas composition Contrary to expectations, as pneumatocysts of Nereocystis develop and become larger in volume, overall gas composition does not change significantly (Fig. 21). The same relative proportions of gases are added/subtracted to pneumatocysts as they grow larger, and therefore the overall amount of gas increases with an increase in pneumatocyst volume (Fig. 22). Through in-situ lab trials, Foreman (1976) showed that the total amount of CO increases almost proportionally to increased wall thickness. This result could be extrapolated to all pneumatocyst gases since it is well known that the pneumatocyst wall becomes thicker with increased volume (Nicolson 1970) and regression lines fitted in Fig. 22 shows an almost proportional increase in mmol of CO, CO2, O2, and estimated N2  per 1 ml of gas. This suggests that the rate of input and output of gases could be the same, and thus the same relative proportion of gases being added at all stages of development. Pneumatocysts that are larger and have been increasing volume for a longer period of time have a greater amount of CO than younger, smaller pneumatocysts. A larger amount of CO present in older pneumatocysts suggests that CO is produced during all developmental stages.  The most recent study examining the presence of CO in Nereocystis suggested that CO production is a product of cell degradation/tearing (autolysis) from the rapidly expanding pneumatocyst (Foreman 1976). Foreman (1976) argued his study provides evidence towards this hypothesis because the input of CO had slowed or subsided when the pneumatocyst stopped increasing volume (Foreman 1976), as stipe growth slowed near the water’s surface (Duncan 1973). Therefore individuals 	 62	sampled once stipe growth has slowed could have lower CO concentrations than individuals undergoing stipe elongation (Foreman 1976). This study supports these findings since Fig. 22 depicts an increase in the total amount of CO (mmol) with increased size.  Earth’s	atmosphere	contains	only	a	small	amount	of	CO	(~0.000025%)	whereas	pneumatocysts	contain	an	average	concentration	of	1.6%	(Fig.	23).	A	study	conducted	by	Landgon	(1917)	determined	whether	or	not	the	concentration	of	CO	was	at	a	toxic	level	by	exposing	pneumatocyst	gases	to	animals	and	measuring	their	physiological	effects.	Subsequently,	the	statement	familiar	to	most	phycologists,	that	the	pneumatocysts	of	Nereocystis	have	enough	CO	“to	kill	a	chicken”	was	a	product	of	Langdon	(1917).	Without	harming	any	animals,	data	collected	during	this	study	can	further	support	this	statement.	1.6%	CO	is	a	potentially	toxic	amount	given	that	concentrations	of	CO	greater	than	100	ppm	(0.01%)	could	kill	or	render	a	person	unconscious	(Suner	et	al.	2008).		Given	that	an	average	adult	male	has	a	lung	capacity	of	5800	ml	and	the	largest	recorded	pneumatocyst	in	this	study	(725	ml)	had	a	CO	concentration	of	1.6%,	if	an	average	sized	man	inhaled	the	gas	inside	the	largest	sampled	pneumatocyst,	then	in	one	breath	he	would	ingest	1500	ppm	of	CO,	15-times	greater	than	the	maximum	concentration	a	person	could	tolerate	before	passing	out.																																													 The	CO2	concentration	in	the	pneumatocyst	is	generally	about	0.6%	(Fig.	21).	The	concentrations	of	CO2	measured	were	similar	to	previous	studies,	which	stated	that	about	0.5	to	1.7%	of	CO2	was	found	in	the	pneumatocyst	(Rigg	and	Swain	1941;	Langdon	and	Galley	1920).		Previous	studies	have	found	that	pneumatocysts	contain		 63	approximately	21-25%	O2	(Langdon	and	Galley	1920;	Rigg	and	Swain	1941).	Contrary	to	earlier	studies,	this	study	found	that	there	is	an	average	of	60%	O2	in	pneumatocysts	(Table	3).	Differences	in	the	methodology	of	collecting	and	storing	gas	samples	between	this	current	study	and	Rigg	and	Swain	(1941)	could	attribute	to	the	discrepancies	between	the	measured	concentrations	of	O2.	Rigg	and	Swain	(1941)	extracted	and	stored	pneumatocyst	gas	in	wax	sealed	syringes	prior	to	analyses,	which	may	have	been	prone	to	leaking.	The	septum-sealed	Exetainers	used	to	store	gases	in	this	study	have	been	shown	to	minimize	air	contamination	and	gas	leakage	up	to	5	weeks	after	the	sample	is	deposited	(Glatzel	and	Well	2008).		In	this	particular	study,	all	gases	were	analyzed	with	in	3	days	of	being	extracted	from	the	pneumatocyst.	Therefore,	I	believe	using	the	Exetainers	used	in	this	study	could	have	yielded	more	accurate	O2	storage	and	measurements.		Estimated	concentrations	of	N2	in	the	pneumatocyst	averaged	about	40%	(Table	3).		These	estimates	were	generally	lower	than	previous	studies	which	suggested	that	78	to	80%	of	the	pneumatocyst	was	comprised	of	N2	and	similar	to	the	concentration	in	air	(Langdon	1917;	Langdon	and	Galley	1920).		Though	N2	was	only	estimated	in	this	particular	study,	higher	measured	concentrations	of	O2	yielded	these	estimations	to	be	less	than	previously	measure	concentrations.	N2	is	an	inert	gas	and	does	not	play	an	integral	role	in	cellular	processes	such	as	photosynthesis	and	respiration.	If	gas	composition	changed	as	a	function	of	SA:V,	larger	pneumatocysts	would	have	relative	gas	concentrations	similar	to	air.	These	estimations	for	relative	concentrations	of	N2	further	suggest	that	gas	stored	in	the		 64	pneumatocyst	is	not	directly	influenced	by	a	decrease	in	SA:V	as	pneumatocysts	grow	larger.		 2.4.2 Pressure and CO2 fluctuations  Though Rigg and Swain (1941) demonstrated that internal pressure of adult pneumatocysts fluctuates as a response to changes in pneumatocyst gas composition, this study suggests that the magnitude of these fluctuations do not significantly change during pneumatocyst development. This experiment therefore demonstrates that internal pressure and CO2  fluctuate similarly across all pneumatocyst sizes. Contrary to expectations, the decrease in surface area to volume ratio (SA:V) as pneumatocysts become larger does not influence pressure or gas fluctuations (Fig. 24). It is possible that cells in smaller pneumatocysts do not utilize the total pneumatocyst surface area to produce gases and cells in larger pneumatocysts over use their allocated surface area.  Calculations used to estimate SA:V generally represent the true SA:V of a pneumatocyst.  Pneumatocysts have an odd geometry, making it difficult to accurately calculate surface area using generic equations formatted for specific geometric shapes, such as a sphere. Pneumatocysts change geometry as they increase volume. Generally, younger, smaller pneumatocysts are spherical until they reach a volume greater than 100 ml. At this point in development, the pneumatocyst extends into the stipe creating a hollowed cylinder to further hold gas and increase total volume. SA:V in the spherical apex of the pneumatocyst shown in Fig. 24A will increase by a factor of 3 divided by the sphere’s inner radius. The changes in SA:V of the added cylinder is not much different since SA:V will increase by a factor of 2 divided by the cylinder’s inner radius. 	 65	Furthermore, changes in SA:V are independent of cylinder height since radius is increasing at a rate much greater than the stipe being hollowed.  This in-situ experiment showed that internal pressure decreases at night, as the concentration of CO2 increases accordingly, and internal pressure increases throughout the day, as the concentration of CO2  declines (Fig. 25). These findings further support the claim made by Rigg and Swain (1941) that the concentration of CO2 is less when it is utilized to produce O2 via photosynthesis during the day, and the concentration of CO2 is high when O2 is utilized via cellular respiration during the night, leaving additional CO2 as a byproduct.  Thus, the levels of CO2 measured in this experiment are reflecting the changes in O2 diurnally due to photosynthesis and respiration. Furthermore, since this study also suggests that O2 takes up more than half of the total gas present in the pneumatocyst, the utilization of O2 during cellular respiration at night is more likely to influence the decrease in overall pressure of the pneumatocyst than CO2 being added.   2.4.3 Pneumatocyst buoyancy  Surprisingly, the buoyant force from the pneumatocyst is eventually outpaced by the weight of growing thalli, and the risk of sinking increases. As individuals become larger with increased pneumatocyst volume, the overall weight of thalli also increases (Fig. 26). When the sporophytes grow and develop, their stipes begin to elongate, increasing light availability for their blades (Duncan 1976).  This growth period inevitably results in heavier thalli with blades that can weigh up to 20 kg in air, creating a downward force that acts against the buoyant pneumatocyst (Denny et al. 1997). The total buoyant force does not significantly change with thallus size and pneumatocyst 	 66	volume (Fig. 26C), suggesting that increased pneumatocyst volume and buoyancy barely offsets the weight of the thalli.  Although it is expected that a larger pneumatocyst volume would result in a larger buoyant force to offset the increased blade weight, the pneumatocyst material becomes thicker and the stipe becomes longer. Thus, as pneumatocysts increase in volume there is more thallus tissue, creating a greater mass that prevents the pneumatocyst from pushing upwards as much as might be expected. With increased thallus weight and no change in total buoyant force, the overall net buoyancy decreases as the pneumatocyst grows larger in volume (Fig. 26B).  Buoyant safety factor (BSF) decreases as thalli become heaver (Fig. 27). Alexander (1988) considered that the optimal safety factor for a biological structure could be estimated by analyzing the probability of failure (in this case, sinking), cost of structural maintenance, and the cost of structural failure. In the case of the pneumatocyst on Nereocystis, cost of structural failure is high in sporophytes since there is only one pneumatocyst and it cannot be regenerated.  Without pneumatocysts, thalli could not remain upright, and would be left growing near the substrate, near hungry herbivores, and in low light, ultimately decreasing fitness (Chenelot and Konar 2007). During the early stages of Nereocystis sporophytes, pneumatocysts have a high BSF (Fig. 27). A high safety factor suggests that the probability of pneumatocysts sinking is low. In this case, there would also be an associated cost of maintenance in having a pneumatocyst buoyant force much greater than the weight it is trying to support.                                         On the contrary, the calculated BSF for older, reproductive sporophytes is low. To maintain buoyancy, the pneumatocyst would have to increase buoyant force as the thallus grows larger and becomes heavy. Taking into consideration the annual life history of 	 67	Nereocystis, the cost of failure in the pneumatocyst is low once the sporophyte has produced reproductive sori during late summer. Nereocystis stipes also have an environmental safety factor that decreases with age (Johnson and Koehl 1994). The cost of breakage is higher in younger thalli and lower in older thalli after sori production (Johnson and Koehl 1994). Both safety factors suggest a reduction in mechanical maintenance of large sporophytes since the thalli will most likely be dislodged and no longer viable during early winter storms.                                                                                                                               The BSF is generally low for the five largest recorded thalli (Fig. 27; Table 4).  Table 4 shows the calculated BSF values for these samples, revealing that all except one sample have a BSF less than 2. Pneumatocyst size is correlated with age and can be used as a proxy to roughly estimate total thallus size (weight) (Duncan 1973; Nicolson 1970).  Therefore, the largest possible sporophyte that can remain upright (BSF=1) would have a pneumatocyst that is roughly 1.3 L (Fig. 27). If this sporophyte were to add any more blade weight, the 1.3 L pneumatocyst would no longer be able to keep the thallus upright, and the thallus would sink. The largest collected pneumatocyst in this study was roughly 1.2 liters (see Ch.1), just 100 ml less than the aforementioned estimate.   2.4.4 Conclusion                                                                                                               In conclusion, the relative proportions of CO, CO2, O2, and estimated N2 gases do not significantly change as pneumatocysts grow and increase volume. Unlike what was found in previous studies, pneumatocyst concentrations of O2, N2, CO, and CO2 were 59%, 40%, 1.6%, and 0.6% respectively. Furthermore, internal pneumatocyst pressure fluctuations do not change in magnitude, as pneumatocysts grow larger. These data 	 68	suggests that pneumatocyst SA:V does not influence the exchange in physiological gases over a diurnal light period.                                                                                                  As thalli grow larger, they also become heavier, causing the net buoyant force of pneumatocysts to decrease. Net buoyancy decreases because the total buoyant force of the pneumatocyst does not change as thalli become heavier. These results suggest that the total buoyant force is eventually outpaced by the weight of growing thalli, and the risk of the pneumatocyst sinking increases. Buoyant safety factor (BSF) decreases as thalli become heaver, suggesting that larger individuals are just buoyant enough to offset the thallus weight. Given trends in BSF, I predict that the largest possible sporophyte that could remain upright (BSF=1) would have a pneumatocyst that is roughly 1.3 liters, closely matching observed maximum size of Nereocystis in the field (1.2 L).              	 69	Conclusion Maintaining buoyancy with pneumatocysts is essential for subtidal seaweeds with long flexible thalli, such as Nereocystis luetkeana, 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 serves to keep the developing sporophyte upright in the water.  In Chapter 1, I demonstrated that pneumatocyst volume does not change passively with decreasing hydrostatic pressure, and increases by a factor of 400. Furthermore, pneumatocyst internal pressure does not passively change with hydrostatic pressure. These results indicate that pneumatocysts are not passive but rather actively regulated, and biological processes such as photosynthesis, respiration, and gas composition likely regulate internal pressures. Pneumatocysts are constantly exposed to a positive pressure gradient and experience compression. The difference in external hydrostatic pressure and internal pneumatocyst pressure becomes greater with depth and therefore smaller pneumatocysts experience the greatest loads. Pneumatocysts do not adjust geometry or material properties to reduce wall stress, which suggests that pneumatocysts are designed to tolerate compressive loads. Since the contribution of pneumatocyst geometry, material properties and the difference in force applied by internal and hydrostatic pressure does not reduce material stress, pneumatocysts found at depth are at greatest risk of buckling. Small pneumatocysts with inner radius of 0.8-0.9 cm and wall thickness of 0.2 cm were 	 70	found to be at a critical size, where the risk of buckling was greatest, since they are found at depth with high material stresses when compressive forces are greatest. These findings indicate that sporophytes reach a critical survival point early in their development, where success in growing to adulthood depends on the ability of small pneumatocysts to resist buckling.  Previous studies have suggested that light attenuation limits the maximum depth of sporophytes in the field to 35 m. However, considering spherical geometry and material properties, small pneumatocysts are also predicted to buckle at 35 m depth, close to the maximum depth observed in the field. Thus, the present study demonstrates that hydrostatic pressure, not just light attenuation, might attribute to the lower limit of Nereocystis in the field.  In Chapter 2, I explored changes in pneumatocyst gas composition and buoyancy through sporophyte development. This study demonstrated that the composition of gases in the pneumatocyst do not change as they develop and grow. The rate of input and output of gases is almost proportional to changes in pneumatocyst volume, suggesting that the same relative proportion of gases is being added at all stages of development.  Pneumatocyst gas composition is very different than the composition of atmospheric air, indicating that pneumatocysts are actively regulated, and biological processes such as photosynthesis and respiration likely contribute to the total gas composition found in pneumatocysts. Particularly, a pneumatocyst has enough CO to not only kill a chicken, but also an adult man.  Contrary to previous studies, pneumatocyst concentrations of O2, N2, CO, and CO2 were 59%, 40%, 1.6%, and 0.6% respectively. Furthermore, internal pneumatocyst pressure fluctuations do not change in magnitude, as 	 71	pneumatocysts grow larger, suggesting that pneumatocyst SA:V does not influence the exchange in physiological gases over a diurnal light period. Since O2 takes up more than half of the total gas present in the pneumatocyst, the utilization of O2 during cellular respiration is more likely to influence the decrease in overall pressure of the pneumatocyst than the increase in CO2 .  As total buoyant force is steadily outpaced by the weight of growing thalli, Adult pneumatocysts are ultimately at risk of sinking. There is a reduction in buoyancy maintenance of the pneumatocyst when sporophytes become massive in the fall, and they are dislodged during storms. Given the decrease in buoyant safety factor over time, the maximum size of adult sporophytes was predicted to be limited by a maximum pneumatocysts volume of 1.3 L, where any extra added weight (i.e. blades) would cause the thallus to sink. The largest collected pneumatocyst was roughly 1.2 liters, just 100 ml less than this prediction.  Given that buoyancy is essential for Nereocystis, data presented here may help to explain how pneumatocysts maintain buoyancy throughout development, highlighting the resilience of the pneumatocyst and its contribution to the growth and development of sporophytes.        	 72	Future directions People have been studying kelp and pneumatocysts for over 100 years (MacMillan 1899; Sykes 1908; Zeller and Neikirk 1915; Frye 1915), yet there are still many questions about their biology. Specifically, research has been conducted on pneumatocysts investigated their gas composition and development (Rigg and Swain 1941; Langdon and Gailey 1920; Langdon 1917; Foreman 1976), but until this thesis, there has been little research on how internal pressure, gas composition, and biomechanical properties change as they grow and develop. Although this thesis discussed how the pneumatocyst of Nereocystis resists breaking under hydrostatic loads, there is little knowledge how other subtidal kelps cope with hydrostatic pressure (Ch.1). More specifically research has mainly focused on intertidal seaweeds with pneumatocysts tolerating fluctuations in hydrostatic pressure as the tide changes (Dromgoole 1981; Brackenbury et al 2006).  Although this study answered many questions regarding Nereocystis pneumatocysts, it also opened doors to more questions. In particular, not all material properties were tested due to time limitations and some values in estimating critical breaking depth need to be verified. Changes in material volume and Poisson’s ratio should be measured to understand how the pneumatocyst material responds to compressive forces. Pneumatocysts were also collected no deeper than 10 m, and young pneumatocysts at 35 m depth should be collected for geometry, material properties, and material stress measurements. This will clarify if the calculated buckling wall stress is similar to what deeper pneumatocysts actually experience.  Research in Chapter 2 suggested that the exchange of physiological gases in the pneumatocysts was not driven 	 73	by SA:V ratio.  Results from this study suggested that the exchange of CO2 in large pneumatocysts must be higher than predicted and might not be scaling with surface area. Therefore, further research is needed to understand how gas is exchanged in the pneumatocyst.     Macrocystis pyrifera (Macrocystis), develops pneumatocysts similar to that of Nereocystis, but instead of one pneumatocyst at the end of a single stipe, there are many pneumatocysts located along the length of each stipe, subtending each blade, and there are multiple stipes per individual. Unlike Nereocystis, pneumatocysts produced by Macrocystis stay at a fixed location throughout development along a given stipe. Therefore, these structures experience a constant hydrostatic load and this pressure differs depending on where the pneumatocyst develops along the stipe.  Future research should be conducted on how and if Macrocystis changes pneumatocyst material properties, geometry, and internal pressure to resist buckling under pressure. Like Nereocystis, Macrocystis pneumatocysts could have materials capable of tolerating high wall stresses. The spherical portion of the pneumatocyst of Nereocystis was at greatest risk of buckling. Unlike Nereocystis, Macrocystis creates pneumatocysts that are oblong, and possibly could have higher wall stresses, since hoop stress is 2 times greater in cylindrical objects compared to spherical objects. After showing that the net buoyancy of Nereocystis changes in accordance to its annual nature, I would expect that Macrocystis buoyancy does not significantly change annually. Macrocystis is not an annual, and has been recorded to have sporophytes that persist for up to 7 years, thus pneumatocyst buoyant force would need to increase as the thallus increases biomass and weight. Furthermore, instead of one stipe, Macrocystis has many, 	 74	which suggests that the loss of one stipe would have minimal consequences, to the survival of the sporophyte. Therefore, I predict that the BSF of Macrocystis would not change as the thallus increases size. It is likely that each pneumatocyst only needs to support the tissue between the pneumatocyst below it, and therefore any loss in tissue would not significantly change the sporophytes’ buoyancy.  Another curious kelp, also closely related to Nereocystis, is Pelagophycus porra (Pelagophycus). Pelagophycus has a very similar morphology to Nereocystis except it is a deep-water species found between 25 m and 45 m. The pneumatocyst of Pelagophycus are capable of reaching a total volume similarly to Nereocystis at depth. As the pneumatocyst of Nereocystis increases in volume, the sporophyte increases stipe length, reducing hydrostatic pressure. Unlike Nereocystis, Pelagophycus develops a large pneumatocyst under hydrostatic loads that are much greater. Stipe elongation does not occur in Pelagophycus and thus the pneumatocyst develops while stationary at depth.  The internal pressure, material properties, and structural design of Pelagophycus pneumatocysts are under-studied. We know that Nereocystis pneumatocysts have an internal pressure that is less than atmospheric pressure since volume increases faster than the gas being added. Pelagophycus pneumatocyst growth rates and internal pressure are unknown. However, it is possible that growth is slower since the pneumatocysts are increasing volume under high hydrostatic loads. If this were the case, pneumatocysts would have an internal pressure less than hydrostatic pressure, but not nearly as low as the pressures measured in Nereocystis. 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